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Overview ======== High-throughput studies revealed that the transcriptome does not always predict the proteome ([@B59]; [@B85]; [@B95]), highlighting the need for a better understanding of post-transcriptional regulation in order to explain this discrepancy. Post-transcriptional regulation is comprised of a complex and diverse set of processes that represent various maturation steps and regulatory modalities for mRNAs including (but not limited to): splicing, mRNA export, stability, polyadenylation, and translation ([@B45], [@B46]). This complexity gives rise to the question: How does the cell coordinate metabolism and regulation of mRNAs encoding proteins in the same biological process so that the proteins can be coordinately produced? In answer to this question, Keene and colleagues proposed the RNA regulon model ([@B48]; [@B47]; [@B45]), where mRNAs encoding functionally related proteins (i.e., involved in the same biochemical processes) contain the same RNA elements, known as USER codes (Untranslated Sequence Elements for Regulation). USER codes can be based on primary, secondary or tertiary elements in the RNA. These USER codes are recognized by RNA binding proteins (RBPs) or regulatory RNAs (such as microRNAs, siRNAs, or snRNAs) which can recruit mRNAs to various machineries for appropriate types of processing ([@B41]; [@B12]; [@B90]). Typically, a given mRNA contains multiple USER codes which would enable coordinated and combinatorial regulation. The combinatorial effect of the USER codes and the context (the sequence context which can influence folding of neighboring USER codes and availability of RBPs and regulatory RNAs) will ultimately affect which kind of machinery will be recruited to a particular mRNA. In this way, the RNA regulon serves as an elegant model to understand how groups of mRNAs can be co-regulated in combination as they flux through the various RNA metabolism steps ultimately allowing coordinated production of their physiologically active forms, proteins. RNA regulons are inherently dynamic, and enable cells to adapt to environmental stresses and cues in a rapid and effective manner. Operation and control of regulons are mediated through targeting RBPs which act as nodes or center-points for these networks. Factors that modulate the localization or activity of these RBPs or that modify the USER codes (such as RNA methylation) ultimately influence the activity of a given regulon. A key control step is the interaction between specific RBPs and their cognate USER codes in the groups of RNAs to be regulated. Here, we suggest the possibility some transcripts may require a two-tier system of USER codes which allow their correct channeling to the appropriate machinery. Here, we provide examples of single and multi-tier systems as a launch point for this notion. Havoc ensues when RNA regulons become dysregulated contributing to a variety of diseases including cancer. Dysregulation of regulons can occur because of dysregulation of RBPs or mutation in the USER codes. Consistent with this, RBPs involved in all levels of mRNA metabolism were found dysregulated or mutated in cancers ([@B44]; [@B30]; [@B15]; [@B75]; [@B84]; [@B89]). Further, many oncogenic pathways involved in malignant transformation, metastasis and drug resistance are regulated by various RNA regulons ([@B20]; [@B12]; [@B93]; [@B90]; [@B11]; [@B78]). In this review, we focus on the eukaryotic translation initiation factor eIF4E, the splicing factor SRSF3 and the Upstream of N-Ras protein (UNR), as examples of RNA regulons which contribute to malignancy. Further, these provide examples of different modalities in terms of the employment of regulatory factors and USER codes, single or multi-tier USER codes systems and the diverse levels of mRNA metabolism that can be affected. The Eukaryotic Translation Initiation Factor eIF4E ================================================== eIF4E is traditionally defined as a factor key to global translation initiation. eIF4E binds the 5′-methyl-7-guanosine (m^7^G) cap on RNAs to recruit these to the translation machinery, thereby increasing the number of polysomes per transcript, i.e., their translation efficiency. Over time it has become clear that eIF4E regulates the translation of only a subset of capped transcripts ([@B19]; [@B27]; [@B83]). For instance, eIF4E overexpression increases the translation of ornithine decarboxylase (*Odc1*) and *Myc* mRNAs but not that of *Gapdh* or *Cyclin D1* ([@B72]); conversely, eIF4E reduction only suppresses *Odc1*, *Myc, Bcl-2*, *Edn1* (Endothelin-1), *Fth1* (Ferritin heavy chain) translation but not β*-Actin* or *Gapdh* ([@B34]; [@B27]; [@B83]). In addition, 25 years ago eIF4E was found localized in the nucleus as well as the cytoplasm where it played a role in the export of selected transcripts ([@B56]; [@B72]). In this way, eIF4E can increase the levels of transcripts available to the translation machinery and thus the protein levels in the absence of increased translation efficiency or increased RNA levels. More recently, ∼10 years ago, eIF4E was found in cytoplasmic P-bodies which appear to be involved in protecting RNAs from turnover ([@B4]; [@B33]). Not all mRNAs are targeted by these pathways and further, being an eIF4E target for one level of regulation does not imbue sensitivity to other processes *a priori*. While eIF4E associates with mRNAs through binding the common m^7^G cap structure, other USER codes act in recruiting necessary co-factors to dispatch mRNAs to the specific export, translation and/or stability machinery. Thus, eIF4E serves as an excellent example of a two-tier (or perhaps multi-tier) USER code system, as described below. There are multiple USER codes defined for export and translation to date. The ∼50 nucleotide eIF4E sensitivity element (4ESE) in the 3′UTR required for export of its target transcripts is one of the best understood eIF4E USER codes. The 4ESE is defined by its secondary structure comprised of paired stem loops as determined by nuclease mapping experiments, and is necessary for export. For instance, *lacZ-4ESE* chimeric mRNAs are sensitive to eIF4E dependent mRNA export while *lacZ* is not ([@B21], [@B22]). At the translation level, USER codes are less well defined but can be found in both the 5′ or 3′UTRs of mRNAs. The 5′UTRs of eIF4E-sensitive mRNAs at the translational level tend to be long and GC-rich, i.e., with complex tertiary structure and this comprises the translation USER codes ([@B38]; [@B19]; [@B55]). Other sequences have been identified, such as the CERT (Cytosine-Enriched Regulator of Translation) ([@B83]), but further studies are needed to determine if this is sufficient to drive translation. Importantly, for both mRNA export and translation, eIF4E targets must also retain the m^7^G cap. Thus, there is a two-tier USER code system, with the m^7^G cap for eIF4E:mRNA binding and a 4ESE or translation USER code which direct mRNAs to their particular post-transcriptional machineries (Figure [1A](#F1){ref-type="fig"}). {#F1} Biochemical studies of the eIF4E-mRNA export complex elucidated the mechanisms by which the 4ESE directs mRNAs to this level of control ([@B87]). Here, the Leucine-rich Pentatricopeptide Repeat Protein (LRPPRC) simultaneously binds both the 4ESE USER code in the 3′UTR of mRNA and eIF4E bound to the mRNA through the cap. Then, the nuclear export receptor CRM1 binds this complex through direct interactions with LRPPRC. In this way, the USER code recruits the export machinery to the given mRNA directing it through this non-canonical export pathway. In the cytoplasm, eIF4E interacts with an alternative set of proteins to act in either translation or recruitment of mRNAs to P-bodies, whether there is a USER code for P-bodies is not yet known ([@B4]; [@B77]). Through these activities eIF4E can elicit biological responses (Figure [1B](#F1){ref-type="fig"}). For instance, RIP-Seq analysis in lymphoma cells indicated that nuclear eIF4E binds over 3000 mRNAs that encode proteins acting in lymphoma-sustaining pathways such as B-cell receptor signaling (Bcl2, Bcl6) and DNA methylation/epigenetic regulation (DNMT1, DNMT3A, HDAC1) ([@B26]). In AML and osteosarcoma cells, eIF4E coordinately increases the export of transcripts encoding all the proteins involved in hyaluronan synthesis ([@B94]). Hyaluronan is a large polysaccharide with traditional roles in building the extracellular matrix, and more recently was found to encapsulate some tumor cells ([@B76]; [@B10]; [@B49]). Indeed, Hyaluronan (HA) production was found to be required for the metastatic and invasive properties associated with eIF4E, and thus serves as the first case where this HA coat was shown to contribute to the oncogenic phenotype ([@B94]). Indeed, inhibition of this regulon with RNAi to eIF4E or treatment with the cap competitor ribavirin impaired the export of the RNAs encoding the HA machinery, reduced HA production and decreased the invasive and metastatic activities of these cells. Indeed, eIF4E overexpression in the presence of RNAi knockdown to *Has3* (hyaluronan synthase 3) mRNA, similarly reduced invasion and metastatic potential indicating that the HA pathway is critical for these eIF4E-driven activities ([@B94]). eIF4E can also reprogramme the cellular machinery to enhance its mRNA export activity and its nuclear import both of which are associated with an increase its oncogenic potential. For instance, eIF4E alters the composition of the nuclear pore complex, allowing it to facilitate export of its target mRNAs ([@B24]). Specifically, eIF4E overexpression leads to downregulation and relocalization of Nup358/RanBP2, redistribution of Nup214 from the nuclear rim and increased levels of RanBP1 through elevated mRNA export of *RanBP1* transcripts. Reduction in RanBP2 with concomitant elevation of RanBP1 likely enhances efficiency of mRNA cargo release on the cytoplasmic side thereby enhancing eIF4E mRNA export efficiency. The effects of eIF4E on RanBP2 are required for its oncogenic activities *in vitro*. eIF4E also enhances the mRNA export of *Gle-1* and *DDX19* mRNAs which encode proteins acting in the release of bulk mRNA cargoes ([@B50]; [@B23]). Interestingly, even these workhorses of the bulk mRNA export pathway have additional functions in stress granule formation and translation ([@B1]; [@B7]; [@B63]). Further, beyond common mRNA targets, these export regulators have their own distinct target transcripts, which results in differing cellular phenotypes observed upon their depletion ([@B69]). In all, this provides an example of how eIF4E can re-wire the nuclear pore to enhance export of its target transcripts while simultaneously modulating the machinery for bulk mRNA export. One obvious way to alter the activity of a regulon is to alter the localization of its key components. eIF4E modulates its own subcellular localization through its interaction with and effects on Importin 8. Importin 8 directly binds and imports eIF4E into the nucleus, enabling eIF4E to be quickly recycled after each round of mRNA export ([@B86]). Importin 8 only associates with eIF4E when eIF4E is not bound to capped mRNAs, providing an interesting surveillance mechanism to inhibit import of actively translating eIF4E or of eIF4E which has not yet released its mRNA cargo from an export cycle. Depletion of Importin 8 impairs nuclear entry of eIF4E, eIF4E-dependent mRNA export and oncogenic activities. eIF4E nuclear entry can also be impaired by addition of m^7^G cap analogs or ribavirin triphosphate (RTP). In this case, the cap or ribavirin analogs prevent association of eIF4E with Importin 8, correlating with reduced nuclear entry of eIF4E, reduced mRNA export and reduced oncogenic activity. Interestingly, Importin 8 also provides evidence of a feedback mechanism whereby eIF4E promotes the export of *Importin 8* mRNAs to increase production of this protein and thus its own nuclear entry ([@B86]). Thus, like its effects on the nuclear pore, eIF4E can modulate a variety of its control points and the machinery it engages. eIF4E expression is also controlled by HuR/ELAV1, a factor involved in many levels of RNA metabolism, the most well described being mRNA stability. HuR increases the stability of eIF4E transcripts thereby interconnecting the HuR/ELAV1 and eIF4E regulons ([@B82]). Indeed, HuR is amongst the first RNA regulons to be described and the eIF4E-HuR overlap provides a case whereby regulons intersect ([@B80]; [@B48]). Indeed, many mRNA stability targets of HuR such as *cyclin D1*, are also mRNA export targets of eIF4E ([@B72]; [@B80]). It is also interesting to note, that eIF4E can directly contact RNAs beyond the m^7^G cap ([@B13]). As described above, the sequence context can alter the activity of a USER code. For instance, a 4ESE-like element found in the coding region of histone mRNAs recruited eIF4E-in cap-independent manner ([@B60]). While the affinity of eIF4E for the 4ESE element is lower than for m^7^G cap, in non-replicative histone H4 it is important for translation. In the nucleus, it seems that the ability of eIF4E to bind the 4ESE in the 3′UTR might be used to inhibit export of uncapped mRNAs, and in this way acts as a surveillance mechanism ([@B87]). Another type of USER code are the Cap-Independent Translational Elements (CITEs) found in the 3′UTR of plant viruses such as *Panicum mosaic virus* and *Pea enation mosaic virus 2* translation enhancers (PTE), and the I-shaped structures (ISS) from *Maize necrotic spot and Melon necrotic spot viruses* ([@B65]). The PTE directly binds eIF4E and initiates translation without using the m^7^G cap ([@B65]). In all, there are multiple USER codes to engage eIF4E and further, the same USER code in different contexts can have alternative functions. Coordinated regulation implies that nodes in RNA regulons could also be valuable therapeutic targets as well as important control points for regulation of normal cellular physiology. eIF4E expression is elevated in wide variety of cancers ([@B27]; [@B14]). The first clinical studies targeting eIF4E in humans used ribavirin, a cap competitor of eIF4E, and thus an inhibitor of all of eIF4E's cap-dependent activities ([@B51], [@B52]; [@B88]). These studies led to clinical responses including remissions in refractory and relapsed AML patients ([@B8], [@B9]), patients with prostate cancer ([@B53]), lymphoma ([@B73]), and head and neck cancers ([@B29]). Consistent with these clinical observations, eIF4E activity was impaired and levels of eIF4E target proteins were reduced in responding AML patients ([@B8], [@B9]). Indeed, AML patients have highly elevated nuclear levels of eIF4E, consistent with elevated Importin 8 levels ([@B86]). In AML patients, ribavirin therapy was associated with reduced nuclear levels of eIF4E and impaired RNA export during response; and at relapse, eIF4E nuclear levels increased as did its mRNA export activity ([@B8]). In this way, reprogramming the eIF4E regulon by preventing nuclear entry led to therapeutic benefit at least in this context. The Serine and Arginine Rich Splicing Factor 3 SRSF3 ==================================================== SRSF3 (also known as SRp20) provides another example of a protein which turns out to function beyond its traditional roles. SRSR3 associates with the spliceosome and was thought to act in the splicing of all intron-containing RNAs ([@B20]). However, recent identification of SRSF3 targets using iCLIP-seq (individual-nucleotide resolution crosslinking and immunoprecipitation sequencing) suggests that specific transcripts are targeted by this factor rather than all intron-containing mRNAs ([@B71]). SRSF3 controls establishment and maintenance of pluripotency through its functions in alternative slicing and 3′ end mRNA processing, mRNA export and mRNA stability ([@B68]; [@B18]; [@B71]). For instance, SRSF3 increases the export of *Nanog* mRNA, which encodes one of the master regulators of pluripotency maintenance. According to iCLIP studies, SRSF3 binds a consensus pentanucleotide element found in RNA segments including exons and introns of both coding and non-coding transcripts. Many pre-mRNAs encoding pluripotency factors contain SRSF3 binding-sites including *Nanog*, *Sox2*, *Kif4*, and *Myc*, and their levels were downregulated in SRSF3-depleted cells ([@B71]). SRSF3 also binds mRNAs encoding various RBPs with previously established roles in pluripotency and reprogramming including the MBNL2 splicing factor ([@B35]) and the polyadenylation factor FIP1 ([@B54]). Indeed, RNAi knockdown of SRSF3 led to failure to induce pluripotency in OKSM MEFs (OCT4, KLF4, SOX2, and Myc overexpressing Mouse Embryonic Fibroblasts) as well as loss of pluripotency and differentiation in iPSC (induced pluripotent stem cells) indicating that this regulon is important for cell reprogramming and maintenance of pluripotency. Aside from its role in splicing, ∼400 transcripts were predicted to be SRSF3 nuclear export targets including *Nanog* mRNA, a key factor in stem cell pluripotency ([@B67]). This export activity of SRSF3 occurred even in intronless *Nanog* constructs indicating that this was a splicing-independent activity of SRSF3. Further, deletion of the SRSF3 binding sites impaired the ability of the bulk mRNA export factor NXF1 to bind *Nanog* mRNA suggesting that SRSF3 association is required to form this export complex ([@B71]). Consistent with this notion, NXF1/TAP directly binds SRSF3 proteins ([@B40]; [@B67]). SRSF3 affects alternative splicing of many RNAs, including its own, and its depletion increases exon skipping and intron retention ([@B5]; [@B71]). Interestingly, a significant proportion of SRSF3 consensus binding-sites were found in introns of target mRNAs, including detained introns (DI). Indeed, SRSF3 is involved in retention of *Nxf1* intron 10 affecting isoform expression and potentially impacting on the export of many mRNAs ([@B57]; [@B67]; [@B71]). DIs with SRSF3 consensus sequences were found in mRNAs encoding other RBPs, including *Fip1/1* and *Mbnl2*. Further, nearly half of NMD-regulated transcripts contained SRSF3-binding sites suggesting that this factor could also play a role in mRNA stability ([@B71]). However, further studies are needed as its effects may be limited to distinct NMD-sensitive transcript variants. Only a single USER code, or one tier system, has been reported for SRSF3 despite the fact it recruits mRNAs to different machineries. The features that allow recruitment to the appropriate machinery are not yet known, so it is possible that a second USER code(s) is required. More studies into the minimal domains required to imbue SRSF3 sensitivity are important to understand how this USER code enables recruitment of different complexes to act in splicing, export and/or stability (Figure [2](#F2){ref-type="fig"}). {#F2} Through its role as a center-point in a RNA regulon, SRSF3 has been implicated in cellular senescence, cell adhesion and migration, proliferation, resistance to apoptosis, as well as establishment and maintenance of pluripotency (Figure [2](#F2){ref-type="fig"}). For instance, *Nanog*, *Sox2*, *Kif4*, and *Myc* are SRSF3 targets ([@B71]). SRSF3 regulates the global chromatin state of pluripotent cells by controlling mRNAs coding chromatin modifiers such as components of Polycomb repressive complex 2 (*PRC2*), *Ezh2*, and *Epop* ([@B96]) and DNA methyl-transferase 3A (Dnmt3a) also involved in gene silencing ([@B71]). Additionally, by regulating other RBPs (FIP1, MBNL2, NXF1) and its own mRNA, SRSF3 is a part of interconnected network which coordinately regulates pluripotency gene expression program. SRSF3 also regulates *FoxM1* transcripts (Forkhead box transcription factor M1, transcriptional regulator involved in regulation of cell cycle and proliferation), and the transcriptional targets of FOXM1 including Cdc25B (member of CDC25 family of phosphatases, required for mitosis) and PLK1 (Polo like kinase 1, highly expressed during mitosis, and frequently elevated in cancers) to control cell cycle progression and proliferation. Depletion of SRSF3 in cancer cells induced G2/M arrest, growth inhibition and apoptosis, while SRSF3 overexpression in rodent fibroblasts induced cell transformation and tumor formation and growth in nude mice ([@B42]). Additionally, through regulation of *TP53* alternative splicing SRSF3 is implicated in cellular senescence. Indeed, downregulation of SRSF3 induced cellular senescence in human fibroblasts ([@B79]). All these activities can contribute to human diseases including cancer. Given its affects on cell physiology it is not surprising that SRSF3 protein expression is elevated in a variety of cancers ([@B42]), while its mRNA levels are downregulated in *de novo* diagnosed AML patients ([@B58]) suggesting that SRSF3 levels could be crucial for maintaining normal cellular homeostasis in that context. Upstream of N-Ras UNR ===================== Upstream of N-Ras, also known as CSDE1 in mammals, is an RBP comprised of five cold-shock domains which bind single-stranded RNAs ([@B62]). Global studies using iCLIP-Seq, RNA-Seq and ribosome profiling revealed that many target mRNAs and a wide variety of RNA processes are potentially impacted by UNR ([@B91]). A majority of the 1532 RNAs found by iCLIP were mature mRNAs, with the UNR consensus binding-site most often located in the CDS or 3′UTR. Bioinformatic analysis suggested that UNR has a preference for unstructured and/or single-stranded RNAs. UNR binds its own mRNA at the 5′UTR, consistent with previously reported translational inhibition from its own IRES ([@B74]). A comparison of the iCLIP and RNA-Seq data after UNR depletion indicated that there are ∼100 direct targets regulated by UNR at the stability level with many of these mRNAs being indirect targets of UNR. While UNR does not affect global translation, ribosome profiling experiments revealed that UNR regulates specific transcripts preferentially (451 genes), with 127 of these being direct targets of UNR ([@B91]). A subgroup of mRNAs regulated by UNR at the level of translational initiation showed preferential UNR binding in the 5′UTR, possibly representing novel IRESs given previously reported roles for UNR in IRES translation ([@B32]; [@B66]; [@B74]). However, these studies suggested possible roles for UNR in elongation and termination of translation for the majority of these transcripts, with other stages of RNA metabolism possibly affected ([@B91]). Like SRSF3, UNR seems to use a single-tier strategy to associate with RNAs and modulate disparate steps in RNA processing. Interestingly, its can have opposing effects on the same processes, e.g., UNR inhibits translation of its own IRES ([@B74]), but stimulates IRES translation for *cMyc* and *Apaf-1* mRNAs ([@B32]; [@B66]). This suggests some context specific features are also at play, whether these are RNA elements or protein co-factors is not yet known. Further, even with the same partner proteins like PABP, UNR can have disparate effects, such as *c-fos* mRNA decay ([@B16]), and translational repression of *pabp* mRNA ([@B70]). Studies in *Drosophila* showed that UNR binds its targets either alone, e.g., *roX2* lnRNA ([@B64]), or with co-factors, as in case of *msl-2* mRNA where USER code recognition is achieved by cooperative complex formation with SXL proteins ([@B37]; Figure [3A](#F3){ref-type="fig"}). Bioinformatic analyses suggests that there may be different binding modes for UNR depending on the location of the consensus motif within the transcript ([@B91]). This suggests that UNR either binds several types of motifs or needs additional RBPs to aide in binding to mRNAs which do not contain UNR consensus binding sites (Figure [3B](#F3){ref-type="fig"}). Thus, UNR may well have a multi-tier system, at least for some mRNAs to dispatch them to their appropriate pathway. {#F3} As expected of an oncogenic RNA regulon protein, UNR controls a series of RNAs involved in metastasis and invasion, particularly in melanoma ([@B91]). UNR protein levels are elevated in a high percentage of primary and metastatic melanoma specimens and cell lines, and its depletion reduced the oncogenic potential of melanoma cells *in vitro* and in mice ([@B91]). Overall, UNR is a major node in a melanoma regulon, where it is thought to regulate over 60% of the transcripts considered to be involved in development of this malignancy. Additionally, UNR is highly expressed in human embryonic stem cells where it coordinatively regulates multiple nodes of networks essential for maintaining pluripotency ([@B43]). UNR stimulates the translation of *RAC1* (Ras-related C3 botulinum toxin substrate 1, guanosine triphospatase belonging to the Ras superfamily), *VIM* (Vimentin, component of intermediate filaments important for mechanical integrity of cells during invasion, and also marker of epithelial-to-mesenchymal transition) and *TRIO* (Rho guanine nucleotide exchange factor which activates RAC1, implicated in uveal melanoma), and increases the stability of *SDC4* (*trans*-membrane receptor which activates RAC1 to transduce signals from extracellular matrix to the cytoskeleton and modulate adhesion and migration), *TNC* (extracellular matrix protein which interacts with SDC4 and is involved in regulation of cell adhesion) and *CTTN* (Cortactin, actin binding protein, implicated in tumor cell invasion and metastasis). Overexpression of VIM and RAC1 can overcome UNR depletion and fully restore colony growth of melanoma cells ([@B91]). UNR regulates the stability of the tumor suppressor *PTEN* and the inflammatory factor *CCL2* transcripts which are downstream effectors of c-Jun, a proto-oncogene hyperactivated in malignant melanoma. Thus, through its combinatorial affects on the melanoma pathway, UNR contributes to this oncogenic phenotype (Figure [3B](#F3){ref-type="fig"}). Conclusion ========== Here, we focussed on eIF4E, SRSF3, and UNR as examples of RNA regulons involved in cancer progression. There are clearly many other physiologically important regulons, such as those centered upon HuR and ARE elements ([@B80]; [@B48]; [@B61]; [@B81]; [@B11]), IFN response and GAIT elements ([@B3]; [@B6]), and others which we could not cover due to space restrictions. The described regulons not only highlight their biological relevance, but also the utility of exploiting these therapeutically. RBPs acting in these regulons are mutated and/or aberrantly expressed in a variety of cancers ([@B92]; [@B25]; [@B36]; [@B15]; [@B84]). Disrupted RBP activity has been reported for nearly every step of mRNA metabolism including splicing (such as U2AF1, SRS2, ZRSR2, SR3B1, SRSF3), export (including THO, ALYREF, Luzp4, GANP, CRM1, eIF4E, SRSF3, UNR), nuclear pore (e.g., Nup88, Nup96/98, Nup214, TPR), and translation (eIF4E, UNR, eIF4A, eIF3), etc., Interestingly, mutations in spliceosome factors are frequent in hematological malignancies but rare in solid tumors ([@B30]; [@B15]), highlighting their contextual importance in driving specific pathways in malignant transformation. Clearly, versatile modes of molecular recognition by RBPs are highly dependent on the context, where RNA structure complexity, available partner RBPs and co-factors as well as potential inhibitors or modulators of binding (regulatory RNAs, signalling molecules, etc.), all contribute to the biological outcome. Indeed, depending on a cell type, CSDE1/UNR may promote or inhibit differentiation and apoptosis ([@B28]; [@B31]; [@B39]). Thus, deeper insight into the workings of regulon networks in healthy and malignant cells could provide information on critical nodes that can be exploited in cancer. From the RNA biology perspective, utilization of the same USER codes and their readers-RBPs in multiple complexes, suggest that RBPs become escorts for mRNAs with certain USER code(s). In this way, RBPs can act in multiple steps in RNA metabolism by virtue of their function as defined by the recognition of specific RNA binding motifs. In this way, RBPs may be much broader actors in RNA metabolism thereby facilitating the wiring of RNA regulons in the cell. Further, given the RNA world theory, while it has been posited that RNA regulons can recapitulate transcriptional programs, perhaps it is possible that RNA regulons came first. Interestingly, analysis of ancestral stem cells revealed that RBPs are more evolutionarily conserved than transcription factors suggesting that RNA regulons have played a key role in animal stem cell biology for millions of years, even playing roles in sponges and premetazoans ([@B2]). Indeed, RNA regulons are employed by single celled organisms such as yeast and across kingdoms being present in plants as well as animals ([@B48]; [@B17]). Further dissection of the regulons themselves and their intricate feedback systems will undoubtedly be central in developing our understanding of oncogenesis. Author Contributions ==================== All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Conflict of Interest Statement ============================== The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. **Funding.** This work was supported by grants from LLS Canada, LLS USA, CIHR, and NIH to KLBB. She holds a Canada Research Chair. [^1]: Edited by: Chiara Gamberi, Concordia University, Canada [^2]: Reviewed by: Fátima Gebauer, Centre de Regulació Genòmica (CRG), Spain; Scott A. Tenenbaum, University at Albany, United States; Jack D. Keene, Duke University, United States [^3]: This article was submitted to RNA, a section of the journal Frontiers in Genetics

INTRODUCTION {#sec1-1} ============ The incidence of diaphragmatic hernia is 1:2000--1:4000 live births. Its presentation is rare in the adults. The presentation in infancy is due to signs and symptoms of respiratory distress. However, in adults, the presentation is mostly asymptomatic or might present with respiratory and abdominal symptoms. Here, the authors would like to report an unusual case of Bochdalek\'s hernia in a 75-year-old male who presented with pain abdomen and slight dyspnoea. This patient was treated with minimally invasive technique i.e., laparoscopic repair of the posterolateral defect in the diaphragm. CASE REPORT {#sec1-2} =========== A 75-year-old man came to our outpatient department with a history of pain abdomen, vomiting and slight dyspnoea with the onset of all these symptoms since 4 days from his presentation. His medical and family history was unremarkable. The physical examination was noncontributory. Ultrasound revealed prostatomegaly. His chest X-ray revealed well-defined radiopacity in the left lower zone completely obscuring left heart border and left hemidiaphragm s/o left moderate pleural effusion. Multiple curvilinear gaseous shadows were noted in the left lower lung field \[[Figure 1](#F1){ref-type="fig"}\].To confirm the diagnosis, a contrast-enhanced computed tomography (CECT) abdomen pelvis with high-resolution computed tomography (HRCT) of lower lung fields was done which revealed, Eventration/left diaphragmatic hernia with intrathoracic extension of bowel loops causing mediastinal shift toward the right side. Dilated fluid filled proximal small bowel loops with collapsed distal small and large bowel loops, suggesting small bowel obstruction \[[Figure 1](#F1){ref-type="fig"}\]. It was diagnosed to be a Bochdalek\'s hernia, and it was planned to be operated laparoscopically. The patient underwent laparoscopic repair and had positive outcomes with better and early recovery.We decided to operate the patient for laparoscopic repair of diaphragmatic hernia. The patient was operated under general anaesthesia with single lung ventilation. The contents of hernia were stomach, colon and small bowel. They were reduced into the abdomen, and lower peritoneal adhesiolysis was done. The defect was closed primarily, and then a composite mesh was placed to strengthen the defect \[[Figure 2](#F2){ref-type="fig"}\]. The patient had an intercostal drainage tube placed on the operated site which was removed on post-operative day 3. He had an uneventful recovery with spirometry exercises and chest physiotherapy. His 3-month follow-up was asymptomatic with no complications. {#F1} {#F2} DISCUSSION {#sec1-3} ========== Bochdalek\'s hernia was first described in 1848 by the Czechoslovakian anatomist, Vincent Alexander Bochdalek.\[[@ref1]\] A Bochdalek hernia is a congenital defect of the diaphragm located in the posterior insertion. This is caused by a lack of closure of the pleuroperitoneal cavity by incomplete diaphragmatic development before the intestine returns to the abdomen from the yolk sac between 8-10 weeks of gestation. In adults, this defect is uncommon, the lung in most cases develop normally, and therefore, symptoms are rare.\[[@ref2]\] In this age group, there are two typical clinical presentations: (a) an incidental finding in chest X-ray performed for symptoms not related to the hernia\[[@ref3][@ref4]\] or (b) when symptoms develop as a result of incarceration, strangulation and visceral perforation inside the chest cavity. Symptoms vary according to the affected organ: digestive symptoms include intermittent abdominal pain, vomiting and dysphagia while respiratory symptoms include chest pain and dyspnoea.\[[@ref3][@ref5]\] The most commonly displaced organ is the stomach followed by the colon, spleen, small intestine and ureter.\[[@ref2][@ref6][@ref7]\] Chest X-ray, CECT abdomen pelvis and HRCT of chest will be helpful in diagnosing the condition and help in planning the mode of treatment. However, an emergency situation might not provide the time for a detailed imaging. The surgical approach for this pathology depends on the presence of visceral complications. In an elective setting, most surgeons recommend the laparoscopic approach; however, when there are septic complications, the abdominal approach is preferred. The current trend is to use minimal invasive surgical techniques such as laparoscopy and thoracoscopy, which has been satisfactorily performed in adults.\[[@ref8]\] CONCLUSION {#sec1-4} ========== The incidence of Bochdalek\'s hernia is rare in adults and might present both symptomatically as well as asymptomatically. These patients mostly present in infancy. In adults, they present with symptoms due to complications of hernia. The knowledge of its incidence in adults is vital for its diagnosis and treatment, as it is treated surgically to avoid complications or to treat them if the patient presents with them. Declaration of patient consent {#sec2-1} ------------------------------ The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patients understand that name and initials will not be published and due efforts will be made to conceal identity, but anonymity cannot be guaranteed. Financial support and sponsorship {#sec2-2} --------------------------------- Nil. Conflicts of interest {#sec2-3} --------------------- There are no conflicts of interest. I would like to thank my parents, department, my teachers for their constant support in drafting this unusual case report.

SummaryMore than half of patients who are treated for multiple brain metastases with whole brain radiation therapy (WBRT) will develop intracranial progression. This multi-institutional pooled analysis included 205 patients from 9 centers who received 2 courses of whole brain radiation therapy, both for multiple metastases. In this cohort, the application of the recursive partitioning analysis score was not predictive of median survival, leading to the construction of a new index, the reirradiation (ReRT) score. Introduction {#sec1} ============ Brain metastases (BM) occur in approximately one-quarter of patients with cancer.[@bib1] They are more common in patients with lung and breast cancers and melanoma.[@bib2], [@bib3], [@bib4] BM are associated with significant symptoms, including headache, weakness, cognitive disturbance, and seizures. If untreated, they result in a median survival (MS) of 1 to 2 months.[@bib5], [@bib6] The choice of treatment approach depends on patient factors (eg, age, performance status), tumor factors (eg, size, number of metastases, primary histology, and status of extracranial disease), and the availability of different therapeutic modalities.[@bib7], [@bib8], [@bib9] Treatment options for BM have expanded in recent years from surgery and conventional external beam whole brain radiation therapy (WBRT) to include high-dose conformal treatment to 1 or more lesions, or a resection cavity, via stereotactic radiosurgery (SRS) or stereotactic fractionated radiation therapy.[@bib9], [@bib10], [@bib11] Although the addition of an SRS boost after WBRT for patients with a single metastasis improved survival, local control, performance status, and steroid dependence in a pivotal phase 3 randomized trial for patients with 2-3 metastases, there was no survival advantage or impact on the rate of neurological death.[@bib12] SRS alone for patients with 1 to 3 BM is an appealing choice, with recent randomized evidence that demonstrates no difference in MS between SRS or SRS and WBRT, in light of the detrimental neurocognitive effects at 3 months for patients who receive both modalities.[@bib13] Although SRS is being actively explored, there are technical challenges for implementation in patients with multiple metastases, and it is considered investigational for those with \>4 lesions.[@bib14] Approximately 50% of patients with BM present with multiple metastases.[@bib1], [@bib15], [@bib16] For this group, WBRT remains the standard of care, with a reported MS after treatment ranging from 2.3 to 7.1 months.[@bib10], [@bib16] Symptomatic improvement after WBRT occurs in 60% to 90% of cases[@bib7], [@bib15] with 1-year local control rates of approximately 70%.[@bib8], [@bib12], [@bib17] However, intracranial progression after WBRT is common and has been reported as the cause of death in one-third to one-half of patients.[@bib2], [@bib3], [@bib4] Radiologically uncontrolled BM are associated with symptom recurrence or progression, along with cognitive decline, with an average decrease of 6.3 points on the mini-mental status examination.[@bib5], [@bib6] Options for salvage at the time of progression after WBRT are limited, especially for those who progress in the form of multiple metastases. Improvements in neurologic signs or symptoms have been reported in up to 80% of patients after repeat WBRT.[@bib7], [@bib8], [@bib9] Predicting which patients may benefit from reirradiation (ReRT) versus best supportive care (BSC) would guide treatment recommendations and discussions on the goals of care. Both the Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis (RPA) and the graded prognostic assessment (GPA) are validated tools that can be used to predict survival at the time of initial BM presentation. The RPA is based on age, status of extracranial disease, and performance status,[@bib10] whereas GPA includes primary histology.[@bib18], [@bib19] However, the use of these tools at the time of ReRT for in-brain recurrence or progression have been reported sparingly.[@bib20], [@bib21] In this study, we aimed to evaluate the factors that are associated with survival after a second course of WBRT for multiple BM, determine the utility of RPA in this setting, and design a specific index to predict survival after ReRT. Methods and materials {#sec2} ===================== Study design {#sec2.1} ------------ A literature search (MEDLINE, to October 2013) was undertaken to identify all reported studies of repeat WBRT for patients with BM ([Appendix](#appsec1){ref-type="sec"}). We identified 11 peer-reviewed publications that met the search criteria. Two groups agreed to provide individual patient data. Of Canadian centers with dedicated palliative radiation oncology clinics and routine prospective data collection strategies, data from 6 additional centers that treated patients between 2000 and 2010 were added to our own. The eligibility criteria included patients \>18 years old with any solid primary tumor who had received 2 courses of WBRT, both for the indication of multiple BM, defined as \>2 lesions. Those undergoing prophylactic cranial radiation, SRS (boost or salvage), or partial cranial radiation therapy were excluded. Biopsy or resection (subtotal or gross total) of a dominant lesion was not an exclusion criterion. Patient characteristics (age, sex, Karnofsky Performance Status \[KPS\]), radiation therapy dose and fractionation, and disease characteristics (primary histology, status of extracranial metastases, control of primary) were abstracted where available or determined retrospectively when possible. KPS was assigned retrospectively for 77.3% of patients at the time of first WBRT and prior to ReRT for 80.4%. KPS was converted from the Eastern Cooperative Oncology Group performance status for 33.3% of patients at the time of first WBRT and for 29.4% prior to ReRT. For the remainder, KPS was assigned retrospectively by 1 of 2 reviewers on the basis of documented oncologic history and physical examination. RTOG neurologic function score was determined retrospectively. An RTOG neurologic function score of 1 indicates absent/minimal neurologic findings, 2 indicates neurologic impairment that does not require nursing care, 3 reflects impairment that requires nursing care, and 4 indicates inability to communicate/comatose state.[@bib15] The interval between courses was calculated from the date of the first fraction of the first course to the first fraction of the second course. RPA class as described by Gaspar et al[@bib10] was assigned where possible for both the first and second course of WBRT. GPA as described by Sperduto et al[@bib18], [@bib19] could not be calculated for this cohort due missing data (eg, for primary breast and lung histologies) and due to the inclusion of primary histologies for which GPA does not exist presently (eg, carcinoma of unknown primary, ovary). Institutional review board approval for data collection and sharing was obtained in accordance with the requirements of each center. Statistical analysis {#sec2.2} -------------------- Summary statistics were calculated, including medians and ranges for continuous variables and frequencies and proportions for categorical variables. MS was analyzed from the first day of each course of WBRT to the date of death using the Kaplan Meier method with 95% confidence intervals. Log rank tests compared survival curves by RPA class and primary histology. Cox proportional hazard analysis described factors associated with survival. Factors that were significant at the *P* \< .10 level on univariate analysis (UVA) were incorporated into multivariate analysis, with hazard ratios and 95% confidence intervals reported. The final multivariate model included factors that were significant at the *P* \< .05 level, which were then used to construct a prognostic index (the ReRT score). A score of 1 was assigned to each variable for ease of clinical use. MS was determined for each ReRT score group (scores of 0-2, 3, or 4-5) using the Kaplan-Meier method and compared using the log rank test. UVA was also performed using a logistic binary regression analysis to explore factors associated with very short survival (≤30 days). A two-sided *P*-value of \<.05 was considered significant. All statistical analyses were conducted using IBM SPSS Statistics Version 15 (IBM Corp., Armonk, NY). Results {#sec3} ======= Demographics and radiation therapy {#sec3.1} ---------------------------------- Of 205 patients from 9 centers, the majority were female (68.3%) with non-small cell lung cancer (NSCLC) as the main histology (40.5%; [Table 1](#tbl1){ref-type="table"}). The median interval between courses was 9.1 months (range, 0.5-68.3 months). The median KPSs were 70 and 60, respectively. At the time of first WBRT, the RPA class was 1 for 14 patients, 2 for 105 patients, 3 for 73 patients, and not assignable for 13 patients. The most frequently used dose and fractionation schedule was 20 Gy in 5 fractions for both courses, with a total dose that ranged from 12 to 48 Gy for the first WBRT and 4 to 30.6 Gy for the second. Twenty-nine patients (14.1%) underwent surgical intervention (biopsy, subtotal or gross total resection) in the interval prior to ReRT.Table 1Demographics of patients (n = 205) at the time of reirradiationDemographicsN (%)Sex Female140 (68.3) Male65 (31.7)Age, median (range), y55 (25-83)Primary histology NSCLC83 (40.5) Breast64 (31.2) SCLC33 (16.1) Other[a](#tbl1fna){ref-type="table-fn"}25 (12.2)RPA Class 110 (4.9) Class 275 (36.6) Class 3120 (58.5)Extracranial metastases Yes25 (12.2) No137 (66.8) Unknown43 (21.0)KPS ≥8040 (19.5) \<80164 (80.0) Unknown1 (0.5)Interval between RT courses ≥9 mo107 (52.2) \<9 mo98 (47.8)Primary Controlled65 (31.7) Uncontrolled131 (63.9) Unknown9 (4.4)RTOG neurological function status 1132 (64.4) 253 (25.8) Unknown20 (9.8)[^1][^2] The MS was 14.5 months (range, 3.0-85.0 months) measured from the first WBRT and 3.6 months (range, 0.2-45.0 months) starting from ReRT. There were significant differences depending on primary histology ([Fig 1](#fig1){ref-type="fig"}). Patients with small cell lung cancer (SCLC) had a risk of death after repeat WBRT that was 2.5 times higher than those with breast cancer (MS: 2.6 vs 5.0 months; *P* \< .001).Figure 1Median survival by primary histology (\*Ref = breast primary). MS, median survival; NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer. Application of the recursive partitioning analysis score at the time of reirradiation {#sec3.2} ------------------------------------------------------------------------------------- At the time of repeat WBRT, more than half of patients were RPA class 3 ([Table 1](#tbl1){ref-type="table"}). The MS by RPA class that was calculated from the first day of ReRT demonstrated a significant difference when comparing class 1 and class 3 (7.5 vs 2.9 months; *P* = .003; [Fig 2](#fig2){ref-type="fig"}). On multivariate analysis, the presence of extracranial metastases, KPS \<80 (the median value of the cohort), \<9-month interval between courses (the median value), uncontrolled primary, and SCLC histology were independently associated with lower survival ([Table 2](#tbl2){ref-type="table"}). Neither age nor RTOG score were predictive.Figure 2Survival by recursive partitioning analysis (RPA) class after repeat WBRT. (\*Ref = RPA class 1). CI, confidence interval; MS, median survival.Table 2Univariate and multivariate analysis for predictors of survival at the time of ReRTFactorUVAMVAHR*P* valueHR*P* valuePresence of extracranial metastases1.89\<0.0012.270.002KPS \<801.72\<0.0011.850.005Interval between courses \<9 mo1.59\<0.0011.530.02Primary uncontrolled1.63\<0.0011.520.03Primary histology[a](#tbl2fna){ref-type="table-fn"} Breast1.00 NSCLC1.150.421.420.11 SCLC2.50\<0.0012.69\<0.001 Other1.190.471.210.55RTOG neuro function score [\>]{.ul}21.380.051.160.51Age ≥65 y1.110.44[^3][^4] Reirradiation score {#sec3.3} ------------------- The aforementioned 5 factors were combined to generate a new prognostic scoring system, the ReRT score. For 156 of 205 patients for whom all data were available, 1 point was assigned for each of the following poor prognostic factors: SCLC; presence of extracranial metastases; KPS \<80, interval between radiation therapy courses \<9 months; and uncontrolled primary. The sum ranged from 0 to 5; however, no patients in this study scored 0. Of 156 patients, 54 (34.6%) scored 1-2, 54 (34.6%) scored 3, and 48 (30.8%) scored 4-5. Survival based on ReRT score as measured from the first day of ReRT demonstrated a significant difference between ReRT score groups. (*P* \< .001; [Fig 3](#fig3){ref-type="fig"}). When comparing classification by ReRT versus RPA score, 45 patients (28.8%) were categorized into a group with a higher expected survival, 34 (21.8%) had a lower expected survival, and the remainder had a similar survival regardless of the index used.Figure 3Survival after repeat whole brain radiation therapy by reirradiation (ReRT) score group (\*Ref = ReRT score 1-2). Very short survival {#sec3.4} ------------------- Twenty-eight patients (13.7%) survived 30 days or fewer (as measured from the first day of ReRT). None of the factors listed in [Table 2](#tbl2){ref-type="table"} predicted very short survival (all *P* \> .11 on UVA). For 20 of these 28 patients, the ReRT score could be calculated: 2.6% scored 2, 35.7% scored 3, and 32.1% scored 4-5. Discussion {#sec4} ========== In this pooled analysis examining the applicability of RPA classifications to predict survival at the time of ReRT, there was a significant difference in MS between classes 1 and 3 (7.5 vs 2.9 months, respectively), but class 2 patients could not be distinguished from class 3 patients. The American College of Radiology advocates consideration of "patient characteristics and preferences, previous treatments employed, and potential risks and toxicities of treatment" to determine the optimal therapeutic approach in this clinical setting.[@bib16] An adaptation of the RPA scoring system may thus be helpful to guide treatment selection in patients with intracranial progression after WBRT, especially as the incidence of BM continues to increase.[@bib16] The results of the Quality of Life after Treatment for Brain Metastases (QUARTZ) trial, which randomized patients with NSCLC who were not eligible for SRS or surgery into cohorts of WBRT or BSC, revealed no difference between treatment arms.[@bib22] However, the patients accrued to that particular clinical study were expected to have a poor prognosis at baseline and are therefore not likely generalizable to the cohort in this study.[@bib23] Early reports of outcomes after repeat WBRT described survivals on the order of 8 weeks.[@bib7], [@bib15] Subsequent studies suggested an MS of 3 to 5 months, with some favorable symptomatic responses and minimal complications.[@bib8], [@bib17], [@bib24] Recent reviews have indicated that ReRT may be beneficial in carefully selected patients with longer predicted survival.[@bib25], [@bib26], [@bib27], [@bib28], [@bib29] The general principles remain that the goals of care, tolerance to prior course of radiation therapy, tumor biology, actionable mutations, and eligibility for clinical trial must also be integrated into the decision-making process when considering ReRT. Similar to other studies ([Table 3](#tbl3){ref-type="table"}), we found the following to be associated with shorter survival: extracranial metastases,[@bib17] KPS,[@bib26], [@bib27], [@bib28], [@bib29] interval between courses,[@bib26], [@bib27] uncontrolled primary,[@bib8], [@bib28] and SCLC histology.[@bib28] An RTOG neurologic function status score of \>2 was found to be significant by other groups[@bib24], [@bib25] but not in the present study, which could be due to the retrospective nature by which it was assigned. Interestingly, lung histology (both NSCLC alone and combined SCLC and NSCLC) correlated with higher MS elsewhere.[@bib29]Table 3Summary of repeat WBRT studiesReferenceN≥2 MetastasesMedian Dose, First WBRT (Range)Median Dose, Second WBRT (Range)MS (95% CI)Predictors of Poor Prognosis on UVAPredictors of Poor Prognosis on MVAAktan et al[@bib27]34100%30 Gy\ (25-30 Gy)25 Gy\ (20-30 Gy)5.3 mo\ (4.1-6.6 mo)NR•KPS ≤70•Severe symptomsScharp et al[@bib28]134NR30 Gy\ (30-40 Gy)20 Gy\ (2-30 Gy)2.8 mo\ (0-28 mo)•SCLC•Male•RPA 3•KPS \<70•Primary uncontrolled•SCLC•KPS \<70•Primary uncontrolledOzgen et al[@bib26]2886%30 Gy\ (20-30 Gy)25 Gy\ (20-30 Gy)3 mo\ (1.8-4.1 mo)NR•KPS ≥60•Interval ≥9.5 mo•Lung histologySon et al[@bib8]17100%35 Gy\ (28--40 Gy)21.6 Gy\ (14--30 Gy)5.2 mo\ (1.3-8.7 mo)•Primary uncontrolledNRAkiba et al[@bib29]31NR30 Gy\ (26-42 Gy)30 Gy\ (3-40 Gy)4.0 mo\ (NR)•KPS \<70•Breast•KPS \<70Karam et al[@bib20][a](#tbl3fna){ref-type="table-fn"}37NRNRNR\ (15-20 Gy)6.9 mo\ (NR)•RPA class 3•\>3 metastases•Histologic gradeNRSadikov et al[@bib25]7286%20 Gy\ (20-30 Gy)NR4.1 mo\ (NR)•RTOG neurologic function score 2-3•ECOG 2-3•Clinically stable or progressive CNS disease after first RTNRAbdel-Wahab et al[@bib30][b](#tbl3fnb){ref-type="table-fn"}15NR30 Gy\ (30-55 Gy)30 Gy\ (30-35 Gy)3.2 mo\ (NR)NRNRWong et al[@bib17]8655%30 Gy\ (1.5-50.6 Gy)20 Gy\ (8-30.6 Gy)4.0 mo\ (0.25-72 mo)•Presence of extracranial metastases•Multiple brain metastases•ReRT dose \<20Gy•Presence of extracranial metastasesCooper et al[@bib24]5245%NRNR16.3 wk\ (NR)•Multiple brain metastases•RTOG neurologic function score 2-4NRHazuka et al[@bib7]44NR30 Gy\ (30-36 Gy)25 Gy\ (6-36 Gy)8 wk\ (NR)NRNRKurup et al[@bib31]56NRNR\ (18-30 Gy)20 Gy\ (20 Gy)3.5 mo\ (0.25-16 mo)NRNRShehata et al[@bib32]35NRNRNRNRNRNR[^5][^6][^7] In an attempt to more precisely estimate survival at the time of repeat WBRT, we developed the ReRT score. Patients with an ReRT score of 1 to 2 are likely to have as long a survival time as those with RPA class 1 at initial diagnosis of BM and should be considered for ReRT. Patients with an ReRT score of 4 to 5 have an estimated survival of approximately 2 months, and BSC with hospice enrollment is likely to be more appropriate. For patients with an ReRT score of 3, the expected potential benefits versus risks of ReRT in relation to BSC should be discussed. Comparing the ReRT score with the RPA, the presence of extracranial metastases and uncontrolled primary remained strongly predictive for both indices. KPS emerged with a modified cut-point (≥80) separating patients. Age was not a significant predictor for MS after ReRT; however, the interval to treatment was significant and may be a surrogate for the underlying biology of the disease and/or responsiveness to treatment. WBRT within 30 days of death has recently been discussed in the setting of quality care indicators.[@bib33] Patients with very short predicted survival are better suited to BSC than to radiation therapy, from which they are not likely to live long enough to benefit, especially in the case of protracted fractionation. However, we failed to find significant factors that were associated with very short survival. Although this is the largest patient cohort reported, conclusions remain limited by retrospective calculation of the GPA, the proportion of patients for whom KPS was retrospectively assigned, and heterogeneous primary histologies. Prior to adoption into routine practice, prospective validation is required. Recently, the impact of biomarkers on prognostication for patients with BM from primary breast and NSCLC has led to modifications of the GPA.[@bib34], [@bib35] This information is not available within the current cohort, which may also be a limitation; however, in practice, these data may either be unavailable or rendered irrelevant once patients have progressed through all available lines of systemic therapy. Additionally, we were unable to abstract quality of life, steroid dependence, setting of care, and KPS after completion of repeat WBRT. Specific symptoms have been reported previously as correlating with prognosis[@bib17], [@bib24], [@bib25], [@bib27] but could not be examined in this cohort. Although we attempted to collect information on toxicity, this was rarely available. Finally, this review did not include patients who received SRS. As more centers use SRS for salvage in patients with \>4 metastases, future work should focus on the potential clinical utility of the ReRT score for this population. Conclusions {#sec5} =========== In the largest reported cohort to receive repeat WBRT, application of the RPA score was not independently predictive of MS for all patients. The new ReRT score, based on histology, KPS, stability of the primary, interval between courses, and absence of extracranial metastases, is a simple tool that is based on readily available clinical information and has been shown to be predictive of survival after repeat WBRT in this pooled population. However, internal and external prospective validation is required to confirm robustness prior to routine clinical implementation. Supplementary data {#appsec1} ================== Appendix Sources of support: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflicts of interest: None. Supplementary material for this article ([http://dx.doi.org/10.1016/j.adro.2017.05.010](10.1016/j.adro.2017.05.010){#intref0010}) can be found at [www.advancesradonc.org](http://www.advancesradonc.org){#intref0015}. [^1]: KPS, Karnofsky Performance Status; NSCLC, non-small cell lung cancer; RPA, recursive partitioning analysis; RT, radiation therapy; RTOG, Radiation Therapy Oncology Group; SCLC, small cell lung cancer. [^2]: Includes head and neck, gynecologic, melanoma, lymphoma, and colorectal. [^3]: HR, hazard ratio; KPS, Karnofsky Performance Status; MVA, multivariate analysis; neuro, neurological; NSCLC, non-small cell lung cancer; RTOG, Radiation Therapy Oncology Group; SCLC, small cell lung cancer; UVA, univariate analysis; WBRT, whole brain radiation therapy. [^4]: Ref=Breast primary. [^5]: CI, confidence interval; CNS, central nervous system; KPS, Karnofsky Performance Status; MS, median survival; MVA, multivariate analysis; NR, not reported; ReRT, reirradiation; RPA, recursive partitioning analysis; RT, radiation therapy, RTOG, Radiation Therapy Oncology Group; SCLC, small cell lung cancer; UVA, univariate analysis; WBRT, whole brain radiation therapy. [^6]: Included breast histology only. [^7]: Included patients who received partial WBRT.

1. Introduction =============== Paraneoplastic neurologic syndromes (PNSs) can be the first symptom of a covert malignancy. Early treatment of PNS may improve the morbidity and mortality rates of cancer patients. Advances in antibody-detection techniques have enabled definitive diagnoses in patients with paraneoplastic and non-paraneoplastic autoimmune encephalitis. However, antibodies are absent in most cases of PNS associated with lymphomas, especially non-Hodgkin lymphoma (NHL).^\[[@R1]\]^ Anti-Ma2-associated encephalitis is a rare immune-mediated PNS with preferential involvement of the limbic system, diencephalon, and upper brain stem. It has been mainly described in adult men with testicular germ cell tumors.^\[[@R2]\]^ Herein, we report a newly diagnosed case of anti-Ma2-associated PNS with coexisting chronic inflammatory demyelinating polyneuropathy (CIDP) in a patient with NHL. 2. Case presentation ==================== 2.1. On admission ----------------- A 74-year-old man was admitted to our hospital because of acute worsening of body aches, numbness of all four limbs, and weakness of the lower limbs. He reported that all of these symptoms had been slowly progressing during the past 5 months. During this period, he had sustained 2 falls during this period and felt that he was slow to react. He had also occasionally experienced visual hallucinations, and had lost almost 5 kg in 1 month. Brain computed tomography (CT) showed lacunar infarction. Magnetic resonance imaging (MRI) of the cervical, thoracic, and lumbar spine showed degenerative changes. His sodium and chloride ion levels were slightly decreased, whereas his high-sensitivity C-reactive protein (hs-CRP) level had increased to 6.5 times the normal value (normal range, 0--3.5 mg/L), and his β~2~ microglobulin level had increased to 3 times the normal value (normal range, 0.7--1.8 mg/L). Routine blood examination and a coagulation profile revealed no abnormalities. Tests for anti-streptolysin O and rheumatoid factor were negative. On admission to our clinic, further aggravation of the clinical symptoms was observed, and right blepharoptosis and limited eyeball movements were detected, indicating right oculomotor paralysis. On examination, the patient appeared drowsy, slow to react, and poorly oriented in time and place. Triparesis involving both lower limbs and the right upper limb was detected, the tendon reflexes had disappeared in all 4 limbs, and hypalgesia in 4 limbs was found in this patient. A physical examination revealed that the patient could not complete the finger-to-nose test and heel-knee-tibia test with his right limbs owing to weakness. He scored 24 points on the Mini-Mental State Examination of cognitive function (orientation, 2; attention and calculation, 1; recall, 1; language, 1; ability to follow simple commands, 1). 2.2. Investigations ------------------- Serological hs-CRP was 34.50 mg/L (normal 0--3.5), erythrocyte sedimentation rate was 86 mm/hour (normal 0--15), and β~2~ microglobulin level was 5.28 mg/L (normal 0.7--1.8). The serum IgA was 6.7 g/L (normal 0.7--4.0), IgG was 16.7 g/L (normal 7.0--16.0), and anticardiolipin IgG antibody level was 16 U/mL (normal 0--10). In addition, tests for IgG antibodies to herpes simplex virus, rubella virus, and cytomegalovirus were positive. The serum titers of IgG antibody of Epstein--Barr virus core antigen was 4.165 s/co (normal \<1.100). The serum levels of creatine kinase and lactate dehydrogenase were normal. Serological testing for syphilis and human immunodeficiency virus was negative. A lumbar puncture revealed albuminocytologic dissociation in the cerebrospinal fluid (CSF), specifically, the cell count was 4 × 10^6^ cells/L (normal 0--8) with 2 lymphocytes and without any abnormal cells, and the protein concentration was 1.32 g/L (normal 0.15--0.45). Anti-PNMA2 (Ma2/Ta) was suspected to be positive in the CSF and weakly positive in the serum (antibody expression was graded as follows: ≤25% positive cells, suspected to be positive; \>25% and ≤50% positive cells, weakly positive; \>50% and ≤75% positive cells, positive; and \>75% positive cells, strongly positive). The serum total prostate-specific antigen was mildly increased; other tumor markers were negative. A lung CT scan showed an old lesion in the superior lobe of the right lung. Abdominal Doppler ultrasonography showed prostatic hyperplasia with calcification. Scrotal Doppler ultrasonography showed a spermatocele in the head of the left epididymis. A prostatic MRI showed prostatic hyperplasia and slight enlargement of the lymph nodes in the inguinal regions and around the iliac vessels. Positron emission tomography (PET)/CT showed that lymph nodes in multiple regions were mildly enlarged with increased metabolism, indicating a high possibility of lymphoma. Histopathological examination of the inguinal lymph nodes was suggestive of angioimmunoblastic T-cell lymphoma (AILT) or peripheral T-cell lymphoma. Immunohistochemical examination yielded the following results: Ki-67 (+60%), CD2 (+), CD3 (+), CD43 (+), CD5 (+), CD20 (−), PAX-5 (−), CD10 (−), Bcl-6 (scattered +), CD21 (DC+), PD-1 (−), Bcl-2 (+), CD15 (−), CD30 (a few scattered +), CD7 (+), CD43 (+), CD4 (+), CD8 (partially +), granzyme B (−), TIA-1 (a few scattered +), CD56 (−), EMA (−), ALK (−), CD30 (scattered +), CD15 (−), EBER (a few scattered+). The immunohistochemical results are shown in Figures [1](#F1){ref-type="fig"}--[4](#F4){ref-type="fig"}. {#F1} {#F2} {#F3} {#F4} Brain MRI was performed twice; both MRI scans showed multiple lacunar infarctions with no suspicious signals in the brain stem and diencephalon (Fig. [5](#F5){ref-type="fig"}). Brain magnetic resonance angiography showed partial disappearance of the left vertebral artery, suggesting blockage. Electromyography showed that peripheral nerve conduction in all 4 limbs was significantly impaired, indicating a preponderance of demyelination (Fig. [6](#F6){ref-type="fig"}). A 4-hour electroencephalogram showed irregular sharp slow waves and δ waves in both temporal regions. {#F5} {#F6} 2.3. Treatment and outcome -------------------------- A definitive diagnosis of CIDP was made based on the symptoms and results of auxiliary examinations. The patient\'s clinical condition stabilized after a cycle of enhancing immunity (intravenous immunoglobulin) and neuronutrition (edaravone injection). There were no signs of CIDP progression. In the following days, a final diagnosis of anti-Ma2-related PNS associated with lymphoma was made. The patient was treated with intravenous dexamethasone (15 mg/day) for 3 days, and all the central and peripheral nervous system symptoms improved. The patient was then transferred to the oncology department to receive systemic chemotherapy. 3. Discussion ============= The diagnosis of PNS represents a clinical challenge, especially when it is associated with lymphoma, which is rare. The frequency of PNS associated with lymphomas was analyzed by the PNS Euronetwork Consortium, which includes 20 European centers. Between 2000 and 2008, the Consortium identified 53 patients with PNS, 29 had NHL. Furthermore, of the 53 patients, only 11 had demyelinating neuropathies, and 9 of these were associated with NHL.^\[[@R3]\]^ Patients with NHL can present with typical CIDP or with predominantly sensory neuropathies, which are probably caused by monoclonal IgM antibodies against myelin-associated glycoproteins or gangliosides.^\[[@R4],[@R5]\]^ Anti-Ma2-associated encephalitis is a rare disease. As a PNS, it develops gradually, and it usually affects the limbic system, diencephalon, and/or brain stem.^\[[@R2]\]^ According to a recent review and analysis, only 68 cases of anti-Ma2-associated neurological syndromes have been reported to date.^\[[@R6]\]^ Most cases occurred in adult men with testicular germ cell tumor or non-small-cell lung cancer, whereas some were associated with tumors such as lymphoma, prostatic adenocarcinoma, gastric adenocarcinoma, and recurrent cervical cancer.^\[[@R7],[@R8]\]^ Ma2 is an intracellular onconeural protein; anti-Ma2 antibodies recognize 2 antigens---PNMA1 and PNMA2 ^\[[@R9]\]^---which are mainly expressed in neurons in the brain, spinal cord, dorsal root ganglia, and tumors.^\[[@R2],[@R10]\]^ The mechanisms underlying selective neuronal injury in anti-Ma2-associated neurological syndromes seem to involve a cytotoxic T-cell-mediated response. Experimental data have showed that the adoptive transfer of T-helper 1 CD4+ T-cells specific for PNMA1 induces encephalomyelitis in rats.^\[[@R9]\]^ Clinically, patients with anti-Ma2-related limbic encephalitis manifest not only the typical symptoms of limbic encephalitis---such as subacute onset of short-term memory loss, epilepsy, and psychiatric symptoms^\[[@R2],[@R11]\]^---but also symptoms of diencephalon and upper brain stem involvement, such as excessive daytime sleepiness and vertical ophthalmoparesis.^\[[@R12],[@R13]\]^. Anti-Ma2 neurological disorders have been shown to even affect the peripheral nervous system, causing muscle atrophy.^\[[@R14]\]^ In our patient, the clinical presentation and results of auxiliary examinations were consistent with the criteria for atypical CIDP according to the European Federation of Neurological Societies/Peripheral Nerve Society guidelines published in 2010.^\[[@R15]\]^ However, the symptoms of slow reaction time, depression, and cognitive impairment are not the usual presentation of CIDP and indicate that the patient had temporal lobe involvement. Furthermore, the CSF was suspected to be positive for anti-PNMA2 (Ma2/Ta) antibodies, indicating coexisting limbic encephalitis. The brain MRI, however, was normal. Similar cases have been described in the literature.^\[[@R16]\]^ The major characteristics of this case can be summarized as follows. First, our report shows that NHL can be associated with not only demyelinating neuropathy as a PNS but also anti-Ma2-associated limbic encephalitis. The simultaneous central and peripheral nervous system involvement observed in our patient is an extremely rare presentation of anti-Ma immunity, with only 2 reported (non-NHL-related) cases thus far.^\[[@R17]\]^ (2) Another interesting aspect of this case was the absence of lesions on brain MRI. In 2009, Blanc et al reported that brain MRI can be normal in some patients with limbic encephalitis.^\[[@R18]\]^ (3) This case shows that PET/CT is an effective and dependable examination for the diagnosis of PNS. A rapid and accurate diagnosis of PNS is essential because any delay can significantly influence clinical management, and the illness can be lethal without proper treatment.^\[[@R19]\]^ An intriguing report showed that in some cases of non-paraneoplastic limbic encephalitis with undetectable antibodies and negative findings on MRI, PET/CT may be highly suggestive of the disease.^\[[@R20]\]^ 4. Conclusion ============= In conclusion, clinical presentations suggestive of an immunological reaction to specific antigens should raise the possibility of a paraneoplastic syndrome. The identification of autoantibodies can guide the clinical search for a specific tumor. PET/CT and biopsy can complement the diagnostic work-up and help to confirm the diagnosis. Finding and treating the tumor are essential to improve the prognosis of patients with PNS. Abbreviations: CIDP = chronic inflammatory demyelinating polyneuropathy, MRI = magnetic resonance imaging, NHL = non-Hodgkin lymphoma, PET-CT = Positron-emission tomography/computed tomography, PNSs = Paraneoplastic neurologic syndromes. WJ and BQ contributed equally to this work. Consent: The Institutional Review Board of Jilin University approved the publication of this case report, and informed consent has been obtained from the patient. Funding: This work was supported by the National Natural Science Foundation of China (No.81500911). The authors report no conflicts of interest.

The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus. Introduction ============ The incidence of community-acquired pneumonia (CAP) among adults in the United States is 16 to 23 per 1000 persons per year, with approximately 30% of these patients requiring hospitalization \[[@REF1],[@REF2]\]. *Streptococcus pneumoniae* is the most commonly detected bacterial species in patients with CAP \[[@REF3],[@REF4]\]. Rates of bacterial pneumonia are 10 times higher in patients infected with human immunodeficiency virus (HIV) than in healthy individuals, despite the former receiving antiretroviral therapy. Bacterial pneumonia is currently the most frequent cause of overall and pulmonary infections in HIV-infected patients, as well as the most frequent diagnosis at hospital admission \[[@REF5]-[@REF7]\]. Pneumonia in HIV-infected patients is frequently caused by simultaneous infection with multiple organisms, making pneumonia management very challenging in these patients. Opportunistic infections in HIV-infected patients can include bacterial species in 60% of cases, mycobacteria in 18%, viruses in 5%, and fungi, including *Pneumocystis pneumonia* in 20% \[[@REF7]\]. Infections with all of these microorganisms must be considered to optimize treatment.  Case presentation ================= A 29-year-old man with a previous history of alcohol use disorder and injection drug use presented to our hospital with fever, cough, and shortness of breath, along with chronic diarrhea lasting for a few months. The patient reported feeling ill for two days prior to presentation but subsequently felt feverish and sweaty, prompting him to visit the emergency department. On examination, the patient was febrile with a temperature of 39.1 °C (102.3 °F) and tachycardic with a heart rate of 101 beats per minute. His blood pressure was borderline, 95/60 mmHg; he was hypoxic with 89% breathing in room air, and he was in a moderate degree of distress. Examination of his lungs revealed decreased air entry on the right side with some crackles, whereas examination of his mouth showed oral thrush. The findings of his abdominal examination were unremarkable, with no evidence of organomegaly or tenderness to abdominal palpation. Biochemical and hematological investigations revealed a healthy leukocyte count of 7,500 cells/µL (neutrophils 94%), low hemoglobin of 10.4 gm/dL (healthy mean corpuscular volume of 99), a healthy creatinine level of 0.4 mg/dL, an elevated aspartate transaminase level of 216 U/L (reference range is \<39 U/L), a healthy alanine transaminase level of 49 U/L (reference range is \<52 U/L), and an elevated total bilirubin level of 1.5 mg/dL (reference range is \<1.00 mg/dL). A chest x-ray showed focal consolidation in the right middle lobe (Figure [1](#FIG1){ref-type="fig"}) . Blood cultures ,* S. pneumoniae* urine antigen and *Legionella pneumophila*urine antigen were performed, and the patient was started on empirical treatment with ceftriaxone and azithromycin. Further assessment of the patient revealed that he was in a monogamous relationship with a male partner and regularly participated in unprotected anal intercourse. A fourth-generation rapid HIV antigen-antibody test was, therefore, performed. {#FIG1} On the second day of admission, the patient continued to experience febrile episodes, was still coughing and experiencing shortness of breath, and was still hypoxic. Results of urinalysis showed that he was positive for *S. pneumoniae* antigen, and rapid HIV testing was also positive with a CD4 count of 18 cells/mm3. He was preliminary diagnosed with pneumonia due to *S. pneumoniae*. Treatment with ceftriaxone was therefore continued, but all other antibiotics were discontinued. On the third day, however, his condition deteriorated, and he developed respiratory failure. The patient was intubated with ventilator settings that included a respiratory rate of 18 breaths/minute, a tidal volume of 460 mL, a fraction of inspired oxygen of 60%, and a positive end-expiratory pressure of 5 cm H~2~O. His blood culture was positive for pan-susceptible S. *pneumoniae*, and repeated chest x-rays showed bilateral infiltrates (Figure [2](#FIG2){ref-type="fig"}). Military tuberculosis was ruled out as three sputum samples were negative. Vancomycin was added, and bronchoscopy was performed to determine the presence of opportunistic infections. His bronchoalveolar lavage fluid was positive for *Pneumocystis jirovecii* and methicillin-resistant *Staphylococcus aureus* . He was therefore started on trimethoprim-sulfamethoxazole and corticosteroids. Two days later, his condition improved, and he was extubated to a high-flow nasal cannula (Figure [3](#FIG3){ref-type="fig"}). He continued to improve and was discharged after being hospitalized for two weeks (Figure [4](#FIG4){ref-type="fig"}). {#FIG2} {#FIG3} {#FIG4} Discussion ========== This report describes a young homosexual man with a history of injection drug use and unprotected intercourse who presented with community acquired pneumonia due to *S. pneumoniae*. However, the lack of response to antibiotics and newly diagnosed AIDS required further investigation, which revealed concurrent pulmonary infection with *Pneumocystis jirovecii* and methicillin-resistant *Staphylococcus aureus*. Bacterial pulmonary infections occur frequently in persons infected with HIV. The development of highly active antiretroviral therapy has led to advances have in the care and treatment of patients with HIV, altering the presentation of HIV-associated pulmonary diseases. Pneumonia caused by bacterial infections, particularly *S. pneumoniae*, remain commonplace, whereas opportunistic infections with agents such as *P. jirovecii* remain a concern in patients without adequate access to optimal medical care \[[@REF8]\]. The annual incidence of bacterial pneumonia in HIV-seropositive patients ranges from 5.5 to 29 per 100, compared with 0.7 to 10 per 100 in HIV-seronegative patients \[[@REF9]-[@REF11]\]. Although bacterial pneumonia can occur throughout the course of HIV infection, it is more frequent in individuals with advanced immunosuppression \[[@REF10],[@REF12]\]. Moreover, the incidence of bacterial pneumonia was shown to directly correlate with CD4 count \[[@REF11]\]. Other traditional risk factors that may be associated with pneumonia include pre-existing lung disease (e.g., bronchiectasis or chronic obstructive pulmonary disease), heavy alcohol use, injection drug use, neutropenia, and severe malnutrition. Bacterial pneumonia in HIV-infected patients is associated with a permanent decline in pulmonary function \[[@REF13]\] and a two-fold to five-fold increase in long-term mortality compared with CD4-matched controls \[[@REF11],[@REF13],[@REF14]\]. Respiratory symptoms and suspected pneumonia are evaluated in HIV infected individuals to establish a definitive diagnosis, thereby allowing the initiation of appropriate treatment. However, definitive diagnoses may require invasive diagnostic procedures such as bronchoscopy, as well as sophisticated laboratory techniques. The clinical and radiographic presentations of HIV-associated opportunistic pneumonias overlap, and HIV infected individuals may present concurrently with pneumonia caused by more than one agent. Bacterial pneumonia may be the first manifestation of underlying HIV infection. Thus, HIV infection should be suspected in any patient presenting with bacterial pneumonia, especially in patients with no other risk factors for pneumonia and in patients with recurrent pneumonia. Similar to findings in non-HIV infected individuals, *S. pneumoniae* and *Haemophilus* species are the most frequently identified causes of CAP. *Pseudomonas aeruginosa* and *Staphylococcus aureus* are more frequent causes of CAP in patients with than without HIV infection \[[@REF15]\]. The clinical and radiological presentations of lower respiratory tract infections in HIV-infected patients are quite variable. Clinical presentations are more severe, and radiological imaging more atypical in severely than in mildly immunosuppressed HIV infected individuals \[[@REF16]\]. Chest radiography is the first step in the diagnostic evaluation of persons with suspected pneumonia. Specific findings on chest radiography, along with CD4 cell count, often suggest a differential diagnosis and plan for management and treatment. Selected laboratory tests may be indicated to assess for specific diseases or disease severity (e.g., arterial blood gas). In some cases, computed tomography of the chest may be indicated. Where feasible, further evaluation should seek to establish a definitive diagnosis, with microbiologic evaluation of sputum, including via staining and culturing, as well as bronchoscopy in selected patients \[[@REF15]\]. A suboptimal response to empiric therapy or to the treatment of an identified pathogen in patients with HIV-related pulmonary diseases suggests the need for a definitive or additional diagnosis. It is also essential to exclude pulmonary tuberculosis in HIV-seropositive patients with CAP who fail to respond appropriately to initial antibiotic therapy, even if another etiological pathogen has been identified \[[@REF15]\]. Conclusions =========== This report describes a young man who presented with what seemed like a straightforward CAP due to*S. pneumoniae*. He was found to be infected with HIV and to have a low CD4 count of 18 cells/mm3 but failed to respond to appropriate CAP treatment. The lack of improvement may have been due to complications of pneumonia, including abscess and acute respiratory distress syndrome, or to simultaneous infection with multiple organisms. These findings suggest that simultaneous infection with multiple organisms may be responsible for CAP in HIV-infected patients. The authors have declared that no competing interests exist. Consent was obtained by all participants in this study

Briefings in Bioinformatics (2016) doi: 10.1093/bib/bbw145 In the article above, the following errors have been addressed: • In Table 1, the SHS formula was incorrectly given as ${SHS} = U_{R}\text{diag}\left( {\xi_{1} + \delta,\cdots,\xi_{n} + \delta} \right)U_{R}^{T}$. This has been corrected to ${SHS} = U_{R}\text{diag}\left( {\lambda_{1} + \delta,\cdots,\lambda_{n - q} + \delta} \right)U_{R}^{T}$.• The running title has been changed from 'Methodological implementation of mixed linear model' to 'Methodological implementation of mixed linear models'.• Reference 22 has been corrected to indicate a page range of '525--6' from '525'.The corrections have been made online and in print. The publisher apologizes for these errors.

The COVID-19 viral pandemic is bearing down on the United States. Thousands have died in China and in Italy, and every country in the world is or will be affected. At home every sniffle or cough elicits a brief pang of fear. To fight the deadly virus, we are turning to methods that proved partly effective in the flu epidemic of 1918. We have learned a new term--social distancing. Oddly at a time when the interdependence of humans all over the world is most exposed, we are asked to isolate ourselves. To do the best for the most vulnerable among us, the right response is to shut the doors and hunker down. It\'s just the opposite of the best medical intentions to reach out, to touch, and to help. Yet the time will come, and for some the time is now, when those intentions rise again to action. Perhaps we can take a lesson from these days of isolation to remember how human flourishing depends on social life. There is another pandemic called atherosclerosis that claims millions of lives annually worldwide. It\'s so familiar that we are apt to forget it in these extraordinary days. Yet we can point to signs of success. I have 2 brothers whose livelihood has depended partly on performing coronary bypass surgery. I recall my surprise early in my medical career when this complex operation became the most commonly performed surgery in the US. By 2000 one brother told me that the pace of performing coronary bypass procedures was declining about 1% per year. That pace has accelerated. Since 2003 the annual number of bypass procedures has dropped by 50%. The annual number of percutaneous coronary interventions (PCI) by cardiologists is a little more than twice the rate of bypass procedures. PCI volume has likewise dropped by half since 2003.[@bib1] So we find ourselves facing an old type of communicable disease today, yet gaining substantial success in battling the most prevalent modern deadly pathology in both developed and emerging economies in the world. In particular, medical, dietary, and lifestyle interventions are working to diminish the impact of atherosclerosis, signaled by far less need for invasive procedures. Preventive cardiology and lipidology are rising. Joint efforts and international cooperation make the mission particularly satisfying. In this issue of JCL we focus in 2 editorials and the Roundtable on atherosclerotic risk in South Asians, defined as the nations of India, Pakistan, Nepal, Bhutan, Bangladesh, Sri Lanka, and Maldives as well as the worldwide diaspora emanating therefrom. South Asians comprise 25% of world population, but suffer 50% of atherosclerotic cardiovascular events. Dr Raman Puri and colleagues have founded the India Lipid Association, and this issue marks the publication in JCL of their online-only, open access expert consensus document applicable to LDL cholesterol goals in India. They discuss potential expanded use of PCSK9 inhibitors to meet LDL cholesterol goals as low as ≤30 mg/dL.[@bib2] You will also find in these pages a discussion of the sphingolipid ceramide as an atherosclerotic risk factor, illustrative cases including a woman with intermittent autoimmune chylomicronemia, advice on how to differentiate familial from multifactorial chylomicronemia, a suggestion that ABCG5/G8 heterozygous pathogenic variants may contribute to familial hypercholesterolemia, ascertainment of hurdles facing cascade screening and pediatric cholesterol screening, and several other new aspects of clinical lipidology. Disclosures {#appsec1} =========== Dr Guyton has received research grants from 10.13039/100014384Amarin, 10.13039/100009857Regeneron, and 10.13039/100004339Sanofi.

As we approach the second anniversary of the launch of *PLoS Pathogens*, it is evident that the journal has rapidly established itself as an important publication in the field of pathogen--host interactions (Image 1). We attribute this success to the commitment and motivation of our talented editorial board, to a receptive community ([Table 1](#ppat-0030145-t001){ref-type="table"}) who sees this as a leading open-access journal, and to a highly competent and helpful staff at the Public Library of Science. ###### Top Ten Downloaded Papers from *PLoS Pathogens*, September 2005 to July 2007  Our initial impact factor, recently assigned by Thomson Scientific (formerly Thomson ISI), is 6.056. For an initial metric calculated from just four months of data, this is a strong start, and we fully expect it to rise rapidly as more information becomes available. But, we also caution about reading too much into impact factor values because of inherent flaws that exist with using this number to measure the impact of any given paper. We recognize that journal impact factors are so often used as a surrogate measure of the quality of a given scientist and his or her work, influencing hiring, promotion, and even grant funding decisions, and for these reasons we think it is important to raise awareness about what impact factors measure. The journal impact factor for any given year is calculated based on information obtained from the preceding two years. For example, for 2007: It is self-evident that this formula fails to reveal the significance of any given paper. Instead, the calculated number represents an average number of citations for a paper in the journal. In other words, the highest impact papers are given an artificially low ranking by this system, whereas papers with the least impact are assigned a greater impact value than they deserve. Confounding this problem, review articles, which are highly visible and citable, artificially inflate the "impact" of the lesser-cited research articles in the same journal. Also, the method by which "citable articles" are calculated in the denominator of this equation is unclear and needs to be made transparent so that the community can become more confident about the nature of the differences that exist between the impact of distinct journals. Clearly, a more scientifically rigorous methodology must be developed if we are to accurately quantify the true impact of a given paper. Several alternative mechanisms have been proposed, including measuring the number of downloads of a paper over the Internet, and we will not elaborate further on these ideas here. However, if you would like to learn more, we encourage you to read the articles cited at the end of this editorial \[[@ppat-0030145-b001]--[@ppat-0030145-b004]\]. In closing, we are grateful to you for establishing *PLoS Pathogens* as a leading open-access journal. On behalf of the editorial board we thank you, the community, for your confidence in the journal and for your continued support.  {#ppat-0030145-g001} John Young is with the Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California, United States of America. Kasturi Haldar is with the Department of Pathology and Microbiology--Immunology, Northwestern University Medical School, Chicago, Illinois, United States of America. **Author contributions.** JY and KH wrote the paper. **Funding.** The authors received no specific funding for this article. **Competing interests.** The authors have declared that no competing interests exist.

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ Conversion of common murine models to an immune-compromised state has become highly desirable for translational research. The creation of immune-compromised mouse strains allows for the preclinical efficacy of human cell transplantations and gene therapy strategies to be tested in small rodent systems before moving forward to human clinical trials. Recently, we have utilized the *W*^*sh*^*/W*^*sh*^ mouse strain to examine human neural crest cell contribution during embryonic and post-natal mouse development \[[@pone.0194443.ref001]\]. In order to increase human cell incorporation into the *W*^*sh*^*/W*^*sh*^ mice, as well as provide a future system for human disease modeling, we used CRISPR (clustered regularly interspaced short palindromic repeat) and Cas (CRISPR-associated) proteins to knockout interleukin 2 receptor subunit gamma (*Il2rγ*) and recombination activating gene-2 (*Rag2*) in the *W*^*sh*^*/W*^*sh*^ mice. The conversion of the *W*^*sh*^*/W*^*sh*^ mice to an immune-compromised state led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. *Escherichia coli (E*. *coli)* strains have been frequently isolated from rodents, but are not routinely or completely characterized; these bacteria are commonly considered commensals and are not currently excluded from specific pathogen-free mouse colonies. However, pathogenic *E*. *coli* can encode various virulence factors, which are classified into different subtypes, such as enteropathogenic *E*. *coli* (EPEC), enterohemorrhagic *E*. *coli* (EHEC), enterotoxigenic *E*. *coli* (ETEC), enteroinvasive *E*. *coli* (EIEC), enteroaggregative *E*. *coli* (EAggEC), diffusely adhering *E*. *coli* (DAEC) and adherent-invasive *E*. *coli* (AIEC) \[[@pone.0194443.ref002]\]. Another group called extraintestinal pathogenic *E*. *coli* (ExPEC) belongs to the B2 *E*. *coli* phylogroup and are associated with human cases of meningitis, septicemia, and urinary tract infections (ExPEC) \[[@pone.0194443.ref003],[@pone.0194443.ref004]\]. The polyketide synthase (*pks)* is a 54-kb genomic island that encodes colibactin (Clb) gene cluster \[[@pone.0194443.ref005],[@pone.0194443.ref006]\]. The genotoxic metabolite colibactin acts as a cyclomodulin that induces DNA damage and cell cycle arrest in mammalian cells. \[[@pone.0194443.ref005]--[@pone.0194443.ref009]\]. The *pks* genomic island is highly conserved in *Enterobacteriaceae* and has been isolated from commensal *E*. *coli* strains (B2 and Nissle 1917 *E*. *coli*), *Citrobacter koseri*, *Klebsiella pneumoniae*, and *Enterobacter aerogenes* \[[@pone.0194443.ref005],[@pone.0194443.ref007]\]. A study identified that IL10^-/-^ mice with experimentally-induced chronic lower bowel inflammation were colonized with a specific *E*. *coli* (O7:H7:K1) strain of phylogenetic group B2 that encodes high number of virulence associated genes \[[@pone.0194443.ref010]\]. The tumor promoting effects of *pks+* NC101 *E*. *coli* strain (O2:H6/41) were identified in germfree IL10^-/-^ mice carcinogenicity studies Further, carcinogenicity studies in germfree IL10^-/-^ mice monoassociated with the *pks+* NC101 *E*. *coli* strain (O2:H6/41) identified its tumor promoting effects \[[@pone.0194443.ref011],[@pone.0194443.ref012]\]. The *pks+* NC101 *E*. *coli* strain associated promotional events were specifically attributed to the *pks* genomic island and excluded the role of inflammation in the formation of invasive carcinoma \[[@pone.0194443.ref011]\]. IL10^-/-^ mice monoassociated with a murine *E*. *coli* strain from wildtype mice (later identified as NC101) induced typhlitis due to increased synthesis of interferon γ and IL4 by CD4^+^ T cells \[[@pone.0194443.ref013]\]. *pks+ E*. *coli* strains have also been utilized experimentally in rodent models to study urosepsis and septicemia \[[@pone.0194443.ref014],[@pone.0194443.ref015]\]. We recently identified *pks+ E*. *coli* strains from the gastrointestinal tract of commercially available mice, as well as mice maintained in a large biomedical research institute, and demonstrated the cytotoxic effects of colibactin produced by the isolated *pks+ E*. *coli* in a cell culture system \[[@pone.0194443.ref016]\]. These *pks+ E*. *coli* strains, which colonize asymptomatic humans, are also associated with inflammation, septicemia, meningitis, and urinary tract infections \[[@pone.0194443.ref003],[@pone.0194443.ref004]\], as well as being commonly isolated in colon cancer patients \[[@pone.0194443.ref017]\]. The purpose of this report is to describe acute morbidity and mortality in immunocompromised mice, from which colibactin producing *E*. *coli* was isolated from blood, genitourinary tract, and brain. As genome-engineering technologies have allowed for an increase in the efficiency and reduction in the time it takes to generate knockout mouse models, many murine models used in human research are becoming immune-compromised in order to test human cell transplantations and viral gene therapies for translational research. The abrupt increase in mortality and morbidity associated with *pks+ E*. *coli* in immune-compromised mice entails routine and complete characterization of these bacteria to ensure exclusion of colibactin-producing *pks+ E*. *coli* strains from specific pathogen-free mouse colonies. Materials and methods {#sec002} ===================== Ethics statement {#sec003} ---------------- All animal experiments were approved by the Massachusetts Institute of Technology Committee on Animal Care (Protocol \#0916-058-19: Gene Disease, Cancer, and Mammalian Development) and all animal procedures were performed following the National Institute of Health guidelines. Our facility conforms to Federal guidelines and has a PHS Approved Animal Welfare Assurance (\#A3125-01). Mice were monitored at least twice daily, and any adverse conditions (e.g. difficulty with ambulation, hunched posture, body condition score (BCS) \< 2, ruffled fur) were immediately brought to the attention of the veterinary staff. The mice for this study displayed normal behavior and health until suddenly showing signs of morbidity, and would succumb within 24 hours. Mice that became moribund/severely ill during this study were euthanized with CO~2~ exposure followed by cervical dislocation, and tissues were collected for analysis. Production of Cas9 mRNA and sgRNA {#sec004} --------------------------------- Bicistronic expression vector px330 expressing Cas9 and sgRNA \[[@pone.0194443.ref018]\] was digested with BbsI and treated with Antarctic Phosphatase, and the linearized vector was gel purified. A pair of oligos ([Table 1](#pone.0194443.t001){ref-type="table"}) for each targeting site was annealed, phosphorylated, and ligated to the linearized vector. T7 promoter was added to the Cas9 coding region by PCR amplification using primers Cas9 F and R ([Table 1](#pone.0194443.t001){ref-type="table"}). The T7-Cas9 PCR product was gel purified and used as the template for *in vitro* transcription (IVT) using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). The T7 promoter was added to sgRNA templates by PCR amplification using the primers listed in [Table 1](#pone.0194443.t001){ref-type="table"} of a previously published paper by the Jaenisch Lab \[[@pone.0194443.ref019]\]. The T7-sgRNA PCR product was gel purified and used as the template for IVT using the MEGAshortscript T7 kit (Life Technologies). Both the Cas9 mRNA and the sgRNAs were purified using MEGAclear kit (Life Technologies) and eluted in RNase-free water. 10.1371/journal.pone.0194443.t001 ###### Oligonucleotides used in this study. {#pone.0194443.t001g} Gene Oligonucleotide Cloning Step --------- ------------------------------------------- -------------------------- *Rag2* `CACCGTATTGTGGGTGGTTATCAGC` sgRNA *Rag2* `CACCGCCCTCAGCAGGAGCAGCTGA` sgRNA *Il2rγ* `AAACGCTGATAACCACCCACAATAC` sgRNA *Il2rγ* `AAACTCAGCTGCTCCTGCTGAGGGC` sgRNA *Rag2* `TTAATACGACTCACTATAGTATTGTGGGTGGTTATCAGC` *in vitro* Transcription *Il2rγ* `TTAATACGACTCACTATAGCCCTCAGCAGGAGCAGCTGA` *in vitro* Transcription *Rag2* `GGAAAAGCATGGGTGTTCTC` RFLP Assay *Rag2* `TCCTGGTATGCCAAGGAAAA` RFLP Assay *Il2rγ* `TCTCCCTGGGGACTTAGCTT` RFLP Assay *Il2rγ* `AGGGGCAGAGTAGGAGCACT` RFLP Assay One-cell embryo injection {#sec005} ------------------------- Mice were obtained from the Jackson Laboratory and maintained in the Whitehead Institute animal facility. *W*^*sh*^*/W*^*sh*^ female mice on a C57BL/6 background and ICR mice were used as embryo donors and foster mothers, respectively. Superovulated female *W*^*sh*^*/W*^*sh*^ mice (7--8 weeks old) were mated to *W*^*sh*^*/W*^*sh*^ stud males, and fertilized embryos were collected from oviducts. Cas9 mRNAs (from 20 ng/μL to 200 ng/μL) and sgRNAs (from 20 ng/μL to 50 ng/μL), as previously described \[[@pone.0194443.ref019],[@pone.0194443.ref020]\], were injected into the cytoplasm of fertilized eggs with well-recognized pronuclei in M2 medium (Sigma). Per previous methods, the injected zygotes were cultured in KSOM with amino acids at 37°C under 5% CO~2~ in air until blastocyst stage by 3.5 days. Thereafter, 15--25 blastocysts were transferred into the uterus of pseudo-pregnant ICR females at 2.5 days post-coitum \[[@pone.0194443.ref019]\]. Following blastocyst transfer, all pseudo-pregnant females and resulting *W*^*sh*^*/W*^*sh*^ immunocompromised mice were housed in autoclaved, sterile environments. Surveyor assay and RFLP analysis for genome modification {#sec006} -------------------------------------------------------- The Surveyor assay was performed as described \[[@pone.0194443.ref021]\]. Genomic DNA from targeted and control mice or blastocysts was extracted and PCR was performed using gene-specific primers ([Table 1](#pone.0194443.t001){ref-type="table"}) under the following conditions: 95°C for 5 min; 35 × (95°C for 30 s, 60°C for 30 s, 68°C for 40 s); 68°C for 2 min; hold at 4°C. PCR products were then denatured, annealed, and treated with Surveyor nuclease (Transgenomics). The DNA concentration of each band was measured on an ethidium-bromide-stained 10% acrylamide Criterion TBE gel (BioRad) and quantified using ImageJ software. For RFLP analysis, 10 μL of *Il2rγ* and 10 μL of *Rag2* PCR product were digested with PvuII. Digested DNA was separated on an ethidium-bromide-stained agarose gel (2%). For sequencing, PCR products were cloned using the Original TA Cloning Kit (Invitrogen), and mutations were identified by Sanger sequencing. Flow cytometry {#sec007} -------------- Blood was collected via the submandibular vein using a sterile Goldenrod animal lancet (Medipoint Inc.) and collected into a BD Microtainer MAP containing 1.0mg of K2 EDTA (Becton, Dickinson and Company). Additionally, mice were euthanized with CO~2~ according to institutional guidelines and their spleens were harvested and processed. Single-cell suspensions were generated from the blood and spleen samples and cells were stained using empirically determined concentrations of antibodies for 25 min on ice. Flow cytometry was used to separate T-cells (TCR-Beta, APC-Cy7), B cells (IgM, Alexa Fluor 488), Natural Killer (NK) cells (NK1.1, DsRed) and lymphocytes (FSC/SSC) from either blood or spleen samples. Flow cytometry data analysis was performed using FlowJo software. Tumor assay {#sec008} ----------- Human breast carcinoma cell line MCF7 was cultured (a gift from the laboratory of Dr. Robert Weinberg, Whitehead Institute for Biomedical Research) \[[@pone.0194443.ref022]\] and 1x10^5^ MCF7 cells were resuspended in 250 μL of Dulbecco's Modified Eagle's Medium (DMEM; Sigma) containing 10% fetal calf serum (FCS, Sigma). The MCF7 cell suspension was co-injected subcutaneously with 250 μL of matrigel into the flank of NOD SCID gamma mice, *W*^*sh*^*/W*^*sh*^ mice, and *W*^*sh*^*/W*^*sh*^ mice with CRISPR deletions of *Rag2* and *Il2rγ*. Before injection, mice were anesthetized with isoflurane, and allowed to recover to full ambulation immediately after injection. Mice were monitored 2x per day for any signs of adverse health and/or tumor growth. Tumors developed within 6 weeks of injection and animals were euthanized before tumor size exceeded 3 cm in diameter, or inhibited the ability of the mouse to ambulate, following institutional guidelines. Necropsy {#sec009} -------- A total of 15 *W*^*sh*^*/W*^*sh*^ adult mice with knockouts of *Rag2* and *Il2rγ*, comprising 12 female and 3 males from the breeding colony housed in autoclaved cages with autoclaved food, water, and hardwood bedding were submitted for necropsy because of unanticipated increased incidence of morbidity and mortality. Culture {#sec010} ------- At necropsy, brain, blood, kidney, and uterus were harvested for culture using sterile techniques. Tissue samples were plated on chromID™ CPS® agar plates (Biomérieux) \[[@pone.0194443.ref023]\]. *E*. *coli* colonies growing on CPS or MacConkey agar plates were initially identified by their ability to ferment lactose. Cultures were characterized as *E*. *coli* by API® 20 E (Biomérieux). Feces from an additional 17 immunocompromised mice without clinical signs were cultured for the presence of *pks+ E*. *coli*. Nine rederived mice (6 female and 3 male, *Rag2* and *Il2rγ W*^*sh*^*/W*^*sh*^) were also cultured for *pks+ E*. *coli*. DNA extraction and PCR amplification {#sec011} ------------------------------------ Thirteen *E*. *coli* isolates were collected with sterile plastic loops placed in sterile PBS in microfuge tubes and used for DNA extraction. The Roche High Pure PCR Template Preparation Kit was used for bacterial DNA extraction. DNA concentration was measured using NanoDrop 2000c (Thermo). Serotyping {#sec012} ---------- Four *E*. *coli* isolates from affected mice were submitted to the *E*. *coli* Reference Center at Pennsylvania State University for complete typing which included: O and H typing and analyses and PCR for heat-labile toxin (LT), heat-stabile toxin (*estA* and *estB*), Shiga-type toxin 1 and 2 (*Stx1* and *Stx2*), intimin gamma (*eae*), and cytotoxic necrotizing factor 1 and 2 (*cnf1* and *cnf2*). *Clb* genes and phylogenetic group identified by PCR {#sec013} ---------------------------------------------------- PCRs were performed to detect *pks (clbA* and *clbQ*) and *cdtB* genes with primers JPN42 and JPN46 for *clbA*, JPN55 and JPN56 for *clbQ*, and cdt1 and cdt2 for *cdtB* gene, respectively \[[@pone.0194443.ref016]\]. Phylogenetic groups of the isolates were determined with a set of primers published previously \[[@pone.0194443.ref016]\]. Cell culture conditions and gentamicin protection assay for colibactin cytotoxicity {#sec014} ----------------------------------------------------------------------------------- *E*. *coli* strains used for cytotoxicity assay included K12 (negative control), NC101 (colibactin positive control), and novel mouse isolates 1512290008 \[[@pone.0194443.ref024]\], 1512290026 \[[@pone.0194443.ref024]\], 1601050009, and 1601060011. The cell culture assay for colibactin cytotoxicity was performed as described previously with modifications \[[@pone.0194443.ref005]\]. HeLa S3 cells (ATCC CCL2.2) were grown and maintained in Eagle\'s Minimum Essential Medium (EMEM, ATCC) containing fetal calf serum (FCS, Sigma) and 1% Antibiotic-Antimycotic solution (Gibco) at 37°C with 5% CO~2~. Fifty-thousand cells were seeded onto 12-well cell culture plates and incubated at 37°C with 5% CO~2~ for 24 h. Overnight cultures of *E*. *coli* strains were grown for 2 h at 37°C and then adjusted to a multiplicity of infection (MOI, number of bacteria per cell at the onset of infection) of 25 and 100. Following inoculation, plates were centrifuged at 200 g for 10 minutes to facilitate bacteria interaction and then incubated at 37°C with 5% CO~2~ for 4 h. Cells were then washed with EMEM and replaced with EMEM containing 10% fetal calf serum (FCS, Sigma) and 200 μg/mL gentamicin (Gibco). Following 72 h incubation, plates were stained with Diff-quick stain (ThermoScientific). Cells were then inspected under a microscope for confluence and morphological changes. Images were captured with Axiovert-10 microscope (Zeiss) using Image Pro-Plus software version 7.0 at 20x magnification. Cell viability was estimated in the Diff-quick stained plates by quantifying signal intensity at 700 nm using an Odyssey CLx plate reader (LI-COR) and Image Studio version 5.2 software (LI-COR). Histopathology and peptic nucleic acid (PNA) fluorescent in situ hybridization {#sec015} ------------------------------------------------------------------------------ Tissue sections of kidney, heart, lung, brain, spinal cord, ear, and reproductive tract from clinically affected mice were fixed in 10% formalin, embedded in paraffin, sectioned at \~4 μm, and stained with hematoxylin and eosin (H&E). The tissue sections from these clinically affected mice were subjected to PNA fluorescent in situ hybridization by *GNR Traffic Light*^*TM*^ PNA FISH^®^ probe to detect *E*. *coli* \[[@pone.0194443.ref016]\]. The tissue sections were incubated with the *E*. *coli* specific PNA FISH probe (AdvanDx, Inc., Woburn, MA) for hybridization at 55°C for 105 min and examined for fluorescence with a Zeiss Axioskop 2 plus microscope. Digital images were acquired with a QImaging-QIClick camera (QImaging, Surrey, BC, Canada). Rederivation of *Rag2* and *Il2rγ W*^*sh*^*/W*^*sh*^ mice {#sec016} --------------------------------------------------------- Mice were rederived by embryo transfer using standard techniques and recipient female Swiss mice free of *pks+ E*. *coli* and other murine pathogens. These mice have been maintained in a separate room under barrier conditions, which consists of maintaining the mice in autoclaved caging with autoclaved bedding, water, and pelleted diet. Statistical analyses {#sec017} -------------------- Differences in experimental groups were determined by the Student's t test as appropriate. p values \<0.05 were considered significant. Results {#sec018} ======= CRISPR/Cas9 deletion of *Rag2* and *Il2rγ* in the *W*^*sh*^*/W*^*sh*^ mouse background {#sec019} -------------------------------------------------------------------------------------- Deletion of the host immune response is crucial for survival of human cells after transplantation into murine models, as well as allowing for gene therapy and other clinical approaches to be applied to the animal model without risk of an adverse systemic immune response to the therapy being tested for efficacy. The onset of efficient and rapid genome editing technologies has increased the ability for different mouse strains to become immune-compromised for translational research studies. As an example, CRISPR and Cas proteins have been demonstrated as an efficient gene targeting technology \[[@pone.0194443.ref018], [@pone.0194443.ref019]\]. In our study, capped polyadenylated Cas9 mRNA was produced by *in vitro* transcription and co-injected into pro-nuclear stage one-cell *W*^*sh*^*/W*^*sh*^ mouse embryos with sgRNAs to knockout *Rag2* ([Fig 1A](#pone.0194443.g001){ref-type="fig"}) and *Il2r****γ*** ([Fig 1D](#pone.0194443.g001){ref-type="fig"}). *Rag2* encodes a protein that is involved in the initiation of V(D)J recombination during B and T cell development while *Il2r****γ*** encodes a protein involved in the growth and maturation of T-cells, B-cells, and NK cells. Therefore, CRISPR/Cas9-induced in-dels or insertions in *Il2r****γ*** and *Rag2* that cause a loss of function of the protein should create an immunocompromised *W*^*sh*^*/W*^*sh*^ mouse strain for further research studies. {#pone.0194443.g001} RFLP assays on DNA from post-natal mice displayed 3 out of 8 (37.5%) mice had altered *Rag2* alleles ([Fig 1B](#pone.0194443.g001){ref-type="fig"}), while 4 out of 8 (50%) mice had altered alleles for *Il2r****γ*** ([Fig 1E](#pone.0194443.g001){ref-type="fig"}). Two males (\#1: WT for *Rag2* and homozygous mutant for *Il2r****γ***; and \#2: homozygous mutant for *Rag2* and unknown, likely a large deletion mutant, for *Il2r****γ***) and two females (\#3: homozygous mutant for both *Rag2* and *Il2r****γ***; and \#5: homozygous mutant for both *Rag2* and *Il2r****γ***) were chosen for continued breeding to create further generations of *Rag2* and *Il2r****γ*** knockout mice ([Fig 1B and 1E](#pone.0194443.g001){ref-type="fig"}, asterisks). Sanger sequencing of the F1 generation displayed four mutant alleles in the population of *Rag2* ([Fig 1C](#pone.0194443.g001){ref-type="fig"}) and 3 mutant alleles in the population for *Il2r****γ*** ([Fig 1F](#pone.0194443.g001){ref-type="fig"}) that were maintained through generations in the colony. Loss of the B, T, and NK cells {#sec020} ------------------------------ Blood samples were collected from *W*^*sh*^*/W*^*sh*^ mice and first generation (F1) *W*^*sh*^*/W*^*sh*^ mice with double allelic mutations and heterozygous mutations in *Rag2* and *Il2r****γ***. As loss-of-function of *Rag2* and *Il2r****γ*** should deplete lymphocytes, and in particular the lymphocyte-derived B-, T-, and NK cells, blood cells were sorted by flow cytometry to quantify the numbers of lymphocytes in the F1 generation compared to control mice ([Fig 2A](#pone.0194443.g002){ref-type="fig"}). There was a significant reduction in the number of lymphocytes (47.85% for control mice, 4.30% for mutant mice; p = 0.0002) in the blood from the F1 generation (N ≥ 4) compared to the control mouse cohort (N ≥ 2). Closer examination of the blood to ascertain specific lymphocyte-derived cell types revealed a significant reduction in both T-cells (17.26% for control mice, 0.96% for mutant mice; p = 0.0297) and B-cells (59.29% for control mice, 10.91% for mutant mice; p \< 0.0001) in the samples from the F1 generation in comparison to control mice ([Fig 2B](#pone.0194443.g002){ref-type="fig"}). NK cells were not present via staining with NK1.1 antibody in any of the F1 generation mice (N = 6; data not shown). Thus, within the first generation of mice, loss of *Rag2* and *Il2r****γ*** even in heterozygotes led to a reduction in the presence of B-, T-, and NK cells. {#pone.0194443.g002} *W*^*sh*^*/W*^*sh*^ F2 mice carrying double allelic mutations in *Rag2* and *Il2r****γ*** were euthanized and spleen samples were taken for further quantification of immune cells. No lymph nodes were detectable for dissection in any of the F2 generation mice (N = 3; data not shown). Flow cytometry was performed on all three F2 mutant mice compared to two controls, and no visible populations of T-cells, B-cells, NK cells, or NKT cells were detected ([Fig 2C](#pone.0194443.g002){ref-type="fig"}). Percentages of each of the B-cells, T-cells, NK cells, NKT-cells, and other, non-immune spleen cells from the flow cytometry analysis are displayed in pie charts for the control mouse (top right) and immune-compromised mutant mouse (bottom right; only a small background of B-cells are visible; [Fig 2C](#pone.0194443.g002){ref-type="fig"}). Thus, F2 mice carrying mutations in both alleles of *Rag2* and *Il2r****γ*** have a complete loss of B-, T-, and NK cells as expected with loss-of-function of *Rag2* and *Il2r****γ***. A functional immunocompromised state in the *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice {#sec021} ------------------------------------------------------------------------------------------------ To ensure that the loss of B-, T-, and NK cells led to a functional immunocompromised state, MCF7 cells were injected subcutaneously into the flank of 5 (3 males and 2 females) *W*^*sh*^*/W*^*sh*^ mice with mutations on both alleles of *Rag2* and *Il2r****γ***. Additionally, MCF7 cells were injected subcutaneously into the flank of 1 NOD SCID gamma mouse, a known immunocompromised mouse model, as well as 4 (2 males and 2 females) *W*^*sh*^*/W*^*sh*^ mice with functional immune systems. Six weeks post-injection, tumors developed in all mice (100%, N = 5) with different *Rag2* and *Il2r****γ*** knockout mutant alleles and a tumor was also noted in the NOD SCID gamma control mouse with a defective immune system which allows proliferation of human mammary carcinoma cells([Fig 2E and 2F](#pone.0194443.g002){ref-type="fig"}, arrows). No tumors formed in any of the *W*^*sh*^*/W*^*sh*^ mice at 7 weeks post-injections, as expected in mice with a functional immune system ([Fig 2D](#pone.0194443.g002){ref-type="fig"}). The rejection of MCF7 cells in the parental strain (*W*^*sh*^*/W*^*sh*^ mice) without CRISPR deletion of *Rag2* and *Il2rγ* and proliferation of MCF7 cells in *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice suggests that these mice are in an immune-compromised state. *E*. *coli* isolation from the *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice {#sec022} ----------------------------------------------------------------------------------- A random survey of the feces from 17 immunocompromised mice being maintained in the colony indicted that 100% of the mice were colonized with *pks+ E*. *coli* ([S1 Fig](#pone.0194443.s001){ref-type="supplementary-material"}). All 17 pks+ *E*. *coli* isolates belonged to phylogenetic group B2 ([S2 Fig](#pone.0194443.s002){ref-type="supplementary-material"}). A sudden increase in morbidity and mortality developed within the *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice, where mouse colony numbers dramatically declined and it was uncertain whether or not the colony could be maintained. This rapid increase in morbidity and mortality warranted an investigation, as these mice were housed in sterile, autoclaved environments within the barrier animal facility and were not removed at any time. *E*. *coli*, *Enterococcus faecalis*, *Enterococcus faecium*, and *Staphylococcus xylosus* were isolated from multiple tissues and blood of the mice submitted for evaluation. The presence of *E*. *coli* was confirmed in 93% of the cases either by culture or FISH analysis. *Enterococcus* spp. (40%) and *S*. *xylosus* (7%) were concurrently isolated along with *E*. *coli* from these affected mice ([Table 2](#pone.0194443.t002){ref-type="table"}). 10.1371/journal.pone.0194443.t002 ###### Characterization of *E*. *coli* isolates from *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice. {#pone.0194443.t002g} Isolate ID Serotype Culture Sample Phylogenetic Group *clbA* *clbQ* *cdtB* CYT ------------ ---------- ---------------- -------------------- ---------- ---------- ---------- ---------- 1512290008 O2:H6 Blood B2 Positive Positive Negative Positive 1512290026 O2:H6 Blood B2 Positive Positive Negative Positive 1601050009 O2:H6 Blood B2 Positive Positive Negative Positive 1601060011 O2:H6 Blood B2 Positive Positive Negative Positive 1601190003 NT Uterus B2 Positive Positive Negative NT 1601190003 NT Kidney B2 Positive Positive Negative NT 1601260012 NT Uterus B2 Positive Positive Negative NT 1601260012 NT Kidney B2 Positive Positive Negative NT 1602030009 NT Uterus B2 Positive Positive Negative NT 1606150011 NT Kidney B2 Positive Positive Negative NT 1608160086 NT Kidney B2 Positive Positive Negative NT 1609230019 NT Uterus B2 Positive Positive Negative NT 1609290016 NT Uterus B2 Positive Positive Negative NT CTY, Colibactin cytotoxicity assay; NT, not tested Serotyping and analyses of other virulence factors in *E*. *coli* {#sec023} ----------------------------------------------------------------- Four isolates of *E*. *coli* from four mice were determined to be serotype O2:H6 ([Table 2](#pone.0194443.t002){ref-type="table"}). The 4 isolates were positive for *pks (clbA* and *clbQ*) and colibactin cytotoxicity assay. None of these 4 isolates were positive for heat-labile toxin (*elt*), heat-stable enterotoxin a and b (*estA* and *estB*), Shiga-like toxin types 1 and 2 (*stx1* and *stx2*), intimin-*γ* (*eae*), and cytotoxic necrotizing factors 1 and 2 (*cnf1* and *cnf2*). Rederivation of *Rag2 and Il2rγ knockout W*^*sh*^*/W*^*sh*^ *mice* {#sec024} ------------------------------------------------------------------ Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of *pks+ E*. *coli*. Six adult female and 3 adult male *Rag2* and *Il2rγ W*^*sh*^*/W*^*sh*^ mice were cultured for the presence of *pks+ E*. *coli* strains using standard culture methods. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. *Clb* genes and phylogenetic group identified by PCR {#sec025} ---------------------------------------------------- Thirteen *E*. *coli* strains from ten mice were positive for *clbA* and *clbQ* genes and negative for *cdtB* ([Table 2](#pone.0194443.t002){ref-type="table"} and [Fig 3](#pone.0194443.g003){ref-type="fig"}). All isolates belong in phylogenetic group B2 ([Table 2](#pone.0194443.t002){ref-type="table"} and [Fig 4](#pone.0194443.g004){ref-type="fig"}). {#pone.0194443.g003} {#pone.0194443.g004} *Clb*-encoding mouse *E*. *coli* isolates exert colibactin cytotoxicity {#sec026} ----------------------------------------------------------------------- To confirm cytotoxic colibactin activity in the clb-encoding novel mouse *E*. *coli* isolates, HeLa cells were transiently infected with live bacteria at MOI 25 and 100. Novel mouse isolates 1512290008, 1512290026, 1601050009, and 1601060011 induced dose-dependent cytotoxicity. About 30--40% of cells survived after MOI 25, while less than 20% survived after MOI 100 ([Fig 5A](#pone.0194443.g005){ref-type="fig"}). Surviving cells appeared megalocytic and were phenotypically indistinguishable to that caused by Clb-encoding NC101 infection ([Fig 5B](#pone.0194443.g005){ref-type="fig"}). Media and K12 negative controls maintained similar cell confluence and morphology ([Fig 5B](#pone.0194443.g005){ref-type="fig"}). {#pone.0194443.g005} Histopathological evaluation {#sec027} ---------------------------- A total of fifteen *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice comprising 12 females and 3 males were submitted for post mortem examination. Both males and females became moribund during our study, usually before 1 month of age; however, breeding females became ill at a rapid rate, either during the final week of pregnancy or within 1 week of nursing pups, and therefore more severely ill female mice were available for analysis. [Table 3](#pone.0194443.t003){ref-type="table"} summarizes the major organ systems affected, nature of the lesion and bacteria isolated and identified in different organ systems by microbial culture and FISH assay. The three affected male mice had pale tan shrunken kidneys with irregularly pitted capsular surface. Histological changes in the kidneys from the affected male mice were consistent with severe necrotizing granulocytic pyelonephritis with abundant bacteria and tubular degeneration and necrosis. Further characterization was performed on tissues/organs from the 12 female mice, to avoid any confounding factors related to gender. 10.1371/journal.pone.0194443.t003 ###### Pathology of spontaneous infection by *E*. *coli* in *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice. {#pone.0194443.t003g} ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Accession \# Sex Microbial Culture Histologic lesions FISH- *E*. *coli* -------------- -------- ------------------------------------------------------------------------------------------------------------------------------ --------------------------------------------------------------------------------- --------------------- ----- ------------------------------------------------------------------------------------------------------------------------- ------------------------------------ 1512290008 Male bl,n,lu,s,k l n Pyelonephritis and interstitial pneumonia ND 1512290026 Male bl,n,lu,k bl Pyelonephritis Kidney 1601050009 Female bl Metritis with macerated fetus, pyelonephritis, pneumonia,\ Uterus and placenta otitis media (bilateral), and epicarditis 1601190003 Female lu, li, k,u Placentitis, metritis, and pneumonia Uterus and placenta 1601260012 Female lu,li,k,u lu,k,u Metritis, vaginitis myocarditis, meningoencephalitis, otitis media(unilateral), and\ Kidneys, uterus, vagina, and heart pneumonia 1602030009 Female lu,u lu Metritis, vaginitis, myocarditis, meningoencephalomyelitis, necrotizing hepatitis, unilateral otitis media and rhinitis Uterus and placenta 1606150011 Male k, br,bl k, br\ br Nephropathy (intralesional bacteria) Kidneys 1608160086 Female k Metritis and necrotizing suppurative pyelonephritis Kidneys 1609230019 Female u Metritis, nephritis, interstitial pneumonia, and meningitis Uterus 1609290016 Female k\ k,u k,u Pyelonephritis and interstitial pneumonia ND ND16-00081 Female No bacterial isolation was performed, as the animals were found dead\ Metritis with abundant intralesional bacteria and macerated fetus and pneumonia Uterus and placenta ND16-00082 Female Cystitis and vaginitis with intralesional bacilli\ Bladder and urogenital tract ND16-00083 Female Metritis and unilateral otitis media with intrahistiocytic bacteria Uterus ND16-00094 Female Endometritis with macerated fetus and unilateral otitis media ND ND16-00164 Female Pyelonephritis\ Uterus cystitis, endometritis, bilateral otitis media, rhinitis with intralesional bacteria, interstitial pneumonia, and meningitis ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- blood-bl, brain-br, kidneys-k, lungs-lu, liver-li, nares-n, spleen-s and uterus-u and ND: Not detected. On gross examination, 4 of the 12 female mice were diagnosed with vaginal septum and pyometra. The vaginal orifice of the affected female mice was longitudinally bisected by a thin dorsoventral band of tissue. The urogenital tract and uterus contained pale tan to yellow, turbid, viscous material. Histopathological examination of the uterus and urogenital tract of 11 female mice revealed intraluminal cellular debris admixed with degenerate granulocytes, mononuclear cells, and myriad of bacteria ([Fig 6A](#pone.0194443.g006){ref-type="fig"}). The endometrium and underlying myometrium were also infiltrated by inflammatory aggregates, in a multifocal pattern. {#pone.0194443.g006} The ear canal was affected by mild to moderate unilateral or bilateral otitis media in 6 out of 12 female mice. The otitis media was characterized by loss of ciliated epithelial lining and the lumen contained cellular debris, pale eosinophilic material, and low numbers of granulocytes and histiocytes admixed with extracellular and intrahistiocytic bacilli ([Fig 6B](#pone.0194443.g006){ref-type="fig"}). The heart was affected by necrotizing granulocytic myocarditis ([Fig 6C](#pone.0194443.g006){ref-type="fig"}) and epicarditis with mural septic thrombi in 3 out of 12 female mice. The aortic valve was focally disrupted by necrotizing valvulitis characterized by loss of fibrous connective tissue and cartilage, with granulocytic and mononuclear cell infiltrates admixed with bacterial colonies. The aorta was completely occluded by septic thrombi and transmurally, the aortic wall was infiltrated by granulocytes and mononuclear cells ([Fig 6D](#pone.0194443.g006){ref-type="fig"}). In 5 out of 12 female mice, the renal architecture was multifocally disrupted by necrosis and inflammation. The renal pelvis was dilated and contained hemorrhage and inflammatory cells ([Fig 7A](#pone.0194443.g007){ref-type="fig"}). The affected renal tubules were lined by attenuated epithelium and occasionally the tubular epithelial cells with intact basement membrane were lost. The tubular lumen contained cellular debris and abundant bacteria. The tubular interstitium was multifocally infiltrated by granulocytes and mononuclear cells ([Fig 7B](#pone.0194443.g007){ref-type="fig"}). {#pone.0194443.g007} In 4 out of 12 female mice, the brain and spinal cord were variably affected by necrohemorrhagic meningoencephalomyelitis. The affected brain and spinal cord parenchyma were disrupted by minimal amounts of hemorrhage and vacuolations. These affected areas were infiltrated by granulocytes and mononuclear infiltrates and the meningeal blood vessels were disrupted by necrotizing vasculitis and fibrin thrombi ([Fig 7C and 7D](#pone.0194443.g007){ref-type="fig"}). Other significant findings in these affected mice included interstitial pneumonia, rhinitis, necrotizing granulocytic hepatitis, and mild granulocytic ureteritis and cystitis. Since all of the above mentioned organs showed significant findings and signs of disease, we hypothesized that these tissues may have been infected with *E*. *coli*. Thus, representative tissue sections from the uterus, kidneys, urogenital tract, heart, and ear were subjected to *E*. *coli* specific PNA-FISH assay \[[@pone.0194443.ref016]\]. The PNA-FISH assay revealed *E*. *coli* positivity by the presence of multifocal and discrete clumps of intra luminal bacilli in the tissue sections from the above mentioned organs ([Fig 6A](#pone.0194443.g006){ref-type="fig"}). Further, four rederived mice (3 males and 1 female), confirmed negative for *pks+ E*. *coli*, were analyzed by a detailed gross necropsy followed by histopathological evaluation of the liver, spleen, pancreas, mesenteric lymph node, kidneys, heart, lungs, urinary bladder, reproductive organs (testes, epididymis, prostate or uterus, ovaries, vagina and vulva). No significant pathological findings were noted other than incidental findings of sparse to minimal perivascular interstitial lympho-plasmacytic inflammatory aggregates in the all the renal medulla and/or pelvis of all animals. The female mouse had a gravid uterus. Discussion {#sec028} ========== Although *E*. *coli* infections in laboratory mice have been previously reported \[[@pone.0194443.ref010],[@pone.0194443.ref013],[@pone.0194443.ref025]--[@pone.0194443.ref029]\], a description of spontaneous disease which includes biochemical, genetic, and phenotypic characterization of *pks+ E*. *coli* isolates has not been undertaken. In one of the previously reported studies, natural infection with uncharacterized *E*. *coli* was associated with urogenital lesions, pneumonia, and septicemia in mice \[[@pone.0194443.ref026]\]. Characterization of *pks+ E*. *coli* NC101 in experimentally infected IL10^-/-^ mice and the natural and experimental characterization of O7:H7:K1 in IL10^-/-^ mice indicated the pathogenic potential of Clb-encoding *E*. *coli* in experimentally infected susceptible animals \[[@pone.0194443.ref010],[@pone.0194443.ref011],[@pone.0194443.ref013],[@pone.0194443.ref030]\]. The current study characterized *pks+ E*. *coli* in clinically affected mice with genitourinary infection, septicemia, and meningitis. The mouse *E*. *coli* isolates encoding two Clb genes (*clbQ* and *clbA*), were cytotoxic to cells *in vitro*. The isolates in this study were serotype O2:H6, which is the same serotype as that of NC101 \[[@pone.0194443.ref012]\]. Serotype O2 in humans is also commonly associated with UTIs, septicemia, and meningitis \[[@pone.0194443.ref031]\]. Importantly, barrier-maintained rederived mice without *pks+ E*. *coli* in their intestinal tracts have produced multiple litters without adverse health effects, nor have lesions noted in the original outbreak observed. The isolates in the current study were cytotoxic to cells *in vitro* suggesting that mouse Clb-encoding *E*. *coli* have the potential to induce clinical disease in laboratory animals, as noted in this study. In a rodent model of septicemia, 8--9 week old female C57BL/6J mice were injected subcutaneously with a *pks+ E*. *coli* colibactin producing 018:K1:H7, ExPEC strain S15 isolated from a meningitis case in a newborn infant \[[@pone.0194443.ref014]\]. The *E*. *coli* induced a profound lymphopenia in septicemic mice, which was attenuated when an isogenic mutant lacking colibactin genotoxin activity was injected into the mice \[[@pone.0194443.ref014]\]. The authors argued that the production of colibactin by *E*. *coli* exacerbates lymphopenia associated with septicemia, reducing the chances that the mice, and by association humans, would survive *pks+ E*. *coli* induced sepsis \[[@pone.0194443.ref014]\]. The prolonged immunosuppression often referred to as immunoparalysis, in sepsis cases, predisposes the patients to nosocomial infections and reactivation of latent viruses \[[@pone.0194443.ref032],[@pone.0194443.ref033]\]. Isolation of commensals such as *Enterococcus* spp. and *S*. *xylosus* along with *pks+ E*. *coli* from these affected mice in our study may be attributed to immunosuppression. *E*. *faecalis* has been shown to exacerbate pathogenic effects induced by Gram-negative bacterial infection \[[@pone.0194443.ref034]\]. However, *E*. *coli* was isolated in 93% of cases in this study either with or without concurrent isolation of *Enterococcus* spp. and *S*. *xylosus*. Recent studies have demonstrated the importance of colibactin in intestinal colonization and invasion of other organs by *pks+ E*. *coli* that leads to systemic infection in neonates \[[@pone.0194443.ref015]\]. Hence, there is a distinct possibility that *pks+ E*. *coli* infection facilitates colonization and/or co-infection by other commensal bacteria such as *Enterococcus* spp. and *S*. *xylosus*. Furthermore, our findings of *pks+ E*. *coli* strains responsible for urosepsis in C57BL/6 *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice reinforce the susceptibility of immuno-compromised mice to opportunistic pathogens. The breeding colony of *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice also presented with a variable incidence of vaginal septa. The vaginal septal defect apparently has not been noted to interfere in the overall breeding performance in this colony. Our results however, suggest that the vaginal defect may have predisposed the female mice to *pks+ E*. *coli* urosepsis. The congenital vaginal septal defect has been reported previously and has been associated with compromising successful breeding and parturition, in both mice and rats \[[@pone.0194443.ref035],[@pone.0194443.ref036]\]. The vaginal defect has been linked to metritis in rats used on reproductive toxicology studies \[[@pone.0194443.ref036]\]. Although rats with vaginal septal defects were screened for rat microbial pathogens, none were noted. However, the authors mention that bacterial cultures obtained from abnormal uterine contents identified the presence of abundant *E*. *coli*. Unfortunately, further molecular identification of putative virulence factors was not undertaken \[[@pone.0194443.ref036]\]. Investigators have also capitalized on the use of neonatal rats to explore the pathogenicity of colibactin expressing *E*. *coli*. Two day old Wistar rats were orally dosed with *pks+ E*. *coli* which efficiently colonized the intestinal tract and translocated across the immature GI tract resulting in sepsis \[[@pone.0194443.ref015]\]. Inactivation of the *clbA* and *clbP* genes responsible for colibactin production in *pks+ E*. *coli* strains, significantly reduced the capacity of the *E*. *coli* strain A192PP to colonize the intestine, translocate, induce septicemia, and cause the death of the neonatal rats \[[@pone.0194443.ref015]\]. This neonatal rat model replicates the age dependency seen in newborn children where ExPEC B2 strains of *pks+ E*. *coli* are noted to express colibactin and cause significant disease \[[@pone.0194443.ref037]\]. Indeed, ExPEC infections are associated with urosepsis, bacteremia, and neonatal meningitis in infants \[[@pone.0194443.ref015],[@pone.0194443.ref038],[@pone.0194443.ref039]\]. *E*. *coli* isolates from human prostatitis cases also have been characterized as ExPEC and some encode Clb \[[@pone.0194443.ref040]\]. Previously, we reported the draft genomes of two *E*. *coli* isolates from this study that were recovered from the blood of mice (accession numbers 1512290008 and 1512290026)\[[@pone.0194443.ref024]\]. Both genomes contained a complete \~54-kb PKS pathogenicity island required for colibactin synthesis\[[@pone.0194443.ref024]\]. Cytolethal distending toxin (*cdt*), cytotoxic necrotizing factor (*cnf*), and cycle inhibiting factor (*cif*) genes known to cause megalocytosis were not present in either of these genomes, which substantiated our PCR results, suggesting the megalocytic cytotoxicity observed in our *in vitro* infection experiments was the result of colibactin activity. Aside from PKS, both of our novel *pks+ E*. *coli* isolate genomes also contained homologous virulence genes that regulate colonization, adherence, immune evasion, cytotoxicity, and iron acquisition at intestinal, vascular, and renal sites by EPEC and ExPEC strains. These genes included enterobactin siderophore receptor protein\[[@pone.0194443.ref041],[@pone.0194443.ref042]\], s-fimbriae minor subunit\[[@pone.0194443.ref043]\], glutamate decarboxylase\[[@pone.0194443.ref044],[@pone.0194443.ref045]\], per-activated serine protease autotransporter enterotoxin\[[@pone.0194443.ref046]--[@pone.0194443.ref048]\], and iss/bor protein precursor\[[@pone.0194443.ref049],[@pone.0194443.ref050]\]. The genomic presence of *pks* and other virulence genes supports our hypothesis that these novel mouse *E*. *coli* isolates have the pathogenic potential to infect and induce clinical diseases in susceptible hosts. The genetic profile of the *Rag2* and *Il2rγ* knockout *W*^*sh*^*/W*^*sh*^ mice predisposes them to progressive kidney disease. The underlying mutation in the *W*^*sh*^*/W*^*sh*^ strains is an inversion mutation in the upstream transcriptional regulatory elements of the c-kit gene that also disrupts the corin gene \[[@pone.0194443.ref051]--[@pone.0194443.ref053]\]. Corin is a trypsin-like transmembrane serine protease that activates atrial natriuretic peptide, which regulates salt-water balance and blood pressure by promoting renal natriuresis. In humans, abnormalities in corin expression result in hypertension \[[@pone.0194443.ref054]\], heart failure \[[@pone.0194443.ref055]\], and chronic kidney disease \[[@pone.0194443.ref056]\]. In our study, the tubular lesions noted in the mice closely resemble obstructive nephropathy. However, there was no evidence for obstruction of renal tubules, urinary bladder, and urethra by crystals, proteinaceous plugs, and associated inflammation. The observation from our study and findings reported in previous studies suggests decreased expression of c-kit and corin could have resulted in the chronic progressive nephropathy observed in these mice due to impaired sodium excretion. However, the exact mechanism by which c-kit and corin exert this detrimental effect on murine renal tubular epithelial cells and whether this defect predisposes the mice to urosepsis remains to be elucidated. In summary, the pks pathogenicity island is present in a high percentage of extraintestinal pathogenic B2 strains of *E*. *coli* and exhibits an increased predilection of causing bacteremia \[[@pone.0194443.ref003]\]. This current finding of *pks+ E*. *coli* strains in cases of urosepsis, as well as *pks*+ *E*. *coli* in the feces of clinically normal *Rag2* and *Il2rγ*^-/-^ mice, and our recent characterization of *E*. *coli* strains from clinically normal and clinically affected laboratory mice indicates that mice can be colonized with Clb-encoding cytotoxic and pathogenic *E*. *coli* \[[@pone.0194443.ref016]\]. Phylogenetic B2 *pks+ E*. *coli* strains have a high likelihood of persistently colonizing infants and are commonly isolated in adults. Given that *pks+ E*. *coli* B2 isolates are present within 6-12-month-old mice obtained from a mouse colony endemically infected with *pks+ E*. *coli*, this supports the hypothesis that mice can also be persistently infected with this colibactin-producing *E*. *coli* \[[@pone.0194443.ref016]\]. The epidemiology and virulence determinants of mouse *pks+ E*. *coli* strains should be investigated further. Also, studies examining their impact in mouse models of inflammation, both intestinal and extraintestinal should be conducted. Importantly, our findings of urosepsis in immunocompromised mice caused by *pks+ E*. *coli* strain O2:H6 represents an opportunity to develop experimental mouse models to investigate the pathogenic potential of *pks+ E*. *coli* and cytotoxin-associated induction of urogenital disease and septicemia noted in humans. Many murine models used in human research are genetically engineered to be immune-compromised in order to test human cell transplantations and viral gene therapies for translational research. This report is the first to describe acute morbidity and mortality associated with *pks+ E*. *coli* urosepsis and meningitis in immunocompromised mice. As *E*. *coli* strains have been frequently isolated from rodents, but are not routinely or completely characterized, our study highlights the importance of routine and complete characterization of *pks+ E*. *coli*. Also, health surveillance strategies to ensure that these *E*. *coli* strains are excluded from specific pathogen-free mouse colonies should be instituted. Supporting information {#sec029} ====================== ###### Amplification of *clb*A and *clb*Q in DNA from 17 mouse *E*.*coli* isolates. Top row: *clb*A gene, bottom row: *clb*Q gene. Lane 1 to lane 17, 17 E.coli isolates from mice fecal samples; line 18, NC101 (positive control); line 19, no DNA control; line 20, 1 Kb plus molecular marker. (PDF) ###### Click here for additional data file. ###### Phylogenetic group determination of *E*.*coli*. Lane 1 to lane 17, 17 E.coli isolates from mice fecal samples; line 18, NC101 (B2 group positive control); line 19, no DNA control; line 20, 1 Kb plus molecular marker. (PDF) ###### Click here for additional data file. We wish to thank the DCM and Whitehead staff for their assistance, as well as Alyssa Pappa for her assistance with manuscript preparation. [^1]: **Competing Interests:**The authors have declared that no competing interests exist.

More than 13 000 new cases of rectal cancer are diagnosed every year in the UK ([Office for National Statistics, 2003](#bib38){ref-type="other"}; [Northern Ireland Cancer Registry, 2007](#bib36){ref-type="other"}; [Welsh Cancer Intelligence and Surveillance Unit, 2007](#bib49){ref-type="other"}). At least 10% of these cancers are inoperable because of involvement of, or penetration through, the mesorectal fascia and implication of other organs ([Devita *et al*, 2001](#bib10){ref-type="other"}). For patients with inoperable disease, prognosis is poor. Radiotherapy and, more recently, combined chemo-radiotherapy (CRT) regimens have been shown to be able to reduce the stage and size of advanced tumours, making them amenable to resection. The ideal combined-modality pre-operative regimen is, however, yet to be determined. To date, phase I/II trials have assessed the effect of radiation in combination with 5-fluorouracil (5-FU) or another fluoropyrimidine with or without leucovorin (to enhance the actions of 5-FU) ([Videtic *et al*, 1998](#bib48){ref-type="other"}; [Rodel *et al*, 2000](#bib40){ref-type="other"}). Post-operative chemotherapy is also sometimes included in the CRT regimen ([Minsky *et al*, 1993](#bib32){ref-type="other"}). Overall, results from these studies have been favourable, resulting in the widespread adoption of CRT regimens, mostly based on 5-FU, in the neoadjuvant treatment of inoperable rectal cancer. Recently, several novel therapies have emerged with activity in rectal cancer. These include raltitrexed, irinotecan, oxaliplatin and oral fluoropyrimidines, including uracil-tegafur and capecitabine ([de la Torre *et al*, 1999](#bib9){ref-type="other"}; [Kalofonos *et al*, 2003](#bib24){ref-type="other"}; [Fernandez-Martos *et al*, 2004](#bib14){ref-type="other"}; [Gambacorta *et al*, 2004a](#bib17){ref-type="other"}, [2004b](#bib18){ref-type="other"}; [Hofheinz *et al*, 2005](#bib22){ref-type="other"}; [Klautke *et al*, 2005](#bib25){ref-type="other"}, [2006](#bib27){ref-type="other"}). Despite the advent of oral fluoropyrimidines, intravenous infusion remains popular in Europe and the United States, because nausea, vomiting and diarrhoea can affect compliance and absorption of oral drugs ([Minsky *et al*, 1993](#bib32){ref-type="other"}; [Rodel *et al*, 2000](#bib40){ref-type="other"}; [Mohiuddin *et al*, 2006](#bib34){ref-type="other"}). For metastatic colorectal cancer, the addition of irinotecan to treatment with 5-FU and leucovorin significantly improves progression-free and overall survival ([Douillard *et al*, 2000](#bib11){ref-type="other"}; [Saltz *et al*, 2000](#bib44){ref-type="other"}). This finding has resulted in an interest in developing its use as part of a combined protocol for the treatment of locally advanced disease. Several phase I and phase II trials have investigated the use of irinotecan as a radiation sensitizer in CRT regimens for locally advanced rectal cancer; however, results are mostly published as meeting abstracts, with short-term follow up only. Irinotecan has been administered as monotherapy combined with radiation, but most of the studies have assessed the CRT regimen of irinotecan plus 5-FU and radiotherapy ([Minsky *et al*, 1999](#bib31){ref-type="other"}; [Klautke *et al*, 2001](#bib26){ref-type="other"}, [2005](#bib25){ref-type="other"}, [2006](#bib27){ref-type="other"}; [Kalofonos *et al*, 2003](#bib24){ref-type="other"}; [Mehta *et al*, 2003](#bib30){ref-type="other"}; [Navarro *et al*, 2003](#bib37){ref-type="other"}, [2007](#bib35){ref-type="other"}; [Mitchell *et al*, 2004](#bib33){ref-type="other"}; [Sebag-Montefiore *et al*, 2005](#bib45){ref-type="other"}). Overall, the results of these preliminary studies suggest promising activity of irinotecan-containing regimens. Six small studies are published as full papers ([Kalofonos *et al*, 2003](#bib24){ref-type="other"}; [Mehta *et al*, 2003](#bib30){ref-type="other"}; [Klautke *et al*, 2005](#bib25){ref-type="other"}; [Mohiuddin *et al*, 2006](#bib34){ref-type="other"}; [Glynne-Jones *et al*, 2007](#bib20){ref-type="other"}; [Willeke *et al*, 2007](#bib50){ref-type="other"}). To conclude, the combined RCT regimen irinotecan plus 5-FU with radiation therapy appears to be a tolerable adjuvant therapy ([Kalofonos *et al*, 2003](#bib24){ref-type="other"}) that results in a good tumour response when given pre-operatively to patients with rectal cancer in whom resection is either possible or uncertain ([Mehta *et al*, 2003](#bib30){ref-type="other"}; [Klautke *et al*, 2005](#bib25){ref-type="other"}). This current study was undertaken to rigorously assess and provide long-term follow-up data for the use of irinotecan in combination with 5-FU and radiotherapy for inoperable, locally advanced (T3/T4) rectal tumours. The aims of this study are two fold: (a) to establish a safe dose of intravenous (iv) irinotecan for administration in combination with a standard course of 5-FU and concomitant pelvic radiotherapy to patients with locally advanced non-resectable rectal cancer and (b) to assess the effectiveness of the regimen, in terms of post-treatment resectability of tumours, and disease-free and overall survival. MATERIALS AND METHODS ===================== Participants ------------ Patients with unresectable rectal cancer were recruited between September 2001 and December 2003 from four centres in the UK: the Christie Hospital NHS Trust, Manchester; Clatterbridge Hospital, Liverpool; North Wales Cancer Treatment Centre, Rhyl; and the Royal Preston Hospital, Preston. The tumour stage of all patients was determined clinically after examination under general anaesthetic and following computed tomography (CT) of the chest, abdomen and pelvis, or magnetic resonance imaging (MRI) of the abdomen or pelvis. In cases of discrepancy, the highest tumour stage was used. All recruited patients were diagnosed as having non-resectable disease, defined as involving the edge of the mesorectal fascia or adherence of the tumour to an adjacent structure or organ, preventing an attempt at resection with a negative circumferential resection margin (CRM). The patients had a WHO performance score of less than two and adequate hepatic and renal function (bilirubin, creatinine, aspartate aminotransferase or alanine aminotransferase \<2.5 × upper limit of normal) and bone marrow reserve (absolute neutrophil count \>2.0 × 10^9^; platelets \>100 × 10^9^; haemoglobin \>10.0 g l^−1^). Patients were excluded if they had received previous radiotherapy or chemotherapy, had been diagnosed with metastatic disease or had a past or current malignancy at other sites (with the exception of adequately treated *in-situ* carcinoma of the cervix uteri and non-melanoma carcinoma of the skin). All patients provided written informed consent and the study was performed in accordance with the ethical principles outlined in the declaration of Helsinki. Ethical approval for this study was obtained from the ethics committees of all participating centres. Procedure --------- Radiotherapy was given by planned target volume to the pelvic area, treating with 25 fractions at 1.8 Gy/fraction via four fields, with all fields treated daily (45 Gy total dose). The planned target volume was defined using simulator or CT planning. The planned target volume was to include: 3 cm superior, inferior and lateral of the extent of the gross tumour volume, but no higher than the sacral promontory; the posterior border of the most posterior aspect of the sacrum; and 2 cm anterior to the tumour or the anterior rectal wall, whichever was the most anterior. Treatment was given from Monday to Friday for 5 weeks. 5-FU 200 mg m^−2^ per day was administered by continuous iv infusion 7 days/week throughout radiotherapy. Irinotecan (50--70 mg m^−2^) was administered on days 1, 8, 15 and 22 via a 30-min iv infusion (day 1 being the first day of radiotherapy; [Figure 1](#fig1){ref-type="fig"}). During phase I of the study, cohorts of three patients received irinotecan in a dose escalating between 50 and 70 mg m^−2^. Development of grade III and IV toxicity guided the calculation of the maximum-tolerated dose. Toxicity assessments were made according to the National Cancer Institute Common Toxicity Criteria version 3.0. In the event of non-haematological toxicity, the dose was modified as follows: for grade I toxicity, no treatment modification was made; for grade II toxicity, 5-FU and irinotecan were interrupted until resolution to grade 0, then resumed at full dose; for grade III toxicity, 5-FU and irinotecan were interrupted until resolution to grade 0, then resumed at 75% of the initial dose. For patients with grade II or grade III toxicity, radiotherapy was continued, but reviewed daily. For those with grade IV toxicity, 5-FU and irinotecan were discontinued and radiotherapy was interrupted; if symptoms did not resolve to grades 0--I within 2 weeks, radiotherapy was permanently discontinued. Total mesorectal excisional (TME) surgery was planned 6--8 weeks after the completion of CRT in all patients. Note that only 28 of 31 had surgery, however, since two developed metastatic disease and one had a myocardial infarction prior to surgery. Tumour response --------------- Every patient\'s treatment response was measured clinically and pathologically. Tumours were staged clinically, according to the TNM (tumour, node, metastasis) system from both pre-surgical and post-surgical CT/MRI scans. Tumour downstaging was considered complete when all lesions disappeared, and partial when tumour size decreased by more than 30%. Disease was considered to be progressive if tumour size increased by more than 20% (maximum transaxial or craniocaudal dimension). Stable disease was defined as tumours that decreased by less than 30% or increased by less than 20%, using the RECIST criteria (response evaluation criteria in solid tumours) ([Therasse *et al*, 2000](#bib46){ref-type="other"}). Patients were seen at least once a week during CRT, then weekly for 6 weeks after the end of radiotherapy. Tumours that were resected after CRT were also classified by histopathology. The presence of residual disease macroscopically at surgery and microscopically in terms of tumour involvement (or clearance) at the CRM was used to classify the surgical resection as R~2~ (macroscopic residual disease), R~1~ (microscopic residual disease---CRM involved or disease within 1 mm of the CRM) or R~0~ (confirmed clear margins---CRM negative). A pathological complete response was recorded when no tumour was seen. Tumours in which only a few abnormal cells were seen without a residual tumour mass were considered indicative of microfocal residual disease. Pathological Dukes\' staging was also recorded. After surgery, patients were followed up by the treating surgeon, according to the local protocol, at a maximum interval of every 6 months; serial carcinoembryonic antigen estimation was performed for up to 5 years. CT scanning of the abdomen and pelvis was also carried out at 6, 12 and 24 months following surgery. Statistical analysis -------------------- The probabilities of disease-free and overall survival were calculated using the Kaplan−Meier method. Disease-free and overall survival rates were calculated from the date of registration to the time of death or relapse. Disease-free survival was defined as the absence of relapse or recurrence of the primary cancer or death from any cause. RESULTS ======= Dose escalation and patient characteristics ------------------------------------------- Twelve patients were recruited for phase I of the study. In the first cohort of three patients, who received irinotecan 50 mg m^−2^, there was one episode of grade III diarrhoea. Three more patients were enrolled for treatment with this dose, and none experienced a grade III or grade IV dose-limiting toxicity (DLT). There was also no DLT in the cohort of three patients who received irinotecan 60 mg m^−2^. However, two of the three patients in the cohort receiving irinotecan 70 mg m^−2^ experienced a DLT. For phase II of the study, data were collected for 31 patients (23 (74%) men, eight (26%) women) aged 37--76 years (mean 61.8 years), with inoperable rectal cancer. All had threatened CRM and clinically fixed tumour as judged by examination under anaesthetic and CT and/or MR imaging. In all 20 patients had tumours 0--5 cm from the anal verge, six 5--10 cm and five 10--15 cm. They were treated with the maximum tolerated dose of irinotecan, which was judged to be 60 mg m^−2^ on the basis of results from the phase I trial. CRT administration and tolerability ----------------------------------- One patient requested discontinuation of chemotherapy after 4 weeks of treatment, having previously required reductions in the irinotecan dose, but completed radiotherapy and subsequently underwent surgery. The other thirty (97%) patients received the prescribed four doses of irinotecan. Patients who completed the treatment protocol received a mean of 34.3 days (range, 28--35 days) of continuous iv 5-FU infusion. The 5-FU dose was reduced in three (10%) patients; in two of these patients, the dose was reduced because of adverse events (pyrexia and diarrhoea) and in one the dose reduction was the result of an administration error. A mean radiation dose of 44.7 Gy (range, 37.8--45.0 Gy) was administered to patients who completed treatment. Only two patients had their total radiotherapy dose reduced. The toxicity observed was generally mild. The most frequent grade III toxicities experienced by the cohort were diarrhoea (*n*=4 (13%)) and constipation (*n*=2 (6%)). Only one (3%) patient experienced grade IV toxicity, which was fever without infection. The incidence of grade III and grade IV toxicities experienced by patients during treatment is given in [Table 1](#tbl1){ref-type="table"}. One patient died from an unrelated myocardial infarction approximately 4 weeks post-treatment and two patients were diagnosed with metastatic disease shortly after completing the full treatment protocol. Twenty-eight patients underwent surgery. One patient died in the immediate post-operative period from an anastomotic leak; 27 patients were evaluable for follow-up after resection. CRT efficacy ------------ All of the patients recruited underwent staging before treatment (*n*=31). In 21 (68%) patients the tumour was classified as T3, and in 10 (32%) as T4. Eleven (35%) patients had no nodal metastases (N0), 15 (48%) were staged as N1 and five (16%) were staged as N2. Before surgery, 24 of 28 (86%) patients underwent repeat staging with pelvic MRI. Of these, seven (29%) were judged to have a complete clinical response, 12 (50%) a partial clinical response and four (17%) stable disease. One (4%) patient showed progressive disease on this MRI scan. Therefore, 19 of 24 (79%) patients assessed by MRI before surgery had downsized tumours on clinical staging. In total, 28 of 31 (90%) patients underwent surgical resection and 27 of 31 (87%) had complete macroscopic resections. Surgery was performed 6--8 weeks after the end of radiotherapy. From the macroscopic and microscopic histopathology reports, patients\' residual disease was summarised as R~0~ in 22 of 27 (81%) patients, R~1~ in four (15%) patients and as R~2~ in one (4%) patient. Histological staging of the evaluable patients after surgery revealed five of 27 (19%) patients in whom there was a pathological complete response. Four of the 27 (15%) patients had minimal residual (microfocal) disease. Eight of the 27 (30%) patients had Dukes\' stage B disease and 10 (37%) patients had Dukes\' stage C disease. After a median follow-up of 24 months (range, 8--43 months), 11 of 31 (35%) patients who were enrolled had experienced disease progression (two of these occurred pre-operatively) and three (10%) patients had died of their disease. There were five local recurrences in the pelvis---two with distant spread beyond the pelvis and three with local recurrence. Six (19%) patients relapsed with metastatic disease only. Of the patients who died, one death was the result of local progression of rectal cancer without distant spread, and two deaths were due to metastatic disease without local relapse. The estimate for 3-year relapse-free survival was 65% ([Figure 2](#fig2){ref-type="fig"}), and the estimate for 3-year overall survival was 90% ([Figure 3](#fig3){ref-type="fig"}). DISCUSSION ========== The results of this study suggest that even the most locally advanced rectal cancer can be downstaged and rendered operable, or even cured, by use of an appropriate CRT regimen. Furthermore, the short-term and long-term morbidity of irinotecan in combination with 5-FU and radiotherapy is acceptable, and follow-up has revealed excellent local and distant disease control. Other studies have not produced such good results with such low toxicity; however, this is a phase I/II study with carefully selected patients, and further work is needed to confirm these preliminary findings. Of 31 patients with non-resectable stage T3/T4 rectal cancer, 28 (90%) underwent TME surgical resection of their tumour following treatment with irinotecan, 5-FU and radiotherapy. Complete surgical resection (R~0~ or R~1~) was achieved in 26 patients (84% of the initial 31, or 96% of all those who were assessable post-surgery). The R~0~ rate for all patients was 22/31 (71%), or 22/27 (81%) of those assessable post-surgery. Phase II trials rarely include a comparator group, making the results from a single study difficult to assess in isolation. While comparisons between studies are limited because of inherent variability in patients, disease severity and treatments, an assessment of the relative efficacy and tolerability of a treatment is nevertheless helpful for determining whether a regimen offers potential benefits, justifying further study. Prior to this study, it was thought that the problematic side effect of diarrhoea associated with each of irinotecan, 5-FU and radiation would cause severe toxicity should the agents be combined, rendering the regimen too toxic for routine use. However, the rate of grade III/IV toxicity (10%) noted in this study is comparable with that reported for oxaliplatin regimes (18--30%) ([Alonso *et al*, 2004](#bib2){ref-type="other"}, [2007](#bib1){ref-type="other"}; [Casado *et al*, 2004](#bib6){ref-type="other"}; [Tucci *et al*, 2004](#bib47){ref-type="other"}; [Fakih *et al*, 2005](#bib13){ref-type="other"}; [Machiels *et al*, 2005](#bib29){ref-type="other"}; [Avallone *et al*, 2006](#bib4){ref-type="other"}; [Rutten *et al*, 2006](#bib42){ref-type="other"}; [Rödel *et al*, 2007](#bib41){ref-type="other"}). Indeed, the findings of this study suggest a safety profile for irinotecan, 5-FU and radiotherapy that is comparable with previously reported findings with capecitabine and radiotherapy (rate of grade III/IV diarrhoea 12--28%) ([Mehta *et al*, 2003](#bib30){ref-type="other"}; [Navarro *et al*, 2003](#bib37){ref-type="other"}; [Mitchell *et al*, 2004](#bib33){ref-type="other"}; [Mohiuddin *et al*, 2006](#bib34){ref-type="other"}; [Willeke *et al*, 2007](#bib50){ref-type="other"}), but using a lower radiation dose (45 Gy as opposed to 50.4 Gy in the capecitabine regimen). Furthermore, the resection rate achieved in this study is higher than that observed in studies using radiotherapy alone (range, 64--75%) ([Emami *et al*, 1982](#bib12){ref-type="other"}; [Frykholm *et al*, 2001](#bib16){ref-type="other"}) or in some studies of CRT regimens (62--95%) ([de la Torre *et al*, 1999](#bib9){ref-type="other"}; [Frykholm *et al*, 2001](#bib16){ref-type="other"}; [Klautke *et al*, 2006](#bib27){ref-type="other"}; [Glynne-Jones *et al*, 2007](#bib20){ref-type="other"}), and it is comparable to that cited in other reports of research with irinotecan (90--100%) ([Minsky *et al*, 1999](#bib31){ref-type="other"}; [Mitchell *et al*, 2004](#bib33){ref-type="other"}; [Klautke *et al*, 2005](#bib25){ref-type="other"}; [Sebag-Montefiore *et al*, 2005](#bib45){ref-type="other"}). It is worth noting, that this current trial included only patients with T3/T4 cancers---that is patients with more advanced disease than those included in previously published studies. Clinical staging in this study was performed by CT/MRI in a subset of patients after CRT treatment. At the time that the study was conducted, CT/MRI was used for investigational purposes only and was, therefore, not undertaken in all of the patients. Clinical staging revealed a complete clinical response rate of 29, and 79% of patients were judged to have downstaged tumours on MRI after treatment. Few studies have reported MR downstaging following CRT, although in a previous study of 31 patients treated with 5-FU and radiotherapy the tumour volume was decreased by at least half in 77% of patients ([Glynne-Jones *et al*, 2007](#bib20){ref-type="other"}). The proportion of patients obtaining a pathological complete response (19%) was similar to that reported for some other irinotecan-based protocols (11--25%) ([Mitchell *et al*, 2004](#bib33){ref-type="other"}; [Klautke *et al*, 2005](#bib25){ref-type="other"}; [Sebag-Montefiore *et al*, 2005](#bib45){ref-type="other"}). Considering all irinotecan-based protocols together, the pathological complete response seems to be greater than that observed with radiotherapy alone (4%) ([Frykholm *et al*, 2001](#bib16){ref-type="other"}) and comparable with most other fluoropyrimidine CRT regimens (12--28%) ([de la Torre *et al*, 1999](#bib9){ref-type="other"}; [Freyer *et al*, 2001](#bib15){ref-type="other"}; [Frykholm *et al*, 2001](#bib16){ref-type="other"}; [Carraro *et al*, 2002](#bib5){ref-type="other"}; [Gérard *et al*, 2003](#bib19){ref-type="other"}; [Aschele *et al*, 2005](#bib3){ref-type="other"}; [Loi *et al*, 2005](#bib28){ref-type="other"}; [Avallone *et al*, 2006](#bib4){ref-type="other"}; [Klautke *et al*, 2006](#bib27){ref-type="other"}; [Pucciarelli *et al*, 2006](#bib39){ref-type="other"}; [Ryan *et al*, 2006](#bib43){ref-type="other"}). Four (15%) patients in the current study had microfocal residual disease, meaning overall that nine (29% of the phase II patient population) patients had a complete, or almost complete, response to treatment. Considering CRM involvement, 81% of patients in this study who underwent TME surgery were classed as R~0~ after surgery (CRM-negative). In studies that report data on the number of CRM-negative cases, results range from 61--74% with radiotherapy ([Emami *et al*, 1982](#bib12){ref-type="other"}; [Frykholm *et al*, 2001](#bib16){ref-type="other"}) to 72--89% with 5-FU-based protocols ([de la Torre *et al*, 1999](#bib9){ref-type="other"}; [Frykholm *et al*, 2001](#bib16){ref-type="other"}; [Avallone *et al*, 2006](#bib4){ref-type="other"}; [Klautke *et al*, 2006](#bib27){ref-type="other"}; [Glynne-Jones *et al*, 2007](#bib20){ref-type="other"}) or 88--89% when irinotecan is given as part of the CRT regimen ([Klautke *et al*, 2005](#bib25){ref-type="other"}; [Sebag-Montefiore *et al*, 2005](#bib45){ref-type="other"}). The addition of irinotecan to CRT regimens does not, therefore, seem to increase the high rate of CRM-negative surgery reported previously, but CRT certainly seems to be more successful than radiotherapy alone. It should be noted that many of the studies mentioned above did not use CT/MRI to assess CRM involvement pre-treatment, but relied on clinical examination alone. Patients with early-stage tumours may, therefore, have been classified as CRM-negative, resulting in overestimation of this parameter. A negative CRM may provide an early surrogate test of efficacy and a predictor of local recurrence, enabling the rapid evaluation of the many new treatment regimens currently being investigated ([Cunningham *et al*, 2002](#bib8){ref-type="other"}). Patients who respond to pre-operative chemo-radiation have improved disease-free and overall survival compared with non-responders ([Janjan *et al*, 2001](#bib23){ref-type="other"}). Therefore, the histological complete response rate and the impact of neoadjuvant therapy on clinical staging are other potential short-term end points that may be early indicators of disease-free and overall survival. Comparison of the disease-free and overall survival results observed here with those of previous studies is complicated by the disease severity at recruitment, the duration of follow-up and whether second colorectal cancers or deaths due to causes other than rectal cancer are included. The only previous study of patients with rectal cancer receiving CRT with irinotecan and 5-FU reporting survival results was in patients with locally advanced rectal cancer who received neoadjuvant therapy. The 4-year disease-free and overall survival rates were 73 and 66%, respectively ([Klautke *et al*, 2005](#bib25){ref-type="other"}). CRT in patients with non-resectable rectal cancer has been shown to significantly improve disease-free survival compared with radiotherapy alone, and may increase overall survival ([Frykholm *et al*, 2001](#bib16){ref-type="other"}). Overall survival rates following CRT treatment with 5-FU have been reported to range from 26 to 73% ([de la Torre *et al*, 1999](#bib9){ref-type="other"}; [Frykholm *et al*, 2001](#bib16){ref-type="other"}; [Avallone *et al*, 2006](#bib4){ref-type="other"}; [Klautke *et al*, 2006](#bib27){ref-type="other"}; [Glynne-Jones *et al*, 2007](#bib20){ref-type="other"}). Reported rates of disease-free or relapse-free survival range from 54 to 74% ([de la Torre *et al*, 1999](#bib9){ref-type="other"}; [Frykholm *et al*, 2001](#bib16){ref-type="other"}; [Avallone *et al*, 2006](#bib4){ref-type="other"}; [Klautke *et al*, 2006](#bib27){ref-type="other"}; [Glynne-Jones *et al*, 2007](#bib20){ref-type="other"}), but again the studies vary in duration of follow-up. The overall and disease-free survival rates observed in the current study are, on the whole, greater than those seen previously. A prospective, randomised trial is now needed to confirm in larger numbers the impact on survival of adding irinotecan to CRT with 5-FU in patients with non-resectable rectal cancer. Further work also continues to investigate whether using oral fluoropyrimidine capecitabine in place of 5-FU in the regimen can produce acceptable toxicity rates, especially with respect to diarrhoea ([Gollins *et al*, 2006](#bib21){ref-type="other"}). Once complete, a prospective, randomised phase III study is proposed, comparing irinotecan 5-FU and radiotherapy with (i) irinotecan, capecitabine and radiotherapy and (ii) oxaliplatin, 5-FU and radiotherapy. This randomised trial will identify the most effective regimen and provide important information on whether R~0~ resection rates and pathological complete remission rates predict long-term survival. When combined with 5-FU and radiotherapy, irinotecan appears to be both effective and safe for the neoadjuvant treatment of rectal cancer. The findings published here suggest that irinotecan has a place in CRT regimens for locally advanced rectal cancer. The regimen should now be compared with other non-irinotecan-based CRT regimens in a randomised phase III trial. Aventis provided irinotecan. Abbie Pound (Succinct Healthcare Communications and Consultancy) provided editorial support for this article funded by an educational grant from Pfizer. {#fig1} {#fig2} {#fig3} ###### Grade III and Grade IV toxicities experienced by patients during treatment **Toxicity** **Grade III, *n* (%)** **Grade IV, *n* (%)** --------------------------- ------------------------ ----------------------- Diarrhoea 4 (13) 0 (0) Constipation 2 (6) 0 (0) Skin sores 1 (3) 0 (0) Lethargy 1 (3) 0 (0) Infection 1 (3) 0 (0) Abdominal cramping 1 (3) 0 (0) Chest pain 1 (3) 0 (0) Fever (without infection) 0 (0) 1 (3) Grade III/IV neutropenia 0 (0) 0 (0)

Files are available from the Open Science Framework platform (<https://osf.io/c3ztu/>). Introduction {#sec001} ============ To execute a hand reaching movement, the central nervous system needs to localize the target with respect to the hand. Its position can be derived from inputs provided by one or multiple sensory modalities such as vision, audition or somatosensation. Multisensory integration is referred to as the combination of information arising from different sensory modalities to form a unified and coherent representation of our environment and body. Accordingly, the brain combines all the relevant sensory information about the object of interest in order to decrease the variance (the uncertainty) and build a more reliable representation of that object \[[@pone.0199627.ref001],[@pone.0199627.ref002]\]. Indeed, it has been shown that spatial localization was less variable for visual-auditory targets than for targets specified by vision or audition only \[[@pone.0199627.ref003],[@pone.0199627.ref004]\]. These findings suggest that the more sensory information available about the target, the more accurate its estimate. When pointing to unseen parts of our own body (e.g. the opposite index finger), the position of the target is derived from proprioceptive signals. Proprioception corresponds to the sense of our body position in space. Consistent with the principles of multisensory integration, it has been found that participants better matched the position of their index finger when they could see their opposite arm during movement than when being blindfolded during the task \[[@pone.0199627.ref005],[@pone.0199627.ref006]\]. The localization of the fingertip was more precise in the presence of both vision and proprioception than when using visual or proprioceptive signals only. These results provide evidence that fingertip localization can be more precise if another sensory modality, in addition to proprioception, provides further information about the finger position. Unlike vision, touch may not provide additional information about finger position in space, since fingertip tactile information theoretically remains the same irrespective of the postural configuration of the upper limb. However, touch can be regarded as a possible source of additional information for position sense, since touch and proprioception, although considered as separate modalities, have been shown to closely interact with each other. Behavioral studies have shown that tactile perception can be modulated by changes in proprioceptive signals, induced by active changes in hand posture \[[@pone.0199627.ref007]\] or tendon vibration \[[@pone.0199627.ref008]\]. Conversely, a finger-position matching task has been reported to be affected by nerve block and cutaneous anesthesia \[[@pone.0199627.ref009]\], indicating that cutaneous afferents may provide a crude position sense for the fingers. Moreover, it has been shown that the localization of a proprioceptive target (i.e., the fingertip) was improved when participants contacted a surface with their target fingertip, which provides them with tactile feedback \[[@pone.0199627.ref010]--[@pone.0199627.ref012]\]. Similarly, accuracy in pointing movements was enhanced when endpoint contact occurred with the effector fingertip \[[@pone.0199627.ref013]\]. In contrast, digital anesthesia resulted in impaired fingertip localization \[[@pone.0199627.ref011]\] as well as decreased movement accuracy during typing \[[@pone.0199627.ref014]\]. This relationship between touch and proprioception is likely to be explained by the convergence of proprioceptive and tactile signals at the cortical level; electrophysiological recordings in monkey have shown that neurons in the hand representation of the primary somatosensory cortex code both tactile and proprioceptive modalities during a reach-to-grasp task \[[@pone.0199627.ref012],[@pone.0199627.ref015]\]. It has also been established that neurons in the somatosensory cortex have both cutaneous and proprioceptive receptive fields \[[@pone.0199627.ref016],[@pone.0199627.ref017]\]. Taken together, these findings suggest that tactile afferent information may contribute to proprioception and improve the accuracy of the hand proprioceptive estimate. The skin contains several mechanoreceptors, including Meissner and Pacinian corpuscles. Meissner corpuscles are located in the superficial layers of the skin and are sensitive to light touch while Pacinian corpuscles are found in deeper layers and respond to deep skin pressure and vibration. The properties of these two receptors suggest that they might be activated by fingertip contact; Pacinian and Meissner corpuscles are fastadapting receptors which are both sensitive to abrupt but not sustained stimuli \[[@pone.0199627.ref018]\], such as when a finger makes or breaks contact with an object. Therefore, it is difficult to distinguish the relative contributions of Pacinian and Meissner corpuscules to the enhancement of proprioception following fingertip contact with a surface \[[@pone.0199627.ref010]--[@pone.0199627.ref013]\]. However, these two types of mechanoreceptors show different responses to cutaneous vibrations. Meissner corpuscles respond to low frequencies, 10--80 Hz, whereas Pacinian corpuscles are sensitive to vibrations at higher frequencies, 80--450 Hz \[[@pone.0199627.ref019]\]. Consequently, by stimulating either of these receptors, it would be possible to know which one contributes to the enhancement of proprioceptive localization. It has been shown that the ability to detect flexion and extension movements imposed at the interphalangeal joints of a finger was impaired when 300 Hz vibrations were applied to the adjacent or the test digit. In contrast, vibrotactile stimuli at 30 Hz did not alter proprioception in the finger \[[@pone.0199627.ref020],[@pone.0199627.ref021]\]. The detection of passive finger movements at the interphalangeal joints is thus impaired by the specific activation of Pacinian, but not Meissner, afferents. These results demonstrate that vibrotactile stimulation can modulate proprioceptive acuity in a passive perceptual task, in which no action is involved. However, to our knowledge this has not been tested in a motor task, such as reaching, where the target location corresponds to the position of the fingertip. The goal of the present study was to investigate the influence of vibrotactile information on the proprioceptive localization of the finger in a motor context. To this purpose, we asked participants to perform reaches to proprioceptively defined targets. They reached with the right index finger (reaching finger) to the unseen left index finger (target finger), which was passively displaced to different locations. Tactile vibrations at 30 or 300 Hz were delivered to the target index fingertip prior to movement onset. When vibrations are applied, the left index finger receives tactile information, in addition to existing proprioceptive information, about its location in space. In order to reach accurately, we presume that the brain constructs a reliable estimate of the target finger position using all the sensory information available. As suggested by previous studies \[[@pone.0199627.ref019]-- [@pone.0199627.ref021]\], high- and low-frequency vibrations are more likely to activate Pacinian and Meissner corpuscles, respectively. We thus used 30 and 300 Hz vibrotactile stimulations to determine if one of these two mechanoreceptors contribute more than the other to touch-proprioception integration, or whether they both contribute to finger proprioceptive localization. We measured reach endpoint accuracy and precision to assess the effect of vibrations. We found that vibrotactile stimulations delivered at low and high frequencies improved both accuracy and precision of finger localization in a proprioceptive reaching task. A control condition in which the vibration was applied elsewhere on the body showed that this improvement in proprioceptive localization cannot be attributed to a global arousal enhancement induced by the tactile stimulus. Methods {#sec002} ======= Participants {#sec003} ------------ Nineteen participants took part in this study (12 females, mean ± SD age = 25.3 ± 10.7 years). They were all right-handed, as assessed by the Edinburgh Handedness Inventory and all had normal or corrected-to-normal vision. Participants were administered a questionnaire to ensure that they did not suffer from neurological, sensory or motor deficits, which may have interfered with their performance. All gave informed written consent to participate in this experiment which conformed to the Declaration of Helsinki (2008) for experiments on human subjects. All experimental procedures were approved by the health research ethics committee in France (CPP Nord-Ouest I, Lyon, 2017-A02562-51) and at the University of Montreal (17-034-CERES-D). Apparatus {#sec004} --------- Participants sat in a dark room on a height-adjustable chair in front of a slanted table. Their head was held steady on a chin rest, aligned with their body midline. A wide-screen OLED monitor (55 inches diagonal, 1920 x 1080 pixels, LG) was placed facing downwards above the table and a half-reflecting mirror was positioned in between the screen and the table so that the screen was projected onto the tabletop surface. The half-reflecting mirror prevented participants from seeing their hands unless there was light underneath the mirror; in that case vision of the hand was possible. Participants performed a proprioceptive pointing task. They were asked to reach with the right index finger (reaching finger) to the unseen left index finger (target finger). Participants' left forearm was resting on a platform in such a way that when the left index finger was aligned with the body midline, the elbow was located on average 17.5 cm on the left relative to the center ([Fig 1A](#pone.0199627.g001){ref-type="fig"}). The left forearm was positioned at an angle of 47°. The forearm platform was motorized and could move laterally (left or right) to different target positions. The target locations for the left index finger were at -10, -5, 0, +5 and +10 cm with respect to the body midline. A tactor was positioned on the left index fingertip and connected to an amplifier (TactAmp 4.2, Dancer Design, England, United Kingdom) that delivered tactile vibrations at a frequency of either 30 or 300 Hz. Eye movements were monitored using an EyeLink 1000 Plus (SR Research, Mississauga, Ontario, Canada) at a sampling rate of 1000 Hz. The positions of both left and right index fingers were measured using an Optotrak 3D Investigator recording system (NDI, Waterloo, Canada). This system recorded the position of two infrared emitting diodes, each one attached to the tip of each index finger. The movement of the infrared markers was tracked and sampled at a rate of 500 Hz. {#pone.0199627.g001} Procedure {#sec005} --------- The sequence of a trial is depicted in [Fig 1B](#pone.0199627.g001){ref-type="fig"}. At the beginning of each trial, a light was switched on for 1500 ms so that participants could see their hands. At the same time, a red dot aligned with the body midline and 15 cm distant from the torso was displayed also for 1500 ms. The red dot served as a start position and participants were asked to align their right index fingertip with the red dot and to keep it in this position until they began reaching. They kept their right fingertip balanced in the air above the table (no surface contact). As soon as the start position disappeared, a white fixation cross was displayed and participants were required to fixate the cross until the end of the trial. The fixation cross was aligned with the body midline above the target positions. After two seconds, the motorized platform moved the left target index finger to one of the 5 possible target locations. To prevent participants from learning proprioceptive target positions across trials, the platform made several back-and-forth movements (from 1 to 5) before stopping on a target location. Then, a vibrotactile stimulation was applied to the left target index fingertip for 1000 ms. Vibrations could be delivered at 0 (no vibration condition), 30 or 300 Hz. After the tactile stimulation, a first auditory tone signaled to the participants that they could begin reaching with their right hand. Participants had 2 s to complete their reach before a second auditory tone instructed them to return to the start position. Participants were instructed to reach to a location just above their left fingertip, pause in the air, then return to the start position. Specifically, participants were asked to reach to where they thought their left index fingertip was as accurately as possible and to avoid contacting their left target index finger with their right hand. To ensure that participants performed the task properly, they first did a practice block and were asked to report when finger-finger contact occurred during the experiment. The next trial began after 750 ms. Each block was composed of 15 trials (3 vibration frequencies x 5 target positions). Each of the possible combinations of target and vibration frequency was presented in a random order. Each participant completed between 10 and 20 blocks to obtain at least 4 trials for each combination of target and vibration frequency. To test for a possible effect of the fingertip vibration by arousal enhancement, participants performed 2 additional control blocks in which the location of the vibrotactile stimulus was varied. The trials were identical to those in the main experiment except that the vibration was delivered to the left shoulder. The order of the blocks (control and main experiments) was counterbalanced across participants. Data analysis {#sec006} ------------- In this proprioceptive reaching task, errors were defined as the difference between the positions of the left (target) index finger and the right (reaching) index finger at the end of the movement. Since the position of the target hand was varied in the horizontal axis, we only considered reaching errors in the *x*-direction. Errors in the *x*-direction were computed for each trial by subtracting the *x*-position of the target hand from the *x*position of the right-hand endpoint. The constant *x*-error was expressed in mm and corresponded to the mean error in the *x*-direction for each target; this measure provides an estimate of the accuracy of the localization of the fingertip position. We used dispersion error as a measure of reach precision \[[@pone.0199627.ref022]\]. The dispersion error corresponded to the surface area of the endpoints around each corresponding target, it was expressed in mm^2^ and computed with the following formula: SD~x~ × SD~y~ × π. With SD~x~ and SD~y~ corresponding to the standard deviations of reach endpoints in the x- and y-direction, respectively. Dispersion error provides an estimate of the precision of the localization of the fingertip position. Constant *x*-errors and dispersion errors were first calculated for each participant, vibration condition and target position, then averaged across target positions and participants. To test the influence of the vibration frequency on constant *x*errors and dispersion errors, a one-way repeated measures ANOVA was performed for each type of error separately. Similarly, one-way repeated measures ANOVAs were performed on constant x-errors and dispersion errors for each of the two attention control experiments. Tukey HSD tests were used for post-hoc comparisons of the means. The threshold for statistical significance was set at 0.05 for all analyses. Results {#sec007} ======= Reach endpoints relative to the five possible proprioceptive target locations are depicted in [Fig 2](#pone.0199627.g002){ref-type="fig"} for one participant. Endpoints are represented for all three experimental conditions: the no vibration condition, the 30 Hz and the 300 Hz vibration conditions (in red, green and blue, respectively). The one-standard-deviation ellipses correspond to the dispersion of reach endpoints and the center of ellipses represents the mean reach error. For this participant, reach endpoints were overall more scattered when no tactile stimulus was delivered to the target index fingertip (in red) compared to the 30 Hz and the 300 Hz vibration conditions (in green and blue). {#pone.0199627.g002} Constant *x*-errors {#sec008} ------------------- A one-way repeated measures ANOVA was performed on the reach errors in the *x*direction. It revealed a significant effect of the vibration frequency (*F*~2,36~ = 7.56, *p* = 0.002, *η*^*2*^ = 41.30). Constant *x*-errors were equal to 12.3 ± 2.5 mm (mean ± SE) when no vibration was applied prior to the proprioceptive reach onset ([Fig 3](#pone.0199627.g003){ref-type="fig"}). Constant *x*-errors in the 30 Hz and the 300 Hz vibration conditions were 8.9 ± 2.1 mm and 9.1 ± 2.2 mm, respectively. Post-hoc tests showed that, compared to the no vibration condition, constant *x*-errors were significantly reduced when either a 30 Hz (mean ± SE of the difference = 3.4 ± 1.2 mm, *t*~18~ = 3.29, *p* = 0.004) or a 300 Hz vibration (3.2 ± 1.0 mm, *t*~18~ = 3.09, *p* = 0.006) was delivered to the left target index finger. However, constant *x*-errors were not significantly different between the 30 Hz and the 300 Hz vibration conditions (-0.2 ± 0.7 mm, *t*~18~ = 0.02, *p* \> 0.05). {#pone.0199627.g003} Dispersion errors {#sec009} ----------------- The one-way repeated measures ANOVA on the dispersion errors was also significant (*F*~2,36~ = 4.61, *p* = 0.017, *η*^*2*^ = 46.83). As depicted in [Fig 4](#pone.0199627.g004){ref-type="fig"}, the greatest dispersion errors are observed in the no vibration condition (923.1 ± 91.3 mm^2^), followed by dispersion errors in the 30 Hz (819.7 ± 82.3 mm^2^) and then in the 300 Hz vibration condition (805.6 ± 68.8 mm^2^). Post-hoc tests showed that these errors significantly decreased when vibrotactile stimuli were delivered at 30 Hz (103.3 ± 41.6 mm^2^, *t*~18~ = 2.10, *p* = 0.050) and 300 Hz (117.5 ± 44.0 mm^2^, *t*~18~ = 2.49, *p* = 0.023). Dispersion errors between the 30 and 300 Hz vibration conditions did not significantly differ from each other (14.2 ± 37.6 mm^2^, *t*~18~ = 0.08, *p* \> 0.05). {#pone.0199627.g004} Control experiment {#sec010} ------------------ We found that both high- and low-frequency vibrations applied to the target fingertip reduced constant *x*-errors and dispersion errors, suggesting that tactile information was combined with proprioception and improved spatial localization of the left target finger. It could be due to an effect of the vibration by arousal enhancement. To test for this, we investigated whether constant *x*-errors and dispersion errors changed when the vibration was delivered elsewhere. Thus, participants performed a control experiment where the vibrotactile stimulus was applied to the left shoulder. If reduced errors consecutive to the vibration of the left fingertip result from an effect of arousal, they should also be observed in this control condition. If they rather result from a specific spatial multi-sensory integration, then stimulation on the shoulder should not improve constant or dispersion errors compared to the no vibration condition. The constant *x*-errors and the dispersion errors when the vibration was applied on the left shoulder are shown in [Fig 5A and 5B](#pone.0199627.g005){ref-type="fig"}, respectively. Constant *x*-errors in the no, 30 Hz and 300 Hz vibration conditions were equal to 8.1 ± 3.5 mm, 13.4 ± 3.1 mm and 11.3 ± 2.8 mm, respectively ([Fig 5A](#pone.0199627.g005){ref-type="fig"}). The one-way repeated measures ANOVA on the constant *x*-errors showed that the vibration frequency effect was significant (*F*~2,36~ = 3.7, *p* = 0.035, *η*^*2*^ = 41.30). Post-hoc tests showed that constant *x*-errors were specifically increased when vibrotactile stimulation was delivered at 30 Hz (-5.2 ± 2.2 mm, *t*~18~ = 2.39, *p* = 0.028) and not when delivered at 300 Hz (-3.2 ± 1.8 mm, *t*~18~ = 1.22, *p* \> 0.05). However, constant *x*-errors were not different between low- and high-frequency vibrotactile stimulations (2.0 ± 1.6 mm, *t*~18~ = 0.61, *p* \> 0.05). As for dispersion errors, there was no significant effect of vibration frequency when the left shoulder was stimulated (*F*~2,36~ = 0.26, *p* \> 0.05; [Fig 5B](#pone.0199627.g005){ref-type="fig"}). These findings suggest that the improved spatial localization of the left target finger following vibrotactile stimulus on the fingertip is unlikely due to global arousal effect of the vibration. {#pone.0199627.g005} Discussion {#sec011} ========== In the context of multisensory information, it is acknowledged that the brain combines all the available sensory information to build a precise and robust representation of the world \[[@pone.0199627.ref001],[@pone.0199627.ref023]\]. For instance, accurate reaching movements require precise target localization prior to motor execution and several studies have shown that this localization was better when more than one sensory modality provided information about the target position \[[@pone.0199627.ref003],[@pone.0199627.ref004],[@pone.0199627.ref024]\]. When pointing to our body parts (e.g. the opposite index finger), the target is proprioceptively defined and reaches tend to be more variable than those directed to visual targets \[[@pone.0199627.ref025]\]. This might be related to a greater uncertainty in the localization of proprioceptive versus visual targets \[[@pone.0199627.ref026]\]. Hence, proprioceptive reaching might be improved if a second sensory modality provides additional information about the spatial location of the target. The interaction between touch and proprioception that has been reported in previous studies \[[@pone.0199627.ref007]--[@pone.0199627.ref009]\] suggests that tactile information could be used as a second source of sensory information to improve the localization of a proprioceptive target. The goal of this study was to investigate the influence of tactile information on the proprioceptive localization of the index finger in a motor context. In order to do so, we had participants perform a position-matching task in which they were asked to make reaches with the right index finger to a proprioceptive target (i.e., the opposite left index finger). No visual feedback of the hand was provided during reach execution and 30 or 300 Hz vibrotactile stimulations were applied on the left target index fingertip prior to movement onset. Trials in which no tactile vibration was delivered to the left index finger were also included. Constant *x*-errors and dispersion errors were measured and compared across all three experimental conditions. Constant *x*-errors represent the reach accuracy, that is to say how close the right reaching finger is from the left target finger; the smaller the constant *x*-error, the greater the reach accuracy. Dispersion errors refer to reach precision which reflect how consistent reach endpoints are when repeated; the smaller the dispersion error, the greater the reach precision. We found that reach accuracy and precision, measured as constant and dispersion errors respectively, were both affected by the application of vibrotactile stimulations on the left target index fingertip. Indeed, both the constant and the dispersion errors were reduced when 30 or 300 Hz vibrations were delivered, as compared to the no vibration condition. Thus, it seems that cutaneous vibrations at either low or high frequencies provided the nervous system with additional (though slightly different) tactile information about the left index finger position. As a result, the spatial localization of the proprioceptive target was enhanced and both the accuracy and the precision of reaching were improved relative to the condition with no tactile stimulation. These results suggest that tactile information from the cutaneous vibrations is integrated with proprioceptive information about the position of the target index finger. In accordance with multisensory integration principles, the congruent proprioceptive and tactile information enhanced the finger proprioceptive localization, and ultimately improved proprioceptive reach performance. The finding that both 30 and 300 Hz vibrotactile stimulations similarly improve reaching performance does not allow us to conclude about the specific contributions of Meissner and Pacinian corpuscles to touch-proprioceptive integration. According to previous studies, low- and high-frequency cutaneous vibrations appear to have distinct effects on proprioceptive acuity \[[@pone.0199627.ref020],[@pone.0199627.ref021]\]. Performance in a passive finger movement detection task was impaired when stimulations at 300 Hz were delivered to the finger. In contrast, the application of 30 Hz vibrations did not alter task performance. However, in our study we found similar results when either 30 or 300 Hz vibrotactile stimulation was applied to the proprioceptive target of the reach (i.e., the left index finger). Both high- and low-frequency tactile stimulations led to an improvement in reach accuracy and precision when pointing to the left index finger. These discrepancies might be explained by the fact that the tasks used in these studies were fundamentally different. Participants in Weerakkody's studies \[[@pone.0199627.ref020],[@pone.0199627.ref021]\] performed a perceptual task in which they reported whether the movement imposed to their finger was a flexion or an extension. In contrast, in our study, participants were asked to localize a proprioceptive target and match its position by reaching with the opposite index finger. It has been proposed that somatosensory, and thus proprioceptive and tactile information is processed differently for perception and for action \[[@pone.0199627.ref027]\]. Similar to the two cortical processing streams described in the visual system \[[@pone.0199627.ref028]\], the "ventral" pathway is concerned with conscious somatosensory perception and object recognition while the "dorsal" pathway is relevant for guidance of action. The functional dissociation between the two somatosensory pathways has been established by studies in brain-damaged patients showing that they could perform motor actions towards somatosensory targets which were not consciously perceived \[[@pone.0199627.ref029]--[@pone.0199627.ref031]\]. These two separate somatosensory streams might explain why vibrotactile information is processed differently in perceptual and motor tasks. Nevertheless, it has been reported that separating the different tactile afferent fibers is challenging. It does not only depend on the stimulus frequency, but also on other parameters such as skin temperature \[[@pone.0199627.ref032]\]. Moreover, Meissner and Pacinian corpuscles are likely to have partially overlapping sensitivities, and thus detection thresholds which are relatively close to each other \[[@pone.0199627.ref019],[@pone.0199627.ref032],[@pone.0199627.ref033]\]. In the present study, it is therefore possible that the two vibrotactile frequencies delivered to the left target index finger might have activated both Meissner and Pacinian corpuscles. That could also explain why we did not observe difference between the 30 and 300 Hz vibration conditions. In the present study, we found that tactile information provided on fingertip was integrated with proprioception, resulting in an improved spatial localization of the target fingertip during proprioceptive reaching. It could be that this improvement in spatial localization produced by the tactile stimuli was due to arousal enhancement related to the presence of an additional signal (i.e., the vibration). However, we found in a control experiment that putting the same vibration on the left shoulder did not improve reach precision (dispersion errors) as it did when the finger was vibrated, and while it produced changes to reach accuracy (constant *x*-errors), these were in the opposite manner as expected. Indeed, there was a decrease in accuracy rather than an increase as would be expected by increased arousal. Furthermore this effect was not consistent across the two vibration frequencies. Alternatively, enhanced spatial localization of the left target finger following vibrotactile stimuli could be explained by spatial attentional cueing effects. The vibration would act as a cue driving attention to the left index finger. If this was the case, a cue from another sensory modality (e.g. audition) delivered nearby the hand should improve fingertip localization as well. We believe that this is unlikely to account for our results since it has recently been shown that auditory cueing does not modulate hand localization accuracy \[[@pone.0199627.ref034]\]. Thus, we can rule out arousal and spatial attentional cueing effects. Our effect results from a specific spatial integration of tactile and proprioceptive information. However, the exact mechanisms underlying this multisensory integration remain to be determined. According to the classic view of somatosensory processing, although both ascending through the dorsal column-medial lemniscal pathway, tactile and proprioceptive inputs remain segregated and are transmitted to distinct areas of the primary somatosensory cortex (S1) \[[@pone.0199627.ref035]\]. Somatosensory signals are not merged together until they reach higher-order somatosensory areas, such as the posterior parietal cortex. This integration is thought to be mediated by area 5 in the intraparietal cortex, where both tactile and proprioceptive inputs converge \[[@pone.0199627.ref036]\]. However, electrophysiological recordings (mainly in areas 3b, 1 and 2) have provided evidence that some neurons in S1 respond to both tactile and proprioceptive signals \[[@pone.0199627.ref016],[@pone.0199627.ref017],[@pone.0199627.ref037]\]. These findings support an alternative but not exclusive hypothesis that multimodal interaction, and thus integration, between touch and proprioception might also occur at the level of S1, presumably in all sub-areas. Indeed, about half of S1 neurons, located in multimodal areas 1 and 2 but also in the previously thought modality-specific areas 3a (proprioception) and 3b (cutaneous), showed responses to both proprioceptive and tactile stimuli \[[@pone.0199627.ref038]\]. Further research is needed to elucidate the mechanisms underpinning touch-proprioceptive integration and determine how tactile inputs influence the processing of proprioceptive information. The authors would like to thank Romain Fournet for technical assistance. [^1]: **Competing Interests:**The authors have declared that no competing interests exist.

Introduction {#S1} ============ Despite the effectiveness of antiretroviral therapy (ART) to suppress viral loads and decrease HIV-related mortality, HIV remains a global epidemic. The World Health Organization (WHO) estimates that of the 36.7 million people currently living with HIV worldwide, only 48% are being treated.^[@R1]^ Early diagnosis of HIV decreases mortality and morbidity by initiating early patient treatment.^[@R2]^ HIV screening is currently performed using commercially-available rapid diagnostic tests (RDT), typically based on lateral flow immunoassay (LFIA) technology that detects HIV antibodies from oral fluid or capillary blood. The low sensitivity during the pre-seroconversion phase of the first four weeks of infection and frequent false negatives require that antibody-detecting RDT results are confirmed by a second or even third laboratory-based assay.^[@R3]^ Even the fourth and fifth generation assays that combine antibody and HIV p24 antigen detection in a combo RDT are less sensitive than laboratory-based assays.^[@R4]^ However, the delay of diagnosis due to laboratory-based testing significantly impairs an HIV-positive patient\'s prompt treatment.^[@R5]^ Point-of-care (POC) nucleic acid-based diagnostic tests could expedite treatment response for vulnerable and newly infected individuals through early detection of the HIV virus. Reverse transcription polymerase chain reaction (RT-PCR) has been performed in microfluidic-based sample-to-answer devices to amplify HIV RNA spiked into saliva samples. However, the complexity of manufacturing a device to perform both sample preparation and cyclical heating often makes it prohibitively expensive for low-resource settings.^[@R6]^ These sensitive detection systems are not cost-effective for early screening and POC testing because they require expensive supporting sample preparation units, cold-chain storage of reagents, off-chip pumps, and trained users.^[@R3],[@R7]^ To address these shortcomings, recent efforts have been focused towards developing integrated sample-to-answer nucleic acid analysis devices that can be used by minimally-trained personnel.^[@R8],[@R9]^ While these integrated nucleic acid analysis devices minimize user steps and costs, to-date we are unaware of any such devices capable of analyzing viral HIV RNA from whole blood samples. There are a few commercial tools for near-patient detection of HIV including Cepheid Xpert Qual Assay, Alere q HIV-1/2 Detect, and Diagnostics for the Real World\'s Samba II. Although these tests are able to integrate and automate sample preparation, they all require cost-prohibitive (\>\$17 000 for the instrument and \>\$17 for the cartridge) benchtop instruments that need stable electrical power supply or consumable batteries.^[@R10]^ Recent advances in technologies for point-of-care molecular detection of HIV include several isothermal nucleic acid amplification techniques that could reduce the complexity and therefore cost of a fully integrated testing device.^[@R11]^ One such isothermal amplification method, loop-mediated isothermal amplification (LAMP), provides specific and efficient amplification of target nucleic acids by targeting 8 unique sequences.^[@R12]^ The isothermal heating (most efficiently between 65 and 72 °C)^[@R13],[@R14]^ of LAMP both lyses many pathogens and robustly amplifies DNA even in the presence of complex sample matrices, further reducing sample processing and instrumentation requirements.^[@R15]-[@R17]^ To expedite sample preparation steps, such as reverse transcription (RT) of HIV RNA targets to amplifiable DNA, several groups have demonstrated that RT can be performed using the same assay conditions as LAMP.^[@R18]-[@R21]^ Gurrala *et al*. used RT-LAMP to amplify HIV-1 RNA and produce a pH change that can be measured with their device.^[@R22]^ The lack of reagent storage and integrated sample preparation, however, decreases the translatability of this and other RT-LAMP devices. Here we report a fully-integrated sample-to-answer platform ([Fig. 1](#F1){ref-type="fig"}) that leverages paper membranes\' wicking abilities and size discriminating pores to a) isolate HIV viral particles from human blood cells, b) amplify RNA from the viral particles using pre-dried RT-LAMP reagents that target the highly conserved *gag* gene of HIV-1, and c) automatically transport RT-LAMP amplicons to an integrated LFIA for simple, visual interpretation of results within 90 minutes of sample application. This microfluidic rapid and autonomous analysis device (microRAAD) demonstrates the potential for simple and low-cost HIV detection at the point of care. Experimental {#S2} ============ Reagents {#S3} -------- Reagents necessary for the RT-LAMP assay included six primers (Integrated DNA Technologies, Skokie, IL), Bst 3.0 polymerase (NEB, Ipswich, MA), deoxynucleotide triphosphates (dNTPs) (Agilent Technologies, Santa Clara, CA), isothermal buffer II (NEB, Ipswich, MA), betaine (Millipore Sigma, Burlington, MA), EvaGreen (VWR International, Radnor, PA), ROX (Thermo Fisher Scientific, Waltham, MA), diethyl pyrocarbonate (DEPC) water (Invitrogen, Carlsbad, CA), and human whole blood collected in sodium citrate (Innovative Research, Novi, MI). Template used in the experiments below included purified genomic RNA from HIV-1 (ATCC, Manassas, VA), non-infectious HIV-1 virus diluted in AccuSpan plasma (AccuSpan Linearity Panel, SeraCare Life Sciences, Milford, MA), purified genomic RNA from dengue virus (DENV) type 1 (BEI resources, Manassas, VA), and purified RNA from chikungunya virus (CHIKV) S-27 (BEI resources, Manassas, VA). *Sph*I and *Pst*I restriction enzymes (NEB, Ipswich, MA) and phosphate buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA) are additional reagents used. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay {#S4} ---------------------------------------------------------------------------- Purified genomic RNA from HIV-1 or non-infectious HIV-1 virus was used at specified concentrations as the template in preliminary testing and in experiments thereafter. LAMP primers were devised using PrimerExplorer v5 to target the *gag* gene of HIV-1, and loop primers were labeled with 6-carboxyfluorescein (FAM) and biotin for detection *via* commercial LFIA (Ustar Biotechnologies, Hangzhou, China). The primer sequences are provided in [Table S1.†](#SD1){ref-type="supplementary-material"} To allow for both reverse transcription and amplification of the HIV-1 target, we used Bst 3.0 polymerase, which includes reverse transcriptase capabilities, and the buffers and dyes listed in [Table S2†](#SD1){ref-type="supplementary-material"} for a 25 μL reaction. 2--4 μL of template (RNA or virus) or negative control (DEPC water or AccuSpan plasma) were added directly to the RT-LAMP master mix prior to heating. A range of temperatures, 58--77 °C, were tested to determine the optimal assay temperature. After heating at indicated temperatures, RT-LAMP amplicons were added to LFIAs for analysis. Hereafter, RT-LAMP was performed at 65 °C for 60 minutes using an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA). The specificity of the target sequence to HIV-1 was verified by BLAST as well as experimentally using 10^5^ copies per reaction of genomic RNA from DENV type 1 and CHIKV S-27. Further, to confirm the identity of the amplified product, the RT-LAMP amplicons were subjected to restriction enzyme digest with *Sph*I and *Pst*I for 1 hour at 37 °C and the digested segments were confirmed *via* 2% agarose gel electrophoresis. For limit of detection (LOD) experiments, RNA or virus template was prepared by performing 10-fold serial dilutions in either DEPC water (RNA) or AccuSpan plasma (virus). For analysis of RT-LAMP in biological sample matrices, increasing amounts of human whole blood was added into the master mix. Real-time fluorescence data of EvaGreen intercalating dye and ROX reference dye was monitored to confirm the amplification progress. RT-LAMP amplicons were characterized *via* LFIA and confirmed *via* gel electrophoresis using a 2% agarose gel run at 100 V for 60 minutes, stained with ethidium bromide, and imaged using an ultraviolet light gel imaging system (c400, Azure Biosystems, Dublin, CA). RT-LAMP reagent drying and storage {#S5} ---------------------------------- Reagents and conditions for drying and storage were modified and adapted from a published protocol.^[@R23]^ The RT-LAMP reagents were deposited, dried, and stored at room temperature, eliminating the need for cold-chain storage (−20 °C) and improving the portability of the device. The primer mixture ([Table S3†](#SD1){ref-type="supplementary-material"}) containing primers, sucrose, glycerol, and Triton X-100 was deposited by hand on 1 cm wide polyethylene terephthalate (PET) film (Apollo, Lake Zurich, IL) in two parallel lines ([Fig. S1†](#SD1){ref-type="supplementary-material"}) at approximately 1.83 μL cm^−1^. After drying in a sterile biosafety cabinet under continuous air flow for 60 minutes at room temperature, the enzyme mixture containing Bst 3.0 polymerase, sucrose, and dNTPs was deposited directly on top of the dried primers in parallel lines at approximately 1.19 μL cm^−1^ and set out to dry for another 60 minutes. The PET with dried reagents was cut into 1 × 1 cm pieces, corresponding to one 25 μL reaction. The reagents for the stability studies were packaged after deposition and initial drying and stored in opaque Mylar bags with silica gel desiccant (Uline, Pleasant Prairie, WI) at room temperature for 3 weeks. The dried RT-LAMP reagents were rehydrated with buffer ([Table S3†](#SD1){ref-type="supplementary-material"}) and virus (positive samples) or DEPC water (negative controls) in tubes or in the polyether sulfone (PES) amplification zone within the integrated device. Blood separation and virus capture in paper membranes {#S6} ----------------------------------------------------- As an initial proof of concept of size-based separation, two sizes of fluorescent particles (Bangs Laboratories, Fishers, IN) were used in vertical flow filtration to allow quantification of the membrane capture efficiency. The 0.11 μm diameter Dragon Green (Ex480/Em520 nm) particles represented HIV-1 virus and the 7.32 μm diameter Suncoast Yellow (Ex540/Em600 nm) particles represented red blood cells. The fluorescent particles were diluted in PBS according to manufacturer\'s instructions. A calibration curve correlating particle concentration to fluorescence was created by serially diluting both particle solutions and measuring dilutions in a SpectraMax M5 microplate reader (Molecular Devices LLC, San Jose, CA) at excitation of 480 nm or 540 nm for the 0.11 μm and 7.32 μm particles, respectively. A 7 mm hole punch was used to cut pieces of blood separation membrane (MF1, GE Healthcare, Chicago, IL) and amplification membrane (0.22 μm PES, Millipore Sigma, Burlington, MA). The cut membranes were sandwiched between two O-rings and placed into a commercial miniprep spin column ([Fig. S2†](#SD1){ref-type="supplementary-material"}) (Qiagen, Hilden, Germany). The spin column was then placed into a clear 2 mL collection tube. One hundred fifty (150) μL of either the 0.11 μm or 7.32 μm particles was pipetted into the spin column containing the membrane of interest. The tubes were then centrifuged for 60 seconds at 0.5 rcf and the fluorescence of the eluent was measured and compared to the calibration curve intensities. Fluorescence of unfiltered particles was measured and used as the baseline to calculate the proportion of particles that passed through the membrane. MF1 and 0.22 μm PES membranes were then used to show size-based capture in a lateral flow format. The 0.22 μm PES (2.5 cm × 1 cm) was overlapped with the MF1 membrane (1 cm × 1 cm) to form the amplification and filtering segments of the integrated device ([Fig. S10†](#SD1){ref-type="supplementary-material"}). First, a 100 μL solution containing approximately 7 × 10^5^ of 0.11 μm particles, 230 of 7.32 μm particles, and deionized water were mixed to form the particle mixture. Higher concentrations of the 7.32 μm particles oversaturated the fluorescence measurements. Thirty (30) μL of the particle solution was pipetted onto the assembled MF1/PES membrane, followed by a 30 μL PBS wash. The particles in the membranes were immediately imaged at 40× magnification with an inverted Axio Observer Z1 Fluorescent microscope and ZenPro software (Carl Zeiss Microscopy, Thornwood, NY) using a rhodamine dye filter cube for the 7.32 μm particles and an Alexa Fluor 488 dye filter cube for the 0.11 μm particles. To demonstrate nucleic acid amplification of viral particles after separation from blood cells in the lateral flow format, MF1 and 0.22 μm PES membranes were overlapped as in the particle lateral flow. Next, 1.2 μL of 2.5 × 10^5^ virus copies per μL HIV-1 was mixed with 12 μL of human whole blood and deposited onto the MF1 membrane of the MF1/PES assembly, followed by a 61.8 μL wash of rehydrating mixture (final concentration of 4 × 10^6^ virus copies per mL of reaction volume) ([Table S3†](#SD1){ref-type="supplementary-material"}). After 1 minute of capillary flow, the PES was removed from the assembly and added into a PCR tube with 23 μL of the enzyme and primer mixtures. The samples and a positive amplification control (reaction without blood or membrane) were amplified for 60 minutes at 65 °C. Amplification was confirmed by placing the PES membranes and control into wells of an agarose gel and performing gel electrophoresis. The remaining solution in the PCR tube that had not saturated the PES membrane was added to a LFIA followed by 40 μL of wash buffer. Resistive heating {#S7} ----------------- Resistive heating elements were fabricated by printing JSB40G Nanosilver Ink (Novacentrix, Austin, TX) onto a Kapton substrate (Kapton HN Semi-Clear Amber Film, 5 mm × 125 μm) using a Dimatix Materials Printer DMP-2850 (Fujifilm Dimatix Inc., Santa Clara, CA) with a drop spacing of 35 μm and a platen temperature of 50 °C. Optimization and characterization were performed previously. The printed traces were then cured in an oven at 400 °C for 10 minutes. Individual traces were measured post-curing with a handheld multimeter to determine the average electrical resistance of the heating element. The design in [Fig. S3†](#SD1){ref-type="supplementary-material"} provided even heating of the amplification zone with an average resistance of 5 Ohms and required an average of 240 mW to reach 65 °C, the temperature necessary for the RT-LAMP assay. The heating elements used to actuate the wax valves also had an average resistance of 5 Ohms and heated to 80 °C using an average of 440 mW. The heat produced by the silver trace dissipates through the Kapton substrate and into the membranes above. The insulation required to maintain steady heating is provided by an acrylic lid and plastic housing pictured in [Fig. 1A](#F1){ref-type="fig"}. Temperature control circuit {#S8} --------------------------- In the integrated microRAAD, temperature regulation of the resistive heating elements was achieved using a miniaturized, custom-designed electronic device. It was constructed to individually monitor and regulate the temperature of each of three heating zones: one heating zone was dedicated to amplification and two heating zones were dedicated to actuating the wax valves. The temperature control circuit was equipped with a microcontroller (ATMEGA328P-AUR, Microchip Technology, Chandler, AZ), three non-contact infrared (IR) temperature sensors (MLX90614ESF-BAA-000-TU, Melexis Technologies NV, Ypres, Belgium), and three transistor-based current drivers (IRLML6244TRPBF, Infineon Technologies, Neubiberg, Germany) driven by three separate 10 bit digital-to-analog converters (DAC6311, Texas Instruments, Dallas, TX) for precise monitoring and control of current delivered to the resistive heating elements. Six pogo pins (0907-1-15-20-75-14-11-0, Mill-Max Manufacturing Corporation, Oyster Bay, NY), two for each resistive heating element, were used to complete the circuit between the current drivers and the resistive heating elements placed between the circuit board and the microfluidic paper analytical device (μPAD, [Fig. 1A](#F1){ref-type="fig"}). The microcontroller was loaded with an Arduino bootloader (Arduino Pro Mini, 3.3 V, 8 MHz, SparkFun Electronics, Boulder, CO) and programmed using the Arduino development environment (Arduino IDE v.1.8.8) over a universal serial bus (USB) connection through a future technology devices international (FTDI) serial-to-USB interface (FTDI Serial TTL-232 USB Cable, Adafruit Industries, New York City, NY). All electronic components were sourced from Digi-Key Electronics (Thief River Falls, MN). The circuit boards were fabricated by PCBWay (Shenzhen, China) and were assembled in-house using standard soldering equipment. The microcontroller was programmed to implement a proportional integral differential (PID) algorithm for maintaining temperature of the amplification zones within the user-specified set points (65 °C for the amplification and 80 °C for each wax valve). PID algorithms are popular closed-loop feedback mechanisms due to their simplicity and effectiveness, making them the *de facto* choice for portable, low computing power electronic devices.^[@R24],[@R25]^ The microcontroller sampled the temperature of each zone (sampling at 16.2 Hz for the amplification zone and 13.5 Hz for the valves) using the non-contact IR sensors and compared the measured temperature to the user-specified set point in order to determine the error value, *e*(*t*) (the difference between the measured value and the set point as depicted in [eqn (1)](#FD1){ref-type="disp-formula"}). The device then computes the proportional, integral, and derivative terms of the PID algorithm. The proportional term represents the difference between the set point and the measured value, multiplied by the proportional gain, *K*~p~ = 0.05. The integral term is the cumulative error and is computed by summing the integral of the current error with the previous errors, multiplied by the integral gain, *K*~i~ = 0.0001. The derivative term represents the change in the error since the last measurement, multiplied by the derivative gain, *K*~d~ = 0.1. u ( t ) = K p e ( t ) \+ K i ∫ 0 t e ( τ ) d τ \+ K d d d t e ( t ) The microcontroller then adjusts the current delivered to the resistive heating elements accordingly. The algorithm is designed to achieve accurate temperature regulation within 0.7 °C of the set point, while minimizing overshoot (1 °C). A serial Arduino interface was enabled between the circuit and a computer allowing real-time monitoring of experimental parameters, including temperature of each zone, time elapsed, and power consumption. We verified the temperature with both an infrared camera (FLIR Systems, Wilsonville, OR) and K-type thermocouple measurement of the top and bottom surfaces of the μPAD using a portable temperature data logger (RDXL4SD, OMEGA Engineering, Norwalk, CT). In the final implementation, the temperature control circuit was powered through a USB port using a USB on-the-go (USB OTG) enabled cellphone (Samsung Galaxy J3 Luna, Android Version 7.0), which removed the need for the computer, increased portability, and ensured fully automated control of the integrated device without the need for user intervention. Integrated microRAAD {#S9} -------------------- The microRAAD for HIV detection is composed of the reusable temperature control circuit and silver ink resistive heating elements and a single-use laminated μPAD, all contained in a plastic housing ([Fig. 1A](#F1){ref-type="fig"}). The base of the plastic housing was designed in SolidWorks and 3D printed on a Fortus 380c printer (Stratasys, Eden Prairie, MN). The 0.08″ thick acrylic lid of the housing (model \#11G0670A, Shape Products Menomonie, WI) and components of the single-use μPAD ([Table S4†](#SD1){ref-type="supplementary-material"}) were designed in Adobe Illustrator and cut with a CO~2~ laser (VLS 3.5, Universal Laser Systems, Scottsdale, AZ). Valve strips were prepared by printing 1.25 mm wide lines of solid wax-ink (Black ColorQube ink, Xerox, Norwalk, CT) onto cellulose membranes (Chr1, GE Healthcare, Pittsburgh, PA) using a Xerox ColorQube 8570 (Norwalk, CT). Membranes were then heated for twelve minutes at 85 °C in a table-top oven (VWR, Radnor, PA) and cut into strips with the laser cutter to create closed valve strips.^[@R26]^ Commercially available LFIAs were modified for microRAAD by cutting off the sample pad. PET squares were sterilized with 70% ethanol. Once prepared, all components were hand assembled ([Table S4](#SD1){ref-type="supplementary-material"} and [Fig. S4†](#SD1){ref-type="supplementary-material"}) and laminated with pressure sensitive self-seal adhesive (GBC, Lake Zurich, IL) to minimize evaporation during the assay. Seventy-five (75) μL of prepared RT-LAMP master mix or rehydrating mixture ([Table S3†](#SD1){ref-type="supplementary-material"}) containing HIV-1 virus (at a final concentration of 4 × 10^6^ virus copies per mL of reaction volume) were loaded into the sample inlet of the μPAD and sealed with a 1 × 1 cm square of self-seal prior to assembling the μPAD into the plastic housing with the temperature control circuit. This method was used to minimize contamination between experiments, however, we also verified that the sample can be added to the sample inlet once the μPAD is assembled in the plastic housing, which is how we anticipate the device would be used at the point of care. The assembled microRAAD was then loaded with wash buffer and subjected to localized heating of the amplification zone and valves. When testing whole blood samples spiked with HIV-1 virus, 1.2 μL of 2.5 × 10^5^ virus copies per μL HIV-1 was mixed with 12 μL of human whole blood and loaded into the sample inlet, followed by a 61.8 μL addition of rehydrating mixture (final concentration of 4 × 10^6^ virus copies per mL of reaction volume). The loaded μPAD was then adhered to the acrylic lid with double-sided adhesive at the wash inlet. Resistive heating elements were adhered to the backside of the μPAD, aligned with the two valves and amplification zone, and faced such that the silver traces would contact the pogo pins of the temperature control circuit inside the plastic housing. Two plastic brackets were slid over the acrylic lid and plastic housing to ensure proper contact within microRAAD. Finally, 130 μL of green food coloring solution (for visualization of flow) were added to the wash inlet and sealed. Heating was initiated *via* the serial interface between the computer and the temperature control circuit: 1) 65 °C for the middle resistive heating element (amplification) for 60 minutes and 2) 80 °C for the outer resistive heating elements (valves) for 2 minutes. After 30 minutes of development (1.5 hours after initiating the heating), the LFIA was imaged using a desktop scanner for analysis (Epson, Suwa, Japan). LFIA quantification and statistical analysis {#S10} -------------------------------------------- All RT-LAMP amplicons were characterized *via* LFIA. The LFIAs were scanned at least 30 minutes after initial sample addition using an Epson V850 Pro Scanner. The test band was quantified using a custom MATLAB script that averages the grey-scale pixel intensity of the test band and subtracts the average background pixel intensity 25 pixels below the test band.^[@R27]^ To determine the limit of detection, a one-way ANOVA *post hoc* Dunnett\'s was performed with multiple comparisons of the LFIA test bands of each concentration against the test bands of no template negative controls with a 95% confidence interval. A Student\'s unpaired, two-sided *t*-test with a 95% confidence interval was used when comparing the negative control and positive samples during the initial testing of 21 day dried reagents and to determine significance between control and positive samples detected in the integrated device. Results and discussion {#S11} ====================== RT-LAMP assay design and optimization {#S12} ------------------------------------- Several published HIV LAMP primer sets yielded slow amplification in our hands which we attribute to differences in reverse transcriptase and polymerase activity and the HIV subtype used to design the primers.^[@R18]-[@R20]^ Moreover, while some primers produced amplicons easily observed by real-time fluorescence and gel electrophoresis, they produced poor LFIA signals possibly due to steric hindrance of the labeled primers at the test band. Therefore, we designed novel primers (sequences provided in [Table S1†](#SD1){ref-type="supplementary-material"}) to specifically target a 201 base pair sequence in the *gag* gene in HIV-1 subtype B. There is one copy of the *gag* gene in the HIV-1 genome and two copies of the genome per viral particle.^[@R28]^ Optimized assay conditions are shown in [Table S2†](#SD1){ref-type="supplementary-material"} and provided the greatest amplification time difference between positive and negative samples, as observed by real-time fluorescence (data not shown). The optimal RT-LAMP assay temperature for these primers is 65 °C as this temperature yielded the strongest test band intensity on LFIAs illustrated in [Fig. S5.†](#SD1){ref-type="supplementary-material"} To assess specificity of the RT-LAMP assay, primers were also tested with RNA from DENV and CHIKV. As seen in [Fig. S6,†](#SD1){ref-type="supplementary-material"} there was no cross-reactivity of the HIV LAMP primers with these other RNA-based viral pathogens. Further confirmation of specificity was provided by performing enzymatic digestions of the amplified product with either *Sph*I or *Pst*I. As predicted by LAMP restriction digest fragment analysis,^[@R29]^ digestion with either of these enzymes resulted in smaller fragments compared to the undigested product, with the *Pst*I digested product collapsing to the shortest fragment seen on the agarose gel in [Fig. S7.†](#SD1){ref-type="supplementary-material"} Taken together, these experiments suggest that the designed primer set targets the intended region in the *gag* gene and that this target amplification is specific to HIV-1. RT-LAMP assay performance {#S13} ------------------------- We first evaluated the newly designed RT-LAMP assay using purified HIV-1 RNA at concentrations ranging from 10^1^--10^6^ RNA copies per reaction (*n* = 3). As expected, the real-time fluorescence data displayed faster amplification for samples with higher initial concentrations of RNA (data not shown). Agarose gel electrophoresis confirmed amplification of the RNA after the 60 minute assay ([Fig. 2A](#F2){ref-type="fig"}). The RT-LAMP amplicons were also added to LFIAs and the test band intensity was quantified to determine the LOD. While 10^1^ copies of RNA was detectable in 1 of 3 replicates, statistically significant differences between the test band intensity of the negative control (0) compared to 10^2^--10^6^ RNA copies per reaction (*p*-value \< 0.01) ([Fig. 2A](#F2){ref-type="fig"}) demonstrate an assay LOD of 100 copies of HIV-1 RNA per reaction. Other groups have reported a comparable LOD, ranging from 30--250 RNA copies per reaction for their HIV RT-LAMP assay.^[@R18],[@R20],[@R30]^ Next, we conducted the RT-LAMP assay with whole HIV-1 virus to ensure sufficient viral lysis at the 65 °C assay temperature. Viral lysis is necessary to release the RNA for amplification. Since the osmotic pressure gradient would prematurely lyse the virus, amplification of virus diluted in water was not tested during characterization studies, as was done with the RNA. Instead, we performed RT-LAMP of HIV-1 virus spiked into reactions containing 16% plasma at concentrations of 10^1^--10^6^ virus copies per reaction (*n* = 3). We used AccuSpan plasma, because the virus stock was supplied in this solution. LFIA analysis showed a statistically significant difference between the test band intensity of 10^4^, 10^5^, and 10^6^ virus copies per reaction compared to the negative control (0) (*p*-value \< 0.05 and *p*-value \< 0.001, respectively) ([Fig. 2B](#F2){ref-type="fig"}) and the agarose gel supported this conclusion. Therefore, the limit of detection of the assay with HIV-1 virus in 16% plasma is 10^4^ virus copies per reaction. While this LOD is 100× higher than purified RNA in water, when RNA is spiked into 16% plasma, we observed no amplification (data not shown). We suspect that the RNA within viral particles is partially protected from inhibitory factors in plasma until the factors are deactivated and the virus is thermally lysed, but further investigation is necessary to delineate the cause of the loss in sensitivity. Still, this is the first demonstration to our knowledge of an HIV RT-LAMP assay without separate RNA extraction and purification, vastly simplifying the sample preparation required for detection of viruses in complex matrices. We then evaluated the robustness of the RT-LAMP assay in the more complex matrix of human whole blood. HIV-1 virus spiked into RT-LAMP reactions at a concentration of 10^5^ virus copies per reaction with increasing percentages of whole blood (0--30%) was detectable *via* gel electrophoresis in up to 15% whole blood whereas LFIA detected HIV-1 in up to 20% whole blood ([Fig. S8†](#SD1){ref-type="supplementary-material"}). In agreement with our observations, other groups have also noticed that LFIA visualization can be more sensitive than gel electrophoresis.^[@R31]^ In the integrated microRAAD device, a blood separation membrane, MF1, captures the red and white blood cells, allowing the virus to flow into the amplification zone. Therefore, while whole blood is not intended to be present in the amplification zone, our results indicate an assay tolerance up to 20% in case of poor MF1 capture efficiency or blood cell hemolysis. RT-LAMP assay modified for ambient temperature storage {#S14} ------------------------------------------------------ Given that RT-LAMP assays currently require cold-chain storage, we set out to develop a reagent drying method to enable ambient-temperature stable reagents. Others have used LAMP reagent vitrification in PCR tubes to enable ambient-temperature storage,^[@R23]^ however, we were concerned that the requirement to separate the enzyme and primers might result in diffusion-limited amplification in the porous membrane of our μPAD. Therefore, prior to experimentation, we calculated the molecular diffusivity of reconstituted reagents into the PES amplification zone using the Stokes--Einstein equation^[@R32]^ and Renkin equation.^[@R33],[@R34]^ The hydrodynamic radius, *α*, was calculated for the largest molecule in both the primer and enzyme mixture, which was the FIP primer and Bst 3.0 polymerase, respectively. Using [eqn (2)](#FD2){ref-type="disp-formula"}, we then calculated the bulk diffusivity, *D*, for each molecule where *R* is the gas constant, *T* is temperature, *η* is viscosity, and *N*~A~ is Avogadro\'s number. D = R T 6 π η α N A Since the diffusion occurs in a porous membrane, we then calculated pore diffusivity, *D*~m~, using [eqn (3)](#FD3){ref-type="disp-formula"}. $$D_{m} = DK(\alpha \slash r)\omega_{r}(\alpha \slash r)$$ where *r* is the pore radius, *K*(*α/r*), the partition coefficient, is equal to (1 -- *α/r*),^2^ and the hydrodynamic drag, ω~r~(*α/r*), is equal to \[1 -- 2.1(*α/r*) + 2.09(*α/r*)^3^ -- 0.95(*α/r*)^5^\]. The predicted pore diffusivity indicates that the time for the reconstituted reagents to diffuse 1 mm through porous PES membrane is approximately 10 minutes. We decided upon layered parallel lines to minimize the distance which the reconstituted reagents would have to diffuse to ensure proper mixing for subsequent amplification. Confident that these assays could work in principle, we experimentally evaluated the robustness of the assay using 21 day dried RT-LAMP reagents. We reconstituted the dried RT-LAMP reagents with rehydrating mixture and HIV-1 at a concentration of 10^5^ virus copies per reaction in water. Positive and negative control reactions using freshly prepared reagents were heated simultaneously. After the 60 minute amplification, samples and controls were analyzed *via* LFIA (*n* = 6, [Fig. 3A](#F3){ref-type="fig"}). The LFIA test band intensity of positive samples using 21 day dried reagents was not statistically significantly different than that of the test band of the freshly prepared positive controls, indicating that the drying process did not damage enzyme or primer activity. As expected, LFIA results of positive samples were statistically differentiable from the negative samples for both the dried and fresh reagent groups (*p*-value \< 0.001) ([Fig. 3A](#F3){ref-type="fig"}). To compare the amplification efficiency of dried and freshly prepared reagents, the LOD of HIV-1 in 16% plasma was determined using 21 day dried RT-LAMP reagents (*n* = 3). There was a statistically significant difference between the test band intensity of the 10^5^ and 10^6^ virus copies per reaction compared to the negative control (0) (*p*-value \< 0.05 and *p*-value \< 0.01, respectively) ([Fig. 3B](#F3){ref-type="fig"}). While not significant, 10^4^ and 10^3^ copies of virus did amplify in some cases; two of three repeats for 10^4^ and one of three repeats for 10^3^ virus copies per reaction. There is a slight loss in sensitivity when using the dried reagents (LOD of 10^5^ *versus* 10^4^ virus copies per reaction), however, the LOD can likely be improved with further assay optimization such as RT and polymerase enzyme selection and primer design. Another study reported a loss of reaction efficiency using lyophilized HIV RT-LAMP reagents stored at ambient temperature for several hours when compared to freshly prepared controls.^[@R21]^ However, Hayashida and colleagues established that vitrified, not lyophilized, LAMP reagents designed for DNA targets have the same sensitivity as freshly prepared reagents after seven months of storage at room temperature.^[@R23]^ These findings in combination with our preliminary evaluation of limited HIV RT-LAMP reagents stored for five months, shown in [Fig. S9,†](#SD1){ref-type="supplementary-material"} give us reason to believe that we can increase the storage time of the HIV RT-LAMP reagents at room temperature far beyond 21 days. Blood separation and virus capture in paper membranes {#S15} ----------------------------------------------------- As our RT-LAMP assay was shown to be robust in up to 20% whole blood, we required the removal of the majority of blood cells that would inhibit the reaction. We developed a simple quantitative method to experimentally test the size-based capture of blood cells and virus in membranes. As seen in [Table 1](#T1){ref-type="table"}, the 7.32 μm particles, representative of blood cells, were captured in the MF1 membrane at an efficiency of 98.6% while 30% of the 0.11 μm particles, representative of the virus, were captured by MF1 (*n* = 3). This implies that MF1 can be used for size-based separation of blood cells from the virus, although some virus will remain in the MF1 membrane, thereby reducing detection sensitivity. Further, 47.6% of the 0.11 μm particles were trapped in the PES membrane ([Table 1](#T1){ref-type="table"}, *n* = 3), indicating that the PES could localize nearly half of the smaller particles within the amplification zone. Despite the PES membrane having a reported 0.22 μm pore diameter, we suspect that a fraction of smaller diameter particles were trapped in the PES and the MF1 membranes due to a combination of properties. Because of the membrane heterogeneity, a portion of the pores may be smaller than the nominal pore size, allowing particles to be physically trapped. Furthermore, the tortuosity of the membranes may prevent particle migration through the membrane.^[@R35]^ Lastly, the proprietary surface chemistries of both membranes may create a slight charge-based attraction that causes particles to adhere to the membranes. Our experimental results indicate a future design that could leverage these factors; extending the PES membrane could enable a filtration and localization of the virus for more sensitive detection from higher sample volumes. After quantifying the particle separation in columns, we verified successful particle capture in a lateral format by imaging fluorescent particles applied to an MF1/PES membrane assembly, depicted in [Fig. S10.†](#SD1){ref-type="supplementary-material"} [Fig. S11†](#SD1){ref-type="supplementary-material"} demonstrates that some of the 0.11 μm particles were trapped in the MF1 membrane, but the majority were dispersed throughout the PES membrane (*n* = 3), aligning with the vertical flow experiment results and indicating that the virus can be separated from the blood cells and localized to the PES amplification zone. After the characterization with particles, we confirmed that blood cells would be trapped in the MF1 membrane while the virus would flow into the PES membrane for subsequent amplification. We spiked HIV-1 virus into human whole blood and added the mixture onto the MF1 membrane which overlapped with the PES membrane and chased the sample with rehydrating mixture. After removing the PES from the MF1/PES assembly and amplifying the trapped virus in the PES membrane, the amplicons were analyzed *via* LFIA. As depicted in [Fig. S12,†](#SD1){ref-type="supplementary-material"} the test band intensity is strong, implying that the virus is indeed dispersed throughout the PES (*n* = 3) yet accessible for amplification. Interestingly, when HIV-1 virus diluted in blood was pre-mixed with the rehydrating buffer prior to adding the combined solution to the assembly, amplification was inconsistent and sometimes completely inhibited because more red blood cells seemingly migrated to the PES ([Fig. S12†](#SD1){ref-type="supplementary-material"}). Our successful membrane amplification results are consistent with previous findings that have also shown that LAMP and other isothermal amplification methods can be performed within the PES membrane.^[@R36]^ MF1 membranes inhibited the amplification assay and products extracted from the MF1 were not visible in either the agarose gel or LFIA (data not shown). Integration of microRAAD {#S16} ------------------------ We verified flow in microRAAD with dyed solutions deposited into the sample and wash inlets and then heated the amplification zone and valves. This initial testing indicated 130 μL of wash buffer and 75 μL of sample were required. With too little volume, there was an insufficient pressure gradient to drive flow past the opened valves, while too much volume caused the fluid to prematurely leak past the valves. The wash solution is necessary to sufficiently drive the RT-LAMP amplicons from the PES amplification zone into the LFIA for detection. Initial fluidic designs transported the wash solution directly to the PES, however, given the PES\'s small pore size and slow wetting, this caused frequent "short-circuiting" of the wash to the LFIA. By directing the wash through the MF1, rather than to the PES directly, we ensured that amplicons in the PES migrated to the LFIA before the wash ([Fig. 4](#F4){ref-type="fig"} and [ESI† Video](#SD2){ref-type="supplementary-material"}). Moreover, we verified that no capillary leaks occurred between the membranes and lamination by confirming the flow rates of sealed and unsealed membranes were equivalent (data not shown). During heating, we observed that the amplification zone reached 65 °C within seconds of initiation and remained at 65 ± 2 °C throughout the 60 minute heating period which is adequate for efficient amplification. In separate experiments, we determined that even low concentrations of template can amplify at temperatures between 62 °C and 71 °C ([Fig. S14†](#SD1){ref-type="supplementary-material"}). The temperature control circuit automatically terminated the amplification zone heating and initiated simultaneous heating of the wax valves. A 1.25 mm width valve was experimentally verified to sufficiently constrain the wash buffer from the amplification zone; thinner valves leaked wash buffer, diluting the amplicons and potentially decreasing assay sensitivity. We have previously reported that only 41 °C is required to open wax-ink valves prepared in chromatography paper,^[@R26]^ however, here we subjected the valves to 80 °C to accelerate their opening. Upon initiation of valve heating, the green wash buffer flowed past valve 1 to the MF1 and the heated sample flowed past valve 2 into the LFIA portion of the μPAD. Within 5--10 minutes of the valves opening, test and control bands were consistently observed on the LFIAs and quantification, in [Fig. S13,†](#SD1){ref-type="supplementary-material"} revealed strong test band intensities. We found that both a laptop and a cellphone with USB OTG provided sufficient current to power the temperature control circuit for the duration of the assay and yielded comparable results ([Fig. 1B](#F1){ref-type="fig"}). To verify the amplification functionality of microRAAD, we initiated the automated detection using 21 day dried RT-LAMP reagents and rehydrating buffer containing HIV-1 virus osmotically lysed in water. Samples containing as few as 3 × 10^5^ virus copies per reaction resulted in unequivocally positive test bands and samples containing no template (0) yielded negative test results (*p*-value \< 0.05) ([Fig. 5B](#F5){ref-type="fig"}). The test band intensity at a concentration of 3 × 10^5^ virus copies per reaction using the dried reagents in microRAAD was comparable to the test band intensity of the same concentration in a tube reaction with the dried reagents ([Fig. 5B](#F5){ref-type="fig"} and [3A](#F3){ref-type="fig"}). Finally, we performed the detection in the integrated microRAAD using 21 day dried amplification reagents and HIV-1 virus in whole blood. As expected and seen in [Fig. 5A](#F5){ref-type="fig"}, the red blood cells remained in the MF1 directly below the sample inlet while the remaining plasma and buffer solution with virus migrated to the PES for amplification. Following amplification, we visually observed positive test bands on the LFIAs within 5--10 minutes after valves opened. There was a statistically significant difference between the test band intensity of the 3 × 10^5^ virus copies per reaction compared to the negative control (0) (*p*-value \< 0.01) ([Fig. 5A](#F5){ref-type="fig"} and [C](#F5){ref-type="fig"}). Notably, the sensitivity using microRAAD for HIV-1 viral detection in blood using dried reagents is comparable to the sensitivity of standard tube reactions with similar conditions ([Fig. 5C](#F5){ref-type="fig"} and [3B](#F3){ref-type="fig"}). Given that the PES membrane absorbs about 20 μL, which is a comparable volume to tube reactions, we expect that diffusion limitations were minimal, further confirmed by our calculations of diffusivity within the PES membrane. Previous groups have similarly reported only 5 to 10-fold reductions in sensitivity when translating manual assays into automated sample-to-answer devices.^[@R8],[@R37],[@R38]^ Liu *et al*. designed a device to detect viral RNA from oral fluid samples in real time down to 12.5 virus copies per reaction, however, viral lysis is required before sample addition and is followed by four more manual steps prior to initiation of the RT-LAMP assay.^[@R39]^ Damhorst *et al*. developed a microfluidic chip for blood cell lysis and modified a smartphone for real-time detection of HIV-1 virus with an LOD of 1.7 × 10^4^ virus copies per reaction.^[@R21]^ However, the user is required to transfer the lysed blood and freshly prepared RT-LAMP reagents to the reaction chamber for amplification.^[@R21]^ Even though this platform is 10-fold more sensitive than microRAAD, we believe that the full automation of microRAAD, which reduces sample handling and exposure to bloodborne pathogens, makes it an advantageous system for rapid HIV testing at the POC. Our initial studies of this integrated sample-to-answer device demonstrate its potential to provide simple, affordable, and rapid detection of HIV from blood samples at the point of care. The consumable components of microRAAD (membranes, LFIA, adhesive, reagents) cost only \$2.23 per assay ([Table S4†](#SD1){ref-type="supplementary-material"}) while the reusable components (temperature control circuit and housing) are \$70.08 and expected to decrease with increased production ([Table S5†](#SD1){ref-type="supplementary-material"}). The price of components is comparable to other rapid HIV tests developed for resource-limited settings and will decrease as we scale-up the manufacturing of the device.^[@R40]^ While low component cost does not guarantee a low price point for consumers, it remains a critical feature of research and development that we considered.^[@R41]^ Even though microRAAD has many advantages over comparable diagnostic tools, there remain some limitations. The sensitivity of this integrated prototype is 3 × 10^5^ virus copies per reaction, or 2.3 × 10^7^ virus copies per mL of whole blood, which falls at the high end of the clinical range, 10^7^ virus copies per mL at peak infection at day 17.^[@R42]^ This reduced sensitivity may be due to microRAAD\'s performance of thermal lysis within the device that bypasses nucleic acid purification and concentration steps. However, the automation of virus analysis significantly streamlines testing and potentially reduces user error. We expect that additional improvements in primer design, RT and polymerase enzyme selection, and the addition of virus concentration from larger volumes of sample could further improve the device\'s sensitivity and enhance clinical utility. Specifically, the incorporation of a smaller pore-sized membrane could enable size-based capture of virus and isolation from inhibiting blood components. Additionally, including an internal amplification control into microRAAD could differentiate negative from invalid results.^[@R37]^ Incorporating these improvements along with extended device storage and usability studies will enable clinically relevant detection and early diagnosis of HIV at the POC. Conclusions {#S17} =========== We have demonstrated microRAAD, an autonomous and fully integrated sample-to-answer device, for the specific detection of HIV-1 virus from human whole blood. After sample addition, the LFIA can be visualized within 90 minutes. Moreover, the user is required to perform only four steps to initiate the testing: load sample and rehydrating mixture, add wash buffer, seal the inlets with adhesive, and initiate the temperature control circuit by connecting a power source such as a computer, cellphone, or portable battery. One of the most noteworthy aspects of microRAAD is the complete automation from blood-in to results-out; minimizing sample preparation and time-critical steps by the user. Furthermore, we have developed a novel RT-LAMP assay and demonstrated that reagents can be dried and stored at room temperature for three weeks before use in the integrated device. The ability to dry reagents eliminates the need for cold chain storage and increases the usability and portability of the device, especially in resource-limited settings. The sensitivity of this integrated prototype is 3 × 10^5^ virus copies per reaction, or 2.3 × 10^7^ virus copies per mL of whole blood, which is comparable to clinically reported HIV-1 concentration at the peak of infection.^[@R42]^ MicroRAAD has the potential to serve as a platform for detection of other pathogens. By modifying the LAMP primers to target a new gene of interest and adjusting sample preparation depending on the pathogen target and sample matrix, this platform could be used for other viruses (*e.g*. DENV, CHIKV) and even bacteria (*e.g. Escherichia coli, Vibrio cholerae,* or *Bordetella pertussis*) and parasitic (*e.g. Plasmodium falciparum*) pathogens. This rapid, integrated, and automated device lends itself to use in low-resource areas where clinics and laboratories are scarce and gold-standard testing can take up to one week.^[@R43]^ Moreover, microRAAD requires only \$2.23 worth of consumable components, making it an affordable detection tool. MicroRAAD combines robust and selective molecular techniques with elegant capillary fluidics and resilient heating controls into a single, portable platform for rapid pathogen detection at the POC. Supplementary Material {#SM1} ====================== This work was funded by the Grand Challenges Explorations Program (OPP1150806), an initiative of the Bill & Melinda Gates Foundation, the National Science Foundation Graduate Research Fellowship Program (DGE-1333468) (EAP), the National Institute of Allergy and Infectious Diseases (R61AI140474), Purdue University\'s Shah Family Global Innovation Lab, and the Purdue Institute for Inflammation, Immunology and Infectious Disease (PI4D). Electronic supplementary information (ESI) available. See DOI: [10.1039/c9lc00506d](10.1039/c9lc00506d) Conflicts of interest There are no conflicts, financial or otherwise, to declare. {#F1} {#F2} {#F3} {#F4} {#F5} ###### Efficiency of membrane capture of fluorescent particles *n* = 3 Particle size Membrane Fluorescence (RFU) Capture efficiency --------------- ------------- -------------------- -------------------- **0.11 μm** None 4007.6 ± 165.0 **0%** MF1 2810.9 ± 193.0 **30.0%** 0.22 μm PES 1986.8 ± 103.2 **47.6%** **7.32 μm** None 275.1 ± 12.2 **0%** MF1 2.3 ± 1.3 **98.6%** 0.22 μm PES 0.9 ± 0.2 **81.9%** [^1]: Author contributions E. A. P., T. J. M., and J. C. L. designed experiments. E. A. P., T. J. M., K. F. K. E., and L. A. J. designed and optimized the RT-LAMP assay and drying protocol. E. A. P., T. J. M., L. A. B., and J. B. B., characterized and developed the fluidic transport and separation of sample components. K. M. B., L. K. L., and L. A. S. designed and fabricated the resistive heating elements. O. S. H., E. A. P., and K. M. B. engineered the temperature control circuit. E. A. P. and T. J. M. tested the fully-integrated microRAAD. E. A. P. and T. J. M. analyzed results and generated figures and tables. J. C. L. conceived of and supervised the project. E. A. P. and T. J. M. wrote the manuscript with review and input from all authors. [^2]: Indicates equal contribution of this work.

Introduction ============ The International Study of Asthma and Allergies in Childhood (ISAAC), created in 1990 to maximize the value of epidemiological studies of asthma and allergic diseases, established a standardized method that facilitated international collaboration, from the establishment of a protocol used worldwide^(^ [@B01] ^)^. Its specific points were: a) to describe the prevalence and severity of asthma, rhinitis, and eczema in children living at different locations and to draw comparisons between them and between countries; b) obtain baseline measures to assess future trends in the prevalence and severity of this diseases; c) provide support for further etiologic studies in genetics, lifestyle, medical care, and air pollution, that may affect these diseases^(^ [@B01] ^)^. The ISAAC was conceived to be carried out in three successive and dependent phases. In the first phase, the study of the compulsory core was developed to assess the prevalence and severity of asthma and allergic diseases in selected populations, using a standardized questionnaire; in the second phase, possible etiological factors were investigated, especially those suggested by the results in the previous phase; in the third phase, the first phase was repeated after a minimum period of 5 years, evaluating the evolutionary trend in the prevalence of asthma and allergic diseases in a given period, including centers that participated in phases I and III simultaneously. In addition, the prevalence of other countries was determined, which, although not involved in phase I, had interest in measuring the prevalence of asthma and its severity, as well as the risk factors associated with the development of the disease, both environmental and of lifestyle, specific to each community^(^ [@B01] ^)^. Collections instruments ======================= The instruments of data collection are the ISAAC video questionnaire (VQ) and the written questionnaire (WQ). The VQ consists of five scenes, where several individuals are presented with pictures of asthma in different intensities and situations, to be answered by adolescents^(^ [@B01] ^)^. The WQ is composed of three modules, which address asthma, rhinitis, and atopic eczema, respectively. The WQ is presented in two versions: one for children aged from 6 to 7 years, answered by parents and/or guardians, and another answered by the adolescents themselves. The question \"wheezing in the last year\" is the one that gathers higher sensitivity and specificity for the diagnosis of asthma, being universally used to identify individuals with active asthma^(^ [@B01] ^)^. Originally written in English, the ISAAC WQ was validated to be used worldwide. In localities where the language was different, it was recommended that it be translated and validated for its appropriate use^(^ [@B01] ^)^. Thus, in our location, the ISAAC WQ was translated into Brazilian Portuguese, back-translated into English, and then validated by criterion and constructively, confirming that their diagnostic characteristics were not modified^(^ [@B02] ^)^. More recently, the ISAAC directive committee published a study that highlights the importance of cultural norms that must be considered in the evaluation of back-translation into English, often allowing linguistic deviations in order to maintain the original meanings^(^ [@B03] ^)^. Although the VQ was designed to overcome barriers imposed by language and possible distortions arising from translations of the WQ, its application in the world did not supplant that of the WQ. In Brazil, the WQ was the only one to be used. However, due to the various denominations that asthma receives, some authors added questions with synonyms usually used to define the disease in the WQ, in order to improve the diagnostic power of the instrument^(^ [@B04] ^)^. Named by many as bronchitis, the inclusion of the question did not improve the diagnostic power of the ISAAC WQ in children^(^ [@B04] ^)^. Taking as a base the structure employed in other national population surveys, for instance, the survey on weight, Valle *et al*validated the ISAAC WQ, as well as its reproducibility, administering it through telephone interview. Parents/caregivers of children (6-7 years old) with or without asthma were interviewed in their homes on two occasions, with an interval of 15 days, and responded to the ISAAC WQ by telephone, evaluating the reproducibility of responses to the questions, given by the same respondent. The question \"wheezing in the last year\" had a perfect concordance index, enhancing the reproducibility of the instrument administered via telephone^(^ [@B05] ^)^. ISAAC Phase I ============= The compilation of worldwide data from ISAAC phase I gathered a significant number of children (6-7 years; n=257,800; 91 centers in 38 countries) and adolescents (13-14 years; n=463,801; 155 centers in 56 countries) never previously evaluated and showed great variability in rates between the different participating centers^(^ [@B06] ^)^. In Brazil, the first ISAAC phase, completed in 1996, was a real watershed in knowledge of the prevalence of asthma and allergic diseases in the country. Before, the Brazilian epidemiological data available were restricted to small population samples, especially in large urban centers and educational institutions, without any standardization in their production, which made it very difficult to compare them. In Brazil, seven centers that enabled obtaining reliable data on the prevalence of asthma, allergic rhinitis, and atopic eczema in children and adolescents participated in this phase. The comparative analysis with all the global data obtained showed that the average prevalence of asthma in children and adolescents is high ([Table 1](#t01){ref-type="table"}), being the eighth among the centers with the highest prevalence - English-speaking countries and Latin America^(^ [@B07] ^)^. As for asthma, in this first phase, it was observed that the use of the medical diagnosis as epidemiological criterion for identifying patients with asthma would induce an underdiagnosis, since the prevalence of the active disease (wheezing in the last year) reached twice as much as the medical diagnosis^(^ [@B08] ^)^. This fact is corroborated, as in the validation of the WQ, it was found that only 50% of adolescents with asthma, regularly followed in a specialized service, identified themselves as having asthma^(^ [@B02] ^)^. Table 1Prevalence of active asthma (wheezing in the past 12 months) and physician-diagnosed asthma in school children from official centers and other Brazilian centers that used the protocol of the International Study of Asthma and Allergies in Childhood (ISAAC) - Phase 1 Other researchers, even without the recognition of the central committee of the ISAAC, used the standard WQ and the protocol, obtaining the prevalence data summarized in [Table 1](#t01){ref-type="table"}. The mean prevalence of active asthma was 23.3% for children and 22.7% for adolescents ([Table 1](#t01){ref-type="table"}). Taking the data from around the world, there was a 20-fold variation in the prevalence of asthma and related symptoms (ranging between 1.8 and 36.7%), being the environmental factors the main responsible for this variation^(^ [@B14] ^)^. English-speaking countries and centers in Latin America were among those with the highest prevalence. Next, to check the possible factors involved in differences in prevalence observed in the various centers involved in ISAAC Phase I, ecological studies were conducted. Anderson *et al* evaluated the relationship of national immunization rates against tuberculosis, diphtheria, pertussis, and tetanus (DPT) and measles with the prevalence of symptoms of asthma, rhinitis, and atopic eczema. A negative and significant association was observed between adolescents and immunization against measles and DTP, but not against tuberculosis^(^ [@B15] ^)^. In another study, we observed a significant inverse relationship between humidity and mean annual temperature variation and prevalence of symptoms of asthma and allergic diseases^(^ [@B16] ^)^. The use of antibiotics in early life and the prevalence of symptoms of asthma, rhinoconjunctivitis and atopic eczema were also reasons of study. These indexes, adjusted for gross domestic product showed no association between the two variables^(^ [@B17] ^)^. Air pollution is identified as a risk factor for the development of asthma and related symptoms, especially in regards to particulate matter (PM), resulting from burning oil. Anderson *et al* investigated the effect of environmental PM on the prevalence of asthma and allergic diseases, analyzing satellite data by estimates and adjustments according to the gross domestic product of the countries involved. Although the data suggested a relationship, there was no statistically significant confirmation between exposure to particulate matter from 10Î1/4 (PM10) and prevalence rates^(^ [@B18] ^)^. On the other hand, Rios *et al*, assessed the prevalence of asthma and related symptoms in adolescents from two localities in the state of Rio de Janeiro, with different degrees of air pollution (PM10) - the municipality of Seropédica, little polluted, and the municipality of Duque de Caxias, very polluted - and found a significant association between exposure to higher levels of PM10 and greater prevalence of active asthma, as well as its intensity^(^ [@B12] ^)^. ISAAC phase II ============== Performed only by a few centers in the world, this phase investigated possible etiological factors, especially those suggested by the results of the first phase, and explored new etiological hypotheses regarding the development of asthma and allergic diseases^(^ [@B19] ^)^. Therefore, this research protocol included the ISAAC WQ, complementary questionnaires including information on disease management, child\'s contacts, examination for flexural dermatitis, immediate skin tests with a battery of common aero-allergens, evaluation of bronchial responsiveness to hypertonic saline, blood collection and storage for the determination of total serum IgE and genetic analysis, besides the WQ on risk factors^(^ [@B19] ^)^. The latter was translated into Portuguese and started to be used in our country by other researchers, to evaluate the possible risk factors associated with the development of asthma. In Brazil, only one center participated in the second phase of ISAAC. ISAAC phase III =============== Seven years after the completion of phase I, phase III was performed^(^ [@B20] ^)^, when the number of participating centers increased significantly, both for children from 6 to 7 years (144 centers in 61 countries), and for adolescents aged 13-14 years (233 centers in 97 countries), reaching 1,187,496 students assessed. Around the world, there was a slight increase in the mean prevalence of current asthma among adolescents (13.2 to 13.7%; mean annual growth of 0.06%) and among students from 6 to 7 years (11.0 to 11.6%; mean annual increase of 0.13%). In Latin America, these mean increases were higher: 0.32% per year for adolescents and 0.07% per year for children aged from 6 to 7 years^(^ [@B21] ^)^. In Brazil, there was an increase in the number of participating centers to 21, as well as in the number of children (n=23,422) and adolescents (n=58,144) interviewed, with centers representing the different regions of the country^(^ [@B19] ^)^. The mean prevalence of asthma was 24.3% (ranging from 16.5 to 31.2%) for children and 19% (ranging from 11.8 to 30.5%) for adolescents, without relation with the socioeconomic status^(^ [@B22] ^)^. To evaluate the temporal trend of prevalence of asthma\'s symptoms and atopic diseases, we performed the reapplication of the ISAAC WQ in centers that participated in phase I. However, some basic rules had to be followed to ensure the comparability of the method used^(^ [@B23] ^)^. The analysis of the centers involved showed that most of the participants of the two phases reapplied the ISAAC protocol properly, which was verified by the careful documentation with the use of standardized tools and checking^(^ [@B23] ^)^. The analysis of data from the participating centers of the two phases showed that, in this range, the mean prevalence of asthma among adolescents fell from 22.7 to 19.9% (-0.4% per year). Nevertheless, it still remained among the highest averages^(^ [@B24] ^)^. Knowing the temporal evolution of allergic diseases in our location, we evaluated the interference of some infectious diseases over them, in the birth of adolescents participating in phase III of ISAAC. Thus, when assessing the possible relationship between the fall in the incidence of tuberculosis and measles, observed over the years, and the increase in allergic diseases, no relationship between them was documented^(^ [@B25] ^)^, contrary to what was observed by other investigators^(^ [@B26] ^)^. The increase in the number of centers that participated in phase III allowed various studies to find reasons for the different rates observed in the country. One of the first ideas was to relate them to the socioeconomic status of the populations evaluated, not documenting association^(^ [@B22] ^)^, as it had already been done by the general coordinators of the projects involving data from phase I^(^ [@B07] ^)^. When evaluating the centers of the northeast region involved in phase III, there were very different levels of prevalence of asthma, i.e., 14.8% in Maceió, Alagoas, and 30.5% in Vitória da Conquista, Bahia. Would there be any reason for this difference? With this question in mind, these centers - Natal (Rio Grande do Norte), Recife and Caruaru (Pernambuco), Maceió (Alagoas), Aracaju (Sergipe), Salvador, Feira de Santana, and Vitória da Conquista (Bahia) -, evaluated the following parameters: latitude, mean annual temperature, altitude, human development index, GINI index, index of social exclusion (deprivation of water, sewer service, and garbage collection), illiteracy rate in people over 10 years and the percentage of households with minimum daily income below one U.S. dollar. The relationship between tropical climate and the high prevalence of asthma was associated with water deprivation and latitude^(^ [@B27] ^)^. Living in a rural environment is a factor that has been identified as a protective factor for the development of asthma and allergic diseases. Evaluating two centers of different states - Rio Grande do Sul and Pernambuco - differences in prevalence rates of asthma and allergic diseases among populations inhabiting urban and rural region in these locations were confirmed^(^ [@B28] ^)^. One of the very common problems when comparing populations that inhabit rural environments is that living in a rural environment does not necessarily mean having habits of rural life. In the Amazon region, children of two islands in Belém were evaluated, one with a better standard of hygiene (Outeiro) and other located in a riparian zone (Combú), in which there is a rural lifestyle. It was found that the prevalence of asthma was significantly higher in Outeiro (30.5 *versus* 16.5%, respectively)^(^ [@B29] ^)^. To be born and grow in a farm environment has been identified as a protective factor against the development of asthma and allergic diseases. However, the analysis of all data obtained with the ISAAC Phase III, regarding exposure to farm animals during pregnancy and in the 1st year of life, documented positive association with increased symptoms of rhinoconjunctivitis, asthma and eczema in children from 6 to 7 years living in poor countries, a fact that was not observed in children living in affluent countries^(^ [@B30] ^)^. On the other hand, exposure to domestic animals such as dogs and cats, has been documented as a risk factor for the development of asthma in children of less affluent countries. In adolescents, exposure to dogs was a risk factor for asthma symptoms, worldwide^(^ [@B31] ^)^. There is evidence that intestinal parasites, especially helminths, have a protective effect on the development of allergic diseases. In Campina Grande, Silva *et al^(^* [@B32] ^)^ evaluated the prevalence of asthma in children living in a community with a low socioeconomic status and found a prevalence of 56.3% and 59.7% for ascariasis and asthma, respectively, without association between the two of them. These data were corroborated more recently by Freitas *et al* in Belém, while assessing children living in riparian regions^(^ [@B29] ^)^. As the ISAAC WQ is standardized, available, and of free access, several national researchers used the protocol and increased the number of locations that had the prevalence of asthma, which were incorporated into the data centers officially recognized by ISAAC in Auckland , New Zealand. [Table 2](#t02){ref-type="table"}, presents the data of students from 6 to 7 years, and [Table 3](#t03){ref-type="table"}, the data of the adolescents^(^ [@B33] ^-^ [@B44] ^)^. Table 2Prevalence of active asthma (wheezing in the past 12 months), severe asthma (wheezing so intense able to stop to say two words in a row in the last 12 months) and asthma diagnosed by a physician in school children (6-7 years old) from official centers and other Brazilian centers that used the protocol of the International Study of Asthma and Allergies in Childhood (ISAAC) - Phase 3 Table 3Prevalence of active asthma (wheezing in the past 12 months), severe asthma (wheezing so intense able to stop to say two words in a row in the past 12 months) and asthma diagnosed by physician in school children (13-14 years old) of official centers and other Brazilian centers that used the protocol of the International Study of Asthma and Allergies in Childhood (ISAAC) - Phase 3 Another controversial topic, not fully explained by the ISAAC phase I, is the influence of air pollution, especially of photochemical agents, in the prevalence of asthma and allergic diseases. In Brazilian centers West São Paulo (SPO), South São Paulo (SPS), Santo André (SA), Curitiba (CR), and Porto Alegre (PoA), where there was monitoring of gaseous pollutants - ozone (O~3~), carbon monoxide (CO), nitrogen dioxide (NO~2~), and sulfur dioxide (SO~2~) -, their relationship with the prevalence of symptoms of asthma, rhinitis and atopic eczema in adolescents was evaluated. The levels of O~3~ in SPO, SPS, and SA and of CO in SA were higher than the acceptable. Although there was not a characteristic pattern for all assessed symptoms or a specification with a given air pollutant, the data obtained suggested a relationship between greater exposure to photochemical pollutants and high prevalence or risk of symptoms of asthma, rhinitis, and atopic eczema^(^ [@B45] ^)^. Allergic sensitization among patients identified as active asthmatic is variable. Data obtained by the ISAAC phase II in our location pointed to the prevalence of positive skin tests for immediate hypersensitivity (SPT) to aero-allergens of 13.3% in school children from the municipality of Uruguaiana, state of Rio Grande do Sul, and 5.4% were identified as having atopic asthma (wheezing in the last year with a positive skin prick test to at least one allergen) and 20.9% as having non-atopic asthma (wheezing in the last year and negative skin prick tests)^(^ [@B46] ^)^. These data are opposite to that observed by other investigators. Pastorino *et al*, assessing adolescents, found 46.8% of sensitization, with that by *D. pteronyssinus* observed in 79.1% of atopic patients. The risk of sensitization was 2.16 times higher in adolescents with asthma^(^ [@B47] ^)^. These data are consistent with those from a national study involving children and adolescents followed in specialized allergy services and evaluated by determination of specific serum IgE, verifying levels of positivity of 66.7% for *D. pteronyssinus*, of 64.5% for *D. farinae*, 55.2% for *Blomia tropicalis*, 32.8% for cockroach and 12% for cat^(^ [@B48] ^)^. On the other hand, Sarinho *et al*, studying asthmatic and non-asthmatic adolescents from the municipality of Caruaru, state of Pernambuco, northeastern Brazil, observed a 54.0% prevalence of sensitization for asthmatics, with prevalence of sensitization to cockroach allergens^(^ [@B49] ^)^. Previous findings point to the need for local studies, with the aim of identifying the most relevant allergens and guiding more targeted therapeutic actions. The analysis of all data from ISAAC phase III revealed that exposure to paracetamol in the 1st years of life and at present are significantly related to a higher risk of asthma, rhinoconjunctivitis, and eczema^(^ [@B50] ^)^, as observed by Kuschnir *et al* in Brazil^(^ [@B51] ^)^. In this study, multivariate analysis confirmed the association of recent and persistent use of paracetamol with increased risk of symptoms of active asthma, rhinoconjunctivitis, and eczema^(^ [@B50] ^)^. Despite its widespread use in Brazil, dipyrone is the most widely used analgesic and antipyretic. Another controversial topic mentioned in several studies is the use of antibiotics in the 1st year of life. These data were examined taking all centers participating in the ISAAC phase III, including Brazil, complementing the work done with centers of phase I^(^ [@B17] ^)^. There was an association between the use of antibiotics in the 1st years of life and current symptoms of asthma, rhinoconjunctivitis, and eczema in children from 6-7 years old. Nevertheless, it is noteworthy that more research is needed to determine whether the observed associations are real, casual, indication bias or reverse causality^(^ [@B52] ^)^. According to the initiative Allergic Rhinitis and its Impact on Asthma (ARIA), asthma and rhinitis should be viewed as a single disease, considering the high frequency of association between them^(^ [@B53] ^)^. Studies show rhinitis as a risk factor for asthma. Analyzing Brazilian data obtained in ISAAC Phase III in Brazilian adolescents, it was found that the association between asthma and rhinitis was high (r=0.82), as well as with rhinoconjunctivitis (r=0.75). The presence of current rhinitis and rhinoconjunctivitis was associated with increased risk of developing asthma and severe asthma^(^ [@B54] ^)^. This fact becomes more evident when atopic dermatitis is associated with rhinitis^(^ [@B55] ^)^. The association between asthma and overweight/obesity is also increasingly common. The systemic inflammation determined by obesity, due to the production of adipokines, the triggering of symptoms during physical activity, and dietary shifts culminate into an inappropriate physical conditioning that accentuates a sedentary lifestyle, which, alone, aggravates the functional conditions of these patients. In the southern region of the country, obesity was evaluated as a risk factor for higher prevalence of asthma and related symptoms in adolescents. The analysis of nutritional parameters showed, for girls, a positive and significant relationship between the prevalence of obesity and the diagnosis of asthma, as well as severe asthma^(^ [@B56] ^)^. A controversial topic regarding asthma in adults is its relationship with cardiovascular disease, especially hypertension. In adolescents in the municipality of Aracaju, state of Sergipe, there was a relationship between blood pressure levels and the prevalence of asthma and related symptoms; however, such association was not confirmed^(^ [@B57] ^)^. Using a similar method, the study of possible risk factors associated especially with asthma and related symptoms was performed in some Brazilian centers, using the complementary questionnaire of the ISAAC phase II^(^ [@B32] ^,^ [@B35] ^-^ [@B38] ^)^. The risk/ protective factors^(^ [@B04] ^,^ [@B58] ^-^ [@B60] ^)^ are shown in [Table 4](#t04){ref-type="table"}, in which it can be verified that being preterm, having a smoker mother, living in a house with humidity, and having a history of other allergic diseases were common factors in at least two of the population samples evaluated. Table 4Risk/protection factors associated to asthma and related symptoms in adolescents from different Brazilian centers: logistic regression analysis Conclusions =========== The ISAAC has definitely demonstrated that asthma is a disease of high prevalence and impact in Brazilian children and adolescents, and it should be seen as a real problem of public health. In the present analysis there were important regional variations, not yet fully understood, as well as various risk factors. The serial assessments conducted by the study suggested that the prevalence of asthma is stable in Brazil, but additional measurements are needed to confirm this trend. Despite the numerous studies performed since its inception, there is still much to investigate about asthma and allergic diseases in Brazil. The great miscegenation of the Brazilian population is certainly one of the factors that interfere with the clarity of the results and the relations between the studied variables. Do we have in Brazil, one or many asthmas? Instituição: Escola Paulista de Medicina da Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brasil Artigos de Revisão A asma na criança e no adolescente brasileiro: contribuição do International Study of Asthma and Allergies in Childhood (ISAAC) Objetivo: ========= Avaliar a asma na população pediátrica brasileira pelo protocolo do *International Study of Asthma and Allergies in Childhood* (ISAAC), internacionalmente padronizado e validado. Fontes de dados: ================ O ISAAC, idealizado para maximizar o valor de estudos epidemiológicos em asma e doenças alérgicas, estabeleceu um método padronizado (questionário escrito autoaplicável e/ou vídeo-questionário) capaz de facilitar a colaboração internacional. Concebido para ser realizado em três fases sucessivas e dependentes, o ISAAC reuniu uma casuística até então inimaginável no mundo e no Brasil. Nesta revisão, reuniram-se os dados de centros brasileiros oficiais do ISAAC e de outros que empregaram o seu método. Síntese dos dados: ================== Finalizada a primeira fase, a prevalência de asma entre escolares brasileiros foi documentada como a oitava em magnitude entre todos os centros participantes do estudo. Os poucos centros envolvidos na segunda fase investigaram possíveis fatores etiológicos, especialmente aqueles sugeridos pelos resultados da primeira fase, e geraram muitas especulações. A terceira fase, repetida após sete anos, avaliou a tendência evolutiva da prevalência de asma e das doenças alérgicas nos centros participantes simultaneamente das fases I e III e determinou a prevalência em outros não envolvidos na fase I. Conclusões: =========== No Brasil, o ISAAC demonstrou, de forma definitiva, que a asma é uma doença de alta prevalência e impacto em crianças e adolescentes, devendo ser encarada como problema de Saúde Pública. Encontraram-se importantes variações regionais, ainda não bem esclarecidas, assim como diversos fatores de risco, o que traz a questão: há no Brasil uma ou muitas asmas? asma epidemiologia criança adolescente fatores de risco Introdução ========== O *International Study of Asthma and Allergies in Childhood*(ISAAC), idealizado em 1990 para maximizar o valor dos estudos epidemiológicos em asma e doenças alérgicas, estabeleceu um método padronizado que facilitou a colaboração internacional, a partir do estabelecimento de protocolo utilizado no mundo todo^(^ [@B01] ^)^. Seus pontos específicos foram: a) descrever a prevalência e a gravidade de asma, de rinite e de eczema em crianças que habitam diferentes centros e realizar comparações entre eles e entre os países; b) obter medidas basais para assessorar futuras tendências na prevalência e na gravidade dessas doenças; c) fornecer subsídios para estudos etiológicos posteriores em genética, estilo de vida, cuidados médicos e poluição atmosférica, capazes de afetar essas doenças^(^ [@B01] ^)^. Concebeu-se o ISAAC para ser realizado em três fases sucessivas e dependentes. Na primeira fase, o estudo do núcleo obrigatório foi desenvolvido para avaliar a prevalência e a gravidade de asma e de doenças alérgicas em populações selecionadas, utilizando-se questionário padronizado; na segunda fase, investigaram-se possíveis fatores etiológicos, especialmente aqueles sugeridos pelos resultados da fase anterior; na terceira fase, repetiu-se a primeira após um período mínimo de cinco anos, avaliando-se a tendência evolutiva da prevalência de asma e de doenças alérgicas em determinado período, incluindo centros que participaram das fases I e III. Além disso, determinou-se a prevalência de outros países que, embora não envolvidos na fase I, tivessem interesse em medir a prevalência de asma e de sua gravidade, bem como os fatores de risco associados ao desenvolvimento da doença, tanto ambientais como de estilo de vida, específicos para cada comunidade^(^ [@B01] ^)^. Instrumentos de coleta ====================== Os instrumentos de coleta dos dados do ISAAC são o vídeo-questionário (VQ) e o questionário escrito (QE). O VQ é composto por cinco cenas, em que diversos indivíduos apresentam-se com quadros de asma em diferentes intensidades e situações, devendo ser respondido por adolescentes^(^ [@B01] ^)^. O QE é composto por três módulos, nos quais se abordam a asma, a rinite e o eczema atópico, respectivamente. O QE é apresentado em duas versões: uma para crianças de seis a sete anos, respondida pelos pais e/ou responsáveis, e outra respondida pelos próprios adolescentes. A questão \"ter sibilos no último ano\" é a que reúne maiores sensibilidade e especificidade para o diagnóstico de asma, sendo empregada universalmente para identificar os indivíduos com asma ativa^(^ [@B01] ^)^. Originariamente escrito em inglês, o QE do ISAAC foi validado para ser empregado em todo o mundo. Em localidades onde o idioma era diferente, recomendou-se que o mesmo fosse traduzido e validado para a sua utilização apropriada^(^ [@B01] ^)^. Assim, em nosso meio, o QE do ISAAC foi traduzido para o português brasileiro, vertido novamente para o inglês e, em seguida, validado por critério e construção, confirmando-se que suas características diagnósticas não foram modificadas^(^ [@B02] ^)^. Mais recentemente, o comitê diretivo do ISAAC publicou um estudo que ressalta a importância das normas culturais a serem consideradas na avaliação de *back-translation* para o inglês, permitindo-se, muitas vezes, desvios linguísticos para que se mantenham os significados originais^(^ [@B03] ^)^. Embora o VQ tenha sido idealizado para vencer barreiras impostas pela língua e possíveis distorções decorrentes das traduções do QE, sua aplicação no mundo não suplantou a do QE. No Brasil, o QE foi o único a ser utilizado. Entretanto, por conta das várias denominações que a asma recebe, alguns autores acrescentaram perguntas com sinônimos usualmente utilizados para definir a doença no QE, a fim de melhorar o poder diagnóstico do instrumento^(^ [@B04] ^)^. Denominada por muitos como bronquite, a inclusão da pergunta não melhorou o poder diagnóstico do QE do ISAAC em crianças^(^ [@B04] ^)^. Tomando-se como base a estrutura empregada em outros levantamentos populacionais nacionais, por exemplo, o inquérito sobre peso, Valle *et al* validaram o QE do ISAAC, assim como sua reprodutibilidade, administrando-o por entrevista telefônica. Pais/cuidadores de crianças (de seis a sete anos) com ou sem asma foram entrevistados em seus domicílios em duas ocasiões, com intervalo de 15 dias, e responderam ao QE do ISAAC pelo telefone, avaliando-se a reprodutibilidade das respostas às questões do questionário, dadas pelo mesmo entrevistado. A questão \"sibilos no último ano\" teve índice perfeito de concordância, reforçando a reprodutibilidade do instrumento administrado via telefone^(^ [@B05] ^)^. ISAAC fase I ============ A compilação dos dados mundiais do ISAAC fase I reuniu um número expressivo de crianças (seis a sete anos; n=257.800; 91 centros de 38 países) e de adolescentes (13-14 anos; n=463.801; 155 centros de 56 países) jamais avaliado anteriormente e mostrou grande variabilidade nas taxas entre os diferentes centros participantes^(^ [@B06] ^)^. No Brasil, a primeira fase do ISAAC, concluída em 1996, foi um verdadeiro divisor de águas no conhecimento da prevalência de asma e de doenças alérgicas no país. Antes, os dados epidemiológicos brasileiros disponíveis eram restritos a amostras populacionais pequenas, principalmente de grandes centros urbanos e de instituições educacionais, sem qualquer padronização em sua obtenção, o que dificultava muito a sua comparação. No Brasil, participaram dessa fase sete centros que viabilizaram a obtenção de dados confiáveis sobre a prevalência de asma, de rinite alérgica e de eczema atópico em crianças e adolescentes. A análise comparativa com todos os dados mundiais obtidos mostrou que a prevalência média de asma nas crianças e nos adolescentes é elevada ([Tabela 1](#t05){ref-type="table"}), sendo a oitava entre os centros de maior prevalência - países de língua inglesa e da América Latina^(^ [@B07] ^)^. Quanto à asma, nessa primeira fase, observou-se que o emprego de diagnóstico médico como critério epidemiológico para identificar os pacientes com possível asma induziria a um subdiagnóstico, uma vez que a prevalência da doença ativa (sibilos no último ano) atingiu o dobro da do diagnóstico médico^(^ [@B08] ^)^. Esse fato é corroborado, pois, na validação do QE, verificou-se que apenas 50% dos adolescentes com asma, regularmente acompanhados em serviço especializado, identificaram-se como tendo asma^(^ [@B02] ^)^. Tabela 1Prevalência de asma ativa (sibilos nos últimos 12 meses) e de diagnóstico médico de asma em escolares de centros oficiais e de outros centros brasileiros que utilizaram o protocolo do International Study of Asthma and Allergies in Childhood - Fase 1 Outros pesquisadores, mesmo sem terem o reconhecimento do comitê central do ISAAC, utilizaram o QE padrão e empregaram o protocolo, obtendo os dados de prevalência sumarizados na [Tabela 1](#t05){ref-type="table"}. A prevalência média brasileira de asma ativa foi de 23,3% para as crianças e de 22,7% para os adolescentes ([Tabela 1](#t05){ref-type="table"}). Tomando-se os dados obtidos no mundo todo, houve variação de 20 vezes na prevalência de asma e de sintomas relacionados (oscilando entre 1,8 e 36,7%), sendo os fatores ambientais os principais possíveis responsáveis por essa variação^(^ [@B14] ^)^. Países de língua inglesa e centros da América Latina ficaram entre os de maior prevalência. A partir daí, para verificar os possíveis fatores envolvidos nas diferenças das prevalências observadas nos diversos centros envolvidos no ISAAC fase I, realizaram-se estudos ecológicos. Anderson *et al* avaliaram a relação das taxas nacionais de imunização contra tuberculose, difteria, pertussis e tétano (DTP) e sarampo com a prevalência de sintomas de asma, rinite e eczema atópico. Observou-se associação negativa e significante entre os adolescentes e a imunização contra sarampo e DTP, mas não contra a tuberculose^(^ [@B15] ^)^. Em outro estudo, observou-se relação significante e inversa entre umidade e variação da temperatura média anual e prevalência de sintomas de asma e de doenças alérgicas^(^ [@B16] ^)^. A utilização de antibióticos nos primeiros anos de vida e a prevalência de sintomas de asma, rinoconjuntivite e de eczema atópico também foram motivos de estudo. Esses índices, ajustados para o produto interno bruto, não mostraram associação entre as duas variáveis^(^ [@B17] ^)^. A poluição atmosférica é apontada como fator de risco para o desenvolvimento de asma e de sintomas relacionados, sobretudo quanto ao material particulado (PM) decorrente da combustão do petróleo. Anderson *et al* investigaram o efeito do PM ambiental sobre a prevalência de asma e de doenças alérgicas, analisando os dados fornecidos via satélite por estimativas e com ajustes pelo produto interno bruto dos países envolvidos. Embora os dados sugerissem relação, não houve confirmação estatisticamente significante entre exposição ao material particulado de 10Î1/4 (PM10) e as taxas de prevalência^(^ [@B18] ^)^. Por outro lado, Rios *et al*, ao avaliarem a prevalência de asma e de sintomas relacionados em adolescentes de duas localidades do estado do Rio de Janeiro, com diferentes graus de poluição atmosférica (PM10) - Seropédica, pouco poluída, e Duque de Caxias, muito poluída -, verificaram associação significante entre exposição a níveis mais elevados de PM10 e maior prevalência de asma ativa, assim como da sua intensidade^(^ [@B12] ^)^. ISAAC fase II ============= Realizada por apenas alguns centros no mundo, essa fase investigou possíveis fatores etiológicos, especialmente os sugeridos pelos resultados da primeira fase, e explorou novas hipóteses etiológicas quanto ao desenvolvimento da asma e das doenças alérgicas^(^ [@B19] ^)^. Para tanto, constaram desse protocolo de investigação o QE do ISAAC, questionários complementares com inclusão sobre o manejo da doença, contatos da criança, exame para dermatite flexural, testes cutâneos de leitura imediata com bateria padronizada de aeroalérgenos, avaliação da hiperresponsividade brônquica à salina hipertônica, coleta sanguínea e estocagem para determinação de IgE sérica total e análise genética, além do QE sobre fatores de risco^(^ [@B19] ^)^. Este foi traduzido para o português e passou a ser empregado em nosso meio por outros pesquisadores para avaliar possíveis fatores de risco associados ao desenvolvimento da asma. No Brasil, apenas um centro participou da segunda fase do ISAAC. ISAAC fase III ============== Passados sete anos da conclusão da fase I, ocorreu a fase III^(^ [@B20] ^)^, quando o número de centros participantes aumentou significativamente, tanto para as crianças de seis a sete anos (144 centros de 61 países), como para os adolescentes de 13-14 anos (233 centros de 97 países), atingindo 1.187.496 estudantes avaliados. Em todo o mundo, houve aumento discreto da prevalência média de asma atual entre os adolescentes (13,2 a 13,7%; incremento médio anual de 0,06%) e entre os escolares de seis a sete anos (11,0 a 11,6%; incremento médio anual de 0,13%). Na América Latina, esses incrementos médios foram mais elevados: 0,32% ao ano para os adolescentes e 0,07% ao ano para as crianças de seis a sete anos^(^ [@B21] ^)^. No Brasil, houve aumento do número de centros participantes para 21, assim como do número de crianças (n=23.422) e de adolescentes entrevistados (n=58.144), tendo-se centros representantes das diferentes regiões do país^(^ [@B19] ^)^. A prevalência média de asma foi de 24,3% (oscilando de 16,5 a 31,2%) para crianças e de 19,0% (oscilando de 11,8 a 30,5%) para adolescentes, sem relação com o nível socioeconômico^(^ [@B22] ^)^. Para avaliar a tendência temporal das taxas de prevalência dos sintomas de asma e de doenças atópicas, efetuou-se a reaplicação do QE do ISAAC nos centros que haviam participado da fase I. Entretanto, algumas regras essenciais deveriam ser obedecidas para assegurar a comparabilidade do método empregado^(^ [@B23] ^)^. A análise dos centros envolvidos mostrou que a maioria dos participantes das duas fases reaplicou o protocolo ISAAC adequadamente, o que se verificou pela documentação cuidadosa com o uso de ferramentas e checagem padronizadas^(^ [@B23] ^)^. A análise dos dados oriundos dos centros participantes das duas fases mostrou que, nesse intervalo, a prevalência média de asma entre os adolescentes caiu de 22,7 para 19,9% (-0,4% ao ano). Apesar disso, ainda se manteve entre as mais elevadas do mundo^(^ [@B24] ^)^. Conhecendo-se a evolução temporal das doenças alérgicas em nosso meio, avaliou-se a interferência de algumas doenças infecciosas, no nascimento dos adolescentes participantes da fase III do ISAAC. Assim, ao se avaliar a possível relação entre a queda na incidência de tuberculose e de sarampo, observada no decorrer dos anos, e a evolução das doenças alérgicas, não se documentou qualquer relação entre ambas^(^ [@B25] ^)^, ao contrário do que foi previamente observado por outros pesquisadores^(^ [@B26] ^)^. O aumento do número de centros que participaram da fase III possibilitou vários estudos a fim de encontrar justificativas para as diferentes taxas observadas no país. Uma das primeiras ideias foi relacioná-las ao nível socioeconômico das populações avaliadas, não se documentando associação^(^ [@B22] ^)^, como já havia sido realizado pelos coordenadores gerais do projeto envolvendo os dados da fase I^(^ [@B07] ^)^. Ao se avaliarem os centros da região nordeste envolvidos na fase III, verificaram-se níveis de prevalência de asma muito díspares, ou seja, 14,8% em Maceió, Alagoas, e 30,5% em Vitória da Conquista, Bahia. Haveria alguma razão para essa diferença? Com esse questionamento em mente, nesses centros - Natal (Rio Grande do Norte), Recife e Caruaru (Pernambuco), Maceió (Alagoas), Aracaju (Sergipe), Salvador, Feira de Santana e Vitória da Conquista (Bahia) -, avaliaram-se os seguintes parâmetros: latitude, temperatura média anual, altitude, índice de desenvolvimento humano, índice GINI, índice de exclusão social (privação de água, de esgoto e de coleta de lixo), índice de analfabetismo em maiores de dez anos e percentagem de domicílios com renda mínima diária inferior a um dólar americano. Não se confirmou a relação entre o clima tropical e a alta prevalência de asma, havendo associação com a privação de água e a latitude^(^ [@B27] ^)^. Viver em ambiente rural é um fator que tem sido apontado como protetor para o desenvolvimento de asma e de doenças alérgicas. Avaliando-se dois centros de regiões distintas do país - Rio Grande do Sul e Pernambuco -, confirmaram-se diferenças nas taxas de prevalência de asma e de doenças alérgicas entre populações que habitam centros urbanos e a região rural nessas localidades^(^ [@B28] ^)^. Um dos problemas muito comuns ao se comparar populações que habitam ambientes rurais é que viver em ambiente rural não significa, necessariamente, ter hábitos de vida rural. Na região amazônica, avaliaram-se crianças de duas ilhas do município de Belém, uma com melhor padrão de higiene (Outeiro) e a outra ribeirinha (Combú), nas quais há um estilo de vida rural. Verificou-se que a prevalência de asma foi significantemente mais elevada na ilha do Outeiro (30,5 *versus* 16,5%, respectivamente)^(^ [@B29] ^)^. Nascer e crescer em fazenda tem sido identificado como fator protetor para o desenvolvimento de asma e de doenças alérgicas. Entretanto, a análise de todos os dados obtidos com o ISAAC fase III, quanto à exposição a animais de fazenda durante a gravidez e no primeiro ano de vida, documentou associação positiva com o aumento dos sintomas de rinoconjuntivite, asma e eczema em crianças de seis a sete anos, moradoras de países não afluentes, fato não observado nas moradoras de países afluentes^(^ [@B30] ^)^. Por outro lado, a exposição a animais domésticos, como cães e gatos, foi documentada como fator de risco para o desenvolvimento de asma em crianças de países menos afluentes. Em adolescentes, a exposição a cães foi fator de risco para sintomas de asma, em todo o mundo^(^ [@B31] ^)^. Há evidências de que as parasitoses intestinais, sobretudo as helmintíases, teriam efeito protetor no desenvolvimento de doenças alérgicas. Em Campina Grande, Silva *et al^(^* [@B32] ^)^ avaliaram a prevalência de asma em crianças moradoras de uma comunidade de baixo nível socioeconômico e verificaram prevalência de ascaridíase de 56,3% e de asma de 59,7%, sem associação entre ambas. Esses dados foram corroborados mais recentemente por Freitas *et al*em Belém, ao avaliarem crianças moradoras de regiões ribeirinhas^(^ [@B29] ^)^. Como o QE do ISAAC é padronizado, disponível e de livre acesso, vários pesquisadores nacionais empregaram o protocolo e ampliaram o número de localidades em que se obteve a prevalência de asma, as quais foram incorporadas aos dados dos centros oficiais reconhecidos pelo ISAAC em Auckland, Nova Zelândia. Na [Tabela 2](#t06){ref-type="table"}, apresentam-se os dados dos escolares de seis a sete anos e, na [Tabela 3](#t07){ref-type="table"}, os dos adolescentes^(^ [@B33] ^-^ [@B44] ^)^. Tabela 2Prevalência de asma ativa (sibilos nos últimos 12 meses), asma grave (sibilos tão intensos capazes de impedir de dizer duas palavras seguidas nos últimos 12 meses) e asma diagnosticada por médico em escolares (6-7 anos de idade) de centros oficiais e de outros centros brasileiros que utilizaram o protocolo do International Study of Asthma and Allergies in Childhood - Fase 3 Tabela 3Prevalência de asma ativa (sibilos nos últimos 12 meses), asma grave (sibilos tão intensos capazes de impedir de dizer duas palavras seguidas nos últimos 12 meses) e asma diagnosticada por médico em escolares (13-14 anos de idade) de centros oficiais e de outros centros brasileiros que utilizaram o protocolo do International Study of Asthma and Allergies in Childhood - Fase 3 Outro tópico controverso, não totalmente esclarecido pelo ISAAC fase I, é a influência da poluição atmosférica, sobretudo de agentes fotoquímicos, na prevalência da asma e de doenças alérgicas. Nos centros brasileiros São Paulo Oeste (SPO), São Paulo Sul (SPS), Santo André (SA), Curitiba (CR) e Porto Alegre (PoA), onde havia monitoramento de poluentes gasosos - ozônio (O~3~), monóxido de carbono (CO), dióxido de nitrogênio (NO~2~) e dióxido de enxofre (SO~2~) -, avaliou-se a sua relação com a prevalência de sintomas de asma, de rinite e de eczema atópico em adolescentes. Os níveis de O~3~ em SPO, SPS e SA e de CO em SA foram superiores aos aceitáveis. Apesar de não se detectar um padrão característico para todos os sintomas avaliados ou uma especificação com determinado poluente do ar, os dados obtidos sugerem relação entre a maior exposição aos poluentes fotoquímicos e a alta prevalência ou risco de sintomas de asma, rinite e eczema atópico^(^ [@B45] ^)^. A sensibilização alérgica entre pacientes identificados como asmáticos ativos é variável. Dados obtidos pelo ISAAC fase II em nosso meio apontam para a prevalência de testes cutâneos positivos de hipersensibilidade imediata (TCHI) a aeroalérgenos de 13,3% em escolares de Uruguaiana, Rio Grande do Sul, sendo que 5,4% foram identificados como tendo asma atópica (sibilos no último ano com TCHI positivo a pelo menos um alérgeno) e 20,9% como tendo asma não atópica (sibilos no último ano e TCHI negativos)^(^ [@B46] ^)^. Esses dados se contrapõem ao observado por outros investigadores. Pastorino *et al*, avaliando adolescentes, verificaram 46,8% de sensibilização, sendo aquela por *D. pteronyssinus* observada em 79,1% dos atópicos. O risco de sensibilização foi 2,16 vezes maior nos adolescentes com asma^(^ [@B47] ^)^. Tais dados corroboram os observados por estudo nacional envolvendo crianças e adolescentes acompanhados em serviços especializados de alergia e avaliados pela determinação dos níveis séricos de IgE sérica específica, com índices de positividade de 66,7% para *D. pteronyssinus*, de 64,5% para *D. farinae*, de 55,2% para *Blomia tropicalis*, de 32,8% para barata e de 12% para gato^(^ [@B48] ^)^. Por outro lado, Sarinho *et al*, ao estudarem adolescentes asmáticos e não asmáticos da cidade de Caruaru, nordeste brasileiro, observaram prevalência de sensibilização de 54,0%para os asmáticos, com predomínio de sensibilização aos alérgenos da barata^(^ [@B49] ^)^. Os achados anteriores apontam para a necessidade de estudos locais, com o intuito de se identificarem os alérgenos de maior relevância e orientar condutas terapêuticas mais bem direcionadas. A análise de todos os dados do ISAAC fase III revelou que a exposição ao paracetamol no primeiro ano de vida e na atualidade é significantemente relacionada ao maior risco de asma, rinoconjuntivite e de eczema^(^ [@B50] ^)^, conforme observado por Kuschnir *et al* no Brasil^(^ [@B51] ^)^. Nesse estudo, a análise multivariada confirmou a associação de uso recente e persistente de paracetamol com risco maior de sintomas de asma ativa, assim como de rinoconjuntivite e eczema^(^ [@B50] ^)^. Outro tópico controverso e apontado por vários estudos é o uso de antibióticos no primeiro ano de vida. Esses dados foram examinados tomando-se todos os centros participantes do ISAAC fase III, incluindo o Brasil, em complementação ao trabalho realizado com os centros da fase I^(^ [@B17] ^)^. Houve associação entre o uso de antibióticos no primeiro ano de vida e os sintomas atuais de asma, rinoconjuntivite e eczema em crianças de seis a sete anos de idade. Apesar disso, vale ressaltar que são necessárias mais pesquisas para determinar se as associações observadas são reais, casuais ou ainda vieses de indicação ou causalidade reversa^(^ [@B52] ^)^. Segundo a iniciativa *Allergic Rhinitis and its Impact on Asthma* (ARIA), a asma e a rinite devem ser vistas como uma doença única, considerando-se a elevada frequência da associação entre ambas^(^ [@B53] ^)^. Estudos apontam a rinite como fator de risco para asma. Analisando os dados brasileiros obtidos na fase III do ISAAC em adolescentes brasileiros, verificou-se que a associação entre a asma e a rinite foi elevada (r=0,82), assim como com a rinoconjuntivite (r=0,75). A presença de rinite e rinoconjuntivite atual foi associada a risco elevado de desenvolver asma e asma de maior gravidade^(^ [@B54] ^)^. Tal fato fica mais evidente quando a dermatite atópica se associa à rinite^(^ [@B55] ^)^. A associação da asma com sobrepeso/obesidade também é cada vez mais comum. A inflamação sistêmica determinada pela obesidade, em decorrência da produção de adipocinas, o desencadeamento de sintomas durante atividade física e os desvios alimentares culminam para um condicionamento físico inapropriado que acentua o sedentarismo e, por si só, agrava as condições funcionais desses pacientes. Na região sul do país, avaliou-se a obesidade como fator de risco para maior prevalência de asma e de sintomas relacionados em adolescentes. A análise de parâmetros nutricionais mostrou, para as meninas, relação positiva e significante entre a prevalência de obesidade e o diagnóstico médico de asma, assim como de asma grave^(^ [@B56] ^)^. Um tópico controverso quanto à asma em adultos é a sua relação com doenças cardiovasculares, sobretudo a hipertensão arterial. Em adolescentes de Aracaju, Sergipe, investigou-se a relação entre níveis pressóricos e a prevalência de asma e de sintomas relacionados, não sendo confirmada tal associação^(^ [@B57] ^)^. Empregando método semelhante, realizou-se, em alguns centros brasileiros, o estudo de possíveis fatores de risco associados sobretudo à asma e aos sintomas relacionados por meio do questionário complementar do ISAAC fase II^(^ [@B32] ^,^ [@B35] ^-^ [@B38] ^)^. Os fatores de risco/proteção^(^ [@B04] ^,^ [@B58] ^-^ [@B60] ^)^ encontram-se na [Tabela 4](#t08){ref-type="table"}, na qual se verifica que ter nascido prematuramente, ter mãe tabagista, habitar domicílio com umidade e ter antecedentes de outras doenças alérgicas foram fatores comuns em, pelo menos, duas das amostras populacionais avaliadas. Tabela 4Fatores de risco/proteção associados à asma e sintomas relacionados em adolescentes de diversos centros brasileiros: análise de regressão logística expressa em Odds Ratio (intervalo de confiança 95%) Conclusões ========== O ISAAC demonstrou definitivamente que a asma é uma doença de alta prevalência e impacto em crianças e adolescentes brasileiros, devendo ser encarada como um problema real de Saúde Pública. Importantes variações regionais, ainda não bem esclarecidas, foram encontradas, assim como diversos fatores de risco. As avaliações seriadas realizadas pelo estudo sugerem que a prevalência de asma encontra-se estável no Brasil, mas mensurações adicionais são necessárias para confirmar tal tendência. Apesar dos vários estudos realizados, ainda há muito a investigar sobre a asma e as doenças alérgicas no Brasil. A grande miscigenação da população brasileira é certamente um dos fatores que interferem na clareza dos resultados e das relações entre as variáveis estudadas. Temos, no Brasil, uma ou muitas asmas? [^1]: Conflito de interesse: nada a declarar

1. Introduction {#sec1-ijerph-17-03529} =============== Sleep paralysis (SP) is a common condition that affects approximately 7.6% of the general population during their lifetime \[[@B1-ijerph-17-03529]\]. The episodes happen either during sleep onset or offset and entail gross motor paralysis coupled with full consciousness of the situation. Although the pathophysiology of SP is not well understood, the occurrence of SP is related to a desynchrony in the architecture of rapid eye movement (REM) sleep \[[@B2-ijerph-17-03529],[@B3-ijerph-17-03529],[@B4-ijerph-17-03529]\]. In polysomnography, this state is marked by plentiful alpha EEG or a mixed pattern of alpha waves and low voltage slow waves and persistence of muscle atonia \[[@B5-ijerph-17-03529]\]. Body paralysis during SP is triggered by the pons and ventromedial medulla that inhibit the motor neurons during REM sleep by γ-aminobutyric acid and glycine \[[@B2-ijerph-17-03529]\]. SP is usually accompanied by somatic sensations, such as chest pain, shortness of breath, heart palpitations, feeling of choking, sweating, trembling, light-headedness, and nausea. These experiences are variously classified and labelled by researchers, for example, panic symptoms \[[@B6-ijerph-17-03529]\], somatic symptoms \[[@B7-ijerph-17-03529]\], or vegetative symptoms \[[@B8-ijerph-17-03529]\]. SP is often accompanied by visual, tactile, as well as auditory hallucinations \[[@B9-ijerph-17-03529],[@B10-ijerph-17-03529]\]. SP hallucinations are commonly classified into three types: (1) the "intruder" associated with a sense of a menacing presence, (2) the "incubus" associated with a feeling of tightness on the chest, and (3) unusual bodily sensations such as feelings of levitation and other types of OBEs (out-of-body experiences) \[[@B9-ijerph-17-03529]\]. SP hallucinations have generated different interpretations that vary depending on the culture in which they occur. In China, SP is understood as "ghost oppression" \[[@B11-ijerph-17-03529]\]. In Japan, it is thought to be caused by one of the Buddhist deities referred to as Fudoh-Myohoh ("Kanashibari") \[[@B12-ijerph-17-03529]\]. In the United States, it is sometimes interpreted as alien abduction \[[@B13-ijerph-17-03529]\]. In Egypt, it is described as an attack by the evil genies (i.e., the jinn) \[[@B14-ijerph-17-03529]\]; in Turkey, as a Karabasan attack (spirit like creatures) \[[@B15-ijerph-17-03529]\]; in Newfoundland, as an old witch ("old hag phenomenon") \[[@B16-ijerph-17-03529]\]; in Italy, as a pandafeche attack (witches and cat-like creatures) \[[@B17-ijerph-17-03529]\]; in South Africa, as an attack by the Thokoloshe \[[@B18-ijerph-17-03529]\]; in Brazil, as a crone with long fingernails who tramples on the chest ("Pisadeira") \[[@B19-ijerph-17-03529]\]; and in Cambodia, as an attack by a ghost, with SP called "the ghost pushes you down" \[[@B6-ijerph-17-03529]\]. These cultural explanations of SP may affect the rate and severity of SP episodes. SP is often associated with severe anxiety \[[@B1-ijerph-17-03529],[@B9-ijerph-17-03529],[@B20-ijerph-17-03529],[@B21-ijerph-17-03529],[@B22-ijerph-17-03529]\], and cultural interpretations of SP (e.g., as a supernatural experience) seem to exacerbate its severity. In Egypt, where SP is regarded as a supernatural experience, 50% of respondents reported fear of death. However, in Demark, where SP is interpreted as a psychological phenomenon, only 17% reported such fears \[[@B23-ijerph-17-03529]\]. Thus, the geographic and cultural bases of SP can have differing affects across different cultures. SP is more common in people with narcolepsy and other psychiatric disorders, especially in those with anxiety disorders such as panic disorders (between 20.8% to 30.6%) \[[@B24-ijerph-17-03529]\], social phobias (22.2%) \[[@B24-ijerph-17-03529]\], or generalized anxiety disorders (15.8%) \[[@B24-ijerph-17-03529]\]. Some studies show an increased incidence of SP in people with post-traumatic stress disorder (PTSD) \[[@B14-ijerph-17-03529],[@B25-ijerph-17-03529]\], 67% in a Cambodian sample \[[@B6-ijerph-17-03529]\]. Research also indicates that depression is strongly associated with SP in a multiple predictor model \[[@B26-ijerph-17-03529]\] and that people who experience SP have higher symptoms of depression compared to people who have never experienced SP \[[@B27-ijerph-17-03529],[@B28-ijerph-17-03529]\]. Sharpless et al., 2010, find a significant correlation between depression symptoms and recurrent fearful SP \[[@B29-ijerph-17-03529]\]. Body position during sleep onset seems relevant as SP is more common when sleeping in a supine position \[[@B9-ijerph-17-03529]\]. Moreover, SP prevalence rates are significantly higher in the student population (28.3%) compared to the general population (7.6%) \[[@B1-ijerph-17-03529]\]. SP that occurs independently of sleep disorders is referred to as isolated sleep paralysis \[[@B30-ijerph-17-03529]\]. The International Classification of Sleep Disorders (ICSD) classification distinguished Recurrent Isolated Sleep Paralysis (RISP) from regular sleep paralysis, placing it in the group of parasomnias related to the REM sleep phase \[[@B31-ijerph-17-03529]\]. Moreover, in the latest version of the International Classification of Diseases (ICD-11), RISP was defined and classified under the code 7B01.1 \[[@B32-ijerph-17-03529]\]. In the fifth edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-5), RISP is not included, although it can be marked 307.40 (unspecified sleep-wake disorder) or 307.49 (other specified sleep-wake disorders) \[[@B33-ijerph-17-03529]\]. To date, no published studies have examined SP in Poland. The aim of the current study is to assess (1) the prevalence of SP in a Polish student population, (2) the occurrence of SP-related somatic and clinical symptoms, and (3) the factors contributing to the occurrence of somatic and clinical symptoms among SP experiencers. 2. Materials and Methods {#sec2-ijerph-17-03529} ======================== 2.1. Participants {#sec2dot1-ijerph-17-03529} ----------------- The study was designed as a cross-sectional questionnaire-based descriptive study. The questionnaire was sent via Facebook to 2500 randomly selected students from various universities (and different years of study) in the Lublin Province of Poland. All data were collected from March 2018 to August 2018. The survey was completed by 439 students: 328 female students (75%) and 111 male students (25%) aged 18 to 50 years ([Table 1](#ijerph-17-03529-t001){ref-type="table"}). All participants completed a battery of online questionnaires: (1) a demographic questionnaire, (2) the Sleep Paralysis Experiences and Phenomenology Questionnaire (SP-EPQ), (3) The PTSD Checklist (PCL), (4) The State-Trait Anxiety Inventory (STAI-T), and (5) The Penn State Worry Questionnaire (PSWQ). 2.2. Materials {#sec2dot2-ijerph-17-03529} -------------- ### 2.2.1. Demographic Questionnaire {#sec2dot2dot1-ijerph-17-03529} A demographic questionnaire was designed for the current study to collect relevant personal data. The questionnaire assessed two domains: (1) Personal Data included gender, age, height, weight, size of the city in which they live, university profile; (2) lifestyle data included cigarette smoking (the number of cigarettes smoked during the day), the number of hours of sleep during 24 h period (daytime and night-time), alcohol consumption (type of alcohol and frequency of intake during the month), the use of dietary energizers (caffeine and caffeine-containing products), sports (number of hours per week devoted to physical activity). ### 2.2.2. Sleep Paralysis Experiences and Phenomenology Questionnaire {#sec2dot2dot2-ijerph-17-03529} The Sleep Paralysis Experiences and Phenomenology Questionnaire (SP-EPQ), was designed by Baland Jalal and Devon Hinton to assess the frequency of SP episodes, the presence of psychological and somatic symptoms, the prevalence indices, and the level of knowledge about the experience \[[@B23-ijerph-17-03529]\]. The questionnaire has been used in Italy and Turkey \[[@B15-ijerph-17-03529],[@B17-ijerph-17-03529],[@B34-ijerph-17-03529]\]. It is an extended version of the Sleep Paralysis Questionnaire (SPQ) previously used in Cambodia \[[@B6-ijerph-17-03529]\], Nigeria \[[@B35-ijerph-17-03529]\], China, America \[[@B36-ijerph-17-03529],[@B37-ijerph-17-03529]\], Egypt, and Denmark \[[@B14-ijerph-17-03529],[@B23-ijerph-17-03529]\]. To adapt the questionnaire for the Polish student demographic, the questionnaire was translated into Polish by a professional translator, verified by a second professional translator, edited by a native speaker with a medical degree, and subsequently checked by a proof-reader. The SP-EPQ consists of 17 open and closed questions regarding the frequency of SP episodes (e.g., lifetime, last year and month), the average duration of SP episodes, and emotions felt during the episode. The questionnaire also contains items assessing somatic symptoms accompanying SP, the nature of hallucinations during SP, causal explanations of SP, and measures taken to prevent further episodes of SP \[[@B23-ijerph-17-03529]\]. The first question of the questionnaire is formulated as follows: \"Some people experienced an event in which they could not move their arms, legs or speak during sleep or waking up, even though they wanted to do it: Have you ever had such an experience?\" If participants answered yes to this question, they were asked to describe the episode, thus allowing confirmation that the experience was in fact SP. ### 2.2.3. The PTSD Checklist (PCL-5) {#sec2dot2dot3-ijerph-17-03529} The PTSD Checklist (PCL-5) measure used in the current study is the latest Polish version of the PCL questionnaire developed for the DSM-5 \[[@B38-ijerph-17-03529],[@B39-ijerph-17-03529],[@B40-ijerph-17-03529]\]. The PCL-5 consists of 20 items pertaining to the severity of PTSD symptoms assessed on a 5-point scale from 0 (not at all) to 4 (very strongly). The Cronbach's alpha coefficient for this study is 0.92. ### 2.2.4. The State-Trait Anxiety Inventory (STAI) {#sec2dot2dot4-ijerph-17-03529} In the study, we used the Polish version of STAI of the Psychological Test Laboratory of the Polish Psychological Association \[[@B41-ijerph-17-03529],[@B42-ijerph-17-03529]\]. We used the fear anxiety subscale as an attribute (X-2 subscale), consisting of 20 items. On a 5-point Likert-scale, the respondents mark to what extent the behaviour described by the statement is typical for them. The measure is scored by summing all the items. The Cronbach's alpha coefficient for this study is 0.88. ### 2.2.5. The Penn State Worry Questionnaire (PSWQ) {#sec2dot2dot5-ijerph-17-03529} The PSWQ \[[@B43-ijerph-17-03529]\] of Janowski's adaptation \[[@B44-ijerph-17-03529]\] consists of 16 statements describing various manifestations of worry. The respondents indicate on a 5-point Likert-scale to what extent the behaviour described by the statement is typical for them. Responses range from 1 (not at all typical for me) to 5 (very typical for me). The minimum theoretical score is 16 with a maximum of 80 in which higher scores indicate a higher tendency to worry. The Cronbach's alpha coefficient for this study is 0.94. 2.3. Statistical Analysis {#sec2dot3-ijerph-17-03529} ------------------------- The statistical calculations were performed using Statistica (STATISTICA, version 12; StatSoft, Inc, Tulsa, OK, USA). Descriptive statistics were used to describe the group characteristics. We applied a chi-squared test (χ2) to compare demographic factors in the examined groups and a Shapiro--Wilk test to assess the distribution of quantitative variables. To examine the null hypothesis, the Mann--Whitney test was used. We assessed the relationship between not normally distributed continuous and ordinal variables using Spearman's rank-order correlation test. For analyses, *p* ≤ 0.05 was considered statistically significant. The project was approved by the Ethics Committee of Medical University of Lublin (the project identification code: KE-0254/125/2017) and performed according to the Declaration of Helsinki guidelines (<http://www.nil.org.pl>). 3. Results {#sec3-ijerph-17-03529} ========== 3.1. Frequency of Occurrence and Symptomatology of SP {#sec3dot1-ijerph-17-03529} ----------------------------------------------------- Among the 439 students, 140 individuals (31.89%; 95% CI: 27.5--36.3%) experienced at least one SP episode in their lifetime. The 140 students who experienced SP ((SP+) 31.89%), and the remaining 299 students who did not experience SP ((SP−) 68.11%) did not differ significantly in terms of age or sex. For a comparison of demographic characteristics of SP experiencers and non-SP experiencers, see [Table 1](#ijerph-17-03529-t001){ref-type="table"}. Among people who suffered from SP, 77% (*n* = 108) have experienced at least one episode in the last year, 34% (*n* = 47) have experienced it 4 times or more in the last year, 29% (*n* = 41) at least one time in the last month. In the SP+ group, 93% (*n* = 130; 95% CI: 87.6--96.7%) reported being afraid during the course of the SP, and 46% (*n* = 64; 95% CI: 36.7--53.3%) felt fear of death. The majority of respondents, 66% (*n* = 92; 95% CI: 57.8--73.7%), confirmed the occurrence of SP− induced hallucinations involving different sensory modalities ([Table 2](#ijerph-17-03529-t002){ref-type="table"}). Ninety-four percent (*n* = 132) of those who had SP (*n* = 140; 95% CI: 90.4--98.2%), experienced at least one type of somatic symptom ([Table 3](#ijerph-17-03529-t003){ref-type="table"}). 3.2. The Relationship between SP and the Severity of Anxiety Symptoms {#sec3dot2-ijerph-17-03529} --------------------------------------------------------------------- The severity of anxiety symptoms, the occurrence of PTSD, and a tendency to worry did not differ between the SP sufferers and individuals who had not experienced SP (*p* \> 0.05). Among males who experienced SP 4 or more times, the severity of STAI symptoms was higher than among males who experienced SP less than 4 times (*p* \< 0.05). Interestingly, this correlation was not found among females experiencing SP; the number of episodes did not correlate with the severity of anxiety symptoms (*p* \> 0.05). 3.3. The Relationship between Frequency of SP and Lifestyle Variables {#sec3dot3-ijerph-17-03529} --------------------------------------------------------------------- There was no correlation between the number of SP episodes experienced and lifestyle variables such as body mass index (BMI), amount of coffee consumed, number of cigarettes smoked, or time spent engaged in physical activity (*p* \> 0.05). However, SP frequency and sleep duration revealed significant gender differences. Among males, the amount of time devoted to sleep during the academic year was positively correlated with the number of lifetime SP episodes (R = 0.34, *p* \< 0.05). Among women, a negative correlation was found between the amount of sleep during the academic year and the number of SP episodes in the last year (R = −0.24, *p* \< 0.05) and lifetime rates of SP (R = −0.20, *p* \< 0.05). Body position during sleep onset had some effect. Fifty-four percent of subjects experienced SP while sleeping on their backs (*n* = 75). Only 8% experienced it while sleeping on the stomach (*n* = 11), and 38.5% stated that sleep position was not important (*n* = 54). Among females, 51.5% (*n* = 53; 95% CI: 42.6--62.4%) experienced SP while sleeping on the back, 9.7% on the stomach (*n* = 10; 95% CI: 4.0--15.8%); among males 59.5% on the back (*n* = 22; 95% CI: 41.4--76.3%) and on the stomach 2.7% (*n* = 1; 95% CI: −3.0--9.0%). However, these differences were not statistically significant. Body position during sleep offset was also recorded. Post hoc analyses revealed that those subjects who stated that sleep position was not important experienced more SP episodes compared to subjects who slept on their back. The differences remained significant even after a Bonferroni adjustment (Z = 2.99, *p* = 0.0085). 3.4. Factors Affecting the Severity of Somatic and Psychopathological Symptoms in People with SP {#sec3dot4-ijerph-17-03529} ------------------------------------------------------------------------------------------------ Among women, the number of somatic symptoms during SP was positively correlated with the number of cigarettes smoked (R = 0.25, *p* \< 0.05) and negatively correlated with time allocated to sleep during the academic year (R = 0.21, *p* \< 0.05). Among males, the number of such somatic symptoms experienced during SP was positively correlated with the number of cigarettes smoked (R = 0.37, *p* \< 0.05) and the amount of coffee consumed (R = 0.45, *p* \< 0.05). However, when looking specifically at hallucinations, there was no relationship between the number of SP-related hallucinations and lifestyle variables (the amount of sleep, coffee drinks consumed, cigarettes smoked). A positive association was found between somatic symptoms and the severity of PTSD symptoms (R = 0.20, *p* \< 0.05). The correlation between lifestyle-related SP risk factors and the number of SP symptoms for all study participants is shown in [Table S1](#app1-ijerph-17-03529){ref-type="app"}. The severity of somatic symptoms was also associated with anxiety symptoms. People that have a fear of death during SP had greater severity of somatic symptoms compared to people who did not fear death (*p* \< 0.05). However, the experience of visual hallucinations was not associated with the fear of death or the severity of other anxiety-related symptoms (*p* \> 0.05). 4. Discussion {#sec4-ijerph-17-03529} ============= Our study is the first of its kind to assess the prevalence of SP in Poland. With this study, we sought to better define SP symptomatology and assess possible causal factors that affect the frequency and severity of SP episodes. We analysed a student population because current research reveals that SP occurs significantly more frequently in student populations as compared to the general population. We found that the frequency of SP episodes in the student population of Lublin, Poland is 32% and is slightly higher than the average rate of SP prevalence in the world which is estimated at 28.3% \[[@B1-ijerph-17-03529]\]. In addition, 34% of the subjects in our study have experienced SP at least four times in the last year, which meets the criteria of recurring isolated sleep paralysis (RISP) \[[@B45-ijerph-17-03529]\]. The prevalence of SP episodes for student populations around the world varies to some extent. The highest SP prevalence was recorded in Peru where 55.8% of students experienced at least one episode of SP \[[@B46-ijerph-17-03529]\]; in Canada, 29--41.9% of students experienced SP \[[@B9-ijerph-17-03529],[@B47-ijerph-17-03529]\]; in Japan, 38.9--43% \[[@B12-ijerph-17-03529],[@B13-ijerph-17-03529],[@B14-ijerph-17-03529],[@B15-ijerph-17-03529],[@B16-ijerph-17-03529],[@B17-ijerph-17-03529],[@B18-ijerph-17-03529],[@B19-ijerph-17-03529],[@B20-ijerph-17-03529],[@B21-ijerph-17-03529],[@B22-ijerph-17-03529],[@B23-ijerph-17-03529],[@B24-ijerph-17-03529],[@B25-ijerph-17-03529],[@B26-ijerph-17-03529],[@B27-ijerph-17-03529],[@B28-ijerph-17-03529],[@B29-ijerph-17-03529],[@B30-ijerph-17-03529],[@B31-ijerph-17-03529],[@B32-ijerph-17-03529],[@B33-ijerph-17-03529],[@B34-ijerph-17-03529],[@B35-ijerph-17-03529],[@B36-ijerph-17-03529],[@B37-ijerph-17-03529],[@B38-ijerph-17-03529],[@B39-ijerph-17-03529],[@B40-ijerph-17-03529],[@B41-ijerph-17-03529],[@B42-ijerph-17-03529],[@B43-ijerph-17-03529],[@B44-ijerph-17-03529],[@B45-ijerph-17-03529],[@B46-ijerph-17-03529],[@B47-ijerph-17-03529]\]; in Egypt, 43% \[[@B25-ijerph-17-03529]\]; in Nigeria, 26.2--44.2% \[[@B48-ijerph-17-03529],[@B49-ijerph-17-03529]\]; in Kuwait, 28.8%; in Sudan, 29.9% \[[@B50-ijerph-17-03529]\]; and in the USA, 24.5--25% \[[@B50-ijerph-17-03529],[@B51-ijerph-17-03529]\]. Despite the significant environmental, economic, social, and cultural differences among these countries, the prevalence of SP remains significantly higher in the student population as compared to the general population. In Europe, only one study estimated the prevalence of SP in a group of students was carried out. Previous research reveals that 19.9% of Irish students experienced SP at least once \[[@B52-ijerph-17-03529]\]. Our study reveals that 32% of Polish students experienced SP at least once---a figure that is significantly higher. This is surprising given the geographic proximity and cultural similarity shared between Ireland and Poland. Our study confirmed that SP is associated with feelings of intense fear. Ninety-three percent of study participants reported that they experienced fear during SP, and 46% reported a specific fear of death. The most similar result was obtained in a Canadian study in which 90% of participants reported anxiety \[[@B21-ijerph-17-03529]\]. For comparison, in Ireland, fear was reported by 82% and fear of death by 39.8% \[[@B52-ijerph-17-03529]\]. In the USA, the Paradis et al. study reported that over 50% of respondents felt fear, and 52% felt fear of death \[[@B51-ijerph-17-03529]\]. In the Sharpless and Grom study, 75.64 % felt fear \[[@B20-ijerph-17-03529]\]. Many other studies confirm that the SP is accompanied by extreme fear \[[@B6-ijerph-17-03529],[@B22-ijerph-17-03529],[@B53-ijerph-17-03529],[@B54-ijerph-17-03529],[@B55-ijerph-17-03529],[@B56-ijerph-17-03529]\]. Our study participants reported both the presence of somatic symptoms and psychological hallucinations. Respondents reported both hypnagogic and hypnopompic hallucinations, i.e., hallucinations before falling asleep and before waking up. The incidence of any hallucinations was 66%. The most common types of hallucinations were visual, 37%. This finding aligns with similar findings reported in Ireland, 53%; in the Czech Republic, 11%; and in Nigeria, 32.6% \[[@B48-ijerph-17-03529],[@B52-ijerph-17-03529],[@B57-ijerph-17-03529]\]. In our study, the reported symptoms were dominated by physical sensations at 92% compared to 66% hallucinations. The most frequently reported somatic symptom was perceived feelings of the heart beating faster (76% of respondents). Other studies have also reported the presence of symptoms such as feelings of pressure \[[@B9-ijerph-17-03529],[@B21-ijerph-17-03529],[@B29-ijerph-17-03529],[@B51-ijerph-17-03529]\], difficulty breathing, pain, light-headedness, and dizziness \[[@B21-ijerph-17-03529]\], smothering, numbness, vibrating, tingling sensations and feeling numbness \[[@B29-ijerph-17-03529]\]. Hinton et al. 2005, described the presence of multiple somatic symptoms such as palpitations and shortness of breath during sleep paralysis among Cambodian refugees that include extremely high rates of panic \[[@B6-ijerph-17-03529],[@B53-ijerph-17-03529]\]. In most studies conducted around the world, the focus has mostly been on hallucinations and has largely omitted the physical symptomatology of SP. The questionnaire we used contained 12 questions about somatic symptoms that may accompany SP. In the commonly used Waterloo Unusual Sleep Experiences Questionnaire VIIIa \[[@B58-ijerph-17-03529]\], the feeling of pressure on the chest was classified as a type of hallucination coded as SP− "incubus" \[[@B58-ijerph-17-03529]\]. The type "incubus" refers to feelings of pressure on the chest, shortness of breath, or the impression that some creature is sitting on the chest of the person experiencing SP \[[@B9-ijerph-17-03529]\]. There is no consensus on the categorization of chest pressure as a type of psychological hallucination or physical symptom associated with stress or impaired breathing during SP \[[@B9-ijerph-17-03529],[@B47-ijerph-17-03529],[@B51-ijerph-17-03529],[@B53-ijerph-17-03529]\]. According to Jalal's "panic-hallucination (PH) model of sleep paralysis," somatic experiences during SP, such as impaired breathing or chest tightness, may be related to disruptions of the REM sleep phase: hypoxia; hypercapnia; occlusion of the airways; and rapid, shallow breathing \[[@B7-ijerph-17-03529]\]. The presence of these symptoms in combination with the paralysis of the body during SP can lead to increased fear and fear for one's own life. Stress associated with experiencing these negative emotions can trigger the so-called "reaction of fight or flight" of the amygdala and exacerbate feelings of panic \[[@B7-ijerph-17-03529]\]. When attempting to move the limbs during an SP episode, in the absence of suppression of proprioceptive feedback, there may be an increase in the sensation of tension and bodily pressure and even pain and limb contraction \[[@B59-ijerph-17-03529]\]. Additionally, SP-related somatic symptoms also occur during panic attacks and are included in the ICD-11 criteria for this diagnosis \[[@B32-ijerph-17-03529]\]. Thus, these factors combined paint a complicated picture of SP-related hallucinations as perhaps both psychological and somatic. Among the surveyed students, 52% of them felt pressure on the chest during SP. In Ireland, this was close to 48% \[[@B52-ijerph-17-03529]\]. In studies conducted outside Europe, the frequency of this symptom was lower, e.g., Kuwait, 19.2%; Sudan, 20.7%; Canada, 28.9% \[[@B50-ijerph-17-03529]\]; and Japan, 19.7% \[[@B47-ijerph-17-03529]\]. Some factors common to Europe, or at least common to Poland and Ireland, may contribute to this, but it is unclear if it may be a cultural factor or due to some other element. The most important factor we have noticed vis-à-vis the prevalence of SP was the length of sleep. This relationship is shaped quite differently for both sexes. For women, we observed a negative correlation between having SP and the number of hours slept during the academic year, while in men the correlation was positive. Our research is in line with the results of other authors \[[@B54-ijerph-17-03529],[@B59-ijerph-17-03529],[@B60-ijerph-17-03529],[@B61-ijerph-17-03529]\], which showed that among Japanese teenagers, the prevalence of SP was higher in those who slept less than 5 h or more than 9 h per 24 h period \[[@B60-ijerph-17-03529]\]. The relationship between the occurrence of SP and sleep duration is not clear. According to some researchers, sleep duration and SP are not related \[[@B62-ijerph-17-03529],[@B63-ijerph-17-03529],[@B64-ijerph-17-03529]\]. Due to inconsistent results on this topic, further studies are needed to explore possible predisposing factors. Other research stresses predisposing factors for SP: poor subjective assessment of sleep quality \[[@B54-ijerph-17-03529],[@B59-ijerph-17-03529],[@B60-ijerph-17-03529],[@B61-ijerph-17-03529],[@B64-ijerph-17-03529],[@B65-ijerph-17-03529]\], sleep deprivation \[[@B5-ijerph-17-03529]\], symptoms of insomnia \[[@B26-ijerph-17-03529],[@B66-ijerph-17-03529]\], napping during the day \[[@B54-ijerph-17-03529],[@B59-ijerph-17-03529],[@B60-ijerph-17-03529]\]. Another important factor that affects the course of SP is the position of the body. Our research confirms that the position of the body when lying on one's back promotes the occurrence of SP \[[@B67-ijerph-17-03529],[@B68-ijerph-17-03529]\]. In a study carried out by Spanos et al. (1995), as many as 70% of students reported having experienced SP when lying on their back \[[@B69-ijerph-17-03529]\]. The results of Fukuda et al. (1998) showed that 58% of Canadian and 84% of Japanese students experienced SP in a position on the back, while in the entire study population 4% of Canadians and 41% of Japanese declared that they usually sleep on their backs \[[@B47-ijerph-17-03529]\]. The relationship between SP-related anxiety symptoms and post-traumatic stress symptoms demonstrated in previous studies has not been confirmed in our study. In the majority of cases studying SP and anxiety or post-traumatic stress symptoms, the research was focused on psychiatric patients, most of whom lived in countries with significant cultural differences from Poland \[[@B6-ijerph-17-03529],[@B53-ijerph-17-03529],[@B55-ijerph-17-03529]\]. The strong relationship between PTSD and anxiety symptoms was shown among Cambodian refugees who were treated in psychiatric clinics and among patients of psychiatric hospitals in China and in the USA \[[@B6-ijerph-17-03529],[@B53-ijerph-17-03529],[@B55-ijerph-17-03529]\]. In Cambodia, among psychiatric patients, the prevalence of SP is high, with 49% having experienced SP in the last year, with the occurrence of the phenomenon almost always attributed to the action of supernatural beings such as ghosts and demons \[[@B6-ijerph-17-03529],[@B14-ijerph-17-03529],[@B53-ijerph-17-03529]\]. Here, the significant cultural differences may come into play as in Poland, and SP is mostly interpreted as a condition caused by stress, fatigue, poor sleep hygiene, and other lifestyle factors---not as a cultural phenomenon. In studies conducted by Jalal and colleagues among Egyptian students experiencing SP, the severity of PTSD symptoms, anxiety symptoms, and the tendency to worry was higher compared to peers who do not experience it \[[@B25-ijerph-17-03529]\]. Among the participants of the study, 11% explained the phenomenon of SP as a genie attack. In the general population of this country, as much as 41% interpreted the phenomenon as the effect of the genie \[[@B15-ijerph-17-03529]\]. In our study, men who experienced SP four or more times had a higher severity of anxiety symptoms on the STAI scale, compared to men who experienced SP less than 4 times (*p* \< 0.05). This suggests that the high levels of anxiety as a trait may be a risk factor for the occurrence of SP episodes among men. The greater severity of anxiety symptoms was associated with a greater number of somatic symptoms present during SP. Additionally, a positive relationship between somatic symptoms and the severity of symptoms of PTSD measured by the PLC-5 scale was also demonstrated among participants. The results obtained by us suggest a relationship between anxiety symptoms and PTSD symptoms and the course and occurrence of SP. The course of SP and the severity of somatic symptoms may be affected by cigarette smoking and caffeinated beverage consumption. Cigarette smoking is associated with a higher incidence of SP \[[@B60-ijerph-17-03529]\]. However, other studies do not confirm this relationship \[[@B54-ijerph-17-03529],[@B64-ijerph-17-03529]\], and the influence of caffeine on the frequency of SP was ruled out by current studies \[[@B61-ijerph-17-03529]\]. Furthermore, our research sheds new light on this problem. Until now, the influence of these psychoactive substances on the severity of SP symptoms has not yet been studied. Our finding seems to be worth considering due to the fact that nicotine \[[@B70-ijerph-17-03529],[@B71-ijerph-17-03529],[@B72-ijerph-17-03529]\] and caffeine \[[@B73-ijerph-17-03529]\] have a negative effect on the quality of sleep. Other such lifestyle variables should be studied in the future, especially in countries where SP is interpreted as caused by lifestyle issues rather than supernatural forces. Additionally, studies involving genetic, neurophysiological, and neuropsychological analysis in different populations could be helpful in further distinguishing among possible factors. Limitations of this study include the sample being limited to university students from Lublin province and reliance on online self-report measures alone. It should be taken into account that SP sufferers were more willing to respond to the survey than people who did not experience this phenomenon. This could have an impact on the prevalence of SP among Polish students. The study had a limited selection of causative factors. We assume that there are other risk factors affecting the occurrence of SP in the Polish student population. In the future, it would be reasonable to take into account other potential causal factors be it behavioural (e.g., naps during day, medicines), psychological (e.g., stress coping capacity, sleep quality) or chronic disorder symptoms (e.g., narcolepsy, depression). Further research is necessary to determine the risk factors for SP in the Polish population. The results of our study indicate that a significant proportion of students experience recurrent isolated sleep paralysis and that this phenomenon is associated with great fear and physical discomfort, which may further exacerbate such anxiety. Frequent and recurrent SP episodes can significantly reduce the quality of life of people experiencing them and can also negatively impact students' lives. The scale of the phenomenon revealed in the study requires a deeper analysis. In addition, it seems that scientific awareness about this disorder is not widely spread in Poland. There are no evidence-based (i.e., empirically supported) available forms of therapy for people experiencing SP. Therefore, further research on SP including determining the effectiveness of available forms of therapy should be encouraged (e.g., Jalal, 2016, 2017) \[[@B7-ijerph-17-03529],[@B74-ijerph-17-03529]\]. 5. Conclusions {#sec5-ijerph-17-03529} ============== 1\. The incidence of SP in the Lublin, Poland, student population was 32% and was slightly higher than the average prevalence in the world, which is estimated at 28.3%. 2\. The clinical picture of SP was dominated by somatic symptoms, which were reported by 94% of respondents (most often tachycardia, 76%), 93% fear (most often fear of death, 46%), and 66% hallucinations (most often visual hallucinations, 37%). 3\. The number of episodes of SP was related to the length of sleep and the position of the body during its course and, in the absence of correlation with BMI, the amount of coffee consumed, the number of cigarettes smoked, and the time spent on physical activity. 4\. The severity of somatic symptoms during SP correlated with lifestyle variables such as sleep duration, smoking habits, and coffee consumption as well as with increased anxiety-related symptoms. The authors thank Wojciech Salamon for his remarkable assistance with data collection. The following are available online at <https://www.mdpi.com/1660-4601/17/10/3529/s1>, Table S1: Correlation between lifestyle related SP risk factors and the number of SP symptoms. ###### Click here for additional data file. P.W.-K., H.K.-J., M.F., and B.J. designed the study and/or wrote the protocol. B.J. commented on the protocol. P.W.-K., M.F., and J.R. collected data. P.W.-K., M.F., and P.B. processed and entered data and conducted a literature review. J.R. undertook the statistical analysis. P.W.-K., H.K.-J., and M.F. wrote the first draft of the manuscript. D.E.H. revised the manuscript. All the authors commented on the manuscript. All authors have read and agreed to the published version of the manuscript. This study did not receive any specific grant from any funding agencies in the public, commercial, or not-for-profit sectors. The authors declare no conflict of interest. ijerph-17-03529-t001_Table 1 ###### Participants' demographic characteristics. Sample Type *N* \% Female (*n*) \% Male (*n*) Age ------------- ----- ----------------- --------------- ----------- -------- SP + 140 31.4 (103) 33.3 (37) 22 (3.92) 18--50 No SP 299 68.6 (225) 76.7 (74) 22 (4.36) 18--50 Note. SP+ = participants with at least one lifetime episode of SP; No SP = individuals who have not experienced SP. ijerph-17-03529-t002_Table 2 ###### The frequency of occurrence of hallucinations during sleep paralysis (SP) in the studied group of students. ---------------------------------------------------------------------------------------------------------------- Type of Hallucinations Visual Auditory Tactile Olfactory ------------------------ -------- -------------- --------- -------------- ---- -------------- --- -------------- Group SP+ 52 37\ 44 31\ 35 25\ 3 2\ (29.0--45.3) (23.6--39.2) (17.7--32.3) (−0.29--4.6) ---------------------------------------------------------------------------------------------------------------- Note. SP+ = participants with at least one lifetime episode of SP; N = number of responders; 95% CI = 95% confidence interval. ijerph-17-03529-t003_Table 3 ###### The frequency of occurrence of somatic symptoms during SP in the studied group of students. Sample Type Group SP+ Females SP+ Males SP+ Differences Between Females and Males ---------------------------- ----------- ----------------- ----------- --------------------------------------- ---- ---- ------------- At least one symptom 132 94 (57.8--73.7) 97 94 35 95 *p \> 0.05* Pressure on chest 72 51 (43.0--59.8) 53 51 19 51 Unable to breathe 51 35 (28.4--44.5) 41 40 10 27 Chest pain/discomfort 54 39 (30.4--46.7) 41 40 13 35 Feeling of chocking 18 13 (7.2--18.5) 16 16 2 5 Nausea, abdominal distress 7 5 (1.4--8.7) 5 5 2 5 Feeling dizzy, unsteady 44 31 (23.6--39.2) 34 33 10 27 Sweating 71 51 (42.3--59.1) 53 51 18 49 Trembling/shaking 51 36 (28.4--44.5) 36 35 15 41 Heart palpitations 106 76 (68.5--82.9) 80 78 26 70 Chills or hot flushes 48 34 (26.3--42.2) 38 37 10 27 Note. SP+ = participants with at least one lifetime episode of SP; N = number of responders; 95% CI = 95% confidence interval; *p* = *p*-value.

GENOME ANNOUNCEMENT {#h0.0} =================== Bacterial neonatal meningitis, one of the most devastating infections in the early period of human life, accounts for high mortality and morbidity among infants ([@B1]). *Escherichia coli* is the second most common pathogen associated with neonatal meningitis, and it accounts for 10% to 30% of these high mortality and morbidity rates, as well as adverse consequences in surviving neonates ([@B1]). Even though neonatal meningitis-causing *E. coli* (NMEC) has been considered one of the major pathogens associated with meningitis during the early period of human life, its pathogenesis has not been fully elucidated ([@B2], [@B3]). The NMEC strain RS218 (O18:H7:K1, ST95) was isolated from the cerebrospinal fluid of a patient with neonatal meningitis in 1974 ([@B4], [@B5]) . Over the past few decades, this strain of *E. coli* has been used extensively in the studies relevant to NMEC pathogenesis and is considered the prototypic strain of NMEC ([@B3][@B4][@B5]). Although some contigs of *E. coli* RS218 have been released, the complete and fully annotated genome of the RS218 strain is still not available ([@B5]). In the present study, the sequence of the genome of *E. coli* RS218, including its plasmid, was fully closed and completely annotated. Genomic DNA of *E. coli* RS218 was isolated using the Promega Genomic Wizard kit (Madison, WI). Genome sequencing was performed with Ion Torrent PGM sequencing technology (Life Technologies, Grand Island, NY) at the Genomics Core Facility of The Pennsylvania State University (University Park, PA) using a 318 sequencing chip. The genome was assembled with both *de novo* and reference-guided assemblies using the DNASTAR SeqMan NGen v. 11.0.0 and Lasergene Suite (Madison, WI). The gaps were closed with primer walking, and the final assembly was anchored to an optical map generated by OpGen, Inc. (Gaithersburg, MD). Annotation was performed using both Rapid Annotation using Subsystem Technology and the NCBI Prokaryotic Annotation Pipeline ([@B6], [@B7]). Analysis of the *E. coli* RS218 genome revealed that it consists of a circular chromosome of 5.087 Mb in size and a 114-kbp plasmid (pRS218) with an average G+C content of 50.6%. The sequence of pRS218 and its involvement in NMEC pathogenesis have recently been published ([@B8]). The chromosome contains 4,658 coding sequences, 88 transfer RNAs, 22 ribosomal RNAs, 1 clustered regularly interspaced short palindromic repeats array, and 5 noncoding RNAs. Additionally, it encodes secretory systems type I to VI (except the type III system), 8 fimbrial clusters, 6 iron acquisition systems, toxins, metabolic pathways, and several putative or hypothetical adhesins and invasins among other proteins. In comparison to the laboratory strain of *E. coli* K-12, the genome of RS218 contains 51 genomic islands which encode many known and potential virulence traits. These genomic data will be useful in future studies to broaden the current understanding of NMEC pathogenesis by identifying novel genes involved in initial colonization of mucosal epithelia as well as penetration of the intestinal mucosal barrier and blood-brain barrier by NMEC. Nucleotide sequence accession numbers. {#s1} -------------------------------------- The complete annotated chromosome and the plasmid of RS218 were deposited in NCBI GenBank under the accession numbers [CP007149](CP007149) and [CP007150](CP007150), respectively. **Citation** Wijetunge DSS, Katani R, Kapur V, Kariyawasam S. 2015. Complete genome sequence of *Escherichia coli* strain RS218 (O18:H7:K1), associated with neonatal meningitis. Genome Announc 3(4):e00804-15. doi:10.1128/genomeA.00804-15. We thank Deb Grove and her colleagues for sequencing performed at the Genomics Core Facility of The Pennsylvania State University. This study was supported by the Department of Veterinary and Biomedical Sciences at The Pennsylvania State University.

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Introduction ============ Hepatocellular carcinoma (HCC) accounts for up to 90% of all malignant primary liver cancer worldwide and represents a major health threat.[@b1-ijn-13-1621] Currently, apart from surgery, patients with liver cancer are mainly treated by chemotherapy.[@b2-ijn-13-1621] However, chemotherapy is limited by a low response rate and severe systemic toxicity due to low specificity and resistance mechanisms of the chemotherapeutic agents toward cancer cells.[@b3-ijn-13-1621] Therefore, novel drug delivery systems are urgently needed to enhance the selective action of cytotoxic drugs to HCC and therefore minimize systemic toxicity to noncancerous tissues. Glycyrrhetinic acid (GA), a pentacyclic triterpenoid, is widely present in the licorice plant and is the active aglycone of glycyrrhizin (GL). Abundant GA receptors have been confirmed on the cellular membrane of hepatocytes.[@b4-ijn-13-1621] The protein kinase C-α was reported as the target binding protein of GA, which had much more expression in HCC cells than in the adjacent nontumor liver cells.[@b5-ijn-13-1621] The amount of GA receptors in tumor tissue has been found to be 1.5- to 5-fold more than that in normal tissue.[@b6-ijn-13-1621] Additionally, GA showed the selective toxicity toward tumor through the downregulation of glutathione.[@b7-ijn-13-1621] Recently, some GA-modified liposomes have been developed with higher drug accumulation in the liver and better anti-HCC activity.[@b5-ijn-13-1621]--[@b9-ijn-13-1621] We have confirmed GA receptors on HCC cells formerly according to the binding effect between fluorescence-labeled GA and GA receptors.[@b10-ijn-13-1621] Therefore, it could be expected that GA-modified liposomes could target selectively to HCC cells and tissues. GA exists in two stereoisomers, including *trans* form 18α- and the *cis* form 18β-GA, lying in the spatial orientation of hydrogen atom of C~18~. Different configurations of GA exhibit various stabilities, solubilities[@b11-ijn-13-1621] and pharmacological effects. 18β-GA exerted protective effects against cyclophosphamide-induced hepatotoxicity.[@b12-ijn-13-1621] 18β-GA also showed anti-HCC proliferation effects, which could induce the HCC cells' apoptosis via modulation of inflammatory markers and inhibit HCC development by reversing hepatic stellate cell-mediated immunosuppression.[@b13-ijn-13-1621],[@b14-ijn-13-1621] 18β-GA could reduce the amount of glucose release induced by glucagon in rat primary cultured hepatocytes, while 18α-GA did not.[@b15-ijn-13-1621] Nevertheless, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a kind of microsomal enzyme belonging to the short-chain dehydrogenase/reductase family, which is highly expressed in many glucocorticoid target tissues, such as the liver, adipose tissue, skeletal muscle and macrophages. 18α-GA selectively inhibited 11β-HSD1, but 18β-GA had no selectivity.[@b16-ijn-13-1621] 18α-GA increased peroxisome proliferator- activated receptor γ expression and decreased nuclear factor-κB DNA-binding activity, inhibiting the proliferation of activated hepatic stellate cells.[@b17-ijn-13-1621] 18α-GA was reported to target prostate cancer cells by downregulation of inflammation-rated genes in DU-145 cells.[@b18-ijn-13-1621] Ishida et al have proved that a carrier-mediated transport system participated in the uptake of GL into isolated rat hepatocytes and the affinity site of the transport carrier bound to GA.[@b19-ijn-13-1621] GA is a hydrolytic product of GL with the differences of hydroxyl or glycosyl group at C~3~. The removal of 11-carbonyl in the ring structure of GA not only eliminated pseudoaldosterone effect but also improved its anti-inflammatory, antiulcer and antiallergic activities.[@b20-ijn-13-1621] 11-deoxy-18β-GA (11-Deo-GA), performing a similar action of 18α-GA, also selectively (and significantly) acted on 11-β-HSD1.[@b21-ijn-13-1621] As for anticancer properties, 11-Deo-GA induced gastric cancer cell apoptosis by upregulation of p21, downregulation of cdc2 and cyclin B1 and association with BID (a BH3 domain-only agonist) translocation from the nucleus to the mitochondria.[@b22-ijn-13-1621] These deduced that for GA's main ring structure, the hydroxyl group at C~3~ and the carbonyl group at C~11~ had certain effect on the liver or HCC cell targeting. Thus, in this study, we focused on the targeting effect of different GA configurations and groups to HCC cells. As shown in [Figure 1](#f1-ijn-13-1621){ref-type="fig"}, 18β-GA, 18α-GA, 3-acetyl-18β-GA (3-Ace-GA) and 11-Deo-GA were obtained, and fluorescein isothiocyanate-labeled 18β-GA (FITC-GA) was synthesized according to reported method.[@b23-ijn-13-1621] The binding site competition to HCC cells of different GA derivatives was studied. The long-circulation phospholipids with potential targeting molecular were synthesized by the GA derivatives linked with DSPE-PEG~2000~-NH~2~. Coumarin 6 (Cou6) and 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) liposomes were prepared to evaluate the targeting effect of GA's configurations and groups in vitro and in vivo. Materials and methods ===================== Materials --------- 18β-GA (purity 98%), 18α-GA (purity ≥98%) and Cou6 (purity 98%) were obtained from J&K Scientific Ltd. (Beijing, China). 3-Ace-GA, 11-Deo-GA and FITC-GA were synthesized and characterized in our laboratory. DSPE-PEG~2000~ (DSPE-PEG, purity ≥97%) and DSPE-PEG~2000~-NH~2~ (DSPE-PEG-NH~2~, purity ≥95%) were bought from AVT Pharmaceutical Co., Ltd (Shanghai, China). Soybean phospholipid (for injection, phosphatidylcholine ≥85%) and cholesterol (purity ≥98%) were from Tywei Pharmaceutical Co. (Shanghai, China). DiR was from AAT Bioquest Int. (Sunnyvale, CA, USA). RIPA lysis buffer, BCA protein assay kit and Hoechst 33258 were from Beyotime Institute of Biotechnology (Shanghai, China). Human HCC (HepG2) cells and mouse ascites hepatoma (H22) cells were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China). HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). H22 cells were cultured and passaged in Kunming mouse ascites. Male BALB/c nude mice (20±2 g), supplied by the Department of Experimental Animals, Shenyang Pharmaceutical University (Shenyang, China), were acclimated under specific-pathogen-free conditions in the central animal facility of the university. All animal experiments were approved by the Ethics Committee of the Shenyang Pharmaceutical University and were carried out in accordance with the guidelines evaluated and approved by the ethics committee of Shenyang Pharmaceutical University. Competition of binding sites ---------------------------- Approximately 2.5×10^6^ HepG2 cells were seeded into 6-well culture plates. After the cells have covered the plates, the medium was removed and the cells were washed with PBS and DMEM (without FBS) successively. The cells were treated with a series concentrations of 18β-GA, 18α-GA, 3-Ace-GA, 11-Deo-GA and FITC-GA in DMEM for 2 h at 37°C. Then, FITC-GA was added to a final concentration of 100 nM. After co-incubation for 2 h at 37°C, the cells were washed with chilled PBS and lysed with 100 μL of lysis buffer in ice bath. The cell lysate was harvested, shaken and centrifuged (1.2×10^4^ rpm for 10 min) at 4°C. The fluorescence intensity of the supernatant was measured (λ~ex~ =490 nm, λ~em~ =520 nm) with the Varioskan Flash Spectral Scanning Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA). The fluorescence intensity was normalized with respect to the cells' protein content, which was determined with a BCA protein assay kit. The fluorescence intensity of different GA derivatives with FITC-GA samples and the only FITC-GA samples normalized with the content of protein in the cells was calculated as the specific binding (B) and maximum binding (B~0~), respectively. The competitive curve fitting was constructed by GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA) software with concentration as x-axis and specific binding ratio (B/B~0~) as y-axis. Synthesis and identification of DSPE-PEGylated GA derivatives ------------------------------------------------------------- The GA derivatives and DSPE-PEG were linked together with amidation ([Figure 2](#f2-ijn-13-1621){ref-type="fig"}).[@b24-ijn-13-1621] In total, 235.5 mg 18β-GA, 235.5 mg 18α-GA, 256.2 mg 3-Ace-GA and 228.2 mg 11-Deo-GA (0.5 mMol) were dissolved in anhydrous acetone with an amount of pyridine. In total, 143.8 mg EDC⋅HCl (0.75 mMol), 86.3 mg NHS (0.75 mMol) and 1,100 mg DSPE-PEG were then added sequentially to the solution with stirring for 48 h under nitrogen protection at ambient temperature. The products were obtained by recrystallization from the condensed reaction solutions, which were mixed with an amount of ice-cold anhydrous ether. The precipitate was dissolved in water and dialyzed for 24 h. The DSPE-PEGylated GA derivatives were obtained by freeze-drying at last. The ^1^H-NMR (DMSO-*d*~6~) spectrum of DSPE-PEGylated GA derivatives was characterized at 300 Hz with an ARX-300 (600) NMR spectrometer (Bruker Optik GmbH, Ettlingen, Germany). Preparation and characterization of Cou6 liposomes -------------------------------------------------- The liposomes were prepared by a modified film dispersion method according to our previous report.[@b23-ijn-13-1621] Briefly, 18.2 mg DSPE-PEG, 21.2 mg 18β-GA-PEG-DSPE, 21.2 mg 18α-GA-PEG-DSPE, 21.4 mg 3-Ace-GA-PEG-DSPE or 21.0 mg 11-Deo-GA-PEG-DSPE (6.6 μM) with 50 mg phospholipid (about 1:10 molar ratio), 20 mg cholesterol and 2 mg Cou6 was dissolved in 10 mL of ethanol-dichloromethane (2:1). A thin lipid film was produced by rotary evaporation and hydrated in 10 mL of water at 60°C, followed by stirring at ambient temperature. The mixture was sonicated for 10 min at 200 W. The colloidal solution obtained was then centrifuged at 1.2×10^4^ rpm/min for 10 min and filtered through 0.22 μm Millipore filters. All the procedures were conducted in darkness. The Cou6 liposomes mixed with DSPE-PEG (PEG-Cou6-Lip), 18β-GA-PEG-DSPE (18β-GA-Cou6-Lip), 18α-GA-PEG-DSPE (18α-GA-Cou6-Lip), 3-Acel-GA-PEG-DSPE (3-Ace-GA-Cou6-Lip) and 11-Deo-GA-PEG-DSPE (11-Deo-GA-Cou6-Lip) were prepared. The particle sizes, polydispersity index (PDI) and zeta potentials of the liposomes were measured with a Nano Analyzer (ZS90; Malvern Instruments, Malvern, UK). The entrapment efficiency (EE), loading capacity (LC) and leakage (LK) of liposomes were determined with mini-column centrifuge-fluorescence spectroscopy (λ~ex~ =497 nm, λ~em~ =523 nm) methods. Analysis of liposomal membrane stability ---------------------------------------- To evaluate the stability of the liposomal membranes, the resistance of the liposomes against nonionic surfactant Triton X-100 was investigated.[@b25-ijn-13-1621] A series concentration (0.5%, 1%, 2%, 5%, 10%, 20%, 50%, 100%) of Triton X-100 solutions was prepared. The Triton X-100 solutions (10 μL) were added to the GA derivative-mediated liposomes (190 μL) in the 96-well plates in triplicate. The plates were shaken for 15 min at 25°C. The absorbance at 590 nm was measured by Multimode Reader. The turbidity was recorded as A/A~0~ (590 nm), where A is the absorbance of samples with a predetermined concentration of Triton X-100 addition and A~0~ is the initial absorbance of samples. Cytotoxicity assay in vitro --------------------------- Effect of the liposomes on HepG2 cells growth was assessed by MTT assay. In total, 5×10^3^ HepG2 cells were seeded into the 96-well culture plates in quintuplicate and incubated overnight. The medium was then replaced with the GA derivative-mediated liposomes at gradient Cou6 concentrations of 2 μg/mL (low), 10 μg/mL (medium) and 50 μg/mL (high). After incubation for 6 h, the cultured medium was then discarded and refreshed with DMEM. In total, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well for additional 4 h of incubation. Formazan was dissolved in 150 μL of DMSO, and the absorbance at 570 nm was measured using the Multimode Reader. The inhibition was calculated according to the absorbance. Confocal observation -------------------- Laser scanning confocal microscopy was used to visualize the subcellular localization of different Cou6 liposomes.[@b26-ijn-13-1621] HepG2 cells were cultured on sterile microscope slides in the 6-well culture plates (2×10^5^ cells/well) and incubated overnight. The media were replaced by the five liposomes in DMEM (without FBS), and the cells were further incubated for 0.25, 1 and 6 h. Then, the media were removed and the cells were washed 3 times with cold PBS. Hoechst 33258 (10 μg/mL, 10 min) was used to localize nuclei. Finally, the cells were fixed with 4% formaldehyde at ambient temperature and captured by laser scanning confocal microscope (Olympus FV1000-IX81; Olympus Corporation, Tokyo, Japan). The content of Cou6 in each sample was measured accordingly as we reported previously.[@b10-ijn-13-1621] In vivo targeting ability study ------------------------------- The DiR liposomes containing DSPE-PEG (PEG-DiR-Lip) and mediated by different GA derivatives (18β-GA-DiR-Lip, 18α-GA-DiR-Lip, 3-Ace-GA-DiR-Lip and 11-Deo-GA-DiR-Lip) were prepared according to the previously mentioned method. Male BALB/c mice were inoculated subcutaneously in the right axillary fossa with 0.2 mL of H22 cell suspension (2×10^6^ cells/mL). Once the tumor reached a volume of 150--200 mm^3^, the H22 tumor-bearing mice were obtained. The mice were divided into five groups and intravenously injected with 0.2 mL of various DiR liposomes. In the period of 12 h postinjection, the mice under anesthetic state were scanned using a Kodak In Vivo Imaging System FX Pro (Carestream Health Inc., Rochester, NY, USA) at different time. Statistical analysis -------------------- All the experiments were performed in triplicate, and the values are presented as mean ± standard deviation. The data were analyzed by applying one-way ANOVA, followed by the methods of Student's *t*-test. *p*-value \<0.05 indicated statistically significant differences. Results and discussion ====================== Competitive effect on binding sites ----------------------------------- According to the differences in the configurations and in the main modified parts at C~3~-hydroxyl and C~11~-carbonyl groups of GA,[@b27-ijn-13-1621] the compounds including 18β-GA, 18α-GA, 3-Ace-GA and 11-Deo-GA were obtained. As is known, receptor competition is increasingly common to discovery of new drugs[@b28-ijn-13-1621] and screen for targeting ligands. Higher binding effect means stronger physiological activity and longer signaling pathway influence. The results on the competitive effect of GA derivatives with FITC-GA in HepG2 cells are shown in [Figure 3](#f3-ijn-13-1621){ref-type="fig"}. The reversible binding of receptors and ligands accounted for 70% of maximum binding (B~0~) of FITC-GA after the inhibitor (18β-GA, 18α-GA, 3-Ace-GA or 11-Deo-GA) was added. EC~50~ values of different GA derivative competition to binding sites in HepG2 cells were calculated. Among them, 18β-GA decreased the specific binding ratio (B/B~0~) significantly. The EC~50~ of 18β-GA was 100.1 nM. B/B~0~ value decreased to 10% of B/B~0~ when 200 nM of 18β-GA was added. The competitive curve of 18α-GA with FITC-GA was flatter and the EC~50~ was 563.1 nM indicating the weak binding effect of GA receptors and competitive ability with FITC-GA. It demonstrated that the 18-H configuration of GA influenced the binding effect greatly. The addition of 3-Ace-GA gave a similar competitive curve of 18β-GA and 111.7 nM of EC~50~, indicating a little influence of 3-OH of GA. The competitive curve of 11-Deo-GA lay between 18α-GA and 3-Ace-GA, with 121.2 nM of EC~50~ demonstrating the competitive effect with FITC-GA. 11-Carbonyl group influenced the targeting action of GA. Characterization of GA derivative-PEG-DSPE ------------------------------------------ The DSPE-PEGylated GA derivatives were synthesized by grafting the carboxyl group of GA onto the amino group of aminated DSPE. The ^1^H-NMR spectrum in [Figure 4](#f4-ijn-13-1621){ref-type="fig"} shows that the signals at δ 0.68--1.11 ppm belonged to the methylene and the methyl groups of the pentacyclic triterpenoid structure of GA. The peaks at δ 1.30--1.40 ppm were 19-H and at δ 5.30--5.50 ppm were 12-H of the GA rings. The peaks at δ 1.20--1.35 ppm and δ 3.30--3.60 ppm mainly attributed to the -CH~2~ protons of stearic acid groups of DSPE and the glycol blocks of PEG~2000~, respectively. In the ^1^H-NMR spectrum, a single peak with one unit area at δ 7.40--7.80 ppm was found (18β-GA-PEG-DSPE 7.78 ppm, 18α-GA 7.63 ppm, 3-Ace-GA 7.50 ppm, 11-Deo-GA 7.47 ppm), which belonged to the --CO--NH-- proton when --COOH of GA derivatives acetylated with the --NH~2~ of DSPE-PEG-NH~2~. The integration value ratio for the peak of amide group and the peak of methyl (δ 0.75--0.83) in stearyl group was about 1:6. Therefore, the long-circulation phospholipids with GA derivatives have been successfully synthesized. The yield of 18β-GA-PEG-DSPE, 18α-GA-PEG-DSPE, 3-Ace-GA-PEG-DSPE and 11-Deo-GA-PEG-DSPE was 69.3%, 53.2%, 75.0% and 66.4%, respectively. Pharmaceutical characters of liposomes -------------------------------------- Liposome size is an important factor that can influence drug dosage, uptake, targeting, clearance and lysosomal accumulation.[@b29-ijn-13-1621] The liposomal characters are shown in [Table 1](#t1-ijn-13-1621){ref-type="table"}. The particle sizes of the five liposomes were less than 180 nm, which were analyzed by dynamic light scattering. The sizes of Cou6 liposomes with DSPE-PEGylated GA derivatives were smaller than those of PEG-Cou6-Lip. The possible reason might be that the hydrophobicity of GA derivatives wrapped the PEG chain tightly and squeezed the particles further. The PDI (0.182--0.213) demonstrated relatively narrow distribution of the liposomes. The zeta potential of the five liposomes was negative ranging from −10 to −18 mV. With the addition of DSPE-PEGylated GA derivatives, the zeta potential was 8 mV or so lower. A greater zeta potential is necessary to inhibit liposomal aggregation.[@b30-ijn-13-1621] The LK of the Cou6 liposomes was lower (\<7%, 14 d). The GA derivative-mediated liposomes showed higher stability with lower potential and lower LK. As a regulatory requirement, EE is a greatly significant parameter to control the quality of the liposomes.[@b31-ijn-13-1621] Results showed that the values of EE and the LC of the five liposomes were all higher than 65% and 17.5 mg/g. 3-Ace-GA-Cou6-Lip and 11-Deo-GA-Cou6-Lip showed the highest and lowest values of EE, respectively. The DSPE-PEGylated GA derivatives had no significant effect on the EE and LC of Cou6. Therefore, the liposomes exhibited good preparation characteristics. Because the optimum size of PEG-liposomes for prolonged circulation is 160--220 nm,[@b32-ijn-13-1621] we could expect a good long-circulation effect of these GA derivative-modified liposomes. Liposomal membrane stability ---------------------------- Triton X-100, a polyoxyethylene nonionic surfactant, has been used to determine the membrane stability of different Cou6 liposomes. Three relevant transitions would take place when Triton X-100 was added into the liposomal suspension. First, lower concentration led to a slight increase in the size of liposome, which might be attributed to the incorporation of surfactant monomers in the lipid bilayer. Second, increasing surfactant amounts led to a progressive decrease in intensity for mixed liposomes, which corresponded to the saturation of Triton X-100 in bilayer and the formation of lipid micelles. The size of micelles was far less than that of liposomal particles. Finally, higher concentration made a slight fall in the particle size, which might be attributed to the progressive enrichment in Triton X-100 of the mixed micelles formed.[@b33-ijn-13-1621] The variations of relative turbidity (RTU) for the suspension vs Triton X-100 concentration were plotted in logarithmic coordinates ([Figure 5](#f5-ijn-13-1621){ref-type="fig"}). The spots showed the inverted *S*-type distribution when a series concentration of Triton X-100 was added into the liposomal suspensions. The RTU expressed horizontal distribution when lower (\<0.15‰) and higher (\>0.60‰) concentrations of Triton X-100 were added because of the less membrane action and thorough membrane destroy, respectively. When a medium concentration of Triton X-100 was added, the liposomal membrane was destroyed in a concentration-dependent manner and the RTU decreased sharply; a straight line (linear regression) was fitted for the relationship of RTU to logarithm concentration of Triton X-100. When half of the liposomal membrane was destroyed (RTU =0.5), the destruction concentration of Triton X-100 (DC~50~) was calculated ([Table 1](#t1-ijn-13-1621){ref-type="table"}) to evaluate the stability of the membrane. The results showed that the membrane of PEG-Cou6-Lip was extremely stable and the membrane stability of GA derivative-modified liposomes decreased slightly. Cytotoxicity assay ------------------ The cytotoxic effects of PEG-Cou6-Lip, 18β-GA-Cou6-Lip, 18α-GA-Cou6-Lip, 3-Ace-GA-Cou6-Lip and 11-Deo-GA-Cou6-Lip on HepG2 cells in 6 h were investigated by MTT assay. As shown in [Figure 6](#f6-ijn-13-1621){ref-type="fig"}, different concentrations (2, 10, 50 μg/mL) of Cou6 used in different liposomes showed certain cytotoxicity against HCC cells. No apparent cytotoxicity was observed at low concentration (IR \<2.0%). Compared to PEG-Cou6-Lip, there were no significant differences in cytotoxicity at 10 μg/mL of Cou6 in four GA derivative-mediated liposomes. High concentration action showed significant differences in cell viability. As a result, 10 μg/mL of Cou6 in liposomes was chosen as the optimal concentration in the further trial and 6 h was taken as the longest action time. Cellular localization --------------------- Cou6 with high quantum yield has been widely used in cellular localization of nano-drug delivery system. Most cells have no background influence in the fluorescence spectrum range of Cou6. In addition, raw Cou6 cannot be directly internalized by the cells.[@b34-ijn-13-1621] In other words, the fluorescence measured in the uptake samples reflects the liposomes taken up by the cells but not the released Cou6. [Figure 7](#f7-ijn-13-1621){ref-type="fig"} shows the confocal laser scanning microscopy (CLSM) images and quantitative analysis of different liposomes loading Cou6 after 15 min, 2 h and 6 h in HepG2 cells. For 18β-GA-Cou6-Lip and 3-Ace-GA-Cou6-Lip, much more green fluorescence intensity was aggregated around the HepG2 cells after incubation for 15 min, demonstrating a good affinity to the cytomembrane. Meanwhile, weak fluorescence was observed near and in the cells when the other three liposomes were added. After co-incubation for 2 h, the green fluorescence was internalized and gathered in the cells. The HCC cells treated with 18β-GA-Cou6-Lip and 3-Ace-GA-Cou6-Lip showed distinct cell outlines with strong fluorescence intensity. However, the cells treated with PEG-Cou6-Lip, 18α-GA-Cou6-Lip and 11-Deo-GA-Cou6-Lip showed obscure outlines. The green fluorescence intensity was enhanced with increasing the incubation time for all the liposomes. Furthermore, after co-incubation for 6 h, most fluorescence was gathered in the cells and all the liposomes were internalized by cells. It was difficult to distinguish the binding sites of the cells. The results indicated that GA-modified nano-carriers showed liver and HCC targeting effects. The C~3~-hydroxyl group of GA had little influence on the targeting ability, which was consistent with the reports of Tian et al.[@b35-ijn-13-1621] The spatial configuration and C~11~-carbonyl group affected the HCC cell targeting of GA. Tumor targeting in vivo ----------------------- The in vivo biodistribution and targeting ability of the liposomes in mice were evaluated using a near-infrared fluorescence image system. DiR was chosen as a fluorescent marker, which is a type of near-infrared fluorescent dye with an excitation spectrum of 750 nm and an emission spectrum of 782 nm. As a lipophilic tracer, DiR allowed the noninvasive tracking of cells for several days in vivo.[@b36-ijn-13-1621] [Figure 8](#f8-ijn-13-1621){ref-type="fig"} shows the real-time images in H22 tumor-bearing nude mice at 0.5, 2 and 12 h after intravenous (caudal-vein) injection of PEG-DiR-Lip, 18β-GA-DiR-Lip, 18α-GA-DiR-Lip, 3-Ace-GA-DiR-Lip and 11-Deo-GA-DiR-Lip. All the liposomes had a time-dependent liver and tumor accumulation in the mice. Most DiR accumulated in the liver at 0.5 h postadministration of the liposomes, and then there was a decrease in the fluorescence intensity from 2 to 12 h. With the prolongation of time, the fluorescence intensity in the tumor region was increased. 18β-GA- and 3-Ace-GA-modified liposomes revealed distinct uptake, which were transported more quickly to the HCC tumors after 2 h of injection. The highest accumulation in an H22 xenograft tumor model after 12 h of administration was found, which was consistent with CLSM results of the two liposomes in HCC cells. As to PEG-DiR-Lip and 18α-GA-DiR-Lip, only a little fluorescence was measured in the tumor tissues. However, the fluorescence intensity in the liver decreased significantly. The targeting effect of 11-Deo-GA-DiR-Lip differed from 18β-GA-DiR-Lip and 3-Ace-GA-DiR-Lip, as well as PEG-DiR-Lip and 18α-GA-DiR-Lip. Part of liposome was transported and accumulated in the HCC tumors. There was no obvious fluorescent signal appearing in kidney tissue. This suggested that the metabolism in the liver was the main elimination route of GA derivative-modified liposomes. The tumor-targeting results indicated that the liposomes were transferred quickly in the liver. There were no significant differences whether GA derivatives modified or not. Different GA derivative ligands showed different HCC tumor-targeting effects. The configuration and groups of GA influenced different degrees on the targeting effects. Conclusion ========== GA receptor has been deemed to be a promising receptor to target HCC attributing to its safety and pharmacological potential. Focusing on the differences of structure configurations and active groups, four GA derivatives (18β-GA, 18α-GA, 3-Ace-GA and 11-Deo-GA) and mediated liposomes (18β-GA-Lip, 18α-GA-Lip, 3-Ace-GA-Lip and 11-Deo-GA-Lip) were developed to show different abilities to target the HCC cells and HCC tumors. 18β-GA and 3-Ace-GA showed significantly competitive effects with FITC-GA on HepG2 cells. The effect decreased in turn for 11-Deo-GA and 18α-GA-Lip. The liposomes modified with GA derivatives showed higher loading ability and better stability. Compared to common long-circulation liposome (PEG-Lip), more 18β-GA- and 3-Ace-GA-modified liposomes were aggregated around HepG2 cells in vitro in short time and were transferred into HCC tumors in vivo for a longer time. There were no significant differences between 18α-GA-Lip and PEG-Lip on HCC targeting. In addition, 11-Deo-GA-Lip showed certain targeting effect. Therefore, it can be concluded that GA shows the targeting action to HCC cells and HCC tumors. The β-configuration hydrogen atom at C~18~ position of GA contributes the most targeting effect. C~11~-carbonyl and C~3~-hydroxy groups of GA have certain and little influences on the targeting action to HCC, respectively. This work was supported by the grants from the Science and Technology Department of Liaoning Province (2013022064, 201602312). **Disclosure** The authors report no conflicts of interest in this work. {#f1-ijn-13-1621} {#f2-ijn-13-1621} {#f3-ijn-13-1621} {#f4-ijn-13-1621} {#f5-ijn-13-1621} {#f6-ijn-13-1621} {#f7-ijn-13-1621} {#f8-ijn-13-1621} ###### Characteristics of GA derivative-mediated liposomes Liposomes Particle size (nm) PDI Zeta potential (mV) EE (%) LC (mg/g) LK (%) DC~50~ (‰) -------------------- -------------------- ------------- --------------------- ------------ ------------ ----------- ------------ PEG-Cou6-Lip 177.3±26.6 0.182±0.012 −10.03±0.93 70.33±2.70 18.75±0.72 6.87±2.07 0.566 18β-GA-Cou6-Lip 162.2±20.0 0.196±0.017 −18.82±0.65 75.14±2.15 20.04±0.57 5.76±1.27 0.380 18α-GA-Cou6-Lip 169.9±14.1 0.207±0.022 −18.41±0.43 76.50±1.02 20.40±0.27 6.05±0.84 0.354 3-Ace-GA-Cou6-Lip 158.0±28.3 0.209±0.008 −18.20±1.07 82.40±0.80 21.97±0.21 5.96±1.10 0.207 11-Deo-GA-Cou6-Lip 152.4±10.2 0.213±0.026 −18.48±0.78 66.18±1.17 17.65±0.31 6.37±0.45 0.253 **Note:** Data expressed as mean ± standard deviation (n=3). **Abbreviations:** GA, glycyrrhetinic acid; PDI, polydispersity index; EE, encapsulation efficiency; LC, loading capacity; LK, leakage; DC~50~, Triton X-100 concentration destroyed 50% liposomal membrane; PEG, polyethylene glycol; Cou6, coumarin 6; Lip, liposome; 18β-GA, 18β-glycyrrhetinic acid; 18α-GA, 18α-glycyrrhetinic acid; 3-Ace-GA, 3-acetyl-18β-glycyrrhetinic acid; 11-Deo-GA, 11-deoxy-18β-glycyrrhetinic acid.

Xie L, Peng Y, Huang K, Wu Y, Wang S. Predictive value of iron parameters in neurocritically ill patients. Brain Behav. 2018;8:e01163 10.1002/brb3.1163 1. INTRODUCTION {#brb31163-sec-0005} =============== Iron has many different roles in the body, acting in carrying oxygen, delivering electron, and catalyzing many biochemistry reactions. After being absorbed from the microvilli of enterocytes, most of the iron in the body keeps a sequestered condition by binding with transferrin in the circulation and is stored in the form of ferritin in the tissues (Andrews & Schmidt, [2007](#brb31163-bib-0001){ref-type="ref"}). In contrast, the non‐transferrin‐bound iron is toxic because it can generate free radicals via participating in the Fenton/Haber--Weiss reaction. These reactive oxidative species could cause lipid peroxidation and bring damages to proteins and DNA (Brissot, Ropert, Le Lan, & Loreal, [2012](#brb31163-bib-0004){ref-type="ref"}; Koskenkorva‐Frank, Weiss, Koppenol, & Burckhardt, [2013](#brb31163-bib-0011){ref-type="ref"}). Under normal conditions, there is a balance of iron metabolism that preserves the biological function of iron while preventing an excess that would cause oxidative stress. Under pathological conditions, however, the iron metabolism shifts and the alteration could cause it to become a defense mechanism. The processes of iron metabolism involve a number of specific proteins (Malyszko, Malyszko, Pawlak, & Mysliwiec, [2006](#brb31163-bib-0015){ref-type="ref"}). Hepcidin, a peptide mainly synthesized by the liver, is regarded as the master regulator modulated in response to hypoxia, iron deficiency, anemia, or inflammation. Iron metabolism disturbance occurs frequently in intensive care unit (ICU) patients, whereby some of the iron parameters reported to be useful in predicting the prognosis of these patients. Nevertheless, these studies are mainly performed in surgical or general ICU. Moreover, which iron parameters could be used to predict reliably with prognosis of critical illness is controversial (Bobbio‐Pallavicini et al., [1989](#brb31163-bib-0003){ref-type="ref"}; Leaf, Rajapurkar, Lele, Mukhopadhyay, & Waikar, [2014](#brb31163-bib-0012){ref-type="ref"}; Tacke et al., [2016](#brb31163-bib-0023){ref-type="ref"}). In terms of neurologic disorders, several studies (Davalos et al., [1994](#brb31163-bib-0007){ref-type="ref"}; Millan et al., [2007](#brb31163-bib-0017){ref-type="ref"}; Millerot et al., [2005](#brb31163-bib-0018){ref-type="ref"}; Simon et al., [2015](#brb31163-bib-0022){ref-type="ref"}; Zuliani et al., [2006](#brb31163-bib-0027){ref-type="ref"}) have suggested that baseline ferritin was related to poor outcome and may represent a marker of disease severity, especially in stroke and brain trauma. However, whether iron parameters, especially ferritin, could be used to predict the prognosis in neurocritically ill patients has not been proved yet. Here, we performed this study to evaluate the predictive value of iron parameters in neurocritically ill subjects. 2. METHODS {#brb31163-sec-0006} ========== 2.1. Subjects {#brb31163-sec-0007} ------------- We retrospectively collected data from a prospectively designed cohort of consecutive patients admitted to a neurocritical care unit (NCU) of a tertiary university‐affiliated academic hospital (Nanfang Hospital, Southern Medical University, Guangzhou, China), from August 2016 to January 2017. The inclusion criteria were Glasgow Coma Scale (GCS) (Teasdale & Jennett, [1974](#brb31163-bib-0024){ref-type="ref"}) ≤12 and/or admission Acute Physiology and Chronic Health Evaluation (APACHE) II score (Knaus, Draper, Wagner, & Zimmerman, [1985](#brb31163-bib-0010){ref-type="ref"}) \>15; and/or demand for intensive care or life‐support measures. Patients younger than 18 years old, with iron‐related disorders in history, pregnant, or required intensive care for \<72 hr were excluded. The critical stroke patients with severe neurologic deficits, without altered consciousness, and being classified as "demand for intensive care or life‐support measures," were included in the study. We excluded patients "who required intensive care for \<72 hr" because our NCU and our stroke unit worked as the same unit. The patient group with acute mild to moderate intracranial hemorrhage within 24 hr and superacute ischemic stroke with thrombolysis and/or intravascular treatment that required transient close monitoring but transferred out of NCU within 72 hr was eliminated from the neurocritically ill patient group. Several literatures have reported that iron metabolism may be affected by the renal function and renal function is connected to mortality in ICU patients (Beier et al., [2011](#brb31163-bib-0002){ref-type="ref"}; Cartin‐Ceba, Afessa, & Gajic, [2007](#brb31163-bib-0005){ref-type="ref"}; Malyszko et al., [2006](#brb31163-bib-0015){ref-type="ref"}), indicating that the renal function might have influence on outcome in this study. Therefore, the enrolled patients were divided into two subgroups for further analysis, based on estimated glomerular filtration rate (eGFR) (Levey et al., [2009](#brb31163-bib-0013){ref-type="ref"}), with eGFR ≥ 60 ml/min/1.73 m^2^ defined as normal (National Kidney Foundation, [2002](#brb31163-bib-0020){ref-type="ref"}). 2.2. Data collection {#brb31163-sec-0008} -------------------- Electronic medical records were carefully reviewed to collect the patient information of demographics, diagnoses, vital signs, GCS scores, APACHE II scores, Sequential Organ Failure Assessment (SOFA) scores (Vincent et al., [1998](#brb31163-bib-0025){ref-type="ref"}), laboratory values, length of NCU stay, and duration of mechanical ventilation. Laboratory data of iron parameters included serum iron, ferritin, transferrin, transferrin saturation (TS), total iron‐binding capacity (TIBC), and unsaturated iron‐binding capacity (UIBC). All data mentioned above were referred to the patients' baseline information within 24 hr after admission. Nobody had prior transfusion. GCS scores were extracted from the first neurological examination at NCU admission. The total scores of APACHE II and SOFA were obtained according to the corresponding parameters within the first 24 hr of NCU admission. Primary endpoints were short‐term (30‐day) all‐cause mortality and long‐term (6‐month) poor outcome, with the latter defined as modified Rankin Scale (mRS) of 4--6. The outcome information was acquired in a medical follow‐up system, in which all patients admitted to NCU were followed up through face‐to‐face or telephone interviews by a trained personnel blinded to the present study. 2.3. Statistical analysis {#brb31163-sec-0009} ------------------------- Continuous data were presented as mean ± standard deviation (*SD*) or median (25%‐75% interquartile range) and compared by Student\'s *t* test or Mann--Whitney *U* test, as appropriate. Differences in proportions among categorical data were assessed using chi‐squared tests and Fisher\'s exact tests for multiple groups. The prognostic value of iron parameters was first assessed by univariate analysis. Then, the significant variables were further included in multivariate models for adjustment. The 95% confident intervals reported for the logistic regression odds ratios were calculated by the maximum‐likelihood estimation (forward selection). Correlations between variables were determined with the Spearman\'s rank correlation test. *p* \< 0.05 was considered statistically significant. All statistical analyses were performed using SPSS, version 20.0 (SPSS, Chicago, IL). 2.4. Ethic statement {#brb31163-sec-0010} -------------------- The study proposal was approved by the Medical Ethics Committee of Nanfang Hospital. Informed consents were signed by all the patients or their surrogates before data collection. 3. RESULTS {#brb31163-sec-0011} ========== 3.1. Overall analysis {#brb31163-sec-0012} --------------------- Of 131 patients screened for eligibility, 103 satisfied inclusion and exclusion criteria (Figure [1](#brb31163-fig-0001){ref-type="fig"}). The etiology of the patients included acute ischemic stroke (38.8%, *N* = 40), intracranial hemorrhage (19.4%, *N* = 20), central nervous system infection (13.6%, *N* = 14), and other neurologic disorders (28.2%, *N* = 29). {#brb31163-fig-0001} For short‐term mortality, higher baseline ferritin (*p* = 0.017) and lower baseline transferrin (*p* = 0.018) and TIBC (*p* = 0.037) showed statistical significance. For long‐term prognosis, baseline transferrin was the only iron parameter showed statistical significance (*p* = 0.043) (Table [1](#brb31163-tbl-0001){ref-type="table"}). In multivariate logistic regression model, none of the iron parameters showed statistical significance (not shown). ###### Characteristics of studied NCU patients Parameters Short‐term mortality Long‐term poor functional outcome ----------------------------------------- ---------------------- ----------------------------------- ------------------------------------------------- --------------------- --------------------- ------------------------------------------------- Age (years, median, IQR) 57.0 (43.0,67.0) 65.5 (38.8,72.8) 0.087 54.0 (38.5,66.5) 61.5 (43.5,71.0) 0.057 Male (*n*, %) 49 (60.5) 14 (63.6) 0.789 28 (62.2) 35 (60.3) 0.846 Primary NCU diagnosis (*n*, %) 0.202 0.266 Stroke 48 (59.3) 12 (54.6) 25 (55.6) 35 (60.4) CNS infections 13 (16.0) 1 (4.5) 9 (20.0) 5 (8.6) Other neurologic disorders 20 (24.7) 9 (40.9) 11 (24.4) 18 (31.0) Prior history (*n*, %) Hypertension 46 (56.8) 14 (63.6) 0.564 25 (55.6) 35 (60.3) 0.625 Diabetes 19 (23.5) 9 (40.9) 0.103 9 (20.0) 19 (32.8) 0.149 Length of NCU stay (days, median, IQR) 5.0 (3.0,12.0) 7.5 (4.5,16.3) 0.277 4.0 (3.0,6.0) 8.5 (4.8,17.0) \<0.001[\*](#brb31163-note-0003){ref-type="fn"} APACHE II score (mean ± *SD*) 11.6 ± 5.6 18.0 ± 6.9 \<0.001[\*](#brb31163-note-0003){ref-type="fn"} 10.0 ± 5.5 15.2 ± 6.1 \<0.001[\*](#brb31163-note-0003){ref-type="fn"} SOFA score (median, IQR) 4.0 (3.0,6.0) 10.0 (8.8,12.0) \<0.001[\*](#brb31163-note-0003){ref-type="fn"} 3.0 (2.0,5.5) 8.0 (5.0,10.0) \<0.001[\*](#brb31163-note-0003){ref-type="fn"} eGFR (ml/min/1.73 m^2^, mean ± *SD*) 86.3 ± 33.9 67.6 ± 38.8 0.009[\*](#brb31163-note-0003){ref-type="fn"} 94.0 ± 28.4 73.2 ± 38.1 0.002[\*](#brb31163-note-0003){ref-type="fn"} C‐reactive protein (mg/dl, median, IQR) 15.3 (5.2,58.4) 42.3 (8.8,132.0) 0.070 8.9 (3.7,54.5) 32.7 (10.6,94.3) 0.009[\*](#brb31163-note-0003){ref-type="fn"} Procalcitonin (μg/L, median, IQR) 0.123 (0.054,0.494) 0.461 (0.118,2.560) 0.006[\*](#brb31163-note-0003){ref-type="fn"} 0.098 (0.050,0.366) 0.201 (0.075,0.830) 0.024[\*](#brb31163-note-0003){ref-type="fn"} Iron parameters Ferritin (ng/ml, median, IQR) 322.5 (170.9,634.6) 583.2 (294.7,1066.2) 0.017[\*](#brb31163-note-0003){ref-type="fn"} 336.8 (169.1,597.4) 393.3 (215.6,866.5) 0.107 Serum iron (μmol/L, median, IQR) 7.0 (4.3,10.0) 5.4 (4.0,8.0) 0.198 8.0 (4.2,12.0) 6.0 (4.2,8.0) 0.070 UIBC (μmol/L, mean ± *SD*) 31.83 ± 9.38 28.55 ± 13.67 0.299 32.28 ± 9.25 30.23 ± 11.29 0.326 TIBC (μmol/L, mean ± *SD*) 39.77 ± 10.30 35.43 ± 12.55 0.037[\*](#brb31163-note-0003){ref-type="fn"} 41.02 ± 10.81 37.16 ± 10.76 0.074 TS (%, median, IQR) 17.6 (12.2,25.7) 16.7 (11.8,25.3) 0.661 19.0 (13.2,26.8) 16.7 (11.5,24.3) 0.277 Transferrin (g/L, median, IQR) 1.82 (1.51,2.14) 1.39 (1.20,2.15) 0.018[\*](#brb31163-note-0003){ref-type="fn"} 1.85 (1.63,2.14) 1.62 (1.32,2.15) 0.043[\*](#brb31163-note-0003){ref-type="fn"} APACHE: Acute Physiology and Chronic Health Evaluation; CNS: central nervous system; eGFR: estimated glomerular filtration rate; IQR: interquartile range; NCU: neurocritical care unit; *SD*, standard deviation; SOFA: Sequential Organ Failure Assessment; TIBC: total iron‐binding capacity; TS: transferrin saturation; UIBC: unsaturated iron‐binding capacity. ^\*^ *p* \< 0.05. John Wiley & Sons, Ltd The correlation analysis showed that the increase in ferritin, as well as the reduction in transferrin and TIBC, was paralleled with the increase in duration of NCU stay, APACHE II scores, SOFA scores, C‐reactive protein, and procalcitonin levels (Table [2](#brb31163-tbl-0002){ref-type="table"}). ###### Correlations between iron parameters and selected variables in total neurocritically ill patients Parameters Duration of NCU stay APACHE II score SOFA score C‐reactive protein Procalcitonin ------------- ---------------------- ----------------- ------------ -------------------- --------------- --------- -------- --------- -------- --------- Ferritin 0.229 0.020 0.281 0.004 0.378 \<0.001 0.453 \<0.001 0.331 0.001 Serum iron ‐ NS −0.244 0.013 −0.204 0.038 −0.527 \<0.001 −0.317 0.001 UIBC ‐ NS −0.233 0.018 −0.258 0.009 −0.321 0.001 −0.356 \<0.001 TIBC −0.200 0.043 −0.316 0.001 −0.323 \<0.001 −0.508 \<0.001 −0.437 \<0.001 TS ‐ NS ‐ NS ‐ NS −0.270 0.006 ‐ NS Transferrin −0.196 0.048 −0.348 \<0.001 −0.362 \<0.001 −0.516 \<0.001 −0.488 \<0.001 APACHE: Acute Physiology and Chronic Health Evaluation; NCU: neurocritical care unit; NS: not significant; SOFA: Sequential Organ Failure Assessment; TIBC: total iron‐binding capacity; TS: transferrin saturation; UIBC: unsaturated iron‐binding capacity. John Wiley & Sons, Ltd 3.2. Subgroup analysis {#brb31163-sec-0013} ---------------------- To eliminate the confounding of renal function, we performed subgroup analysis in patients with eGFR ≥ 60 ml/min/1.73 m^2^ (*N* = 75). 3.3. 30‐day mortality of patients with eGFR ≥ **60 ml/min/1.73 m^2^** {#brb31163-sec-0014} --------------------------------------------------------------------- Compared with short‐term survivors (63 patients), the victims were more likely to have underlying diabetes, higher APACHE II and SOFA scores at admission, whereas there were no differences in any iron parameters (Supporting information Table [S1](#brb31163-sup-0001){ref-type="supplementary-material"}). In multivariate logistic analysis, ferritin was independently associated with 30‐day mortality (OR: 1.002; 95% CI: 1.000--1.003; *p = *0.008) (Table [3](#brb31163-tbl-0003){ref-type="table"}). ###### Univariate and multivariate logistic regression analysis for iron parameters on adverse outcomes in NCU patients with eGFR ≥ 60 ml/min/1.73 m^2^ Short‐term mortality Long‐term poor functional outcome ----------------------- ---------------------- ----------------------------------- ---------------------- ------- Univariate analysis Ferritin 1.002 (1.000--1.003) 0.022 1.001 (1.000--1.003) 0.044 Serum iron ‐ 0.689 ‐ 0.391 UIBC ‐ 0.383 ‐ 0.629 TIBC ‐ 0.291 ‐ 0.366 TS ‐ 0.592 ‐ 0.937 Transferrin ‐ 0.188 ‐ 0.311 Multivariate analysis Ferritin 1.002 (1.000--1.003) 0.008 1.002 (1.000--1.004) 0.031 These factors were adjusted in multivariate regression models: age, gender, etiology, diabetes, and Acute Physiology and Chronic Health Evaluation (APACHE) II score. eGFR: estimated glomerular filtration rate; NCU: neurocritical care unit; TIBC: total iron‐binding capacity; TS: transferrin saturation; UIBC: unsaturated iron‐binding capacity. John Wiley & Sons, Ltd 3.4. 6‐month functional outcome of patients with eGFR ≥ 60 ml/min/1.73 m^2^ {#brb31163-sec-0015} --------------------------------------------------------------------------- Increased APACHE II score, SOFA score, length of NCU stay, and serum C‐reactive protein levels were observed in patients with poor functional outcome at 6 months compared to those with good functional outcome (39 patients), while there were no differences in any iron parameters (Supporting information Table [S1](#brb31163-sup-0001){ref-type="supplementary-material"}). In multivariate logistic analysis, ferritin was independently associated with 6‐month functional outcome (OR: 1.002; 95% CI: 1.000--1.004; *p = *0.031) (Table [3](#brb31163-tbl-0003){ref-type="table"}). 4. DISCUSSION {#brb31163-sec-0016} ============= In this study, we verified serum ferritin as a predictor of clinical outcome in a cohort of neurocritically ill patients and found that serum ferritin was independently associated with 30‐day mortality and 6‐month poor functional outcome in neurocritically ill patients with eGFR ≥ 60 ml/min/1.73 m^2^. Our results showed that neurocritically ill patients who died within 30 days tended to have higher baseline serum ferritin as well as lower transferrin and TIBC, and those with poor functional outcome at 6 months presented with a lower transferrin level at admission. These results were in line with former studies. Darveau, Denault, Blais, & Notebaert ([2004](#brb31163-bib-0006){ref-type="ref"}) found that elevated ferritin and diminished serum iron, TS, and transferrin levels are generally observed in more critically ill patients. Bobbio‐Pallavicini et al. ([1989](#brb31163-bib-0003){ref-type="ref"}) investigated 51 postoperative critically ill patients and found that the increase in ferritin concentration correlated with worsening of the clinical status. In a prospective observational cohort study of 121 critically ill patients, plasma catalytic iron levels on ICU day 1 were significantly associated with hospital mortality and 30‐day mortality (Leaf et al., [2014](#brb31163-bib-0012){ref-type="ref"}). These results could be explained by the common inflammation, especially infectious inflammation, in critically ill patients. In the presence of inflammation, a great amount of hepcidin is released, which subsequently degrades the cellular iron exporter ferroportin on enterocytes and macrophages and limits the availability of serum iron, resulting in decreased TS. Pro‐inflammatory cytokines, such as interleukin (IL)‐1β, IL‐6, and tumor necrosis factor, induce the transcription and translation of ferritin, resulting in increased ferritin. Intracellular iron content modulates the binding affinity of cytoplasmic iron regulatory proteins (IRP)‐1 and IRP‐2 with iron‐responsive elements and rapidly reduces the mRNA expression of transferrin receptors causing transferrin concentrations decreased. Our study also demonstrated the serum iron, transferrin, UIBC, TIBC, and ferritin levels had significant correlation with APACHE II scores and infectious biomarkers at admission. In subgroup analysis, we demonstrated serum ferritin was an independent predictor of short‐term mortality and long‐term poor outcome, in agreement with previous researches in ICU patients (Leaf et al., [2014](#brb31163-bib-0012){ref-type="ref"}; Munoz et al., [2005](#brb31163-bib-0019){ref-type="ref"}; Simon et al., [2015](#brb31163-bib-0022){ref-type="ref"}; Tacke et al., [2016](#brb31163-bib-0023){ref-type="ref"}). And in neurologic disorders, Millan et al. ([2007](#brb31163-bib-0017){ref-type="ref"}) studied 134 consecutive patients with acute ischemic stroke and found that ferritin levels before thrombolytic treatment were higher in patients with poor outcome (modified Rankin scale, mRS \> 2) at 90 days. It was suspected that increased ferritin could be in part the result of a neuroprotective mechanism with the aim of sequestering toxic‐free iron in the ischemic brain. Another study performed in patients with intracerebral hemorrhage also demonstrated that serum ferritin at baseline was markedly higher and iron as well as transferrin was lower in patients with poor outcome (mRS ≥ 3) at 90 days (Yang et al., [2016](#brb31163-bib-0026){ref-type="ref"}). A prospective study enrolled 69 male patients who suffered from severe traumatic brain injury, and the results showed that higher ferritin concentrations were significantly associated with fatal outcome (Simon et al., [2015](#brb31163-bib-0022){ref-type="ref"}). Ferritin is known as an acute‐phase protein in response to inflammation, a common pathological state in critically ill patients (Marshall, [2001](#brb31163-bib-0016){ref-type="ref"}). Here, we also observed that the ferritin levels were moderately correlated with the infectious biomarkers and APACHE II and SOFA scores in this study. Infectious inflammation stimulates the upregulated expression of hepcidin and ferritin, resulting in relative iron deficiency in bloodstream (Drakesmith & Prentice, [2012](#brb31163-bib-0008){ref-type="ref"}). Upon that the ferritin level as the hallmark of iron homeostasis fluctuation probably reflects the severity of inflammatory response and risks of multiple organ dysfunction and anemia, complications that contribute to the increased mortality in critical illness. In this study, we found for each 10 ng/ml raise in ferritin, there was 2% increase in odds of short‐term mortality. Since early evaluation of neurocritically ill patients at risk of mortality may benefit the management of their treatment strategies, the protective effect of intervention in disturbances of iron homeostasis in neurocritical illness might worthy to be further investigated (Ganz, [2013](#brb31163-bib-0009){ref-type="ref"}; Ma, You, & Hao, [2012](#brb31163-bib-0014){ref-type="ref"}; Selim, [2009](#brb31163-bib-0021){ref-type="ref"}). Several limitations of this study should be noted. First, the retrospective nature of this study makes it susceptible to selection and information bias. Second, we were unable to assess all potentially relevant variables. Third, the sample size of this study was relatively small. Further studies should be done to validate the results, and the impact of treatment strategies for iron metabolism on the prognosis is promising. In conclusion, our study demonstrated that elevated ferritin level was independently associated with increased short‐term mortality and long‐term poor outcome in neurocritically ill patients with eGFR ≥ 60 ml/min/1.73 m^2^. Future studies with larger sample size should be conducted to confirm our conclusions, and the protective effect of intervention on iron metabolism among neurocritically ill patients might deserve further investigation. CONFLICT OF INTEREST {#brb31163-sec-0018} ==================== None declared. Supporting information ====================== ######   ###### Click here for additional data file. None. [^1]: These authors contributed equally to the manuscript.

Introduction {#S0001} ============ Vesicoureteral reflux (VUR) is an important disorder in children because of high association with urinary tract infection (UTI) and permanent renal damage (scar)^\[[@CIT0001]\]^. Reflux--associated nephropathy is one of the most important causes of end stage renal disease and kidney transplantation in children and adults^\[[@CIT0002],\ [@CIT0003]\]^. Currently most cases of VUR are not diagnosed until the patients presented with urinary tract infection, the condition that increases risk of renal damage. The advent of prenatal ultrasonography has enhanced the early detection of various urinary tract abnormalities such as VUR. Reflux was detected in 15-30% of infants with abnormal prenatal ultrasound findings^\[[@CIT0004]--[@CIT0007]\]^. Controversy exists regarding the natural history and treatment of VUR diagnosed antenatally and those detected later in life, usually after urinary tract infections. Prenatal diagnosis should be an ideal opportunity to detect VUR earlier and prevent later renal damage but some authors suggested that fetal vesicoureteral reflux is essentially benign and need less aggressive investigation and management^\[[@CIT0008],\ [@CIT0009]\]^. Conversely others have opposite idea^\[[@CIT0010],\ [@CIT0011]\]^. They believe that there is no difference between two forms of VUR according to natural history and outcome. The aim of this study was to assess the natural history and outcome of vesicoureteral reflux in infants less than 1 year and compare prenatally detected with those detected later during the first year of life. Subjects and Methods {#S0002} ==================== This prospective study was carried out in BooaliSina university hospital, Sari, IRAN, from September 2004 to March 2012. The study was approved by the Research committee of Mazandaran University of medical sciences. All parents were given written informed Consent before enrolling the infants into the study. All infants less than 12 months old with VUR were enrolled in this study.VUR was diagnosed in the follow up process of antenatal diagnosed hydronephrosis or postnatal conditions such as urinary tract infections. Reflux was diagnosed by cystography and classified as grade one to five according to severity. All infants with any grade of reflux enrolled in study. Infants with reflux associated with any other pathological condition and those with incomplete follow up were excluded. We divided children into two groups. Group 1 consisted of patients with antenatal hydronephrosis that VUR was detected on postnatal investigation. In group 2 there were infants that had normal prenatal ultrasound but VUR was diagnosed during the first year of life following the workups for UTI. We followed all infants with prenatal hydronephrosis that were referred to our clinic. All infants were studied by urinary tract ultrasonography performed at first and six weeks of age. voiding cystourethrogram (VCUG) was performed in infants who had persistent hydronephrosis on both postnatal sonographies. We ordered VCUG for children less than one year old with urinary tract infection. Reflux grade was classified at first VCUG according to the system proposed by International Reflux Study Committee. All patients received prophylactic antibiotics until resolution of reflux or improvement to lower nondilating grades of reflux. We used cephalexin, cotrimoxazole, amoxicillin for prophylaxis. We prospectively followed patients at least for six months for assessment of defined outcomes including somatic growth, need for surgical intervention, reflux resolution, formation of scar, hypertension and ultimately episodes of UTI. For assessment of somatic growth we considered Height for age Z score (HAZ) at 12±2 months of age. Reflux resolution was defined based on results obtained by follow up VCUG at 12-18 months later as: no change, improvement of less than 50% in severity of VUR, improvement of more than 50% in severity and cure (normal cystography on follow up). Hypertension was defined as values persistently above the 95^th^ percentile for age, gender and height on three consecutive visits. Scar was defined as permanent change in renal outline demonstrated by dimercaptosuccinic acid (DMSA) scintigraphy performed at the age of six months or later in prenatal group and at least six months after infection in the other group. The Scar severity was classified as grade 1 to 4 according to international classification as follows: normal = 0; focal scarring in one region = 1; scarring involving two regions = 2; scarring involving all three regions = 3; and generalized reduction in cortical mass = 4. The number of confirmed UTI was considered as a prognostic factor. UTI was defined as growth of at least 100,000 colony forming unit/ml in urine obtained by bag, 1000 colonies in catheter sample or any colony in urine obtained by suprapubic aspiration. We considered more than two episodes of infections as recurrent UTI. Neither of specialists that performed radiographic or scintigraphic imaging was aware of the infants\' data. Data analysis was done by SPSS12 software. Statistical analysis of the differences between groups was determined by χ^2^ and Fisher exact test and Wilcox on signed-rank test. *P*-value \<0.05 was considered statistically significant. Findings {#S0003} ======== A total of 152 patients was enrolled in the study (70 boys and 82 girls). Sixty seven infants presented with antenatally detected hydro-nephrosis and 85 infants presented with UTI and other complaints after birth. Demographic and basic clinical features of patients are presented in [Table 1](#T0001){ref-type="table"}. As expected, most of patients of prenatal group were male, whereas the female sex was more common in postnatal group. The reflux severity was slightly higher in postnatal group ([Tables 1](#T0001){ref-type="table"} and [2](#T0002){ref-type="table"}). There were 68 patients with unilateral and 84 patients with bilateral VUR, a total of 236 refluxing renal units were included in analysis, 98 in group 1 and 138 in group 2. ###### Demographic and basic clinical features of patients Prenatal group Postnatal group *P*-value ------------------------------------------------ -------------- ---------------- ----------------- ----------- **Number of patients** 67 85 NA **Number of renal units** 98 138 NA **Sex** **Male (%)** 45 (67.2) 25(70.6) \<0.001 **Female (%)** 22 (32.8) 60(29.4) **Length of follow up (months) \[Mean (SD)\]** 29.17(19.04) 36.03(18.34) 0.7 NA: not applicable; SD: Standard deviation ###### Severity of reflux in prenatal and postnatal groups Grade of reflux Prenatal group (%) Postnatal group (%) *P*-value ----------------- -------------------- --------------------- ----------- **One** 4(4) 2(1) 0.03 **Two** 20(20) 41(30) **Three** 36(37) 60(44) **Four** 14(14) 20(15) **Five** 24(25) 15(11) The average length of follow up was 33±19 (range; 6-94) months with no significant difference between two groups. Outcome {#S20004} ------- UTI: There were 16(10.5%) patients with recurrent UTI, four patients of group 1 and 12 infants of group 2. There was no significant difference in the occurrence of recurrent UTI between two groups (*P*=0.1). Renal scar: Renal scars developed in 46 (19.5%) of renal units including 25 units in group 1 and 21 units in group 2. There was no significant difference between two groups (*P*=0.3). Severity of scar developed in the two groups is shown in [Table 3](#T0003){ref-type="table"}. Somatic growth: At approximately 1 year of age, the mean height-for-age Z score was 0.03 (±1.13) and --0.09 (±1.20) for groups 1 and 2 respectively (*P*=0.5). ###### UTI development, scar severity and reflux resolution in two sex groups Factor Male Number (%) Female Number (%) *P*-value ----------------------- ---------------- ----------------- ------------------- ----------- **Recurrent UTI** 4 (5.7) 12 (14.6) 0.07 **Scar severity** **Grade0** 81(75) 109(85) 0.1 **Grade1** 10 (9) 8(6) **Grade2** 3 (3) 5 (4) **Grade3** 8 (7) 2(2) **Grade4** 6 (6) 4(3) **Reflux resolution** **Un-changed** 11(14) 14(11) 0.9 **\<50% improvement** 8(10) 11(9) **\>50% improvement** 10(13) 17(14) **Cure** 51(64) 81(66) UTI: Urinary tract infection Reflux Resolution: Reflux was unchanged in 7 (16.7%) and cured in 20 (49%) of renal units in group 1 patients at 12-18 months of age, these values were respectively 12 (10.3%) and 93 (73%) of renal units for group 2 ([Table 4](#T0004){ref-type="table"}). There was a significant difference in resolution of VUR (*P*\<0.001). Reflux in infants of postnatal group tends to resolve spontaneously more than that in prenatal group. The sex of patient had no significant influence on outcome of VUR ([Table 5](#T0005){ref-type="table"}). ###### Severity of scar in renal units in prenatal and postnatal groups Scar severity Prenatal group Postnatal group *P*. value --------------- ---------------- ----------------- ------------ **No scar** 73 (75) 117 (85) 0.3 **Grade 1** 9 (9) 9 (7) **Grade 2** 5 (5) 3 (2) **Grade 3** 5 (5) 5 (4) **Grade 4** 6 (6) 4 (3) ###### Patterns of reflux resolution in two prenatal and postnatal groups Resolution pattern Prenatal group Postnatal group *P*. value ---------------------------------- ---------------- ----------------- ------------ **No change (%)** 7 (11) 18 (13) \<0.001 **Less than 50% resolution (%)** 11 (17) 8 (6) **More than 50% resolution (%)** 16 (24) 11 (8) **Full improvement (cure) (%)** 32 (49) 100 (73) As predicted, higher grades of reflux were associated with less chance of spontaneous resolution of VUR. Reflux resolution in end of study for grades one to five was 80%, 82.7%, 70.6%, 60.7% and 24.2% respectively. Surgery: Antireflux procedures were performed in 4 (6%) infants of group 1 and also 7 (8.2%) of group 2 patients, the values with no statistically significant difference (*P*=0.6). The primary indications of surgery were scar in 9 (81.8%) cases, and no improvement of higher grades VUR after 12-24 months therapy in 2 (18.2%) other cases. Discussion {#S0005} ========== Prenatal ultrasound has changed the detection and management of many urologic abnormalities. VUR, found in 15-30% of cases of prenatal hydronephrosis, is one of the most common abnormalities^\[[@CIT0002],\ [@CIT0003],\ [@CIT0012],\ [@CIT0013]\]^ Early detection and management of reflux with antenatal ultrasound could improve long term outcome^\[[@CIT0014],\ [@CIT0015]\]^, but controversy exists regarding the natural history and management of this entity. Some authors have reported that prenatal VUR could lead to renal damage (like other forms of primary VUR) while other studies suggest that fetal VUR is relatively benign^\[[@CIT0010],\ [@CIT0011],\ [@CIT0016],\ [@CIT0017]\]^. In our study 152 infants with VUR were studied in two separate groups with antenatal and postnatal diagnosis. Similar to other studies, the sex predominance belonged to boys in antenatal and girls in postnatal group. This sex difference seems initially a conflicting factor but there was no significance difference between the two sexes in terms of most prognostic factors studied. The severity of reflux was slightly higher in postnatal group relative to antenatal group. This is not unexpected because we enrolled symptomatic infants in postnatal group. It is noteworthy that 39% of asymptomatic infants had high grade reflux. Farhat et al found these grades of VUR in 48% of patients^\[[@CIT0009]\]^ and Upadhyay et al showed that 39% of renal units had VUR grade 4 or 5^\[[@CIT0018]\]^. This significantly high percentage of dilating reflux emphasizes the opinion that screening is mandatory for all neonates with history of antenatal hydronephrosis^\[[@CIT0004]\]^. What is the long term course of VUR? We followed our patients for an average of 33 months. VUR resolved spontaneously in 49% of our antenatal VUR patients and 73% of postnatal patients, whereas 29% and 19% of patients had no significant improvement, respectively. It is notable that overall resolution rate of prenatal VUR was significantly lower compared to postnatal VUR pointing that prenatal VUR may persist at least similar to and even more than the reflux observed in any other time of life. Ismaili et al reported that 69% of their 67 refluxing renal units resolved within 24 months^\[[@CIT0019]\]^. Upadhyay et al showed 52% cure and 24% persistence of VUR^\[[@CIT0018]\]^. In our and two other studies the severity of VUR has direct influence on persistence of VUR. Sjostrom et al followed 115 infants with grade 3-5 reflux and reported that spontaneous resolution occurred in only 27% of their patients and 12% of them improved to grade 2 and 1 reflux^\[[@CIT0020]\]^. In our opinion, this persistence rate of VUR is important and we need to follow infants until complete resolution of reflux, but another important question is: What is the relation between VUR and renal damage? Renal scar was observed without any statistical difference in 25% of antenatal VUR and 15% of postnatal VUR and near half of them were severely damaged. Farhat and coworkers detected renal scarring in 12 out of 54 renal units^\[[@CIT0009]\]^. In a similar study, Sjostrom and coauthors found that half of their children had generalized renal scar and one- quarter had focal damage^\[[@CIT0020]\]^. In study of Upadhyay et al, scar was seen in 23% of renal units prior to study but only two new scars developed in 14 patients that were followed^\[[@CIT0018]\]^. Near to other studies we found no case of CRF and HTN in our patients^\[[@CIT0018],\ [@CIT0019],\ [@CIT0021]\]^. Sjostrom wrote that from 84 refluxing infants with history of antenatal hydronephrosis, 27 had chronic kidney disease (CKD) stage 2 and three had CKD stage 3 and there was a significant difference between prenatal and postnatal group^\[[@CIT0020]\]^. We found no significant difference between two groups although this conclusion is not strictly appropriate in this relatively short time of follow up. And finally, one helpful item is the frequency of urinary tract infections in both groups. Recurrent UTI was observed in only 16 infants with no significant difference between the two groups. Recurrent UTI was observed with 15-25% frequency in some mentioned studies^\[[@CIT0009],\ [@CIT0018],\ [@CIT0019]\]^. Surprisingly, Estrada et al reported a very low rate of UTI. UTI developed in only1.3% of the patients who were screened and did not have VUR and, therefore, were not receiving antibiotics. Of the screened patients with VUR who were receiving prophylactic antibiotics, UTI developed in 1.6% at a mean age of 9.4 months. In 363 patients who did not undergo an initial voiding cystourethrogram, a febrile urinary tract infection developed in 16 (4.4%) patients at a mean age of 9.3 months. They concluded that in patients with a history of prenatal hydronephrosis, identification of VUR and use of prophylactic antibiotics significantly reduces the risk of febrile UTI^\[[@CIT0010]\]^. Based on ours and some other reports, infection episodes were seen in a considerable rate and need attention and appropriate prophylaxis. Our study has one limitation: we did not have baseline scintigraphic data about renal damage immediately after birth and therefore some reported scars in both groups may belong to congenital events. It was not our routine protocol to perform DMSA in first two months of life due to physiologically decreased renal function. Conclusion {#S0006} ========== Our study confirmed that VUR diagnosed in the setting of prenatal hydronephrosis has relatively similar importance and outcome. The findings of our study emphasize the importance of early diagnosis and management of the prenatal VUR after birth. This study was the postgraduate thesis of Dr S. Mohammadi. It was supported by a grant from Research Deputy of Mazandaran University of Medical sciences. Conflict of Interest {#S0007} ==================== None

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INTRODUCTION {#sec1-1} ============ According to international registry of heart--lung transplant, the annual cardiac transplant rate worldwide varies between 5000 and 10,000 and this number is expected to increase.\[[@ref1]\] These patients may require anaesthesia for elective or emergency surgery in hospitals where specialised anaesthesiologists may not be available. Therefore, the anaesthesia team must be aware of the physiological effect of denervation, the unique anaesthetic implications of a transplanted heart, potential patient risks such as rejection and infection as well as the effect of immunosuppressants. This review elaborates the anaesthetic management of a post-heart transplant patient who can present for various surgeries. A Medline search for heart transplant, anaesthesia, adult, paediatric and surgery was conducted and 38 relevant literatures are added to this review. CONDITIONS FOR WHICH A POST-HEART TRANSPLANT PATIENT MAY REQUIRE ANAESTHESIA AND SURGERY {#sec1-2} ======================================================================================== The heart transplant recipient may require surgery for procedures related to the transplant or to non-transplant-associated conditions \[[Table 1](#T1){ref-type="table"}\]. The need for surgery for different problems has been documented from 2 h to \>10 years post-transplant. The incidence of significant general surgical complications developing within the 30-day post-transplant period has been reported between 4.8% and 7%.\[[@ref2]\] ###### Conditions for which a post-heart transplant patient may require anaesthesia and/or surgery  PHYSIOLOGY OF THE TRANSPLANTED HEART {#sec1-3} ==================================== Heart transplant involves the removal of the diseased heart, in which the aorta and main pulmonary arteries (PA) are transected, the cardiac plexus is interrupted, and the heart is partially denervated. The atria of recipients remain innervated, but conduction does not occur across the atrial suture line. In spite of the fact that the transplanted heart is a denervated organ, the intrinsic cardiac mechanisms are preserved. The heart is extremely sensitive to changes in loading conditions, and the Frank-Starling pressure volume relationship becomes paramount in adjusting contractility. It is commonly said to be 'preload dependent' as cardiac output (CO) depends on the venous return. In comparison to normal, the transplanted heart has a higher resting heart rate (HR) (90--110 beats/min), similar maximum HR, higher minimum HR and reduced HR variability in a 24 h Holter monitoring study.\[[@ref3]\] This is due to the absence of parasympathetic innervation. Most of the transplanted heart recipients have normal sinus rhythm with an increased refractory period of the sinus node; thus, many have first-degree heart block and a higher rate of pacemaker implantation. The possible reasons of heart block include biatrial anastomosis, organ rejection, nodal ischaemia and inadequate myocardial preservation. Clinically, significant atrial and ventricular arrhythmias are infrequent although ectopic beats are common. Presence of fatal ventricular arrhythmias usually indicates severe acute rejection or allograft coronary artery disease.\[[@ref2]\] The resting electrocardiogram (ECG) is usually altered showing two *P* waves; one is from recipients\' own sino-atrial (SA) node and other is from donor\'s SA node. Tachycardia in response to physiological stress, for example, pain and hypovolaemia is blunted as it depends on circulating catecholamines. Carotid sinus massage and Valsalva manoeuvre have no effect on HR.\[[@ref4]\] These patients are at higher risk of developing atrial flutter or fibrillation a few years later. This is because of the onset of some degree of reinnervation. Complete neuronal control has been described 15 years after transplantation. This explains the frequent complaint of angina, vasovagal episodes and cardiac arrest after neostigmine administration in these patients.\[[@ref5]\] Coronary autoregulation remains intact. Immediately after transplant, left ventricular dysfunction is due to anoxic injury during graft transfer, acute withdrawal of sympathetic support or after load mismatch. There is a rapid improvement of ventricular function, and CO becomes normal within few days. The PA pressure and pulmonary capillary wedge pressure remain elevated during the 1^st^-month after transplant and become stable by 1 year. The systemic vascular resistance is frequently elevated; however, a 15% increase in blood volume following transplant may explain the high normal CO through the Frank-Starling mechanism of increasing preload in the setting of cardiac denervation.\[[@ref2]\] There is well-preserved systolic function and mild diastolic dysfunction. Mild-to-moderate mitral and tricuspid valve regurgitation may be present. The myocardial function is subnormal during stress and exercise with a low peak HR, low peak CO and maximum oxygen uptake. This is expected to result from lack of efferent cardiac innervations, either due to HR change alone or together with a submaximal inotropic response.\[[@ref6]\] PERIOPERATIVE CONSIDERATIONS {#sec1-4} ============================ This can be discussed in the following headings: Immunosuppressants and their interaction with commonly used drugs in the perioperative period {#sec2-1} --------------------------------------------------------------------------------------------- The commonly used immunosuppressive agents are cyclosporine A, azathioprine, antilymphocyte globulin, monoclonal antibodies and corticosteroids. Recently, tacrolimus and mycophenolate have replaced cyclosporine and azathioprine, respectively, in some immunosuppression protocols.\[[@ref7]\] The blood level of both cyclosporine and tacrolimus must be kept within the indicated therapeutic range to get the desired effect. The perioperative fluctuation of the plasma level of these two drugs should be strictly monitored as there is a significant reduction of blood level by dilution with volume infusion or cardiopulmonary bypass.\[[@ref8]\] Both these drugs are metabolised by cytochrome P-450 system of liver, and therefore many of the drugs administered perioperatively can affect their plasma levels\[[@ref9][@ref10]\] \[[Table 2](#T2){ref-type="table"}\]. Data on cyclosporine A and tacrolimus interaction with major anaesthetic agents are lacking. Fever, anaemia, leucopenia, thrombocytopenia, hypertension, diabetes, renal dysfunction, neurotoxicity, osteoporosis leading to high rate of fractures and anaphylaxis are some major side effects of immunosuppressants which have some impact on perioperative management and choice of anaesthetic agents. Withdrawal of azathioprine in patients taking warfarin may precipitate bleeding.\[[@ref11]\] Although the exact mechanism is not known, it is assumed that 6-mercaptopurine, a metabolite of azathioprine, induces hepatic microenzymes that metabolise warfarin. Azathioprine and allopurinol combination can lead to serious adverse effects (severe bone marrow suppression, pancreatitis and hepatotoxicity) that can be reduced with close monitoring of metabolites and blood levels.\[[@ref12]\] Prednisone has a similar side effect profile like tacrolimus. However, its action is different because it has anti-inflammatory actions on organ systems. Mycophenolate mofetil has similar efficacy and side effects as azathioprine with the added advantage of lower incidence of fungal infection.\[[@ref13]\] A better understanding of pharmacokinetic changes with age now allows a reduction of dose of immunosupressants in old age while maintaining the therapeutic level.\[[@ref14]\] [Table 3](#T3){ref-type="table"} describes the effect of various anaesthetic agents on immunosupressants and vice versa. ###### Drugs that interact with cyclosporine A and tacrolimus  ###### Effect of anaesthetic agents on various chemotherapeutic agents in heart transplant patients and vice versa  Knowledge regarding post-transplant complications and their implications in anaesthetic practice {#sec2-2} ------------------------------------------------------------------------------------------------ Apart from the burden of old age and co-morbidities, post-transplant patients have a high incidence of the following problems which need to be identified and managed perioperatively. Infection: A high incidence of post-operative wound infection is observed in patients receiving tacrolimus. Strict aseptic techniques during handling of such patients, minimum use of indwelling catheters and earliest removal of invasive lines are mandatory. Fungal infection may need prolonged treatment. The patients should receive cytomegalovirus (CMV) negative blood transfusion. Microbiology advice should be strongly sought for prevention as well as strict control of infection. Any infection should be treated preoperatively. It is important to realise that immunocompromised patients may not present with typical signs and symptoms of infection i.e., fever and leucocytosis. A high index of suspicion is essential and microbiological tests can rule out the diagnosis\[[@ref19][@ref20]\]Rejection: Allograft rejection may occur at any time during the post-transplant period, especially with discontinuation of immunosuppressants. Unexplained weight gain, fever, dyspnoea and peripheral oedema are the usual features of rejection. Urgent endocardial biopsy is needed to confirm the diagnosis;\[[@ref21]\] however, a negative biopsy does not exclude rejection. The episodes of acute rejection necessitate, emergency management with increased immunosupression, for example, intravenous (IV) immunoglobulin and plasmapheresis. At times, the patient may need mechanical circulatory support. The presence of any degree of rejection should be ruled out and managed preoperatively, as post-operative morbidity rate is high if it remains untreated before surgery\[[@ref22]\]Allograft vasculopathy: Cardiac allograft vasculopathy is the atherosclerotic obstructive disease of coronary vessels. It may result from a variety of causes, for example, immune-mediated vascular injury, ischaemic endocardial injury before transplant, immunosuppressive agents, CMV infection, hyperlipidaemia, smoking and hypertension. It occurs within 1^st^ year of transplantation in 10%--20% of patients and in nearly 50% cases within 5 years\[[@ref2][@ref23][@ref24]\]Even in angiographically normal coronary arteries, luminal narrowing may develop insidiously. The lack of afferent innervations renders episodes of myocardial ischaemia silent in these patients. Intravascular ultrasound is the most sensitive technique to detect early changes. Angioplasty is used for focal proximal lesions. At times, disease is wide spread, and distal revascularisation by any means is impossibleMiscellaneous: Apart from the above said specific problems, post-transplant patients may also suffer from diabetes, epilepsy, hypertension (50% patients with cyclosporine A therapy), cholelithiasis and pancreatitis.\[[@ref25][@ref26][@ref27][@ref28]\] Because haemodynamic changes during stress and exercise are dependent on circulating catecholamines, beta blockers are best avoided in these patients for treatment of hypertension.\[[@ref29]\] Pre-operative assessment and premedication {#sec2-3} ------------------------------------------ The transplant team as well as the attending anaesthesiologist and surgeon should have a good coordination during perioperative period of a major surgical procedure. The following investigations should be available preoperatively. ECG (to assess graft function and arrhythmias if any). About 5% of the patients may present with pacemakerEndomyocardial biopsy (to rule out the evidence of rejection)Echocardiography (ventricular function assessment and detection of allograft vasculopathy)\[[@ref30]\]Coronary angiography: Reserved for patients with suspected allograft vasculopathy\[[@ref31]\]Laboratory parametersComplete haemogram (rule out bone marrow depression)ElectrolytesRenal function tests: In therapeutic doses, both cyclosporine and tacrolimus may cause dose-related decrease in renal blood flow and glomerular filtration rate due to renal vasoconstriction\[[@ref32]\]Liver function testsBiomarkers: Brain natriuretic peptide may have a role in the detection of allograft rejection and coronary vasculopathy.\[[@ref33]\] These patients tolerate similar premedication as those without a transplant. However, dose adjustment for some drugs as well as adjuvants is needed \[[Table 2](#T2){ref-type="table"}\]. Assess stress test findings to establish patients exercise tolerance and if necessary obtain a review from cardiologist. The dose of immunosuppressants should not be altered and should be continued post-operatively to reduce the risk of rejection. Daily monitoring of the steady state blood level is recommended. Oral cyclosporine should be administered 4--7 h before surgery to maintain therapeutic blood levels. The alteration of dose of other immunosuppressive drugs is not required unless the route of administration need to be changed from oral to iv. The oral and iv dose of azathioprine is approximately equivalent, and oral dose of prednisolone is equal to the similar iv dose of methylprednisolone. Supplemental steroids are not necessary for stress coverage except in post-transplant recipients in whom steroids are recently withdrawn. Intraoperative concerns and anaesthesia techniques {#sec2-4} -------------------------------------------------- A variety of anaesthetic techniques (local, regional, neuroleptic and general) have been used successfully in these patients. Between general anaesthesia (GA) and regional anaesthesia, no technique has been demonstrated to be better as long as care is given to maintain the preload.\[[@ref34]\] GA is usually preferred by many as there is a possibility of impaired response to hypotension after spinal or epidural anaesthesia. The type of intraoperative monitoring depends on the type of surgery as well as the availability of monitoring. In case of major surgery, invasive blood pressure, urinary catheter and transoesophageal echocardiography may be required to monitor vital organ and volume therapy. Oral endotracheal intubation is always preferred to nasotracheal intubation because of the potential risk of infection caused by nasal flora. Gingival hyperplasia at times is present in patients taking cyclosporine. This may lead to bleeding and aspiration during airway manipulation. Airway obstruction may be encountered in patients with diabetes and lymphoproliferative disorders. Avoid hyperventilation in patients taking cyclosporine and tacrolimus because of a decrease in seizure threshold with these two drugs. There is a loss of sympathetic response to laryngoscopy and intubation.\[[@ref35]\] Laryngeal mask airway is not contraindicated. The denervated heart has a blunted HR response to inadequate anaesthetic depth or analgesia. Non-steroidal anti-inflammatory drugs should be avoided for pain control because of the risk of bleeding. In a post-transplant heart, the catecholamine response is different from that of normal heart because intact sympathetic nerves are required for the normal uptake and metabolism of catecholamines. The receptor density, however, remains unchanged, and the transplanted heart can respond to direct acting drugs, for example, sympathomimetics. Dopamine is a less effective inotrope, whereas isoprenaline and dobutamine have similar effects in both transplanted and normal heart. Because atropine has no effect on a transplanted heart, isoprenaline and epinephrine should be readily available to manage bradycardia and hypotensive emergencies.\[[@ref35]\] [Table 4](#T4){ref-type="table"} summarises the haemodynamic response of some commonly used drugs for resuscitation. ###### Response of denervated heart to various cardiovascular agents drug  Post-operative care {#sec2-5} ------------------- In addition to the routine care as those for non-transplant recipients, increased attention should be paid to the preload status, renal function and prevention of infection. The immunosuppressants should be continued postoperatively and blood level is to be monitored. ANAESTHESIA MANAGEMENT IN SPECIAL CASES {#sec1-5} ======================================= Pregnancy and delivery {#sec2-6} ---------------------- Pregnancy is possible in heart transplant recipients without affecting allograft survival. Currently used immunosuppressants are not teratogenic and need not be discontinued during pregnancy. The risk of preeclampsia, eclampsia, premature labour and allograft rejection is high in these patients; therefore, there is a need for more caution. No matter what anaesthesia technique is followed, maintenance of haemodynamic stability is important. Neuraxial block is preferred by many authors because it produces less impact on baby compared to GA.\[[@ref36]\] The following points need to be remembered during neuraxial blockade: control appropriate block level because\[[@ref37]\] too high a level of block may inhibit sympathetic nerves and cause vasodilatation which is unfavourable for a transplanted heart; too low a level is not suitable for surgery as resultant pain may lead to increased myocardial oxygen consumption. Strict maintenance of preload is mandatory, and one should not forget that too much of fluid is also deleterious for the denervated heart as it can lead to heart failure. Phenylephrine is the vasoconstrictor of choice to maintain haemodynamic stability. Strict asepsis and antibiotic prophylaxis should be used for all operative, and instrumental delivery and immunosuppressants continued postoperatively. Laparoscopic surgery {#sec2-7} -------------------- Pneumoperitoneum is well tolerated. The occasional hypertensive response to pneumoperitoneum can be tackled by increase in analgesic dose.\[[@ref38]\] CONCLUSION {#sec1-6} ========== A good understanding of the changes in physiology of a heart transplant recipient is essential for the best perioperative management. Some important factors must be addressed including changes in haemodynamic status, pharmacological management of denervated heart and prevention of cardiac allograft rejection postoperatively. Financial support and sponsorship {#sec2-8} --------------------------------- Nil. Conflicts of interest {#sec2-9} --------------------- There are no conflicts of interest.

Introduction ============ The neural mechanisms that regulate and coordinate breathing and respiratory-related behaviors such as coughing are not well understood. This lack of knowledge hampers elucidation of pathophysiological deficits in airway protection and impedes development of new therapeutic approaches for dystussia that occur with neurological disorders (Suárez et al., [@B80]; McCool, [@B51]). Computational neural network models for breathing and the neurogenesis of cough inferred from *in vivo* experiments have iteratively aided prediction and refinement of hypotheses for further *in vivo* testing (Shannon et al., [@B72], [@B71]; Baekey et al., [@B5]; Rybak et al., [@B67]; Poliaček et al., [@B58]). Such data-driven models, based in part on elements and connectivity inferred from simultaneous extracellular recordings of many brainstem neurons (e.g., Segers et al., [@B68]; Ott et al., [@B55]), have largely been evaluated in either open loop conditions or with feedback of lung volume simply represented as a filtered version of the motor output (Lindsey et al., [@B44]). While useful, these approaches have precluded model-based assessment of the potential influence of mechanical properties of the musculoskeletal system on respiratory motor pattern generation and cough effectiveness (Smith et al., [@B78]). More generally, neuromechanical models can provide a framework for estimating and predicting the extent to which motor patterns are constrained and influenced by mechanical properties and muscle synergies (Chiel et al., [@B15]). A model that relates a respiratory neural output to mechanical outputs has been available for some time (Riddle and Younes, [@B63]; Younes and Riddle, [@B84]; Younes et al., [@B85]) and remains an important element in contemporary models of the respiratory system (Cheng et al., [@B13]; Cheng and Khoo, [@B14]). However, the Younes--Riddle model with its single inspiratory neural output and single state variable (lung volume) lacks features essential for model-based assessment of the respective contributions and interactions of neural and biomechanical mechanisms during cough. We have developed a respiratory neural network model with inspiratory (phrenic), expiratory (lumbar), and laryngeal neural outputs and required a mechanical model with corresponding inputs to control the abdominal, diaphragm, and laryngeal muscles. Moreover, it is well known that a given lung volume can be achieved with different configurations of the rib cage and abdomen (Konno and Mead, [@B39]; Younes and Riddle, [@B84]), and with separate neural control of the diaphragm and abdominal wall muscles as in our network model, all of these configurations can potentially be achieved. Thus, the first aim of the work reported here was to develop a model of the mechanical respiratory system that includes separate muscle models for the diaphragm, abdominal wall, and larynx, and two state variables to represent the thoracoabdominal configuration. Our second aim was to link the resulting mechanical subsystem to an enhanced integrate and fire (IF) neural network model of the brainstem network for respiratory motor pattern generation and to assess the integrated system's behavior with muscle activation parameters for eupneic conditions. A third related objective was to extend the simulations to include evoked coughs in order to evaluate the model's performance in response to defined perturbations that enhance or reduce inspiratory drive. This latter goal was motivated in part by evidence that changes in the inspiratory or "operating" volume can influence airflow during the expulsive phase of the cough (Smith et al., [@B78]). An additional impetus for the third aim came from results of recent model simulations, which suggested that elevated systemic arterial blood pressure -- such as may occur during coughing (Sharpey-Schafer, [@B74]) -- attenuates cough inspiratory drive, a result supported by coordinated *in vivo* experiments (Poliaček et al., [@B58]). The network model used in the present work builds upon that and other recent prior efforts (Rybak et al., [@B67]). The network incorporates multiple circuit paths and operations for tuning inspiratory drive that have been inferred from spike train cross-correlation feature sets (Lindsey et al., [@B43]; Shannon et al., [@B71]; Segers et al., [@B68]; Ott et al., [@B55]). These circuits include parallel channels for modulation of inspiratory phase activity in "tonic" expiratory neurons that inhibit premotor inspiratory bulbospinal neurons and drive. In the course of sequentially eliminating sources of inspiratory drive for cough in the neuromechanical model, we also noted a contribution of tonic expiratory neuron activity to modulation of inspiratory phase drive during cough. This disinhibitory regulation predicted from the modeling results was subsequently supported by an analysis of *in vivo* data as described in a companion report (Segers et al., [@B69]). Materials and Methods ===================== Neural circuit components were derived from previously described respiratory network models of discrete "IF" populations after MacGregor ([@B47]) and a "hybrid IF burster" population with Hodgkin--Huxley style equations after Breen et al. ([@B12]). These models were developed iteratively with *in vivo* experiments that both guided model development and tested model predictions, as detailed in Rybak et al. ([@B67]) and Poliaček et al. ([@B58]). The enhanced network model used herein is described further in the Results. Biomechanical model elements were developed using parameters derived from published work as described in Results. Of particular importance was the work of Grassino et al. ([@B26]), who measured transdiaphragmatic pressure and diaphragm activation while controlling the thoracoabdominal configuration, making it possible to estimate the effect of rib cage motion on the abdominal volume. Our abdominal wall model is based on measurements of the curvature of the abdomen by Song et al. ([@B79]) taken during insufflation for laparoscopic surgery. The rib cage, lung, and diaphragm volumes are derived from the measurements of Cluzel et al. ([@B17]), who measured them from MRI's. The thoracoabdominal configuration at extreme and resting supine lung volumes are from Konno and Mead ([@B39]). Models were implemented using a program package written in the C language for the UNIX environment. Simulations were run on 64-bit Intel multiprocessor-based computers under the Linux operating system. The GNU Scientific Library was used to solve the differential equations of the biomechanical model, to find the roots of the implicit model equations, to do the abdominal volume integration, and for a spline approximation of the abdominal volume function. For each condition of the linked neural network and biomechanical model, four trials were run with different random number seeds for the stochastic network model. A pairwise two-sided *t*-test with non-pooled SD was used for each variable, and the *p*-values were adjusted for multiple testing (Holm, [@B32]). A difference was considered significant if the adjusted *p*-value was less than 0.05. Results ======= The results are presented in two main parts. Section ["Mechanical Model Implementation: Respiratory Muscles, Chest Wall, and Lungs"](#s1){ref-type="sec"} details the biomechanical model. Section ["Brainstem Network Model Architecture and System Performance When Linked to the Biomechanical Model"](#s2){ref-type="sec"} describes the linkage between the biomechanical and neural network models and neuromechanical system behavior during various perturbations of the network. Mechanical model implementation: Respiratory muscles, chest wall, and lungs {#s1} --------------------------------------------------------------------------- The biomechanical model described below converts respiratory neural outputs in the form of spike trains representing lumbar, phrenic, expiratory laryngeal, and inspiratory laryngeal motor neuron activity generated by a stochastic model of the brainstem respiratory network deterministically into mechanical outputs such as lung volume, tracheal flow, and alveolar pressure for a supine male human. Lung volume is fed back to the network model to simulate pulmonary stretch receptors. The mechanical model components include (i) three-element Hill muscle models of the diaphragm and abdominal muscles (Hill, [@B31]), (ii) a model of the larynx based on the results of Tully et al. ([@B81], [@B82]) and Rohrer's ([@B65]) equation, and (iii) lung/diaphragm/ribcage/abdomen volume relationships modeled on the data of Grassino et al. ([@B26]) and the analysis of Mead and Loring ([@B52]). The first two equations represent the entire mechanical model. Each term is a function of the motor outputs of the network model (*u*~di~, *u*~ab~, and *u*~lm~), and the diaphragm and abdominal wall volumes (*V*~di~ and *V*~ab~) and their time derivatives ($\overset{˙}{V}$~di~ and $\overset{˙}{V}$~ab~), and is defined by the subsequent equations. The parameters referenced in the model equations are listed in Table [1](#T1){ref-type="table"}. ###### **Parameters used in the biomechanical model**. Parameter Definition Value Units Source --------------------------------------------------------- --------------------------------------------------------------------------------------------------- -------- --------------- --------------------------------------------------------------------------------------------- **FREE PARAMETERS USED IN THE MODEL** *C*~1~ Rib cage contribution to abdominal volume 0.369 Dimensionless Derived from Grassino et al. ([@B26]) *C*~ab~ Compliance of the abdominal wall 0.108 L/cmH~2~O Derived from Estenne et al. ([@B21]) *C*~L~ Compliance of the lung 0.201 L/cmH~2~O Derived from Permutt and Martin ([@B57]) *c*~t~ Transverse chord of the abdominal wall 0.32 m Derived from Song et al. ([@B79]) *D* Diameter of the trachea 18 mm Derived from Baier et al. ([@B6]) and Kamel et al. ([@B36]) $f_{\text{a}}^{\text{TLC}}$ Obligatory ring fraction 0.15 Dimensionless Mead and Loring ([@B52]) *F*~CEmax~ Maximal force capacity of the external oblique 33 N Ratnovsky et al. ([@B60]) *F*~di~ Fraction of the diaphragm pressure expanding the rib cage via insertional forces 0.15 Dimensionless Derived from Loring and Mead ([@B45]) *k* Conversion factor from force to surface tension in abdominal muscle 0.68 m cmH~2~O/N Derived from De Troyer et al. ([@B18]) and Ratnovsky et al. ([@B60]) L~CEO~ Length of human transversus abdominis 19.1 cm Gaumann et al. ([@B23]) $P_{\text{ica}}^{\text{abTLC}}$ Maximal expiratory pressure due to intercostal and accessory muscles at total lung capacity (TLC) −135 cmH~2~O Derived from Ratnovsky et al. ([@B59]) *R*~ab~ Passive resistance of the abdominal wall 1.5 cmH~2~O/(L/s) Derived from Barnas et al. ([@B7]) *R*~di~ Passive resistance of the diaphragm 6 cmH~2~O/(L/s) Derived from Barnas et al. ([@B7]) *R*~rc~ Passive resistance of the rib cage 2.7 cmH~2~O/(L/s) Derived from Barnas et al. ([@B7]) *V*~c~ Mediastinal plus lung blood and tissue volume 1.756 L Derived from Cluzel et al. ([@B17]) *V*~CEmax~ Maximal contractile velocity of the external oblique 34.7 cm/s Ratnovsky et al. ([@B60]) $V_{\text{di}}^{\text{FRC}}$ Volume under diaphragm at functional residual capacity (FRC) 2.967 L Derived from Cluzel et al. ([@B17]) ${\overset{˙}{V}}_{\text{di}}^{\max}$ Maximal rate of change of volume under diaphragm 2.449 L/s Derived from Goldman et al. ([@B25]) and Chow and Darling ([@B16]) **FREE PARAMETERS USED IN CALCULATED PARAMETERS** $V_{\text{L}}^{\text{FRC}}$ Volume of the lung at FRC 2.29 L Cluzel et al. ([@B17]) $V_{\text{rc}}^{\text{kmFRC}}$ Volume of the rib cage at FRC as a fraction of VC relative to residual volume (RV) 0.1282 Dimensionless Konno and Mead ([@B39]), Figure 14 $V_{\text{ab}}^{\text{kmFRC}}$ Volume of the abdominal wall at FRC as a fraction of VC relative to RV 0.0400 Dimensionless Konno and Mead ([@B39]), Figure 14 $V_{\text{ab}}^{\text{kmTLC}}$ Volume of the abdominal wall at TLC as a fraction of VC relative to RV 0.3391 Dimensionless Konno and Mead ([@B39]), Figure 14 VC Vital capacity 5.370 L Roca et al. ([@B64]) $\sigma_{\text{di}}^{\text{RV}}$ Passive recoil pressure of the diaphragm at RV 20 cmH~2~O Derived from Agostoni et al. ([@B2]), Grassino et al. ([@B26]) and Siafakas et al. ([@B75]) $V_{\text{rc}}^{\text{FRC}}$ Volume of the rib cage at FRC 7.013 L Derived from Cluzel et al. ([@B17]) $V_{\text{rc}}^{\text{kmTLC}}$ Volume of the rib cage at TLC as a fraction of VC relative to RV 0.6609 Dimensionless Konno and Mead ([@B39]), Figure 14 *f*~di~ Ratio of diaphragm length at TLC to RV 0.65 Dimensionless Smith and Bellemare ([@B77]) *C*~rc~ Compliance of the rib cage 0.110 L/cmH~2~O Derived from Gilroy et al. ([@B24]) **CALCULATED PARAMETERS USED IN CALCULATED PARAMETERS** $V_{\text{ab}}^{\text{RV}}$ Volume behind abdominal wall at RV L Eqs [32](#E25){ref-type="disp-formula"} and [33](#E26){ref-type="disp-formula"} $V_{\text{L}}^{\text{RV}}$ Volume of the lung at RV L Eq. [34](#E27){ref-type="disp-formula"} $\sigma_{\text{L}}^{\text{RV}}$ Passive recoil pressure of the lung at RV cmH~2~O Eq. [35](#E28){ref-type="disp-formula"} $\sigma_{\text{ab}}^{\text{RV}}$ Passive recoil pressure of the abdominal wall at RV cmH~2~O Eq. [36](#E29){ref-type="disp-formula"} $V_{\text{ab}}^{\text{FRC}}$ Volume behind abdominal wall at FRC L Eq. [37](#E30){ref-type="disp-formula"} $V_{\text{di}}^{\text{RV}}$ Volume under diaphragm at RV L Eq. [38](#E31){ref-type="disp-formula"} $V_{\text{ab}}^{\text{TLC}}$ Volume behind abdominal wall at TLC L Eq. [39](#E32){ref-type="disp-formula"} $V_{\text{L}}^{\text{TLC}}$ Volume of the lung at TLC L Eq. [40](#E33){ref-type="disp-formula"} $\sigma_{\text{L}}^{\text{TLC}}$ Passive recoil pressure of the lung at TLC cmH~2~O Eq. [41](#E34){ref-type="disp-formula"} $\sigma_{\text{ab}}^{\text{TLC}}$ Passive recoil pressure of the abdominal wall at TLC cmH~2~O Eq. [42](#E35){ref-type="disp-formula"} $\sigma_{\text{di}}^{\text{TLC}}$ Passive recoil pressure of the diaphragm at TLC cmH~2~O Eq. [43](#E36){ref-type="disp-formula"} $\sigma_{\text{rc}}^{\text{TLC}}$ Passive recoil pressure of the rib cage at TLC cmH~2~O Eq. [44](#E37){ref-type="disp-formula"} $\sigma_{\text{rc}}^{\text{RV}}$ Passive recoil pressure of the rib cage at RV cmH~2~O Eq. [45](#E38){ref-type="disp-formula"} $f_{\text{a}}^{\text{RV}}$ Fraction of the rib cage exposed to abdominal pressure at RV Dimensionless Eq. [46](#E39){ref-type="disp-formula"} **CALCULATED PARAMETERS USED IN THE MODEL** *V*~sum~ Sum of diaphragm, rib cage, and abdominal wall volume contributions L Eq. [47](#E40){ref-type="disp-formula"} *V*~ab0~ Volume behind abdominal wall at zero passive tension L Eqs [32](#E25){ref-type="disp-formula"} and [33](#E26){ref-type="disp-formula"} *V*~L0~ Volume of the lung at zero passive tension L Eq. [48](#E41){ref-type="disp-formula"} $K_{\text{di}}^{\text{psv}}$ Coefficient of passive diaphragm recoil pressure cmH~2~O/L^2^ Eq. [49](#E42){ref-type="disp-formula"} $L_{\text{di}}^{\text{min}}$ Ratio of diaphragm length at zero volume to resting length Dimensionless Eq. [50](#E43){ref-type="disp-formula"} $P_{\text{ica}}^{\text{abRV}}$ Maximal expiratory pressure due to intercostal and accessory muscles at TLC cmH~2~O Eq. [51](#E44){ref-type="disp-formula"} $P_{\text{ica}}^{\text{diTLC}}$ Maximal inspiratory pressure due to intercostal and accessory muscles at TLC cmH~2~O Eq. [52](#E45){ref-type="disp-formula"} $\sigma_{\text{di}}^{\text{max}}$ Maximum active recoil pressure of the diaphragm cmH~2~O Eq. [53](#E46){ref-type="disp-formula"} $\sigma_{\text{rc}}^{\text{add}}$ Passive rib cage recoil pressure midway between volume limits cmH~2~O Eq. [54](#E47){ref-type="disp-formula"} $\sigma_{\text{rc}}^{\text{mul}}$ Rib cage sigmoid compliance coefficient cmH~2~O Eq. [55](#E48){ref-type="disp-formula"} *V*~di0~ Volume under diaphragm at zero tension L Eq. [56](#E49){ref-type="disp-formula"} $V_{\text{di}}^{\text{TLC}}$ Volume under diaphragm at TLC L Eq. [57](#E50){ref-type="disp-formula"} $V_{\text{rc}}^{\max}$ Upper limit of rib cage volume L Eq. [58](#E51){ref-type="disp-formula"} $V_{\text{rc}}^{\min}$ Lower limit of rib cage volume L Eq. [59](#E52){ref-type="disp-formula"} $V_{\text{rc}}^{\text{RV}}$ Volume of the rib cage at RV L Eq. [60](#E53){ref-type="disp-formula"} $V_{\text{rc}}^{\text{TLC}}$ Volume of the rib cage at TLC L Eq. [61](#E54){ref-type="disp-formula"} *V*~rc0~ Volume of the rib cage at zero tension L Eq. [62](#E55){ref-type="disp-formula"} ### Pressure balance on the rib cage In Eq. [1](#E1){ref-type="disp-formula"}, *P*~pl~ is the pleural pressure seen by the interior surface of the rib cage, *P*~ab−pl~ corrects for the fact that the rib cage sees abdominal pressure in the zone of apposition, *F*~di~σ~di~ is the equivalent pressure due to the insertional forces of the diaphragm on the lower ribs, *P*~ica~ is the equivalent pressure due to the intercostal and accessory muscles, and σ~rc~ is the recoil pressure of the rib cage that balances the sum of the other pressures. P pl \+ P ab \- pl \+ F di σ di \+ P ica = σ rc ### Pressure balance on the diaphragm In Eq. [2](#E2){ref-type="disp-formula"}, σ~ab~ is the abdominal pressure, the excess of which over the pleural pressure must be balanced by σ~di~, the recoil pressure of the diaphragm. σ ab \- P pl = σ di ### Pleural pressure In Eq. [3](#E3){ref-type="disp-formula"}, tracheal flow ($\overset{˙}{V}$~L~) is the derivative of lung volume. *R*~rs~ is the resistance of the airway, which when multiplied by $\overset{˙}{V}$~L~ gives the pressure drop, by the hydraulic analog of Ohm's law. σ~L~ is the recoil pressure of the lung. P pl = \- R rs V ˙ L \- σ L ### Lung volume Lung volume is a function of *V*~di~ and *V*~ab~. Diaphragm volume, *V*~di~, is the volume above the level of the diaphragm insertions on the rib cage and below the dome of the diaphragm. Abdominal wall volume, *V*~ab~, is the volume between the abdominal wall and a frontal plane that coincides with the contracted position of the abdominal wall. It has been commonly assumed that the abdominal contents are incompressible and that the abdominal cavity has only two movable walls -- the diaphragm and the abdominal wall -- and that therefore a displacement in one must be met by an equal and opposite displacement of the other (Grimby et al., [@B27]; Grassino et al., [@B26]; Macklem et al., [@B48]; Loring and Mead, [@B45]; Lichtenstein et al., [@B42]; Fitz-Clarke, [@B22]); in other words, that *V*~di~ + *V*~ab~ = *V*~sum~ (*V*~sum~ constant). Under this assumption, the abdominal volume completely determines the volume under the diaphragm, *V*~di~. Following Lichtenstein et al. ([@B42]), *V*~di~ determines the static contractile pressure generated by the diaphragm at a given activation. However, experimental data (Grassino et al., [@B26], Figure [4](#F4){ref-type="fig"}) show that both rib cage volume and abdominal volume affect this pressure. Therefore, in our model, we added a term, *C*~1~*V*~rc~, to the equation, allowing the rib cage and the abdominal wall to independently alter the volume under the diaphragm, effectively adding a third movable wall to the abdominal container, as discussed by Mead and Loring ([@B52]). The value of *C*~1~ was determined by fitting our model to published data (Grassino et al., [@B26], Figure [4](#F4){ref-type="fig"}) giving diaphragm pressure as a function of rib cage and abdominal volume at a fixed diaphragm activation. Given a value of *C*~1~, our model equations will calculate the diaphragm pressure from the volumes. We then found the value of *C*~1~ that was the best fit of the calculated pressure values to the measured values. The resulting equation is *V*~di~ + *C*~1~*V*~rc~ + *V*~ab~ = *V*~sum~, which together with Eq. [20](#E15){ref-type="disp-formula"}, gives us Eq. [4](#E4){ref-type="disp-formula"} for lung volume (*V*~L~). V L = V sum \- 1 \+ C 1 V di \- V ab \- C 1 V c C 1 ### Airway resistance Rohrer's equation was used to calculate airway resistance (Rohrer, [@B65]; Hey and Price, [@B30]). The equation Pressure = *K*~1~ · Flow + *K*~2~ · Flow^2^ or, dividing through by flow, Resistance = *K*~1~ + *K*~2~ \|Flow\| was applied twice, once for laryngeal resistance (*k*~1~ + *k~2~*\|$\overset{˙}{V}$~L~\|) and once for the resistance of the oropharynx and lower airway (0.72 + 0.44\|$\overset{˙}{V}$~L~\|), to give Eq. [5](#E5){ref-type="disp-formula"}. We used a value of *K*~2~ = 0.44 for the oropharynx and lower airway based on the assumption that *K*~2~ is 0 for the oropharynx (Renotte et al., [@B62]; Eq. [8](#E5){ref-type="disp-formula"}), and 0.44 for the lower airway (Renotte et al., [@B62], Table [2](#T2){ref-type="table"}). Assuming *K*~1~ for the larynx is negligible during quiet breathing (see below), we used 0.34 for the lower airway and 0.38 for the oropharynx (Renotte et al., [@B62], Table [2](#T2){ref-type="table"}), for a total of 0.72. The values of *k*~1~ and *k*~2~ for the larynx depend on the diameter of the glottis, as shown in Eqs [6](#E5){ref-type="disp-formula"}--[8](#E5){ref-type="disp-formula"}. The parameter *D* is the diameter of the human trachea. The variable *u*~lm~ is the net laryngeal muscle activation, ranging from −1 (closed glottis) to 1 (open glottis). The variable *d* is the diameter of the glottis, or more precisely, the diameter of a circle with the same area as the opening of the glottis. The resting diameter (when *u*~lm~ = 0) is taken to be 10.9 mm (Baier et al., [@B6]; Brancatisano et al., [@B10]; D'Urzo et al., [@B20]), and is assumed (see Eq. [8](#E5){ref-type="disp-formula"}) to change in proportion to *u*~lm~ (Tully et al., [@B81], [@B82]). The coefficient *k*~2~ given by Eq. [7](#E5){ref-type="disp-formula"} is calculated using the equation for flow through an orifice (Simpson, [@B76], Table II). The value of *k*~2~ for the upper airway is different for inspiration and expiration (Renotte et al., [@B62], Table [2](#T2){ref-type="table"}), which we assume is due to changes in *d*. Assuming equal excursions from the resting diameter, we solved for the coefficient that would give us the reported values of *k*~2~, and got 0.167. This approach resulted in a resting value for *k*~2~ of 0.681. We calculated the ratio of the mean resting value of *k*~1~ to the mean resting value of *k*~2~ (Tully et al., [@B81], Table [2](#T2){ref-type="table"}), and multiplied the ratio by 0.681 to get a resting value of *k*~1~ of 0.0035 for the larynx, which is small relative to *K*~1~ for the rest of the airway, justifying our assumption above that *K*~1~ for the larynx is negligible during quiet breathing. The Darcy--Weisbach equation for pressure loss in a pipe and the Darcy friction factor for laminar flow tell us that the resistance is proportional to 1/*d*^4^ (Kreith et al., [@B40]), which we use in Eq. [6](#E5){ref-type="disp-formula"} for *k*~1~. Plugging in the resting value of *k*~1~ determined above and the resting value of *d*, we solved for the coefficient, which gives us 49.6. R rs = k 1 \+ 0 . 72 \+ k 2 \+ 0 . 44 V ˙ L \(5\) k 1 = 49 . 6 d 4 \(6\) k 2 = 0 . 167 D 2 d 4 \+ d 2 D 2 \- 1 \(7\) d = D , u lm \> 71 109 10 . 9 1 \+ u lm , \- 1 ≤ u lm ≤ 71 109 0 , u lm \< \- 1 \(8\) ### Abdominal pressure on the rib cage A fraction *f*~a~ of the rib cage is exposed to abdominal pressure rather than pleural pressure. The recoil pressure of the diaphragm, σ~di~, is the difference between abdominal pressure and pleural pressure, so *P*~ab − pl~ adjusts the pressure seen by the rib cage for this difference. P ab \- pl = f a σ di ### Abdominal fraction of the rib cage At total lung capacity (TLC), none of the diaphragm is apposed to the rib cage (Mead and Loring, [@B52]), so we assumed that at all lung volumes, a portion of *V*~di~ equal to *V*~di~ at TLC $\left( V_{\text{di}}^{\text{TLC}} \right)$ does not contribute to the zone of apposition. The remainder $\left( {V_{\text{di}} - V_{\text{di}}^{\text{TLC}}} \right)$ is divided by the remainder plus the lung volume $\left( {V_{\text{di}} - V_{\text{di}}^{\text{TLC}} - V_{\text{L}}} \right)$ to give an estimate of the fraction of the rib cage surface above the diaphragm insertions that is exposed to abdominal pressure. The "obligatory ring" below the diaphragm insertions, which is about 15% of the rib cage surface (Mead and Loring, [@B52]), is always exposed to abdominal pressure, and is represented by $f_{\text{a}}^{\text{TLC}}$ in Eq. [10](#E7){ref-type="disp-formula"}. Our rib cage volume, *V*~rc~, is the volume above a plane through the diaphragm attachments (Cluzel et al., [@B17], Figure [3](#F3){ref-type="fig"}). The estimate of 15% of the rib cage surface was in the context of a different definition of rib cage volume in which a change in rib cage volume is equal to the change in lung volume with the abdominal wall held still (Konno and Mead, [@B39]). This alternative definition implies a larger volume for the rib cage because it includes parts below the diaphragm insertions. From our volume equations, this means that rib cage volume is larger than ours by a factor of (1 + *C*~1~), so we divided the previously calculated fraction of the smaller rib cage by (1 + *C*~1~) to turn it into a fraction of the larger rib cage before adding it to $f_{\text{a}}^{\text{TLC}}.$ f a = 1 1 \+ C 1 V di \- V di TLC V di \- V di TLC \+ V L \+ f a TLC ### Recoil pressure of the lung Equation [11](#E8){ref-type="disp-formula"} assumes a linear relationship between lung volume and recoil pressure. *C*~L~ is lung compliance. *V*~L0~ is the lung volume at zero recoil pressure, which we took to be equal to the residual volume (RV) after a maximal exhalation, the small recoil pressure remaining at RV being close enough to zero for our purposes (Permutt and Martin, [@B57]; Harris, [@B28]). σ L = V L \- V L0 C L ### Recoil pressure of the diaphragm In Eq. [12](#E9){ref-type="disp-formula"}, the term $u_{\text{di}}\sigma_{\text{di}}^{\text{max}}F_{\text{fl}}^{\text{di}}F_{\text{fv}}^{\text{di}}$ corresponds to the Hill muscle model (Ratnovsky et al., [@B60], Eq. A6), except that $\sigma_{\text{di}}^{\text{max}}$ is a pressure rather than a force and $F_{\text{fl}}^{\text{di}}$ and $F_{\text{fv}}^{\text{di}}$ are functions of volume and its derivative (flow), respectively, rather than length and velocity. We substituted pressure for force because, by Laplace's ([@B41]) Law, the pressure is proportional to the force when the curvature is constant, and the curvature of the human diaphragm dome does not change significantly with volume (Braun et al., ([@B11]). Moreover, there is a constant ratio between diaphragm force and pressure in the dog (Kim et al., [@B37]). Our substitution of volume for length (with an offset) is supported by the observation that the relationship between diaphragm pressure and length is not clearly different from linear when measured at RV, functional residual capacity (FRC), and TLC (Cluzel et al., [@B17]). To the extent that the action of the diaphragm resembles that of a piston (Kim et al., [@B37]), this linearity is expected. There is precedent for a Hill-style model in terms of pressure and flow for the respiratory system (Younes and Riddle, [@B84]). In Eq. [12](#E9){ref-type="disp-formula"}, *u*~di~ is phrenic activation of the diaphragm; $\sigma_{\text{di}}^{\text{max}}$ is static diaphragm recoil pressure at optimum length and maximum activation; *R*~di~$\overset{˙}{V}$~di~ is the pressure due to the passive resistance of the diaphragm. σ di = u di σ di max F fl di F fv di \+ σ di psv \+ R di V ˙ di ### Passive recoil pressure of the diaphragm In Eq. [13](#E10){ref-type="disp-formula"}, $\sigma_{\text{di}}^{\text{psv}}$ is the passive transdiaphragmatic pressure as a function of diaphragm volume. This pressure is taken to be zero at resting diaphragm volume $V_{\text{di}}^{\text{FRC}}$ (Agostoni and Rahn, [@B3]) and below, and quadratic above (Reid et al., [@B61]). σ di psv = K di psv . V di \- V di FRC 2 , V di \> V di FRC 0 , V di ≤ V di FRC ### Volume-pressure relationship of the diaphragm In Eq. [14](#E11){ref-type="disp-formula"}, $F_{\text{fl}}^{\text{di}}$ is the static pressure-volume relationship of the diaphragm (corresponding to Ratnovsky et al., [@B60], Eq. A7 with the relative length replaced by a linear function of volume as described above). The parameter *V*~di0~ is the volume under the diaphragm at the resting length. This is taken to be equal to the diaphragm volume at RV, based on the observation that the "highest Pdi twitch amplitude was recorded at RV" (Smith and Bellemare, [@B77]). The parameter $L_{\text{di}}^{\min}$ is the length of the diaphragm at zero volume (i.e., when the diaphragm is flat) divided by the resting length, and is calculated by assuming that the length of the diaphragm at TLC is 65% of that at RV (Smith and Bellemare, [@B77]). F fl di = exp \- 0 . 5 1 \- L di min V di0 V di \+ L di min \- 1 . 05 0 . 19 2 ### Pressure-flow relationship of the diaphragm In Eq. [15](#E12){ref-type="disp-formula"}, $F_{\text{fv}}^{\text{di}}$ is the pressure-flow relationship of the diaphragm, with the velocity replaced by flow as discussed above (Hatze, [@B29]; Rosen et al., [@B66]; Artemiadis and Kyriakopoulos, [@B4]). The variable $\overset{˙}{V}$~di~ is the time derivative of the volume under the diaphragm. The maximum rate of change of diaphragm volume, ${\overset{˙}{V}}_{\text{di}}^{\text{max}},$ was derived from data which gives transdiaphragmatic pressure as a function of flow at several levels of diaphragm activation up to 45% (Goldman et al., [@B25], Figure [6](#F6){ref-type="fig"}). Because the rib cage was held still, the flow represents the rate of change of diaphragm volume. Fitting the curves to a Hill-style relation between pressure and flow (Younes and Riddle, [@B84]), there is a maximum flow (where the pressure goes to zero) for each level of diaphragm activation. Experimental results suggest that the maximum flow increases somewhat linearly to 80% activation and then levels off (Chow and Darling, [@B16]). We used that assumption together with the results for the maximum flow at lower activations to compute a maximum flow at 100% activation, which we use for ${\overset{˙}{V}}_{\text{di}}^{\text{max}}.$ F fv di = 0 . 1433 0 . 1074 \+ exp \- 1 . 409 sinh 3 . 2 V ˙ di V ˙ di max \+ 1 . 6 ### Pressure of the intercostal and accessory muscles In Eq. [16](#E13){ref-type="disp-formula"}, *P*~ica~ is the effective pressure generated by the intercostal and accessory muscles; positive values act to expand the rib cage. In Eq. [17](#E13){ref-type="disp-formula"}, $P_{\text{ica}}^{\text{di}}$ is the pressure due to the action of the inspiratory intercostals, which are assumed to be inactive when the diaphragm volume is above $V_{\text{di}}^{\text{FRC}}$ (low lung volumes). Below $V_{\text{di}}^{\text{FRC}},$ the pressure exerted by the inspiratory intercostals is assumed to be proportional to the activation of the diaphragm (*u*~di~), and the proportionality constant itself is assumed to scale linearly from 0 at $V_{\text{di}}^{\text{FRC}}$ to its maximum value of $P_{\text{ica}}^{\text{diTLC}}$ at $V_{\text{di}}^{\text{TLC}}.$ The parameter $P_{\text{ica}}^{\text{diTLC}}$ was calculated as the pressure necessary to complete the pressure balance on the rib cage at TLC. In Eq. [18](#E13){ref-type="disp-formula"}, $P_{\text{ica}}^{\text{ab}}$ is the pressure due to the action of the expiratory intercostals, which is assumed to be proportional to the abdominal muscle activation (*u*~ab~); the proportionality constant itself is assumed to scale linearly with the rib cage volume, changing from $P_{\text{ica}}^{\text{abRV}}$ at RV to $P_{\text{ica}}^{\text{abTLC}}$ at TLC. In Eq. [18](#E13){ref-type="disp-formula"}, $P_{\text{ica}}^{\text{abTLC}}$ is the pressure due to the expiratory intercostals at TLC and maximal abdominal activation. This parameter's value was calculated by taking the mean male maximal mouth pressure at TLC (from Ratnovsky et al., [@B59], Table [1](#T1){ref-type="table"}) and subtracting it from the rib cage recoil pressure at TLC. $P_{\text{ica}}^{\text{abRV}}$ is the pressure due to the expiratory intercostals at RV and maximal abdominal activation. We calculated this parameter by solving the model equations for *P*~ica~ while assuming RV conditions. This gives us the intercostal pressure necessary to reach RV. P ica = P ica di \+ P ica ab \(16\) P ica di = u di P ica diTLC V di \- V di FRC V di TLC \- V di FRC , V di \< V di FRC 0, V di ≥ V di FRC \(17\) P ica ab = u ab P ica abRV \+ V rc \- V rc RV V rc TLC \- V rc RV P ica abTLC \- P ica abRV \(18\) ### Recoil pressure of the rib cage The volume of the rib cage is assumed to be a sigmoid function of the recoil pressure of the rib cage, σ~rc~. With increasing pressure the volume asymptotically approaches a maximum $\left( V_{\text{rc}}^{\text{max}} \right),$ and with decreasing pressure it asymptotically approaches a minimum $\left( V_{\text{rc}}^{\text{min}} \right).$ A generalized logistic function is used for the sigmoid, giving *V*~rc~ as a function of σ~rc~; that equation is solved for σ~rc~ to give the first part of Eq. [19](#E14){ref-type="disp-formula"}. The final term of Eq. [19](#E14){ref-type="disp-formula"} is the pressure due to the passive resistance of the rib cage (*R*~rc~) and the rate of change of its volume ($\overset{˙}{V}$~rc~) The parameter $\sigma_{\text{rc}}^{\text{mul}}$ controls the maximum slope of the sigmoid; the slope is the compliance of the rib cage. It is calculated to make the compliance equal to *C*~rc~/(1 + *C*~1~). The factor of (1 + *C*~1~) appears because *C*~rc~ is for a rib cage volume defined differently than *V*~rc~. *C*~rc~ is the compliance of the rib cage, an average of values for three sitting subjects (Gilroy et al., [@B24], Table [1](#T1){ref-type="table"}). σ rc = σ rc mul log V rc max \- V rc V rc \- V rc min \+ σ rc add \+ R rc V ˙ rc ### Volume of the rib cage The rib cage volume (*V*~rc~) is the sum of the lung volume (*V*~L~), the volume under the diaphragm (*V*~di~), and the volume of the mediastinum and the lung blood and tissue (*V*~c~). V rc = V L \+ V di \+ V c ### Recoil pressure of the abdominal wall The abdominal wall is modeled as a surface with a circular segment cross-section in each transverse plane, all with the same radius, and a circular segment cross-section in each sagittal plane, all with another radius. The volume behind the abdominal wall, *V*~ab~, is bounded by this surface and by a frontal plane. The values for the sagittal (*r*~s~) and transverse (*r*~t~) radii were derived from measurements taken during insufflation for laparoscopic surgery in humans (see Figure [3](#F3){ref-type="fig"}, Song et al., [@B79]). We fit exponential curves to the data points and the resulting relationship between the fitted sagittal and transverse radii was found to be well approximated by a linear function: *r*~s~ = 8.00 *r*~t~ − 1.10. The length of the longest transverse chord in the bounding frontal plane (*c*~t~) was found which gave the volume change stated in the paper. In Eq. [21](#E16){ref-type="disp-formula"}, $u_{\text{ab}}F_{\text{CEmax}}F_{\text{fl}}^{\text{ab}}F_{\text{fv}}^{\text{ab}}$ is the Hill muscle model equation (Ratnovsky et al., [@B60], Eq. A6); *u*~ab~ is the activation of the diaphragm by the lumbar motor neurons; *F*~CEmax~ is the maximal force capacity of the contractile element for a 1.5 cm^2^ cross-section of canine external oblique muscle (Ratnovsky et al., [@B60], Table [1](#T1){ref-type="table"}). The constant *k* converts from a force to a surface tension, and (1/*r*~s~ + 1/*r*~t~) converts the surface tension to a pressure, using the Law of Laplace (Laplace, [@B41]). The second term on the right, (*V*~ab~ − *V*~ab0~)/*C*~ab~, is the passive recoil pressure of the abdominal wall. *V*~ab0~ is the volume behind the abdominal wall at which the recoil pressure is 0. This was taken to be equal to $V_{\text{ab}}^{\text{FRC}},$ since we assume a supine position. *C*~ab~ is the compliance of the abdominal wall. The final term is the pressure due to the passive resistance of the abdominal wall (*R*~ab~) and the rate of change of abdominal volume ($\overset{˙}{V}$~ab~). σ ab = u ab F CEmax F fl ab F fv ab . k r t \+ k r s \+ V ab \- V ab0 C ab \+ R ab V ˙ ab In Eq. [22](#E17){ref-type="disp-formula"}, $F_{\text{fl}}^{\text{ab}}$ is the static force-length relationship of the abdominal wall (Ratnovsky et al., [@B60], Eq. A7); *L*~CE~ is the length of the transversus abdominis; *L*~CE0~ is its resting length. F fl ab = exp \- 0 . 5 L CE L CE0 \- 1 . 05 0 . 19 2 In Eq. [23](#E18){ref-type="disp-formula"}, $F_{\text{fv}}^{\text{ab}}$ is the force-velocity relationship of the abdominal wall muscles (Hatze, [@B29]; Rosen et al., [@B66]; Artemiadis and Kyriakopoulos, [@B4]). The variable ${\overset{˙}{L}}_{\text{CE}}$ is the velocity of the contractile element (the time derivative of *L*~CE~) and the parameter *V*~CEmax~ is the maximal contractile velocity of canine external oblique muscle (Ratnovsky et al., [@B60], Table [1](#T1){ref-type="table"}). F fv ab = 0 . 1433 0 . 1074 \+ exp \- 1 . 409 sinh 3 . 2 L ˙ CE V CEmax \+ 1 . 6 ### Length-volume relationship of the abdominal wall Equations [24](#E19){ref-type="disp-formula"} through 31 calculate the length of the abdominal muscle (*L*~CE~) from the volume behind the abdominal wall (*V*~ab~). Eqs [26](#E19){ref-type="disp-formula"} through 31 calculate *V*~ab~ as a function of *r*~t~ (the transverse radius); Eq. [25](#E19){ref-type="disp-formula"} uses the inverse of the resulting function to calculate *r*~t~ from *V*~ab~; Eq. [24](#E19){ref-type="disp-formula"} calculates *L*~CE~ from *r*~t~. In practice, the function *V*~ab~(*r*~t~) is pre-calculated, and approximated and inverted with a spline, and the spline is used to evaluate *r*~t~(*V*~ab~) during simulation. L CE = 100 r t sin \- 1 c t 2 r t \(24\) r t V ab = V \- 1 V ab , V ab \> 0 0 . 5 c t , V ab ≤ 0 \(25\) V r t = ∫ \- c s ∕ 2 c s ∕ 2 A r t , y d y \(26\) In Eq. [27](#E20){ref-type="disp-formula"}, the Pythagorean Theorem is applied in the midsagittal plane to get *c*~s~, the length of the chord that connects the ends of the abdominal wall in the frontal plane that serves as a boundary of the abdominal wall volume. c s = 2 r s 2 \- h 0 \- r s 2 In Eq. [28](#E21){ref-type="disp-formula"}, the Pythagorean Theorem is applied in a transverse plane to get *h*~0~, the distance from the peak of the abdominal wall to the frontal plane that serves as a boundary of the abdominal wall volume. h 0 = r t \- r t 2 \- c t 2 2 In Eq. [29](#E22){ref-type="disp-formula"}, the formula for the area of a circular segment is applied in the transverse plane at a distance *y* from the peak of the abdominal wall to get the area between the abdominal wall and the boundary frontal plane. A = r t 2 2 2 cos \- 1 1 \- h r t \+ sin 2 cos \- 1 h r t \- 1 In Eq. [30](#E23){ref-type="disp-formula"}, the Pythagorean theorem is applied in the midsagittal plane to get *h*, the distance from the abdominal wall to the boundary frontal plane at a distance *y* in the craniocaudal direction from the peak of the abdominal wall. h = r s 2 \- y 2 \- r s 2 \- c s 2 2 Equation [31](#E24){ref-type="disp-formula"} is the relation between the sagittal radius (*r*~s~) and the transverse radius (*r*~t~) derived from the results in Song et al. ([@B79]). r s = 8 . 00479 r t \- 1 . 10158 ### Equations for calculated parameters σ ab u ab = 1 , V ab RV , V ˙ ab = 0 , V ab0 = σ ab RV \(32\) V ab0 = V ab RV \+ V ab kmFRC ⋅ VC \(33\) V L RV = V L FRC \- V rc kmFRC \+ V ab kmFRC ⋅ VC \(34\) σ L RV = V L RV \- V L0 C L \(35\) σ ab RV = σ di RV \- σ L RV \(36\) V ab FRC = V ab RV \+ V ab kmFRC ⋅ VC \(37\) V sum = V di FRC \+ C 1 V rc FRC \+ V ab FRC \(38\) V di RV = V sum \- V ab RV \- C 1 V rc RV \(39\) V ab TLC = V ab FRC \+ V ab kmTLC \- V ab kmFRC ⋅ VC \(40\) V L TLC = V rc TLC \- V di TLC \- V c \(41\) σ L TLC = V L TLC \- V L0 C L \(42\) σ ab TLC = V ab TLC \- V ab0 C ab \(43\) σ di TLC = σ L TLC \- σ ab TLC \(44\) σ rc TLC = σ rc mul log V rc max \- V rc TLC V rc TLC \- V rc min \+ σ rc add \(45\) σ rc RV = σ rc mul log V rc max \- V rc RV V rc RV \- V rc min \+ σ rc add \(46\) f a RV = 1 1 \+ C 1 V di RV \- V di TLC V di RV \- V di TLC \+ V L RV \+ f a TLC \(47\) V L0 = V L RV \(48\) K di psv = σ di RV V di RV \- V di FRC 2 \(49\) L di min = V di TLC \- f di V di RV V di TLC \- V di RV ∕ 1 . 05 \(50\) P ica abRV = σ L RV \+ σ rc RV \- f a RV \+ F di σ di RV \(51\) P ica diTLC = σ L TLC \+ σ rc TLC \- f a TLC \+ F di σ di TLC \(52\) σ di u di = 1 , V di TLC , V ˙ di = 0 , σ di max = σ di TLC \(53\) σ rc add = σ rc mul log V rc0 \- V rc min V rc max \- V rc0 \(54\) σ rc mul = V rc max \- V rc min 1 \+ C 1 4 C rc \(55\) V di0 = V di RV \(56\) V di TLC = V sum \- V ab TLC \- C 1 V rc TLC \(57\) V rc max = V rc TLC \+ 0 . 05 V rc TLC \- V rc RV \(58\) V rc min = V rc RV \- 0 . 99 V rc TLC \- V rc RV \(59\) V rc RV = V rc FRC \- V rc kmFRC ⋅ VC 1 \+ C 1 \(60\) V rc TLC = V rc FRC \+ V rc kmTLC \- V rc kmFRC ⋅ VC 1 \+ C 1 \(61\) V rc0 = V rc FRC \(62\) Brainstem network model architecture and system performance when linked to the biomechanical model {#s2} -------------------------------------------------------------------------------------------------- The computational model of the pontomedullary respiratory network (Figure [1](#F1){ref-type="fig"}) instantiated the hypothesis (Shannon et al., [@B72]; Rybak et al., [@B67]) that airway cough receptors affect several neuron populations in the ventral respiratory column (VRC) and pontine respiratory group (PRG) via cough 2nd order neurons. Evoked changes reconfigured the respiratory network to produce the cough motor pattern through the same VRC neurons involved in providing drive to respiratory muscles during normal breathing. The model incorporated recent enhancements (Poliaček et al., [@B58]) and additional neuron populations and other changes as detailed in Tables [2](#T2){ref-type="table"}--[4](#T4){ref-type="table"}. ![**Schematic of the raphé-pontomedullary respiratory network model used in this study**. The model extends that used in Poliaček et al. ([@B58]) and Rybak et al. ([@B67]) and follows labeling conventions enumerated therein. Parameters for the represented cell populations (large colored circles) and connections (see Key) are listed in Tables [2](#T2){ref-type="table"} and [4](#T4){ref-type="table"}. Parameters for the I-Driver population with conditional bursting pacemaker properties were as described previously (Rybak et al., [@B67]; Poliaček et al., [@B58]). Abbreviations of brainstem regions or "compartments": pre-BötC, pre-Bötzinger complex; VRC or VRG, ventral respiratory column or group; PRG, pontine respiratory group. Abbreviations of most populations were as enumerated in Table [1](#T1){ref-type="table"} of Rybak et al. ([@B67]): Aug and Dec: neurons with augmenting or decrementing activity patterns, respectively, during the indicated phase (I-inspiratory; E-expiratory) of maximum firing rate. BS, bulbo-spinal; ELM, expiratory laryngeal motoneurons; EI, neurons with a peak firing rate during the E-to-I phase transition; IE, neurons with a peak firing rate during the I-to-E phase transition; ILM, inspiratory laryngeal motoneurons; NRM, non-respiratory-modulated neurons. Two phrenic motor neuron populations with different threshold ranges innervated the diaphragm (PHR, PHR-HT: high threshold); two lumbar motor neuron populations activated the abdominal muscle (LUM, LUM-HT: high threshold). I-Dec_2, second inhibitory I population in the VRC (e.g., see Ott et al., [@B55]); Lung Def_1s, Lung Dis_1s, Deflation 2nd order: lung deflation-sensing neuronal circuit elements. See text for further discussion.](fphys-03-00264-g001){#F1} ###### **Population parameters for network model with adjusted (Δ) and additional (+) neuronal populations modified from Poliaček et al. ([@B58])**. Population name Size Resting threshold (mV) THO variability (mV) Membrane time constant Post-spike increase in G~K+~ Post-spike *G*~K+~ time constant (ms) Adaptation threshold increase Adaptation (ms) Noise amplitude DC (mV) ------------------------------------ ------ ---------------------------------------------------------------------- ---------------------- ------------------------ ------------------------------ --------------------------------------- ------------------------------- ----------------- ----------------- --------- I-pons 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.03 2.0 rostral IE-pons 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.3 5.0 caudal IE-pons 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.3 5.0 E-pons 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.3 13.0 EI-pons 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.3 20.0 NRM-pons 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.03 25.0 NRM-BötC 300 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.03 25.0 ΔI-DRIVER 300 See Table A2 in Rybak et al. ([@B67]) for I-Driver neuron properties ΔI-Dec 300 10.0 1.0 6.0 75.0 8.5 0.9 1500.0 0.2 20.0 +I-Dec_2 300 10.0 1.0 6.0 75.0 8.5 0.8 1200.0 0.2 5.0 ΔI-Aug 300 12.0 3.0 6.0 75.0 5.0 0.0 5000.0 0.6 18.0 ΔVRC IE 99 18.0 2.0 9.0 50.0 5.0 0.0 1000.0 0.075 0.0 ΔE-Dec-Phasic 300 9.0 1.0 6.0 75.0 8.5 0.9 1500.0 0.1 4.0 ΔE-Dec-Tonic 300 8.0 1.0 9.0 50.0 3.8 0.8 2000.0 0.3 0.0 E-Aug-early 300 10.0 1.0 6.0 27.0 2.5 0.0 500.0 0.3 30.0 E-Aug-late 300 10.0 1.0 9.0 27.0 2.5 0.0 500.0 0.1 27.0 ΔE-Aug-Cough 300 12.0 2.0 9.0 75.0 7.5 0.0 500.0 0.2 0.0 +E-Aug (+) 300 10.0 2.0 6.0 75.0 7.5 0.1 1000.0 0.5 20.0 ΔE-Aug-BS (+) 300 12.0 3.0 6.0 100.0 6.0 0.1 1000.0 0.5 0.0 ΔPump (+) 300 5.0 0.5 6.0 25.0 3.8 0.08 500.0 0.1 0.0 ΔPump (-) 300 5.0 0.0 6.0 25.0 3.8 0.08 500.0 0.1 0.0 ΔI-Aug-BS 300 12.0 3.0 6.0 75.0 5.0 0.0 5000.0 0.5 0.0 +Phrenic 210 10.0 2.0 5.0 200.0 6.0 0.08 500.0 0.5 0.0 +Phrenic-HT 70 16.0 2.0 60.0 200.0 5.0 0.08 500.0 0.5 0.0 ΔLumbar 210 15.0 2.0 6.0 75.0 7.5 0.08 500.0 0.5 0.0 +Lumbar-HT 70 18.0 2.0 30.0 200.0 7.5 0.1 500.0 0.5 0.0 ΔILM 300 20.0 1.0 6.0 25.0 3.8 0.08 500.0 0.1 2.0 ΔE-Dec-pre-ELM 300 11.0 0.0 6.0 100.0 6.0 0.8 500.0 0.5 1.0 ΔELM 300 18.0 2.0 6.0 100.0 6.0 0.9 100.0 0.5 0.0 ΔLUNG PSRs 300 11.0 1.0 9.0 20.0 7.0 0.0 500.0 0.5 0.0 +Lung deflation receptors (Def_1) 300 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.5 0.0 +Lung distortion receptors (Dis_1) 300 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.5 0.0 \>Cough 2nd order 100 10.0 1.0 9.0 20.0 7.0 0.3 500.0 0.1 0.0 ΔDeflation 2nd order 300 8.0 1.0 9.0 27.0 2.5 0.5 1000.0 0.3 0.0 Raphé 8 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.01 0.0 Raphé 28 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.1 0.0 Raphé 29 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.5 0.0 Raphé 30 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.5 0.0 Raphé 31 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.1 10.0 Raphé 32 100 10.0 1.0 9.0 20.0 7.0 0.0 500.0 0.1 10.0 *Variable names used by MacGregor ([@B47]) are in italics. All values representing voltages are relative to the resting potential, which is considered equal to zero. *N* is the number of neurons simulated in each population. *THO*, the resting threshold, is normally distributed in the population around the value of THO with a standard deviation equal to the "THO variability" value. *TMEM* is the membrane time constant. *B* is the amplitude of the post-spike increase in potassium conductance. *TGK* is the time constant of the potassium conductance decay following an action potential. *C* and *TTH* define the change in threshold associated with spike adaptation. *C* is the ratio of the threshold increase to the membrane potential increase; its value is between 0 and 1. *TTH* is the time constant of the rise in threshold with spike adaptation. *Noise Amplitude*: each cell has an internal noise generator that acts like two synapses, one with an equilibrium potential of 70 mV above resting and the other with −70 mV. Each acts like it has an incoming firing probability of 0.05 per time step, and a synapse time constant of 1.5 ms. This parameter is the conductance that gets added to the synapse conductance on each (virtual) spike. *DC*: an injected current will raise the membrane potential by an amount that is inversely proportional to the membrane conductance. Instead of being specified directly as a current, this parameter is specified in mV, and it is interpreted as the current that is required to raise the membrane potential by the specified number of mV when the membrane conductance has its resting value. The effect on the membrane potential at other membrane conductances will be inversely proportional to the conductance. Note also that as in other types of IF neuron models, our neuron models do not actually generate action potential-like spikes but only identified moments of spikes, so "spiking" shown in all neuron simulations are represented graphically by assigning vertical spike-like lines at computed times of threshold crossing. The population "E-Aug-Late-HT" in Rybak et al. ([@B67]) has been renamed to "E-Aug-Cough" in the base model for this simulation. \>, neuron populations that relay perturbations of the network model. A fiber population composed of 100 fibers, each with a firing probability of 0.05 at each simulation time step and 100 type Ex_1 excitatory synaptic terminals (synaptic strength 0.03), represented cough receptor excitation. These fibers excited the Cough 2nd order neuron population. For abbreviations, see list of abbreviations and legend of Figure [1](#F1){ref-type="fig"} in the main text*. ###### **Synaptic parameters for the network model**. Synapse name Synapse type Synapse equilibrium potential (mV) Synapse time constant (ms) -------------- ------------------------------------ ------------------------------------ ---------------------------- Ex_1 Excitatory 115.0 1.5 Inh_4 Inhibitory −25.0 1.5 Inh_7 Inhibitory −25.0 2.0 Inh_10 Inhibitory −25.0 1.5 Ex_13 Excitatory 115.0 1.5 Pre-ex_13 Inhibitory (pre-synaptic to Ex_13) 0.0 1.5 Ex_19 Excitatory 115.0 5.0 Inh_22 Inhibitory −25.0 5.0 Inh_25 Inhibitory −25.0 4.5 Ex_28 Excitatory 115.0 1.5 Pre-ex_28 Inhibitory (pre-synaptic to Ex_28) −25.0 3.5 ###### **Connectivity for the network model modified from Poliaček et al. ([@B58])**. Source population Target population Synaptic type Conduction times No. of terminals Synaptic strength Source pop. N Target pop. N Divergence Mean no. of terminals Convergence ------------------------ ------------------- --------------- ------------------ ------------------ ------------------- --------------- --------------- ------------ ----------------------- ------------- ---------------- I-Driver I-Dec ex_1 2 6 100 0.006 300 300 84.99 ± 3.14 1.18 84.99 ± 7.54 I-Driver I-Aug ex_1 2 6 100 0.01 300 300 84.93 ± 3.00 1.18 84.93 ± 7.73 I-Driver I-Driver ex_1 0 4 50 0.003 300 300 46.34 ± 1.76 1.08 46.34 ± 5.84 E-Dec-Phasic I-Driver inh_22 2 6 50 0.03 300 300 46.16 ± 1.77 1.08 46.16 ± 9.24 E-Dec-Phasic E-Aug-early inh_4 0 2 150 0.012 300 300 118.24 ± 4.01 1.27 118.24 ± 8.86 E-Dec-Phasic E-Aug-late inh_4 2 4 150 0.04 300 300 118.10 ± 3.94 1.27 118.10 ± 9.61 E-Dec-Phasic VRC-IE inh_4 0 2 50 0.1 300 99 39.33 ± 2.31 1.27 119.18 ± 7.22 E-Dec-Phasic I-Dec inh_4 0 2 200 0.2 300 300 146.07 ± 4.42 1.37 146.07 ± 8.71 +E-Dec-Phasic I-Aug-BS inh_4 2 6 100 0.15 300 300 85.21 ± 3.08 1.17 85.21 ± 7.27 E-Dec-Phasic I-Aug inh_4 2 6 50 0.1 300 300 46.19 ± 1.72 1.08 46.19 ± 5.43 E-Dec-Phasic rostral IE-pons ex_13 2 4 100 0.001 300 100 63.13 ± 2.98 1.58 189.39 ± 6.74 E-Dec-Phasic caudal IE-pons ex_13 2 4 100 0.001 300 100 63.23 ± 3.19 1.58 189.70 ± 9.56 +E-Dec-Phasic I-Dec_2 inh_4 2 6 150 0.1 300 300 118.39 ± 4.42 1.27 118.39 ± 8.69 +E-Dec-Phasic E-Aug (+) inh_4 2 6 100 0.03 300 300 85.18 ± 2.99 1.17 85.18 ± 10.46 +E-Dec-Phasic E-Aug-Cough (−) inh_22 2 6 100 0.025 300 300 85.20 ± 3.05 1.17 85.20 ± 9.59 I-Dec E-Aug-early inh_7 2 6 115 0.5 300 300 95.67 ± 3.39 1.20 95.67 ± 7.14 I-Dec E-Dec-Phasic inh_25 2 6 200 0.2 300 300 145.80 ± 5.20 1.37 145.80 ± 9.31 I-Dec I-Aug inh_25 2 6 120 0.025 300 300 99.27 ± 3.48 1.21 99.27 ± 8.70 I-Dec E-Aug-late inh_7 2 6 115 0.5 300 300 95.71 ± 3.51 1.20 95.71 ± 7.84 I-Dec VRC-IE inh_25 0 4 33 0.025 300 99 28.15 ± 1.85 1.17 85.29 ± 6.01 I-Dec E-Dec-Tonic inh_25 2 6 100 0.001 300 300 84.90 ± 2.96 1.18 84.90 ± 9.43 I-Dec ILM ex_1 0 3 50 0.002 300 300 46.35 ± 1.71 1.08 46.35 ± 6.00 I-Dec E-Aug-BS (+) inh_7 0 4 100 0.05 300 300 85.44 ± 3.02 1.17 85.44 ± 9.96 I-Dec EI-pons ex_13 2 4 100 0.001 300 100 63.33 ± 3.16 1.58 190.00 ± 8.78 I-Dec I-pons ex_13 2 4 100 0.0005 300 100 63.50 ± 3.23 1.57 190.51 ± 8.03 I-Dec I-pons inh_7 2 4 100 0.0005 300 100 63.66 ± 3.21 1.57 190.98 ± 8.42 I-Dec rostral IE-pons inh_4 2 4 100 0.0001 300 100 63.55 ± 3.04 1.57 190.65 ± 8.05 I-Dec caudal IE-pons inh_7 2 4 100 0.0001 300 100 63.27 ± 3.06 1.58 189.80 ± 8.43 I-Dec Lumbar inh_7 0 4 100 0.1 300 210 79.70 ± 3.14 1.25 113.86 ± 8.97 I-Dec E-Dec-pre-ELM inh_4 2 6 200 0.06 300 300 146.12 ± 4.70 1.37 146.12 ± 8.41 +I-Dec E-Aug (+) inh_4 0 2 130 1.0 300 300 105.84 ± 3.65 1.23 105.84 ± 7.43 +I-Dec ELM inh_4 0 5 200 0.06 300 300 146.22 ± 4.96 1.37 146.22 ± 9.62 +I-Dec Lumbar-HT inh_7 2 6 100 0.1 300 70 53.32 ± 2.84 1.88 228.53 ± 6.63 +I-Dec I-Dec inh_25 2 6 140 0.0125 300 300 112.11 ± 3.97 1.25 112.11 ± 9.09 +I-Dec E-Aug-Cough (−) inh_7 2 6 115 0.16 300 300 95.71 ± 3.51 1.20 95.71 ± 7.84 I-Aug I-Aug ex_1 0 5 50 0.025 300 300 45.99 ± 1.77 1.09 45.99 ± 5.31 I-Aug caudal IE-pons inh_7 2 4 100 0.0001 300 100 63.43 ± 3.19 1.58 190.28 ± 9.05 I-Aug I-Aug-BS ex_1 2 6 100 0.06 300 300 85.20 ± 3.01 1.17 85.20 ± 7.27 I-Aug ILM ex_1 2 6 70 0.035 300 300 62.72 ± 2.32 1.12 62.72 ± 6.93 I-Aug VRC-IE ex_1 2 6 16 0.0015 300 99 14.82 ± 0.97 1.08 44.92 ± 6.80 I-Aug I-pons ex_13 2 4 100 0.0025 300 100 63.59 ± 2.94 1.57 190.76 ± 7.71 I-Aug rostral IE-pons inh_4 2 4 100 0.0001 300 100 63.43 ± 2.95 1.58 190.29 ± 8.57 +I-Aug I-Dec_2 ex_28 2 6 200 0.01 300 300 146.05 ± 4.72 1.37 146.05 ± 9.65 E-Aug-early E-Dec-Phasic inh_4 2 6 110 0.007 300 300 92.32 ± 3.45 1.19 92.32 ± 7.95 E-Aug-early I-Dec inh_4 0 5 100 0.06 300 300 85.13 ± 2.98 1.17 85.13 ± 7.70 E-Aug-early I-Aug inh_4 2 6 100 0.135 300 300 85.44 ± 3.23 1.17 85.44 ± 7.76 E-Aug-early VRC-IE inh_4 0 2 24 0.05 300 99 21.46 ± 1.29 1.12 65.02 ± 6.96 E-Aug-early I-Aug-BS inh_4 0 2 150 0.001 300 300 118.31 ± 4.21 1.27 118.31 ± 7.57 E-Aug-early E-Aug-late inh_10 0 2 50 0.001 300 300 46.01 ± 1.81 1.09 46.01 ± 6.57 E-Aug-early E-pons ex_13 2 4 100 0.002 300 100 63.08 ± 2.85 1.59 189.25 ± 7.68 E-Aug-early I-pons inh_7 2 4 100 0.0005 300 100 63.08 ± 2.85 1.59 189.25 ± 7.68 +E-Aug-early I-Dec_2 inh_4 2 6 150 0.08 300 300 118.31 ± 4.21 1.27 118.31 ± 7.57 +E-Aug-early Phrenic inh_4 0 2 150 0.001 300 210 107.57 ± 4.12 1.39 153.68 ± 8.34 +E-Aug-early Phrenic-HT inh_4 0 2 150 0.001 300 70 61.97 ± 2.40 2.42 265.57 ± 5.37 +E-Aug-early ELM inh_4 2 6 175 0.007 300 300 132.52 ± 4.21 1.32 132.52 ± 9.21 E-Aug-late E-Aug-early inh_10 0 2 200 0.04 300 300 145.91 ± 4.71 1.37 145.91 ± 9.07 E-Aug-late I-Dec inh_4 2 6 55 0.2 300 300 50.33 ± 1.87 1.09 50.33 ± 6.68 E-Aug-late I-Aug inh_4 2 6 100 0.1 300 300 85.27 ± 3.04 1.17 85.27 ± 7.41 E-Aug-late E-Dec-Phasic inh_4 2 6 120 0.015 300 300 98.86 ± 3.48 1.21 98.86 ± 9.67 E-Aug-late I-Aug-BS inh_4 2 6 150 0.06 300 300 118.14 ± 4.18 1.27 118.14 ± 8.44 E-Aug-late VRC-IE inh_4 0 2 24 0.02 300 99 21.32 ± 1.39 1.13 64.62 ± 7.03 E-Aug-late E-Dec-Tonic inh_4 0 2 100 0.05 300 300 85.18 ± 3.01 1.17 85.18 ± 6.53 E-Aug-late E-Dec-pre-ELM inh_22 2 6 115 0.05 300 300 95.69 ± 3.35 1.20 95.69 ± 7.07 E-Aug-late ELM inh_7 2 6 200 0.1 300 300 145.91 ± 4.71 1.37 145.91 ± 9.07 +E-Aug-late I-Dec_2 inh_4 2 6 150 0.075 300 300 118.14 ± 4.18 1.27 118.14 ± 8.44 +E-Aug-late Phrenic inh_4 4 8 150 0.06 300 210 107.09 ± 3.99 1.40 152.98 ± 7.22 +E-Aug-late Phrenic-HT inh_4 0 2 150 0.06 300 70 61.83 ± 2.37 2.43 265.00 ± 5.23 +E-Aug-late ILM inh_4 0 2 115 0.04 300 300 95.59 ± 3.32 1.20 95.59 ± 8.06 VRC-IE I-Dec inh_7 0 4 200 0.035 99 300 146.65 ± 4.99 1.36 48.39 ± 5.40 Raphé 8 Raphé 29 ex_1 0 3 50 0.0125 100 100 39.73 ± 2.28 1.26 39.73 ± 5.01 Raphé 28 Raphé 30 ex_1 0 3 50 0.0125 100 100 39.51 ± 2.47 1.27 39.51 ± 4.93 +I-Aug-BS Phrenic-HT ex_1 3 6 18 0.05 300 70 15.99 ± 1.24 1.13 68.51 ± 7.54 +I-Aug-BS Phrenic ex_1 3 6 50 0.05 300 210 44.95 ± 2.01 1.11 64.21 ± 6.33 NRM-BötC rostral IE-pons inh_7 0 1 100 0.002 300 100 63.41 ± 3.22 1.58 190.23 ± 7.92 NRM-BötC caudal IE-pons inh_7 0 1 100 0.002 300 100 63.37 ± 3.02 1.58 190.12 ± 7.75 NRM-BötC I-pons ex_13 0 1 100 0.002 300 100 63.25 ± 3.00 1.58 189.74 ± 8.31 +E-Aug-Cough (−) E-Aug (+) inh_22 2 6 100 0.05 300 300 85.39 ± 3.06 1.17 85.39 ± 7.75 E-Aug-Cough (−) E-Aug-BS (+) inh_22 2 6 100 0.5 300 300 85.34 ± 3.04 1.17 85.34 ± 7.22 +E-Aug-Cough (−) I-Dec_2 inh_22 0 3 200 0.05 300 300 145.65 ± 4.67 1.37 145.65 ± 8.84 E-Aug-Cough (−) E-Dec-pre-ELM inh_4 2 6 100 0.1 300 300 85.22 ± 3.02 1.17 85.22 ± 7.02 +E-Aug-Cough (−) I-Aug-BS inh_4 0 3 200 0.025 300 300 145.65 ± 4.67 1.37 145.65 ± 8.84 +E-Aug-Cough (−) ILM inh_4 0 3 200 0.025 300 300 146.45 ± 4.63 1.37 146.45 ± 8.96 Lung PSRs Pump (+) ex_1 0 3 75 0.015 300 300 66.50 ± 2.43 1.13 66.50 ± 6.72 Lung PSRs Pump (−) ex_1 0 3 50 0.015 300 300 46.23 ± 1.78 1.08 46.23 ± 9.43 caudal IE-pons I-Driver ex_1 0 5 100 0.001 100 300 85.68 ± 2.78 1.17 28.56 ± 4.45 Pump (--) E-pons pre-ex_13 0 4 100 0.99 300 100 63.19 ± 2.88 1.58 189.58 ± 7.28 +Pump (--) I-Dec_2 inh_4 0 2 25 0.0035 300 300 24.04 ± 0.88 1.04 24.04 ± 5.11 Pump (--) I-Dec inh_4 0 2 25 0.0035 300 300 23.98 ± 0.90 1.04 23.98 ± 5.97 Pump (--) I-pons pre-ex_13 0 4 100 0.99 300 100 63.53 ± 2.94 1.57 190.58 ± 7.02 Pump (--) EI-pons pre-ex_13 2 4 100 0.99 300 100 63.55 ± 2.95 1.57 190.64 ± 7.44 Pump (--) Lung Def_1s inh_4 0 4 100 0.02 300 300 85.22 ± 3.09 1.17 85.22 ± 7.46 Pump (--) rostral IE-pons pre-ex_13 0 4 100 0.99 300 100 63.63 ± 3.02 1.57 190.88 ± 9.09 Pump (--) caudal IE-pons pre-ex_13 0 4 100 0.99 300 100 63.63 ± 3.02 1.57 190.88 ± 9.09 Pump (+) E-Dec-Phasic ex_1 0 2 100 0.01 300 300 85.47 ± 2.95 1.17 85.47 ± 8.14 Pump (+) VRC-IE ex_1 2 6 100 0.01 300 99 63.10 ± 2.99 1.58 191.20 ± 11.20 Pump (+) I-Aug ex_1 0 2 25 0.0 300 300 24.07 ± 0.91 1.04 24.07 ± 4.15 Pump (+) E-Dec-T ex_1 0 2 100 0.002 300 300 85.12 ± 3.16 1.17 85.12 ± 6.94 +Pump (+) I-Dec_2 ex_1 2 6 100 0.005 300 300 85.20 ± 3.05 1.17 85.20 ± 9.59 E-Dec-T Raphé 29 inh_4 0 3 100 0.001 300 100 63.59 ± 3.05 1.57 190.76 ± 7.20 E-Dec-T I-Aug-BS inh_4 2 6 100 0.03 300 300 84.99 ± 3.14 1.18 84.99 ± 7.54 E-Dec-T rostral IE-pons ex_13 2 4 100 0.001 300 100 63.26 ± 3.11 1.58 189.77 ± 11.78 E-Dec-T I-pons ex_13 2 4 100 0.0005 300 100 63.33 ± 3.02 1.58 189.98 ± 9.72 E-Dec-T I-pons inh_7 2 4 100 0.0005 300 100 63.20 ± 3.13 1.58 189.60 ± 7.79 E-Dec-T rostral IE-pons inh_4 2 4 100 0.0005 300 100 63.57 ± 3.16 1.57 190.70 ± 7.57 E-Dec-T caudal IE-pons ex_13 2 4 100 0.001 300 100 63.20 ± 3.18 1.58 189.61 ± 7.72 E-Dec-T caudal IE-pons inh_7 2 4 100 0.0005 300 100 63.40 ± 3.17 1.58 190.20 ± 9.74 E-Dec-T ELM inh_4 0 4 100 0.04 300 300 85.04 ± 3.30 1.18 85.04 ± 8.24 E-Dec-T I-Aug inh_4 2 6 100 0.0075 300 300 85.12 ± 3.35 1.17 85.12 ± 8.17 +E-Dec-T I-Dec_2 pre-ex_28 2 6 100 0.2 300 300 84.99 ± 3.14 1.18 84.99 ± 7.54 rostral IE-pons EI-pons inh_4 2 4 100 0.03 100 100 63.79 ± 3.24 1.57 63.79 ± 4.98 rostral IE-pons VRC-IE ex_1 0 1 100 0.001 100 99 62.90 ± 3.28 1.59 63.54 ± 4.65 rostral IE-pons E-Dec-Phasic ex_1 0 5 100 0.02 100 300 85.06 ± 2.76 1.18 28.35 ± 4.15 EI-pons rostral IE-pons ex_1 2 4 100 0.002 100 100 63.47 ± 3.20 1.58 63.47 ± 4.78 EI-pons caudal IE-pons ex_1 2 4 100 0.002 100 100 63.36 ± 3.47 1.58 63.36 ± 4.41 EI-pons VRC-IE ex_1 0 4 50 0.0003 100 99 39.46 ± 2.35 1.27 39.86 ± 5.11 EI-pons E-Dec-T ex_1 0 4 100 0.01 100 300 85.16 ± 3.33 1.17 28.39 ± 5.10 E-Dec-pre-ELM ELM ex_19 2 6 250 0.0125 300 300 169.56 ± 5.08 1.47 169.56 ± 7.81 Def 2nd (−) E-Dec-Phasic inh_4 2 6 100 0.04 300 300 85.17 ± 3.11 1.17 85.17 ± 9.16 +E-Aug (+) E-Aug-BS (+) ex_19 2 6 100 0.02 300 300 85.16 ± 2.97 1.17 85.16 ± 7.43 Raphé 8 Raphé 31 inh_4 0 3 50 0.005 100 100 39.38 ± 2.09 1.27 39.38 ± 6.21 Raphé 8 Raphé 32 inh_4 0 3 50 0.005 100 100 39.51 ± 2.47 1.27 39.51 ± 4.93 Raphé 8 E-Aug-BS (+) inh_22 0 3 400 0.0 100 300 221.45 ± 4.77 1.81 73.82 ± 4.47 Raphé 29 Raphé 30 ex_1 0 3 50 0.01 100 100 39.51 ± 2.47 1.27 39.51 ± 4.93 Raphé 29 E-Dec-T ex_19 0 3 100 0.15 100 300 84.74 ± 3.12 1.18 28.25 ± 4.23 Raphé 29 E-Dec-Phasic ex_19 0 3 100 0.2 100 300 84.74 ± 3.12 1.18 28.25 ± 4.23 Raphé 30 Raphé 29 inh_4 0 3 50 0.01 100 100 39.51 ± 2.47 1.27 39.51 ± 4.93 Raphé 32 Raphé 31 inh_4 0 3 50 0.005 100 100 39.51 ± 2.47 1.27 39.51 ± 4.93 Raphé 32 E-Dec-Tonic inh_22 0 3 100 0.01 100 300 85.28 ± 3.13 1.17 28.43 ± 4.65 Raphé 32 E-Dec-Phasic inh_22 0 3 100 0.01 100 300 84.74 ± 3.12 1.18 28.25 ± 4.23 Raphé 32 E-Dec-pre-ELM inh_22 0 3 100 0.01 100 300 84.74 ± 2.97 1.18 28.25 ± 4.67 \>+Cough 2nd order (+) I-Aug-BS ex_1 2 6 100 0.02 100 300 85.25 ± 2.83 1.17 28.42 ± 5.15 \>Cough 2nd order (+) I-Aug ex_1 2 6 100 0.0045 100 300 85.25 ± 2.83 1.17 28.42 ± 5.15 \>Cough 2nd order (+) I-Dec ex_1 2 6 100 0.0045 100 300 85.27 ± 2.89 1.17 28.42 ± 4.68 \>+Cough 2nd order (+) I-Dec_2 ex_1 2 6 100 0.05 100 300 85.54 ± 3.07 1.17 28.51 ± 4.72 \>Cough 2nd order (+) E-Aug-late ex_1 2 6 100 0.005 100 300 85.25 ± 2.83 1.17 28.42 ± 5.15 \>Cough 2nd order (+) E-Aug-early ex_1 0 3 100 0.01 100 300 85.07 ± 3.05 1.18 28.36 ± 4.19 \>Cough 2nd order (+) VRC-IE inh_4 0 3 100 0.2 100 99 63.13 ± 3.05 1.58 63.77 ± 5.20 \>Cough 2nd order (+) caudal IE-pons ex_1 0 3 100 0.001 100 100 63.59 ± 3.21 1.57 63.59 ± 5.84 \>Cough 2nd order (+) rostral IE-pons ex_1 0 3 100 0.001 100 100 63.59 ± 3.21 1.57 63.59 ± 5.84 \>Cough 2nd order (+) I-pons ex_1 0 3 100 0.001 100 100 63.59 ± 3.21 1.57 63.59 ± 5.84 \>Cough 2nd order (+) E-pons ex_1 2 6 100 0.001 100 100 63.59 ± 3.21 1.57 63.59 ± 5.84 \>Cough 2nd order (+) EI-pons ex_1 0 3 100 0.001 100 100 63.59 ± 3.21 1.57 63.59 ± 5.84 \>Cough 2nd order (+) E-Dec-pre-ELM ex_19 2 6 100 0.004 100 300 85.14 ± 3.06 1.17 28.38 ± 3.97 \>+Cough 2nd order (+) E-Aug (+) ex_1 2 6 100 0.05 100 300 85.25 ± 2.83 1.17 28.42 ± 5.15 \>+Cough 2nd order (+) E-Aug-Cough (−) ex_1 2 6 100 0.04 100 300 85.25 ± 2.83 1.17 28.42 ± 5.15 \>+Cough 2nd order (+) ILM ex_1 2 6 100 0.001 100 300 84.92 ± 3.23 1.18 28.31 ± 4.63 E-pons rostral IE-pons inh_4 2 4 100 0.0001 100 100 63.17 ± 3.15 1.58 63.17 ± 5.22 E-pons caudal IE-pons inh_4 2 4 100 0.0001 100 100 63.47 ± 3.13 1.58 63.47 ± 5.60 E-pons I-Dec inh_4 0 1 100 0.008 100 300 85.14 ± 3.03 1.17 28.38 ± 4.11 NRM-pons I-pons ex_1 0 4 100 0.015 100 100 63.26 ± 3.28 1.58 63.26 ± 4.40 NRM-pons I-pons inh_4 0 4 100 0.05 100 100 63.62 ± 3.11 1.57 63.62 ± 4.61 NRM-pons I-Driver ex_1 2 6 100 0.11 100 300 85.10 ± 3.00 1.18 28.37 ± 4.51 NRM-pons VRC-IE ex_1 0 1 100 0.01 100 99 63.02 ± 2.53 1.59 63.66 ± 4.67 NRM-pons I-Aug ex_1 0 1 100 0.01 100 300 85.21 ± 2.94 1.17 28.40 ± 4.81 NRM-pons E-Aug-early ex_1 0 4 100 0.025 100 300 85.82 ± 3.10 1.17 28.61 ± 4.20 NRM-pons E-Aug-late ex_1 0 4 50 0.003 100 300 45.82 ± 1.89 1.09 15.27 ± 3.67 NRM-pons E-Dec-Phasic ex_1 0 1 100 0.01 100 300 84.87 ± 3.22 1.18 28.29 ± 4.18 NRM-pons E-Dec-Tonic ex_1 0 1 100 0.1 100 300 85.35 ± 3.04 1.17 28.45 ± 4.06 NRM-pons NRM-BötC inh_4 0 1 100 0.001 100 300 85.11 ± 2.96 1.17 28.37 ± 5.07 E-Aug-BS (+) Lumbar ex_1 6 10 25 0.03 300 210 23.59 ± 1.14 1.06 33.70 ± 5.45 +E-Aug-BS (+) Lumbar-HT ex_1 3 6 10 0.05 300 70 9.34 ± 0.75 1.07 40.03 ± 3.18 I-pons rostral IE-pons ex_1 0 4 100 0.005 100 100 62.93 ± 2.89 1.59 62.93 ± 5.56 I-pons VRC-IE ex_1 0 5 100 0.005 100 99 63.61 ± 3.41 1.57 64.25 ± 4.84 I-pons I-Aug ex_1 0 4 50 0.005 100 300 46.17 ± 1.67 1.08 15.39 ± 3.39 I-pons caudal IE-pons ex_1 0 4 100 0.005 100 100 63.67 ± 2.85 1.57 63.67 ± 4.57 +I-Dec_2 E-Dec-Tonic inh_4 2 6 125 0.1 300 300 102.25 ± 3.60 1.22 102.25 ± 7.81 +I-Dec_2 E-Dec-pre-ELM inh_4 2 6 100 0.01 300 300 85.11 ± 3.10 1.17 85.11 ± 7.52 +I-Dec_2 E-Aug-BS (+) inh_4 2 6 50 0.00005 300 300 46.04 ± 1.73 1.09 46.04 ± 8.13 +I-Dec_2 ELM inh_4 2 6 100 0.02 300 300 85.61 ± 3.37 1.17 85.61 ± 7.12 +I-Dec_2 E-Aug (+) inh_4 2 6 100 0.02 300 300 85.19 ± 3.09 1.17 85.19 ± 8.08 +Lung Def_1s Def 2nd (−) ex_1 0 3 35 0.03 300 300 33.04 ± 1.38 1.06 33.04 ± 6.68 +Lung DIS_1s ELM ex_1 0 3 100 0.09 300 300 84.97 ± 2.93 1.18 84.97 ± 7.40 *+, Connection added to the network in Poliaček et al. ([@B58]). \>, Connection relaying a perturbation to the network model. Connections between individual neurons were made according to a sequence of pseudorandom numbers calculated from a unique seed number for each source-to-target connection. Targets were chosen with replacement. This table includes the means ± SD of the number of neurons in each target population innervated by each source neuron in each population. Corresponding values are also shown for source neurons that innervated each target neuron in each population. These data indicate the extent of divergence and convergence, respectively. Most neurons in each source population made a single terminal connection with each target neuron. *Mean No. of Terminals*, the mean number of terminals from each source neuron innervating each target neuron. The efficacy of connections between populations of neurons was influenced by the change in conductance associated with each action potential at a synapse (*Synaptic Strength*) and the number of terminals for each axon. *Synaptic types* were distinguished by their equilibrium potentials and time constants. The time constant of some synapses was slightly longer than others because troughs in cross-correlograms from which the particular synaptic connections were inferred tended to have longer durations. 11 types of synapses were used in the simulation (see Table [3](#T3){ref-type="table"}). If the value of the presynaptic modulatory strength parameter (*Synaptic Strength*) was \<1.0, the strength of the connection it modulated was reduced to the product of the presynaptic Synaptic Strength parameter and target synapse conductance. If the presynaptic Synaptic Strength parameter was \>1.0, the amount by which it was greater than 1 was added to its target synapse's conductance. *Minimum and maximum conduction times* are expressed in 0.5 ms simulation clock ticks for each source-to-target axon population. *No. of Terminals*, number of terminals from source neuron*. ### Linking the neural network and biomechanical models {#s3} The diaphragm received input from two phrenic motor neuron populations (PHR, PHR-HT) with different threshold ranges to generate motor unit diversity and facilitate an ordered recruitment during increased inspiratory drive. Diaphragm activation was based on the mean instantaneous firing rates (*P*, *P*~1~), of the two populations by the expression (0.3*P* + 0.7*P*~1~)/*X* where *X* is the firing rate for maximum diaphragm activation; values of 50--200 spikes/s were used (Nail et al., [@B54]) to approximate the plot for diaphragm activation in Figure [1](#F1){ref-type="fig"} of Mantilla and Sieck ([@B49]). Similarly, two lumbar motor neuron populations (LUM, LUM-HT) activated the abdominal muscle with *X* set to 80. Inspiratory laryngeal motor (ILM) and expiratory laryngeal motor (ELM) neuron populations regulated laryngeal resistance over a range between fully open (+1) and fully closed (−1), inclusively. Lung afferent populations were regulated by injected currents defined at each simulation step by evaluation of expressions that included model lung volume. Pulmonary stretch receptors (PSRs) became more active with increasing lung volume *V* (membrane bias = 0.5*V* mV/%VC) and mediated the Hering--Breuer reflex. Deflation-sensitive lung receptors were also implemented (Paintal, [@B56]; Luck, [@B46]; Wei and Shen, [@B83]; Iscoe and Gordon, [@B35]; Bergren and Peterson, [@B8]; Matsumoto et al., [@B50]; Yu et al., [@B86]). A low threshold population (Def_1, membrane bias = − 0.225(*V* − 70) mV/%VC) and its afferent pathway introduced in Poliaček et al. ([@B58]) was used to represent a class of possible network mechanisms for generating an inhibitory bias on E-Dec neurons. Simulated "vagotomy" (elimination of the effects of lung afferents) in the present model removed this inhibition, contributing to the observed prolongation of the expiratory phase (Te increased from 2.76 s to 3.15 s (*p*-value = 0.0004, two-sided *t*-test). Vagotomy also removed the influence of the PSRs and increased inspiratory phase duration (Ti) from 1.94 s to 2.61 s, (*p*-value = 4 × 10^−7^; see references and discussion in Dick et al., [@B19]). A higher threshold "distortion" (Dis_1) receptor population (cf. Iscoe and Gordon, [@B35]) excited the ELM population when lung volume was below FRC (membrane bias = −1.75(*V* − 10) mV/%VC if *V *\< 10, 0 otherwise). We note that the synaptic strength and firing rates of this speculative Dis_1 population, added for development purposes in other work (Hutchison and Lindsey, [@B33]), resulted in negligible modulation of ELM population activity under the conditions of the present study (see ["Influence of Some Added Network Connections"](#s5){ref-type="sec"}). ### Additional enhancements to the current network model {#s4} The "I-Dec_2" population was added to provide a second inhibitory VRC inspiratory neuron population for tuning inspiratory drive as proposed in Ott et al. ([@B55]) for central chemoreceptor modulation of breathing. In some previous models (Rybak et al., [@B67]; Poliaček et al., [@B58]), the "E-Aug-late" population inhibited numerous target populations, but also served to excite the VRG bulbospinal E-Aug-BS (+) population that drives expiratory lumbar motor neurons. A new "E-Aug (+)" population was added to facilitate differential regulation of the lumbar motor neurons and expiratory drive modulation as proposed in the literature (Iscoe, [@B34]; Shannon et al., [@B73]; Molkov et al., [@B53]). Other parameters were adjusted and populations added in anticipation of linking the network model to the biomechanical model derived from data from human subjects. In the antecedent model (Poliaček et al., [@B58]), the I-Aug-BS population output served both a premotor function and represented the "phrenic" output. The inhibitory connections from the VRC-IE population to the I-Aug-BS population were eliminated in the new model; E-Dec-P inhibition of the I-Aug-BS population was retained. The resulting eupneic respiratory cycle frequency (12.7 cycles/min) was within the range for resting breathing in the human adult (A.D.A.M. Medical Encyclopedia, [@B1]). ### Eupneic motor pattern and "baseline" cough The joint neuromechanical model generated a eupneic motor pattern and an evoked cough. Figure [2](#F2){ref-type="fig"} shows membrane potential records from simulated neurons in representative PRG, raphé, and VRC neuron populations and the six types of motor neurons. The "IF" neuron populations do not generate action potential-like spikes; instances of threshold crossings are indicated graphically by corresponding vertical spike-like lines. Additional traces include integrated population activity of the three lung afferent populations and biomechanical system metrics, including lung volume, tracheal flow, and alveolar pressure. The three phases of the cough cycle (Bolser et al., [@B9]) are highlighted. {ref-type="fig"}); see Table [2](#T2){ref-type="table"} for properties of this population and Table [4](#T4){ref-type="table"} for details of connections with other populations. See text for further discussion.](fphys-03-00264-g002){#F2} The inspiratory phase of the cough was characterized by increased activation of the diaphragm and enlargement of upper airway via activation of the ILM (abductor) motor neuron population, resulting in an increased lung volume (43% VC), inspiratory flow, and transdiaphragmatic and transpulmonary pressures. The subsequent compressive phase included activation of the ELM (adductor) motor neurons with transient laryngeal closure, together with activation of lumbar motor neurons and abdominal expiratory muscles. During this phase, tracheal airflow stopped and there was an increase in alveolar and abdominal pressure. In the following expulsive phase, high air flow velocity (72.2% VC/s) resulted from the opening of the larynx during continued abdominal muscle activation. ### Cough behavior with changes in inspiratory drive Two series of simulations with complementary perturbations of cough inspiratory drive were made to assess model behavior during the phases of cough. Different sets of random number seeds were used for each simulation to generate variability in model output by altering the thresholds of individual neurons in each population and the convergent and divergent connectivity patterns among populations within ranges defined by the initial baseline parameter settings. In the first series, the activation strengths for the connections between phrenic motor neuron populations and the diaphragm were increased by factors of 2 and 4. Figure [3](#F3){ref-type="fig"} shows the integrated activity of each motor neuron population together with lung volume, tracheal flow, alveolar pressure, and abdominal pressure for baseline conditions (left) and a trial with four times the baseline activation strength (right). {ref-type="fig"} and [2](#F2){ref-type="fig"} for definitions) during eupneic and "baseline" cough motor patterns before (A) and after (B) increasing the gain of diaphragm activation by a factor of 4 (see center schematic inset); otherwise, cough stimulus parameters as in Figure [2](#F2){ref-type="fig"}**. Scales on right are for **(A,B)**. See text for further details.](fphys-03-00264-g003){#F3} Outputs from four trials for each amplified condition were compared with each other and with the baseline results. Figure [4](#F4){ref-type="fig"} shows the means (±95% confidence limits) of selected biomechanical outputs measured during baseline cough (1×) and the two conditions of increased activation gain (2×, 4×). Pairs of symbols connected by a line indicate no significant difference. Successively larger peak expiratory flow rates and abdominal pressures were respectively associated with greater lung volumes during preceding inspiratory phases of the evoked coughs, even though abdominal drive did not change. This result established that differences in flow with the generated changes in inspiratory (operating) volumes were the consequence of the modeled biomechanical system. {#F4} Mean respiratory cycle frequencies measured during pre-cough eupneic intervals for each condition were also evaluated. The respiratory frequency increased with the highest (4×) inspiratory drive, a change associated with changes in feedback from lung afferents under the "closed loop" conditions evaluated. In the second series of simulations, inspiratory drive was altered only during the cough cycle by changing synaptic strengths of Cough 2nd order neuron inputs to selected model populations. The top panels in Figures [5](#F5){ref-type="fig"}A~1--C~1~~ show schematics of a subset of the model network and sites where synaptic strengths were changed relative to the baseline conditions represented in and described for Figure [3](#F3){ref-type="fig"}A. Corresponding panels in Figures [5](#F5){ref-type="fig"}A~2--C~2~~ show integrated traces of motor neuron population activities and biomechanical model outputs for the respective perturbations; arrows highlight significant changes in the indicated metrics (further enumerated in Figure [6](#F6){ref-type="fig"}). {ref-type="fig"}A. Corresponding panels (**A~2~--C~2~**) show integrated traces of motor neuron population activities and biomechanical model outputs (left labels) for the respective perturbations of inspiratory drive. Arrows mark changes. See text for further details.](fphys-03-00264-g005){#F5} {#F6} First (Figure [5](#F5){ref-type="fig"}A), synaptic strengths from the Cough 2nd order population to the I-Aug and I-Aug-BS populations were doubled. The highlighted segment of the inset (Figure [5](#F5){ref-type="fig"}A~1~) shows integrated traces for the I-Aug-BS, I-Dec_2, and E-Dec-Tonic populations during a eupneic cycle and the subsequent evoked cough. I-Aug-BS activity increased under this condition (asterisk). Next (Figure [5](#F5){ref-type="fig"}B), cough inspiratory drive was decreased relative to baseline by deletion (synaptic strength = 0.0) of the excitatory connections between the Cough 2nd order population and both the I-Aug and I-Aug-BS populations. The excitation of the I-Dec_2 population by 2nd order cough neurons remained, partially suppressing the recurrent inhibition of the I-Dec_2 and two I-Aug populations. The highlighted segment of the inset (Figure [5](#F5){ref-type="fig"}B~1~) shows reduced I-Aug-BS activity during the evoked cough under this condition (asterisk). The third perturbation further reduced cough inspiratory drive by also transiently blocking I-Dec_2 neuron inhibition of the E-Dec-Tonic population during the cough cycle. The elimination of this influence resulted in increased E-Dec-Tonic inhibition of the I-Aug and I-Aug-BS populations during the cough (asterisk in highlighted segment of Figure [5](#F5){ref-type="fig"}C~1~). Figure [6](#F6){ref-type="fig"} plots (from the top) the means of peak values (±95% confidence limits) for lung volume, expiratory tracheal flow, alveolar pressure, abdominal drive, and abdominal pressure measured under conditions of pre-cough eupnea (Eupnea), baseline cough (Base), and the three conditions represented in Figure [5](#F5){ref-type="fig"}. The differences in peak lung volumes during cough under the three conditions (A--C) confirm functional roles for both the excitatory and disinhibitory influences on inspiratory drive in the model. Deletion of the excitatory component of cough inspiratory drive (B) caused peak expiratory tracheal flow to decrease relative to the previous baseline and enhanced coughs (Base, A). However, peak abdominal drive and abdominal pressure did not change. A further reduction in peak lung volume to levels below eupneic control due to transiently increased E-Dec-Tonic inhibition of inspiratory drive (C) during cough resulted in no further change in expiratory flow, although peak abdominal pressure was reduced relative to baseline cough values. A third series of simulations was done with the isolated biomechanical model. Figure [7](#F7){ref-type="fig"} plots the peak expiratory flow in four coughs simulated at different operating volumes but equal peak abdominal pressure of 26.5 cmH~2~O. In each cough, the diaphragm and abdominal activations were first controlled to produce the desired operating volume, then the laryngeal muscles were controlled to close the airway, then the abdominal activation was controlled to produce an abdominal pressure of 26.5 cmH~2~O, and finally the laryngeal muscles were controlled to open the airway. Note that no statistics were done on these runs because the biomechanical model is deterministic. As in the first series of simulations, successively larger peak expiratory flow rates were associated with greater lung volumes during preceding inspiratory phases of the simulated cough, but unlike the first series, the peak abdominal pressure was the same in each cough. This result established that differences in flow with the generated changes in inspiratory (operating) volumes were not entirely due to the differences in pressure seen in the first series. {#F7} ### Comparisons with behaviors of antecedent models Table [5](#T5){ref-type="table"} shows means of inspiratory and expiratory phase durations during eupnea and cough and peak firing rates of motor neuron populations common to the present model and those described in Rybak et al. ([@B67]) and Poliaček et al. ([@B58]). The current model has lower firing rates, similar to those observed *in vivo* (Iscoe, [@B34]; Mantilla and Sieck, [@B49]), and longer respiratory phase durations; inspiratory phase durations are also more variable (see coefficients of variation, Table [5](#T5){ref-type="table"}). ###### **Comparison with previous models**. Variable Unit Rybak Poliaček No speculative Current ------------ ------- ------- ---------- ---------------- --------- ------- -------- ------ -------- ------- -------- ------ ------ ------- ------ **EUPNEA** ELM spk/s 170 0.00\* 0.10 0.00\* 231 0.00\* 0.08 0.00\* 19 1.00 0.23 1.00 19 0.23 ILM spk/s 83 0.00\* 0.06 0.00\* 49 0.00\* 0.07 0.00\* 26 0.62 0.29 1.00 19 0.47 LUMBAR spk/s 24 0.00\* 0.05 0.07 135 0.00\* 0.08 1.00 10 1.00 0.08 1.00 11 0.08 PHRENIC spk/s 109 0.00\* 0.10 1.00 129 0.00\* 0.11 1.00 62 0.33 0.08 1.00 56 0.13 Ti s 0.663 0.00\* 0.04 0.00\* 1.506 0.00\* 0.07 1.00 1.744 0.03\* 0.07 1.00 1.939 0.10 Te s 1.053 0.00\* 0.12 1.00 1.396 0.00\* 0.10 1.00 2.905 1.00 0.12 1.00 2.760 0.11 **COUGH** ELM spk/s 302 0.00\* 0.33 0.04\* 521 0.00\* 0.22 0.86 55 1.00 0.07 1.00 55 0.09 ILM spk/s 238 0.00\* 0.11 1.00 154 0.00\* 0.14 1.00 37 1.00 0.10 1.00 36 0.10 LUMBAR spk/s 235 0.00\* 0.07 1.00 536 0.00\* 0.10 1.00 76 1.00 0.11 1.00 76 0.06 PHRENIC spk/s 348 0.00\* 0.14 0.16 705 0.00\* 0.16 0.07 98 1.00 0.04 1.00 97 0.05 Ti s 0.494 0.00\* 0.20 1.00 0.503 0.00\* 0.23 1.00 2.471 1.00 0.09 1.00 2.302 0.12 Te s 0.564 0.00\* 0.24 1.00 0.476 0.00\* 0.19 1.00 3.490 1.00 0.19 1.00 3.242 0.15 *The behaviors of the networks in Rybak et al. ([@B67]) and Poliaček et al. ([@B58]) and a model without recently added speculative connections are compared with the model in this paper. The mean of the peak (in each respiratory cycle) firing rates for four motor populations and the mean inspiratory (Ti) and expiratory (Te) phase durations are shown. Each mean for each previous model is followed by the *p*-value from a two-sided *t*-test with non-pooled SD for the difference in means between the previous model and the current model. The cycle counts for Rybak, Poliaček, No speculative, and Current, respectively, were 67, 40, 24, and 24 for eupnea and 56, 60, 10, and 10 for cough. The table also shows the coefficient of variation (CV) for each firing rate or phase duration for assessment of differences in variability between the models. Each CV is followed by the *p*-value from an *F*-test for the ratio of variances of the observations divided by their mean between the previous model and the current model. *p*-Values have been adjusted for multiple testing (Holm, [@B32]); significant values (at the 0.05 level) are marked with an asterisk*. These antecedent variants of the present neuronal network model were designed without a linked biomechanical system. The new joint neuromechanical model aids tuning of phase-timing relationships and the scaling of model motor outputs. To illustrate this model feature, we linked the earlier network models to the new biomechanical model. We note that the phrenic and lumbar motor neuron activities from the Rybak and Poliaček models are not strictly comparable because the current model has a second population of each type of motor neuron to model recruitment with increased drive. Lung stretch receptor inputs to the previous network models remained filtered versions of the phrenic motor output (i.e., there was no feedback from the mechanical models to the network). Figure [8](#F8){ref-type="fig"} shows records of lung volume, alveolar pressure, tracheal flow, and laryngeal muscle activation from the current neuromechanical model (Figure [8](#F8){ref-type="fig"}A) and for the two earlier models when connected to the biomechanical system (Figures [8](#F8){ref-type="fig"}B,C). The scaling and activation of the laryngeal muscles caused airway closure prior to each eupneic expiration when using the older models' outputs (lma = −1, flow flattens at 0). During cough in the previous models, the next inspiration started before the previous expiration was complete, resulting in a progressive increase in lung volume from cough to cough. ![**Outputs of the mechanical model when linked to the current network model (A) and the networks in (B) (Rybak et al., [@B67]) and (C) (Poliaček et al., [@B58])**. The earlier networks were designed without a mechanical model, but were connected to the current mechanical model to generate these plots. The plots are lung volume, alveolar pressure, tracheal flow, and laryngeal muscle activation (lma). The value of lma is 1 for a maximally open glottis, 0 for the resting diameter, and −1 for a closed glottis. The first few cycles are eupneic cycles, and the rest are coughs. The time scale is different for the current model because it was designed with slower cycles to match human respiration.](fphys-03-00264-g008){#F8} ### Influence of some added network connections {#s5} As noted in Sections ["Linking the Neural Network and Biomechanical Models"](#s3){ref-type="sec"} and ["Additional Enhancements to the Current Network Model,"](#s4){ref-type="sec"} the current network model includes lung afferents responsive to lung deflation and presynaptic inhibition by E-Dec-Tonic neurons of excitatory inputs from the I-Aug population to I-Dec_2 neurons. The latter feature was added to prolong I-Dec_2 neuron activity when E-Dec-Tonic neuron I-phase activity is reduced. Removal of these three speculative model elements resulted in shorter inspiratory phase durations (Table [5](#T5){ref-type="table"}, "No speculative" and "Current" columns). Discussion ========== The new biomechanical model system detailed in the Results incorporates several features developed using measures from human subjects. These include a model of the abdominal volume that captures the interaction of the diaphragm, rib cage, and abdominal wall based on Grassino et al. ([@B26]), an abdominal wall model based on measurements of the curvature of the abdomen by Song et al. ([@B79]) taken during insufflation for laparoscopic surgery, and a model of the larynx using results from Tully et al. ([@B81], [@B82]). The mechanical model was linked to an enhanced version of a previously described computational network model (Rybak et al., [@B67]; Poliaček et al., [@B58]) with IF neuron populations, connections, and other properties measured or inferred from *in vivo* and *in vitro* studies of mammalian brainstem circuits for breathing and cough (Shannon et al., [@B71]; Segers et al., [@B68]; Lindsey et al., [@B44]). The joint neuromechanical system is a prototype for study of the neural control of breathing and airway defensive behaviors. To our knowledge, computational neural network models of cough generation have been evaluated previously using measures of motor neuron burst sequences, phase durations, and the time varying firing rates of neuron populations that only indirectly reflect possible muscle activation patterns and their attendant biomechanical consequences. The new model generated eupneic breathing and cough motor patterns together with corresponding alterations in lung volume, tracheal air flow, and other relevant metrics of cough mechanics. The present results also show the utility of the model as an aid for tuning the motor pattern and as a tool to evaluate the efficacy of phase-timing relationships. A specific goal of this project was to assess model output during cough under conditions of altered inspiratory drive. We were motivated in part by the recent observation that lung operating volume at the onset of the compressive phase of cough influences subsequent air flow velocities during the expulsive phase (Smith et al., [@B78]). Inspiratory drive was altered by two distinct approaches: (i) increased gain of phrenic motor neuron activation of the diaphragm, and (ii) sequential modulation or deletion of synaptic inputs to inspiratory premotor populations. Both perturbations altered cough inspiratory volume. We also found changes in expulsive phase air flow associated with corresponding changes in peak abdominal pressure attributable to cough mechanics, results that could not have been achieved by measures of the motor pattern output alone. In the first protocol, higher end inspiratory volumes resulted in greater tracheal air flow during the subsequent expulsive phase under the same abdominal expiratory motor drives. Under the second protocol, the difference in operating volumes between the enhanced drive and reduced excitatory drive states was associated with corresponding reductions in expiratory flow and peak abdominal pressure. Discrepancies with experimental results and model limitations ------------------------------------------------------------- The discrepancy between the present results and those of Smith et al. ([@B78]) is noteworthy. The latter study found changes in expulsive flow rates during voluntary coughs from different operating volumes in the absence of significant alterations in thoracic or abdominal pressures, whereas we found changes in flow associated with changes in abdominal pressure, despite no change in abdominal drive. The change in expiratory pressure in the model is due to the action of the intercostal and accessory muscles; the expiratory pressure increases because the pressure from those muscles in the model increases with rib cage volume at constant activation. Our model calculates the expiratory pressure generated by the intercostal and accessory muscles at TLC and full abdominal activation necessary to produce maximal expiratory pressure, and at RV to complete the pressure balance on the rib cage (a much smaller number; see ["Additional Enhancements to the Current Network Model"](#s4){ref-type="sec"}). The model assumes that the expiratory pressure generated by the intercostal and accessory muscles scales linearly with rib cage volume between RV and TLC, and linearly with abdominal activation, leading to higher pressures at higher rib cage volumes with equal activation. The experimentally observed increase in maximal expiratory pressure with rib cage volume could be due to increased activation of the intercostals or improved mechanical advantage at larger volumes or a combination of the two. If improved mechanical advantage is a factor, the brainstem would have to reduce drive at higher volumes during cough to avoid higher pressures, suggesting that it may be sensing the generated pressures and adjusting drive accordingly (see e.g., discussion in ["Tonic Expiratory Neurons: Model Results and Predictions"](#s6){ref-type="sec"}). A refined configuration to accommodate separate intercostal muscles, intercostal motor neuron populations, and muscle afferents (Shannon, [@B70]) and their control of the chest wall would be useful in this regard. When we ran the biomechanical model in isolation with reduced abdominal drive at higher volumes in order to maintain an equal peak pressure (see Figure [7](#F7){ref-type="fig"}), we saw peak flow rate changes comparable in magnitude to those seen by Smith et al. ([@B78], Figure [3](#F3){ref-type="fig"}B), due to the increasing recoil pressure of the lung at higher volumes. The peak flow rates were comparable despite the fact that the peak abdominal pressure in our simulation was less than half that observed by Smith et al. This lower resistance is likely due to the fact that we did not model airway collapse, which is known to limit peak flow rates (Knudson et al., [@B38]). We found that increased flow during cough at higher lung volume is primarily due to increased lung recoil pressure. The lung recoil pressure certainly increases with lung volume, but the accuracy of the resulting flow in the model may be affected by certain known limitations of the model: (i) airway collapse during cough is not modeled, resulting in an underestimate of airway resistance; (ii) the lung compliance is assumed to be constant in the model, whereas it is thought to vary with lung volume *in vivo*; (iii) the model does not take into account hysteresis in the lung flow-volume curve; (iv) volume changes due to blood shift out of the trunk during cough are not modeled; and (v) volume changes due to gas compression are not modeled (see Smith et al., [@B78] for data on volume changes due to blood shift and gas compression). Nevertheless, our model suggests the hypothesis that the increased flow during cough is primarily due to increased lung recoil pressure. Tonic expiratory neurons: Model results and predictions {#s6} ------------------------------------------------------- The model incorporated multiple target sites for cough drive modulation, a feature of the network architecture based on correlational linkage maps of functional connectivity and associated neuronal responses to stimuli that either enhance or suppress inspiratory drive *in vivo* (Lindsey et al., [@B43]; Shannon et al., [@B71]; Poliaček et al., [@B58]; Ott et al., [@B55]). Deletion of excitatory mechanisms for cough inspiratory drive resulted in reductions in peak lung volume and a subsequently diminished peak air flow relative to baseline during the expulsive phase (Figure [6](#F6){ref-type="fig"}B). Although removal of the disinhibitory component of the drive enhancement mechanism mediated by the E-Dec-Tonic population did not further reduce expulsive phase air flow velocity, it did lead to both an additional decrease in inspiratory phase lung operating volume and a reduced expulsive phase peak abdominal pressure relative to baseline values, even though peak abdominal drive did not change. We have previously proposed the hypothesis that tonic expiratory neurons provide a reservoir for inspiratory drive modulation. Suppression of their inspiratory phase activity during central chemoreceptor-mediated drive and spike train cross-correlation analyses both suggest that VRC tonic E neuron inhibition of premotor inspiratory neurons is reduced in high drive states, at least in part, by increased I-Dec neuron inhibition (Ott et al., [@B55]). The present model included a network "module" previously introduced (Poliaček et al., [@B58]) for baroreceptor modulation of breathing. That circuit, inferred from spike train correlational linkages and neuron responses to baroreceptor stimulation (Lindsey et al., [@B43]), operated via excitatory and disinhibitory raphé neuron influences acting upon VRC E-Dec-Phasic and E-Dec-Tonic neuron populations. Simulations of baroreceptor activation using prior models (Poliaček et al., [@B58]; Lindsey et al., [@B44]) with circuits inferred from *in vivo* observations (see references in Lindsey et al., [@B43]; Poliaček et al., [@B58]; Ott et al., [@B55]) generated prolongation of expiration and reduced inspiratory drive during both eupneic respiratory cycles and evoked cough. Collectively, these data support the hypothesis that inhibition of the E-Dec-Tonic population in the cough network amplifies inspiratory drive via disinhibition. Experimental data consistent with this hypothesis is presented in a companion paper (Segers et al., [@B69]). Modulation of tonic expiratory neuron activity could also operate in a push-pull mechanism in which cough drive is balanced against the potentially suppressive influences of blood pressure changes caused by cough mechanics. Future directions ----------------- The present model provides a framework for integrating respiratory network model development with respiratory mechanics and will guide and facilitate scaling and timing of motor neuron activity patterns and functionally antecedent connectivity for the generation of breathing, cough, and swallow. The simulations of cough and breathing suggest that an important area of focus for future modeling efforts will be reconciliation of known differential effects of pulmonary volume-related feedback on breathing and airway defensive behaviors such as coughing and the expiratory reflex. Specific components of the model that are proposed to have the greatest effect on its potential for prediction are the gain of pulmonary volume-related feedback and the interaction of this feedback with cough-related sensory information. Future models should also guide experiments targeting the control of behavior that must be tightly coordinated with breathing, such as sniffing, swallowing, and vocalization. Conflict of Interest Statement ============================== The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by NIH grants HL89104, HL89071, and HL103415 from NHLBI, and grant NS19814 from NINDS. [^1]: Edited by: Raimond L. Winslow, The Johns Hopkins University, USA [^2]: Reviewed by: Raimond L. Winslow, The Johns Hopkins University, USA; Silvina Ponce Dawson, Universidad de Buenos Aires, Argentina [^3]: This article was submitted to Frontiers in Computational Physiology and Medicine, a specialty of Frontiers in Physiology.

1. Introduction {#sec1-vaccines-07-00065} =============== Pertussis is a globally endemic and highly infectious disease that can cause respiratory, nutritional and neurological complications, and death \[[@B1-vaccines-07-00065]\]. Infants and young children have the highest risk of pertussis sequelae and death during the window of vulnerability (between birth and the first infant pertussis vaccination) \[[@B2-vaccines-07-00065]\]. Since the introduction of whole-cell pertussis vaccines (wP) in the 1940s, global pertussis-related morbidity and mortality have decreased substantially, particularly in high-income countries \[[@B3-vaccines-07-00065]\]. However, despite vaccination, an estimated 16 million pertussis cases and 195,000 child deaths occur globally every year, with the greatest burden in low and middle-income countries \[[@B4-vaccines-07-00065]\]. Furthermore, many high-income countries with high and stable vaccination rates, such as New Zealand, Australia and the United States, have increasing pertussis burden and regular epidemics are not being prevented \[[@B1-vaccines-07-00065]\]. Acellular pertussis vaccines (aP) are now used instead of wP vaccines in most high-income countries. In comparison to wP vaccines, aP vaccines are less reactogenic, yet have a lower effectiveness \[[@B5-vaccines-07-00065],[@B6-vaccines-07-00065],[@B7-vaccines-07-00065],[@B8-vaccines-07-00065]\]. Estimates of aP vaccine effectiveness (VE) vary substantially with age, number of doses, outcome (hospitalisation/non-hospitalised notification/all cases), local epidemiology and health care service policies. For example, a New Zealand nested case-control study estimated a 93% VE against pertussis hospitalisation in the first year of life for a three-dose primary pertussis vaccination series (DTaP, Infanrix^®^-Hexa, GlaxoSmithKline, Rixensart, Belgium) \[[@B9-vaccines-07-00065]\]. However, an Australian case-control study estimated 85% VE against pertussis hospitalisation in the first year of life for a three-dose primary pertussis vaccination series (DTaP combination vaccines, GlaxoSmithKline, Rixensart, Belgium) \[[@B10-vaccines-07-00065]\]. Both studies used national-level registry data but the lower VE in the Australian study may in part be due to the high polymerase chain reaction (PCR) testing for *Bordetella pertussis* in Australian health services compared with New Zealand \[[@B9-vaccines-07-00065]\]. As aggressive PCR testing for pertussis is not commonplace, it is likely that aP VE is lower than previously reported by observational studies. It follows that pertussis vaccination failure may be more common, particularly because aP are known to attenuate clinical presentation making vaccinated individuals more difficult to diagnose with pertussis \[[@B11-vaccines-07-00065],[@B12-vaccines-07-00065],[@B13-vaccines-07-00065]\]. AP vaccines likely have a multimodal model of failure (leaky, primary and secondary) and are known to protect best against morbidity and mortality early in life \[[@B14-vaccines-07-00065]\]. As such, in the 0--4 age group, the VE of a three-dose aP primary series is highest against pertussis hospitalisation in infants less than one-year-old (93%) and lowest against non-hospitalised notifications in three-year-olds (84%) \[[@B9-vaccines-07-00065]\]. Pertussis vaccination failure is the occurrence of pertussis, caused by *B. pertussis* in an individual vaccinated in accordance with the national immunisation schedule \[[@B15-vaccines-07-00065]\]. When applied to maternal pertussis vaccination in pregnancy, pertussis vaccination failure is understood as pertussis occurring in her infant within the first six weeks of life (before the infant's first pertussis vaccination). Vaccination failure is a complex concept and has four broad categories of causes: Vaccinee-related (e.g., immunocompromise), schedule-related (e.g., poor timing and number of doses recommended), vaccine-related (e.g., poor coverage of pathogen strains or serotypes), and usage-related (e.g., cold chain storage failure, suboptimal administration) \[[@B15-vaccines-07-00065]\]. Pertussis vaccination failure requires two conditions: Suboptimal vaccine-induced protection and exposure to *B. pertussis*. One risk factor may increase the risk of one or both conditions. Studies of pneumococcal and varicella vaccination failure have begun by investigating known risk factors for pneumococcal and varicella; we share this approach \[[@B16-vaccines-07-00065],[@B17-vaccines-07-00065]\]. Risk factors for pertussis disease have been outlined previously, although risk factors for pertussis vaccination failure is an area of little prior research \[[@B18-vaccines-07-00065],[@B19-vaccines-07-00065],[@B20-vaccines-07-00065],[@B21-vaccines-07-00065],[@B22-vaccines-07-00065],[@B23-vaccines-07-00065],[@B24-vaccines-07-00065],[@B25-vaccines-07-00065],[@B26-vaccines-07-00065]\]. A number of factors have been associated with susceptibility to *B. pertussis* infection and disease; a brief and nonexclusive list includes low birth weight, prematurity, asthma, immunocompromised conditions and socioeconomic deprivation \[[@B18-vaccines-07-00065],[@B19-vaccines-07-00065],[@B20-vaccines-07-00065],[@B21-vaccines-07-00065],[@B22-vaccines-07-00065],[@B23-vaccines-07-00065],[@B24-vaccines-07-00065],[@B25-vaccines-07-00065],[@B26-vaccines-07-00065]\]. The literature on pertussis vaccination failure is limited and is almost exclusively published from the Niakhar Studies and Senegal Pertussis Trials \[[@B27-vaccines-07-00065],[@B28-vaccines-07-00065],[@B29-vaccines-07-00065],[@B30-vaccines-07-00065]\]. The context of these findings (lower middle income country) likely do not fully apply to high-income countries \[[@B29-vaccines-07-00065],[@B30-vaccines-07-00065]\]. The risk factors reported for pertussis vaccination failure from the Senegal Pertussis Trial included high birth rank, underweight and stunting \[[@B29-vaccines-07-00065],[@B30-vaccines-07-00065]\]. Vaccine-related reasons for failure are dealt with by extensive ongoing biomedical and pharmaceutical research efforts \[[@B31-vaccines-07-00065],[@B32-vaccines-07-00065],[@B33-vaccines-07-00065],[@B34-vaccines-07-00065],[@B35-vaccines-07-00065]\]. Therefore, a strategic use of resources is to focus on improving the effectiveness of current aP vaccines through changes to the national immunisation schedule \[[@B36-vaccines-07-00065]\]. Limited consideration has been given to biological and social variations in vaccinee circumstances and their interaction with national immunisation schedules that may contribute to vaccination failure. This study pursues an understanding of pertussis vaccination failure in relation to New Zealand′s current national immunisation schedule. To our knowledge, no studies have investigated pertussis vaccination failure in a high-income country. The main aim of this study is to describe pertussis vaccination failure cases and investigate candidate risk factors for pertussis vaccination failure in the New Zealand context. The identification of risk factors and subsequently higher risk groups is an important step in manipulating the national immunisation schedule for better protection of vulnerable groups; one that aligns with public health goals such as maximising overall health and reducing disparities between groups. 2. Materials and Methods {#sec2-vaccines-07-00065} ======================== The Health and Disability Ethics Committees (a national-level committee) waived the need for ethics approval (17 January 2017). However, this study received approval from the University of Auckland Human Participants Ethics Committee (UAHPEC) (reference number 018664) on 31 March 2017. Minor amendments to the original UAHPEC application were approved by UAHPEC on 19 December 2017 (reference number 018664). The research questions for this study are: What evidence is there for risk factors of pertussis vaccination failure? (a)Between birth and first pertussis immunisation in infants born to mothers who received maternal pertussis vaccination during their pregnancy;(b)in fully vaccinated infants and young children before their four-year pertussis booster; In order to answer these questions, this study has two components and four sub-studies (see [Figure 1](#vaccines-07-00065-f001){ref-type="fig"}). Study objectives:(1)Describe infants born to mothers Tdap vaccinated during pregnancy with pertussis disease before their first pertussis vaccination, from birth to first pertussis vaccination using Ministry of Health national administrative data;(2)Identify risk factors for pertussis disease in infants of women Tdap vaccinated during pregnancy by determining associations between risk factors identified in Objective 1 and pertussis disease in infants born to women Tdap vaccinated during pregnancy between birth and first pertussis vaccination;(3)Describe fully vaccinated infants and young children with pertussis vaccination failure from birth to four year booster vaccination or four years old using Ministry of Health national administrative data and national notification data;(4)Identify risk factors of pertussis vaccination failure in fully vaccinated infants and young children by determining associations between risk factors identified in Objective 3 and pertussis vaccination failure. 2.1. Study Design {#sec2dot1-vaccines-07-00065} ----------------- The study setting is New Zealand. For each study population, one case series study will be conducted to describe the pertussis vaccination failure cases and one retrospective cohort study will be conducted to identify risk factors for pertussis vaccination failure ([Figure 1](#vaccines-07-00065-f001){ref-type="fig"}). The study population characteristics for each objective are described below. ### 2.1.1. Objective One Inclusion Criteria {#sec2dot1dot1-vaccines-07-00065} All single live birth infants with a gestation ≥28 weeks whose mothers were administered funded pertussis vaccine (Tdap) during pregnancy in New Zealand and with a pertussis notification identified in the national notifiable diseases database or pertussis hospitalisation identified in the national minimum dataset in the period between birth and first infant pertussis vaccination between 1 January 2013 and 31 December 2016 inclusive. ### 2.1.2. Objective Two Inclusion Criteria {#sec2dot1dot2-vaccines-07-00065} All single live birth infants with a gestation ≥28 weeks whose mothers were administered funded pertussis vaccine (Tdap) during their pregnancy in New Zealand between 1 January 2013 and 31 December 2016 inclusive. ### 2.1.3. Objective Three Inclusion Criteria {#sec2dot1dot3-vaccines-07-00065} All infants/young children registered in the national immunisation register who meet the study definition of "fully vaccinated" and have a pertussis notification identified in the national notifiable diseases database or pertussis hospitalisation identified in the national minimum dataset occurring between greater than 35 days after the third dose of the primary course and before the four-year pertussis booster dose or four years of age, whichever comes first. These events must have occurred between 1 January 2006 and 31 December 2016 inclusive. ### 2.1.4. Objective Four Inclusion Criteria {#sec2dot1dot4-vaccines-07-00065} All infants/young children registered in the national immunisation register who meet the study definition of "fully vaccinated" between 1 January 2006 and 31 December 2016. The birth cohort is defined as those born between 1 January 2006 and 31 December 2016, inclusive, using the date of birth from the national health index dataset. The study population will include 11 sub-cohorts, eight of which include children who reached the maximum of four years, nine sub-cohorts reached 2--3 years, 10 reached 1--2 years and 11 reached 0--1 year. Six national administrative datasets were used for all the objectives in this study (see [Table 1](#vaccines-07-00065-t001){ref-type="table"}). ### 2.1.5. Study Definitions {#sec2dot1dot5-vaccines-07-00065} **Maternal pertussis vaccination:** The administration of a Tdap booster (Boostrix^®^, GlaxoSmithKline, Rixensart, Belgium) to a pregnant woman between 28 and 38 weeks of gestation. The 28--38-week timeframe relates to the current funding guidelines for the maternal pertussis booster in pregnancy vaccination \[[@B1-vaccines-07-00065]\]. The New Zealand schedule recommends a maternal pertussis immunisation for every pregnancy \[[@B1-vaccines-07-00065]\]. The pregnancy during which the mother was vaccinated is the pregnancy during which the infant in the dataset was carried. **Fully vaccinated**: The on-time interval-adjusted administration of the primary pertussis course with either DTaP-IPV (Infanrix^®^-IPV, GlaxoSmithKline, Rixensart, Belgium), funded in 2006--2008 for the primary course or DTaP-IPV-HepB/Hib (Infanrix^®^-Hexa, GlaxoSmithKline, Rixensart, Belgium), funded for the primary course since 2008 \[[@B36-vaccines-07-00065]\]. This definition involves the integration of principles of immunology \[[@B43-vaccines-07-00065]\], guidelines from the Advisory Committee on Immunization Practices \[[@B44-vaccines-07-00065]\], and the New Zealand definition of timeliness for the primary pertussis series \[[@B45-vaccines-07-00065]\] (see [Figure 2](#vaccines-07-00065-f002){ref-type="fig"}). The following conditions must be met to be considered fully vaccinated:(a)First pertussis-containing vaccination administered no earlier than 4 days (inclusive) before 6 weeks of age, and no later than 10 weeks of age;(b)Second pertussis-containing vaccination no later than six weeks after the scheduled age of 3 months;(c)Third pertussis-containing vaccination no later than six weeks after the scheduled age of 6 months;(d)Each pertussis-containing vaccination administered at least 3 weeks apart. **Maternal pertussis vaccination failure**: One or more pertussis hospitalisation(s) and/or notification(s) occurring between birth and first pertussis vaccination in infants whose mothers met the study criteria for maternal pertussis vaccination. As there is a difference in the level of certainty in the diagnosis, clinically confirmed and clinically suspected cases will be reported separately. (a)Clinically confirmed maternal pertussis vaccination failure in the infant:The occurrence of pertussis hospitalisation (ICD-AM 10 code A37.0 only) and or notification ("confirmed" status only) in the infant between birth and first pertussis vaccination.(b)Clinically suspected maternal vaccination pertussis vaccination failure in the infant:The occurrence of pertussis hospitalisation (ICD-AM 10 code A37.9 only) and or notification ("probable" and "suspected" status only) in the infant between birth and first pertussis vaccination. **Primary vaccination series pertussis vaccination failure**: One or more pertussis hospitalisation(s) and/or notification(s) occurring within the follow-up period in fully vaccinated infants/young children. As there is a difference in the level of certainty in the diagnosis, clinically confirmed and clinically suspected cases will be reported separately. (a)Clinically confirmed primary series pertussis vaccination failure:The occurrence of pertussis hospitalisation (ICD-AM 10 code A37.0 only) and or notification ("confirmed" status only) more than 35 days after the last vaccination in the primary series but before four years of age or the receipt of the four-year pertussis booster vaccination. This timeframe aimed to capture only the individuals considered fully vaccinated at the time of exposure to *B. pertussis* who subsequently developed disease. Thirty-five days allowed 14 days for immune response to the pertussis vaccine and 21 days for the incubation period of pertussis.(b)Clinically suspected primary series pertussis vaccination failure:The occurrence of pertussis hospitalisation (ICD-AM 10 codes A37.8 and A37.9 only) and or notification ("probable" and "suspected" status only) more than 35 days after the last vaccination in the primary series but before four years of age or the receipt of the four-year pertussis booster vaccination. **Pertussis notification**: any case of pertussis reported to EpiSurv by clinicians or through electronic laboratory alerts with a case classification of 'suspect', 'probable' and 'confirmed' ([Table 2](#vaccines-07-00065-t002){ref-type="table"}). Notifications were limited to non-hospitalised notifications to prevent duplication of the same event and to indicate mild (non-hospitalised notified) versus severe (hospitalised) cases. Notification events were determined using the EpiSurv database, and events must have occurred between 1 January 2006 and 31 December 2016. **Pertussis hospitalisation**: a principle or other diagnosis on hospital discharge with any of the pertussis related ICD-10-AM codes in [Table 3](#vaccines-07-00065-t003){ref-type="table"}. Hospitalisation events were determined using the national minimum dataset, and events must have occurred between 1 January 2006 and 31 December 2016 inclusive. Both primary and secondary diagnoses for hospitalised cases were included; these will not reported separately. 2.2. Multiple Notification and Hospitalisation Events {#sec2dot2-vaccines-07-00065} ----------------------------------------------------- Multiple pertussis hospitalisations and notifications will be treated as separate events if at least 60 days has lapsed since the preceding pertussis hospitalisation/notification. Sixty days was the estimated length of a pertussis disease event; this allowed for 14 days of incubation (range between 5 and 21 days), 11 days for the catarrhal stage (range between 1 and 2 weeks), 21 days for the paroxysmal stage (range between 1 and 6 weeks) and 14 days for the convalescent stage (range between 1 and 4 weeks) \[[@B47-vaccines-07-00065]\]. 2.3. Statistical Analysis Plan {#sec2dot3-vaccines-07-00065} ------------------------------ All data analyses will use SAS statistical software (SAS Institute Inc., Cary, NC, USA). An alpha of 0.05 for statistical significance will be used in all analyses. The frequencies and percentages will be reported for categorical data. Pearson′s chi-square test or Fishers exact test will be used to analyse associations between categorical data. The data will be tested for normally distributed data, *t*-tests will be used to test the differences between the means for normally distributed data, Mann--Whitney or Kruskal--Wallis tests will be used for non-normally distributed data. The continuous data will be reported as means and standard deviations, unless there are more appropriate statistics to report (for example, median). A pattern analysis will be used to investigate possible risk factor patterns in the cohort study. The variables included in this analysis will be those deemed relevant as per existing literature and case series study findings. The thresholds will be based on the distribution of the data or other relevant published information (epidemiological, clinical and statistical). As part of Objective 1, the number and the percentage of infants born to women who did not receive pertussis immunisation during their pregnancy will be reported. Appropriate statistical modelling methods will be used to test the predictive power of the potential risk factors for pertussis. A survival analysis where the first event was pertussis diagnosis will be used. The covariates will include age, sex, ethnicity, deprivation and geographical area. The additional covariates for Objective 2 will include variables that influence the efficacy of maternal/infant antibody transfer e.g., time between maternal vaccination and delivery and number of previous maternal pertussis pregnancy vaccinations. ### Power Statement for Objective 4 As the sample size is fixed, the power available to detect a statistically significant difference (α = 0.05) between the exposure groups was calculated using OpenEpi \[[@B48-vaccines-07-00065]\]. As the exposure variable selection had not occurred yet, socioeconomic deprivation was chosen as an example for power calculation because there is a known association between socioeconomic deprivation (measured by the New Zealand Deprivation Index 2013 (NZDep13)) and infectious respiratory diseases in New Zealand \[[@B49-vaccines-07-00065],[@B50-vaccines-07-00065]\]. Hospitalised and notified cases are unlikely to be homogenous and as the hospitalised cases are likely the smallest group (approximate *n* = 85), the hospitalised cases are the basis for the power calculation. The sample size between the 2006 and 2016 study period is estimated to be 504,984 infants and young children. There was an approximate 40% exposure to socioeconomic deprivation (NZDep13 deciles 7--10) and a 0.02% rate of pertussis vaccination failure events during the follow up period in the deprived group and 0.01% in the not-deprived group. It was estimated that this study had 84% power to detect a difference for Objective 4 at a significance level of 0.05% ([Table 4](#vaccines-07-00065-t004){ref-type="table"}). 3. Discussion {#sec3-vaccines-07-00065} ============= Pertussis vaccination failure research is pauce with critical knowledge gaps. One such gap is whether there is evidence for high-risk groups for pertussis vaccination failure (beyond the immunocompromised). High-risk groups are significant for the pursuit of equity in public health and for strategic interventions for disease control and prevention. This study aims to improve the use of the currently available aP vaccine by investigating 'who' may be at greater risk for pertussis vaccination failure under the current primary immunisation schedule in New Zealand. A better understanding of the relationship between risk factors and pertussis occurring between birth and first pertussis immunisation in infants born to mothers pertussis immunised during pregnancy may also help to develop pertussis prevention strategies for this group. Finally, estimating how common pertussis vaccination failure is in infants and young children and how common pertussis is between birth and first pertussis vaccination are important epidemiological information required for better pertussis control. The use of large linked administrative datasets offers significant advantages, such as enabling the study of rare events like pertussis vaccination failure and detailed, verifiable and longitudinal information. The use of national administrative data also limits the choice of variables to that which is available and of a reasonable quality. Some study limitations will therefore be related to the validity of the variables chosen for study purposes and the quality and reliability of these variables over time. The case series studies are exploratory in nature, however, it is a valuable and necessary starting point for further research. A comprehensive description of demographic, clinical and pharmaceutical variables in relation to pertussis vaccination failure has not been published before. The complimentary use of exploratory and hypothesis testing study designs is a significant strength of this study, as this design minimises potential issues with the case series studies and maximises the potential of the cohort studies by making available high-quality information based on more than just investigative hunches. This study will begin to address a major gap in paediatric pertussis prevention literature. We are grateful to Nikki Turner for her support and feedback in the designing of this study. We also would like to thank Lottery Health Research New Zealand for the PhD in health scholarship for H.C. Conceptualization, H.C., A.H., E.B. and H.P.-H.; methodology, H.C., A.H., E.B. and H.P.-H.; software, H.C., A.H.; data curation, A.H.; writing---original draft preparation, H.C.; writing---review and editing, H.C., A.H., E.B. and H.P.-H.; supervision, A.H., E.B. and H.P.-H.; project administration, H.C. This research received no external funding. The authors declare no conflict of interest. {#vaccines-07-00065-f001} {#vaccines-07-00065-f002} vaccines-07-00065-t001_Table 1 ###### Data sources. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Data Source Information Relevant Fields ------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------- National Health Index database The national health index (NHI) database holds static demographic information such as the NHI number, sex, date of birth, socioeconomic deprivation and ethnicity \[[@B37-vaccines-07-00065]\]. Some demographic information such as address, level of socioeconomic deprivation and ethnicity are changeable and therefore amenable to regular updates in real-time (at every presentation to health care services). The NHI number is a unique randomly assigned alphanumeric identifier that enables accurate identification of individuals for medical care and administrative records in New Zealand. The NHI number links all patient information from national and regional health services \[[@B37-vaccines-07-00065]\]. Encrypted NHI number;\ Date of birth;\ Sex;\ New Zealand Deprivation Index 2013 (NZDep13) decile;\ Ethnicity National Immunisation Register An electronic collection of all registered immunisation enrolments and events of children in New Zealand and from 2005, these are updated weekly \[[@B38-vaccines-07-00065]\]. This collection does not necessarily contain all infants and young children who were vaccinated, parents or legal guardians can withdraw their child′s information from the national immunisation register collection. Encrypted NHI number;\ Vaccine type;\ Antigen type;\ Vaccination date;\ Vaccination status; National Minimum Dataset A health statistic collection dataset containing clinical and other information about public and private hospital discharges for inpatients and day patients \[[@B39-vaccines-07-00065]\]. Encrypted NHI;\ Admission and discharge date;\ Length of stay;\ ICD-10 diagnosis code(s) Maternity collections A collection of the demographic and clinical features of women in New Zealand using publicly funded maternity/new-born services from 9 months before birth to 3 months after \[[@B40-vaccines-07-00065]\]. Encrypted NHI;\ Birth weight;\ Gestational age;\ Other inpatient and day-patient event data across pregnancy, birth and postnatal periods pertaining to both fetus/infant and mother Pharmaceutical collection A system used for management of subsidised pharmaceuticals in New Zealand \[[@B41-vaccines-07-00065]\]. Encrypted NHI;\ Anatomical Class;\ Chemical name;\ Duration of supplied pharmaceuticals National Notifiable Diseases Database (EpiSurv) EpiSurv is a web assessable application operated by the Institute of Environmental Science and Research built using the Surveillance information New Zealand framework \[[@B42-vaccines-07-00065]\]. EpiSurv holds the national notifiable disease database and allows the real time recording of notifiable disease cases across New Zealand from public health services in New Zealand. EpiSurv is under contract from the Ministry of Health to collect demographic, clinical and risk factor information of reported cases. Encrypted NHI;\ Disease;\ Notification status;\ Risk factors for disease;\ Report date;\ Onset date; -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- vaccines-07-00065-t002_Table 2 ###### Environmental Sciences and Research Limited Notification Classification System and criteria \[[@B46-vaccines-07-00065]\]. Classification Definition ---------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Suspect Idiopathic presentation of any paroxysmal cough with whoop, vomit or apnoea Probable Presentation clinically compatible with pertussis with a high *B. pertussis* IgA test or a significant (fourfold increase in titres) in antibody levels between paired sera at the same laboratory Confirmed Clinically compatible presentation with either laboratory confirmed pertussis infection (*B. pertussis* only) or epidemiologically linked to a confirmed case vaccines-07-00065-t003_Table 3 ###### ICD10 AM diagnostic codes for pertussis. ICD10-AM Code Description --------------- -------------------------------------------- A37.0 Whooping cough due to *B. pertussis* A37.8 Whooping cough due to *Bordetella* species A37.9 Whooping cough, unspecified vaccines-07-00065-t004_Table 4 ###### Power calculation. Exposed (Deprived, NZDep13 Deciles 7--10) Not-Exposed (Not Deprived, NZDep13 Deciles 1--6) ----------------- ------------------------------------------- -------------------------------------------------- Sample size 219,814 285,800 Risk of disease 53 cases ÷ 219,814 × 100 = 0.02% 32 cases ÷ 285,800 × 100 = 0.01% Power based on normal approximation without continuity correction = 84% \[[@B48-vaccines-07-00065]\].

Introduction {#Sec1} ============ Regenerative myogenesis has emerged as arguably one of the most powerful paradigms to investigate a variety of processes involving stem cells and tissuegenesis. Adult skeletal muscle satellite (stem) cells emerge from a proliferative population of myogenic cells that reversibly exit the cell cycle asynchronously during perinatal growth.^[@CR1],[@CR2]^ They are located between muscle fibres and the basement membrane ensheathing it and since their initial identification in the frog,^[@CR3]^ genetic and cell lineage strategies led to detailed analysis of their properties. Notably, critical regulators of quiescence, commitment and self-renewal have been identified, while exposing underlying heterogeneities in myogenic cell states.^[@CR1],[@CR4]^ In vertebrates, genetic and embryological studies have shown that the bHLH myogenic regulatory factors (MRFs) *Myf5*, *Mrf4*, *Myod* and *Myogenin* have crucial roles in governing striated muscle cell fate and differentiation. Mice triple mutant for *Myf5*, *Myod* and *Mrf4* lack skeletal muscles and their progenitors pointing to these genes as critical determination factors, whereas *Myogenin* and *Mrf4* act during differentiation.^[@CR4]--[@CR7]^ In the adult, *Myf5* is expressed in quiescent and activated satellite cells,^[@CR8]^ whereas MYOD protein expression is a hallmark of an activated satellite cell.^[@CR4]--[@CR6]^ Upstream transcription factors include *Pax3*, *Tbx1*, *Six1/4* and *Pitx2* and they act in different locations in the embryo to establish the founder muscle stem cell population.^[@CR9]^ The properties of the paraxial mesoderm from which head and body muscles arise are also different. All body muscles and some located in the head arise from transient structures called somites, and these are under the regulation of the paired/homeobox transcription factors *Pax3* in the embryo, and later, *Pax7*.^[@CR10],[@CR11]^ In contrast, head muscles are derived from cranial paraxial mesoderm and are *Pax3*-independent, but regulated in the embryo by *Tbx1* and *Pitx2* among other genes.^[@CR9],[@CR12]^ From mid-embryonic stages, virtually all stem/progenitor cells throughout the body are marked by *Pax7* expression. These cell-intrinsic differences observed during embryogenesis occur in the context of a heterogeneous extrinsic milieu. Indeed, an important consideration is the role of stromal cells that constitute the stem cell niche, and that also arise from distinct embryological origins in the head and the body. Their impact on muscle stem/progenitor cell fates remains largely unexplored. Some of these issues will be discussed in this review. The interplay between satellite and stromal cells in skeletal muscle {#Sec2} ==================================================================== Although fewer studies have focused on the role of interstitial cells (Fig. [1](#Fig1){ref-type="fig"}), their critical roles in homoeostasis and regeneration has been highlighted in several reports. For example, fibroadipogenic progenitors (FAPs) promote myoblast differentiation and participate in fibrosis following muscle damage.^[@CR13],[@CR14]^ Another cell type, called PICs (Pw1 + interstitial cell) was also identified as residing outside the basement membrane of the muscle fibre. *Pw1* is an imprinted gene that is involved in stress regulation.^[@CR15],[@CR16]^ The transplantation of PICs into injured muscle results in their contribution to regenerating fibres.^[@CR16]^ Mesoangioblasts that are associated with blood vessels were also reported to contribute to skeletal muscles.^[@CR17]^ Interestingly, mesoangioblasts isolated from mouse, dog and human express high levels of *Pw1*, where this gene was shown to confer the myogenic potential of mesoangioblasts, and their ability to cross the vessel wall.^[@CR18]^ Recently, an interstitial cell type that is marked by the basic-Helix-loop-Helix transcription factor *Twist2* (*Dermo1*), was reported to be *Pax7*-negative during homoeostasis and following muscle injury.^[@CR19]^ Intriguingly, these cells contribute specifically to type IIb/x myofibres during adulthood and muscle regeneration, and their genetic ablation causes wasting of type IIb (fast glycolytic) myofibres.^[@CR19]^ How these different cell types are related to so-called "mesenchymal stem cells" remains obscure. FAPs can be isolated by cytometry using PDGFRα/Sca1^[@CR20]^ whereas PICs (PW1+/PDGFRα− fraction with myogenic capacity) were reported to be a sub-population of interstitial cells.^[@CR21]^ As the field tries to resolve these different cell types further, it is interesting to note that a detailed study of mesenchymal "stem" cells pointed to a substantial heterogeneity in this population depending on their tissue of origin,^[@CR22]^ therefore a concerted effort is clearly needed to further characterise stromal cells in different tissues and address the potentially misleading designation of stromal cells generically as "mesenchymal stem cells". Given these findings, and the potential misappropriation of cell populations, single cell mass spectrometry and single cell RNAseq of the entire muscle resident cell population was done to resolve some of these discrepancies.^[@CR23]^ Of note a total of 10 different cell types were identified, including known populations (satellite cells, FAPs, endothelial cells, etc) and previously uncharacterised resident tenocyte-like cells and smooth muscle/mesenchymal cells with myogenic potential were identified.^[@CR23]^Fig. 1The satellite cell and stromal cell niche. Satellite cells states are regulated through their interactions with their microenvironment. While direct interactions (M-cadherin, Notch pathway)^[@CR38],[@CR46]^ and communication (FGF2-FGFR1 pathway)^[@CR48]^ between muscle fibres and satellite cells have been identified, muscle stem cells also interact with a variety of components of the extracellular matrix (e.g. Collagens VI and V, Laminin, Fibronectin, SDC3/4)^[@CR45],[@CR79]^ and diffusable cytokines and growth factors (e.g. Angiopoietin-Tie2 receptor)^[@CR37]^. In addition to satellite cells, several cell types contribute to muscle growth, homoeostasis and regeneration, including pericytes, mesenchymal stromal cells (e.g. Pw1+ Interstitial Cells, FibroAdipogenic Progenitors, Twist2+ cells)^[@CR16],[@CR19],[@CR21]^, immune cells (e.g. resident or infiltrating macrophages)^[@CR156]^ as well as connective tissue cells. These interactions are remodelled during ageing, notably with increased FGF2 production from muscle fibres and decreased expression of FGFR1 in satellite cells, driving satellite cells to break quiescence^[@CR39]^, and decreased levels of fibronectin^[@CR45]^, which weakens satellite cell adhesion capacity and increases their susceptibility to apoptosis by anoikis More detailed studies on FAPs have shown that TNFα-mediated apoptosis of this population is critical for normal regeneration. During chronic injury, as with dystrophic *mdx* mice, continued expression of transforming growth factor β1 (TGF β1) results in persistence of FAPs and fibrosis. Pharmacological inhibition with the tyrosine kinase inhibitor Nilotinib, which has potent antifibrotic activity, blocks TGF β1 activity and results in reduced fibrosis.^[@CR20]^ However, Nilotinib treatment also blocks expansion of FAPs and compromises regeneration through a non-cell-autonomous anti-proliferative effect on satellite cells.^[@CR24]^ These studies and others cited below highlight the dynamic nature of regeneration, and the importance of determining when to intervene for a desired outcome. Stromal cells that have myogenic capacity have been considerably less well characterised compared to satellite cells, and their contributions to muscle or self-renewal into a satellite cell position are limited, or not demonstrated, compared to bona fide satellite cells. Significantly, elimination of satellite cells by selective diphtheria toxin ablation results in failed regeneration,^[@CR25]--[@CR27]^ indicating that in the short term, non-satellite cells do not contribute to muscle regeneration. Similar ablation studies should be extended to all interstitial cell types. It is interesting to note that satellite cells have significantly distinct genetic requirements in different anatomical locations as indicated above (e.g. *Tbx1/Pitx2* in head; *Pax3* in body). It is therefore likely that stromal cells, which can be of mesodermal or neural crest origin, might impact differentially on the fate of myogenic cells in relation to their anatomical location.^[@CR4]^ Importantly, emerging satellite cells continue to proliferate until about 2 weeks postnatally, yet they are ensheathed under a basement membrane from mid-late foetal stages.^[@CR1],[@CR28]^ Therefore, contact with extracellular matrix proteins in the basal lamina of the basement membrane is not sufficient to trigger cell cycle exit. In addition, enseathment under the basal lamina results in pre-quiescent and post-quiescent satellite cells being physically separated from stromal cells and in contact only with the myofibre, until its disruption following injury (Fig. [2](#Fig2){ref-type="fig"}). How sporadic interactions with stromal cells prior to this confinement affect the fate of myogenic cells is an open question. Given that the transcriptome profiles of *Pax7*+ myogenic cells change significantly during prenatal and postnatal development,^[@CR29]--[@CR31]^ stem cell and niche cell interactions need to be explored further in different contexts when direct satellite and niche cell contacts can occur (Fig. [2](#Fig2){ref-type="fig"}).Fig. 2Developmental, adult, ageing and diseased skeletal muscle niches. During development and regeneration, stromal and myogenics cells can be in direct contact, and myogenic cells can be exposed to stromal-derived extracellular matrix proteins, whereas in the late foetal to postnatal stages, muscles stem cells are separated from stromal cells by a basement membrane (from mid-foetal stages)^[@CR1],[@CR28]^. Stromal cells have distinct embryonic origins depending on their anatomical location---those in the head are of neural crest and mesoderm origin, whereas those in the limbs are mostly of mesodermal origin^[@CR4]^. In addition to this spatial character, niche cells evolve during development and postnatal life thereby introducing a temporal dimension to the regulation of muscle stem cells as they give rise to quiescent satellite cells and age. The postnatal niche is disrupted following chronic (e.g. myopathies) and acute (chemical, physical) injury. In the former, there is an asynchronous response of infiltrate and in the latter, more phasic appearance and disappearance of neutrophils and macrophages is noted Identification of the quiescent state {#Sec3} ===================================== Non-cycling cells have been characterised in different states: quiescent, terminally differentiated, apoptotic/necrotic, and senescent. In contrast to apoptosis, differentiation and senescence---cellular quiescence is a reversible cell state representing a non-cycling cell. In multicellular organisms, postnatal tissue homoeostasis and regeneration upon trauma are mediated by adult tissue specific stem cells that are generally quiescent. Following injury, and in response to diffusible and mechanical cues, adult satellite cells activate to generate transit amplifying myoblasts that will in turn fuse to form multinucleated myofibres. A subset of the transit amplifying precursors renews the stem cell pool by returning to the quiescent state. In vitro models can recapitulate this process to some extent where a pool of 'reserve' cells is associated with differentiated myotubes.^[@CR32]^ Efforts have also been made to artificially recapitulate cellular quiescence or the niche environment ex vivo.^[@CR33]--[@CR35]^ In the yeast *Saccharomyces cerevisiae*, quiescence is induced in response to nutrient limitation.^[@CR36]^ In multicellular organisms, cellular quiescence might also act as a protective mechanism, against accumulation of mutations due to proliferation, but also against various environmental stresses. Deregulation of quiescence can lead to precocious differentiation, senescence, or apoptosis, and it is associated with impaired tissue regeneration.^[@CR37]--[@CR51]^ In the context of therapies, a promising strategy is the transplantation of tissue stem cells that have been corrected in vitro. To date, a major challenge has been overcoming the loss of stemness properties following ex vivo expansion,^[@CR52],[@CR53]^ and diverse strategies,^[@CR54]--[@CR56]^ including isolation of transplantable myogenic cells from teratomas,^[@CR57]^ show some promising results. Furthermore, retaining quiescence properties might be a means to survive the transplantation process,^[@CR58],[@CR59]^ thereby underscoring the importance of characterising this cell state. Cellular quiescence, or G0, is defined as a transient and reversible cell cycle arrest during the G1 phase (Fig. [3](#Fig3){ref-type="fig"}). It is characterised by a decrease in cell size, increased nucleocytoplasmic ratio, 2N genome content, low RNA and protein synthesis, altered metabolism and gene expression profile.^[@CR60]^ Quiescence had been considered for some time to be a default state corresponding to the absence of proliferation and differentiation. It is now clear that cellular quiescence is an actively maintained state^[@CR61]^ with new functional markers continually being identified.Fig. 3Quiescence to proliferation transition in satellite cells. **a** During homoeostasis, adult satellite cells are maintained in a reversible non-proliferating quiescent G0 state by regulators including Calcitonin receptor, Collagen V, Notch pathway, FGF signalling and effectors of the RNAi machinery^[@CR39],[@CR40],[@CR46],[@CR51],[@CR79]^. However, specific quiescence markers are still lacking. Satellite cells within a healthy tissue respond to a distant injury by transiting from deep quiescence to a quiescent G0/G1 or 'G(alert)' state^[@CR85]^, with increased proliferative capacity and regenerative potential. This transition is under the control of mammalian target of rapamycin (mTOR) signalling, which in turns controls mitochondrial metabolism (see text). Following an acute tissue injury or chronic mild degeneration of muscle fibres, satellite cells exit from their quiescent state and proliferate. This transition is accompanied by a metabolic shift from fatty acid oxidation to glycolysis.^[@CR147]^ Some cells irreversibly exit the cell cycle to differentiate into mononuclear myocytes that eventually fuse to regenerate muscle fibres, while others self-renew and return to quiescence. Entry into quiescence is poorly characterised, and an activation marker is MYOD. **b** Isolation of adult quiescent satellite cells involves repeated mechanical and enzymatic dissociation of the tissue. These procedures invariably lead to satellite cell activation, as shown by the rapid upregulation of FOS and JUN, and phosphorylation of p38^[@CR65]^ Considerable effort has been put into identifying quiescence-specific markers, notably for satellite cells^[@CR31],[@CR41],[@CR45],[@CR62]−[@CR67]^ (Fig. [3a](#Fig3){ref-type="fig"}). Of those, *Calcr* (Calcitonin receptor) and *Odz4* are of interest as they encode membrane-associated proteins that could potentially be used to isolate quiescent muscle stem cells by fluorescence activated cell sorting (FACS).^[@CR68],[@CR69]^ Interestingly, *Odz4* is re-expressed earlier than *Calcr* in satellite cells during regeneration,^[@CR69]^ and proliferating neonatal satellite cells express *Odz4*, but not *Calcr*, indicating that some quiescence genes are differentially expressed during homoeostasis, muscle regeneration and postnatal growth. Until recently, studies focused on identifying quiescence-specific transcripts in adult muscle stem cells have required purification procedures based on mechanical and enzymatic dissociation followed by satellite cell isolation by FACS. These involve dissociating satellite cells from their microenvironment, which invariably leads to their activation (Fig. [3b](#Fig3){ref-type="fig"}). In addition, it is now clear that satellite cell activation occurs more rapidly than previously thought; for example phosphorylated p38 appears within 30 min after isolating satellite cells on myofibres.^[@CR70]^ Hence, some highly dynamic quiescence-specific or activation-specific genes, non-coding RNAs, and epigenetic states might have been missed in previous studies^[@CR31],[@CR65],[@CR67]^ (Fig. [3b](#Fig3){ref-type="fig"}). Accordingly, it has been known for decades that the Fos/Jun stress response pathway activates within minutes during the G0 to G1 transition or in response to stress^[@CR31],[@CR71]^ (Fig. [3b](#Fig3){ref-type="fig"}). Although some markers allow the identification of deeply quiescent satellite cells and of actively proliferating myoblasts, in vivo dynamic markers to follow early activation or re-entry into quiescence during regeneration are largely lacking. Quiescent muscle stem cells {#Sec4} =========================== Molecular regulation of quiescence {#Sec5} ---------------------------------- Notch signalling has a critical role in maintaining quiescence of satellite cells, and the expression of canonical Notch targets (e.g. *Hes*, *Hey*) is markedly reduced before cells enter the cell cycle, thereby allowing MYOD protein to accumulate.^[@CR20],[@CR28]^ Later during regeneration, Notch signalling increases, both in differentiating and in self-renewing myoblasts.^[@CR72]^ During homoeostasis, quiescent satellite cells display high levels of Notch pathway activity, relayed by its intracellular effector *Rbpj*. Satellite cell-specific deletion of *Rbpj* during homoeostasis results in loss of satellite cells, their differentiation, and fusion to existing myofibers.^[@CR38],[@CR46]^ Intriguingly, in this case the majority of satellite cells boycott S-phase and do not enter the cell cycle (absence of BrdU uptake). Deletion of *Rbpj* also leads to an impaired regeneration capacity following injury.^[@CR46]^ Nonetheless, *Rbpj* null satellite cells can undergo a normal activation and transit amplification after muscle injury, indicating different contextual roles for Notch signalling in maintenance of quiescence and initial cell cycle progression through G1/S in muscle homoeostasis or regeneration.^[@CR72]^ Another role for Notch was proposed, this time through regulation of E3 ubiquitin ligase *Mib1* (Mind bomb 1) which triggers endocytosis of ligands that interact with Notch receptor.^[@CR73]^ Remarkably, sex hormones were reported to act during puberty, and after regeneration, to induce *Mib1* in myofibres, thereby resulting in increased Notch activation in satellite cells, and their entry into quiescence. This model might appear counterintuitive, as it suggests that satellite cell quiescence in different muscle masses is under systemic regulation. Moreover, Notch activation inhibits expression of *Myod* and sustains the expression of *Pax7* while allowing the proper homing of satellite cells to their niche, thereby participating in the maintenance of the non-committed state of satellite cells.^[@CR46],[@CR74],[@CR75]^ As mentioned above, *Odz4* is specifically expressed in neonatal and adult quiescent satellite cells. The ODZ proteins are type II transmembrane proteins, for which protein cleavage sites have been demonstrated in the extracellular and transmembrane domains, and cleaved intracellular ODZ2 acts as a transcription factor.^[@CR76]^ Although the mode of action of *Odz4* specifically in satellite cells remains to be determined, *Odz4* germline null mice display a reduced body weight and size, decreased muscle mass and satellite cell pool size, both during homeostasis and after injury-mediated regeneration. Satellite cells isolated from these animals show prolonged *in vitro* proliferation and enhanced differentiation.^[@CR77]^ In addition, a satellite cell-specific deletion of *Calcr* sensitises satellite cells to apoptosis, which eventually decreases the size of the stem cell pool and impairs regeneration efficiency upon injury.^[@CR51]^ *Calcr* encodes a G-protein coupled receptor (GPCR), known to regulate calcemia, and CALCR and its ligand calcitonin inhibit bone resorption in osteoclasts.^[@CR78]^ In muscle, *Calcr*^−/−^ satellite cells express higher levels of the cell cycle marker KI67 and other cell-cycle-related genes (*Ccna2*, *Ccnd1*), without any increase in expression of myogenic genes such as *Myod*, and without impacting on myogenic differentiation.^[@CR51]^ Interestingly, *Calcr* was reported to regulate satellite cell quiescence through cAMP-PKA signalling and thereby position satellite cells within their niche through cAMP-Epac signalling.^[@CR51]^ Intriguingly, the ligand for CALCR, calcitonin, is produced by the thyroid, therefore, as for the regulation of *Mib1* indicated above, this suggests that maintenance of satellite cell quiescence is also under systemic control. However recent evidence from our laboratory suggests that this may not be the case following identification of a novel *Notch*/*ColV*/*Calcr* axis where Collagen V, expressed by satellite cells, can act as a surrogate ligand for CALCR to maintain the quiescent state cell-autonomously.^[@CR79]^ Interestingly, intron 4 of *Calcr* contains the microRNA miR489, which is necessary for their maintenance of the quiescent state by post-transcriptionally repressing the oncogene *Dek*.^[@CR40]^ In addition, miR-195/497 was shown to induce cell cycle arrest^[@CR80]^ suggesting that other miRs could have critical roles in maintenance of quiescence. Indeed, satellite cell loss of *Dicer*, which is a key mediator of miRNA processing, resulted in depletion of this pool, upregulation of KI67, and failure to regenerate following injury.^[@CR40]^ Other signalling pathways have been explored in the context of satellite cell quiescence. For example, Angiopoietin 1 (ANG1) and its receptor TIE-2 regulate the quiescent state where TIE-2 is expressed by a subset of quiescent satellite cells and ANG1/TIE-2 signalling, through ERK1/2 pathway favours cell cycle exit.^[@CR37]^ Furthermore, previous studies had suggested that c-MET and its ligand hepatocyte growth factor could regulate the quiescence to activation transition, however, conditional ablation of the *c-Met* receptor gene in satellite cells showed no requirement for satellite cell activation, myoblast proliferation, or myocyte differentiation, but a role in muscle fusion and regeneration.^[@CR51]^ Oncostatin M, which belongs to the interleukin-6 family of cytokines, also regulates satellite cell quiescence as depletion of its receptor leads to loss of this stem cell population and impaired regeneration after injury.^[@CR81]^ Although some transcripts of activated and differentiated cells are detected in quiescent satellite cells, their proteins are generally detected after activation. As indicated above, MYOD is one such example where appearance of the protein identifies, and can promote satellite cell activation. In this context, repression of translation can be critical for maintaining satellite cell quiescence. This is mediated by phosphorylation of translation initiation factor eIF2a, resulting in stabilisation of the quiescent state, as inhibition of eIF2a phosphorylation results in exit from quiescence, activation of the myogenic programme and compromised self-renewal.^[@CR82]^ Intriguingly, a decrease in the satellite cell pool can be associated^[@CR40],[@CR46],[@CR51]^ or not^[@CR48]^ with impaired regeneration capacity upon injury. Indeed, a recent report indicated that the dynamics and extent of regeneration, both at the stem cell population and histological levels, vary depending on the method used to induce muscle injury^[@CR43]^ (snake venom myotoxins, chemicals or physical procedures). Furthermore, the number of satellite cells can increase several fold with some injury procedures raising further questions on how stem cell numbers are regulated locally, and globally. Other factors, such as the myofibre, can impact on satellite cell numbers. For example, transgenic mice overexpressing *Tead1* (TEA domain transcription factor binds to Hippo signalling effectors YAP/TAZ) in myofibres have 6X more quiescent satellite cells.^[@CR83]^ Although this transgenic mouse ameliorated muscle regeneration in a dystrophic *mdx* background, it is not clear if satellite cell numbers have a role. Taken together, these reports raise the question---how many stem cells are required to assure proper muscle function during regeneration and ageing? Distinct states of quiescence {#Sec6} ----------------------------- Until recently, cellular quiescence was considered to be a passive and homogenous cellular state with reduced metabolic activity. Of note, recent reports indicate that quiescent satellite cells can exist in different states, more or less primed for commitment,^[@CR84]^ or poised for activation.^[@CR85]^ Satellite cells expressing high levels of *Pax7* are less primed for commitment, display a lower metabolic activity and a delayed first mitosis upon activation in vivo and in vitro compared to cells expressing lower levels of *Pax7*. PAX7^Hi^ cells were therefore proposed to be in a deeper quiescent, or "dormant" cell state, compared to the remaining population that is more primed for cell-cycle entry.^[@CR84]^ Hence, the quiescent state exists as a continuum from PAX7^Hi^ to PAX7^Low^ cells. During extreme stress, such as death, where cells are exposed to tissue necrosis, acidosis and lack of oxygen, the majority of satellite cells adopt a dormant cell state^[@CR86]^ similar to that noted in a sub-population (PAX7^Hi^) in living mice.^[@CR84]^ Therefore, satellite cells can modulate their physiological and metabolic status to adapt to changing microenvironments. It is likely that satellite cells that are primed would be first-responders to mild-injury, and that dormant satellite cells would be mobilised under severe trauma, however, this remains to be proven formally. By analogy, yeast that are subjected to nutrient deprivation alter their metabolic and transcriptome profiles significantly depending on the type of starvation regime employed.^[@CR36]^ Therefore, one possibility is that dormancy vs. primed quiescence are determined by anatomical position. It is interesting to note that about 85% of satellite cells are located in close proximity to a blood vessel, whereas the remainder are distal, and likely in a more nutrient-deprived hypoxic niche,^[@CR87]^ raising the possibility that these distally located cells might be in a dormant state. Interestingly, quiescent satellite cells that are distant from a site of muscle injury (ex. contralateral muscle) can transit from G0 to the G0/G1 state (also called 'G(alert)'), characterised by an increased cell size, mitochondrial activity, gene expression profile, improved differentiation and enhanced regeneration capacity^[@CR85]^ (Fig. [2a](#Fig2){ref-type="fig"}). Although the BaCl~2~ that was used to promote muscle injury could have diffused to distal muscles, some of the observed phenotypes were confirmed with physical injury suggesting a systemic relay mechanism. This G0-G(alert) transition state is reversible and was shown to be dependent on the mTor signalling pathway.^[@CR85]^ Taken together, these findings indicate that adult satellite cells exist in different quiescent states within which they can transit in response to environmental cues. Following exit from quiescence, the fate a satellite cell can vary according to the context: apoptosis,^[@CR40],[@CR51]^ direct differentiation^[@CR46]^ or senescence.^[@CR41],[@CR49]^ These cell states can be altered significantly during ageing, leading to a decline in satellite cell number and function^[@CR39],[@CR49],[@CR88],[@CR89]^ as a result of defects in self-renewal, maintenance of quiescence, regenerative capacity or increased susceptibility to apoptosis and senescence.^[@CR39],[@CR41],[@CR45],[@CR49]^ Deregulation of satellite cell function: ageing and senescence {#Sec7} ============================================================== Pathologies associated with ageing involve cell-autonomous as well as non-cell-autonomous mechanisms, including cellular senescence. Cellular senescence represents an irreversible cell cycle exit state that is often associated with pathologies where cancerous, DNA-damaged or ageing cells withdraw from the cell cycle and suppress apoptotic mechanisms while engaging tumour suppressors such as P53, P16/P19.^[@CR90]--[@CR92]^ An important signature of senescent cells that undergo a stress response such as oxidative, replicative and genotoxic stress, is the so-called "senescence-associated secretory phenotype" (SASP) where several growth factors, inflammatory molecules (chemokines, cytokines), proteases, and extracellular matrix components modify the fate of nearby cells. Currently, markers specific for senescent cells are lacking; commonly used markers include senescence-associated β-galactosidase (SA-β-Gal) activity in lysosomes, the level of lysosomal content, and elevated levels of P16.^[@CR90],[@CR91]^ Although the pathological consequences of cellular senescence have been well documented where suppression of senescent cells can have dramatic consequences including increase of lifespan, its potential beneficial effects have only been reported recently for tissue remodelling during development and regeneration^[@CR91],[@CR93]--[@CR98]^ (Fig. [4](#Fig4){ref-type="fig"}). Suppression of senescence during prenatal stages does not promote overt phenotypes, however, removal of senescent endothelial and fibroblast cells was associated with less efficient wound healing of skin.^[@CR97]^ These studies raise the notion of a beneficial role for cellular senescence, likely through the SASP (Fig. [4](#Fig4){ref-type="fig"}). Similarly, senescent cells were reported to have a role during limb regeneration in salamander following amputation.^[@CR98]^ Furthermore, mutation of the endocytic adaptor proteins *Numb:Numblike* resulted in senescence of myogenic cells and compromised regeneration. This phenotype was rescued in a *p53*-null context, and by administration of an antioxidant.^[@CR93]^ Notably, this *Numb*-mediated senescence was distinct from senescence observed in endothelial cells during early regeneration that was independent of *p53* and oxidative stress.^[@CR93]^ It is interesting to note that cellular senescence also has a role in cell plasticity where the generation of teratomas following induction of pluripotency factors (*Oct4*, *Sox2*, *Klf4* and *Myc*) in vivo and ex vivo was modulated, at least in part, by senescent cells.^[@CR95]^Fig. 4Cellular senescence in different contexts. Although senescence has been extensively reported in pathological contexts^[@CR90]--[@CR92]^, recent studies have reported that cellular senescence is associated with developmental and regenerative processes, suggesting a beneficial role^[@CR91],[@CR93]--[@CR98]^. Senescence observed during muscle regeneration was not altered on a *p53*-null background, however, *Numb:Numblike* mutants that exhibit a higher level, and persistent cell senescence during regeneration, are rescued on a *p53-null* background and with antioxidants suggesting that senescence can be modulated differentially in this mutant compared to wild-type mice^[@CR93]^ Recent studies pointed to functional differences between aged and 'geriatric' mice where in the latter, the regenerative capacity of satellite cells decreases even further during extreme ageing, after 28 months of age in mice. Here, satellite cells lose their quiescent state to become pre-senescent, due to de-repression of the senescence marker *p16*^*INK4a*^ following loss of the PRC1-mediated repressive H2A-lysine 119 ubiquitination mark. Upon injury, they fail to activate (absence of *Myod* expression) and transit to a full senescent state that is marked by increased *p16*^*INK4a*^ expression, increased number of γ-H2AX foci indicating DNA double strand breaks, and appearance of a senescence-associated β-galactosidase activity. Interestingly, this pre-senescent phenotype could be reverted by silencing of *p16*^*INK4a*^ but not by exposure to a youthful environment, underscoring the cell-autonomous nature of this pre-senescent phenotype.^[@CR49]^ More recently, it was shown that aged satellite cells display decreased autophagy and mitophagy activities, leading to increased intracellular reactive-oxygen species.^[@CR41]^ Notably, scavenging ROS restored *p16*^*INK4a*^ repression by re-establishing its repressive H2A-lysine 119 modification, decreased senescence and increased proliferation potential, while restoring autophagy reversed the senescence phenotype and restored the regenerative capacity of geriatric satellite cells.^[@CR41],[@CR92]^ More generally, it appears that cellular quiescence protects satellite cells from ROS through upregulation of genes involved in the antioxidant response, such as *Thioredoxin reductase 1*, *Sulfiredoxin* and *Glutathione peroxidase 3*.^[@CR66]^ The FGF2/SPRY1 axis appears to be deregulated in aged muscles, yet it remains unclear how this signalling effects its action. In one study, maintenance of aged myofibres in basal medium resulted in compromised proliferation of satellite cells unless FGF2 was added exogenously.^[@CR88]^ Satellite cells express FGF receptors 1 and 4, and FGFR1 appears as their functional FGF receptor, as *Fgfr4* did not rescue satellite cell proliferation upon *Fgfr1* deletion.^[@CR99]^ Additional FGF ligands might be involved in the regulation of satellite cell proliferation in vivo, such as FGF6.^[@CR100]^ In contrast, a decline in satellite cell numbers in the aged niche was attributed to increased FGF2 production and decreased *Spry1* expression.^[@CR39]^ *Spry1* is a tyrosine kinase receptor that is expressed in quiescent satellite cells, and it antagonises FGF2 signalling. Satellite cell deletion of *Spry1* during regeneration prevents their return to quiescence, resulting in a decrease in the quiescent satellite cell pool after regeneration.^[@CR48]^ Moreover, aged human satellite cells exhibit higher levels of DNA methylation (mC), and in this context, the *SPRY1* promoter region was reported to have higher levels of mC compared to myogenic cells isolated from young individuals, thereby linking DNA methylation to increased sensitivity to higher levels of FGF2 in the aged niche.^[@CR39],[@CR101]^ Impaired response to FGF2 signalling that results in elevated levels of p38α and p38β diminishes self-renewal capacity of aged satellite cells, and pharmacological inhibition of this pathway,^[@CR54],[@CR55]^ in combination with culture on soft hydrogel substrates^[@CR55]^ leads to rescue of this phenotype. Similarly, it has been observed that satellite cells attach to their surrounding microenvironment preferentially via fibronectin/integrin interactions. Aged muscles display substantially lower levels of fibronectin, leading to a weaker adhesion of satellite cells and an increased susceptibility to anoikis,^[@CR45]^ a phenotype similar to loss of β1-integrin function in SCs.^[@CR47]^ Some of the decline in satellite cell function has also been attributed to deregulation of the Janus kinases (JAKs, via cytokine receptors), and Signal Transducer and Activator of Transcription proteins (STATs, relay of JAKs to activate nuclear targets) pathway, where STAT3 is stimulated by inflammatory cytokine associated with disease and ageing in muscle. Knockdown of *Jak2* or *Stat3* results in increased satellite stem cell divisions, and transient pharmacological inhibition of this pathway ameliorates transplantation potential and muscle force following injury.^[@CR102],[@CR103]^ An apparent contradiction arises from the above-mentioned studies, regarding whether aged satellite cells can be rejuvenated in a non-cell-autonomous manner. Indeed, geriatric satellite cells could not reverse their pre-senescent state when transplanted into a young recipient muscle,^[@CR49]^ whereas decreasing niche-derived FGF activity^[@CR39]^ or tissue complementation with exogenous fibronectin^[@CR45]^ in aged muscles prevented the loss of satellite cells and restored a youthful muscle regeneration respectively. These findings suggest that the aged muscle phenotype can be reversed by exposure to a 'young' environment, however, the pre-senescent state of geriatric satellite cells might represent a point of no return. The most compelling evidence for youthful restoration, and the negative impact of circulating molecules in old mice, come from heterochronic parabiosis experiments, where young donor constituents (blood, secreted molecules, etc) transferred to an old host were shown to restore a rejuvenated phenotype in muscle and the nervous system.^[@CR104],[@CR105]^ The identification of effector molecules that potentially mimic this response is an active area of research. The TGBβ member GDF11, which is homologous to Myostatin (GDF8, inhibitor of myogenesis), is one putative candidate, however its precise role remains under debate.^[@CR106]--[@CR108]^ To what extent is the muscle stem cell population heterogeneous? {#Sec8} ================================================================ Do proliferating and quiescent cell states each reflect a range of properties that individual myogenic cells can assume, or instead do myogenic cells represent distinct subpopulations each with fixed deterministic potential (Fig. [5)](#Fig5){ref-type="fig"}? In this context, it is interesting to note that heterogeneity has been suggested to be a feature of hematopoietic stem cells (HSCs) where at the top of the hierarchy, sub-classes of α and β HSC subpopulations exist with equivalent myeloid potential, whereas α-HSCs have a reduced ability to produce mature lymphoid cells.^[@CR109],[@CR110]^ Cellular plasticity can also underlie heterogeneity where endogenous cells are endowed with greater fate plasticity following their isolation and transplantation, for example, in the blood, skin, and muscle lineages.^[@CR111]--[@CR114]^Fig. 5Models for regulation of satellite cells during regeneration. **a** Deterministic model (top), where PAX7^Hi^ cells retain this state through proliferation and return to the quiescent state. Here PAX7^Lo^ cells would derive from PAX7^Hi^ quiescent and proliferating cells and would be poised for commitment. This hierarchical model suggests intrinsic mechanisms as driving forces for maintenance of these relative states and would result in vulnerability if only a subset of the cells in the population have long-term stem-like properties. In the stochastic model (bottom), PAX7^Hi^ and PAX7^Lo^ cells are interchangeable states, presumably due to fluctuations in gene expression, and obedience to extrinsic signals. This model proposes that all satellite cells have the potential to assume a stem-like or committed state. **b** Satellite cells undergo rounds of exit and entry into the niche to assume a quiescent state, and during ageing, exit from the niche is suggested to occur more frequently without replenishment, thereby resulting in declining numbers of muscle stem cells. We entertain the possibility that re-entry into the niche could reset or rejuvenate the stem cell and endow it with properties for long-term persistence As indicated above, heterogeneity was also noted among quiescent satellite cells, suggesting that they can explore different levels of quiescence and metabolic states, perhaps driven by local niche signals. Our recent finding that satellite cells from different muscles exhibit different metabolic profiles^[@CR115]^ suggests that at least some of the heterogeneities reported may be attributed to differences in myofibre niche properties. Another feature of myogenic cells that is modulated during lineage progression is their response to DNA double strand induced stress. Recent findings have reported that adult stem cells are remarkably proficient in repairing DNA strand breaks compared to their committed progeny.^[@CR116]--[@CR118]^ Specifically, satellite cells were reported to repair with greater efficiency and accuracy following irradiation-induced DNA damage, compared to myoblasts and differentiated cells, and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs (DNA-dependent protein kinase, catalytic subunit).^[@CR118]^ It remains to be determined if this resistance to DNA damage mediated stress persists in myopathies where satellite cells experience repeated bouts of quiescence and activation, or during ageing, and how this property affects mutational load and genome integrity over time. Although heterogeneities have been reported among stem cell populations in different tissues, it remains unclear if this reflects a heterogeneity that is associated with distinct cell states, or if the population is uniform, but exhibits a wide range of behaviours. At the organismal level, individuals can survive extreme conditions of temperatures, dehydration and starvation, and some amphibians can undergo multiple consecutive freeze-thaw cycles with no apparent adverse effects.^[@CR119]^ This can also be the case for individual cells thereby providing the opportunity to adapt to changing environments. However, empirical methods often do not expose these potentials. When cells are analysed, generally post-fixation, their properties several hours before or after the isolation procedure are eclipsed. Single cell transcriptomics can circumvent some of these shortcomings by documenting the range of behaviours in a population, as for example in intestinal and hematopoietic stem and committed cells.^[@CR120],[@CR121]^ Heterogeneity among proliferating cells has been investigated to a limited extent. For example, myogenic cells perform symmetric (SCD) and asymmetric (ACD) cell divisions following muscle injury or after their isolation on single myofibres.^[@CR35],[@CR40],[@CR84],[@CR122]--[@CR129]^ It remains unclear what determines this choice, whether it is predetermined, and if SCD and ACD are obligate, or if switching in modes occurs in successive cell cycles. Several types of ACDs have been reported for muscle including non-random DNA segregation (NRDS), where old and new DNA strands segregate to distinct daughter cells,^[@CR35],[@CR40],[@CR84],[@CR122],[@CR128]^ transcription factors that include PAX7 (stem), MYOD (committed) and MYOGENIN (differentiated),^[@CR35],[@CR84],[@CR122],[@CR127]^ cytoplasmic proteins including NUMB,^[@CR122],[@CR130]^ p38,^[@CR129]^ and Par proteins and dystrophin.^[@CR125]^ In addition, several signalling pathways and extracellular molecules have been reported to influence the balance between SCD and ACD including fibronectin and WNT7a.^[@CR126],[@CR131]^ It is clear that cell fates can be governed by extrinsic and intrinsic cues, and studies in *Drosophila* have shown the importance of extrinsic cues in neuroblast and germline cell divisions.^[@CR35],[@CR132]^ Some insights also come from examining individual adult myogenic cells on micropatterns that represent artificial niches, where their shape can be designed to promote SCD and ACD.^[@CR35],[@CR133],[@CR134]^ In this scenario, the frequency of NRDS and the asymmetric distribution of PAX7 and MYOGENIN were increased on fibronectin/fibrinogen coated micropatterns with an asymmetric motif compared to those that were placed on a symmetric motif.^[@CR35]^ Therefore, extrinsic cell adhesion cues can have a major impact on at least two readouts for ACD. If these findings can be extrapolated to the in vivo situation, they suggest that the topology of the immediate microenvironment might dictate cell fate decisions in muscle. However, these observations pose a conundrum. Although during muscle regeneration the microenvironment is highly dynamic, as far as cell types and extracellular matrix are concerned, a symmetric niche does not clearly exist in vivo; albeit it can be argued that a satellite cell undergoing planar cell divisions on a myofibre during homoeostasis might experience symmetrically distributed extrinsic cues. However, in absence of the myofibre, during G2/M when the axis of cell division will be determined, the resulting daughter cells will most likely experience distinct cues at opposite poles of the division axis in a regenerating tissue. If extrinsic cues have a predominant role in dictating cell fates, then one would expect that all divisions should be asymmetric---but this does not appear to be the case. How can in vitro and in vivo observations be reconciled? One possibility is that cells respond to threshold levels of extrinsic cues, and that an asymmetric response is triggered only if the differential in cell adhesion cues or signalling molecules at opposite poles of the division axis are sufficiently marked. Alternatively, intrinsic differences among cells might lend some to be more responsive to extrinsic asymmetric cues. Beyond the notion of cellular heterogeneity, it is interesting to ask whether cellular memory persists over consecutive cell divisions. In other words, would a PAX7^Hi^ cell assume a PAX7^Hi^ cell state after one or several cycles of injury and return to homoeostasis, or alternatively, is the internal clock reset with each cell cycle thereby providing stochastic access to both self-renewal and commitment fates to each cell in the population (Fig. [5](#Fig5){ref-type="fig"}). Empirical evidence for cell equivalence is lacking in muscle, although this model is currently favoured for stem cells in other tissues.^[@CR135]--[@CR137]^ Muscle regeneration---can it be improved? {#Sec9} ========================================= Altering genetic or epigenetic functions can lead to compromised regeneration raising the possibility that regeneration in wild-type mice might be improved if these processes can be manipulated. Regenerative myogenesis is non-uniform among mice of distinct genetic backgrounds; SJL/J mice regenerate faster than BALB/c mice following muscle injury or whole muscle engraftments.^[@CR138]^ Moreover, a milder phenotype is observed with *Mdx* mice on a 129/sv background compared to DBA2 or C57BL/6 backgrounds.^[@CR139],[@CR140]^ Nevertheless, dystrophic mice are generally less severely affected compared to the DMD condition in human.^[@CR141]^ Strikingly, the MRL multi-strain mouse has been reported to be a "super-regenerator" compared to C57BL/6 for tissue recovery following an ear punch assay.^[@CR142],[@CR143]^ When crossed with γ-Sarcoglycan null mouse, fibrosis and regeneration were reduced in skeletal and cardiac muscle, a phenotype that was mapped in part to a region on Chromosome 2.^[@CR144]^ Metabolism was suggested to be a driver of this phenotype as the MRL mouse relies more on aerobic glycolytic energy metabolism, increased glutamate oxidation, and reduced fatty acid oxidation compared to C57BL/6 mice.^[@CR142]^ A notable example of how metabolism can impact disease is provided by the role of Nicotinamide adenine dinucleotide (NAD^+^). Sirtuins consume NAD^+^ and generate nicotinamide for deacetylation of proteins. Additionally, PARP (poly\[adenosine 5′-diphosphate (ADP)-ribose\] polymerase) proteins consume NAD^+^ during poly(ADP)-ribosylation of proteins. Interestingly, repletion of NAD^+^ was shown to provide protection from metabolic diseases, mitochondrial dysfunction and necrosis, and resulted in improved skeletal and cardiac function in *Dystrophin* and *Dystrophin:Utrophin* mutant mice.^[@CR145]^ This is thought to occur by countering increased PARP consumption of NAD^+^, thereby leading to the recovery of NAD^+^-dependent sirtuin signalling. In another study, treatment of mice with the NAD^+^ precursor nicotinamide riboside was shown to induce the mitochondrial unfolded protein response, and this resulted in reduced senescence and increased lifespan in mice.^[@CR146]^ Furthermore, satellite cells undergo a metabolic reprogramming upon in vitro activation after isolation, transiting from fatty acid oxidation to glycolysis as a main source of energy production.^[@CR147]^ This is accompanied by decreased levels of NAD^+^, which directly regulates the activity of the histone deacetylase SIRT1. This in turn results in increased levels of histone acetylation and a subsequent activation of muscle gene transcription. Satellite cell-specific inactivation of *Sirt1* led to their precocious activation and differentiation.^[@CR147]^ In another study, examination of foetal, perinatal, quiescent (young, post-mortem, aged) and regenerating myogenic stem cells identified striking differences in metabolic requirements.^[@CR115]^ Specifically, aged satellite cells were shown to exhibit a compromised oxidative phosphorylation response, relying more on glycolysis for ATP production. In addition, proliferating foetal and perinatal myogenic cells have a low respiration demand, relying more on glycolysis compared to proliferating cells isolated from regenerating muscle, thereby underscoring the impact of the microenvironment on satellite cell metabolic response. The AMP-activated protein kinase (AMPK) has several roles in metabolism including catabolism and activation of mitochondrial biogenesis. Interestingly, the key metabolic regulator *AMPKα1* was shown to have a major role in controlling the balance between self-renewal and differentiation during muscle regeneration.^[@CR148]^ Satellite cell-specific inactivation of *AMPKα1* not only increased their glycolytic activity but also drastically enhanced their self-renewal, leading to impaired muscle regeneration. This study places metabolism as a key parameter controlling satellite cell fate decisions. As the metabolic activity of stem and progenitors is explored in more detail, it is important to note that this data is generally obtained from cells examined ex vivo, where culture conditions potentially alter metabolic profiles. Although relative comparisons have been highly informative, a key future objective is to generate in vivo readouts to assess directly the metabolic demands of stem and niche cells. Reactive-oxygen species (ROS) have also been linked to a certain extent to pathological processes in regeneration. As indicated above the regeneration deficit when *Numb:Numbl* are conditionally inactivated in satellite cells can be rescued by administering the antioxidant *N*-acetyl cysteine (NAC). However, exposure to NAC does not improve the regenerative process in wild-type mice suggesting that oxidative stress is neither significantly promoting, nor deleterious to the regeneration process at early stages.^[@CR93]^ During muscle differentiation, there is a higher demand for oxidative metabolism requiring mitochondrial biogenesis and increased ROS production.^[@CR92]^ In another example, adult long-term-haematopoietic stem cells (LT-HSCs) use hypoxia induced glycolysis preferentially,^[@CR149]^ perhaps to reduce ROS since these cells, as well as neural stem cells that do so through the action of *FoxO*,^[@CR150]^ are sensitive to ROS levels that promote differentiation and apoptosis. It has been suggested that elevated levels of ROS drives stem cells out of quiescence in hypoxic conditions, and into proliferation when in normoxia, and that oxidative phosphoryation is low in LT-HSCs.^[@CR151]^ Conditional inactivation of the transcription factors *Pitx2:Pitx3* that act downstream of *Pax3* and *Pax7* results in elevated levels of ROS and compromised prenatal myogenesis, a phenotype that can be rescued by administration of NAC.^[@CR152]^ The generation of ROS alone does not account for all of the oxidative stress. Indeed reactive nitrogen species (RNS; reaction of nitric oxide (NO) with superoxide (O~2~^−^) to produce peroxynitrite (ONOO^−^)) is often ignored when considering overall oxidative stress (combined ROS + RNS). The combined actions of ROS and RNS results in modifications of protein structure, lipids, signalling and cytoskeletal elements and DNA damage.^[@CR92]^ Autophagy is also intimately linked with metabolic processes. Here, damaged proteins and organelles are degraded by lysosomes to assure quality control. In conditions of limiting nutrients, as well as in quiescent cells, breakdown products resulting from autophagy can provide fuel for cellular activities.^[@CR41],[@CR92],[@CR153]^ It is clear that autophagy, as well as mitophagy, impact on cell and tissue response during stress in undamaged and injured muscles.^[@CR41]^ In satellite cells, impairment of autophagy results in loss of proteostasis and increased oxidative stress due to mitochondrial dysfunction, resulting in senescence entry.^[@CR41]^ These processes need to be examined in stromal cells to ascertain their global impact on muscle physiology. In the context of myopathies where chronic cycles of degeneration and regeneration prevail, one key aim has been to improve the efficiency of the regeneration process. However, recent findings on *Nfix* mutant alone or on the myopathic *α-Sarcoglycan* null model showed that delaying the regeneration process and shifting to slow myogenesis has a dramatic positive outcome in functional tests including extensive running.^[@CR154],[@CR155]^ Although *Nfix* deletion could act through other pathways unrelated to regeneration speed *per se*, these findings suggest that multiple pathways, including metabolic processes, could be targeted to temper rather than accelerate muscle regeneration. The immune response in regeneration and disease {#Sec10} =============================================== The immune response during regeneration has become another central focus in recent years where monocytes, macrophages, neutrophils and T cells impact on the regeneration process. The critical role of macrophages in wound repair and muscle regeneration has been documented extensively.^[@CR156]^ A pro-inflammatory response following production of macrophages from infiltrating monocytes, as well as resident macrophages, is associated with phagocytosis and clearing of debris in injured muscle. A second phase of inflammation that follows after a few days is associated with increased myofibre differentiation, angiogenesis, matrix remodelling and subsequently homoeostasis. The distinction between yolk sac and foetal liver derived macrophages raises the question of their respective roles and their dynamics during homoeostasis and regeneration.^[@CR157],[@CR158]^ Furthermore, chronic myopathies are characterised by a non-synchronous regeneration process resulting in the biphasic macrophage response being disrupted, and the coexistence of these populations contributing to the pathophysiology.^[@CR156]^ Eosinophils and regulatory T cells also infiltrate the damaged muscle where they affect FAP and myogenic cell proliferation, respectively.^[@CR159]--[@CR161]^ Notably, regulatory T cells (Treg) produce Amphiregulin that was reported to improve the regeneration process by acting on myogenic cells.^[@CR159]^ The inflammatory cytokine Prostaglandin E2 (PGE2) was also shown to stimulate satellite cell proliferation and enhance muscle regeneration by promoting the cAMP/phosphoCREB pathway and activation of the NURR1 transcription factor.^[@CR162]^ Intriguingly, in addition to their pro-myogenic effects, restorative macrophages were shown to stimulate myogenesis/angiogenesis through secretion of Oncostatin M,^[@CR163]^ whereas this compound is secreted by muscle fibres and functions to maintain satellite cell quiescence.^[@CR81]^ As for myogenic cells, AMPKα1 has a key role in macrophages where it is required for the anti-inflammatory/restorative inflammatory phenotype during skeletal muscle regeneration. Loss of function in myeloid cells results in lasting inflammation and defects in muscle regeneration due to a block in the phenotypic transition of macrophages.^[@CR161]^ In summary, recent studies on regenerative myogenesis have underscored the flexibility and wide range of responses exhibited by muscle satellite cells to a variety of stresses, by regulating their depth of quiescence, mode of proliferation and modulation of metabolic processes. Whether return to the quiescent state allows stem cells to reset their state, or perhaps even restore their potential, needs to be explored (Fig. [5b](#Fig5){ref-type="fig"}). As the study of quiescence takes more prominence, we note that the mirtron miR-708 was recently shown to regulate quiescence and self-renewal by antagonizing cell migration through targeting the transcripts of the focal-adhesion associated protein Tensin3^[@CR164]^. Thus, the G0 state exhibits multiple safeguards that merit future attention. Furthermore, the nature of the niche stromal cells is distinct spatially (ex. head vs. trunk) and temporally, pointing to a dynamic niche during prenatal life, adult, disease and ageing. These factors will likely have a significant impact on muscle stem cell properties, as well as their regenerative potential. Future research in these areas should provide valuable information on how to optimise regeneration and boost stem cell potential. **Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. We acknowledge support from the Institut Pasteur, Association Française contre les Myopathies, Agence Nationale de la Recherche (Laboratoire d'Excellence Revive, Investissement d'Avenir; ANR-10-LABX-73), and the European Research Council (Advanced Research Grant 332893). We thank Marina Thizeau for illustrations of Figs. [1](#Fig1){ref-type="fig"}, [2](#Fig2){ref-type="fig"}, and [3a](#Fig3){ref-type="fig"}. All authors researched, collated, and wrote this paper. Competing interests {#FPar1} =================== The authors declare no competing interests.

Background ========== Perinatal mortality in the Netherlands -------------------------------------- Perinatal mortality and morbidity in the Netherlands is relatively high compared to other countries in Europe, shown by Peristat I (data of 1999) \[[@B1]\] and Peristat II (data of 2004) \[[@B2]-[@B4]\]. Initiated by the Dutch Minister of Health, a Committee Project group Pregnancy and Birth was started in 2008, just after publication of Peristat II. The main goal was to improve quality of obstetric care in the Netherlands. Beside several implementations such as regional Obstetric Cooperatives and the Dutch Perinatal Audit, a nation-wide research programme on pregnancy and birth of the Netherlands Organization for Health, Research and Development (ZonMw) was developed. Recently, the data of the third Euro-Perinatal European Perinatal Health Report (data of 2010) were launched \[[@B5]\]. Perinatal mortality in the Netherlands has declined with 14% between 2004 and 2010, however the current mortality rate still represents a poor international position, which is even more remarkable considering that the Netherlands was ranked second highest in Europe concerning welfare \[[@B6]\]. In 2004, the Netherlands featured the third highest perinatal mortality (out of 26 countries). In 2010, the Netherlands ranked the sixth highest perinatal mortality out of 29 European countries. The perinatal mortality in the Netherlands should decrease faster than in other European countries in order to be ranked in the top. Dutch system ------------ The Netherlands has an estimated population of 16.7 million. In the Netherlands, around 175,000 children are born yearly, of which around 1,500 babies die (perinatal mortality). Obstetric care is organised in low-risk primary care, medium-risk secondary, and high-risk tertiary care (Figure [1](#F1){ref-type="fig"}). Primary care concerning low-risk pregnancies is represented by independent midwives (and general practitioners). A low-risk pregnant woman has the possibility of planning her delivery either at home or in a primary care hospital setting, both under responsibility of her own independent midwife. The Netherlands has a high rate of home delivery, although rate is declining from 26% home deliveries in 2007 to 15.6% in 2012 \[[@B7]\]. Secondary care is regionally organised in 92 hospitals of which 10 hospitals provide tertiary care in a perinatology centre facilitating a Neonatal Intensive Care Unit (NICU) and an Obstetric High Care unit (OHC). Secondary and tertiary care is provided by obstetric nurses, secondary care (hospital) midwives, residents and obstetricians, working together in teams. During pregnancy and/or delivery a pregnant woman may evolve from low-risk to medium- or high-risk, followed by a referral from primary to secondary or tertiary care. Indications for referral are defined in the 'Obstetric Indication List' \[[@B8]\]. Perinatal data of 2007 show a huge shift between primary and secondary/tertiary care: the intention (42%) and reality (26%) to deliver at home, the intention (42%) and reality (16%) to deliver in primary care in hospital, and the intention (16%) and reality (61%) to deliver in secondary care in hospital \[[@B9]-[@B14]\]. This shift between primary and secondary care is getting more extensive when interpreting recent perinatal data of 2012: about 85% of pregnancies starts in primary care and 15% in secondary/tertiary care. Finally, 30% will give birth in primary care and 70% in secondary/tertiary care. This means that about 65% of all pregnancies will be referred from primary to secondary/tertiary care, during pregnancy or delivery \[[@B7]\]. This extensive shift between the different care levels results in multiple medical handovers, potentially causing errors in communication and process management. {#F1} Risk of home delivery --------------------- A recent Dutch study showed a higher risk of delivery related perinatal mortality among women with planned delivery in primary care (at home or in hospital) compared to women who started delivery in secondary care. An even higher risk of perinatal mortality was found in women who were referred from primary to secondary care during delivery \[[@B15]\]. Another Dutch study did not find a significant difference between a planned home and hospital delivery among low-risk women in primary care \[[@B16]\]. However, the results of these two Dutch studies cannot be compared because different groups and different comparisons were studied: the first study compared planned primary care delivery with planned secondary care delivery while the second study compared planned home delivery with planned hospital delivery in primary care. The British Birthplace cohort study concluded that nulliparous low risk women with a planned home delivery have an increased incidence of adverse perinatal outcome. For multiparous women, there were no significant differences in adverse perinatal outcome by planned place of birth. Interventions during delivery were substantially lower in all non-obstetric unit settings \[[@B17]\]. Causes of perinatal death ------------------------- Analysis of Dutch data showed that 85.2% of perinatal mortality is caused by one or more of the four following disorders, together the so called Big 4: small for gestational age (SGA: birth weight below 10^th^ percentile), preterm delivery before 37 weeks of gestation, congenital anomaly and low Apgar score (Apgar score below 7). Big 4 disorders are overlapping each other often, creating a multiple diagnosis. Accumulation of Big 4 disorders obviously increases mortality rate. The group with exclusively one Big 4 disorder causing perinatal death is small. Of all pregnancies, 16.3% represents a Big 4 disorder. Of all Big 4 pregnancies, 29% starts delivery in primary care. This indicates that risk selection is inadequate. \[[@B9],[@B18]-[@B20]\]. These data suggest that evaluation and improvement of process management of pregnancies complicated by a Big 4 disorder will be beneficial for perinatal outcome. Process parameters and communication audit ------------------------------------------ Analysis of all term perinatal death cases in 2010 by the Dutch Perinatal Audit revealed one or more substandard factors (SSF) in 52% of the cases. In 56% of the cases with SSF, multiple care providers were involved. In 44% of the cases with SSF there was a possible or (very) probable relation with perinatal death. International research described a possible or (very) probable relation with perinatal death in 25-30% of all perinatal death cases with substandard care \[[@B21]\]. The Dutch Perinatal Audit has recommended the following: develop uniform care paths, focus on standardised communication and handovers based on the SBAR system (Situation, Background, Assessment, Recommendation), and organise team trainings \[[@B22]\]. It has become clear that within the entire obstetric collaborative network process parameters can be improved. Communication between obstetric care providers within one discipline as well as between different disciplines is important to guarantee an optimal referral process. Moreover, adequate and uniform communication towards the patient (and partner) is important for positive perception \[[@B9],[@B18],[@B22]\]. Quality of care as perceived by patients ---------------------------------------- During the last decade, there has been growing interest in quality of care as perceived by patients. With increasing attention to patient-centered care, indicators of care quality more and more involve perceived quality of care and patient satisfaction \[[@B23]-[@B26]\]. Measuring patient-reported outcomes is a common strategy used to monitor quality of care in a number of countries. Because of the unique obstetric care system in the Netherlands with different care levels, pregnant women often see different care providers \[[@B27]\]. Recently published data showed that patients who had been referred from primary to secondary care report lower quality of care \[[@B28]\]. These patients received care in more than one institution, from several care providers. Referral during pregnancy and delivery may have a negative effect on a systematic way of communication towards the patients and might cause inconsistency in advice, information, and protocols \[[@B28]\]. Simulation-based team training ------------------------------ Team training in obstetric emergencies reduces poor perinatal outcome as was shown by a British retrospective cohort study \[[@B29]\]. Recently, a systematic review has concluded that medical simulation is effective for medical education \[[@B30]\]. A meta-analysis showed that simulation-based medical education (SBME) with deliberate practice (DP) is superior in improving medical skills to traditional clinical medical education such as the Halstedian approach (see one, do one, teach one) \[[@B31]\]. DP reflects a life-long period of deliberate effort to improve performance in a specific domain. There are nine elements of DP: 1) high motivation and concentration, 2) well-defined learning objects, 3) appropriate level of difficulty, 4) focused, repetitive practice, 5) rigorous, reliable measurements, 6) feedback, 7) monitoring, error correction, 8) evaluation and performance that may reach a master standard, 9) advancement to the next task \[[@B32]\]. Crew resource management (CRM) has been defined as 'error management capability to detect, avoid, trap or mitigate the effects of human error and therefore prevent fatal accidents'. It was developed primarily for improving air safety \[[@B33]\]. CRM is a training system that focuses on interpersonal communication, leadership and decision-making. It focuses on the ability of each team member to see, analyse and react, and thereby reducing potential errors. It pursues an open culture where the freedom to respectfully question authority is encouraged. Learning goals of CRM are: enhanced situational awareness, self-awareness, leadership, assertiveness, decision-making, flexibility, adaptability and communication. CRM in team training has shown to improve those competences and results in a positive attitude of trainees towards team building and communication \[[@B34]\]. A positive attitude of trainees, as represented by the first level of Kirkpatrick's model for evaluation of a training, should result in a learning effect (Kirkpatrick level two) and behavioural change (level three) and finally be translated in patient outcomes (level four) (Figure [2](#F2){ref-type="fig"}) \[[@B35]\]. A recent study concluded that simulation-based obstetric team training using the concepts of CRM results in a significant improvement in team performance and application of essential medical skills \[[@B36]\]. In our study, multiprofessional teams will be trained in a medical simulation centre with simulated settings that resemble reality as closely as possible. Commercially available high fidelity patient simulators (Noelle™ and Newborn Hal™, Gaumard, Miami, Florida, USA) are used. {#F2} Methods ======= Design ------ The proposed research concerns a transmural multiprofessional simulation-based obstetric team training regarding process management of the Big 4 causes of perinatal mortality. The obstetric collaboration network consisting of ambulance staff, maternity nurses, primary care midwives, obstetric nurses, hospital midwives, residents and obstetricians will be trained. The Ethical committee did agree that specific ethical approval is not required for this type of study in The Netherlands. Because the study does not interfere with patient care, written informed consent of the participants of the training (care givers) is not indicated. The study is planned to be implemented in a sub-region of the Netherlands (Zuidoost-Brabant) consisting of over one million inhabitants. In this area, around 120 independent midwives are providing primary care. Parallel to this project, in the same region the Regional Consortium Obstetrics Brabant has been founded. This consortium is an association of a perinatology centre (tertiary care: Máxima Medical Centre, Veldhoven), adjacent hospitals (secondary care: Jeroen Bosch hospital Den Bosch, Catharina hospital Eindhoven, Bernhoven hospital Uden, Elkerliek hospital Helmond, St Anna hospital Geldrop) and surrounding primary care midwives. Each hospital with its referring midwifery practices (regional Obstetric Cooperative) is considered to be a separate study group. A stepped wedge trial design will be employed. A stepped wedge trial is a cluster-randomised trial in which all study groups (clusters) receive the intervention by a sequential roll out of the trainings over a number of time periods. Computerised randomisation will define the sequence of the study groups. This design was chosen primarily for logistical reasons and because of the fact that all study groups will eventually receive the team training. Recent literature shows that the stepped wedge trial design has several advantages over a randomised trial, and can offer a number of opportunities. All clusters start in the control condition. The clusters will switch to the intervention at consecutive time points, where the time of the switch is randomised for every cluster. Eventually, all clusters will receive the intervention (Figure [3](#F3){ref-type="fig"}). The stepped wedge design is useful when the intervention is thought to have a beneficial effect. With a classic cluster trial, randomisation would withdraw the intervention from a part of the study groups. In addition, there are other advantages of the stepped wedge design. First, the clusters act as their own controls because they receive both the control and intervention conditions. Therefore, the intervention effect can be estimated from both between- and within-cluster comparisons. This results in more statistical power and smaller required sample sizes than in a parallel group design. The stepped wedge design is also useful where phased implementation is preferable because of logistical, practical or financial constraints \[[@B37]-[@B39]\]. {#F3} ### Focus groups Prior to the intervention, focus group interviews were performed. This resulted in insight in topics relevant to patients and care providers, concerning adequate communication and process management. The input of the focus group interviews was used for development of questionnaires for patients and care providers. The focus groups were organised separately in the following categories: ● The pregnant women and women who recently gave birth. These focus group interviews were used to explore what is important to women regarding care during pregnancy and delivery. Based on these focus groups, the Pregnancy and Childbirth Questionnaire (PCQ), to measure quality of care as perceived by women who recently gave birth, has recently been developed and validated \[[@B28]\]. The PCQ will be used for an assessment of all women who recently gave birth before and after the training, concerning the entire Consortium of Brabant. ● The primary care (independent) midwife. The midwives were interviewed to evaluate most common problems in communication. This implies referral of patients to secondary/tertiary care but also aspects of communication when a client is referred back to primary care (during pregnancy/after delivery). The most relevant items were used to construct a questionnaire that will be sent out to all independent midwives for an assessment before and after the training. ● The 'maternity nurse'. This care provider was interviewed in the focus group together with the primary care midwife. The maternity nurse assists the community midwife during a home delivery. Besides that, after the delivery, she provides care to mother and child at home for about five until eight days. The communication between this nurse, the primary midwife and the patient is very important. ● Secondary care (hospital) midwife. This is an important target group because these midwives are often the first person to contact in case of referral from primary to secondary/tertiary care. Moreover, she is (together with the resident) the first person to contact the obstetrician to inform about the patient. ● The obstetrician and obstetric resident. In case of referral these medical doctors are the finals responsible for the follow-up of pregnancy/delivery. This group was interviewed to evaluate which aspects are crucial in the communication during medical handovers. Recruitment and intervention ---------------------------- All obstetric care providers being part of the Consortium Brabant were invited for participating in the study. The Consortium Brabant consists of six hospitals with in total 60 obstetricians. The surrounding primary care consists of approximately 120 primary care midwives organised in about 45 independent midwifery practices. One hospital, St Anna hospital Geldrop with lowest annual delivery rate of around 1.000, decided not to join the team trainings because of logistic reasons. The region of the Máxima Medical Centre was used for a pilot study, leaving four study groups with annual around 9,000 deliveries for this study project. Every hospital with its regional Obstetric Cooperation accounts for one study group. Within the study group, training teams will be formed consisting of ambulance staff (two per team), maternity nurses (one or two per team), primary care midwives (two to five per team), obstetric nurses (two per team), secondary care midwives (one or two per team), residents (one to three per team) and obstetricians (one to three per team), representing the entire obstetric collaborative network with a total of 12--18 care providers per team. There will be two instructors/facilitators per training: one medical instructor (obstetrician) and one communication expert. An expert panel, consisting of representatives of all obstetric care levels, designed obstetric scenarios for the team training, taking into account the topics that have resulted from the focus groups. Training will focus on process management of Big 4 disorders. The focus will mainly be on non-technical skills such as CRM, communication tools and using SBAR, and less on medical technical skills. The team training will take place at the medical education and simulation centre in Eindhoven, the Netherlands (Medsim) \[[@B40]\]. The medical simulation centre pursues a safe learning environment for trainees. The following four scenarios will be trained: 1\. Unexpected home delivery of fetus in breech presentation 2\. Extreme preterm delivery starting at home 3\. Home delivery with fetal distress and unexpected SGA 4\. Unexpected resuscitation of newborn with unexpected congenital heart abnormality at home. The scenarios are based on national and international guidelines \[[@B41]-[@B52]\]. Prior to the training, teams will receive an explanation concerning the equipment and environment. Each trainee will participate actively in at least one scenario and often more. Each scenario will start with an introductory briefing video. Thereafter, the team moves to the simulation room where they manage the simulated patient. State of the art high fidelity patient simulators will be used (Noelle™ and Newborn Hal™, Gaumard, Miami, Florida) and patient actresses. All scenarios will be videotaped (using B-Line Medical® software, Washington, DC). After each scenario a debriefing with reviewing the video recordings will be provided. The instructors will provide feedback on teamwork and skills (medical technical and non-technical) using video recordings. Learning goals based on CRM will be evaluated during the debriefing, such as: attention situational awareness, self-awareness, leadership, assertiveness, decision-making, flexibility, adaptability, and communication tools. There will be a focus on standardised communication and handovers based on the SBAR system (Situation, Background, Assessment, Recommendation). The nine elements of DB will, if achievable, be applicated to the training by: 1\) A syllabus concerning communication tools, CRM and medical knowledge about Big 4 disorders has been written and will be handed out prior to the training. To stimulate the motivation and concentration of the trainee a multiple choice exam prior to the training will be performed. 2\) Learning objects: a\. prior to the training, all trainees will be asked to define an individual learning goal b\. learning goals will be defined per specific scenario and will be evaluated during the debriefing c\. take home messages will be hand over to the teams at the end of the training. The teams will work on implementing learning goals in their Obstetric Cooperation. 3\) This training will not sufficiently be able to focus on an (individual) appropriate level of difficulty. In general, the scenarios have an increasing difficult level. 4\) This training is a one-time training making focused and repetitive practice not achievable. However, to achieve repetitive awareness and use of communication tools based on CRM and SBAR, the study groups will use pocket charts with communication tools in daily practice. 5\) Rigorous, reliable measurements: the knowledge of the trainees will be assessed by a multiple-choice exam before and after the training. 6\) Feedback will be provided during the debriefing. After each scenario a debriefing session will take place. The debriefing will exist of three phases: reaction of trainees, analysis of performance and take home messages. By reviewing video recordings, feedback will focus on predefined learning goals, on team performance and application of medicals skills. 7\) Monitoring and error correction will be performed by reviewing videotaped performance during the debriefing. 8\) Evaluation and performance that may reach a master standard: this is not achievable, since there is no definition of what the master standard would be 9\) Advancement to the next task: this is not realistic with a one-day training. After the training, all trainees will fill in an evaluation form about their experiences concerning the training in which they will score (0--5) for 36 different items. ### In situ simulation Four months after the intervention, the effect of training on team performance will be measured by so-called unannounced (as far as possible) in situ simulations, during which care providers are assessed on their teamwork within their own working environment. The in situ simulation will consist of one or two scenarios which will be managed by a team consisting of primary and secondary care providers, located at a delivery room in the hospital. The in situ simulation will be videotaped and analysed by independent experts. Hypothesis ---------- Multiprofessional simulation-based obstetric team training, using CRM and elements of DP, will improve perinatal outcome, team performance, quality of care as perceived by patients and collaboration of care providers. Questions to be answered: 1\. Does multiprofessional simulation-based obstetric team training improve perinatal outcome? 2\. Does multiprofessional simulation-based obstetric team training improve team performance as assessed by an unannounced in situ simulation? 3\. Does multiprofessional simulation-based obstetric team training improve quality of care as perceived by patients? 4\. Does multiprofessional simulation-based obstetric team training improve collaboration of care providers? Outcome measures ---------------- Primary outcome will be a composite adverse perinatal outcome as defined by perinatal mortality and/or NICU admission. Data on the primary outcome will be obtained from the Netherlands Perinatal Registry (PRN). Secondary outcomes will be: 1\. 1.Team performance. For measuring team performance, an independent panel of experts will evaluate the videotaped team training sessions and calculate the Clinical Teamwork Scale (CTS) \[[@B53]\]. 2\. Quality of care as perceived by patients. This will be measured before and after the training by using a questionnaire consisting of the validated PCQ and some additional questions regarding pregnancy, delivery and the first postpartum week \[[@B28]\]. 3\. Care providers' satisfaction with teamwork and collaboration between and within the different levels of care. This will be measured before and after the training using a questionnaire which is partly based on the validated Doctors' Opinions on Collaboration (DOC) questionnaire for general practitioners and medical specialists and adjusted to the obstetric care field \[[@B54]\]. 4\. Incidence of: a\. Big 4 disorders defined as \[[@B18]\]: Small for gestational age, defined as a birth weight below the 10^th^ percentile Preterm delivery before 37 weeks Congenital anomalies Five minute Apgar score below 7 b\. number of Big 4 pregnancies starting delivery in primary care c\. perinatal mortality d\. fetal mortality rate e\. neonatal mortality rate f\. NICU admission g\. admission to neonatology unit (non-NICU) h\. caesarean section i\. ventouse or forceps delivery j\. episiotomy k\. hemorrhage postpartum (\>1000 ml of blood loss) l\. third or fourth degree perineal trauma Sample size calculation ----------------------- In 2010 perinatal mortality rate was 0.9% and the NICU admission rate 2.3%. To avoid double telling, a composite rate of mortality and NICU admission will be around 3%. \[[@B5]\]. The sample size for the study was calculated by using the formula as proposed by Woertman and De Hoop \[[@B38]\]. This formula calculates the design effect required on top of the sample size calculation for a standard randomised clinical trial (RCT). To show a reduction in perinatal mortality and NICU admission rate from 3% to 1.65%, with an alpha of 0.05 and a power of 80%, a total of 4,000 deliveries would be needed for a simple RCT design. The design effect was calculated assuming an intracluster correlation (ICC) of 0.05, a cluster size of 1,800 deliveries per year, and four clusters or study groups. Taking into account the design effect, we need 565 deliveries per measurement period per cluster. To achieve this number we need 16 weeks for each period, adding up to a total study period of 82 weeks including a 16-week control period before the first training. A mixed effects model will be used to model the data and test the hypothesis of no effect from team training to accommodate cluster effects and time effects. Statistical significance will be accepted at a two-sided p-value \< 0.05. In the study region 9,000 deliveries occur per year, with a minimum of around 1,806 deliveries per year per study group (cluster) and a maximum of around 3,500. Discussion ========== As far as we know now, transmural multiprofessional simulation-based obstetric team training, using CRM and elements of DP, integrating the entire obstetric collaborative network, has never been studied before. We hypothesise that this obstetric team training improves perinatal outcome, team performance, quality of care as perceived by patients, and collaboration between care providers. The current project fits well within one of the main goals of the Dutch government to set up research that can prevent avoidable perinatal mortality and morbidity. Management of obstetric scenarios, based on the Big 4 causes of perinatal mortality, will be practiced in a medical simulation centre by teams with representatives of the obstetric collaborative network. The innovative aspect of the current project is the focus on non-technical skills (CRM, SBAR) rather than technical skills with the application of the elements of DP and defining learning goals based on CRM and the fact that different care providers will be trained together in one integrative cooperating team. Because team training and communication training has shown to be effective in secondary obstetric care \[[@B36]\], there is no reason to believe that this will not work within team training with integrating care providers from primary, secondary and tertiary care. To achieve a better ranking position concerning perinatal mortality rates in Europe, it is necessary to intensify an integrative organisation of obstetric care in the Netherlands in which all different care levels will integrate, in which uniform care paths will be developed forming 'patient centered care'. This is in line with the recent letter of the Minister of Health which has been sent to the House of Parliament in which she focuses on the development of an integrative obstetric health system. Abbreviations ============= CRM: Crew resource management; CTS: Clinical teamwork scale; DOC: Doctors' opinion on collaboration; DP: Deliberate practice; Medsim: Medical education and simulation centre Eindhoven; NICU: Neonatal Intensive Care Unit; OHC: Obstetric high care; PRN: National Dutch Perinatal Registry; SBAR: Situation, Background, Assessment, Recommendation; SBME: Simulation-Based Medical Education; SGA: Small for gestational age; SSF: Substandard factors; ZonMw: the Netherlands Organization for Health, Research and Development. Competing interests =================== The authors declare that they have no competing interests. Authors' contributions ====================== All authors were involved in conception and design of the study. All authors participated in the design of the study during several meetings and are local investigators at the participating centers. All authors drafted the manuscript. All authors edited the manuscript and read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6920/14/175/prepub> Acknowledgements ================ Funding ------- This study is funded by ZonMw, the Netherlands Organization for Health, Research and Development, project number 2009020010.

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Introduction ============ Chemical molecules, especially volatile ones, are the vessel of crucial information that may determine an animal's eventual survival and reproductive success. Perhaps for this reason, the sense of chemoreception is ubiquitously represented in the animal kingdom (Ache and Young, [@B2]). The role of the olfactory system is to decode the complex eddies of molecules in the environment and shape them into pieces of relevant information that will allow the animal to make decisions and engage in adapted behaviors. Major tasks of the olfactory system are for instance the identification of food sources, the detection of possible dangers (such as fire or predators), the recognition of potential mates as well as allowing social interactions. How the nervous system operates this transformation from the detection of chemical molecules via the formation of neural representations until the creation of percepts has been the focus of intense research especially in vertebrates (Lledo et al., [@B134]; Mori et al., [@B148]; Leon and Johnson, [@B131]; Mandairon and Linster, [@B135]) and in insects (Galizia and Menzel, [@B64]; Laurent, [@B129]; Galizia, [@B60]; Masse et al., [@B137]). A general finding of these studies is that the basic rules underlying olfactory processing in these different classes of animals are highly similar (Hildebrand and Shepherd, [@B96]; Ache and Young, [@B2]). For the most part, this resemblance is thought to result from evolutionary convergence due to similar constraints (Eisthen, [@B45]). Olfaction consists in a series of transformations from the chemical world of odor molecules into spatiotemporal patterns of neural activity in the animal's brain, eventually giving rise to a perceptual odor representation. Odor molecules exist in a myriad of chemical compositions, three-dimensional shapes, and vibration properties, to name but a few of their characteristics. They cannot be easily described based on simple dimensions like the wavelength and intensity of stimulus light when studying color vision. Therefore, only multiple descriptors can adequately describe an odorant molecule. In olfaction, the first transformation is thus the detection of particular features of the molecules by dedicated receptor (and associated) proteins, leading through a transduction of the signal to the activation of a subset of receptor cells (Touhara and Vosshall, [@B215]). This combinatorial code will then be conveyed to a series of structures in the brain and will undergo intense processing leading to a reformatting of the odor representation that will allow the extraction of the most relevant information for the system (Laurent, [@B129]; Kay and Stopfer, [@B111]). This processing will then give rise to a perceptual representation used for behavioral decision, and may link odor quality with hedonic value and learned relationships between odor and probable outcomes. For a century now, the honeybee *Apis mellifera* L. has been a key insect model in which behavioral, neuroanatomical, and neurophysiological approaches have been performed to unravel the basis of olfaction and olfactory learning. Honeybees are social insects which present a wide range of behaviors relying on olfaction both within and outside of the colony (Winston, [@B223]; Seeley, [@B194]). Moreover, the study of olfaction is easily amenable to the laboratory, since dedicated protocols have been developed in which bees show rapid and robust odor learning abilities (Menzel, [@B142]; Giurfa, [@B79]). In addition, the olfactory pathway of the honeybee brain has been extensively described (Kenyon, [@B114]; Mobbs, [@B146]; Strausfeld, [@B206]; Kirschner et al., [@B115]) and the bee brain is easily accessible to neurophysiological experiments like electrophysiological or optical imaging recordings (Galizia and Menzel, [@B64]; Sandoz et al., [@B181]). We will discuss in turn these different aspects. Olfactory Behavior in the Honeybee ================================== Role of pheromones in social life --------------------------------- Honeybees employ a rich repertoire of pheromones to ensure intraspecific communication in many behavioral contexts (Free, [@B58]; Slessor et al., [@B199]; Sandoz et al., [@B181]). The social organization of a honeybee colony is determined by chemical signals produced by the queen, but also by workers. Most honeybee pheromones are complex blends of many substances which are most effective when all components are present in appropriate ratios in the blend. The most important pheromonal components, which were sometimes used in olfactory learning experiments, are detailed below. The queen, the only fertile female in the colony, communicates her presence mostly by means of a mixture of substances released from her mandibular glands. The queen mandibular pheromone (QMP) was originally considered to be a unique substance, 9-oxo-(E)-2-decenoic acid (9-ODA) (Barbier and Lederer, [@B11]; Butler et al., [@B25]), but later studies revealed that the actual pheromone contains several additional components (Slessor et al., [@B198]; Keeling et al., [@B112]). The queen pheromone reinforces social cohesion, by attracting workers and enticing them to groom the queen. It also has a physiological effect on workers, inhibiting their ovarian development (Hoover et al., [@B97]) and modifying gene expression (Grozinger et al., [@B85]). An interesting aspect of this pheromone is that it acts on different receivers. The queen component 9-ODA thus also acts on males (drones) and plays a crucial role for in-flight mating, attracting them from as far as 60 m (Free, [@B58]). The second major source of pheromones is workers, who perform different tasks depending on their age (Winston, [@B223]). Aggregation pheromones are used by workers to mark and elicit attraction of other workers to important locations (profitable food source, potential nest site, etc.). This pheromone is a complex blend comprising many volatiles among which geraniol and citral are principal components (Pickett et al., [@B167]). On the other hand, alarm pheromones are released when confronting potential enemies (Breed et al., [@B22]). The main alarm pheromone is released near the sting and consists of more than 40 highly volatile compounds, among which the major component isopentyl acetate (IPA; Boch et al., [@B19]; Collins and Blum, [@B30]; Pickett et al., [@B166]). Release of this pheromone attracts other bees and causes them to sting and attack. Another alarm pheromone, 2-heptanone, is released by workers' mandibular glands (Shearer and Boch, [@B195]) and exerts a repellent action on potential intruders and robbers from other hives. Additionally, it is used by foragers to mark recently depleted flowers to avoid immediate revisit (Giurfa and Núñez, [@B81]). Role of floral odors in food search ----------------------------------- When reaching 2--3 weeks of age, workers engage in foraging for nectar or pollen outside the hive (Seeley, [@B193]). Honeybees are generalist pollinators and are not bound to a limited number of plants for gathering food. However, at the individual level, they are "flower constant," memorizing the features of a given floral species, and exploiting it as long as profitable (Grant, [@B82]; Chittka et al., [@B29]). Floral cues include color, odor, shape, and texture, but among those, odors play the most prominent role, being most readily associated with nectar or pollen reward (von Frisch, [@B219]; Menzel et al., [@B144]). The scent of a flower is a mixture of many volatile compounds that varies with respect to genotype, stage of development, and local environmental conditions (Pham-Delègue et al., [@B163]; Dobson, [@B40]; Dudareva and Pichersky, [@B42]). Flowers of the same plant may show differences in volatile compounds according to the time of day and with respect to their pollination status (Tollsten and Bergström, [@B214]; Schiestl et al., [@B187]). To maximize their profit from foraging, honeybees have to show good *olfactory discrimination* capacity. In other words, they have to be able to distinguish between fine differences in the volatile emissions of the visited flowers, to choose flowers whose volatile blend indicates good forage (Menzel, [@B141]). Indeed, honeybees are able to differentiate between very subtle differences in odor blends, as for instance between two genotypes of the same species or between flowering stages (Pham-Delègue et al., [@B163]; Wright et al., [@B226]). On the other hand, many of the variations in volatile emissions displayed by flowers are not indicative of any difference in reward quality, and therefore, another key ability is *olfactory generalization*. This ability corresponds to extending a behavior learned for a given stimulus to other, novel, stimuli, which are perceived as different, but sufficiently similar, to the learned one (Shepard, [@B196]). As for many lines of work about honeybee behavior and sensory capacities, both of these abilities were first recognized experimentally by Karl von Frisch. In a pioneering investigation, von Frisch ([@B218]) trained free-flying bees to visit an artificial feeder presenting several essential oils (odor mixtures). Using a set of 32 such odors, von Frisch observed that after learning that one odor was associated with sucrose solution, bees tended to prefer this odor over others, clearly discriminating among odors, although they also sometimes visited other odors that were, to the human nose, similar to the rewarded one, thus displaying clear generalization behavior. This work laid the ground to a plethora of experimental studies on the olfactory detection, perception, and learning capacity of honeybees with odors. Olfactory learning protocols in freely flying and restrained bees ----------------------------------------------------------------- Many experiments have been performed with free-flying bees visiting scented feeders (e.g., Kriston, [@B119], [@B120]; Pham-Delègue et al., [@B162]; Laska et al., [@B128]). These experiments have the advantage of providing an ecologically relevant context, but many variables of the experiment, like bees' physiological status, or the time intervals between learning trials, cannot be precisely controlled. Moreover, the search for the neural basis of olfaction needs the use of neurophysiological methods to monitor the bee brain while it processes and learns odors (Menzel, [@B142]; Giurfa, [@B79]). For these reasons, an experimental protocol allowing the study of olfactory learning on restrained individuals was developed, the conditioning of the proboscis extension reflex (PER; Figure [1](#F1){ref-type="fig"}A). The PER was initially described by Minnich ([@B145]) in flies and Frings ([@B59]) in bees. When the antennae, mouthparts, or tarsi of a hungry bee are touched with sucrose solution, the animal reflexively extends its proboscis to suck the sucrose. This response was later conditioned by Kuwabara ([@B123]) and Takeda ([@B213]), by associating visual and olfactory stimuli respectively with a sucrose reward. Perfecting the olfactory version of this protocol, Bitterman et al. ([@B17]) also showed that it corresponds to a case of associative Pavlovian conditioning. Odors to the antennae do not usually release a PER in naive animals. If an odor is presented immediately before the sucrose solution (forward pairing), an association is formed and the odor will subsequently trigger the PER in a following test (Figures [1](#F1){ref-type="fig"}A,B). Thus, the odor can be viewed as the conditioned stimulus (CS) and sucrose solution as the reinforcing unconditioned stimulus (US). This association is thought to recapitulate the final phase of the foraging behavior, when bees drink nectar from an odorous flower. ![**The study of olfactory learning and generalization in bees**. **(A)** Conditioning of the proboscis extension reflex (PER) on restrained bees. The PER is a reflex shown by bees when their antennae, tarsi, or mouthparts are contacted with sucrose solution. During conditioning, an odor (conditioned stimulus, CS) is presented in temporal association with sucrose solution to the antennae and to the proboscis (unconditioned stimulus, US), so that the odor progressively gains control over the PER \[see acquisition in **(B)**\]. After conditioning, presentation of the odor alone triggers the PER. **(B)** Generalization experiment. During acquisition, bees learn to associate the CS with the sucrose reinforcement and respond with a PER to the CS. In a test phase, bees are presented in a random order with the CS and novel odorants 1 and 2 (respectively NO1 and NO2). The perceptual similarity between the CS and each novel odor can be measured as the response level to this novel odor relative to responses to the CS. In this example, NO1 would be considered as more perceptually similar to the CS for bees, than NO2. **(C)** Frontal view of the bee head showing a three-dimensional model (from Brandt et al., [@B21]) of the brain. Olfactory processing follows three main steps. First, odors are detected at the level of the antenna. Information is conveyed to the antennal lobe (AL) for primary processing. Processed information is then relayed by different pathways to higher-order centers, the mushroom bodies (MB), and the lateral horn (LH), creating multiple olfactory representations in the bee brain (see Figure [4](#F4){ref-type="fig"}C).](fnsys-05-00098-g001){#F1} More recently, another type of Pavlovian conditioning protocol on restrained individuals was developed, which is based on aversive associations. The sting extension reflex (SER) is a defensive response of bees to potentially noxious stimuli (Breed et al., [@B22]), which can be elicited experimentally by delivering a mild electric shock to the thorax (Núñez et al., [@B152], [@B151]; Balderrama et al., [@B9]). During conditioning, harnessed bees learn to associate an initially neutral odor (CS) with the electric shock (US; Vergoz et al., [@B217]; Giurfa et al., [@B80]; Roussel et al., [@B174]). While PER conditioning is appetitive and induces attraction toward the CS in a choice test (Sandoz et al., [@B183]; Chaffiol et al., [@B27]; Carcaud et al., [@B26]), SER conditioning is aversive and bees will accordingly avoid the CS (Carcaud et al., [@B26]). Hence, olfactory processing, detection and learning capacities of honeybees can now be studied and compared with respect to different reinforcement modalities. Learning of odors with different biological meanings ---------------------------------------------------- The olfactory abilities and behavior of honeybees are the fruit of millions of years of co-evolution between hymenoptera and angiosperms. One could imagine that it would be beneficial to bees to not only be able to learn the features of rewarding flowers, but also to "know" in advance the sensory characteristics of a potential food source (Menzel, [@B141]). Do bees have an "innate search image"? It was initially expected that bees would only be able to learn floral odorants in an appetitive context, but the extreme plasticity of their olfactory learning behavior was soon recognized (von Frisch, [@B218]). While certain odorants are clearly attractive to bees prior to foraging (essential oils, aggregation pheromones), others are also clearly repulsive (propanol, 3-methyl indole). However, bees can still learn to associate these stimuli with sucrose reward, both in free-flying (Kriston, [@B119], [@B120]) and in restrained conditions (Vareschi, [@B216]). Nevertheless, some odorants will be learned more quickly than others (above studies), and after learning, may produce stronger or longer responses (Smith and Menzel, [@B203]). However, it is difficult to interpret such differences as truly "innate," since bees already learn odors within the hive (Farina et al., [@B51]) and may not be truly naïve when used in conditioning experiments. Interestingly, bees can even learn to associate pheromonal odors with sucrose reinforcement. This has been proven with aggregation pheromones (citral, geraniol, Getz and Smith, [@B75]; Smith, [@B200]; Laska et al., [@B128]), and even more surprisingly with alarm pheromones (IPA and 2-heptanone, Smith and Menzel, [@B203]; Laska et al., [@B128]; Sandoz et al., [@B184]). However, even though learning does take place, social pheromones do not seem to be treated like general odors (Getz and Smith, [@B77]; Sandoz et al., [@B184]). For instance alarm pheromones (IPA and 2-heptanone) produce very high generalization to other odors (Sandoz et al., [@B184]). Most puzzling, although these two molecules do not have a similar structure, very high generalization was observed between them, suggesting that bees may have also associated their biological value (here alarm) with the appetitive reward and used this information to generalize. Odor discrimination and generalization -------------------------------------- As explained above, stimulus discrimination and generalization are two crucial abilities for bees. To study discrimination, researchers use *differential* *conditioning* procedures: bees are repeatedly presented with two odors, one (CS+) that is associated with reinforcement, while the other (CS−) is presented without reinforcement. If bees respond significantly more to the CS+ than to the CS−, it can then be concluded that they can discriminate between them. To study generalization, bees are simply conditioned to one odorant (CS) and are then presented with novel odorants without reinforcement (Figure [1](#F1){ref-type="fig"}B). The perceived similarity between the CS and each novel odorant is measured as the level of response to this odorant relative to the CS (amount of generalization, Figure [1](#F1){ref-type="fig"}B). An important question in sensory neuroscience is along which dimensions animals measure similarity among stimuli (Shepard, [@B196]). Vareschi ([@B216]) was the first to use PER conditioning to study the discrimination capacities of honeybees with a wide range of odors. He used a kind of differential conditioning, with one rewarded odor (CS) and 27 non-rewarded odors presented in-between CS trials. Bees were found to differentiate the odors from \>95% of the 1816 tested odor pairs. The same high discrimination ability is also found in free-flying bees (97% of 1848 tested odor pairs, Laska et al., [@B128]). In the bee, as in vertebrates (Mori et al., [@B148]; Johnson and Leon, [@B108]), aliphatic odor molecules have attracted the interest of researchers because they can be described by two main characteristics: their chemical group and the length of their carbon chain. Bees generalize more often between odors with similar carbon chain lengths or belonging to the same functional group, as found with restrained (Smith and Menzel, [@B203]) and with free-flying bees (Getz and Smith, [@B76]; Laska et al., [@B128]). Recently, Guerrieri et al. ([@B88]) systematically studied the generalization behavior of bees with 16 odorants presenting all combinations of four possible functional groups (primary and secondary alcohols, aldehydes, ketones) and chain lengths (six to nine carbons). These authors found that generalization is not always symmetrical, so that generalization from odor A to odor B is not always the same as from B to A. Strikingly, learning an aldehyde induced low generalization to other odors, while bees often responded to aldehydes after learning other odorants. In this study, the first factor determining honeybees' generalization behavior was a molecule's chain length, followed by the chemical group. This was the demonstration on a simple set of odor molecules that chemical dimensions are somehow encoded in the brain of honeybees and determine their behavior (Guerrieri et al., [@B88]). However the bees' natural environment provides an incredible wealth of possible odor molecules and we are still far from knowing the encoding dimensions for all these molecules. Odor concentration ------------------ The fact that honeybees are able to learn absolute odor concentrations was recognized by Kramer ([@B117]), who trained individual workers in simulated odor gradients using a locomotion compensator with feedback control of odor concentration. The bees were reinforced with sucrose solution at a particular concentration of an odor, and were then placed at different concentrations. They showed a typical upwind walk when placed in a range of concentrations relatively close to the learned one (20--180%), but walked downwind when placed outside of these boundaries. Moreover, bees showed a particular alerting behavior at about 85--90% of the learned concentration (Kramer, [@B117]). Similarly, free-flying bees visiting a vertical odor array choose the right odor at the right concentration and reject higher or lower concentrations (Ditzen et al., [@B39]). In contrast to the freely moving situation, differential conditioning with two concentrations of the same odor is difficult in harnessed bees (Bhagavan and Smith, [@B14]; Pelz et al., [@B159]). Honeybees' sensory capacity and motivation may be different in these two situations. In the visual modality, for instance, honeybees easily associate colors or patterns with sucrose reward when flying freely, but show much lower performance when restrained (especially when the antennae are not cut, Hori et al., [@B98]; Mota et al., [@B149]). Concentration strongly influences the salience of olfactory stimuli. Generally, odors are learned more quickly at higher concentration (Bhagavan and Smith, [@B14]; Wright et al., [@B225]), and support better memory consolidation (Pelz et al., [@B159]). Moreover, conditioned responses to a high concentration are produced more quickly, suggesting that the olfactory system needs less time to determine odor quality at high than at low concentration (Wright et al., [@B225]). The discrimination power between different odorants also increases with their concentration (Getz and Smith, [@B77]; Wright and Smith, [@B227]). Lastly, bees generalize more from low to high concentrations, than from high to low concentrations (Marfaing et al., [@B136]; Getz and Smith, [@B77]; Bhagavan and Smith, [@B14]; Pelz et al., [@B159]). However, in some instances bees generalize more between different odors at the same concentration, than between different concentrations of the same odor (Wright et al., [@B228]). To summarize, odor identity is not totally invariant as a function of concentration, so that it is both possible for bees to differentiate between concentrations of an odorant, but also to show high generalization between different concentrations of this odorant. Such versatile capacities may be crucial when foraging for identifying and locating floral sources. The case of olfactory mixtures ------------------------------ Natural floral odors encountered by foraging bees are not single molecules but complex mixtures (Knudsen et al., [@B116]). Honeybees are thus confronted to the problem of discriminating among complex blends but also of recognizing the same floral source although its blend composition varies. Some authors have attempted to understand complex mixture processing in learning experiments with whole floral extracts (Pham-Delègue et al., [@B164]; Le Métayer et al., [@B130]) or with synthetic mixtures of six to 14 components (Pham-Delègue et al., [@B162]; Wadhams et al., [@B221]; Blight et al., [@B18]; Reinhard et al., [@B169]). A general finding of these experiments is that when bees learn a mixture and are afterward tested with the individual components, they usually respond to some components more strongly than to others. Such components have been termed key-compounds (or key-components, Wadhams et al., [@B221]; Laloi et al., [@B127]; Reinhard et al., [@B169]). What determines that a component is a key-component? Neither relative quantity nor volatility are predictive (Wadhams et al., [@B221]; Le Métayer et al., [@B130]; Reinhard et al., [@B169]). Rather, the perceptual salience of a component appears to be important, as measured by the conditioning success with this odor presented alone (Laloi et al., [@B127]). Additionally, whether a component will be learned in a mixture depends on the identity of the other components (Laloi et al., [@B127]; Reinhard et al., [@B169]). Thus, the processing of different odorants simultaneously produces unpredictable outcomes, a phenomenon termed "mixture interaction." Due to the apparent complexity of mixture processing, research on mixture interactions has focused on binary mixtures (Getz and Smith, [@B75], [@B76], [@B77]; Chandra and Smith, [@B28]; Smith, [@B201]; Deisig et al., [@B36]). Generally an odor is better learned when presented alone, than when together with a second odorant (Smith, [@B201]). Usually, when learning a mixture AB, bees can recognize the components (Getz and Smith, [@B75], [@B76]). However, one component is often learned better than the other, a phenomenon called "overshadowing." Using three odors presented in the form of binary mixtures, Smith ([@B201]) found that overshadowing depended on which odors were in a pair, so that overshadowing was difficult to predict. Mixture interactions may also depend on the sequence of experiences the bee has had with the stimuli. In the phenomenon of "blocking," initial learning of an odorant A blocks learning of odorant B when the mixture AB is subsequently trained. Although this effect has been observed in different studies (Smith and Cobey, [@B202]; Linster and Smith, [@B133]; Hosler and Smith, [@B99]), it remains controversial, as it only rarely appeared when possible confounding variables were controlled (Gerber and Ullrich, [@B74]; Guerrieri et al., [@B87]). On a theoretical level, concepts from psychophysical theories have been used to attempt to understand how a mixture is represented in the bee brain (Chandra and Smith, [@B28]; Deisig et al., [@B36]; Lachnit et al., [@B126]). Two widely differing theories have been put to the test. First, the elemental approach assumes that a compound AB will be represented in the brain as two elements, A and B, each of which can be associated with the US (Rescorla and Wagner, [@B172]). In other terms, "the whole equals the sum of its parts." On the other hand, configural approaches propose a radically different view by assuming that the representation of AB is a different entity from those of A and B ("the whole is different from the sum of its parts," Pearce, [@B156], [@B157]). In fact both accounts were shown to be wrong using the so-called patterning experiments (Chandra and Smith, [@B28]; Deisig et al., [@B36]). In such experiments, bees have to differentiate between two single odorants A and B and the mixture AB. In negative patterning, the single elements are both reinforced when presented alone (A+, B+), while the mixture is non-reinforced (AB−). Conversely, in positive patterning, the two elements are non-reinforced when presented alone (A−, B−), while the mixture is reinforced (AB+). Honeybees can be trained to solve both tasks with odors (Deisig et al., [@B36], [@B35]). The elemental approach can explain positive patterning but not negative patterning, because when each component is reinforced, a mixture would elicit, through elemental summation, twice as much responding as each component. On the other hand, in its principle, the configural approach could cope with both patterning tasks, as compound and elements are associated with reinforcement independently (Pearce, [@B157]). However, it ran into problems when analyzing response summation between elements and mixtures at the beginning of conditioning (Deisig et al., [@B37]). Experiments in bees thus suggested that the best model for explaining mixture learning was an expansion of elemental models, called the unique cue hypothesis (Rescorla, [@B170], [@B171]; Whitlow and Wagner, [@B222]). In addition to the representations of the elements, the compound would give rise to a supplementary (internal) representation, the *unique cue*. During the negative patterning problem, the unique cue U would build inhibitory associations with the US, while the elements A and B would build excitatory associations. When A and B are presented alone, the excitatory association would thus trigger behavioral responses, but during AB presentations, the added inhibitory strength of the unique cue would hamper the response. Using different types of patterning tasks, it was possible to show that a version of the unique cue hypothesis best coped with all the experimental results (Deisig et al., [@B36], [@B35], [@B37]; Lachnit et al., [@B126]). For simplicity, all experiments described above considered that mixture composition is stable in time, which is not the case in nature, as floral aroma changes throughout the day and the plant's state. Honeybees seem to focus on those components which remain relatively constant in their concentration (Wright and Smith, [@B227]). Such ability may be beneficial for bees in order to recognize the same floral species in spite of fluctuations in the composition of its odor blend. Neuroanatomy of the Honeybee Olfactory System ============================================= An advantage of the bee model for understanding olfaction and olfactory learning is that the neuroanatomy of its olfactory pathway is known in great detail (e.g., Kenyon, [@B114]; Pareto, [@B155]; Suzuki, [@B210]; Mobbs, [@B146]; Abel et al., [@B1]; Strausfeld, [@B206]; Kirschner et al., [@B115]). Olfactory processing follows different steps, from the detection of molecules at the periphery, via primary processing by antennal lobe (AL) networks, until the establishment of olfactory representations in higher-order brain centers (Figure [1](#F1){ref-type="fig"}C). A simplified model of the different neuron types involved in olfactory processing is provided in Figure [2](#F2){ref-type="fig"}. {#F2} Peripheral odor detection: the antenna -------------------------------------- Peripheral odor detection starts at the level of olfactory receptor neurons (ORNs), which are located below cuticular structures on the antennae, called sensilla (Kaissling, [@B110]). Different morphological types of sensilla exist on the insect antenna, but sensilla placodea (pore plate sensilla) are the main olfactory sensilla in the honeybee (Esslen and Kaissling, [@B47]). A sensillum placodeum is formed by an oval-shaped (9 μm × 6 μm) thin cuticular plate with numerous minute pores and is innervated by five to 35 ORNs (Schneider and Steinbrecht, [@B188]; Esslen and Kaissling, [@B47]; Kelber et al., [@B113]). Odorant molecules reach the dendrites of ORNs by diffusing through an extracellular fluid, called the receptor or sensillum lymph, filling the sensillum cavity (Kaissling, [@B110]; Masson and Mustaparta, [@B139]). In this fluid, odorant binding proteins (OBPs) may help transporting odorants to the ORNs but very little is known about them in bees. When reaching the ORN membrane, the odorant molecule interacts with the olfactory receptor protein (OR). Insect ORs belong to a family of highly divergent proteins with seven-transmembrane domains, which are different from the vertebrate OR family (Benton, [@B12]; Touhara and Vosshall, [@B215]). The functional receptor is a heteromeric complex of an OR and a broadly expressed co-receptor AmOr2, which is the honeybee ortholog to the co-receptor Or83b of *Drosophila* (Benton et al., [@B13]; Robertson and Wanner, [@B173]). Honeybees present a remarkable expansion of the insect odorant receptor family relative to the repertoires of the fly *Drosophila melanogaster* and the mosquito *Anopheles gambiae*, which respectively possess 62 and 79 ORs, with a total of 170 OR genes including seven pseudogenes (Robertson and Wanner, [@B173]). The primary olfactory center: the antennal lobe ----------------------------------------------- ORN axons form the antennal nerve and project to a primary olfactory center in the brain, the AL (Figure [2](#F2){ref-type="fig"}). The bee AL is compartmentalized in 165 spheroidal neuropile units called glomeruli. Glomeruli are the anatomical and functional units of the AL and constitute the first site of synaptic interaction between ORNs and other neuron types. Glomeruli can be recognized based on their relative position, size, and shape, using an anatomical atlas of the AL (Flanagan and Mercer, [@B55]; Galizia et al., [@B62]). In *Drosophila* axons of ORNs expressing the same odorant receptor converge onto the same glomerulus (Vosshall et al., [@B220]; Dahanukar et al., [@B32]). Thus, the array of AL glomeruli corresponds to an array of OR types. Noticeably, the number of 163 potentially functional ORs in bees coincides with the number of glomeruli in the AL (\~165). This would thus support the one-receptor/one-ORN/one-glomerulus hypothesis in bees. Within each glomerulus, ORNs release acetylcholine (ACh), the primary excitatory transmitter of the insect brain (Bicker, [@B15]). Thus doing, they activate local neurons (LNs) connecting different glomeruli and projection neurons (PNs), which relay the olfactory message processed at the level of the AL to higher-order centers such as the lateral horn (LH) and the mushroom bodies (MBs). Local neurons are neurons whose branching patterns are restricted to the AL (Figure [2](#F2){ref-type="fig"}). The \~4000 LNs can be classified in two main types. One type innervates most if not all glomeruli in a uniform manner, and are therefore called homogeneous LNs (homo-LNs; Flanagan and Mercer, [@B56]; Fonta et al., [@B57]). Neurons of the second type innervate only a small subset of glomeruli and are called heterogeneous LNs (hetero-LNs). They have one dominant glomerulus with very dense innervation and a few other glomeruli with very sparse processes (Flanagan and Mercer, [@B56]; Fonta et al., [@B57]). Hetero-LNs branch in the core of the sparsely innervated glomeruli but branch in the whole (core and cortex) of their densely innervated glomerulus (Fonta et al., [@B57]; Abel et al., [@B1]). LNs are thought to carry out the first processing of olfactory information, with two different functions of Homo-LNs and Hetero-LNs, respectively global inhibition for gain control and asymmetrical lateral inhibition between glomeruli for refining odor representation and allowing better discrimination among olfactory representations (Sachse and Galizia, [@B177]). Local neurons in bees are mostly inhibitory and may use many different neurotransmitters. About 750 LNs are GABAergic (Schäfer and Bicker, [@B185]) and functional data indicate that GABA is indeed inhibitory in the AL (Stopfer et al., [@B204]; Sachse and Galizia, [@B177]; Dupuis et al., [@B43]). In addition, glutamate (for review see Bicker, [@B15]) and histamine (only about 35 neurons, Bornhauser and Meyer, [@B20]) have been identified in the AL. Several lines of evidence indicate that glutamate (Barbara et al., [@B10]; El Hassani et al., [@B46]) and histamine (Sachse et al., [@B179]) also act as inhibitory neurotransmitters in the bee brain. Lastly, the AL houses many, often small, subpopulations of LNs which each express characteristic peptides, including allatostatins, allatotropin, tachykinins, FMRFamide, and other RFamide peptides (Galizia, [@B60]; Kreissl et al., [@B118]). Projection neurons connect the AL with higher-order brain areas (Figure [2](#F2){ref-type="fig"}), following five different pathways, called antenno-protocerebral tracts (APTs; Mobbs, [@B146]; Abel et al., [@B1]). From their morphology, PNs can also be classified in two types. Uniglomerular projection neurons (uPNs) branch in a single glomerulus within the AL and have axons that project to the MBs and to the LH using the two major APT tracts (see below). On the other hand, multiglomerular projection neurons (mPNs) branch in most glomeruli and are therefore potentially capable, in contrast to uPNs, to extract combinatorial information across glomeruli. Their axons follow the three lesser tracts, the medio-lateral (ml) APTs, leading not to the MBs, but to other areas of the protocerebrum, surrounding the α-lobe of the MB or extending toward the LH (Abel et al., [@B1]; Kirschner et al., [@B115]). The more numerous uniglomerular PNs (\~800) form two roughly equal tracts toward higher-order brain centers, the lateral (l-APT), and medial (m-APT) tract. The l-APT leaves the AL dorsally and then runs on the lateral side of the protocerebrum, forming collaterals in the LH and then continuing on to the MB calyces. The m-APT runs along the brain midline first toward the MBs where collaterals enter into the calyces, and then travels laterally to end in the LH (Abel et al., [@B1]; Kirschner et al., [@B115]). The current understanding of this anatomical organization is that the honeybee brain may arbor two parallel olfactory subsystems, as excitatory transmission of the olfactory message follows essentially two independent pathways in each brain hemisphere toward higher-order brain centers. L- and m-APT neurons take their information from two non-overlapping groups of 84 and 77 glomeruli respectively (Abel et al., [@B1]; Kirschner et al., [@B115]). Following the current hypothesis that each glomerulus is the projection center for all ORNs expressing a given OR, one may thus say that PNs from the l- and m-APT each transmit information about two independent portions of the honeybee odor detection repertoire. Moreover, the central projection areas of the two PN tracts are segregated in the MB calyces and in the LH with only partial overlap (Abel et al., [@B1]; Kirschner et al., [@B115]). How are the two subsystems connected through local networks? Several hypotheses are possible, from almost total segregation of the subsystems with hetero-LNs providing lateral inhibition only within each system, to an equal and symmetrical weight of lateral inhibition between both subsystems (Galizia and Rössler, [@B67]). However, no data are available yet concerning this question. Second-order olfactory centers: the mushroom bodies and the lateral horn ------------------------------------------------------------------------ Olfactory information leaving the AL takes several routes to the MBs and LH (Figure [2](#F2){ref-type="fig"}). While the function of the LH is still unclear, the MBs are thought to be involved in a sparsening of olfactory representation, in olfactory learning and memory, as well as the integration of olfactory information with other sensory modalities (Menzel et al., [@B143]; Menzel, [@B142]; Giurfa, [@B78], [@B79]). MB-intrinsic neurons are the Kenyon cells (KCs; Kenyon, [@B114]), which form two cup-shaped regions called calyces in each hemisphere. MB calyces are anatomically and functionally subdivided into the lip, the collar, and the basal ring (Mobbs, [@B146], [@B147]; Gronenberg, [@B84]; Strausfeld, [@B206]). The lip region and the inner half of the basal ring receive olfactory input, whereas the collar and outer half of the basal ring receive visual input (Gronenberg, [@B84]), in addition to input from mechanosensory and gustatory pathways (Strausfeld, [@B206]; Schröter and Menzel, [@B191]). The projections of individual PNs extend in most parts of each calyx (Müller et al., [@B150]). PN boutons form multisynaptic microcircuits in the MB lips, with GABAergic input and KC output connections arranged to form particular structures termed microglomeruli (Ganeshina and Menzel, [@B70]). KC axons project in bundles into the midbrain, forming the peduncle and the vertical and horizontal lobes (also called α and β lobes). The calyx is topologically represented in the lobes (Mobbs, [@B146]; Strausfeld et al., [@B207]; Strausfeld, [@B206]). About 55 GABAergic feedback neurons from the MB output lobes project back to the calyces (Bicker et al., [@B16]; Figure [2](#F2){ref-type="fig"}). Due to the parallel arrangement of intrinsic KCs, most subcompartments in the calyx receive feedback from their corresponding layer in the α lobe (Grünewald, [@B86]). Most KCs provide bifurcating axons to both α and β lobes. In the bee, about 800 PNs diverge onto a major proportion of the 170,000 KCs of each MB (i.e., onto olfactory KCs). Each PN contacts many KCs and each KC receives input from many PNs. If the figures calculated for the locust *Schistocerca americana* (Jortner et al., [@B109]) were to apply to the honeybee, each KC would contact about 400 PNs (i.e., 50% of the total PN count). This organization appears ideal for a combinatorial readout across PNs (Laurent, [@B129]). The second major target area of both the m- and l-APT uPNs is the LH. In addition to the uPN innervation, the LH receives input from mPNs via the ml-APTs (Fonta et al., [@B57]). Similarly to the olfactory input of the MB calyx, the LH shows a PN tract-specific compartmentalization, with at least four subcompartments: one receives exclusively projections of m-APT uPNs, while others receive mixed input from m- and l-APT PNs, from l-APT and ml-APT PNs, or from the latter type alone (Kirschner et al., [@B115]). Possible local computations within this structure as well as the connectivity between PNs and other neurons are still mostly unknown. Mushroom body output -------------------- A number of neuron populations project from the MBs toward other brain centers (Figure [2](#F2){ref-type="fig"}), with two major output regions being the α and β lobes (Mobbs, [@B146]). About 400 extrinsic neurons (ENs) from the α lobe have been studied in details (Rybak and Menzel, [@B175]). Some are unilateral neurons with projection fields restricted to the ipsilateral protocerebrum, while others are bilateral neurons connecting both α lobes, or projecting from one lobe to the contralateral protocerebrum around the α lobe (Rybak and Menzel, [@B175]). A single conspicuous neuron in each MB, called Pe-1, forms a major output pathway from the peduncle of the MBs (Mauelshagen, [@B140]). This neuron arborizes extensively in the peduncle and projects to the lateral protocerebral lobe, and more specifically to the LH where it synapses directly or via interneurons onto descending neurons involved in behavior. Although the anatomical description of projections to the LH is good, knowledge of the neurons leaving the LH and of descending pathways involved in behavioral output is still scarce in bees. Some anatomical descriptions of descending neurons in other insects, like cockroaches, suggest that their dendrites are distributed mainly in the lateral and medial protocerebrum, which are major termination areas of MB output neurons, but not in the AL, MBs, or regions of the LH receiving PN input (Okada et al., [@B154]). Thus it is possible that both MB output neurons and yet unknown LH output neurons contact descending neurons and can therefore modulate behavior. Investigation of descending neurons and the neural pathways involved in behavioral control in bees may help bridge this gap (Ibbotson and Goodman, [@B103]; Ibbotson, [@B102]; Schröter et al., [@B190]). Aversive and appetitive reinforcement information ------------------------------------------------- The olfactory pathway also receives input from different modulatory systems. Of special importance are the reinforcement systems necessary for the formation of neural associations between odors and particular outcomes. Such associations rely on the co-activation of two neural pathways, the olfactory pathway and a pathway representing the specific reinforcement. As in other insects, appetitive reinforcement in bees depends on octopamine (Hammer and Menzel, [@B94]; Farooqui et al., [@B52]) and aversive reinforcement on dopamine (Vergoz et al., [@B217]). A single, putatively octopaminergic, neuron in the bee brain, VUM-mx1 (Figure [2](#F2){ref-type="fig"}), was shown to represent a neural substrate of the sucrose US pathway (Hammer, [@B93]), because the forward (but not backward) pairing of an odor CS with an artificial depolarization of VUM-mx1 produces an associative memory trace. VUM-mx1, has its cell body in the suboesophageal ganglion (SOG), and converges with the olfactory pathway in both brain hemispheres at three sites, in the AL, in the MB calyces, and in the LH. Another neuron with a similar projection pattern has been found with its cell body in another neuromere of the SOG (VUM-md1, Schröter et al., [@B190]). On the other hand, many dopaminergic neurons are found in the bee brain (Schäfer and Rehder, [@B186]; Schürmann et al., [@B192]) but until now, none of them could be shown to provide aversive reinforcement. Neurophysiological Study of Olfactory Processing and Learning ============================================================= Peripheral odor detection: the antenna -------------------------------------- The search for the neural correlates of olfactory detection and processing has started at the periphery, using extracellular recordings of single placodes (e.g., Lacher and Schneider, [@B125]; Lacher, [@B124]; Vareschi, [@B216]; Akers and Getz, [@B3], [@B4]). The first demonstration that placode sensilla were responsible for olfactory detection was provided by Lacher and Schneider ([@B125]) with individual placode recordings showing responses to benzylacetate in workers, and caproic acid in drones, but no answers to light, sound, water vapor, or CO~2~. ORNs show little spontaneous activity, but respond to odors mostly with a spike frequency increase in a phasic-tonic manner. Sometimes, they also show inhibitory responses or off-responses (response at the end of the stimulus). Because a single placode houses many ORNs it is difficult to segregate the responses of individual cells based on spike amplitude. Using statistical techniques to attempt such segregation, Akers and Getz ([@B3]) found that units with similar odor-response spectra were more likely to be found in different placodes than within the same placode. This observation fits with anatomical data showing that ORNs from a placode innervate different glomeruli (Kelber et al., [@B113]). The complex organization of ORNs in the honeybee antenna has strongly hindered efforts to study peripheral odor detection in this species. Olfactory processing in the antennal lobe ----------------------------------------- Thanks to the technique of *in vivo* calcium imaging, it was however possible to record neural activity at the glomerular level (Joerges et al., [@B107], Figure [3](#F3){ref-type="fig"}). This recording technique uses fluorescent dyes to measure the increase of intracellular calcium (coming from the extracellular medium and/or released from internal stores) following neuronal excitation (Joerges et al., [@B107]; Galizia and Vetter, [@B69]). In the most simple form of this technique, bees are fixed in a recording chamber, and the head capsule is carefully opened (Figures [3](#F3){ref-type="fig"}A,B). Membranes and tracheas covering the brain are removed, and a calcium-sensitive fluorescent dye (for instance, Calcium Green 2-AM) is bath-applied onto the brain (Figures [3](#F3){ref-type="fig"}C,D). After about 1 h incubation, during which the dye has penetrated AL cells, the brain is rinsed with saline solution and the bee is placed under an upright fluorescence microscope in front of an odor stimulation device (Figure [3](#F3){ref-type="fig"}A). This bath-application of Calcium Green 2-AM allows recording a composite calcium signal which could potentially come from all cell populations of the AL: ORNs, LNs, PNs, and glial cells (Joerges et al., [@B107]). Due to the numerical preponderance of ORNs and because odor-induced signals have a very stereotypical time course and do not show spontaneous activity or inhibitory responses (the hallmark of LNs and PNs, see below), these recordings are thought to emphasize presynaptic calcium variations from ORNs (Galizia et al., [@B66]; Sachse and Galizia, [@B178]), with a possibly significant contribution from glial cells surrounding each glomerulus (Galizia and Vetter, [@B69]). The compound signal has therefore been long interpreted as representative of sensory input (Sachse and Galizia, [@B178]; Deisig et al., [@B33], [@B34]; Sachse et al., [@B179]). ![**Optical imaging of odor representations in the bee brain**. Thanks to *in vivo* calcium imaging, odor representation can be recorded in the bee brain. **(A)** Bees are placed under an epifluorescence microscope in front of an odor-delivery device delivering a permanent airflow. **(B)** Their head capsule is opened revealing the brain, which is then kept under saline solution at all times, while the antennae are maintained in the airflow. **(C)** Example view of the brain surface after bath-application of a calcium dye (method 1, below). The recording can be restrained to the region corresponding to the antennal lobe (square). **(D)** Using different staining techniques, odor representation was recorded at different levels of olfactory processing. On the left the staining technique and the imaged neuronal population are shown, while on the right, activity maps evoked by two sample odorants (1-hexanol and linalool) as well as an exemplary time course are presented. (1) Using bath-application of a calcium-sensitive dye (Calcium-Green 2-AM), a compound signal can be recorded in the antennal lobe in response to odors (Joerges et al., [@B107]). This signal is thought to represent mostly olfactory input from the ORN population (see text). Different odors induce different, but overlapping, multiglomerular activity patterns. Bath application signals are temporally slow and biphasic. (2) Using retrograde staining with a migrating dye (Fura-2 dextran), projection neurons can be selectively stained (Sachse and Galizia, [@B177]). A dye-coated electrode is inserted into the PNs axon tract (arrow number 2). The dye is taken up by the neurons and migrates back to their dendrites in AL glomeruli. Such staining allows the selective recording of AL output information sent to higher-order centers. Odors also induce multiglomerular activity patterns, but these are scarcer (less glomeruli are activated) and more contrasted than the compound signals. The time course is mostly phasic-tonic, but also presents some complex temporal patterns and inhibitions. (3) Inserting the dye-coated electrode into the ventral part of the vertical lobe allowed recording activity from Kenyon cell dendrites and somata (Szyszka et al., [@B211]). Olfactory representation becomes even sparser in the MBs as few KCs respond to each odorant. Responses are phasic and often present off-responses at stimulus offset. (Recordings 1 and 2 from Deisig et al., [@B33], [@B34] -- Recordings 3 from Szyszka et al., [@B211]).](fnsys-05-00098-g003){#F3} Optical imaging experiments showed that odors elicit combinatorial activity patterns across glomeruli (Joerges et al., [@B107]; see Figure [3](#F3){ref-type="fig"}D). Combining imaging recordings with anatomical staining allowed assigning activity patterns to identified glomeruli using the published anatomical AL atlas (Galizia et al., [@B62]). This showed that odor quality is represented in the AL according to a specific distributed code conserved between individuals (Galizia et al., [@B63]; Sachse et al., [@B180]). Each glomerulus -- representing an ORN type expressing a given OR -- shows a rather broad molecular receptive range (Galizia et al., [@B63]). Because the optical imaging technique allows recording activity only at the tissue surface, only a small part of the 165 glomeruli could be accessed (up to 38 glomeruli; Sachse et al., [@B180]). The question therefore arose whether the signals recorded in this subpart of the AL had any significance with regards to odor representation and olfactory behavior. To answer this question, Guerrieri et al. ([@B88]) studied the generalization behavior of honeybees among a panel of 16 odorants for which the activity patterns in these glomeruli were known (Sachse et al., [@B180]). As mentioned above, these authors built a complete generalization matrix among the 16 odorants differing according to their functional group and their carbon chain length (Figure [4](#F4){ref-type="fig"}A). Importantly, this work demonstrated for the first time a significant correlation between the similarity among odors in the behavior and in the neurophysiological recordings (Figure [4](#F4){ref-type="fig"}B). Thus, calcium signals in this subpart of the AL could to some extent allow predicting bees' generalization behavior. As shown on the figure, the data showed however some scatter and the question whether extending the neurophysiological recordings to more glomeruli, or to other parts of the brain may ameliorate this prediction remains unanswered. In theory, the bee brain contains different sets of olfactory representations in its different olfactory structures, which each can be characterized by an odor-similarity matrix based on combinatorial activity of its neuronal units (Figure [4](#F4){ref-type="fig"}C). It will be the goal of future research to compare the capacity of these different levels for predicting bees' olfactory behavior. {ref-type="fig"}B, it was possible to measure the perceptual similarity among all possible pairs of 16 aliphatic odorants. The table presents the amount of generalization between any two of these odors. Odors used for conditioning are presented vertically, while odors used in the generalization tests are presented horizontally. Bees respond preferentially to the learned odor (main diagonal), but also to other -- perceptually similar -- odors. For instance, they generalize between odors sharing the same carbon chain length (smaller diagonals) or the same functional group (boxes along diagonals, see for instance the high generalization among aldehydes). **(B)** Similarity among odors at the neural level (measured in the AL using bath-application of the calcium dye, method 1 in Figure [3](#F3){ref-type="fig"}) significantly correlates with similarity at the behavioral level, as measured in **(A)**. Thus, the AL contains a neural representation of odors which allows predicting to some extent the bees' olfactory behavior. **(C)** At its different processing levels, the bee brain is thought to contain multiple odor representations, which can be characterized by different odor-similarity matrices. Sequential and/or parallel transformation of olfactory information shapes odor representations in higher-order centers that would eventually determine olfactory behavior. Thus, higher-order representations should correlate more strongly with behavioral output than more peripheral representations \[like AL input, see **(B)**\]. As shown in Figure [3](#F3){ref-type="fig"}, we have access to representations at the level of ORNs, PNs, and KCs corresponding to the l-APT subsystem. There, a sparsening of olfactory representation is found, but its influence on predicting behavior is still unknown. In addition, representations in the m-APT subsystem, as well as in the LH are still unknown and should be studied in future work.](fnsys-05-00098-g004){#F4} AL neurons are involved in the processing of incoming odor information provided by ORNs. Intracellular recordings of LN and PN responses provided some insights about this processing (Flanagan and Mercer, [@B56]; Sun et al., [@B209]; Stopfer et al., [@B204]; Abel et al., [@B1]; Müller et al., [@B150]; Krofczik et al., [@B122]). LNs are odor-specific, responding in a differential manner to different odors. They can show excitatory responses to some odors and inhibitory responses (i.e., a reduction of spiking activity relative to background) to others (Sun et al., [@B209]). Staining of hetero-LNs allows identifying the glomerulus in which this LN most intensively branches (Galizia and Kimmerle, [@B61]). Generally, the response profile of the recorded LN corresponded to the known response profile of the innervated glomerulus, suggesting that hetero-LNs take their input in this glomerulus. LNs tend to show a shorter latency than PNs, which allows them to rapidly and efficiently inhibit the firing of PNs (Krofczik et al., [@B122]). Projection neuron responses are the product of direct excitation from ORNs, direct inhibition from LNs and possibly disinhibition from LN--LN connections and can therefore be temporally complex (Sun et al., [@B209]; Müller et al., [@B150]). PNs are usually spontaneously active and can change their responses upon odor presentations in an either excitatory or inhibitory manner (respectively increasing or decreasing firing rate; Abel et al., [@B1]; Müller et al., [@B150]). However, PNs belonging to the two anatomical tracts conveying information to MBs and LH may have different response properties. In contrast to initially thought (Müller et al., [@B150]), there does not seem to be very clear-cut differences between PN pathways in their propensity to respond to odorants, all PNs responding rather non-specifically to many odors (Krofczik et al., [@B122]). On average, the dynamic response profiles of l- and m-APT neurons were found to be similar so that in both systems odor identity would be encoded both in the pattern of response latencies and in the subset of activated PNs. However, even though responses to single odors may be similar, this work and a recent imaging study (Yamagata et al., [@B230]) both showed that the two subsystems may treat odor mixtures differently. Moreover, the two systems seem to respond differently to odor concentration (Yamagata et al., [@B230]). While intracellular recordings precisely describe the temporal response patterns of individual AL neurons to odors, imaging methods allow recording the combinatorial responses of many PNs simultaneously. This was possible using back-tracing of PN processes with the calcium dye Fura-dextran (Figure [3](#F3){ref-type="fig"}D). By placing a high concentration of dye into the axonal tract of l-APT PNs on their way to the MBs, the dye is taken up by the neurons and transported retrogradely to the soma near the AL, and to the dendrites within AL glomeruli (Sachse and Galizia, [@B177]). In agreement with electrophysiological recordings, the imaging recordings showed that PN odor-response patterns are temporally more complex than the input activity (bath application), and can show both excitation and inhibition phases. These calcium responses seem to follow -- although with a lower temporal resolution -- the spiking activity of the neurons, as shown by consecutive intracellular electrophysiology and optical imaging of the same AL neurons (Galizia and Kimmerle, [@B61]). Imaging of PN population activity allowed comparing glomerular activity patterns between the input (Sachse et al., [@B180]) and PN output representation, even within the same animal (Sachse and Galizia, [@B178]). Such comparison showed that most glomeruli which are intermediately or weakly active in the compound signal, do not present any calcium increase in PNs (Sachse and Galizia, [@B177]). Thus, PN patterns are sparser than input patterns. Moreover, it was found that AL networks improve the separability of odor representations, both with single odors over a wide concentration range (Sachse and Galizia, [@B178]) and with mixtures (Deisig et al., [@B34], see below). From this, AL processing appears to perform mainly two operations: *gain control*, which quantitatively controls the overall amount of PN activity and *contrast enhancement* which qualitatively modifies the activity patterns. These two properties can be attributed to the action of inhibitory LN networks, in particular GABAergic ones. Indeed, application of GABA onto the brain blocks spontaneous activity and totally abolishes calcium response to odors (Sachse and Galizia, [@B177]). Conversely, application of a GABA~A~-like receptor antagonist, picrotoxin, stimulates spontaneous activity, and increases the number of activated glomeruli upon odor presentation, also modifying the time course of the signals. Picrotoxin also abolishes network oscillation dynamics (Stopfer et al., [@B204]). Imaging recordings confirmed the existence of (at least) two different inhibitory networks, following the anatomical features of the bee AL (Sachse and Galizia, [@B177]). The first one would be a global inhibitory network driven by all glomeruli and affecting all glomeruli, corresponding to homo-LNs. Based on the above results, it would be sensitive to picrotoxin and have a gain control function. The second network would be an asymmetrical inhibitory network driven by one glomerulus and affecting mainly another glomerulus, corresponding to hetero-LNs. The neurotransmitter for the hetero-LN network involved in contrast enhancement is still unknown. Glutamate (Barbara et al., [@B10]) or histamine (Sachse et al., [@B179]) may play such a role, but this has not been demonstrated to date. Which rule underlies the inhibition relationships of hetero-LNs between individual glomeruli? Comparison of the result of computational modeling with imaging experiments established that the transformation of odor representation between AL input and output is best achieved by an interglomerular inhibition based on functional similarity between glomeruli and less so by inhibition based on anatomical neighborhood relationships or random connections (Linster et al., [@B132]). Odor representation in the mushroom bodies ------------------------------------------ After AL processing, l-APT and m-APT PNs convey information to the MB calyces. In honeybees, KCs, the MB-intrinsic neurons, are too small to perform intracellular recordings. Data from locusts suggest that whereas PNs respond to odors with trains of spikes, KCs often respond with a single or very few spikes (Perez-Orive et al., [@B161]). KCs do not show any spontaneous activity, and respond to very few odorants, i.e., representation at the KC level is highly sparse. Optical imaging recordings used Fura-dextran forward- and back-fills of PNs and KCs respectively, to study this transformation of odor representation from the AL to the MB (Szyszka et al., [@B211], Figure [3](#F3){ref-type="fig"}D). This study showed that the proportion of cells responding to only one odor out of a four-odor panel increased at each level, respectively 55, 70, and lastly 92%. Thus, olfactory representation would follow a series of transformations with a progressive sparsening of odor representation (Figure [4](#F4){ref-type="fig"}C). The last step of sparsening, which takes place at the level of the MB calyx involves several mechanisms. First, the low synaptic strength between PNs and KCs would imply that coherent input from many PNs at the same time is needed to excite a KC (Perez-Orive et al., [@B161]). Second, KCs would detect coincidence among many PNs thanks to odor-driven inhibition produced by LH inhibitory neurons locked in anti-phase to PN oscillations (Perez-Orive et al., [@B161], [@B160]). Third, local microcircuits involving GABA processes in the MB microglomeruli would also shape KC responses (Ganeshina and Menzel, [@B70]; Szyszka et al., [@B211]). Odor representation at the KC level is thus highly sparse, and each KC represents a particular pattern of PN inputs, possibly for a particular concentration of an odorant (Stopfer et al., [@B205]) or a particular composition of a mixture (Broome et al., [@B23]). Therefore, they are thought to be the ideal representation of a particular odorant for storing associative memories, i.e., storing the information that one particular odor has been associated with a sucrose reward or with a noxious stimulus (Heisenberg, [@B95]; Gerber et al., [@B73]). Mushroom body output and the lateral horn ----------------------------------------- The most studied MB output neuron is the Pe-1 neuron, which is recognizable by a characteristic firing pattern in doublets or triplets of action potentials (Mauelshagen, [@B140]; Rybak and Menzel, [@B176]; Okada et al., [@B153]). This wide-field neuron does not only respond to odors, but also to other sensory modalities (visual, mechanosensory). Moreover, it changes its responses during conditioning (Okada et al., [@B153]). At this level of the olfactory pathway, odor information is thus integrated with other modalities, and the function of neurons such as Pe-1 might not be to represent specific information about the learned odor like its identity, concentration, or multimodal context, but rather that this particular stimulus combination has been learned. Practically nothing is known about odor processing and representation in the honeybee LH. In *Drosophila*, recent neuroanatomical work could reconstruct putative maps of olfactory input to the LH (Jefferis et al., [@B106]). In this species, the response spectra of individual ORNs to odors are known (Hallem et al., [@B92]; Hallem and Carlson, [@B91]; Galizia et al., [@B65]) and glomeruli receiving input from ORNs carrying each receptor have been mapped (Couto et al., [@B31]; Fishilevich and Vosshall, [@B54]). Moreover, the projection patterns of uniglomerular PNs from identified glomeruli have been retraced to the higher-order centers. The putative olfactory maps at the level of the LH predict a clear segregation between candidate pheromone responsive PNs and fruit odor responsive PNs (Jefferis et al., [@B106]). Such functional segregation was not apparent in the MBs, although PNs from different glomeruli also project there in at least 17 different areas (Jefferis et al., [@B106]). Thus, in *Drosophila*, particular subregions of the LH may code the biological nature of olfactory stimuli. If a similar organization of the olfactory circuit exists in the honeybee, one could expect the honeybee LH to exhibit pheromone processing regionalization. Anatomical and electrophysiological work in ants also confirms this idea (Yamagata and Mizunami, [@B229]). Moreover, because the LH receives input from associative neurons like Pe-1, it was proposed that it may represent a pre-motor center for both innate biological behavior (pheromones) and acquired behavior (associative learning). Future research should invest more efforts in anatomical and physiological experiments for addressing this question. Concentration coding -------------------- Odor concentration strongly affects the odor map in the AL as the number of activated glomeruli increases with increasing concentrations of the odor (Sachse and Galizia, [@B178]). Thus, neurons integrating the overall excitation over many glomeruli, like multiglomerular PNs, may be adequate for monitoring absolute stimulus concentration. But how can odor-specific concentration coding as well as concentration invariance be achieved given the changing nature of the odor representation with concentration? The identity of an odorant is combinatorial and resides more in the *relative* activation of different glomeruli (or PNs) than in the absolute activation of individual glomeruli (Galizia and Szyszka, [@B68]). Therefore, neurons recognizing a particular pattern of inputs, such as KCs, could perform both operations, as was shown in locusts (Stopfer et al., [@B205]): while some KCs were found to be tuned to a narrow concentration range of one particular odorant, other KCs recognized the same odorant on a wide concentration scale. Some concentration invariance can be achieved earlier in the olfactory pathway, mainly through gain control mechanisms. Imaging experiments showed that processing in the AL makes odor representation more reliable over a broader concentration range (Sachse and Galizia, [@B178]). Moreover, the two PN subsystems may provide differential information to higher-order centers. Imaging recordings of PN boutons in the MB lips showed that while l-APT neurons display low concentration dependency (i.e., concentration invariant representation), m-APT neurons show a clear concentration effect and change their response quickly with concentration (Yamagata et al., [@B230]). Thus, concentration coding and concentration invariance may be extracted by differential processing at the level of PNs, and/or differential readout by KCs. Mixture processing ------------------ *In vivo* calcium imaging at the AL input showed that usually a glomerulus is activated by a mixture when at least one of its components activates this glomerulus (Joerges et al., [@B107]; Deisig et al., [@B33]). A putative presynaptic inhibition process induces a gain control at the system's input, so that complex mixtures do not saturate the capacity of the olfactory system. The more components a mixture contains, the more *suppression* phenomena were observed (Deisig et al., [@B33]), i.e., cases in which the response to a mixture was lower than to the components (Duchamp-Viret et al., [@B41]). Taking into account all measured glomeruli, the whole mixture representation follows essentially an elemental rule, because it can be predicted linearly from the responses to the components: the more a component activates the AL when presented alone (in number of activated glomeruli, for instance), the more present it is in the mixture representation (Deisig et al., [@B33]). The situation was slightly different at the PN level, as AL processing via LN networks increased the number of suppression cases, allowing the emergence of synthetic properties, i.e., the appearance of a representation that cannot be predicted based only on component information (Deisig et al., [@B34]). Indeed, similarity relationships between mixtures and their components were more homogeneous than at the input with a more equal representation of weak- and strong-components in the mixture. These recordings showed that reformatting by LNs in the AL increases separability among odor mixture representations, probably facilitating olfactory mixture discrimination by bees (Deisig et al., [@B34]). How mixture representation further transforms along the olfactory pathway is mostly unknown. Recordings at the level of PN boutons in the MB lips confirmed an important proportion of suppression effects in l-APT PNs, but showed that such mixture non-linearities are mostly absent in m-APT PNs, providing an additional hypothesis for the functional role of this dichotomous system: one system would be involved in synthetic processing, while the other would conserve component information (Yamagata et al., [@B230]). The strong sparsening of odor representation from PNs to KCs and their coincidence detection properties could be the basis for mixture-specific units. Olfactory Plasticity ==================== In bees, olfactory processing is not a static phenomenon, but is subject to plasticity as a function of both age and experience. This plasticity is manifested by structural and functional changes of olfactory circuits. Developmental plasticity ------------------------ The olfactory system of bees goes through intensive remodeling during the pupal stage and metamorphosis. The compartmentations of AL and MB calyces first take place during the beginning of pupal development (Menzel et al., [@B143]; Hähnlein and Bicker, [@B90]). At pupal stage 1, the AL neuropil is still homogeneous without any trace of the first spherical neuropil regions, called "preglomeruli" which appear starting at pupal stage 3 (Masson and Arnold, [@B138]). During subsequent stages the number of preglomeruli progressively increases, so that at pupal stage 7, all glomeruli appear adult-like (Gascuel and Masson, [@B71]). At the MB level, a small, homogeneously structured neuropil that is not yet divided into subcompartments appears at the prepupal stage (Menzel et al., [@B143]). Then, starting at pupal stage 3, the calyces gradually become separated from each other, with the lip, collar, and basal ring regions being clearly developed at pupal stage 6 (Hähnlein and Bicker, [@B90]). Interestingly, PNs achieve their adult arborization pattern within their main output region (MB lip) earlier during development (pupal stage 1) than their dendritic processes within their input region, the AL (pupal stage 2; Schröter and Malun, [@B189]). The olfactory system remains highly plastic throughout adulthood: an age-dependent increase in neuropil volume is observed for most of the MB, but the lip (olfactory), and collar (visual) regions show both age-related and experience-dependent volume increases (Withers et al., [@B224]; Durst et al., [@B44]; Fahrbach et al., [@B50]; Ismail et al., [@B104]). Moreover, the density of microglomerular complexes in the lips also undergoes changes with both age and experience (Groh et al., [@B83]; Krofczik et al., [@B121]). The hive is a highly odorous environment, and bees at all ages are subject to constant olfactory stimulation from honey and pollen stores and from pheromones produced by the queen, workers, and brood (Winston, [@B223]). This olfactory environment can have a significant effect on the maturation of the olfactory system of young bees. A number of experiments attempted to understand the effect of a passive olfactory exposure on honeybees' behavior. Some studies showed an increase in orientation toward a prior passively exposed odor, both in bees walking in an olfactometer (Pham-Delègue et al., [@B165]) and in free-flying bees visiting an artificial feeder (Jakobsen et al., [@B105]). In contrast, in the PER conditioning procedure, no effect of passive exposure was found, or if it was found, exposed bees tended to learn the exposure odor less efficiently than naïve bees (Getz and Smith, [@B77]; Gerber et al., [@B72]; Sandoz et al., [@B183]). At the same time exposed bees were found to spend more time than controls in this odor in a four-armed olfactometer (Sandoz et al., [@B183]). As control bees tended to avoid the odor, this increased time spent in the exposure odor field was interpreted as a reduced sensitivity of bees after passive exposure. Several processes may explain this effect. For example, constant passive exposure could have induced sensory adaptation of the bees' olfactory system. This would decrease a spontaneous avoidance by bees of the pure compound in the olfactometer, and make it a less salient compound to be learnt in a PER conditioning procedure. As sensory neurons continue to mature until 8 days after emergence (Masson and Arnold, [@B138]; Allan et al., [@B5]), exposure at an early age may permanently alter bees' olfactory sensitivity. In another series of experiments, odors were provided mixed with a sucrose solution for different periods during young adulthood (Arenas and Farina, [@B6]). Bees clearly associated the odor with the sucrose reward and showed long-term memory performance in a PER test at a later stage (17 days). Interestingly, the odor associated with sucrose reward when bees are 5--8 days old resulted in better olfactory retention at the adult stage than when the same exposure was performed before (1--4 days old) or -- more surprising -- after this critical period (9--12 days old; Arenas and Farina, [@B6]). *In vivo* calcium imaging showed that precocious olfactory experience increased general odor-induced activity as well as the number of glomeruli activated by the learned odor in the adult AL, but also affected qualitative odor representations (Arenas et al., [@B8]). Thus early olfactory experiences inside the hive may have long-lasting effects, reflected in behavioral responses to odorants and concomitant neural activity in the adult olfactory system. Fitting with the idea of developmental plasticity, bees were found to memorize novel odor-sucrose associations more efficiently after such early experience than controls (Arenas et al., [@B7]). Neural correlates of olfactory learning --------------------------------------- During the adult stage, honeybee foragers experience odors in the context of food search, and learn to associate floral odorants with sucrose reward (see above). A number of studies have searched for possible structural and functional plasticity of the olfactory pathway during or at different moments after the formation of an odor-sucrose association. Usually, in such experiments, differential conditioning is used so that changes in neural responses to a reinforced odorant (CS+) can be compared to changes observed to a non-reinforced odorant (CS−). Doing so, several studies found learning-correlated changes in odor-evoked patterns in the AL, taking place either shortly (10--30 min) after differential conditioning (Faber et al., [@B48]) or later (2--5 h, Rath et al., [@B168]; 24 h, Sandoz et al., [@B182]; Fernandez et al., [@B53]). At short-term, the amplitude of calcium responses to the CS+ were found to increase (Faber et al., [@B48]). Electrophysiologically, increases and decreases in PN spike rates were found in response to odors after conditioning, with a strongest effect for the CS+ (Denker et al., [@B38]). Later, between 2 and 6 h after training, differential increases and decreases in the responses of individual glomeruli were found (Rath et al., [@B168]), which was not the case at shorter-term (Peele et al., [@B158]). Lastly, at 24 h, PN calcium signals were found to increase to the CS+ (Fernandez et al., [@B53]). A general observation of these studies was that the similarity between the patterns of the CS+ and of the CS− was decreased after learning, suggesting that olfactory learning improves the discrimination of the learned odorant from other ones (Faber et al., [@B48]; Fernandez et al., [@B53]; Rath et al., [@B168]). On a structural level, olfactory experience during foraging was shown to induce glomerular volume and structure changes (Sigg et al., [@B197]; Brown et al., [@B24]). It was long unclear whether such changes were actually due to olfactory experience *per se*. Recently, however, a specific glomerular volume increase was demonstrated in a subset of glomeruli as a result of the formation of a long-term appetitive olfactory memory after 72 h (Figures [5](#F5){ref-type="fig"}A,B; Hourcade et al., [@B101]). It thus seems that in the AL, learning-induced plasticity takes different forms at different moments after the associative event. ![**Structural plasticity related to olfactory long-term memory**. **(A)** Structural changes in olfactory circuits of the bee brain were evaluated as a result of the formation of a long-term appetitive olfactory memory. Bees conditioned to an odor CS (paired bees) are compared to bees subjected to pseudoconditioning in which CS and US are presented explicitly without temporal association (unpaired bees). After 72 h, paired but not unpaired bees show strong behavioral olfactory long-term memory. At that time, the brains were prepared and either the volume of olfactory glomeruli in the antennal lobe **(B)** or the number of microglomeruli in the mushroom body calyx **(C)** were measured. **(B)** Volumetric analysis of 17 identified glomeruli in the antennal lobe, based on neutral red staining and 3D reconstruction. A global increase in glomerular volume was found in paired bees relative to unpaired bees. For each of the learned odors (here 1-hexanol), three glomeruli showed a significant volume increase (data from Hourcade et al., [@B101]). **(C)** Counts of microglomeruli numbers in the MB calyx, based on synapsin/phalloidin double staining. Olfactory long-term memory induced an increase in microglomeruli numbers in the lip region (olfactory) compared to unpaired or naive bees. This long-term plasticity relies on transcription as injection of Actinomycin D blocked the effect. This structural plasticity related to olfactory long-term memory was logically found only in the calyx lip (olfactory input region) and not in the collar (visual input region). (Data from Hourcade et al., [@B100]).](fnsys-05-00098-g005){#F5} Likewise, modified odor-evoked responses to a learned odor were found in the MB calyces shortly after conditioning (10--30 min, Faber and Menzel, [@B49]; Szyszka et al., [@B212]). In particular, specific imaging of KC activity showed that repeated presentation of an odor induces a reduction of the evoked response (interpreted as habituation), while appetitive training induced a recovery from this decrease (Szyszka et al., [@B212]). On a structural level, a long-term olfactory memory trace 72 h after training was revealed as an increase in the density of microglomeruli in the MB lips (Figures [5](#F5){ref-type="fig"}A,C, Hourcade et al., [@B100]). MB output neurons are also subject to changes through associative learning, as exemplified by the Pe-1 neuron (Okada et al., [@B153]), by recurrent PCT neurons (Hähnel and Menzel, [@B89]) or by other ENs (Strube-Bloss et al., [@B208]). In some cases too, specific changes are found in responses to the CS+ and response differences between CS+ and CS− were increased (Strube-Bloss et al., [@B208]). Thus many electrophysiological, functional imaging, or neuroanatomical studies find strong neural plasticity within olfactory circuits, especially after associative conditioning. However, it is often difficult to relate such neural plasticity to its exact function. Are the observed changes related to modifications of odor processing, modulating the neural representation of the learned odors so that it can be better distinguished from environmental background? Or are they related to an "engram," revealing the storage of odor-reinforcement associations in the brain? Currently we think that the AL is mostly responsible for the former, while the MB would be crucial for the latter, but considerable work is still needed to confirm this hypothesis. Future neurobiological studies will need a combination of approaches, asking in particular whether the observed cells (and their plasticity) are necessary and/or sufficient for the expression of olfactory plasticity at the behavioral level (Gerber et al., [@B73]). Conclusion ========== One century of experiments have provided extensive data on the olfactory behavior of honeybees, on the neuroanatomical organization of their olfactory pathway as well as on the neural representation of odors within these circuits. All these experiments concur to show that the honeybee olfactory system is tuned for performing a number of operations that are crucial for meeting the demands of social life, food search, and mating. This system thus allows to (1) detect and identify odor stimuli, allowing graded responses to increasingly similar odors; (2) measure stimulus concentration allowing both concentration invariant and concentration-specific odor recognition; (3) detect components within a mixture as well as extract mixture-unique properties; (4) constantly adapt to the odorous environment; and (5) learn relationships between almost any odor and appetitive or aversive outcomes. Although our understanding of odor representation at the different levels of the bee brain has greatly improved in the last years thanks to state-of-the-art recording techniques, entire brain regions have yet to be explored. The most prominent are the m-APT dependent parts of AL and MBs, as well as the utterly unstudied LH. Thanks to optical imaging, our understanding of the spatial representation of odors has greatly improved, but temporal aspects are still poorly understood. Even in such a simple system, as compared to vertebrates, olfactory coding involves complex interactions between different neuron types, so that only computational approaches feeding on comprehensive sets of experimental data may help understanding the dynamics and processing rules of the olfactory system. Lastly, plasticity appears in multiple regions of the olfactory pathway, but their respective implications for tuning the olfactory system or for storing outcome-related memories is still unknown. It shall be the goal of future research to progress in these questions, so that a comprehensive model of olfactory detection, processing, and learning in the honeybee can be constructed, the ultimate goal of sensory neuroscience. Conflict of Interest Statement ============================== The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. I would like to thank M. H. Pham-Delègue, R. Menzel, M. Giurfa, and C. G. Galizia for their intellectual contribution to the ideas developed in this review. Tilman Franke produced the pictures in Figures [2](#F2){ref-type="fig"}A,B. This research is financed by the CNRS and by grants from the National Research Agency (ANR-NEURO-2007 and ANR-BLANC-2010) as well as from NERF and R2DS Ile-de-France Programs. [^1]: Edited by: Milagros Gallo, University of Granada, Spain [^2]: Reviewed by: Milagros Gallo, University of Granada, Spain; Monique Gauthier, University Paul Sabatier Toulouse 3, France

Introduction {#Sec1} ============ Inflammatory disorders can affect virtually any organ and are a frequent cause of morbidity. Recently, immunotherapies emerged as highly efficient treatments in specific inflammatory diseases. Since their prescription is rapidly increasing, their cost represents a growing burden for healthcare systems. It is therefore highly desirable to guide the use of such expensive therapies by dedicated biomarkers. The high level of glucose consumption during inflammatory processes has prompted the use of 2-deoxy-2-\[^18^F\]fluoro-D-glucose (\[^18^F\]FDG) positron emission tomography (PET) in the management of a wide variety of inflammatory disorders^[@CR1]^. \[^18^F\]FDG however suffers from poor specificity since glycolysis is not limited to aseptic inflammation but is also present in infection, cancer and their related inflammatory processes. Recently, the need to develop biomarkers targeting specifically the host immune response in cancer has emerged^[@CR2]^. In addition, \[^18^F\]FDG is avidly taken up by organs such as brain or heart in physiological conditions thereby hampering its diagnostic value. Inflammation involves surface expression of endothelial adhesion molecules, platelet adhesion and activation of leukocytes resulting in leukocyte transmigration into tissues^[@CR3]--[@CR5]^. An example of such adhesion molecules is the CD31 receptor also known as PECAM-1, a transmembrane homophilic receptor^[@CR6]--[@CR8]^ which is constitutively and exclusively expressed by endothelial cells, platelets and leukocytes. CD31 is highly concentrated at the intercellular junctions between endothelial cells^[@CR9]--[@CR11]^ and plays an important role in the homeostasis of the endothelial barrier function. In addition, CD31 plays a determinant role in the regulation of leukocyte and platelet activation^[@CR12]--[@CR18]^. The extracellular portion of CD31 is organized into six Ig-like domains. Domain 1 mediates the trans-homophilic CD31-CD31 binding when two CD31+ cells interact^[@CR19],[@CR20]^. This trans-homophilic binding induces a cis-homodimerisation of CD31 juxta-membrane portion^[@CR19]^ and triggers a specific inhibitory tyrosine phosphate-dependent signaling through the CD31 cytoplasmic domain. By these mechanisms, CD31 appears as a central regulator of inflammatory responses as it modulates the recruitment and extravasation of immune cells at sites of platelet-coated, inflamed microvessels^[@CR21]^. Importantly, the CD31-mediated regulation is determined by its molecular integrity. In pro-inflammatory conditions, the CD31 extracellular portion is cleaved so that recently activated cells express a truncated form of CD31 (cleaved CD31). The loss of the trans-homophilic domain of CD31 invalidates its inhibitory function and allows the full activation of cleaved CD31 cells^[@CR16],[@CR22]^. We hypothesized that the cleaved molecular form of CD31 might be a suitable target for molecular imaging because it is present in large amounts at the surface of the three major cell types at stake at sites of active inflammation (leukocytes, platelets and endothelial cells). To target cleaved CD31, we selected an octapeptide (H-RVFLAPWK-OH) derived from the immunosuppressive CD31 peptide^[@CR23]^ and further engineered as the retro-inverso analogue, i.e. the reverse sequence with D-amino acids^[@CR24]^. The resulting functional mimic of the parent peptide (H-kwpalfvr-OH) is a good ligand candidate because it is stable *in vivo* and it specifically binds to the cleaved cis-homophilic juxta-membrane sequence of the CD31 ectodomain (aa 551--574) common to all cleaved CD31 cells^[@CR22],[@CR24]^. Herein, we designed and prepared a bioconjugate of D-P8RI coupled to 6-Hydrazinonicotinic acid (HYNIC) as a bifunctional complexing agent for technetium-99m (^99m^Tc)^[@CR25]^. We then assessed the performance of the resulting radiotracer ^99m^Tc-HYNIC-D-P8RI for *in vivo* non-invasive imaging of inflammatory cells in an experimental rat model comparatively to its counterpart with L-Proline (L-P8RI) and to the well-established radiolabeled glucose analogue 2-deoxy-2-\[^18^F\]fluoro-D-glucose (\[^18^F\]FDG) as controls. Results {#Sec2} ======= Design, synthesis and physico-chemical characterization of the Tc-labeled D-P8RI {#Sec3} -------------------------------------------------------------------------------- The bioconjugate radiotracer is composed of (i) the D-P8RI peptide sequence which targets the cleaved CD31 molecular species, (ii) the HYNIC group enabling ^99m^Tc chelation, (iii) a PEG linker between both moieties to reduce steric hindrance, preserve the P8RI binding capacity and maintain hydrophilic properties, and iv) the radioisotope ^99m^Tc for SPECT imaging. Results of the synthesis of HYNIC-D-P8RI are detailed in Supplementary Material (see Fig. [1a](#Fig1){ref-type="fig"} (R = R1) for structural formula, and Fig. [1b](#Fig1){ref-type="fig"} for RP-HPLC profile).Figure 1(**a**) P8RI-based conjugates: HYNIC-D-P8RI (R = R1) and putative structure of its corresponding EDDA complexes after labeling: \[^99m^Tc(HYNIC-D-P8RI)(EDDA)\] (R = R2) and \[^99m^Tc(HYNIC-D-P8RI)(EDDA~2~)\] (R = R3). (**b**--**e**) Optimization of the radiolabeling process. **b** RP-HPLC chromatograms of: HYNIC-D-P8RI (UV detection); (**c**--**e**) ^99m^Tc-HYNIC-D-P8RI (radio detection) using tricine as coligand, EDDA as coligand and tricine/EDDA exchange strategy, respectively. The chemical or radiochemical purity (CP or RCP), as well as the retention time (t~R~) are indicated on each panel. This peptide derivative was further labeled using different coligands. The profile obtained with tricine was heterogenous and the RCP was \<88% as assessed by RP-HPLC (Fig. [1c](#Fig1){ref-type="fig"}) whereas EDDA and tricine/EDDA exchange labeling yielded a dominant radiolabeled species profile with RCP \<74% and ≥94%, respectively (Fig. [1d,e](#Fig1){ref-type="fig"}). Retention factors (Rf) of radiolabeled HYNIC-D-P8RI evaluated with TLC in the different mobile phases were: Rf = 0 in methylethylketone (MEK) and Anticoagulant Citrate Dextrose Solution (ACD), Rf = 0.8--1 in CH~3~CN/H~2~O (3/2) allowing to estimate radiolabeling yields at 92.6, 71.1, and 95.3% for tricine, EDDA and tricine/EDDA experiments, respectively (data not shown). Radiolabeling impurities found in TLC corresponded to non-peptide bound species such as ^99m^Tc-coligands and ^99m^TcO4^−^, no ^99m^Tc-colloid was detected. The tricine/EDDA exchange labeling strategy was chosen for the following experiments based on the high yield, on the high RCP obtained and the high specific activity over 114 GBq/µmoL. To identify the coordination state of the complex by MS, ^99^Tc-HYNIC-D-P8RI was prepared using the tricine/EDDA process except that ^99m^Tc was replaced by a larger amount of ^99^Tc. A summary of HPLC/MS analyses is presented (Table [1](#Tab1){ref-type="table"}). When compared with the control unlabeled peptide, the mass spectrum of the labeled conjugate showed an increase of 270 Da and 446 Da, which can be assigned to coexisting Tc complexes coordinated with one and two EDDA, respectively (Fig. [1a](#Fig1){ref-type="fig"}, R = R2, R3). Peptide species containing tricine were not present to any significant degree.Table 1Selected HPLC/MS data for HYNIC-D-P8RI and its corresponding Tc conjugates. Calculated *m/z* values are based on the exact molecular mass using MassLynx calculator (Waters, France). M refers to the unlabeled and uncharged peptide conjugate HYNIC-P8RI.Compound*m/z* calculated*m/z* observedAssignmentHYNIC-D-P8RI1354.7641354.718\[M + H\]^+^\[^99^Tc(HYNIC-D-P8RI)(EDDA)\]1624.7501625.116\[(M + ^99^Tc + EDDA - 5 H) + H\]^+^\[^99^Tc(HYNIC-D-P8RI)(EDDA~2~)\]1800.8301801.226\[(M + ^99^Tc + 2 EDDA - 5 H) + H\]^+^ *In vitro* properties {#Sec4} --------------------- Percentage of ^99m^Tc-HYNIC-D-P8RI bound to plasma protein were: 7.16 ± 0.44, 4.80 ± 1.36, 3.75 ± 1.12, 4.84 ± 1.17, 5.68 ± 0.75, 5.83 ± 0.51 and 1.23 ± 0.55 for 0, 0.5, 1, 2, 3 and 4 h of incubation and control (plasma replaced by PBS), respectively (Fig. [2a](#Fig2){ref-type="fig"}). Percentage of intact ^99m^Tc-HYNIC-D-P8RI referring to plasma stability was: 93.02 ± 0.04, 91.05 ± 0.02, 89.07 ± 0.02 for 1, 2 and 3 h of incubation, respectively (Fig. [2b](#Fig2){ref-type="fig"}). The octanol-water partition coefficient Log P value was: −2.92 ± 0.04.Figure 2*In vitro* assessment of ^99m^Tc-HYNIC-D-P8RI in human plasma (**a**,**b**). (**a**) Percentage of human plasma protein binding over 4 h (mean ± SD) evaluated by size exclusion chromatography. Control (grey bar) refers to the same experiment performed in PBS instead of human plasma. (**b**) Percentage of intact ^99m^Tc-HYNIC-D-P8RI in human plasma over 4 h, referred as RCP and assessed by RP-HPLC. (**c**) Biodistribution (%ID/g, n = 3) of ^99m^Tc-HYNIC-D-P8RI in male Wistar rats at 15, 30, 90 min and 24 h post-injection. Columns, mean; bars, SD. (**d**) RP-HPLC chromatogram (radio detection) of rat urine 90 min p.i; radiochemical purity (RCP) and retention time (t~R~) are indicated. *In vivo* properties {#Sec5} -------------------- ### Biodistribution and *in vivo* stability {#Sec6} Results of biodistribution in animals at 15, 30, 90 min and 24 h p.i. are summarized in Fig. [2c](#Fig2){ref-type="fig"}. Almost no uptake from most organs and rapid clearance through renal excretion was observed. The accumulated activity was always \<0.2% ID/g in heart, thyroid, spleen and brain. Urinary clearance was predominant (14.04 ± 2.93% ID/g for kidney and 61.38 ± 13.42% ID/g for bladder 90 min p.i.) concomitant to a rapid clearance from blood (9.74 ± 1.21% ID/g 15 min p.i and 0.04 ± 0.02% ID/g 24 h p.i). Based on biodistribution results, images acquisition in animal injected with Turpentine has been set 1 h p.i. because of the very low activity remaining in blood and muscle. Urine RP-HPLC exhibited a single radiolabeled species profile similar to the injected peptide (t~R~ = 10.0 min and RCP 93.1%) as shown in Fig. [2d](#Fig2){ref-type="fig"}. ### SPECT/CT and PET/MRI imaging {#Sec7} SPECT/CT and PET/MRI mainly showed a diffuse uptake of the different radiotracers in the turpentine injected hindlimb (TIH) that was more visible compared to the contralateral saline injected hindlimb (SIH) (Fig. [3](#Fig3){ref-type="fig"}). Mean signal ratios (TIH/SIH) were 4.49 ± 0.43 (n = 23), 4.05 ± 0.47 (n = 19) and 2.10 ± 0.68 (n = 9) for \[^18^F\]FDG, ^99m^Tc-HYNIC-D-P8RI and ^99m^Tc-HYNIC-L-P8RI, respectively (Fig. [4](#Fig4){ref-type="fig"}). One way Anova (F Ratio = 4.49, p \< 0.02) followed by means comparison exhibited a significantly different mean signal ratio between: \[^18^F\]FDG and ^99m^Tc-HYNIC-L-P8RI (p \< 0.01), ^99m^Tc-HYNIC-D-P8RI and ^99m^Tc-HYNIC-L-P8RI (p \< 0.03) but not between \[^18^F\]FDG and ^99m^Tc-HYNIC-D-P8RI (p \> 0.48).Figure 3Axial, sagittal and coronal views of rats left (saline) and right (Turpentine oil) hind limbs (pink and green regions of interest, respectively) obtained with ^99m^Tc-HYNIC-D-P8RI (**a**), ^99m^Tc-HYNIC-L-P8RI (**b**) SPECT/CT, and \[^18^F\]FDG PET/MR (**c**). ^99m^Tc-HYNIC-D-P8RI and \[^18^F\]FDG uptake was associated with local inflammation compared with a low signal of ^99m^Tc-HYNIC-L-P8RI. All radiotracers displayed a very low uptake in the contralateral hindlimb (control). Bladder is pointed with a white arrow in (**a**).Figure 4Mean signal ratios (Mean signal inflammatory muscle/Mean signal contralateral muscle) calculated on scintigrams from the \[^18^F\]FDG, ^99m^Tc-HYNIC-D-P8RI and ^99m^Tc-HYNIC-L-P8RI injected animals. \*p \< 0.05, \*\*p \< 0.01, NS: Non significant. ### Histology and autoradiography {#Sec8} Masson's trichrome staining of the rat right gastrocnemius muscle, two days after turpentine injection, was characterized by a massive infiltration of leukocytes as compared with the preserved left muscles injected with saline (Fig. [5](#Fig5){ref-type="fig"}). Autoradiography of the same samples allowed to co-localize ^99m^Tc-HYNIC-D-P8RI uptake and inflammatory cell infiltration (Fig. [5c](#Fig5){ref-type="fig"}). Conversely, uptake in the contralateral muscle was low (Fig. [5d](#Fig5){ref-type="fig"}). This result was partially verified using rhodamine labelled D-P8RI which accumulated at the border of the turpentine oil injection site (Fig. [6a,b](#Fig6){ref-type="fig"}) on cells mainly composed of CD68+ leucocytes at the immediate vicinity of the damaged tissue (Fig. [6c,d](#Fig6){ref-type="fig"}).Figure 5Masson's trichrome staining (**a,b,e**--**l**) and autoradiography (**c**,**d**) of gastrocnemius muscles 2 days after intramuscular injection of saline (control, left hind limb) or turpentine oil (right hind limb). Animals were sacrificed 1 hour after intravenous ^99m^Tc-HYNIC-D-P8RI administration. Masson's trichrome staining in (**a**) and (**b**) corresponds to the same slices of autoradiography in (**c**) and (**d**). Turpentine injected muscle (**a)** displays evidence of inflammation with edema and major leukocyte infiltration (**e,g,i,k**: x1.25, x2.5, x10, x20 respectively), co-localized with a high uptake of ^99m^Tc-HYNIC-D-P8RI. The representative micrographs (**b**) corresponds to a control muscle matching with a low uptake of ^99m^Tc-HYNIC-D-P8RI (**d**). Higher magnifications of the control show no evidence of inflammation (**f,h,j,l)**.Figure 6Fluorescence microscopy of the rat right gastrocnemius muscle, 2 days after turpentine oil injection. (**a**) Rhodamine labeled D-P8RI positive signal (black) is concentrated at the border of the turpentine oil injection. (**b**) Higher magnification in the blue inset reveals that D-P8RI labeled cells are enriched at the immediate vicinity of the damaged tissue. (**c**) Immunofluorescence of another rat right gastrocnemius muscle after turpentine oil injection. (**d**) The inner layer of the cells concentrated around the site of turpentine oil injection is mainly composed of CD68+ leucocytes (Fuchsia). (**e**) The mononuclear and polynuclear leukocytes (identified by the DAPI staining) at the immediate vicinity of the damaged tissue (red inset) display a cleavage of CD31 as revealed by the relative negative extracellular CD31 (eCD31) signal (green) in spite of a consistent intracellular CD31 (iCD31) staining (red). (**f**) The cleavage of CD31 was instead rather limited at the surface of the leukocytes located at the periphery of the inflammatory site (green inset). Of note, in parallel with a lesser CD31 cleavage phenomenon, the D-P8RI signal appears weaker in such an outer layer of the inflammatory site, as documented in (**b**). Remarkably, in tissue-infiltrated leukocytes, the amount of extracellular CD31 was lower than that of intracellular CD31 (Fig. [6e,f](#Fig6){ref-type="fig"}), pointing at the cleavage of the molecule on these activated cells. Discussion {#Sec9} ========== In the present work, we developed a completely novel approach for inflammation-specific imaging based on a synthetic peptide ligand targeting cleaved CD31 that is expressed upon activation by both inflammatory and endothelial cells. ^99m^Tc-HYNIC-D-P8RI was stable *in vivo* and allowed to detect acute sterile inflammation with a high specificity. Histological analysis proved the co-localization of ^99m^Tc-HYNIC-D-P8RI signal and leukocyte accumulation in tissues and suggested that the labeling is indeed mediated by cleaved CD31. Initially considered as a cell adhesion molecule, CD31 is actually highly expressed at endothelial intercellular borders where it especially prevents leukocyte extravasation^[@CR26]^. The native (non cleaved) CD31 is constitutively and exclusively expressed by platelets, leukocytes and endothelial cells at high levels (approximately one million copies of CD31 are present on the surface of endothelial cells)^[@CR27]^. When native CD31 is engaged on interacting cells, it delivers a mutual 'leave-me alone' signal that mute cell activation^[@CR28]^. However, when the activating stimuli is strong enough to overcome the activation threshold set by CD31, the function of the latter is invalidated by a proteolytic cleavage to allow full cell activation. The cleavage of CD31 occurs close to the cell membrane but leaves a small sequence of the truncated molecule lingering at the surface of recently activated cells. The D-P8RI specifically binds to this truncated molecular species and not to full length CD31. Since CD31 is encoded by a constitutive gene, in the absence of further stimulation, full CD31 molecules comes back within 3 hours at the surface of cells returning to baseline, to which P8RI cannot bind. Therefore, molecular imaging based on P8RI-mediated targeting of cleaved CD31 represents a relevant strategy to localize ongoing inflammatory processes. The sequence of D-P8RI^[@CR24]^ (patent WO2013190014A1) was designed based on the results of a functional screening of a large library of peptides derived from the parent 23 aa sequence corresponding to the epitope of the LYP21 antibody (aa 551--574), located within the cleaved cis-homophilic juxta-membrane sequence of the CD31 ectodomain^[@CR22],[@CR23]^. Herein, we described the method to modify the P8RI sequence at the alpha position of the N-ter D-Lysine. A previous study has shown that N-ter modification of a P8RI containing peptide does not adversely affect its properties and inhibits lymphocyte activation *in vitro* and *in vivo*^[@CR22]^. Also, D-P8RI was designed as a retro-inverso peptide with all-D-amino acids allowing to maintain bioactivity and to confer resistance towards plasma proteases^[@CR24],[@CR29],[@CR30]^. Indeed, urine RP-HPLC analysis demonstrated that the tracer is eliminated unchanged indicating that most of it is not subjected to a catabolic degradation. Quite a big spread in the mean signal ratio of ^99m^Tc-HYNIC-D-P8RI injected animals was observed. This may be explained by the model variability and especially difficulty to control turpentine oil diffusion at the site of injection which could be verified by a biodistribution study on turpentine oil injected animals. Also,the small size of the imaging agent is likely to facilitate diffusion in inflamed tissues. This feature ensures high sensitivity but may lead to decreased specificity because of possible non-specific retention of the tracer. To address this issue, we compared the signals derived from ^99m^Tc-HYNIC-D-P8RI and from its counterpart with L-Proline as a control. We showed that ^99m^Tc-HYNIC-D-P8RI had a significantly higher uptake in the inflammatory area compared to ^99m^Tc-HYNIC-L-P8RI, suggesting signal specificity. On the other hand, capillary permeability remains limited during early steps of acute inflammation or inflammation at a chronic stage. The wide spectrum of P8RI that includes binding sites on activated endothelium is likely to provide a high number of targets without the need to cross the capillary wall. *In vitro* and *in vivo* biodistribution assessment of ^99m^Tc-HYNIC-D-P8RI showed the absence of significant uptake in tissues other than those related to urinary elimination. Compared to other imaging agents such as \[^18^F\]FDG which is taken up by brain and myocardium, this absence of physiological background activity in most tissues is expected to improve the contrast of the signal. Taken together, these characteristics enable to anticipate a good sensitivity of detection in a wide range of clinical settings. In addition the uptake of ^99m^Tc-HYNIC-D-P8RI in our acute model of aseptic inflammation was comparable to that obtained with the well validated \[^18^F\]FDG. Currently, \[^18^F\]FDG PET/CT is the modality of choice for the clinical diagnosis of inflammatory diseases and the treatment monitoring. The limitations related to such an approach relying on glycolytic activity are well acknowledged regarding both the lack of specificity and the lack of signal relevance in brain and to some extent in the myocardium. ^99m^Tc-HYNIC-D-P8RI presents interesting features to support future developments for translational imaging research such as: specific targeting, favorable biodistribution, *in vivo* stability, relatively straightforward production process and radiolabeling procedure and swift blood clearance that make the tracer suitable for early imaging with good contrast in a wide variety of indications, including those in which \[^18^F\]FDG is not optimal. In particular, D-P8RI may be an interesting tool to develop molecular imaging probes for the longitudinal monitoring of therapies. For instance, it may be suitable in the field of autoimmune diseases in which \[^18^F\]FDG failed to reliably monitor inflammation such as arthritis in which there is a need to select patients that could be eligible for innovative anti-inflammatory immunotherapies^[@CR31],[@CR32]^. Conclusions {#Sec10} =========== ^99m^Tc-HYNIC-D-P8RI constitutes a new tool for the detection and localization of inflammatory sites. Our work suggests that targeting cleaved CD31 is an attractive strategy for the specific *in vivo* imaging of inflammatory cells. Methods {#Sec11} ======= Three different radiotracers were used in this study. First, ^99m^Tc-HYNIC-D-P8RI, the candidate radiotracer was prepared and evaluated. Second, its homologue with L-Proline, ^99m^Tc-HYNIC-L-P8RI, was used as a control and dedicated to target specificity evaluation. Finally, \[^18^F\]FDG was employed as a positive control. Reagents were purchased from Sigma/Aldrich (St. Louis, MO, USA), except when otherwise stated. HYNIC-P8RI synthesis, reverse-phase (RP) high performance liquid chromatography (HPLC), electrospray ionization (ESI) mass spectrometry (MS), chemical and radiochemical purity (CP and RCP), thin layer chromatography (TLC), Log P value and fluorescence microscopy are available in Supplementary Data. Radiotracers {#Sec12} ------------ \[^18^F\]FDG was obtained from Advanced Accelerator Applications (Saint Genis Pouilly, France) and used as received. HYNIC-D-P8RI and HYNIC-L-P8RI were synthesized in-house (as described in Supplementary Data). Radiolabeling {#Sec13} ------------- In order to select the optimal radiolabeling approach, ^99m^Tc labeling of HYNIC-D-P8RI was assessed using different co-ligands: 1/Tricine as coligand: in a rubber-sealed N~2~ purged vial, HYNIC-D-P8RI (20 µg) was incubated with a tricine solution (250 μL, 40 mg/mL in PBS buffer pH 7.2), tin(II) chloride dihydrate solution (20 μL, 1 mg/mL in 0.1 N HCl), 1 GBq sodium pertechnetate (Na^99m^TcO4) and saline for a total volume of 1 mL. The mixture was incubated for 30 min at room temperature (RT); 2/Ethylenediamine-N,N′-diacetic acid (EDDA) as coligand: the tricine procedure was followed but with EDDA (250 µL, 20 mg/mL in 0.1 N NaOH) instead of tricine; 3/Tricine-EDDA coligands exchange labeling: the tricine procedure was followed except that EDDA (250 µL, 20 mg/mL in 0.1 N NaOH) was added to the reaction vial. The mixture was then incubated for 10 min at 100 °C. The radiochemical purity (RCP) was determined as described in the Supplementary Material. Protein binding {#Sec14} --------------- Protein binding of the purified radiolabeled D-P8RI peptide (final concentration 100 pmol/mL) was determined in triplicate after 0, 0.5, 1, 2, 3 and 4 h of incubation in fresh human plasma (collected in citrate tubes) at 37 °C and analyzed after size exclusion chromatography (illustra Microspin G-50 Columns, Sephadex G-50, GE Healthcare, UK). Simultaneously, radiolabeled peptide was incubated in PBS at 37 °C as a control. G-50 columns were prespun at 2000 × g for 1 min then 20 μL of mixture was added and the column was centrifuged at 2000 × g for 2 min. Columns and eluates were counted on a Wallac Wizard 3″ 1480 (Perkin Elmer, MA, USA), then protein binding was calculated using the following activity (A) ratio: A eluate/(A column + A eluate). Plasma stability *in vitro* {#Sec15} --------------------------- Plasma stability was assessed in triplicate at 1, 2 and 4 h after incubation of the radiolabeled D-P8RI peptide (final concentration 100 pmol/mL) in human plasma at 37 °C with RCP ≥94%. After precipitation of plasma proteins with acetonitrile and centrifugation (15000 × g, 10 min), the supernatant was analyzed by RP-HPLC as described in Supplementary Data. Plasma stability was expressed in RCP (%). Animal model {#Sec16} ------------ Two to 3-month-old adult male Wistar rats, weighing 250--300 g were used. The animals were housed in a controlled environment. The procedures and animal care complied with the principles formulated by the National Society for Medical Research. All methods were performed in accordance with the guidelines of the French Directorate of Veterinary Services (authorization: No. 75-214). All methods were approved by the animal ethics committee of the Claude Bernard Institute (APAFIS \#14821, Paris, France). Aseptic inflammation was obtained according to a standardized procedure consisting of turpentine oil injection characterized by a massive local influx of activated inflammatory cells^[@CR33],[@CR34]^. In all *in vivo* imaging experiments, animals were treated with both 0.15 mL of turpentine by intramuscular (I.M.) injection into the right gastrocnemius muscle (hind-limb) and 0.15 mL of sterile saline in the left contralateral muscle. Biodistribution and *in vivo* stability {#Sec17} --------------------------------------- Biodistribution of ^99m^Tc-HYNIC-D-P8RI was performed 15, 30, 90 min and 24 h post-injection. Animals (n = 3 for each time point) were injected intravenously through penis vein with 30 ± 3 MBq of ^99m^Tc-HYNIC-D-P8RI. They were sacrificed by isoflurane overdose and blood, heart, liver, thyroid, spleen, kidney, brain, intestine and muscle tissues were harvested and counted using a well counter. Results were expressed as the percentage of injected dose per gram of tissue (% ID/g). To characterize radiotracer elimination and potential degradation, rat urine was collected 90 min p.i, passed through a 0.22 µm PVDF filter (Durapore^TM^ 13 mm, Merck), and analyzed using RP-HPLC as described in Supplementary Data. *In vivo* imaging procedures {#Sec18} ---------------------------- Imaging procedures of ^99m^Tc-HYNIC-P8RI (D and L) were performed using Single Photon Emission Computed Tomography coupled to X-ray computed tomography (SPECT/CT). Acquisitions were performed two days after turpentine injection, on a dedicated small-animal hybrid camera (NanoSPECT/CT, Mediso, Hungary) equipped with multi-pinhole high-resolution collimators, (256 × 256 matrix), and a 20% energy window centered on 140 keV. *In vivo* scintigraphy was performed 1 h post intravenous injection (p.i.) through the penis vein of 74 ± 7 MBq of ^99m^Tc-HYNIC-P8RI under isoflurane anesthesia. Prior to scintigraphic acquisition, a whole-body high-resolution CT scan (45 keV/145 mAs; matrix size 256 × 256) was performed for anatomic localization. Iterative reconstruction of SPECT acquisitions was performed using Tera-Tomo software (Mediso) with ordered-subsets expectation maximization (4 iterations, 8 subsets) and Butterworth 3-dimensional post-filtering (cutoff frequency, 1.3 cycle/pixel; order, 5). Reconstructed slices were visually assessed in 3 planes (axial, coronal, and sagittal). \[^18^F\]FDG PET/MRI scans were acquired using a Mediso NanoScan PET/MRI (1 T MRI, Mediso Ltd., Budapest, Hungary). After retro-orbital injection of the radiotracer (≈20 MBq; 100--150 μL), PET acquisitions were performed 1 h post-injection (p.i.) followed by MRI acquisition for anatomic localization. PET data were reconstructed using Tera-Tomo™ 3D software (Mediso Ltd., Budapest, Hungary), a 3D-OSEM Monte Carlo based algorithm with attenuation and scattering corrections (voxel size equal to 0.3 × 0.3 × 0.3 mm^3^). Intensity value in the voxels was calibrated in kilo Becquerel per cubic centimeter (kBq.cm-3) corrected for decay and positron branching ratio of ^18^F. Imaging data analysis {#Sec19} --------------------- Three-dimensional Regions of Interests (3D-ROIs) were placed on the inflammatory muscles and the mean signal per volume unit (kBq/mL) was measured. Assuming a tissue density of 1 g/mL, the radioactivity contained in the ROI was divided by the dose administered to the animal and the volume of the ROI to obtain an imaging ROI-derived % ID/g. The same ROI was applied to the contralateral control muscle. The mean signal ratio was then calculated by dividing the mean signal from the inflammatory ROI by the mean signal from the contralateral ROI. *Ex vivo* quantification of ^99m^Tc-HYNIC-P8RI uptake {#Sec20} ----------------------------------------------------- Autoradiography. TURP rats injected with ^99m^Tc-HYNIC-P8RI (D and L series) or \[^18^F\]FDG (74 MBq) were sacrificed 1 h post-injection. The posterior hindlimbs muscles were dissected, harvested en bloc and quickly frozen in OCT (Sakura, Torrance, CA, USA) at −80 °C for 15 min. A Leica cryomicrotome (Leica Microsystems Inc, Buffalo Grove, IL, USA) was used to obtain 20 μm-thick sections for autoradiography. ^99m^Tc-HYNIC-P8RI uptake on tissue slices was quantified using a calibrated bio-image analyzer CR-35 Bio (Elysia-Raytest, Liège, Belgium) after exposing sections for 24 h on dedicated phosphor imaging plates. Histology and fluorescence microscopy {#Sec21} ------------------------------------- Masson's trichrome staining was performed on 10 µm-thick cryosections. Slices were air dried then fixed on Bouin's fixative. Then slices were digitalized with a slide scanner (Nanozoomer 2.0RS, Hamamatsu, Japan) to correlate radiotracer uptake on autoradiography with anatomical landmarks. Immunohistochemistry was performed on paraffin embedded samples using: mouse anti rat CD68 monoclonal antibody (Antibody \#MCA341R from Bio-Rad) and DAPI staining to identify mononuclear and polynuclear leukocytes. CD31 cleavage was evaluated using an anti-extracellular CD31 antibody (goat anti-rat antibody, \#AF3628 from R&D systems) and an anti-intracellular CD31 antibody (rabbit anti-human antibody, \#119-15418 from RayBiotech). Fluorescent tracking of D-P8RI was performed immediately before the sample harvesting using rhodamine labeled D-P8RI peptide injected through the left ventricular apex (bolus, 5 mg/kg) followed by perfusion of 10 mL saline to wash out all unbound peptide. Statistical *a*nalysis {#Sec22} ---------------------- Continuous variables were expressed as mean ± standard deviation and compared by use of one way ANOVA test followed by a post-hoc analysis (3 groups). Statistical significance between experimental groups was assessed using the JMP software v14.3 (SAS institute, France) with 95% confidence level. A value of p \< 0.05 was considered significant. Supplementary information ========================= {#Sec23} DATASET 1 **Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Jonathan Vigne, Sylvie Bay, Giuseppina Caligiuri and Francois Rouzet. Supplementary information ========================= is available for this paper at 10.1038/s41598-019-56163-x. This work was supported by INSERM, RHU iVASC and by a grant from MSDAVENIR (Merck Sharp and Dohme Avenir, project "Save-Brain"). This work was performed on a platform member of France Life Imaging network (grant ANR-11-INBS-0006). We thank Frédéric Bonhomme for help with mass spectrometry by direct infusion. J.V. carried out the radiochemistry experiments, evaluated the radiotracers, participated in the study design and coordinated and co-drafted the manuscript. S.B. and C.G. carried out peptide chemistry. J.S., A.T., N.A., R.A.L., G.P. and G.R. participated in animal experiments and *in vivo* imaging experiments. G.E., F.A., M.L.B. participated in histology experiments. A.N., D.L.G., G.C. and F.R. participated in the study design, performed statistical analysis and helped to draft the manuscript. Part of the data presented in this work are comprised in a recently filed patent: Inventor Sylvie Bay, Antonino Nicoletti, Dominique Le Guludec, Jonathan Vigne, Giuseppina Caligiuri; "CD31shed as a molecular target for imaging of inflammation". PCT/EP2017/060574. No other potential conflicts of interest relevant to this article exist.

Melioidosis causes severe sepsis and death in the Top End of Northern Australia during the monsoonal wet season.[@bib1] The wet season (melioidosis season) is defined to capture the seasonal presentation in the tropical wet season (November to April),[@bib2] with average monthly rainfalls of 100 to 500 mm in the 6 months ([Figures 1](#fig1){ref-type="fig"} and [Supplementary Figure S1](#appsec1){ref-type="sec"}) and high humidity of \> 80%.[@bib3] Melioidosis is caused by the saprophytic Gram-negative bacterium and Tier 1 select agent *Burkholderia pseudomallei,* which naturally occur in tropical soil and water.[@bib4] *Burkholderia pseudomallei* is widespread in Northern Australia and Southeast Asia and is increasingly recognized as being endemic in other tropical regions globally.[@bib1], [@bib4], [@bib5], [@bib6] The Darwin Prospective Melioidosis Study (DPMS) is a long-running, large, prospective observational study started in October 1989 that aims to understand the clinical and microbiological aspects of melioidosis in the Top End of the Northern Territory (NT), and to use this information to lessen the burden of the disease through earlier diagnosis and improved treatment. The study has documented all cases of melioidosis in the Top End of the NT since 1 October 1989,[@bib7], [@bib8] with around 85% of cases occurring during the tropical wet season (November to April)[@bib2] ([Figure 1](#fig1){ref-type="fig"}).Figure 1Correlation between cases of melioidosis managed at Royal Darwin Hospital in 2009--2010 and rainfall at Darwin airport.Reproduced with permission from Parameswaran U, Baird RW, Ward LM, et al. Melioidosis at Royal Darwin Hospital in the big 2009--2010 wet season: comparison with the preceding 20 years. *Med J Aust.* 2012;196:345--348. Copyright © 2012 The Medical Journal of Australia. Chronic kidney disease (CKD) is an independent risk factor for melioidosis, and CKD is associated with a higher mortality rate whenever melioidosis occurs.[@bib9], [@bib10] Other factors associated with high risk for melioidosis include diabetes mellitus, hazardous alcohol use, chronic lung disease, rheumatic heart disease and cardiac failure, and immune-suppressive medications, most notably the use of corticosteroids. Age and indigenous ethnicity are also independent predictors for melioidosis. These factors are also common among adult patients dependent on dialysis in this region.[@bib11] In our region, we have previously reported staggering higher incidence rates of melioidosis among adults dependent on dialysis than among those without dialysis-dependent CKD (988.8/100,000 vs. 24.0/100,000 patient-years), equating to a crude relative risk for melioidosis among adults dependent on dialysis of 38.4 (95% confidence interval \[CI\] = 25.7--57.5).[@bib11] As observed in some previous wet seasons, during the 2011 to 2012 wet season, we observed a higher frequency of melioidosis among the dialysis cohort.[@bib12] Rates of melioidosis are lower among our renal transplant cohort. Our routine practice to specifically mitigate wet season−associated melioidosis for the renal transplant and immunosuppressed cohort includes consideration of trimethoprim+sulfamethoxazole (TMP+SMX) prophylaxis treatment, at a dose higher than usually used for *Pneumocystis jirovecii* pneumonia (PJP) prophylaxis.[@bib13] Pharmacodynamic and pharmacokinetic studies of TMP+SMX for the treatment of melioidosis indicate that high doses of oral TMP+SMX are required for eradication after an initial intensive treatment with i.v. ceftazidime or meropenem.[@bib10], [@bib14], [@bib15], [@bib16], [@bib17], [@bib18] There are no published data on the prophylactic use of TMP+SMX (or any other antibiotics) to reduce melioidosis in high-risk groups, although TMP+SMX has been used as postexposure prophylaxis for *Burkholderia pseudomallei* infection among laboratory staff.[@bib19] Therefore, following the increase in both the number of melioidosis cases observed in the 2011 to 2012 wet season, and the concomitant increase in the size of the prevalent dialysis patient cohort, we undertook a prospective open-label intervention by implementing a prophylaxis guideline for hemodialysis patients in the Top End of the Northern Territory over the wet season (1 November 2014 to 30 April 2015), using oral trimethoprim+sulfamethoxazole (TMP+SMX), 160/800 mg daily. The aim of this study was to determine the efficacy and safety of prophylaxis with daily TMP+SMX for melioidosis in hemodialysis patients from the Top End of Northern Australia during the wet season from 1 November 2014 to 30 April 2015. Materials and Methods {#sec1} ===================== Study Design {#sec1.1} ------------ The study was a prospective, open-label, interventional study carried out as part of the larger Darwin Prospective Melioidosis Study, which documents all cases of melioidosis and treatment in the Top End of the Northern Territory.[@bib20] Study Population {#sec1.2} ---------------- All patients ≥18 years of age who had been on maintenance hemodialysis for ≥3 months were offered the prophylactic treatment. All eligible hemodialysis patients throughout the Top End received daily TMP+SMX, excluding persons with known hypersensitivity to trimethoprim and/or sulfamethoxazole, lipoamides, or any other ingredients in the formulations of the tablets, severe hepatic failure, marked liver parenchymal damage or jaundice, or serious hematological disorders (thrombocytopenia \< 80,000 platelets/μl, leukopoenia \< 3.5 × 10^9^/l (neutrophil count \< 2.7 × 10^9^), and porphyria, and any other contraindications to TMP+SMX. Those who declined the treatment were also excluded from the prophylaxis treatment. The cohort could therefore be described categorically as those who received the intervention and a control group of those who were ineligible for the intervention (or TMP+SMX-group \[prophylaxis\] vs. nonprophylaxis group). All patients received the usual wet season advice on melioidosis prevention.[@bib14] Definitions {#sec1.3} ----------- Patients' ethnicity was entered as indigenous if they were Aboriginal and/or Torres Strait Islander in their demographical data entry in the clinical records. Abnormal liver function was defined by any rise in liver transaminases and bilirubin. Neutropenia was defined as a neutrophil count of \<2.7 × 10^9^/l and thrombocytopenia as a platelet count of \<150,000 platelets/μl. For the purpose of assessing safety, we also defined categories of thrombocytopenia, based on the protocol that we have developed with platelet counts of \<80,000, ≥80,000 to \<150,000, and ≥150 000, and based on the conventional definition of platelet counts by severity of \<50,000, ≥50,000 to \<150,000, and ≥150 000. Dosing of Trimethoprim+Sulfamethoxazole {#sec1.4} --------------------------------------- There were no guidelines for dosing of TMP+SMX for prophylaxis of melioidosis in hemodialysis patients, so we initially used the standard dosage that has been safely used as eradication treatment for persons on a dialysis dose of 1 double-strength tablet of TMP+SMX 160 mg/800 mg once a day.[@bib21] On dialysis days, the patients received the drug after dialysis. All patients on the prophylaxis also received folic acid 5 mg once a day to avoid TMP+SMX−induced folate deficiency. Treatment Rollout {#sec1.5} ----------------- All 261 patients who were eligible and were not receiving treatment for melioidosis were offered treatment. The medication rollout was undertaken from 1 November 2014, with the last patient receiving the first dose on 11 January 2015. Clinical Safety and Laboratory Monitoring {#sec1.6} ----------------------------------------- The majority of our patients receive hemodialysis within satellite dialysis units at least 3 times a week, achieving a minimum of 12 hours of treatment per week,[@bib22] and have blood tests performed monthly (at the start of every month or whenever clinically indicated) as part of routine care. The renal pharmacist provided in-services to primary dialysis nurses across all dialysis units, who then routinely asked questions of each patient pertaining to any medication complications. Patients were asked to report signs of nausea, vomiting, and skin reactions at each dialysis session and whenever they attended a nephrologist's clinic appointment. Patients concurrently were specifically monitored for the development of neutropenia, thrombocytopenia, and abnormal liver function at each monthly blood test throughout the treatment phase and for another 2 months after completion. Statistical Analysis {#sec1.7} -------------------- A descriptive analysis was undertaken. Data are described as frequency and percentage for categorical variables, and continuous data were reported as mean (SD) with 95% CIs for normally distributed data and as median (interquartile range) for data that were not normally distributed. For comparisons, we used a 2-sample Student *t* test for continuous normally distributed data and the Mann−Whitney *U* test for non−normally distributed data. Comparisons between categorical data were performed using the χ^2^ and Fisher exact tests as appropriate. Data were analyzed using intention-to-treat analysis. Statistical significance was determined by a 2-tailed *P* value of \< 0.05 and 95% CIs where appropriate. Treatment Cessation {#sec1.8} ------------------- The treatment was stopped in \> 95% of patients on 30 April 2015. In the rest of the patients, treatment was stopped within a few days after 30 April 2015. Ethical Considerations {#sec1.9} ---------------------- The Darwin Prospective Melioidosis Study is a prospective study approved by the Human Research Ethics Committee of the Northern Territory Department of Health and the Menzies School of Health Research (Approval \# HREC 02/38).[@bib20] Results {#sec2} ======= Baseline Characteristics and Risks of Melioidosis {#sec2.1} ------------------------------------------------- A total of 269 patients were receiving maintenance hemodialysis on 1 November 2014. Eight patients (3%) developed melioidosis and were already receiving appropriate therapy prior to the commencement of the prophylaxis intervention, and were thus excluded from this analysis. These 8 patients received the minimum 2-week intensive treatment with i.v. antibiotics (ceftazidime or meropenem), followed by the 3-month eradication therapy with TMP+SMX. They then continued on prophylaxis (dose similar to that of the eradication therapy) to the end of the wet season, and were not included in the overall analysis ([Figure 2](#fig2){ref-type="fig"}).Figure 2Melioidosis intervention study flow diagram. The analysis describes the remaining 261 patients (97%), divided into the TMP+SMX prophylaxis group (who received prophylaxis, n = 169 \[64.8%\]) and the nonprophylaxis group (who did not receive prophylaxis, n = 92 \[35.2%\]). Of the 92 patients who did not receive the prophylaxis (92/262 \[35.2%\]), the reasons were history of allergy to the drug (n = 10), remoteness and other logistical reasons (n = 60), poor attendance for dialysis (n = 11), and refusal of treatment (n = 11). Overall, the main comorbidities included diabetes (66.7%), history of cardiovascular disease (70.5%; including cardiac arrhythmias, ischemic heart disease, congestive cardiac failure, and rheumatic heart disease), hypertension (66.7%), and chronic lung disease (36.8%). None of the 269 patients were on any immunosuppressive therapy. There was no significant difference in the age of participants by group (TMP+SMX-prophylaxis group vs. nonprophylaxis group, 54.7 \[11.3\] years vs. 54.3 \[11.2\] years; *P* = 0.751). The proportion of indigenous patients in each group was also similar at ∼ 85% There was no statistically significant difference in the baseline platelet counts (180.8 \[76.7\], 95% CI = 156.0−205.7, vs. 178.4 \[75.2\], 95% CI = 154.0−202.8, *P* = 0.898) and neutrophil counts (5.0 \[2.2\], 95% CI = 4.2−5.7, vs. 5.9 \[2.3\], 95% CI = 5.1−6.7, *P* = 0.205) at the start of the study between the groups. [Table 1](#tbl1){ref-type="table"} provides the details of the comparison in the baseline characteristics and the risks of melioidosis between the 2 groups.Table 1Comparison of baseline characteristics and risk factors for melioidosis between the TMP+SMX prophylaxis group and the nonprophylaxis groupComorbidityProphylaxis (n = 169)\ n (%)Nonprophylaxis (n = 92)\ n (%)*P* valueGender (male)94 (55.6)52 (56.5)0.897Ethnicity (indigenous)143 (84.6)78 (84.7)1.000Diabetes mellitus113 (66.9)61 (66.3)0.728Ischemic heart disease/heart failure119 (70.4)65 (70.7)0.840Chronic obstructive pulmonary disease62 (36.7)34 (37.0)0.635Chronic liver disease6 (3.6)4 (4.3)0.670Atrial fibrillation104 (61.5)56 (60.9)0.648Dyslipidemia118 (69.8)66 (71.7)0.216Hypertension114 (67.5)63 (68.5)0.917Thyroid disease7 (4.1)4 (4.3)1.000History of latent tuberculosis5 (3.0)3 (3.3)0.157Obesity8 (4.7)5 (5.4)0.517Pulmonary hypertension6 (3.6)4 (4.3)0.768Rheumatic heart disease11 (6.5)9 (9.7)0.547Systematic lupus erythematosus4 (2.4)2 (2.2)0.270Secondary hyperparathyroidism12 (7.1)8 (8.7)0.768Dialysis adequacy KT/V \> 1.4150 (88.8)82 (89.1)0.675 URR \> 70152 (90.0)81 (88.0)0.586[^1] Adherence to Treatment {#sec2.2} ---------------------- Of the 169 patients who received the treatment, 159 patients (94%) received the full treatment through the study period. Of the remaining 10 patients (5.9%), 7 patients (4.1%) received treatment at least 2 times a week mainly due to missing some dialysis sessions, and 3 patients (1.8%) stopped the treatment due to adverse drug reactions. Efficacy of TMP+SMX Prophylaxis and Cases of Melioidosis {#sec2.3} -------------------------------------------------------- During the period from 1 November 2014 to 30 April 2015, a total of 16 documented cases of culture-positive melioidosis (6%) were observed among the 261 hemodialysis patients eligible for this analysis. This corresponds to a melioidosis event frequency of 0% in the TMP+SMX prophylaxis group and 17.4%in the nonprophylaxis group (0/169 vs. 16/92, *P* \< 0.001). Mortality {#sec2.4} --------- Thirteen patients (5%) died during the 6-month period of the treatment. Six of 169 patients (3.6%) were from the TMP+SMX prophylaxis group, and 7 of 92 patients (7.6%) were from the nonprophylaxis group (*P* = 0.231). One patient among the 8 patients who were receiving treatment for melioidosis at the time of starting the study died of complications related to a myocardial infarction but had completely recovered from the infection at the time of death. The 6 patients in the TMP+SMX prophylaxis group died of complications related to cardiovascular disease. In the nonprophylaxis group, 4 of the 7 patients died of melioidosis, and the other 3 patients died of cardiovascular complications. The remaining 12 patients who had melioidosis (75%) were successfully treated as per protocol.[@bib16], [@bib17] Safety of the Trimethoprim/Sulfamethoxazole Prophylaxis {#sec2.5} ------------------------------------------------------- There was a higher incidence rate of non−clinically significant thrombocytopenia noted in the TMP+SMX-prophylaxis group ([Tables 2](#tbl2){ref-type="table"} and [3](#tbl3){ref-type="table"}); however, the majority of cases were not severe enough to warrant the withdrawal of treatment. Three patients (1.8%) were withdrawn from the TMP+SMX-prophylaxis group due to significant side effects. These patients included 1 patient with drug reaction with eosinophilia and systemic symptoms syndrome (DRESS) and thrombocytopenia and 2 patients with skin rash.Table 2Comparison between the 2 groups of the proportion of patients in the categories with platelet count of 50,000/μl as the minimum during the study and 1 month after stopping treatmentMonth[a](#tbl2fna){ref-type="table-fn"}Platelet count category[b](#tbl2fnb){ref-type="table-fn"}Group*P* value[c](#tbl2fnc){ref-type="table-fn"}Prophylaxis n (%)Nonprophylaxis n (%)112 (1.0)2 (2.2)0.613272 (42.8)19 (20.6)395 (56.2)71 (77.2)212 (1.2)2 (2.2)0.613269 (40.8)16 (17.4)398 (58.0)74 (80.4)314 (2.4)2 (2.2)1.000268 (40.2)15 (16.3)397 (57.4)74 (81.5)413 (1.8)2 (2.2)1.000267 (40.2)14 (16.3)397 (58.0)72 (81.5)513 (1.8)1 (1.1)1.000254 (33.1)19 (21.7)3107 (65.1)66 (77.2)6[d](#tbl2fnd){ref-type="table-fn"}14 (2.4)1 (1.1)0.660249 (30.2)15 (17.4)3110 (67.5)69 (81.5)[^2][^3][^4][^5][^6]Table 3Comparison between the 2 groups of the proportion of patients in categories with platelet count of 80,000/μl as the minimum during the study and 1 month after stopping treatmentMonth[a](#tbl3fna){ref-type="table-fn"}Platelet count category[b](#tbl3fnb){ref-type="table-fn"}Group*P* value[c](#tbl3fnc){ref-type="table-fn"}Prophylaxis n (%)Nonprophylaxis n (%)1110 (5.9)5 (5.4)1.000264 (37.9)16 (17.2)395 (56.2)71 (77.2)2114 (8.3)4 (4.4)0.309257 (33.7)14 (15.2)398 (58.0)74 (80.4)3114 (8.3)3 (3.3)0.187258 (34.3)14 (15.2)397 (57.4)74 (81.5)4113 (7.7)3 (3.3)0.185257 (34.3)13 (15.2)397 (58.0)72 (81.5)5113 (8.3)4 (4.3)0.309244 (26.6)16 (18.5)3107 (65.1)66 (77.2)6[d](#tbl3fnd){ref-type="table-fn"}110 (5.9)4 (4.4)0.776243 (26.6)12 (14.1)3110 (67.5)69 (81.5)[^7][^8][^9][^10][^11] One patient had a severe skin reaction, which improved after stopping the TMP+SMX. The second patient in the TMP+SMX prophylaxis group who developed DRESS also had multiorganism culture−positive severe sepsis. She was on peritoneal dialysis (PD) and developed severe PD peritonitis requiring PD catheter removal. She was converted to hemodialysis as a bridging treatment pending complete recovery from the PD peritonitis. She had a short period dialysing via a temporary internal jugular catheter. Upon complete recovery from the infections, the catheter was converted to a tunneled catheter. She was then commenced on the TMP+SMX as prophylaxis for melioidosis. Four weeks later, she was admitted with a presumed infection of the right-sided internal jugular vein tunneled dialysis catheter and a skin rash. She was commenced on several antibiotics including vancomycin, meropenem, and fluconazole, as blood cultures were positive for multiple organisms which included *Pseudomonas eruginosa*, *Staphylococcus capitis*, *Staphylococcus epidermidis,* and *Candida* species. The TMP+SMX was immediately stopped on admission due to the rash, but the patient continued to deteriorate and subsequently died. The cause of death in this patient was attributed to a combination of sepsis with severe drug reaction. It was not possible to exclude other antibiotics as the cause of DRESS. Nevertheless, the temporal relationship between the initial appearance of the rash prior to commencement of the other antibiotics suggested that TMP+SMX was the most likely cause. Neutropenia was more common in the TMP+SMX prophylaxis group than the nonprophylaxis group. However, this was generally not severe enough to cease the prophylaxis in any patient ([Table 4](#tbl4){ref-type="table"}).Table 4Comparison between the 2 groups of the proportion of patients in neutrophil count categories during the study and 1 month after the studyMonth[a](#tbl4fna){ref-type="table-fn"}Neutrophil count category[b](#tbl4fnb){ref-type="table-fn"}Group*P* value[c](#tbl4fnc){ref-type="table-fn"}Prophylaxis proportion, n (%)Nonprophylaxis proportion, n (%)1115 (8.9)2 (2.2)0.0382154 (91.1)90 (97.8)2114 (8.3)5 (5.4)0.4642155 (91.7)87 (94.6)3115 (8.9)2 (2.2)0.0382154 (91.1)89 (97.8)4115 (9.0)2 (2.2)0.0392152 (91.0)86 (97.8)5118 (11.2)3 (3.3)0.0092146 (88.8)83 (96.7)6[d](#tbl4fnd){ref-type="table-fn"}113 (7.7)3 (3.3)0.1852150 (92.3)82 (96.7)[^12][^13][^14][^15] There was no difference in abnormal liver function between the 2 groups. There was no impact on hemoglobin and requirements for erythropoiesis-stimulating agents. Thrombocytopenia and neutropenia had improved in all patients in the 2 months after ceasing the prophylaxis. [Figure 2](#fig2){ref-type="fig"} shows a flow diagram of how patients were evaluated, by TMP+SMX prophylaxis group and nonprophylaxis group, and presentation of selected events. Discussion {#sec3} ========== We completed a prospective interventional study to determine the safety and efficacy of daily oral TMP+SMX prophylaxis to prevent melioidosis among a cohort of maintenance hemodialysis patients living in a region with endemic *Burkholderia pseudomallei* and melioidosis-related harms. The key findings are as follows: (i) daily oral TMP+SMX prophylaxis was probably efficacious against melioidosis in hemodialysis patients; (ii) daily dosing was relatively safe, although there was a tendency for increased episodes of nonsevere thrombocytopenia and neutropenia, without hepatic adverse events; (iii) logistical difficulties with administration of a daily dosing regimen and patient factors resulted in a number of patients being excluded from the prophylaxis; and (iv) the incidence rate of melioidosis of 17.4% in the nonprophylaxis group was higher than in our recent publication[@bib11]; however, this is not unusual, as it has been shown in previous work in which rates as high as 29% have been recorded during some wet seasons[@bib12], [@bib23] ([Figure 1](#fig1){ref-type="fig"}). The predominant reason for some patients not receiving prophylaxis was remoteness and logistical reasons. Our dialysis patients from remote areas perform self-care dialysis and dialysis in remote satellite units. By selection, they tend to be the healthiest patients, who can manage self-care and require minimal support. They receive regular follow-up by their nephrologists. However, these logistical issues have been addressed in the subsequent wet seasons, and patients now receive the prophylaxis regardless of where they have dialysis. In this high-risk population,[@bib11] the prophylaxis intervention was effective when provided in combination with wet season personal preventive advice,[@bib20] as no cases of melioidosis were observed in the TMP+SMX prophylaxis group. These measures include minimizing contact with the bacteria (covering open skin wounds, avoiding contact with soil and standing water, and wearing covered footwear) and using standard contact precautions (mask, gloves, and gown where appropriate) to help prevent infection.[@bib14] All cases of melioidosis occurred in patients who either developed the infection before the prophylaxis was introduced, or did not receive the prophylaxis, which supports the policy in our region of routinely providing melioidosis prophylaxis during the wet season. The nonprophylaxis group included patients who had nonmodifiable reasons (including TMP+SMX allergy, the frequency of which was similar to that in other reports of TMP+SMX),[@bib24] and reasons that may be modifiable. Potentially modifiable causes of ineligibility for the prophylaxis group included client preferences to decline a prophylactic treatment, and timely supply of the drug to home-based patients in regions remote from the treatment center. Thrombocytopenia and neutropenia among participants in the TMP+SMX-group were observed ([Table 2](#tbl2){ref-type="table"}, [Table 3](#tbl3){ref-type="table"}, [Table 4](#tbl4){ref-type="table"}), but were not severe enough to warrant withdrawal of treatment. It is important to note that a number of patients in the sample had low platelet and neutrophil counts that were not related to the TMP+SMX, as evidenced by rates of thrombocytopenia and neutropenia in the nonprophylaxis group ([Table 2](#tbl2){ref-type="table"}, [Table 3](#tbl3){ref-type="table"}, [Table 4](#tbl4){ref-type="table"}). Withdrawal of TMP+SMX was required only in the event of a severe drug reaction. Our data show the safe and efficacious use of TMP+SMX to mitigate melioidosis in our hemodialysis cohort, which has been developed into a comprehensive protocol for the next wet season (2015−2016) ([Supplementary Material S1](#appsec1){ref-type="sec"}). To address logistic supply issues and pill burden and to improve medication adherence, we adapted the prophylaxis intervention, using TMP+SMX 1 double-strength tablet supervised post-dialysis 3 times a week in the wet season from 1 November 2015 to 30 April 2016. There were no identified cases of melioidosis during this period in those on the 3 times a week prophylaxis, and there were no significant adverse events. Following the success of this treatment, a comprehensive educational and awareness program led by the Northern Territory Centre for Disease Control has been undertaken to increase staff and patient education and awareness. We have produced a patient information video[@bib25] and are addressing medication prescribing and dispensing and monitoring issues. In the current incomplete wet season (1 November 2016 to 30 April 2017), there have been 3 cases of melioidosis in patients who had not started the prophylaxis due to various logistic issues. To date, we have not yet implemented a prophylaxis program for our peritoneal dialysis cohort, as antibiotic use can be linked to the development of fungal peritonitis. A number of limitations of this study are acknowledged. It is a prospective observational study of an antibiotic intervention that required voluntary participation, which introduces the potential for bias. However, the study has clearly shown efficacy and safety of the administration of TMP+SMX in hemodialysis patients. There have been no events to date of TMP+SMX-resistant melioidosis in this cohort, but careful monitoring is required to ensure ongoing surveillance and drug stewardship. This prophylaxis program has been successful as part of a comprehensive multiagency strategy to mitigate harm in our patients. Patients within our hemodialysis cohort are regularly engaged in clinical care, which enables a greater responsiveness to side effects or adverse events. It is unknown whether this protocol may be effective in groups with less access to health care and monitoring, such as the wider diabetic population in melioidosis-endemic regions. Finally, potential confounding factors, especially the adherence to preventive measures for melioidosis, were not adjusted for in the study, although we have no reason to believe that this was different between the 2 groups. In conclusion, the use of 1 double-strength tablet of TMP+SMX postdialysis in hemodialysis patients was shown to be effective and safe in the prevention of melioidosis. We recommend the use of this approach in tropical Northern Australia and other regions where melioidosis is prevalent. Disclosure {#sec5} ========== All the authors declared no competing interests. Supplementary Material {#appsec1} ====================== Supplementary Material S1Prophylaxis guideline for melioidosis in hemodialysis patients.Figure S1Average monthly rainfall in millimeters (mm) at Darwin Airport for the years 1999 to 2015.Figure S2Consolidated Standards of Reporting Trials (CONSORT) flow diagram: melioidosis intervention study. We acknowledge our colleagues in the microbiology laboratory at Royal Darwin Hospital for their expertise in the laboratory diagnosis of melioidosis, and our clinical colleagues at Royal Darwin Hospital for management of the patients. JH was supported by NHMRC Fellowship 1092576 and a Jacquot Research Establishment Award. The Darwin Prospective Melioidosis study is supported by grants from the Australian National Health and Medical Research Council, including project grants 1098337 and 1131932 (the HOT NORTH initiative). **Supplementary Material S1.** Prophylaxis guideline for melioidosis in hemodialysis patients. **Figure S1.** Average monthly rainfall in millimeters (mm) at Darwin Airport for the years 1999 to 2015. **Figure S2.** Consolidated Standards of Reporting Trials (CONSORT) flow diagram: melioidosis intervention study. Supplementary material is linked to the online version of the paper at [www.kireports.org](http://www.kireports.org){#intref0010}. [^1]: URR, urea reduction ratio. [^2]: There was no statistically significant difference in platelet counts at baseline between the 2 groups. [^3]: Month after start of treatment. [^4]: Platelet count categories (per μl): 1: ≤ 50,000; 2: 51,000−150,000; 3: ≥150,000. [^5]: Fisher exact test. [^6]: One month after stopping treatment. [^7]: There was no statistically significant difference in platelet counts at baseline between the 2 groups. [^8]: Month after start of treatment. [^9]: Platelet count categories (per μl): 1: ≤80,000; 2: 81,000−150 000; 3: ≥150,000. [^10]: Fisher exact test. [^11]: One month after stopping treatment. [^12]: Months from start of treatment. [^13]: Neutrophil count categories (× 10^9^/l): 1: \< 2.7; 2: ≥ 2.7. [^14]: Fisher exact test. [^15]: One month after stopping treatment.

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Introduction ============ To obtain system-level descriptions of biological and cellular events, transcriptome profiling, including recent RNA-sequencing approaches,^[@bib1]^ has generated large, unmanageable high-throughput data sets.^[@bib2]^ To make sense of these gene expression data sets, researchers have developed various gene set analysis (GSA) tools, such as DAVID^[@bib3]^ and GSEA,^[@bib4]^ in combination with prior knowledge-based databases, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG),^[@bib5]^ Gene Ontology,^[@bib6]^ BioCarta, PANTHER,^[@bib7]^ MetaCyc,^[@bib8]^ Molecular Signatures Database^[@bib4]^ and RegulonDB.^[@bib9]^ Despite these diligent research efforts, the biologically meaningful interpretation of findings from high-throughput gene expression data remains a bottleneck.^[@bib2]^ The persistence of this congestion arises from the challenge of exploring the complex relationships between cellular components,^[@bib10]^ especially in the context of functional molecular pathways. Pathway information as it relates to a phenotype of interest (for example, a disease) necessarily implies that a key molecular target should be considered within the framework of its network. A network focus enables us to more effectively infer key transcriptional changes related to the specific phenotype by examining multiple downstream (or cross-talk) effectors of the target. However, the current GSA^[@bib11]^ tools utilize over-representation analysis,^[@bib12]^ which reports the enrichment of functional groups (for example, gene sets) for the genes of interest. They compromise the connectivity in favor of computational simplicity that is based on cellular components and not their connectivity.^[@bib3],[@bib4]^ In other words, current tools do not analyze the wiring diagram of the interactions (for example, activation, inhibition) in a functional molecular network.^[@bib13]^ We have developed PATHOME (*path*way and transcript*ome* information), a novel computational algorithm for identifying differentially expressed subpathways. Methodologically, PATHOME has two benefits: It analyzes the regulation information between nodes in the biological pathways and is applicable to a small number of samples. PATHOME is not a permutation-based approach that requires more samples in order to obtain a null distribution for a statistical test. We demonstrated the utility of PATHOME by applying it to gene expression data of gastric cancer (GC), thereby identifying tumor-related dysregulated pathways and novel therapeutic targets. Based on a reference set of known cancer-related pathways, PATHOME showed greater sensitivity and robustness than other leading methods in detecting differential molecular signals. For the WNT signaling pathway revealed only by PATHOME, we validated its involvement in gastric carcinogenesis through experimental studies of both cell lines and animal models. Our results further revealed a potential therapeutic target, WNT5A. Thus, PATHOME represents a powerful tool for inferring biologically interpretable patterns from gene expression data. Results ======= Overview of PATHOME algorithm and study design ---------------------------------------------- PATHOME takes the gene expression profiles of two comparison groups (for example, cancer vs non-cancer tissue) and related biological pathways from prior knowledge. In this study, we used the KEGG pathway database as the source of prior knowledge. PATHOME first decomposes the pathways into linear paths (subpathways) from the top nodes to leaf nodes, and then employs simple statistical tests to evaluate the significance of differential expression patterns along the subpathways ([Figures 1a and b](#fig1){ref-type="fig"}). The interaction property between pathway members (for example, activation or inhibition) is also considered ([Figure 1c](#fig1){ref-type="fig"}). A detailed description of the PATHOME algorithm is provided in Materials and methods. To evaluate the performance and demonstrate the utility of PATHOME, we applied it to publicly available gene expression data sets of Asian GC ([Table 1](#tbl1){ref-type="table"}). [Figure 2](#fig2){ref-type="fig"} summarizes our study strategy, which consists of two stages: a discovery stage and a validation stage. The primary aim of the discovery stage is to compare the performance of PATHOME with those of DAVID^[@bib3]^ and GSEA,^[@bib4]^ two leading GSA tools based on a reference set of known cancer-related pathways. The data sets we used in this stage were one Korean GC data set (GSE13861)^[@bib14]^ and one Japanese GC data set (GSE15081).^[@bib15]^ A second aim of this two-stage strategy was to select potential therapeutic targets in GC. Thus, based on differential subpathways inferred from the discovery stage, we further identified robust gene signatures by using independent data sets at the validation stage. The data sets we used in this stage were another Korean GC data set (GSE36968)^[@bib16]^ and one Chinese GC data set (GSE27342).^[@bib17]^ Finally, through hierarchical clustering analysis, transcription factor-binding site (TFBS) analysis and expression pattern analysis from ArrayExpress,^[@bib18]^ we identified the target gene/pathway for experimental validation. Performance comparison of PATHOME with other algorithms ------------------------------------------------------- The comparison sample groups in the first gene expression data set (GSE13861)^[@bib14]^ were 65 primary gastric adenocarcinoma frozen tissue samples and 19 normal appearing gastric tissue samples. With PATHOME, we identified 113 810 subpathways that belong to 27 KEGG pathways ([Supplementary Table 1](#sup1){ref-type="supplementary-material"}) at a false discovery rate (FDR)* *\<0.05. To the same data set, we applied DAVID and GSEA, using default parameter settings (the results are shown in [Supplementary Tables 2 and 3](#sup1){ref-type="supplementary-material"}, at FDR\<0.3). The comparison groups in the second gene expression data set (GSE15081)^[@bib15]^ were the samples from 18 patients with GC who experienced peritoneal relapse and 38 who did not experience peritoneal relapse. At FDR\<0.05, PATHOME reported 126 095 subpathways that belong to 15 KEGG pathways ([Supplementary Table 4](#sup1){ref-type="supplementary-material"}). The significant pathways identified by DAVID and GSEA are shown in [Supplementary Tables 5 and 6](#sup1){ref-type="supplementary-material"} (FDR\<0.3), respectively. To evaluate the performance of PATHOME, we used a set of known cancer-related pathways^[@bib19]^ as a reference standard for comparing the three methods (see [Table 2](#tbl2){ref-type="table"}). PATHOME used a lower significance cutoff (FDR\<0.05) compared with that of the two methods (FDR\<0.3 for both methods). Despite the lower cutoff, our method detected more differential cancer-related pathways. Selection of potential therapeutic targets in the WNT signaling pathway ----------------------------------------------------------------------- Although other tools assess the overall enrichment of genes of interest in a given pathway, the strategy of PATHOME is to thoroughly inspect all possible paths in the pathway. Path decomposition in [Figure 1a](#fig1){ref-type="fig"} allows PATHOME to detect a pathway for which the enrichment analyses of other tools cannot report significance. To further demonstrate the utility of our method, applied PATHOME to the identification of potential therapeutic targets from within the results we had obtained. For this purpose, PATHOME reported significant subpathways relating to WNT signaling, MAPK signaling, insulin signaling, focal adhesion and chemokine signaling ([Table 2](#tbl2){ref-type="table"}) in East Asians. Among these identified pathways, we selected the WNT pathway as identified uniquely by PATHOME for further cell line and animal studies for accuracy validation. Although the two discovery data sets were constructed from different comparisons of GC patients, the PATHOME results strongly suggested that subpathways of the WNT pathway were involved in the development of primary GC and peritoneal cancer relapse. Using Cytoscape,^[@bib20]^ we combined the significant subpathways related to the WNT pathway from the two data sets and visualized the combined subpathways of 62 genes ([Figure 3a](#fig3){ref-type="fig"} and [Supplementary Table 7](#sup1){ref-type="supplementary-material"}). At the validation stage, we investigated whether the expression patterns in the combined subpathways inferred from the discovery stage were reproducible in another two independent data sets: our published RNA-seq GC data set of Korean patients (GSE36968: 24 primary cancer tissue samples vs 6 non-cancer samples)^[@bib16]^ and another microarray data set (GSE27342: 160 samples of paired gastric tumor and adjacent normal tissues).^[@bib17]^ We found that the expression changes in the 62 genes of the combined subpathways showed great concordance in terms of up- or downregulation (relative to non-cancer samples) between GSE13861 and GSE36968 (Fisher\'s exact text, *P*\<0.007, [Supplementary Table 8](#sup1){ref-type="supplementary-material"}) or between GSE13861 and GSE27342 (Fisher\'s exact text, *P*\<0.018, [Supplementary Table 8](#sup1){ref-type="supplementary-material"}), highlighting the reproducibility of differential WNT subpathways identified. We arbitrarily assigned the four data sets into either the discovery stage or the validation stage. Because of the considerable concordance among the data sets ([Supplementary Table 8](#sup1){ref-type="supplementary-material"}), this assignment yet enabled us to work in the current study design in spite of the heterogeneity of the origins of the data sets. To pinpoint individual genes for further experimental valuation, we performed hierarchical clustering analysis, TFBS analysis and expression pattern analysis from ArrayExpress.^[@bib18]^ The hierarchical clustering analysis ([Figure 3b](#fig3){ref-type="fig"}) found that eight genes showed up- or downregulation consistently across all four data sets used in both stages ([Figure 3b](#fig3){ref-type="fig"}, indicated by the red arrows), among which five genes (*WNT5A*, *VANGL1*, *SFRP2*, *FZD1* and *PLCB1*) were upregulated. We next focused on these five upregulated genes (as potential drug-inhibition candidates) and evaluated their TFBSs. Using the ENCODE transcription factor ChIP-Seq data^[@bib21]^ and the evolutionarily conserved TFBSs,^[@bib22]^ we examined the TFBSs within upstream 2-kb regions from the transcription start sites of the five genes. Interestingly, VANGL1 has two binding sites for hepatocyte nuclear factor 4 alpha (HNF4α) according to the ChIP-Seq data; whereas WNT5A and PLCB1 have binding sites for HNF4α in terms of conserved TFBSs ([Supplementary Figure 1A](#sup1){ref-type="supplementary-material"}). Thus, VANGL1, WNT5A and PLCB1 are potential target genes in the WNT pathway through HNF4α-mediated transcriptional regulation. To further confirm the potential regulation of HNF4α with these three genes at the transcriptional level, we examined the expression patterns of the four genes (*HNF4α* and *VANGL1*, *WNT5A*, *PLCB1*) in various experimental data sets in a public expression warehouse, ArrayExpress.^[@bib18]^ We found that 27 out of 166 data sets reported by ArrayExpress showed co-expression of the four genes ([Supplementary Figure 1B](#sup1){ref-type="supplementary-material"}), which was statistically significant (binomial distribution, *P*\<1.2 × 10^−14^). [Supplementary Figure 3](#sup1){ref-type="supplementary-material"} shows that gene expression of *WNT5A, FZD1, PLCB1* and *VANGL1* was detected in eight GC cell panels. Our previous study showed that the loss of the energy-sensing protein AMPK is a critical event in the initiation of GC; and that both mRNA and protein levels of HNF4α can be inhibited by metformin-mediated AMPKα activation in GC cells.^[@bib16]^ Thus, the above analyses revealed HNF4α-WNT5A as having a cross-talk function between the AMPKα energy-sensing pathway and the WNT signaling pathway. We hypothesized that a metformin effect on HNF4α by an AMPKα-mediated mechanism could transcriptionally downregulate the WNT pathway. To confirm this link, we selected WNT5A for further experimental validation (described in the following section). Involvement of WNT pathway and associated antitumor activity in GC ------------------------------------------------------------------ We first examined the basal protein expression level of several WNT family members (including WNT5A) and WNT-related downstream genes in 14 gastric tumor cell line panels ([Figure 4a](#fig4){ref-type="fig"}). Although WNT1 and WNT3 protein expression levels varied between the GC cell lines, WNT5A showed a similar protein expression level between gastric cell lines through immunoblotting measurement. In addition, we examined the basal protein expression level of WNT5A in xenograft tumors of 16 gastric tumor cell lines, and found similar protein expression levels between each xenograft model through immunohistochemistry ([Figure 4b](#fig4){ref-type="fig"}). As described above, WNT5A contains several highly conserved HNF4α-binding sites in its promoter region. Upon small interfering RNA (siRNA)-HNF4α treatment, the WNT5A gene expression level by real-time reverse transcription--PCR (RT--PCR) was downregulated by greater than 50% and 85% on days 2 and 3, respectively ([Figure 4c](#fig4){ref-type="fig"}), and the *TCF4* gene expression level was 25% downregulated on day 2. Meanwhile, the WNT5A protein level showed 21% and 52% inhibition on days 2 and 3, respectively ([Figure 4d](#fig4){ref-type="fig"}); whereas there was no difference in the protein level of β-catenin, and HIF1α was downregulated by 70% ([Figure 4d](#fig4){ref-type="fig"}). These results indicate that HNF4α as a transcription factor could regulate the transcription of WNT5A. However, different quantitation of knockdown and downregulation of gene expression level and protein level for HNF4α and WNT5A is observed in our experiments. To investigate the functional relevance of WNT5A in gastric tumors, we performed metformin treatment in the tumor cell lines. Upon metformin treatment, both the NCI-N87 and AGS gastric tumor cell lines showed anti-proliferation activities ([Figure 4e](#fig4){ref-type="fig"}). While there were small changes in WNT1 and WNT3 protein levels, we observed 42% inhibition of WNT5A and 58% inhibition of β-catenin protein expression level, with indifferences seen for glycogen synthase kinase (GSK)3 protein levels in both the NCI-N87 and AGS cell lines ([Figure 4e](#fig4){ref-type="fig"}), also, detected loss of transcription factor (TCF) and lymphoid enhancer-binding factor (LEF) protein expression level on days 2 and 3 ([Figure 4e](#fig4){ref-type="fig"}). The WNT canonical signaling pathway acts by regulating cell differentiation, cell growth and cell proliferation. We observed the regulation of cyclin D1 and cyclin D2 protein levels through WNT5A ([Figure 4e](#fig4){ref-type="fig"}). Moreover, we detected minimal effects on the phosphorylated-calcium/calmodulin-activated protein kinase (pCAMKII) protein level in WNT non-canonical signaling pathway, as we observed 5 out of 14 gastric cell lines to have confirmed pCAMKII protein detection. Potentially in GC, WNT5A does not activate a CAMKII-dependent signaling cascade. However, we observed different level of β-catenin protein expression inhibition in comparison between metformin and knockdown of HNF4α through siRNA. But in both metformin and siHN4α, decreased level of HIF1α protein expression level was observed in our previous^[@bib16]^ and present studies, where β-catenin potentiates transcription of HIF1α. Further studies are warranted to confirm this supposition. We silenced WNT5A expression using siRNA knockdown on the two GC cell lines (NCI-N87 and AGS) and performed cell proliferation assay. As shown in [Figures 5a and b](#fig5){ref-type="fig"}, siRNA-WNT5A affected cell growth through apoptosis. As shown in [Figure 5c](#fig5){ref-type="fig"}, siRNA-WNT5A affected protein translation of cell growth regulator cyclin D1 in both cell lines. To further evaluate our findings in a more biologically relevant context, we performed xenograft experiments in mouse models. Consistently, we found that the metformin treatment introduced strong antitumor activity ([Figure 6a](#fig6){ref-type="fig"}, NCI-N87 and MKN-45 cell lines, respectively). Importantly, we found the regulatory relationship to be consistent with that observed in the cell line studies. Upon metformin treatment vs non-treatment, the protein level of WNT5A decreased in eight out of nine NCI-N87 xenograft animals and in six out of eight MKN-45 xenograft animals. Also, the detected β-catenin protein level decreased more than 45% in the NCI-N87 and 25% in the MKN-45 animal models compared with that in the mice without metformin treatment, taking the mean of the ratio of protein expression levels ([Figures 6b and c](#fig6){ref-type="fig"}). Furthermore, immunohistochemistry revealed a loss of WNT5A protein expression in mice treated with metformin ([Figure 6d](#fig6){ref-type="fig"}). In summary, these *in vivo* studies confirmed the involvement of WNT5A signaling in the WNT pathway in gastric tumorigenesis through HNF4α, highlighting the potential of WNT5A as a therapeutic target in GC. Discussion ========== We developed a novel algorithm, PATHOME, for sensitively detecting differentially expressed pathways. Biological pathways consist of nodes and edges representing gene information and regulation information (for example, activation, inhibition), respectively. Current GSA methods for pathway (or gene set) analysis, such as DAVID,^[@bib3]^ GSEA,^[@bib4]^ GoMiner,^[@bib23]^ Onto-Tools^[@bib24]^ and GeneTrail,^[@bib25]^ consider nodes only. In contrast, our method is able to consider both types of information stored in the pathways, thereby improving the sensitivity for detecting differential signals. Disease occurs as a consequence of the dysregulation of a complex interdependency among biological components.^[@bib10]^ The identification of biologically targetable components requires the consideration of interdependency in terms of measurable statistical significance. Statistical significance is a metric by which researchers can rank or sort the importance of connections within biological cascades (equivalent to subpathways in our study). Along that line, PATHOME considers all subpathways from KEGG pathways, and tests each subpathway that is associated with differential biological phenotypes (cancer vs normal tissue). Other known tools consider whole pathways; whereas we have designed PATHOME to inspect a greater part of the biological cascade, given a set of pathways. As in [Figure 1a](#fig1){ref-type="fig"}, popular tools perform a single test for the toy pathway, removing interconnectivities among biological components. In contrast, PATHOME considers five tests while assessing the interconnectivities for the pathway, which indicates a more thorough search of the biological pathways. By applying PATHOME to GC gene expression data sets, we demonstrated its advantages. First, based on a reference pathway set, PATHOME detected more known cancer pathways than the alternative tools we assessed (at a lower FDR). Second, focusing on the WNT pathway that was uniquely detected by PATHOME, we provided strong evidence for the involvement of this pathway in gastric tumorigenesis through perturbation experiments in both cell line and animal studies. Combining the evidence from perturbation experiments in cell lines and animal models through metformin, our results revealed a cross-link between the AMPK metabolic pathway and the WNT signaling pathway, with HNF4α-WNT5A regulation having a key interaction in this link. A study performed by Kato *et al.*^[@bib26]^ suggested an antitumor effect of metformin on cell-cycle regulation in MKN-1, -45 and -74 GC cell lines. We also report a potential metformin inhibitory effect in GC cells and xenograft models. WNT5A is expressed in a variety of human tumors, including those of the esophagus, stomach, pancreas, colon and rectum, breast, lung, prostate gland, endometrial uterus and embryo, as well as in melanoma, osteosarcoma, Ewing sarcoma, neuroblastoma, skin basal cell carcinoma, skin squamous cell carcinoma and leukemia.^[@bib27]^ Kurayoshi *et al.*^[@bib28]^ reported that the WNT5A protein is highly expressed in advanced stages of GC and that its expression correlates with a poor prognosis. In addition, HNF4α and WNT signaling pathways are active members of the same machinery that controls the transcription of differentially zoned HNF4α-dependent genes in the liver, pancreas and biliary tract.^[@bib29]^ Even though WNT5A and HNF4α have been independently reported to be associated with cancer,^[@bib27]^ the link between HNF4α and WNT5A, to our knowledge, was first suggested in GC. Combining the evidence from the computational and perturbation experiments in cell lines and animal models, we demonstrated the oncogenic activity of WNT5A in GC and suggested WNT5A as a potential therapeutic target. Further study is warranted regarding the development of metformin therapeutic options and WNT5A targeted therapies to address GC progression within Asian patient populations. In [Figure 6e](#fig6){ref-type="fig"}, we propose cross-talk between HNF4α/WNT pathway. Despite successful application of PATHOME to GC, PATHOME solely analyzes the linear decomposition of pathways into paths. In the future, loops and loop-backs in the network need to be considered in statistical network analysis. We also note that as the HNF4α-WNT5A link is not annotated in the KEGG pathway database, PATHOME consequently does not pick up the HNF4α-WNT5A link. Materials and methods ===================== PATHOME algorithm ----------------- ### Overview The goal of this algorithm is to identify a set of subpathways that differentiate two experimental groups (for example, cancer vs non-cancer) by considering both prior knowledge about mutual regulations and experimental gene expression data. Here, we used the KEGG pathways as prior knowledge, and assumed the KEGG pathways as a tree structure for the PATHOME application. For computational simplicity, as shown in [Figure 1a](#fig1){ref-type="fig"}, we broke down the KEGG pathway maps into each possible path (subpathway) from the top node to the leaf node.^[@bib30]^ A depth-first search algorithm was used to decompose the pathway maps into all possible paths. As our previous study indicated that the number of possible linear paths (subpathways) was huge (\~0.13 billion possible paths),^[@bib31]^ we used a selection step before the statistical significance test step to reduce the number of tests ([Figure 1](#fig1){ref-type="fig"}). ### The selection step (selection of candidate subpathways) As shown in [Figure 1c](#fig1){ref-type="fig"}, the rule is defined to associate the regulation information of adjacent entries with their expression correlation in terms of the sign information of the Pearson product-moment correlation coefficients. If two adjacent entries are connected by an edge that denotes activation (arrow-headed edge), the expression correlation between the two entries is assumed to be positive; if the two entries are connected by an edge that denotes inhibition (blunt-ended edge), the expression correlation between the two entries is assumed to be negative. This rule is applied separately to each experimental group. In each group, we identify the consecutive segment starting from the leaf node of each subpathway so that all the edges of the segment should satisfy the association rule. That leads to the determination of the segment (in the subpathway) that is to be statistically evaluated in the test step. Assuming a subpathway with the number of nodes (genes) *p*, the leaf node is assigned to index 1 (*G*~1~) and the root node to index p (*G*~p~). The two experimental groups are denoted as *k* (*k*=1 or 2). If the edge (*e*~*i,i*+1~) between adjacent genes *G*~i~ and *G*~*i*+1~ is denoted as an activation edge in KEGG, *e*~*i,i*+1~ is encoded as +1. If the edge is represented as an inhibition edge in KEGG, *e*~*i,i*+1~ is set to −1. The Pearson correlation coefficient between the adjacent genes, *G*~i~ and *G*~i+1~, is denoted as *r^k^*~*i,i*+1~ in group *k* (for example, *k*=1 for one experimental group; *k*=2 for the other group). Thus, the association rule assumed in [Figure 1c](#fig1){ref-type="fig"} simply satisfies that *e*~*i,i*+1~x *r^k^*~*i,i*+1~ is positive. The length of the segment (*l^k^*) of the *k*-th group in the subpathway under the rule is mathematically represented as follows: where *sgn(·)* is the sign function, and *I(·)* is the indicator function. The first term −*I(·)* inspects the satisfaction of the association rule, keeping *l*^k^ progressing toward upstream. Inspecting the association rule from the leaf node to the root node, the penalizing term *R(·)* stops progressing when the association rule is not satisfied. Given the subpathway, we obtain the two consecutive segments from the two groups (see details about *l^k^* in [Supplementary Figure 2](#sup1){ref-type="supplementary-material"}). If both lengths of the two segments are greater than three, we move to the test step for the subpathway. PATHOME analyzes the interconnectivity between two adjacent nodes. The interconnectivity measure, the Pearson product-moment correlation coefficient, is obtained even in three samples in a group. PATHOME can be applied to a small number of samples, such as three samples in a group. Summarizing the first step, a candidate subpathway for the next step should satisfy the following two conditions: (i) the two experimental groups agree with the association rule between the expression correlation and the edge information for the adjacent entries along the path; and (ii) both consecutive segments for the two groups have at least four elements (three consecutive edges) in order to filter a subpathway with short segments. ### The test step (statistical significance test) In this step, we identify the subpathway for which the correlation difference between the two consecutive segments for the two experimental groups is statistically significant. As all the consecutive correlation coefficients, *r^k^*~*i*,*i*+1~s, in the two *l* consecutive segments meet their corresponding regulation information ([Figure 1](#fig1){ref-type="fig"}), we do not need to further consider the sign information of the Pearson correlation coefficient in this step. To improve the normality approximation, we transform the absolute value (\|*r^k^*~*i*,*i*+1~\|) of the Pearson correlation coefficient into (0,∞) by taking the Fisher transformation, as follows: If the lengths of the two segments are different, we set the minimum (say, *l*^min^) of *l^k^*s as the length of the two segments to be compared. As a result, we obtain {*c^k^*~*i*,*i*+1~\| *k*=1, and *i*=1,..,*l*^min^−1} and {*c^k^*~*i*,*i*+1~\| *k*=2, and *i*=1,..,*l*^min^−1} in the given subpathway. Let *μ^k^* represent the mean of *c^k^*~*i*,*i*+1~ s in group *k*. We test the significance under the null hypothesis: H~0~: *μ*^1^=*μ*^2^. That is, the alternative hypothesis suggests that the global mean of the correlations of the expressions between the two groups are different. We used a *z*-test statistic to measure significance. We also considered multiple comparisons, and set the FDR at 0.05.^[@bib32]^ Computational analysis of GC gene expression data sets ------------------------------------------------------ We obtained four GC data sets^14--17^ from Gene Expression Omnibus (http://[www.ncbi.nlm.nih.gov/geo/](http://www.ncbi.nlm.nih.gov/geo/)). We compared our method with two popular GSA methods, DAVID^[@bib3]^ and GSEA.^[@bib4]^ For performance comparison, we used the cancer-related pathways proposed by Vogelstein and Kinzler^[@bib19]^ as a reference standard. We used the three methods (PATHOME, DAVID and GSEA) to examine the agreement between the reference standard and the reported pathways. To examine the TFBSs in the genes of interest, we extracted a 2-kb upstream region of each gene from the UCSC Genome Browser, and obtained the *HNF4A-*binding site information from the TFBS Conserved track and the Txn Factor ChIP track in the UCSC Genome Browser. Biological experiments ---------------------- ### Cells and reagents Human NCI-N87 and AGS derived from primary tumors GC cells were obtained from the American Type Culture Collection (ATCC; <http://www.atcc.org/>); and the MKN-1 and MKN-45 cell lines derived from primary tumor and tumor site of liver metastasis, respectively, were made available from Yonsei Cancer Center. SNU-1, -484 and -719 cell lines derived from primary tumor, SNU-5, -16, -620, -601, -638, -668 cell lines derived from ascites and SNU-216 cell line derived from tumor site of lymph node metastasis were made available from the Korean Cell Line Bank (<http://cellbank_snu.ac.kr/>). Monoclonal antibodies to human HIF-1α and β-catenin were purchased from BD Transduction Laboratories (BD Biosciences, San Jose, CA, USA); HNF4α, WNT1, WNT3, GSK, TCF, LEF, pCAMKII (Thr 286), cyclin D1, cyclin D2 and β-actin were purchased from Cell Signaling Tech. Inc. (Boston, MA, USA). WNT5A was purchased from Abgent (San Diego, CA, USA) and metformin was purchased from Sigma-Aldrich (St Louis, MO, USA). ### Cell culture Human NCI-N87, AGS, HS 746T, MKN-1, 45, SNU-1 -5, -16, -620, -216, -484,-601, -638, -668, -719 GC cell line studies were analyzed within 6 months of tissue resuscitation; the tissues were cultured in RPMI-1640 (CellGro, Manassas, VA, USA) and 10% fetal calf serum (FCS; Hyclone, ThermoScientific, Waltham, MA, USA) at 37 °C in 5% CO~2~. ATCC used short tandem repeat profiling. Cells (2.5 × 10^5^) were seeded and incubated under normoxic conditions to 70--80% confluence and then incubated in the presence or absence of metformin at 10 m[M]{.smallcaps} concentration for up to 5 days according to the required time in the study. ### Western blotting Cells were grown under hypoxic conditions in the presence or absence of 10 m[M]{.smallcaps} metformin. The cells were washed twice in a phosphate-buffered saline solution and western blotting was conducted, as previously described.^[@bib33]^ The tumors collected from xenograft mouse models were divided into two pieces for immunoblotting and immunohistochemistry, as previously described.^[@bib33]^ The blots were quantified using ImageQuant software (Molecular Dynamics/GE Healthcare Biosciences, Sunnyvale, CA, USA). ### Real-time RT--PCR analysis Total RNA was isolated from cell lysates using the PARIS kit (Ambion/Applied Biosystems, Foster City, CA, USA) according to the manufacturer\'s protocol. Next, TaqMan quantitative RT--PCR was performed on the ABI 7300 system using the TaqMan one-step RT--PCR Master Mix kit and predesigned primer/probe pairs for *HNF4α, WNT5A, TCF4* and β~2~-microglobulin (Applied Biosystems). Normalization procedures and analyses were carried out with β~2~-microglobulin using the 2(-delta-delta C(T)) method as the internal reference,^[@bib34]^ and using Applied Biosystems GeneAmp 5700 SDS software. All measurements were performed in triplicate. ### siRNA transfection siRNA SMARTpool sequences were obtained from Dharmacon/Thermo Fisher Scientific (Waltham, MA, USA); and the cells were transfected with 25 n[M]{.smallcaps} siRNA-HNF4α, siRNA-WNT5A and a siRNA nontargeting control using Dharma-FECT 1 lipid transfection reagent. The transfection medium was removed after 24 h and replaced with fresh medium, and the cells were grown in 5% CO~2~ at 37 °C for an additional 48--72 h. RT--PCR and/or western blot analyses were performed to confirm target knockdown by siRNA. The transfected cells were treated with metformin and cultured under hypoxic conditions for an additional 18 h. ### Immunohistochemical staining Hematoxylin and eosin-stained slides were reviewed and representative areas were selected for tissue microarray. Normal mucosa and cancer tissues were selected, respectively. In the experiment, 2-mm diameter cores were taken from archival paraffin-embedded blocks using a trephine apparatus (Superbiochips Laboratories, Seoul, Republic of Korea). Tissue microarray blocks were sectioned at a thickness of 3 μm, and these sections were then dried for 1 h at 56 °C. Immunohistochemical staining was performed with the automated staining instrument BenchMark XT (Ventana Medical Systems, Inc., Tucson AZ, USA) as follows: the sections were deparaffinized and rehydrated with EZ Prep (Ventana Medical Systems, Inc.) and washed with Tris-buffered saline. The antigens were retrieved with heat treatment in pH 8.0 Tris-EDTA buffer (CC1, Ventana Medical Systems, Inc.) at 95 °C for 30 min for WNT5A. Endogenous peroxidases were blocked with 3% H~2~O~2~ for 10 min at room temperature. Nonspecific-binding blocking was done with a ready-to-use protein blocker solution (Ventana Medical Systems, Inc.) for 20 min at RT. A primary antibody was applied to the slide section at 42 °C (WNT5A for 30 min at 1:5000 dilutions (clone 6F2, Abgent, AO1264a)). Then the sections were incubated with HPR multimer labeled secondary antibody (ultraView Universal DAB detection kit, Ventana Medical Systems, Inc.) for 20 min at RT and stained using ultraView universal DAB kit (Ventana Medical Systems, Inc.) for 8 min and hematoxylin counterstain. ### Interpretation of immunohistochemistry We defined the intensity WNT5A stain as follows: if no signal or only a faint equivocal signal was observed at × 100 power, it was regarded as negative, 0; if more than 10% of tumor cells showed clear nuclear signals at × 100 power, similar to those of the foveolar epithelial cells, it was weakly positive, 1; if more than 10% of tumor cells showed obviously stronger signals, at × 40 power, similar to those of the chief cells for WNT5A, it was strongly positive, 2. The positivity of the WNT5A stain was measured by the percentage of weakly or strongly positive tumor cells. The stain was scored as 0 (negative) if weakly or strongly positive cells were ⩽50% and as 1 (positive) if they were \>50%. Representative results of WNT5A immunostaining for GC are shown in [Figure 5c](#fig5){ref-type="fig"}. ### *In vivo* antitumor study Approximately 10^7^ NCI-N87 and MKN-45 GC cells in log cell growth were injected and subcutaneously suspended in 0.2 ml phosphate-buffered saline in the flanks of severe combined immunodeficient (*scid*) mice. The animals were weighed weekly and tumor diameters were measured twice weekly at right angles (*d*~short~ and *d*~long~) with electronic calipers and converted to volume by the formula volume=\[(*d*~short~)2 × (*d*~long~)\]/2. When the tumors reached volumes between 150 and 300 mm^3^, the mice were stratified into groups of 10 animals, total 40 animals having approximately equal mean tumor volumes, and the administration of metformin commenced at 250 mg/kg per oral daily for 25 days. Control animals received vehicle (water) alone. Twenty-four hours after the last metformin administration commenced, tumors were collected for western blot analysis and for immunohistochemistry preparation. When the tumor volume reached ⩾1500 mm^3^ or became necrotic, the animals were euthanized. ### Statistical analysis For the experiments on individual genes in this section, *P*\<0.05 was statistically significant based on a Student\'s *t-*test or *z*-test to compare the experimental group with the control group. This study was supported by grants from the National Cancer Center of the Republic of Korea, NCC-1210350--2 (to YHK), NCC-1210460 (to SN); the National Research Foundation of Korea (NRF) grant funded by the Republic of Korea (MSIP) 2012R1A3A2026438, 2008--0062618, 2013M3A9C4078158 (to TP); grants from the US National Institutes of Health, CA95060, CA129616, CA17094 and CA98920 (to GP), CA143883 (to HL); the University of Texas MD Anderson Cancer Center--GS Hogan Gastrointestinal Research Fund and the Lorraine Dell Program in Bioinformatics for Personalization of Cancer Medicine (to HL). The Korea Institute of Science and Technology Information provided SN with a high-performance computing resource. We thank LeeAnn Chastain for editorial assistance. **AUTHOR CONTRIBUTIONS** HL, TP and YHK conceived and designed the experiments; SN and TP designed the algorithm; SN, DH, HL and YHK analyzed the data; HRC, K-TK, M-CK, CHK, HRJ, HSP, GP, YHK performed the experiments; SN, HL, TP and YHK wrote the paper. All authors have read and approved the manuscript for publication. [Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies this paper on the Oncogene website (http://www.nature.com/onc) The authors declare no conflict of interest. Supplementary Material {#sup1} ====================== ###### Click here for additional data file. {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} {#fig6} ###### Summary of gastric cancer-related transcriptome-wide expression data sets used in this study *Data set* *Ethnic group* *Comparison group* *Profiling platform* -------------------- ---------------- ------------------------------- ------------------------------------ GSE13861^[@bib14]^ Korean 65 Tumor vs 19 non-tumor Illumina Human WG-6 v3.0 GSE15081^[@bib15]^ Japanese 18 Relapse vs 38 relapse-free Human Oligo Chip 30K GSE36968^[@bib16]^ Korean 24 Tumor vs 6 non-tumor SOLiD Single-read RNA-seq GSE27342^[@bib17]^ Chinese 80 Tumor vs 80 non-tumor Affymetrix Human Exon 1.0 ST array ###### Performance comparison of three methods based on terms of cancer-related pathways identified in Vogelstein and Kinzler^[@bib19]^ *Reference standard*^[@bib19]^ *KEGG pathway* *Title* *GSE13861 data set*^[@bib14]^ *GSE15081 data set*^[@bib15]^ -------------------------------- ---------------- ---------------------- ------------------------------- ------------------------------- --- --- --- --- HIF1 HSA04150 mTOR signaling X X X X X X   HSA05200 Pathways in cancer 0 X X X X X   HSA05211 Renal cell carcinoma X X X X X X P53 HSA04115 P53 signaling X X X X X X RB (cell cycle) HSA04110 Cell cycle X X 0 X X X Apoptosis HSA04210 Apoptosis X X X X X X GLI HSA04340 Hedgehog signaling X X X X X X APC HSA04310 Wnt signaling 0 X X 0 X X RTK HSA04012 ERBB signaling X X X X X X   HSA05200 Pathways in cancer 0 X X X X X SMAD HSA04350 TGF-β signaling X X X X X X PI3K HSA04012 ERBB signaling X X X X X X   HSA05200 Pathways in cancer 0 X X X X X   HSA04150 mTOR signaling X X X X X X   HSA04010 MAPK signaling 0 X X 0 X X   HSA04910 Insulin signaling 0 X X X X X   HSA04510 Focal adhesion 0 0 X 0 X X   HSA04062 Chemokine signaling 0 X X 0 X X   HSA04370 VEGF signaling X X X 0 X X Abbreviations: APC, antigen-presenting cell; ERBB, v-erb-b avian erythroblastic leukemia viral oncogene; GLI, glioma-associated oncogene; HIF1, hypoxia inducible factor; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide-3-kinase; RB, retinoblastoma; RTK, protein-tyrosine kinase; TGF, transforming growth factor; VEGF, vascular endothelial growth factor. Note: X (not detected), 0 (detected). [^1]: These authors contributed equally to this study.

1. Introduction {#sec1} =============== Psoriatic arthritis (PsA) is a multifactorial chronic inflammatory joint disease associated with psoriasis, usually with no detectable rheumatoid factor (RF), and belongs to the seronegative subgroup of spondyloarthritis \[[@B1]\]. Clinically, patients with PsA present with different manifestations such as mild mono-/oligoarthritis, destructive polyarthritis, dactylitis, enthesitis, and spondylitis \[[@B2]\]. PsA is estimated to be present in 6--42% of patients with skin psoriasis \[[@B3]\], but unlike rheumatoid arthritis (RA) PsA is equally common in men and women \[[@B1]\]. Both environmental and genetic factors are important for the development of PsA \[[@B1]\]. Whilst the pathogenesis is not fully understood, the immunological process, occurring in the skin of patients with psoriasis, resembles the events occurring in the joints when synovial cells begin to proliferate \[[@B4]\]. PsA has many similarities with RA in addition to separate and specific clinical properties. In comparison to RA, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels are not as diagnostically reliable when measured in samples from patients with PsA as both, or either, are often within the normal range despite the presence of an advanced joint disease. Madland et al. found that S-calprotectin related to radiographic changes rather than disease activity in patients with low disease activity but in the study they also found that S-calprotectin was associated with patients with moderate/high activity measured as physicians\' global assessment and more than three swollen joints \[[@B5]\]. Calprotectin, a member of the family of S100 leukocyte proteins secreted primarily by neutrophilic granulocytes and monocytes, is a calcium binding protein \[[@B6]\] with antimicrobial properties. The calprotectin heterocomplex consists of two different proteins, S100A8 and S100A9 (MRP14/MRP8 or calgranulin A/B) \[[@B6]\], encoded by the S100A8/S100A9 gene located on chromosome 1q21 \[[@B7]\]. The presence of calcium induces conformational changes in the heterodimer, thereby allowing the binding of other proteins. Moreover, calprotectin contains zinc-binding domains involved in an antibacterial activity \[[@B8]\]. In the cytosol of neutrophilic granulocytes, calprotectin has been estimated to account for more than 40% of the total protein content \[[@B9], [@B10]\]; conversely calprotectin is not usually present in lymphocytes \[[@B9]\]. Calprotectin is also an important mediator of many regulatory functions such as chemotactic activity, deactivation of macrophages, and inhibition of immunoglobulin synthesis \[[@B10]\]. Elevated levels of calprotectin have been identified at sites of inflammation and in the extracellular fluid in patients with RA, cystic fibrosis, Sjögren\'s syndrome, and abscesses \[[@B10]\]. In plasma from patients with RA, the concentration of calprotectin is known to be increased compared with that in healthy individuals. Measurement of faecal calprotectin levels is a diagnostic tool, and a biomarker, for inflammatory activity in bowel diseases such as ulcerative colitis and Crohn\'s disease \[[@B11]\]. A high concentration of calprotectin has been detected in the synovial fluid from patients with PsA and RA \[[@B12]\]. Production of calprotectin is also expressed in the epidermal keratinocytes of patients with psoriasis \[[@B13]\] whereas the normal epidermis from healthy individuals shows very low levels. In addition to its biological effects, calprotectin is involved in epidermal proliferation, differentiation, and inflammatory cell migration \[[@B13]\]. Numerous proinflammatory cytokines and chemokines have been found in skin lesions, blood, and synovial fluid of patients with inflammatory conditions such as arthritis and psoriasis, although their etiological significance is not fully understood \[[@B14]--[@B24]\]. Cytokines such as interleukin (IL)-17A and IL-22 have been suggested to be involved in hyperproliferative and inflammatory reactions in the psoriatic epidermis based on the therapeutic effects of cytokine antagonists as a part of the treatment in patients with PsA and plaque psoriasis \[[@B15], [@B16]\]. Interleukin-17A is also known to increase the production of IL-16 \[[@B17]\] and to induce the expression of CCL20 in primary cultures of keratinocytes \[[@B18]\]. An overexpression of IL-12 and IL-23 has been reported to occur in the psoriatic process \[[@B15]\], whilst it has been proposed that IL-15 and IL-18 participate in the pathogenesis of RA \[[@B19], [@B20]\]. High concentrations of IL-22 have been detected in the synovial fluid from patients with PsA and RA, and a correlation between the levels of IL-22 and area of the psoriatic lesion(s) and severity index (PASI) score has been identified \[[@B21]\]. In an early phase of PsA development, elevated levels of CXCL10 have been found, whilst a decrease has been observed in long lasting PsA \[[@B22]\]. A recent study revealed a correlation of IL-33 concentrations between the synovial fluid and serum of patients with RA \[[@B23]\]. The high expression of CXCL12 in the synovium of RA patients is believed to attract CD4+ memory T cells \[[@B24]\]. The aim of this cross-sectional study was to analyse different inflammatory markers, that is, S100A8/S100A9 (calprotectin) and cytokines in blood in patients with PsA in order to identify a potential marker for PsA or the clinical subtypes of PsA. 2. Patients and Methods {#sec2} ======================= 2.1. Patients and Controls {#sec2.1} -------------------------- Blood samples were collected from 65 patients with PsA (33 males/32 females, age 50.5 ± 14.2 years (mean ± SD)). The patients with PsA were compared with 31 healthy controls matched for age and gender. S-calprotectin, high sensitivity- (hs-) CRP, and cytokines in plasma were analysed in both patient and control groups whilst the measurement of ESR was only performed for the patients with PsA. All of the patients fulfilled the CASPAR criteria \[[@B25]\] and/or the Moll and Wright criteria \[[@B1]\] for PsA. Of the patients, 32 fulfilled the Moll and Wright criteria for mono-/oligoarthritis and 33 patients fulfilled the criteria for polyarthritis at the time of the study. All patients in this study were examined clinically for inflammatory joint manifestations and skin involvement. The number of tender and swollen joints, with a duration of more than 6 weeks, was assessed using 66-joint count. Mono-/oligoarthritic disease pattern was defined when four or less tender and swollen joints were present at the time of the medical examination, and polyarthritic disease pattern was diagnosed when more than four tender and swollen joints were present. Using a five-point scale, the affected skin area was graded from "no actual lesion" to "extensive involvement," and the activity of skin involvement (erythema, induration, and scaling) was graded using four grades, that is, "no activity," "mild," "moderate," and "severe activity." The study was approved by the Regional Research Ethics Committee of Umeå University. All participants gave their informed consent. 2.2. Laboratory Measurement {#sec2.2} --------------------------- Calprotectin in serum (ng/mL) was analysed using the PhiCal Calprotectin ELISA kit (*Immundiagnostik, Bensheim, Germany*). Erythrocyte sedimentation rate (mm/h, Westergren method) was measured using routine laboratory methods. Analysis of high sensitivity CRP (hs-CRP) in serum was performed using immunoturbidimetry (*Cobas 6000/8000, Roche Diagnostics, USA*). The cytokines were measured in plasma samples (pg/mL) using multiplex detection kits from Bio-Rad (Hercules, CA, USA). A 7-plex kit was used to measure the concentrations of IL-12, IL-15, IL-17A, IL-22, IL-23, IL-33, and CCL20, a 2-plex kit was used for IL-16 and IL-18, and single kits were used for measurements of CXCL10 and CXCL12. The assays were performed according to the manufacturer\'s protocols and analyzed with a Bio-Plex 200 System using Bio-Plex Manager 6.1 software (Bio-Rad, Hercules, CA, USA). 2.3. Statistical Analysis {#sec2.3} ------------------------- Differences between the collected data were tested for using the Kruskal Wallis test and/or Mann-Whitney test, and for identifying correlations the Spearman rank-order equation was used. To assess the utility of S-calprotectin and hs-CRP as inflammatory markers for PsA, sensitivity and specificity were calculated. The receiver operating curve (ROC) was used to identify the relationship between the sensitivity and specificity. For multivariate analysis, logistic regression enter method was performed with variables showing significant differences in the association analyses, with PsA as independent variable. All *P* values refer to a two sided test and a *P* value ≤ 0.05 was considered statistically significant. ESR values were not included in the multivariate analysis because laboratory measurements of ESR were only available for the patients with PsA. 3. Results {#sec3} ========== S-calprotectin and hs-CRP levels were significantly higher in patients with PsA compared with controls (*P* \< 0.001, resp.). The serum levels of calprotectin were significantly higher in patients with mono-/oligoarthritis as well as polyarticular disease than in healthy controls (*P* \< 0.001, resp.), and the levels in the polyarticular group were higher than in patients with mono/oligoarticular disease (*P* = 0.017) ([Table 1](#tab1){ref-type="table"}). In addition, hs-CRP levels were significantly higher in patients with mono-/oligoarthritis compared with the control group (*P* = 0.026), and patients suffering from polyarthritis have significantly higher levels of hs-CRP than both the control and patient groups with mono-/oligoarthritis (*P* \< 0.001 and *P* = 0.012, resp., [Table 1](#tab1){ref-type="table"}). ESR was significantly higher in patients with polyarthritis compared with individuals suffering from mono-/oligoarthritis (*P* = 0.002; [Table 1](#tab1){ref-type="table"}). The levels of S-calprotectin correlated with hs-CRP (*P* \< 0.001; *r* ~*s*~ = 0.441), swollen joint count (*P* = 0.002, *r* ~*s*~ = 0.397), and CXCL10 (*P* = 0.046, *r* ~*s*~ = 0.678) ([Table 2](#tab2){ref-type="table"}) but not with any of the other cytokines. CXCL10 correlated with S-calprotectin (*P* = 0.046, *r* ~*s*~ = 0.678), hs-CRP (*P* = 0.05, *r* ~*s*~ = 0.400), ESR (*P* = 0.008, *r* ~*s*~ = 0.347), and swollen join count (*P* = 0.033, *r* ~*s*~ = 0.278) ([Table 2](#tab2){ref-type="table"}). None of the cytokines was associated with PsA or its clinical subtypes nor did they correlate with the number of swollen joints. However, several of the cytokines correlated with tender joints (IL-12 (*P* = 0.039, *r* ~*s*~ = 0.270), IL-15 (*P* = 0.014, *r* ~*s*~ = 0.319), IL-17A (*P* = 0.007, *r* ~*s*~ = 0.348), IL-22 (*P* = 0.046, *r* ~*s*~ = 0.260), IL-33 (*P* = 0.026, *r* ~*s*~ = 0.290), and CCL20 (*P* = 0.018, *r* ~*s*~ = 0.306)), and correlations were found between the cytokines IL-12, IL-15, IL-17A, IL-22, IL-23, IL-33, and CCL20 (*P* \< 0.001; data not shown). The ROC curve was used to identify the optimal cut-off for S-calprotectin and hs-CRP. The ROC curve indicated 475.00 ng/mL to be the optimal cut-off value for S-calprotectin (area under the curve (AUC) = 0.866, OR = 42.00, and 95% CI = 8.023--197.683, *P* \< 0.001) with a sensitivity of 75.0%, a specificity of 93.3%, and a positive predictive value of 95.7% ([Figure 1](#fig1){ref-type="fig"}). When analysing hs-CRP, the ROC-curve indicated 3.75 mg/L as the optimal cut-off (AUC = 0.751, OR = 6.88, 95% CI = 1.88--25.190, *P* = 0.004, sensitivity = 43.3%, specificity = 90%, and positive predictive value 89.7%) ([Figure 2](#fig2){ref-type="fig"}). In multiple logistic regression analysis, S-calprotectin, hs-CRP, and CXCL10 were included. S-calprotectin was the only variable significantly associated with psoriatic arthritis (*P* = 0.002, OR = 1.006, and 95% CI = 1.002--1.010). No significant difference was found for the S-calprotectin level between either the area of the affected skin or activity of the skin disease, and there were no correlations between age, gender, or the cytokines analysed apart from CXCL10 (data not shown). 4. Discussion {#sec4} ============= Identification of a marker for the diagnosis and/or predicting a prognosis of PsA, as well as supporting the evaluation of disease activity, has been a long-term target for rheumatologists. There is no known, clinically useful marker, and laboratory parameters, such as ESR and/or CRP, are not always increased in patients with PsA, even those suffering from a diagnosed and clinically active disease. Until recently, calprotectin was known to be increased in the faeces of patients with inflammatory bowel disease and was primarily used as a marker for the diagnosis and evaluation of inflammatory bowel diseases (i.e., Crohn\'s disease and ulcerative colitis). An increased level of calprotectin in serum is found in patients with other inflammatory diseases, such as RA and psoriasis, and a high concentration of S100 proteins has been detected in the inflamed synovial tissue from patients with PsA and RA \[[@B10]\]. Several cytokines are known to be elevated in inflammatory diseases, including PsA, and studies on treatment options that modify cytokine regulation are ongoing \[[@B14]--[@B24]\]. The primary aim of this study was to examine the levels of S-calprotectin, hs-CRP, and cytokines in relation to PsA, the peripheral disease pattern, and the disease activity in patients with PsA and, secondly, to evaluate the utility of these variables as markers of inflammation. S-calprotectin levels were found to be significantly higher both in patients with mono-/oligoarthritis and in those with polyarthritis compared with controls, thus showing that, even in patients with a disease pattern usually associated with less inflammation, a laboratory detectable parameter could be measured. There was also a correlation of S-calprotectin with ESR, hs-CRP, and the number of swollen joints (using a 66-joint scoring system) confirming that S-calprotectin can be used as an inflammatory marker. When evaluating the utility of S-calprotectin and hs-CRP as disease markers, S-calprotectin was found to be a better predictor for PsA than hs-CRP at the optimum cut-off value based on analysis of the ROC-curve. In a multiple logistic regression model including S-calprotectin, hs-CRP, and CXCL10, S-calprotectin was found to be the best predictor for PsA which further strengthens the proposal of S-calprotectin being a potential marker for PsA. Madland et al. have reported a correlation between S-calprotectin and radiographic changes rather than disease activity of PsA in patients with low disease activity \[[@B5]\]. We have not evaluated radiographic findings in our study as we wanted to study possible differences between disease patterns, but the findings of associations between S-calprotectin and moderate/high disease activity and more than three swollen joints are in line with our findings. Further studies are needed to confirm the results. The cytokines analysed in this study did not associate with PsA or any of the clinical subtypes of PsA. This is contrary to other published studies and could be a result of the small number of patients included in this study. S-calprotectin did show strong association with PsA, despite the small number of patients, and our findings indicate that S-calprotectin reflects the burden of the joint disease, even in those patients with only a few joints involved and who often have normal ESR and/or CRP. S-calprotectin is reported to be increased in several inflammatory diseases. This study generated promising results since it was possible to detect an increased serum level of S-calprotectin in patients with PsA and also to identify it as a better predictor of ongoing disease than hs-CRP or any of the proinflammatory cytokines IL-12, IL-15, IL-16, IL-17A, IL-18, IL-22, IL-23, IL-33, CCL20, CXCL10, and CXCL12. Although the results are very promising the number of patients in this study is small, which could be reflected in the quite low OR, and further investigation of the relationship between PsA and S-calprotectin levels should be undertaken in order to confirm the results. The authors thank Professors Solbritt Rantapää Dahlqvist, Kristina Nilsson, and Ingegerd Söderström for technical support and The Swedish Psoriasis Association and The Swedish Rheumatism Association for unrestricted grants. Conflict of Interests ===================== The authors declare that they have no conflict of interests. {#fig1} {#fig2} ###### Characteristics of patients and controls. ---------------------------------------------------------------------------------------------------------------------------   Control\ Mono-/oligoarthritis\ Polyarthritis\ *P* value group 1, *N* = 31 group 2, *N* = 32 group 3, *N* = 33 ---------------------------------------------- ------------------- ----------------------- ------------------- ------------ Mean age, years, mean (±SD) 52.5 (±15) 50.9 (±12.7) 50.0 (±15.7) ns Gender (female/male) 15/16 17/15 15/18 ns Duration of skin disease, years, mean (±SD) --- 24.9 (±13.3) 22.7 (±16.0) ns Duration of joint disease, years, mean (±SD) --- 16.4 (±15.5) 15.2 (±10.8) ns ESR, mm/h, mean (±SEM) --- 10.2 (±1.25) 18.5 (±2.1) 0.002 hs-CRP, mg/L, mean (±SEM) 1.6 (±0.37) 3.17 (±0.58) 6.5 (±1.07) 0.026^1^ \ 0.012^2^ \ 0.000^3^ S-calprotectin ng/mL, mean (±SEM) 308.85 (±20.82) 705.05 (±85.5) 1471.93 (±403.9) 0.000^1^ \ 0.017^2^ \ 0.000^3^ --------------------------------------------------------------------------------------------------------------------------- ns: not significant. ^1^Comparison between group 1 and group 2. ^2^Comparison between group 2 and group 3. ^3^Comparison between group 1 and group 3. ###### Correlations between S-calprotectin, hs-CRP, ESR, swollen joint count (SJC), and CXCL10 analysed with Spearman rank-order correlation. -----------------------------------------------------------------------------------------------------------   S-calprotectin\ hs-CRP\ ESR\ SJC\ CXCL10\ *P* (*r* ~*s*~) *P* (*r* ~*s*~) *P* (*r* ~*s*~) *P* (*r* ~*s*~) *P* (*r* ~*s*~) ----------------- ----------------- ----------------- ----------------- ----------------- ----------------- S-calprotectin\ ---\ \<0.001\ 0.056\ 0.002\ 0.046\ *P* (*r* ~*s*~) (1.000) (0.441) (0.255) (0.397) (0.678) hs-CRP\ \<0.001\ ---\ \<0.001\ 0.045\ 0.05\ *P* (*r* ~*s*~) (0.441) (1.000) (0.571) (0.260) (0.216) ESR\ 0.056\ \<0.001\ ---\ 0.001\ 0.008\ *P* (*r* ~*s*~) (0.255) (0.571) (1.000) (0.400) (0.347) SJC\ 0.002\ 0.045\ 0.001\ ---\ 0.033\ *P* (*r* ~*s*~) (0.397) (0.260) (0.400) (1.000) (0.278) CXCL10\ 0.046\ 0.05\ 0.008\ 0.033\ ---\ *P* (*r* ~*s*~) 0.678 (0.216) (0.347) (0.278) (1.000) ----------------------------------------------------------------------------------------------------------- [^1]: Academic Editor: Clive Liu

Introduction ============ Tuberculosis (TB) is one of the ancient and deadliest disease of mankind, still posing a major health, social and economic burden at a global level and primarily in low and middle income countries.[@b1-mjhid-5-1-e2013070] The lack of an effective vaccine, the long and expensive drug regimens, the few diagnostic tools available in countries where TB is endemic and the dismantlement in several nations of the health systems and control measures that so effectively contributed to control TB throughout most of the XX century, led to the reemergence of TB as a global pandemic. The last twenty years have seen a renewed interest on TB by health authorities and governments which resulted in halving TB deaths. However, it is widely accepted that only a better understanding of the pathogenic processes associated with infection and disease will lead to the development of effective tools capable of conquering this ancient scourge. TB is one of the first and most studied infectious disease, as classically highlighted by the seminal work of R. Koch more than 100 years ago, but we have yet to answer many key questions on the mechanisms of pathogenesis and on the immunological correlates, if any, associated with protection from developing disease such as those posed by E.L. Trudeau more than a century ago.[@b2-mjhid-5-1-e2013070] Mycobacterium tuberculosis -------------------------- ### Evolution TB is caused by members of the specie *Mycobacterium tuberculosis* complex (MTBC), which includes: *Mycobacterium tuberculosis* (*Mtb*), the etiologic agent of TB in humans; *M. africanum*, that causes TB in humans only in certain regions of Africa; *M. bovis*, *M. caprae* and *M. pinnipedii*, causing TB in wild and domesticated mammals; *M. microti*, that causes TB only in voles. Deciphering the ≅ 4 Mbp genome provided a new understanding of the biology of the tubercle bacillus, with the identification of new and somehow unexpected properties[@b3-mjhid-5-1-e2013070] and allowed the reconstruction of the history of *Mtb* as a global human infectious agent.[@b4-mjhid-5-1-e2013070]*Mtb* emerged as a human pathogen in Africa around 70.000 years ago and then spread out of the continent following human migrations.[@b5-mjhid-5-1-e2013070],[@b6-mjhid-5-1-e2013070] It is now widely accepted that the ancients *Mtb* strains originated from environmental mycobacteria (smooth tubercle bacilli),[@b7-mjhid-5-1-e2013070] that can still be isolated from immunocompromised patients in certain parts of east Africa, are unable to cause chronic persistent infection in the immune-competent host and are not transmitted among humans. These ancient *Mtb* strains evolved, through a genetic bottleneck, so to persist in low density populations, causing disease reactivation following long period of latent infection.[@b8-mjhid-5-1-e2013070] Following domestication, humans were able to transmit the disease to animals and *M. bovis* emerged as a pathogen of domesticated and wild animals.[@b4-mjhid-5-1-e2013070] The introduction of agriculture, civilization and the increase in human population density in urban areas led to the selection of *Mtb* strains with enhanced virulence and transmissibility that are named modern *Mtb* strains.[@b9-mjhid-5-1-e2013070],[@b10-mjhid-5-1-e2013070] The modern *Mtb* strains spread throughout the world causing the TB epidemics that ravaged mankind for centuries and these strains are responsible for most of the TB cases nowadays.[@b11-mjhid-5-1-e2013070] ### The Bacillus *Mtb* is a slow growing mycobacteria with a doubling time of 12--24 h under optimal conditions. A major feature of *Mtb* is the peculiar cell wall structure, that provides an exceptionally strong impermeable barrier to noxious compounds and drugs and that plays a fundamental role in virulence. The classical view of the mycobacterial cell wall structure has been revised thanks to the introduction of a new electron microscopy technique, cryo-electron tomography on vitreous section, that preserves cell wall organization by avoiding sample dehydration.[@b12-mjhid-5-1-e2013070],[@b13-mjhid-5-1-e2013070] Thanks to these advancements it was shown that mycobacteria possess an outer membrane, functionally similar to what seen in gram-negative bacteria, consisting in an asymmetric lipid bilayer made of long fatty acids in the inner leaflet (mycolic acids) and of glycolipids and waxy components on the outer layer. The outer and inner membrane form a periplasmic space, with the presence of a thin layer of peptidoglycan in the innermost side covalently linked to arabinogalactan and lipoarabinomannan which in turn are bound to mycolic acids. Isoniazid and ethambutol, two of the most effective anti-TB drugs, target the synthesis of the mycolic acids and arabinogalactan, respectively, highlighting the importance of the mycobacterial cell wall in *Mtb* biology. Protein secretion systems are the main virulence factors of pathogenic bacteria and in *Mtb* five type 7 secretion systems were identified (ESX1-5)([Figure 1](#f1-mjhid-5-1-e2013070){ref-type="fig"}).[@b14-mjhid-5-1-e2013070] The best characterized of these is ESX1, which is missing in the attenuated *M. bovis* vaccine strain Bacille Calmette and Guerin.[@b15-mjhid-5-1-e2013070],[@b16-mjhid-5-1-e2013070] ESX1 is required for the full virulence of *Mtb*, which uses this secretion system to translocate from the phagosome into the cytosol of infected macrophages where it may persist in a protected environment.[@b17-mjhid-5-1-e2013070]--[@b19-mjhid-5-1-e2013070] ESX1 secretes among many antigens, ESAT-6 and CFP-10, two small highly immunogenic proteins that form the basis of the immunological diagnosis of *Mtb* infection in the interferon-gamma release assays (IGRAs).[@b20-mjhid-5-1-e2013070] Since BCG lacks ESX1 and does not express ESAT-6 and CFP-10, IGRAs can be used to detect *Mtb* infection even in subjects previously immunized with BCG, which may not be otherwise distinguished with the classical Mantoux intradermal reaction. ESX3 is involved in the acquisition of iron and zinc by *Mtb* and is essential for growth also in culture.[@b21-mjhid-5-1-e2013070] ESX5 is found only in MTBC, *M. marinum* and *M. ulcerans* and it may represents a secretion systems specifically evolved to interact with a complex immune system such as that of mammals.[@b22-mjhid-5-1-e2013070] While the role and function of ESX2 and ESX4 are still debated, the elucidation of the ESX systems on TB pathogenesis is certainly one of the major advancements of the last decade in the TB field, providing a new understanding of the host-pathogen interaction and very rewarding in terms of new diagnostics and potentially capable of providing new therapeutics and vaccines in the near future. The characterization of other *Mtb* surface constituents such as the mycobacterial adhesin HBHA[@b23-mjhid-5-1-e2013070] and PE_PGRS proteins[@b24-mjhid-5-1-e2013070],[@b25-mjhid-5-1-e2013070] is starting to shed light on the molecular mechanisms involved in the interaction between the bacilli and host cells, and may lead to the development of "smart" tools capable of interfering with *Mtb* pathogenesis. Another group of proteins known to play an important role in pathogenesis are those under the control of the dormancy survival regulon (Dos), which controls expression of more than 50 genes responsible for the *Mtb* hypoxic response.[@b26-mjhid-5-1-e2013070],[@b27-mjhid-5-1-e2013070]*Mtb* senses the harsh environment in macrophages and granulomas, characterized by low oxygen and nutrient depletion, and responds by activating a dormant state, whereby the bacilli stops multiplying, down-regulate central metabolism and activate anaerobic metabolism, with induction of stress proteins that provide *Mtb* with unique biological and immunological features.[@b28-mjhid-5-1-e2013070] These metabolically active but not replicating dormant bacilli can persist for a long time *in vivo* and may revert to an active state thanks to the resuscitating promoting factors (rpf), which act on the peptidoglycan to trigger a cascade of events that promotes bacterial growth.[@b29-mjhid-5-1-e2013070],[@b30-mjhid-5-1-e2013070] Hence, *Mtb* persists in host tissues under different metabolic states, with important implications from a pathogenetic and clinical practical perspectives, since dormant bacteria are susceptible only to certain antibiotics (pyrazinamide, rifampin and metronidazole) but resistant to other such as isoniazid.[@b31-mjhid-5-1-e2013070] TB Pathogenesis =============== *Mtb* infection occurs when few tubercle bacilli dispersed in the air from a patient with active pulmonary TB reach the alveoli of the host. Here, *Mtb* is quickly phagocytized by professional alveolar macrophages that most often can kill the entering bacteria thanks to the innate immune response ([Figure 2](#f2-mjhid-5-1-e2013070){ref-type="fig"}).[@b32-mjhid-5-1-e2013070] If the bacilli can survive this first line of defense, it starts actively replicating in macrophages, diffuse to nearby cells including epithelial and endothelial cells, reaching in few weeks of exponential growth a high bacterial burden.[@b33-mjhid-5-1-e2013070] During these early steps of infection, *Mtb* can diffuse to other organs through the lymphatics and by haematogenous dissemination where it can infect other cells.[@b34-mjhid-5-1-e2013070] Thereafter, once the adaptive immune response kicks in, migration to the site of primary infection of neutrophils, lymphocytes and other immune cells form a cellular infiltrate that later assume the typical structure of a granuloma.[@b35-mjhid-5-1-e2013070] Fibrotic components cover the granuloma that becomes calcified such that bacilli remain encapsulated inside and protected by the host immune response. This primary lesion, classically termed the Ghon complex,[@b36-mjhid-5-1-e2013070] was thought to be the "sanctuary" of *Mtb* during latent infection, with bacilli persisting in a dormant, non-metabolically active state, for years, decades, or most often for lifetime. In this scenario, when, during latent infection, for unknown reasons, bacilli would start replicating inside this primary lesion, active disease would ensue.[@b37-mjhid-5-1-e2013070] A major corollary of this hypothesis, with relevant pathophysiological and clinical implications, was that reactivation of TB originated from this very primary site of infection. This hypothesis was challenged since the early 20^th^ century, when it was shown that viable and infective bacilli were found in unaffected portion of lung tissues of infected guinea pigs or human necropsy rather than from the central core of the tuberculous lesions.[@b37-mjhid-5-1-e2013070],[@b38-mjhid-5-1-e2013070] Despite these early findings, only in 2000 Hernandez-Pando et al,[@b39-mjhid-5-1-e2013070] using normal lung tissues isolated at necropsy from patients who had died for causes other than TB in a TB endemic country, were able to detect by in situ PCR *Mtb* DNA in non-phagocytic cells, fibroblasts and endothelial cells, clearly suggesting that in latent TB subjects *Mtb* bacilli can persists in tissues and cells not associated with the granuloma or the Ghon complex. Using similar experimental settings, *Mtb* was detected in the fat tissue surrounding several organs, residing intracellularly in adipocytes, where it can survive protected from the host immune response.[@b40-mjhid-5-1-e2013070] All these evidences suggest that during LTBI *Mtb* can reside in different organs, tissues and cell types, not associated with the site of primary infection and lacking any sign of the typical granulomatous lesions. Studies carried out in the non-human primate model of TB further corroborated these findings indicating that during latent infection *Mtb* is metabolically active and replicates in host tissues despite the lack of any clinical sign or symptom of disease.[@b41-mjhid-5-1-e2013070],[@b42-mjhid-5-1-e2013070] Interestingly, in a single monkey with active TB it was possible to observe many different type of lesions, ranging from liquefied cavities with massive loads of bacilli, to necrotic or caseous hypoxic lesions with variable number of bacteria, to sterile lesions.[@b43-mjhid-5-1-e2013070] A similar scenario was observed in patients with pulmonary TB, where diverse lesions were observed simultaneously and with lesions responding differently to chemotherapy,[@b43-mjhid-5-1-e2013070] suggesting that they represent distinct *Mtb* subsets in different microenvironments. Based on the new understanding of the biology of *Mtb*, its different metabolic states, the dynamic host immune responses occurring during infection and on the spectrum of conditions that are observed during infection, it has been proposed that during latent infection most bacilli persist in a dormant state with fewer *Mtb* found in an active replicating state. These replicating bacilli, named "scouts" are processed and killed by the host immune defenses and as a result they are responsible for the induction of the large number of effector/memory T cells directed against *Mtb* antigens that are found in the peripheral blood.[@b44-mjhid-5-1-e2013070] Hence, during latent TB dormant bacteria constantly replenish the bulk of actively replicating bacilli readily killed by the host. When, for any reason, host immune responses fail to control these scouts, uncontrolled bacterial replication promotes diseases manifestations and active disease ensues.[@b45-mjhid-5-1-e2013070] Classical examples are highlighted by HIV infection that affects CD4 T cells that play a pivotal role in controlling *Mtb* replication;[@b46-mjhid-5-1-e2013070] treatment with biological therapies with anti-TNF that are known to increase the risk of developing TB disease up to 25 times in latent TB subjects as a result of the disruption of granuloma organization and depletion of certain populations of CD8 T cells known to play a role in controlling *Mtb*;[@b47-mjhid-5-1-e2013070],[@b48-mjhid-5-1-e2013070] treatment with corticosteroids, vitamin D deficiency and any other condition affecting T cell function are also known to increase the risk of active TB in latent TB subjects, underscoring the clinical implications that any event capable of perturbing the host-pathogen dynamic equilibrium can have. Cancer patients, including those with haematological diseases, are also at increased risk of developing TB and in these patients clinical outcomes are usually very aggressive, may present as systemic infections with a high fatality rate and diagnosis is usually delayed.[@b49-mjhid-5-1-e2013070],[@b50-mjhid-5-1-e2013070] The context of the TB spectrum, with the immunological and biological implications previously discussed, clearly highlight the risk that an infection usually controlled by the host immune response with no clinical signs or symptoms, can reactivate once the subtle balance affecting the dynamic equilibrium between the host and the bacilli occurs. Hence, it is very important to deploy proper and effective diagnostic protocols capable of detecting latent infection in these high risk groups and very sensitive assays to identify active disease when TB is suspected. TB Diagnosis ============ Direct diagnosis ---------------- Definitive diagnosis of TB requires the detection of *Mtb* from the biological sample by at least one of the current microbiological techniques: microscopical analysis, isolation in culture or molecular methods. These assays form the basis for the microbiological diagnosis of TB and the clinicians may require detection of *Mtb* in one or more specimens depending on the clinical symptoms, if any.[@b51-mjhid-5-1-e2013070] High sensitivity and specificity has been observed in the detection of *Mtb* in specimens such as sputum, bronchoalveolar lavage or induced sputum for the diagnosis of pulmonary TB.[@b52-mjhid-5-1-e2013070] The introduction of new, highly sensitive, fully automated molecular assays for the detection of *Mtb* has been recognized as a major achievement of the last decades,[@b53-mjhid-5-1-e2013070] though it is important to remind that molecular diagnosis should not be ordered routinely when the clinical suspicion of TB is too low.[@b54-mjhid-5-1-e2013070]--[@b56-mjhid-5-1-e2013070] Non-pulmonary forms of TB may be more problematic to diagnose because of the difficulties in identifying the proper specimens and the lower sensitivity of the microbiological assays in the non-pulmonary specimens, probably resulting from a lower bacterial concentration. Detection of *Mtb* in the urine or stools could be used to detect systemic infections and recently new assays capable of detecting mycobacterial components (lipoarabinomannan, LAM) in the urine were shown to be helpful to diagnose TB in HIV-infected subjects and immunocompromised patients, but not to diagnose pulmonary TB in immunocompetent subjects.[@b57-mjhid-5-1-e2013070],[@b58-mjhid-5-1-e2013070] Detection of *Mtb* in clinical specimens has been observed in HIV patients not showing any clinical sign or symptom of the disease[@b59-mjhid-5-1-e2013070] and in a recent report on the diagnosis of TB in children it was shown that ≈25% of children positive for *Mtb* did not show any clinical sign or symptom.[@b60-mjhid-5-1-e2013070] These results highlight the challenges associated with TB diagnosis and provide clinical evidences for the TB spectrum concepts.[@b43-mjhid-5-1-e2013070] Immunological Diagnosis ======================= The immunological diagnosis of TB has been historically performed by the Mantoux test or tuberculin skin test (TST) and the introduction in the last decade of the interferon-gamma release assays (IGRAs), that measure T cell responses directed against *Mtb* specific antigens in peripheral whole blood, has provided a new and valuable tool in the diagnosis of *Mtb* infection. Discussion of the immunological diagnosis of TB is beyond the scope of this review, but it is worth mentioning that TST and IGRAs are aimed at detecting *Mtb* infection but cannot distinguish between LTBI subjects with non signs or symptoms of disease and active TB patients.[@b61-mjhid-5-1-e2013070],[@b62-mjhid-5-1-e2013070] Despite many efforts, the prognostic value of IGRAs was shown to be insufficient and while many experimental assays are being devised and tested with the attempt to improve the current RD1-based assays,[@b63-mjhid-5-1-e2013070] it is important to remind that IGRAs can be used only as an "aid" in the diagnosis of TB and cannot be used alone to rule out TB nor to make conclusive diagnosis of TB. The concept of the TB spectrum discussed in this review provides the biological and immunological framework to support this statement. This work was funded by MIUR PRIN (project number 2009N98ST4_001) awarded to MS and GF. **Competing interests:** The authorshave declared that no competing interests exist. {#f1-mjhid-5-1-e2013070} {#f2-mjhid-5-1-e2013070}

Introduction {#sec1} ============ Low-molecular-weight gelators have started to evoke considerable interest in recent years on account of their application in cosmetics,^[@ref1]^ food industry,^[@ref1]^ controlling/triggering drug release,^[@ref2],[@ref3]^ tissue engineering,^[@ref3],[@ref4]^ sensors,^[@ref5]^ template materials, dye-sensitized solar cells,^[@ref6]^ and so on. The one-dimensional self-assembly of the gelling agent with a fiberlike structure eventually entangles to produce a three-dimensional network followed by immobilization of solvent molecules via capillary force, leading to gelation in a particular solvent. Low-molecular-weight gelators based on carbohydrates,^[@ref7]\ ,[@ref8]^ amino acids,^[@ref9]^ and several other organic building blocks^[@ref10]^ and their applications are well known. We have designed and synthesized a series of isoxazole-based low-molecular-weight gelators and shown their applications in the separation of bisphenol from water and the recovery of oil spills. Bisphenol is one of the highest volume chemicals produced in the industry as a starting material.^[@ref11]^ It often leaches from plastic and food containers as well as plastic water pipes and has an adverse and pervasive effect on humans and wildlife. Bisphenol as an endocrine disruptor that binds with some bisphenol-binding proteins^[@ref12]−[@ref14]^ and hormone receptors^[@ref15]−[@ref17]^ and shields their normal functionality in cells.^[@ref18]^ This could cause an abrupt and dramatic alteration of cell functionality,^[@ref19]−[@ref21]^ human reproductivity,^[@ref22]^ brain adipose tissue,^[@ref23]^ and so on. With growing demand and an increase in the production of plastic, human exposure to bisphenol has increased significantly over the years. There exist certain methods for the separation of bisphenol from water. They are mostly based on chromatographic techniques^[@ref24]−[@ref26]^ or reverse osmosis.^[@ref27]^ However, the practical implementation of these methods is questionable as they fail to quantitate separation of bisphenol from water and are largely uneconomic. Thus, there is a constant demand for an effective, alternative, and an economical way to separate bisphenol from water. We have now synthesized a series of isoxazole-based gelators by a systematic alteration of the hydrocarbon chain length (from C~8~H~16~ to C~16~H~31~) in the lipophilic part of the molecule and reveal their potential for efficient and quantitative separation of bisphenol from water. Besides that, these classes of isoxazole-based gelators were used for the cleanup of oil spills owing to the phase-selective gelation of these gelators. Marine oil spills were caused by leakage and release of more than 5 million tons of crude oil and petroleum products (refined fuel oils) into the ocean over the period of 1965--2010 in the Gulf of Mexico.^[@ref28],[@ref29]^ It has become a major threat to marine life and ocean ecosystems. There is an immediate and earnest need for the effective and efficient development of smart materials and technologies for oil spill control and recovery for combating oil spills.^[@ref30],[@ref31]^ Certain methods like bioremediation,^[@ref32]^ use of dispersants,^[@ref33]^ adsorption,^[@ref34]^ and use of solidifiers^[@ref8]^ and sorbents^[@ref35],[@ref36]^ have also been reported for the cleanup of oil spills. However, the problems associated with these existing methods are the release of toxic residues. Also, they are noneconomic, time-consuming, and allow only poor recovery. We have thus used isoxazole-based low-molecular-weight gelators for the removal of oil spills from water. Results and Discussion {#sec2} ====================== Syntheses of Gelators {#sec2.1} --------------------- [Scheme [1](#sch1){ref-type="scheme"}](#sch1){ref-type="scheme"} shows the syntheses of gelators **Ga--c**. For this, 3,4-dihydroxybenzaldehyde was treated with alkyl bromide (compounds **2a--c**) containing different hydrocarbon chains under basic conditions to obtain compounds **3a--c**. **3a--c** were reacted with hydroxylamine hydrochloride in ethanol for 2 h to afford compounds **4a--c**, and these were treated further with compound **1** in the presence of NaOCl in dry DCM at room temperature for an hour to obtain gelators **Ga--c** (for details, see [Scheme S1](http://pubs.acs.org/doi/suppl/10.1021/acsomega.0c00004/suppl_file/ao0c00004_si_001.pdf) in the Supporting Information, SI). {#sch1} Gelation Properties {#sec2.2} ------------------- The gelation abilities of these compounds were determined by the simple method of being stable to the inversion of the container,^[@ref8]^ and gelation is considered to have occurred while the gel is stable once the vial is turned upside down after cooling. The gelation abilities of all of these compounds in various solvents are depicted in [Table S1](http://pubs.acs.org/doi/suppl/10.1021/acsomega.0c00004/suppl_file/ao0c00004_si_001.pdf) in the Supporting Information. All gelators are versatile organic gelators as they induce gelation in not only ethanol but also a series of oils like castor oil, olive oil, etc., and ambidextrous gelation properties of these gelators could be useful for the preparation of hybrid material in different solvents and also for sensing and tissue engineering application purposes.^[@ref3],[@ref37]^ It should be noted that all of these gels are thermoreversible since they turn into liquid state upon heating and revert to the gel state upon cooling (see [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A). [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A depicts the thermoreversible nature of the gel obtained from compound **Ga** in ethanol as it undergoes a change to the solution state while heating and reverts to the gel state again after cooling. {#fig1} Rheology Study of the Gel {#sec2.3} ------------------------- The rigidity, viscoelastic property, and mechanical stability of the gel were determined by rheological property measurements of the gels derived from 1% (w/v) gelators of compounds **Ga--c** in castor oil and ethanol. All gels were cured for a period of 12 h before the rheology measurements ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}B,C). Rheology data of the gels in castor oil ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}B) and ethanol ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}C) demonstrate that for all of these gels, storage modulus (*G*′) is higher than loss modules (*G*″), implying the viscoelastic nature of these gels.^[@ref38]^ Notably, for all of these gels derived from castor oil, both *G*′ and *G*″ are almost independent of the measured range of frequency of oscillation at a constant strain. The dependence of the mechanical strength of the gel on the alkyl chain length is illustrated.^[@ref39]^ The variation of the mechanical strength of the gel with the hydrocarbon chain can be illustrated by considering its tangent of the phase angle value (tan δ),^[@ref40]^ which is the ratio of (*G*′) over (*G*″). In the case of an isoxazole-based gel derived from castor oil with C16 hydrocarbon chain length, the value of tan δ is much lower than for C10 (**Gb**) and for C8 (**Ga**) ([Table S2](http://pubs.acs.org/doi/suppl/10.1021/acsomega.0c00004/suppl_file/ao0c00004_si_001.pdf), Supporting Information). This clearly indicates that the gel derived from the C16 hydrocarbon chain gel is more mechanically stable than gels with shorter chains. The variation of tan δ value with the hydrocarbon chain length is reversed in the case of a gel derived from ethanol compared to the gel derived from castor oil. This signifies different gelation mechanisms and different orientations of the gel fibers in the three-dimensional (3D) structure of the gel network in these two organic solvents.^[@ref41]^ Morphology of the Gels {#sec2.4} ---------------------- To visualize the morphology of the organogel, field emission scanning electron microscopy (FESEM) experiments were carried out using xerogel of compounds **Ga**, **Gb**, and **Gc** obtained from organogels of compounds **Ga**, **Gb**, and **Gc** in ethanol, respectively. [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}A--C depicts the FESEM images of xerogels derived from gels **Gc**, **Gb**, and **Ga** in ethanol, respectively. The FESEM image of the xerogel of **Gc** ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}A) revealed that the organogel of **Gc** consists of numerous fibrous networks with a length of several nanometers and a diameter of 100--300 nm, which are entangled with each other to eventually give a self-assembled woven structure, whereas the FESEM images of xerogels of **Ga** and **Gb** ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}B,C) reveal a thin, flat, and ribbon-like structure, implying that there might be some relation among gel strength, morphology, and the 3D structure of the gel with the hydrocarbon chain length of the lipophilic part of the molecule. {#fig2} Investigation of Driving Forces Leading to the Formation of the Gel {#sec2.5} ------------------------------------------------------------------- To investigate the nature of intramolecular forces constituting the self-assembly processes leading to the formation of gel in organic solvents, temperature-dependent ^1^H spectroscopy measurements were carried out by using 0.5% of organogel of **Gb** in methanol-*d*~4~. [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}D depicts variable-temperature ^1^H spectra of the gel obtained from **Gb**. At low temperatures, due to the formation of a rigid supramolecular gel network, signals for protons are broad and unresolved due to high relaxation time owing to the strong gel network.^[@ref42]^ With a gradual increase in temperature, the proton signals become sharper compared to the signals at 15 °C due to the disordering of the self-assembled rigid gel network.^[@ref43]^ Temperature-dependent NMR spectra clearly reveal that the proton at the isoxazole ring plays a vital role in intermolecular H bonding, which is one of the key parameters in inducing the gelation process to make a self-assembled structure in the organic solvent, as the proton of the isoxazole ring suffers an enormous upfield shift (shown by the red arrow in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}D) as the temperature increases, leading to a disruption of intermolecular H bonding of the gel network. The fact that H bonding plays a vital role during the gelation process in the organic solvent was further confirmed by FTIR spectroscopic studies on **Gb**. [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}E represents the extended region of the FTIR spectra of the gelator **Gb** in chloroform solution (red curve) and ethanol gel (black curve). In the case of a chloroform solution of **Gb**, the band appearing at 1665 cm^--1^ can be assigned to the stretching vibration of the C=N bond^[@ref44]^ of the isoxazole backbone, which is shifted to 1672 cm^--1^ in ethanol gel, clearly suggesting the existence of H bonding on the gel network in the self-assembled gel state.^[@ref45]^ Separation of Bisphenol Using Isoxazole-Based Gel {#sec2.6} ------------------------------------------------- The organogel in castor oil was used for the separation of bisphenol from water ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}A). A specific volume of castor oil was added into a standard stock solution of bisphenol in water, followed by the addition of the gelator to the mixture under mechanical shaking, heating, and bath sonication for a short period. The gelator exclusively indulges in gelation with the upper oil layer. The water layer is untouched and can be discarded. Notably, during the process of gelation as well as the formation of entangled fibrous gel network under mechanical shaking and bath sonication, the gel fibers engross, entrap, and immobilize all of the biphenolic substances due to supramolecular interaction (probably due to π--π\* stacking). This causes a quantitative removal of the biphenolic substances from water into the upper oil gel, which was confirmed by the UV--visible spectroscopic study. [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}C depicts the UV--visible spectra of bisphenol in water before and after separation. The black curve shows the corresponding absorption spectrum of bisphenol in water before separation with an absorption maximum at 276 nm, and the red curve reflects the remaining bisphenol concentration in water after the addition of castor oil and gelator. The separation of the upper gel by simple tweezers allows a quantitative removal of bisphenol from water. To the best of our knowledge, this is the first example of the separation of a biphenolic substance using such a supramolecular gel system. {#fig3} Phase-Selective Gelation and Recovery of Oil from Oil--Water Mixture {#sec2.7} -------------------------------------------------------------------- **Ga--c** were also used for the separation of oil from an oil--water mixture ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}A--D) due to their excellent ability to allow robust cogelation in a wide variety of oils, including crude mineral oil from an oil--water mixture, which is an essential requirement for their recovery from oil spills.^[@ref29]^ The rheology data in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}B reflects that the value of *G*′ in the castor oil gel of **Gc** is around 3500 Pa, which signifies high gel strength (stiffness) of the gel in castor oil and therefore **Gc** was used for oil separation and recovery from the oil--water mixture. For the recovery of oil spills, 10 mg of gelator **Gc** was scattered over the upper oil layer of the binary mixture of tap water and diesel in a 20:1 ratio (v/v) and heated at 40 °C for a few minutes. While cooling to room temperature, the upper diesel layer stopped swirling but not the lower water layer, which confirms selective gelation of the top diesel layer ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}B). The as-formed gel was strong enough and was scooped out by a spatula and placed in a round-bottom flask ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}C), followed by distillation to recover the diesel in another round-bottom flask ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}D). Heating a binary mixture of seawater in the presence of a gelator is not economical. Thus, in a modified approach, a tetrahydrofuran (THF) solution of the gelator **Gc** was sprayed over the binary mixture of tap water and diesel in a 20:1 ratio (v/v), leading to instant gelation within a few seconds, making this approach more useful and practical for oil spill recovery. To make this approach closer to a real scenario, the above oil spill recovery procedure was carried out using artificial seawater, prepared according to the literature procedure,^[@ref46]^ and the same result was obtained, implying that the phase-selective gelation properties of these isoxazole-based gelators were not hampered in the presence of a high salt concentration in seawater. To investigate the maximum volume of water that can be used for the recovery of oil spills in a binary mixture of oil and water, a recovery experiment was carried out using a water and diesel mixture in a 1000:1 ratio (v/v) in the presence of THF solution of gelator **Gc**. Gelator **Gc** was still able to carry out phase-selective gelation of upper diesel layer. This further supports the utility of this method for real applications. {#fig4} Conclusions {#sec3} =========== In summary, we have synthesized a series of isoxazole-based low-molecular-weight gelators for the efficient separation of bisphenol A from water, owing to their robust gelation properties with excellent mechanical strength. The phase-selective cogelation allows their use for a wide range of applications, including the cleanup of oil spills. These classes of gelators can be easily prepared and offer fast and efficient gelation. Experimental Section {#sec4} ==================== Materials {#sec4.1} --------- 1-bromohexadecane, 1-bromodecane, 1-bromoctane, 3,4-dihydroxybezaldehyde, 3-nitrophenol, and hydroxylamine hydrochloride were purchased from Sigma-Aldrich and used without further purification. Structure determinations were carried out by a Brucker AscendTM 400 MHz spectroscope. Field emission scanning electron microscopy images were recorded using Supra 55 (Carl Zeiss). Xerogels for FESEM were prepared by slow evaporation of the gel samples, which were drop-casted on a 1 × 1 cm^2^ glass plate and dried overnight in the air inside a desiccator. The xerogel samples were then sputter-coated with Au and subjected to a FESEM study. UV--visible spectroscopic experiments of the bisphenol solutions were conducted using a PerkinElmer Lambda 365 spectrophotometer. ### Infrared (IR) Spectroscopy {#sec4.1.1} IR spectroscopic analysis of the gel was performed using a Cary 660 FTIR spectrophotometer in ATR mode. Samples were prepared using 0.5% (w/v) of compound **Gb** in ethanol or chloroform and cured overnight. The experiments were carried out by placing a small amount of the gel sample on the crystal of ATR, and the data was recorded. For a solution sample, a small drop of a solution of compound **Gb** was placed on the crystal of ATR. ### Gelation Method {#sec4.1.2} Typically, gelation tests were done by adding the gelator (10 mg) to the required solvent (1 mL) in a sealed 3 mL vial and heated until the solid was dissolved entirely. Subsequently, the solution was slowly allowed to cool to room temperature, and the gelation was visually observed. The gel sample was produced, which did not show any gravitational flow in the inverted tube. All gels found were thermally reversible. ### Rheological Studies {#sec4.1.3} Rheological studies of gel samples were carried out by a Bohlin Gemini-2 Malvern rheometer using parallel plates (25 mm, stainless steel). The gap between the parallel plates was 500 μm. Gel samples were prepared by taking 1% of the corresponding gelators (w/v) in the desired solvents and cured for 12 h before measurements. A small portion of the gel was placed on the smooth plate of the rheometer by a spatula, and the gel was allowed to equilibrate for 10 min before starting the experiment. Measurements were performed in frequency sweep (0.01--50 Hz) mode. All experiments were repeated twice. ### Oil Spill Recovery {#sec4.1.4} 10 mg of compound **Gc** was dissolved in 100 μL of THF, and the resultant solution was sprayed onto the test tube containing a binary mixture of 1 mL of diesel and 20 mL of water. The upper diesel phase of this biphasic mixture was immediately converted to gel within a minute. The aqueous phase remained. There was no movement of the gel layer in the presence of the aqueous layer observed in an inverted test tube, which indicated that the gel in the test tube was robust enough and supported its utility on a large scale for real use. The presence of a small amount of THF does not change the gelation ability of gelator in diesel, petrol, kerosene, and crude mineral oil. Phase-selective gelation was not affected in the presence of different salts (NaCl, KCl, MgSO~4~), acid, and base in the water medium. This property is strongly inspiring for the practical application of these gelators, forming a gel even in the oil phase in the presence of an oil--saltwater mixture such as seawater. After the formation of diesel gel, it was successfully collected from the diesel--water mixture using tweezers/spatula. After that, we recovered the gelator **Gc** using vacuum distillation at above 125 °C. Gelation studies have found that the gelation ability of recovered **Gc** from the gel phase by vacuum distillation is the same as in the original state. ### Separation of Bisphenol from Water {#sec4.1.5} A stock solution of bisphenol was prepared by adding 3 mg of bisphenol to 25 mL of water, forming a series of standard stock bisphenol solutions, with the concentrations 0.12, 0.06, 0.03, and 0.015 mg/mL prepared by dilution. The corresponding absorbances of these stock solutions were measured and plotted against concentrations. 1 mL of 0.12 mg/mL bisphenol solution was taken in a vial, and to this, 1 mL of castor oil was added, followed by simultaneous sonication, heating, and the addition of 10 mg of gelator **Gc**. The mixture was cooled to room temperature and kept overnight in a refrigerator, causing a clear separation between the top oil gel and the bottom water layer. The top gel layer was scooped out by a spatula to get a clear water layer, which was then subjected to UV--visible spectroscopic studies to check the remaining bisphenol concentration in water. The Supporting Information is available free of charge at [https://pubs.acs.org/doi/10.1021/acsomega.0c00004](https://pubs.acs.org/doi/10.1021/acsomega.0c00004?goto=supporting-info).Gelation table; ^1^H and ^13^C NMR spectra of all compounds; rheology data for isoxazole-based gels extracted from Figure 2B--C; procedure of the synthesis of compounds **3a--c**, **4a--c**, and **Ga--c** ([PDF](http://pubs.acs.org/doi/suppl/10.1021/acsomega.0c00004/suppl_file/ao0c00004_si_001.pdf)). Supplementary Material ====================== ###### ao0c00004_si_001.pdf The authors declare no competing financial interest. S.N. gratefully acknowledges the DST-INSPIRE Faculty Grant \[DST/INSPIRE/04/2017/000961\].

1. Introduction =============== Human cytomegalovirus (HCMV) is predominantly an opportunistic pathogen, causing clinically significant disease in people who have inadequate immune responses, including neonates, transplant recipients, and people with uncontrolled HIV infections \[[@B1-viruses-06-00476]\]. Viral replication initiates with the expression of the immediate early genes, the best characterized of which are IE1 and IE2. IE1 and IE2 initiate the cascade of viral gene expression, regulating the expression of the remainder of the viral genome, resulting in the expression of early and then late genes, and ultimately the production of new virions. HCMV has a very large genome of \~235 kb and encodes an estimated 176 genes \[[@B2-viruses-06-00476]\]. Despite the large genome size, only \~50 genes are required for HCMV replication in cell culture, suggesting that the remaining 70% of the genes contribute to replication and latency in the human host \[[@B3-viruses-06-00476],[@B4-viruses-06-00476]\]. The essential genes of HCMV can be grouped according to function: attachment of the virus to the target cells, transcriptional regulation, viral DNA replication, virion formation, and virion egress. Although there are a relatively small number of essential HCMV genes, to date the functions of several of the essential genes, including UL34, have been minimally characterized. The UL34 gene is essential for viral replication as determined by Dunn *et al.* \[[@B3-viruses-06-00476]\] and Yu *et al*. \[[@B4-viruses-06-00476]\] in their global analyses of the HCMV genome. The UL34 gene is transcribed throughout the viral replication cycle, resulting in the expression of early and late transcripts \[[@B5-viruses-06-00476]\]. The early transcript becomes abundant by 3 hours post-infection (hpi) while the late transcript predominates from 48 hpi throughout the remainder of the viral replication cycle. Two highly related proteins are encoded by the early and late transcripts, with the late protein (UL34b) identical to the early protein (UL34a) except for the absence of 21 amino terminal amino acids. Both UL34 proteins localize to the nucleus and are sequence-specific DNA-binding proteins that act as transcriptional repressors; the interaction of UL34 proteins with the UL34 binding sites in the US3 and US9 genes down-regulates their expression \[[@B6-viruses-06-00476],[@B7-viruses-06-00476]\]. In addition to the UL34 binding sites within the US3 and US9 genes, there are 12 to 13 additional binding sites located in the viral genome. Six of the UL34 binding sites are located within protein coding regions, 3 or 4 binding sites (the number is strain dependent) are in a region flanking the lytic origin of replication, and the remaining 3 binding sites are located 5\' of protein coding regions \[[@B6-viruses-06-00476]\]. The positions of the UL34 binding sites relative to coding regions suggests that UL34 proteins may be multifunctional, contributing not only to transcriptional repression but also contributing to viral replication in as yet unidentified ways. The experiments described here were undertaken to identify the contributions of each of the UL34 proteins to viral replication and to examine the intracellular localization pattern of UL34 proteins during infection. 2. Results and Discussion ========================= 2.1. Both UL34 Proteins Are Essential for Viral Replication ----------------------------------------------------------- Yu *et al.* \[[@B4-viruses-06-00476]\] and Dunn *et al.* \[[@B3-viruses-06-00476]\] identified UL34 as essential for viral replication in their global analyses of the HCMV genome. We extended their results by constructing and studying recombinant viruses using the bacterial artificial chromosome (BAC) that contains the HCMV AD169 genome, pHB5 \[[@B8-viruses-06-00476]\]. HCMV-BACs that either entirely lacked UL34 (ΔUL34), contained UL34 with a mutation in the ATG initiating translation of the early protein \[ATG mutated to ATC (methionine to isoleucine), ΔE mutant\], contained UL34 with a mutation in the ATG initiating the late protein \[ATG mutated to GTG (methionine to valine), ΔL mutant\], or had the UL34 open reading frame restored (UL34 rescue, RUL34). The ability of each of the recombinant viruses to replicate was assayed following electroporation of the HCMV-BACs into primary human fibroblasts, along with a plasmid expressing the tegument protein, pp71. Following electroporation, cells were observed for plaque formation for 4 weeks. The parental BAC, pHB5, and the UL34 rescue BAC (RUL34) gave rise to plaques by 8 days post-transfection. No plaques developed in the cells receiving the ΔUL34 mutant, the ΔE UL34 mutant or the ΔL UL34 mutant BAC during a 4 week observation period. From these results, we concluded that the expression of both UL34 proteins is essential for viral replication. 2.2. Reduced Viral Gene Expression in the Absence of UL34 Proteins ------------------------------------------------------------------ To examine the defect in viral replication associated with the absence of UL34 proteins, semi-quantitative RT-PCR reactions were performed on RNA samples extracted following the electroporation of the UL34-HCMV BACs into human fibroblasts. Levels of expression for the essential genes UL32, UL37, UL44, UL46, UL84 and UL123 (IE2) were assayed as were levels of expression of the non-essential UL36 and UL69 genes. IE2, UL36, and UL37 are immediate early genes; UL44 and UL84 are early genes; UL69 is an early/late gene and UL32 and UL46 are late or presumed late genes ([Figure 1](#viruses-06-00476-f001){ref-type="fig"}A). Levels of expression were analyzed at 6 and 8 days post-transfection; time points that correspond approximately to early and late times of infection, based on the time when plaques are visible. Viral transcript levels were normalized to the transcript levels of the cellular gene, glyceraldehyde phosphate dehydrogenase (GAPDH). At 6 and 8 days post-transfection, transcript levels for all genes asssayed were decreased in the UL34 mutant viruses when compared to the UL34 rescued virus (RUL34, [Figure 1](#viruses-06-00476-f001){ref-type="fig"}B,C). Six days post-transfection, expression of the major immediate early (mIE) gene, IE2, was decreased in the absence of the early, late or both UL34 proteins. Similar to the reduction in the level of IE2 transcripts, levels of UL44, UL84, UL32, and UL46 transcripts were detected at a reduced level for all of the UL34 mutant viruses at 6 days post-transfection ([Figure 1](#viruses-06-00476-f001){ref-type="fig"}B). In contrast, no UL69 or UL37 expression was detected for the UL34 mutant viruses; and UL36 expression was detected only in the UL34 mutant virus expressing the late UL34 protein (UL34b). At 8 days post-transfection, only UL44 transcripts were detectable for the UL34 deleted-BACs, albeit at a much reduced level compared to the UL34 rescued virus ([Figure 1](#viruses-06-00476-f001){ref-type="fig"}C). {#viruses-06-00476-f001} There is no UL34 binding site within the major immediate early gene, and in studies utilizing transient expression assays, UL34 has no activating or repressing effect on the mIE promoter \[[@B9-viruses-06-00476]\]. Consequently, the reduction in IE2 transcript levels seen in the absence of UL34 proteins suggests that UL34 proteins have a general effect on the level of IE2 transcripts. This is supported by the reduction in transcript levels for all viral genes assayed ([Figure 1](#viruses-06-00476-f001){ref-type="fig"}B,C). UL32 and UL37 contain UL34 binding sites within their open reading frames; however, the diminution in transcript levels is consistent for all genes tested, regardless of the presence of a UL34 binding site. Intriguingly, the inability to detect UL69 and UL37 transcripts in the absence of either UL34 protein suggests that both proteins are required for their expression, contrasting with UL36 expression, where transcripts are not detectable in the absence of the late UL34 protein, suggesting that the late UL34 protein (UL34b) is required for UL36 expression. At 8 days post-transfection, of the genes assayed, only UL44 transcripts were detected for the UL34 mutant viruses, in contrast to the transcripts detected for the UL34 rescued virus ([Figure 1](#viruses-06-00476-f001){ref-type="fig"}C). The lack of either UL34 protein results in a decrease in expression of IE2 and an absence of UL37 expression; proteins critical for later gene expression. These data suggested that the effects of the UL34 mutations were cumulative, that is, a reduction in the expression of essential genes earlier in the infection cycle (day 6) results in very little viral gene expression by 8 days post-transfection, corresponding to the defect in viral replication. Some variations in transcript levels were seen when comparing the UL34 mutant HCMV BACs. Expression of only the late UL34 protein resulted in detectable levels of UL36 and an increase in the level of UL32 expression, relative to the other UL34 mutant viruses, suggesting that the late protein plays a significant role in the expression of these two genes. There are UL34 binding sites located within the coding regions of UL37 and UL32 that may directly influence gene expression. UL34 proteins have a known transcriptional effect, repressing expression of the US3 and US9 genes \[[@B6-viruses-06-00476],[@B7-viruses-06-00476]\]. However, the data presented in [Figure 1](#viruses-06-00476-f001){ref-type="fig"} suggests that UL34 has a more general effect on viral gene expression given the lack of UL34 binding sites within the other assayed genes, coupled with the decrease in gene expression. Both forms of the UL34 protein are required for viral replication, with the early and late proteins differentially affecting the expression of other viral genes. 2.3. UL34 Localizes to Viral DNA Replication Centers ---------------------------------------------------- The UL34 proteins localize to the nucleus when visualized using a tag of enhanced green fluorescent protein (EGFP) or using indirect immunofluorescence \[[@B5-viruses-06-00476]\]. Here, we examined the intracellular location of UL34 proteins during the viral replication cycle, using polyclonal rabbit anti‑sera to UL34 and indirect immunofluorescence. Monoclonal antibodies to other viral proteins were used simultaneously to co-label the same infected cell population. Primary human fibroblasts were infected with HCMV strain Towne and fixed and stained at 3, 24, 48, 72 and 96 hours post‑infection (hpi). As UL34 proteins are detectable by 4 hpi using either immunoprecipitation or western blot analyses \[[@B5-viruses-06-00476]\], we initially compared the intracellular pattern of UL34 expression to that of the immediate early protein, IE2. At 3 hpi, UL34 was detected in a punctate staining pattern ([Figure 2](#viruses-06-00476-f002){ref-type="fig"}). Co-labeling with an antibody to IE2 demonstrated that UL34 colocalized with IE2 in the punctate dots at 3 hpi. UL34 continued to colocalize with IE2 throughout the remainder of the viral replication cycle, with the staining of UL34 and IE2 increasing as viral replication progressed ([Figure 2](#viruses-06-00476-f002){ref-type="fig"}). At 3 hpi, UL34 was not detectable in all cells positive for IE2, as expected, with IE2 expression activating the expression of other viral genes. {#viruses-06-00476-f002} As IE2 is found in viral DNA replication structures \[[@B10-viruses-06-00476]\], the colocalization of UL34 with IE2 suggested that UL34 localizes to viral DNA replication centers. To further examine the intracellular location of UL34, additional immunofluorescence experiments were performed, comparing the intracellular location of UL34 to that of UL44. UL44 encodes a viral DNA polymerase processivity factor; antibody labeling of UL44 is commonly used to identify viral DNA replication centers \[[@B11-viruses-06-00476],[@B12-viruses-06-00476]\]. UL44 was detectable at 24 hours post-infection, localizing as bright nuclear foci with a low level of diffuse nuclear staining ([Figure 3](#viruses-06-00476-f003){ref-type="fig"}). UL34 colocalized with UL44 in the bright nuclear foci. The pattern of colocalization continued throughout the viral replication cycle, with UL34 colocalizing extensively with UL44. Although UL34 colocalizes with UL44 in viral DNA replication centers, UL34 was not identified in the proteins complexing with UL44 \[[@B11-viruses-06-00476]\], suggesting that UL34 does not interact directly with UL44. {#viruses-06-00476-f003} The similar staining patterns of UL34, IE2 and UL44 again suggested that UL34 localizes predominantly to viral replication centers. To compare the intracellular location of UL34 with that of another viral protein found in the nucleus but not in viral DNA replication centers, infected cells were labeled with antisera to UL34 and with a monoclonal antibody to ICP22. The US22 gene encodes ICP22, a tegument protein that is found in the nucleus early in infection and in both the nucleus and cytoplasm at later stages of viral replication \[[@B13-viruses-06-00476]\]. As shown in [Figure 4](#viruses-06-00476-f004){ref-type="fig"}, little colocalization of UL34 with ICP22 is seen, suggesting that UL34 specifically colocalizes with IE2 and UL44 in viral DNA replication centers. {#viruses-06-00476-f004} IE2 accumulates at the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs) \[[@B10-viruses-06-00476]\], becoming incorporated into viral DNA replication centers, along with the core proteins including UL44, as the viral replication cycle continues. Our data demonstrate that UL34, along with IE2 and UL44, is also found in viral DNA replication centers. The contribution of UL34 to viral replication is potentially mediated through the UL34 binding sites located in the viral genome near the origin of lytic replication. The interaction of pUL34 with the ori-lyt region and the localization of UL34 to viral DNA replication center along with the two essential proteins, IE2 and UL44, suggests that UL34 may contribute to the efficiency of viral DNA replication. 2.4. UL34 Colocalizes with Nucleolin ------------------------------------ At late stages of infection (96 hpi), UL34 was found widely distributed in the nucleus. The circular areas within the nucleus that did not contain UL34 (See [Figure 2](#viruses-06-00476-f002){ref-type="fig"}, 72 hpi as an example) were identified as nucleoli using an antibody to label the nucleolar protein, fibrillarin (data not shown). However, UL44 colocalizes partially with the cellular protein nucleolin, and the interaction of nucleolin with UL44 is essential for viral replication \[[@B11-viruses-06-00476]\]. The colocalization of UL34 and UL44 suggested that UL34 would colocalize with nucleolin. To examine the interaction of UL34 and nucleolin, infected cells were costained with antibodies to UL34 and to nucleolin. Early in infection (24 hpi), UL34 accumulated in bright nuclear foci adjacent to the nucleolin staining ([Figure 5](#viruses-06-00476-f005){ref-type="fig"}). However, at late times of infection, UL34 partially colocalized with the ring of nucleolin detected at the perimeter of the nucleoli ([Figure 5](#viruses-06-00476-f005){ref-type="fig"}, see inset). These data suggested that nucleolin and nucleoli contribute to organization of UL34 in the nucleus and to the formation and organization of viral DNA replication compartments. Strang *et al*. \[[@B11-viruses-06-00476],[@B12-viruses-06-00476]\] found nucleolin predominantly dispersed in the nucleus following viral infection (using HCMV strain AD169), and further found that UL44 partially colocalized with nucleolin at the perimeter of viral replication centers. In contrast, using a monoclonal antibody to nucleolin rather than polyclonal antisera, we detected no dispersal of nucleolin. The difference seen in nucleolin distribution following infection may be a result of utilizing different antisera, or alternatively, strain specific differences, with the localization data presented here obtained following infection with HCMV strain Towne. In summary, UL34 proteins are localized in close proximity to nucleolin early in infection; late in infection, UL34 proteins partially colocalize with nucleolin. {ref-type="fig"}; the DAPI-stained image was not included in the merged image for the 96 hpi timepoint. The arrowhead indicates the association of UL34 with nucleolin early in infection. The inset shows an enlargement of the colocalization of UL34 and nucleolin at 96 hpi.](viruses-06-00476-g005){#viruses-06-00476-f005} 3. Experimental Section ======================= 3.1. Virus, Cells and Immunofluorescence ---------------------------------------- HCMV strain Towne was propagated in primary human fibroblasts in Dulbecco's minimal essential medium supplemented with glutamine, penicillin, streptomycin and 10% Nuserum. For immunofluorescence studies, primary human fibroblasts were plated on glass coverslips and infected with HCMV strain Towne at a moi of 1--2 pfu/cell. Prior to antibody staining, cells were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde and permeabilized using 0.2% Triton X-100 in PBS. Following four washes with PBS, cells were incubated with 10% normal human serum at 37 °C for 1 hour. Primary antibodies were diluted to the recommended concentrations in PBS containing 3% bovine serum albumin; cells were incubated with the primary antibodies for 1 hour at room temperature. Secondary fluorescent dye-tagged antibodies to mouse or rabbit antibodies were incubated with the cells for 30 minutes at room temperature. Nuclei were stained with 4\',6-diamidino-2-phenylindole (DAPI); cells were visualized using a Nikon fluorescence microscope and photographed using a SPOT camera. The following primary antibodies were used: monoclonal antibodies to UL44 (ICP36) and to US22 (ICP22) were obtained from Virusys Corporation, Taneytown, MD, USA; the monoclonal antibody to IE2 was obtained from Millipore, Billerica, MA, USA; the monoclonal antibody to nucleolin was obtained from Abcam, Cambridge, MA, USA, and the fluorescent dye-tagged secondary antibodies were obtained from Life Technologies, Grand Island, NY, USA. 3.2. Recombinant Virus ---------------------- The BAC plasmid, pHB5, which contains the HCMV AD169 genome was kindly provided by U. Koszinowski \[[@B8-viruses-06-00476]\]. A mutated BAC lacking UL34 was constructed as described by Datsenko and Wanner \[[@B14-viruses-06-00476]\] using primers (285, 5\'CAGAACCCGTCGCCATTTCCCCTCATA-TACGGTACACGTCCCC CTGATCTGTGTAGGCTGGAGCTGCTTC 3\' and 286, 5\' AAACCAGAGCGGAACTTGAGAAATCAACGCTTTATTGTTCTCCAGTGACGCATAT-GAATATCCTCCTTAG 3\') which contain sequences complementary to the ends of UL34 and to the kanamycin gene \[[@B14-viruses-06-00476]\]. Briefly, the primers were used to amplify the kanamycin and frt sequences from pKD13; the PCR product was then introduced into pHB5 using pKD46, which expresses the lambda Red genes. Following insertion of the FRT-kanamycin cassette and deletion of the UL34 open reading frame, the kanamycin gene was removed from the BAC using pCP20 which expresses the Flp recombinase. Recombinant BACs were then constructed by replacing the deleted UL34 gene with either the wild type sequence or with a UL34 gene containing mutations of either the early or late translation initiation codons for the early and the late proteins. pBJ545, which contains the HindIII H fragment of HCMV strain Towne in pST76K_SR, was mutated using oligo pairs 328 and 329 (328, 5\' CCTGCGAGCCGCCGAGGTGCGTGACAACGTGGC 3\'; 329, 5\' GCCACGTTGTCACGC ACCTCGGCGGCTCGCAGG 3\') or 330 and 331 (330, 5\' ACCGCCCCACCGCCGTCGTCGTCAT CAACTTCATCATCACCACC 3\'; 331, 5\' GGTGGTGATGATGAAGTTGATGACGACGAC GGCGGTGGGGCGGT 3\') and the Quickchange mutagenesis kit (Stratagene, Santa Clara, CA, USA) giving rise to pBJ598 (early ATG mutated to ATC) and pBJ599 (late ATG mutated to GTG). pBJ598 and 599 were electroporated into bacteria containing the ΔUL34-BAC and pKD46. Resulting colonies were screened using PCR to identify BACs containing the replaced UL34 sequences. 3.3. Quantitative Reverse Transcriptase Polymerase Chain Reactions ------------------------------------------------------------------ The following pairs of oligonucleotides were used to amplify a portion of the indicated gene transcript: UL37 (368, 5\' TCAGACGATCCGATGAACGT 3\' and 369, 5\' TCTCCTCCGAGCCAAAAGTC 3\'), UL44 (370, 5\' CTAGCCGCACTTTTGCTTCT 3\'and 371, 5\' ACGGTCTTTCCTCCAAGGAA 3\'), UL69 (372, TTAGTCATCCATATCATCGC 3\' and 373, GAGCTTAACTTGATGACGCC 3\'), UL122 (374, GAGACTTGTTCCTCAGGTCC 3\', and 375, 5\' CAACATGATCATCCACGCTG 3\'), UL36 (376, 5\' TCAGTTGTTCATGTAAACGT 3\', and 377, ACCACTTTGAACTCTCCTAC 3\'), glyceraldehyde phosphate dehydrogenase (GAPDH; 39, 5' AGAGACATCATCCCTGCCTCT 3\', and 391, 5\' TTTTTCTAGACGGCAGGTCA 3\'), UL84 (398; 5\' GCAGACCATGGCTAAAGTGA 3\' and 399, 5\' TTAACCGTACTGGTGAGCGA 3\') and UL32 (20, 5\' TGCAGTTTATCGGTCTACAGCG 3\'; and 21, 5\'CGRATCCTTGAGGTGCACAAAG 3\'). RNA was extracted from human fibroblasts that had been electroporated with the indicated HCMV-BAC 6 and 8 days post-transfection. RNA was treated with RQ1 DNAse followed by DNAse stop solution. PCR reactions were performed following reverse transcription reactions or in the absence of reverse transcription to confirm the absence of contaminating DNA. Amplimers were quantified following gel electrophoresis. 4. Conclusions ============== The experimental results presented here demonstrate that both UL34 proteins play an essential role in viral replication. Viruses containing in frame point mutations substituted for the initiation codons for the early and the late proteins are replication defective. The absence of UL34 protein expression resulted in a diminution of viral gene expression, and most notably, the absence of UL37 and UL69 transcripts. With the exception of UL34 repression of US3 and US9 expression \[[@B6-viruses-06-00476],[@B7-viruses-06-00476]\], we have been unable to identify any direct effects of UL34 expression on viral gene transcription. This suggests that UL34 proteins are multi-functional, not only specifically repressing the expression of some viral genes, but also having a general effect on viral gene transcription. We postulate that the effects of the UL34 proteins may be to facilitate the establishment of an environment in the nucleus favorable for viral gene expression and viral DNA replication. This notion is supported by the localization of UL34 proteins to viral DNA replication centers. Furthermore, the proximal localization of UL34 to nucleolin suggests that UL34-nucleolin interactions may facilitate the intranuclear positioning of the replication centers. In summary, this work demonstrates the significance of both UL34 proteins, and raises questions about the viral and cellular proteins that UL34 proteins interact with. We thank Yingguang Liu for constructing the initial UL34 deletion. We thank the American Heart Association for funding. RR and BJB designed and conducted the experiments; BJB prepared the manuscript. All authors read and approved the manuscript. The authors declare no conflict of interest.

**W**ilhelm Hasselbach (1921--2015) was born in Falkenstein, a small village north of Frankfurt/Main, Germany. For most of his life he worked and lived in Heidelberg, where he was a director at the Max Planck Institute for Medical Research. He is probably best known for the discovery of the *calcium pump* of the sarcoplasmic reticulum (Hasselbach and Makinose [@CR12], [@CR14]), which is known to be responsible for muscle relaxation. To be sure it was agreed then that the particulate (vesicular) fraction of the fragmented sarcoplasmatic reticulum was a "*relaxing factor*" (c.f. also Ebashi and Lipmann [@CR4]). But before 1961, it was not at all clear whether the vesicular particles (Hasselbach's "relaxing granules", c.f. Nagai et al. [@CR19]) produced a soluble relaxing factor inhibiting the interaction of actin and myosin or whether they "acted by binding calcium", as already suggested by Annemarie Weber (quoted by Dorothy Needham in her seminal book "Machina Carnis"). And indeed, "free" calcium in the medium was found to be *actively* removed by the particulate "factor"---in fact by transporting calcium ions across an osmotic gradient into the vesicles of the particulate fraction, as beautifully demonstrated by Hasselbach and Makinose ([@CR11], [@CR12]). About the same time, Setsuro Ebashi ([@CR2]) also found that the "relaxing material" of muscle removed calcium from the medium in vitro. His independent finding was, however, not fully published until the following year (Ebashi and Lipmann [@CR4]). Clearly Hasselbach's discovery paved the way for the later detailed elucidation of active calcium transport mechanisms and their role in the relaxation of skeletal, smooth and cardiac muscle (c.f. Hasselbach [@CR7]; Hasselbach and Oetliker [@CR16]). For instance, Hasselbach and Makinose ([@CR13]) were able to show that calcium transport and (Ca^2+^-activated) ATP splitting, as well as the so called ATP--ADP exchange reaction, were tightly coupled, suggesting the transient formation of an intermediate phosphorylation of a putable calcium transporter protein. Also, this process was found to be higly efficient: for every molecule of ATP split two ions of calcium were actively transported across the membrane surrounding the vesicles of the reticulum. Furthermore, by manipulating the experimental conditions, the calcium pump could be reversed, resulting in ATP synthesis rather than in breakdown, as well as in passive calcium efflux from the calcium storing vesicles. Thus Makinose and Hasselbach ([@CR18]) arrived at the conclusion "that the calcium translocation across the sarcoplasmic membranes is reversibly connected with the phosphoryl transfer reaction giving rise to a splitting of ATP when calcium moves inward and to an ATP synthesis when calcium moves outward". As later noted by Hasselbach and others, several drugs, including caffeine, also cause calcium efflux, but this was not found to be related to the reversal of the pump but rather to the passive release of stored calcium through calcium ion permeable channels, which could be blocked by ryanodine. Later experimental studies by Hasselbach and his long-term collaborator Andrea ("Andy") Migala ([@CR15]) confirmed "that the caffeine sensitive calcium channel functions as an assembly of at least four ryanodine binding sites whereby the occupation of one site suffices to abolish \[caffeine induced\] calcium release". This, of course, fitted well into current schemes of the role and mechanism of ryanodine receptors in excitation contraction coupling. While these findings were interesting, Hasselbach's most outstanding achievement was certainly his major contribution to the discovery and molecular elucidation of an ATP-driven active ion transport across a biological membrane. *He had discovered the Ca* ^*2*+^-*ATPase (SERCA) of the sarcoplasmatic reticulum*! Surely this success ought to be seen in the context of Jens Christian Skou's discovery (Nobel Prize in 1997) of the Na/K-ATPase in 1957, which was then proposed to be *possibly* related to the active ion transport across the nerve membrane (Skou [@CR21]). And most importantly, Peter Caldwell and colleagues (Caldwell et al. [@CR1]) already suggested that "the *presence* of ATP may be required for the normal operation of (an) active transport mechanism"! Because of his numerous achievements, Wilhelm Hasselbach was awarded the prestigious Feldberg Prize in 1963, and the Paul Morawitz Prize of the German Society of Cardiology in 1986. He was elected a member of the German Academy of Science Leopoldina (Deutsche Akademie der Naturforscher Leopoldina) in 1969, a member of the Brasilian Academy of Science in 1975, and an honorary member of the German Physiological Society in 1991. In 1961, Hasselbach became a scientific member of the Max Planck Society, and as from 1966, he was a director at the Heidelberg Max Planck Institute for Medical Research until his retirement in 1989. Besides, he was an honorary professor at Heidelberg University whose lectures on applied physiology were highly appreciated by medical students. **B**y all standards, Professor Hasselbach was a kind, helpful and modest man. Moreover he was always grateful for the help he received from others, and in particular in those difficult times after World War II when he was wounded. In 1998, looking back to his life-long experimental work, he stated: "The discovery of the ATP-driven calcium pump in the sarcoplasmic reticulum membranes reaches back to the postwar \[World War II\] years and would not be possible without the generous support by the American scientific community...; these pre- and postwar relations helped to establish the calcium pump as a physiologically relevant mechanism in all kinds of cells" (Hasselbach [@CR9]). Ironically, Hasselbach had his first opportunity to visit the United States rather late. John Gergely had invited him, H. H. Weber and me to a meeting taking place in May 1962 on the occasion of the opening of the Retina Foundation Institute in Boston. We all met at Dedham, Massachusetts, in a luxurious New England mansion, together with other visitors from Japan, England, Canada, Italy, Belgium, Sweden and Poland, where we were kindly received by young and old members of the American muscle community. We had long and unrestricted discussions, and those on the nature of the "relaxing factor" were somewhat heated. It was even proposed that the calcium pump could not cause relaxation of contractile systems per se. Rather it was suggested that the "true" relaxing factor might be a soluble inhibitor released from the sarcoplasmic reticulum but inactive unless myoplasmic free calcium was removed by the pump. In general, Hasselbach was a remarkable discussant. As Avril Somlyo once recalled, "he had an intensity and enthusiasm that made him special to interact with... always good discussions"! After the Dedham meeting, Hasselbach and I visited Annemarie Weber's Lab in New York. He then visited Fritz Lipmann, and I went on to work for a couple of weeks with David Bohr in Ann Arbor, Michigan. Soon the concept of the "soluble relaxing factor" was abandoned when Annemarie Weber found that many phenomena concerning the inhibition of actomyosin ATPase and of contraction by the tentative factor could be explained simply by the removal of calcium ions contaminating crude preparations of actomyosin and myofibrils (Weber et al. [@CR23]), which on the other hand could be activated by traces of free calcium binding cooperatively to the myofibrillar proteins (Weber and Herz [@CR22]). As later pointed out by Hasselbach ([@CR8]), "the 'soluble relaxing factor' finally became obsolete, when Ebashi ([@CR3]), Ebashi et al. ([@CR5]) showed that the 'contractile proteins' themselves contain a complex proteinsystem (originally called "native tropomyosin" or simply "Ebashi factor") which in the absence of calcium prevents the interactions between ATP, myosin and actin". In retrospect, the meetings in Boston and Dedham were most memorable events for both of us. In fact this was our first journey to the United States---both of us travelling together on the old ocean liner "Hanseatic". Actually I knew Wilhelm ("Willi") Hasselbach long before this, in fact already since the summer of 1957, when he visited Dorothy Needham, Samuel Victor Perry and Kenneth Bailey, my research supervisor in Cambridge. I was then working on catch muscles of clams, and Hasselbach kindly showed me how to prepare skinned (i.e. glycerinated) smooth muscle fibre bundles and possibly use them to study contraction. And then he added, "perhaps later in Heidelberg...", and indeed I became his colleague later on. Naturally, Hasselbach was *the* expert as he had just shown that, unlike skeletal muscle, skinned smooth muscle preparations had a rather high magnesium requirement for contraction (Hasselbach and Ledermair [@CR10]). Hasselbach was then already well known to the muscle community, as he had devised a method (the famous "Hasselbach-Schneider solution") by which myosin could be selectively extracted from skeletal muscle, thus leaving actin behind. Remember, his method had allowed Jean Hanson and Hugh Huxley to show that the myosin component of muscle resided exclusively in the A band, the length of which stayed constant in muscle contraction and relaxation as proposed by the sliding filament hypothesis. This in fact is a telling story that I knew first "from hearsay", and much later also from Hasselbach's personal account in a book chapter (Hasselbach [@CR8]). In 1949, and after completing his doctoral thesis in medicine, Hasselbach joined the Team of H. H. Weber in Tübingen, where he met Hildegard Portzehl, Weber's daughter Annemarie, and Gerhard Schneider. Together with Schneider, he was meant to extract globulin X, which Weber had described in the thirties as a major protein constituent of muscle. Much to the dismay of "boss" Weber, they did not succeed. Instead they observed that myosin was selectively extracted from striated muscle such that its band structure disappeared. Importantly, they used salt solutions containing pyrophosphate substituting for the then barely available ATP (Hasselbach and Schneider [@CR17]). These solutions were later called Hasselbach-Schneider solution by Jean Hanson and Hugh Huxley, when they used it to selectively remove myosin from the A band of myofibrils. With this work and the subsequent electron microscope studies, Hasselbach earned international recognition (Hasselbach [@CR6]). It was Jean Hanson who took the initiative to promote further collaboration and visited him in early 1954, shortly after the Weber group had moved to the Max Planck Institute in Heidelberg. Wilhelm (Willi) Hasselbach was a devoted scientist but also a kind-hearted family man. Friends of the family wrote, "he seemed like a great person and father". Wilhelm Hasselbach died in Heidelberg at age 94. As he had wished, he was buried in Falkenstein, the village where he was born. I am very grateful to Avril Somlyo for many helpful comments and to Drs. Klaus-Wilhelm and Hans-Joachim Hasselbach for kindly providing reprints and much further information. [^1]: Handling editor: Mathias Gautel

Background ========== Down syndrome (DS) is a complex disorder caused by trisomy of the entire or a critical portion of chromosome 21 (HSA21); it represents the most frequent genetic cause of mental retardation, with a frequency of about 1/1000 new-borns, and is associated with a huge number of congenital heart defects \[[@B1]\]. DS individuals have also an increased risk of early-onset Alzheimer disease \[[@B1]\]. Immunological and autoimmune disturbances, with high rates of infections and malignancies, are recurrent phenomena in DS pathogenesis \[[@B2]\], and infections still represent major cause of death in DS \[[@B3],[@B4]\]. Despite the increased risk of leukaemia, DS patients have a low incidence to develop solid tumors \[[@B5],[@B6]\], and a reduced incidence of diabetic retinopathy, suggesting, at least in part, a common angiogenesis\' suppression \[[@B5],[@B7]\]. Impaired endothelial function at a young age, possibly due to increased oxidative stress and yet unknown mechanisms, is a common DS feature \[[@B8]\]. DS phenotype results from a dosage imbalance of HSA21 genes, although expression analyses have reported conflicting results \[[@B9],[@B10]\]. The over-expression of chromosome 21 genes greatly varies across the trisomic tissues \[[@B11],[@B12]\], and analyzing specific cell type/tissue, in easy-accessible and non-invasive manner, may be more productive \[[@B13],[@B14]\]. Growing interest is emerging on circulating endothelial progenitor cells (EPCs) and their pivotal role in the maintenance of endothelium integrity, repair after injury and postnatal neovascularization \[[@B15]-[@B17]\]. Many studies are providing encouraging insights into the use of EPCs in the clinical setting \[[@B18],[@B19]\]. Indeed, accumulating evidences indicate a reduced availability, and/or impaired EPC function, in cardiovascular and metabolic diseases \[[@B17],[@B20],[@B21]\]. EPCs number was recently shown to be impaired in DS fetuses and children \[[@B22],[@B23]\] and CD34+ haematopoietic progenitors exhibited a marked growth decrease in Ts65Dn - a DS mouse model - accounting, at least in part, for DS vascular anomalies and defective immune response to pathogens \[[@B24]\]. Bacterial toxins may trigger pathogenic events through the over-production of cytokines and chemokines, leading to the alteration of endothelial function and capillary leakage \[[@B25]\]. Particularly, we recently demonstrated \[[@B26]\] that *Bartonella henselae*, a gram-negative intracellular bacteria responsible of vasoproliferative disorders in immunocompromised individuals \[[@B27],[@B28]\], adheres to and invades EPCs. The present study was designed to pursue the molecular mechanisms contributing to immune, vascular and haematopoietic defective DS phenotypes, by investigating the number and functions of DS EPCs compared to euploid cells, also focusing on bioinformatics analysis of differentially expressed genes. Moreover, by using the previously described *B. henselae*model, we investigated the susceptibility of DS progenitors to this pathogen infection, also performing a detailed analysis of deregulated genes after Bartonella infection, with particular attention to angiogenesis and immune response pathways. Methods ======= Subjects -------- DS and euploid donors were recruited at the Institute of General Pathology, Section of Clinical Pathology, Faculty of Medicine, University of Milan, and at the Second University of Naples, and an approval statement was obtained by the ethics\' review boards of both Institutions. Informed consent was obtained from all persons involved in all clinical investigation of this study according to the principles expressed in the Declaration of Helsinki. All subjects recruited for EPC isolation were free of infection, and no individual was taking any medication known to affect immune system/response. DS and euploid individuals were 65% males and 35% females as gender and 28 ± 9 as mean age. The experiments, where not specified, were performed on at least six DS and age-matched euploid individuals. Plasma samples were obtained from 50 DS individuals and 30 age matched euploids subdivided into three age subgroups (young 0-20 y.o.; adult 21-40 y.o.; old 41-60 y.o.) as described elsewhere \[[@B29]\]. EPC Isolation ------------- EPCs were isolated from non-institutionalized individuals with DS and age-matched euploid donors. EPCs were isolated as previously described \[[@B30]\]. Briefly, PBMCs were isolated by density gradient centrifugation of peripheral blood samples on Histopaque-1077 (Sigma). Cells were washed twice with PBS and counted. PBMCs were plated on culture dishes pre-coated with gelatin and fibronectin and maintained in endothelial basal medium-2 (EBM2; Cell Systems). Cells were cultured at 37°C with 5% CO2 in a humidified atmosphere. After four days, non-adherent cells were removed and adherent cells were used for further analyses. Bacterial strains and growth conditions --------------------------------------- The *B. henselae*strain ATCC 49882 (LGC Promochem) was grown on Columbia agar supplemented with 5% defibrinated sheep blood (Oxoid) in a humidified atmosphere at 37°C and 5% CO2. For production of bacterial stock suspensions, bacteria were harvested after 7 days of culture until they reached the mid-exponential phase of growth (109 bacteria/ml), resuspended in Tryptone Soya Broth USP (Oxoid) containing 10% glycerol, and stored at -80°C. The number of viable bacteria in the frozen stocks was determined as previously described \[[@B26]\]. B. henselae infection --------------------- For infection, Bartonella stock solutions were thawed, washed and suspended in antibiotic-free cell culture medium, and sedimented onto cultured EPCs at different multiplicity of infection (MOI) of 50, 100, 250, 500 and 1000 \[[@B26]\]. The MOI for infections was confirmed by plating serial dilutions of the infection inoculum. Assays were performed three times in triplicate. Confocal immunofluorescence microscopy -------------------------------------- EPCs were dual stained with Dil-Ac-LDL and lectin from *Ulex europaeus*and counted both by fluorescence microscopy and flow cytometry as previously described \[[@B26],[@B30]\]. Images were obtained by Zeiss LSM 510 with plan-apochromat X 63 (NA 1.4) oil immersion objective. EPCs images were used to measure cell size with ImageJ (<http://rsb.info.nih.gov/ij/>). SDF-1α plasma levels -------------------- Commercially available SDF-1α ELISA kit (Quantikine, R&D Systems) was used to determine plasma SDF-1α levels. Tests were carried out at RT on freshly thawed plasma samples of 50 DS individuals and 30 age matched euploids subdivided into three age subgroups (young 0-20 y.o.; adult 21-40 y.o.; old 41-60 y.o.) \[[@B29]\]. Concentration was determined by comparison with a standard curve, following manufacturer\'s instruction. Transmission electron microscopy -------------------------------- After a short incubation with Trypsin/EDTA, DS and euploid EPCs, both infected and uninfected with *B. henselae*, were harvested, centrifuged and washed in PBS. After centrifugation at a speed of 400 g for 7 min, cells were fixed in 4% glutaraldehyde as described \[[@B31]\]. Postfixation, dehydratation of specimen, semithin (2 μm) and ultrathin (80 nm) sections were performed as previously described \[[@B26]\]. Semithin sections were analysed with a light microscope (Polivar Reichert-Jung). Ultrathin sections were examined with Leo 912 AB transmission electron microscope operating at 80 kV. C11-BODIPY581/591 fluorescence ------------------------------ Oxidation-sensitive fluorescent probe, C11-BODIPY581/591 (C11-BO, Invitrogen), was loaded (2 μM final concentration) into the cells 30 min. before oxidative treatment. The samples were aliquoted in triplicate wells of a 24-well microplate, and fluorescence was determined with confocal laser microscopy at different times (0, 1, and 6 hours) from oxidant treatment. Between times, plates were maintained at 37°C in 5% CO2. To determine red fluorescence each microplate was excited at 543 nm (emission at 590 nm); for green fluorescence, microplates were excited at 488 nm (emission at 526 nm). Blank wells were also evaluated as well as C11-BO alone. Microarray analysis and quantitative RT-PCR ------------------------------------------- Total RNA (10 μg) was isolated as previously described \[[@B32]\] \[Additional file [1](#S1){ref-type="supplementary-material"}: Supplementary Methods\]. A pool of three samples (a total 15 μg of cRNA) was used for each hybridization - 2 pools of three infected and three uninfected euploid and DS - on the Affymetrix U133 2.0 probe array cartridge as described elsewhere \[[@B26]\]. Microarray data were submitted to Array Express (<http://www.ebi.ac.uk/arrayexpress;> provisional accession number E-MTAB-312). Results were validated by qRT-PCR and semi-qRT-PCR, performed as described \[[@B32]\], using primer pairs listed in \[Additional file [2](#S2){ref-type="supplementary-material"}: Supplemental Table S1\]. In silico significant pathway identification -------------------------------------------- Analysis of over-represented genes was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) \[[@B33],[@B34]\] and the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System \[35 \[Additional file [1](#S1){ref-type="supplementary-material"}: Supplementary Methods\]. Statistical analysis -------------------- EPCs number, cell size differences, SDF-1α plasma levels, fluorescence intensity of C11-BO and qRT-PCRs data were reported as mean values, and results analysed by paired Student t test. *P*value \< 0.05 was considered statistically significant \[Additional file [1](#S1){ref-type="supplementary-material"}: Supplementary Methods\]. Results ======= EPC number and phenotype ------------------------ We established that the number of EPCs isolated from peripheral blood of young and adult (mean age 28 ± 9) DS was significantly lower than age-matched euploid individuals (*P*\< 0.0001 vs euploid EPCs; see Figure [1A](#F1){ref-type="fig"}). Phase contrast fluorescent microscopy \[Additional file [3](#S3){ref-type="supplementary-material"}: Supplemental Figure S1A\] and FACS analysis (data not shown) were used to identify double-positive cells for Dil-Ac-LDL and lectin \[[@B26],[@B30]\]. By confocal microscopy and TEM we also observed early signs of cytoplasmatic disruption in DS progenitors, and cell size increase compared to euploid. In particular, by using ImageJ, we measured the cell size of DS EPCs, showing a significant increase compared to euploid cells (Figures [1B](#F1){ref-type="fig"} and Additional file [3](#S3){ref-type="supplementary-material"}: Supplemental Figure S1B\]). We also measured cell cycle progression of DS progenitors vs euploid cells and we did not find any significant difference \[Additional file [3](#S3){ref-type="supplementary-material"}: Supplemental Figure S1C\]. Moreover, the ultrastructural examination revealed an increased number of phagolysosomes and vacuolization of DS progenitors compared to euploid cells (Figure [1C](#F1){ref-type="fig"}). {#F1} SDF-1α plasma levels -------------------- EPC number is known to correlate to chemokines\' plasma levels, such as SDF-1α (stromal derived factor-1α). Thus, we first measured by ELISA its plasma levels in peripheral blood of 30 euploid and 50 DS individuals, collected in three age subgroups \[[@B29]\]. Then, comparing mean SDF-1α values we found a significant decrease of SDF-1α plasma levels in young and adults DS compared to age-matched euploid individuals (*P*= 0.02) (Figure [1D](#F1){ref-type="fig"}). Moreover, we measured by quantitative RT-PCR the expression of *SDF-1α*encoding gene, *CXCL12*, and of its membrane receptor *CXCR4*. A significant decrease in the expression of *CXCL12*(5-fold; *P*\< 0.05) and *CXCR4*(2-fold; *P*\< 0.05) was observed in DS endothelial progenitors compared to euploid cells (Figure [1E](#F1){ref-type="fig"}). *B. henselae*infection of endothelial progenitors ------------------------------------------------- EPCs isolated from young euploid and DS individuals were infected after 3 days of culture with *B. henselae*at different MOI as described elsewhere \[[@B26]\] (Figure [2A](#F2){ref-type="fig"}). TEM examination confirmed that *B. henselae*is internalized by endothelial cells as bacterial aggregates within invasomes or as single bacteria by protrusions of the cells. Interestingly, the EPC number was dramatically impaired in both DS and euploid after bacteria internalization \[Additional file [3](#S3){ref-type="supplementary-material"}: Supplemental Figure S1D\]. In contrast, by confocal microscopy at high magnifications (x630), a more detrimental effect was observed in infected DS progenitors, showing some morphological major differences compared to euploid EPCs when the same non-lethal MOI of Bartonella was used (Figure [2A](#F2){ref-type="fig"}). Ultrastructural analysis revealed that infected DS progenitors have increased intracellular accumulation of bacteria, forming invasomes, compared to euploid cells infected at the same MOI of 100 (Figure [2B](#F2){ref-type="fig"}). Cytoplasmic protrusions of cell membranes were also observed in both samples following adherence of the bacteria to the host cells. Moreover, after infection at higher MOI (250), DS progenitors showed larger invasomes, also displaying, in some cases, invasome and cell membrane rupture with subsequent bacteria outflow (Figure [2B](#F2){ref-type="fig"}). In contrast, infected euploid cells showed significant lower number of invasomes. The number of infected DS cells was estimated to be significantly higher compared to euploid cells at both MOI used (Figure [2C](#F2){ref-type="fig"}). We did not use infection at MOI ≥500 of *B. henselae*since they were not compatible with DS progenitors\' survival. {#F2} Oxidative stress in DS progenitors ---------------------------------- To determine oxidant activities in living cells, membrane lipid peroxidation (LP) of isolated EPCs was measured by using C11-BO, a fatty acid analogue. We cultured both DS and euploid EPCs in the presence of a fixed concentration of hydroperoxide (200 nM) \[[@B36]\], and performed a time-course (0, 1 and 6 hours) experiment measuring the fluorescence emission by confocal microscopy. Particularly, in 6-hours treated DS samples, we observed a significant shift (Figure [3A](#F3){ref-type="fig"} upper panel) in the fluorescence emission from red towards green (590 nm to 520 nm) compared to untreated DS EPCs (*P*\< 0.01). This shift was not observed in euploid hydroperoxide-treated EPCs vs untreated, as already described \[[@B36]\]. {#F3} To evaluate the protective effects of *B. henselae*, known to induce - albeit at low MOI (50) - long-term endothelial cell survival and proliferation \[[@B37],[@B38]\], the same experiment was performed on DS and euploid progenitors infected with a low MOI (about 50) of Bartonella. No differences in LP were observed in both DS and euploid infected cells compared to uninfected, in the absence of hydroperoxide treatment (data not shown). More interestingly, a significant decrease in LP was observed in Bartonella-infected DS progenitors vs uninfected (p \< 0.01) (Figure [3A](#F3){ref-type="fig"} lower panel). This finding is in accordance to the previously reported beneficial effect of infection at low MOI of *B. henselae*\[[@B37],[@B38]\]. Moreover, it is known that a constitutive increase in *S100B*, due to HSA21 trisomy, is likely to induce ROS generation, leading to increased oxidative stress in DS \[[@B39]\], and the over-expression of *SOD1*gene has been suggested to be responsible of oxidative damage to neurons \[[@B40]\]. Thus, we measured the expression levels of both *S100B*and *SOD1*genes in DS and euploid isolated EPCs, showing their significant over-expression in DS derived cells (Figure [3B](#F3){ref-type="fig"}). Chromosome 21 expression profile -------------------------------- We chose a user fold-change of 2 and a P-value cut-off of 0.005 for selecting a list of differentially expressed genes, and we first focused on the HSA21 genes according to GenBank annotation. Thus, to evaluate the impact of an extra copy of chromosome 21 on DS progenitors we performed a detailed analysis of HSA21 genes in DS vs eupolid EPCs. We observed that only 109 out of a total of 386 genes annotated on HSA21 (NCBI RefSeq 36.3), were detected in this microarray analysis. Furthermore, 52 (about 14% of total HSA21 annotated genes) showed an evidence of differential expression in DS EPCs compared to euploid cells. In particular, 37 genes were up- and 15 down-regulated (72% up- and 28% down-regulated, respectively) \[Additional file [4](#S4){ref-type="supplementary-material"}: Supplemental Table S2\]. Database searches based on GO classification, revealed that differentially expressed genes were mostly associated to immune response (GO:0006955) and transcription regulation (GO:0045449) (Figure [4A](#F4){ref-type="fig"}). Particularly, crucial genes involved in the immune response, such as interferon receptors (*IFNAR1*and *IFNAR2*), and oxidative stress, such as SOD1, S100B and APP - recently implicated in DS neurotoxicity from elevated expression of free radicals \[[@B39]\] - were highly up-regulated in DS vs euploid EPCs (Figure [3B](#F3){ref-type="fig"}; Additional file [4](#S4){ref-type="supplementary-material"}: Supplemental Table S2). {#F4} Although we observed relatively small up-regulation of chromosome 21 genes, significant changes in gene expression were not limited to HSA21 genes. Indeed, we observed a global and pronounced deregulation overall the chromosomes, possibly explained by dosage imbalance of HSA21 genes encoding transcriptional factors or gene expression modulators (Figure [4A](#F4){ref-type="fig"}; Additional file [4](#S4){ref-type="supplementary-material"}: Supplemental Table S2). A less frequently explored gene characteristic for microarray analysis is the chromosomal location of the genes, especially when studying diseases caused by genome alterations. First we demonstrated, by using chi-squared association tests, that differentially expressed genes are not uniformly distributed along the chromosomes. We observed that in DS EPCs the chromosomes 21 - as expected - and 19 were enriched for differentially expressed genes compared to the other chromosomes (α 0.001; *P*= 6.7E-0.6; Additional file [5](#S5){ref-type="supplementary-material"}: Supplemental Figure S2). As a proof of the robustness of our findings, the analysis was repeated with different fold-change and *P*cut-offs, reporting similar qualitative findings. Moreover, by using positional gene enrichment (PGE) approach \[[@B41]\] to map a set of genes to the exact location on chromosome \[Additional file [6](#S6){ref-type="supplementary-material"}: Supplemental Figure S3\], we observed that chromosome 19 has the highest percent of deregulated genes \[Additional file [3](#S3){ref-type="supplementary-material"}: Supplemental Figure S1A\] and, in particular, that the enriched p12 band contains most of the genes encoding for transcription factors of the zinc finger protein superfamily. Gene pathways\' perturbation in DS progenitors ---------------------------------------------- By using the same selection parameters, the comparison of global DS and euploid expression profiles revealed that, after filtering (\"Materials and Methods\"), 2913 genes (2489 of them with single probe and 424 with multiple probes) were differentially expressed, on a total of 11327 distinct genes considered. By using DAVID and PANTHER Classification System, differentially expressed genes were categorized according to their known or hypothetical biological function, and the enrichment for specific gene pathways was evaluated. The analysis revealed that most of the deregulated genes were involved in \"angiogenesis\", \"inflammation mediated by cytokines and chemokines\", \"integrins\" and \"interleukines\" signaling pathways (Figure [4B](#F4){ref-type="fig"}). A particular enrichment was also observed for cell cycle and cell cycle-related genes (Figure [4C](#F4){ref-type="fig"}). Interestingly, angiogenesis inhibitors encoding genes - such as *CXCL10*and other interferon-stimulated genes - were up-regulated in DS-derived progenitors, whereas, on the opposite, pro-angiogenic genes (*VEGFA*, *CXCL12*, *EDN1, CASP8*) were dramatically down-regulated (Table [1](#T1){ref-type="table"}). ###### Differential expression of angiogenesis-related genes in DS vs C endothelial progenitors *Gene name* *Gene symbol* *RefSeq* *Fold change* *Function* ------------------------------------------------ --------------- -------------- --------------- --------------------------------------------------------------- Chemokine (C-X-C motif) ligand 10 *CXCL10* NM_001565 6.7 I Antiangiogenic chemokine Interferon stimulated gene 20 *ISG20* NM_002201 4.4 I Angiogenesis inhibitor SAM domain- and HD domain-containing protein 1 *SAMHD1* NM_015474 3.0 I [Interferon-γ stimulated; angiogenesis inhibitor]{.smallcaps} Caspase 8 *CASP8* NM_001228 3.5 D Adhesion and homing of EPC Chemokine (C-X-C motif) ligand 12 *CXCL12* NM_199168 10.2 D; 5.7 D Mobilization and recruitment of EPCs Chemokine (C-X-C motif) receptor 4 *CXCR4* NM_001008540 3.0 D Endothelial cells migration and homing Chemokine (C-X-C motif) receptor 7 *CXCR7* NM_020311 4.3 D Endothelial cells migration and homing Endothelin 1 *EDN1* NM_001955 13.6 D; 3.5 D Promotes migration and proliferation of endothelial cells Endothelin receptor type B *EDNRB* NM_000115 2.8 D Migration, proliferation of endothelial cells Vascular endothelial growth factor A *VEGFA* NM_001025367 3.6 D Angioproliferative Stanniocalcin 1 *STC1* NM_003155 56.0 D VEGF-mediated angiogenic response Since evidences indicate that chemokines, cytokines and soluble factors affect the mobilization and recruitment of endothelial progenitors \[[@B42],[@B43]\], we evaluated the expression of some crucial genes by quantitative RT-PCR (Figure [1E](#F1){ref-type="fig"} and [4D](#F4){ref-type="fig"}). Particularly, we observed a down-regulation of *CXCR4*receptor and of its ligand, encoded by *CXCL12*gene, in DS progenitors compared to euploid cells (Figure [1E](#F1){ref-type="fig"}), the over-expression of *RCAN1*gene (or *DSCR1*) and the down-regulation of *CXCR7*receptor (Figure [4D](#F4){ref-type="fig"}), which play a key role in endothelial cells migration and homing. No significant differential expression was observed for *IL8*and *DYRK1A*genes in DS progenitors compared to euploid (Figure [4D](#F4){ref-type="fig"}). *B. henseale*-induced gene expression variations ------------------------------------------------ A similar approach for gene list selection was used to investigate the genetic response of DS-EPCs to *B. henselae*infection at a 100 MOI. GO term enrichment analysis \[[@B38]\] showed that a considerable number of induced/repressed genes belong to immune and inflammatory response pathways \[Additional file [7](#S7){ref-type="supplementary-material"}: Supplemental Figure S4\], with the majority of genes annotated within the \"Jak/STAT\" and \"Cytokine-cytokine receptor interaction\" pathways. Furthermore, we categorized the most prominently up-regulated genes in DS infected EPCs in two related functional classes: \"Interferons related/induced genes\" and \"cytokines and chemokines\", consisting of 19 and 32 genes, respectively. The same approach was then used for infected euploid cells. Transcriptional levels of related genes, observed by microarray, are shown in \[Additional file [8](#S8){ref-type="supplementary-material"}: Supplemental Table S3\]. We focused our interest toward the cluster of differentially expressed genes of the Jak/STAT pathway (Figure [5](#F5){ref-type="fig"}). The analysis revealed that DS infected EPCs have a very distinct \"molecular signature\" compared to infected euploid progenitors, mostly characterized by the up-regulation of interferon-stimulated genes (ISGs). Particularly, a large subset of differentially expressed crucial genes was also confirmed by semi-quantitative RT-PCR \[Additional file [7](#S7){ref-type="supplementary-material"}: Supplemental Figure S4\]. As a reflection of the robust induction of ISGs, \'IFN signaling\' was identified as the top scored pathway induced in DS progenitors after infection of *B. henselae*. {#F5} Other deregulated genes - involved in cell cycle regulation and gene expression - were further identified, many of which are known to be targets of interferon action as a consequence of the anti-proliferative effects. Discussion ========== Alteration of EPC number has been described in a wide range of conditions, such as cardiovascular, inflammatory, immune, and infectious diseases \[[@B21],[@B44]\]. A decreased *in vitro*growth capacity of bone marrow-derived progenitors in DS mouse model Ts65Dn \[[@B24]\] and a reduced number of CD34+ in DS fetuses and children were also reported \[[@B22]\]. More recently, Diller et al. (2008) reported an impairment of CD34+/AC133+/KDR+ cells in a small subset of DS individuals affected by Eisenmenger syndrome \[[@B23]\]. Despite the use of different experimental approaches and the limited number of individuals, the reduced number of circulating progenitors is an important common finding. These evidences may possibly account for the differences in angiogenesis, inflammatory and immune response reported in DS \[[@B14]\]. Here, we have shown that DS patients exhibit a marked reduction of ≈ 40% in the number of EPCs, also displaying a significant increase in cell size and major detrimental morphological changes (i.e., cell vacuolization and high number of active lysosomes). The reduced number of progenitors could be associated with alterations in the cell cycle; however, we did not find any significant difference between the groups \[Additional file [3](#S3){ref-type="supplementary-material"}: Supplemental Figure S1C\]. Thus, we investigated other possible mechanisms responsible for the observed EPCs impairment, such as mobilization/homing and oxidative stress susceptibility. Growing evidences indicate that chemokines and cytokines, such as SDF-1α and its receptor CXCR4, play a crucial role in the mobilization and homing of EPCs from bone marrow \[[@B45],[@B46]\], also affecting cell proliferation \[[@B41],[@B42]\]. Our findings of a significant decrease in SDF-1α plasma levels in young DS - compared to age-matched euploids - and a strong decrease of *CXCL12*and *CXCR4*gene transcription in their EPCs, suggest a link with the reduced number of circulating progenitors and the angiogenesis suppression observed in DS. These results are strengthened by microarray analysis, indicating that DS progenitors have a pronounced perturbation, at least at transcriptional level, in the angiogenesis and cell cycle pathways. Indeed, by using this approach we demonstrated the transcriptional deregulation of *CXCR7*, *IL8*and *RCAN1*genes, crucial factors involved in endothelial cells\' migration, homing and angiogenesis \[[@B47]-[@B49]\]. Moreover, we found a down-regulation of *CASP8*, which has been demonstrated to have a novel apoptosis-unrelated role in proangiogenic cells \[[@B50]\], although this gene was found to be associated with breast cancer by gene-based association study, and its down-regulation has also been reported in breast cancer \[[@B51]\]. We recently demonstrated the relevance of oxidative stress on the number and function of progenitor cells \[[@B52]\]. Besides, it is known that oxidative stress is a crucial issue in the pathogenesis of DS, especially due to the high incidence of Alzheimer-like disease at young age \[[@B53]\]. It has been also suggested that constitutive expression of trisomic genes, *S100B*and *SOD1*, is likely to represent a leading cause of ROS generation and increased oxidation in DS neurons \[[@B39],[@B40]\]. Oxidative stress relevance in DS fetuses was also highlighted by microarray analysis of uncultured amniotic fluid \[[@B54]\]. Here, we have assessed, by both experimental evidences and microarray analysis, that DS progenitors are more susceptible to hydroperoxide-induced LP and significantly over-express *S100B*and *SOD1*genes. These findings strengthen the hypothesis of their involvement in the susceptibility to oxidation observed in DS endothelial progenitors. In our study we have infected DS progenitors with a human pathogen, *B. henselae*, responsible of vasoproliferative diseases such as bacillary angiomatosis and peliosis in immunocompromised patients \[[@B27],[@B28]\], previously demonstrated to adhere to and invade EPCs \[[@B26]\]. Particularly, we investigated the effect of infection on DS progenitors\' number, morphology and oxidative stress response. After hydroperoxide treatment, we observed a significant LP decrease in Bartonella-infected DS progenitors compared to uninfected DS cells. This finding confirms the beneficial effect of *B. henselae*infection at low MOI on mature endothelial cells\' survival \[[@B37],[@B38]\]. In contrast, a reduced cell number in both DS and euploid groups was observed after Bartonella infection at higher MOIs; however, detrimental changes were visible only in DS EPCs, displaying a higher number of invasomes and infected cells compared to euploid cells. The molecular basis of such Bartonella-induced detrimental effect on endothelial progenitors of DS was investigated at a transcriptional level by microarray. Significant up-regulation of the Jak/STAT pathway was observed only within infected DS progenitors, whereas, on the opposite, infected euploid cells displayed a significant down-regulation. These findings strengthen the hypothesis that transcriptional analysis of EPCs is clearly of major interest in the context of this syndrome. Indeed, the activation of ISGs, involved in the immune response against infections and in tumor surveillance \[[@B55]\], also inhibits angiogenesis by decreasing the production of angiogenic factors such as VEGF and IL8 \[[@B56]\]. Our results clearly show that a similar ISGs activation occurs in infected DS progenitor cells, and, albeit at lower levels, in uninfected DS progenitors (data not shown). Pathologic immune and inflammatory responses are regulated by the cross-talk between interferons and TNFα \[[@B26]\], as well as deregulation of chemokines/cytokines greatly affects the mobilization and recruitment of endothelial progenitors. The imbalance between ISGs and other molecules might be of great immunological relevance concerning the well-known DS haematological defects. Conclusions =========== Physiological angiogenesis plays a central role in the embryogenesis and placental development; on the other hand, pathological angiogenesis represents a critical issue in the progression of many diseases, such as solid tumor growth and retinopathy. Individuals with Down syndrome, due to decreased incidence of angiogenesis-dependent diseases, have been postulated to be a systemic anti-angiogenesis model. Indeed, they exhibit a significantly increased anti-angiogenic surveillance, possibly due to increased expression of anti-angiogenic regulators on chromosome 21 \[[@B57]\]. However, it has been shown a complex regulation of gene expression not only related to gene copy number, with several genes escaping the rule of \"increased transcription proportional to the gene copy number\" \[[@B58],[@B59]\]. This findings suggest that many pathological traits observed in DS may be controlled by other more complex and, above all tissue-specific, regulatory mechanisms \[[@B59]\]. Our study shows that circulating endothelial progenitors are reduced in patients with DS, possibly correlating to the low SDF-1α plasma levels, to a reduced expression of its membrane receptor in these cells, and to their higher oxidative stress and pathogen infection susceptibility compared to euploid cells. A significant perturbation in the angiogenesis and inflammation gene pathways was also observed by microarray analysis, highlighting that gene expression analysis is a crucial issue for the study of common diseases. Endothelial dysfunction, angiogenesis\' suppression and infection recurrence are hallmarks of DS, and the impairment in the number and function of circulating progenitors may account for some of their pathological features. Further studies are needed to understand possible therapeutic implications of circulating EPCs in Down syndrome. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= CV, LS, ACa, BA, PS, ACi and CN designed research. CV, LS, ACa, RCo, VM, LM, CF, MR and MP carried out experimental research. VM and BA performed Transmission electron microscopy. CV and MR performed microarray analysis and quantitative RT-PCR. CV and CAn assisted with statistical analysis. BF, MMC, BS and RCa participated in the participant recruitment. CV, LS, ACa, RCo, CAn, VM, LM, BF, AG, CF, MR, MP, BA, MMC, BS, RCa, PS, ACi and CN analyzed data. CV, LS, ACa, CAn, BA, PS, ACi and CN wrote the paper. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1755-8794/3/40/prepub> Supplementary Material ====================== ###### Additional file 1 Supplementary Methods ###### Click here for file ###### Additional file 2 Table S1: Primer pairs used for quantitative and semi-quantitative RT-PCR ###### Click here for file ###### Additional file 3 **Figure S1: Impaired EPC number and function**. A) Representative photomicrographs of merged double-positive Dil-Ac-LDL/Lectin cells isolated from euploid (left panel) and DS (right panel) subjects (100X magnification). B) Fluorescence micrographs of EPCs labeled for 30 min with C11-BO in euploid and DS subjects. C) EPC number expressed as percentage in the different phases of cell cycle obtained by FACS. D) Curves indicate the percentage of EPC number infected with *B. henselae*in euploid and DS individuals. Results are representative of five different experiments in duplicate. ###### Click here for file ###### Additional file 4 Table S2: Chromosome 21 genes differentially expressed in DS vs euploids ###### Click here for file ###### Additional file 5 **Figure S2: Distribution of differentially expressed genes along the human chromosomes (DS vs euploids)**. A) Bar graph showing the empirical frequency distribution of differentially expressed genes along the autosomes of DS progenitors vs euploids. Asterisks indicate the significantly deregulated chromosomes. B) Representation of the robustness of our findings shown in A. The left column shows the different user-defined fold-change. For each á value used in the analysis are shown the relative p-values. C) Bar graph showing the percent of differentially expressed genes along the DS autosomes. ###### Click here for file ###### Additional file 6 **Figure S3: Positional gene mapping of differentially expressed genes (DS vs euploids)**. Graphic representation of positional gene enrichment (PGE) approach used to map differentially expressed genes in DS vs euploids EPCs to the exact location on the chromosome. ###### Click here for file ###### Additional file 7 **Figure S4: *B. henseale*-induced gene expression in DS EPCs**. A) Bar graph showing the top-scored deregulated gene pathways after infection in DS progenitors. Ratio indicates the percent of differentially expressed genes within the related pathway. B) Semiquantitative RT-PCR of Jak/STAT genes deregulated after *B. henseale*infection. ###### Click here for file ###### Additional file 8 Table S3: Differentially expressed genes after *B. henselae*infection ###### Click here for file Acknowledgements ================ We thank the patients and individuals recruited as controls for their participation. This work was supported by Legge 5, Regione Campania 2008 to Prof. A. Ciccodicola; PRIN 2006, Foundation Jerome Lejeune, and Regione Campania 2008 to Prof. C. Napoli; and Progetto di Rilevante Interesse Nazionale Ministero Italiano Università e Ricerca 2006 \[grant number 2006068944_001\] \"Basi genetiche e molecolari della patogenicità batterica\" to Prof. P. Salvatore.

The electronic version of this article is the complete one and can be found at: <http://f1000.com/prime/reports/b/5/27> Introduction {#s01} ============ Heart failure is a leading cause of death in the United States and is mentioned in one out of nine death certificates \[[@bib-001]\]. Survival after diagnosis has improved over time; however, the death rate remains high with \~ 50% mortality within 5 years. There is no cure for heart failure short of a heart transplant, which is an option only for a few select individuals. Thus, there is a great need for new therapies. Heart failure, and here we focus on systolic heart failure or heart failure with reduced ejection fraction, results from damage to the heart that prevents it from meeting its primary function to deliver oxygenated blood to the body. The most common causes of heart failure are coronary artery disease and hypertension, which can compromise contractility by reducing oxygen delivery to the heart muscle (or by adverse remodeling of the heart in the case of hypertension) or cause muscle damage outright through infarction. Ironically, cardiac muscle damage leading to heart failure may also result from reperfusion-injury following percutaneous coronary intervention (PCI), which is performed to increase oxygen delivery to cardiac muscle \[[@bib-002]\]. With reduced contractility, increased neurohormonal drive kicks in, causing genetic and structural changes to the heart that further reduce the ability of the heart to function as a pump \[[@bib-003],[@bib-004]\]. Moreover, the increased metabolic and oxidative stress and inflammation that develop during the progression of heart failure elicit additional structural damage to the heart that also compromises pump function \[[@bib-005],[@bib-006]\]. Thus, the trajectory of heart failure is multipronged and exponential, which makes therapeutic treatment difficult. At best, medicines today slow the progression of heart failure. An alternative strategy to treat heart failure is to restore the function of the heart as a pump using gene therapy. Here, we focus on successful preclinical trials that used viral gene delivery to enhance cardiac myocyte contractility by improving calcium handling, some of which have already progressed to clinical trials. Calcium handling in the failing heart {#s02} ===================================== Contraction of cardiac muscle is initiated by the influx of Ca^2+^ via L-type Ca^2+^ channels that open as a result of membrane depolarization brought about by the action potential. This Ca^2+^ activates the sarcoplasmic reticulum Ca^2+^ release channels known as ryanodine receptor 2 (RyR2), leading to a large increase in sarcoplasmic Ca^2+^. The rise in sarcoplasmic calcium causes contraction by Ca^2+^ binding to troponin C, which relieves constraints on actin-myosin interaction and cross-bridge formation. With relaxation, 60%--80% of the sarcoplasmic Ca^2+^ is actively transported into the sarcoplasmic reticulum lumen by the sarco(endo)plasmic reticulum Ca^2+^-ATPase pump (SERCA2a), while the remainder exits the cardiac myocyte by way of the Na^+^-Ca^2+^ exchanger \[[@bib-007]\]. With heart failure, Ca^2+^ uptake into the sarcoplasmic reticulum is impaired due to decreased expression of SERCA2a \[[@bib-007]\]. Activity of SERCA2a is decreased as well, due to an increased association of SERCA2a with its inhibitory regulator phospholamban for two reasons: first, normally, phosphorylation of phospholamban relieves SERCA2a inhibition by causing its dissociation from SERCA2a, but, in heart failure, there is less phosphorylation of phospholamban due to increased protein phosphatase 1 (PP1) activity \[[@bib-008]\]; second, the decreased SERCA2a/phospholamban ratio means that there is more phospholamban relative to SERCA2a, thus favoring SERCA2a inhibition. In addition, the sarcoplasmic reticulum becomes leaky in heart failure due to CaMKII-dependent phosphorylation of RyR2 \[[@bib-009]\]. Thus, in the failing heart, the sarcoplasmic reticulum does not function as well in removing Ca^2+^ from the sarcoplasm, which has two consequences: (1) there is an increase in resting sarcoplasmic Ca^2+^ that contributes to reduced relaxation and diastolic dysfunction; and (2) there is less Ca^2+^ released from the sarcoplasmic reticulum during contraction, which means that the force of contraction is reduced \[[@bib-007]\]. Strategies to improve or restore sarcoplasmic reticulum function would be predicted to reverse heart failure. Viral gene therapy for heart failure {#s03} ==================================== AAV delivery {#s03_1} ------------ Recombinant AAVs (adeno-associated viruses \-- serotypes 1, 6, 8, and 9 in particular) are currently the most widely used vectors for gene delivery to the heart, mainly due to their relative selectivity for cardiac myocytes, efficient long-term transgene expression, and low immunogenicity and rates of insertional mutagenesis \[[@bib-010]-[@bib-014]\]. However, restricted packaging capacity and inability to evade neutralizing antibodies limit their potential therapeutic effect and usage. Strategies are currently underway to overcome these issues. Cell-specific promoters can be used to further enhance expression of the transgene in cardiac myocytes \[[@bib-015]\]. While numerous vector delivery techniques have been developed, intravascular is the least invasive but effective approach for AAV delivery to the heart, largely due to the ability of the virus to cross the capillary endothelium. To avoid post-intravenous (IV) systemic neutralization and non-cardiac tissue transduction and subsequent toxicity and titer dilution, direct and indirect intracoronary injections are preferred and proven to be more efficient \[[@bib-016]\], and these are the current mode of administrating AAV in heart failure clinical trials. Finally, although AAV-mediated gene expression is relatively long lived, the recombinant vectors used do not integrate into the genome, making it likely that follow-up injections will be needed \[[@bib-017]\]. For more information about the different gene delivery technologies the reader is referred to an excellent recent review by MG Katz et al. \[[@bib-018]\]. SERCA2a on trial: a new era for heart failure management {#s03_2} -------------------------------------------------------- Impaired SERCA2a activity is associated with high diastolic but low systolic Ca^2+^ levels, which correlate with poor disease prognosis \[[@bib-019]\]. Overwhelming preclinical evidence from animal models has shown the importance of SERCA2a in heart failure and the power of treating heart failure by increasing SERCA2a expression by means of viral transduction of a SERCA2a transgene \[[@bib-020]-[@bib-023]\]. In 2007, Celladon Corporation sponsored the first Phase I/II human clinical trial (\[[@bib-024]\] CUPID; NCT00454818) in which a SERCA2a transgene was transferred, via an AAV1 Vector (MYDICAR), by percutaneous intra-coronary infusion. No safety concerns were revealed by the Phase I open label and sequential dose escalating part of the trial involving nine heart failure patients. A small Phase II, 3-doses, randomized, double-blind, placebo-controlled trial enrolling 39 heart failure patients followed. In addition to supporting the earlier safety findings, this trial reported significant clinical improvement of cardiac remodeling and heart failure symptoms. These results were accompanied by a marked reduction in cardiovascular hospitalization and clinical events that persisted for 6 to 12 months post high-dose treatment \[[@bib-025]\]. A larger Phase II, randomized, double-blind, placebo-controlled, multinational and multicenter clinical trial *A Study of Genetically Targeted Enzyme Replacement Therapy for Advanced Heart Failure* (CUPID-2b) is currently underway (NCT01643330). This trial will hopefully overcome the limitations of the small studies and confirm the efficacy of gene therapy in the management of heart failure. Details on the clinical trials involving AAV-mediated SERCA2a gene therapy are summarized in [Table 1](#tbl-001){ref-type="table"}. ###### Clinical trials on SERCA2a for heart failure  --------------------------------------------------------------------------------------------------------------------------------------- Clinical Trial ---------------------- ---------------------------- ------------------------- ---------------------------- ---------------------------- **NCT** NCT00454818 NCT00534703 NCT00454818 NCT01643330 **Acronym** CUPID NA CUPID CUPID-2b **Sponsor** Celladon Corporation Imperial College London Celladon Corporation Celladon Corporation **Study design** OL/SDE R/DB/PC R/DB/PC R/DB/PC **Outcome measures** Safety\ Safety\ Safety\ Efficacy Efficacy Efficacy Efficacy **Drug/Vector** MYDICAR (AAV1-CMV-SERCA2a) AAV6.-CMV-SERCA2a MYDICAR (AAV1-CMV-SERCA2a) MYDICAR (AAV1-CMV-SERCA2a) **Doses (DRP)** 1.4 × 10^11^\ 5 × 10^12^ 6 × 10^11^\ 1 × 10^13^ 6 × 10^11^\ 3 × 10^12^\ 3 × 10^12^ 1 × 10^13^ **Delivery** AECAI AECAI AECAI AECAI **Phase** PI PI/PII PII PII **N-Status** 9 patients-completed 16 patients-unknown 39 patients-completed 200-recruiting **Results** Positive NA Positive NA **PMID** 19327618 NA 21709064 NA --------------------------------------------------------------------------------------------------------------------------------------- AECI, antegrade epicardial coronary artery infusion; DB, double-blinded; DRP, DNase resistant particles; NA, not applicable; OL, open label; PI, Phase I; PII, Phase II; PC, placebo-controlled; R, randomized; SDE, sequential dose escalation. Other potential targets {#s03_3} ----------------------- In addition to SERCA2a, many calcium cycling proteins are potential therapeutic targets in heart failure ([Figure 1](#fig-001){ref-type="fig"}) \[[@bib-017]\]. Preclinical gene therapy studies targeting these molecules, directly or indirectly, support their safety and efficacy in improving cardiac function ([Table 2](#tbl-002){ref-type="table"}). Their assessment as targets for heart failure management in clinical trials is likely. {#fig-001} ###### Summary of preclinical trials targeting calcium handling protein by viral gene therapy that showed benefit for treating heart failure  ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Targets Role Vector/Animals Objective Status/Comments -------------------------------------------- ------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------ ----------------------------------------- ----------------------------------- Adenylyl cyclase 6 AC6 Indirect increase in SERCA2a activity Ad-mouse \[[@bib-031],[@bib-032]\]/pig \[[@bib-033],[@bib-034]\] Upregulation Ad5.h-AC6 PI/II clinical trial G protein-coupled receptor kinase (GRK2) Desensitize β-adrenergic receptor Ad-rabbit \[[@bib-047],[@bib-048]\]\ Inhibition Very promising AAV6-sheep \[[@bib-049],[@bib-050]\]/rat \[[@bib-051]\] Phospholamban (PLN) Inhibits SERCA2a activity Ad-Hamster \[[@bib-052]\]/sheep \[[@bib-053]\] AAV-mouse \[[@bib-054]\]/hamster \[[@bib-055]\]/rat \[[@bib-056],[@bib-057]\] Downregulation and/or limiting activity Promising (Optimization required) Protein phosphatase 1 (PP1) Indirect inhibition of SERCA2a activity via PLB dephosphorylation Ad-hamster \[[@bib-058]\]\ Downregulation/Inhibition Promising (more studies required) AAV9-mouse \[[@bib-026]\] S100 calcium-binding protein A1 (S100A1) Enhances SERCA2a/RyR2 and SR Ca^2+^ cycling proteins activity Ad-rat \[[@bib-039]\]\ Upregulation Very promising AAV6-rats \[[@bib-040]\]\ AAV9-pigs \[[@bib-041]\] Small ubiquitin-related modifier 1 (SUMO1) Promotes SERCA2a protein stability and activity AAV9-mouse \[[@bib-037]\] Upregulation Promising (more studies required) SR Ca^2+^-ATPase (SERCA2a) Ca^2+^ storage during diastole AAV1-rat \[[@bib-020]\]/pig \[[@bib-021]\]\ Upregulation AAV1- SERCA2a PII clinical trial AAV2-sheep \[[@bib-022],[@bib-059]\]\ AAV6-sheep \[[@bib-060]\] ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Ad, adenovirus; AAV, adeno-associated virus; PI, Phase I; PII, Phase II. ### Protein phosphatase 1 {#s03_3_1} Protein phosphatase (PP1)β is the catalytic subunit isoform of PP1 that primarily suppresses sarcoplasmic reticulum Ca^2+^ uptake by dephosphorylating phospholamban. Knockdown of the PP1β using short-hairpin (sh)RNA improved cardiac function and reduced cardiac remodeling and interstitial fibrosis in the muscle LIM protein-deficient mouse model of heart failure \[[@bib-026],[@bib-027]\]. shRNA was delivered with an AAV9 vector under the control of a B-type natriuretic protein (BNP) promoter to achieve heart failure-inducible gene expression. ### Adenylyl cyclase 6 {#s03_3_2} β-Adrenergic enhancement of contraction of the heart is mediated in part through adenylyl cyclase 6 (AC6), which converts ATP to cAMP and, thus, activates protein kinase A (PKA). PKA phosphorylates phospholamban to enhance SERCA2a activity and cardiac troponin I to enhance the force of contraction and relaxation \[[@bib-028]-[@bib-030]\]. In AC6 knockout hearts, phospholamban phosphorylation and SERCA2a activity are reduced \[[@bib-028]\]. Viral delivery of an AC6 transgene has been shown effective in alleviating heart failure symptoms in both small and large animal models of heart failure, due to improved sarcoplasmic reticulum Ca^2+^ function and storage resulting from increased phospholamban phosphorylation and decreased PP1 expression \[[@bib-031]-[@bib-034]\]. AC6 may also decrease phospholamban expression independently of cAMP formation \[[@bib-028]\]. ### GRK2 {#s03_3_3} The serine/threonine kinase GRK2 (G-protein-coupled receptor kinase) attenuates β-adrenergic signaling in the heart due to receptor desensitization and downregulation \[[@bib-035]\]. Levels and activity of GRK2 are increased in heart failure due to over-activation of the compensatory sympathetic drive. Viral-mediated knockdown of GRK2 in cardiac muscle is, thus, a viable strategy for enhancing cardiac contractility, while its knockdown in the adrenal gland helps reduce the increased circulating levels of catecholamines observed in heart failure \[[@bib-035]\]. ### Phospholamban {#s03_3_4} Phospholamban is a small, 52 amino acid protein that binds SERCA2a as a monomer and inhibits its activity \[[@bib-036]\]. Phosphorylation of phospholamban causes dissociation from SERCA2a and favors the formation of pentamers that are functionally inactive towards SERCA2a. Several strategies have been used to target phospholamban in models of heart failure using viral delivery, including expression of a chicken antibody-derived protein targeting phospholamban, an inhibitory phospholamban peptide, antisense, a zinc-finger transcriptional repressor, and a pseudophosphorylated mutant of phospholamban. Improvements in contractility and Ca^2+^ handling of cardiac myocytes have been reported. ### SUMO1 {#s03_3_5} Sumoylation is posttranslational modification of SERCA2a that was recently identified as highly relevant to development and potential treatment of heart failure \[[@bib-037]\]. SERCA2a is sumoylated by SUMO1 on two specific lysine residues in the heart and levels of SERCA2a sumoylation are reduced in the failing human heart and animal models of the failing heart, as are levels of SUMO1 and SERCA2a. Restoration of SUMO1 during transverse aortic constriction-induced heart failure in the mouse by recombinant AAV9 improved cardiac function. Evidence was provided that sumoylation increases both SERCA2a ATPase activity and protein stability. Although SUMO1 seems like a promising target for treating heart failure, its broad-based action in cardiac myocytes may prove an insurmountable limitation \[[@bib-038]\]. ### S100A1 {#s03_3_6} This is a Ca^2+^ binding protein that is highly expressed in cardiac myocytes, where it localizes at the sarcoplasmic reticulum, mitochondria, and myofilaments \[[@bib-036]\]. S100A1 interacts with SERCA2a, phospholamban, and RyR2. Exact details are not defined, but S100A1 seems to function as a Ca^2+^-sensitive chaperone that enhances the activity of other proteins. End-stage failing human hearts have reduced levels of S100A1 protein, and a number of gene therapy studies have shown the utility of increasing expression of S100A1 to improve cardiac function in heart failure \[[@bib-039]-[@bib-041]\]. A cautionary note {#s03_4} ----------------- Results of clinical trials are needed before the utility of gene therapy to treat heart failure can be adequately assessed. A negative outcome in the absence of any serious adverse events may slow, but not deter, further research into this approach. For one thing, there is a certain attractiveness to the simplicity of the idea of treating heart failure by restoring cardiac muscle performance. Of course that begs the question of whether it makes sense to "flog" an already "sick" cardiac muscle cell to make it work harder. Gene therapy to do so may turn out simply to be a way of buying additional time for true myocardial regeneration to take place using cardiac or induced-pluripotent stem cells. The reader is referred to several recent review articles dealing with stem cell therapy in heart failure \[[@bib-042]-[@bib-045]\]. Summary and future perspectives {#s04} =============================== Heart failure is a major health issue with a high rate of crippling morbidity and mortality. In the USA, the total annual cost of heart failure is estimated to be greater than 30 billion dollars and is expected to more than double over the next decade, due to an aging population \[[@bib-046]\]. Thus, there is a substantial need for new strategies to prevent the progression of heart failure. Until now, heart failure was seen as an incurable disease. For the first time, results of recent clinical trials using a viral-delivery gene therapy approach have offered the promise of an effective therapy for reversing systolic heart failure. These trials are the outgrowth of the methodical approach of basic research in identifying viable targets to enhance cardiac contractility. Time will tell whether a single target approach is sufficient to restore heart function and prevent deterioration or whether multiple gene targets are needed along with stem cell therapy to eventually replace the injured myocardium. Using a viral-delivery gene therapy approach to treat heart failure by enhancing contractility is not yet a reality, but substantial progress in that direction has been made in the last few years. This work was supported by grant R01 HL088101 from The National Heart, Lung, and Blood Institute (NHLBI) to George W. Booz. Disclosures =========== The authors declare that they have no disclosures. AAV : adeno-associated virus AC : adenylyl cyclase GRK2 : G-protein-coupled receptor kinase 2 IV : intravenous PKA : protein kinase A PP1 : protein phosphatase 1 RyR2 : ryanodine receptor 2 SERCA2a : sarco(endo)plasmic reticulum Ca^2+^-ATPase pump

1. Introduction {#sec1-ijerph-14-00773} =============== Humans evolved into their current form over 6--7 million years, beginning with our ancestors' evolution from a subset of primates \[[@B1-ijerph-14-00773]\]. This period represents 99.99% of the span of human evolution, during which time we lived in a natural environment. It is therefore considered that human physiological functions are best adapted to natural environments \[[@B2-ijerph-14-00773]\]. The considerable urbanization and artificialization that have occurred since the Industrial Revolution and the rapid spread of modern information technology contribute to the increased "stress state" experienced by humans in modern societies \[[@B3-ijerph-14-00773]\]. In 1984, American clinical psychologist Craig Brod coined the term "technostress" \[[@B4-ijerph-14-00773]\]. In 2008, it was reported that more than 3.3 billion people---half of the globe's population---live in an urban environment \[[@B5-ijerph-14-00773]\]. In response to these stressful situations, scientific evidence supporting physiological relaxation by exposure to natural stimuli from forests and urban green spaces has accumulated \[[@B6-ijerph-14-00773]\]. However, there have been few studies on the relaxation effect derived from familiar, naturally derived stimuli in daily life. Therefore, in the current study, we focused on contact with wood, which has been historically used in indoor environments in Japan. Wood is a familiar, natural material that has been used in houses and furniture, and it is empirically known to have a relaxation effect on humans. The interest in and expectations of the relaxation effect of wood on humans have increased in recent years, and scientific evidence of this effect is required. In a review outlining the current state of research regarding the physiological effects of wood-derived stimulation on humans \[[@B7-ijerph-14-00773]\], it was reported that data on the physiological effects of olfactory stimulation have continued to accumulate, following the development of physiological measurement technology in recent years. The oldest report, published in 1992, focused on the physiological effect of olfactory stimulation with Taiwan cypress oil \[[@B8-ijerph-14-00773]\], and several other reports have been published since. In recent years, the physiological relaxation effects of olfactory stimulation by stimuli such as air-dried Japanese cypress wood chips \[[@B9-ijerph-14-00773]\], α-pinene \[[@B10-ijerph-14-00773]\], and D-limonene \[[@B11-ijerph-14-00773]\], which are components derived from wood, have been reported. Several studies on visual stimulation have also been conducted using rooms with different designs and proportions of wood \[[@B12-ijerph-14-00773],[@B13-ijerph-14-00773],[@B14-ijerph-14-00773]\] and large wall panels \[[@B15-ijerph-14-00773]\]. In contrast, there have been extremely few reports on tactile stimuli. Morikawa et al. \[[@B16-ijerph-14-00773]\] reported the differential effects on blood pressure when touching plates of Japanese cypress wood vs. artificial material. Sakuragawa et al. \[[@B17-ijerph-14-00773]\] examined differences in the effects of tactile stimulation on human physiology that resulted from materials at different temperatures (cool, room temperature, and warm). They found the following: (1) touching an aluminum plate increased blood pressure, but the increase was inhibited when the aluminum was warmed; (2) touching an acrylic plastic plate increased blood pressure, and a greater rate of increase was observed when the acrylic was chilled; and (3) blood pressure did not change in response to touching objects made of Japanese cypress, Japanese cedar, or oak, and did not increase even when the oak was chilled. These reports are pioneering studies on the physiological effects of tactile stimulation with wood on humans. However, they have limitations in that they only examined blood pressure, which is an index of autonomic nervous activity, as a measure of the physiological response to stimuli. Therefore, in our previous research, we examined the physiological effects on brain activity and autonomic nervous activity of touching uncoated wood vs. other materials with the palm of the hand \[[@B18-ijerph-14-00773]\]. In this previous study, as an indicator of brain activity, oxyhemoglobin (oxy-Hb) concentrations were measured in the left and right prefrontal cortices using near-infrared time-resolved spectroscopy (TRS). Heart rate variability (HRV) was used as an indicator of autonomic nervous activity. The results indicated that touching uncoated white oak wood with the palm calmed prefrontal cortex activity and increased parasympathetic nervous activity more than the other materials (marble, tile, and stainless steel), thereby inducing physiological relaxation \[[@B18-ijerph-14-00773]\]. As a next step, it is necessary to clarify the influence of touching wood with various coatings on the physiological response, as much of the wood used in our everyday lives is coated. Several studies have reported on the psychological effects of contact with coated wood. For example, Bhatta et al. \[[@B19-ijerph-14-00773]\] investigated various coated wood (pine and oak) surfaces through the lateral motion of active fingertip exploration. The results indicated that natural and smooth-finished wood surfaces were perceived as more comfortable and relaxing than varnished and wax coated materials. In addition, Berger et al. \[[@B20-ijerph-14-00773]\] examined the subjective responses of touching interior wooden materials with the palm of the hand and sole of the foot. These results showed that oil-finished wood was evaluated as being subjectively preferable to lacquer-coated material and sheet laminated board material. However, there have been no reports on the effects of tactile (i.e., palm of the hand) stimulation with wood with various coatings on the physiological response. This study therefore aimed to clarify the effects of touching wood with different coatings with the palm on left and right prefrontal cortex activity, assessed using TRS, and on autonomic nervous activity, assessed using HRV and heart rate. 2. Materials and Methods {#sec2-ijerph-14-00773} ======================== 2.1. Participants {#sec2dot1-ijerph-14-00773} ----------------- The study participants were 18 female university students (mean age, 21.7 ± 1.6 years). We excluded smokers, those currently in treatment for disease, and those with menstrual period during the study period. All the participants were informed about the aim and procedures of the experiment before providing written informed consent to participate. This study was performed in accordance with the regulations of the Ethics Committee of the Center for Environment, Health and Field Sciences, Chiba University, Japan (Project Identification Code Number: 5). 2.2. Study Protocol {#sec2dot2-ijerph-14-00773} ------------------- Physiological measurements were performed in a chamber with an artificial climate maintained at 25 °C, 50% relative humidity, and 230-lux illumination. In the waiting room, the participants received a description of the experiment and then moved into the chamber. After sensors for physiological measurement were fitted to the participants' forehead, they received a description of the measurement procedure while in a seated position. Next, they practiced touching a stimulus with their palm using a dummy sample (sheet laminated flooring). The experimental procedure was as follows. Participants rested with their eyes closed for 60 s ([Figure 1](#ijerph-14-00773-f001){ref-type="fig"}, left). Upon receiving instructions from the experimenter, they moved their right forearm using their elbow as a fulcrum and placed the palm on the stimulus (hereafter referred to as "material") for 90 s ([Figure 1](#ijerph-14-00773-f001){ref-type="fig"}, right). After touching the material for 90 s, they returned their hand to the previous position upon the experimenter's instruction ([Figure 1](#ijerph-14-00773-f001){ref-type="fig"}, left). The experimenter then replaced the current material with the subsequent material, hid it with a cloth, and instructed participants to open their eyes. Subsequently, participants completed the subjective evaluation test. [Figure 2](#ijerph-14-00773-f002){ref-type="fig"} shows the experimental schedule. Materials were presented in a counterbalanced order to eliminate any effects due to the order of tactile stimulation. Physiological responses were measured continually. 2.3. Tactile Stimulation {#sec2dot3-ijerph-14-00773} ------------------------ White oak (*Quercus alba*) wood was used as the base material. Five laminae without vertical joints (size of each lamina = length 300 × width 60 × thickness 15 mm) were mutually bonded along the width of each wood plate. To prevent bending, a second bonding was performed using Japanese cedar plywood (length 300 × width 300 × thickness 28 mm); the total thickness of the material was 43 mm. The surface of the white oak material was finished by brushing with a stainless-steel wire brush. Additionally, four samples were prepared: (1) uncoated white oak ("uncoated," [Figure 3](#ijerph-14-00773-f003){ref-type="fig"}A); (2) plant oil with an open pore finish extracted from perilla or flax seed applied once as a topcoat to the white oak using a roll coater ("oil finish," [Figure 3](#ijerph-14-00773-f003){ref-type="fig"}B); (3) ordinary temperature glass-coating agent with an open pore finish applied twice as an undercoat and once as a topcoat to the white oak using a brush ("vitreous finish," [Figure 3](#ijerph-14-00773-f003){ref-type="fig"}C); and (4) nitrocellulose lacquer with a semi-open pore finish applied once as an undercoat and two-component polyurethane paint applied once as a topcoat to the white oak using a spray gun ("urethane finish," [Figure 3](#ijerph-14-00773-f003){ref-type="fig"}D). These types of coating were selected as materials representative of those used to coat interior building materials (e.g., floors or walls). Finally, a sample (5) with a strong coating, similar to that used on the surface of a piano, was prepared. The surface of the white oak was sanded with an abrasive-band machine (sandpaper 240 grid; Hitachi Koki Co., Ltd., Tokyo, Japan). This material was coated with nitrocellulose lacquer once as an undercoat using a spray gun. After drying, the surface was polished with an abrasive-band machine (sandpaper 320 grid; Hitachi Koki Co., Ltd., Tokyo, Japan). The above processes (undercoat and polishing treatments) were repeated five times each. Finally, polyurethane resin paint with a closed pore finish was applied once as a topcoat using a spray gun ("mirror finish," [Figure 3](#ijerph-14-00773-f003){ref-type="fig"}E). All materials were kept at room temperature. The physical properties of the five coating materials are shown in [Table 1](#ijerph-14-00773-t001){ref-type="table"}. 2.4. Physiological Measurement {#sec2dot4-ijerph-14-00773} ------------------------------ ### 2.4.1. TRS {#sec2dot4dot1-ijerph-14-00773} As an indicator of brain activity, TRS, which is a near-infrared spectroscopy method, was used. Sensors were mounted on the subject's forehead, and oxy-Hb concentrations in the prefrontal cortex were measured (TRS-20 system; Hamamatsu Photonics K.K., Shizuoka, Japan) \[[@B23-ijerph-14-00773],[@B24-ijerph-14-00773],[@B25-ijerph-14-00773]\]. Oxy-Hb concentrations in the left and right prefrontal cortex were measured before participants touched the materials (pre-measurement) and during the 90 s in which participants touched the materials (post-measurement). Because most data were measured at approximately 1.0--1.2 s, they were transformed by linear interpolation. In addition, all data were calculated as the difference between the averages of the 10-s values before touching. ### 2.4.2. HRV and Heart Rate {#sec2dot4dot2-ijerph-14-00773} HRV and heart rate were used as indicators of autonomic nervous activity. HRV was analyzed during the periods between consecutive R waves (R--R intervals) on electrocardiograms measured with a portable electrocardiograph (Activtracer AC-301A; GMS, Tokyo, Japan) \[[@B26-ijerph-14-00773],[@B27-ijerph-14-00773]\]. The power levels of the low-frequency (LF; 0.04--0.15 Hz) and high-frequency (HF; 0.15--0.40 Hz) components of HRV were calculated using the maximum-entropy method (MemCalc/Win; GMS, Tokyo, Japan). HF power reflected parasympathetic nervous activity, and the LF/HF ratio reflected sympathetic nervous activity \[[@B28-ijerph-14-00773],[@B29-ijerph-14-00773]\]. To normalize HRV parameters across participants, natural logarithmic-transformed values were used in the analysis \[[@B30-ijerph-14-00773]\]. The values of ln(HF), ln(LF/HF), and heart rate represented the changes that occurred in each 30 s and the overall mean during the 90 s in which participants touched the samples. In addition, all data were calculated as the difference between the averages of the 30-s pre-measurement values. 2.5. Psychological Measurement {#sec2dot5-ijerph-14-00773} ------------------------------ The modified semantic differential (SD) method \[[@B31-ijerph-14-00773]\] was used to evaluate the psychological effects of touching the five samples. The SD method tests the subjective evaluations of participants through a questionnaire with opposing adjectives, each of which is evaluated on a 13-point scale. Six pairs of adjectives were assessed: "comfortable--uncomfortable," "natural--artificial," "relaxed--awakening," "warm--cold," "uneven--flat," and "dry--moist." 2.6. Statistical Analysis {#sec2dot6-ijerph-14-00773} ------------------------- SPSS software version 21.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Paired *t*-tests with the Holm correction were used to compare physiological responses (1) before vs. after touching each material (i.e., pre- vs. post-measurement) and (2) among the five samples (uncoated, oil finish, vitreous finish, urethane finish, and mirror finish). Wilcoxon signed-rank tests with the Holm correction were applied to analyze the differences in psychological indices (1) before vs. after touching each material (i.e., pre- vs. post-measurement) and (2) among the five samples (uncoated, oil finish, vitreous finish, urethane finish, and mirror finish). In all cases, the significance level was set at *p* \< 0.05. 3. Results {#sec3-ijerph-14-00773} ========== 3.1. Physiological Effects {#sec3dot1-ijerph-14-00773} -------------------------- ### 3.1.1. TRS {#sec3dot1dot1-ijerph-14-00773} [Figure 4](#ijerph-14-00773-f004){ref-type="fig"} shows the changes in oxy-Hb concentration per second in the left and right prefrontal cortices while touching the five samples. The mean baseline 10-s oxy-Hb concentration in the left prefrontal cortex did not significantly differ among the five samples before touching the stimuli (uncoated: 43.23 ± 0.95 µM (mean ± standard error); oil finish: 43.38 ± 0.92 µM; vitreous finish: 43.53 ± 0.92 µM; urethane finish: 43.36 ± 0.87 µM; and mirror finish: 42.97 ± 0.89 µM; *p* \> 0.05). There was also no significant difference in the baseline 10-s oxy-Hb concentration in the right prefrontal cortex (uncoated: 43.76 ± 1.23 µM; oil finish: 43.13 ± 1.14 µM; vitreous finish: 42.83 ± 1.17 µM; urethane finish: 43.41 ± 1.12 µM; and mirror finish: 43.13 ± 1.13 µM; *p* \> 0.05). Oxy-Hb concentrations in the left and right prefrontal cortices immediately decreased after touching uncoated wood with the palm and remained lower than the pre-measurement value until the end of contact. With mirror finish, oxy-Hb concentrations gradually increased during contact. With oil, vitreous, and urethane finishs, a change was observed between uncoated and mirror finish. [Figure 5](#ijerph-14-00773-f005){ref-type="fig"} shows a comparison of the average differential (post- to pre-measurement) oxy-Hb concentration value in the left and right prefrontal cortices while touching the five samples. Oxy-Hb concentrations in the left prefrontal cortex were as follows: uncoated: −0.20 ± 0.12 µM; oil finish: −0.02 ± 0.08 µM; vitreous finish: −0.11 ± 0.09 µM; urethane finish: 0.12 ± 0.13 µM; and mirror finish: 0.24 ± 0.08 µM ([Figure 5](#ijerph-14-00773-f005){ref-type="fig"}, left). Comparing the pre- and post-measurement values, the oxy-Hb concentration after touching wood with mirror finish was significantly increased compared with the pre-measurement value ([Figure 5](#ijerph-14-00773-f005){ref-type="fig"} left, ^☨^ *p* \< 0.05). Comparing the five samples, touching uncoated, oil-finished, and vitreous-finished wood significantly decreased oxy-Hb concentrations in the left prefrontal cortex compared with touching mirror-finished wood ([Figure 5](#ijerph-14-00773-f005){ref-type="fig"} left, \* *p* \< 0.05). Oxy-Hb concentrations in the right prefrontal cortex were as follows: uncoated: −0.34 ± 0.13 µM; oil finish: −0.03 ± 0.10 µM; vitreous finish: −0.07 ± 0.08 µM; urethane finish: 0.07 ± 0.09 µM; and mirror finish: 0.19 ± 0.13 µM ([Figure 5](#ijerph-14-00773-f005){ref-type="fig"}, right). Comparing the pre- and post-measurement values, the oxy-Hb concentration after touching uncoated wood was significantly decreased compared with the pre-measurement value ([Figure 5](#ijerph-14-00773-f005){ref-type="fig"} right, ^☨^ *p* \< 0.05). Comparing the five samples, touching uncoated wood significantly decreased oxy-Hb concentrations in the left prefrontal cortex compared with touching urethane-finished and mirror-finished wood ([Figure 5](#ijerph-14-00773-f005){ref-type="fig"} right, \* *p* \< 0.05). ### 3.1.2. HRV and Heart Rate {#sec3dot1dot2-ijerph-14-00773} [Figure 6](#ijerph-14-00773-f006){ref-type="fig"}A shows changes in the ln(HF) value, which reflects parasympathetic nervous activity, while touching the five samples. The mean baseline values of ln(HF) at 30 s before touching the stimuli did not significantly differ among the five samples (uncoated: 5.56 ± 0.23 lnms^2^; oil finish: 5.69 ± 0.21 lnms^2^; vitreous finish: 5.67 ± 0.19 lnms^2^; urethane finish: 5.80 ± 0.19 lnms^2^; and mirror finish: 5.65 ± 0.22 lnms^2^; *p* \> 0.05). The ln(HF) value immediately increased after contact with uncoated wood and remained higher than the pre-measurement value or that when touching the four other samples until the end of contact. [Figure 6](#ijerph-14-00773-f006){ref-type="fig"}B shows the average differential (post- to pre-measurement) ln(HF) value while touching the five samples. The ln(HF) values were as follows: uncoated: 0.49 ± 0.14 lnms^2^; oil finish: 0.25 ± 0.09 lnms^2^; vitreous finish: 0.02 ± 0.14 lnms^2^; urethane finish: 0.08 ± 0.08 lnms^2^; and mirror finish: 0.09 ± 0.17 lnms^2^. Comparing the pre- and post-measurement values, the ln(HF) values after touching uncoated and oil-finished wood were significantly increased compared with the pre-measurement values (^☨^ *p* \< 0.05). Comparing the five samples, touching uncoated wood significantly increased the ln(HF) value compared with touching vitreous-finished, urethane-finished and mirror-finished wood (\* *p* \< 0.05). However, there was no significant difference in ln(LF/HF), which is an index of sympathetic nervous activity, among the five samples (uncoated: −0.69 ± 0.31; oil finish: 0.15 ± 0.23; vitreous finish: −0.05 ± 0.18; urethane finish: −0.40 ± 0.20; and mirror finish: 0.14 ± 0.25; *p* \> 0.05). [Figure 7](#ijerph-14-00773-f007){ref-type="fig"}A shows the changes in heart rate while participants touched the five samples. The mean baseline heart rate 30 s before touching the stimuli did not significantly differ among the five samples (uncoated: 72.81 ± 2.30 beats/min; oil finish: 72.12 ± 2.28 beats/min; vitreous finish: 73.01 ± 2.05 beats/min; urethane finish: 72.07 ± 2.06 beats/min; and mirror finish: 71.72 ± 2.11 beats/min; *p* \> 0.05). Heart rate immediately increased after contact with mirror-finished wood and remained higher than the pre-measurement value and that of the other four samples until the end of contact. However, in uncoated, oil-finished, vitreous-finished, and urethane-finished wood, heart rate decreased after contact and remained lower than the pre-measurement values until the end of contact. [Figure 7](#ijerph-14-00773-f007){ref-type="fig"}B shows the average differential (post- to pre-measurement) heart rate value while touching the five samples. Heart rates were as follows: uncoated: −1.62 ± 0.60 beats/min; oil finish: −1.54 ± 0.46 beats/min; vitreous finish: −1.48 ± 0.49 beats/min; urethane finish: −1.31 ± 0.74 beats/min; and mirror finish: 0.77 ± 0.87 beats/min. Comparing the pre- and post-measurement values, heart rates after touching uncoated, oil-finished and vitreous-finished wood were significantly decreased compared with pre-measurement values (^☨^ *p* \< 0.05). Comparing the five samples, touching uncoated, oil-finished and vitreous-finished wood significantly decreased heart rate compared with touching mirror-finished wood (\* *p* \< 0.05). 3.2. Psychological Effects {#sec3dot2-ijerph-14-00773} -------------------------- The results of subjective evaluation by the modified SD method are shown in [Figure 8](#ijerph-14-00773-f008){ref-type="fig"}. Reports of "comfortable" ([Figure 8](#ijerph-14-00773-f008){ref-type="fig"}A) and "relaxed" ([Figure 8](#ijerph-14-00773-f008){ref-type="fig"}B) feelings did not significantly differ among the five samples (*p* \> 0.05). With regard to the "natural" feeling, uncoated and oil-finished wood, which were perceived as "indifferent to slightly natural," were considered significantly more "natural" than mirror-finished wood, which was perceived as "slightly to moderately artificial" (*p* \< 0.05, [Figure 8](#ijerph-14-00773-f008){ref-type="fig"}C). In addition, reports of the "natural" feeling were significantly lower after contact with mirror-finished wood compared with vitreous-finished and urethane-finished wood (*p* \< 0.05, [Figure 8](#ijerph-14-00773-f008){ref-type="fig"}C). With regard to the "warm--cold" feeling, participants reported feeling "indifferent to slightly cold" after contact with uncoated and oil-finished wood, and reported feeling "slightly to moderately cold" after touching mirror-finished wood, representing a significant difference (*p* \< 0.05, [Figure 8](#ijerph-14-00773-f008){ref-type="fig"}D). With regard to "uneven--flat" feeling, uncoated, oil-finished, and vitreous-finished wood, which were perceived as "slightly uneven," and urethane-finished wood, which was perceived as "indifferent to slightly uneven," were considered significantly more uneven than mirror-finished wood, which was perceived as "slightly to moderately flat" (*p* \< 0.05, [Figure 8](#ijerph-14-00773-f008){ref-type="fig"}E). A significant difference was observed between uncoated and urethane-finished wood on the "uneven--flat" feeling (*p* \< 0.05, [Figure 8](#ijerph-14-00773-f008){ref-type="fig"}E). Finally, with regard to the "dry--moist" feeling, uncoated and oil-finished wood, which were perceived as "indifferent to slightly dry," were considered significantly drier than mirror-finished wood, which was perceived as "indifferent to slightly moist" (*p* \< 0.05, [Figure 8](#ijerph-14-00773-f008){ref-type="fig"}F). 4. Discussion {#sec4-ijerph-14-00773} ============= This study aimed to clarify the effects of touching wood with various coatings with the palm on left and right prefrontal cortex activity, assessed using TRS, and on autonomic nervous activity, assessed using HRV. The results were as follows: (1) Tactile stimulation with uncoated wood calmed prefrontal cortex activity (vs. urethane-finished and mirror-finished wood), increased parasympathetic nervous activity (vs. vitreous-finished, urethane-finished, and mirror-finished wood), and decreased heart rate (vs. mirror-finished wood). These results demonstrate a physiological relaxation effect of wood on humans. Further; (2) tactile stimulation with oil- and vitreous-finished wood also calmed left prefrontal cortex activity and decreased heart rate relative to mirror-finished wood; (3) In terms of subjective evaluations, we observed differences among the various coated materials in terms of "natural feeling," "warm-cold feeling," "uneven-flat feeling," and "dry-moist feeling," and these psychological findings were shown to be consistent with physiological responses. However, for "comfortable" and "relaxed" feelings, which have great value in the subjective evaluation, there was no statistical difference among various types of coated wood. These results clarified the utility of physiologically evaluating the effects of wood on humans. In this study, touching uncoated, oil-finished, and vitreous-finished wood significantly decreased oxy-Hb concentrations in the left prefrontal cortex compared with touching mirror-finished wood. However, in the right prefrontal cortex, touching uncoated wood significantly decreased oxy-Hb concentrations compared with touching urethane-finished and mirror-finished wood. A difference in left and right prefrontal cortex activity was found. Several previous studies have examined the effect of nature-derived stimuli on prefrontal cortex activity. In these studies, which examined olfactory stimulation with rose, orange \[[@B32-ijerph-14-00773]\], and Japanese cypress leaf \[[@B33-ijerph-14-00773]\] oils, it was observed that oxy-Hb concentration decreased significantly in the right prefrontal cortex, but there was no significant difference in the left prefrontal cortex. Regarding visual stimulation with real foliage plants \[[@B34-ijerph-14-00773]\] and 3D images of the water lily \[[@B35-ijerph-14-00773]\], it was also observed that oxy-Hb concentration decreased significantly in the right prefrontal cortex, but there was no change in left prefrontal cortex activity. In contrast, regarding tactile stimulation, it has been shown that touching uncoated Japanese cypress wood significantly decreased oxy-Hb concentrations in the left prefrontal cortex, whereas there was no difference in the right prefrontal cortex \[[@B36-ijerph-14-00773]\]. In the current study, there was a significantly greater influence on the left than the right prefrontal cortex; this finding is partially consistent with the results of previous studies on tactile stimulation with wood \[[@B36-ijerph-14-00773]\]. These results suggested that tactile stimulation greatly influenced the left prefrontal cortex. However, the underlying mechanism remains unknown. In the future, it will be necessary to accumulate further data and to clarify the difference in left vs. right prefrontal cortex activity. Regarding the physiological effects of tactile stimulation of wood with the palm, Ikei et al. \[[@B18-ijerph-14-00773]\] examined the effects of tactile stimulation with uncoated white oak wood on brain activity and autonomic nervous activity in comparison with other materials (marble, tile and stainless steel). Participants touched the surface of each material for 90 s while sitting with their eyes closed. The results indicated that tactile stimulation with white oak significantly (1) decreased oxy-Hb concentration in the left and right prefrontal cortices relative to marble, tile, and stainless steel and (2) increased ln(HF)-reflected parasympathetic nervous activity relative to marble and stainless steel, thereby inducing physiological relaxation \[[@B18-ijerph-14-00773]\]. In addition, Sakuragawa et al. \[[@B17-ijerph-14-00773]\] reported differences in the effects of tactile stimulation with several materials, such as wood, on blood pressure. Participants touched the surface of each material for 60 s with their eyes closed. The results indicated the following: (1) Blood pressure increased transiently just after touching oak, Japanese cypress, and Japanese cedar; however, the change did not persist; (2) Blood pressure was high even after the transient increased while touching artificial materials (aluminum or acrylic plastic). Several reports have been prepared on the physiological effects caused by olfactory stimulation with wood, which are consistent with the findings of our research. Olfactory stimulation with air-dried Japanese cypress wood calmed prefrontal cortex activity compared with high-temperature-dried wood \[[@B9-ijerph-14-00773]\]. The effects of olfactory stimulation with Japanese cypress leaf oil on brain activity and autonomic nervous activity have also been investigated \[[@B33-ijerph-14-00773]\]. Olfactory stimulation with Japanese cypress leaf oil calmed prefrontal cortex activity and increased parasympathetic nervous activity compared with the control condition (air), indicating that olfactory stimulation with Japanese cypress leaf oil can induce physiological relaxation \[[@B33-ijerph-14-00773]\]. Inhalation of α-pinene \[[@B10-ijerph-14-00773]\] and D-limonene \[[@B11-ijerph-14-00773]\], which are major odor components contained in Japanese cedar and Japanese cypress, increased parasympathetic nervous activity and decreased heart rate compared with control condition (air), indicating physiological relaxation. Our findings on tactile stimulation with uncoated wood are therefore consistent with those of previous studies on tactile and olfactory stimulation by wood or wood-derived materials, demonstrating a physiological relaxation effect of touching natural wood with the palm of the hand. In the current study, tactile stimulation by uncoated, natural wood and also that by oil- and vitreous-finished wood calmed left prefrontal cortex activity and decreased heart rate relative to mirror-finished wood. This finding can be partially attributed to the differing properties of the coatings tested \[[@B37-ijerph-14-00773],[@B38-ijerph-14-00773]\]. In terms of physical properties (e.g., surface roughness and heat flow rate), oil- and vitreous-finished wood were similar to uncoated wood. Oil and vitreous finish retain the texture of natural (i.e., uncoated) wood, and it is suggested that this is why tactile stimulation by oil- or vitreous-finished wood induced physiological relaxation. We believe that tactile stimulation via touching uncoated wood with the palm brought about physiological relaxation effects because physiological functions of modern day humans are best adapted to natural environments or materials. Living in highly urbanized and artificial environments therefore places modern day humans in a state of stress. Accordingly, we enter a relaxed state when exposed to nature-derived stimulation such as wood, which brings us closer to our original natural state. This concept is known as the "back-to-nature" theory \[[@B2-ijerph-14-00773],[@B39-ijerph-14-00773]\]. In this study, we investigated the effect of contact with coated wood, which is generally used in the built environment, on the human body. Results indicated that touching natural wood or near-natural coated wood with the palm of the hand induced physiological relaxation. We conclude that our finding can serve as a scientific basis for the use of wood, assuming the actual building environment. However, for "comfortable" and "relaxed" feelings, there was no difference among the five samples. This suggests the progress and utility of physiological measurements in biological impact assessment. In sum, our results revealed a physiological relaxation effect of tactile contact with natural wood (uncoated) or near-natural wood (oil and vitreous finish). However, the study has certain limitations. First, although we clarified the effect of touching wood with the palm of the hand, we did not examine the effect of contact with other body surfaces. Future research should examine the effect of wood on physiological response when touched with the sole of the foot because wood is often used as a flooring material. Second, this study only measured the physiological effects with wood by placing the palm of the hand on the stimulus. It is necessary to clarify the impact of more active forms of contact on the physiological response, such as stroking the surface of the wood with the hand. Finally, the study participants were all female university students in their twenties. Studies on other samples, such as males, children, and the elderly, are therefore required. 5. Conclusions {#sec5-ijerph-14-00773} ============== The following conclusions were drawn: (1) Tactile stimulation with uncoated wood calmed prefrontal cortex activity (vs. urethane-finished and mirror-finished wood), increased parasympathetic nervous activity (vs. vitreous-finished, urethane-finished, and mirror-finished wood), and decreased heart rate (vs. mirror-finished wood), demonstrating a physiological relaxation effect of wood on humans; (2) Tactile stimulation with oil- and vitreous-finished wood calmed left prefrontal cortex activity and decreased heart rate relative to mirror-finished wood; However, (3) for the "comfortable" and "relaxed" feelings, there was no difference among the five samples. This work was supported by MARUHON, Inc. and JSPS KAKENHI Grant Number JP16K18200. Harumi Ikei contributed to data acquisition, statistical analysis, and interpretation of the results. Chorong Song contributed to data acquisition, statistical analysis, and interpretation of the results. Yoshifumi Miyazaki played an important role in this research, particularly in the study design and interpretation of the results. All authors contributed to manuscript preparation and are responsible for the final editing and approval of the manuscript. The authors declare no conflict of interest. {#ijerph-14-00773-f001} {#ijerph-14-00773-f002} {#ijerph-14-00773-f003} {#ijerph-14-00773-f004} {#ijerph-14-00773-f005} {#ijerph-14-00773-f006} {#ijerph-14-00773-f007} {#ijerph-14-00773-f008} ijerph-14-00773-t001_Table 1 ###### Details of materials. *h*: thickness of materials; *λ*: thermal conductivity; *Ra*: arithmetic average roughness; *JCP*: Japanese cedar plywood. Coating Type *h* (mm) λ (W/(m-K)) ^1^ *Ra* (µm) ^2^ Application Rate (g/m^2^) ^3^ ----------------- --------------- ----------------- --------------- ------------------------------- Uncoated 15 (+ JCP 28) 0.120 57.10 -- Oil finish 15 (+ JCP 28) 0.128 56.80 30 Vitreous finish 15 (+ JCP 28) 0.122 57.72 100 Urethane finish 15 (+ JCP 28) 0.119 36.75 644 Mirror finish 15 (+ JCP 28) 0.119 0.12 1400 ^1^ A heat flow meter (HFM 436 Lambda; NETZSCH, Selb, Germany), tuned according to ASTM C518-10 \[[@B21-ijerph-14-00773]\] and ISO8310 \[[@B22-ijerph-14-00773]\], was used. The direction of heat flow was vertically downward. The temperatures of the high- and low-temperature heat plates were 35 °C and 15 °C, respectively. The thermal conductivity at an average material temperature of 25 °C was calculated. Test specimens were used with the cedar plywood attached; ^2^ A contact-surface roughness profilometer (SE3500; Kosaka Laboratory Ltd., Tokyo, Japan) with a diamond needle was used. The evaluation length was 50 mm. The central portion of the samples was measured five times with 50 mm spacing, and the average value was calculated; ^3^ This refers to the attachment mass of the paint per unit area of the surface to be painted. [^1]: These authors contributed equally to this work.

A significant fraction of the organic carbon produced in the surface sunlit ocean through photosynthesis is exported into the deep ocean as sinking particles ([@r1], [@r2]), supporting a globally relevant carbon flux fueling the generally starved bathypelagic microbial communities ([@r3], [@r4]). Although the carbon reaching the dark ocean is ultimately controlled by the activity of the prokaryotic communities that attach to these particles ([@r5], [@r6]), so far no study has investigated whether sinking particles may also constitute dispersion vectors of viable prokaryotic diversity into the deep ocean. Particles formed in surface waters are rapidly colonized by prokaryotes, and particle-attached communities are often more metabolically active ([@r7][@r8]--[@r9]) and phylogenetically diverse (e.g., refs. [@r10][@r11]--[@r12]) than suspended (free-living) assemblages. Although it has been shown that particle-attached prokaryotic communities change compositionally with depth (e.g., refs. [@r13][@r14]--[@r15]), it is not clear whether these changes are due to an ecological succession of taxa on the degrading particle ([@r16]) or continuous colonization of the particles during sinking. Assemblages attached to sinking particles could influence the structure of deeper prokaryotic communities if dormant or slow-growing surface taxa thrive when reaching a certain depth or as the nature of the particle changes. In this case, sinking particles would provide a continuous supply of viable immigrants to the deep ocean. Assessing the relevance of such dispersal process would require comparing particle-attached communities between surface and bathypelagic waters, yet this has never been attempted, not even at local scales. Indeed, the vertical connectivity between communities from surface to bathypelagic waters remains poorly understood because spatial surveys focusing in the vertical dimension often describe the communities from each depth without assessing their potential linkages throughout the water column (e.g., refs. [@r17][@r18]--[@r19]). Only a few recent studies have shown that communities from surface and deep waters may be connected through advection or sinking of water masses ([@r20][@r21]--[@r22]), but they did not consider the role of sinking particles as a vector supporting vertical connectivity. In addition, because large particles sink faster than small particles---which might even remain buoyant ([@r23][@r24]--[@r25])---communities associated with larger, fast-sinking particles are likely to have greater chances to reach the deepest layers due to higher dispersal rates. To date, however, most literature on particle-attached prokaryotes has been restricted to the dichotomous exploration of free-living versus attached populations (e.g., refs. [@r26][@r27]--[@r28]), yet it is known that particles occur along a continuum of sizes that can be colonized by different microbial populations ([@r29]). Furthermore, this continuum of sizes is strongly related to the composition of the particle, which also indicates the particle origin: larger particles are younger and more labile and are originated in the surface ([@r30]), whereas smaller particles tend to be older and more recalcitrant ([@r31]), originating from the degradation of larger particles ([@r32]). Given that the origin and composition of particles in surface waters vary spatially ([@r25], [@r33][@r34]--[@r35]), it could be hypothesized that the microbial diversity patterns in the deep ocean mirror to some extent such surface heterogeneity. In fact, it was recently shown that deep-sea particle-attached communities present a much clearer biogeography than their free-living counterparts ([@r36]), but whether these patterns are established locally in the deep sea or transferred from surface via sinking particles remains unexplored. Although the importance of hydrologically mediated microbial dispersal for shaping local assemblages has been clearly demonstrated in freshwater ecosystems ([@r37][@r38]--[@r39]) and more recently also in oceanic waters through movement of water masses ([@r20][@r21]--[@r22], [@r40]), the role of particle-driven vertical dispersion in explaining the biogeographic patterns of deep-sea prokaryotes has not yet been assessed. Here we explore whether sinking particles represent a dispersal vector of prokaryotes into the deep ocean, contributing to the vertical connectivity of the marine microbiome, and test the hypothesis that particle size influences vertical connectivity. We investigated the composition of free-living prokaryotic communities as well as that of those attached to particles of different sizes (ranging from 0.8 to 200 µm) in eight stations across the global tropical and subtropical ocean, assessing changes in their composition from surface (3 m) to bathypelagic waters (4,000 m). Specifically, we test the hypothesis that communities attached to the largest particles show a strong vertical similarity due to their assumed faster sinking rates (i.e., higher dispersal) and that free-living prokaryotic assemblages are more different across the water column than their vertically connected particle-attached counterparts. Moreover, if part of the sinking diversity were to detach from particles when reaching the bathypelagic layer, thus becoming part of the free-living community, a percentage of the prokaryotes attached to particles in surface waters should also be present as free-living in bathypelagic communities. Finally, we test the hypothesis that if vertical connectivity is a globally relevant phenomenon, then deep ocean biogeographic patterns should resemble those of the surface particle-attached communities. Results {#s1} ======= Taxonomic Composition of Prokaryotic Assemblages. {#s2} ------------------------------------------------- We studied stations spanning a broad longitudinal gradient across the tropical and subtropical Pacific, Atlantic, and Indian Oceans ([*SI Appendix*, Fig. S1*A*](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). The depths sampled within each station presented pronounced vertical physicochemical and biological variation ([*SI Appendix*, Fig. S1*B*](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). Accordingly, the studied prokaryotic communities were clearly structured along the water column, differing mostly between the photic versus the aphotic realms, and presented also distinct biogeographic signatures ([Fig. 1 *B* and *C*](#fig01){ref-type="fig"} and [*SI Appendix*, Table S1](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). Whereas communities from the largest particles (≥3.0 µm) clustered together regardless of depth, communities from the two smallest fractions differed greatly between surface and deeper layers ([Fig. 1 *A* and *B*](#fig01){ref-type="fig"}). Consequently, vertical community differences were higher for the smallest size fraction (PERMANOVA *R*^2^ = 0.37, *P* \< 0.001) than for the largest size fractions (*R*^2^ = 0.09, *P* = 0.597) ([*SI Appendix*, Table S2](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)), whereas horizontal differences, i.e., between stations, were higher in the largest size fractions (*R*^2^ = 0.54, *P* \< 0.001) than in the smallest size fraction (*R*^2^ = 0.21, *P* \< 0.753) ([*SI Appendix*, Table S2](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). As indicated by the RC~Bray~ (Raup-Crick metric based on Bray Curtis distances), the observed beta diversity patterns differed significantly from those expected by chance (i.e., ecological drift) in ∼92% of cases \[following Stegen et al. ([@r41])\], and this suggests that other processes (like selection and limited or high dispersal) rather than random community assembly (drift) generated the observed beta diversity patterns. {#fig01} Operational Taxonomic Units (OTUs) richness was highly variable among size fractions and depths (range 136--1,044 OTUs per sample, average 580), but in general, richness decreased toward larger size fractions in all depths ([*SI Appendix*, Fig. S2](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). In terms of taxonomic composition at the phylum or class level, the three fractions larger than 3.0 µm were in general more similar among each other than they were to the smallest size fractions ([*SI Appendix*, Fig. S3](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). The distribution of Particle-Association Niche Index (PAN-Index) values, used to identify preferences of OTUs for particular size fractions, showed two modes around values 1.5 and 3.5 ([*SI Appendix*, Fig. S4](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). We, therefore, differentiated OTUs enriched in small size fractions (PAN-Index \< 2.7) from those enriched in large size fractions (PAN-Index ≥ 2.7). We found that the preference for one lifestyle or the other seemed to be phylogenetically conserved at the order level (but in some cases not at the class nor at the phylum level). Whereas some orders such as SAR11, SAR324, or Acidimicrobiales showed preference for small size fractions, other orders like Rhizobiales, Alteromonadales, or Planctomycetales were preferentially enriched in large size fractions (PAN-Indexes ≥ 2.7; [*SI Appendix*, Fig. S5](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). Vertical Connectivity Between Oceanic Prokaryotic Communities. {#s3} -------------------------------------------------------------- To determine the vertical connectivity between prokaryotic communities, we explored whether OTUs present at one depth could be detected in the other depths. To do so, all OTUs were categorized into four depth groups: surface (SFC), deep-chlorophyll maximum (DCM), mesopelagic (MESO), and bathypelagic (BATHY), defined by the depth where they were first detected, assuming a directionality from surface to bathypelagic waters and considering all stations together. For example, if an OTU was detected in any of the surface samples, it was categorized as SFC, but if an OTU was first detected in mesopelagic waters but not in the previous depths (surface and DCM), it was categorized as MESO, and so on. For this categorization, the nonrarefied OTU table was used, so that we could detect the largest number of OTUs per sample. This analysis showed that even though new OTUs appeared continuously when moving from one depth to the next one ([Fig. 2](#fig02){ref-type="fig"}), communities from all depths and size fractions were largely dominated by OTUs present in surface waters (SFC OTUs). When this categorization of OTUs was done considering each station separately, we observed a similar pattern, but in some stations, there was a larger contribution of OTUs not present in surface waters in deep layers, particularly in the free-living fraction ([*SI Appendix*, Fig. S6](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). This indicates that some of the DCM, MESO, or BATHY OTUs in some stations were not present at the surface of these particular stations but were present in surface waters at other sites. In any case, bathypelagic communities of all stations were still numerically dominated by surface sequences, pointing to a high vertical connectivity of the open ocean microbial communities. This implies that community changes across depths ([Fig. 1*B*](#fig01){ref-type="fig"}) are to a large extent due to shifts in the relative abundances of taxa present throughout the water column (e.g., rare surface taxa that become abundant in deeper layers). {ref-type="sec"} for details).](pnas.1802470115fig02){#fig02} The vertical differentiation among communities of a given size fraction varied gradually from the free-living prokaryotic communities toward those in the largest particles ([Fig. 3](#fig03){ref-type="fig"}), and differences from the small to the largest size fraction were statistically significant (Wilcoxon test, *P* value \< 0.001). We found the highest beta diversity and OTU turnover among depths in the 0.2--0.8 µm fraction ([Fig. 3 *A* and *B*](#fig03){ref-type="fig"}), indicating a higher replacement of OTUs within the free-living communities across depths compared with communities attached to the largest particles. Conversely, communities from the largest size fraction showed higher nestedness ([Fig. 3*C*](#fig03){ref-type="fig"}), and OTUs were present across more depths ([Fig. 3*D*](#fig03){ref-type="fig"}) compared with those in smaller size fractions, suggesting that communities attached to larger particles are more connected throughout the water column than those free-living or attached to small particles. We then divided the SFC OTUs (i.e., OTUs detected in any of the surface stations; see above) into those enriched in small size fractions (PAN-Index \< 2.7) and those enriched in large size fractions (PAN-Index ≥ 2.7) and compared their distribution along the water column in small (and suspended, i.e., \<3.0 µm) and large (and sinking, i.e., ≥3.0 µm) size fractions ([Fig. 4](#fig04){ref-type="fig"}). We found that surface OTUs present in the smallest surface size fractions (SFC OTUs with PAN-Index \< 2.7) accounted for a decreasing proportion of the communities toward deeper waters in the small suspended size fractions ([Fig. 4*A*](#fig04){ref-type="fig"}) and composed around 25% of abundance in the largest size fractions along the water column ([Fig. 4*B*](#fig04){ref-type="fig"}). Conversely, most bathypelagic communities were composed by OTUs present in the surface in association to large particles (SFC OTUs with PAN-Index ≥ 2.7), which composed about 80% of bathypelagic sequences in the largest size fractions ([Fig. 4*B*](#fig04){ref-type="fig"}) and more than 50% of bathypelagic sequences in the small suspended size fractions ([Fig. 4*A*](#fig04){ref-type="fig"}). This suggests that deep-sea communities are largely populated by surface microbes arriving via the largest particles and that this dispersal influences mostly the bathypelagic particle-attached communities ([Fig. 4*B*](#fig04){ref-type="fig"}) but also the deep-sea free-living assemblages ([Fig. 4*A*](#fig04){ref-type="fig"}). The colonization of the bathypelagic free-living communities by sinking particles is also supported by the observation that free-living communities from the bathypelagic are more similar to attached communities from the bathypelagic than to any other communities ([Fig. 1](#fig01){ref-type="fig"}). On the contrary, surface free-living communities contribute much less to the bathypelagic diversity likely due to their more limited vertical dispersal. {ref-type="sec"} for more details.](pnas.1802470115fig03){#fig03} {ref-type="sec"} for further details.](pnas.1802470115fig04){#fig04} Transfer of Biogeographic Patterns from Surface to the Deep Sea. {#s4} ---------------------------------------------------------------- Given the higher vertical transport of the microbes associated to larger particles, we would expect that spatial differences (i.e., differences between stations) among communities from the largest particles are maintained vertically, whereas suspended communities are expected to be more vertically isolated, and thus, their surface biogeographic patterns are not expected to be transferred across depths. We tested this inference by comparing, for each size fraction, spatial differences between surface communities and mesopelagic or bathypelagic communities using Mantel tests ([Table 1](#t01){ref-type="table"}). We found that the dissimilarities between suspended communities from the surface and the mesopelagic or bathypelagic waters were not significantly correlated. In contrast, the dissimilarities between particle-attached communities from the surface and the deep waters presented a high significant correlation ([Table 1](#t01){ref-type="table"}), suggesting that the compositional differences among stations of deep-sea particle-attached communities were caused at least partially by the biogeographic patterns of surface particle-attached assemblages. ###### Comparisons of community structure between depths Surface vs. mesopelagic Surface vs. bathypelagic ------------- ------------------------- -------------------------- ------- ------- 0.2--0.8 µm −0.18 0.752 −0.18 0.829 0.8--3.0 µm −0.14 0.675 0.29 0.11 3.0--5.0 µm 0.58 \*\* 0.73 \*\* 5.0--20 µm 0.82 \*\* 0.73 \*\* 20--200 µm 0.87 \*\*\* 0.64 \*\* R coefficients of the Mantel correlations between the taxonomic dissimilarity matrices from surface and mesopelagic communities and surface and bathypelagic communities, for each of the five size fractions. Higher R values mean that the compositional differences between communities at a given depth were highly correlated (and thus were similar) to the differences between communities from a different depth. Significance of the correlations is stated as follows: \*\*\**P* \< 0.001, \*\**P* \< 0.01, and \**P* \< 0.05. The results presented support the hypothesis that the biogeography of surface particle-attached prokaryotes is transferred to deeper waters via particle sinking, but the fact that communities from the photic and aphotic realms were very different in terms of taxonomic composition ([*SI Appendix*, Fig. S3](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)) suggests that this vertical dispersal of particle-attached microbes must be accompanied by large changes in their abundances during sinking. For example, the growth of taxa that were rare (and perhaps dormant) in surface waters but can thrive in deeper depths could explain such compositional shifts. We identified at each station the OTUs that potentially grew during particle sinking by choosing the surface OTUs prevalent in the larger size fractions (SFC OTUs with PAN-Index ≥ 2.7) that increased in relative abundance (dominance) toward deeper waters, named here as "seed" OTUs (see details in [*Materials and Methods*](#s6){ref-type="sec"}). We identified 90 seed OTUs in total, which contributed to ∼6% of sequences at the surface, and showed clear increases in relative abundance toward deeper waters with up to 55% (average ∼35%) of the total abundance in bathypelagic particle-attached communities ([Fig. 5](#fig05){ref-type="fig"}). The pool of taxa behaving as seeds differed between stations, and different dominant seed groups were found in each station (e.g., *Oceanospirillales* dominate at station 20, *Sphingobacteriales* at station 77, and *Corynebacteriales* at station 94) ([Fig. 5](#fig05){ref-type="fig"}), Moreover, when we inspected the community structure of only these seed OTUs, we observed that they clustered according to the eight stations ([*SI Appendix*, Fig. S7](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). This geographic signature was less clear for the smallest size fractions (smaller symbols) in some cases, but still it points to the high relevance of the surface particle colonization processes in determining the structure of communities from deeper layers. {ref-type="sec"} for details). Pie charts indicate the taxonomic composition at the order level (in percentage of sequences) of the seed OTUs at each station. Note the different scales in the graphs.](pnas.1802470115fig05){#fig05} Discussion {#s5} ========== Our results support the hypothesis of strong particle-mediated prokaryotic connectivity between the surface and the deep ocean, a hypothesis suggested earlier (e.g., refs. [@r42] and [@r43]) and tested here. Most of the dominant prokaryotes from the deep-ocean can also be detected in surface waters, and this vertical connectivity is higher in communities associated with the larger size fractions (i.e., larger particles), likely due to their higher sinking rates. Our results demonstrate that particle sinking constitutes a dispersal vector of viable microbial diversity from surface waters to the bathypelagic zone that ultimately determines the biogeography of deep-sea prokaryotes. They also suggest that sinking particles may indeed be a relevant seed bank of viable taxa for the deep ocean, in line with previous studies suggesting the existence of an oceanic reservoir of dormant diversity ([@r44][@r45]--[@r46]). Although the vertical differentiation of communities from photic to aphotic realms is well documented (e.g., refs. [@r47][@r48]--[@r49]), we show here that this difference varies with particle size, being greatest among free-living assemblages and prokaryotic communities associated to the smallest size fractions ([Fig. 1](#fig01){ref-type="fig"} and [*SI Appendix*, Fig. S3 and Table S1](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). Our results also concur with previous studies indicating that prokaryotic communities strongly differ between the free-living (suspended) and attached fractions in epipelagic ([@r29], [@r43], [@r50]) and also in bathypelagic ([@r10], [@r51], [@r52]) waters, supporting that niches present on particles are distinct from the dissolved phase. Others have evidenced that the composition of the larger particles is different from that of the smaller particles (and also from the dissolved phase), with larger particles being younger and more labile and the smaller particles being older and more recalcitrant ([@r31], [@r32]). The finding that all communities, including those inhabiting the bathypelagic layer, were numerically dominated by OTUs present in surface waters supports a strong vertical connection between surface and bathypelagic communities at the local ([*SI Appendix*, Fig. S6](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)) and the global ([Fig. 2](#fig02){ref-type="fig"}) scales. Given the variability across depths in the structure of the communities ([Fig. 1](#fig01){ref-type="fig"} and [*SI Appendix*, Fig. S3](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)), the vertical changes in prokaryotic assemblages are likely driven by changes in the relative abundance of taxa present through several depths during sinking, i.e., rare surface taxa that become abundant in deeper waters. Only a few recent studies have assessed the vertical connectivity of marine prokaryotic communities along the water column, indicating that advection and convection of water masses can shape the structure of surface and deep microbial communities by promoting their transport and new habitat colonization ([@r20][@r21]--[@r22]). However, these studies have focused only on the free-living assemblages and vertical water mass transport processes as the driving mechanism. Here we provide evidence that the export of sinking particles represents an important dispersal pathway of diversity from surface to bathypelagic waters. Although this process could be considered as unidirectional from surface to deep waters, supported by evidence of rapid-sinking particles from surface to the deep ocean along the stations sampled by the Malaspina expedition ([@r53]), it is also possible that upwelling events of deep-water masses transport viable deep-sea prokaryotes back into surface waters. This possibility should be tested in future research in upwelling zones. The hypothesis that vertical prokaryotic connectivity is due to the dispersion of taxa from surface to deeper waters driven by sinking particles is further supported by the observation that the connectivity throughout the vertical column is higher for communities associated to the largest particles than for those attached to the smaller ones, as expected from the faster sinking of larger particles (e.g., sinking rates of \<1 m⋅d^−1^ for small particles and \>1,000 m⋅d^−1^ for large aggregates, reviewed in ref. [@r3]). For example, the community composition of the smaller size fractions was more variable between depths (i.e., higher beta diversity) than those of the largest size fractions ([Fig. 3*A*](#fig03){ref-type="fig"} and [*SI Appendix*, Table S2](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). Also, the highest OTU replacement (turnover) across depths was found between communities from the smallest particles, and OTUs from communities associated with largest particles were the most ubiquitous across depths ([Fig. 3*C*](#fig03){ref-type="fig"}). Altogether, this points to a more intense vertical dispersal of taxa between communities from the largest size fractions, whereas prokaryotic communities from the smallest size fractions present more restricted depth distributions, i.e., are more isolated vertically, due to their more limited connectivity. In addition, we observed that the OTUs with preference for large particles in surface waters also dominate the mesopelagic and bathypelagic communities ([Fig. 4](#fig04){ref-type="fig"}). The fact that the deep communities in the small size fractions were also composed to a large extent of surface particle-attached prokaryotes ([Fig. 4*A*](#fig04){ref-type="fig"}), and that free-living communities from the bathypelagic layer are more similar to attached communities from the bathypelagic layer than to any others ([Fig. 1](#fig01){ref-type="fig"}), suggests that large particles are indeed vectors transporting viable prokaryotes from the surface to the bathypelagic, some of which can thrive in the free-living fraction of the bathypelagic realm. Thus, the transport of prokaryotes from surface to deep waters occurs mostly via large particles, which act as a source of potential immigrants (or inoculum) to the suspended community living in the deep ocean. These results agree with a previous study indicating that particular taxa can change their preference from large particles to small particles through depth ([@r51]), probably responding to environmental conditions. Particles have a highly heterogeneous organic and inorganic composition that is altered during sinking but that is mainly determined by the environmental conditions of the surface waters where they formed ([@r25], [@r34], [@r35]). Such particle composition determines the colonization and the structure of the initial microbial community (reviewed in ref. [@r54]). It follows from this that the pool of particle-dispersed taxa that sinks and has the potential to thrive when arriving to deeper layers will depend on local conditions and particle origin in surface waters and should differ across stations. We tested this hypothesis by identifying the pool of surface particle-attached prokaryotes that increased their relative abundances toward deeper waters, those acting as seed OTUs. These OTUs belonged to different taxonomic groups in the different stations examined, likely indicating the effect of the initial surface particle colonizers in determining deep ocean microbial communities locally. This implies that sinking particles transport diverse communities, yet some of these taxa \[probably dormant or slowly growing ([@r55])\] have the potential to grow and dominate deep-ocean communities when the surrounding environmental conditions or the nature of the particles change as they are transported toward deeper waters. Additionally, other taxa can decrease their relative abundance with depth as they find unfavorable conditions when moving from the surface to the deep ocean ([@r51]). A similar process has also been observed in other ecosystems, such as the river-to-lake freshwater continuum, where the transport and growth of rare bacteria from terrestrial source environments was shown to strongly determine the structure of the receiving aquatic communities ([@r39]). These results concur in highlighting the need to take potential dispersal sources into account to understand observed biogeographic patterns. Focusing on free-living prokaryotes, Wilkins et al. ([@r20]) suggested that advection of seawater masses can shape microbial community structure by increasing opportunities for colonization. However, given the limited deep vertical water transport in the open ocean, sinking particles are likely to play a key role in determining and shaping the vertical connectivity of oceanic microbial communities across much of the global ocean by allowing continuous dispersal of viable organisms into the mesopelagic and bathypelagic realms. Despite a general perception of a homogeneous dark ocean, genomic approaches have unveiled the enormous and dynamic genetic variability of the deep sea microbial communities (reviewed in ref. [@r3]). Indeed, a recent global survey of prokaryotic communities in the bathypelagic realm showed that they differed between oceanic basins and that this biogeographic signal was stronger for the particle-attached members (0.8--20 µm size fraction) than for their free-living counterparts (0.2--0.8 µm size fraction) ([@r36]). This agrees with our observation that at all depths, communities from the largest particles showed much clearer differences between stations than the free-living communities ([*SI Appendix*, Table S2](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)). However, we observed that the compositional differences between surface stations of the particle-attached communities were correlated with the compositional differences between deep-sea stations and that the strength of this correlation increased with increasing particle size, whereas no such pattern was observed for surface vs. deep-layer free-living communities ([Table 1](#t01){ref-type="table"}). Altogether, this indicates that the biogeography of deep-sea communities mirrors that of the overlaying surface communities as a consequence of particle-mediated dispersal and is thus partially determined by that of the attached prokaryotic community originating in the surface. Salazar et al. ([@r36]) suggested that submarine mountains that divide the deep ocean into basins might act as ecological barriers for prokaryotic communities, thus favoring their differentiation. However, this explained a limited fraction of the variance in community composition. Our results further suggest that taxonomic differences of the pool of taxa arriving via sinking particles may also be a major mechanism explaining the observed biogeographic patterns of deep-sea prokaryotes. In our study design based on the comparison of prokaryotic community structure along vertical profiles, we described the variability of community composition along the water column, in a vertical gradient defined by the four depths sampled in each station. We are aware that the described processes are not directly connected along a vertical line, because horizontal transport velocities can be greater than sinking rates. Hence, the sampling points of each depth must be considered as representative samples of a wider area (i.e., they should not be interpreted as points located directly on top of each other but rather as vertical profiles with samples representative of a larger area). Still, similar patterns were observed when analyzing each station separately ([*SI Appendix*, Fig. S6](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)) and all stations together ([Fig. 2](#fig02){ref-type="fig"}). In addition, the observed similarity in biogeographic patterns between surface and deep particle-attached communities ([Table 1](#t01){ref-type="table"}), as well as the station-specific signature in the pool of taxa able to grow on sinking particles ([*SI Appendix*, Fig. S7](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)), seems to support that there is indeed vertical connectivity occurring within the area represented by each sampled station. In summary, we show that the global ocean prokaryotic microbiome exhibits a strong vertical connectivity through the entire water column. This connection occurs via particle sinking, which highlights the role of particles as microbial vectors that introduce viable surface taxa into the deep ocean. In addition, our results indicate that all local communities seem to be dominated by OTUs that are already present in surface waters; that there are OTUs from the surface that thrive at depth; and that the biogeography of the bathypelagic realm is influenced, to some extent, by particle colonization events occurring in surface waters. The contributions of both particles and their attached communities to the bathypelagic realm seem therefore crucial. To the existing evidence that sinking particles are an essential source of carbon and nutrients for the development of heterotrophic life in the deep ocean ([@r3], [@r4]), our results add that communities attached to particles provide a source of viable diversity to deeper ocean layers. Given that the bathypelagic realm is constituted mostly by slow sinking or buoyant particles ([@r4]), which are resource-rich habitats for microbes ([@r56]), and that deep-sea prokaryotes are more adapted to the attached lifestyle than surface ones ([@r57][@r58][@r59]--[@r60]), deep-sea microbial activity and life must be concentrated on particles ([@r3], [@r6], [@r57]). Therefore, this particle-driven dispersal of microbes likely constitutes a fundamental mechanism influencing the structure, assembly, and functioning of deep ocean microbial communities across the global ocean. Concluding, we describe here the particle-driven vertical dispersion of prokaryotes from the surface to the deep ocean, and our findings suggest a plethora of additional questions that could be explored in the future. Do microbes that sink ever return to the surface ocean, or is there a dispersal mechanism that brings them back to the surface? If the transported microbes depend directly on the processes occurring at the surface, how does variability in particle composition and sinking rates affect the communities in the deep ocean? Is the dispersal of organisms arriving from the surface the main process determining the biogeography of the deep ocean? Here we unveil the importance of particle-driven vertical dispersion, but the relevance of other mechanisms of dispersion in structuring ocean microbial communities at the global scale remains to be assessed, mechanisms that could explain, e.g., how organisms considered endemic of vent systems can colonize geographically and potentially isolated distant hydrothermal habitats ([@r45]). The postulated existence of a global seed bank in the ocean ([@r44]) requires identifying the various prokaryotic mechanisms responsible for dispersal. Our results provide evidence of one such mechanism occurring at a global scale. Materials and Methods {#s6} ===================== Study Area and Sampling. {#s7} ------------------------ We selected a total of eight stations of those sampled during the Malaspina 2010 Expedition ([@r61]) between December 2010 and July 2011. The selected stations were distributed across the global tropical and subtropical ocean (latitudes between 30° N and 40° S): three in the Atlantic Ocean, two in the Indian Ocean, and three in the Pacific Ocean. At each station, four depths were sampled corresponding to the surface (SFC, 3 m), the deep chlorophyll maximum (DCM, 48--150 m), the mesopelagic (MESO, 250--670 m), and the bathypelagic waters (BATHY, 3,105--4,000 m). Surface water was sampled with a Niskin bottle, and water from the other depths was sampled with a rosette of Niskin bottles attached to a conductivity--temperature--depth (CTD) profiler. Vertical profiles of salinity, potential temperature, and dissolved oxygen were recorded continuously with the CTD sensors installed in the rosette sampler. Nutrients (nitrate, phosphate, and silica) were determined using standard procedures as explained in Catalá et al. ([@r62]). Bacterial abundance and size were determined by flow cytometry, and bacterial heterotrophic production was estimated using the ^3^H-leucine incorporation method ([@r63]) as described in detail in ref. [@r64]. Prokaryotic biomass from different size fractions was collected by prefiltering the water through a 200-µm net mesh and sequentially filtering 10 L through 20-, 5.0-, 3.0-, 0.8-, and 0.2-µm pore-size filters, all 47-mm polycarbonate filters (20-μm pore-size filter from GE Water and Process Technologies and the rest of the filters from Millipore), using a peristaltic pump, resulting in five different size fractions (0.2--0.8, 0.8--3.0, 3.0--5.0, 5.0--20, and 20--200 µm). Filter clogging or particle dislodging may affect the taxonomic composition observed in each size fraction ([@r65]), but we tried to minimize these issues by filtering at very low speed and pressure and by changing frequently the filters. The filters were flash-frozen in liquid N~2~ and stored at −80 °C until DNA extraction. We assume that the 0.2--0.8 µm size fraction will harbor mostly free-living prokaryotic communities that remain suspended in the water column, and the rest of the size fractions will comprise prokaryotes associated to different kinds of particles (e.g., gels, organic and inorganic particles, and living or not living organisms) of variable sizes that will influence their sinking velocities. We define 0.2--0.8 and 0.8--3.0 µm as small size fractions (or small particles, suspended particles), whereas 3.0--5.0, 5.0--20, and 20--200 µm are defined as large size fractions (or large particles, sinking particles). DNA Extraction, Sequencing, and Sequence Processing. {#s8} ---------------------------------------------------- The DNA was extracted with a phenol-chloroform protocol (as described in ref. [@r66]). The hypervariable V4--V5 region of the 16S rRNA gene was PCR amplified with primers 515F-926R ([@r67]) and sequenced in an Illumina MiSeq platform using 2 × 250 bp paired-end approach at the Research and Testing Laboratory facility ([rtlgenomics.com/](http://rtlgenomics.com/)). Computing analyses were run at the Marbits bioinformatics platform of the Institut de Ciències del Mar. The amplicons were processed through a protocol (detailed in ref. [@r68]) based on Uparse ([@r69]). Briefly, reads were assembled with PEAR (Paired-End reAd mergeR) ([@r70]), and those with \>100 nucleotides were selected. Quality check, dereplication, OTU clustering (99%), and reference-based chimera filtering (using Silva database v.119) were processed with Usearch ([@r71]). Taxonomic assignment of OTUs was generated by BLAST (Basic Local Alignment Search Tool) searches of representative sequences against Silva database v.123. Nonprokaryotic OTUs (eukaryotes, chloroplast, and mitochondria), as well as singletons, were removed. To allow comparisons between samples, the OTU table was randomly subsampled to the number of reads present in the sample with the lowest amount of reads (which was *n* = 5,390), and a total of 13,567 OTUs were obtained. Data Analysis. {#s9} -------------- Statistical analyses and plots were done in R ([www.r-project.org](http://www.r-project.org/)) using the *vegan* ([@r72]), *simba* ([@r73]), *spaa* ([@r74]), *betapart* ([@r75]), and *BiodiversityR* ([@r76]) packages. The OTU richness of each size fraction and at each depth was calculated using the rarefied OTU table, and the Bray--Curtis metric was used as an estimator of community dissimilarity. To check if the communities were structured stochastically or not, the RC~Bray~ index was calculated. The index consists of the Raup--Crick metric ([@r77]) using Bray--Curtis dissimilarities ([@r41]). A total of 1,000 randomizations were performed for OTUs with \>100 reads in the entire dataset. Communities were clustered using nonmetric multidimensional scaling (nMDS) analyses based on Bray--Curtis distances. Statistical differences among categories such as size fraction, station, and depth were explored with permutational multivariate analyses of variance (PERMANOVA) tests (adonis function, R *vegan* package). To elucidate the connectivity between communities throughout the water column of a given size fraction, a set of parameters was calculated considering each station separately: Vertical beta diversity (i.e., community differentiation) was calculated using the *trudi* function from R package *simba*. Vertical OTU turnover (i.e., dissimilarity due to species replacement) and nestedness (i.e., dissimilarity due to species loss) were estimated using the *beta.multi* function in the R *betapart* package and were based on the Sorensen index. The vertical niche breadth of each OTU was calculated using the *niche.width* function in the R *spaa* package and applying the Levins ([@r78]) index. Niche breadth was defined as the number of different depths where an OTU appeared (i.e., OTUs with niche breadth values of 4 were present across the four depths, whereas OTUs with niche breadth values of 1 were present in only one depth), and an average of all of the niche breadth values of the OTUs of each station was calculated. To test if differences from small to large size fractions were statistically significant in beta diversity, turnover, nestedness, and niche breadth parameters, a Wilcoxon ranked sign test was performed comparing the 0.2--0.8 µm and the 20--200 µm size fractions. Correlations between communities from different size fractions and depths were calculated using Mantel tests (based on Bray--Curtis distances). To differentiate between the OTUs dominant in (i.e., enriched in, with preference for) smaller or larger size fractions, we defined the PAN-Index. The PAN-Index indicates in which size fraction an OTU is more abundant (and therefore more dominant over the other OTUs) and was calculated using the abundance-weighted mean of each OTU among the five size fractions. This PAN-Index defines the size fraction preference of every OTU in the continuum of sizes (i.e., in which size fraction an OTU is more dominant) and is a modification of the PAN-Index presented in Salazar et al. ([@r52]) where only two size fractions (free-living vs. attached) were considered. Values of PAN-Index ranged from 1 to 5, and each number reflects the size preference of a given OTU as follows: 1, preference for the size fraction 0.2--0.8 µm; 2, preference for 0.8--3.0 µm; 3, preference for 3.0--5.0 µm; 4, preference for 5.0--20 µm; and 5, preference for 20--200 µm size fraction. The PAN-Index values presented a bimodal distribution ([*SI Appendix*, Fig. S4](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental)), and we consequently divided them in two groups: OTUs with PAN-Index ≥ 2.7 (i.e., those with preference for large particles) and OTUs with PAN-Index \< 2.7 (i.e., those with preference for small particles). To identify the pool of taxa potentially growing in sinking particles, we first selected among the surface OTUs with PAN-Index ≥ 2.7 those showing the largest shifts in relative abundance by calculating the Euclidean distance of their relative abundances between all pairs of samples and choosing the OTUs with a mean distance \>10, following Ruiz-González et al. ([@r39]). We then identified those that increased in relative abundances (dominance) toward deeper layers (i.e., showing higher mean relative abundances in mesopelagic and/or bathypelagic waters than in the surface and/or the DCM). This was done for each station, and these OTUs were named seed OTUs because they might represent taxa seeding deeper communities. Supplementary Material ====================== We thank all the scientists and crew for their support during sample collection in the Malaspina 2010 cruise and especially E. Borrull, C. Díez-Vives, E. Lara, D. Vaqué, G. Salazar, and F. Cornejo-Castillo for DNA sampling. C. Antequera kindly provided laboratory assistance during DNA extraction. We are also grateful to G. Salazar for his help in adapting the PAN-Index to a multiple size fractionation. We thank the Marbits bioinformatics platform of the Institut de Ciències del Mar in Barcelona and especially Pablo Sánchez for computing support. M.M. was supported by a Consejo Superior de Investigaciones Científicas-Junta para la Ampliación de Estudios Grant (CSIC-JAE-Predoc Grant) and by the Ministry of Labor, Employment and Social Security. R.L. was supported by a Ramón y Cajal fellowship (RYC-2013-12554, Ministerio de Economía y Competitividad (MINECO), Spain), and C.R.-G. was supported by a Juan de la Cierva fellowship (IJCI-2015-23505). This is a contribution to the Malaspina 2010 Expedition, funded by the Spanish Ministry of Economy and Competitiveness through the Consolider-Ingenio program (Reference CSD2008-00077). The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: Prokaryotic reads were deposited in the European Nucleotide Database (<http://www.ebi.ac.uk/ena>; accession nos. [ERP109198](http://www.ebi.ac.uk/ena/data/view/ERP109198), [ERS2539749](http://www.ebi.ac.uk/ena/data/view/ERS2539749)--[ERS2539903](http://www.ebi.ac.uk/ena/data/view/ERS2539903)). This article contains supporting information online at [www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental](http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802470115/-/DCSupplemental). [^1]: Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved May 30, 2018 (received for review February 9, 2018) [^2]: Author contributions: M.M., C.M.D., J.M.G., and M.M.S. designed research; M.M. performed research; M.M. and C.R.-G. analyzed data; R.L. analyzed genomic sequences; M.M., C.R.-G., R.L., C.M.D., J.M.G., and M.M.S. wrote the paper.

Background {#Sec1} ========== Comprehensive care for people diagnosed with psychosis includes access to adjunctive evidence based psychosocial interventions \[[@CR1], [@CR2]\]. Cognitive remediation therapies for psychosis are recommended in the recently updated Australian and New Zealand College of Psychiatrists clinical practice guidelines \[[@CR1]\]. Translating clinical guideline recommendations for psychosocial interventions into routine practice has been a challenge \[[@CR3], [@CR4]\]. Implementation science provides a framework to guide the translation of evidence based therapies into routine care \[[@CR5]\]. This approach champions external validity and engagement with real world clinical settings and practice. In an implementation framework program implementation outcomes (fidelity, feasibility, reach, acceptability, maintenance, and costs) are delineated as a distinct outcome category that influences service and clinical program outcomes \[[@CR6]\]. Improved clinical outcomes are seen as arising from the marriage of interventions with robust evidence with effective implementation strategies \[[@CR7]\]. Fidelity refers to the delivery of the intervention as proven to be effective and can be measured at an individual therapist level or at a program level. Feasibility assesses if the intervention has utility for routine use in usual care settings and can be assessed at an individual therapist/ participant level and/or an organisational level. Reach is the level of dissemination within an organisation and is related to treatment access and can be assessed via auditing programs within a population. Acceptability relates to satisfaction with the program and can be assessed at an individual provider or participant level. Maintenance of programs refers to the ongoing use of a program after the initial implementation and depends on the degree to which the program becomes embedded within the usual business of the service and can be assessed via regular audits. Cost of implementing new programs relates to new labour and non-labour establishment and recurrent costs and is of particular concern to the financial management of the provider organisation. Cognitive impairment is common in people diagnosed with schizophrenia \[[@CR8], [@CR9]\]. The deficits are global and impact on both neuro cognition (attention, memory, planning) and social cognition (difficulties perceiving and processing emotions) \[[@CR9], [@CR10]\]. Approaches to remediation of these deficits have included integrated programs with a combined approach to neuro cognition and social cognition and also stand-alone programs focussing either on neuro cognition or social cognition \[[@CR11], [@CR12]\]. There is evidence from a meta-analysis of randomized controlled trials for the benefit of Cognitive Remediation therapy (CR) on global cognition (Effect Size ES =0.45) with greater benefit if CR is combined with some form of rehabilitation (ES = 0.59) \[[@CR11]\]. The core components of CR appear to be: intensity of practice (2--4 times a week); the combination of drill and practice of tasks with strategy training and a context that facilitates use of new skills learnt. In the meta-analysis, 14 different CR programs were examined with no particular program having superior effect \[[@CR11]\]. There is also evidence from a meta-analysis of the effectiveness of interventions remediating social cognitive deficits in schizophrenia \[[@CR12]\]. Social Cognitive Interaction Training (SCIT) is one example of a social cognition remediation program. It is comprised of three phases (Introduction & Emotions, Figuring out Situations, Checking it out) over 20 to 24 sessions. It is administered in a group format \[[@CR13]\]. It has been delivered in weekly hour-long sessions (20--24 weeks), and weekly 2-h sessions (10-12 weeks) \[[@CR14]\]. Four CR programs and four SCIT programs had been established in Brisbane's Princess Alexandra Hospital (PAH), and the associated health district prior to this study commencing. These programs commenced following staff training in CR in 2008 and SCIT in 2010. In 2012 the service restructured into teams based on diagnosis or model of service (e.g. rehabilitation) to encourage the development of core diagnostically relevant evidence based practices and to more equitably distribute resources. This restructure gave organisational impetus to apply implementation theory to the issue of limited implementation and dissemination of cognitive therapies for psychosis. Staff from the service involved in psychosis care, Psychosis Academic Clinical Unit (ACU) and Rehabilitation ACU in which the study was carried out developed a plan to guide implementation of CR and SCIT in the 3 geographically defined hospital health districts of the service. The implementation plan was organised around the stages of implementation: exploration/adoption; initial implementation; full implementation and program maintenance \[[@CR7]\]. The exploration/adoption stage had been completed with pilot studies in one hospital site \[[@CR15], [@CR16]\]. The baseline organisational factors were assessed \[[@CR17]\]. The initial implementation focussed on the implementation drivers with the appointment of implementation champions, the formation of the implementation team, the securing of infrastructure for each sites and the establishment of referral pathways and regular training and supervision of staff. The full implementation stage involved annual program audits, and annual program fidelity monitoring. The CR program fidelity for programs implemented in the first year of the study has previously been reported \[[@CR18]\].The maintenance stage involved securing ongoing program funding support, regular training and the development of a pool of program specialists who were involved in providing supervision and iterative program improvements. This study reports on the implementation outcomes of program fidelity, feasibility, reach, acceptability, maintenance and cost, three years into the implementation process. Method {#Sec2} ====== Design and setting {#Sec3} ------------------ This evaluation study was conducted over three years from 2013 to 2015 and collected information on the implementation outcomes (fidelity, feasibility, reach, acceptability, maintenance, and costs) of CR and SCIT programs implemented. The study setting was the community mental health services of a metropolitan health service (MHS) with a population of around 920,000 in Brisbane, Australia. The health service comprises three hospital health districts (PAH, Logan and Bayside). It has been estimated that approximately three thousand people residing in this area have severe and persistent mental illness with complex needs (Greater Metro South Brisbane Medicare Local Schedule 10, 2013). The area has high rates of socioeconomic disadvantage with 19.8% of people postponing mental health care because of cost (Greater Metro South Brisbane Medicare Local Schedule 10, 2013). The geographic area covers inner metropolitan suburbs to semi-rural areas. There are three hospitals in the geographically defined health districts. Population {#Sec4} ---------- ### Program facilitators {#Sec5} Both programs were facilitated by a multidisciplinary group of therapists who had identified an interest in becoming CR and SCIT therapists and had completed the requisite training and credentialing \[[@CR18]\]. The therapy was undertaken as part of their general role in the service with therapy time quarantined and case weighting altered to facilitate staff capacity to undertake this work. There were 12 cognitive remediation therapists (4 psychologists, 2 occupational therapists, 2 psychiatrists, 3 mental health nurses and 1 social worker). There were 10 SCIT therapists (5 psychologists, 2 psychiatrist, 2 nurses and 1 social worker). ### Program participants {#Sec6} People diagnosed with a schizophrenia spectrum disorder who were being treated in the community, aged between 18 and 65, and who did not have an intellectual impairment or active substance abuse disorder were eligible to participate. Service users could participate in the program and not consent to the evaluation research project. ### Cognitive rehabilitation therapies implemented {#Sec7} The characteristics of the programs implemented are listed in Table [1](#Tab1){ref-type="table"}. The specific CR interventions used in this study were a computer based program, CIRCuiTS (Computerized Interactive Remediation of Cognitive and Thinking Skills) \[[@CR19]\] and a program based on educational software using computers, Neuro psychological Educational Approach to Cognitive Remediation (NEAR) program \[[@CR20]\]. Both programs run twice a week for around an hour. CR is run as an open group with 4 participants to one therapist.Table 1CR and SCIT program componentsCRSCITComponents• Computer program\ • 15 min bridging group• Manualised therapyFrequency• Twice a week• Once a weekDuration• 10--20 weeks• 10--12 weeksIntensity• 60 min• 120 minMode of delivery• Computer session 45 min + 15 min bridging small group\ • Open group• Small group = 8\ • 2 therapists\ • Closed groupMaterials• Computer\ • Bridging manual• Facilitators Manual\ • Participants Manual SCIT is based on a manual and uses some resources available freely online. SCIT is run as a closed small group of a maximum of 8 participants and 2 facilitators. The program is structured to build skills over 3 phases focussing in phase 1 on emotional processing; phase 2 focussing on thinking skills in social situations; and phase 3 integrating these skills into real life social situations experienced by participants. Measures {#Sec8} -------- ### Data sources {#Sec9} Implementation outcomes were assessed using data from multiple sources including yearly staff surveys, program audits, and measures of program feasibility. ### Yearly staff surveys (Additional file [1](#MOESM1){ref-type="media"}) {#Sec10} This survey asked questions about staff interest, training and current practice with 8 questions about cognitive behavioural therapy (with SCIT classified as a CBT derived therapy) and 5 questions related to CR. The survey was distributed yearly for the three years of the study to clinical staff in the community psychosis and rehabilitation teams. ### Annual program audits {#Sec11} Program audits were conducted annually by the program co-ordinator VGJ. For CR the number of programs running in each service, the maintenance of programs established, the number and retention of facilitators, the number of computers and funding was recorded. For SCIT the number of programs conducted and number and retention facilitators was noted. ### Annual fidelity checks {#Sec12} No fidelity instrument had been developed for CIRCuiTS. Program fidelity was therefore measured using an instrument developed by Alice Medalia and colleagues for the Neuro psychological Educational Approach to Cognitive Remediation (NEAR) program which was adapted for the CIRCuiTS and SCIT programs (Medalia and Freilich, 2008). There were 12 criteria for CR programs: clinician credentials and training; supervision; software used and selection (NEAR only); referral process; assessment; personalised folders; application of assessment; specific CR techniques used; intrinsic motivation; time on each program (NEAR only); software features (NEAR only) and communication with referring team. The 7 criteria for SCIT program fidelity were: clinician credentials and training; supervision; manual adherence; referral process; assessment process; application of assessment; and demonstration of specific SCIT techniques. Each criterion was rated by FD on a 5 point scale: from not achieved; occasionally achieved; frequently achieved; infrequently not achieved; and 100% adherence. ### Acceptability measures {#Sec13} A three-item measure of transportability, acceptability and feasibility was used by the SCIT program facilitators at the end of the program. This measure had been used in a previous study by the SCIT program developers \[[@CR21]\]. The items asked if program participation was useful, if it helped thinking in social situations and helped to relate to others. Participants and referring clinical staff were asked to rate their responses on a 7 point (1 not helpful, 4 unsure and 7 very helpful) Likert scale. This measure was administered post-treatment. This measure has not been previously used in CR studies and was not used to evaluate the feasibility or acceptability of CR in this study. The number of people referred to CR who commenced, whether consenting to the research or not, and reasons for discontinuing the program was used as a proxy measure of acceptability of the program. Program costs {#Sec14} ------------- Cost outlays for the programs included labour and non-labour expenses over the three years. Labour costs were calculated based on an average therapist's hourly award wage. Therapists spent 2 h per week conducting the groups with SCIT being held once a week for 2 h and CR 1-h sessions twice a week. The costs of acquiring new non-labour items were included in the calculation. For CR this included the cost of purchasing computers and internet connection. Data analysis {#Sec15} ------------- Descriptive statistics were used to report the implementation outcomes. Results {#Sec16} ======= Program population {#Sec17} ------------------ The consenting participants who commenced CR (*n* = 40) were aged between 26 and 60 with a mean age of 42 (SD = 10.4). They had between 6 and 15 years of education with a mean of 10.4 years (SD =2.0). More males (67%) than females (33%) consented to the study. Twenty-five percent were living in Department of Housing accommodation; 25% were living in private dwellings; 40% in residential rehabilitation units; and 10% where the accommodation was unspecified. Of the 19 participants who completed 10 or more sessions, 68% were male and 32% female. The age range of participants consenting and who commenced SCIT (*n* = 33) was 19 to 50 years with a mean of 30.9 years (SD = 8.6 years). They had between 6 and 15 years of education with a mean of 10.8 years (SD = 2.2 years). More males (67%) than females (33%) consented to the study. Fifty- 1 % lived in a residential rehabilitation unit; 46% in a private dwelling; and 3% accommodation was unspecified. Of the 22 participants who completed 16 or more sessions, 64% were male and 36% female. Fidelity {#Sec18} -------- All CR program facilitators had completed the two day training and were observed to be competent by an experienced facilitator. A clear referral and assessment process was followed by all staff. All programs were rated as having frequent fidelity to the specific CR techniques with no ratings on any occasion falling below frequently achieved. Each program had different processes for feedback to treating teams with no standard process. Monthly centralised supervision was accessed routinely by facilitators from only 1 program. All staff facilitating SCIT were trained and experienced facilitators were coupled with less experienced facilitators. Fidelity to the treatment manual was discussed and reviewed by the facilitators following each session to minimize "therapeutic drift". All programs were rated as having frequent fidelity to the specific SCIT techniques with no ratings on any occasion falling below "frequently achieved". Each program had different processes for feedback to treating teams (e.g. informal verbal feedback to case managers, regular feedback in team meetings). Feasibility {#Sec19} ----------- All 3 hospitals in the geographically defined health districts participated. The estimated total population of patients diagnosed with schizophrenia spectrum disorders in the service at the time of the study was 1660. From this population 105 people were referred to the CR programs and 53 were referred for the SCIT programs. All sites had staff from multiple disciplines trained and competent to deliver both therapies. CR was run as an open group. The SCIT groups were run as a closed group once or twice a year. Three SCIT programs were conducted in the hospital district that had previously run pilot SCIT programs and 1 program ran at each of the other two hospital districts. Reach {#Sec20} ----- Each hospital district was serviced by at least 1 CR and 1 SCIT program. The inner city hospital district had 4 CR programs established and 3 SCIT programs were conducted. CR was conducted at the residential rehabilitation units but accepted referrals from the community clinics in Logan and Bayside Hospitals and districts. The referring community clinics have made requests for access to programs that were at full capacity necessitating the creation of waiting lists. Based on this unmet demand a further 2 CR programs and 2 SCIT programs are estimated to be needed for the community teams. Acceptability {#Sec21} ------------- Of the 89 people referred to CR, 52 consented to participating in the evaluation research. Twelve of the 52 consenting failed to commence the program. Of the 40 participants consenting and commencing the CR program, 21 completed at least 10 sessions. Nineteen of the research participants discontinued CR before completing 10 sessions. Seven participants had time constraints, usually due to family responsibilities; 3 were hospitalised due to acute deterioration in mental state; 4 moved area and had trouble accessing the program and for one participant no reason for discontinuation was given. All cases of acute deterioration in mental health were considered by their clinical team as not relating to the programs. Where possible an interview of participants on exiting the program early was used to explore satisfaction with the CR program. Only 4 participants (10%) stopped attending CR due to loss of interest in the program. Of the 53 eligible people referred to SCIT, 39 consented to be involved in the study. Six of the 39 did not commence. Of the 33 commencing SCIT, 24 completed more than 16 sessions which was considered as having completed the program. Eight failed to complete the program due to acute deterioration requiring hospitalisation and no reason was recorded in one case. All cases of acute deterioration in mental health were considered by their clinical team as not relating to the programs. Program acceptability was recorded for SCIT using a three item measure (Table [2](#Tab2){ref-type="table"}). On average, based on a scale of 1 to 7 both participants and staff considered the program useful in relation to social thinking and relating. The participants' average scores were above 5 with a range of 4 to 7 for "program participation was useful" and "program participation helped in thinking in social situations" criteria and 3--7 for "program participation helped me to relate to others".Table 2Participant and therapist acceptability and feasibility ratings SCITParticipant\ Mean RangeClinicians\ Mean RangeParticipation in the group was useful to me/my client5.1 (4--7)5.2 (3--7)The group helped me/ my client think about social situations5.6 (4--7)5.3 (2--7)The group helped me/ my client to relate to other people5.2 (3--7)5.4 (4--7) Maintenance {#Sec22} ----------- Over the 3 years of the study (2013--2015) the number of CR programs doubled to 8. The 3 programs running in the PAH district and 1 program running at Logan hospital district at baseline were still active at the end of the study period. In 2014 an additional 2 programs were established (1 Logan, and 1 Bayside). Another 2 programs were established in Bayside in 2015. All established programs were maintained at the last audit in December 2015. In 2013 there were 10 program facilitators. These facilitators were retained and 3 new trained facilitators commenced in 2014. The 13 trained facilitators were retained at the end point audit in December 2015. In 2014 ongoing funding was secured to maintain internet connection and 12 new computers were purchased to replace old computers and to support the new programs. SCIT programs were run each six months in the PAH hospital district (2013--2015) and one program at Bayside and Logan in the last year of the project. The PAH had previous experience with SCIT and the Bayside and Logan programs were run at the residential rehabilitation services that had only opened in 2015. These programs are ongoing following the end of the study and scheduled to run every six months depending on demand. Ten therapists were trained in the mental health service and were still running groups at the last audit in 2015. Costs {#Sec23} ----- The total expenditure on CR programs including labour and non-labour was \$32,848 over the three years (2013--2015). The use of some donated computers lowered costs for purchasing computers. The SCIT programs involved only labour costs of \$11,520 (Table [3](#Tab3){ref-type="table"}).Table 3Total program cost outlays and cost offsets over three years (2013--2015)CR Total costsSCIT Total costsComputers\$4000N/RInternet\$1200N/RStaff ×  2 (estimated therapy hours x rate of pay) \$48/h\$27,648/3 years\$11,520/3 yearsExpenditure\$32,848\$11,520 Discussion {#Sec24} ========== Studies of the implementation of cognitive rehabilitation therapies into routine mental health care using implementation theory are beginning to emerge \[[@CR17], [@CR18], [@CR22]--[@CR24]\]. The implementation plan described in this paper resulted in CR and SCIT programs being disseminated throughout a large health service comprising three hospital districts. There was organisational executive support for the development of evidence based programs with the service reorganisation in 2012. Organisational support has been found to be necessary but not sufficient for successful program implementation \[[@CR7]\]. The organisational support and previous exploration of the cognitive rehabilitation therapies of interest, resulted in a plan to systematically implement CR and SCIT. CR and SCIT had been run in one hospital district since 2008 and 2010 respectively with no programs existing in two other hospital districts \[[@CR15], [@CR16]\]. This is consistent with the implementation literature that has found passive diffusion of innovations are not as successful as planned and staged implementation \[[@CR7]\]. The growth in the number of programs over the years of the study is also consistent with the 2 to 4 year project plan required for most implementation projects \[[@CR7]\]. The current evaluation study found the programs were feasible within services more distal to the largest hospital in the area. External factors to an organisation may determine the feasibility of decentralized or centralising programs. Centralized well-resourced program "hubs" may be acceptable and accessible if transport is convenient and affordable. Program maintenance and quality can be a challenge with dissemination to smaller, less resourced centres. In this study the programs were disseminated out from the largest hospital (PAH) that had established the initial programs to the other two hospitals (Logan and Bayside) due to the large geographical area and the relative expense of public transport for this population. Support for these programs was provided from the central "hub" via supervision either face to face or via teleconferencing. Four participants who moved areas failed to complete CR implying that lack of local access can influence the feasibility of attending programs. For new programs to become embedded in routine care, the organisational fit, feasibility and acceptability of the programs to staff need to be considered \[[@CR25]\]. In this study, findings regarding maintenance of programs and retention of facilitators were interpreted as proxy measures indicating these programs demonstrated an acceptable organisational fit. Reasons for discontinuing CR early were collected. The overall attrition rate for those consenting to the evaluation was high (55%). Wykes in a meta-analysis found the overall attrition rate for CR to be 11% with a range of 0--47.5% (Wykes et al., 2011). In this study in relation to acceptability, once initiated only 10% ceased the CR program because of loss of interest. In both programs acute illness resulted in early exit from the programs reflecting the relapsing nature of schizophrenia. Accurate assessment of the utilization of the CR program was not possible due to the low rates of participants consenting to research (i.e., 37, 42%attended CR but did not consent to the evaluation). Previous pilot studies of SCIT found lower attrition in participants living in residential settings \[[@CR15]\]. A significant number of participants were recruited for SCIT from residential settings (40% CR and 51% SCIT participants). In this study 65% of non-completers of SCIT were living in the community. The support from staff in the residential settings to remind and to help motivate participants to attend may contribute to these lower attrition rates. This also supports current efforts to increase service user involvement in design and production of research, which has been shown to improve recruitment \[[@CR26]\]. In the maintenance phase the programs need to become embedded into clinical governance procedures where utilization can be monitored as part of routine quality assurance practices. A formal economic analysis was not included in the protocol. Cost outlays are higher for CR than for SCIT due to more sessions being conducted and the use of computers and internet (Table [3](#Tab3){ref-type="table"}). Previous studies suggest that cognitive remediation programs may result in service cost savings \[[@CR27]\]. Limitations {#Sec25} ----------- Implementation studies are inherently context specific making extrapolation to other services difficult. These results are particularly relevant to the Australian public mental health sector. The higher program participation, while not consenting to research, also biases the results, limiting extrapolation to the population of people who attended the programs. The 3 year implementation timeframe for this study is brief given the complexity of the interventions. The study site had experience in the programs (exploration phase of implementation) and this needs to be considered in relation to the overall time required to implement these programs. The study did not involve a formal cost-effectiveness analysis. This information is needed to help prosecute the case for funding the implementation of these therapies in the context of increasingly constrained health budgets. Conclusions {#Sec26} =========== Implementation theory and practice can help guide the implementation of evidence based therapies recommended into routine, comprehensive psychosis care. Having a staged plan over time is required to address the complex workforce, organisational and quality issues that are inherent in delivering cognitive therapies for psychosis. Service user involvement in the design and running of psychosocial programs is recommended. Additional file =============== {#Sec27} Additional file 1:SSPARS BMC Staff survey. (DOC 28 kb) ACU : Academic Clinical Unit CBT : Cognitive Behaviour Therapy CIRCuiTS : Computerised Interactive Remediation of Cognitive Thinking Skills CR : Cognitive Remediation Therapy ES : Effect Size MHS : Metropolitan Health Service NEAR : Neuropsychological Educational Approach to Cognitive Remediation PAH : Princess Alexandra Hospital SCIT : Social Cognitive Interaction Training **Electronic supplementary material** The online version of this article (10.1186/s12913-018-3240-5) contains supplementary material, which is available to authorized users. Funding {#FPar1} ======= This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Availability of data and materials {#FPar2} ================================== The datasets analysed during the current study are not publicly available as this is not part of our ethical approval. The data is available from the corresponding author on reasonable request and with the ethics committee approval. Authors FD, MH, VGJ, EN, and HW all contributed to the conception and design, acquisition of data, analysis and interpretation of data. FD, MH and HW have been involved in drafting the manuscript and revising it critically for important intellectual content, Authors FD, MH and HW have given final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== This study was approved by the Metro South Health Human Research Ethics Committee (HRE/13/QPAH/16). The study was explained to each participant, and a written informed consent was obtained from all participants prior to conducting the study. Competing interests {#FPar4} =================== The first author is a director of rehabilitation in the study site and was on the writing committee of the recently published Australian and New Zealand Schizophrenia Clinical Practice Guidelines. The authors alone are responsible for the content and writing of the paper. Publisher's Note {#FPar5} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

1. Introduction {#sec1-marinedrugs-17-00119} =============== Most naturally occurring fatty acids have an unbranched chain consisting of an even number of carbon atoms ranging from 4 to 28. Depending on the nature of the hydrocarbonated chain, the fatty acids can be saturated, monounsaturated, or polyunsaturated \[[@B1-marinedrugs-17-00119]\]. Many fatty acids can be synthesized by humans, however not some polyunsaturated fatty acids (PUFAs), such as omega-3 (n−3) and omega-6 (n−6) fatty acids \[[@B2-marinedrugs-17-00119]\]. The parent constituents of omega-3 and omega-6 fatty acids are α-linolenic acid (C~18:3~ n−3, ALA) and linoleic acid (C~18:2~ n−6, LA), respectively. Omega-6 fatty acids, such as arachidonic acid (C~20:4~ n−6; AA), can be synthesized by humans from LA, whereas the essential omega-3 fatty acids, such as eicosapentaenoic acid (C~20:5~ n−3; EPA), docosapentaenoic acid (C~22:5~ n−3, DPA), and docosahexaenoic acid (C~22:6~ n−3, DHA), can be synthesized from ALA. However, the conversion rate of ALA to EPA, DPA, and DHA is very low \[[@B3-marinedrugs-17-00119]\]. Consequently, both omega-6 and omega-3 PUFAs have to be taken up through the diet, preferably at a ratio of 5:1 or less \[[@B4-marinedrugs-17-00119]\]. Presently, the most common source of omega-3 PUFAs is represented by fish of the Salmonidae, Scombridae, and Clupeidae families which contain a high percentage of DHA and EPA \[[@B3-marinedrugs-17-00119]\]. The ever-rising global demand for omega-3 PUFAs cannot be met by fish oil due to diminishing fish stocks and pollution of marine ecosystems \[[@B5-marinedrugs-17-00119]\] which has led to increased interest in alternative sustainable sources. Vegetable oils from genetically engineered plant oilseeds and microorganisms are two potential alternatives to fish oil, even though omega-3 PUFAs are highest in the latter. *Brassica juncea*, *Arabidopsis thaliana*, and *Camelina sativa* are some examples of genetically engineered plant species with potential to accumulate omega-3 PUFAs \[[@B6-marinedrugs-17-00119]\]. Although transgenic plants present numerous advantages, their production is dependent on seasonal and climatic conditions and the availability of arable land. Moreover, there are public concerns regarding the cultivation of transgenic crops in open ecosystems. These, together with regulatory issues, restrict the large-scale production of genetically modified crops \[[@B7-marinedrugs-17-00119]\]. Microorganisms are known natural producers of microbial oils similar to those obtained from plants and animals and a possible source of nutritionally important omega-3 PUFAs \[[@B8-marinedrugs-17-00119]\]. The use of microorganisms benefits from the ability to use biochemical engineering to improve microbial growth rates, low nutrient requirement to achieve growth, easily controllable culture conditions, and availability of well-annotated genomes and metabolic pathways that allow their genetic manipulation \[[@B9-marinedrugs-17-00119]\]. Moreover, microbial oils usually contain a significant amount of natural antioxidants, such as carotenoids and tocopherols, which play an important role in protecting omega-3 PUFAs from oxidation and therefore improve their storage stability. The first commercial product obtained from microbial oil was a gamma-linolenic acid (C~18:3~ n-6)-rich oil produced using the filamentous fungus *Mucor circinelloides* and its production lasted from 1985 until 1990 \[[@B10-marinedrugs-17-00119]\]. Oleaginous microalgae constitute microscopic bio-factories that are capable of producing elevated amounts of oil which can be used as feedstock for omega-3 PUFAs \[[@B11-marinedrugs-17-00119]\]. A number of algal species, such as *Nitzschia*, *Navicula*, *Nannochloropsis*, *Phaeodactylum*, and *Porphyridium*, have been identified as producers of omega-3 PUFAs \[[@B6-marinedrugs-17-00119],[@B12-marinedrugs-17-00119]\]. The utilization of oleaginous microalgae for microbial oil production has many advantages over other non-conventional sources; they attain higher lipid productivity than plants, allow for easy scale-up of upstream and downstream processing, and are less influenced by seasonal or climatic variations. In addition, oleaginous microalgae can utilize numerous low-cost renewable substrates for growth and lipid accumulation \[[@B13-marinedrugs-17-00119]\]. Finally, as opposed to fungi, which form mycelia, microalgae are made of single cells, facilitating their handling in large-scale cultivations \[[@B14-marinedrugs-17-00119]\]. Diatoms are one of the most productive classes of microalgae and can easily adapt to environmental changes \[[@B15-marinedrugs-17-00119]\]. Almost all diatom species can grow photoautotrophically, with a few of them able to grow heterotrophically. *Phaeodactylum tricornutum* utilizes glucose in the presence of light and glucose makes up to 90% of the carbon assimilated into biomass under exponential growth \[[@B16-marinedrugs-17-00119]\]. Transcriptome analysis revealed no correlation between the expression of membrane glucose transporters and light or glucose exposure. Hence, the inability of *P. tricornutum* to grow on glucose in the dark was attributed to the low expression of glucose transporters \[[@B16-marinedrugs-17-00119]\]. As a result, *P. tricornutum* represents a facultative mixotroph that cannot grow heterotrophically on organic carbon in the absence of light. More recently, *P. tricornutum* was shown to grow heterotrophically in the dark on various organic carbon sources following the engineered introduction of glucose transporters \[[@B17-marinedrugs-17-00119]\]. The production of biomass and lipids from microalgae is strongly affected by the mode of cultivation as microalgae can be cultivated photoautotrophically, mixotrophically, heterotrophically, and photoheterotrophically \[[@B18-marinedrugs-17-00119]\]. Heterotrophic and mixotrophic conditions are considered advantageous over photoautotrophic cultivation due to the higher productivity, lipid concentration, and lipid content that can be achieved \[[@B19-marinedrugs-17-00119]\]. However, the high cost associated with organic carbon sources is a major bottleneck for the commercialization of the above process. The use of non-edible lignocellulosic materials and industrial waste as sources of sugars could reduce overall production costs, thereby aiding the transition to large-scale lipid production \[[@B13-marinedrugs-17-00119]\]. Among the various alternatives, wood biomass is an excellent option for countries such as Sweden with as high as 53.1% of its land covered by forests, a thriving forest-based industry, and sustainable forest management \[[@B20-marinedrugs-17-00119]\]. Norway spruce (*Picea abies*) and birch (*Betula pendula* and *Betula pubescens*) account for 40.8% and 12.4%, respectively, of the total standing volume of forest (3490 million m^3^sk) \[[@B20-marinedrugs-17-00119]\]. As these two tree species represent characteristic examples of hardwood and softwood, we developed a novel hybrid organosolv--steam explosion method that yields fractionated, pretreated solids with high cellulose content \[[@B21-marinedrugs-17-00119],[@B22-marinedrugs-17-00119]\]. The ensuing hydrolysates were effectively utilized for the heterotrophic growth of *Auxenochlorella protothecoides* by our group, producing very high lipid yields \[[@B23-marinedrugs-17-00119]\]. Here, we intended to evaluate the use of these hydrolysates for the production of fatty acids of nutraceutical value as an alternative and novel approach. To this end, we developed a cost-effective process for the cultivation of *P. tricornutum*, a species capable of producing EPA-rich lipids, on forest hydrolysates. To the best of our knowledge, this is the first report of forest biomass being used for the production of nutraceutical fatty acids from microalgae. 2. Results and Discussion {#sec2-marinedrugs-17-00119} ========================= 2.1. Effect of Various Initial Glucose Concentrations on the Growth and Lipid Accumulation of P. tricornutum under Mixotrophic Cultivation {#sec2dot1-marinedrugs-17-00119} ------------------------------------------------------------------------------------------------------------------------------------------ Photoautotrophic cultivation requires light as an energy source, CO~2~ as an inorganic carbon source, and nutrients to support microalgae growth \[[@B24-marinedrugs-17-00119]\]. However, some microalgae show enhanced growth under mixotrophic cultivation whereby photoautotrophic and heterotrophic metabolism are simultaneously active, allowing the use of organic carbon sources and, consequently, higher lipid and biomass productivity \[[@B25-marinedrugs-17-00119]\]. To compare the effect of photoautotrophic versus mixotrophic cultivation on biomass and lipid accumulation by *P. tricornutum*, different initial levels of glucose ranging from 0 g/L (control) to 10 g/L were added to basal F/2 medium with yeast extracted as an organic nitrogen source, while the photoautotrophic cultivation was performed with sodium nitrate as an inorganic nitrogen source instead of yeast extract. The C/N ratio was adjusted to 20 and each tube was inoculated with a 10% volume of exponentially growing seed culture. The results for cell dry weight, biomass yield, total lipid concentration, lipid yield, and lipid content of *P. tricornutum* grown on various initial concentrations of glucose are presented in [Table 1](#marinedrugs-17-00119-t001){ref-type="table"}. The highest cell dry weight (4.32 ± 0.32 g/L) and biomass productivity (0.332 ± 0.009 g/L/d) along with total lipid concentration (1.16 ± 0.23 g/L) and lipid productivity (0.089 ± 0.002 g/L/d) were observed when *P. tricornutum* was supplemented with 10 g/L glucose. Overall, biomass concentration increased from 3.38 ± 0.16 g/L to 4.32 ± 0.32 g/L and biomass productivity increased from 0.260 ± 0.003 g/L/d to 0.332 ± 0.009 g/L/d when microalgae were shifted from 2 g/L to 10 g/L glucose ([Table 1](#marinedrugs-17-00119-t001){ref-type="table"}). However, biomass yield based on glucose consumption followed an opposite trend, decreasing from 1.69 ± 0.19 g/g~substrate~ (2 g/L glucose) to 1.34 ± 0.19 g/g~substrate~ (10 g/L glucose). Moreover, an increase in glucose concentration above 2 g/L resulted in a significant portion of it (up to 68% of the initial input) to remain untouched ([Table 2](#marinedrugs-17-00119-t002){ref-type="table"}). Lipid concentration increased with increasing glucose concentration from 0.88 g/L (2 g/L glucose) to 1.08 g/L (4 g/L glucose), while lipid yield was significantly reduced from 0.44 ± 0.09 g/g~substrate~ to 0.34 ± 0.08, respectively; beyond 4 g/L of glucose, any further increase had only a minor impact on lipid concentration ([Table 1](#marinedrugs-17-00119-t001){ref-type="table"}). The lower biomass and lipid yields, together with the unconsumed glucose had a negative impact on the economics of the process and thus the addition of 2 g/L was chosen for further experiments. The addition of even this small amount of glucose increased the cell dry weight and lipid concentration by approximately 1.34 and 1.54 times, respectively, compared to the culture without any sugar source (glucose). The result obtained with inorganic nitrogen in the photoautotrophic cultivation showed a lower amount of cell dry weight (0.89 ± 0.11 g/L) and lipid concentration (0.20 ± 0.06 g/L) than those obtained with yeast extract as an organic nitrogen source in mixotrophic cultivation without glucose ([Table 1](#marinedrugs-17-00119-t001){ref-type="table"}). Mixotrophic growth of the obligate photoautotrophic microalga *P. tricornutum* in the presence of various organic carbon sources has been described previously \[[@B15-marinedrugs-17-00119],[@B19-marinedrugs-17-00119],[@B26-marinedrugs-17-00119]\]. Liu et al. (2009) reported that when *P. tricornutum* was cultivated photoautotrophically, maximum biomass was 460 ± 3 mg/L which was increased to 555 ± 10 mg/L, 587 ± 8 mg/L, and 713 ± 11 mg/L under mixotrophic growth on 100 mM glucose, acetate, and glycerol, respectively, however, the cultivation conditions were different from this study \[[@B19-marinedrugs-17-00119]\]. In another study, when *P. tricornutum* was cultivated on the soluble fraction of raw potato under a semi-continuous mode, cell productivity was 2.4 times higher than with photoautotrophic cultivation \[[@B27-marinedrugs-17-00119]\]. 2.2. Effect of Various C/N Ratios on the Growth and Lipid Accumulation of P. tricornutum {#sec2dot2-marinedrugs-17-00119} ---------------------------------------------------------------------------------------- Lipid synthesis in oleaginous microorganisms depends on cultivation conditions and, most prominently, on the metabolic accessibility of nutrients. These microorganisms can synthesize elevated quantities of lipids under different stress conditions, including abiotic stress \[[@B28-marinedrugs-17-00119]\]. The most common abiotic stress is deficiency of a key nutrient together with excess amounts of carbon, which then triggers lipid accumulation pathways \[[@B29-marinedrugs-17-00119]\]. In general, lipid accumulation is significantly affected by the quality and quantity of key nutrients such as carbon, nitrogen, phosphorus, and sulfur as well as their ratios (C/N, C/P, C/S) \[[@B29-marinedrugs-17-00119]\]. Accordingly, after optimizing the initial concentration of glucose in the medium for maximum biomass yield (g/g), the lipid concentration was optimized by varying the concentration of nitrogen and subsequently the C/N ratio ([Figure 1](#marinedrugs-17-00119-f001){ref-type="fig"}). An increase in the C/N ratio had a negative impact on the cell dry weight, which decreased from 3.38 ± 0.16 g/L (C/N 20) to 2.32 ± 0.13 g/L (C/N 100) ([Figure 1](#marinedrugs-17-00119-f001){ref-type="fig"}), while the lipid concentration increased from 0.88 ± 0.11 g/L (C/N 20) to 1.21 ± 0.22 g/L (C/N 60). However, any further increment in the C/N ratio resulted in a decrease in lipid concentration ([Figure 1](#marinedrugs-17-00119-f001){ref-type="fig"}). Given that the highest lipid content (38.53 ± 0.87% *w*/*w*) was attained at C/N 60, this ratio was selected as optimal for the subsequent batch cultivation of the algae using wood hydrolysates. An analogous positive effect of lower nitrogen concentration on lipid production was observed previously during photoautotrophic growth of *P. tricornutum* \[[@B30-marinedrugs-17-00119]\]. Nitrogen starvation is regarded as the best strategy to enhance lipid accumulation in oleaginous microorganisms \[[@B31-marinedrugs-17-00119]\]. Under nitrogen-limiting conditions, microalgae undergo rapid metabolic remodelling as nitrogen plays an important role in the synthesis of proteins, chlorophyll, and nucleic acids. However, the molecular synthesis of fatty acids differs from species to species and depends mainly on cultivation mode \[[@B30-marinedrugs-17-00119]\]. Diatoms are heterokont algae and are believed to have originated from secondary endosymbiotic events whereby a photoautotrophic alga was engulfed by a heterotrophic eukaryotic host \[[@B32-marinedrugs-17-00119]\]. Nitrogen uptake in diatoms is similar to that in other photoautotrophs. The assimilated nitrogen in the form of nitrate is reduced to nitrite by a cytosolic NADH-dependent nitrate reductase and transported to the chloroplast where it is reduced further to ammonium by a cyanobacterium-like ferredoxin-dependent nitrite reductase \[[@B33-marinedrugs-17-00119],[@B34-marinedrugs-17-00119]\]. Ammonium is then assimilated into amino acids and other nitrogenous compounds by the joint action of plastid-localized glutamine synthetase (GSII) and glutamate synthase (GOGAT) \[[@B34-marinedrugs-17-00119]\]. In diatoms, carbon metabolism and nitrogen assimilation are linked via the tricarboxylic acid (TCA) cycle through 2-oxoglutarate and oxaloacetate \[[@B35-marinedrugs-17-00119]\]. Amino acid catabolism provides several intermediates for the TCA cycle which generates acetyl CoA which is used in fatty acid elongation \[[@B35-marinedrugs-17-00119]\]. 2.3. Mixotrophic Cultivation of P. tricornutum on Wood Hydrolysates {#sec2dot3-marinedrugs-17-00119} ------------------------------------------------------------------- *P. tricornutum* can assimilate organic carbon through mixotrophic cultivation \[[@B36-marinedrugs-17-00119]\]. The use of glucose derived from a renewable and non-edible resource such as lignocellulosic biomass can serve as an excellent way to promote low-cost commercial scale PUFA production. Here, we selected two sources of forest biomass to study the mixotrophic cultivation of *P. tricornutum* for EPA production, namely birch and spruce woodchips. To overcome the natural recalcitrance of lignocellulosic biomass, we improved the release of monomeric sugars by applying a pretreatment hybrid organosolv--steam explosion step \[[@B21-marinedrugs-17-00119],[@B22-marinedrugs-17-00119]\] prior to enzymatic hydrolysis. As a result, 77.07 g/L and 64.70 g/L of glucose were produced after enzymatic hydrolysis of birch and spruce, corresponding to 89% and 80.9% cellulose hydrolysis \[[@B23-marinedrugs-17-00119]\], respectively. [Figure 2](#marinedrugs-17-00119-f002){ref-type="fig"} shows a time-course of *P. tricornutum* grown mixotrophically using pure glucose (GFM; glucose, 2 g/L; C/N 60), birch (BH; glucose, 2 g/L; C/N 60), or spruce hydrolysate (SH; glucose, 2 g/L; C/N 60) and a comparison with the cultivation where no sugar source (i.e., glucose) was used. Cell dry weight, lipid concentration, and lipid content were 3.15 ± 0.53 g/L, 1.21 ± 0.19 g/L and 38.41 ± 0.21% *w*/*w*, respectively, when this alga was grown in pure glucose (GFM; glucose, 2 g/L; C/N 60) (see [Table 2](#marinedrugs-17-00119-t002){ref-type="table"}). These values were higher than those obtained with mixototrophic cultivation without any sugar source (glucose): 2.55 ± 0.24 g/L, 0.59 ± 0.25 g/L and 23.13 ± 0.32% *w*/*w*, respectively ([Figure 2](#marinedrugs-17-00119-f002){ref-type="fig"}A). The highest cell dry weight and lipid concentrations were 3.31 ± 0.28 g/L and 1.29 ± 0.18 g/L, respectively, for spruce hydrolysate and 3.23 ± 0.32 g/L and 1.26 ± 0.11 g/L, respectively, for birch hydrolysate ([Table 2](#marinedrugs-17-00119-t002){ref-type="table"}). The results obtained with birch and spruce hydrolysates were similar to those attained with pure glucose, demonstrating the superiority of the hybrid organosolv--steam explosion method as it results in readily fermentable hydrolysates. The highest biomass (0.254 ± 0.007 g/L/d) and lipid (99.23 ± 1.09 mg/L/d) productivities were obtained when *P. tricornutum* was grown on spruce hydrolysate ([Table 2](#marinedrugs-17-00119-t002){ref-type="table"}). 2.4. EPA and DHA Production under Photoautotrophic and Mixotrophic Cultivation {#sec2dot4-marinedrugs-17-00119} ------------------------------------------------------------------------------ *P. tricornutum* has substantial potential for the synthesis of long-chain omega-3 PUFAs of nutraceutical value and particularly EPA \[[@B37-marinedrugs-17-00119]\]. Generally, EPA content is dependent on the carbon source provided in mixotrophic culture \[[@B36-marinedrugs-17-00119]\]. The fatty acid profile of lipids extracted from *P. tricornutum* grown photoautotrophically and mixotrophically on glucose, birch, and spruce hydrolysates is presented in [Table 3](#marinedrugs-17-00119-t003){ref-type="table"}. Under photoautotrophic conditions, the fatty acid profile consists mainly of C~14:0~ (8.24%), C~16:0~ (15.39%), C~18:0~ (4.32%), C~16:1~ (17.23%), and C~18:1~ n9t (15.21%), with EPA (C~20:5~) and DHA (C~22:6~ n3) amounting to 13.43% and 1.65%, respectively ([Table 3](#marinedrugs-17-00119-t003){ref-type="table"}). The fatty acid profile was changed when the cultivation was carried out with yeast extract as organic nitrogen without using any sugar source under mixotrophic condition in which the main fatty acids were C~16:0~ (13.62%), C~16:1~ (13.62%), and C~18:1~ n9t (18.3%), with EPA (C~20:5~) and DHA (C~22:6~ n3) amounting to 14.0% and 1.71%, respectively. A switch of cultivation at C/N 20 with glucose (2 g/L) had a positive impact on EPA and DHA content which increased to 16.76% and 2.82%, respectively ([Table 3](#marinedrugs-17-00119-t003){ref-type="table"}). The shifting of cultivation from autotrophic mode to mixotrophic mode showed increment in the amount of PUFAs from 16.40% to 18.91% while declining the amount of saturated fatty acids (SFAs) from 30.27% to 25.56%. The overall quantities of saturated (SFAs) and monounsaturated fatty acids (MUFAs) were similar in both mixotrophic cultivation modes (with and without sugar source), whereas the PUFAs content increased from 18.91% to 22.12% in mixotrophic cultivation with 2 g/L glucose (GFM; C/N 20) ([Table 3](#marinedrugs-17-00119-t003){ref-type="table"}). Under nitrogen-limiting conditions (C/N 60), EPA and DHA contents increased further to 18.38% and 3.56%, respectively, as did MUFAs and PUFAs compared to C/N 20 ([Table 3](#marinedrugs-17-00119-t003){ref-type="table"}). A possible explanation for the high amount of MUFAs in nitrogen-limiting conditions is the elevated synthesis of SFAs in the early stationary phase; these SFAs (stearic acid; C~18:0~) are then converted to MUFAs (oleic acids, C~18:1~) in later stages by Δ9 desaturase, which adds double bonds \[[@B38-marinedrugs-17-00119],[@B39-marinedrugs-17-00119]\]. The addition of extra Δ9 bonds by the specific monooxygenase system requires 1 mole of O~2~ to desaturate one mole of C~18:0~ \[[@B40-marinedrugs-17-00119]\]~.~ This adaptation prevents the accumulation of reactive oxygen species under stress conditions \[[@B41-marinedrugs-17-00119]\]. The antioxidative property was verified by overexpressing Δ9 fatty acid desaturase in *Cryptococcus curvatus* (CBS 570) \[[@B42-marinedrugs-17-00119]\], whereas the oxidative process was proved using a Δ9 desaturase inhibitor which prevents the conversion of C~18:0~ to C~18:1~ \[[@B43-marinedrugs-17-00119]\]. *P. tricornutum* was cultivated under various conditions of salinity, temperature, nitrogen concentration, and light intensity to evaluate the growth, fatty acid profile, and DHA/EPA ratio \[[@B44-marinedrugs-17-00119]\]. The results were similar to the ones reported here, showing that nitrogen limitation in *P. tricornutum* increased the amount of SFA and MUFAs yet lowered PUFAs \[[@B44-marinedrugs-17-00119]\]. In contrast to our work, Yodsuwan et al. (2017) reported that *P. tricornutum* synthesized more EPA under nitrogen sufficient conditions \[[@B30-marinedrugs-17-00119]\]. The fatty acids profiles that were obtained on BH (C/N, 60) and SH (C/N, 60) were similar to those obtained on GFM at C/N 60 ([Table 3](#marinedrugs-17-00119-t003){ref-type="table"}). A low DHA/EPA ratio (\<0.50) was proposed to have a beneficial effect on mitigating high-fat diet-induced liver damage in mice, whereas a DHA/EPA ratio of 0.50 could alleviate inflammatory risk factors \[[@B45-marinedrugs-17-00119]\]. In the present study, the effect of various modes of cultivations on the DHA/EPA ratio are presented in [Table 3](#marinedrugs-17-00119-t003){ref-type="table"}. The DHA/EPA ratio increased when cultivation was shifted from photoautotrophic to mixotrophic. The DHA/EPA ratio was highest when *P. tricornutum* was grown in spruce hydrolysate (0.25) as compared to phototrophycally grown cultures (0.12). As DHA and EPA are required to maintain membrane function in microalgae, an increased DHA/EPA ratio might reflect a response to oxidative stress \[[@B44-marinedrugs-17-00119]\]. The DHA/EPA ratio has many regulatory effects on animal metabolisms such as visual, neurological, and the cardiovascular system \[[@B45-marinedrugs-17-00119],[@B46-marinedrugs-17-00119]\], while there are only few reports on the effect of the DHA/EPA ratio in microalage. The concentration, yield, and productivity of EPA and DHA by *P. tricornutum* cultivated under photoautotrophic and mixotrophic conditions on various substrates are presented in [Table 4](#marinedrugs-17-00119-t004){ref-type="table"}. The highest EPA concentration (256.32 mg/L), yield (77.43 mg/g~dry\ biomass~), and productivity (19.69 mg/L/d) were obtained on spruce hydrolysates and were higher than the corresponding values achieved via mixotrophic cultivation without glucose (79.80 mg/L, 31.66 mg/g~dry\ biomass~, and 6.14 mg/L/d, respectively) and also with photoautotrophic cultivation (26.86 mg/L, 30.17 mg/g~dry\ biomass,~ and 2.44 mg/L/d, respectively) ([Table 4](#marinedrugs-17-00119-t004){ref-type="table"}). Similar results were obtained with DHA concentration (63.08 mg/L), yield (19.05 mg/g~dry\ biomass~), and productivity (4.85 mg/L/d), all of which were higher than under mixotrophic growth without glucose (9.75 mg/L, 3.86 mg/g~dry\ biomass~, and 0.75 mg/L/d, respectively) and under photoautotrophic cultivation (3.30 mg/L, 3.70 mg/g~dry\ biomass~, and 0.30 mg/L/d, respectively). Thus, a switch from photoautotrophic to mixotrophic conditions with glucose had a significant impact on the productivity of EPA and DHA, representing an important step towards the commercial production of EPA and DHA from *P. tricornutum*. In another study, *P. tricornutum* was cultivated mixotrophically on fructose and glycerol in fed-batch and semi-continuous modes. The highest biomass and EPA productivities (1.5 g/L/d and 40 mg/L/d, respectively) were observed in semi-continuous cultures on glycerol \[[@B15-marinedrugs-17-00119]\]. In another work, EPA productivity was 54.8 mg/L/d using the same strain grown photoautotrophically in a 200-L horizontal photobioreactor \[[@B36-marinedrugs-17-00119]\]. Cultivation conditions other than carbon and nitrogen sources have also been seen to affect EPA content in *P. tricornutum*. For example, the addition of light (photosynthetically active radiation) with UV at 8.22 W/m^2^ to photoautotrophically cultivated *P. tricornutum* CS29 increased EPA and DHA from 16.59% and 1.28% to 19.84% and 1.35%, respectively \[[@B47-marinedrugs-17-00119]\]. *P. tricornutum* UTEX 640 cultivated in an airlift tubular photobioreactor outdoors reached a maximum concentration and productivity of EPA of 423 mg/L and 13 mg/L/d, respectively \[[@B48-marinedrugs-17-00119]\]. Hamilton et al. (2016) created a transgenic heterotrophic strain of *P. tricornutum* by co-expressing a ∆5 elongase from *Ostreococcus tauri* and glucose transporter from the moss *Physcomitrella patens* to enhance the accumulation of long-chain omega-3 PUFAs. They reported that the transgenic strains (Pt_Elo5) and (Pt_HElo5_5) those grown in the presence of light and 1% glucose resulted in a lower EPA content (15.9% and 19.0%, respectively) yet a higher DHA content (6.3% and 7.5%, respectively) than the wild-type strain (24.7% and 1.8%) \[[@B17-marinedrugs-17-00119]\]. Moreover, strain Pt_HElo5_5 synthesized little EPA (3.4 µg/mg dry weight) and DHA (1 µg/mg dry weight) when grown heterotrophically on 1% glucose \[[@B17-marinedrugs-17-00119]\]. From these results, we can conclude that mixotrophic cultivation is the most suitable mode of cultivation for the synthesis of high amounts of EPA and DHA, especially when employing wild-type strains. 2.5. Pigment Composition in the Lipids Obtained during Mixotrophic Cultivation of P. tricornutum on Wood Hydrolysates {#sec2dot5-marinedrugs-17-00119} --------------------------------------------------------------------------------------------------------------------- Besides EPA and DHA, photosynthetic pigments produced by marine microalgae are also of strong interest to researchers for their medicinal or health benefits in antioxidative responses, immunomodulation, retinal degradation, muscular dystrophy, and cancer prevention \[[@B49-marinedrugs-17-00119]\]. *P. tricornutum* is a known producer of fucoxanthin which induces G1 cell-cycle arrest and can be utilized to treat cancer \[[@B49-marinedrugs-17-00119]\]. However, the presence of chlorophyll, carotenoids, tocopherols, and phospholipids in edible oils has been shown to increase their shelf-life when stored in the dark and prevent them from autooxidation due to antioxidant and prooxidant activity \[[@B50-marinedrugs-17-00119]\]. A high ratio of carotenoid to total chlorophyll, including a high ratio of *chl* a to *chl* b, in microalgae grown under nitrogen starvation conditions is responsible for a reduction in light-harvesting complexes and the accumulation of lipids to protect cells against oxidative stress \[[@B51-marinedrugs-17-00119]\]. *Chl* a, *chl* b, and carotenoids were determined in *P. tricornutum* grown photoautotrophically and mixotrophically on GFM, BH, and SH ([Figure 3](#marinedrugs-17-00119-f003){ref-type="fig"}). *P. tricornutum* cultivated under photoautotrophic conditions had the highest amount of *chl* a (42.37 µg/mL) and *chl* b (22.02 µg/mL), with the corresponding values in mixotrophically grown algae being considerably lower ([Figure 3](#marinedrugs-17-00119-f003){ref-type="fig"}). In contrast, the amount of carotenoids was similar under both autotrophic and mixotrophic conditions, ranging from 16.47 µg/mL to 18.12 µg/mL ([Figure 3](#marinedrugs-17-00119-f003){ref-type="fig"}). *Chl* a, *chl* c, and carotenoids are the main photosynthetic pigments, with *chl* a in particular acting as a reaction center for the light harvesting complex of *P. tricornutum* \[[@B52-marinedrugs-17-00119]\]. Similarly to our present results, previous photoautotrophic cultivation of *P. tricornutum* yielded higher amounts of *chl* a and *chl* b than mixotrophic cultivations with glucose, glycerol, and acetate \[[@B19-marinedrugs-17-00119]\]. Glucose has been reported to lower the evolution of photosynthetic O~2~ as a result of reduced activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as well as photosystem II reaction centre protein D1 in *Galdieria sulphuraria* \[[@B53-marinedrugs-17-00119]\]. Moreover, limited nitrogen availability has been shown to reduce the synthesis of photosynthetic pigments and proteins in *P. tricornutum* CCMP 2561 \[[@B54-marinedrugs-17-00119]\] and similar results were observed in our study during mixotrophic cultivation of *P. tricornutum* under the nitrogen-limited condition (see [Figure 3](#marinedrugs-17-00119-f003){ref-type="fig"}). 3. Materials and Methods {#sec3-marinedrugs-17-00119} ======================== 3.1. Materials {#sec3dot1-marinedrugs-17-00119} -------------- *P. tricornutum* SAG 1090-6 was procured from the Culture Collection of Algae (international acronym SAG) at Göttingen University, Germany. All the chemicals, including cultivation media and Guillard's (F/2) marine water enrichment solution (G0154), were purchased from Sigma Aldrich (St. Louis, MO, USA). Artificial seawater was prepared using reed aquarium salts (Tetra marine sea salt, Melle, Germany) in distilled water. Yeast extract (total N 11.6% and amino N 6.2%) used as an organic nitrogen source (Cat. No. 92144) was procured from Sigma, Aldrich, (St. Louis, MO, USA). All solvents and reagents were of HPLC grade. 3.2. Medium and Culture Conditions {#sec3dot2-marinedrugs-17-00119} ---------------------------------- The green microalga *P. tricornutum* was maintained at 16 °C on F/2 agar plates containing tetracycline (20 µg/mL) to eliminate bacterial contamination. Once new colonies appeared on the plate, they were streaked on new agar plates without antibiotic. Initially, the microalga was grown photoautotrophically in a photobioreactor (Multi-Cultivator MC 1000-OD; Photon Systems Instruments, Drasov, Czech Republic) on F/2 marine water enrichment solution and sodium nitrate as inorganic nitrogen source under 14/10-h light/dark regimen (intensity of 100 μmol/m^2^/s^1^) at 20 ± 1 °C. Salinity and pH were adjusted to 28 ppt and 8.0 prior to sterilization, respectively, and aeration was provided by air bubbling at atmospheric pressure. The F/2 marine water enrichment solution had the following composition (mg per liter): NaNO~3~, 75; NaH~2~PO~4~, 4.411; FeCl~3~. 6H~2~O, 3.15; CuSO~4~.5H~2~O, 0.01; ZnSO~4~.7H~2~O, 0.022; MnCl~2~.4H~2~O, 0.18; Na~2~MoO~4~.2H~2~O, 0.006; CoCl~2~.6H~2~O, 0.01; biotin, 0.005; EDTA disodium.2H~2~O, 4.36; thiamine.HCl, 0.1; vitamin B12, 0.005 mg. The culture conditions that were used in this study, such as light intensity, salinity, temperature, and pH, were optimized earlier for the maximum biomass and lipid accumulation by Qiao et al. (2016) \[[@B44-marinedrugs-17-00119]\]. 3.3. Optimization of Biomass and Lipid Production under Mixotrophic Cultivation {#sec3dot3-marinedrugs-17-00119} ------------------------------------------------------------------------------- For mixotrophic cultivation, cells were harvested from the photoautotrophically grown cultures by centrifugation at 3000× *g* for 10 min under aseptic conditions. The algal pellet was washed with 0.9% saline (NaCl) to remove residual medium and cell density was adjusted to 6.9--9.2 × 10^8^ cells/mL using 0.9% saline. All mixotrophic cultivations were carried out in a photobioreactor (see [Section 3.2](#sec3dot2-marinedrugs-17-00119){ref-type="sec"}) consisting of eight test tubes of 85 mL capacity. Cultivation conditions were akin to the photoautotrophic ones except for the addition of glucose as a carbon source to F/2 medium. To examine the effect of initial glucose on biomass and lipid accumulation under the mixotrophic mode, five different glucose concentrations, ranging from 2 g/L to 10 g/L, were added at 2 g/L steps to basal F/2 medium (designated as glucose F/2 medium, GFM). The C/N ratio was adjusted to 20 by adding an appropriate amount of yeast extract on the basis of total N present in yeast extract (11.6% of total nitrogen with 6.2% amino N) and total C of the sugar source. One experiment was also performed without the addition of any sugar source in yeast extract-based F/2 medium and was considered the control experiment for mixotrophic cultivation without sugar source (glucose). After optimization of biomass production under different glucose concentrations, lipid production was optimized at varying C/N ratios (20, 40, 60, 80, and 100) by adjusting the amount of yeast extract. 3.4. Batch Cultivations of P. tricornutum Using Hydrolysates from Organosolv-Pretreated Birch and Spruce Woodchips {#sec3dot4-marinedrugs-17-00119} ------------------------------------------------------------------------------------------------------------------ Birch and spruce woodchips (milled to \<1 mm) were pretreated using a hybrid organosolv--steam explosion method, as described previously \[[@B21-marinedrugs-17-00119],[@B22-marinedrugs-17-00119]\]. More specifically, birch was pretreated at 200 °C for 15 min with 60% *v*/*v* ethanol and 1% w/w~biomass~ H~2~SO~4~ for 15 min, whereas spruce was pretreated at 200 °C for 30 min with 52% *v*/*v* ethanol and 1% w/w~biomass~ H~2~SO~4~. At the end of pretreatment, the solids were separated from the liquor, washed with ethanol, and air-dried. Enzymatic hydrolysis of pretreated solids was carried out as described previously \[[@B23-marinedrugs-17-00119]\]. Briefly, 100 g of a 10% *w*/*w* solids solution in 50 mM citrate-phosphate buffer (pH 5) was prepared in 500 mL Erlenmeyer flasks. The commercial Cellic CTec2 solution (Novozymes A/S, Bagsværd, Denmark) was applied at an enzyme load of 20 FPU/g~solids~ and hydrolysis was performed for 48 h at 50 °C and 180 rpm. At the end of hydrolysis, the liquid was separated from the solids by centrifugation and glucose concentration was determined by HPLC \[[@B23-marinedrugs-17-00119]\]. Subsequently, birch (BH) and spruce (SH) hydrolysates were mixed with basal F/2 medium to achieve the glucose concentration known to be optimal for maximum biomass and lipid yield. Yeast extract was used to achieve the desired C/N ratio. Batch mixotrophic cultivations were carried out in a 1.9-L flat-panel airlift-photobioreactor (Labfors 5, Infors AG, Basel, Switzerland). Light intensity was adjusted to 100 µmol/m^2^/s^1^ by an LED panel (adjustable 260 water-cooled high-power LEDs) under a 14/10-h light/dark regimen. The other culture conditions such as pH and salinity of medium were adjusted to 8.0 and 28 ppt while the temperature was adjusted to 20 ± 1 °C. Sterile air was sparged at atmospheric pressure throughout the experiments. Samples from the photobioreactor were taken on a regular basis to determine cell density, cell dry weight, lipid content, and residual glucose in the medium. 3.5. Analytical Methods {#sec3dot5-marinedrugs-17-00119} ----------------------- During cultivation in the Multi-Cultivator MC 1000-OD, cell growth was monitored every 10 min by in situ density measurements at 680 nm and 730 nm. Cell dry weight (g/L) was determined gravimetrically after centrifuging 10 mL of culture broth at 4000× *g* for 10 min. The cell pellet was washed twice with distilled water to remove residual medium and dried in an oven at 55 °C until a constant weight was attained. Biomass productivity was calculated by the following equation:$$B = \frac{\left( {W2 - W1} \right)}{\left( {T2 - T1} \right)}$$ where *B* is biomass productivity (g/L/day) and *W*1 and *W*2 are cell dry weights (g/L) on days *T*1 (start point of cultivation) and *T*2 (endpoint of cultivation), respectively. The lipids were extracted from the dried biomass according to the protocol described in Patel et al., 2018. Briefly, the dried biomass from 25 mL of culture broth was mechanically crushed using a mortar and pestle to form a fine powder, followed by extraction with a solvent mixture (chloroform:methanol, 2:1 *v*/*v*) overnight at room temperature, with constant shaking. The slurry was filtered using a 0.22-μm filter and the solvent containing the lipids was transferred to pre-weighed glass vials. The glass vials were dried under vacuum and weighed to estimate the total lipid concentration (g/L). Lipid content (% *w*/*w*) on the basis of cell dry weight (g/L) was calculated by the following equation: $$L = \frac{TLC}{CDW}$$ where *L* is lipid content (% *w*/*w*) and *TLC* and *CDW* are total lipid concentration (g/L) and cell dry weight concentration (g/L), respectively. Lipid productivity (*P*), expressed in mg/L/day, was calculated by the following equation: $$P = \frac{L \times B}{100}$$ Residual glucose from the mixotrophic cultivations was measured by HPLC (PerkinElmer, Waltham, MA, USA) equipped with a refractive index detector and a Bio-Rad Aminex HPX-87N column (BioRad, Hercules, CA, USA). The column was maintained at 85 °C and 0.01 M Na~2~HPO~4~ was used as the mobile phase at a flow rate of 0.6 mL/min. Sugar consumption was calculated with the following equation:$$S = \frac{Gt1 - Gt2}{Gt1} \times 100$$ where *S* is the % sugar consumption, *Gt*1 is the amount of initial sugar added (g/L), and *Gt*2 is the residual sugar left at each sampling time. Photosynthetic pigments (*chlorophyll a*, *chlorophyll b*, and *carotenoids*) were analyzed by harvesting 2 mL of culture (wet biomass) followed by the addition of methanol (2 mL) and incubation at 45 °C for 24 h. Cell debris were removed by centrifugation and the supernatant was used to measure absorbance at 665.2 nm, 652.4 nm, and 470 nm with a UV/visible spectrophotometer. The amounts of pigment were determined using the following equations: where *A* is absorbance at a particular wavelength \[[@B55-marinedrugs-17-00119]\]. The lipid profile of this alga grown on various substrates was estimated on the basis of its transesterified products by GC-FID. Total extracted lipids were transesterified with an acid catalyst (8 mL of 6% methanolic H~2~SO~4~), as described previously \[[@B23-marinedrugs-17-00119]\]. The obtained fatty acid methyl esters (FAMEs) were analyzed by GC-FID (Agilent, Santa Clara, CA, USA) using a capillary column (Select FAME; dimensions 50 m × 0.25 mm ID and 0.25 μm film thickness) under previously reported operating conditions \[[@B23-marinedrugs-17-00119]\]. 3.6. Statistical Analysis {#sec3dot6-marinedrugs-17-00119} ------------------------- Data are expressed as means ± standard deviation of three independent recorded values as all experiments were repeated three times. One-way analysis of variance (ANOVA) using Microsoft Office Excel 2016 (Microsoft, Redmond, WA, USA) with *p* \< 0.05 was used for data acceptance. 4. Conclusions {#sec4-marinedrugs-17-00119} ============== The present study shows that mixotrophic growth of *P. tricornutum* led to higher EPA and DHA productivity than photoautotrophic growth. The use of nitrogen-limiting conditions further increased EPA and DHA content in the obtained lipids. Birch and spruce hydrolysates can serve as an excellent source of glucose for *P. tricornutum*, enabling approximately 3.11- and 3.2-times higher EPA productivity, respectively, than during photoautotrophic cultivation. To the best of our knowledge, this is the first report where forest biomass is used for microalgae growth and production of nutraceutical lipids. Accordingly, forest biomass can serve as a novel low-cost renewable resource for microalgae PUFA production. We would like to acknowledge Kentaro Umeki and Albert Bach Oller from Energy Engineering, Division of Energy Science, Department of Engineering Sciences and Mathematics, Luleå University of Technology, Luleå, Sweden for providing the GC-FID apparatus. Conceptualization, A.P., U.R., P.C., and L.M.; Methodology, A.P., K.H., and L.M.; Experimental work, A.P., K.H., and L.M.; Analysis of the results, A.P. and L.M.; Writing---Original Draft Preparation, A.P.; Writing---Review and Editing, U.R., P.C., and L.M. The current research was funded by Bio4Energy, a strategic research environment appointed by the Swedish government. The authors declare no conflict of interest. {#marinedrugs-17-00119-f001} {#marinedrugs-17-00119-f002} {#marinedrugs-17-00119-f003} marinedrugs-17-00119-t001_Table 1 ###### Comparison of photoautotrophic and mixotrophic cultivations and the effect of different initial concentrations of glucose on cell dry weight (g/L), total lipid concentration (g/L), lipid content (% *w*/*w*), biomass yield (g/g~substrate~), lipid yield (g/g~substrate~), and residual glucose (g/L) of *P. tricornutum* after 312 h of culture. Initial Glucose Concentration (g/L) in GFM Cell Dry Weight (g/L) Biomass Productivity ^\#^ (g/L/d) Lipid Concentration (g/L) Lipid Content (%, *w*/*w*) Lipid Productivity ^\#^ (g/L/d) Biomass Yield (g/g~substrate~) Lipid Yield (g/g~substrate~) Residual Glucose Concentration (g/L) -------------------------------------------- ----------------------- ----------------------------------- --------------------------- ---------------------------- --------------------------------- -------------------------------- ------------------------------ -------------------------------------- Photoautotrophic cultivation 0.89 ± 0.11 0.081± 0.001 0.20 ± 0.06 22.47 ± 0.23 0.018 ± 0.001 \- \- \- 0 (control) 2.52 ± 0.14 0.193 ± 0.002 0.57 ± 0.09 22.62 ± 0.28 0.043 ± 0.007 \- \- \- 2 3.38 ± 0.16 0.260 ± 0.003 0.88 ± 0.11 26.03 ± 0.45 0.067 ± 0.009 1.69 ± 0.19 0.44 ± 0.09 0.00 ± 0.00 4 4.10 ± 0.21 0.315 ± 0.005 1.08 ± 0.12 26.34 ± 0.21 0.083 ± 0.001 1.31 ± 0.21 0.34 ± 0.08 0.86 ± 0.17 6 4.14 ± 0.31 0.318 ± 0.008 1.12 ± 0.21 27.05 ± 0.71 0.086 ± 0.002 1.29 ± 0.13 0.35 ± 0.07 2.80 ± 0.23 8 4.24 ± 0.19 0.326 ± 0.004 1.15 ± 0.17 27.12 ± 0.87 0.088 ± 0.001 1.19 ± 0.21 0.32 ± 0.04 4.45 ± 0.41 10 4.32 ± 0.32 0.332 ± 0.009 1.16 ± 0.23 26.85 ± 0.76 0.089 ± 0.002 1.34 ± 0.19 0.36 ± 0.09 6.78 ± 0.37 *^\#^* The total biomass productivity and lipid productivity were calculated when the cell dry weight reached its highest value. marinedrugs-17-00119-t002_Table 2 ###### Quantitative estimation of cell dry weight, total lipid concentration, lipid content, biomass productivity, and lipid productivity of *P. tricornutum* cultivated under photoautotrophic and mixotrophic mode on various substrates. Parameters Photoautotrophic Cultivation GFM (C/N, 60) BH (C/N, 60) SH (C/N, 60) ----------------------------------- ------------------------------ --------------- --------------- --------------- Cell dry weight (g/L) 0.89 ± 0.11 3.15 ± 0.53 3.23 ± 0.32 3.31 ± 0.28 Biomass Productivity ^\#^ (g/L/d) 0.081± 0.001 0.242 ± 0.005 0.248 ± 0.004 0.254 ± 0.007 Lipids concentration (g/L) 0.20 ± 0.06 1.21 ± 0.19 1.26 ± 0.11 1.29 ± 0.18 Lipid content (%, *w*/*w*) 22.47 ± 0.23 38.41 ± 0.21 39.00 ± 0.23 38.97 ± 0.43 Lipids productivity ^\#^ (mg/L/d) 18.18 ± 0.34 93.07 ± 0.68 97.00 ± 0.85 99.23 ± 1.09 *^\#^* The total biomass productivity and lipid productivity were calculated when the cell dry weight reached its highest value. marinedrugs-17-00119-t003_Table 3 ###### Analysis of fatty acids profile of *P. tricornutum* cultivated under photoautotrophic and mixotrophic mode on various substrates. Fatty Acids (%) in Total Lipid Photoautotrophic Cultivation Mixotrophic Cultivation Without Glucose GFM; C/N 20 GFM; C/N 60 BH; C/N 60 SH; C/N 60 ------------------------------------------ ------------------------------ ----------------------------------------- ------------- ------------- ------------ ------------ ----------- ------- ----------- ------- ----------- ------- ----------- **Saturated Fatty Acids (SFAs)** (C~14:0~) 8.24 **30.27** 7.18 **25.56** 2.60 **24.62** 2.9 **26.33** 3.1 **27.1** 2.65 **26.13** (C~16:0~) 15.39 13.62 12.53 11.01 13.23 12.32 (C~18:0~) 4.32 2.96 1.72 3.8 2.89 2.65 (C~20:0~) 2.32 1.80 3.54 3.65 3.43 3.87 (C~24:0~) \- \- 4.23 4.97 4.45 4.64 **Mono Unsaturated Fatty Acids (MUFAs)** (C~16:1~) 17.23 **34.56** 13.62 **34.23** 15.99 **34.48** 17.17 **38.78** 17.65 **39.12** 17.87 **39.61** (C~18:1~ n9t) 15.21 18.3 15.37 16.96 16.34 16.87 (C~18:1~ n9c) 2.12 2.31 3.12 4.65 5.13 4.87 **Poly Unsaturated Fatty Acids (PUFAs)** (C~18:2~ n6c) 1.32 **16.40** 3.20 **18.91** 2.54 **22.12** 2.8 **24.74** 2.56 **26.68** 2.71 **27.47** (C~18:3~ n3) \- \- \- \- \- \- (C~20:5~ n3) EPA 13.43 14.0 16.76 18.38 19.80 19.87 (C~22:6~ n3) DHA 1.65 1.71 2.82 3.56 4.32 4.89 **DHA/EPA** 0.12 0.12 0.17 0.19 0.22 0.25 **Total fatty acids** 81.23 78.70 81.22 89.85 92.9 93.21 marinedrugs-17-00119-t004_Table 4 ###### EPA and DHA concentration (mg/L) and productivity (mg/L/d) by *P. tricornutum* cultivated under photoautotrophic and mixotrophic cultivation mode on various substrates. Parameters Photoautotrophic Mode of Cultivation Mixotrophic Mode of Cultivation ------------------------------------ -------------------------------------- --------------------------------- -------- -------- -------- -------- **Total EPA concentration (mg/L)** 26.86 79.80 147.48 222.39 249.48 256.32 **EPA yield (mg/g~dry\ biomass~)** 30.17 31.66 43.63 70.60 77.23 77.43 **EPA productivity (mg/L/d)** 2.44 6.14 11.31 17.07 19.15 19.69 **Total DHA concentration (mg/L)** 3.30 9.75 24.82 43.08 54.43 63.08 **DHA yield (mg/g~dry\ biomass~)** 3.70 3.86 7.34 13.67 16.85 19.05 **DHA productivity (mg/L/d)** 0.30 0.75 1.91 3.32 4.18 4.85

Background {#Sec1} ========== Pest activities are one of the major problems associated with farming. The animal rearing and creation, as well the management of farming lands, disrupts the ecological stability that regulates potential pest species \[[@CR1]\]. Insects and other arthropods are particularly problematic pests worldwide. In Brazil, where agriculture is the main source of income, insect-pests cause an average annual loss of 7.7% in crop production (US\$ 17.7 billion), resulting in the reduction of approximately 25 million tons of food, fiber, and biofuels \[[@CR2]\]. Chemical pesticides are still the usual method for arthropod-pests control causing great concern, in view of the known negative side effects to humans, animals and the environment. Thus, the development of safer and environmentally compatible new pest control tools is pivotal \[[@CR3]\]. Entomopathogenic fungi are complex organisms that use a myriad of strategies to achieve a successful infection and can be used to control the major arthropod pests of agriculture, as well as vectors of diseases. Among the most commonly entomopathogenic fungi applied in biological control are the species from *Metarhizium* genus, particularly *Metarhizium anisopliae* \[[@CR2]\]. The infection cycle of *M. anisopliae* begins when viable conidia attach to the host cuticle. Under favorable conditions, the conidia germinate and develop the appressorium, a specialized infection structure, in order to transpose the host cuticle barrier. Once into the host hemocoel, hyphae differentiate into blastospores, unicellular infection structures that help in host colonization by fungal dispersion, leading the host to death. After host death, the fungus switches for a saprophytic state, in order to consume the host body and produce new conidia \[[@CR4]\]. In recent years, genome sequencing, RNA-seq, and comparative genomic analyses have been used for an exploratory view of the genomes and for the discovery of new virulence determinants in *Metarhizium* spp. \[[@CR5]--[@CR7]\]. However, the still limited knowledge about *Metarhizium*-host interactions is one of the factors that limit in-depth entomopathogenic application for control of economic important arthropods species. DNA methylation of cytosine bases is a heritable epigenetic mark and an important mechanism to control gene expression. DNA methylation is regarded as a key and stable mechanism to repress gene transcription \[[@CR8]\]. Striking, different isoforms of DNA methyltransferases (DNMTs) are enrolled in the process. These enzymes catalyze the transfer of methyl groups to cytosine bases, leading to the formation of 5-methylcytosine (5mC) \[[@CR8]\]. The presence and genome pattern distribution of 5mCs have been explored in several fungal species, including the *Metarhizium robertsii* \[[@CR9]\]. Remarkably, 5mC patterns in fungal genomes fluctuate from low levels (1.8% *Ganoderma sinense* \[[@CR10]\]) to almost undetectable levels in *Magnaporthe oryzae* (0.22% \[[@CR11]\]) and *M. robertsii* (0.38 to 0.42%). However, these lower levels of DNA methylation still significantly affect the fungal fitness. In *M. oryzae*, DNMT null mutant strains showed defects in asexual reproduction. In addition, such strains displayed an imbalance of transposable elements silencing \[[@CR11]\]. Moreover, *M. robertsii* DNMT knockout strains showed similar defects in asexual reproduction (e.g., defects in conidial production), vegetative growth, and virulence \[[@CR12]\]. In view of DNA methylation importance in several organisms, including species in the *Metarhizium* genus, it is reasonable to expect that this epigenetic mark can regulate major steps, as well as virulence determinants, during entomopathogenic infection. Thus, we explored DNA methylation patterns in *M. anisopliae* during two very distinct conditions: fungal growth over cattle-tick cuticles (i. e., mimicking an infection condition that have been useful for induction of virulence determinants) and in complete rich medium (i. e., a saprophytic growth condition with abundance of nutrients). Additionally, we compared the Bisulfite sequencing (BS-Seq) results with previous RNA-seq data obtained in the same experimental conditions and the results were further confirmed employing quantitative reverse transcription PCR (RT-qPCR) in the presence or absence of DNMT inhibitor. The results here demonstrate that more regions are methylated under the mimicked infection condition. Additionally, we suggest a putative role for DNA methylation repressing putative virulence factors during the transition between virulent and saprophytic states during *M. anisopliae* infection cycle. Results {#Sec2} ======= Global mapping of DNA methylation in rich medium (saprophytic-like condition) and tick cuticles (mimicked infection condition) {#Sec3} ------------------------------------------------------------------------------------------------------------------------------ In order to understand the impact of DNA methylation in *M. anisopliae* distinct lifecycles, a BS-seq was conducted using a mimicked infection condition (*M. anisopliae* growth in Tick Cuticles; 48hTC) and a control, saprophytic-like condition (*M. anisopliae* growth in Rich Medium; 48hRM). The experiments herein analyzed followed the recommendations of the Standards and Guidelines for Whole Genome Shotgun Bisulfite Sequencing of the NIH Roadmap Epigenomics Mapping Consortium, which suggests the use of at least two biological replicates with an average coverage of at least 30 times \[[@CR13]\]. Two biological replicates were used from each condition and, after trimming and performing quality controls, an average of 6.5 million and 3.36 million clean paired-end reads were obtained for 48hRM and 48hTC, respectively. Mapped sequencing coverage had an average of 51 times for 48hRM and 31 times for 48hTC. The cytosines present in genome were detected with a high coverage (91.03% for 48hRM and 85.17% for 48hTC). Notably, a higher proportion of the identified methylated sites was found in the 48hTC condition (0.89% of total cytosines detected) compared to the 48hRM condition (0.60% of total cytosines detected). For the 48hRM condition, most methylated sites were found at CHH residues (60.44%), followed by CpG sites (21.25%) and CHG sites (18.31%). For the 48hTC condition, a similar scenario was found, with 61.88% of methylated sites occurring at CHH residues, followed by CpG (20.23%) and CHG (17.89%) sites (Table [1](#Tab1){ref-type="table"}). Table 1Patterns of putative 5mCs sites distribution in the conditions evaluatedCpGCHGCHHTOTAL48hTC0.50%\* (20.23%\*\*)0.55%\* (17.89%\*\*)1.15%\* (61.88%\*\*)0.89%\*\*\*48hRM0.40%\* (21.25%\*\*)0.45%\* (18.31%\*\*)1.85 %\* (60.44%\*\*)0.60%\*\*\*\*Percentage of putative 5mCs sites across the genome normalized by the total number of Cs in a context-dependent fashion;\*\*Percentage of residues predominance among the putative 5mCs sites identified;\*\*\* Percentage of putative 5mCs sites across the genome normalized by the total number of Cs in genome Identification and functional prediction of putatively methylated mRNA genes {#Sec4} ---------------------------------------------------------------------------- A stringent criterion was used to evaluate potentially methylated genes. It consisted in the identification of 5mCs in the open reading frames (ORFs) of each gene and their respective 500 bp flanking regions. Only sequences spanning an average of 20 5mCs identified were considered methylated. In both conditions (48hTC and 48hRM), a total of 670 protein-coding genes attended such criteria (Fig. [1](#Fig1){ref-type="fig"}a and Additional file [1](#MOESM1){ref-type="media"}). Accordingly, besides more methylated sites, the 48hTC condition showed more putative methylated genes (i. e., 390 mRNA genes were uniquely methylated in the 48hTC condition) when compared with 48hRM (i. e., 135 mRNA genes were uniquely methylated in the 48hRM) with 145 mRNA genes methylated in both conditions (Fig. [1](#Fig1){ref-type="fig"}a). However, no differences could be found in the content of methylation in these 145 putatively methylated genes when the two conditions were compared. Fig. 1Putatively methylated mRNA genes and GO enrichment analysis. **a** Venn diagram depicting the set of methylated genes in 48hTC and 48hRM. **b** Seventy-three GO terms were over-represented, with 55 GO terms in the 48hTC condition, 9 GO terms in the 48hRM condition and 9 GO terms in both conditions. **c** Venn diagram depicting the set of enriched GO terms in 48hTC and 48hRM To functionally characterize the set of mRNA genes putatively methylated, the predicted proteins were analyzed for the presence of conserved domains using the NCBI Conserved Domain Database (CDD). A small fraction (\~ 5.1%) of the putatively methylated protein coding genes did not presented an associated predicted domain (Additional file [2](#MOESM2){ref-type="media"}). Furthermore, the three most abundant domains refer to Adenylate forming domain (cl17068), ABC ATPase superfamily (cl 25,403), and Acyl transferase domain (cl08282), all of which related to synthesis of secondary metabolites (Additional File [2](#MOESM2){ref-type="media"}). Gene Ontology (GO) enrichment analysis revealed that 73 GO terms were over-represented among the methylated mRNA genes (Fig. [1](#Fig1){ref-type="fig"}b and Additional file [3](#MOESM3){ref-type="media"}). Notably, 55 GO terms were uniquely found in the 48hTC condition, 9 GO terms were uniquely found in the 48hRM condition and 9 GO terms were found in both conditions (Fig. [1](#Fig1){ref-type="fig"}b and c). There were several GO terms over-represented in 48hTC linked to cell remodeling (GO:0071554, GO:0071555, GO:0071852, and GO:0031505), and regulation of response to stimulus (GO:0065007, GO:0050794, GO:0050789, GO:0031326, GO:0070887, GO:0060255, GO:0050896, GO:0031323, GO:0019222, GO:0003006, GO:0051234, GO:0051716, GO:0042221, GO:0044419, and GO: 0009605 (Fig. [1](#Fig1){ref-type="fig"}b). Furthermore, GO terms linked to cytoskeleton morphogenesis appeared on both conditions: actin process and organization in 48hTC (GO:0030029 and GO:0030036) and microtubule organization in 48hRM (GO:1902850, GO:0000226, GO:0007017 and GO:0007018), although the GO terms are not shared between both condition (Fig. [1](#Fig1){ref-type="fig"}b). Thus, the results indicate that DNA methylation can regulate genes related to fungal cell morphogenesis and stimuli processing in *M. anisopliae* and DNA methylation can, potentially, affect the transition between specialized infection structures during arthropod colonization. DNA methylation and secondary metabolite backbone genes {#Sec5} ------------------------------------------------------- Secondary metabolites (SMs) are small molecules with a myriad of biological activities and applications. In fungi, the genes required for biosynthesis of SMs are usually found arranged in co-regulated biosynthetic gene clusters (BGCs), which contains backbone genes (e. g., polyketide synthases \[PKS\], non-ribosomal peptide synthetases \[NRPS\], hybrids \[PKS-NRPS\] and terpene cyclases \[TCs\]), as well as adjacent genes that assist in metabolite maturation \[[@CR14], [@CR15]\]. Notably, GO terms associated to SM biosynthesis were found in 48hTC (GO:0019438 \[aromatic compound biosynthetic process\], GO:0044281 \[small molecule metabolic process\], GO:0017144 \[drug metabolic process\], GO:1901362 \[organic cyclic compound biosynthetic process\] and GO:1901360 \[organic cyclic compound metabolic process\]). Thus, the methylation pattern of the backbone genes from 73 BGCs found in *M. anisopliae* strain E6 was inferred from the BS-Seq results (Fig. [2](#Fig2){ref-type="fig"}; BGC/Backbone gene nomenclature follows our previous report \[[@CR7]\]). Additionally, since backbone gene decreased expression led to decreased compound synthesis \[[@CR16]\], the exploration of the methylated pattern of the backbone gene, as well as transcription activity of the backbone gene can be a indicator of BGC active/inactive state. In the 48hTC condition, 14 backbone genes were putative methylated, while 9 backbone genes were putative methylated in 48hRM condition and 21 backbone gene were putative methylated in both conditions, which correspond to near 60% of the total of BGCs found in *M. anisopliae* genome (Fig. [2](#Fig2){ref-type="fig"}a). As we previously generated RNA-seq data using the same experimental design used to acquire the BS-seq data (i. e., 48hRM and 48hTC, each in biological duplicates) \[[@CR6]\], the expression of the putative methylated backbone genes was inferred from the RNA-seq data, looking for possible correlations between methylated state and expression profile. Ten out of 44 BGCs ( 22.7%) displayed detectable expression in the RNA-seq data (RPKM ≥2), but there were no statistical differences (ND) between conditions (Fig. [2](#Fig2){ref-type="fig"}b and Additional file [4](#MOESM4){ref-type="media"}). Three out of 44 ( 6.8%) putative methylated BGCs were down-regulated (Down) in the RNA-seq data (Fig. [2](#Fig2){ref-type="fig"}b and Additional file [4](#MOESM4){ref-type="media"}). Additionally, from those 73 BGCs originally identified, 15 BGCs were up-regulated (Up) in the comparison 48hRM x 48hTC, indicating a bigger expression in the 48hTC condition, as previously described \[[@CR7]\]. Six out of 44 (13.6%) putative methylated BGCs were among those up-regulated in the RNA-seq data (Fig. [2](#Fig2){ref-type="fig"}b and Additional file [4](#MOESM4){ref-type="media"}). Noteworthy, 25 out of 44 BGCs (56.8%) did not have detectable expression (NE) in the RNA-seq data in both conditions (RPKM \< 2) (Fig. 2B and Additional file 4). However, it is important to notice that nearly half of the BGCs (among the 73 identified) were silent under the conditions evaluated in the RNA-seq \[[@CR7]\]. In this way, the results suggest that DNA methylation can be important to regulate the silent state of these biosynthetic pathways. Fig. 2The impact of DNA methylation on secondary metabolite backbone genes. **a** Venn diagram depicting the set of putatively methylated SM backbone genes in 48hTC and 48hRM. **b** Expression and differential expression profile of the 44 putatively methylated backbone genes on the comparison 48hRM x 48hTC performed by \[[@CR6]\]. BGC/backbone gene nomenclatures were extracted from \[[@CR7]\]. Up: up-regulated; Down: down-regulated; ND: no difference; NE: not expressed Pattern of expression of the putative methylated mRNA genes inferred from the RNA-seq data {#Sec6} ------------------------------------------------------------------------------------------ We extended the evaluation of the patterns of expression using the RNA-seq data for all putative methylated protein coding genes found. To be classified as differentially expressed, the genes must display a fold change of at least of 1 with FDR corrected *p*-value lower than 0.01, when considered the comparison between the conditions herein analyzed (48hRM and 48hTC). A total of 474 out of 670 (\~ 70%) putative methylated protein coding genes displayed detectable expression in the RNA-seq data (RPKM ≥2), but there were no statistical differences between conditions (48hRM x 48hTC) (Additional file [4](#MOESM4){ref-type="media"}). This contrasts with the subset of putative methylated BCGs backbone genes, which did not display detectable expression in the RNA-seq data (Fig. [2](#Fig2){ref-type="fig"}b). Noticeably, the methylated genes that fall in the ND category are abundant in all conditions (\~ 73% for protein coding genes only methylated in the 48hTC condition, \~ 66% for protein coding genes only methylated in the 48hRM condition and \~ 67% for protein coding genes methylated in both conditions) (Additional file [4](#MOESM4){ref-type="media"}). Additionally, a total of 79 out of 670 (\~ 12%) putative methylated protein coding genes did not have detectable expression in the RNA-seq data (RPKM \< 2), a total 40 out of 670 (\~ 6%) putative methylated mRNA genes were down-regulated in the RNA-seq data and 77 out of 670 (\~ 11%) putative methylated mRNA genes were up-regulated in the RNA-seq data (Additional file [4](#MOESM4){ref-type="media"}). Although, the vast majority of putative methylated mRNA genes were expressed, a clear pattern of down-regulation or up-regulation linked to DNA methylation could not be observed. As previously reported by Li and coworkers (2017) for *Metarhizium robertsii*, DNA methylation at the putative promoter or gene ORFs does not always imply transcriptional changes. Additionally, promoter methylation can even enhance gene expression \[[@CR9]\]. Evaluation of putative methylated genes expression using RT-qPCR {#Sec7} ---------------------------------------------------------------- To validate the results from BS-seq, we selected seven genes to further analyze by RT-qPCR. These genes belong to three different categories: (I) genes putatively methylated; (II) genes with methylation sites but under the established cut-off of 20; and (III) DNMT genes from *M. anisopliae* genome (whose orthologs were previously functionally characterized in *M. robertsii* \[[@CR12]\]). The putatively methylated mRNA genes have been chosen based on the RNA-seq data and putative importance on *Metarhizium* biology. MANI_024437 is the backbone gene for the destruxin BGC (MaNRPS1) \[[@CR7]\], which was strongly up-regulated in the comparison 48hRM x 48hTC (Fig. [2](#Fig2){ref-type="fig"}b and Additional file [4](#MOESM4){ref-type="media"}) and it was putatively methylated in 48hRM and 48hTC conditions (Additional file [1](#MOESM1){ref-type="media"}). MANI_023437 is another backbone gene, which codes for a protein putatively enrolled in the biosynthesis of a xenolozoyenone-like metabolite \[MaNRPS-PKS3\]) \[[@CR7]\]. MANI_023437 was down-regulated in the comparison 48hRM x 48hTC (Fig. [2](#Fig2){ref-type="fig"}b) and it was putative methylated in 48hRM and 48hTC conditions(Additional file [1](#MOESM1){ref-type="media"}). MANI_111160 codes for a collagen-like protein (*Mcl1*), a known virulence determinant \[[@CR17]\] and MANI_026638 codes for a putative chitin synthase enrolled in cell wall morphogenesis. Both genes (MANI_111160 and MANI_026638) did have detectable expression in the RNA-seq data (RPKM ≥2), but there was no statistical difference between the experimental conditions (Additional file [4](#MOESM4){ref-type="media"}). Additionally, while MANI_026638 was putative methylated in 48hRM and 48hTC, MANI_111160 was only putative methylated in the 48hTC condition (Additional file [1](#MOESM1){ref-type="media"}). As a control for the methylation cut-off, MANI_017257, which codes a putative exo-beta-1,3-glucanase from family 17 of glycoside hydrolases, was included in the analysis. To gain information on how the DNA methylation can affect gene expression when the fungus was grown on tick cuticles as the sole carbon source (the condition with the greatest number of putative methylated mRNA genes), DNMT activity was inhibited by adding 5-Azacytidine (5-AZA) to the cultures. The gene expression patterns of the seven chosen genes were explored using three incubation periods (24, 48, and 72 h), which spans the period between the early interaction between fungal cells and tick cuticles to the establishment of the infection. The results obtained with 5-AZA treatment support the BS-Seq results (Fig. [3](#Fig3){ref-type="fig"}). For all chosen identified methylated genes, 5-AZA treatment led to increased expression in, at least, one of the incubation times analyzed (Fig. [3](#Fig3){ref-type="fig"}). For MANI_111160, 5-AZA treatment led to increased expression in 24 h (Fig. [3](#Fig3){ref-type="fig"}). For MANI_024437 and MANI_111160, 5-AZA treatment led to increased expression in 48 h (Fig. [3](#Fig3){ref-type="fig"}). Strikingly, for MANI_023437, 5-AZA treatment led to increased expression in all times explored (Fig. [3](#Fig3){ref-type="fig"}). Remarkably, no statistically significant differences were found for MANI_017257 with and without 5-AZA treatment (Fig. 3) supporting the cut-off previously established. Moreover, both DNMTs analyzed did not show statistically significant expression differences with the 5-AZA's treatment, showing that, at least, when the fungus is grown with tick cuticles as the sole carbon source, a potential negative feedback, induced by DNA methylation, may not happen (Fig. [4](#Fig4){ref-type="fig"}). Fig. 3The impact of 5-Azacytidine treatment on methylated genes expression. Quantitative real time RT-PCRs of MANI_024437 (Destruxin synthetase); MANI_023437 (Xenolozoyenone-like polyketide synthase); MANI_111160 (Collagen-like protein *Mcl1*); MANI_026638 (Class 2 chitin synthase) and MANI_017257 (GPI-anchored cell wall beta-1,3-endoglucanase) were performed after growth of *M. anisopliae* E6 with *R. microplus* cuticles, as the sole carbon and nitrogen source, for 24, 48 and 72 h with and without 200 mM of 5-azacytidine (an DNMT inhibitor) supplementation. The results were processed according to 2^-ΔCt^ method and relative transcript levels were normalized with beta-tubulin (MANI_018534). Data are shown as the mean ± SD from three experimental replicates of three biological replicates. \* *p* \< 0.05; \*\* *p* \< 0.01 Fig. 4The impact of 5-Azacytidine treatment on DNMTs expression. Quantitative real time RT-PCRs of MANI_011878 (DNA cytosine-5-methyltransferase) and MANI_017005 (RID1 DNA methyltransferase) were performed after growth of *M. anisopliae* E6 with *R. microplus* cuticles, as the sole carbon and nitrogen source, for 24, 48 and 72 h with and without 200 mM of 5-azacytidine (an DNMT inhibitor) supplementation. The results were processed according to 2^-ΔCt^ method and relative transcript levels were normalized with beta-tubulin (MANI_018534). Data are shown as the mean ± SD from three experimental replicates of three biological replicates Discussion {#Sec8} ========== Entomopathogenic fungi and arthropod-pathogenic fungi from *Metarhizium* genus are cosmopolitan species that can survive on soil (as a saprophyte) as well as infect arthropods (as a pathogen) and plants (as an endophyte) \[[@CR4], [@CR18]\]. The adaptation to different niches and hosts needs different repertoires of genes, effector molecules and cellular structures. During a saprophytic growth under a carbon and nitrogen rich medium (e. g., MCc), the expression of virulence determinants, only required for host infection, should be, theoretically, not induced. On the other hand, during pathogen-arthropod interaction, to attain a successful infection, *Metarhizium* spp. should switch between different specialized infection structures, up-regulate virulence determinants as well as keep a tight control of endogenous resources to avoid death by starvation. The impact of the epigenetic machinery, specifically DNA methylation, in the lifecycle of fungal species from *Metarhizium* genus have started to be addressed in *M. robertsii* \[[@CR9]\]. Exploring the changes in the methylation pattern between the conidia and mycelia stages, Li and coworkers (2017) showed that approximately 0.38% of the total number of cytosines were putatively methylated in conidia, while 0.42% of the total number of cytosines were putatively methylated in mycelia \[[@CR9]\]. However, the impact of DNA methylation in the infection process was not evaluated in *M. robertsii*. In the experiments conducted here, we started to address this problem, employing a mimicked infection condition that has been used before and a saprophytic-like condition as a control \[[@CR6], [@CR19]--[@CR21]\]. Noteworthy, previous results have shown that virulence determinants were up-regulated in the mimicked infection condition \[[@CR6], [@CR7]\]. Remarkably, when compared to the results of Li and coworkers (2017), more putatively methylated sites were found in *M. anisopliae* strain E6 (0.60--0.89%), suggesting that different conditions can greatly influence the methylation patterns. Furthermore, species-specific factors can also influence methylation, as previously observed, the methylation pattern between different species can differ markedly \[[@CR22]\]. Noteworthy, although more putative methylated sites were found in *M. anisopliae*, the proportion of putative methylated sites in the CHH, CpG and CHG residues were similar between the *Metarhizium* spp., with \~ 57, 23 and 20% of the methylation sites in CHH, CpG and CHG residues, respectively, in *M. robertsii*; compared to \~ 61, 21 and 18% in *M. anisopliae*. Furthermore, the BS-Seq results support the impact of DNA methylation modification on the modulation of *M. anisopliae* virulence. It is assumed that, in the presence of glucose, other catabolic pathways and virulence determinants should be repressed in *M. anisopliae*, as there is no need to express these genes in a nutrient rich condition. Whereas, in the infection condition, these pathways would be available, in view of host's/nutrient's complexity. However, what we found was the contrary of that hypothesis, with more genes putatively methylated in the infection condition. Among the methylated genes, two well-known virulence determinants were found: the destruxin backbone gene and the collagen-like protein MCL1. MCL1 cotes blastospores and is enrolled in evasion from host immune responses \[[@CR17]\]. It is important to note that MCL1 expression is tight controlled during the infection cycle of *M. anisopliae,* with the higher expression occurring during hemolymph colonization \[[@CR23]\]. Destruxins are important metabolites produced by fungi from *Metarhizium* genus \[[@CR24]\]. These compounds have shown insecticidal activity against several arthropod hosts \[[@CR24]--[@CR26]\] and are essential molecules for infection of some *Metarhizium* species \[[@CR7], [@CR27]\]. Moreover, the production of destruxin, evaluated by the expression of the destruxin BGC (MaNRPS1), have previously displayed a temporal pattern of regulation during the mimicked infection condition \[[@CR7]\]. In this way, it is feasible to propose that the patterns of 5mCs in virulence genes can be modulated during the infection cycle. However, more experiments are needed to confirm such suggestion. The influence of chromatin structure and epigenetic machinery on SM biosynthesis is well-known \[[@CR28], [@CR29]\]. As several backbone genes enrolled in the biosynthesis of SM are putatively methylated, it is possible that *M. anisopliae* employs DNA methylation as an additional mechanism to control SM-associated gene expression. In line with this assumption, the treatment of fungal cultures with drugs that affects the epigenetic machinery have led to isolation of new SMs in several fungal species \[[@CR29]\]. Moreover, the results herein shown that employing 5-AZA led to the up-regulation of at least two SM backbone genes. Collectively, the results suggest that culture treatment with 5-AZA may drive the isolation and characterization of new SMs from *M. anisopliae*. As the infection cycle progresses, *M. anisopliae* switches through different specialized infection structures (e.g., appressorium and blastospore) and lifestyle states (e. g., entomopathogenic and saprophytic) \[[@CR4]\]. These changes imply in transcriptional reprogramming to best-fit the different fungal requirements. Chitin synthesis and degradation are essential steps during fungal cell morphogenesis. Among the methylated genes, a chitin synthase gene was chosen for RT-qPCR evaluation. As the results showed, 5-AZA treatment led to increased expression. Moreover, the importance of the DNA methylation on cell morphogenesis is further supported by the GO analysis, reiteratively demonstrating the importance of this epigenetic modification in the lifecycle and infection of *M. anisopliae*. Conclusions {#Sec9} =========== Although several layers of gene regulation exist in *Metarhizium* spp., mimicked infection models (e. g., tick cuticles or hind wings) have been valuable conditions to induce virulence determinants expression and specialized infection structures formation \[[@CR7], [@CR30]--[@CR32]\]. In view of complexity of factors that can influence a successful outcome for *Metarhizium* spp. in the infection process, the expression of virulence determinants can be crucial even when the virulence determinants are potentially energetically expensive, as SMs \[[@CR33]\]. In the absence of host's stimuli, the energetic expenditure should be reallocated. In this scenario, the DNA methylation may be an important mechanism. Furthermore, the DNA methylation can also be important to ensure that a given gene will not be expressed outside a specific phase of infection or specific cellular structure. The results here presented provide an overview of methylation dynamics during a mimicked *Metarhizium*-host interaction, contributing with the already established foundation for this epigenetic mark in *Metarhizium* species. Methods {#Sec10} ======= Fungal culture and maintenance, samples treatment and DNA extraction {#Sec11} -------------------------------------------------------------------- *M. anisopliae* strain E6 was originally isolated from *Deois flavopicta* collected in Espírito Santo State, Brazil \[[@CR34]\]. This strain was maintained at 28 °C in solid Cove's Complete Medium (MCc), which contains (w/v) 1% glucose, 0.6% NaNO~3~, 0.15% casein hydrolysate, 0.05% yeast extract, 0.2% peptone and 1.5% Agar. After sterilization, 2% (v/v) of filter-sterilized Salts Solution (2.6% KCl, 2.6% MgSO~4~•7H~2~O and 7.6% KH~2~PO~4~ (w/v)) and 0.04% (v/v) of filter-sterilized Trace Elements Solution (0.04% Na~2~Ba~4~O~7~•7H~2~O, 0.4% CuSO~4~•5H~2~O, 0.01% FeSO~4~, 0.8% Na~2~MbO~4~•7H~2~O, 0.8% MnSO~4~•7H~2~O and 0.8% ZnSO~4~•7H~2~O (w/v)) were added to the sterilized medium prior to use (modified from \[[@CR35]\]). *Rhipicephalus microplus* cuticles were prepared by dissection of ingurgitated females, extensively washed with sterile water and sterilized by autoclaving \[[@CR31]\]. Spore suspension (5 × 10^6^ spores per mL^− 1^) was used to inoculate the cuticles by immersion for 30 s. The inoculated cuticles were disposed over 1% water agar plates and maintained for 48 h at 28 °C (mimicked infection condition, Tick Cuticle: 48hTC). Each of the two biological replicates consisted of mycelium grown over approximately 5 g (wet weight) of tick cuticles, which are disposed onto 5 Petri dishes. The comparative control condition was conducted with two biological replicates of *M. anisopliae* grown in 100 ml liquid MCc for 48 h at 28 °C (Control condition, Rich Medium: 48hRM). The resulting mycelia obtained on such conditions were grounded to powder in liquid nitrogen and DNA extraction was carried out through a standard protocol \[[@CR36]\]. Extracted DNA was subjected to RNAse treatment and stored at − 20 °C. Bisulfite conversion, library construction and sequencing {#Sec12} --------------------------------------------------------- Before bisulfite conversion, one ug of each DNA sample, referring to the two biological replicates from the two conditions herein analyzed (48hTC and 48hRM) were cleaned up with Agencourt AMPure beads XP (Beckman Coulter). Samples were quantified using a Qubit 2.0 fluorometer (Invitrogen) to estimate the DNA yield. *Escherichia coli* strain ER2925 Non-methylated Genomic DNA (Zymo Research) was spiked in each sample (1 ng of control per 200 ng of sample) to determine the bisulfite conversion efficiency and as a quality control for bisulfite treatment. Bisulfite conversion was carried out with EZ DNA Methylation-Gold™ Kit (Zymo Research) following manufacturer's instructions. The only exception was the 64 °C incubation step, which was performed for only 1.5 h instead of 2.5 h, in order to avoid DNA degradation. After bisulfite conversion, the samples were quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). The sequencing of the libraries was performed with TrueSeq DNA Methylation kit (Illumina) using as input the four bisulfite-treated single-stranded DNA samples. All the steps of the protocol were carried out following manufacturer's instructions. Quality control of the distinct libraries was performed using the 2100 Bioanalyzer System with the Agilent High Sensitivity DNA Kit (Agilent). The libraries were individually quantified via qPCR using a KAPA Library Quantification Kits for Illumina platforms (KAPA Biosystems). Using adaptors containing distinct indexes, a total of four libraries were pooled together in equimolar amounts and sequenced in a MiSeq sequencing system (Illumina) using MiSeq Reagent Kits to obtain 250 bp paired-end reads. Data treatment {#Sec13} -------------- The total output reads were deindexed (separation of reads) based upon the unique bar-codes using the BaseSpace pipeline into distinct fastQ files, corresponding to each of the four conditions herein analyzed (two biological replicates for 48hTC and two biological replicates for 48hRM). The raw paired-end reads were visually inspected with FastQC \[[@CR37]\], trimmed and subjected to quality control using Trimmomatic \[[@CR38]\] . Reads were processed to remove adapter and other illumina-specific sequences (ILLUMINACLIP:TruSeq2-PE.fa:2:20:10), the first 20 nucleotides were cropped (HEADCROP:20), the reads were limited to a size of 225 nucleotides (CROP:225), reads with Phred quality scores lower than 20 in the beginning and 25 in the end were cropped (LEADING:20 TRAILING:25) and only reads greater than 30 were considered (MINLEN:30). The resulting reads were aligned to the *M. anisopliae* strain E6 \[[@CR6]\] and *E. coli* strain ER2925 (K12 derivative) genome sequences using Bismark/ Bowtie2 \[[@CR39], [@CR40]\]. Genome coverage from each library was calculated using Coverage/Read Count Calculator (<http://apps.bioconnector.virginia.edu/covcalc/>). In order to distinguish between 5mCs and background, the observed methylation in *E. coli* ER2925 DNA was used as a background control, to provide a false-positive measurement of bisulfite treatment. The error rates, for each replicate, were determined as 0.005, 0.003, 0.006, and 0.005 for 48hRM R1, 48hRM R2, 48hTC R1 and 48hTC R2, respectively. These values represent the probability *p* (false discovery rate) on the binomial distribution *p* (*n*, *p*). Based in such probabilities, the *p*-values were calculated, and the hypothesis' tests were performed to determine the methylated cytosines, being the significance threshold set to 0.02 \[[@CR41]\]. For each sample the bisulfite conversion rate was \> 97.4%. After this procedure, the positions of 5mCs was verified in *M. anisopliae* strain E6 genome annotation \[[@CR6]\] in order to identify putative methylated genes. A generic definition of gene was used here, which expanded the ORF and the surrounding 500 bp upstream and downstream. Genes were classified as methylated if they had reads mapping for at least in average, from both biological replicates, of 20 5mCs, as previously demonstrated \[[@CR22]\]. A diagram, representing the pipeline, is presented in Additional file [7](#MOESM7){ref-type="media"}: Fig. S1. All scripts were present in as Additional file [5](#MOESM5){ref-type="media"}. Domain annotation, gene ontology and RNA-seq data {#Sec14} ------------------------------------------------- Genes identified as methylated were evaluated for the presence of conserved domains in their predicted amino acid sequences, using the Conserved Domain Database (CDD) with default parameters (only hits with *e*-value lower than 0.01 were accepted) \[[@CR42]\]. Gene Ontology (GO) terms annotation and enrichment was performed using Blast2GO v 5.0 with default parameters: (I) Blast step: *e*-value cut-off 1 × 10^− 10^; the database was the Fungi section of Swiss-Prot; recover of the 10 best hits; (II) Annotation step: cut-off 55; GO weight 5; *e*-value 1 × 10^− 6^; hit filter 500; (III) Enrichment step: only hits with false discovery rate corrected *p*-value smaller or equal to 0.05 were accepted \[[@CR43]\]. Veen diagrams were generated with Venny \[[@CR44]\]. RNA-seq data (including RPKM values for 48hRM and 48hTC) as well as differential expression (log2-fold change; 48hRM x 48hTC) were recovered from our previously published data \[[@CR6]\]. The same experimental design used for the determination of methylation pattern in saprophytic-like (48hRM) and the mimicked infection (48hTC) conditions was employed to determine the genome wide gene expression pattern \[[@CR6]\]. Ribosomal RNA-depleted RNA samples isolated from two independent biological replicates from each condition (48hRM and 48hTC) were submitted to RNA-Seq and the differential expression analysis was conducted using HT-Seq and edgeR \[[@CR6]\]. For expression analysis, genes with RPKM values ≥ 2 were considered with detectable expression. Genes were considered differentially expressed if the corresponding log2-fold change ratios were ≥ 1 or ≤ − 1, with a 5% false discovery rate corrected *p*-value smaller or equal to 0.01. Culture conditions, RNA extraction, sample treatment and RT-qPCR {#Sec15} ---------------------------------------------------------------- In order to evaluate the influence of 5mCs presence on gene expression, another independent experiment was conducted. In this experiment, *M. anisopliae* was grown in two different conditions for RNA isolation, employing three different times (24, 48 and 72 h) and three biological replicates for each time. Fungal conidia (5 × 10^6^ spores per mL^− 1^) were inoculated in 100 ml of liquid medium, which consist of *R. microplus* (1% (w/v)), as the sole carbon and nitrogen source, amended or not with 200 mM 5-azacytidine (an DNMT inhibitor). The resulting fungal math was grounded to powder in liquid nitrogen and RNA extraction was carried out using TRIzol reagent protocol (Invitrogen) and treated with RQ DNAse (Promega). Reverse transcription and cDNA synthesis were performed using ImProm-II Reverse transcriptase (Promega) using oligo-dT. The relative expression of four putative methylated genes (MANI_024437 \[Destruxin synthetase\]; MANI_026638 \[Class 2 chitin synthase\]; MANI_111160 \[Collagen-like protein *Mcl1*\]; MANI_023437 \[Xenolozoyenone-like polyketide synthase\]); one gene (MANI_017257 \[GPI-anchored cell wall beta-1,3-endoglucanase\]) in which the number of methylation sites did not reach the cut-off (\< 20 methylation sites); and the 2 DNMTs found in the genome of *M. anisopliae* strain E6 (MANI_011878 and MANI_017005) were determined by RT-qPCR (StepOne Real-Time PCR System) with an initial step of 95 °C for 10 min, followed by 50 cycles of 95 °C for 15 s, 55 °C for 15 s, and 60 °C for 60 s. A melting curve analysis was performed at the end of the reaction to confirm the presence of single PCR products. The results were processed according to the 2^-ΔCt^ method \[[@CR45]\] and relative transcription levels were normalized with beta-tubulin (MANI_018534) gene transcript levels. The primer sequences used in RT-qPCR analysis are listed in Additional file [6](#MOESM6){ref-type="media"}. RT-qPCR statistical analysis {#Sec16} ---------------------------- Data were expressed as mean ± standard deviation (SD) of replicates. All assays were performed in three experiment conditions, with technical triplicate repetitions. The Student's *t*-test was employed to test for significance between values. Comparisons of means with *p*-values ≤0.05 were considered statistically different. Supplementary information ========================= {#Sec17} **Additional file 1.** Putatively methylated genes. **Additional file 2.** CDD analysis. **Additional file 3.** GO enrichment analysis. **Additional file 4.** Expression and differential expression profile of the 670 putatively methylated mRNA genes extracted from previously published RNA-seq data. **Additional file 5.** BS-seq scripts. **Additional file 6.** The primer sequences used in RT-qPCR analysis. **Additional file 7: Figure S1.** Flowchart describing the whole BS-seq pipeline. 48hRM : Control condition, Rich Medium 48hTC : Mimicked infection condition, Tick Cuticle 5-AZA : 5-azacytidine 5mC : 5-methylcytosine BGC : Biosynthetic Gene Cluster BS-seq : Bisulfite sequencing CDD : Conserved Domain Database DNMT : DNA methyltransferase Down : Down-regulated GO : Gene Ontology MCc : Cove's Complete Medium ND : No Difference NE : Not Expressed NRPS : Non-ribosomal peptide synthetases PKS : Polyketide synthase PKS-NRPS : Hybrids (Polyketide synthase/ Non-ribosomal peptide synthetases) RPKM : Reads Per Kilobase Million RT-qPCR : quantitative reverse transcription PCR SD : Standard Deviation SM : Secondary Metabolite TC : Terpene Cyclase Up : Up-regulated **Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Nicolau Sbaraini and Reinaldo Bellini contributed equally to this work. Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1186/s12864-019-6220-1. The authors would like to thank to Luis Willian Pacheco Arge and LNCC's staff for support through experiments and manuscript preparation. Conceived and designed the experiments: NS, RB, RLMG, MHV, ATRV, AS and CCS. DNA extraction and sample treatment: NS. Bisulfite conversion, library construction and sequencing: ALG. Data treatment: RB. Domain annotation, Gene Ontology and RNA-seq data: NS, RB, RLMG and CCS. RNA extraction: NS and ABP. RT-qPCR: ABP and AWAG. Contributed with reagents/materials/analysis tools: MHV, ATRV, AS and CCS. Wrote the article: NS, RB, ATRV, AS and CCS. The manuscript has been read and approved by all named authors. This project was funded by the Advanced Network of Computational Biology (RABICÓ -  Biocomputacional Grant  23038.010041/2013-13) from CAPES. AS was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - 401983/2016--1 and 302637/2017--6). ATRV was supported by grants from CNPq (303170/2017--4) and from Fundação de Amparo a Pesquisa do Estado do RJ (FAPERJ - 26/202.826/2018). CCS was supported by CNPq grants (404141/2016--1 and 309897/2017--3). The datasets generated during the current study are available in the NCBI's repository, BioSample accessions SAMN11941230, SAMN11941231, SAMN11941232, SAMN11941233. Not Applicable. Not Applicable. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Introduction {#s0001} ============ For advanced chronic kidney disease patients who will be initiated on HD therapy soon, the type of vascular HD access remains a big challenge. Guidelines from different countries strongly recommend native arteriovenous fistula (AVF) as the best method for dialysis amongst patients who are undergoing hemodialysis \[[@CIT0001]\]. It is well established that AVF had the superiority over other types of vascular access: central venous catheter (CVC) and arteriovenous graft (AVG) since it provides the best longevity, less likely rates of infection and least association with mortality and morbidity in the majority of patients \[[@CIT0012]\]. Despite these advantages of AVF, the number of patients undergoing dialysis through CVC or AVG is high \[[@CIT0016]\]. In 2003, The Fistula First breakthrough initiative (FFBI) which was a National Access Improvement Initiative to encourage the use of AVF as vascular access in HD population. This initiative was established as a collaboration with the Centers for Medicare & Medicaid Services (CMS), the end stage renal disease (ESRD) Networks, and the entire renal community \[[@CIT0010]\]. The FFBI initial goal was to increase the percentage of native AVF use to 44%, in 2009 the percentage of HD patients having AVF was 65% which exceeded the initial goal \[[@CIT0017]\]. Meanwhile, the overall proportion of prevalent AVF utilization increased from 33% in all HD patients in 2003 to 62.7% by the mid of 2016 \[[@CIT0010],[@CIT0016]\]. However, in 2015 United States Renal Data System (USRDS) annual data reported that the percentage of patients receiving HD therapy through CVC was 80% \[[@CIT0016]\]. Achieving optimal AVF access is a complicated process and many barriers have been described, including hospital systems, HD patients, and and provider-related \[[@CIT0018]\]. According to the 2017 annual health report of the Palestinian Ministry of Health, the overall number of HD patients has increased from 1014 patients in 2015 to 1119 patients in 2016 from different 12 dialysis centers in the West Bank, Palestine \[[@CIT0018]\]. To the best of our knowledge, this is the first study in Palestine to investigate the vascular access relevant issues amongst HD patient including perceptions and barriers to AVF use. The aim of this study is to explore patients' perceptions of advantages and perceived barriers that impede AVF utilization as a first vascular access choice. Material and methods {#s0002} ==================== Study design and setting {#s0003} ------------------------ In this cross-sectional study, we investigate the attitudes toward AVF and the perceived barriers to its creation among Palestinian HD patients. We recruited all adult participants aged 18--85 years, receiving HD as outpatients from August-December of 2018 at Palestinian Medical Complex Hospital in Ramallah, Palestine which is considered one of the largest Ministry of Health dialysis units in Palestine as per the total number of patients who undergo hemodialysis weekly. Participants {#s0004} ------------ We screened 198 participants who had the diagnosis of ESRD, undergoing regularly scheduled HD sessions of Saturday--Monday--Wednesday or Sunday--Tuesday--Thursday. Exclusion criteria included pediatric age group (less than 18 years), acute dialysis; major mental or neurological illness that precludes their ability to be recruited with fully consenting; refusal to participate; death before completing their data or those who were unavailable at the time of the study. This study was carried out in accordance with the recommendations of the Al-Quds University Research Ethics Committee with written informed consent from all subjects. The protocol was approved by the Al-Quds University Research Ethics Committee. Data collection {#s0005} --------------- All participants underwent in-person interviews either before, after, or during the HD session using structured questions. Patients' medical records were all reviewed to collect their demographics and characteristics information. Demographic data collected included age, sex, weight, height, body mass index (BMI). The presence of comorbidities including diabetes mellitus, hypertension, dyslipidemia, coronary artery disease, or cerebrovascular disease was recorded. Data pertaining to the cause of ESRD: Diabetes mellitus, hypertension, polycystic kidney disease, glomerulonephritis, other, or unknown were also obtained. Information was collected regarding HD initiation, access, and attitudes toward fistula creation and use, including time in months from HD initiation, current access method and whether vein mapping was done before vascular access creation. In addition, data was gathered if patients previously received sufficient education about AVF and for those patients who did not have a fistula or had a delay in its creation, perceived barriers were explored in detail. Furthermore, patients were asked whether they recommend AVF as the preferred access to others, the reasons for their recommendation as well as the characteristics of those who refused fistula. Statistical methods {#s0006} ------------------- Data were summarized by calculating means and standard deviation (*SD*) or medians and range for quantitative variables and percentages for categorical variables. Descriptive terms were used where appropriate. The reported attitudes and perceived barriers were analyzed as categorical variables. Results {#s0007} ======= We screened 198 Palestinian patients who had the diagnosis of ESRD, and undergoing regularly scheduled HD therapy during the study period that extends from August to December of 2018. Out of them, 156 were included in our study and 42 were excluded (three were pediatric age group (less than 18 years); 2 refused to participate; 22 died before completing their data; and 15 were unavailable at the time of the study). Patient's age ranged from 18 to 85 years (*M* = 55; *SD* = 15), gender (92 males and 64 females, 59% and 41%, respectively), and 29 (19%) were smokers. Average BMI (*M* = 26; *SD* = 6). At the time of the study, patients had an average time since starting dialysis of 24 months ranged (1 to 216). Detailed demographics characteristics including the cause of ESRD and major associated comorbidities were shown in ([Table 1](#t0001){ref-type="table"}). The current access method for hemodialysis based on age group showed that AVF is highly used in patient's groups who are younger than 55 and between the age of 67 and 79. While, 60% of patients who are between 55 and 66 years use permanent CVC ([Table 2](#t0002){ref-type="table"}). Patient attitudes and perceived barriers toward AVF creation are presented in [Tables 3](#t0003){ref-type="table"} and [4](#t0004){ref-type="table"}. ###### Baseline demographics and characteristics of study participants. Patient characteristics Overall *n* = 156 ----------------------------------------- ------------------- Baseline demographics   Age (years) mean ± *SD* 55 ± 15 Gender    Male, *n* (%) 92 (59)  Female, *n* (%) 64 (41) Weight (kg) mean ± *SD* 74.2 ± 16.6 Height (m) mean ± *SD* 1.66 ± 8.5 BMI (kg/m^2^) mean ± *SD* 26 ± 6 Smoker *n* (%) 29 (19) Cause of ESRD   Diabetes mellitus *n* (%) 68 (44%) Hypertension *n* (%) 23 (15)% Adult polycystic kidney disease *n* (%) 8 (5%) Glomerulonephritis *n* (%) 21 (13%) Other *n* (%) 19 (12%) Unknown *n* (%) 17 (11%) Associated comorbidities   Diabetes mellitus *n* (%) 87 (56%) Hypertension *n* (%) 108 (69%) Dyslipidemia *n* (%) 60 (38%) Coronary artery disease *n* (%) 67 (43%) Cerebrovascular disease *n* (%) 11 (7%) Peripheral vascular disease *n* (%) 34 (22%) BMI: Body Mass Index; ESRD: End stage renal disease. ###### Current access method and duration of HD based on age group.   Age group ---------------------------------------------------- --------------- --------------- --------------- ---------- Current access method         Temporary CVC *n* (%) 1 (1.5%) 3 (5.5%) -- 1 (100%) Permanent CVC *n* (%) 21 (31.3%) 33 (60%) 14 (42.4%) -- AVF *n* (%) 43 (64.2%) 19 (34.5%) 19 (57.6%) -- AVG *n* (%) 2 (3%) 0 -- -- Time in months since HD initiation, median (range) 37.9 (1--216) 25.1 (2--108) 38.6 (1--216) -- HD: Hemodialysis; CVC: Central venous catheter; AVF: arteriovenous fistula; AVG: arteriovenous graft. ###### Perceived barriers toward AVF creation based on age group.   Age group ---------------------------------------------------------- ------------ ------------ ----------- ---------- Reported outcome         Perceived barrier to AVF[^a^](#TF3){ref-type="table-fn"}         Late referral to surgical evaluation 21 (38.2%) 14 (27.5%) 7 (25.9%) -- Refusal to undergo AVF surgery 27 (49.1%) 28 (54.9%) 17 (63%) 1 (100%) Too long to surgical appointments after referral 7 (12.7%) 9 (17.6%) 3 (11.1%) -- ^a^Out of 134 patients with non-AVF dialysis access or delayed AVF creation. ###### Attitudes toward AVF creation. Reported outcome *n* (%) ----------------------------------------------------------------- ------------ Previously received sufficient education about AVF?    Yes 72 (46)  No 84 (54) Previous Vein mapping done?    Yes 87 (56)  No 69 (44) Would you recommend AVF to other HD Patients    Yes 115 (73.7)  No 26 (16.7)  Not reported/Not certain 15 (9.6) If answer to above question is Yes, why would you recommend it?  Less infection 71 (60.2)  Easier to care for 12 (10.2)  Easier showering 10 (8.5)  Better hygiene 2 (1.7)  All above 20 (16.9)  Other/unspecified 3 (2.5) The most common cause for no AVF was the refusal to undergo AVF surgical procedures in 73 patients (54.5%) and there was no difference among those patients based on their age group, followed by late referral to a surgical evaluation in 42 patients (31.3%) and time too long to surgical appointments after referral in 19 (14.2%; [Figure 1](#F0001){ref-type="fig"}). Out of the patients who refused to undergo surgical procedures, 31 (42.5%) patients were concern about the surgical procedure itself, 17 (23.3%) have a poor understanding of their disease and access needs, 11 (15.1%) they fear of needles, 13 (17.8%) denial their disease or even their need for HD, and 1 (1.4%) patient due to other causes including cosmetics ([Table 5](#t0005){ref-type="table"}). {#F0001} ###### Characteristics of those who refused fistula or do not recommend it. Characteristics of those who refused fistula *N* (%) -------------------------------------------------------- ----------- Total number of those who refused/do not recommend AVF 73 (54.5) Reason for refusing AV fistula    Concern about the surgical procedure/refused surgery 31 (42.5)  Poor understanding of disease/access needs 17 (23.3)  Fear of needles 11 (15.1)  Denial of disease or need for HD 13 (17.8)  Others include Cosmetics reasons 1 (1.4%) HD: Hemodialysis; AVF: arteriovenous fistula. Of the overall group, 72 (46%) reported they received sufficient education and information about AVF prior to creation, on the other hand, 84 patients (54%) thought that was not the case. Vein mapping is done for 87 patients (56%) prior to an attempt for fistula creation. One hundred fifteen patients (73.7%) would strongly recommend AVF to other HD patients as a method of vascular access. Patients attributed their preferences and recommendations for AVF to many reasons including decreased risk of infection 71 (60.2%), easier to care for 12 (10.2%), emphasis on easier shower 10 (8.5%), better associated hygiene 2 (1.7%), three of the patients (2.5%) reported unspecified causes and 20 (16.9%) all of these reasons in combination ([Figure 2](#F0002){ref-type="fig"}). Overall, 26 patients (16.7%) did not recommend AVF as the method of HD access to other patients ([Table 4](#t0004){ref-type="table"}). {#F0002} Discussion {#s0008} ========== Current clinical practice guidelines from different countries strongly advocate AVF as the best vascular access since it has been considered to have the lowest risk of complications and need for interventions, best long-term patency, and superior patient survival \[[@CIT0020]\]. Having an AVF prior to the commencement of HD is not only associated with lower morbidity and mortality but it is also associated with better patient-reported quality of life and lower health care expenditure \[[@CIT0025],[@CIT0026]\]. Despite this, many patients maintained on HD therapy use CVC \[[@CIT0027]\]. A recent study conducted in the same area to investigate the rate of pre-dialysis nephrology care and AVF usage amongst 156 chronic HD patients showed a high incidence of CVC use and a relatively large portion of HD did not have any pre-dialysis nephrology care. Furthermore, a low incidence of AV utilization found even in patients who received pre-dialysis care \[[@CIT0028]\]. Investigations regarding the system, physicians and patients\' characteristics that may be responsible for delays in AVF creation remain an ongoing challenge. To the best of our knowledge, no previous studies have presented data that help address the attitudes and perceived barriers to timely AVF creation and utilization amongst HD patients in Palestine, which is the aim of this analysis. Our study found that most of HD patients believe that AVF is the best choice of vascular access for many reasons; the vast majority of them would recommend it to their fellow patients who are newly starting dialysis because the existing patients believe it is prone to lower risk of infection compared with CVC. Other reported advantages are related to quality of life, including easier care, better hygiene and easier for showering. These patients' attitudes toward AVF could be attributed to their personal experience with CVC related complications in particular infection when they used it as the method of initial HD vascular access, they may have been also influenced by the experiences of other patients who suffered from the drawbacks associated with CVC. A previous study of a national random sample of 1563 HD patients conducted in the United States to investigate the relationship of initial HD vascular access type with patient-reported health status and quality of life scores at the time of HD initiation and at day 60. Their results showed that patients with AVF at initiation and at day 60 reported perceived greater physical activity and energy, emotional and social well-being, fewer symptoms, less effect of dialysis and burden of kidney disease, and better sleep compared with patients with persistent CVC use \[[@CIT0025]\]. In addition, Do Hyoung Kima et al. investigated the effects of vascular access types on the survival and quality of life and depression in the incident hemodialysis patients among 1461 patients who newly initiated HD. The primary outcomes were all-cause mortality and HRQOL and depression. The secondary outcome was all-cause hospitalization. Kidney Disease Quality of Life Short Form 36 (KDQOL-36) and Beck's depression inventory scores were measured to assess HRQOL and depression. In the survival analysis, patients with AVF had a better survival and low hospitalization rates, and the patients with AVF or AVG showed both higher HRQOL and lower depression scores than those with CVC \[[@CIT0029]\]. In another cohort study, preferences and concerns regarding HD vascular access were reviewed by asking 128 HD patients and 64 of dialysis nurses, technicians, HD access surgeons, and nephrologists, found that the access preferred by patients was of the utilization of a superficial access in the forearm which was easy to cannulate, had minimal effect on their appearance, provided quick hemostasis after dialysis and enabled arm comfort during dialysis, whereas from their point of view, the most common problem was pain during needle insertion \[[@CIT0030]\]. In our study, we found that the most reported perceived barriers for those who have not been dialyzing through an AVF, or who had a delay in AVF creation were the refusal to undergo AVF surgery, late referral to surgical evaluation and too long to surgical appointments after referral. In a study of a cohort of 319 HD patients conducted in nine nephrology centers in New Zealand and Australia, barriers to timely AVF creation were investigated. Their results revealed that lack of formal policies for patient referral, absence of patient database for access purposes that could facilitate the management, and also long wait times to surgical evaluation and access creation were the perceived barriers to access creation \[[@CIT0031]\]. These barriers were previously implicated by nephrologists and primary care providers in a qualitative study to identify modifiable challenges to adequate preparation of patients for renal replacement therapy \[[@CIT0032]\]. With regard to the patients who refused to undergo AVF which was the main barrier in our sample (54.5%), more details were explored to find the reasons behind their refusal. Our study revealed that concern about the surgical procedure (42.5%), poor understanding of the disease or access needs, fear of needles, denial of disease or need for HD and cosmetic reasons were the most cited barriers related to the patient. In a related systematic review of qualitative studies, aiming to understand the attitudes, beliefs, preferences, and values of 375 patients who refused AVF creation or use, Xi et al., performed interviews with those patients investigating the rationale for decision making \[[@CIT0033]\]. Three main reasons that affected the decision to refuse AVF were identified: Poor previous experience with the fistula such as issues with cannulation, bleeding, or appearance, issues with knowledge transfer and informed decision making, and patient acceptance of current status quo without a desire for change. Patients can have a strong preference for the status quo and are disappointed to switch from an acute start CVC access to a long-term AVF may explain why a large number of patients in our sample refuse AVF \[[@CIT0034]\]. In contrast to our study, decreasing infection rate was not the focus of the 375 patients who refused AVF creation or use in the study of Xi et al. \[[@CIT0033]\]. The same was seen in another study that investigated patients' perspectives on complications of vascular access-related interventions and found that infectious complications were not reported as a major concern by patients when the access modalities are compared. On the other hand, physical complications which manifested as fear and pain associated with cannulation were more likely a cause of patients' dissatisfaction with AVF compared to CVC access \[[@CIT0035]\]. In another study, fear of painful and difficult cannulation and patients trust in their ability to manage complications of CVC were the reasons to avoid AVF \[[@CIT0036]\]. Nearly half of our patients reported they received insufficient education about different types of HD access and the pros and cons of each one. It was previously noted that patients with less dialysis knowledge were found to be less likely to use arteriovenous access for dialysis at initiation and transitioning to AVF after starting hemodialysis, since the poor understanding of the AVF is an important aspect regarding the barriers related to the patient \[[@CIT0037]\]. In fact, 23% of our sample reported a poor understanding of vascular access and this is a modifiable challenge which by improving patient's education may facilitate the AVF utilization \[[@CIT0038]\]. Several factors contribute to the heterogeneity of AVF prevalent use and the distribution that include the age (young vs. old) \[[@CIT0039]\]. In our study, the largest percentage of patients under the age of 55 and between 67 and 76 currently uses AVF. Conclusion {#s0009} ========== In this study among dialysis patients in Ramallah/Palestine, most participants would recommend an AVF as the mode of access. Barriers to AV use were found to be classified into three major categories; provider-related in which there is a late referral to surgical evaluation, system-based including too long to surgical appointments after referral, and issues pertaining to the patient who may refuse or be reluctant to undergo AVF surgery. The reasons that stand behind the patient's refusal of AVF were: concern about the surgical procedure, poor understanding of the disease or access needs, denial of disease or need for HD, and fear of needles. These results suggest the need for a systematic evaluation of the attitudes that precede AVF creation, to identify potential targets for care improvement such as timely referral to a surgical evaluation in addition to facilitating sufficient education about HD access methods may allow for better AVF utilization in HD patients in Palestine. Furthermore, engaging patients in care planning and decision making may improve patient knowledge about treatment options and adherence. Geolocation information {#s0010} ======================= This study conducted at the national dialysis center in Ramallah, West Bank, Palestine, which is considered one of the largest Ministry of Health dialysis units in Palestine as per the total number of patients who undergo hemodialysis weekly. Disclosure statement {#s0011} ==================== The authors declare no conflict of interest.

Introduction {#s1} ============ Aphids (Insecta: Hemiptera), a group of economically important insect pests that consume plant phloem sap, cause substantial losses of crop yield by direct feeding on host plants and by vectoring plant viruses (Dixon, [@B22]). More than 450 species within Aphididae attack agricultural and horticultural plants, of which over 100 are categorized as significant and economically important pests (Blackman and Eastop, [@B13]). While some aphids are specific to plant species in a single taxonomic family, others have an exceptionally broad host range across many plant families. Green peach aphid (*Myzus persicae*) is a generalist with a host range comprising 40 different plant families including Brassicaceae, Solanaceae, and Fabaceae. Moreover, it is the most versatile viral vector, capable of transmitting more than 100 plant viruses (Ramsey et al., [@B56]). In contrast, pea aphid (*Acyrthosiphon pisum*) feeds specifically on legumes. Despite different feeding habits, they are both classified in the tribe Macrosiphini within the subfamily Aphidinae (von Dohlen et al., [@B67]). The close relationship between the two aphids is further supported by analysis of mitochondrial and nuclear sequences as well as transcriptomic sequence comparisons (Ramsey et al., [@B56]; Kim and Lee, [@B38]). Due to the difference in host range, green peach aphids most likely ingest toxic metabolites that pea aphids would not normally encounter, such as glucosinolates in Brassicaceae and alkaloids in Solanaceae, necessitating a more complex metabolic system (Ramsey et al., [@B55]). Hemipteran immature nymphs and fully developed adults sometimes differ in their feeding behavior. *Lygus hesperus* nymphs prefer developing cotton squares, whereas adults prefer vegetative structures (Snodgrass, [@B60]). In three spittlebug species (*Aeneolamia varia, A. reducta* and *Zulia carbonaria*), foliage-feeding adults are more capable of feeding upon resistant hybrid crops than root- and stem-feeding nymphs (Cardona et al., [@B15]). Besides host and tissue preferences, quantity of food intake can vary (Banks and Macaulay, [@B6]). Profiling in nymphal and adult transcriptomes could reveal biological properties that are developmental stage-specific. In Asian citrus psyllid (*Diaphorina citri*) for instance, the transcriptome comparison revealed distinct patterns of protein and energy requirements between nymphs and adults (Vyas et al., [@B68]). This approach has also identified differentially expressed resistance/detoxification genes, e.g., cytochrome P450, glutathione S-transferase (GST), and ATP-binding cassette transporter genes from two developmental stages of a thiamethoxam-resistant strain of whitefly (Yang et al., [@B73]). Contrasting gene expression among different insect developmental stages on a large scale can not only shed light on development modulation, reproduction, and developmental stage-specific interaction with host plant, xenobiotics, and invading microbes, but can also facilitate the improvement of pest management strategies (Yang et al., [@B73]; Tian et al., [@B65]; Vyas et al., [@B68]). However, stage-specific gene expression in immature nymphs and fully developed adults has not yet been characterized in aphids. While comparative genomic sequence analysis has furnished tremendous information regarding genetic factors underlying inter-species divergence (Chinwalla et al., [@B19]; Kaufman et al., [@B36]; Kirkness et al., [@B39]; Zdobnov and Bork, [@B77]; Arensburger et al., [@B4]; Bonasio et al., [@B14]; Werren et al., [@B72]), an increasing number of studies have applied RNA-seq for this purpose, particularly in species whose genome sequences are unavailable. For example, transcriptomic comparisons have been performed between different aphids, *A. pisum* vs. *Sitobion avenae* (Wang et al., [@B69]), whitefly (*Bemisia tabaci*) species complexes Middle East-Asia Minor 1 vs. Mediterranean (Wang et al., [@B71]), ranid frogs *Rana chensinensis* vs. *Rana kukunoris* (Yang et al., [@B74]), ornamental primrose species *Primula poissonii* vs. *Primula wilsonii* (Zhang L. et al., [@B79]), and fishes, *Erythroculter ilishaeformis* vs. *Danio rerio* (Ren et al., [@B58]). Comparisons among pea aphid, green peach aphid and grain aphid *(S. avenae)* have enabled investigation of the transcriptome evolution and understanding of the differences in host plant adaptation and insecticide resistance among them (Ollivier et al., [@B50]; Ramsey et al., [@B55]; Wang et al., [@B69]). Between grain aphid and pea aphid 340 gene orthologs are considered to be under positive selection based on the rates of nonsynonymous (Ka) and synonymous (Ks) substitutions (Wang et al., [@B69]). Such orthologs were also identified when Ollivier et al. ([@B50]) compared coding sequences (CDSs) derived from the genome sequence of pea aphid and EST database derived from 5 tissues of green peach aphids reared on 5 host plants (Ramsey et al., [@B56]). Later, Ramsey et al. ([@B55]) sequenced the transcriptome from mixed stages of green peach aphids using 454 pyrosequencing. Besides the reads mapped to the existing ESTs, they obtained 47,832 additional unigenes with a mean length of 160 bp, from which they identified more detoxification genes in green peach aphid than in pea aphid (Ramsey et al., [@B55]). However, limited transcriptomic information may not fully reflect the divergence between the two species. In this study, we performed transcriptomic sequencing of green peach aphid nymphs and adults using Illumina RNA-seq technology, *de novo* assembled sequencing reads, and annotated the resulting unigenes. Gene expression profiling between nymphs and adults identified genes potentially involved in development modulation. Furthermore, comparative transcriptomic analyses identified genes unique to green peach aphid (relative to pea aphid) and orthologous gene pairs under positive selection. Data analysis has helped expose certain genetic factors underlying host plant adaptation by the two destructive aphid species. Materials and methods {#s2} ===================== Plant growth and insect rearing ------------------------------- Arabidopsis ecotype Col-0 plants were grown in LP5 potting medium (Sun Gro Horticulture, Agawam, WA, USA) in an environmental chamber at 23⋅C (day)/21⋅C (night), 65% relative humidity (RH), and a photosynthetic photon flux density of 88 μmol m^−2^ s^−1^ with a 12-h light/12-h dark photoperiod. The green peach aphid (a tobacco-adapted red lineage from Dr. Georg Jander, Boyce Thompson Institute for Plant Research, Cornell University) had been maintained on Col-0 for over 40 generations. Age-synchronized nymphs and adults were subjected to RNA extraction as described below. RNA isolation and transcriptome sequencing ------------------------------------------ Neonate nymphs (within 16 h) were placed on 4-week-old Col-0 plants for 4 or 8 days respectively. Sixty 4-day-old nymphs and 60 8-day-old adults were collected, immediately frozen in liquid nitrogen, and stored at −80⋅C for RNA extraction. Three independent biological replicates were performed for transcriptome sequencing analysis. Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNase-Free DNase (Qiagen, Valencia, CA, USA) was added to remove residual DNA. Samples were then further purified using RNeasy Mini Kit (Qiagen) according to the manufacturer\'s instructions. Purified total RNA samples were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and qualified by Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Transcriptome sequencing was performed on an Illumina HiSeq 2500 platform with 125-nucleotide (nt) paired-end reads at Texas A&M AgriLife Genomics and Bioinformatics Services (College Station, TX, USA). Sequence assembly and annotation -------------------------------- After trimming the adaptor sequences and removing short or low-quality reads (\>5% unknown nucleotides or more than 20% nts with \>10% error rate), the processed reads were assembled using Trinity software (Trinity Software, Inc., Plymouth, NH, USA) and clustered with TGICL Clustering tools (The Institute for Genomic Research, Rockville, MD, USA) (Pertea et al., [@B52]; Grabherr et al., [@B28]). The publically available databases, NCBI non-redundant (Nr), NCBI non-redundant nucleotide (Nt), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Cluster of Orthologous Groups of proteins (COG) were used to perform BLAST analyses to annotate the functions of these assembled unigenes (*E*-value cutoff of 10^−5^). Blast2GO software (<http://www.geneontology.org>) was used for gene ontology (GO) annotations (Conesa et al., [@B20]). Differential gene expression and RT-qPCR confirmation ----------------------------------------------------- Genes differentially expressed between nymphs and adults were identified based on Fragments Per Kilobase per Million mapped reads (FPKM) values, which adjusts the number of fragments mapped to a transcript by the total number of fragments mapped to all unigenes and the length of the transcript (Mortazavi et al., [@B45]; Ji et al., [@B34]). The false discovery rate (FDR) was used for the *P*-values in multiple tests and analyses. A FDR ≤ 0.001 and an absolute value of the log~2~ ratio ≥ 1 provided significance threshold for gene expression differences. To validate the FPKM analysis, expression of 20 selected genes were measured in nymphs and adults by RT-qPCR. For each total RNA sample, 2 μg RNA was used to synthesize cDNAs with random hexamer primers (Invitrogen) and M-MuLV reverse transcriptase (New England Biolabs, Beverly, MA, USA). qPCR reactions were performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer\'s protocol and run on the CFX384^TM^ Real Time System (BioRad, Hercules, CA, USA). Dissociation curve analyses were performed to ensure amplification specificity. Mean fold change in gene expression was calculated as described previously (Chi et al., [@B17]). Primer sequences are provided in Table [S1](#SM1){ref-type="supplementary-material"}. The 18S rRNA gene of green peach aphid (Acc. No. AF487712.1) was amplified as the internal control. Functional analysis of differentially expressed unigenes -------------------------------------------------------- GO enrichment analysis was performed to recognize the main biological functions of differentially expressed unigenes. The hypergeometric test was performed to find significantly enriched GO terms in differentially expressed unigenes compared to the whole reference transcriptome background (Su et al., [@B62]; Ji et al., [@B34]). The *P*-value was calculated with the formula: $$P\, = \, 1\, - {\sum\limits_{i\mspace{2mu} = \mspace{2mu} 0}^{m - 1}\frac{\begin{pmatrix} M \\ i \\ \end{pmatrix}\begin{pmatrix} {N - M} \\ {n - i} \\ \end{pmatrix}}{\begin{pmatrix} N \\ n \\ \end{pmatrix}}}$$ where N and n are defined as the number of genes in the transcriptome and differentially expressed genes with GO annotations, respectively. The variables *M* and *m* represent the gene number in the transcriptome annotated to a certain GO term and differentially expressed genes within the group (M-m ≥ 0), respectively. The calculated *P*-value was subjected to Bonferroni correction. GO terms with corrected *P*-value, i.e., *Q* \< 0.05 were considered significantly enriched. KEGG analyses were performed to identify significantly enriched pathways represented by differentially expressed unigenes. The hypergeometric test was used in a similar way to that for GO enrichment analysis and the terms with *Q* \< 0.05 were determined as enriched pathways. Ka and Ks analyses ------------------ To predict CDS regions, unigenes were first aligned by BLAST analyses with *E*-value cutoff of 10^−5^ to public databases in the priority order of Nr, Swiss-Prot, KEGG, and COG. Coding regions with the best match in BLAST were considered to be the CDS. Unigenes unable to be aligned to any databases were scanned by ESTScan, which may predict some coding regions. The CDSs of pea aphid were predicted from the mRNA sequence data (<https://www.aphidbase.com/aphidbase/content/download/3250/33670/file/aphidbase_2.1b_mRNA.fasta.bz2>). After filtering the redundant CDSs that may result from alternative splicing, predicted CDSs of the two aphid species were used to identify orthologous genes using OrthoMCL (Li et al., [@B41]). Only single-copy ortholog pairs longer than 150 bp were considered as putative orthologous gene pairs. Ka, Ks, and Ka/Ks-values were computed using the YN method implemented in the software KaKs Calculator Version 1.2 (Yang and Nielsen, [@B75]; Wang et al., [@B71], [@B69]). As the sequencing errors were distributed among synonymous and non-synonymous sites at equal frequencies, they were not expected to strongly influence the results of analyses (Tiffin and Hahn, [@B66]; Wang et al., [@B71], [@B69]). Results and discussion {#s3} ====================== Illumina sequencing analysis and *de novo* assembly --------------------------------------------------- High-throughput RNA-seq generated the most extensive current transcriptome for the green peach aphid. After quality checks, about 74.1, 74.0, and 74.5 million reads were obtained from the three replicates of nymphs and 74.6, 76.0, and 74.3 million reads from adults (Table [1](#T1){ref-type="table"}). All reads were deposited in the NCBI Short Read Archive (SRA, the accession number [SRP073458](SRP073458)). The reads were assembled into 89,944, 85,416, and 82,810 contigs with mean lengths of 474, 502, and 460 nt for nymphs and 81,641, 78,710, and 87,354 contigs with mean lengths of 472, 484, and 464 nt for adults (Table [1](#T1){ref-type="table"}). Using paired-end joining and gap-filling, these contigs were finally assembled into a total of 62,627 consensus sequences with a mean length of 1460 nt. GC contents were 39.00% for nymphs and 39.63% for adults, comparable to that of the pea aphid (38.80%) (Wang et al., [@B69]). ###### **Summary of transcriptome parameters of green peach aphid nymphs and adults**. **Nymph** **Adult** ------------------------------ ------------ ------------ ------------ ------------ ------------ ------------ Number of processed reads 74,068,728 74,017,762 74,553,158 74,568,296 76,017,216 74,335,676 Number of contigs 89,944 85,416 82,810 81,641 78,710 87,354 Mean length of contigs (nt) 474 502 460 472 484 464 GC content (%) 39.11 38.89 38.06 39.58 39.69 39.44 Number of unigenes 61,186 55,776 60,271 53,928 52,829 57,758 Mean length of unigenes (nt) 1054 998 957 960 965 986 *Values combined all independent biological replicates*. Functional annotation and classification of the assembled unigenes ------------------------------------------------------------------ Of the 62,627 unigenes, 33,543 were annotated by referencing to the Nr database (Table [S2](#SM2){ref-type="supplementary-material"}); 66.66% of the annotated sequences had very strong homology (*E* \< 10^−60^), 12.02% showed strong homology (10^−60^ \< *E* \< 10^−30^) and the rest 21.32% showed homology (10^−30^ \< *E* \< 10^−5^) to known sequences. With respect to species, 92.30% of the unique annotated sequences matched to pea aphid, 1.45% to *Tribolium castaneum*, 0.49% to *Bombus impatiens*, and 0.41% to *Camponotus floridana*. GO assignments were used to classify the functions of the predicted unigenes; 14,260 sequences were categorized into 46 GO terms consisting of three domains: biological process, cellular component and molecular function (Figure [1](#F1){ref-type="fig"}). The most abundantly expressed genes in "biological process" were involved in cellular process (9028), single-organism process (7075), and metabolic process (6557). In "molecular function," genes involved in catalytic (6894), binding (6678), and transporter (1137) activities were most abundantly expressed (Figure [1](#F1){ref-type="fig"}). {#F1} To better understand the biological pathways that are active in the green peach aphid, we mapped all sequences to the canonical reference pathways in the KEGG database. As a result, 23,695 sequences were assigned to 187 insect-related KEGG pathways (Table [S3](#SM3){ref-type="supplementary-material"}), with 3286 unigenes (15.47%) being involved in metabolic pathways. These annotations could be useful for further investigation of specific processes, functions and pathways. Comparison of gene expression profiles between nymphs and adults ---------------------------------------------------------------- When different developmental stages were compared, 1639 genes showed higher expression in nymphs and 605 higher in adults (Figure [2](#F2){ref-type="fig"}, Table [S4](#SM4){ref-type="supplementary-material"}). We performed RT-qPCR on selected genes to validate these gene expression data. Of the 20 selected genes, 18 were in agreement with RNA-seq results, suggesting good quality of transcriptomic analysis (Table [S5](#SM5){ref-type="supplementary-material"}). To gain insight into the major biological pathways represented by the differentially expressed genes, 21 enriched insect-related pathways (*Q* \< 0.05) were identified using the hypergeometric test (Table [2](#T2){ref-type="table"}); 14 were associated with "metabolism" and 3 with "digestive system," suggesting differential metabolic and digestive activities between nymphs and adults (Banks and Macaulay, [@B6]; Randolph et al., [@B57]). The most enriched pathway being "metabolism of xenobiotics by cytochrome P450" is intriguing because it may reflect developmental stage-specific interaction with the host plant. Presumably, nymphal, and adult aphids ingest different amounts of allelochemicals, given that more detoxification genes, e.g., 16 of the 23 differential P450 genes, and all differential esterase (6) and GST (1) genes, were expressed in higher abundance in adults (Table [3](#T3){ref-type="table"}). Developmental stage-dependent variations in expression patterns have often been observed in the detoxification genes (Harrison et al., [@B29]; Strode et al., [@B61]; Yang et al., [@B73]). High expression of *CYP321B1* is detected in the late larval stage of tobacco cutworm (*Spodoptera litura*) (Wang et al., [@B70]). In *B. tabaci*, relatively high expression of *CYP6CM1* is found in adults, correlating with the observation that specific resistance to neonicotinoid imidacloprid is largely restricted to adults (Nauen et al., [@B47]; Jones et al., [@B35]). Similarly, high expression of *CYP6P9* in adults of *Anopheles funestus*, but not in larvae, explains the adult resistance (Amenya et al., [@B2]). In a pyrethroid resistant strain of *Anopheles gambiae, CYP6Z1* is expressed in adults but undetectable in larvae or pupae (Nikou et al., [@B49]). Direct correlation between expression levels of detoxification genes at different developmental stages and resistance to pesticides is also exemplified by the beet webworm (*Pyrausta sticticalis*) (Leonova and Slynko, [@B40]) and citrus red mite (*Panonychus citri*) (Liao et al., [@B42]; Zhang K. et al., [@B78]). Banks and Macaulay ([@B6]) reported that adult aphids have higher food consumption than nymphs. Ahmad ([@B1]) stated that increased amounts of dietary allelochemicals due to increased food consumption may explain elevated P450-mediated metabolic activity. In parallel, green peach aphid adults likely ingest more plant materials, thus more allelochemicals from host plants, necessitating higher detoxification capacity. {#F2} ###### **Significantly enriched insect-related KEGG pathways represented by the genes differentially expressed between nymphs and adults**. **Pathway** ***Q*-value** ---------------------------------------------- --------------- Metabolism of xenobiotics by cytochrome P450 2.09 × 10^−7^ Steroid hormone biosynthesis 1.14 × 10^−6^ Retinol metabolism 1.34 × 10^−6^ Pentose and glucuronate interconversions 2.76 × 10^−6^ Ascorbate and aldarate metabolism 9.68 × 10^−6^ Glycine, serine and threonine metabolism 9.68 × 10^−6^ Circadian rhythm 2.81 × 10^−5^ Pentose phosphate pathway 1.92 × 10^−4^ Tyrosine metabolism 2.51 × 10^−4^ Glycerophospholipid metabolism 5.00 × 10^−4^ Glycerolipid metabolism 1.59 × 10^−3^ Starch and sucrose metabolism 2.93 × 10^−3^ Notch signaling pathway 6.75 × 10^−3^ Other types of O-glycan biosynthesis 7.06 × 10^−3^ RNA polymerase 7.09 × 10^−3^ Fat digestion and absorption 7.09 × 10^−3^ Insect hormone biosynthesis 1.99 × 10^−2^ Valine, leucine and isoleucine biosynthesis 2.45 × 10^−2^ Protein digestion and absorption 2.67 × 10^−2^ Vitamin digestion and absorption 3.92 × 10^−2^ Dorso-ventral axis formation 4.00 × 10^−2^ ###### **Differentially expressed detoxification and cuticle formation-related genes in adult and nymph**. **Gene ID** **Fold change (log~2~)[^\*^](#TN2){ref-type="table-fn"}** **Nr-annotation** ----------------------------------------------------------- ----------------------------------------------------------- ------------------------------------------------------------ **DETOXIFICATION GENES UP-REGULATED IN ADULT** *[Unigene28862](Unigene28862)* 7.29 Cytochrome P450 4g15-like *[Unigene5938](Unigene5938)* 5.20 Cytochrome P450 4g15-like *[Unigene38834](Unigene38834)* 4.64 Cytochrome P450 4C1-like *[Unigene12004](Unigene12004)* 3.09 Cytochrome P450 6a13-like *[CL1335.Contig8](CL1335.Contig8)* 2.37 Cytochrome P450 *[Unigene21970](Unigene21970)* 2.37 Cytochrome P450 6a13-like *[CL2142.Contig1](CL2142.Contig1)* 2.13 Cytochrome P450 18a1-like *[Unigene8797](Unigene8797)* 2.03 Cytochrome P450 6a14-like *[Unigene13485](Unigene13485)* 1.73 Cytochrome P450 4C1-like *[Unigene17164](Unigene17164)* 1.65 Cytochrome P450 6j1-like *[CL4129.Contig2](CL4129.Contig2)* 1.52 Cytochrome P450 4C1-like *[Unigene18192](Unigene18192)* 1.35 Cytochrome P450 18a1-like *[CL2142.Contig2](CL2142.Contig2)* 1.31 Cytochrome P450 18a1-like *[Unigene30119](Unigene30119)* 1.23 Cytochrome P450 6a2-like *[CL1335.Contig7](CL1335.Contig7)* 1.12 Cytochrome P450 6a13-like *[Unigene8106](Unigene8106)* 1.03 Cytochrome P450 4g15-like *[Unigene11947](Unigene11947)* 2.98 Esterase E4-like *[CL2237.Contig3](CL2237.Contig3)* 1.97 Esterase FE4-like *[Unigene14425](Unigene14425)* 1.89 Esterase FE4-like *[Unigene30909](Unigene30909)* 1.60 Esterase FE4-like *[CL2237.Contig6](CL2237.Contig6)* 1.28 Esterase FE4-like *[CL1600.Contig6](CL1600.Contig6)* 1.14 Carboxylesterase-6-like *[Unigene8449](Unigene8449)* 1.07 Glutathione S-transferase D4-like **DETOXIFICATION GENES UP-REGULATED IN NYMPH** *[CL27.Contig6](CL27.Contig6)* −4.63 Cytochrome P450 4C1-like *[Unigene12432](Unigene12432)* −3.88 Cytochrome P450 4C1-like *[CL27.Contig7](CL27.Contig7)* −3.41 Cytochrome P450 4C1-like *[CL1617.Contig5](CL1617.Contig5)* −2.30 Cytochrome P450 6a14-like *[CL3489.Contig2](CL3489.Contig2)* −1.83 Cytochrome P450 4C1-like *[Unigene13770](Unigene13770)* −1.24 Cytochrome P450 6k1-like *[Unigene24402](Unigene24402)* −1.20 Cytochrome P450 6k1-like **CUTICLE FORMATION-RELATED GENES UP-REGULATED IN ADULT** *[CL2631.Contig2](CL2631.Contig2)* 2.57 Cuticle protein-like precursor *[Unigene31315](Unigene31315)* 2.32 RR1 cuticle protein 5 **CUTICLE FORMATION-RELATED GENES UP-REGULATED IN NYMPH** *[Unigene2111](Unigene2111)* −4.53 Cuticle protein *[CL4114.Contig1](CL4114.Contig1)* −4.17 Cuticular protein-like precursor *[Unigene17916](Unigene17916)* −4.16 Cuticular protein 11 precursor *[Unigene16021](Unigene16021)* −3.72 Cuticular protein 11 precursor *[Unigene7580](Unigene7580)* −3.69 Cuticular protein-like precursor *[Unigene27924](Unigene27924)* −3.53 Cuticular protein 11 precursor *[Unigene25191](Unigene25191)* −3.45 Cuticular protein 16 precursor *[CL4114.Contig2](CL4114.Contig2)* −3.45 Cuticular protein-like precursor *[CL5082.Contig1](CL5082.Contig1)* −3.15 Cuticular protein 21 *[Unigene4987](Unigene4987)* −2.93 Cuticular protein 22 precursor *[CL5082.Contig2](CL5082.Contig2)* −2.85 Cuticular protein 21 *[Unigene21389](Unigene21389)* −2.71 Cuticular protein 21 *[CL6036.Contig9](CL6036.Contig9)* −2.58 Cuticular protein 28 precursor *[Unigene21306](Unigene21306)* −2.45 Cuticular protein 22 precursor *[Unigene24136](Unigene24136)* −2.36 Cuticular protein 62 precursor *[Unigene13474](Unigene13474)* −2.30 Cuticular protein 22 precursor *[Unigene13465](Unigene13465)* −2.25 Cuticular protein 28 precursor *[CL5767.Contig2](CL5767.Contig2)* −2.21 Cuticular protein CPG12-like precursor *[Unigene13461](Unigene13461)* −2.19 Cuticle protein-like *[Unigene9175](Unigene9175)* −2.17 Cuticular protein 23 precursor *[Unigene24655](Unigene24655)* −1.99 Cuticular protein 47 precursor *[Unigene13436](Unigene13436)* −1.91 Cuticular protein 9 precursor *[Unigene13449](Unigene13449)* −1.89 Cuticular protein 28 precursor *[Unigene13417](Unigene13417)* −1.88 Cuticular protein 9 precursor *[CL1419.Contig2](CL1419.Contig2)* −1.87 Cuticular protein 15 precursor *[Unigene8362](Unigene8362)* −1.72 Cuticular protein precursor *[Unigene13478](Unigene13478)* −1.72 Cuticular protein 1 precursor *[Unigene4947](Unigene4947)* −1.71 Cuticular protein 47 precursor *[Unigene11489](Unigene11489)* −1.71 RR1 cuticle protein 7 precursor *[Unigene13482](Unigene13482)* −1.70 Cuticular protein precursor *[Unigene10882](Unigene10882)* −1.70 Cuticular protein 60 precursor *[Unigene11447](Unigene11447)* −1.68 Cuticular protein CPG12-like precursor *[Unigene17255](Unigene17255)* −1.67 Cuticular protein 20 precursor *[CL4704.Contig1](CL4704.Contig1)* −1.66 Cuticular protein 57 precursor *[Unigene13459](Unigene13459)* −1.65 Cuticular protein 37 precursor *[Unigene13431](Unigene13431)* −1.65 Cuticular protein 1 precursor *[Unigene13477](Unigene13477)* −1.63 Cuticular protein 16 precursor *[Unigene13435](Unigene13435)* −1.63 Cuticular protein 16 precursor *[Unigene13480](Unigene13480)* −1.60 Cuticular protein 9 precursor *[Unigene13432](Unigene13432)* −1.59 Cuticular protein 45 precursor *[Unigene13457](Unigene13457)* −1.58 Cuticular protein 16 precursor *[Unigene11389](Unigene11389)* −1.58 Cuticular protein CPG12-like precursor *[CL1419.Contig4](CL1419.Contig4)* −1.58 Cuticular protein 15 precursor *[CL5117.Contig1](CL5117.Contig1)* −1.56 RR2 cuticle protein 2 *[Unigene13440](Unigene13440)* −1.55 Cuticular protein 45 precursor *[Unigene13416](Unigene13416)* −1.55 Cuticular protein 1 precursor *[Unigene13481](Unigene13481)* −1.53 Cuticular protein precursor *[Unigene13443](Unigene13443)* −1.53 Cuticular protein 37 precursor *[CL1419.Contig1](CL1419.Contig1)* −1.50 Cuticular protein 15 precursor *[Unigene13479](Unigene13479)* −1.47 Cuticular protein 28 precursor *[Unigene13452](Unigene13452)* −1.46 Cuticle protein-like *[CL6036.Contig5](CL6036.Contig5)* −1.45 Cuticular protein 28 precursor *[Unigene13484](Unigene13484)* −1.45 Cuticular protein 1 precursor *[Unigene31055](Unigene31055)* −1.42 Cuticular protein 48 *[CL1419.Contig3](CL1419.Contig3)* −1.41 Cuticular protein 15 precursor *[Unigene21674](Unigene21674)* −1.41 Cuticular protein 52 precursor *[Unigene31334](Unigene31334)* −1.40 Cuticular protein 20 precursor *[Unigene13420](Unigene13420)* −1.39 Cuticular protein 37 precursor *[Unigene14603](Unigene14603)* −1.37 Cuticular protein 28 precursor *[Unigene13424](Unigene13424)* −1.35 Cuticular protein 16 precursor *[Unigene14604](Unigene14604)* −1.34 Cuticular protein 28 precursor *[Unigene7739](Unigene7739)* −1.32 Cuticular protein analogous to peritrophins 3-D1 precursor *[Unigene13438](Unigene13438)* −1.26 Cuticular protein 28 precursor *[CL6036.Contig11](CL6036.Contig11)* −1.26 Cuticular protein 28 precursor *[CL6036.Contig6](CL6036.Contig6)* −1.24 Cuticular protein 28 precursor *[Unigene24750](Unigene24750)* −1.23 Cuticle protein precursor *[Unigene13418](Unigene13418)* −1.21 Cuticular protein 45 precursor *[CL6036.Contig10](CL6036.Contig10)* −1.18 Cuticular protein 28 precursor *[Unigene554](Unigene554)* −1.17 Cuticular protein 31 precursor *[Unigene14112](Unigene14112)* −1.14 Cuticular protein 30 precursor *[Unigene13441](Unigene13441)* −1.13 Cuticular protein 9 precursor *[Unigene8067](Unigene8067)* −1.09 Cuticular protein 68 precursor *[Unigene13475](Unigene13475)* −1.09 Cuticular protein 28 precursor *[Unigene13426](Unigene13426)* −1.08 Cuticular protein precursor *[Unigene31278](Unigene31278)* −1.07 RR1 cuticle protein 1 *[CL6048.Contig2](CL6048.Contig2)* −1.05 Cuticular protein precursor *[Unigene1224](Unigene1224)* −1.04 Cuticle protein-like *[Unigene13467](Unigene13467)* −1.03 Cuticular protein 1 precursor *[Unigene24090](Unigene24090)* −1.01 Cuticular protein 58 precursor *[Unigene5077](Unigene5077)* −1.55 Ecdysis triggering hormone *Log~2~ (FPKM-value in adult/ FPKM-value in nymph)*. The differentially expressed genes were also assigned to 20 GO enriched functional groups; ontology distributions are shown in Figure [3](#F3){ref-type="fig"}. Enriched in the "biological process" and "molecular function" include cuticle formation-related groups such as "structural constituent of cuticle," "chitin-based cuticle attachment to epithelium" and "molting cycle, chitin-based cuticle." The insect cuticle, composed of chitin and cuticle proteins, not only supports and maintains the physical structure, but also serves as a natural barrier against adverse external impacts (Andersen et al., [@B3]). Cuticle protein comparisons among insects at different developmental stages show that, rather than being an inert structure, the insect cuticle is developmentally modified (Chihara et al., [@B18]; Dombrovsky et al., [@B23]). Consistent with these findings, among the 81 differentially expressed transcripts of cuticular proteins and their precursors we detected, 79 were highly expressed in nymphs (Table [3](#T3){ref-type="table"}). Insects of this developmental stage repeatedly shed their cuticles and replace them with new layers, thus their cuticle biosynthesis is likely more active. No doubt, hormones play an essential role in insect ecdysis. Enrichment of the "steroid hormone biosynthesis" pathway among the differential genes (Table [2](#T2){ref-type="table"}) supports this notion. The major steroid hormone ecdysone plays an essential role in larval ecdysis, a process mediated by hormones, such as ecdysone and ecdysis triggering hormone (ETH) (Robbins et al., [@B59]; Ewer et al., [@B26]). Interestingly, the ETH-encoding gene *Unigene5077* was highly expressed in green peach aphid nymphs (Table [3](#T3){ref-type="table"}). {#F3} Transcriptomic divergences between green peach aphid and pea aphid ------------------------------------------------------------------ Transcriptome comparisons of different aphid species could provide useful information in understanding transcriptome evolution and the genetic factors underlying the biological divergence of these species. To identify genes specific to green peach aphid (relative to pea aphid), we compared the transcriptome we obtained in this study with publically available mRNA sequence data of pea aphid. tBLASTx identified homologous pea aphid mRNAs for 41,912 of our unigenes, leaving 20,595 having no hits. After removing sequences shorter than 250 bp (too short to be translated into polypeptides meaningful for comparisons) and BLASTn hits from pea aphid mRNAs and Nt databases, the remaining 13,752 were considered green peach aphid-specific unigenes under the rearing conditions described (Table [S6](#SM6){ref-type="supplementary-material"}). Arabidopsis was selected as our host plant because it is readily consumed by green peach aphid. Its short life cycle, abundant genetic resources and well developed RNAi technique (Ramsey et al., [@B56]; Pitino et al., [@B53]; Bhatia et al., [@B12]; Elzinga et al., [@B25]) can greatly facilitate our more in-depth studies of candidate genes derived from the current study. One caveat however, is that choice of hosts may impact aphid gene expression. Few studies have been conducted to compare transcriptome profiles of the same insect species feeding on different host plants, but some information is available on differential gene expression of insect populations reared on different varieties/lines of the same host plant species (Ji et al., [@B34]; Bansal et al., [@B7]; Yu et al., [@B76]). It appears that the vast majority of genes are present (but likely varied in expression level) among different insect populations, and that genes solely expressed in one population are rare. Whether this observation can be extended to insect populations feeding on different host plant species is yet to be determined. The Nr, Nt, Swiss-Prot, COG, KEGG, and GO annotations of green peach aphid-specific unigenes were then performed (Table [S6](#SM6){ref-type="supplementary-material"}). Only 4.52% were predicted to have defined functions (Table [4](#T4){ref-type="table"}), and functions of the remaining sequences need further study in the future. Likewise, KEGG classification identified only 30 unigenes, the most predominant group being "xenobiotics biodegradation and metabolism" (13.33%) (Figure [4](#F4){ref-type="fig"}). This finding correlates well with the fact that green peach aphids feed on a wider variety of plant species, and may have to encounter more types of toxic plant metabolites than pea aphids. ###### **Annotations of green peach aphid-specific unigenes**. **Public database** **Number of annotated unigenes** **Percentage (%)** --------------------- ---------------------------------- -------------------- Nr 85 0.62 Nt 562 4.09 Swiss-Prot 31 0.23 COG 9 0.07 KEGG 30 0.22 GO 10 0.07 {#F4} Ka and Ks analysis between green peach aphid and pea aphid ---------------------------------------------------------- Contrasting with the above analysis where the focus was on genes unique to green peach aphid, here we concentrated on single-copy orthologous genes between the two aphids. From the 33,963 green peach aphid CDSs (mean length, 1275 bp) derived from our RNA-seq, 3989 that had one-to-one orthologs in pea aphid CDSs were identified, and 3824 contained both substitution types, from which Ka/Ks ratios were calculated (Table [S7](#SM7){ref-type="supplementary-material"}). The Ka/Ks ratio provides information about the evolutionary forces operating on a particular coding gene and has been widely used to measure the intensity and mode of selection; Ka/Ks = 1 indicates a neutral evolution; Ka/Ks \< 1 suggests that nonsynonymous mutations are deleterious and purged from the population; Ka/Ks\>1 indicates that nonsynonymous amino acid substitutions offer fitness advantages and are fixed in the population at a higher rate than synonymous substitutions (Hurst, [@B33]). However, this cutoff value for positive selection has recently been adjusted to 0.5 by Swanson et al. ([@B64]). They found that 15 of 16 genes with 0.5 \< Ka/Ks \< 1 showed statistical evidence for adaptive evolution (Swanson et al., [@B64]). Since then, this new value has been adopted for "positive selection" determination in many studies (Kelleher et al., [@B37]; Elmer et al., [@B24]; Yang et al., [@B74]; Zhang L. et al., [@B79]; Ren et al., [@B58]; Cheng et al., [@B16]; Mu et al., [@B46]; He et al., [@B30]; Pereira et al., [@B51]). In our study, a total of 24 pairs of orthologs had a Ka/Ks ratio greater than 1, and 124 had a Ka/Ks ratio between 0.5 and 1 (Table [S8](#SM8){ref-type="supplementary-material"}). Relative to the earlier study by Ollivier et al. ([@B50]), our CDS construction is more complete than that of EST-based (33,963 CDSs, 1275 bp mean length vs. 6652 CDSs, 667 bp mean length), due to improvements in sequencing technology. Nevertheless, some putative orthologs under positive selection were identified by both studies, such as C002 (Table [S8](#SM8){ref-type="supplementary-material"}). Other genes related to insect-plant interactions include those encoding mucins (Ka/Ks = 1.09 and 0.94), the essential components of peritrophic matrix. Fast-evolving mucin proteins presumably contribute to aphid adaptation to different dietary pro-oxidants, phenolic, and lipophilic xenobiotics associated with their respective host plants (Hiraishi et al., [@B32]; Felton and Summers, [@B27]; Barbehenn, [@B8], [@B9]; Barbehenn and Stannard, [@B10]; Hegedus et al., [@B31]). Likewise, the homolog of salivary protein gene *Me17* (Ka/Ks = 0.69), identified in multiple aphid species with dissimilar plant host ranges, is thought to play important roles as the effector in suppressing defense responses in different host plants and in promoting aphid colonization (Atamian et al., [@B5]; Pitino and Hogenhout, [@B54]; Elzinga et al., [@B25]). Nicotinic acetylcholine receptors (nAChR) in insects are often the target sites for naturally occurring and synthetic insecticides (Millar and Denholm, [@B44]; Bass et al., [@B11]). A high mutation rate in the nAChR β-2 subunit (Ka/Ks = 0.55) could help green peach aphids adapt to tobacco and become resistant to nicotine, as is the strain used in this study (Devine et al., [@B21]; Nauen et al., [@B48]). Another interesting ortholog pair encode odorant-binding protein 10 (OBP10) (Ka/Ks = 0.52). Nucleotide and amino acid sequence comparisons between the two species indicated that all substitutions occurred in the predicted mature protein region, and 19 of the 62 substitutions resulted in 12 hydrophilic and hydrophobic amino acid conversions (Figure [S1](#SM9){ref-type="supplementary-material"}). Sun et al. ([@B63]) observed that the two aphid species showed similar as well as dissimilar behavioral responses to certain tested odors. Presumably, fast evolution in OBPs could contribute to the change in their binding activity, which in turn could facilitate host shift or impact host range (Matsuo et al., [@B43]; Sun et al., [@B63]). Conclusions {#s4} =========== Our RNA-seq data have increased molecular resources available for the green peach aphid, a major agricultural pest as well as a biological model for insect-plant interaction studies. The transcriptomic analyses have deepened our understanding of aphid development and aphid-plant interactions. Our results have also provided useful insight into the molecular mechanisms underlying the biological variations in aphids, especially in adaptation to different host plants. Author contributions {#s5} ==================== KZS, JF, and RJ conceived and designed the experiments. RJ performed the experiments. RJ, YW, YC, MZ, HZ, LZ, and KZS contributed to the transcriptome data analysis. RJ, JF, and KZS wrote the manuscript. All authors contributed to the discussion and approved the final manuscript. Funding ======= This research was supported by the National Natural Science Foundation of China (31501636), the China Postdoctoral Science Foundation (2014M551529), Jiangsu Agricultural Science and Technology Independent Innovation Fund (CX(15)1055), Texas A&M Genomics Seed Grant, Texas Invasive Ant Research and Management Seed Grant, and the USDA-AFRI grant (2014-67013-21781). Conflict of interest statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We would like to thank Dr. Ron Salzman for his critical review of the manuscript. We appreciate technical assistance in sample preparation for sequencing from Dr. Charles Johnson and Dr. Richard Metz. Supplementary material {#s6} ====================== The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fpls.2016.01562> ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. [^1]: Edited by: Jyoti Shah, University of North Texas, USA [^2]: Reviewed by: Muthu Venkateshwaran, University of Wisconsin--Platteville, USA; Vamsi J. Nalam, Indiana University--Purdue University Fort Wayne, USA [^3]: This article was submitted to Plant Biotic Interactions, a section of the journal Frontiers in Plant Science

Background ========== Dengue is a mosquito-borne viral infection caused by one of the four dengue virus serotypes (DENV-1 to 4), belonging to genus *Flavivirus*, family *Flaviviridae.* The viruses replicate alternately on the mosquito vector, mainly (*Ae. aegypti*) and human host and are responsible for infections throughout tropical and subtropical regions of the world \[[@B1],[@B2]\]. The rapid global spread of the four DENV serotypes in the last 50 years resulted in the dispersal of genotypes associated with increased disease severity \[[@B3]\]. In Brazil, dengue has been a major public health problem since DENV-1 introduction and spread in 1986 \[[@B4]\], however the introduction of the genotype III of DENV-3, in December 2000, in Nova Iguaçu, State of Rio de Janeiro (RJ), caused one of the most severe epidemics reported in the country in 2002 \[[@B5]-[@B7]\]. Despite the co-circulation of DENV-1, DENV-2 and DENV-3 in that area, DENV-3 was the only serotype detected in pools of *Ae. aegypti* during an entomological surveillance performed \[[@B8]\]. Sequencing of distinct DENV genomic regions has identified five genotypes for DENV-3: Genotypes I to III (GI to GIII) which are responsible for most DENV-3 human infections and have been associated with both dengue fever (DF) and dengue haemorrhagic fever (DHF) epidemics in Southeast Asia, Indian Subcontinent, South Pacific and East Africa and Americas, and Genotypes IV and V (GIV and GV) which were not associated with DHF epidemics and are only represented by few early sequences from Americas, South Pacific and Asia \[[@B9]-[@B13]\]. The DENV genome is composed by a positive single-stranded RNA of approximately 11 kb in length with an open reading frame encoding for the viral polyprotein, which is cleaved into three structural proteins (C, prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4 and NS5) flanked by 5′ and 3′ untranslated regions (UTRs) of about 100 and 400 nucleotides, respectively \[[@B1]\]. The flaviviruses UTRs are predicted to form secondary stem-loop (SL) structures, which are highly conserved and play a role in viral replication \[[@B14]-[@B18]\]. According to predicted secondary structures, the DENV 3′UTR can be divided into three domains \[[@B18]\]. The domain I, which is located immediately after the NS5 stop codon, is considered the most variable region (VR) within the viral 3′UTR, as it shows large heterogeneity in both length and nucleotide sequences \[[@B19]-[@B21]\]. Mutations and deletions within these regions may alter infectivity and reduce efficiency of viral replication \[[@B22],[@B23]\] and differences between strains in these regions may correlate with DENV virulence and pathogenicity \[[@B24]-[@B27]\]. Furthermore, deletions and nucleotide variations were also described in the VR within the same serotype \[[@B28]-[@B30]\]. Domain II is of moderate conservation, comprising several hairpins motifs and where conserved sequence (CS2) and repeated CS2 (RCS2) are present. Domain III is the most conserved region of the 3′UTR with CS1 followed by a terminal stem-loop (3′SL) \[[@B18]\]. Here, aiming to contribute for the studies on human host-virus-vector interactions, we fully sequenced the genome of one DENV-3 isolated from naturally infected field-caught mosquitoes in RJ and characterized the viral 3′UTR in comparison to other sequenced DENV-3 isolated from naturally infected mosquitoes and human hosts. Material and Methods ==================== Ethical Statement ----------------- All human DENV-3 strains belong to a previously gathered collection from the Laboratory of Flavivirus, IOC/FIOCRUZ, RJ, Brazil obtained from acute phase human serum through the passive surveillance system from an ongoing Project approved by resolution number CSN196/96 from the Oswaldo Cruz Foundation Ethical Committee in Research (CEP 274/05), Ministry of Health, Brazil. *Ae. aegypti* examined in this study were collected by the staff of the Dengue Control State Program for the determination of house infestation index, virological and entomological surveillance. No special permission or written consent is required for house entrance for mosquito collection and larval site treatment. Viral strains ------------- The DENV-3 strains isolated from *Ae. aegypti* adult mosquitoes (*n=*4) and human hosts (*n=*10) naturally infected in RJ were collected from epidemics occurred from 2001 to 2008. The first Brazilian DENV-3 strain (BR74886/02) isolated from a human fatal case fully sequenced \[[@B31]\] was used for comparison purposes and detailed information on the strains is provided on Table [1](#T1){ref-type="table"}. ###### Brazilian DENV-3 isolated from naturally infected vectors and human cases analyzed in this study **Strain** **Origin State\*** **Year of isolation** **Source** **GenBank accession number** **Sequence analyzed** **Reference** ------------ -------------------- ----------------------- ------------- ------------------------------ ----------------------- --------------------------- BR73354/01 RJ 2001 Mosquitoes FJ177308 Complete genome This study BR73356/01 RJ 2001 Mosquitoes JN383345 3′UTR This study BR73636/01 RJ 2001 Mosquitoes JN383346 3′UTR This study BR81200/06 RJ 2006 Mosquitoes JN383344 3′UTR This study BR70562/01 RJ 2002 Human serum JN380902 3′UTR This study BR74792/02 RJ 2002 Human serum JN380899 3′UTR This study BR74916/02 RJ 2002 Human serum JN380901 3′UTR This study BR74947/02 RJ 2002 Human serum JN380904 3′UTR This study BR77515/03 RJ 2003 Human serum JN380900 3′UTR This study BR78969/04 RJ 2004 Human serum JN380905 3′UTR This study BR80740/05 RJ 2005 Human serum JN380906 3′UTR This study BR80996/06 RJ 2006 Human serum JN380903 3′UTR This study BR83904/07 RJ 2007 Human serum JN380898 3′UTR This study BR072/08 RJ 2008 Human serum JN380907 3′UTR This study BR74886/02 RJ 2002 Human liver AY679147 Complete genome Miagostovich et al., 2006 \*RJ: Rio de Janeiro. DENV-3 human cases ------------------ From 2001 to 2008, the Laboratory of Flavivirus, as a Regional Reference Laboratory for the Brazilian Ministry of Health, received a total of 16,185 dengue suspected cases for routine diagnosis. Virus isolation was attempted in 9,405 cases and DENV-3 was the infecting serotype in 52.8% of the positive isolates. The samples analyzed in this study were chosen randomly and representative of each year, during and after the 2002 epidemic in RJ. DENV-3 entomological surveillances ---------------------------------- The three DENV-3 strains isolated (BR73354/01, BR73356/01 and BR73636/01) in 2001 from naturally infected *Ae. aegypti* adult mosquitoes used in this study were collected during an entomological survey performed in 35 districts of Nova Iguaçu, RJ, from July 2000 to June 2001. The other DENV-3 strain (BR81200/06) was isolated from naturally infected *Ae. aegypti* adult mosquitoes collected during an entomological survey conducted on 7 districts with different infestation index, randomly chosen in the municipality of Rio de Janeiro, RJ, from March 2005 to February 2006. Briefly, adult mosquitoes were collected twice a week, alternately in the morning and in the afternoon with manual and battery backpack aspirators and with nets, both indoor and in the yards and gardens, close to the dwellings. Mosquitoes were identified using a key as previously described \[[@B32]\], pooled according to gender, date, district of collection and stored in liquid nitrogen at the same day of collection. A total of 503 *Ae. aegypti* mosquitoes (352 females and 151 males) collected in 2000--2001 and 874 *Ae. aegypti* females collected in 2005--2006 were pooled (74 pools of 9--17 mosquitoes/pool in 2000--2001 and 27 pools of 2--10 mosquitoes/pool, jn 2005--2006) and all pools were submitted for virus isolation. Only positive pools were submitted to RT-PCR. for DENV serotype confirmation. Preparation of vectors ---------------------- Mosquitoes' pools were macerated in 1 ml of Leibovitz L-15 medium (Sigma) plus antibiotics (penicillin-streptomycin, 10,000 units - Invitrogen) and centrifuged (6,000 rpm at 4°C for 30 min). Supernatant was transferred to an Eppendorf tube containing 100 mL of streptomycin / fungizone and penicillin, kept in an ice bath for 1 hour and centrifuged (3,000 rpm at 4°C for 15 min). Supernatant was transferred to an Eppendorf tube containing 0.3ml of fetal calf serum (Invitrogen) and frozen (−70°C). Virus isolation --------------- Virus isolation was performed by inoculation into monolayers of C6/36 *Aedes albopictus* cells \[[@B33]\] in Leibovitz L-15 medium (Sigma) supplemented with 2% fetal calf serum (Invitrogen) and 0.2 mM of nonessential amino acids (Invitrogen). Cells were incubated at 28°C for 5 to 7 days and observed for cytopathic effects. Isolates were identified by indirect fluorescent antibody test (IFAT) using serotype-specific monoclonal antibodies \[[@B34]\] and infected supernatant was clarified by centrifugation and virus stocks stored in 1-mL aliquots at −70°C. RNA extraction -------------- Viral RNA was extracted using QIAamp Viral RNA Mini kit (Qiagen) following the manufacturer's instructions and stored at -70°C for DENV typing and sequencing. For the viral 3′UTR sequencing, the RNA was extracted directly from serum and mosquitoes macerate, previously detected by RT-PCR. For the full genome sequencing of the DENV-3 strain BR73354/01, the RNA was extracted from the first passage in cell culture. RT --PCR (Reverse transcriptase- polymerase chain reaction) ----------------------------------------------------------- RT---PCR for detecting and typing DENV was performed as described previously \[[@B35]\]. Sequencing and phylogenetic analysis ------------------------------------ PCR products were sequenced in both directions using the BigDye Dideoxy Terminator sequencing kit (Applied Biosystems). The mosquitoes' DENV-3 full-length genome sequence and 3′UTR sequences obtained in this study were deposited in GenBank ( <http://www.ncbi.nlm.nih.gov>) and are described on Table [1](#T1){ref-type="table"}. Sequence and similarity identity analysis was performed using BioEdit software ( <http://www.mbio.ncsu.edu/bioedit/bioedit.htmL>). The multiple alignments were performed using CLUSTAL W ( <http://www.ebi.ac.uk/clustalw/>) and the phylogenetic analysis by MEGA 5 software ( <http://www.megasoftware.net>), using the Neighbor-joining method, according to the Tamura-Nei model, with a bootstrap of 1,000 replications for the analysis of the complete genome. For the 3′UTR analysis, the Maximum likelihood method, according to the Kimura-2 model was chosen as determined by the best-fit substitution model provided by MEGA 5. Strains representative from the five genotypes available in GenBank ( <http://www.ncbi.nlm.nih.gov>) were used for the comparison, DENV-1 (GenBank accession number \#AF513110), DENV-2 (\#AF489932) and DENV-4 (\# AF326573) strains were used as outgroup to root the tree. Secondary structure analysis ---------------------------- The predicted secondary structures were generated by MFOLD web server ( <http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form>) with default folding parameters and folding predictions at 37^o^C on the VR from the 3' UTR of DENV-3. Results and discussion ====================== The DENV-3 was re-introduced in Latin America in 1994, after an absence of 17 years, being initially isolated in Panama and spreading throughout Central America \[[@B36],[@B37]\] to Caribbean \[[@B38]-[@B40]\] and South America \[[@B6],[@B28],[@B41]-[@B43]\]. However, some phylogenetic studies point to its introduction through Mexico \[[@B44]\] a few years earlier \[[@B13]\]. This introduction caused by the genotype III of DENV-3, originally from the Southeast Asia and characterized by an increased virulence, coincided with the occurrence of a higher number of severe and DHF cases \[[@B37],[@B45]-[@B47]\]. In Brazil, the first DENV-3 was isolated in December of 2000 in Nova Iguaçu, RJ \[[@B5]\] when the *Ae aegypti* infestation level was 8.1% and, 58% of those mosquitoes were resistant to temephos at that time \[[@B8],[@B48]\]. Due to the role of the city of Nova Iguaçu in dengue epidemiology, after the DENV-1 introduction in 1986 \[[@B4]\], field studies were conducted for detection of DENV in field-caught vectors \[[@B8],[@B49]\]. The potential emergence of strains associated with severe disease highlights the need for the surveillance of DENV in human host and vectors, as the detection of DENV in infected field-caught vectors is considered a useful tool for the early prediction of epidemics and detection of new serotypes/genotypes introductions \[[@B50],[@B51]\]. The entomological surveillance performed in the first semester of 2001 in Nova Iguaçu, RJ, resulted in the isolation of three DENV-3 strains from the districts of Santa Efigênia (BR73354/01), California (BR73356/01) and Morro Agudo (BR73636/01) isolated from three pools (9 mosquitoes/pool) of naturally infected *Ae. aegypti* females \[[@B8]\]. In January 2006, one DENV-3 strain (BR81200/06) was isolated from one *Ae. aegypti* pool composed of three females, collected indoors in the Vargem Pequena neighborhood, west region of RJ. In order to access the differences among Brazilian DENV-3, we sequenced and deposited on Genbank (FJ177308) the entire genome sequence of one virus isolated from naturally infected *Ae. aegypti* (BR73354/01) and compared to the Brazilian strain 74886/02 (AY679147), isolated from the liver of a fatal case during the DENV-3 epidemic occurred in 2002 \[[@B31]\]. The nucleotide similarity was 99.3% and the phylogeny based on the analysis of the complete coding region characterized the Brazilian strain as belonging to genotype III (Indian Subcontinent), Figure [1](#F1){ref-type="fig"}. {#F1} Amino acid substitutions were observed throughout the entire coding region, when the Brazilian DENV-3 strains were compared to the prototype PHIL/H87/1956 and strains representative of the other genotypes. Some substitutions were exclusive to the Brazilian DENV-3 strains analyzed (Table [2](#T2){ref-type="table"}) and some were shared among the Brazilian strains and the strain isolated from *Ae. aegypti* in Taiwan in 1998 (TAIWAN/TWmosq/1998), Table [2](#T2){ref-type="table"}. Exclusive substitutions to the DENV-3 Brazilian strains analyzed in this study were observed on the capsid, prM and envelope genes. Besides those others observed were also shared by other genotype III strains previously analyzed \[[@B11]\]. ###### Amino acid differences among selected Brazilian DENV-3 and strains representative of the DENV-3 genotypes, compared to the prototype H87 **Genotypes** **GI**     **GII**   **GI** **GII** **GIV** --------------- ---------- ------- ------------ --------- ------- -------- --------- --------- --- Capsid C ~108~ M **I** **I** **I** **I**       C~112~ T **A** **A** **A** **A**       prMembrane prM~86~ H **R** **R** **R** **R**       Envelope E~81~ I **V** **V** **V** **V**       E~124~ S **P** **P** L L L **P**   E~132~ H **Y** **Y** **Y** **Y**   **Y**   E~169~ A **T** **T** **T** **T** V V V E~270~ T **N** **N** **N** **N**   **N**   E~301~ L **T** **T** **T** **T** S     E~380~ I     **T** **T**       E~383~ K   **N** **N** **N**       E~452~ I **V** **V** **V** **V**       NS1 NS1~84~ I     **V**         NS1~94~ T I   I I I I I NS1~139~ S N   N N N N N NS1~339~ N **S** **S** **S** **S**       NS2A NS2A~37~ L **F** **F** **F** **F**       NS2A~158~ I **M** **M** **M** **M**       NS2A~175~ I **V** **V** **V** **V**   **V**   NS2A~195~ T **A** **A** **A** **A**       NS2B NS2B~60~ V **I** **I** **I** **I**       NS2B~109~ I **V** **V** **V** **V**       NS3 NS3~115~ I **T** **T** **T** **T**       NS3~452~ V **A** **A** **A** **A**   **A**   NS4A NS4A~99~ D **E** **E** **E** **A**       NS4B NS4B~21~ V **I** **I** **I** **I**       NS4B~138~ V **I** **I** **I** **I**       NS5 NS5~97~ V   **L**           NS5~98~ T   **S**           NS5~101~ R   **\*STOP**           NS5~105~ K   **T**           NS5~112~ E   **A**           NS5~114~ V   **R**           NS5~117~ S   **P**           NS5~127~ M   **R**           NS5~131~ D   **E**           NS5~133~ F   **D**           NS5~135~ L   **P**           NS5~229~ S **A** **A** **A** **A**       NS5~233~ M   **R**           NS5~288~ S **N** **N** **N** **N**       NS5~365~ P **S** **S** **S** **S**       NS5~371~ K **R** **R** **R** **R**       NS5~374~ E **G** **G** **G** **G**       NS5~389~ R   **K** **K** **K**   **K**   NS5~422~ R **K** **K** **K** **K**       NS5~429~ E **D** **D** **D** **D**       NS5~585~ K **T** **T** **T** **T**       NS5~639~ L **P** **P** **P** **P**       NS5~763~ T **S** **S** **S** **S** A S A NS5~835~ D **N** **N** **N** **N**         Blank cells indicate amino acid similarities to the prototype strain H87 (GenBank accession number M93130). \*STOP: stop codon. On the NS1 gene only one residue substitution was observed, and substitution on NS2A and NS2B were also reported. On the NS3 gene, a substitution on only NS3~115~ was exclusive to the Brazilian DENV-3. On NS4B, the same substitution was observed on positions NS4B~21~ and NS4B~138~. However, on the NS5 gene, besides the ten substitutions exclusive to the Brazilian DENV-3, substitutions exclusive to the Brazilian DENV-3 isolated from *Ae. agypti* (BR73354/2001) were observed almost consecutively, on NS5~97~ and NS5~98~. On NS5~101~, the substitution resulted in a stop codon and other substitutions throughout NS5 were also observed. Despite this, the original macerate was re-inoculated in C6/36 cells culture and the DENV −3 infection confirmed by the presence of cytopathic effect and a positive RT-PCR. The presence of genome-defective DENV-3 containing either stop codons or deletions *in vivo* has been reported previously \[[@B52]\]. Despite the use of the E gene for DENV phylogenetic and evolutionary studies \[[@B9],[@B12],[@B28],[@B53]-[@B62]\] due to its biological properties and selective pressure imposed by the host immune response, the role of sequences heterogeneity in other genomic regions which includes the non-structural genes and the genome UTRs cannot be excluded \[[@B53]\]. Previous studies have suggested that the sequence and secondary structures of the 5′ and 3′UTR of flaviviruses play an important role in viral replication and differences in these regions may influence viral virulence \[[@B24],[@B26],[@B27],[@B63]\]. Mutations and deletions within these regions may alter infectivity and reduce efficiency of viral replication \[[@B22],[@B23]\]. Furthermore, the domain I from the DENV 3′UTR is considered the most variable region (VR) from the 3′UTR \[[@B18]\] and can serve as a good marker for DENV evolution \[[@B19]-[@B21]\]. The analysis of the 3′UTR of the strain BR73354/01 genome showed an 8- nucleotides deletion within the 11-nucleotides insertion on the VR, previously observed for the Brazilian DENV-3 strain isolated from humans \[[@B31]\] and common to genotype III DENV-3 strains from the Latin America/Caribbean and Sri Lanka regions \[[@B27],[@B64]\]. Nucleotides substitutions exclusive to the BR73354/01 were observed on positions 10,383 and 10,391 from the 3′UTR. One substitution on the RCS2 and one on the CS2 were shared by all Brazilian DENV-3 when compared to the prototype. We additionally analyzed and deposited on GenBank the 3′UTR sequences from other three DENV-3 strains isolated from naturally infected *Ae. aegypti* isolated in 2001 and 2006 in RJ (BR73356/01, BR73636/01 and BR81200/06) and from ten DENV-3 isolated from humans from 2001 to 2008. The strain BR73356/01 presented the same 8-nucleotides deletion observed for the strain BR73354/01. However, the other two strains also isolated from mosquitoes in RJ (BR73636/01 and BR81200/06) presented the 11-nucleotides insertion common to the human strains. The analysis of the 3′UTR from DENV-3 isolates from humans showed that nine out of ten strains also presented the 11-nucleotides previously described. However, one of the strains isolated in Rio de Janeiro in 2002 (BR74792/02) showed the same 8-nucleotides deletion observed on the mosquito strains (Figure [2](#F2){ref-type="fig"}). Previous studies have shown deletions and nucleotide variations in the VR within a same serotype \[[@B28],[@B29]\]. Despite those observations, it was also shown a high conservation on the 3′UTR RCS2, CS1 and CS2 conserved regions (Figure [2](#F2){ref-type="fig"}, gray-shadowed areas) among all the Brazilian strains analyzed. This was quite expected as domain II is of moderate conservation and domain III, the most conserved region of all \[[@B18]\].Therefore, we focused on the VR of the 3′UTR, aiming to better characterize those mutations by predicting the secondary structures of that region. Not all strains presenting the 11-nucleotides insertion were similar in structure (Figure [3A](#F3){ref-type="fig"} and [3B](#F3){ref-type="fig"}). In fact, despite the 11-nucleotides insertion, the strain BR72/2008 presented a unique secondary structure (Figure [3B](#F3){ref-type="fig"}). The only difference from the latter is a nucleotide substitution (G →A) on the 11-nucleotides insertion region, when compared to all other Brazilian sequences analyzed (Figure [2](#F2){ref-type="fig"}). The strain BR80996/2006, also showed a unique secondary structure due to the nucleotides substitution presented in the VR, despite the presence of the insertion shared with the other strains. The slight difference presented by the strain BR83904/2007, was due to a substitution (C→T) exclusive to this sequence. Furthermore, all three  sequences with  the  8-nucleotides  deletion  (BR73354mosq/2001, BR73356mosq/2001 and BR74792/2002) presented the same secondary structure (Figure [3C](#F3){ref-type="fig"}). {#F2} {#F3} Phylogenetic studies based on the 5′and 3′UTR have shown to be very useful for molecular epidemiological studies \[[@B19],[@B21],[@B27],[@B28],[@B65]\]. The Maximum-Likelihood phylogenetic tree of Brazilian DENV-3 strains isolates from naturally infected *Ae. aegypti* mosquitoes and humans based on the 3′UTR sequence analysis places those strains as belonging to genotype III, corroborating the findings of the full-length genome analysis (data not shown). Conclusions =========== Here, we analyzed the coding region and the 3′UTR of DENV-3 from both human host and mosquitoes and described insertions, deletions and a substitution leading to stop codon formation. The majority of DENV-3 in this study was characterized by the 11-nucleotide insertion in the 3′UTR, despite the observation of strains carrying the 8-nucleotide deletion. In spite the presence of distinct viral variants, it is suggested that the major variant is transmitted. However, how those distinct viral populations are maintained or transmitted is not fully understood, therefore the availability of viruses isolated from both hosts are crucial for the better comprehension of the vector-virus-human host interactions and for quasispecies investigations. Furthermore, the analysis of those distinct viral populations in experimentally infected mosquitoes may help to elucidate those observations. Competing interest ================== The authors have no conflict of interest. Authors' contributions ====================== FBS, RMRN and RLO designed the study. MGC, FNB performed the experiments, MGC, FBS and RLO wrote the paper. All authors read and approved the final manuscript. Financial support ================= CNPq, CAPES, FIOCRUZ, FIOCRUZ/PAPES V and FAPERJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgements ================ We thank to the field staff of the Sanitary District of Nova Iguaçu/Vector Control Program for its help on the vectors collection. To Mauro Menezes, Wellington Silva, Anielly Ferreira, José da Costa Farias Filho, Jaqueline Simoes and Clarice R Santos for technical assistance.

1.. Introduction   {#sec1} ================== Precipitation or aggregation of proteins is often problematic when handling protein solution for research and commercial use. Developing methods to inhibit undesired aggregation of proteins is one of the critical issues in the biopharmaceutical field (Shire *et al.*, 2004[@bb16]; Yang *et al.*, 2010[@bb21]). To our knowledge, in protein design studies, we often come across water-insoluble artificial proteins (Isogai *et al.*, 2005[@bb10]; Imamura *et al.*, 2012[@bb8]). In contrast, a mechanism which prevents an uncontrolled aggregation is likely to exist in naturally occurring proteins (Isogai, 2006[@bb9]; Monsellier & Chiti, 2007[@bb14]). Understanding physicochemical mechanisms governing self-association of proteins will be useful for future protein engineering. In the present study, we used a small-angle X-ray scattering (SAXS) technique to analyze protein's interparticle interference, which yields an intermolecular interaction of a protein in solution through use of the theory for liquids (Tardieu *et al.*, 1999[@bb18]). We employed myoglobin as a model protein. Removal of heme is regarded as a kind of simple protein engineering. Interestingly, it was reported that the apomyoglobin, in which the heme is removed, is prone to aggregate, while holomyoglobin, in which the heme is binding, is highly water-soluble (Fändrich *et al.*, 2001[@bb2], 2003[@bb3]). Crystallization of proteins would be controlled by protein's self-interaction. To date, a three-dimensional structure of apomyoglobin by X-ray crystal analysis has not been uploaded to the Protein Data Bank, while holomyoglobin was the first success in the X-ray crystal analysis of the protein structures (Kendrew *et al.*, 1958[@bb12]). Unravelling how removal of heme changes the protein--protein interaction of myoglobin will be a step toward understanding key factors for the aggregation and the solubility. 2.. Materials and methods   {#sec2} =========================== 2.1.. Sample preparation   {#sec2.1} -------------------------- Holomyoglobin from equine skeletal muscle was purchased from Sigma. Apomyoglobin was prepared according to the following methods (Teale, 1959[@bb19]; Hapner *et al.*, 1968[@bb7]). 10 mg ml^−1^ aqueous solution of myoglobin was adjusted to pH ∼1.5 with HCl at 297 K. An equal volume of ice-cooled 2-butanone was mixed with the solution, and then the organic phase containing the extracted heme was discarded. This procedure was repeated three times. The aqueous phase was dialyzed against sodium bicarbonate (0.05 g l^−1^) and then ultrapure water (Milli-Q, Millipore). The apomyoglobin solution was finally lyophilized. The solutions of holomyoglobin and apomyoglobin for the measurements were prepared by dissolving the powders into the ultrapure water. The pH of the holomyoglobin and apomyoglobin solutions were 7.5 and 6.7, respectively. The concentrations of both the proteins were determined by using an ∊~410nm~ of 1.6 × 10^5^ for holomyoglobin and ∊~280nm~ of 1.43 × 10^4^ for apomyoglobin (Goto *et al.*, 1990[@bb5]). 2.2.. Small-angle X-ray measurement   {#sec2.2} ------------------------------------- SAXS was measured using a SAXSess camera (Anton Paar; Graz, Austria) with a sealed-tube X-ray source (PANalytical, The Netherlands), operating at 40 kV and 50 mA, and line collimation. X-ray intensities were recorded using an imaging plate (100 µm × 100 µm pixel size; Fuji Film, Japan), which was read using a Cyclone scanner (PerkinElmer, USA). The X-ray wavelength, λ, was 1.542 Å (Cu *K*α); the camera length was 264.5 mm. The image data were integrated into one-dimensional scattering intensities using the program *ImageJ* (developed by Wayne Rasband, National Institute of Health, Bethesda, MD, USA) with the macros in Utah SAXS Tools written by Professor David P. Goldenberg (University of Utah, USA). The scattering parameter *q* is defined as *q* = 4πsinθ/λ, where 2θ is the scattering angle of X-rays. The width of the integration area (detector slit width) was 10 mm. Protein solution and water were measured at 293 K in 1 mm-diameter glass capillaries for 1 h. 2.3.. Calculation of a structure factor from an intermolecular potential   {#sec2.3} -------------------------------------------------------------------------- The SAXS intensity of a protein, *I*(*q*), is described bywhere *c* is the protein concentration, *k* is a constant, *P*(*q*) is a form factor (protein's self-scattering) and *S*(*q*) is a structure factor (protein's interparticle interference). The SAXS profiles of the dilute protein solutions (0.5 wt%), at the concentration of which the interparticle interferences are negligibly small, yields *kP*(*q*) by an indirect Fourier transform program *GNOM* (Svergun, 1992[@bb17]) involving correction of the slit smeared effect. *S*(*q*) can be connected to the intermolecular potential of a protein molecule, *V*(*r*), by solving the Ornstein--Zernike equation with an appropriate closure. In the present analysis, we used the closure obtained from a random phase approximation, under which *S*(*q*) is described by(Hansen & McDonald, 1976[@bb6]; Narayanan & Liu, 2003[@bb15]), where *S* ~0~(*q*) is the structure factor of the reference system, *n* is the protein's number density, *k* ~B~ is the Boltzmann constant and *V*(*q*) is the Fourier transform of *V*(*r*). *S* ~0~(*q*) has been evaluated using the empty core model (Croxton, 1975[@bb1]; Kelkar *et al.*, 1992[@bb11]). The present study employed the Derjaguin--Laudau--Verwey--Overbeek (DLVO) model potential (Verwey & Overbeek, 1948[@bb20]), in which *V*(*r*) is expressed aswhere *r* is the distance between the protein molecules. The term *V* ~HS~(*r*) is the hard sphere potential given bywhere σ is the diameter of the protein. The terms *V* ~C~(*r*) and *V* ~AY~(*r*) are the screened repulsive Coulomb potential and the attractive potential, respectively \[here, *V* ~AY~(*r*) is assumed to be a Yukawa-type potential\], where *Z* is the net charge on the protein, *e* is the elementary charge, ∊ is the dielectric constant of the medium, κ is the reciprocal Debye--Hückel screening length, *J* is the depth of attractive potential at *r* = σ, and *d* is the range of the attractive potential. *S*(*q*) can be simulated, according to the equations described above and the given values for the parameters (*Z*, σ, *J* and *d*). In the present study, we used *Z* = 1 and σ = 41.2 Å, the values of which were from the literature (Longeville *et al.*, 2003[@bb13]). The procedure for the present analysis was performed by using *IGOR Pro* version 6.22A (Wavemetrics, USA). 3.. Results and discussion   {#sec3} ============================ Fig. 1[▶](#fig1){ref-type="fig"} shows the observed SAXS profiles of holomyoglobin and apomyoglobin at 0.5 and 6.3 wt%, where the intensities are normalized by the protein concentrations. For both the 6.3 wt% solutions, the intensities at low scattering vectors (*q* \< ∼0.1 Å^−1^) were depressed compared with that for 0.5 wt% solutions, which are due to the interparticle interference, *i.e.* the structure factor. We analysed the structure factor component in the SAXS intensities based on the DLVO model interaction potential and the equations above. A slit-smeared SAXS intensity, *I* ~s~(*q*), was simulated from *I*(*q*), which is from the determined *kP*(*q*) and the theoretically derived *S*(*q*) with equation (1)[](#fd1){ref-type="disp-formula"}, and the instrumental beam profile used as a weighting function (Glatter & Kratky, 1982[@bb4]). To find the values of the parameters (*J* and *d*), which reproduce the experimental *I* ~s~(*q*), we compared the experimental *I* ~s~(*q*) with the calculated *I* ~s~(*q*), varying the values of *J* (0.01--9 *k* ~B~ *T*) and *d* (1--15 Å). The maps of the residual sum of squares are shown in Figs. 2(*a*) and 2(*b*)[▶](#fig2){ref-type="fig"}. It is found that smaller *J* and larger *d* tend to reduce the residuals for holomyoglobin, while larger *J* and smaller *d* tend to reduce the residuals for apomyoglobin. The protein--protein interaction potentials, *V*(*r*), calculated with the optimal values of *J* and *d*, which give the best fits, are depicted in Fig. 2(*c*)[▶](#fig2){ref-type="fig"}. At small *r* (*r* \< ∼45 Å), the potential of apomyoglobin is more attractive than that of holomyoglobin, due to the larger *J* value. This could explain the irreversible aggregation-prone property of apomyoglobin (Fändrich *et al.*, 2003[@bb3]). In addition, the attractive potential of holomyoglobin is relatively small negative at small *r* (*r* \< ∼45 Å), but still remains at larger *r* (*r* \< ∼55 Å) due to the larger *d* value. The attractive potential allows the proteins to be close to each other, but to be separated because the magnitude is comparable with thermal energy (∼*k* ~B~ *T*) at short range. This seems to underlie the highly water-soluble and less irreversible aggregation-prone properties of holomyoglobin. 4.. Summary   {#sec4} ============= In the present study we investigated the role for the heme in the self-interaction of myoglobin molecules by SAXS. From the fitting of the experimental SAXS data with the simulated SAXS intensities based on the DLVO model and the equations in the liquid theory, it is suggested that change in the SAXS profile by removal of the heme could be explained by an increase in the intermolecular attractive interaction potential at short range from the protein molecule. We remark that the potential we can obtain depends on the selected model. Therefore, we progress a study developing a method to determine the intermolecular potential of proteins from experimental structure factors without assuming any model potential by applying an integral equation theory. This work was supported in part by the Global Center-of-Excellence Program 'Advanced School for Organic Electronics' supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to HI). {#fig1} {ref-type="fig"} (full line: holomyoglobin; dashed line: apomyoglobin).](s-20-00919-fig2){#fig2}

Background ========== Across all healthcare settings, clinicians perceive particular patients as \'difficult\' \[[@B1]\]. High users of medical services, these patients are generally unsatisfied with the care they receive \[[@B2]-[@B6]\] and may evoke strong negative emotions in clinicians \[[@B1],[@B7]\]. Although clearly a subjective and imprecise term, the perception of patients as \'difficult\' may result in worse care for patients involved \[[@B8],[@B9]\] and increased stress and burn-out among professionals \[[@B10],[@B11]\]. In the scarce empiric research into patients perceived as difficult in psychiatric services, incidence varies between 6 and 28%\[[@B12],[@B13]\]. Earlier, we found that especially patients who do not comply with the obligations of the sick role as defined by sociologist Parsons \[[@B14]\], run the risk to be perceived as \'difficult\' \[[@B6]\]. People have the right to be relieved from their routine social obligations and not be held accountable for their illness, if only they seek and accept professional help, and do their utmost best to restore good health as soon as possible \[[@B14]\]. Among patients perceived as \'difficult\', patients with long-term non-psychotic disorders may be seen as not complying with the latter obligation. Unlike patients with psychotic disorders - who are more obviously out of contact with reality - they may be held accountable for their behaviours \[[@B6]\]. Among long-term non-psychotic patients, no particular psychiatric diagnosis is associated with difficulty, while the number of psychosocial problems, psychiatric service use, and ways in which clinicians perceive these patients are \[[@B13]\]. Clinician variables, such as a dominant focus on medical problems over interest in psychosocial issues, however, repeatedly have been found to be associated with perceived difficulty \[[@B2]-[@B4],[@B13]\], clearly showing that \'difficult\' is defined within the relationship of patient and clinician. Although substantial research into the patient-clinician alliance has taken place \[[@B15]\], the perspectives of patients in general and long-term non-psychotic patients in particular have hardly been explored \[[@B16]\]. Also we are aware of only one (small) study that explored the care experiences of \'difficult\' patients \[[@B17]\]. Here, we focussed on the alliance between the perceivedly \'difficult\' patient and the clinician with the purpose to understand why certain patients - according to their accounts of receiving care - come to be perceived as \'difficult\'. Thus, we hoped to shed a different light on the labelling of patients as \'difficult\' and the possibly poor patient-clinician interactions resulting from it. We stated three research questions: (1) which difficulties do patients who are perceived as \'difficult\' experience in their contact with psychiatric clinicians, (2) which explanations do they have for these difficulties, and (3) what changes should be made to decrease these difficulties? Methods ======= Design ------ To answer the research questions we used a qualitative Grounded Theory \[[@B18]\] research design with individual interviews of long-term non-psychotic patients perceived as \'difficult\' by clinicians. Grounded Theory is a qualitative research method developed for social scientific research, that aims to develop theory grounded in empirical data. It is also widely used in health sciences, mostly - like other qualitative methods - in areas in which current (theoretical) knowledge is limited. Grounded Theory is considered particularly useful in the study of roles and interpersonal processes due to its origin in symbolic interactionism \[[@B19]\]. Participants ------------ We included patients in public psychiatric care meeting the following requirements, based on a widely accepted definition of severe mental disorder \[[@B20]\]: (1) being in psychiatric care for at least two years, (2) having high psychiatric symptomatology and low social functioning (Global Assessment of Functioning \[GAF\] score ≤50 \[[@B21]\]), (3) suffering from a non-psychotic disorder on DSM Axis I and/or a personality disorder on DSM Axis II. One subjective criterion regarding difficulty as perceived by treating clinicians was added. Participants had to have had disagreement over form or content of treatment with two or more professionals at least once in the past two years, as assessed by at least two clinicians. A similar criterion has been used in earlier studies \[e.g. \[[@B12]\]\] and, as imperfect as it is, adds concretization (disagreement), quantity (at least once in past two years), and intersubjectivity (two clinicians). Procedure --------- We selected 12 outpatient departments in three mental health institutes in The Netherlands, striving for a differentiated sample of locations, according to degree of treatment specialization, nature and severity of psychopathology, and geographical dispersion. Key figures of these departments were informed about the research project and were asked to invite clinicians to participate. Treating clinicians (community psychiatric nurses, psychiatrists, psychologists, and social workers) introduced the research to eligible patients as an investigation into difficult relations between psychiatric patients and clinicians. After patients gave consent to establish contact, the first author checked their eligibility with the clinician and then called or e-mailed the patients to arrange an individual interview at their preferred location. After getting acquainted and having explained the project, informed consent, basic socio-demographic and clinical data were obtained prior to the interview. Each participant received a gift certificate to the equivalent of €35/£30. Data collection --------------- Two experienced qualitative researchers (BK & JvO) carried out open-ended interviews between March 2008 and September 2009. The research team (BK, JvO, RP, BvM, AK) spent two instructional meetings to immerse in the subject, to design the interview structure and to practice its application. A topic guide, based on a literature search of relevant databases and patient literature was flexibly used \[additional file [1](#S1){ref-type="supplementary-material"}\]. In the first series of eight interviews, participants were asked after certain topics if they had not mentioned them at all. In the following series of interviews, these checking questions were replaced by questions originating from the analysis of previous interviews. Participants were invited to start their account by the general question: \"Which problems do you experience in contact with psychiatric clinicians, both now and in the past?\". Next, the interviewers invited participants to tell in detail about each of these problems and suggest possible explanations for them. Patients were also invited to suggest solutions or alternatives for the present care. All interviews were electronically recorded and transcribed verbatim. Transcripts were analysed in their original language, Dutch, while relevant quotations were translated into English for this paper. Data analysis ------------- Data analysis took place between March 2008 and October 2009 in an iterative process, typical to the Grounded Theory-method of constant comparison \[[@B18]\]. Each member of the research team independently coded two out of the first four interviews and checked it against coding by the others \[[@B22]\]. This procedure was followed to construct a mutually agreed on initial code tree, from which further coding could be done by one person (BK), using MAXQDA-software \[[@B23]\]. The research team met after respectively 4, 8, 11, 14 and 21 interviews to discuss progress, monitor interviewers\' techniques and congruence, evaluate and conceptually analyze coded interviews, select and explore emerging categories and the mutual relationships, and design theoretical sampling strategies for following interviews. After eight interviews, six main large descriptive categories were constructed to order data. Each category fell apart in three to seven sub-categories. After 11 interviews, a tentative theoretical model of the care process was constructed and a preliminary core category (\'incongruous expectations and perceptions of needs\') was identified. After 14 interviews, an extensive thick description of data was written, structured according to the six descriptive categories. It was discussed and commented on in the research team, resulting in a number of additional questions used in the following interviews to clarify, refine, and expand the categories. Also after 14 interviews, intermediate results were sent to the participants interviewed for a member check, and were accepted as they were. In addition to the existing questions, in interviews 15 through 21 the tentative model was presented to participants and their feedback was elicited. A summary of the research findings and the final theoretical model was discussed in the final meeting after 21 interviews. Methods and results were discussed with external supervisors (AS & GH) after 8, 14 and 21 interviews. An example of the analytical process is the *in vivo*(1^st^order) code \'clinician feels offended\', that was categorized under \'clinicians\' accountability\', then under \'clinicians\' professional characteristics\', that finally became part of one of the six main categories \'professionals\'. Furthermore, because of the both personal and professional qualities of this characteristic of clinicians which was believed relevant to further analysis, a memo (called \'mixing up of personal and professional characteristics\') was added to this fragment. Next, other clinician characteristics were explored and coded in detail, paying attention to for instance causes and consequences (*axial coding*). When clinicians\' characteristics became part of the central theme of this research, it was further explored in relation to the model later reported on (*selective coding*). As posited by Lincoln and Guba \[[@B24]\], qualitative research should show sufficient rigour, or \'trustworthiness\' in their words. In order to enhance this project\'s credibility and dependability, member checking was used to validate intermediate findings. Also, peer debriefing was done with the external supervisors, and a thick description was made to allow co-researchers to assess the research\' transferability. A detailed log book, consisting of memo\'s about data collection, analysis, and interpretation, was kept to ensure confirmability. Ethical approval was obtained from the Institutional Review Board of the organisation the 1^st^author is affiliated with. Informed consent was obtained from all participating patients. Results ======= In total, 29 patients recruited by clinicians were approached by the researchers. Eight refused (lack of time, lack of interest, or too much stress), 21 were interviewed (duration 26-75 minutes, mean 61 minutes). Almost all participants were socially isolated: living alone, having no (paid) work, having very few meaningful social contacts, and having several psychosocial problems (table [1](#T1){ref-type="table"}). ###### Characteristics of participants n \% ---------------------------------------------------------------------------------------------------------------------------- ---------------------- ------ **Age**(mean, sd and range) 38.6 (9.8) \[22-60\] **Gender**  male 10 47.6  female 11 52.4 **Living arrangement**  Alone 19 90.5  With partner 2 9.5  Else 0 \- **Housing arrangement**  Rental 17 81.0  Owned 2 9.5  Living with others 2 9.5  Else 0 \- **Day-time activity**  Work 2 9.5  Volunteer work 5 23.8  Education/college 0 \-  None 14 66.7  Else 0 \- **Number of significant and supportive contacts**(mean, sd, range) 1.7 (1.2) \[0-4\] **Present mental health contact**  None 1 4.7  Outpatient 18 85.7  Day treatment or inpatient 2 9.5 **Years of mental health contact**(mean, sd, range) 15.2 (7.6) \[3-31\] **Number of psychosocial problem areas**(DSM Axis IV; e.g. family issues, housing or financial problems) (mean, sd, range) 3.2 (2.0) \[0-5\] **Diagnosis** *Axis I* Chronic depression/dysthymia 5 23.8 Post Traumatic Stress Disorders 5 23.8 Bipolar Disorder II 3 14.3 Attention Deficit Hyperactivity Disorder 1 4.7 Any substance abuse disorder 3 14.3 *Axis II* Borderline Personality Disorder 12 57.1 Personality Disorder Not Otherwise Specified 7 33.3 Axis I only 2 9.5 Axis II only 7 33.3 Both 12 57.1 From the 17^th^interview we did not collect data that added significantly to our findings. Thus, we carried out four additional interviews (18-21) to ensure that we reached theoretical saturation, and concluded data collection after interview 21. Overall, interviews proceeded relatively smoothly. Some patients expressed substantial grief, anger, or despair about current or past mental health contacts. The interviewers then paused, validated these emotions, and inquired whether the participants wanted to terminate the interview - which did not happen in any instance. Our qualitative analysis was guided by six large categories of which four referred to actors: patients, clinicians, psychiatric services, and the patient\'s social system. Two other categories referred to interpersonal processes: contact between patient and professional, and treatment of the patient\'s problems by the clinician. These six categories are used to structure the answering of the three research questions in the results below, and specifically to construct a model of the patient-professional interaction in the second part of the results-section. Difficulties experienced by \'difficult patients\' -------------------------------------------------- Almost all participants described themselves as being \'difficult\' for professionals, either because they knew they were perceived as such or because they said that they were not \'regular customers\'. Participants described (1) challenging behaviours exhibited by themselves towards clinicians and services, (2) difficulties in contact with individual psychiatric clinicians, and (3) difficulties with mental health care services. Patients described behaviours that could be perceived as \'difficult\' in quite some detail. These varied from not showing up on or walking away from appointments, to disqualifying and offending professionals, to shopping around for help, or claiming, threatening, fighting and stalking professionals. With regard to these behaviours, many acknowledged their heightened sensitivity for interpersonal rejection, personal history of problematic relationships, and high expectations of psychiatric services. These services are a last resort for many of them, often related to the absence of substantial social support. Patients\' sometimes very outspoken expectations of clinicians and services are, in their view, repeatedly not being met. The following citation exemplifies an expectation that may not be particularly high, but clearly very different from what psychiatric clinicians are able or willing to offer. *In the beginning I had this ideal picture of day treatment, that they would comfort me and such things. That did not happen though, instead when I laid down on the couch they said that I could not do so.*\[P15\] *But you do have a preset expectation (\...), like they will start helping me now. You do not think that you will have to do the work, no, you believe they will do it.*\[P19\] The expectation \'to be helped\' is recurrent in many participants\' accounts. Patients feel a strong need for help but actually do not know what can be done. Clinicians in turn, in complex cases, do not know either which tends to culminate in mutual powerlessness. *Can we do anything else for you, they asked. I don\'t know, I said. (..). I mean if I all knew so well than I would not be here, would I??*\[P11\] The second kind of difficulties are those regarding interpersonal contact with clinicians, in which participants differentiate between \'personal characteristics\' and \'professional characteristics\'. On the personal level, participants in particular miss true interest and authenticity. This stretches farther than politeness or professional courtesy, farther than just being listened to. For many participants, clinicians\' merely professional interest seems insufficient, possibly related to their aforementioned high expectations. Some participants make a direct link between their own difficult behaviours within the mental health contact and the lack of \'right interest\' from clinicians. If there is no such true interest, these participants tend to stay away or start acting in a way that may be perceived as \'difficult\'. *When I say something out of personal experience some doctors reply \'well who has went to school for this?\'. Those kind of remarks make me very, very angry.*\[P13\] Professional characteristics participants search for in clinicians, are taking the lead, accepting responsibility, and setting out a clear course of treatment. An empathic and understanding attitude does not suffice, participants also want their clinician to assess them correctly, to look beyond their initial presentation and confront their easy excuses. While the aforementioned personal characteristics (true interest and authenticity) are most important to the interpersonal process of contact, clinicians\' professional qualities are most important for the treatment process. Participants clearly state that these professional characteristics, however important, come into play only when a good-enough contact with the clinician has developed. At the same time, in many of the participants\' accounts, personal and professional characteristics are not so clearly distinguishable. For instance, taking responsibility is not only seen as a strong professional asset but also as a sign of personal involvement, of real interest, and even of warmth. *They decided to take me by the scruff of the neck and help me. They did not give up on me. And that is what I am enormously grateful for now.*\[P2\] In some cases the desire for warmth and responsibility goes as far as one participants wishing for a long-term compulsory admission. *But for a psychiatric patient, who has no-one, an involuntary admission may mean that there is still one person on the earth, even though it is an institution, that at least cares a bit about my fate.*\[P12\] The wish for clinicians\' personal involvement, however, is limited by the extent to which clinicians bring their own emotions into the contact. Clinicians\' strong emotions are perceived as a source of potential difficulties by participants. For instance, one participant described a therapist that addressed the patient\'s noticeable alcohol odour due to drinking the night before. She expressed her personal feelings about the patient coming to their first appointment hung over and kept on repeating her discontent. *She did not ask one single question, all she did was whine about what I had done to her. Yeah, right. Well, now I go home and hang myself - how would*that *make her feel?*\[P3\] In line with this, several participants state that clinicians tend to interpret \'difficult\' behaviours far too easily as personally directed towards them. They want clinicians to be more neutral in such cases, to understand certain behaviours as part of the patient\'s disabled behavioural repertoire and to asses it correctly as meaningful or functional. Yet at the same time participants loathe this neutrality when it turns into a distant, objectifying attitude. This puts the professional in a one-up position which many patients find hard to tolerate. The third kind of difficulties are those with psychiatric services, which tend to hamper access by all kinds of complex organisational procedures, such as low contactibility of clinicians, limitation of care, and high thresholds for certain treatments. Also there are unwritten rules, so they say, considering themes that are apparently not appropriate to discuss or do. These issues are at odds with the involvement participants desire. At a more abstract level, participants note collective negative attitudes in psychiatric clinicians, exemplified by the negation of patients\' positive characteristics and pessimism about recovery opportunities. While participants feel that their illness, deviance, and difficulty is focussed on constantly in psychiatric services, they also experience that in order to maintain their contact or to receive treatment, they should behave as \'good\' patients (i.e. seek and accept help and do their best to get better as soon as possible). *Professionals continuously laid demands on me about what I could or should not do. Never positive about what I could or should do.*That *I can draw strength from. Not from demands or expectations of what I should or could not do.*\[P15\] Participants state that in psychiatric services, patients\' failures and pathology are constantly paid attention to and pointed out. Yet at the same time these pathological behaviours (e.g. using illicit drugs, self-mutilating or attempting suicide) are not tolerated and may be reasons to refer or discharge patients, which may be one of the unwritten rules referred to above. *I came there and could not smoke marihuana, I could not self-mutilate, I could not\... But what I*could *do was unclear to me. I did not understand it.*\[P15\] Another participant tells about her admission to a hospital because of suicidal intentions, where she had to hand in her medication. After refusing this, she was discharged (still in possession of the pills). *That serious they took the problem, they put you back on the street. (\...). Try to keep someone inside and to make contact with where someone\'s at, do not start a struggle over pills or self harm. That I still find so strange that people are put on the street because they do that*\[self harm\]*. No, I find that cruel, truly cruel.*\[P14\] Another such account: *I grew only more suicidal and destructive. All the time I got some sort of slap in my face: you better leave, we can\'t do anything for you. All it was, was a confirmation that I did not belong there, that I was nothing.*\[P19\] Explanations for perceived difficulties: lack of recognition ------------------------------------------------------------ We now move to possible explanations for the difficulties in the patient-clinician relationship. All patients want clinicians to recognize their suffering and their needs. This recognition of needs, however, does not automatically mean that patients want to be seen as *patients in need*. Many find it hard to accept the patient role, or even concur with their given diagnosis. A distant and strictly medical approach (i.e. being offered diagnosis, prognosis and treatment by a skilled doctor) was endorsed by none of the participants. While they believed this to be a necessary but not sufficient element of care, it was once again pointed out that treatment cannot exist without contact. For some, receiving a diagnosis meant recognition of the genuineness of their problems and suffering. *But if you have an appointment with a psychiatrist who does not say what is best for you than you do not have it. You don\'t have that little paper that says what is exactly wrong with you.*\[P5\] *Well, I was happy that I finally could, well, give it a name. That it was truly something. A personality disorder, or whatever you want to name it.*\[P11\] For others, receiving a diagnosis exemplified the inequity of the patient-professional interaction. With personality disorders, participants often resented their given diagnosis since they believed it actually hampered access to health care. Some expressed the wish to receive a diagnosis unburdened with the notion of \'being guilty\' of their behaviour, in order to have better access to services. As such, different notions by patients and professionals of both the function and type of diagnosis may be partly explanatory for difficulties. Independent of diagnosis, all participants expressed a deep need to feel understood, and in some cases, to be cared for by health professionals. The mental health system was described as a far from ideal but still the best environment to have this need met, better than their - so often absent - social system or other helping agencies. In other words, mental health care offers the least bad environment, shown by the statement of a participant who expresses her feeling to be relegated to mental health care. *People don\'t understand that*\[vulnerability\] *at all. It is such a lack of recognition. (\...). Then, psychiatry is the lesser of two evils. That is why I stay there, I believe. I do occasionally have a good conversation, or I am sometimes able to find some relief. Otherwise I only start doing crazy things and become more sad.*\[P14\] From this point of view we may understand difficulty partly as a consequence of patients\' ambivalence towards psychiatric care: needing it without wanting to. This perceived need merits further attention, since in spite of previous negative experiences and expressed discontent with several clinicians\' characteristics, participants do remain in psychiatric care. *It*\[psychiatry\] *does not bring me any further, it does not offer any grip. It is not something one can pull oneself up on like for instance work is. Once again, I will always keep on going there*\[mental health care\] *without wanting to.*\[P8\] They appear to be looking for exceptions to the rule, for the one clinician that does understand them. Some are able to find this person but many are not and keep on fighting the misunderstanding they experience. Many clinicians appear to be unable to truly identify and validate the needs of these patients. At the same time, these needs may be so existential that psychiatric services will never be able to accommodate them, as exemplified below. *I expect, and that appears to be undeliverable, my basic problem is that I just want my mother. But that one simple thing is not available in psychiatry.*\[P12\] Instead of \'tender loving care\', patients get \'distant\' advice and structure. Many deeply resent the \'doctor knows best\'-attitude of some clinicians, and do not want to be told what their life is, or should be like. Such active, but often also strict and formal clinicians, are easily perceived as bringing about a power imbalance that takes away the patient\'s control over the treatment encounter, and even the patient\'s life. Yet, not having to be in control also relieves patients from their obligations and clearly acknowledges their needs and limitations in doing things themselves. Two participants exemplify this paradox in vivid terms. *These power relations feel very safe on the one hand because you just don\'t have anything to say anymore. Really, that security from when you were a child. Everything is being done for you and you just have to do this at that time and nothing else really. But on the other hand, it is not good since you cease to be a person.*\[P4\] *At the moment I am not right, I feel very dependent, really very small. Then I think, oh no, I really need them. Yet, when I feel better, I am annoyed about them and their idea that they can decide what is good for me*\[P13\]. Patients once again appear very ambivalent about truly accepting help and the patient role. They express their difficulties with being either a person who is competent and autonomous, or a patient who is incompetent and dependent, and appear unable to combine those. Yet, according to participants not only patients have difficulties relating to this polarized notion of autonomy and helplessness. Clinicians also have difficulties to tolerate these two sides of one person, and tend to respond paradoxically to patients that display either one of them. Whenever a patient appears able to communicate his or her needs clearly, professionals see this as a sign of good mental health. So, when the patient asks for help in a \'normal\' way, that is without dramatizing, threatening or without visibly being shattered, clinicians tend to believe that help is not actually required. *They said: \'you can articulate it so clearly, we believe that nothing is necessary\'. That I found so bizarre, since I was doing everything to articulate myself clearly since otherwise I could not bring the message across. I would not receive help when I articulated it poorly, nor when I articulated my needs clearly.*\[P11\] Implicit notions about help-seeking behaviour are suggested by these examples. Clinicians expect patients to ask for help in a non-dramatic, rational, but still indigent way. Patients should thus not come up too autonomous or dependent, since clinicians seem to hold unspoken views of what is the right way to ask for help. When the patient is highly autonomous, the clinician appears to be unnecessary and may feel unseen him or herself. When the patient is overly dependent or \'needy\', the clinician sees this as overreacting or even manipulative, and as potential risk of dependency. Patients desire a special kind of understanding and compassion from clinicians, that incorporates both their personal qualities and their difficulties, and not solely focuses is on what is wrong, or easily concludes that nothing is wrong. Clinicians, on the other hand, are easily confused over patients\' presentations and tend to take adequate help-seeking behaviour for the absence of problems and needs. Margins for both patients\' and clinicians\' behaviour appear very narrow, which we will further exemplify in the next paragraph. *I am afraid that it is a mixture of my own paranoia and hostility towards health professionals, and the way I interpret what they say. And the interaction that comes from this. (\...). Plus that they have this panic-like fear for dependency of patients.*\[P12\] Changes in patient-clinician contact: using the \'therapeutic window\' through different stages ----------------------------------------------------------------------------------------------- The narrow margins of \'right\' behaviour of both patients and clinicians described above, returned across many interviews and categories. Also, they were not static entities but changed over time. This closely relates to the core category we came to construct: incongruence of expectations and perceptions of needs. Participants repeatedly described wanting something else than professionals: more or another kind of care, more (or less) personal involvement, or a more structured approach to problems. Combining this with another recurring finding, that of contact and treatment as two separate dimensions, we tentatively constructed a stages model in the contact process with \'required\' clinician behaviour per stage (figure [1](#F1){ref-type="fig"}). In each stage, there is a \'therapeutic window\' of optimal clinician behaviour, and two wider spaces - both below and above the therapeutic dosage - of \'toxic\' behaviour. {#F1} The first three stages of this model (figure [1](#F1){ref-type="fig"}) all concern \'contact\', while the latter three concern \'treatment\'. In the first stage (\'acquaintance\') patient and professional meet and get basically acquainted. Patients expect some basic interest of the professional at this stage, while rapid over-involvement or clear disinterest may be toxic and prevent the patient from returning for a next meeting. The next stage (\'clique/fit\') requires more closeness from the professional, but not over-disclosure of personal information or too much distance. A clique refers to a certain level of personal contact that shows the patient that the clinician cares. *I think it\'s a clique, it has to do with a clique. A clique between professional and patient is very important. Because if it cliques, then you gain trust.*\[P2\] The third stage (\'true contact\') is a crucial one, in which the clinician needs to recognize and genuinely understand the patient with both his or her qualities and shortcomings, as well as the patients\' suffering. In this stage most difficulties tend to arise, since expectations are up from the previous stages. Patient and clinician must navigate themselves through all the ambivalent demands described in the previous paragraphs. Toxic responses by clinicians include over-identification with the patient, and trivialization of problems and needs since this reinforces patients\' earlier experiences of uncaring clinicians. In this stage, toxic clinician behaviour may result in more intense patient responses (e.g. becoming disqualifying, angry, clinging, or threatening) than not returning for another appointment. *I believe that because when you are recognized, you are heard, and then you don\'t start fighting all the time to be heard.*\[P14\] The second three stages all concern \'treatment\'. The fourth stage (\'mutual strategy\'), is the one in which the content of treatment becomes involved. A mutual agreement over goals and a treatment strategy need to be developed. In order to do this, more than just understanding is required, the clinician needs to be active and directive. This solidity should not be too rigid, or be too weak, since both are toxic to patients that look for a clear course. *And then the conversations start to dilute into something I can\'t define any more. (\...). Then I have completely lost track. There is no structure any more, no direction. Yeah, at a certain moment, yeah, you just stop going.*\[P3\] In the next stage (\'active help\') the clinician should show not to be afraid to take responsibility for the patient\'s well-being and show continued involvement. Participants state that it is important that clinicians show their willingness to do some work for their patients. Failure to find a non-toxic level of intervention may result in patients perceiving the clinician as paternalistic or non-committed. *And if there\'s some time left, they ask me if they should join me to social services or anything. And that is really great sometimes, because it makes me more motivated to do start doing such things again by myself.*\[P9\] In the sixth and final stage (\'continuation of fitting help\') clinicians must carefully monitor the care process for recurring or new difficulties in the contact. The clinician needs to be perseverant in focussing on treatment goals, and vigilant for possible breaches in the contact. Too much persistence can result in rigid insistence, which like its opposite - negligence - is toxic to the patient. *So there is little attention for the progress one has made. Is he feeling better, is it right what we are doing here?*\[P5\] We may state that the therapeutic window for interventions with \'difficult\' patients is very narrow. In each stage things can go wrong due to either the lack of, or the excess of this required behaviour by clinicians. In both cases, such behaviour may be toxic to patients who are in substantial need of recognition of their problems and needs as described before. Discussion ========== This research explored the views of patients perceived as \'difficult\' on their contacts with psychiatric clinicians and services, in order to improve our understanding of difficult treatment encounters. We found that patients have difficulties with a variety of clinicians\' and services\' characteristics, of which disinterest, noncommittal, and a general negative view are the most important. The interpersonal process of perceived lack of recognition, grounded in the incongruence of expectations of one another, may be considered the major explanation for difficulties between patients and professionals. We constructed a staged model in which the development of personal contact is most important to patients during the first three stages, and to which substantial treatment is added in the next three stages. The stage in between personal contact and substantial treatment is pivotal and concerns the recognition of patients as both genuinely ill, and valuable human beings with capacities and shortcomings. Substantial findings -------------------- Although the starting point of this research, and the premise of our sampling strategy, it cannot be upheld that \'difficult\' is an attribution that can be objectively made upon patients. The findings of this study thus deserves interpretation on different levels. A first important finding on patient level is that perceived difficulty may partly be explained by the ambivalence of these patients to fully assume the patient role. This appears to be a central feature of all participants and explains why such patients are found among people with quite different diagnoses. Not specific diseases themselves, but the way people perceive them and the way they want health clinicians to respond to them, appears associated with difficulty. Also, it explains why these patients evoke such strong and ambivalent emotions in health professionals. If the patient is unwilling to accept the patient role, a clinician cannot take up the designated role of genuine helper. It is quite well established that any health professional whose help is denied, questioned, ridiculed or whatsoever, feels frustrated \[e.g. \[[@B1],[@B3]\]\]. To a certain extent, the \'difficult\' patient who feels unseen, unheard and unrecognized, is mirrored by the clinician who remains unrecognized as a genuine helper. A second important finding, on professional and services level, is that mental health care does not very well know how to respond to patients that behave different and less predictable than other patients. The response of choice to patients that are ambivalent about being a patient, seems to be an intensification of efforts to make him or her fit the \'normal\' patient frame - which in fact has the opposite effect. For instance, assuming the expert role to convince the patient to behave differently, is exactly what will exacerbate the patient\'s unpreparedness to do so. It may be much more effective for the professional to recognize, voice, and discuss the patient\'s ambivalence. A third finding, that encompasses different levels, is that patients who are perceived as \'difficult\' and their clinicians who perceive them as such, have very different expectations about the contact with one another. The expectations patients have in different stages of the interaction with health professionals have been exemplified in the model. This model offers insight into the various expectations and allows clinicians to discuss these with patients in different treatment stages. Clinicians may thus use this knowledge to explicate mutual expectations and set up mutually agreed on goals and actions. Limitations and strengths ------------------------- There are limitations to our study. First, the results need careful interpretation since they potentially suffer from a self-serving bias of participants. Very much like clinicians in earlier research \[[@B13]\], patients primarily report behaviours of the other they have trouble with. Second, our findings do not apply to psychiatric patients that are sent, or even sentenced, to mental health care. Third, we were unable to use alternative data sources to verify our findings (triangulation \[[@B25]\]). Despite several invitations, none of the participants was willing to attend a focus group discussion to verify intermediate findings and collect new data. Fourth, sampling proved to be complicated during the entire research for which reason selection bias is a risk. Many clinicians did not readily enrol possible participating patients, notwithstanding the description of this project as research into difficult interactions. Also, the requirements of both purposive sampling (to allow variation of socio-demographic characteristics, psychiatric diagnosis and health care settings) and theoretical sampling (following from intermediate analyses) limited the number of suitable participants. Also, initially enrolled patients did not always follow through when the interview date came closer. The period of data collection was therefore substantially extended. Potential undersampling of the most \'difficult\' patients, however, is countered by the fact that participants, who were announced as \'really difficult\' patients by clinicians, proved to be willing and even eager to participate. We believe that refusing research cooperation is not a primary characteristic of this population, thus suggesting the absence of selection bias on these grounds. Although our sample size was smaller than intended, theoretical saturation appeared relatively soon, and was followed by four additional interviews to ensure validity. To our knowledge, this is the first qualitative study into the experiences of \'difficult\' patients using a sufficient sample size and rigorous qualitative methodology. Current and future research --------------------------- Our findings, and especially the model, concur quite well with, and add some detail to, the literature on the importance of the therapeutic alliance in psychiatric treatment and the required focus on bonds, goals, and tasks \[[@B26],[@B27]\]. The importance of true interest in, and recognition of, the patient and his or her suffering, is under different names also found in modern care models for different non-psychotic disorders \[[@B28]-[@B30]\]. More surprisingly, findings from studies of \'difficult\' patients with medically unexplained symptoms in general health care, are quite consistent with ours \[e.g. \[[@B31]\]\]. In this study, patients expectations also differed from those of doctors, while in another study \[[@B32]\] the recognition of suffering, followed by a open discussion of treatment options was a finding comparable to our findings. Future research into difficult alliances may sample pairs of patients (both perceivedly \'difficult\' and \'non-difficult\') and professionals, both investigating their mutual expectations, interactions, and progress over time. Conclusions =========== The incongruence of some patients\' and professionals\' expectations may result in power struggles that may make professionals perceive patients as \'difficult\'. Explication of mutual expectations may be useful in such cases. Additionally, clinicians may first wholeheartedly acknowledge and recognize the needs of such patients, only to proceed with more formal treatment procedures (such as clarification of expectations, setting of goals, and choosing of interventions) from there. The presented model may be helpful to navigate through the different stages of the patient-professional contact. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= BK, BvM, AS and GH devised the idea of the study and designed the methods. JvO conducted, and RP participated in the interviews. BK, BvM, JvO, RP and AK performed analyses of the data and regularly discussed progress. BK led the data collection, analysis, and prepared the manuscript. BvM, AK, AS and GH co-drafted the manuscript. All had full access to all data. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-244X/10/96/prepub> Supplementary Material ====================== ###### Additional file 1 **Literature review of documents written by patients**. search strategy and results of a review of patient documents in the psychiatric literature. ###### Click here for file Acknowledgements ================ This study was funded by ZonMW (The Netherlands Organisation for Health Care Research and Development) \'Geestkracht\'-program (Grant 100-002-031), Altrecht Mental Health Care and Gelderse Roos Mental Health Care. None of these bodies had any role in the study design; the collection, analysis, and interpretation of data; the writing of the paper; or the decision to submit this paper for publication. Ethical approval The Altrecht Mental Health Care Committee on Scientific Research gave permission for this study (reference 2008/JJ/6181), and advised that further ethical approval was not required.

Introduction {#S0001} ============ Prostate cancer is the sixth most frequent cancer in the world (in the number of new cases), the third most common cancer in men, and the most frequent cancer in men in Europe, North America, and some parts of Africa \[[@CIT0001]\]. The most general sites of prostate cancer distant metastasis are bones, regional lymph nodes, lung, liver, brain, and the epidural space \[[@CIT0002]\]. However, metastasis from prostate cancer is an extremely unusual situation with a reported rate of 0.4% to 1% of cases \[[@CIT0003]\]. We report two prostate cancer cases that presented with a supraclavicular bulky mass as the initial and the most bothersome symptom. Case 1 {#S0002} ====== A 78-year-old male who attended the emergency department with a bulky left supraclavicular mass was consulted because of lower urinary tract symptoms. Physical examination revealed a solid, multilobulated mass in the left supraclavicular region, measuring 9 cm at its longest diameter. He reported weight loss of 15 kg over the previous four months and symptoms related to lower urinary tract obstruction for five months. Laboratory investigations in terms of whole blood count, blood chemistry and urinalysis were all in the normal ranges. A thorough genitourinary examination revealed no obvious pathology. Digital rectal examination (DRE) revealed a hard, irregular prostate with a volume of 58 cc in transrectal ultrasonography (TRU). Serum prostate-specific antigen (PSA) level was 171.0 ng/ml. Bone scintigraphic examination revealed a metastatic lesion at the fifth lumbar vertebra. Fine needle biopsy of the left-sided supraclavicular lymph node was performed. Histological examination revealed a lymph node widely replaced by metastatic adenocarcinoma staining positive for PSA and prostate-specific acid phosphatase consistent with metastatic adenocarcinoma of the prostate ([Fig. 1](#F0001){ref-type="fig"}). Therefore, twelve quadrant fine needle biopsies were performed under TRU guidance which revealed a prostate cancer with a Gleason score of 8/10. {#F0001} He was started on cyproterone acetate, 100 mg, three times a day and leuprorelin acetate injections, 3.75 mg, monthly. Ten months later, the patient had significant symptomatic relief with a marked reduction in supraclavicular lymphadenopathy and a decreased prostate-specific antigen level of 12.1 ng/ml. Case 2 {#S0003} ====== A 65-year-old male patient was admitted to the oncology outpatient clinic with complaints of left supraclavicular lymphadenopathy. Physical examination revealed a solid, 5 cm mass in the left supraclavicular region. Laboratory investigations in terms of whole blood count, blood chemistry and urinalysis were all in the normal ranges. A thorough genitourinary examination revealed no obvious pathology. Digital rectal examination (DRE) revealed a hard, irregular prostate with a volume of 64 cc in transrectal ultrasonography (TRU). Serum prostate-specific antigen (PSA) level was \> 1500 ng/ml. Serum free PSA level was \> 25 ng/ml. Bone scintigraphic examination revealed multiple vertebral metastatic lesions. Trucut biopsy of the left-sided supraclavicular lymph node was performed. Histological examination revealed a metastatic adenocarcinoma staining positive for PSA and prostate-specific acid phosphatase consistent with metastatic adenocarcinoma of the prostate. He was started on bicalutamide 50 mg once a day. Discussion {#S0004} ========== Prostate cancer is the most frequent cancer and the second leading cause of cancer death in men \[[@CIT0004]\]. Dissemination of prostate cancer into the adjacent organs (urethra, bladder neck and seminal vesicles) is rare \[[@CIT0005]\]. Prostate carcinoma is known to invade by way of three mechanisms: local, haematogenous spreading, and lymphatic metastasis \[[@CIT0006]\]. Bone represents the principal location of distant metastasis in prostate cancer \[[@CIT0007]\]. Lymphatic spreading occurs most often in regional lymph nodes such as the obturator, internal and external iliac lymph nodes, presacral and para-aortic lymph nodes. Distant superficial lymphadenopathy is a rare symptom on initial presentation in prostate cancer \[[@CIT0008]\]. In terms of a supraclavicular mass discovered during physical examination, most frequently nasopharynx, oral cavity and upper gastrointestinal tract cancer metastases to cervical and supraclavicular lymph nodes are observed \[[@CIT0009]--[@CIT0011]\]. Saeter *et al*. \[[@CIT0012]\] noted that, in 35 patients with non-regional lymphatic invasion from prostate carcinoma, the left supraclavicular fossa was the most common location of metastasis in 69% of cases. Butler *et al*. published 19 patients with prostate cancer presenting at first with supraclavicular lymphadenopathy, in which the diagnosis was verified by prostate biopsy in 14 patients. They reported that only 42% of all patients had an abnormal DRE \[[@CIT0013]\]. Case 1 also had an abnormal DRE. Woo *et al*. described a 76-year-old patient presenting with supraclavicular lymphadenopathy. They noted a normal prostate on DRE. A PSA performed a few days after admission was 326 ng/ml and a fine needle biopsy of the lymphadenopathy confirmed a prostate cancer metastasis \[[@CIT0014]\]. Cho *et al*. reported 26 cases of metastatic prostate cancer in supradiaphragmatic lymph nodes, in which only 7 cases had a history of prostate cancer; they noted that 58% had an abnormal rectal examination \[[@CIT0015]\]. Although prostate cancer is widespread, the prostate is often overlooked as the first site for men presenting with supraclavicular lymph node metastases \[[@CIT0005]\]. Fine needle aspiration biopsy of supraclavicular lymph nodes may be useful for pathological diagnosis of prostate cancer metastasis. In our patients, serum PSA level and immunohistochemical staining for PSA in biopsy material were used in detection of the primary location of the cancer. Two patients had highly elevated serum PSA levels, and bone scintigraphic examination revealed metastatic lesions. It should always be kept in mind that prostate cancer is the most frequent cancer in elderly men, and although very unusual, the presenting finding can be a cervical or supraclavicular lymphadenopathy; thus clinicians should be aware of urological examinations of such cases.

{#sp1 .348} {#sp2 .349}

Introduction {#Sec1} ============ An elastance--resistance model is an effective tool to quantify the systolic pumping mechanics of the heart in in situ, open-chest experiments \[[@CR5], [@CR14], [@CR21], [@CR22]\]. Parameters generated by this model to characterize the cardiac physical processes are maximal systolic elastance (*E*~max~) and theoretical maximum flow (*Q*~max~). Physically, *E*~max~ can represent the intrinsic myocardial contractility in an intact heart because (1) it reflects subtle changes in the contractile status of the left ventricle and (2) it is independent of the preload, afterload, and heart rate for a given cardiac contractile state \[[@CR11], [@CR24]\]. *Q*~max~ is the amount of outflow generated by the ventricle if it were to eject under zero-load condition, and it shares an inverse relationship with the resistive behavior of the left ventricle \[[@CR20], [@CR22]\]. To develop an elastance--resistance model for assessing the cardiac pumping properties, simultaneously recording the left ventricular (LV) pressure and aortic flow (*Q*^m^) followed by recording the isovolumic LV pressure is indispensable \[[@CR5], [@CR14], [@CR21], [@CR22]\]. The ascending aorta-occlusive method must be used to measure the isovolumic LV pressure at the end of diastole under open-chest conditions. Obviously, the technique for recording the isovolumic LV pressure is not permitted in human patients. In 1997, Chang and Kuo \[[@CR7]\] used a high-fidelity multi-sensor catheter to simultaneously measure the LV pressure and aortic flow in anesthetized, closed-chest dogs. A curve-fitting technique, proposed by Sunagawa et al. \[[@CR26]\], was performed to estimate the isovolumic LV pressure by using the recorded instantaneous LV pressure of an ejecting contraction. They discovered that an elastance--resistance model with the estimated isovolumic LV pressure can potentially be used to study the systolic pumping mechanics of the heart. In 1989, Kelly et al. \[[@CR12]\] found that second zero crossing of fourth-order derivative of aortic pressure is close to peak of flow. The approximation of the aortic flow to a triangle (*Q*^tri^) was reported and validated by Westerhof et al. \[[@CR29]\]. In their study, the timing of the peak of the triangle was derived using a fourth-order derivative of the aortic pressure waveform. Using *Q*^tri^, they successfully separated the measured aortic pressure pulse into its forward and backward components. In 2017, Wang et al. \[[@CR28]\] evaluated a method for determining the slope (*E*~es~) of the end-systolic pressure--volume relation (*ESPVR*) on the basis of the measured LV pressure and an assumed *Q*^tri^. In their study, *Q*^tri^ was derived using a fourth-order derivative of the LV (but not aortic) pressure to approximate its corresponding *Q*^m^. The present study elaborated these concepts by determining the LV *E*~max~ and *Q*~max~ by using the measured LV pressure without recording the isovolumic LV pressure and aortic flow signals. A single-beat estimation technique was employed to calculate the *E*~max~ and *Q*~max~ by using an elastance--resistance model based solely on the measurement of the LV pressure and cardiac output. The technique proposed by Sunagawa et al. \[[@CR26]\] was used to estimate the isovolumic LV pressure from the measured LV pressure. An uncalibrated *Q*^tri^ was constructed from the measured LV pressure based on the method proposed by Wang et al. \[[@CR28]\]. The *Q*^tri^ scale was calibrated using the cardiac output. Values of *E*~max~^triQ^ and *Q*~max~^triQ^ obtained using *Q*^tri^ were compared with those of *E*~max~^mQ^ and *Q*~max~^mQ^ obtained from the measured *Q*^m^. Healthy rats (NC), rats with chronic kidney disease (CKD), and rats with type 1 or type 2 diabetes mellitus (DM) were analyzed. If the proposed method works out, the systolic elastic and resistive behaviors of the ventricular pump in human patients can be quantitated by using a minimally invasive measurement on the LV pressure together with non-invasive measurement on cardiac output. Methods {#Sec2} ======= Animals and catheterization {#Sec3} --------------------------- Male Wistar rats aged two months were divided into the following four groups: (1) NC (*n* = 25), (2) CKD (*n* = 14), (3) type 1 DM (*n* = 20), and (4) type 2 DM (*n* = 12). Female healthy rats (FNC; *n* = 7) were also included in the present study. According to the method reported by Floege et al. \[[@CR9]\], CKD was induced through 5/6 subtotal nephrectomy (i.e., right nephrectomy and ligation of two branches of the left renal artery) in rats under anesthesia with sodium pentobarbital (50 mg kg^−1^; intraperitoneal). The levels of serum creatinine and blood urea nitrogen were determined using an autoanalyzer (Model 7070, Hitachi Electronics Co., Ltd., Tokyo, Japan). Type 1 DM was induced through a single-tail vein injection with 55 mg kg^−1^ streptozotocin (STZ; Sigma, St. Louis, MO, USA) in 0.1 M citrate buffer (pH 4.5; Sigma) \[[@CR28]\]. Type 2 DM was induced by intraperitoneally administering 180 mg kg^−1^ nicotinamide (NA) (Sigma, St. Louis, MO, USA) 30 min before an intravenous injection of 50 mg kg^−1^ STZ dissolved in 0.1 M citrate buffer (pH 4.5) \[[@CR15]\]. To confirm hyperglycemia, the blood glucose levels were measured using a SURESTEP Test Strip (Lifescan Inc., Milpitas, CA, USA) in the rats with induced DM. Changes in the systolic mechanical behavior of the ventricular pump were monitored 8 weeks after DM and CKD induction. All the rats were provided ad libitum Purina chow and water and housed under 12-h light--dark cycles. The experiments were conducted according to the Guide for the Care and Use of Laboratory Animals, and our study protocol was approved by the Animal Care and Use Committee of the National Taiwan University \[[@CR28]\]. The cardiodynamic variables were measured in the anesthetized rats, according to previously described general surgical procedures and methods \[[@CR28]\]. Briefly, the rats were anesthetized with sodium pentobarbital (50 mg kg^−1^; intraperitoneal), placed on a heating pad, intubated, and ventilated using a rodent respirator (Model 131, New England Medical Instruments, Medway, MA, USA). The chest was opened at the second intercostal space on the right side. An electromagnetic flow probe (100 series; internal circumference, 8 mm; Carolina Medical Electronics, King, NC, USA) was positioned around the ascending aorta to measure the pulsatile aortic flow. A high-fidelity pressure catheter (Model SPC 320; size, 2F; Millar Instruments, Houston, TX, USA) was inserted through the isolated right carotid artery into the left ventricle to measure the LV pressure. The electrocardiogram (ECG) of lead II was recorded using a Gould ECG/Biotach amplifier (Cleveland, OH, USA). Signals (5--10 beats at steady state) were selected on the basis of the following criteria: (1) recorded beats with optimal LV pressure and aortic velocity profiles; (2) beats with an RR interval less than 5% different from the average value for all recorded beats; and (3) exclusion of ectopic and post-ectopic beats. The selective beats were averaged in the time domain by using the peak R wave of the ECG as a fiducial point. A single-beat estimation technique was used to calculate the *E*~max~ and *Q*~max~ that characterize the systolic pumping mechanics of the heart \[[@CR7]\]. Construction of an isovolumic pressure and a triangular flow from the measured LV pressure {#Sec4} ------------------------------------------------------------------------------------------ The isovolumic LV pressure (*P*~iso~, Fig. [1](#Fig1){ref-type="fig"}b) can be derived from the measured LV pressure of an ejection contraction (*P*~LV~, Fig. [1](#Fig1){ref-type="fig"}a) by Eq. ([1](#Equ1){ref-type=""}) described in Appendix 1. The estimated peak isovolumic pressure (*P*~isomax~) is the pressure sum of the peak developed isovolumic pressure (*P*~idmax~) and the LV end-diastolic pressure (*P*~d~) (Fig. [1](#Fig1){ref-type="fig"}b). The uncalibrated *Q*^tri^ can be constructed by using the measured LV pressure waveform demonstrated in Appendix 2. An inverse Fourier transformation of the fourth-order derivative of the LV pressure with the first 15 harmonics (Fig. [1](#Fig1){ref-type="fig"}c) was used to determine the onset, termination, and peak time points of the triangle (green curve, Fig. [1](#Fig1){ref-type="fig"}d). The *Q*^tri^ scale was calibrated using the cardiac output.Fig. 1Construction of the LV isovolumic pressure (*P*~iso~, **b**) and aortic triangular flow (*Q*^tri^, **d**) from the measured LV pressure waveform (*P*~LV~, **a**) in a CKD rat. LV, left ventricular; *P*~d~, LV end-diastolic pressure; *P*~idmax~, peak developed isovolumic pressure; *P*~isomax~, peak isovolumic pressure; *Q*^m^, measured aortic flow Calculation of the LV end-systolic elastance {#Sec5} -------------------------------------------- The LV end-systolic elastance (*E*~es~) can be calculated from the LV end-systolic pressure--stroke volume relationship (*ESPVsR*) that can be derived from the end-systolic pressure--volume relationship (*ESPVR*) \[[@CR7], [@CR25]\]. Briefly, the pressure-ejected volume loop (green curve, Fig. [2](#Fig2){ref-type="fig"}a, b) was obtained by the measured LV pressure (Fig. [1](#Fig1){ref-type="fig"}a) and the time integration of aortic flow from either *Q*^m^ (black curve, Fig. [1](#Fig1){ref-type="fig"}d) or *Q*^tri^ (green curve, Fig. [1](#Fig1){ref-type="fig"}d). In Fig. [2](#Fig2){ref-type="fig"}a, b, drawing a tangential line from the estimated *P*~isomax~ to the right corner of the pressure-ejected volume loop yields a point referred to as the end-systolic equilibrium point \[[@CR2]\]. The line that connects the estimated *P*~isomax~ to the end-systolic equilibrium point is the *ESPVsR*, which is denoted as the red line. The slope of this red line represents the LV *E*~es~. Calculation of the LV maximal systolic elastance and theoretical maximum flow {#Sec6} ----------------------------------------------------------------------------- The LV *E*~max~ and *Q*~max~ can be generated by using the elastance--resistance model, i.e., Eq. ([2](#Equ2){ref-type=""}), to predict the model-derived LV pressure, which was described in Appendix 3. The signals required to fit the elastance--resistance model were the measured LV pressure, the estimated isovolumic pressure, and the aortic flow from either *Q*^m^ or *Q*^tri^. The parameters (i.e., *Q*~max~ and *V*~eed~) that coincided with the minimum objective function are considered as the model estimates of the systolic pumping mechanics of the heart (green line, Fig. [2](#Fig2){ref-type="fig"}c, d). Thus, the maximal systolic elastance of the left ventricle can be computed using the relationship *E*~max~ = *P*~isomax~ / *V*~eed~; *V*~eed~ is the effective LV end-diastolic volume. The maximal internal resistance of the left ventricle is expressed as *R*~max~ = *P*~isomax~ / *Q*~max~.Fig. 2Calculation of the LV *E*~es~ and *E*~max~ from the measured *P*~LV~ in the same rat, which is shown in Fig. [1](#Fig1){ref-type="fig"}. The LV *E*~es~^mQ^ is the slope of the *ESPVsR* line, as derived from the measured *Q*^m^ (red line, **a**) and the LV *E*~es~^triQ^ from the assumed *Q*^tri^ (red line, **b**). The elastance--resistance model is used to predict the LV pressure data using either the measured *Q*^m^ (green line, **c**) or assumed *Q*^tri^ (green line, **d**), generating their corresponding LV *E*~max~^mQ^ and *E*~max~^triQ^. *E*~es~, end-systolic elastance; *E*~max~, maximal systolic elastance; *ESPVsR*, end-systolic pressure--stroke volume relationship; LV, left ventricular; *Q*~max~, theoretical maximum flow; *Q*^m^, measured aortic flow; *Q*^tri^, calibrated triangular flow; *P*~isomax~, peak isovolumic pressure; *P*~LV~, measured LV pressure Statistical analysis {#Sec7} -------------------- The results are expressed as the median ± interquartile range. For comparing the effect of CKD on serum creatinine and blood urea nitrogen with that of NC, the Mann--Whitney rank-sum test was used to test for a difference between the two groups. However, the Kruskal--Wallis one-way analysis of variance (ANOVA) on ranks was performed to compare the effects of type 1 and type 2 DM on blood sugar with that of NC. The ANOVA on rank was also used to determine the statistical significance of the results for the five-group comparison on the LV pumping mechanics. Statistical significance was assumed at the level of *P* \< 0.05. In cases where the ANOVA results indicated that a cardiodynamic variable differed significantly among groups, Dunn's test was used to identify which group exhibited divergent median value from that of the NC group. The simple linear regression is used to correlate the dependent variable (e.g., *E*~max~^triQ^) with the independent variable (e.g., *E*~max~^mQ^). The linearity of the relationship is reflected in the coefficient of determination. Larger *r*^2^ value indicates that the equation is a good description of the relation between the independent and dependent variables. The smaller *P* value denotes the greater probability that the independent variable can be used to predict the dependent variable. Bland--Altman plots depict the difference between the two methods of measurement on the same subjects, in which good agreement is shown by values that lie close to the 0 mean difference line and between the 95% confidence interval limits of agreement \[[@CR3]\]. The 95% limits of agreement are estimated by mean difference ± 1.96 standard deviation of the difference. Results {#Sec8} ======= Exemplification for constructing an isovolumic pressure and a triangular flow from the measured LV pressure {#Sec9} ----------------------------------------------------------------------------------------------------------- Figure [1](#Fig1){ref-type="fig"} shows the estimated isovolumic LV pressure (b) from the measured LV pressure (a) by using a non-linear least-squares approximation technique in one male CKD rat. Figure [1](#Fig1){ref-type="fig"} also illustrates the construction of a calibrated *Q*^tri^ (green curve, d) by using the filtered fourth-order derivative of the measured LV pressure (c). Exemplification for generating the end-systolic pressure--stroke volume relationship {#Sec10} ------------------------------------------------------------------------------------ Figure [2](#Fig2){ref-type="fig"} depicts the calculation of the LV *E*~es~ from the measured LV pressure in the same rat, which is shown in Fig. [1](#Fig1){ref-type="fig"}. The LV *E*~es~ is the slope of the *ESPVsR* line (red line, 2a and 2b), which was obtained from the measured LV pressure (1a), the estimated isovolumic pressure (1b), and the time integration of aortic flow by using either *Q*^m^ (black curve, 1d) or *Q*^tri^ (green curve, 1d). Exemplification for predicting the LV pressure using the elastance--resistance model {#Sec11} ------------------------------------------------------------------------------------ Figure [2](#Fig2){ref-type="fig"} also demonstrates the calculation of the LV *E*~max~ and *Q*~max~ by using the elastance--resistance model based solely on the measured *P*~*LV*~ in the same rat, which is shown in Fig. [1](#Fig1){ref-type="fig"}. The similarity between the computed (green line, 2c and 2d) and measured (red line, 2c and 2d) pressure data obtained from the measured LV pressure, the estimated isovolumic pressure, and aortic flow either from the *Q*^m^ or *Q*^tri^ is apparent. The coefficient of determination and the standard error of the estimate evaluating the goodness of the model fit using *Q*^m^ were 0.989 ± 0.005 and 2.247 ± 0.742%, respectively, and those from *Q*^tri^ were 0.987 ± 0.004 and 2.395 ± 0.443%, respectively, when all studied rats were taken into account (*n* = 78). LV *E*~max~ versus LV *E*~es~ in all studied rats (*n* = 78) {#Sec12} ------------------------------------------------------------ Although the LV *E*~es~^mQ^ (453.9 ± 117.7) was greater than the LV *E*~max~^mQ^ (445.7 ± 112.5), no statistical significance was found between these two indices describing the intrinsic contractile status of the heart. No significant difference was also observed between the LV *E*~es~^triQ^ (470.9 ± 130.7) and the LV *E*~max~^triQ^ (464.3 ± 110.8). Relation of the LV dynamic parameters obtained using *Q*^m^ with those generated from *Q*^tri^ {#Sec13} ---------------------------------------------------------------------------------------------- Figure [3](#Fig3){ref-type="fig"} displays the relationship between the *Q*~max~, *V*~eed~, and *E*~max~ calculated from the *Q*^m^ (*Q*~max~^mQ^, *V*~eed~^mQ^, and *E*~max~^mQ^, respectively, on the horizontal axes) and the *Q*~max~, *V*~eed~, and *E*~max~ calculated from the *Q*^tri^ (*Q*~max~^triQ^, *V*~eed~^triQ^, and *E*~max~^triQ^, respectively, on the vertical axes). Figure [3](#Fig3){ref-type="fig"}a displays a significant regression line for the *Q*~max~: *Q*~max~^triQ^ = 2.4053 + 0.9767 × *Q*~max~^mQ^ (*r*^2^ = 0.7798; *P* \< 0.0001). Figure [3](#Fig3){ref-type="fig"}b presents the regression equation of the *V*~eed~^triQ^ = 0.0218 + 0.9514 × *V*~eed~^mQ^ (*r*^2^ = 0.8611; *P* \< 0.0001). Figure [3](#Fig3){ref-type="fig"}c illustrates the regression line between the *E*~max~^triQ^ and *E*~max~^mQ^: *E*~max~^triQ^ = 51.9133 + 0.8992 × *E*~max~^mQ^ (*r*^2^ = 0.8257; *P* \< 0.0001).Fig. 3Relationship between the *Q*~max~ (**a**), *V*~eed~ (**b**), and *E*~max~ (**c**) calculated from the measured *P*~LV~ and *Q*^m^ (*Q*~max~^mQ^, *V*~eed~^mQ^, and *E*~max~^mQ^, respectively, on the horizontal axes) and the *Q*~max~, *V*~eed~, and *E*~max~ calculated from the measured *P*~LV~ and assumed *Q*^tri^ (*Q*~max~^triQ^, *V*~eed~^triQ^, and *E*~max~^triQ^, respectively, on the vertical axes). *E*~max~, maximal systolic elastance; LV, left ventricular; *P*~LV~, measured LV pressure; *Q*^m^, measured aortic flow; *Q*^tri^, calibrated triangular flow; *Q*~max~, theoretical maximum flow; *V*~eed~, effective LV end-diastolic volume; NC, normal controls; CKD, rats with chronic kidney disease; type 1 DM, streptozotocin-induced diabetic rats; type 2 DM, streptozotocin--nicotinamide-induced diabetic rats Figure [4](#Fig4){ref-type="fig"} presents the Bland--Altman plot for the *Q*~max~ (a), *V*~eed~ (b), and *E*~max~ (c), with mean differences of 1.5441 (mL s^−1^), −0.0055 (mL), and 3.0672 (mmHg mL^−1^), respectively.Fig. 4Bland--Altman plots of the *Q*~max~ (**a**), *V*~eed~ (**b**), and *E*~max~ (**c**). Dashed lines represent averages; dashed--dotted lines denote 95% confidence intervals. *E*~max~, maximal systolic elastance; LV, left ventricular; *Q*~max~, theoretical maximum flow; *V*~eed~, effective LV end-diastolic volume; NC, normal controls; CKD, rats with chronic kidney disease; type 1 DM, streptozotocin-induced diabetic rats; type 2 DM, streptozotocin--nicotinamide-induced diabetic rats Baseline characteristics in the studied rats {#Sec14} -------------------------------------------- Table [1](#Tab1){ref-type="table"} shows the baseline characteristics of the NC, CKD, and type 1 and type 2 DM groups. Compared with the NC rats, the female healthy rats had decreased body weight associated with no change in blood sugar. The rats with CKD exhibited impaired renal function, as manifested by increased levels of serum creatinine and blood urea nitrogen. No alteration in body weight was observed in the CKD group. The rats with STZ-induced type 1 DM had higher blood glucose levels associated with a decrease in body weight compared with the NC rats. Table [1](#Tab1){ref-type="table"} also reveals that, partially protected by NA, the STZ-NA-induced type 2 DM elicited moderate and stable hyperglycemia and prevented STZ-induced hypoinsulinemia and body weight loss.Table 1Baseline characteristics of NC rats, rats with CKD, and rats with either type 1 or type 2 DMGroupBW (g)BS (mg dL^−1^)BUN (mg dL^−1^)SCr (mg dL^−1^)Female  FNC (*n* = 7)294.7 ± 32.5^\*^104.1 ± 12.1nanaMale  NC (*n* = 25)454.5 ± 56.499.0 ± 11.120.3 ± 5.80.67 ± 0.13  CKD (*n* = 14)416.1 ± 59.8na66.7 ± 13.4^\*^1.72 ± 0.50^\*^  Type 1 DM (*n* = 20)328.8 ± 41.1^\*^465.5 ± 44.8^\*^nana  Type 2 DM (*n* = 12)412.4 ± 50.8158.4 ± 26.7^\*^nanaAll values are expressed as the median ± interquartile range. BW, body weight; BS, blood sugar; BUN, blood urea nitrogen; SCr, serum creatinine; NC, normal controls; CKD, rats with chronic kidney disease; type 1 DM, streptozotocin-induced diabetic rats; type 2 DM, streptozotocin--nicotinamide-induced diabetic rats; na, not applicable\**P* \< 0.05 compared with the male controls Basic hemodynamic measurements in the studied rats {#Sec15} -------------------------------------------------- Table [2](#Tab2){ref-type="table"} presents the basic hemodynamic measurements of the NC, CKD, and type 1 and type 2 DM groups. Compared with the NC group, the female healthy rats had a decline in cardiac output and peak isovolumic pressure, but no change in heart rate. No alterations in the heart rate, cardiac output, and peak isovolumic pressure were observed in the rats with CKD. The type 1 (but not type 2) DM group exhibited a significant reduction in heart rate. However, the type 2 (but not type 1) DM group showed a decline in cardiac output and peak isovolumic pressure.Table 2Basic hemodynamic data of NC rats, rats with CKD, and rats with either type 1 or type 2 DMGroup*HR* (beats min^−1^)*CO* (mL s^−1^)*P*~isomax~ (mmHg)Female  FNC (*n* = 7)378.5 ± 43.11.290 ± 0.594^\*^186.7 ± 61.4^\*^Male  NC (*n* = 25)398.0 ± 38.32.260 ± 0.619263.0 ± 24.6  CKD (*n* = 14)385.2 ± 51.32.231 ± 0.324284.2 ± 34.8  Type 1 DM (*n* = 20)342.7 ± 36.8^\*^2.289 ± 0.441247.7 ± 45.8  Type 2 DM (*n* = 12)384.5 ± 67.41.793 ± 0.670^\*^240.9 ± 53.8^\*^All values are expressed as the median ± interquartile range. *HR*, basal heart rate; *CO*, cardiac output; *P*~isomax~, peak isovolumic pressure; NC, normal controls; CKD, rats with chronic kidney disease; type 1 DM, streptozotocin-induced diabetic rats; type 2 DM, streptozotocin--nicotinamide-induced diabetic rats\**P* \< 0.05 compared with the male controls Effects of sex and CKD and type 1 or type 2 DM on LV pumping dynamics {#Sec16} --------------------------------------------------------------------- Figure [5](#Fig5){ref-type="fig"} illustrates the effects of sex and the experimental induced CKD and type 1 and type 2 DM on the systolic mechanical behavior of the ventricular pump, as derived from either the *Q*^m^ or *Q*^tri^. Compared with the NC group, the female healthy group had decreased *Q*~max~^mQ^ and *V*~eed~^mQ^ but showed no alteration in *E*~max~^mQ^. No statistical significant difference in *Q*~max~^mQ^ was found between the CKD group and the NC group. However, the CKD group showed an increase in the *V*~eed~^mQ^ and a decrease in the *E*~max~^mQ^. The type 2 (but not type 1) DM group exhibited a significantly lower *Q*~max~^mQ^ than the NC group did. Both DM groups showed no alteration in the *V*~eed~^mQ^ but showed a decline in the *E*~max~^mQ^. Meanwhile, the female healthy group, the CKD group, and both diabetic groups exhibited a difference in the *Q*~max~^triQ^, *V*~eed~^triQ^, and *E*~max~^triQ^, which showed similar statistical significance to that of their measured counterparts (i.e., *Q*~max~^mQ^, *V*~eed~^mQ^, and *E*~max~^mQ^, respectively).Fig. 5Effects of sex, CKD, and type 1 and type 2 DM on *Q*~max~^triQ^ (**d**), *V*~eed~^triQ^ (**e**), and *E*~max~^triQ^ (**f**), as derived from the assumed *Q*^tri^, showed similar statistical significance to those of their measured counterparts, i.e., *Q*~max~^mQ^ (**a**), *V*~eed~^mQ^ (**b**), and *E*~max~^mQ^ (**c**), as calculated from the measured *Q*^m^. All values are expressed as the median ± interquartile range. *E*~max~, maximal systolic elastance; LV, left ventricular; *P*~LV~, the measured LV pressure; *Q*^m^, measured aortic flow; *Q*^tri^, calibrated triangular flow; *V*~eed~, effective LV end-diastolic volume; *Q*~max~, theoretical maximum flow; FNC, female NC; NC, normal controls; CKD, rats with chronic kidney disease; type 1 DM, streptozotocin-induced diabetic rats; type 2 DM, streptozotocin--nicotinamide-induced diabetic rats. \**P* \< 0.05 compared with the controls Discussion {#Sec17} ========== The myocardium of the left ventricle is a viscoelastic material whose mechanical properties are reflected in the behavior of the ventricular chamber (i.e., the relationships among chamber pressure, volume, and flow). The relationship between instantaneous ventricular pressure and volume, the so-called pure elastance model, has been described in terms of a time-varying elastance \[[@CR16], [@CR24]\]. For an ejecting beat, the LV end-systolic elastance can be derived from the pure elastance model at the end of systole, the time at which the time-varying elastance becomes maximal \[[@CR16], [@CR23]\]. However, this end-systolic elastance is only an approximation to the maximal systolic elastance that is derived from isovolumic contraction, due to deactivation of myocardial shortening \[[@CR6], [@CR10], [@CR18]\]. Hunter et al. \[[@CR11]\] and Shroff et al. \[[@CR17]\] attempted to identify the mechanical nature of the ejecting ventricle by developing models reasonably able to predict pressure--volume--flow dynamics. Campbell et al. \[[@CR4]\] and Shroff and Motz \[[@CR21]\] arrived at an identical configuration---a series combination of a time-varying elastance and a viscous resistance to formulate the elastance--resistance model. Pure elastance model versus elastance--resistance model {#Sec18} ------------------------------------------------------- The LV systolic elastance can be calculated from either the pure elastance model, indicated as *E*~es~, or the elastance--resistance model, denoted as *E*~max~. However, only the elastance--resistance model has the ability to generate the LV *Q*~max~ having an inverse relationship with the LV internal resistance. At the molecular level, the LV systolic elastance can be determined by the properties of the contractile unit along with the activation process (i.e., availability of Ca^2+^) and extramyocytic components \[[@CR19]\]. However, the ventricular resistance seems to be related to the biochemical alterations of the myocardium, having an inverse relation with percent slow myosin \[[@CR19]\]. The pure elastance model is a model-independent approach; the elastance--resistance model is a model-based approach. The major difference between these two approaches is that the original pure elastance model can accurately predict stroke volume but not instantaneous flow; for a reasonably accurate prediction of instantaneous flow, a series resistance has to be added \[[@CR21]\]. It should be noted that the choice of a model-independent versus model-based approach clearly depends on the specific application. If the framework of an analysis involves mean values of relevant variables (e.g., stroke volume, stroke work), the pure elastance model may be appropriate \[[@CR25]\]. If the analysis goals are such that instantaneous behavior is of significance (e.g., coupling from the perspective of pulsatile energy generated by the left ventricle, effects of arterial wave reflection), the elastance--resistance model may be more appropriate \[[@CR19]\]. Evaluation of systolic pumping mechanics of the heart using a single LV pressure pulse {#Sec19} -------------------------------------------------------------------------------------- As mentioned earlier, the signals required to fit the elastance--resistance model are the simultaneously recorded LV pressure and aortic flow, followed by the isovolumic LV pressure. Obviously, the technique of measuring the isovolumic signal by occluding the ascending aorta limits its applicability in human patients. That may be the reason why a significance of the elastance--resistance model was not appreciated in a field a lot. In the present study, we proposed a technique to generate the essential signals for fitting the elastance--resistance model on the basis of the measured LV pressure alone. The first one estimated the isovolumic LV pressure from the LV pressure pulse of the ejecting beat. The second step generated a triangular flow from the measured LV pressure to approximate its corresponding aortic flow. Construction of an isovolumic pressure and a triangular flow from the measured LV pressure {#Sec20} ------------------------------------------------------------------------------------------ In 1980, Sunagawa et al. \[[@CR26]\] proposed a curve-fitting technique to estimate the isovolumic LV pressure from the instantaneous pressure of an ejecting contraction (Appendix 1). They discovered that the isovolumic LV pressure and its peak value could be estimated with reasonable accuracy within the interval *t*~ej~ \< *t* \< *t*~pisomax~, where *t*~ej~ is the onset of ventricular ejection and *t*~pisomax~ is the time of peak isovolumic pressure. A triangular shape of the aortic flow wave is a reasonable assumption because the triangular approximation is consistent with the general aortic flow envelope obtained from Doppler ultrasound \[[@CR30]\] or electromagnetic \[[@CR29]\] flow measurements. In 2006, Westerhof et al. \[[@CR29]\] provided a novel method for separating the measured aortic pressure wave into its forward and backward components on the basis of a single aortic pressure pulse and an assumed triangular flow. The attractiveness of their study is that calibration of the triangular flow wave derived from the measured aortic pressure is not essential in the analysis. In 2017, Wang et al. \[[@CR28]\] proposed a method to construct an aortic triangular flow by using the measured LV pressure (Appendix 2). To investigate the cardiac contractile mechanics, the assumed triangular flow has to be calibrated with the cardiac output. In the present study, the LV pressure pulse was the only signal measured. Thus, the method proposed by Wang et al. \[[@CR28]\] was employed to generate the *Q*^tri^ for determining the systolic elastic and resistive behaviors of the heart. The similarity between the aortic flow pattern of the assumed *Q*^tri^ and that of the measured *Q*^m^ was demonstrated in Fig. [1](#Fig1){ref-type="fig"}d. Restriction of the LV pressure data on the fitting interval *t*~ej~ \< *t* \< *t*~pisomax~ {#Sec21} ------------------------------------------------------------------------------------------ Hunter et al. \[[@CR11]\] have demonstrated three components in the LV response to a flow pulse; elastance, resistance, and deactivation. Without considering the deactivation factor, our model-based approach was highly dependent on the ventricular elastance and resistance. Although not perfect, the elastance--resistance model can be used to fit the measured LV pressure of an ejecting beat suitably if the fitting interval is *t*~ej~ \< *t* \< *t*~pisomax~ (Appendix 3) \[[@CR5]\]. That is because the deactivation component was virtually absent during early systole, became evident during mid-systole, and was most pronounced during late systole \[[@CR11]\]. In the present study, we demonstrated that the elastance--resistance model with either the *Q*^m^ or *Q*^tri^ could be satisfactorily applied to measure the intrinsic LV systolic mechanics within the specified fitting interval. The LV *E*~max~ and *Q*~max~ calculated using the assumed *Q*^tri^ strongly correlated with the corresponding values derived from the measured *Q*^m^ (*Q*~max~^triQ^ vs. *Q*~max~^mQ^, Fig. [3](#Fig3){ref-type="fig"}a; *E*~max~^triQ^ vs. *E*~max~^mQ^, Fig. [3](#Fig3){ref-type="fig"}c). Physiological meaning of the model-generated parameters for CKD and type 1 or type 2 DM {#Sec22} --------------------------------------------------------------------------------------- As mentioned earlier, the LV *E*~max~ was determined by the ratio of peak isovolumic pressure to the effective end-diastolic volume. Compared with the NC group, the female healthy group had diminished effective end-diastolic volume (*V*~eed~^mQ^, Fig. [5](#Fig5){ref-type="fig"}b; *V*~eed~^triQ^, Fig. [5](#Fig5){ref-type="fig"}e) with a reduction in the peak isovolumic pressure (Table [2](#Tab2){ref-type="table"}), resulting in no alteration in the *E*~max~ (*E*~max~^mQ^, Fig. [5](#Fig5){ref-type="fig"}c; *E*~max~^triQ^, Fig. [5](#Fig5){ref-type="fig"}f). By contrast, a decline in *Q*~max~ (*Q*~max~^mQ^, Fig. [5](#Fig5){ref-type="fig"}a; *Q*~max~^triQ^, Fig. [5](#Fig5){ref-type="fig"}d) was observed in the female healthy rats. In the CKD group, the increased effective end-diastolic volume (*V*~eed~^mQ^ or *V*~eed~^triQ^) without any significant change in the peak isovolumic pressure contributed to a reduction in the *E*~max~ (*E*~max~^mQ^ or *E*~max~^triQ^). These results indicate that the myocardium is incapable of producing the pressure force enough to support *E*~max~ along with the increased effective end-diastolic volume. Thus, the CKD heart can be characterized as a weaker pressure generator. Although no statistical significance was found, the CKD group exhibited an increase in the *Q*~max~ (*Q*~max~^mQ^ or *Q*~max~^triQ^) compared with the corresponding respective values in the NC group. An increase in *Q*~max~ is indicative of a decrease in LV internal resistance, which can enhance ventricular outflow for a given cardiac contractile status and arterial load \[[@CR18]\]. Therefore, the left ventricle of CKD can be characterized as a stronger flow generator. The opposing effects of reduced *E*~max~ and increased *Q*~max~ may negate each other, and, then, the cardiac pumping function of the CKD rats could be preserved before heart failure occurs. In both diabetic groups, there was a trend toward decreasing peak isovolumic pressure (especially the type 2 DM) and increasing effective end-diastolic volume (*V*~eed~^mQ^ or *V*~eed~^triQ^), leading to a reduction in the *E*~max~ (*E*~max~^mQ^ or *E*~max~^triQ^). These results indicate that the myocardium cannot generate the pressure force enough to support *E*~max~ along with the increased effective end-diastolic volume. Thus, the diabetic heart can be characterized as a weaker pressure generator. A decline in *Q*~max~ (*Q*~max~^mQ^ or *Q*~max~^triQ^) was also observed in both diabetic groups (especially the type 2 DM). A decrease in *Q*~max~ is indicative of an increase in LV internal resistance, which can reduce ventricular outflow for a given cardiac contractile state and arterial load \[[@CR18]\]. Therefore, the diabetic heart can be characterized as a weaker flow generator. The decreased *E*~max~ and *Q*~max~ demonstrate a deterioration in systolic pumping function of the diabetic heart. Changes in the *E*~max~ and *Q*~max~, assessed using either the *Q*^m^ or *Q*^tri^, suggested that CKD and DM could modify the systolic elastic and resistive behaviors of the left ventricle. Limitations {#Sec23} ----------- Hunter et al. \[[@CR11]\] have demonstrated that besides elastance and resistance, there are at least two or more processes involved in the description of systolic mechanical behavior of the ventricular pump. These processes may include the volume influence factor and the deactivation factor. Campbell et al. \[[@CR4], [@CR5]\] provided clear evidence of failings of the elastance--resistance model, especially during late systole. However, they demonstrated that the elastance--resistance model could be satisfactorily used to fit the measured LV pressure of an ejecting beat in the fitting interval *t*~ej~ \< *t* \< *t*~pisomax~. Although not perfect, the elastance--resistance model can provide such a succinct representation of the systolic mechanical behavior of the ventricular pump. Furthermore, Shroff et al. \[[@CR20]\] reported that the elastance--resistance model is a useful tool to quantify the systolic pumping mechanics of the left ventricle, provided the limitations of this model are clearly understood. Conclusions {#Sec24} =========== The LV *E*~max~ and *Q*~max~ can be determined by using a minimally invasive measurement on the LV pressure. The aortic flow and isovolumic LV pressure necessary to fit the elastance--resistance model are both derived from the measured LV pressure together with cardiac output. We discovered that the estimated triangular flow can approximately calculate the LV *E*~max~ and *Q*~max~ in healthy rats and rats with CKD and type 1 or type 2 DM, and they had a strong correlation with the corresponding values derived from the measured aortic flow. The left ventricle of CKD can be characterized as a weaker pressure generator (decreased *E*~max~) but stronger flow generator (increased *Q*~max~). By contrast, the diabetic heart can be characterized as a weaker pressure (decreased *E*~max~) and flow (decreased *Q*~max~) generator. We suggest that the single-beat estimation technique is an effective method for calculating the *E*~max~ and *Q*~max~ by using a single LV pressure pulse recording together with cardiac output. Perspectives {#Sec25} ------------ Although the LV pressure and aortic flow can be simultaneously measured using a high-fidelity micromanometer (such as model SVPC-664D, Millar Instruments Inc., USA) in human patients, the catheter and its associated instruments are expensive and not widely available. Moreover, the flow signal in the ascending aorta is not easy to access because of the small magnitude with noise disturbances. Although the aortic flow can non-invasively be obtained by Doppler ultrasound \[[@CR30]\], there is difficulty in "simultaneously recording the LV pressure and aortic flow" for fitting the elastance--resistance model. In the present study, both the aortic flow and isovolumic LV pressure were derived from the only measured LV pressure together with cardiac output. In clinical settings, it is easier and safer to measure cardiac output by using non-invasive impedance cardiography \[[@CR1]\] or echocardiography \[[@CR13]\] to calibrate the triangular flow scale. The novelty of such an approach is that one can compute the ventricular elastance and resistance without any measurements of the ascending aortic flow wave and LV pressure from isovolumic contraction. The single-beat estimation technique can be used to investigate the cardiac pumping mechanics from solely the LV pressure of an ejection contraction obtained over a single cardiac cycle without any perturbations of the loading conditions. Thus, the practical applicability of this study is that one may evaluate the LV systolic mechanical properties in patients by using a single LV pressure measurement together with cardiac output, because the generation of the isovolumic LV pressure and *Q*^tri^ and the calculation of the LV *E*~max~ and *Q*~max~ can be automatically achieved. Appendix 1 {#Sec26} ========== Estimation of the isovolumic pressure from an ejecting contraction {#Sec27} ------------------------------------------------------------------ The isovolumic LV pressure (*P*~iso~, Fig. [1](#Fig1){ref-type="fig"}b) was derived from the measured LV pressure of an ejection contraction (*P*~LV~, Fig. [1](#Fig1){ref-type="fig"}a) by using a non-linear least-squares approximation technique as follows \[[@CR26]\]:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {P}_{\mathrm{iso}}(t)=\frac{1}{2}{P}_{\mathrm{id}\max}\left[1-\cos \left(\omega t+c\right)\right]+{P}_{\mathrm{d}} $$\end{document}$$where *P*~idmax~ is the peak developed isovolumic pressure, *ω* is the angular frequency, *c* is the phase-shift angle of the sinusoidal curve, and *P*~d~ is the LV end-diastolic pressure. The *P*~iso~(*t*) was obtained by fitting the measured *P*~LV~ segments from the end-diastolic pressure point to the peak +*dP*~LV~/*dt* and from the pressure point of the peak −*dP*~LV~/*dt* to the same level as the end-diastolic pressure of the preceding beat \[[@CR27]\]. The peak of the ECG R wave was used to identify the LV end-diastolic point. The estimated peak isovolumic pressure, *P*~isomax~, is the pressure sum of the peak developed isovolumic pressure and the LV end-diastolic pressure (Fig. [1](#Fig1){ref-type="fig"}b). Appendix 2 {#Sec28} ========== Construction of the unknown flow wave by using a triangle {#Sec29} --------------------------------------------------------- The fourth-order derivative of the LV pressure waveform (Fig. [1](#Fig1){ref-type="fig"}c) was filtered to determine the uncalibrated *Q*^tri^ \[[@CR28]\]. This calculation was performed using an inverse Fourier transformation of the fourth-order derivative of the LV pressure with the first 15 harmonics. The *Q*^tri^ onset was identified using the peak of the pink curve near the end of the isovolumic contraction period (first vertical blue line). The *Q*^tri^ termination was identified using the nadir of the pink curve, near the middle of the isovolumic relaxation period (third vertical blue line). The base of the unknown *Q*^tri^ was subsequently constructed using the duration identical to the time interval between the onset and termination of the *Q*^tri^. After the ejection commenced, the first zero crossing from negative to positive (second vertical blue line) determined the peak of the triangle \[[@CR28]\]. The *Q*^tri^ scale was calibrated using the cardiac output. Thus, the unknown flow wave *Q*^tri^ was approximated to a triangle (green curve, Fig. [1](#Fig1){ref-type="fig"}d). Appendix 3 {#Sec30} ========== Prediction of the LV pressure using the elastance--resistance model {#Sec31} ------------------------------------------------------------------- The model-derived LV pressure $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\hat{P}}_{LV}(t) $$\end{document}$ can be predicted using the elastance--resistance model if the model parameters are previously identified \[[@CR5], [@CR20]\]. The relationship among the instantaneous LV pressure (*P*~LV~), aortic flow (*Q*), and isovolumic LV pressure (*P*~iso~) can be written as follows:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\hat{P}}_{LV}(t)=P{}_{iso}(t)\left[1-\frac{V_{ej}(t)}{V_{eed}}\right]\left[1-\frac{Q(t)}{Q_{\mathrm{max}}}\right] $$\end{document}$$where *V*~ej~(*t*) is the instantaneously ejected volume computed by calculating the running integral of the *Q*(*t*) from either the *Q*^tri^ or *Q*^m^ and *V*~eed~ is the effective LV end-diastolic volume, that is, the difference between the LV end-diastolic volume and the dead volume. The dead volume is the theoretical value below which the ventricle cannot generate any supra-atmospheric pressure. *Q*~max~ is the theoretical maximum flow that could be generated under zero load condition, and not the maximum of the flow trace in the ascending aorta. Both the *V*~eed~ and *Q*~max~ are model parameters determined by curve-fitting techniques. Campbell et al. \[[@CR5]\] demonstrated that Eq. ([2](#Equ2){ref-type=""}) can be used to fit the measured LV pressure of an ejecting beat suitably if the fitting interval is *t*~ej~ \< *t* \< *t*~pisomax~, where *t*~ej~ is the onset of ventricular ejection and *t*~pisomax~ is the time of peak isovolumic pressure. Initial values of *V*~eed~ and *Q*~max~ were chosen first. The Nelder--Mead simplex algorithm \[[@CR8]\] was, then, used to iteratively adjust the *V*~eed~ and *Q*~max~ to minimize the root-mean-square error \[[@CR7]\]. The parameters that coincided with the minimum objective function were considered the model estimates of the systolic pumping mechanics of the heart (Fig. [2](#Fig2){ref-type="fig"}c, d). Goodness of the model fit can be reflected in the coefficient of determination (r^*2*^) and the standard error of the estimate (*SEE*), which were calculated from a linear regression of the model-generated pressure on measured pressure values. The LV systolic elastance was computed using the relationship *E*(*t*) = *P*~iso~(*t*) / *V*~eed~, and its maximal value was the maximal systolic elastance (*E*~max~ = *P*~isomax~ / *V*~eed~). The LV internal resistance was expressed as *R* (*P*~iso~) = *P*~iso~(*t*) / *Q*~max~. CKD : chronic kidney disease DM : diabetes mellitus *E*~es~ : end-systolic elastance (mmHg mL^−1^) *E*~es~^mQ^ : *E*~es~ calculated from the LV pressure and *Q*^m^ *E*~es~^triQ^ : *E*~es~ calculated from the LV pressure and *Q*^tri^ *E*~max~ : maximal systolic elastance (mmHg mL^−1^) *E*~max~^mQ^ : *E*~max~ calculated from the LV pressure and *Q*^m^ *E*~max~^triQ^ : *E*~max~ calculated from the LV pressure and *Q*^tri^ LV : left ventricular NA : nicotinamide *Q*^m^ : measured aortic flow wave (mL s^−1^) *Q*^tri^ : assumed triangular flow wave (mL s^−1^) *Q*~max~ : theoretical maximum flow (mL s^−1^) *Q*~max~^mQ^ : *Q*~max~ calculated from the LV pressure and *Q*^m^ *Q*~max~^triQ^ : *Q*~max~ calculated from the LV pressure and *Q*^tri^ STZ : streptozotocin *V*~eed~ : effective end-diastolic volume (mL) *V*~eed~^mQ^ : *V*~eed~ calculated from the LV pressure and *Q*^m^ *V*~eed~^triQ^ : *V*~eed~ calculated from the LV pressure and *Q*^tri^ **Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This study was supported by a grant from the National Taiwan University Hospital, Taiwan (NTUH 106-16 and NTUH 107-s3897), the National Taiwan University Hospital, Hsin-Chu Branch, Taiwan (HCH-104-004), and the National Science Council of Taiwan (MOST 106-2314-B-002-155-MY3). This manuscript was edited by Wallace Academic Editing. C.H.W., R.W.C., E.T.W., and K.C.C. developed the study concept and study design and drafted the manuscript. R.W.C. and C.Y.C. conducted the animal experiments, collected data, and performed statistical analyses. Y.S.C. and M.S.W. provided advice on surgical procedures. H.L.K. and Y.J.C. participated in data interpretation. C.H.W. and K.C.C. supervised this study and critically revised the manuscript. All authors have read and approved the final manuscript. The authors have no conflicts of interest to declare.

1. Introduction {#sec1} =============== Enhancement technology is regarded as one of the most active fields of digital image processing. It improves the quality and appearance for low contrast image, and it can be used in monitoring, imaging systems, human-computer interaction \[[@B1]--[@B3]\], and many other areas \[[@B4]--[@B9]\]. The histogram equalization (HE) technique is simple and easily implemented, which is most extensively utilized for contrast enhancement. HE utilizes the cumulative density function (CDF) of image for transferring the gray levels of original image to the levels of enhanced image. The main drawback of HE is that it tends to change the mean brightness of the image to the middle level of the dynamic range and results in annoying artifacts and intensity saturation effects. This drawback makes HE technique unsuitable for many consumer electronics applications, for example, TV and cameras. In order to overcome the shortcomings mentioned above, many other HE-based methods have been proposed, such as the brightness preserving bihistogram equalization (BBHE) \[[@B10]\], dualistic subimage histogram equalization (DSIHE) \[[@B11]\], and minimum mean brightness error bihistogram equalization (MMBEBHE) \[[@B12]\]. BBHE \[[@B10]\] partitions the histogram based on the image mean while DSIHE \[[@B11]\] uses image median to segment. MMBEBHE \[[@B12]\] recursively divides the image histogram into multiple groups based on mean brightness error (MBE). Although these methods have made great progress, they still have their own drawbacks, including failing with images having nonsymmetric distribution \[[@B10]\], failing to preserve mean brightness \[[@B11]\], producing more annoying side effects \[[@B12]\], and losing structural information \[[@B13]\]. In these techniques, however, desired improvement may not always be achieved, and the difference between input and output image is minimal \[[@B14]\]. Chen and Ramli proposed the method called recursive mean-separate histogram equalization (RMSHE \[[@B15]\]), in which the authors suggested recursive division of histograms based on the local mean. The mean brightness of processed image approaches towards the mean brightness of input image. Wang et al. improved DSIHE \[[@B11]\] into recursive subimage histogram equalization (RSIHE \[[@B16]\]) based on contrast enhancement, by introducing recursive segmentation in the similar manner as Chen and Ramli proposed in \[[@B15]\], although this method is similar to RMSHE \[[@B15]\] but it uses median values instead of mean values to divide histogram into subhistograms. Adaptively modified histogram equalization (AMHE) \[[@B17]\] method is developed by Kim et al., which can modify the probability density function (PDF) of the grayscale as well as apply histogram specification to the modified PDF. Unfortunately, the entire redistribution to the original histogram by those methods can cause overenhancement, underenhancement, and some artifacts appearing in some smooth regions. Although the AMHE \[[@B17]\] does not produce any degradation, it darkens the bright areas of the sky and fails to boost the brightness of the dark regions. In addition, some other methods based on histogram equalization for contrast enhancement with brightness enhancement have also been proposed, such as the dynamic histogram specification introduced by Sun et al., which preserves the shape of the input image histogram but does not enhance it significantly \[[@B18]\]. Tsai et al. suggested a contrast enhancement algorithm for color images \[[@B19], [@B20]\]. Huang et al. proposed an adaptive gamma correction with weighting distribution (AGCWD \[[@B21]\]) to enhance the contrast and preserve the overall brightness of an image; in the method, the gamma correction and a probability distribution for luminance pixels were used. The AGCWD technique may not give desired results when an input image lacks bright pixels since the highest intensity in the output image is bounded by the maximum intensity of the input image, because the highest enhanced intensity will never cross the maximum intensity of the input image \[[@B22]\]. Besides, AGCWD \[[@B21]\] leads to loss of information in processed image due to its sharp increasing resultant transformation curve described below. An image enhancement technique using the idea of exposure value, named image enhancement using exposure-based subimage histogram equalization (ESIHE \[[@B23]\]), was advanced. The method divided the clipped histogram into two parts by using the precalculated exposure threshold \[[@B24]\]. The effects of using intensity exposure in histogram segmentation before histogram clipping were studied in \[[@B25]\]. Through simulation on standard images, low contrast images, and noisy images, the study showed that \[[@B25]\] could yield a certain enhancement results; however, the method usually causes underenhancement. Tang and Mat Isa introduced an algorithm named bihistogram equalization using modified histogram bins (BHEMHB) \[[@B26]\], which segmented the input histogram based on the median brightness and altered the histogram bins before HE is applied, but it made limited improvement for contrast. In order to effectively increase the contras of the input image with brightness and details well preserved, an efficient algorithm named Mean and Variance based Subimage Histogram Equalization (MVSIHE) is developed in this paper. The proposed method is more effective for preserving the mean brightness and details of the enhanced image while improving the contrast compared with some other state-of-the-art methods. According to the experiments based on 100 images for our method, we know that the MVSIHE technique can achieve the multiple objectives of entropy maximization, details, and brightness preservation as well as control on overenhancement. The main contributions of this paper are as follows. Firstly, we introduce the mean and variance based algorithm to divide the histogram of the image. Secondly, a novel transformation called hyperbolic tangent transformation is developed to modify the histogram bins to overcome this domination problem. Thirdly, we put forward a normalization transformation, which can make the brightness component of the output image have a wider dynamic range and the output image look more natural and clearer. Furthermore, results indicate that the proposed method is a better approach compared to the state-of-the-art methods. This paper is organized as follows: [Section 2](#sec2){ref-type="sec"} describes the proposed MVSIHE method. Data samples and performance evaluations are given in [Section 3](#sec3){ref-type="sec"}. [Section 4](#sec4){ref-type="sec"} shows experimental results and comparisons with state-of-the-art methods, and our concluding remarks are included in [Section 5](#sec5){ref-type="sec"}. 2. Proposed Image Enhancement Method {#sec2} ==================================== 2.1. Threshold Calculation Based on Mean and Variance {#sec2.1} ----------------------------------------------------- The histogram of an image is divided into four parts with three thresholds which are adaptive and obtained by the same method. The procedure to obtain the thresholds will be presented in detail as follows. An input image *X* is given; let *H*  \[*l*~low~, *l*~up~\] be the global histogram of the input image *X*, where *l*~low~ and *l*~up~ represent lower and uppermost intensities of the image *X*. *H*(*l*) is the histogram of the gray level *l*, which is described as$$\begin{matrix} {H\left( { l} \right) = n_{l}\mspace{1800mu} for\,\, l = l_{low},\ldots,l_{up},} \\ \end{matrix}$$where *n*~*l*~ is the of gray level *l* in the image *X*, the pdf of the image, pdf(*l*), can be defined as $$\begin{matrix} {pdf\left( { l} \right) = \frac{H\left( l \right)}{\left( {M\ast N} \right)}\mspace{1800mu} for\,\, l = l_{low},\ldots,l_{up},} \\ \end{matrix}$$where *M∗N* is the total number of pixels in the input image *X*. The threshold value for histogram segmentation can be obtained. First, we divide the whole histogram into two parts by an adaptive threshold *k*. Then the two parts can be presented as Sub~0~{0 \~ *k*} and Sub~1~{*k* + 1 \~ *l*~max~}. The probability of each part can be solved by$$\begin{matrix} {{Sub}^{1,2}\text{:}\,\,\omega_{0}\operatorname{} = {\sum\limits_{l = 0}^{k}{pdf\left( { l} \right)}},} \\ \\ {{Sub}^{2,2}\text{:}\,\,\omega_{1}\operatorname{} = {\sum\limits_{l = k + 1}^{l_{max}}{pdf\left( { l} \right)}}.} \\ \\ \end{matrix}$$ Next, the mean value of each part can be calculated by$$\begin{matrix} {{Sub}^{1,2}\text{:}\,\,\mu_{0}\operatorname{} = {\sum\limits_{l = 0}^{k}\frac{l\ast pdf\left( l \right)}{\omega_{0}}},} \\ \\ {{Sub}^{2,2}\text{:  }\mu_{1}\operatorname{} = {\sum\limits_{l = k + 1}^{l_{max}}\frac{l\ast pdf\left( l \right)}{\omega_{1}}}.} \\ \\ \end{matrix}$$ Therefore, the mean of the whole image *X* is described as $$\begin{matrix} {\mu = \mu_{0}\omega_{0} + \mu_{1}\omega_{1}.} \\ \end{matrix}$$ Then, we can seize the variance between the two parts by $$\begin{matrix} {\sigma^{2}\left( { k} \right) = \omega_{0}\left( {\mu_{0} - \mu} \right)^{2} + \omega_{1}\left( {\mu_{1} - \mu} \right)^{2}.} \\ \end{matrix}$$ Then the optimization model can be defined as$$\begin{matrix} {{\max\limits_{k}{\,\sigma^{2}\left( { k} \right)}}.} \\ \end{matrix}$$We can obtain the optimal threshold *k*~opt~ by ([7](#EEq7){ref-type="disp-formula"}), which is utilized to segment the histogram of image. Note that we set *k*~2~ = *k*~opt~; the optimal thresholds *k*~1~ and *k*~3~ of the two parts up and down the threshold *k*~2~ can also be obtained in the same way as the above, respectively. Finally, the histogram *H*  \[*l*~low~, *l*~up~\] is segmented into four subhistograms; that is, $$\begin{matrix} {H\left\lbrack { l_{low},l_{up}} \right\rbrack = {\bigcup\limits_{i = 1}^{4}{sub}^{i,4}}\left\lbrack { l_{low}^{i,4},l_{up}^{i,4}} \right\rbrack,} \\ \end{matrix}$$*l*~low~^*i*,4^ and *l*~up~^*i*,4^ are the boundary values of the luminance range within the *i*th segmentation. Hence, the all subimages are captured by$$\begin{matrix} {{sub}^{i,4} = \left\{ { l\left\{ { i,j} \right\}{\, \mid \,}{l^{i,4}}_{low} \leq l\left\{ { i,j} \right\} \leq {l^{i,4}}_{up},\,\forall\left\{ { i,j} \right\} \in l} \right\},} \\ \end{matrix}$$the pdf of *i*th subhistogram is represented by $$\begin{matrix} {{pdf}_{{sub}^{i,4}} = \frac{h_{{sub}^{i,4}}\left( l \right)}{n_{{sub}^{i,4}}}\mspace{1800mu} for\,\, l = {l^{i,4}}_{low},\ldots,{l^{i,4}}_{up},} \\ \end{matrix}$$where *n* is the number of pixels of the *i*th segmentation. After the segment of input image histogram, the next stage of processing procedure is histogram modification. As mentioned in the introduction, CHE emphasizes the domination of high-frequency histogram bins, thus resulting in loss of details in the image. Low-frequency histogram bins tend to be swallowed by high-frequency bins in the neighborhood. MVSIHE modifies the histogram bins to overcome this domination problem. Histogram bin modification is performed using ([11](#EEq11){ref-type="disp-formula"}) for the subhistogram \[[@B27]\]. $$\begin{matrix} {\operatorname{}new\_{pdf}_{{sub}^{i,4}} = \left( {\frac{e^{{pdf}_{{sub}^{i,4}}} - e^{- {pdf}_{{sub}^{i,4}}}}{e^{{pdf}_{{sub}^{i,4}}} + e^{- {pdf}_{{sub}^{i,4}}}}} \right)} \\ {\mspace{21820mu} for\,\, l = {l^{i,4}}_{low},\ldots,{l^{i,4}}_{up},} \\ \end{matrix}$$where *n*~sub^*i*,4^~ is the total number of pixels in the *i*th subimage. $$\begin{matrix} {\operatorname{}{cdf}_{{sub}^{i,4}}\left( { l} \right) = {\sum\limits_{l = l_{low}}^{l}{new\_{pdf}_{{sub}^{i,4}}}}\left( { l} \right)} \\ {\mspace{21630mu} for\,\, l = {l^{i,4}}_{low},\ldots,{l_{up}}^{i,4}.} \\ \end{matrix}$$ 2.2. Histogram Equalization {#sec2.2} --------------------------- CHE involves mapping an input gray level *L* using transformation function *f*(*l*), which can be defined as $$\begin{matrix} {f\left( { l} \right) = X_{0} + \left( { X_{0} - X_{L - 1}} \right)\ast cdf\left( { l} \right),} \\ \end{matrix}$$where *X*~0~ and *X*~*L*−1~ represent the minimum and maximum gray levels, respectively. As observed in ([8](#EEq8){ref-type="disp-formula"}), the remapping of the input image is within the entire dynamic range \[*X*~0~, *X*~*L*−1~\] after applying CHE. The proposed method equalizes the modified subhistograms by ([14](#EEq14){ref-type="disp-formula"}); thereafter, the equalized subhistograms are integrated to produce the final enhanced output image. $$\begin{matrix} {\operatorname{}f\left( { l} \right)_{{sub}^{i,4}} = {l^{i,4}}_{low} + \left( {{l^{i,4}}_{up} - {l^{i,4}}_{low}} \right)\ast{cdf}_{{sub}^{i,4}}\left( { l} \right)} \\ {\mspace{21560mu} for\,\, l = {l^{i,4}}_{low},\ldots,{l^{i,4}}_{up}.} \\ \end{matrix}$$ 2.3. Normalization of Intensity Levels {#sec2.3} -------------------------------------- In our proposed method, each segment is equalized independently and output image is obtained by adding the equalized subsegments. This may result in saturation of intensities and interference caused by nonuniform light; in order to solve the problems, we utilize the normalization of intensity levels of the processed image. The normalization transformation is defined as$$\begin{matrix} {T\left( { L} \right) = \frac{L - l_{min}}{L_{max} - L_{min}}\left( { L_{up} - L_{low}} \right) + L_{low},} \\ \end{matrix}$$where *L* is a matrix of the input image\'s luminance component and *L*~max~ and *L*~min~ are the maximum and the minimum values of *L*, respectively. *L*~low~ and *L*~up~ are the boundary values of the luminance range within \[*L*~low~, *L*~up~\], without loss of generality, *L*~low~ is set as 0, and *L*~up~ is 255 to obtain a maximum luminance range for 256 gray levels. After normalization of intensity levels, for the sake of getting a more comprehensive and informative information output image, we fuse INT~img~ and INP~img~ together by the following: $$\begin{matrix} {PRC_{img} = \delta\ast INT_{img} + \left( { 1 - \delta} \right)\ast INP_{img},} \\ \end{matrix}$$where INT~img~ is image obtained after applying ([15](#EEq15){ref-type="disp-formula"}), INP~img~ is input image, and PRC~img~ is finally output image. Parameter is between 0 and 1. [Figure 1](#fig1){ref-type="fig"} shows the statistical results (100 test images) with different parameters *δ*. From Figures [1(a)](#fig1){ref-type="fig"}, [1(b)](#fig1){ref-type="fig"}, and [1(c)](#fig1){ref-type="fig"}, we can know that the average values of Peak Signal-to-Noise Ratio (PSNR), Discrete Entropy (DE), and Absolute Mean Brightness Error (AMBE) can obtain optimum value when *δ* is roughly to 0.6. 3. Data Samples and Performance Evaluations {#sec3} =========================================== 3.1. Data Samples {#sec3.1} ----------------- In this paper, we compare the performance of the proposed method with some other state-of-the-art methods: DSIHE \[[@B11]\], RMSHE \[[@B15]\], MMBEBHE \[[@B12]\], RSIHE \[[@B16]\], ESIHE \[[@B23]\], and BHEMHB \[[@B26]\]. The MVSIHE and other HE-based image enhancement methods for comparison are tested by using 100 benchmark images from a public image database named CVG-UGR-Database \[[@B28]\]. 3.2. Subjective Evaluation {#sec3.2} -------------------------- Subjective evaluation of contrast enhancement is necessary as well as objective evaluation. The enhancement results can only be appreciated if the resultant image gives pleasurable effect in appearance. By visual quality inspection the judgment of annoying artifacts, overenhancement, and unnatural enhancement can be done. The visual assessment results are effective quality measures to judge the performance of contrast enhancement algorithm. 3.3. Objective Evaluation {#sec3.3} ------------------------- Qualitative analysis involves visually evaluating the image enhancement results. The quality of the enhanced images determines the capability of the techniques, which are justified by human eyes. Here, a qualitative analysis regarding the amount of details of the image, level of contrast, homogeneity of regions, and naturalness is performed; we can establish numerical justifications by quantitative measurements. However, it is difficult to find an objective measure that is in accordance with the subjective assessment due to the lack of any universally accepted criterion. Here, we evaluate the performance of enhancement techniques using three quality metrics: Peak Signal-to-Noise Ratio (PSNR), Discrete Entropy (DE), and Absolute Mean Brightness Error (AMBE). ### 3.3.1. Evaluation of Contrast Enhancement {#sec3.3.1} The proposed method not only enhances the contrast of the image, but also obtains a natural-looking output image without undesirable artifacts. The noise level should not be amplified during the enhancement process \[[@B29]\]. For this reason, two analyses named PSNR and image contrast function are used. To calculate the PSNR value, MSE is firstly computed by ([18](#EEq18){ref-type="disp-formula"}). PSNR is broadly used to evaluate the quality achievement between the original and output images \[[@B13], [@B30]--[@B34]\] and the degree of contrast enhancement in the image. A large PSNR value which is desired for it means that the processed image is least degraded compared with the original input image.$$\begin{matrix} {MSE\operatorname{} = \frac{1}{MN}{\sum\limits_{m}^{}{\sum\limits_{n}\left| {X\left| {m,n} \right| - Y\left| {m,n} \right|} \right|}}^{2},} \\ \\ \end{matrix}$$$$\begin{matrix} {PSNR\operatorname{} = 10{{\,\log_{10}}\left\lbrack {\frac{\left\lbrack {L - 1} \right\rbrack^{2}}{MSE}} \right\rbrack},} \\ \\ \end{matrix}$$where *X*  (*m*, *n*) is the gray level of the original image at a 2D position (*m*, *n*) and *Y*  (*m*, *n*) is the gray level of the processed image at the same position. Besides PSNR, image contrast function is used to evaluate the contrast improvement as well, as indicated in ([20](#EEq20){ref-type="disp-formula"}) \[[@B35], [@B36]\]. $$\begin{matrix} {\operatorname{}C_{contrast}} \\ {\mspace{1800mu} = \frac{1}{MN}{\sum\limits_{m = 1}^{M}{\,{\sum\limits_{n = 1}^{N}{Y^{2}\left( {m,n} \right) - \left| {\frac{1}{MN}{\sum\limits_{m = 1}^{M}{\,{\sum\limits_{n = 1}^{N}{Y\left| {m,n} \right|}}}}} \right|}}}}^{2},} \\ \end{matrix}$$where *M* and *N* represent the width and height of the image, respectively. The greater *C*~contrast~, the greater dynamic range of gray levels; thus the output image can provide better contrast and additional information contained in the image. *C*~contrast~ is then taken as a logarithm to convert it into decibel (dB) unit by $$\begin{matrix} {{C^{\ast}}_{contrast} = 10{{\,\log_{10}}C_{contrast}}.} \\ \end{matrix}$$ ### 3.3.2. Evaluation of the Richness of Information {#sec3.3.2} Entropy is a measure of the richness of information in the image, and the larger entropy value the image has, the higher the information contained in the output image is. The entropy for the whole image can be defined by$$\begin{matrix} {DE = {\sum\limits_{l = 0}^{L - 1}{e\left( { l} \right)}} = - {\sum\limits_{l = 0}^{L - 1}{p\left( { l} \right){\log_{2}{p\left( { l} \right)}}}}.} \\ \end{matrix}$$ The entropy of the image can achieve maximum value only when *p*(0) = *p*(1) = ⋯ = *p*(*L* − 1) = 1/*L* \[[@B27]\]. This is the scenario when the probability distribution of the image intensity values is uniform, which is the concept behind HE. ### 3.3.3. Evaluation of Brightness Preservation {#sec3.3.3} AMBE is usually used to measure mean brightness preservation, which can be mathematically represented by ([22](#EEq22){ref-type="disp-formula"}) \[[@B37]--[@B39]\]. AMBE exhibits the difference in mean brightness between the input and the output image. Mean brightness of the input and processed images is calculated using ([23](#EEq23){ref-type="disp-formula"}) and ([24](#EEq24){ref-type="disp-formula"}), respectively. Thus, a small AMBE value is desired, and a zero AMBE value is the best result.$$\begin{matrix} {AMBE\operatorname{} = \left| { E\left( { X} \right) - E\left( { Y} \right)} \right|,} \\ \\ \end{matrix}$$$$\begin{matrix} {E\left( { X} \right)\operatorname{} = \frac{1}{MN}{\sum\limits_{m}{\,{\sum\limits_{n}{X\left( { m,n} \right)}}}},} \\ \\ \end{matrix}$$$$\begin{matrix} {E\left( { Y} \right)\operatorname{} = \frac{1}{MN}{\sum\limits_{m}{\,{\sum\limits_{n}{Y\left( { m,n} \right)}}}},} \\ \\ \end{matrix}$$where *E*(*X*) and *E*(*Y*) are the mean brightness of the input and processed images, respectively. 4. Experiment Results and Discussion {#sec4} ==================================== 4.1. Experiment Results {#sec4.1} ----------------------- In this section, the simulation results of the proposed method MVSIHE are compared with existing histogram equalization based methods mentioned. [Table 1](#tab1){ref-type="table"} provides the list of methods with their detailed description. The comparison is from the aspects of contrast enhancement, brightness preservation, naturalness of the image, and ability to preserve details in the image. In this paper, the test images are given names as F16, Bridge, Couple, Fish, Lena, and Plane; they are presented in this study for initial performance evaluation on the proposed MSVIHE. The results obtained for each image are presented in Figures [2](#fig2){ref-type="fig"}[](#fig3){ref-type="fig"}[](#fig4){ref-type="fig"}[](#fig5){ref-type="fig"}[](#fig6){ref-type="fig"}--[7](#fig7){ref-type="fig"}, respectively. Image (a) indicates the input image, while images (b) to (i) represent the respective resultant images after applying other compared methods and the proposed MSVIHE. The quantitative results of these test images are illustrated in Tables [2](#tab2){ref-type="table"}[](#tab3){ref-type="table"}[](#tab4){ref-type="table"}--[5](#tab5){ref-type="table"}. The best value for each analysis is in bold face. For the first test image F16 in [Figure 2(a)](#fig2){ref-type="fig"}, the proposed MVSIHE yields output image with the mean brightness closest to the input image. The overall appearance of the image is very similar to the input image and for the proposed MVSIHE method can get the lowest AMBE value. The proposed method can well preserve most of the details of the image compared with the other methods for it grapes the highest value of entropy. This can be seen from the highlighted area with red boxes. MVSIHE also produces images with homogeneous texture. Most of the image area, particularly the background of the image, appears to have a smooth texture with a few small regions. The largest PSNR value is obtained by the MVSIHE-ed image, which shows that the technique least amplified the noise level in the image during the enhancement process. The proposed MVSIHE can well preserve the brightness of the processed image due to its largest contrast value. For the test image Bridge in [Figure 3(a)](#fig3){ref-type="fig"}, just as the contrast enhancement which is more significant compared with the other techniques, most details of the image are well preserved with its highest value of entropy. This can be seen from the words highlighted with red boxes. Processed images are with relatively good contrast, the value of the contrast by MVSIHE is ranked as second, and the effects of contrast enhancement are not far-off between all the methods. The MVSIHE method least amplifies the noise level in the image during the enhancement process for it can obtain the largest PSNR value. The proposed MVSIHE can simultaneously enhance the overall contrast of the test image Couple to an optimum level and preserve the details of the image, which can be observed on the window area highlighted with a box, as shown in [Figure 4(h)](#fig4){ref-type="fig"}. It is clear that the saturation effect is less apparent and thus the window area can be clearly seen. This saturation effect (i.e., the window area regions become too bright) can be observed in the RSIHE-ed image. Observation on the ability of the proposed MVSIHE to preserve details is supported by the entropy measurement, in which the enhanced image has an entropy value larger than most of the methods, indicating that the information entropy is well preserved. The MVSIHE-ed image has the largest value of PSNR (i.e., 22.6008), showing that BHEMHB least degrades the image during the enhancement process. In addition, the MVSIHE-ed image has the largest contrast measurement, which suggests that the proposed method can well preserve the brightness of the output image. The MVSIHE-ed image has the largest value of PSNR (i.e., 26.401), showing that MVSIHE least degrades the image during the enhancement process. The proposed MVSIHE can simultaneously enhance the overall contrast of the Fish image to an optimum level and preserve the details. This outcome can be observed on the fish scale highlighted with a box, as in [Figure 5(h)](#fig5){ref-type="fig"}. Observation on the ability of the proposed MVSIHE grapes the biggest value of entropy, demonstrating that the information entropy is well preserved. The ability to preserve details comes with a small tolerance in mean brightness preservation. Furthermore, the effects of contrast enhancement are less momentous for all methods, which demonstrates relatively good contrast. The ability of MVSIHE in contrast enhancement is about the same to the other methods. The output image enhanced by MVSIHE, as shown in [Figure 5(h)](#fig5){ref-type="fig"}, also exhibits a natural look, which means that it does not look too artistic after the enhancement process. The input image Lena has the characteristics that regions that are either fully black or fully white are relatively few, as shown in [Figure 6(a)](#fig6){ref-type="fig"}. The resultant image enhanced with the proposed MVSIHE has a clearer contour compared with images using the other methods, as can be seen on regions within boxes. Unlike images enhanced with other techniques, especially RMSHE, the image enhanced with MVSIHE presented fewer saturation effects. The proposed MVSIHE ranked first place for test image Lena in the entropy measurement, a ranking that is slightly less than that of other methods. We can know that MVSIHE is specifically designed to preserve the details; the difference reveals that the performance of MVSIHE is comparable with others in retaining image details. Furthermore, the proposed method can well preserve brightness for its lowest AMBE value. For the test image Plane in [Figure 7(a)](#fig7){ref-type="fig"}, the proposed MVSIHE produces an output image with most of the details well preserved because it possesses the highest entropy value. This result can be seen on regions highlighted with boxes, where the writing does not disappear and small details, such as edges of the plane, can be seen. The shifting effect of mean brightness is pregnant in the DSIHE-ed and RMSHE-ed images, resulting in the loss of naturalness in these images. By contrast, the resultant image enhanced with MVSIHE has a smooth texture, wherein less nonhomogenous regions are observed, especially on the background, compared with other techniques. In addition, the MVSIHE-ed image has the largest contrast measurement. Findings on the performance of the proposed techniques for the six test images, namely, F16, Fish, Plane, and Lena, are satisfactory when compared with those of the seven other methods. Thus, apart from these six test images, the four objective evaluation functions (i.e., entropy, PSNR, AMBE, and Contrast) are employed on the 100 test images to further validate the capability and performance of the proposed MVSIHE. [Figure 8](#fig8){ref-type="fig"} presents the average values of these quantitative analyses for 100 test images. [Figure 8](#fig8){ref-type="fig"} indicates that the proposed method illustrates excellent performance when compared with the other HE-based methods. In average, the MVSIHE-ed image contains the highest amount of information. It can well preserve the richness and details of information in output image due to its highest entropy value, which reaches 7.26 for an average of 100 test images. The proposed MVSIHE outperforms all the other methods, with its largest PSNR value, which shows that the output images enhanced by MVSIHE have a natural appearance with minimum artifacts compared with others. The proposed method can least degrade the image during the enhancement process. 4.2. Discussions {#sec4.2} ---------------- With regard to mean brightness, the MVSIHE-ed image demonstrates high capability, especially when compared with the DSIHE-ed images. DSIHE method yields an image that is too bright when referred to the original image. AMBE values for all the techniques are computed, and MVSIHE can obtain the lowest value compared to all the others. The naturalness of the image is maintained in the MVSIHE-ed image, because the image is enhanced at a sufficient level without introducing an unpleasant look or nonhomogeneous regions while improving the contrast of the input image. The highest PSNR value by the MVSIHE-ed image indicates that MVSIHE enhances the image with minimum noise and artifacts. Contrast measurements show that the MVSIHE can get the largest value mostly, which illustrates that the contrast enhancement performance of MVSIHE is better than others. Moreover, MVSIHE acquires the lowest AMBE value. The AMBE value obtained by MVSIHE is notably better than that obtained by RSIHE technique because RSIHE is specifically designed to maintain the mean brightness of the image. The lowest AMBE value indicates that MVSIHE possesses the highest capability in retaining the mean brightness of the image compared with all the other methods, in which the output images enhanced with MVSIHE typically have a mean brightness closest to the input image. With regard to the overall contrast enhancement, otherwise, the proposed MVSIHE ranked second among the seven methods. The range of contrast measurements is small (i.e., only 1.69 dB), which indicates that despite its outstanding performance in detail preservation and mean brightness preservation, MVSIHE demonstrates comparable performance in contrast enhancement. Both qualitative and quantitative analyses show that the proposed MVSIHE yields promising enhancement results. 5. Conclusion {#sec5} ============= This paper presents a new method referred to as the Mean and Variance based Subimage Histogram Equalization (MVSIHE) with brightness and details preservation. The main idea lies on recursively separating the input histogram based on the mean and variance. The effect of intensity levels normalization and fusion strategy is also investigated in this paper. Unpleasant artifacts and unnatural enhancement may occur due to excessive equalization while enhancing the contrast of an input image, and the ultimate goal of MVSIHE is to allow higher level of brightness and details preservation as much as possible. The contrast of the input image is effectively increased with brightness and details well preserved. All findings are supported by experimental results, which have shown that the proposed method has superior performance to some state-of-the-art methods. In the future, the proposed MVSIHE could be modified so that it can be incorporated in several application areas such as digital photography, video processing, and other applications in consumer electronics. Conflicts of Interest ===================== Liyun Zhuang and Yepeng Guan declare that there are no conflicts of interest regarding the publication of this paper. {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} {#fig6} {#fig7} {#fig8} ###### Properties of the proposed MVSIHE and other HE-based methods. Methods Implementation steps^a^ Main focus --------------------------------------------- ----------------------------------------------------------- ------------------------------------ DSIHE \(1\) HS using probability density function \(1\) Contrast enhancement \(2\) HE \(2\) Detail preservation RMSHE \(1\) HS using mean brightness (*γ* = 2) \(1\) Mean brightness preservation \(2\) HC using the middle gray level \(2\) Detail preservation \(3\) HE   MMBEBHE \(1\) HS using minimum mean brightness error \(1\) Mean brightness reservation \(2\) HE   RSIHE \(1\) HS using median brightness (*γ* = 2) \(1\) Mean brightness preservation \(2\) HE   ESIHE \(1\) HC using the average number of intensity occurrence \(1\) Mean brightness preservation \(2\) HS using exposure threshold \(2\) Enhancement rate restriction BHEMHB \(1\) HS using median brightness (*γ* = 1) \(1\) Mean brightness preservation \(2\) Modification of histogram bins \(2\) Detail preservation \(3\) HE   MVSIHE \(1\) HS using mean and variance brightness (*γ* = 2) \(1\) Mean brightness preservation \(2\) Modification of histogram bins \(2\) Detail preservation \(3\) HE \(3\) Contrast enhancement \(4\) Fuse processed image with input image   ^a^HS indicates histogram segmentation, HC indicates histogram clipping, and HE indicates histogram equalization. ###### PSNR calculated for the test images. Image name DSIHE RMSHE MMBEBHE RSIHE ESIHE BHEMHB Proposed ------------ --------- --------- --------- --------- --------- --------- ------------- F16 20.9870 21.8760 24.4849 22.1130 22.8690 23.9420 **27.0944** Bridge 24.2212 22.9133 23.5086 24.3100 28.4320 26.7650 **31.4561** Couple 19.7178 18.3277 21.0514 20.5600 21.6780 20.4635 **22.6008** Fish 20.1459 19.7673 22.0862 24.9780 25.4060 26.4010 **28.2385** Lena 23.5663 22.1785 22.7730 24.8000 25.7990 26.5950 **29.3948** Plane 28.7635 27.6540 28.3268 17.3390 18.2420 20.6130 **30.3730** ###### DE calculated for the test images. Image name DSIHE RMSHE MMBEBHE RSIHE ESIHE BHEMHB Proposed ------------ -------- ------------ --------- -------- -------- -------- ------------ F16 6.3590 6.0909 6.5023 6.4910 6.6120 6.6660 **7.3898** Bridge 7.2512 7.1588 7.0540 6.5257 7.3680 7.1456 **7.8408** Couple 6.9940 **7.9891** 6.8467 6.3971 7.4100 7.2350 7.6047 Fish 6.1604 6.1689 6.2672 5.9100 5.9850 6.0170 **7.1066** Lena 7.1996 7.0085 7.2195 7.4610 7.4970 7.5620 **7.7539** Plane 6.3447 6.1205 6.2500 6.3050 6.3800 6.4470 **7.1520** ###### AMBE calculated for the test images. Image name DSIHE RMSHE MMBEBHE RSIHE ESIHE BHEMHB Proposed ------------ --------- --------- ------------ --------- --------- ------------ ------------ F16 20.2554 5.7030 0.4496 6.4810 2.8740 1.3340 **0.1322** Bridge 2.3752 3.6479 **2.1850** 3.6592 3.7860 2.3750 3.5623 Couple 4.0264 11.9858 0.7215 5.0718 2.1570 **0.5652** 2.2811 Fish 9.5402 11.7333 8.2674 3.7260 3.8960 4.5760 **3.3199** Lena 6.0463 10.3377 0.8662 5.1090 2.5310 2.4650 **0.8474** Plane 16.6670 13.2179 **3.3521** 13.1660 11.2240 8.1610 4.6093 ###### Contrast calculated for the test images. Image name DSIHE RMSHE MMBEBHE RSIHE ESIHE BHEMHB Proposed ------------ ----------- ------- ----------- ------- ------- ----------- ----------- F16 69.73 68.86 69.98 68.82 69.28 69.07 **70.15** Bridge 66.16 66.30 **66.52** 63.32 63.01 65.16 66.43 Couple 64.56 64.65 63.98 62.61 62.01 64.48 **64.67** Fish 66.13 66.22 67.15 66.85 66.42 **67.60** 67.56 Lena **64.83** 63.22 64.17 64.33 63.66 64.52 64.62 Plane 70.11 69.67 69.21 69.25 69.33 70.06 **70.24** [^1]: Academic Editor: Silvia Conforto

Talin links integrins to the actin cytoskeleton at focal adhesions. On page 43, Morgan et al. show that talin also links synaptic activity to actin rearrangements. FigureRings of actin (green) around synaptic vesicle clusters are lost when talin cannot bind PIPKIγ (right). Synapses are specialized sites of cell adhesion that were recently shown to hold talin. Talin interacts with PIPKIγ, a PI(4,5)P~2~-producing enzyme, which in turn regulates talin and other actin regulatory proteins. The importance of this interaction is now shown at synapses, where PI(4,5)P~2~ also controls clathrin coat dynamics. The authors interfered with talin--PIPKIγ interactions in the axons of giant lamprey neurons. The interference inhibited the recycling of synaptic vesicles on the presynaptic side. Clathrin-coated pits formed but did not bud off the plasma membrane. These changes were associated with reduced synaptic actin networks. The defects are probably due to low levels of PI(4,5)P~2~ resulting from a block in PIPKIγ recruitment to the membrane. The same lab has also recently shown genetically that PI(4,5)P~2~ is necessary for synaptic vesicle trafficking (Di Paolo et al. *Nature*. 431:415--422). As PIP~2~ production is up-regulated by neuronal depolarization, the authors are now studying how the talin-PIPKIγ interaction is affected by the firing of synapses. [^1]: <lebrasn@rockefeller.edu>

The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus. Introduction ============ Venous thromboembolism (VTE) includes both venous thrombosis and pulmonary embolism (PE). It is associated with significant morbidity and mortality with a 30-day death rate of about 3% and 31%, respectively, in those left untreated \[[@REF1]\]. Despite being on therapeutic doses of anticoagulation, patients can still develop recurrent PE, which is appropriately termed "anticoagulation failure." The rate of recurrent PE is up to 4% with low-molecular-weight heparin (LMWH) and 2-4% with vitamin K antagonists (VKA) \[[@REF1]\]. Case presentation ================= A 32-year-old Caucasian female presented to the emergency department with an acute onset of shortness of breath (SOB). Her past medical history was significant for recurrent VTE of unknown etiology with removal of an inferior vena cava filter due to misplacement. She had no family history of thromboembolic disorder and no past medical history of smoking or oral contraceptive usage. Her first episode of PE was spontaneous about six years ago followed by multiple episodes of VTE that required thrombolysis on three separate occasions. Her comprehensive hypercoagulable workup in the past included factor V Leiden mutation, JAK2 V617 mutation, Lupus anticoagulant, antithrombin III activity, PNH flow cytometry, factor II gene mutation, protein C, protein S, anti-cardiolipin antibody, anti-beta-2 glycoprotein-1 antibody, and homocysteine levels, which were all unremarkable. She had recurrent PE on many therapies including warfarin (with therapeutic international normalized ratio of 2.5-3.5), rivaroxaban, apixaban, dabigatran, heparin, and fondaparinux. She also had a history of rash secondary to enoxaparin making management more complicated. Her pertinent physical exam findings on presentation included hypoxia on 2 liters of nasal cannula with oxygen saturation at 96%, respiratory rate of 22 and decreased breath sounds bilaterally in the lung bases with no signs of deep vein thrombosis (DVT). A computed tomography (CT) scan of the chest with contrast on admission showed new pulmonary emboli on the right side. At this point it was challenging to decide the next step in management since she had failed most known anticoagulants in the past. This also caused much physical, psychological, and financial burden on the patient due to recurrent hospitalizations over a short period of time. The hematology service was consulted, and after a thorough discussion with the patient, rivaroxaban 15 mg twice daily was initiated since she had failed the 20 mg once daily dose in the past. Unfortunately, after about three weeks of rivaroxaban treatment she presented with another episode of PE. She was started on therapeutic heparin at this time with activated partial thromboplastin time (aPTT) range between 90-140 seconds. A higher aPTT range was selected given her recurrence. She required large doses of heparin to maintain her aPTT, but after it was maintained at the new therapeutic goal, her breathing eventually improved and she did not require further oxygen supplementation. At this time, the hematology service decided to combine two oral anticoagulants to prevent further episodes of PE since she has had recurrence on heparin. They initially recommended combining a second oral anticoagulant with rivaroxaban such as apixaban or dabigatran, but due to the patient's health insurance coverage issues and the higher price of novel oral anticoagulants, two drugs from this class did not seem feasible. Instead, she was started on warfarin with a therapeutic goal INR range of 2-3 along with rivaroxaban 15 mg bis in die (BID). At the time of writing this manuscript, six months since the patient was seen, she has had no recurrence of PE or signs of bleeding. Discussion ========== Venous thromboembolism (VTE), including DVT of the extremities or pelvis and PE, is associated with a significant morbidity and mortality with approximately 60,000 to 100,000 deaths in the United States every year \[[@REF2]\]. Anticoagulation is the mainstay of treatment in patients with VTE, and aggressive management is essential in decreasing mortality \[[@REF2]\]. The optimal management of anticoagulation failure includes dose escalation, switching to a different anticoagulant or adding an antiplatelet drug \[[@REF3]\]. The literature was reviewed to find the utility of adding an antiplatelet drug such as aspirin in our patient, but it was shown to only be beneficial in antiphospholipid syndrome \[[@REF3]\] and not in other patient groups. We trialled all the aforementioned strategies before ultimately placing the patient on dual anticoagulants including dose escalation of rivaroxaban and using different anticoagulants. Unfortunately, she had recurrence of thromboembolic events in each case. Another approach to anticoagulation failure is by combining anticoagulants \[[@REF2]\]. Two case reports in the literature were found pertaining to dual anticoagulation \[[@REF2]\]. The first case report included a 43-year-old female with clear adenocarcinoma of the ovary who presented with recurrent VTE despite being on a therapeutic dose of dalteparin \[[@REF2]\]. Coumadin was added to dalteparin with a therapeutic INR goal of 2-3, after which there was no recurrence of thromboembolic events \[[@REF2]\]. The second case report was a 57-year-old male with colon adenocarcinoma who was started on dual anticoagulation with warfarin and dalteparin after he failed dalteparin monotherapy \[[@REF2]\]. In both case reports, thromboembolic events were prevented with no bleeding episodes for more than three to four months \[[@REF2]\]. Of note, the patients in these case reports had a diagnosis of cancer, and the utility of dual anticoagulant therapy in patients without a diagnosis of cancer is not well established. We did an extensive literature review and could not find any case reports utilizing dual anticoagulant therapy in the management of VTE in those without a diagnosis of cancer \[[@REF2]\]. There is no evidence to our knowledge to support the practice of combining warfarin with other oral anticoagulants, which was ultimately done successfully in our patient since she failed a large variety of monotherapies. The only evidence we found at this time towards dual anticoagulants is combining LMWH with warfarin. Unfortunately, this could not be done with our patient since she had developed a rash with enoxaparin in the past \[[@REF2]\]. Conclusions =========== In summary, this case report is unique in demonstrating that dual anticoagulation can be used in patients with recurrent VTE especially when monotherapies have failed them. Caution should be exercised in evaluating the benefits and risks (mainly bleeding) before giving this treatment. Further studies are needed to determine the optimal combination of anticoagulation with minimal bleeding risk. The authors have declared that no competing interests exist. Consent was obtained by all participants in this study

1. Introduction =============== Stroke is one of the leading causes of morbidity and mortality worldwide. More than 80% of strokes are ischemic and are caused by obstruction of one or more cerebral arteries \[[@b1-ad-9-5-924], [@b2-ad-9-5-924]\]. Lack of blood supply deprives the brain cells of necessary glucose and oxygen, and disturbs cellular homeostasis, culminating in neuronal death. Currently, tissue plasminogen activator (tPA) is the only effective pharmacological therapy approved by the Food and Drug Administration for acute ischemic stroke since 1996, but its use remains limited due to the narrow therapeutic window \[[@b3-ad-9-5-924]-[@b6-ad-9-5-924]\]. It is imperative to develop novel therapeutic strategies of stroke \[[@b7-ad-9-5-924], [@b8-ad-9-5-924]\]. Mitochondria, as the powerhouse of cell, play a critical role in cell energy homeostasis and are thus inevitably involved in the ischemic neuronal death. After ischemia, due to the reduced blood supply, the energy balance is disrupted and adenosine triphosphate (ATP) synthesis is disturbed. In addition to their fundamental role in energy generation, mitochondria are critical involved in the regulation of such forms of cell death as apoptosis, autophagy and necroptosis. Mitochondrial dysfunction has been regarded as one of the hallmarks of ischemia/reperfusion (I/R) injury which induces neuronal death\[[@b9-ad-9-5-924]\]. Accumulating evidences indicate that the maintaining of the mitochondrial function is crucial for neuron survival and neurological improvement. Therefore, targeting mitochondria is one of the promising neuroprotective strategies for stroke treatment \[[@b10-ad-9-5-924]\]. Recently, it has been demonstrated that mitochondria can be transferred from astrocytes to neurons and served as "help-me signaling" in cell-to-cell communication after cerebral ischemia \[[@b11-ad-9-5-924]\]. In this review, we will discuss current knowledge on the role of mitochondria in cell death and cell survival, and highlight the advance of mitochondria-based therapy in stroke. In particular, we will emphasize the most critical mechanisms responsible for mitochondrial quality control, as well as the recent findings on mitochondrial transfer in acute ischemic stroke. 2. Pathophysiology of ischemic stroke ===================================== Ischemic stroke is a consequence of a critical reduction of regional cerebral blood flow (rCBF), leading to severe oxygen and glucose deprivation. Mitochondrial disfunction induced by oxygen and glucose deprivation occurs within minutes after ischemia, resulting in depletion of ATP production and overproduction of reactive oxidative species (ROS). Compared to other brain cells, neurons have higher energy demand, but their energy reserves are limited. Depletion of ATP is one of the major initiator, which triggers the ischemic cascades such as, membrane ion pump failure, efflux of cellular potassium, influx of sodium, chloride and water, and membrane depolarization \[[@b12-ad-9-5-924]-[@b14-ad-9-5-924]\]. Multiple mechanisms, including excitotoxicity, mitochondrial response, free radical release, acidotoxity, protein misfolding, and inflammation have been extensively studied as the events leading to the cellular death and neuronal loss after stroke \[[@b15-ad-9-5-924]-[@b17-ad-9-5-924]\], which are depicted in [Fig. 1](#F1-ad-9-5-924){ref-type="fig"}. Figure 1.Mechanisms underlying neuronal death in ischemic stroke(1) Mitochondrial response, including excessive ROS production, mitochondrial calcium overloading, and disrupted mitochondria quality control. (2) Excitotoxicity. Excessive glutamate release and impeded reuptake of excitatory amino acids result in the activation of NMDARs, AMPARs and KARs. (3) Acidotoxity. Extracellular acidification leads to ischemic neuronal death by activating acid-sensing ion channel 1a (ASIC1a). (4) Protein misfolding. Protein misfolding and aggregation are observed after brain ischemia. (5) Inflammatory reaction. Microglia are activated and release cytokines and chemokines to induce inflammation reaction. All the factors mentioned above work synergistically to trigger cell death pathways such as apoptosis, necroptosis and autophagy. ROS: reactive oxygen species; AMPAR: α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor; NMDAR: N-methyl-D-aspartate receptor; KAR: kainite receptor; DAPK1: death associated protein kinase 1; PSD95: postsynaptic density protein 95; ASIC1a: acid-sensing ion channel 1a; RIPK1: receptor interacting protein kinase 1; TNF-α: tumor necrosis factor-α; IL-6: Interleukin 6; IL-1: Interleukin 1. ###### Mitochondrial dynamic-related proteins. Mammals Yeast Role in mitochondria dynamics Location -------------------- --------- ------------------------------- ----------------------------------------------------------- Drp1 Dnm1p Fisson cytosolic, and recruited to outer membrane during fission Fis1 Fis1p Fisson Outer mitochondrial membrane Endophilin B1 \- Fisson cytosolic, and recruited to outer membrane during fission Mfn1/2 F201p Fusion Outer mitochondrial membrane Opa1 Mgm1p Fusion Inner mitochondrial membrane KIFs \_ Transport Cytosolic Cytoplasmic dynein Dynclil Transport Cytosolic There are two major zones of injury in ischemic brain: the infarct core and the ischemic penumbra. The "infarct core" is the area with the blood flow decline below the threshold of energy failure (15%-20%). The "infarct core" will be irreversibly damaged during few minutes after stroke and cannot be salvaged \[[@b18-ad-9-5-924]\]. On the contrary, ischemic penumbra is the region with impaired functions, but preserved structure integrity \[[@b19-ad-9-5-924]\]. Cells in penumbra are salvageable if reperfusion is established during the early hours or collateral circulation is adequate to maintain the neuronal demand for oxygen and glucose \[[@b15-ad-9-5-924]\]. Therefore, the penumbra is the pharmacological target for the treatment of acute ischemic stroke. In the infarct core, neuronal cells undergo necrotic alterations, which are accompanied by glutamate release and excitotoxic cell damage to neighboring regions \[[@b20-ad-9-5-924]\]. The penumbra is moderately hypoperfused and energy metabolism is partially preserved, which transiently sustain tissue viability. Timely interventions are effective to avoid the progression of the penumbra into infarction. The molecular mechanisms leading to the cell death in the penumbra differ from those in the infarct area. Several types of regulated cell death such as autophagy, apoptosis, necroptosis, and pyroptosis can be triggered by ischemia and contribute to the post stroke brain injury. To some extent, different kind of cell death can cross-regulate each other \[[@b21-ad-9-5-924], [@b22-ad-9-5-924]\]. Mitochondria can be considered as one of the master regulators of stress signaling \[[@b23-ad-9-5-924]-[@b26-ad-9-5-924]\] ([Fig. 2](#F2-ad-9-5-924){ref-type="fig"}). The impairment of mitochondrial respiratory function and membrane potential activate the cascade of events that leads to neuronal death after ischemia. Depolarized mitochondria initiate excessive ROS production, decreased ATP generation, PTEN-induced putative kinase 1 (PINK1) accumulation, as well as unfolded protein response (UPR) \[[@b27-ad-9-5-924]\]. The increased levels of ROS and overloading calcium open the membrane permeability transition pore allowing for the release of cytochrome c, which activates the effector caspases and the final execution of apoptotic death \[[@b28-ad-9-5-924]\]. PINK1 selectively recruits Parkin and phosphorylates both Parkin and ubiquitin to trigger mitophagy \[[@b29-ad-9-5-924]\]. Interruption of the signaling pathways is apparently able to disrupt, or in some instances greatly delay, the spiral of increasingly vicious reactions that culminate in cell death. Figure 2.Mitochondria play a central role in ischemic neuronal deathIschemia triggers the depolarization of mitochondrial membrane potential (ΔΨm), reduction of ATP production, accumulation of PINK1, recruitment of Parkin, overproduction of reactive oxygen species (ROS), overloading of matrix calcium, and opening of mitochondrial permeability transition pore (mPTP), eventually leading to neuronal death. 3. Structure and function of mitochondria ========================================= Mitochondria are ovoid or rod-shaped organelles with double-membrane structure, which consist of four distinct compartments - the outer membrane, the intermembrane space, the inner membrane, and the matrix \[[@b30-ad-9-5-924]\]. The outer membrane contains a number of pores which form large aqueous channels to permit free diffusion of molecules less than 5000 daltons into the intermembrane space. The intermembrane space contains proteins (e.g. cytochrome c) that play major roles in mitochondrial energetics and apoptosis. In contrast to the outer membrane, the inner membrane has much more restricted permeability, equipped with a variety of ion channels and transporters as well as mitochondrial enzyme systems like the electron transport chain. The matrix contains most of the enzymes that responsible for the citric acid cycle reactions. The size, shape and number of mitochondria vary widely depending on the functional state of the cells \[[@b31-ad-9-5-924], [@b32-ad-9-5-924]\]. And structural reorganization does occur in cases of stress \[[@b33-ad-9-5-924], [@b34-ad-9-5-924]\]. Mitochondria are pivotal regulators of cell survival. The critical role of mitochondria in cell survival is mainly reflected in 3 aspects. (1) Mitochondria are the hubs of cellular calcium signaling \[[@b23-ad-9-5-924]\]; (2) The enzyme complexes located on the mitochondrial inner membrane are essential components for the maintenance of cell energy requirements and metabolic homeostasis \[[@b24-ad-9-5-924]\]; and (3) Cell survival greatly relies on the integrity and functionality of mitochondria, hence dynamic mitochondrial changes in their shapes and populations are critical for mitochondrial quality control \[[@b25-ad-9-5-924], [@b26-ad-9-5-924]\]. Neurons are particularly dependent on mitochondria for calcium signaling and ATP production, which makes them more sensitive to ischemia and hypoxia during stroke. As a consequence, mitochondrial defects have much devastating effects in central nervous system (CNS). 4. Mitochondrial quality control systems and ischemic stroke ============================================================ 4.1 Mitochondria quality control systems ---------------------------------------- Mitochondria control virtually every aspect of cell function, including managing redox status, modulating Ca^2+^ homeostasis, generating ATP, and regulating response to cellular and environmental stresses. Over the last decade, mitochondria have moved to the forefront of cell biology research for their central role in regulation of cell death signaling. In stroke, abnormalities in mitochondrial membrane potential and ROS over-production induce neuronal injury ([Fig. 2](#F2-ad-9-5-924){ref-type="fig"}). Here we focus on the most recent findings on the role of mitochondrial quality control processes especially regulation of dynamics and mitophagy in stroke, highlighting their potential as therapeutic targets. Neuronal survival critically depends on the integrity and functionality of mitochondria. A hierarchical system of cellular surveillance mechanisms protects mitochondria against stress and damage, hence ensures the selective removal of dysfunctional mitochondrial proteins. There are 3 levels of well-conserved mitochondria quality control mechanisms \[[@b35-ad-9-5-924]\]. The first level of defense involves a multilayer network of detoxifying systems capable of fighting oxygen- and aldehyde-mediated mitochondrial toxicity. The second level of defense relies to mitochondrial proteases and chaperones responsible for the maintenance of mitochondrial proteostasis. Toxic folding intermediates or the accumulation of aggregates are deleterious for the cells and it is imperative to correct protein folding and maintain cellular homeostasis. The third level of defense involves the control of mitochondrial morphology, quantity of mitochondria through mitochondrial dynamics (fusion and fission) and mitophagy (mitochondrial clearance). Maintaining mitochondrial integrity and elimination of damaged mitochondria are important avenues to preventing widespread mitochondrial dysfunction, oxidative stress, and cell death in ischemic insult. Hence, mitochondrial dynamics and mitophagy play critical roles in maintaining cellular homeostasis and function. Here we will focus on the third level of defense in response to ischemic stress. 4.2 Mitochondrial dynamics and ischemic stroke ---------------------------------------------- Mitochondria are highly dynamic cellular organelles characterized by their ability to change size, shape and location through highly coordinated processes of fission (separation of a single mitochondrion into two or more daughter organelles), fusion (the opposing reaction) and transport to strategic locations \[[@b36-ad-9-5-924]-[@b38-ad-9-5-924]\]. Healthy mature neurons with established synaptic connections exhibited longer, more stationary mitochondria whereas impaired or immature neurons have smaller, more motile mitochondria. Mitochondrial fusion and fission were first observed in yeast in 1994. By now it is known that these processes play critical roles in maintaining mitochondria integrity and function of cells when experiencing metabolic or environmental stresses \[[@b39-ad-9-5-924]\]. Mitochondrial fusion allows the rapid exchange of mitochondrial membranes, mitochondrial DNA (mtDNA), and mitochondrial metabolites within a mitochondrial network; while damaged mitochondria can be repaired through fusion with healthy mitochondria for integrating of contents and promotes cell survival by complementation. On the other hand, mitochondrial fission enables the segregation of damaged mitochondria and leads to their subsequent elimination via mitophagy \[[@b40-ad-9-5-924]\]. Fission and fusion are active processes which require many specialized large guanosine triphosphatases (GTPases). Dynamin-related protein 1 (Drp1), Fis1and Endophilin B1 are required for mitochondrial fission in mammals; Mitofusin 1 (Mfn1), Mitofusin 2 (Mfn2) and Opa1are responsible for fusion. Kinesin superfamily proteins (KIFs) and cytoplasmic dynein are the main microtubule-based motor proteins ensure targeted trafficking of mitochondria and precise regulation of their mobility \[[@b41-ad-9-5-924]\] ([Table 1](#T1-ad-9-5-924){ref-type="table"}). In neurons, mitochondrial fission is essential for mitochondrial transport to their potential docking sites in axons and dendrites. Defects in mitochondrial fusion, fission or transport lead to impaired mitochondrial motility and function in neurons ([Fig. 3](#F3-ad-9-5-924){ref-type="fig"}). In neuroglial cells, alteration in mitochondrial dynamics contribute to the differential functions of reactive microglia in neurological diseases \[[@b42-ad-9-5-924], [@b43-ad-9-5-924]\]; and in astrocytes, proinflammatory stimuli induced mitochondrial fission, and ultimately bioenergetics to maintain mitochondrial network \[[@b44-ad-9-5-924]\]. Figure 3.Mitochondrial fusion, fission or transport in neuronsDamaged mitochondria can be repaired through fusion with healthy mitochondria, and mitochondrial fission enables the segregation of damaged mitochondria and subsequent elimination via mitophagy. Mitochondria are transported and packed at axonal synapses, and are essential for neuronal transmission and plasticity. Previously the molecular machineries of mitochondrial fission and fusion were studied separately. Currently, more and more researchers are interested in investigating the coordination/balance of mitochondria dynamics. Mitochondrial fission protein Drp1 was observed to affect the activity of fusion. Santel's group found that the depletion of Drp1 resulted in reduction of Mfn1, Mfn2 and Opa1 in Hela and HUVEC cells \[[@b45-ad-9-5-924]\]. Consistent with their work, Saita et al reported that loss of Drp1 led to the degradation of Mfns via the ubiquitin-proteasome system \[[@b46-ad-9-5-924]\]. These studies indicated that in addition to the canonical roles of Drp1 in mitochondrial fission, it also acts as regulatory factors that control mitochondrial fusion. Furthermore, Anand et al reported that expression of mitochondrial fusion protein Opa1 unexpectedly triggered mitochondrial fragmentation in Yme1l^-/-^ cells and HEK293T cells, which suggests that mitochondrial fusion protein Opa1 is associated with fission \[[@b47-ad-9-5-924]\]. The coordination of fusion proteins and fission proteins are responsible for fine-tuning mitochondrial dynamics and cell sensitivity to stress. It is well documented that in ischemic brain injury mitochondrial dynamics are closely linked to their morphology and functions. Mitochondrial fission is an early event required for ischemic neuronal death, which is mediated by Drp1 and Mfn1/2 \[[@b48-ad-9-5-924]-[@b50-ad-9-5-924]\]. In primary cultured cortical neurons, nitric oxide exposure triggered dose-dependent mitochondrial fission and caused ultrastructural damage, which are associated with bioenergetic failure and increased ROS production. After middle cerebral artery occlusion (MCAO) in mice, mitochondrial fission occurs as early as 3 h after reperfusion \[[@b48-ad-9-5-924]\]. In the ischemic penumbra, the protein level of both Drp1 and Opa1 increased at 2 days after MCAO, and returned to the baseline at day 7. In the ischemic core, however, the expression of Drp1 and Opa1 progressive decreased until 2 days after transient MCAO \[[@b51-ad-9-5-924]\]. Hyperglycemia further enhanced mitochondrial fission by increasing protein levels of Drp1 and Fis1, as well as decreasing the level of Opa1 in MCAO mice \[[@b52-ad-9-5-924]\]. By contrast, the expression of mitochondrial fusion protein Mfn2 decreased in both *in vivo* and *in vitro* ischemic models, and reduced Mfn2 expression led to mitochondrial dysfunction and disruption of Ca^2+^ homeostasis \[[@b49-ad-9-5-924], [@b50-ad-9-5-924]\]. Using transgenic mice expressing mitochondria targeted yellow fluorescent protein (mito-eYFP), Owens and his colleagues examined cell-type specific changes of mitochondrial dynamics in hippocampal region following global cerebral ischemia in mice \[[@b53-ad-9-5-924]\]. In cornuammonis 1 (CA1) area of hippocampus, mitochondrial fragmentation started as early as 2 h after the reperfusion and reached the peak at 24 h. Similar observations were done in the CA3 and dentate gyrus (DG) area; however, mitochondria in CA3 and DG neurons started to refuse after 24 h of reperfusion; Astrocytes underwent transient mitochondrial fission from 2 h after ischemia and regained their normal shape at 24 h of recovery \[[@b53-ad-9-5-924]\]. Their data first suggested that ischemia resistant neurons were able to shift their mitochondrial dynamics toward fusion after extensive fragmentation. Recently, using *in vivo* two-photon microscopy, Kislin et al visualized the fragmentation of neuronal mitochondria in the living mouse brain \[[@b54-ad-9-5-924]\]. They induced severe global ischemia by bilateral common carotid artery occlusion, furthermore they induced severe focal stroke injury by Rose Bengal photosensitization and moderate and mild traumatic brain injury by focal laser lesion or mild photo-damage respectively. Mild injury induced transient mitochondrial fragmentation and decreased dendritic spine density, however, severe brain ischemia caused mitochondrial fragmentation and dendritic damage. Their findings showed that alterations of mitochondrial morphology were very sensitive to ischemia and were reversible in moderate and mild injury \[[@b54-ad-9-5-924]\]. ###### Chemical agents targeting mitochondrial dynamics in ischemic stroke Agents Proposed mechanisms Refs. ------------------------ --------------------------------------------------------------------------------------------- ------------------------------------------------------------ Drp1 siRNA Inhibition of fission via DRP1 down-regulation \[[@b55-ad-9-5-924]-[@b57-ad-9-5-924], [@b61-ad-9-5-924]\] Drp1 cysteine mutation Inhibition of fission by preventing S-nitrosylation induced Drp1 activation \[[@b59-ad-9-5-924]\] Ginkgolide K Inhibition of fission by increasing Drp1 phosphorylation and inhibiting Drp1 recruitment \[[@b60-ad-9-5-924]\] mdivi-1 Inhibition of fission by inhibiting the GTPase activity of Drp1 \[[@b55-ad-9-5-924], [@b61-ad-9-5-924]\] P110 Inhibition of fission by inhibiting Drp1 enzyme activity and blocking Drp1/Fis1 interaction \[[@b62-ad-9-5-924], [@b63-ad-9-5-924]\] Mfn2 overexpression Promotion of fusion \[[@b49-ad-9-5-924], [@b50-ad-9-5-924]\] Pharmacological agents or genetic interventions that either inhibiting fission or promoting fusion generally protect neurons from ischemic injury ([Table 2](#T2-ad-9-5-924){ref-type="table"}). Down-regulation or inhibition of Drp1 reduced mitochondrial fission and maintained normal mitochondrial morphology and function in ischemic neurons \[[@b55-ad-9-5-924]-[@b57-ad-9-5-924]\]. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential, and cell death in glutamate toxicity or OGD *in vitro*, and after ischemic brain damage *in vivo* \[[@b57-ad-9-5-924]\]. However, Drp1 knockout caused embryonic lethality in mice \[[@b58-ad-9-5-924]\]. Drp1 was activated by S-nitrosylation and this process can be prevented by cysteine mutation to protect mitochondria from fragmentation \[[@b59-ad-9-5-924]\]. Ginkgolide K, a natural compound found in Ginkgo biloba leaves, attenuated neuronal apoptosis in OGD model by increasing Drp1 phosphorylation at Ser637 and inhibiting Drp1 recruitment \[[@b60-ad-9-5-924]\]. Mitochondrial division inhibitor mdivi-1, a selective inhibitor of mitochondrial fission protein Drp1, protected brain from OGD reperfusion injury and MCAO through suppressing the ROS production and decreasing the expression of cytochrome C \[[@b55-ad-9-5-924], [@b61-ad-9-5-924]\]. P110, a novel and selective peptide inhibitor of mitochondrial fission, inhibited Drp1 enzyme activity and blocked Drp1/Fis1 interaction \[[@b62-ad-9-5-924]\]. It abolished Drp1-dependent p53 stabilization on mitochondria and reduced brain infraction in rats subjected to brain ischaemia/reperfusion injury \[[@b63-ad-9-5-924]\]. Promoting mitochondrial fusion also protects cell from injury. Mfn2 overexpression attenuated mitochondrial dysfunction and restored mitochondrial morphology \[[@b49-ad-9-5-924], [@b50-ad-9-5-924]\]. Interestingly, treadmill exercise pretreatment enhanced mitochondrial fusion via up-regulation of Opa1 after cerebral ischemia \[[@b64-ad-9-5-924]\]. 4.3 Mitophagy and its role in ischemic stroke --------------------------------------------- Mitophagy was first described by Lemasters and colleagues in 2005, who determined that mitophagy is an autophagic response, which is responsible for specifically removal of damaged mitochondria to maintain mitochondria homeostasis \[[@b65-ad-9-5-924]\]. The molecular mechanisms regulating mitophagy were originally characterized in yeast. Uth1p and Aup1p are two major proteins required for mitophagy in nutrient deprivation or stationary phase, respectively \[[@b66-ad-9-5-924], [@b67-ad-9-5-924]\]. However, the corresponding mammalian homologues of Uth1p and Aup1p are yet to be identified. The mechanisms of mitophagy in mammalian cells can be generally classified as ubiquitin (Ub)-dependent and Ub-independent pathways. Theserine/threonine kinase PINK1 and the E3 ligase Parkin pathway is currently the best understood Ub-dependent mechanism of mitophagy for the recognition of damaged mitochondria \[[@b68-ad-9-5-924]\]. Recently mitophagy has received increasing attention in stroke and has been evidenced in *in vivo* models and in cultured neurons. In ischemic stroke, mitophagy could be predominantly mediated by PINK1/Parkin pathway. However, the role of mitophagy in the development of ischemic brain injury remains controversial. Accumulating data have showed that mitophagy is a double-edged sword that can be protective or destructive after experimental stroke \[[@b69-ad-9-5-924]-[@b73-ad-9-5-924]\]. Most studies support the hypothesis that mitophagy favors neurons adapted to the stress by removing impaired mitochondria and suppressing cell death signaling cascades. These studies proposed that mitophagy is a promising therapeutic target for stroke treatment. In permanent MCAO, mitophagy was triggered at as early as 1 hour and involved in the removal of damaged mitochondria and cellular survival in a Drp1-dependent way \[[@b74-ad-9-5-924]\]. Mitophagy was also activated in the reperfusion phase after MCAO and contributed to the inhibition of post stroke apoptosis \[[@b69-ad-9-5-924]\]. Rapamycin treatment improved mitochondrial function through enhancing mitophagy after experimental ischemic stroke \[[@b75-ad-9-5-924]\]. The neuroprotective role of methylene blue in acute cerebral ischemia can also be explained by promoting mitophagy and maintaining the mitochondrial membrane potential, which consequently decreased necrosis \[[@b76-ad-9-5-924]\]. Melatonin-mediated mitophagy attenuated the early brain injury after subarachnoid hemorrhage through inhibition of NLRP3 inflammasome activation \[[@b77-ad-9-5-924]\]. Mitophagy also contributed to the neuroprotection induced by limb remote ischemic conditioning after ischemic stroke \[[@b73-ad-9-5-924]\]. The destructive role of mitophagy after cerebral ischemia has also been reported \[[@b70-ad-9-5-924], [@b71-ad-9-5-924]\]. Inhibition of mitophagy by decreasing p-Drp1 and Parkin mediated the neuroprotective effects of carnosine in rats after MCAO\[[@b70-ad-9-5-924]\], and excessive induction of mitophagy leads to cell death in neonatal stroke\[[@b71-ad-9-5-924]\]. The harmful roles of mitophagy in stroke have not been adequately emphasized. Further investigations are required to understand whether mitophagy is beneficial or detrimental after stroke. It appears that the effects of mitophagy depend on the severity of it. While physiological or mild levels of mitophagy favor the neuronal survival, intensive or excessive levels could be lethal and exacerbate the ischemic brain injury. 5. Intercellular Mitochondrial transfer ======================================= 5.1 Intercellular mitochondrial transfer and potential mechanisms ----------------------------------------------------------------- For a long time, mitochondria were considered to be retained intracellularly for their lifetime. Recently, intercellular exchange of membrane vesicles and organelles through structures such as tunneling nanotubes (TNT) or microvesicles has been evidenced \[[@b78-ad-9-5-924], [@b79-ad-9-5-924]\]. Mitochondrial transfer has been observed in several cardiovascular injury models, and has also been demonstrated in experimental stroke \[[@b11-ad-9-5-924], [@b80-ad-9-5-924], [@b81-ad-9-5-924]\]. In 2006 the horizontal transfer of mitochondria, from human mesenchymal stem cells (MSCs) to somatic cells *in vivo* was shown by Prockop's lab \[[@b82-ad-9-5-924]\]. Furthermore, it has been documented that intercellular mitochondrial transfer occurs in different cell types both *in vitro* and *in vivo* \[[@b11-ad-9-5-924], [@b80-ad-9-5-924], [@b81-ad-9-5-924], [@b83-ad-9-5-924]-[@b87-ad-9-5-924]\]. MSCs are indicated as the most popular donator for mitochondrial transfer. Many studies suggest that transfer of mitochondria from one cell to another is a protective mechanism accountable for rescue of injured cells from mitochondrial dysfunctions in response to stress \[[@b11-ad-9-5-924], [@b88-ad-9-5-924], [@b89-ad-9-5-924]\]. When co-cultured with mitochondria dysfunctional somatic cells, human MSCs developed cytoplasmic projections and steamed mitochondria to rescue the mitochondrial function of the somatic cells \[[@b82-ad-9-5-924]\]. Bone marrow derived stromal cells protected against acute lung injury by restoration of alveolar bioenergetics through Cx43-dependent alveolar attachment and mitochondrial transfer \[[@b85-ad-9-5-924]\]. In I/R injury, MSCs transferred mitochondria to injured endothelial cells through TNT-like structures, and inhibited endothelial cells from apoptosis by rescue of aerobic respiration \[[@b81-ad-9-5-924]\]. All these data corroborate that mitochondrial transfer undertakes metabolic cross-talk between healthy cells and injured cells. The released mitochondria can be taken and reprogrammed by recipients to activate signals for cell survival. Therefore, mitochondrial transfer is greatly expected to be novel therapeutic approaches for mitochondrial diseases or disorders, as well as for stroke. Figure 4.Intercellular mitochondrial transferMitochondria can be released by donate cells and uptake by recipient cells. Stressed or dying cells release mitochondria through tunneling nanotubes (TNTs) or microvesicles. Mitochondrial transfer can occur between same or different cell types. The mechanisms underlying mitochondria release and uptake by recipient cells are still unclear. Stressed cells or dying cells release mitochondria serving as danger warning signals. When stimulated with tumor necrosis factor alpha (TNF-α), mouse embryonic fibroblasts and hepatocytes extruded mitochondria through auto-lysosomal exocytosis \[[@b90-ad-9-5-924]\]. In human T-lymphoblastic leukemia cells and murine fibroblast cells, TNF-α induced necroptosis was accompanied by extracellular release of mitochondria \[[@b91-ad-9-5-924]\]. This is an active process, which occurs before the disruption of the plasma membrane. The mitochondria were intact and no mtDNA was emitted. Several molecular mechanisms have been proposed to regulate intercellular mitochondrial transfer. A study by Ahmad and his colleagues reported that Miro1 promoted mitochondrial transfer from MSCs to epithelial cells, which was well-directed rather than a general exchange of contents \[[@b92-ad-9-5-924]\]. In human acute myeloid leukemia, ROS drives TNTs formation and regulates the transfer of mitochondria from bone marrow stromal cells to leukemia blasts through NADPH oxidase-2 \[[@b93-ad-9-5-924]\]. Astrocytic release of extracellular mitochondria was mediated by a calcium-dependent mechanism involving CD38 and cyclic ADP ribose signaling in ischemic stroke mice, and integrin-mediated Src/Syk signaling might be involved in the endocytosis of mitochondria in neurons \[[@b11-ad-9-5-924]\]. Several other studies suggested that macrophage cells and immune cells recognized the released mitochondria by damage-associated molecular patterns \[[@b27-ad-9-5-924], [@b91-ad-9-5-924], [@b94-ad-9-5-924]\]. However, the health status and the fates of the transferred mitochondria before/after internalization were not assessed in these studies. Recently, using mitochondrial reporter mitoROGFP, which is able to detect oxidative stress, Melentijevic et al showed that stressed or damaged mitochondria were preferentially extruded in adult neurons from Caenorhabditis elegans \[[@b88-ad-9-5-924]\]. Human macrophages and dendritic cells engulfed released mitochondria and triggered the secretion of proinflammatory cytokines \[[@b91-ad-9-5-924]\]. In a co-culture system of MSCs with cardiomyocytes or endothelial cells subjected to oxidative challenge with hydrogen peroxide (H~2~O~2~), mitochondria from suffering cells were engulfed and degraded by MSCs, leading to induction of the cytoprotective enzyme heme oxygenase-1 and stimulation of mitochondrial biogenesis \[[@b95-ad-9-5-924]\]. Such studies have revealed that organelles can functions as a new aspect of transcellular signaling like moleculars, which opens an entirely new perspective on cell-to-cell communication ([Fig. 4](#F4-ad-9-5-924){ref-type="fig"}). Elucidation of the mechanisms of mitochondrial transfer will help to establish therapy-based mitochondrial restoration for human diseases caused by mitochondrial dysfunction. 5.2 Mitochondrial transfer techniques and its application in ischemic stroke ---------------------------------------------------------------------------- In 1982, Clark and Shay first transferred mitochondria with antibiotic-resistant genes to sensitive cells, which enabling the cells to survive in a selective medium \[[@b96-ad-9-5-924]\]. Their observations opened the new field of research on mitochondrial transfer. Various approaches have been developed to replenish the damaged mitochondria in recipient cells and suggest new possible applications. Coincubation is the first and simple approach tested and the efficiency differs in different cell lines \[[@b96-ad-9-5-924]\]. Microinjection and such other invasive techniques as nanoblades can quickly repopulate the resident mtDNA, but they are less efficient than coincubation for the limited cell numbers \[[@b97-ad-9-5-924], [@b98-ad-9-5-924]\]. Additional techniques has been developed to facilitate the mitochondria internalization into the recipient cells, for example, conjugating the mitochondria with cell-penetrating peptide Pep-1, labeling mitochondria with anti-TOM22 magnetic beads, and increase of mitochondrial uptake by MitoCeption technique \[[@b99-ad-9-5-924]-[@b101-ad-9-5-924]\]. In the *in vivo* studies, mitochondria were either directly injected into the ischemic heart or delivered intracoronarlly at the onset of reperfusion in rabbits\[[@b102-ad-9-5-924], [@b103-ad-9-5-924]\]. Both two methods effectively decreased the infarct size and restored the heart functions. In ischemic stroke, direct injection of astrocyte-derived mitochondria into the peri-infarct cortex 3 days after MCAO promoted neuron survival by increasing the level of phosphorylated AKT and BCL-XL \[[@b11-ad-9-5-924]\]. In neurons, mitochondria are concentrated within the presynaptic nerve terminal. Due to the necessity of long-distance transportation mitochondria can not be replenished quickly after ischemia. The transfer of extracellular mitochondria may, therefore, open a new avenue for neuroprotective strategies. The beneficial effects of mitochondrial transfer in stroke were first studied by Po-Jui Huang and his colleagues \[[@b104-ad-9-5-924]\]. Transferring exogenous mitochondria via in-situ injection or systemic administration of exogenous mitochondria mitigated cell death both in MCAO rats and OGD neurons and improved the motor performance of rats after MCAO \[[@b104-ad-9-5-924]\]. The grafted mitochondria were traced by pre-labeled with BrdU for their distribution and were internalized by neurons, astrocytes and microglia. Disrupting electron transport or ATPase synthase of the donor mitochondria significantly attenuated the protective effect. Their data supported that in-situ injected mitochondria can be taken by neurons, astrocytes and microglia in MCAO mice, and the intact respiratory activity is essential for the mitochondrial potency on neural protection \[[@b104-ad-9-5-924]\]. A followed-up study on mitochondrial transfer in stroke was performed in Eng H. Lo's group. Cultured astrocytes were observed to produce functional extracellular mitochondria, which are able to increase the ATP levels and viability of neurons suffered from OGD. Extracellular mitochondria injected in the peri-infarct cortex of MCAO mice were found in neurons. The transplantation of mitochondria resulted in an upregulation of cell-survival-related signals in MCAO mice\[[@b11-ad-9-5-924]\]. The authors suggested that astrocytes have the ability to transfer healthy mitochondria to rescue the damaged neurons after stroke. In their another study, extracellular mitochondria were detected in cerebrospinal fluid (CSF) with decreased mitochondrial membrane potentials in subarachnoid hemorrhage (SAH) rats \[[@b105-ad-9-5-924]\]. In clinic, the similar results were found in SAH patients; and higher membrane potentials of mitochondria in the CSF were correlated with good clinical recovery at 3 months after SAH onset \[[@b105-ad-9-5-924]\]. It is possible that after stroke, neuronal cells transfer mitochondria not only for disposal, but also for recycling and to quickly counteract the energy deficits caused by ischemia. 6. Conclusions and perspectives =============================== Mitochondrial disfunction is an early and initiating events in the pathophysiology of stroke \[[@b106-ad-9-5-924]\]. Defected bioenergenetics, abnormal mitochondrial morphology and structure, and aberrant mitochondrial dynamics play a central role in the activation of death signaling pathways \[[@b107-ad-9-5-924]\]. Interventions targeting mitochondrial quality control and mitochondrial dynamics by pharmacological agents or genetic modifications have been demonstrated neuroprotective in preclinical studies \[[@b49-ad-9-5-924], [@b108-ad-9-5-924], [@b109-ad-9-5-924]\]. Unfortunately, clinical trials using mitochondrial protectants or antioxidants, either alone or in combination with other therapeutic approaches, have been unsuccessful. Targeting mitochondria pharmacologically in the clinic is still challenging. Recently, the evidences of mitochondrial transfer open an entirely new perspective in the intercellular communication. Emerging findings indicate that mitochondria themselves can function as "help-me" signaling in response to diverse extracellular stimulus and recruit adjacent cells to rescue the injured cells. Removing the impaired mitochondria and replenishing healthy mitochondria are promising therapeutic approaches to treat hypoxia/ischemia related diseases, especially in CNS which mitochondria are abundance in the distal axonal synapses and dendritic protrusions \[[@b110-ad-9-5-924]-[@b112-ad-9-5-924]\]. Though clinical application of mitochondrial transplantation has been performed successfully in pediatric patients suffered from myocardial ischemia-reperfusion injury recently, challenge still remains to implement mitochondrial transfer in clinic widely and safely \[[@b113-ad-9-5-924]\]. Great efforts need to be devoted to investigating the detailed mechanisms on mitochondria release from donor cells and recognition by recipients, the generalizability of the beneficial results, and the ethical implications related to artificially mitochondria transferred. Addressing these hurdles underlying mitochondrial transfer will develop novel manipulation targets for the diseases related to mitochondrial dysfunction. This study was supported by the National Natural Science Foundation of China No. 81601013 to Junjia Tang and No. 81671130 to Qin Hu.

Introduction {#Sec1} ============ Uveitis is an infectious or non-infectious inflammatory disease of the uvea and other intraocular tissues including the retina, vitreous, and lens. With an incidence of more than 50/100,000-person years, uveitis is a common cause of legal blindness in the western world \[[@CR1]\]. In infectious uveitis, the etiology is specific, and the presentation may be characteristic of that infection, although not necessarily for syphilis, tuberculosis, and some AIDS-related opportunistic infections which may mimic many other forms of autoimmune non-infectious uveitis. However, the pathophysiology of non-infectious uveitis is not fully understood, and the etiology might be multifactorial---involving an interplay of genetic, immune, and environmental factors. Viral prodrome that is associated with the presentation of many non-infectious uveitis entities could provide a link connecting the two. Similarly, gut commensals provide a signal directly through the retina-specific T cell receptor and cause autoreactive T cells to trigger uveitis. Microbiota may also serve as an "adjuvant" providing innate signals that amplify and direct the host immune response for development of uveitis \[[@CR2]\]. Induction of biologic drugs in the treatment of non-infectious uveitis has introduced a new treatment modality in the field of uveitis. Basic research done in this field has paved way for newer molecules which offer a more targeted and specific therapeutic approach. Anti-TNF-α, considered the first-line biological agent recommended in the treatment of uveitis, has proven remarkably effective. It is by virtue of rapid inflammation control and increased tolerability due to minimal, specific, and manageable side-effects. Despite these benefits, most studies show that approximately 50% of patients with non-infectious uveitis treated with anti-TNF are unresponsive/intolerant \[[@CR3]\]. Both physicians and patients have increased expectations from therapeutic agents and end result is not just a good control of inflammation but achieve total clinical remission early in the disease course, so that minimal sequelae result. Non-responsiveness to TNF blockade and/or residual disease activity, as well as the ongoing, slowly progressing structural damage in a significant proportion of patients treated with TNF inhibitors suggest that---apart from TNF---there are other biological targets involved in the disease process. Interleukin 6 (IL-6) has emerged as a major player in the pathogenesis of autoimmune disease and chronic inflammation. Infection and inflammation cause significant upregulation of IL-6, which features pleiotropic activity and mediates various biological functions. It causes induction of acute-phase reactants in liver, T cell differentiation, regulates inflammatory cells, homeostasis, and healing after tissue injury \[[@CR4]\]. Various systemic autoimmune diseases and certain types of cancers have been associated with dysregulation in IL-6 production \[[@CR5]\]. In cases of diabetic macular edema, retinal vein occlusion, and chronic uveitis, IL-6 has been found to be significantly elevated in ocular fluids \[[@CR6], [@CR7]\]. In the past decades, tocilizumab, a monoclonal antibody (mAB) which targets IL-6 receptor (IL-6R), has been investigated and approved for therapeutic use in a number of immunologic diseases. Several clinical trials have reported it to be effective for the treatment of uveitis and associated macular edema \[[@CR8]--[@CR11]\]. IL-6 biology: mechanism of action {#Sec2} ================================= Human IL-6 is a 26 kDa protein made up of 212 amino acids codified by a gene located in chromosome 7p21 \[[@CR12]\]. IL-6 triggers signal transduction after binding the IL-6 receptor (IL-6R). There are two forms of IL-6R: the 80 kDa transmembrane receptor protein and the 55 kDa soluble form (sIL6-R). It is believed that the pleiotropic effect of IL-6 derives from the broad range of cells expressing gp130. IL-6 plays an important role in protecting the host against environmental insults and sends out warning signals through all systems of body about the occurrence of acute events. Physiological levels of IL-6 are very low (1--5 pg/ml) and hardly detectable in serum, but the levels can increase \> 100,000-fold during acute inflammatory response \[[@CR13]\]. Different cell types in the body produce IL-6, including cells of innate immune system such as neutrophils and monocytes/macrophages. IL-6 is important in the integrated host defense against numerous pathogens including bacteria, fungi, viruses, and mycobacteria \[[@CR14]\]. When these infectious pathogens stimulate Toll-like receptors (TLRs) by producing pathogen-associated molecular patterns (PAMPs), they result in prompt production of IL-6 by monocytes and macrophages. In non-infectious inflammation such as during burns and trauma, the affected cells produce damage-associated molecular patterns (DAMPs) which in turn stimulate TLRs, to produce IL-6 \[[@CR15]\]. Expression of inflammatory cytokines such as IL-6, TNF-α, and IL-1β is upregulated through various signaling pathways initiated by PAMPs and DAMPs. TNF-α and IL-1β in turn can activate transcription factors to synthesize IL-6 \[[@CR16]\]. IL-6 stimulates hepatocytes to induce synthesis of acute-phase reactants like C-reactive protein (CRP), fibrinogen, haptoglobin, alpha-1-antichymotrypsin, and serum amyloid A. CRP is a consistent biomarker of inflammation and is often employed to monitor various inflammatory processes. Its expression mainly depends on IL-6 \[[@CR17]\]. Persistent elevation of alpha-1-antichymotrypsin and serum amyloid A has been associated with the pathogenesis of Alzheimer's disease \[[@CR18]\]. Reduced serum iron levels have been reported secondary to IL-6-mediated hepcidin production; latter exerts antagonistic action on the iron transporter ferroportin-1 in intestinal epithelium. This IL-6-hepcidin interaction might be responsible for the anemia found in chronic inflammatory states \[[@CR19]\]. IL-6 also upregulates the expression of ZIP 14 (zinc importer), which results in low levels of zinc in chronic inflammation. IL-6 downregulates the expression of fibronectin, albumin, and transferrin. This change in acute-phase reactants is routinely used in clinical laboratory tests for detection of inflammation. In addition to its role in host defense, IL-6 mediates various biological functions. IL-6 induces activation of stem cells and helps megakaryocytes' maturation into platelets during hematopoiesis \[[@CR20]\]. In bone marrow, IL-6 upregulates the receptor activator of nuclear factor kappa-B ligand (RANKL), which in turn leads to bone resorption and osteoporosis by activating osteoclasts \[[@CR21]\]. Production of IL-6 in inflamed tissues upregulates vascular endothelial growth factor (VEGF), which results in increased angiogenesis and vascular permeability \[[@CR22]\]. IL-6 promotes proliferation of dermal keratinocytes, and collagen production by fibroblasts, which may contribute to the pathogenesis of diseases like psoriasis, systemic sclerosis, and thyroid eye disease \[[@CR23]\]. IL-6 receptors and signaling pathways {#Sec3} ===================================== Interleukin 6 plays an important role in host defense against environmental stress such as infection and injury. Dysregulated IL-6 production has been implicated in the development of various autoimmune diseases and chronic inflammatory diseases. IL-6 is a prototypical four-helix bundle cytokine that is a member of the neuropoietins, which includes IL-6, IL-11, IL-27, IL-31, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, neuropoietin, and neurotrophin-1 \[[@CR19]\]. These cytokines are structurally related and bind to Class I cytokine receptors. With the exception of IL-31, all IL-6 type cytokines share the membrane glycoprotein gp130 as a common beta receptor and signal transducer subunit \[[@CR24], [@CR25]\]. IL-6 signaling occurs through two cellular pathways: the classical and trans-pathway. In the classical signaling pathway, IL-6 binds to membrane-bound type I receptor complex consisting of the ligand-binding glycoprotein, IL-6α. The expression of this receptor is mostly restricted to leukocytes and hepatocytes. The IL-6/IL-6α complex subsequently associates with gp130 leading to gp130-homodimer formation \[[@CR26]\]. In trans-pathway, IL-6 provides signaling to cells lacking IL-6R via binding to soluble IL-6R (sIL-6R), which is generated by alternative splicing or ectodomain shedding of the membrane-bound IL-6 receptor (Fig. [1](#Fig1){ref-type="fig"}) \[[@CR27]\]. Both classical and trans-signaling pathways are gp130-mediated and activate the same intracellular pathways. Fig. 1IL-6 classic-signaling and IL-6 trans-signaling. IL-6 classic signaling requires membrane bound IL-6R and is restricted to hepatocytes, some epithelial cells and some leukocytes. IL-6 trans-signaling requires sIL-6R and is possible on all cells of the body since all cells express the gp130 protein. Adapted from "IL-6 trans-signaling via the soluble IL-6 receptor: Importance for the pro-inflammatory activities of IL-6." by Rose-John S, Int J Biol Sci 2012; 8:1237-1247 \[Copyright: Ivyspring International Publisher\] After the formation of gp130 homodimer, IL-6 initiates the intracellular signaling by activating the Janus kinase family tyrosine kinases (JAKs) \[[@CR28]\]. Activation of these kinases leads to phosphorylation and activation of signal transducers and activators of transcription 3 (STAT3) and the SH2-domain containing protein tyrosine phosphatase-2 (SHP2) \[[@CR29], [@CR30]\]. Phosphorylated STAT3 translocates to the nucleus and regulates transcription of various genes. SHP2 activates SOS/Ras-Raf-MEK-MAP kinase pathway to regulate genes \[[@CR31]\]. It is important to note that the activation of STAT3 in turn induces the suppressor of cytokine signaling 1 (SOCS1) and SOCS3, which bind tyrosine-phosphorylated JAK and gp130 respectively, to stop IL-6 signaling by means of a negative feedback loop \[[@CR32], [@CR33]\]. There is counter-regulation by a soluble form of gp130 (sgp130), present at high concentrations in serum of healthy individuals. As part of the physiological IL-6 buffer in the blood, this natural inhibitor forms a complex with IL-6/sIL-6R, preventing the binding of IL-6/sIL-6R to membrane-bound gp130. This ensures that IL-6/sIL-6R trans-signaling is tightly regulated \[[@CR34]\]. Various studies have shown that classic signaling via the membrane-bound receptor is regenerative and protects from bacterial infections, whereas trans-signaling via the soluble receptor is proinflammatory \[[@CR35]\]. Therefore, it may make sense to block only the IL-6 trans-signaling alone, which would maintain the regenerative function of IL-6 and specifically suppress only inflammatory arm mediating the disease process (Fig. [2](#Fig2){ref-type="fig"}) \[[@CR27]\]. Fig. 2Signaling, activation, and transduction pathways of IL-6. Adapted from "Hall of Fame among Pro-inflammatory Cytokines: Interleukin 6 Gene and Its Transcriptional Regulation Mechanisms." by Luo Y, Zheng SG. Immunol. 2016; 7:604. \[Copyright: © 2016 Luo and Zheng\] IL-6 and autoimmunity {#Sec4} --------------------- As mentioned earlier, IL-6 is a pleiotropic cytokine that plays important roles in hematopoiesis, immune defense, and oncogenesis \[[@CR36]\]. Historically, IL-6 molecule had been studied under many different names such as B cell stimulatory factor-2 (BSF-2), IFN-β2, Hybridoma/plasmacytoma growth factor, hepatocyte-stimulating factor (HSF), until advancements in molecular testing. In the following sections, we will discuss the role of IL-6 in immune cascades and defense mechanisms, pathological significance of IL-6 signaling in inflammatory autoimmune systemic and ocular diseases, as well as therapeutic implications of IL-6 targeted therapy. Immunomodulatory role of IL-6 {#Sec5} ----------------------------- ### B cells {#Sec6} IL-6 was originally identified and named as B cell stimulatory factor 2 because it promotes the differentiation of activated B cells into plasma cells which are responsible for antibody production \[[@CR36]\]. Interactions between T and B cells during antibody production were first reported in 1968 and it was theorized that certain molecules were released from T cells which stimulate B cells to produce antibodies \[[@CR37]\]. B cells can produce antibodies, but not without having T cells producing those growth and differentiation factors. IL-6 produced by plasmacytoid dendritic cells is critical for this process \[[@CR38]\]. IL-6 also promotes T follicular helper cell differentiation as well as production of IL-21, which also promotes B cell differentiation and increase immunoglobulin synthesis \[[@CR39], [@CR40]\]. Moreover, IL-6 may promote the survival of the plasma blasts that secrete immunoglobulin or pathological autoantibodies, e.g., anti-aquaporin 4 in patients with neuromyelitis optica (NMO) \[[@CR41]\]. IL-6 may act as an autocrine growth factor in some types of multiple myelomas while some others are themselves able to produce IL-6 \[[@CR42]\]. ### T cells {#Sec7} As mentioned, IL-6 was first identified as a B cell function and differentiation factor; however, T cell differentiation and activation is another major action of IL-6 \[[@CR36]\]. IL-6 signaling has been found to control proliferation of resting T cells and reinforcing their resistance against apoptosis by inducing IL-2 production and STAT-3 activation \[[@CR43]\]. IL-6 has also been identified as major regulator between regulatory T cells (Treg) and effector Th17 cells. In combination with transforming growth factor (TGF)-β, IL-6 brings about differentiation into Th17 cells, but inhibits TGF-β-induced Treg development \[[@CR44], [@CR45]\]. This results in an increase of Th17 cell population over Treg cells which may have a role in altered immunological tolerance and resulting in the development of autoimmune inflammatory diseases \[[@CR46]\]. IL-6 also modulates Th1 and Th2 balance. It enhances the production of Th2 cells by promoting IL-4 and IL-13 production \[[@CR47]\]. On the other hand, it inhibits Th1 cell differentiation and interferon-gamma (IFN-γ) production \[[@CR48]\]. IL-6 and acute-phase response {#Sec8} ----------------------------- IL-6 is a major cytokine in the initiation process of acute-phase responses \[[@CR36]\]. In the serum of healthy individuals, the IL-6 level is less than 5 pg/ml; however, IL-6 concentration increases dramatically during infectious and non-infectious events \[[@CR49]\]. IL-6 acts as an important factor in the synthesis of acute-phase proteins by the liver, such as C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen, hepcidin, and α1-antichymotrypsin \[[@CR50]\]. Administration of IL-6 inhibitors completely normalizes the serum levels of CRP and SAA, indicating that their synthesis depends primarily on IL-6 \[[@CR51]\]. These major acute-phase reactants act as an inducer of systemic inflammatory and infectious response. Elevated levels of CRP have been reported in serum of patients with various autoinflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, among others, as well as bacterial and viral infections \[[@CR52]\]. When the emergent stimuli are completely removed from the host, the IL-6-mediated signal transduction cascade is terminated, leading to normalization of the CRP level in serum (Fig. [3](#Fig3){ref-type="fig"}) \[[@CR53]\]. Fig. 3Pro- and anti-inflammatory activities of IL-6. Anti-inflammatory activities of IL-6 include STAT3-dependent regeneration of epithelial cells and the induction of the hepatic acute-phase response. These activities are dependent on the membrane-bound IL-6R. Pro-inflammatory activities of IL-6 include recruitment of inflammatory cells, inhibition of apoptosis of inflammatory cells, and inhibition of regulatory T cell differentiation. Adapted from "Hall of Fame among Pro-inflammatory Cytokines: Interleukin-6 Gene and Its Transcriptional Regulation Mechanisms." by Luo Y, Zheng SG. Immunol. 2016; 7:604. \[Copyright: Ivyspring International Publisher\] IL-6 regulation {#Sec9} --------------- Due to the rapid plasma clearance, IL-6 levels are largely regulated at the transcriptional and post-transcriptional gene expression level \[[@CR49]\]. The molecular aspects of IL-6 regulation comprise of complex interactions between proteins, miRNAs, and IL-6 gene expression, which is beyond the scope of this review. IL-6 in various systemic autoimmune diseases {#Sec10} -------------------------------------------- IL-6 was first associated with disease development in a case of cardiac myxoma, a benign heart tumor \[[@CR54]\]. Subsequently, excessive IL-6 expression patterns were detected in several other autoimmune inflammatory diseases including chronic rheumatoid arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, Adamantiades-Behcet's disease, and systemic sclerosis \[[@CR30], [@CR50], [@CR55], [@CR56]\]. It is also known that the extent of elevation of serum IL-6 level depends on the type and severity of the disease \[[@CR57]\]. On this basis, IL-6 inhibition strategy is currently being pursued to develop novel therapies for inflammatory pathologic conditions. The following sections will focus on IL-6 inhibitors and their usage in systemic and ocular inflammatory diseases. Experimental autoimmune uveitis---animal model {#Sec11} ---------------------------------------------- Several experimental autoimmune uveitis (EAU) studies demonstrated the importance of IL-6 in non-infectious uveitis (NIU). In an animal model of T cell-mediated uveitis (interphotoreceptor retinoid binding protein (IRBP) immunization model), Yoshimura et al. showed that IL-6-deficient mice could not induce Th17 cells and the EAU score was found to be decreased in those mice in the entire time course \[[@CR58]\]. On the other hand, systemic administration of anti-IL-6 receptor antibody reduced uveitic inflammation. This effect in EAU appears to occur via the suppression of both Th1 and Th17 differentiation, both of which are important in this animal model of uveitis. Haruta et al. induced EAU in wild-type (WT) mice and in mice lacking IL-6, IL-17, and IFN-γ and also in IL-6-lacking mice treated with anti-CD 25 monoclonal antibody (mAb) to deplete Treg cells \[[@CR59]\]. IL-6 deficiency resulted in the inhibition of the antigen-specific Th1 response and enhanced the generation of antigen-specific Treg cells. Authors concluded that blockade of IL-6 signaling suppresses not only Th17 but also IRBP-specific Th1 by promoting regulatory T cells in EAU. IL-6 in ocular pathologies and non-infectious uveitis {#Sec12} ----------------------------------------------------- IL-6 is a key cytokine which is strongly upregulated during infection/inflammation and associated with variety of systemic autoimmune diseases. Elevated levels of IL-6 have been detected in many ocular diseases such as glaucoma, central vein occlusion, dry eye disease, chemical burn injuries, corneal infections, allergic eye diseases, and ocular inflammatory diseases \[[@CR56], [@CR60]\]. Murray et al. was the first to demonstrate elevated aqueous humor levels of IL-6 in 24 human subjects with uveitis, including, Fuchs' heterochromic iridocyclitis and toxoplasma uveitis \[[@CR61]\]. Several groups also found IL-6 to be elevated in ocular fluids including vitreous in patients with active or chronic NIU supporting its mainstream role in ocular inflammatory process \[[@CR7], [@CR62], [@CR63]\]. All these evidences, since the first report on the discovery of IL-6 in 1973 by Kishimoto and Ishizaka, have led to a pursuit of new IL-6 inhibitory drugs, for the management of pathologic inflammatory conditions including several types of NIU \[[@CR64]\]. Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of macular edema in central retinal vein occlusion (CRVO) and diabetic retinopathy by enhancing vascular permeability and altering retinal endothelial tight junctions. In the presence of hypoxia, IL-6 plays a major role in VEGF induction. Vitreous levels of IL-6 and VEGF correlate with the severity of ischemia and are found to be significantly elevated in patients with ischemic CRVO and correlated with the severity of disease \[[@CR65], [@CR66]\]. Elevated levels of IL-6 with other cytokines have been shown in proliferative diabetic retinopathy (PDR) wherein the progression may lead to tractional or combined mechanism retinal detachment \[[@CR67]\]. IL-6 has also been implicated in the pathogenesis of proliferative vitreo-retinopathy (PVR) after rhegmatogenous retinal detachment (RRD). IL-6 stimulated the production of matrix metalloproteinases (MMPs) which play a major role in the development of PVR \[[@CR68]\]. IL-6 has also been found to have a role in various other ocular diseases such as allergic conjunctivitis and dry eye disease, infectious keratitis, ocular neovascularization, and posterior capsular opacification \[[@CR69]--[@CR73]\]. ### Non-specific anti-IL-6 drugs {#Sec13} #### Corticosteroids {#FPar1} The mechanism of action of corticosteroids on IL-6 inhibition is not fully understood; however, they are known to inhibit IL-6 production at the transcriptional level \[[@CR57], [@CR74]\]. This process may involve suppression of gene upregulation by nuclear factor-κβ (NF-κβ) or by the occlusion of promotor elements in the IL-6 promotor \[[@CR5]\]. Although corticosteroids are the mainstay treatment in many types of inflammatory diseases and IL-6 inhibition is one of their pleiotropic mechanisms of action, their dose-dependent side effects limit long-term therapeutic usage. These effects are mediated by its specific inhibition of IkB kinase-b, which prevents activation of nuclear factor -kB (NF-kB) #### Tetracyclines {#FPar2} Tetracyclines are broad-spectrum antibiotics that can act against a wide range of microorganisms via inhibition of protein synthesis \[[@CR75]\]. The immunomodulatory and anti-inflammatory properties of tetracyclines suggested that this drug might be effective in the treatment of autoimmune disorders \[[@CR76]\]. Wide spectrum anti-inflammatory effects of these drugs are thought to be partially due to suppression of IL-6 by the blockage of NF-κβ signaling \[[@CR77]\]. ### Targeted biological anti-IL-6 drugs {#Sec14} #### Tocilizumab {#FPar3} Tocilizumab (Actemra®, Roche AG, Basel, Switzerland) is a humanized mouse monoclonal antibody inhibitor of IL-6 receptor. Tocilizumab (TCZ) prevents the binding of IL-6 with its membrane and soluble receptors and antagonizes its action \[[@CR4], [@CR6]\]. It is currently approved for the treatment of rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), and giant cell arteritis (GCA) by the FDA \[[@CR37], [@CR78]\]. Apart from these, many retrospective open-label studies have shown efficacy of TCZ in inflammatory and/or autoimmune diseases refractory to conventional therapy and/or other biologics which included series of other large-vessel vasculitis (Takayasu's arteritis), Adamantiades-Behçet's disease (ABD), adult onset Still's disease, multicentric Castleman disease (approved in Japan), relapsing polychondritis, Cogan's disease, inflammatory myositis, and lupus \[[@CR79]\]. Large randomized controlled trials (RCTs) on TCZ therapy for RA paved way for its usage in other systemic and ocular inflammatory conditions, since it provided valuable information on the efficacy and side effect profile of the drug. TCZ demonstrated therapeutic potential in moderate to severe active RA patients with inadequate response to methotrexate (MTX), or other conventional disease-modifying antirheumatic drugs (cDMARDs) or biological disease-modifying antirheumatic drugs (bDMARDs) like TNF antagonists \[[@CR36], [@CR37], [@CR49], [@CR50]\]. Also, TCZ was found to be better than MTX monotherapy, with rapid improvement in signs and symptoms in patients with active RA, for whom previous treatment with MTX/biological agents had not failed \[[@CR52]\]. On the other hand, two phase 3 studies showed that immediate initiation of TCZ with or without MTX in early progressive RA was also more effective and associated with sustained remission and low disease activity, but with a similar safety profile compared with initiation of MTX alone \[[@CR80], [@CR81]\]. The results of the phase 4 ADACTA study comparing the efficacy of TCZ 8 mg/kg monotherapy versus anti-TNF-α agent adalimumab monotherapy were also in favor of TCZ in terms of reduction of signs and symptoms of RA, in patients for whom MTX was deemed inappropriate \[[@CR82]\]. Successful use of tocilizumab with an acceptable safety profile has been reported in patients with NIU who did not respond to several other treatments. In 2011, Muselier et al. reported the first two cases of refractory uveitis (birdshot chorioretinopathy (BSC) and idiopathic panuveitis) treated by tocilizumab with favorable results and acceptable short-term safety profile \[[@CR83]\]. After that report, several case series have demonstrated efficacy of tocilizumab in the treatment of uveitis refractory to anti-TNF agents \[[@CR8], [@CR84], [@CR85]\]. Papo et al. in their study with eight consecutive unselected patients with severe and refractory non-infectious uveitis including BD, BSC, and idiopathic cases concluded that tocilizumab was safe and promising \[[@CR86]\]. After 8 months of median follow-up, six out of eight patients improved in terms of inflammation control. Mesquida et al. reported on the long-term efficacy and safety of tocilizumab for refractory uveitis-associated macular edema \[[@CR10]\]. All their patients were refractory to cDMARD and at least one bDMARD prior to initiation of tocilizumab. Uveitis diagnoses were BSC (*n* = 3), JIA-associated uveitis (*n* = 3), and idiopathic panuveitis (*n* = 1). After a 15-month follow up, no serious adverse events were observed. Mean central foveal thickness improved significantly from 550 to 274 μm at month 12. Visual acuity also significantly improved from 0.67 to 0.4 at month 12. The same group published 24 months results of quiescent uveitis patients with recalcitrant uveitic macular edema (ME), treated with TCZ \[[@CR9]\]. Diagnoses included patients with BSC, JIA-associated uveitis, idiopathic panuveitis, sympathetic ophthalmia, and ankylosing spondylitis. Sustained inflammatory remission was maintained in all 12 patients. However, an attempt to withdraw TCZ could only be made in five of them because of systemic disease and perceived high risk of visual loss. All five patients in whom TCZ therapy was withdrawn, ME relapsed within 1 to 3 months after cessation. A re-challenge with TCZ infusions in those patients induced recovery. In the study, tocilizumab was generally well-tolerated except one case of dose-dependent neutropenia and another case of pneumonia \[[@CR9]\]. In addition to these encouraging results in small case series and retrospective studies with relatively small number of patients, the first prospective randomized clinical trial STOP-Uveitis was conducted to assess the safety and efficacy of tocilizumab in NIU \[[@CR11]\]. STOP-Uveitis was a 6-month study of 37 patients treated with one of two intravenous doses (either 4 or 8 mg per kg) of tocilizumab for posterior NIU. The majority of the cases were of idiopathic origin (28/37 patients) but subjects who had uveitis secondary to Vogt-Koyanagi-Harada syndrome, sarcoidosis, punctate inner choroiditis, and ABD were also included. Only 20--25% of the study population had history of immunomodulatory therapy usage. Two patients developed low absolute neutrophil counts (ANC) after receiving the first infusion of tocilizumab; one normalized before the second infusion while the other subject exited the study. No ocular adverse events related to the study drug were observed. The STOP-Uveitis study demonstrated that the therapy was well-tolerated and associated with a reduction in vitreous haze and cystoid macular edema at both doses (Fig. [4](#Fig4){ref-type="fig"}) \[[@CR11]\]. Fig. 4A case of an 18-year-old male subject with idiopathic panuveitis and macular edema who was a subject in the STOP-Uveitis Study, demonstrating changes in vitreous haze and central retinal thickness after treatment with intravenous infusions of 4 mg/kg tocilizumab. The subject was not on any other therapy while he was being treated with tocilizumab. \[Adapted from Sepah YJ, Sadiq MA, Chu DS, et al. Primary (month-6) outcomes of the STOP-uveitis study: Evaluating the safety, tolerability, and efficacy of tocilizumab in patients with non-infectious uveitis. *Am J Ophthalmology* 2017; 183:71-80.\] #### Tocilizumab: dose and route of administration {#FPar4} The approved starting dose of TCZ differs in various regions of the world. TCZ also has two approved systemic modes of administration: intravenous (IV) and subcutaneous (SC). In the USA, induction therapy employs 4 mg/kg monthly IV regimen followed by step up to 8 mg/kg monthly based on therapeutic response. However, in Europe, the approved initiating and maintenance dose is 8 mg/kg IV monthly, titrated to 4 mg/kg if side effects occur. SC route of administration has important advantages such as providing more convenient route of administration, including self-application at home, negating the requirement for and associated health care costs of intravenous access, and frequent clinic visits. The efficacy of the SC route over IV route was evaluated in several studies. Two randomized, double-masked, 24-week comparative, phase 3 studies demonstrated the non-inferiority of TCZ-SC to TCZ-IV: SUMMACTA in Europe and MUSASHI in Japan \[[@CR87]--[@CR89]\]. SUMMACTA compared TCZ-SC 162 mg every week to TCZ-IV 8 mg/kg every 4 weeks, and MUSASHI compared TCZ-SC 162 mg every 2 weeks or TCZ-IV 8 mg/kg every 4 weeks, showed safety and non-inferior efficacy of TCZ-SC. BREVACTA, another randomized, double-masked, 24-week comparative trial in the USA, evaluated 162 mg TCZ-SC every 2 weeks versus placebo \[[@CR88]\]. BREVACTA showed that TCZ-SC was superior to placebo. These studies led to the approval of the subcutaneous formulation and the dosing recommendations in the European Union and the USA. Consistent with the respective intravenous labels, in the EU, the approved starting dose for TCZ-SC is 162 mg every week, with a possible decrease in dosage frequency to every 2 weeks. In the USA, the starting dose for TCZ-SC is 162 mg every 2 weeks, with a possible increase in dosing to every week, based on clinical response approved \[[@CR90]\]. In summary, TCZ-SC appears to be as effective as TCZ-IV with comparable safety. #### Tocilizumab: safety and pre-administration evaluation {#FPar5} The safety of TCZ in patients with RA has been evaluated in several phase 3 and 4 RCTs \[[@CR37], [@CR50], [@CR53], [@CR80], [@CR82], [@CR87], [@CR88]\]. Infections such as nasopharyngitis, upper respiratory tract infections, pneumonia, and cellulitis were the most common. Other major adverse events were gastrointestinal perforations (GIPs), neutropenia, and malignancies. Laboratory test abnormalities were also reported with TCZ therapy, including elevated liver enzyme levels, decreased neutrophil counts, and change in lipid levels. The real-world studies, in line with the data from RCTs, also found higher incidence of GIPs in patients treated with TCZ than in those treated with other biologics or cDMARDs \[[@CR91], [@CR92]\]. For every 1000 patients treated with TCZ per year, between one and two additional GIPs might be expected to occur compared with those treated with other TNF inhibitors \[[@CR93]\]. In particular, the risk for lower-intestinal perforations (LIPs) seems to be higher in patients with a history of diverticulitis \[[@CR91], [@CR92]\]. Malignancy and neutropenia are two additional concerns for patients receiving TCZ. The risk for malignancy is potentially greater in patients with RA who use immunosuppressive agents. The current hypothesis is that inflammatory activity associated with RA drives the increased lymphoma risk. Additionally, some evidence suggests that patients with RA treated with biologics are at increased risk for malignancy, specifically non-melanoma skin cancers, compared with the general population \[[@CR94]\]. Despite these inferences, the analysis of the data from phase 3 trials and long-term extension studies did not demonstrate increased risk for overall or site-specific malignancies above the risk expected in patients with RA. The overall malignancy rate observed in tocilizumab, all-exposure population was higher than that in the general population but was consistent with that expected in the RA population and in the geographic regions studied \[[@CR16]\]. Data for neutropenia incidence with TCZ are divergent. De Benedetti et al. reported neutropenia incidences as high as 57% in a cohort of 112 patients with JIA \[[@CR95]\]. A dose-dependent effect on neutropenia-onset was observed in RCTs, where the incidence of neutropenia at 8 mg/kg was almost twice as observed at 4 mg/kg \[[@CR37], [@CR49]\]. In a retrospective cohort study, TCZ was associated with a higher incidence of neutropenia compared with abatacept and infliximab. However, the increased incidence of neutropenia did not result in a higher risk for severe infections \[[@CR96]\]. Also, data from these trials confirm that grade 4 neutropenia (\< 0.5 ANC × 109/l) is extremely uncommon. Adverse events reported in RCTs with TCZ in pediatric age group, treated for JIA, were similar to the adult group and included infections, neutropenia, and abnormalities in liver function test results \[[@CR95], [@CR97]\]. Notably, no or very few cases of macrophage activation syndrome (MAS), which resolved, were reported in those studies. On the other hand, a patient registry post-marketing surveillance (PMS) in Japan, which was conducted to investigate the safety and effectiveness of TCZ in real-world clinical settings, reported slightly higher adverse event rates than those reported in clinical trials of TCZ for systemic JIA \[[@CR98]\]. This dissimilarity was attributed to the fact that RCTs had excluded patients with concurrent medical or surgical conditions; or concomitant diseases of the nervous, renal, endocrine, or hepatic systems. All this data from RCTs and cohort studies highlights the need for careful patient selection when treating with anti-IL-6 agents (i.e., exclusion of individuals with previous diverticulitis, exclusion of infections such as tuberculosis, fungal, etc.). Patients should be closely monitored for the development of signs and symptoms of infection during and after treatment. In order to ensure safe use of TCZ in daily practice, physicians and patients should also be aware that, under TCZ, conditions such as GIP, MAS (in pediatrics) may occur with mild symptoms only and careful examination and testing are crucial. ### Newer IL-6 inhibitors {#Sec15} TCZ was launched as the first biologic drug targeting IL-6 in 2010, which provided a strong alternative to anti-TNF-α agents. Clinical success of TCZ was encouraging, which led pharmaceutical industry to undertake research in discovering other IL-6 blocking pathways. Currently, several IL-6/IL-6R inhibitors are under investigation in various phases of different studies (Table [1](#Tab1){ref-type="table"}). These molecules can be broadly divided into two categories: Table 1Features of various IL-6 inhibitors---available and investigational, \[in RA\]TargetStructureDose and route of administrationCurrent indications (FDA approved)Potential indicationsTocilizumabsIL-6R mIL-6RRecombinant humanized4--8 mg/kg IV q4wk162 mg SC q2wk/q4wkRA, JIA, GCATakayasu's arteritis, Behçet's disease, adult onset Still's disease, multicentric Castleman's disease (approved in Japan), relapsing polychondritis, Cogan's disease, inflammatory myositis, lupus, NIUSarilumabsIL-6R mIL-6RHuman mAb200 mg SC q2wkRARA, NIUSirukumabIL-6Human mAbFDA approval denied for RAn/a--SiltuximabIL-6Chimeric12 mg/kgq3wkCastleman's diseaseMultiple myeloma, currently no studies in NIUClazakizumabIL-6Humanized mAb25--200 mg SC q4wkNot FDA approvedRenal transplant, psoriatic arthritis, RAOlokizumabIL-6Humanized mAb64 mg SC q2wk/q4wkNot FDA approvedRAALX-0061sIL-6R mIL-6RNanobody (heavy chain-only)n/aNot FDA approvedSLE, RAMEDI 5117IL-6Human mAbn/aNot FDA approved--*RA* rheumatoid arthritis, *JIA* juvenile idiopathic arthritis, *GCA* giant cell arteritis, *NIU* non-infectious uveitis, *SC* subcutaneous, *IV* intravenous, *n*/*a* not available, *qnwk* once every "*n*" number of weeks Targeting IL-6 with Sirukumab, Siltuximab, Olokizumab, Clazakizumab, and EBI-031 Targeting IL-6R with Sarilumab and ALX-0061 {#Sec16} =========================================== Sarilumab {#FPar6} --------- Sarilumab (Kevzara®, Sanofi Genzyme, Regeneron Pharmaceuticals, USA) is a fully human anti-IL-6Rα mAb that blocks both classic and trans-signaling pathway by binding to membrane-bound as well as soluble forms of IL-6Rα, with higher affinity as compared to tocilizumab. Sarilumab received its USFDA approval in May 2017 for treatment of RA. It is indicated for the treatment of rheumatoid arthritis and is undergoing clinical trials for use in the management of posterior segment non-infectious uveitis. In the SATURN study, in patients with non-infectious uveitis, reductions in vitreous haze and steroid dosing were seen in treated patients. Visual acuity and central macular thickness also improved with a 200 mg dose administered subcutaneously every 2 weeks. This drug targets a specific inflammatory mediator and has been associated with fewer side effects than other available therapies. Neutropenia and elevated alanine amino-transferase levels were reported as adverse events \[[@CR99]\]. Sirukumab {#FPar7} --------- Sirukumab (Plivensia®, Janssen Biologics, Horsham, PA GlaxoSmithKline, UK) is a fully human mAB that binds IL-6. US Food and Drug Administration's Arthritis Advisory Committee did not recommend sirukumab for the treatment of adults with active rheumatoid arthritis (RA). While the committee unanimously agreed that there is substantial evidence of efficacy of sirukumab for the treatment of these patients, the safety profile is not adequate to support its approval. In phase 3 SIRROUND-D study, the primary adverse events were related to immunosuppression, consistent with those found with other DMARDS; however, there was a trend of increased overall mortality with sirukumab compared with placebo \[[@CR100]\]. Out of a total 35 deaths in study patients, 34 occurred in those taking sirukumab. Prominent causes of death were major cardiac events, infection, and malignancy. Increased risk for serious infection was associated with sirukumab; opportunistic infection and tuberculosis were both reported with its use. Siltuximab {#FPar8} ---------- Siltuximab (Sylvant®) is a chimeric (human-murine) mAB that targets IL-6. It is approved for the treatment of patients with multicentric Castleman's disease by FDA. It can neutralize the IL-6 effect in a number of human malignancies, reducing cancer-related cachexia and anorexia. In a phase I, open-label study, no dose-related or cumulative toxicity was apparent across all disease indications. A dose of 12 mg/kg every 3 weeks was recommended on the basis of the high response rates in Castleman's disease and the sustained CRP suppression. Most common drug-related adverse event was thrombocytopenia (25%). Randomized studies are ongoing in Castleman's disease and multiple myeloma \[[@CR101]\]. There are no studies at present to indicate its use in NIU. Olokizumab {#FPar9} ---------- Olokizumab (R-Pharma, UCB) is a humanized mAB that acts on site-3 of IL-6 and prevents binding of IL-6 to its signaling co-receptor gp130, thereby blocking the assembly of IL-6 signaling complex. It was found effective in a 12-week phase 2b study in RA patients who were refractory to TNF inhibitors. OKZ treatment, at several doses, demonstrated similar efficacy to TCZ across multiple endpoints. Most adverse events were mild or moderate and comparable between OKZ and TCZ treatment groups. OKZ produced significantly greater reductions in DAS28 (CRP) from baseline at week 12 compared with placebo. Reported adverse events were consistent with the safety profile expected of this class of drug without newer safety concerns \[[@CR102]\]. No studies at present suggest the use of olokizumab in NIU. Clazakizumab {#FPar10} ------------ Clazakizumab (Vitaeris®, ALD518, Bothell, USA) is also a humanized anti-IL-6 agent. It showed greater affinity and prolonged half-life in comparison to olokizumab. Clazakizumab proved clinical efficacy by achieving its primary endpoint in treating RA patients, refractory to MTX; however, dose-response effect was lacking. Patients achieved significant improvements in disease activity, including higher rates of remission, as compared with patients receiving only MTX. Clazakizumab treatment groups showed a higher rate of serious adverse events (range 8.3 to 13.6%), compared with 3.3% in MTX group. Pharmacological effects of IL-6 blockade showed consistency with levels of IL-6 measured in laboratory \[[@CR103]\]. Clazakizumab also has not been evaluated in the management of NIU. Conclusion {#Sec17} ========== Future of IL-6 inhibition {#Sec18} ------------------------- Various studies in the literature show association of inflammatory disorders like uveitis, retinal vascular occlusions, and diabetic macular edema with increased levels of IL-6. Such data provides a clue to possible area of research and clinical trials targeting these diseases using IL-6 inhibitors. In a pre-clinical study, EAU was treated using intraocular IL-6 locally; this points toward the development of intravitreal IL-6 therapy which can help to avoid associated systemic side-effects. IL-6 provides an alternative to TNF-α inhibition in the management of NIU. Tocilizumab and secukinumab have shown promising results over established biologics. Various other IL-6 inhibitors are under study and could provide similar therapeutic potentials with added advantages. It has been recognized that the levels of IL-6 were raised in other ocular vascular diseases such as retinal vein occlusion and diabetic macular edema. There are also cases of NIU with macular edema that responded well to tocilizumab therapy but returned after withdrawal of therapy, only to resolve again when treatment was restarted. Further studies are needed to understand the role of IL-6 inhibitors in non-uveitic macular edema, where anti-VEGF therapy remains the standard of care. Subcutaneous administration has been evaluated in various studies and has been found non-inferior to IV route. Thus, SC administration can further decrease infusion-related inconvenience and expenditure. Once the newer molecules of IL-6 inhibitors, which target only the trans-cellular pathway, become more established, as we gain experience in their use, complications of IL-6 inhibition such as sepsis can be minimized. Perspective {#Sec19} ----------- In the current era of pharmacotherapy for uveitis, we can theoretically target TNF, IL-1, IL-12/IL-23, IL-6, IL-17, CTLA4, CD20, etc.---various cytokines that have been identified in the serum and eyes of patients with different types of uveitis. There may be several agents that aim at particular cytokines. As clinicians and scientists gain deeper understanding of the pathophysiology of uveitis, we may be able to target individual cytokines for each disease in each patient, moving toward the goal of precision medicine. As part of future therapeutic plans, patients may undergo biomarker profiling, to be performed on serum and ocular fluids for example, *prior to* the initiation of any therapeutic plans. Based on specific biomarker(s) identified for specific patients, we can employ monotherapy (i.e., IL-6 inhibitor) *or* combination therapy (i.e., IL-1 and IL-6 antagonists or anti-TNF and anti-IL-17 pharmacologic agents) to manage the ocular diseases, being vigilant not to acquire excessive immunosuppression. In summary, increasing number of studies are showing that IL-6 inhibitors can be very effective biologic agents in the management of NIU. Moreover, additional role of IL-6 inhibition may be realized in the future, possibly opening a newer approach to the management of retinal vascular diseases and non-uveitic macular edema. IL-6 : Interleukin 6 IV : Intravenous SC : Subcutaneous NIU : Non-infectious uveitis RVO : Retinal vein occlusion DME : Diabetic macular edema VEGF : Vascular endothelial growth factor TNF : Tumor necrosis factor mAB : Monoclonal antibody TLR : Toll-like receptors PAMPs : Pathogen-associated molecular patterns DAMPs : Damage-associated molecular patterns CRP : C-reactive protein ZIP : Zinc importer RANKL : Receptor activator of nuclear factor kappa-B ligand JAK : Janus kinase STAT3 : Signal transducers and activators of transcription 3 NMO : Neuromyelitis optica TGF : Transforming growth factor IFN-y : Interferon gamma EAU : Experimental autoimmune uveitis PG : Prostaglandin TCZ : Tocilizumab OKZ : Olokizumab RA : Rheumatoid arthritis SLE : Systemic lupus erythematosus GCA : Giant cell arteritis JIA : Juvenile idiopathic arthritis MTX : Methotrexate BSCR : Birdshot chorioretinopathy BD : Behcet's disease cDMARDs : Conventional disease-modifying antirheumatic drugs bDMARDs : Biological disease-modifying antirheumatic drugs MAS : Macrophage activation syndrome USFDA : United States Food and Drug Administration GIP : Gastrointestinal perforation LIP : Lower intestinal perforation Treg : T regulatory cells pg : Picogram **Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Not applicable. Availability of data and material {#FPar11} ================================= Not applicable. SK, MH3, EV, SH, MH2, CP, NN, RA, AT, DV, YJS, and QDN conceptualized the manuscript. SK, MH3, and EV designed the manuscript. All authors read and approved the final manuscript. Authors' information {#FPar12} ==================== QDN is a recipient of a Physician Scientist Award from Research to Prevent Blindness, New York, NY, and serves on the Scientific Advisory Board for AbbVie, Bayer, Genentech, Regeneron, and Santen, among others. QDN also chaired the Steering Committee for the RISE and RIDE studies and was on the Steering Committee for the VISTA Study, and other studies sponsored by Genentech and Regeneron. None. Not applicable. Not applicable. The authors declare that they have no competing interests.

Exotropia in healthy children younger than 1 year of age is rare, and several studies have been conducted on this disease entity \[[@B1][@B2][@B3][@B4][@B5][@B6][@B7]\]. Occlusion therapy alone is not sufficiently effective in reducing exodeviation, and surgery is the most promising treatment for early-onset exotropia as well as intermittent exotropia \[[@B8]\]. Although non-surgical treatments, including occlusion therapy, have limitations as a single treatment for intermittent exotropia \[[@B9]\], surgery in combination with preoperative occlusion therapy has been found to more effective in reducing exodeviation and has shown a superior success rate compared to that of surgery alone \[[@B10]\]. Occlusion therapy is frequently recommended for patients with exotropia to prevent amblyopia or/and suppression \[[@B11]\]. However, no studies have investigated whether preoperative occlusion therapy is effective in reducing recurrence after surgery for early-onset exotropia. Therefore, we investigated the effect of preoperative part-time occlusion therapy on long-term outcome after surgery for early-onset exotropia. Materials and Methods ===================== The study was approved by the ethics committee of Gachon University Gil Hospital, in Korea. We conducted a retrospective review of the medical records of 51 consecutive patients who had undergone surgery for early-onset exotropia in our clinic (Department of Pediatric Ophthalmology, Gachon University Gil Hospital) between March 1998 and December 2010, and who were followed postoperatively for at least 3 years. In this study, early-onset exotropia was defined as either constant or intermittent exotropia with onset prior to 1 year of age. Patients with exotropia who had history of prematurity (defined as a gestational age \<37 weeks), trauma or neurological problems (such as developmental delay, seizures, or cerebral palsy), ocular diseases that could affect vision or ocular alignment, history of previous ocular muscle surgery, and follow-up duration \<3 years after primary exotropia surgery were excluded. All patients were referred to a pediatrician to exclude neurological disorders \[[@B1]\]. Patients data of gender, age at onset, age at diagnosis, preoperative and postoperative angles of deviation, intermittent versus constant phase before surgery, age at surgery for exotropia, follow-up period after surgery, preoperative refractive status, associated inferior oblique muscle weakening procedure performed simultaneously with surgery for exotropia, amblyopia, postoperative stereopsis at the final visit, and compliance with part-time occlusion therapy were recorded. All patients underwent a full initial ophthalmologic and orthoptic examination before surgery and were strongly advised to undergo daily part-time occlusion therapy using adhesive patches during a preoperative follow-up period of at least 6 months. Occlusion therapy was prescribed as follows: monocular patching in the dominant eye or alternate patching in the absence of a dominant eye. Eye dominance was determined using results from the cover--uncover test on each eye and the hole-in-the card test performed repeatedly. For the hole-in-the-card test, patients were instructed to view a distant object through the hole with both eyes open. Then, the examiner determined which eye the patient was using to see through the hole. Occlusion time was decided in consideration with the children\'s ages (1.5 to 2 hours for children younger than 3 years to minimize the risk of occlusion amblyopia and 3 hours for children older than 3 years). The occlusion therapy was advised to be performed at home under the surveillance of parents. Using self-report calendar logs and clinical interviews, compliance with occlusion therapy was calculated as the ratio of the number of hours when occlusion was actually performed (that were estimated by the parents) against the number of hours for which it had been prescribed. Compliance was graded as the following: "1" (for no compliance), "2" (for 1%--25%), "3" (for 26%--50%), "4" (for 51%--75%), or "5" (for 76%--100%). Patients with early-onset exotropia were followed preoperatively every 3 months, and the compliance rate was calculated at every follow-up session and averaged at the end of follow-up. Age at onset was defined as the age at which a family member first observed ocular misalignment. These estimates were confirmed using previous patient photographs or video records. The angle of deviation was measured using an alternate prism cover test at distance (6 m) and at near (1/3 m) with an accommodative target with spectacles. The modified Krimsky test was applied in a few patients in whom the alternate prism cover test had not been possible. Constant exotropia was defined as a clinic control score ≥5, in accordance with the revised Newcastle control score \[[@B12]\]. A score ≥5 was determined when exotropia manifested spontaneously both in near and distant viewing conditions or when exotropia occurred spontaneously in one condition and remained manifest after dissociation/prolonged fixation in the other condition. The preoperative refractive status was defined as the mean spherical equivalent of both eyes measured by cycloplegic refraction with topical administration of 1% tropicamide (Mydriacyl; Alcon, Puurs, Belgium) and 1% cyclopentolate hydrochloride (Cyclogyl; Alcon Laboratories, Fort Worth, TX, USA). Amblyopia was determined using results from recognition acuity tests or regarded as being present when a preverbal child consistently appeared uncooperative in fixation behavior while covering of one eye. Near stereopsis was measured using the Titmus stereoacuity test (Stereo Optical, Chicago, IL, USA) and was graded into four categories: stereopsis of 100 arcsec or better, 140 to 400 arcsec, 800 to 3,000 arcsec, and nil stereopsis. The surgical procedure, which was performed by the senior surgeon (HJP), was primarily bilateral lateral rectus muscle recession, and surgical dosages were applied using a standard table \[[@B13]\]. When there was associated inferior oblique muscle overaction (with/without V pattern) or dissociated vertical deviation, an inferior oblique muscle weakening procedure was performed simultaneously. Among the enrolled patients in this study, there were no cases of surgery on the vertical rectus or superior oblique muscles. Surgical outcome was regarded as being successful when ocular alignment was measured as orthophoria to exodeviation less than 10 prism diopters (PD) both at distance and near. Reoperation was recommended when a cosmetically objectionable deviation (usually exotropia exceeding 20 PD or esotropia exceeding 15 PD) was observed. When consecutive esotropia was observed, patients were followed up for at least 6 months before reoperation was recommended. In order to investigate the effect of preoperative occlusion therapy on the long-term surgical outcome of early-onset exotropia after surgery, we compared the long-term surgical success/recurrence/consecutive esodeviation and the level of postoperative stereopsis between patients with good compliance with part-time occlusion therapy (\>50% adherence rate, grades 4 and 5) and those with poor compliance (≤50% adherence rate, grades 1, 2, and 3). Additionally, in order to investigate the effect of preoperative occlusion therapy on patients with constant exotropia, we compared these factors between patients with good compliance and patients with poor compliance in the constant exotropia patients. We conducted the Fisher\'s exact test, Mann-Whitney *U*-test, and Kaplan-Meier survival analysis using SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA). The *p*-values less than 0.05 were considered statistically significant. Results ======= In all 51 early-onset exotropia patients who underwent surgery during the study period, the mean duration of preoperative occlusion therapy was 10.2 ± 5.4 months (range, 6 to 28 months). The mean follow-up duration after surgery for exotropia was 78.0 ± 28.1 months (range, 36 to 135 months). Overall, the final success rate of surgery for early-onset exotropia was 66.7%. Five patients (9.8%) showed persisting consecutive esotropia and eventually underwent surgical correction for these consecutive esotropia at a mean age of 18.8 months (range, 8 to 40 months) after the primary surgery for exotropia. Meanwhile, 12 patients (23.5%) showed recurrence of exotropia greater than 10 PD, and four patients (7.8%) underwent surgical correction for recurrent exotropia during the follow-up period after the initial surgery. With the exception of three patients who experienced both 1.5 to 2 hours of patching and 3 hours of patching due to continued occlusion treatment around 36 months of age, 38 patients were younger than 3 years and were prescribed 1.5 to 2 hours of patching (compliance rate, 2.7 ± 1.6), and 10 patients were older than 3 years and prescribed 3 hours of pathing (compliance rate, 3.1 ± 1.5). The compliance rates were not different according to patching hours (*p* = 0.626). [Fig. 1](#F1){ref-type="fig"} depicts the compliance rates of the 51 enrolled patients. Twenty-six patients fulfilled part-time occlusion therapy with \>50% adherence rate (grades 4 and 5) and were assigned to the good compliance group. The remaining 25 patients who underwent part-time occlusion therapy with a ≤50% adherence rate (grades 1, 2, and 3) were assigned to the poor compliance group. The demographic and clinical data of these 51 patients are presented in [Table 1](#T1){ref-type="table"}. There were no significant differences between the two study groups except for the improvement-to-dete-rioration ratio of the angle of deviation during the preoperative follow-up period. Improvement in the angle of deviation of more than 10 PD was shown in 10 patients. Among these patients, nine completed the occlusion therapy with a \>50% adherence rate. Meanwhile, no patients in the good compliance group showed deterioration. The success rate of surgery was significantly higher in the good compliance group (21 of 26 patients, 80.8%) than in the poor compliance group (13 of 25 patients, 52.0%) (*p* = 0.040). There were also significant differences between the two study groups in the undercorrection/recurrence rate (*p* = 0.049). The recurrence rate was higher in the poor compliance group (nine of 25 patients, 36.0%) than in the good compliance group (three of 26 patients, 11.5%). The Kaplan-Meier estimates also showed significant differences in the success rate (*p* = 0.049) ([Fig. 2A](#F2){ref-type="fig"}) and the competing risk analysis for undercorrection/recurrence (*p* = 0.046) ([Fig. 2B](#F2){ref-type="fig"}). The overcorrection/consecutive exodeviation rates were similar between the good compliance group (two of 26 patients, 7.7%) and the poor compliance group (three of 25 patients, 12.0%) (*p* = 0.614). Postoperative stereopsis could be measured in 44 of the 51 patients in our cohort. The good compliance group had a better level of stereopsis than the poor compliance group (*p* = 0.045) ([Fig. 3A](#F3){ref-type="fig"}). When comparing the success rate according to compliance with part-time occlusion therapy among the 24 patients who had surgery for constant exotropia, the success rate was greater in the good compliance subgroup (nine of 12 patients, 75.0%) than in the poor compliance subgroup (seven of 12 patients, 58.3%). However, this result was not statistically significant (*p* = 0.448). Meanwhile, the level of stereopsis was significantly better in the good compliance subgroup than in the poor compliance subgroup in the 19 patients who had early-onset constant exotropia and who underwent stereoacuity testing (*p* = 0.020) ([Fig. 3B](#F3){ref-type="fig"}). Discussion ========== Several studies have investigated the factors influencing surgical outcomes of exotropia in patients younger than 1 year of age. However, the factors that predict the long-term surgical success of early-onset exotropia have not been well established \[[@B3][@B4]\]. Although smaller preoperative deviation was found to be associated with more favorable out-comes at 6 weeks after surgery, smaller preoperative deviation did not guarantee the long-term outcome at 1 year following operation, according to results from a study by Yam et al. \[[@B3]\] and Park and Kim \[[@B4]\] also reported that no single preoperative factor affected the surgical outcome of early-onset exotropia at 1 year after surgery. Among patients who showed exotropia within the first year of life, age of onset did not influence the surgical outcome (*p* = 0.488). With 6 months of age at onset as a reference, the number of patients who showed exodeviation before 6 months of age was also not different between groups (nine patients in the poor compliance group and six patients in the good compliance group, *p* = 0.368). In this study, preoperative part-time occlusion therapy was suggested to be a possible factor improving long-term surgical outcome. During the preoperative follow-up period, patients with early-onset exotropia were strongly advised to undergo part-time occlusion therapy and asked to record their compliance with part-time occlusion therapy. Then, we identified higher long-term success and lower recurrence rate in the good compliance group than in the poor compliance group. In addition, with respect to sensory outcomes, fulfillment of occlusion therapy was associated with better postoperative levels of stereopsis. The beneficial effect of occlusion therapy on intermittent exotropia has been reported frequently \[[@B14][@B15][@B16][@B17][@B18]\]. These studies have reported that occlusion therapy decreased the angle of exodeviation in intermittent exotropia or even that occlusion therapy postponed surgical intervention by converting exotropia to orthophoria or exophoria. A recent randomized controlled trial also showed less deterioration of intermittent exotropia in patients who underwent part-time occlusion compared to those who did not (6.1% vs. 0.6%, *p* = 0.004) \[[@B11]\]. Occlusion therapy limits binocular stimulation, avoiding and correcting abnormal retinal correspondence and suppression \[[@B19]\]. Occlusion therapy has both sensory and motor effects on intermittent exotropia by reducing scotoma size and improving fusional vergence amplitude \[[@B20]\]. In addition to these positive effects on intermittent exotropia, part-time occlusion therapy in children with intermittent exotropia helped to solve the concentration problem psychosocially \[[@B21]\]. The results of our study support the previous hypothesis emphasizing the usefulness of occlusion therapy even in early-onset exotropia \[[@B10][@B11][@B14][@B15][@B16][@B17][@B18][@B19][@B20]\]. Among patients with constant exotropia, we failed to find a statistically significant difference in surgical success in the motor aspect (*p* = 0.448). Meanwhile, there was a statistically significant difference in the level of stereopsis among patients with constant exotropia (*p* = 0.020). The level of stereopsis was better in the good compliance subgroup than in the poor compliance subgroup ([Fig. 3B](#F3){ref-type="fig"}). The lack of significant difference in the motor aspect between subgroups may be due to small sample size within each subgroup and may indicate the need for larger sample size to compare the surgical success among early-onset constant exotropia patients. There are several limitations in this study. The design of this study was retrospective in nature. In addition, onset of early-onset exotropia was based on patient history, and this could be affected by recall bias. However, we confirmed these estimates using a number of old photographs or video records provided by patients. As another limitation related with retrospective design, this investigation was an observational study rather than a case-control study. We advised parents to ensure that their children underwent part-time occlusion therapy stringently. Then, we checked the level of compliance with occlusion therapy at every follow-up session and averaged these to obtain more reliable data related to compliance. Third, we assessed and ruled out any children who were suspected to have developmental difficulties that could influence poor compliance and surgical outcomes, with consultation from pediatricians. Although we excluded these patients, a few patients with subtle developmental difficulties may have been included in the enrolled patients, and this might influence the compliance rate with preoperative occlusion therapy and the surgical outcomes of exotropia. One of the biggest limitations was the small number of subjects. However, the small number of subjects in this study is an inherent limitation since exotropia in healthy children younger than 1 year of age is rare \[[@B22]\]. This study included a relatively larger number of subjects compared to previous studies. We suggest that a large, case-control study is needed to confirm the usefulness of preoperative occlusion therapy in early-onset exotropia surgery. Although the effect of preoperative occlusion therapy on the success rate of constant exotropia was insignificant, preoperative occlusion therapy was useful for improving surgical outcomes among patients who underwent surgery for exotropia. In addition, preoperative occlusion therapy also improved postoperative stereopsis in all patients who underwent surgery for early-onset exotropia and even in those who underwent surgery for constant exotropia. Therefore, preoperative part-time occlusion therapy should be proposed and explained to the family during the preoperative follow-up period to improve surgical outcomes. This study was presented at the 112th meeting of Korean Ophthalmology Society in Goyang, Korea. **Conflict of Interest:** No potential conflict of interest relevant to this article was reported. {#F1} {#F2} {#F3} ###### Comparison of demographic and clinical data between both groups according to occlusion compliance in patients who underwent surgery for early-onset exotropia  Values are presented as number (%) or mean ± standard deviation (range). PD = prism diopters; SER = spherical equivalent refraction; D = diopters. ^\*^Fisher\'s exact test; ^†^Mann-Whitney *U*-test.

Introduction {#s1} ============ Postoperative cognitive changes are a common complication, especially in elderly patients who undergo major surgeries, including arthroplasty, laparotomy, and thoracotomy with or without cardiopulmonary bypass (Saczynski et al., [@B47]; Evered et al., [@B9]). Perioperative neurocognitive disorders (PND) are characterised by symptoms such as disturbance of memory, attention, consciousness, information processing, and the sleep-wake cycle, leading to postoperative morbidity and mortality (Evered et al., [@B9]). PND occurs predominantly in elderly patients but may also occur in other age groups (Johnson et al., [@B21]; Backeljauw et al., [@B2]). Multiple factors may be involved in PND (Cibelli et al., [@B6]; Han et al., [@B14]; Li et al., [@B34]); however, the molecular mechanisms underlying this condition are unclear. Under some circumstances, general anaesthetics may cause long-term cognitive impairment (Li et al., [@B33]; Zhong et al., [@B64]; Cao et al., [@B5]) but have also been shown to promote brain protection (Fukuda and Warner, [@B200]), indicating that anaesthetics may play different roles in PND. Cognitive processes are complex. In humans, these processes can be divided into five dimensions: attention, perception, memory, language, and learning. In clinical research, memory impairment after anaesthesia and surgery is a typical symptom and is often used as the main diagnostic criterion for PND (Evered et al., [@B9]). Memory functions involve formation, consolidation, storage, and recall. Brain-derived neurotrophic factor (BDNF) is implicated in several adaptive and pathological processes (Harward et al., [@B16]; Tanqueiro et al., [@B52]; Lima Giacobbo et al., [@B12]; Oh et al., [@B41]). An increasing number of studies have demonstrated that BDNF in the hippocampus, especially in the dorsal CA1 region, plays a key role in regulating cognition and memory (Bambah-Mukku et al., [@B3]; Bekinschtein et al., [@B4]; Reimers et al., [@B44]). A clinical study reported that BDNF protein expression decreased after anaesthesia and surgery in patients with cognitive impairment (Wyrobek et al., [@B60]). Multiple amino acid residues on histone H3 can be modified in cognitive processes (Hyun et al., [@B19]; Kim and Kaang, [@B26]). Lysine 9 in H3 (H3K9) can be acetylated or methylated (Kilpinen et al., [@B25]; Lanouette et al., [@B30]). Theoretically, the deacetylation or hypermethylation of H3K9 can induce chromatin condensation, resulting in long-term gene silencing, and the trimethylation of H3K9 (H3K9me3) mediates transcriptional silencing (Karmodiya et al., [@B23]; Zovkic and Sweatt, [@B65]; Zovkic et al., [@B66]). In contrast, the acetylation and demethylation of H3K9 activate transcription (Ushijima et al., [@B55]). SUV39H is a typical histone methyltransferase involved in H3K9 trimethylation and promotes gene silencing. Moreover, H3K9 trimethylation is critical for cognitive impairment and is involved in the transcriptional repression of the *Bdnf* gene (Kuzumaki et al., [@B28]; Gupta-Agarwal et al., [@B13]; Maddox et al., [@B36]; Karpova, [@B24]). Considering that H3K9 is located near the promotors, modifications in this histone affect DNA methylation and transcription (Du et al., [@B8]; Zhao et al., [@B63]). This study assessed whether the trimethylation of H3K9 was involved in the downregulation of BDNF expression leading to cognitive and memory impairment, and the stage at which memory processing was affected by anaesthesia and surgery. H3K9 trimethylation downregulated BDNF expression and impaired memory formation, but not recall, during anaesthesia and surgery. Therefore, inhibiting H3K9 trimethylation and increasing the expression of BDNF may help prevent PND in a clinical setting. Materials and Methods {#s2} ===================== Animals {#s2-1} ------- Adult male C57BL/6J mice (10--12 months old) and adult male vGLUT1-IRES-CreERT mice (homozygous, C57BL/6J background, 10--12 months old) were obtained from Xuzhou Medical University Animal Center (Xuzhou, China). All mice (five animals per cage) were acclimatised under a 12--12 h light-dark cycle and were allowed *ad libitum* access to food and water. All behavioural experiments were carried out between 8:00 am and 5:00 pm. All protocols were approved by the Animal Welfare Committee of Xuzhou Medical University and complied with Animal Research: Reporting of *in vivo* Experiments guidelines. Drugs and Primers {#s2-2} ----------------- Chaetocin (Sigma-Aldrich, USA; 3 μg in 0.3 μl of 10% DMSO in PBS) was microinjected into the dorsal CA1 region of the hippocampus on each side 2 h before the tests. Recombinant BDNF (R&D Systems, USA; 0.5 μg in 0.3 μl of distilled water) was microinjected into the dorsal CA1 region of the hippocampus on each side 1 h before the tests. The following primers were used in RT-PCR: total BDNF, 5′-AAGGACGCGGACTTGTACAC-3′ (forward), 5′-CGCTAATACTGTCACACACGC-3′ (reverse); GAPDH, 5′-AGGTCGGTGTGAACGGATTTG-3′ (forward), 5'-TGTAGACCATGTAGTTGAGGTCA-3′ (reverse); The primers for chromatin immunoprecipitation (ChIP) IV, 5′-AAAAACGGTCCAAAGACCAC-3′ (forward), 5′-TCACTAAGCCCCCTTCCTCT-3′ (reverse). Anaesthesia and Surgery {#s2-3} ----------------------- Animals were placed on a transparent chamber prefilled with 1.4% isoflurane and 100% oxygen (Peng et al., [@B42]). After a 15-min induction, a simple laparotomy was performed. A 0.5-cm longitudinal midline incision was made from the xiphoid to the proximal pubic symphysis on the skin, abdominal muscles, and peritoneum. Then, the incision was sutured layer by layer. Compound lidocaine cream (2.5% lidocaine and 2.5% prilocaine) was applied to the incision wound every 8 h for 2 days. The surgical procedure lasted approximately 20 min for each mouse, and the animals remained under anaesthesia for up to 2 h. The temperature of the anaesthesia chamber was controlled to maintain the rectal temperature of the mice at 37 ± 0.5°C during anaesthesia/surgery. The concentrations of isoflurane and oxygen and animal breathing were monitored. The animals from the control group remained in a chamber filled with 1.4% isoflurane and 100% oxygen for 2 h but were not subjected to surgery. Open Field Test {#s2-4} --------------- An open field test (OFT) was used to determine the spontaneous locomotor activity of mice according to a previous study (Wang et al., [@B57]). The OFT apparatus consisted of a 50 × 50 cm open arena with 50-cm-high walls, and the floor was divided into nine squares of equal areas. Briefly, the mice were individually placed into the centre of the apparatus and were free to explore the environment for 10 min. Thereafter, the total distance travelled was recorded by ANY-maze software during the following 5-min period. Y-Maze Test {#s2-5} ----------- The maze consisted of three arms (8 × 30 × 15 cm, width × length × height) that were 120° from each other: the start arm (always open), novel arm (closed during training and open during tests), and the other arm (always open). During training, mice were allowed to explore the maze for 10 min. After a 2-h interval, the animals were returned to the start arm for a 5-min exploration with the novel arm open. The number of entries and time spent on each arm were recorded. Novel Object Recognition Test {#s2-6} ----------------------------- A novel object recognition (NOR) test was performed using a 50 × 50 cm open arena with 50-cm-high walls. The arena was divided into four quadrants, and two identical objects were placed diagonally from each other. Mice were individually placed into the centre of the apparatus and were free to explore the environment for 10 min. After that, the animals returned to the cage, and one of the objects was replaced by a novel object with a different shape and colour. The apparatus was cleaned with 75% ethanol. After a 2-h interval, mice were allowed to freely explore the area for 5 min, and the time spent exploring the novel object was measured. Contextual Fear Conditioning Test {#s2-7} --------------------------------- The test was performed as previously reported (Gao et al., [@B11]). Briefly, on the day of training or test, mice were placed in the conditioning room at least 2 h before fear conditioning. Subsequently, the animals were transferred to the training chamber, freely explored the area for 3 min, received a foot shock (0.7 mA) for 2 s, and returned to the conditioning chamber. Age-matched control animals remained in the conditioning chamber for the same period as the conditioned animals but received no foot shocks. Freezing behaviour was measured for 3 min. Stereotaxic Surgery {#s2-8} ------------------- Experimental mice were anaesthetised using 1.4% isoflurane and placed in a stereotaxic instrument according to a previous report (Hu et al., [@B17]). The scalp was cut open, and the skull was exposed using 75% ethanol and 1% H~2~O~2~. Double-guide cannulas (Plastics One, USA) were bilaterally implanted into the dorsal hippocampus (anterior-posterior, −1.5 mm; mediolateral, ±1.0 mm, dorsal-ventral, −2.0 mm) using stereotactic manipulators under aseptic conditions. Each cannula contained a dummy cannula and a dust cap (Plastics One, USA) and was fixed to the skull with dental cement. A single injection was performed into each hemisphere using a 33 G Hamilton microinjector in a volume of 0.3 μl and a flow rate of 0.1 μl/min. Needles were gently removed 10 min after injection. The incisions were sutured, and the mice were transferred to a temperature-controlled room (31 ± 1°C). Chromatin Immunoprecipitation (ChIP) {#s2-9} ------------------------------------ ChIP assays were performed using the EZ-Magna ChIP A/G ChIP Kit (Millipore, USA). dCA1 tissues were homogenised in a protease inhibitor solution, fixed in 37% formaldehyde, and cross-linked at room temperature for 30 min. The reaction was inhibited with glycine, and the isolated chromatin samples were sonicated to shear DNA. The cross-linked protein/DNA was immunoprecipitated with a primary antibody at 4°C overnight. The protein/DNA complexes were eluted and reversed cross-links of protein/DNA complexes to free DNA with proteinase K. The purified DNA was collected for PCR analysis (Ding et al., [@B7]). Western Blot Assays {#s2-10} ------------------- Western blotting was performed as previously reported (Hao et al., [@B15]). Briefly, dissected dCA1 tissues were homogenised in RIPA buffer containing a protease inhibitor cocktail and a protein phosphatase inhibitor cocktail. The lysate was run on SDS-page gel and transferred to nitrocellulose membranes. The membrane was incubated with the following primary antibodies: anti-BDNF (1:1,000; ab108319; Abcam, UK), anti-H3K9me3 (1:1,000; 13969; Cell Signaling Technology, USA), anti-histone H3 (1:1,000; 4499; Cell Signaling Technology, USA), and β-actin (1:1,000; sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000; Beyotime Institute of Biotechnology, Haimen, China) and scanned using an infrared imaging system (Bio-Raid, USA). Statistical Analysis {#s2-11} -------------------- Data are presented as the mean ± standard error of the mean. The *Z*-score was calculated based on the results of duration in the novel arm, entries in the novel arm and discrimination index. The formula was *Z*-score = \[ΔX~surgery~ − MEAN (Δ~control~)\]/SDΔ~control~. All analyses were performed using Prism software. Data normality was assessed using the D'Agostino-Pearson omnibus normality test. Data with a normal distribution were analysed using one-way analysis of variance for multiple comparisons with or without repeated factors, followed by *post hoc* Student--Newman--Keuls (SNK) tests for multiple comparisons. Data without a normal distribution were analysed using non-parametric Kruskal--Willis tests followed by Dunn's multiple comparisons test. *P*-values for multiple comparisons were adjusted. Data from the two study groups were compared using unpaired Student's *t*-test. Statistical significance was set at *p* \< 0.05. Results {#s3} ======= Anaesthesia and Surgery Downregulated BDNF and Impaired Cognition in Middle-Aged Mice {#s3-1} ------------------------------------------------------------------------------------- We assessed whether anaesthesia and surgery could downregulate BDNF in the hippocampal dorsal CA1 area and whether laparotomy under inhalation anaesthesia in middle-aged mice was a well-established postoperative cognitive impairment model. Middle-aged mice aged 10--12 months underwent laparotomy under general anaesthesia with isoflurane (Peng et al., [@B42]). The Y-maze and NOR tests were performed to assess cognitive changes induced by anaesthesia and surgery ([Figure 1A](#F1){ref-type="fig"}). All cognitive behavioural data were collected to calculate *Z*-scores. The total distance travelled in the OFT and Y-maze test, and the total number of entries in all arms in the Y-maze test were collected to determine the effect of abdominal surgery on locomotor activity, and *Z*-scores were calculated to analyse changes in locomotor activity. {#F1} The *Z*-scores indicated the occurrence of significant cognitive deterioration in the surgery groups (*p* \< 0.0001; [Figure 1B](#F1){ref-type="fig"}). However, there were no significant differences in locomotor activity between the surgery and control groups ([Figure 1C](#F1){ref-type="fig"}). These results suggested that anaesthesia and surgery impaired cognition in middle-aged mice but had no significant effect on locomotor activity. A clinical study reported that BDNF protein expression decreased intraoperatively in cognitively impaired patients. We assessed whether BDNF expression contributed to cognitive dysfunction in an animal model. The expression of BDNF in the dorsal CA1 was decreased in the surgery groups from 6 to 72 h after surgery (*p* = 0.0014; [Figure 1D](#F1){ref-type="fig"}, [Supplementary Figure S4A](#SM4){ref-type="supplementary-material"}) and was positively correlated with cognition *Z*-scores (*r*^2^ = 0.2977, *p* = 0.002673; [Figure 1E](#F1){ref-type="fig"}). BDNF mRNA expression in this hippocampal region also decreased in the surgery groups from 6 to 72 h after surgery (*p* = 0.0005; [Figure 1F](#F1){ref-type="fig"}) and was positively correlated with cognition *Z*-scores (*r*^2^ = 0.5750, *p* \< 0.0001; [Figure 1G](#F1){ref-type="fig"}). Furthermore, BDNF mRNA expression was positively correlated with BDNF protein expression (*r*^2^ = 0.2244, *p* = 0.01089; [Figure 1H](#F1){ref-type="fig"}). These results suggested that the decrease in BDNF expression in the dorsal CA1 region affected cognitive impairment induced by anaesthesia and surgery. Postoperative BDNF Downregulation in the Dorsal Hippocampus Impaired Memory Formation but Not Recall {#s3-2} ---------------------------------------------------------------------------------------------------- Cognitive processes are complex. The present results suggested that anaesthesia and surgery affected cognition; however, which cognitive processes are impaired is unclear. Contextual fear conditioning experiments were conducted to determine whether anaesthesia and surgery inhibited novel cognitive formation or other cognitive processes. First, training-surgery-test and surgery-training-test experiments were performed to investigate whether anaesthesia and surgery impaired either memory formation or recall. The results showed that anaesthesia and surgery only affected memory formation because fear conditioning was blocked in mice trained after 6 h (*p* \< 0.0001) and 24 h (*p* = 0.0001) of surgery, but not before surgery (*p* \> 0.9999, *p* = 0.7981; [Figures 2A,B](#F2){ref-type="fig"}). The freezing time in the control group was significantly different than in the iso-train-test group at 6 h (*p* = 0.0441; [Supplementary Figures S1A,B](#SM1){ref-type="supplementary-material"}). {#F2} As previously reported, the BDNF protein expression changes after training represent the fear memory formation. BDNF protein expression changes triggered by foot shock 30 min after training were determined. BDNF expression decreased in mice trained after 6 h and 24 h of surgery (*p* \< 0.0001; [Figure 2C](#F2){ref-type="fig"}, [Supplementary Figure S4B](#SM4){ref-type="supplementary-material"}). In the control group, isoflurane inhalation attenuated BDNF expression triggered by training at 6 h after surgery (*p* \< 0.0001; [Supplementary Figures S1C](#SM1){ref-type="supplementary-material"}, [S4E](#SM4){ref-type="supplementary-material"}). These results suggested that anaesthesia and surgery inhibited BDNF expression and impaired memory formation but not recall. Reversion of the Postoperative Repression of BDNF Expression in the Dorsal Hippocampus Prevented the Impairment of Memory Formation Caused by Anaesthesia and Surgery {#s3-3} --------------------------------------------------------------------------------------------------------------------------------------------------------------------- BDNF was overexpressed in the dorsal CA1 to confirm whether BDNF downregulation impaired memory formation, and to determine the role of BDNF in contextual fear memory ([Figure 3A](#F3){ref-type="fig"}, [Supplementary Table S1](#SM5){ref-type="supplementary-material"}). The genetically modified adeno-associated virus overexpressing BDNF was infused into the dorsal CA1 region 2 weeks before fear conditioning tests. The results showed that BDNF overexpression enhanced fear memory at 6 h (*p* = 0.8205) and 24 h (*p* = 0.8119) after surgery ([Figures 3B,C](#F3){ref-type="fig"}) and the vector did not affect the expression of BDNF ([Supplementary Figures S3A](#SM3){ref-type="supplementary-material"}, [S4G](#SM4){ref-type="supplementary-material"}). {#F3} Subsequently, we assessed whether exogenous BDNF could enhance memory formation, but not memory recall. Recombinant BDNF was infused into the dorsal CA1 to test whether memory recall was impaired ([Figure 4A](#F4){ref-type="fig"}). The infusion of recombinant BDNF after surgery and training and before the test did not enhance fear memory recall (*p* = 0.0003, *p* = 0.0005; [Figure 4B](#F4){ref-type="fig"}), whereas the infusion of BDNF after surgery and before training significantly enhanced fear memory formation (*p* = 0.8296, *p* = 0.77117; [Figure 4C](#F4){ref-type="fig"}). These results demonstrate that BDNF is involved in memory formation but not memory recall. {#F4} H3K9 Trimethylation Caused the Long-Term Downregulation of BDNF in the Dorsal Hippocampus and Impaired Memory Formation {#s3-4} ----------------------------------------------------------------------------------------------------------------------- Considering that BDNF expression was repressed from 6 h to 72 h after anaesthesia and surgery, we investigated the mechanism underlying this long-term downregulation. It is known that histone modification plays essential roles in the long-term regulation of transcription, and H3K9 trimethylation mediates transcriptional silencing. The expression of H3K9me3 was measured in this study. The results indicated that H3K9me3 expression increased significantly in the surgery group at 12 h after surgery (*p* \< 0.0001; [Figure 5A](#F5){ref-type="fig"}, [Supplementary Figure S4C](#SM4){ref-type="supplementary-material"}). In the inhalation-only groups, the alteration of H3K9me3 was not observed at all time points (*p* = 0.9085; [Supplementary Figures S3B](#SM3){ref-type="supplementary-material"}, [S4H](#SM4){ref-type="supplementary-material"}). Next, we assessed the binding of H3K9me3 to the *Bdnf* exon IV promoter, which is widely distributed in the central nervous system and plays a crucial role in cognition and memory. At 24 h after anaesthesia and surgery, the binding of H3K9me3 to the *Bdnf* exon IV promoter was significantly increased (*p* \< 0.0001; [Figure 5B](#F5){ref-type="fig"}), and the expression of the mRNA coded by *Bdnf* exon IV was decreased (*p* \< 0.0001; [Figure 5C](#F5){ref-type="fig"}); moreover, the mRNA coded by *Bdnf* exon IV expression was negatively correlated with H3K9me3 expression (*r*^2^ = 0.5080, *p* = 0.001942; [Figure 5D](#F5){ref-type="fig"}) and ChIP results (*r*^2^ = 0.2673, *p* = 0.04028; [Figure 5E](#F5){ref-type="fig"}) and positively correlated with the overall expression of BDNF mRNA (*r*^2^ = 0.5935, *p* = 0.0004796; [Figure 5F](#F5){ref-type="fig"}), suggesting that H3K9 trimethylation was crucial to the long-term downregulation of BDNF. {#F5} We hypothesised that BDNF downregulation might be divided into two stages: short-term (up to 24 h) and long-term (more than 24 h). Considering that inhalation anaesthetics are reported to play a role in the rapid inhibition of neuronal activity and that this effect can be prolonged after anaesthesia, we hypothesised that the repression of BDNF expression was induced by isoflurane in the short-term in our study. Therefore, the expression of BDNF was measured in control mice, which received inhalation anaesthesia for 2 h but did not undergo surgery. The results indicated that both the protein expression (*p* = 0.0078; [Supplementary Figures S2A](#SM2){ref-type="supplementary-material"}, [S4F](#SM4){ref-type="supplementary-material"}) and total mRNA expression (*p* = 0.0289) of BDNF ([Supplementary Figure S2B](#SM2){ref-type="supplementary-material"}) were decreased at 6 h after anaesthesia. These findings suggested that the short-term downregulation of BDNF occurred by inhibiting neuronal activity after anaesthesia, whereas the long-term downregulation was in different pathway. To further determine the effect of H3K9 trimethylation on BDNF expression, histone methyltransferase SUV39H antagonist chaetocin (Ch) was injected into the dorsal CA1 region, which is involved in H3K9 trimethylation ([Figure 5G](#F5){ref-type="fig"}). The inhibition of H3K9 trimethylation restored BDNF expression and reversed the behavioural changes induced by surgery at 24 h but not at 6 h after surgery (*p* \< 0.0001; [Figures 5H,I](#F5){ref-type="fig"}, [Supplementary Figure S4D](#SM4){ref-type="supplementary-material"}) and the vehicle did not affect the BDNF expression ([Supplementary Figures S3C](#SM3){ref-type="supplementary-material"}, [S4I](#SM4){ref-type="supplementary-material"}). These results suggested that the long-term downregulation of BDNF triggered by H3K9 trimethylation might be the cause of cognitive and memory impairment postoperatively. Discussion {#s4} ========== In this study, we demonstrated that anaesthesia and surgery impaired cognition by repressing the expression of the *Bdnf* gene in the dorsal hippocampus, resulting in impairment of memory formation. General anaesthetics can downregulate BDNF expression in the short-term (within 12 h) by inhibiting neuronal activity, resulting in the short-term impairment of memory formation. Anaesthesia and surgery-induced H3K9 trimethylation and the long-term transcriptional repression of BDNF, resulting in long-term (more than 24 h) memory impairment. Given that PND is considered a long-term cognitive impairment, BDNF downregulation induced by H3K9 trimethylation might be the main cause of impairment of memory formation after anaesthesia and surgery. BDNF is essential for cognition and memory (Bekinschtein et al., [@B4]) and it is able to cross the Brain-Blood Barrier (BBB) freely for its molecular weight (Klein et al., [@B27]). BDNF binds to receptor TrkB and activates the phospholipase C (PLC), PI3K, and MAPK/ERK pathways (Minichiello et al., [@B38]; Yoshii and Constantine-Paton, [@B62]). Furthermore, BDNF enhances the activation of CREB *via* the MAPK/ERK pathway (Sen et al., [@B49]). Arc expression is also dependent on this pathway, which is highly related to neuronal activation (Lalonde et al., [@B29]). Given that memory formation involves the hyperactivity of pyramidal neurons, the downstream activation of BDNF and excitability of pyramidal neurons play a crucial role in cognition and memory. BDNF is known to activate GluA1, a subunit of AMPA receptors, and enhances GluA1 trafficking to the cell surface. BDNF bound to TrkB activates the PLC pathway to enhance calcium signals, consequently activating CaMKII and resulting in GluN2B phosphorylation. Both GluA1 and GluN2B are necessary for long-term potentiation (LTP) and affect cognition and memory. The mechanisms of postoperative cognitive changes are complicated, and it is involved in variety of effects. Multiple reasons such as low temperature, ischemia, stress, inappropriate surgery and anaesthesia might induce the postoperative cognitive changes (Monk et al., [@B39]; Salazar et al., [@B48]; Rundshagen, [@B46]; Robinson et al., [@B45]; Tian et al., [@B53]). Almost all the reasons lead to the alteration of BDNF expression after anaesthesia and surgery. In this study, the repression of BDNF expression impaired memory formation directly, and this finding agrees with previous studies. Epigenetics is reported to be involved in the long-term regulation of cognition and memory without DNA sequence changes. Histone modification is an essential epigenetic mechanism and regulates transcription in cognitive processes (Ding et al., [@B7]; Kim and Kaang, [@B26]). It is reported that several sites on histones H2 to H4 are modified by acetylation, methylation, phosphorylation, ubiquitination, and citrullination. Histone H3 methylation is a common way to modify regulation of transcription. H3 methylation results in chromatin condensation, which can inhibit transcription factors that are recruited to DNA binding sites to induce gene silencing. The pharmacological inhibition of H3K9 trimethylation increases BDNF expression in the hippocampus of aged mice (Snigdha et al., [@B51]). Furthermore, some sites modified in H3, such as H3K9, are located near promotors and cooperate with DNA methyltransferases (DNMTs) to co-repress DNA transcription. H3K9 methyltransferase SUV39H and G9a are also recruited in this complex with DNMT3a or DNMT3b (Fuks et al., [@B10]; Rai et al., [@B43]). The deacetylation of H3K9 and H3K14 is regulated by HDAC2 (Wagner et al., [@B56]; Singh and Thakur, [@B50]; Watts et al., [@B58]), recruited and activated by MeCP2 (Mahgoub et al., [@B37]). The crosstalk between DNA methylation and histone modification allows the stable silencing of the promoters. In our model, some factors promoted the downregulation of BDNF, leading to long-term cognitive impairment after anaesthesia and surgery. Memory processes are complex, and memory formation is essential for cognitive functioning. Multiple factors are involved in memory formation, and BDNF is reported to play a key role in it. Theoretically, BDNF enhances memory formation but impairs memory retention. *Bdnf* transcription depends on multiple factors, and H3 methylation is directly and indirectly involved in this process. H3K9 trimethylation promotes chromosome condensation and represses BDNF expression, and histone methyltransferase can recruit DNMTs to CpG islands located near the *Bdnf* exons promotors and co-silence *Bdnf* transcription indirectly. These factors impair memory formation and retention, consequently inhibiting LTP. General anaesthetics target primarily the GABA~A~ receptor (Jurd et al., [@B22]; Nishikawa and Harrison, [@B40]; Topf et al., [@B54]; Winegar and MacIver, [@B59]; Jia et al., [@B20]; Ying et al., [@B61]; Li et al., [@B34]) and lead to the rapid inhibition of neuronal activity and, ultimately, sedation. BDNF expression was repressed in this study but was short-term, demonstrating that the inhibition of neuronal activity was induced by the activation of GABA~A~ receptors. The prolonged repression of BDNF expression had a significant impact on long-term cognitive impairment induced by anaesthesia and surgery, and this process was highly related to H3K9 trimethylation. Neuroinflammation is a major contributing factor to cognitive dysfunction. H3K9 trimethylation is reported to be involved in IL-6 regulation (Li Z. et al., [@B35]), and IL-6 expression is increased in PND patients (Androsova et al., [@B1]; Li et al., [@B32]). Furthermore, the disruption of the BBB after surgery may allow more inflammatory molecules to cross this barrier (Li M.-F. et al., [@B31]). These inflammatory molecules may trigger changes in histone H3 and result in BDNF transcriptional repression, leading to long-term memory impairment after anaesthesia and surgery. Anaesthesia and surgery and postoperative stress may also induce glucocorticoid receptor phosphorylation (Tian et al., [@B53]), which contributes to postoperative cognitive impairment. Furthermore, H3K9 trimethylation has been reported to be involved in the regulation of stress (Hunter et al., [@B18]). Despite these known effects, additional studies are needed to elucidate the role of these factors. In conclusion, the results indicated that BDNF downregulation induced by H3K9 trimethylation impaired memory formation but not memory recall during anaesthesia and surgery. These findings may help understand the molecular mechanisms for PND, and BDNF may be used to prevent postoperative cognitive impairment in the clinic. Data Availability Statement {#s5} =========================== The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. Ethics Statement {#s6} ================ The animal study was reviewed and approved by Animal Welfare Committee of Xuzhou Medical University. Written informed consent was obtained from the owners for the participation of their animals in this study. Author Contributions {#s7} ==================== TW and CG conceived and designed the study. TW, X-YS, and KT performed the research. XY, LL, J-RH, YG, and JC contributed data and research tools. TW prepared the manuscript. Conflict of Interest {#s8} ==================== The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. **Funding.** This work was supported by the National Natural Science Foundation of China (Grant Nos. 81870852 and 81471101), the Natural Science Foundation of Jiangsu Province (Grant No. BK20181146), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant Nos. 18KJA320007 and 17KJB320018), the Qing Lan Project of Jiangsu Province, and the Postgraduate Research and Practice Innovation Program of Jiangsu Province (Grant Nos. KYCX19_2226 and KYCX19_2243). Supplementary Material {#s9} ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fnmol.2019.00246/full#supplementary-material>. ###### Inhalation of isoflurane was unable to attenuate the cognition and BDNF expression in dCA1 of hippocampus aroused by training. **(A,B)** Contextual fear conditioning behavioral tests for inhalation-only groups. **(A)** Training-inhalation-test results at 6 (6 h vs. Con, *t*~(14)~ = 0.562, *p* = 0.583, *n* = 8) and 24 h (24 h vs. Con, *t*~(14)~ = 0.3374, *p* = 0.7408, *n* = 8) after inhalation anesthesia. **(B)** Inhalation-training-test results at 6 (6 h vs. Con, *t*~14~ = 2.212, *p* = 0.0441, *n* = 8) and 24 h (24 h vs. Con, *t*~(14)~ = 0.7316, *p* = 0.4765, *n* = 8) after inhalation anesthesia. **(C)** BDNF expression changes after training in anesthesia-only groups at 6 h (*F*~(3,\ 12)~ = 24.25, *p* \< 0.0001; Train vs. Con, *p* = 0.0052; 6 h + Train vs. 6 h, *p* = 0.0008; 6 h + Train vs. Train, *p* = 0.0465; 6 h vs. Con, *p* = 0.0066, *n* = 4) and 24 h (*F*~(3,12)~ = 33.39, *p* \< 0.0001; Train vs. Con, *p* \< 0.0001; 24 h + Train vs. 24 h, *p* = 0.0001; 24 h + Train vs. Train, *p* = 0.6215; 24 h vs. Con, *p* = 0.9931, *n* = 4). All results were represented as $\overline{x}$ ± s.e.m; ns, no significance; \**p* \< 0.05, \*\**p* \< 0.01. ###### Click here for additional data file. ###### Inhalation of isoflurane repressed BDNF in dCA1 of hippocampus in short-term. **(A)** Inhalation anesthesia without surgery mice showed changes of BDNF protein at 6 h (*F*~(6,21)~ = 4.019, *p* = 0.0078; 6 h vs. Con, *p* = 0.0429, *n* = 4). **(B)** Total BDNF mRNA was changed at 6 h (*F*~(6,21)~ = 2.980, *p* = 0.0289; 6 h vs. Con, *p* = 0.005, *n* = 4). All results were represented as $\overline{x}$ ± s.e.m; \**p* \< 0.05, \*\**p* \< 0.01. ###### Click here for additional data file. ###### Inhalation of isoflurane, vehicles and vectors did not affect the H3K9me3 and BDNF expressions. **(A)** The vehicles (*F*~(5,18)~ = 13.74, *p* \< 0.0001; Con vs. Con + Veh, *p* = 0.9892; 6 h vs. 6 h + Veh, *p* = 0.9992; 24 h vs. 24 h + Veh, *p* \> 0.9999, *n* = 4) and **(B)** vectors (F~(5,18)~ = 16.98 *p* \< 0.0001; Con vs. Con + Veh, *p* = 0.9977; 6 h vs. 6 h + Veh, *p* = 0.9999; 24 h vs. 24 h + Veh, *p* = 0.9685, *n* = 4) did not alter the BDNF expression. **(C)**Inhalation anesthesia without surgery mice did not show significant changes of H3K9me3 at all time points (*F*~(6,21)~ = 0.3387, *p* = 0.9085). All results were represented as $\overline{x}$ ± s.e.m; ns, no significance. ###### Click here for additional data file. ###### Full-length pictures of the blots and gels presented in [Figure 1](#F1){ref-type="fig"} to [Figure 5](#F5){ref-type="fig"} and [Supplementary Figure S1](#SM1){ref-type="supplementary-material"} to [Supplementary Figure S3](#SM3){ref-type="supplementary-material"}. **(A)** Full-length for [Figure 1D](#F1){ref-type="fig"}. **(B)** Full-length for [Figure 2C](#F2){ref-type="fig"}. **(C)** Full-length for [Figure 5A](#F5){ref-type="fig"}. **(D)** Full-length for [Figure 5H](#F5){ref-type="fig"}. **(E)** Full-length for [Supplementary Figure S1C](#SM1){ref-type="supplementary-material"}. **(F)** Full-length for [Supplementary Figure S2A](#SM2){ref-type="supplementary-material"}. **(G)** Full-length for [Supplementary Figure S3A](#SM3){ref-type="supplementary-material"}. **(H)** Full-length for [Supplementary Figure S3B](#SM3){ref-type="supplementary-material"}. **(I)** Full-length for [Supplementary Figure S3C](#SM3){ref-type="supplementary-material"}. ###### Click here for additional data file. ###### rAAV-Bdnf titer test report. ###### Click here for additional data file. [^1]: Edited by: Hiroyuki Okuno, Kagoshima University, Japan [^2]: Reviewed by: Maiko Hasegawa-Moriyama, Shinshu University, Japan; Diansan Su, Shanghai JiaoTong University, China [^3]: ^†^These authors have contributed equally to this work

For a long time, the prediction of molecular crystal structures from first principles has been one of the most challenging computational problems. Because of the large number of degrees of freedom, methods with low computational effort have to be used. The price for this efficiency is, however, a loss of accuracy. In recent years, important progress has been made in the development of dispersion corrections \[[@B1]-[@B3]\], which raise the accuracy of density functional theory (DFT) calculations to a much higher level. Based on these developments, the software package GRACE \[[@B4]\] was the first program ever to correctly predict all four crystal structures at the 4th crystal structure prediction blind test, organized by G. M. Day and the Cambridge Crystallographic Data Centre in 2007 \[[@B5]\]. GRACE uses dispersion-corrected density functional theory calculations (DFT-D) to generate reference data to which a tailor-made force field (TMFF) is fitted for each respective molecule \[[@B6]\]. Crystal structures are generated with a Monte-Carlo parallel-tempering algorithm that uses the TMFF for the evaluation of lattice energies. Only the most stable crystal structures according to the TMFF are then re-optimized and re-ranked using DFT-D. The DFT calculations are performed using an interface to the two common ab-inito programs VASP \[[@B7]\] and QuantumESPRESSO \[[@B8]\]. In the most recent, 5th blind test, organized in 2010, the excellent agreement between experimental and predicted structures for small, neutral molecules was confirmed. However, it turned out that the crystal structure prediction of molecular salts and hydrates is still a challenging task. The inability of DFT without exact exchange to describe anions properly, as recently published by Jensen \[[@B9]\], suggests that the inclusion of the Hartree-Fock exchange should improve the accuracy for molecular salts and hydrates. Therefore, we will present an assessment of a variety of current density functionals, in which the dispersion-correction parameters were fitted with respect to a minimization of the deviation between experimental and computed crystal structures.

Introduction ============ Gastric cancer (GC) is responsible for over 1,000,000 new cases and around 783,000 deaths in the world annually, making it the 5th most frequently diagnosed cancer and the third leading cause of cancer-related death ([@ref-5]). Surgery with subsequent adjuvant chemoradiotherapy remains the only treatment with curative potential ([@ref-3]), and the prognosis for gastric adenocarcinoma is primarily determined by the TNM classification of staging system ([@ref-54]). The clinical outcome, nonetheless, is notably variable and erratic in individual patient, which firmly implies that a few of the biological determinants of tumor behavior are unidentified. Thus, advances in molecular insight into GC are critically required for improved prognostic stratification and new targeted therapeutic strategies. Recently, with the progress of high-throughput screening, sequencing has enabled a more thorough insight into the molecular identity of GC. An updated classification scheme has been introduced based on comprehensive molecular characterization including tumors infected with Epstein--Barr virus, tumors with microsatellite instability (MSI), and tumors with a distinct degree of aneuploidy, which were termed genomic stability and chromosomal instability. Each subgroup shows peculiar genetic and clinical characteristics ([@ref-6]). Alterations of DNA methylation is a vital event during tumorigenesis, and gastrointestinal cancers show the highest frequency of DNA methylation alterations among the reported tumor types ([@ref-6]). Methylation of the dinucleotides of CpG islands throughout the genome is mediated by DNA methyltransferases ([@ref-10]), and commonly results in gene silencing. Disorder of DNA methylation in cancer affects gene expression and results in the cancer progression ([@ref-53]). CpG island methylator phenotype (CIMP) in tumors, which has been initially described and broadly debated in colorectal cancer ([@ref-21]). Lately CIMP has been described in other tumor types including bladder, breast, glioblastoma, pancreatic and prostate cancers, as well as for gastric adenocarcinomas and is considered to be helpful for predicting prognosis ([@ref-22]; [@ref-36]; [@ref-52]). In GC, conflicting conclusions regarding the prognostic association of CIMP have been scattered among previous studies, owing to the limitation of selected DNA methylation markers and the presence of multiple confounding factors in these studies ([@ref-1]; [@ref-4]; [@ref-39]). Although a meta-analysis of the prognostic value of CIMP status in GC has been performed, an explicit conclusion was not reached ([@ref-42]). In this study, we aimed to use publicly available data to comprehensively analyze CIMP in GC, and to develop a CIMP-related prognostic gene signature. Materials and Methods ===================== Data acquisition ---------------- We downloaded methylation data, which has 408,376 probes and 397 samples, including 395 GC samples and two normal samples, measured by the Illumina HumanMethylation450 platform, from the cancer genome atlas (TCGA)-STAD project (<https://portal.gdc.cancer.gov/>) by using the *TCGA-Assembler 2* package ([@ref-55]). RNA-Seq profiles were obtained from TCGA by virtue of *GDC Data Transfer Tool*. We downloaded two verified microarrays with matched clinical information from the GEO GC database: [GSE13861](GSE13861) ([@ref-8]) (65 GC samples; platform: GPL6884 Illumina HumanWG-6 v3.0 expression beadchip ), [GSE62254](GSE62254) ([@ref-11]) (300 GC samples; platform: GPL570 Affymetrix Human Genome U133 Plus 2.0 Array ). We used the *TCGAbiolinks* package to acquire the mutation data of GC samples ([@ref-9]). We obtained complete and matched clinical information on GC patients from cBioportal, including sex, age, histologic features, pathologic stage, family history and, infection status for *Helicobacter pylori* and Epstein--Barr virus. Our study was performed according to the publication guidelines required by TCGA. Data analysis ------------- The *Minfi* package was adopted to analyze methylation data ([@ref-2]). In view of the distribution of CpG islands and the technical limitations of sequencing, we filtered the probes from the X and Y chromosomes or probes that are known to have common SNPs at the CpG site, and cross-reactive probes. The *DESeq2* package was adopted to analyze the differentially expressed genes (DEGs) between CIMP-related subgroups ([@ref-32]). The criteria to determine DEGs were an adjusted *p*-value \< 0.05 and an absolute value of log2 fold change \>2, and BH method was used for adjustment for multiple testing. In order to identify the different pathways between GC samples with specific CIMP status, we performed GSEA analysis ([@ref-48]). The mRNA expression data downloaded from the Gene Expression Omnibus (GEO) database were normalized and analyzed by the *limma* package ([@ref-44]). The mutation data were summarized and analyzed by the *maftools* package ([@ref-34]). Raw code for analyzing was uploaded in [Data S1](#supp-9){ref-type="supplementary-material"}. Identification of CIMP in GC samples ------------------------------------ To assess the CIMP feature in GC, CpG methylation sites with a relatively high variability of β-values in tumor samples (SD \> 0.2) and relatively low β-values in normal samples (mean β value \< 0.05), were chosen as the representative CpG methylation sites for subsequent clustering analysis, following a previous study ([@ref-28]). The *ConsensusClusterPlus* package was adopted to run unsupervised clustering analysis based on M value of the selected 2,082 probes by means of the *K*-means algorithm ([@ref-56]). Analysis of tumor-infiltrating immune cells ------------------------------------------- The proportions of the 22 types of tumor-infiltrating immune cells were counted by CIBERSORT ([@ref-37]). CIBERSORT is a tool to provide an estimation of cell composition of mixed tissues based on gene expression profiles. We uploaded the modified gene expression data and standard annotation to the CIBERSORT portal and ran the LM22 signature, which contains 547 genes distinguishing 22 human immune cell types, and 1,000 permutations. Final results were normalized to sum up to one and could be assessed straightforwardly as cell fractions for contrast ([Table S1](#supp-4){ref-type="supplementary-material"}). Development and validation of a CIMP-related prognostic signature ----------------------------------------------------------------- Using the *DESeq2* package, 1,072 DEGs were calculated between the CIMP-H and CIMP-L samples ([Table S2](#supp-5){ref-type="supplementary-material"}), which were defined by relative methylation level. We then performed Cox regression to assess the prognostic significance of the DEGs. A suitable prognostic model is assumed to identify a smaller number of genes favorable for clinical practice. We therefore used the least absolute shrinkage and selection operator (LASSO) in combination to diminish the number of CIMP-related prognostic genes ([@ref-15]). The *glmnet* package was used to perform the penalized Cox regression model with the LASSO penalty, and 1,000-times cross-validations were applied to determine the optimal values of the penalty parameter lambda. We selected lambda.min to get six CIMP-related prognostic genes. We then extracted the coefficients from multivariate Cox regression to build a gene signature. We adopted the *survminer* package to determine the cut-off value of the risk score. Then, the patients were divided into high-and low-risk subgroups according to their risk score. We used Kaplan--Meier analysis to compare OS rates between the high- and low-risk group. To identify whether the risk score was an independent factor, we conducted univariate Cox regression and multivariate Cox regression analyses. Statistical significance was inferred where *p* \< 0.05. Validation in GEO dataset ------------------------- To confirm the performance of our prognostic signature, we applied it to two GEO databases, [GSE13861](GSE13861) (*n* = 65) and [GSE62254](GSE62254) (*n* = 300). The mRNA expression data were prepared using the *limma* package. Scale, a generic R function including centering and scaling, was used to scale the GEO mRNA expression data to common range. Next we used a developed prognostic signature to calculate the risk score of every sample and divided patients by the cut-off value. Kaplan--Meier analysis was conducted between the high- and low-risk groups for overall survival. In addition, we conducted receiver operating characteristic (ROC) curve analysis and calculated an AUC for every database. Functional enrichment analysis ------------------------------ We applied the *clusterProfiler* package for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis between different risk score-related subgroups ([@ref-59]). Then we adopted the *GOplot* package to illustrate our GO and KEGG results ([Tables S3](#supp-6){ref-type="supplementary-material"} and [S4](#supp-7){ref-type="supplementary-material"}). Protein--protein interaction analysis was carried out based on the DEGs of high-and low-risk subgroups using the STRING portal (<https://string-db.org>) and Cytoscape software ([@ref-47]). Functional annotation of genes in the module was perform by DAVID database ([@ref-20]). Results ======= Methylation landscape of GC sample ---------------------------------- In this study, we utilized DNA methylation profiles from TCGA database to perform a comprehensive analysis of DNA methylation in GC. We adopted 2,082 methylation sites with high variability as our CIMP signature for downstream analysis. Unsupervised hierarchical clustering analysis of 395 GC samples based on our specific CIMP signature was performed and all the patients were separated into three subgroups as CIMP-L, CIMP-M and CIMP-H ([Figs. 1A](#fig-1){ref-type="fig"} and [1B](#fig-1){ref-type="fig"}; [Table S5](#supp-8){ref-type="supplementary-material"}). The CIMP-L subgroup had the lowest methylation level, while the CIMP-H subgroup had broad hypermethylation across these sites. In addition, we plotted the Delta area and consensus CDF to verify our clustering pattern ([Figs. S1A](#supp-1){ref-type="supplementary-material"} and [S1B](#supp-1){ref-type="supplementary-material"}). To assess the performance of our classification based on CIMP signature, we reclassified GC samples according to a previous DNA methylation clustering analysis ([@ref-6]). A strong concordance was exhibited between these two classification systems ([Fig. 1C](#fig-1){ref-type="fig"}). The C1 cluster, representing an EBV-associated DNA methylation signature with extreme hypermethylation, consisted primarily of CIMP-H samples, while the C4 cluster, representing a hypomethylated subgroup, consisted mainly of CIMP-L. Importantly, to evaluate the correlation between CIMP and prognosis, the overall survival of each subgroup was assessed by the Kaplan--Meier method. The result indicated a significant difference in prognosis among the different CIMP-related subgroups, with the CIMP-H group showing better prognosis and the CIMP-L group showing worse prognosis ([Fig. 1D](#fig-1){ref-type="fig"}). In addition, we investigated the relationship between CIMP and progression-free survival (PFS). However, we found no significant differences in PFS existed among the CIMP-related subgroups ([Fig. S1C](#supp-1){ref-type="supplementary-material"}). {#fig-1} The clinical characteristics of patients with different CIMP statuses were summarized ([Table 1](#table-1){ref-type="table"}). Clinical features, including lymph node metastasis, MSI status and EBV infection, had significant differences between CIMP-related subgroups. No significant difference was found in aspects of age, gender, pathologic tumor classification, Lauren classification, grade or *Helicobacter pylori* infection. Specifically, within the CIMP-H subgroup more patients suffered MSI gastric adenocarcinoma and EBV infection, and less had lymph node metastasis. In addition, we found that in the CIMP-H subgroup, patients with EBV could be well distinguished from patients with MSI ([Fig. S1D](#supp-1){ref-type="supplementary-material"}). 10.7717/peerj.9624/table-1 ###### Clinical and demographic characteristics of GC patients in CIMP-related subgroups.  CIMP-L CIMP-M CIMP-H *p*-Value ----------------------- ------------- ------------- ------------- ----------- Number of patients 179 128 88 Age (Mean (SD)) 64.3 (10.5) 65.6 (10.5) 66.4 (11.3) 0.191 Gender 0.194 Female 70 (39.1%) 38 (29.7%) 28 (31.8%) Male 109 (60.9%) 90 (70.3%) 60 (68.2%) Pathologic_T 0.724 T1 7 (3.9%) 7 (5.5%) 7 (8.0%) T2 39 (21.8%) 24 (18.8%) 15 (17.0%) T3 85 (47.5%) 63 (49.2%) 38 (43.2%) T4 48 (26.8%) 34 (26.6%) 28 (31.8%) Pathologic_N 0.044 N0 51 (28.5%) 36 (28.1%) 37 (42.6%) N1 49 (27.4%) 31 (24.2%) 22 (25.3%) N2 36 (20.1%) 34 (26.6%) 9 (10.3%) N3 38 (21.2%) 27 (21.1%) 18 (20.7%) NX 5 (2.8%) 0 (0%) 1 (1.1%) Pathologic_M 0.115 M0 155 (86.6%) 115 (89.8%) 83 (94.3%) M1 11 (6.1%) 10 (7.8%) 2 (2.3%) MX 13 (7.3%) 3 (2.3%) 3 (3.4%) Pathologic_Stage 0.225 Stage I 23 (12.8%) 14 (10.9%) 16 (18.2%) Stage II 58 (32.4%) 42 (32.8%) 32 (36.4%) Stage III 78 (43.6%) 59 (46.1%) 38 (43.2%) Stage IV 20 (11.2%) 13 (10.2%) 2 (2.3%) Lauren.Class 0.112 Diffuse 49 (27.4%) 36 (28.1%) 18 (20.5%) Intestinal 115 (64.2%) 71 (55.5%) 61 (69.3%) Mixed 15 (8.4%) 21 (16.4%) 9 (10.2%) Grade 0.264 G1 4 (2.2%) 3 (2.3%) 2 (2.3%) G2 76 (42.5%) 41 (32.0%) 25 (28.4%) G3 94 (52.5%) 81 (63.3%) 60 (68.2%) GX 5 (2.8%) 3 (2.3%) 1 (1.1%) *H. pylori* infection 0.779 No 79 (87.8%) 56 (90.3%) 33 (91.7%) Yes 11 (12.2%) 6 (9.7%) 3 (8.3%) MSI.status \<0.001 MSI-H 4 (4.0%) 11 (14.4%) 34 (47.9%) MSI-L 11 (10.9%) 18 (23.7%) 8 (11.3%) MSS 86 (85.1%) 47 (61.9%) 29 (40.8%) EBV.positive \<0.001 Negative 101 (100%) 76 (100%) 46 (64.8%) Positive 0 (0%) 0 (0%) 25 (35.2%) Gene set enrichment analysis in different CIMP-related subgroups ---------------------------------------------------------------- To identify the biological processes or pathways potentially regulated by the CpG island methylation signature, we applied the GSEA analysis between different CIMP-related subgroups based on RNA-seq profiles. We found that the gene signatures of "Rickman metastasis up", "Vesicle localization", "Insulin receptor signaling pathway", "Regulation of glucose transmembrane transport", "Serotonin receptor signaling pathway", "G-protein coupled amine receptor activity" were enriched in CIMP-L subgroup ([Figs. 2A](#fig-2){ref-type="fig"}--[2F](#fig-2){ref-type="fig"}). Importantly, all the pathways have been linked to GC progression. {#fig-2} Tumor-infiltrating immune cells in different CIMP-related subgroups ------------------------------------------------------------------- We assessed the presence of tumor-infiltrating immune cells (TIICs) in CIMP-related subgroups by using CIBERSORT ([Fig. 3](#fig-3){ref-type="fig"}). Obviously, immune cells showed differential infiltration pattern between CIMP-related subgroups. The proportions of B cells, plasma cells, T cells CD4 memory resting, regulatory T cells and resting mast cells were significantly higher in the in the CIMP-L subgroup. Meanwhile, CD8+ T cells, T cells CD4 memory activated, T follicular helper cells, M1 macrophages, and Dendritic cells resting were higher in the CIMP-H subgroup. Other immune cells, including NK cells and monocytes, didn't show significant differences. In addition, we found in most samples no apparent T cells CD4 naïve, T cells gamma delta and Eosinophils were infiltrated. These results indicated that CIMP-L subgroup have a distinct immune phenotype, which is considered to impair and suppress antitumor immunity. {#fig-3} Analysis of DNA somatic mutations in patients with distinct CIMP status ----------------------------------------------------------------------- A distinct set of genetic aberrations between CIMP-related subgroups was evident in our study. We found 350 samples with mutations in a total of 391samples (89.51%), with TTN and TP53 ranking as the most common mutation gene ([Fig. 4A](#fig-4){ref-type="fig"}). Mutation of the TP53 gene was found enriched in the CIMP-L subgroup. At the same time, mutations of TTN and MUC16 were higher in the CIMP-H subgroup. The most common mutations in GC samples were missense mutations, comprising the majority of SNPs, the main SNV classification was C \> T transition, and the number of altered bases in each sample was counted ([Figs. 4B](#fig-4){ref-type="fig"}--[4E](#fig-4){ref-type="fig"}). We then showed the top 10 mutated genes in GC with ranked percentages, including TTN (53%), MUC16 (31%), TP53 (46%), LRP1B (27%), SYNE1 (26%), ARID1A (24%), CSMD3 (23%), FAT4 (19%), FLG (20%), HMCN1 (19%), and summarized the mutation types in GC ([Figs. 4F](#fig-4){ref-type="fig"} and [4G](#fig-4){ref-type="fig"}). We then calculated the tumor mutation burden (TMB), which is considered to correlate with enhanced clinical response to immunotherapy and superior OS. We found TMB was higher in the CIMP-H subgroup ([Fig. 4H](#fig-4){ref-type="fig"}). {#fig-4} Establishment of a CIMP-related prognostic gene signature --------------------------------------------------------- To screen differentially expressed genes (DEGs) in CIMP subgroups, we downloaded RNA-seq data for 208 samples defined as CIMP-H or CIMP-L subgroups from the TCGA database and analyzed the data by using the *DEseq2* package. We identified 1,072 DEGs, which we narrowed down to 147 genes highly associated with OS using univariate Cox regression. To obtain the genes with the highest potential prognostic values, we used least absolute selection and shrinkage operator (LASSO) regression analysis. A prognostic signature comprising six genes, including *cystatin E/M (CST6), solute carrier family 7 member 2 (SLC7A2), RAB3B, member RAS oncogene family (RAB3B), insulin like growth factor binding protein 1 (IGFBP1), V-set and transmembrane domain containing 2 like (VSTM2L)* and *even-skipped homeobox 2 (EVX2)*, was developed ([Figs. 5](#fig-5){ref-type="fig"}, [1A](#fig-1){ref-type="fig"} and [1B](#fig-1){ref-type="fig"}; [Table 2](#table-2){ref-type="table"}). The risk score was calculated as follows: risk score = (0.230 × the normalized expression of *CST6*) + (0.257 × the normalized expression of *SLC7A2*) + (0.156 × the normalized expression of *RAB3B*) + (0.114 × the normalized expression of *IGFBP1*) + (0.024 × the normalized expression of *VSTM2L*) + (0.187 × the normalized expression of *EVX2*). The cutoff value (0.235) was counted by the *survminer* package ([Fig. 5C](#fig-5){ref-type="fig"}). The patients were then divided into high- and low-risk subgroups according to their risk score. We found high-risk patients had more deaths and higher expression levels of CIMP-related prognostic genes ([Figs. 5D](#fig-5){ref-type="fig"} and [5E](#fig-5){ref-type="fig"}). We then found those in the high-risk had a worse OS than those in the low-risk group ([Fig. 5F](#fig-5){ref-type="fig"}). To access the performance of the prognostic signature, time-dependent ROC curves and AUC were printed and counted ([Fig. 5G](#fig-5){ref-type="fig"}). The AUC was 0.664 at 1 year, 0.704 at 3 years and 0.667 at 5 years. The univariate and multivariate Cox regression analyses indicated that the predictive value of risk score for overall survival was independent of CIMP status ([Figs. 5H](#fig-5){ref-type="fig"} and [5I](#fig-5){ref-type="fig"}). {#fig-5} 10.7717/peerj.9624/table-2 ###### The CIMP-related prognostic gene signature based on six genes in TCGA cohort. HR: hazard ratio, CI: confidential interval.  Univariate Cox regression -------- ------- --------------------------- ------------------ ---------- ------- CST6 0.230 1.327 \[1.139--1.547\] 2.80E−04 3.633 SLC7A2 0.257 1.405 \[1.192--1.656\] 5.01E−05 4.055 RAB3B 0.156 1.361 \[1.152--1.607\] 2.76E−04 3.637 IGFBP1 0.114 1.244 \[1.070--1.448\] 4.63E−03 2.832 VSTM2L 0.024 1.233 \[1.053--1.444\] 9.44E−03 2.596 EVX2 0.187 1.221 \[1.084--1.376\] 1.02E−03 3.284 Validation and evaluation of the CIMP-related signature in the GEO cohort ------------------------------------------------------------------------- To further verify the robustness of the six-genes prognostic signature in GC, two verified microarrays with matched clinical information from the GEO GC database were analyzed. In every dataset, patients were stratified into high-or low-risk group according to the cutoff point calculated following the prognostic signature. Consistent with the results from the TCGA cohort, the high-risk group had a worse survival outcome in two datasets ([Figs. 6A](#fig-6){ref-type="fig"} and [6B](#fig-6){ref-type="fig"}). Indicating favorable performance of our prognostic signature, the AUC of the [GSE13861](GSE13861) dataset was 0.638 at 1 year, 0.777 at 3 years, 0.745 at 5 years ([Figs. 6C](#fig-6){ref-type="fig"}). The AUC of [GSE62254](GSE62254) dataset was 0.674 at 1 year, 0.627 at 3 years and 0.615 at 5 years ([Fig. 6D](#fig-6){ref-type="fig"}). We then compared our CIMP-related prognostic signature with two prognostic signatures published previously. We extracted formulae from each study, and the results of ROC curve analysis implied that the CIMP-related prognostic signature was comparable or better at predicting the prognosis in the TCGA cohort ([Figs. 6E](#fig-6){ref-type="fig"} and [6F](#fig-6){ref-type="fig"}). Taken together, the prognostic signature based on CIMP was a reliable prognostic marker in GC.  (*n* = 65) and (B) [GSE62254](GSE62254) (*n* = 300) cohorts. Receiver operating characteristic curve of CIMP-related prognostic signature at different years in (C) [GSE13861](GSE13861) (*n* = 65) and (D) [GSE62254](GSE62254) (*n* = 300) cohorts. (E) and (F) Receiver operating characteristic curves of the other signatures reported in previous studies in the prediction of OS for TCGA cohort.](peerj-08-9624-g006){#fig-6} The risk score developed from the six genes signature as an independent prognostic factor ----------------------------------------------------------------------------------------- We contrasted the prognostic value of the risk score was contrasted with clinical parameters by univariate and multivariate analyses. Clinical parameters included diagnostic age, gender, pathologic TNM, pathologic stage, pathologic grade, Lauren classification, status of *H. pylori* and EB virus infection and MSI status. We found that risk score acted as an independent prognostic factor and had significant effects in both the univariate analysis and the multivariate analysis, with *p* values \< 0.05 ([Fig. 7A](#fig-7){ref-type="fig"}). Furthermore, the risk score had robust prognostic value (with HR = 3.364, 95% CI \[1.906--5.937\]). We then used the risk score as a nomogram to predict patients' outcome ([Fig. 7B](#fig-7){ref-type="fig"}). The Calibration plot indicated that predicted OS and the actual OS rates at 1,3 and 5 years were similar ([Figs. 7C](#fig-7){ref-type="fig"}--[7E](#fig-7){ref-type="fig"}). To verify the role of methylation in the expression of prognostic signature gene, we used Pearson correlation to evaluate the relationship between the methylation levels of the *CST6, SLC7A2, RAB3B, IGFBP1, VSTM2L* and *EVX2* promoters and their expression levels. Consistent results were found among these six genes ([Figs. 8A](#fig-8){ref-type="fig"}--[8F](#fig-8){ref-type="fig"}). Moreover, the expression of signature genes was consistent among the CIMP-related subgroups. Expression levels of the signature genes were higher in the CIMP-L subgroup than those in the other subgroups ([Figs. 8G](#fig-8){ref-type="fig"}--[8L](#fig-8){ref-type="fig"}). {#fig-7} {#fig-8} Distinct biological processes in risk score stratified subgroups ---------------------------------------------------------------- We identified 382 DEGs between the high-risk and low-risk subgroups in GC samples. Then, we carried out GO and KEGG analyses to identify the molecular mechanisms associated with these DEGs. For GO analysis, the top five enriched terms were "cornification", "keratinocyte differentiation", "epidermis development", "keratinization" and "epidermal cell differentiation" ([Fig. 9A](#fig-9){ref-type="fig"}). In KEGG analysis four pathways were enriched, including "neuroactive ligand-receptor interaction", "complement and coagulation cascades", "staphylococcus aureus infection" and "cholesterol metabolism". The "neuroactive ligand-receptor interaction" was shown to be the main associated pathway with 14 genes involved ([Fig. 9B](#fig-9){ref-type="fig"}). In addition, STRING was used to draw 382 DEGs into a PPI network complex, which contained 366 nodes and 1,176 interactions ([Fig. S3](#supp-3){ref-type="supplementary-material"}). Then, Cytoscape was used to identify the most significant module in the PPI network. The most significant module (score = 17.2) recognized by MCODE, a plug-in of Cytoscape, contained 38 nodes and 318 interactions ([Fig. 9C](#fig-9){ref-type="fig"}). Consistent with the results of GO analysis, the genes in the module were found to be related to "keratinocyte differentiation", "keratinization", "peptide cross-linking" and "epidermis development" ([Table 3](#table-3){ref-type="table"}). {#fig-9} 10.7717/peerj.9624/table-3 ###### The enriched GO terms of genes in the most significant module.  ID Term Count *p*-Value -------------- ------------------------------ ------- ----------- <GO:0030216> keratinocyte differentiation 15 5.49E−25 <GO:0031424> keratinization 13 3.49E−23 <GO:0018149> peptide cross-linking 13 6.06E−23 <GO:0008544> epidermis development 11 7.52E−16 <GO:0002576> platelet degranulation 8 1.25E−09 Discussion ========== In the field of cancer research, increasing attention has focused on DNA methylation. Patterns of DNA methylation can predict prognosis and survival of human cancers ([@ref-16]). CIMP refers to promoter CpG island hypermethylation and is well characterized in colorectal cancers. In contrast, the relationships between CIMP and clinicopathological features are controversial in GC. Previous studies on CIMP used different methods to measure methylation, like measuring across several CpG sites of a gene or across several genes. Conflicting conclusions may be drawn, due to variation among studies in methodologies of DNA methylation analysis and CIMP marker panels, which brought the bias of chosen marker panels ([@ref-1]; [@ref-38]; [@ref-39]; [@ref-42]). In the present study, we used the methylation data measured by the Illumina HumanMethylation450K platform to assess the DNA methylation status of 40,8376 CpG sites including CpG sites located at the promoter regions of protein-coding genes in multiple samples simultaneously. We adopted methylation sites with high variability as our CIMP signature for unsupervised hierarchical clustering to comprehensively assess CIMP status in GC. In addition, our study included more patients with methylation data from the same platform than previous study ([@ref-6]). We firstly used this methodology to divide GC samples into three distinct subgroups according to their levels of methylation at selected methylation sites and describe the positive relation between CIMP and prognosis at the global methylation level, in contrast to analyses using chosen markers. Consistent with this, our CIMP-H subgroup showed more favorable clinical characteristics including less lymph node metastasis and lower metastasis status. Promoter hypermethylation is a prominent feature of EBV-associated GC, and we found that samples with EBV infection were enriched in the CIMP-H subgroup in this study ([@ref-24]). Previous studies indicated that GC patients with MSI show a significant longer overall survival compared with those who have MSS and assumed that MSI GC has a better prognosis because of its earlier stage at diagnosis, less lymph node metastasis and intestinal histological type ([@ref-33]; [@ref-41]). Consistent with these researches, samples with MSI were enriched in the CIMP-H subgroup which showed a better prognosis in our study. We then plotted the Kaplan--Meier survival curves according to previous DNA methylation clustering. However, we found no significant differences in this clustering ([Fig. S2A](#supp-2){ref-type="supplementary-material"}). We believed that it was caused by too few cases in the C1 group, which was corresponding to our CIMP-H group. Therefore, we combined C2 and C3 groups, and plotted Kaplan-Meier survival curves between C2 + C3 and C4 groups. We found that the difference in survival was still not statistically significant, but we could see the difference in survival between these two groups ([Fig. S2B](#supp-2){ref-type="supplementary-material"}). We believed it was due to the fact that the previous DNA methylation clustering was based on the merger of two methylation platforms, and that the number of samples included was not as large as ours. In our study, we found that there was no significant relationship between CIMP and PFS in GC. However, considering that some patients lack PFS information, we need to include more patients with complete PFS information to clarify the role of CIMP in PFS of GC in future research. Our study provided insight into the landscapes of molecular features in patients with distinct CIMP statuses. Based on the gene set enrichment analysis, we identified cancer-related oncogenic pathways enriched in the CIMP-L subgroup, including metastasis, vesicle transfer, G-protein coupled receptor, energy transfer. Cancer cell-derived vesicles serve as intercellular communication vehicles and carry pathogenic components, such as proteins, mRNA, miRNA, DNA, lipids and transcriptional factors, that can mediate paracrine signaling in the tumor microenvironment ([@ref-14]). Vesicles mediate the formation of pre-metastatic niches to promote metastasis in tumors, including GC ([@ref-12]; [@ref-23]; [@ref-40]). [@ref-26] reported that Insulin-like growth factor-I (IGF1) regulated the expression of the VEGF ligand to facilitate angiogenesis and lymphangiogenesis in GC cell lines, and blocking IGF1 could enhance the effectiveness of bevacizumab. High glucose conditions were shown to promote GC cell proliferation and reduce susceptibility to chemotherapy ([@ref-61]). In addition, serotonin-induced signaling pathways promoted tumor progression ([@ref-45]). This suggests these pathways could have potential as novel drug targets. Immunotherapy is becoming a routine cancer treatment option, and disparate tumor-infiltrating immune cells profiles were observed among CIMP-related subgroups. CD8+ (cytotoxic) T cells are very important for immune defense and tumor surveillance, and are known to correlate with more favorable outcome in GC ([@ref-17]). In GC, tumor-associated plasma cells are polarized to produce IgG4 and associated with tumor progression and poor prognosis ([@ref-35]). Regulatory T cells (Tregs) are T cells which have a role in regulating or suppressing other cells in the immune system, leading to limiting excessive immune responses. Tregs suppress activation, proliferation and cytokine production of CD4+ T cells and CD8+ T cells. Tregs are thought to suppress B cells and dendritic cells. [@ref-30] revealed that Tregs promoted Lgr5 expression in GC cells via TGF-β1 signaling pathway and was negatively associated with survival. Differences in the levels of the M1 macrophages were also observed. M1 macrophages are an integral cellular component of the immune system, and play a critical role in protection against intracellular pathogens and cancer cells ([@ref-58]). M1 macrophages have been reported to inhibit tumor growth in GC ([@ref-31]). Recently, TMB has been increasingly accepted as a biomarker of response to immunotherapy. High TMB contributes to the synthesis of aberrant and potentially immunogenic mutation-associated neoantigens by the cancer cells, which attract CD8+ CTLs and activated Th1 cells to the tumor microenvironment. In this study, we found higher TMB in the CMIP-H subgroup, potentially indicating a better response to immunotherapy in this group. Consistent with the distribution of TMB, more MSI samples, especially MSI-H, were detected in the CIMP-H subgroup. The prevalence of MSI in GC is relatively high, and as MSI-H GCs are strongly associated with PD-L1 positivity, they could be applicable targets of anti-PD-1 therapies ([@ref-25]). The mutations of TTN and MUC16 were announced to be correlated with better survival result in lung and GCs ([@ref-7]; [@ref-27]). Recent researches have indicated that clinicopathological parameters such as tumor depth, lymph node metastasis, margin status, and metastatic condition are unsatisfactory for accurately predicting patient prognosis. Outcome varies tremendously among patients with comparable clinicopathological features. Therefore, with the advantage of high-throughput sequencing technologies, mRNAs have been used as molecular biomarkers of the cancer diagnosis and prognosis and shown their critical clinical application potential. For examples, [@ref-62] investigated genes relevant to the cell cycle from the TCGA database and described a set of five genes (*MARCKS, CCNF, MAPK14, INCENP* and *CHAF1A*), which were significantly associated with OS. They used this signature to stratify GC patients into two groups with significantly different survival outcomes. Distinct clinical features were also demonstrated between the two groups. In another research, the predictive value of DNA methylation gene for prognosis was determined in GC, and different pathways and biological processes associated with tumorigenesis were found in groups with distinct gene methylation levels ([@ref-19]). In our study, a gene signature including *CST6, SLC7A2, RAB3B, IGFBP1*, *VSTM2L* and *EVX2*, was developed based on CIMP. *CST6* has been reported to play a role in the progression of triple-negative breast cancer (TNBC) and may act as a tumor-promoter gene. High *CST6* expression was also associated with a higher rate of lymph node metastasis ([@ref-29]). *SLC7A2* is essential for transport of L-arginine, lysine and ornithine and genetic polymorphisms in the *SLC7A2* gene are associated with colorectal cancer progression ([@ref-49]). *SLC7A2* has also been found to play a role in radio-resistance of non-small cell lung cancer. *RAB3B*, a member of RAS oncogene family, is shown to be a target of miR-200b, which is supposed to be tumor suppressor in GC ([@ref-51]; [@ref-57]). *RAB3B*, has been shown to be overexpressed in prostate cancer patients and promote prostate cancer cell survival ([@ref-50]). *IGFBP1*, an insulin-like growth factor binding protein, is revealed to be associated with hematogenous metastasis and poor survival in GC. And the expression of *IGFBP1* is positively associated with tumor invasion, lymph node metastasis and vascular invasion ([@ref-46]). *VSTM2L* is reported to be downregulated in the *H. pylori*-positive GC samples ([@ref-18]). However, the role of *VSTM2L* in tumors is rarely reported, and its role requires further studies. *EVX2* is recently revealed to be regulated by methylation and serve as a methylation biomarker for lung cancer ([@ref-43]). In our study, the expression of *EVX2* was higher in CIMP-L subgroup consistent with its mechanism of epigenetic regulation. Our six-genes risk signature was an independent prognostic biomarker of GC, with patients in high-risk groups showing significantly worse prognosis than those in low-risk groups. Our results support the notion that gene risk signature might have more predictive power than traditional prognostic parameters. The prognostic performance of our signature was validated in the TCGA dataset, and the external datasets [GSE13861](GSE13861) and [GSE62254](GSE62254). Further, it compared favorably to two other gene-based signatures ([@ref-13]; [@ref-62]), based on ROC and AUC analyses predicting 1-, 3- and 5-year overall survival. In our study, distinct biological analyses in risk score stratified subgroups indicated that keratinization and keratinization-related processes may play an important role in GC progression. At present, there is no research about the role of keratinization in GC and it needs further studies. In addition, the neuroactive ligand-receptor interaction was shown to be the most significant pathway in stratified subgroups and revealed to be involved in apoptosis and cell proliferation ([@ref-60]). Conclusion ========== In summary, we first identified an accurate and comprehensive association between CIMP and clinical prognosis in GC, where high CIMP indicated better patient prognosis. We then developed and validated a six-genes prognostic signature related to CIMP that can predict the survival of patients with GC, where higher risk score indicated worse patient prognosis. This signature could be an effective tool in clinical practice as a supplement to traditional staging system to indicate progression and predict overall survival of GC. However, our study has some limitations. Firstly, it was based on a retrospective design, so the numbers of patients with the same clinical features in CIMP subgroups were not comparable. Also, the number of datasets used to validate the clinical prognostic signature is not large, and further validation by future prospective studies is desirable. Knowledge of the signature genes in GC development is currently scarce and further experiments are needed to verify their potential molecular mechanisms. Supplemental Information ======================== 10.7717/peerj.9624/supp-1 ###### Unsupervised hierarchical clustering analysis of GC samples. \(A\) Delta area of the consensus clustering. (B) Consensus cumulative distribution function (CDF) of the consensus clustering. (C) Progression-free survival curves of CIMP-H and CIMP-L subgroups. (D) MSI status in patients with EBV in CIMP-H subgroup. ###### Click here for additional data file. 10.7717/peerj.9624/supp-2 ###### The relationship between prognosis and DNA methylation clustering reported by Cancer Genome Atlas Research. \(A\) Kaplan-Meier survival curves of the C1, C2, C3 and C4 groups. The C1 group has the most prevalence of DNA hypermethylation and the C4 group has the lowest methylation level. The C2 and the C3 group have the medium methylation level. (B) Kaplan-Meier survival curves of the C2 + C3 and C4 groups. The C2 + C3 group represents the combination of the C2 and C3 groups. ###### Click here for additional data file. 10.7717/peerj.9624/supp-3 ###### The PPI network of differentially expressed genes between risk score stratified subgroups. The PPI network was analyzed by String software. 366 nodes and 1176 edges are in the PPI network. ###### Click here for additional data file. 10.7717/peerj.9624/supp-4 ###### Tumor-infiltrating immune cells profile. ###### Click here for additional data file. 10.7717/peerj.9624/supp-5 ###### Differentially expressed genes between CIMP-H and CIMP-L groups. ###### Click here for additional data file. 10.7717/peerj.9624/supp-6 ###### The result of GO analysis. ###### Click here for additional data file. 10.7717/peerj.9624/supp-7 ###### The result of KEGG analysis. ###### Click here for additional data file. 10.7717/peerj.9624/supp-8 ###### The result of cluster in TCGA cohort. ###### Click here for additional data file. 10.7717/peerj.9624/supp-9 ###### Raw code. ###### Click here for additional data file. We thank Jie Shen and Liang Liu for comments on this article. We appreciate Ganxun Li and Furong Liu for assistance with the bioinformatics analysis. Additional Information and Declarations ======================================= The authors declare that they have no competing interests. [Zhuo Zeng](#author-1){ref-type="contrib"} performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft. [Daxing Xie](#author-2){ref-type="contrib"} performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. [Jianping Gong](#author-3){ref-type="contrib"} conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft. The following information was supplied regarding data availability: The raw data are available from TCGA project TCGA-STAD and from the NCBI GEO GC database (accession numbers: [GSE13861](GSE13861), [GSE62254](GSE62254)). The code used to analyze the data is available as a [Supplemental File](#supplemental-information){ref-type="supplementary-material"}.

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Introduction ============ Public schools have an obligation to question and refute policies that do not benefit their students and their communities and a corresponding responsibility to protect students, for whom school attendance is mandated, from harm. However, implementation of change in school procedures and policies presents challenges and requires an ethical justification for the change and feasible methods for accomplishing it. The mission of schools is broader than simply teaching academic skills. Schools have long accepted responsibility for supporting the health of their students, for example, by requiring immunizations, providing health screenings, and by offering meal programs that support their students\' nutritional health. Nutritional health is associated with academic performance ([@B1]), and well-nourished students are better able to learn and less likely to miss school for health reasons ([@B2]). Research shows that children from low-income families who participate in school breakfast programs score higher on standardized tests and have better school attendance than similar students who do not participate ([@B3]). Breakfast programs also improve classroom behavior and attentiveness ([@B4]). In 1904, Robert Hunter wrote, \"It is utter folly, from the point of view of learning, to have a compulsory school law which compels children, in that weak physical and mental state . . . to sit at their desks, day in and day out for several years, learning little or nothing . . . because hungry stomachs and languid bodies and thin blood are not able to feed the brain\" ([@B5]). Focusing on nutritional health promotion in schools can support the common good by reducing the impact, including substantial financial costs, of future diet-related disease associated with the childhood obesity epidemic. Furthermore, optimizing nutrition in childhood is critical to learning and future productivity. We must consider whether schools have an ethical obligation to serve the common good in this area, even if the actions they take appear to conflict with the autonomy or freedom of choice of children, parents, and school staff, or the interests of food and beverage companies. The purpose of this article is to present a bioethics framework for justifying stricter regulation of school food, specifically, to determine whether this type of health promotion in schools is ethically justified ([@B6]). To determine whether current school environments meet an ethical threshold or whether these environments fall short and should be altered, we will apply Beauchamp and Childress\'s 4 foundational principles for a discourse on the ethics of a biomedical intervention: autonomy (addressing conflict around individualism), beneficence (addressing the social benefit), nonmaleficence (addressing the issue of doing no harm), and justice (addressing equity in burdens and benefits) ([@B7]). We describe the underlying problem of rapidly increasing incidence rates of childhood obesity and the potential role of schools in altering the trend. Schools\' Roles in Addressing Childhood Obesity =============================================== The National School Lunch Program was established in 1946 to \"safeguard the health and well-being of the Nation\'s children\" as a \"measure of national security\" by preventing the widespread malnutrition that disqualified many military recruits during World War II ([@B8]). Early program participants were served balanced meals to ensure consumption of vegetables, protein, starches, and dairy products according to the best nutrition standards of the time ([@B5]). Participation in the program, which was expanded during the 1960s and 1970s, was demonstrated to improve children\'s diets ([@B9]). The obesity epidemic -------------------- With the tripling in obesity rates among children ([@B10]), schools face new challenges. Approximately 1 in 3 children born in 2000 will develop diabetes in his or her lifetime ([@B11]), and in a large study of children aged 5 to 17 years, 39% of those who were obese had 2 or more risk factors for cardiovascular disease ([@B12]). Poppendieck, in advising policy makers on the benefits of putting money into healthy school foods today to reduce future health care expenditures, calls her recommendations \"Pay now or pay later\" ([@B13]). **Children\'s inadequate nutrition** ------------------------------------ Although children today are consuming sufficient or even excessive food calories, they are not meeting the nutritional requirements described in the federal government\'s Dietary Guidelines for Americans ([@B14]).  Children\'s intake of fruits, vegetables, and whole grains does not come even close to current recommendations. Furthermore, children aged 5 to 18 years consume approximately 720 to 950 empty discretionary calories per day ([@B15]). Calories from added fats and sugars are displacing those from the nutrient-rich foods needed for growth and health. **Schools\' provision of food to students** ------------------------------------------- Children spend up to half their waking hours in school, where they may consume as much as one-third to one-half of their daily calories. Therefore, the school food environment is a logical focus for efforts to encourage healthy dietary behaviors. Today, school food service includes 2 competing arms --- the federally regulated reimbursable National School Lunch and School Breakfast programs ([@B8],[@B16]) and the competitive foods marketplace, which has expanded substantially during recent decades. Competitive foods and beverages are those foods sold throughout schools in vending machines, school stores, snack bars, and at fund-raisers. These are typically foods of low nutritional quality, including sweetened beverages, chips and other salty snacks, and sweets such as cookies and pastries ([@B17],[@B18]). During a typical day in the first 5 years of the 21st century, 55% of high school students and 44% of middle school students consumed competitive foods at school, frequently instead of school meals ([@B19]). Although states and school districts can voluntarily impose restrictions on competitive foods, these agencies are often unaware of the impact of the school food environment on student health. This lack of awareness, coupled with the funding that competitive foods provide to schools, has led to prolonged inaction. However, data now indicate that reductions in competitive food offerings can actually increase meal program participation rates, thereby increasing food service department revenues rather than reducing them as is often feared by school administrators ([@B20]). Efforts to promote and increase access to the meal program can be key to school-based efforts to reduce obesity, benefiting both children and schools. **Recent trends in school food regulation** ------------------------------------------- California in 2005 became the first state to legislate statewide nutrition standards to regulate sale of competitive foods and beverages in grades kindergarten through 12 ([@B21]). Evaluation studies of California\'s implementation of the legislation reveal that schools were successful at eliminating or severely reducing offerings of noncompliant (less nutritious) competitive foods and beverages in schools ([@B20],[@B22]). The food and beverage industry replaced or adapted snack foods to meet the new guidelines mandated by the legislation. For example, sports drinks replaced sodas, baked chips replaced original varieties of chips, and reduced-fat crackers replaced original crackers. Although the new offerings met the letter of the legislation\'s requirements to limit fat, sugar, and calories, they did not substantially increase the availability of such health-promoting foods as fruits, vegetables, whole grains, and low-fat dairy foods. During the past decade, other states and municipal governments have implemented new obesity-prevention policies in schools with respect to competitive food sales. These policies vary considerably by state and locality. Although competitive foods continued to be available in most schools in the latter half of this decade ([@B19]), more than half of all states and several local authorities adopted policies regarding such foods that were more restrictive than those mandated by US Department of Agriculture (USDA) regulations ([@B15]). In recent years, perhaps in response to the variability of state and local requirements, Congress and USDA have been pressured to revisit the issue of school food quality. Furthermore, 2 Institute of Medicine (IOM) reports ([@B15],[@B23]) recommended improvements to both competitive and school meal food offerings on the basis of the strongest scientific evidence available. In December 2010, the Healthy, Hunger-Free Kids Act of 2010 was signed into law ([@B24]). The act requires the Secretary of Agriculture to establish science-based nutrition standards within a year of enactment. The standards apply to all foods and beverages served outside school breakfast or lunch programs anywhere on school campuses. The extent to which these standards will fully meet the Institute of Medicine recommendations is not clear. Furthermore, the Healthy, Hunger-Free Kids Act mandates significant improvements to the National School Lunch and School Breakfast programs whereby school meals will be aligned with dietary recommendations for children as outlined in federal Dietary Guidelines for Americans. As the new standards are implemented, school meals will provide increased offerings of nutritious items (eg, fruits, vegetables, whole grains, 1% or nonfat milk) and decreased offerings of foods high in fat, sugar, and sodium. Meal reimbursement rates will be increased slightly to support the purchase of the nutritious offerings. If these policies are implemented as recommended by IOM, significant improvements in nutrition will be realized. However, the political challenge of effectively limiting the sale of less nutritious foods and the economic challenge of paying for more healthful options could result in more limited improvements than are needed to enable schools to promote children\'s optimal nutritional health. Application of a Bioethics Framework for Change in School Nutrition =================================================================== Opponents of school food regulation argue that people have the right to choose the foods they eat. However, we structure and regulate many student activities in the school setting and do not consider doing so an abridgement of children\'s rights. The argument that a child has the right to choose foods of poor nutritional quality at school conflicts with the societal value of child protection. A child\'s right to freedom from obesity is among the 54 binding standards and obligations of the 1989 United Nations Convention on the Rights of the Child ([@B25]). Beauchamp and Childress\'s ([@B4]) 4 foundational principles of biomedical ethics --- autonomy, beneficence, nonmaleficence, and justice --- can help address the question of whether a mandate to provide nutritious foods to children at school meets bioethics standards that justify regulatory action. 1. **Autonomy.** The conflict between school nutrition interventions and individual rights can be summarized as follows: who is responsible for ensuring that a child eats healthful foods --- the parent, the child, or the school? Children are not autonomous agents at home or at school. Because children do not have the knowledge and experience needed to choose foods on the basis of nutritional quality, responsible parents provide foods in the home from which the child can reasonably select. For example, parents would rarely serve candy alongside vegetables on the dinner table and expect their children to choose the vegetables instead of the candy. Similarly, school authorities are responsible for offering foods from which the child can select but limiting choices to those that provide nutritional benefit rather than harm. 2. **Beneficence.** Core to the mission of the National School Lunch and School Breakfast programs is provision of foods that meet the recommended dietary guidelines for optimal nutrition for children. However, efforts to encourage children to eat nutritionally sound school meals are undermined by provision of snacks and beverages that compete with healthier meals. Even partially regulated snack foods compete with healthier meals. Offering nutritious, appealing foods at school meals without competition from less healthy snack foods optimizes students\' opportunity to consume a health-promoting diet. Furthermore, the provision of a healthful school meal can serve as a model for educating children and parents alike. From its origins, the National School Lunch Program has asserted: \"The educational features of a properly chosen diet served at school should not be under-emphasized. Not only is the child taught what a good diet consists of, but his parents and family likewise are indirectly instructed\" ([@B8]). 3. **Nonmaleficence.** The principle of nonmaleficence is based on the premise that an intervention should not inflict harm. Providing nutritional foods does not cause harm. However, providing easy access to foods of poor nutritional quality should be construed as causing harm. The ongoing situation in which schools and their suppliers profit from sales of unhealthy foods may be purposely disguised by efforts to teach children not to choose the less healthy food that is offered. Observers might argue that children will not eat foods that are held to a higher nutritional standard; however, children will not go hungry. Students at schools providing nutritious food offerings will still have access to a variety of food choices along with the option of bringing foods from home. Furthermore, students have reported a preference for healthy, fresh food choices over other snack foods ([@B26]). 4. **Justice.** The principle of social justice demands that humans be treated fairly, with an equitable distribution of benefits and burdens. Distributive justice takes differences into account and recommends that social and economic inequities are acceptable if they are consistent with just principles and lead to the greatest benefit for the least advantaged ([@B27]). Schools provide an opportunity to address social inequities so that children from disadvantaged families have an equal opportunity to become productive citizens. Marketing of foods and beverages on school grounds (eg, in classroom materials, on sporting equipment, by signage) is a school fund-raising technique. Frequent exposure to this marketing in schools in low-income areas where children are at greater nutritional risk is at odds with fairness and social justice. Children from low-income families often experience more psychosocial stresses; having access to healthful foods may modify the effects of these stresses on children\'s growth and development. Although access to healthful food in schools will be of the greatest benefit to those with the fewest resources, all children benefit from improved nutrition. Doing harm, especially to the most vulnerable children, can never be justified. Selling foods of poor nutritional quality for profit, even if for support of desirable sports or music programs, is an example of such harm. The Ethical Basis for Future Action =================================== Providing foods of poor nutritional quality to finance school programs and profit commercial entities fails to meet society\'s ethical obligation to minimize harm, provide benefit, and protect vulnerable children who are a captive audience. Children are suffering as a consequence of such practices, and children from low-income families, who are most vulnerable to food insecurity, are at greatest risk for damage from consuming empty calories at school. Fostering optimal nutrition not only protects against obesity but is also essential for maximizing cognitive function and academic performance ([@B2]). Although new school policies related to health education, school food offerings, and physical education often have been well-received, they rarely have been of sufficient strength to produce demonstrated changes in child obesity rates. Using a bioethics framework, we can begin to formulate a rationale for interventions that support the crucial role schools play in providing nutritious, appealing meals that help children meet their dietary requirements. Schools can and should model an environment that promotes learning and health. In that context, interventions should limit competitive foods to only those foods that contribute to meeting the Dietary Guidelines for Americans and do not contribute empty calories. Only foods that support children\'s nutritional health should be offered at public schools, and available competitive foods should be equally healthy supplements to the school meal, not less healthy alternatives. These interventions are in children\'s best interests. The precautionary principle ([@B28]) states, \"When an activity raises threats of harm to human health or the environment, precautionary measures should be taken even if some cause and effect relationships are not fully established scientifically.\" The principle implies that society has a responsibility to intervene and protect the public from exposure to harm where scientific investigation identifies a plausible risk. The risk that malnutrition poses to children\'s ability to learn is well-documented ([@B1],[@B2]). Recent studies link provision of improved nutrition and physical activity at school to improved academic performance for students, especially among low-income minority students ([@B29],[@B30]). Providing a healthy diet would help minimize disparities in learning, and children whose families are least able to provide consistent access to adequate food would benefit most substantively. The risk that poor nutrition and obesity pose to children\'s future health (eg, osteoporosis, heart disease, diabetes) is also well-documented. There is no justification for the promotion of diets that increase those risks. Societal will is needed to provide the required resources to help children achieve nutritional health and simultaneously develop healthy lifetime eating habits. The best interests of children and society demand no less. This article highlights ideas generated and conclusions reached at the Symposium on Ethical Issues in Interventions for Childhood Obesity, sponsored by the Robert Wood Johnson Foundation (RWJ) and Data for Solutions, Inc. We gratefully acknowledge RWJ\'s support of this work and thank Sheila Stern and Lauren Goldstein for their editorial assistance. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. *Suggested citation for this article:* Crawford PB, Gosliner W, Kayman H. The ethical basis for promoting nutritional health in public schools in the United States. Prev Chronic Dis 2011;8(5):A98. <http://www.cdc.gov/pcd/issues/2011/sep/10_0283.htm>. Accessed \[*date*\].

INTRODUCTION: {#sec1-1} ============= Surgical intervention to the temporal lobe caries the risk of injury to the optic radiation.\[[@ref4][@ref5][@ref6]\]. The risk of injury varies with each approach, as the extent tissue removal is different form one surgical technique to the other. During surgery it is almost impossible to differentiate the optic radiation from other white fibers.\[[@ref1][@ref2][@ref3]\]. Amygdalohippocampectomy is one of the common surgeries used to treat temporal lobe epilepsy and the operation may be performed with several modifications\[[@ref7][@ref8][@ref9][@ref10][@ref11]&[@ref12]\]. The approach through the middle temporal gyrus is a common modification. We have conducted a cadaveric anatomical study to localize the optic radiation within the temporal lobe, define its anterior and lateral extension and to test if a standardized incision to the middle temporal gyrus damages it. MATERIALS AND METHODS: {#sec1-2} ====================== This is a cadaveric anatomical study. 11 human cerebral hemispheres that were formalinated for 3-6 months were studied. The work was performed at the Department of Neurosurgery, King Edward Memorial Hospital, Parel, Mumbai, India. The optic radiation was dissected out using Klinger\'s fiber dissection method under operating microscope ([Figure-1](#F1){ref-type="fig"}). Dissection was done with a small spatula made of bamboo and a surgical dissector. Dissection was started from lateral and inferior surfaces of cerebral hemisphere. Following removal of gray mater, short and long association fibers, superior longitudinal fascicle, extreme capsule, claustrum, external capsule and lentiform nucleus the internal capsule was reached. Then a 2 cm length anterior-posterior incision was made on middle of middle temporal gyrus extending from 3cm posterior to temporal pole. The incision was continued perpendicular to surface up to temporal horn of lateral ventricle ([Figure-2](#F2){ref-type="fig"}). Roof of the temporal horn was exposed from below by removing the lower part of temporal lobe and lateral part of floor of temporal horn with preservation of hippocampus ([Figure-3](#F3){ref-type="fig"}).Then stepwise dissection of optic radiation from lateral geniculate body to visual area was made. The localization of the optic radiation in the sublentiform and retrolentiform area was confirmed ([Figure-4](#F4){ref-type="fig"}). These white fibers were dissected from lateral geniculate body to the visual area situated on the upper and lower lip of calcarine sulcus. The anterior bundles of optic radiation(Meyer\'s loop) that initially pass anteriorly on the roof of the temporal horn and turning posteriorly to pass along the roof and lateral surface of the temporal horn was dissected out followed by middle and superior fibers. Middle fibers (Macular fibers) from lateral geniculate body pass to the roof of inferior horn and turned posteriorly to pass along the lateral wall of atrium and occipital horn of lateral ventricle to reach visual cortex. The upper (posterior) fibers pass directly backward around the atrium and occipital horn to reach upper lip of calcarine sulcus. Temporal pole, amygdala and hippocampus were preserved. Anterior and lateral extension of optic radiation was studied. Examination was done, to see whether any damage to optic radiation was made by the incision or not. The distances between anterior limit of optic radiation, anterior end of temporal horn ([Figure-2](#F2){ref-type="fig"}) and the temporal pole were recorded. {#F1} {#F2} {#F3} {#F4} RESULTS: {#sec1-3} ======== Anterior end of optic radiation extended 2mm (1-3.5mm) beyond the tip of temporal horn. The distance of optic radiation from temporal pole was 26mm (23-31mm). Most anterior 8.5mm (8-10mm) of the Meyer loop was completely on the roof and there was no extension over lateral wall of the temporal horn. In next posterior 17.5mm (16-20 mm) it extends over lateral wall of temporal horn with gradual progression. The incision that was made over the middle temporal gyrus entered into the temporal horn 2mm (1.5-2.4mm) below and lateral to the optic radiation at its anterior part and 0.9mm (0.75 to 1mm) below the optic radiation at its posterior end. Posterior end of incision is 1.5mm (1-2.5 mm) anterior to optic radiation margin. The incision did not damage optic radiation in any cerebral hemisphere (n=11) and entry point in temporal horn was below the optic radiation. DISCUSSION: {#sec1-4} =========== During dissection of optic radiation, we found that fibers originating from lateral geniculate body can be traced to visual area as three bundles (anterior, middle and posterior) described in other articles.\[[@ref3][@ref13][@ref14]\] In the published articles on the measurement between anterior extension of optic radiation and temporal pole is 21-30mm.\[[@ref1][@ref3][@ref6][@ref15]\] Rubino et al\[[@ref6]\] and Pujari et al.\[[@ref15]\] reported the extension of optic radiation is 2 mm anterior to tip of temporal horn. Rubino et al described that the Meyer\'s loop extended from roof to lateral wall of temporal horn during its way to visual area.\[[@ref6]\] In our study, the 2cm anterior-posterior incision over middle of middle temporal gyrus 3 cm posterior to the temporal pole perpendicular to surface did not damage the optic radiation. So amygdalohippocampectomy through middle temporal gyrus approach with 2m incision 3cm behind the temporal pole is relatively safe in respect of optic radiation damage. But change of direction to upward or posterior extension of incision or dissection is prone to damage of optic radiation. We found that any surgical resection that involves the superior temporal gyrus more than 26 mm from the temporal tip is likely to injure the Meyer\'s loop of the optic radiation. In previously pulished series resections less tha 25-30mm were reported as sade resections\[[@ref1][@ref6][@ref15]\]. CONCLUSION: {#sec1-5} =========== Amygdalohippocampectomy through an incision on the middle temporal gyrus of 2 cm length from 3 cm posterior to the temporal pole, to enter the temporal horn through the lower aspect of the lateral wall is more likely to miss the anatomical Meyer\'s loop. Any entry from the superior aspect of the temporal horn and any temporal lobectomy inclusive of the superior temporal gyrus to enter the temporal horn will produce Meyers loop damage. The findings support the fact that the more inferior the surgical trajectory to the temporal horn of the lateral ventricle, the lover is the risk of visual field damage. Acknowledgments - The authors would like to express the deep appreciation to Prof. Dr. Atul H. Goel., who placed his laboratory facilities at our disposal. He stimulated the technique and introduced the fiber dissection technique in his Microneurosurgical fellowship Course at King Edward Memorial Hospital, Parel, Mumbai, India.

Introduction {#Sec1} ============ The knowledge on how *BRCA1/2* mutation carriers should be counseled and treated has evolved continuously over the last two decades \[[@CR1], [@CR2]\]. Some important issues remain to be solved, though. One of these is if breast-conserving therapy (BCT) followed by postoperative radiotherapy is as good of an alternative to mastectomy (M) for carriers as it is for other breast cancer patients \[[@CR3], [@CR4]\]. Once a carrier has had a breast cancer, the risk of contralateral breast cancer (CBC) is indeed very high \[[@CR5], [@CR6]\]. It is reasonable to assume that the risk of new primary breast cancers in the ipsilateral breast is also high if breast tissue is still there for tumor development. On the other hand, radiotherapy and other adjuvant treatments change the microenvironment in the breast and reduce the number of cancer precursors, and could thereby, in addition to reducing the risk of true recurrences, possibly also reduce the number of new primary tumors in the treated breast. It is not always possible to differentiate an ipsilateral event being a true recurrence or a new primary breast cancer. In the following, both are denominated "local recurrences." Results from cohort studies and case--control studies have been conflicting regarding the risk of local recurrence as first recurrence (LR) in carriers following BCT \[[@CR7]--[@CR17]\]. Importantly, no study to date has shown a survival difference between mutation carriers and noncarriers treated with BCT or between carriers treated with BCT and carriers treated with M. However, BCT can be associated with other disadvantages, such as the requirement of close follow-up for a long time, most likely a recommendation of adjuvant chemotherapy in case of a LR, and an increased cancer-specific distress. Even though generally well tolerated, also M and bilateral mastectomy (BM) can for some patients be associated with disadvantages, like a negative impact on sexuality and body image \[[@CR18]\]. The absolute long-term risk of LR and survival endpoints are of pivotal importance for mutation carriers with newly diagnosed breast cancer to know, in order to be able to make informed decisions about type of surgery. To further evaluate the appropriateness of BCT in carriers, we conducted a cohort study. The aim of the study was to compare LR and survival between carriers treated with BCT and carriers treated with M. Materials and methods {#Sec2} ===================== Study population {#Sec3} ---------------- In an institutional database, where all persons that have undergone mutation analysis of the *BRCA1* and *BRCA2* genes at a single institution in Lund, Sweden, are registered, all women with an invasive breast cancer stage I-III diagnosed between 1975 and 2011 and a pathogenic germline mutation in *BRCA1* or *BRCA2* were selected. Women with variants of uncertain significance in *BRCA1* or *BRCA2* were not included. Out of 204 identified patients, 183 had consented to longitudinal follow-up (or, in case they were dead, their next of kin had consented), the others were excluded. Clinical data were abstracted from medical records and pathology reports and supplemented by information from self-reported questionnaires. TNM stage was classified according to American Joint Committee on Cancer 7th edition. Patients with a diagnosis of ovarian cancer, except for patients with no ovarian cancer recurrences and ≥10 years elapsed after ovarian cancer diagnosis before breast cancer, were excluded (*n* = 2). Another 8 patients were excluded as we were not able to retrieve medical records. Of the remaining 173 patients, 11 were treated with partial mastectomy without postoperative radiotherapy; they were excluded. For the present study, 162 patients thus constituted the study population. Only 9 of these patients had the mutation analysis initiated after their death (on archived tissue); for the others it was initiated while they were alive. Due to a small number or patients, *BRCA1* (*n* = 114) and *BRCA2* (*n* = 48) mutation carriers were grouped together for analyses. Vital status was controlled in the Swedish Census Register. Current analyses were based on follow-up information through January 31, 2012. Study endpoints {#Sec4} --------------- Study endpoints were local recurrence as first recurrence (LR), overall survival (OS), breast cancer death, and distant recurrence, for the pre-specified subgroups of patients treated with BCT and M, respectively. If the final surgery was M within 1 year after breast cancer diagnosis, the patient was allocated to M, even if the first surgical procedure was a partial mastectomy. For the analysis of LR, patients were censored at the date of last follow-up, whereas distant or regional spread of cancer (breast cancer, ovarian cancer, or another type of cancer) and death where treated as competing risks. In other words, local recurrences occurring after a regional or a distant recurrence were not considered. Further, patients treated with BCT were censored at the time of prophylactic mastectomy. All cases of LR were invasive. For women with bilateral breast cancer, we were not able to distinguish death or distant recurrence due to the first primary breast cancer from death or distant recurrence due to the second primary breast cancer. Statistical analyses {#Sec5} -------------------- Differences in patient, tumor, and treatment characteristics between the BCT group and the M group were tested using Fisher's exact test. Cumulative incidence curves were calculated for LR in the presence of other recurrences or death as competing risks, and for breast cancer death and distant recurrence in the presence of death of other cause than breast cancer as competing risk. Kaplan--Meier curves were used to illustrate OS. To compare event rates between the treatment groups, cause-specific log-rank tests and Cox regression analyses were used. The following covariates were selected for multivariable analyses: type of surgery, age at diagnosis, TNM stage, and use of (neo)adjuvant chemotherapy. Age at diagnosis was split at the median to account for nonlinear associations and to make interpretations of the results easier. All tests and confidence intervals were two-tailed. All analyses were conducted using the R statistical package (R 3.1.0), using libraries survival and cmprsk. For the discussion part, *p* values below 0.05 were considered statistically significant. Results {#Sec6} ======= Study population {#Sec7} ---------------- Forty-five patients were treated with BCT and 117 patients had M as final surgery within one year from the breast cancer diagnosis. Patient, tumor, and treatment characteristics are listed and compared between these two groups in Table [1](#Tab1){ref-type="table"}. BCT was common in the time period 1990--1999, M was more common before and after that. Tumors treated with M were more often stage III and less often stage I than tumors treated with BCT. Mean age at diagnosis was 43.3 years in both groups. Patients treated with M more often received (neo)adjuvant chemotherapy (59 vs. 42 %) and adjuvant endocrine therapy (37 vs. 13 %) than patients treated with BCT. M was followed by postoperative radiotherapy in 53 % of the cases. A bilateral prophylactic mastectomy was done by 40 % of the patients in the BCT group, and a contralateral prophylactic mastectomy was done by 44 % of the patients in the M group. In both groups, 67 % underwent a bilateral oophorectomy (Table [1](#Tab1){ref-type="table"}). Out of these 108 oophorectomies, 10 were done prior to breast cancer diagnosis, 39 within two years following breast cancer diagnosis, and 59 at a later date. The mean follow-up for OS was 12.9 years for patients alive at the end of follow-up; 14.9 years in the BCT group and 12.1 years in the M group. Table 1Patient, tumor, and treatment characteristicsVariableBCT (*n* = 45)M (*n* = 117)*p* ^a^Mean follow-up^b^, years14.912.1Mean age at diagnosis, years43.343.3Median age at diagnosis, years43.042.0Year of diagnosis0.019 1975--19898 (18 %)31 (26 %) 1990--199924 (53 %)34 (29 %) 2000--201113 (29 %)52 (44 %)TNM stage0.023 I22 (51 %)33 (29 %) II17 (40 %)53 (47 %) III4 (9 %)26 (23 %) Missing25Tumor grade0.55 I0 (0 %)2 (3 %) II5 (33 %)12 (19 %) III10 (67 %)48 (77 %) Missing3055ER status0.19 Negative25 (78 %)59 (63 %) Positive7 (22 %)34 (37 %) Missing1324PgR status1 Negative23 (74 %)67 (74 %) Positive8 (26 %)24 (26 %) Missing1426(Neo)adjuvant chemotherapy0.054 No26 (58 %)47 (41 %) Yes19 (42 %)69 (59 %)  CMF-like7 (16 %)22 (19 %)  Anthracycline-based11 (24 %)29 (25 %)  Taxane-containing016 (14 %)  Unknown1 (2 %)2 (2 %)Adjuvant endocrine therapy0.004 No39 (87 %)73 (63 %) Yes6 (13 %)43 (37 %)Postoperative radiotherapy No0 (0 %)55 (47 %) Yes45 (100 %)61 (53 %)Oophorectomy No15 (33 %)38 (32 %) Yes30 (67 %)78 (67 %)  Bilateral prophylactic oophorectomy22 (49 %)58 (50 %)  Ovarian cancer8 (18 %)18 (15 %)^c^  Palliative oophorectomy02 (2 %) Missing01 (1 %)Prophylactic mastectomy^d^17 (40 %)51 (44 %)Contralateral breast cancer12 *of* 43^e^26 *of* 117*BCT* breast-conserving therapy, *M* mastectomy, *TNM* tumor node metastasis, *ER* estrogen receptor, *PgR* progesterone receptor, (*Neo*)*adjuvant* = neoadjuvant *or* adjuvant^a^Fisher's exact test for all^b^Mean follow-up for OS for patients alive at end of follow-up^c^Another 3 patients in the M group and none in BCT group were diagnosed with primary peritoneal carcinosis after a prophylactic oophorectomy^d^Bilateral prophylactic mastectomy after BCT and contralateral prophylactic mastectomy after M^e^Information on contralateral breast cancer missing for 2 patients in the BCT group Local recurrence {#Sec8} ---------------- The mean time at risk for LR was 6.0 years in the BCT group and 8.8 years in the M group. The analysis of LR in the BCT group was based on 11 cases of LR diagnosed at a mean time of 7.6 years after breast cancer diagnosis; out of 11 cases of LR, 9 were isolated and 2 were concurrent with a regional recurrence. In the M group, 9 cases of LR were diagnosed at a mean time of 1.9 years after breast cancer diagnosis; out of 9 cases of LR, 6 were isolated, 1 was concurrent with a regional recurrence, and 2 were concurrent with a distant recurrence. Compared to M, BCT was associated with an increased risk of LR in univariable analysis (HR 4.0; 95 % CI 1.6--9.8) and in multivariable analysis adjusting for tumor stage, age, and use of (neo)adjuvant chemotherapy (HR 2.9; CI 1.1--7.8) (Table [2](#Tab2){ref-type="table"}). In this multivariable model, younger age was associated with a higher risk of LR (\<43 vs. ≥43 years: HR 2.7; CI 1.0--7.6), and use of (neo)adjuvant chemotherapy resulted in a point estimate below 1 but a wide confidence interval (HR 0.6; CI 0.2--1.7). Following M, all local recurrences were seen in the first 5 years. As opposed to this, following BCT the rate of LR continued to be high also after the first 5 years. The cumulative risk of LR in the BCT group was 15, 25, and 32 % after 5, 10, and 15 years, respectively. The cumulative risk of LR in the M group was 9 %, after 5, 10 as well as 15 years (Table [3](#Tab3){ref-type="table"} and Fig. [1](#Fig1){ref-type="fig"}). Table 2Univariable and multivariable analysis for LRUnivariable coxMultivariable cox^a^*n*EventsHR95 % CI*P*HR95 % CI*P*16020Type of surgery0.0030.03 M11691.01.0 BCT44114.01.6--9.82.91.1--7.8Age at diagnosis0.060.06 ≥43 years7351.01.0 \<43 years87152.61.0--7.12.71.0--7.6TNM stage0.100.52 I55111.01.0 II7060.350.1--1.00.60.2--1.7 III3020.420.1--1.91.10.2--5.8(Neo)adjuvant chemotherapy0.080.31 No73141.01.0 Yes8660.420.2--1.10.60.2--1.7*LR* local recurrence as first recurrence, *HR* hazard ratio, *CI* confidence interval, *M* mastectomy, *BCT* breast-conserving therapy, *TNM* tumor node metastasis, (Neo)adjuvant = neoadjuvant *or* adjuvant^a^Cox proportional hazards model including all covariates. *n* = 155, *Events* = 19Table 3Five-, ten-, and fifteen-year cumulative incidences (%) of death of any cause, breast cancer death, distant recurrence, and LR split on surgical subgroups, and the corresponding hazard ratiosDeath of any cause = 1-OSBreast cancer deathDistant recurrenceLRMBCTMBCTMBCTMBCT5-year (%)17201420252591510-year (%)32322927313592515-year (%)374229343135932Unadjusted HR (95 % CI)1.01.0 (0.6--1.7)1.01.1 (0.6--2.1)1.01.2 (0.7--2.2)1.04.0 (1.6--9.8)Adjusted HR^a^ (95 % CI)1.01.4 (0.8--2.5)1.01.6 (0.8--3.3)1.01.8 (0.9--3.5)1.02.9 (1.1--7.8)*OS* overall survival, *LR* local recurrence as first recurrence, *HR* hazard ratio, *CI* confidence interval, *M* mastectomy, *BCT* breast-conserving therapy^a^Adjusted for TNM stage, age at diagnosis, and use of (neo)adjuvant chemotherapyFig. 1Cumulative incidence for local recurrence as first recurrence by type of surgery Overall survival, breast cancer death, and distant recurrence {#Sec9} ------------------------------------------------------------- In univariable analysis, no difference in OS, breast cancer death, or distant recurrence was seen between the BCT group and the M group (Table [3](#Tab3){ref-type="table"} and Figs. [2](#Fig2){ref-type="fig"}, [3](#Fig3){ref-type="fig"}[4](#Fig4){ref-type="fig"}). In multivariable analysis, adjusting for tumor stage, age at diagnosis, and use of (neo)adjuvant chemotherapy, the hazard ratios were higher, but remained inconclusive (Table [3](#Tab3){ref-type="table"}). The 5-, 10-, and 15-year cumulative incidences are listed in Table [3](#Tab3){ref-type="table"}. Fig. 2Overall survival by type of surgeryFig. 3Cumulative incidence for breast cancer death by type of surgeryFig. 4Cumulative incidence for distant recurrence by type of surgery Discussion {#Sec10} ========== In this cohort study, we report that the risk of LR was substantially higher after BCT than after M for *BRCA1/2* mutation carriers. The cumulative incidence of LR 15 years after BCT was 32 %, which was significantly higher than after M and is more than two-fold higher than after BCT in the general population, where most tumors are sporadic and the patients on average are older \[[@CR19], [@CR20]\]. Apart from type of surgery, younger age was also associated with an increased risk of LR. This can probably be explained by the fact that carriers who get breast cancer when they are very young are likely to have other predisposing or modifying factors of genetic or environmental nature, and thus an increased risk of new primary breast cancers \[[@CR10], [@CR15], [@CR21]\]. Most of the case--control and cohort studies that have been conducted to compare the outcome of BCT between carriers and sporadic cases have reported point estimates of LR that have been higher for carriers, although the difference in the majority of the studies have not obtained a significant difference at *p* values \<0.05, and it is therefore difficult to draw firm conclusions from them. The largest such study was reported by Pierce et al. in 2006 \[[@CR12]\]. *BRCA1/2* mutation carriers were matched with sporadic cases, all treated with BCT. The 15-year risk of LR was 24 % for carriers and 17 % for sporadic cases (*p* = 0.19). When patients that had done an oophorectomy were excluded from the analysis, carriers had an increased risk of LR (HR 1.9; *p* = 0.03). The same collaborative group published a different type of study in 2010, which is very similar to ours in study design and is the only previous study that has compared BCT and M for *BRCA1/2* mutation carriers \[[@CR13]\]. No difference in survival was seen between BCT and M, but patients treated with BCT had a higher risk of LR: the 15-year risk of LR was 23.5 versus 5.5 %. Interestingly, chemotherapy decreased the risk, so in the subgroup of BCT patients treated with chemotherapy, the risk of LR was only 11.9 % after 15 years; a level of risk comparable to what it is for sporadic cases and very relevant for counseling and treatment of carriers today, since a majority of them will receive chemotherapy if diagnosed with a breast cancer. With the limitations of a smaller number of patients and, therefore, no analyses of modifying factors conducted separately in the BCT subgroup, we also found a trend for a decreased risk of LR after use of chemotherapy. Metcalfe et al. \[[@CR11]\] reported data from a large cohort of carriers treated with partial mastectomy without any group for comparison and found the 15-year risk of LR to be 15.8 %. Use of chemotherapy, oophorectomy, and postoperative radiotherapy decreased the risk. The age at diagnosis and uptake of oophorectomy was similar, but the proportion of patients that received chemotherapy was lower in our cohort than in the above-mentioned studies, which could partly explain the higher absolute risk of LR after BCT seen in our study. A study that reported a higher risk of LR after BCT than what we do, is a retrospective cohort study by Haffty et al., in which the cumulative risk of LR 12 years after BCT was 49 %. Of note, no mutation carriers in that cohort were treated with adjuvant endocrine therapy or oophorectomy, and the mean age at diagnosis was only 34 years \[[@CR9]\]. In a recent meta-analysis by Valachis et al., no significant difference in LR after BCT was seen between *BRCA1/2* mutation carriers and controls; however, a significant difference was observed when the analysis was restricted to studies with a median follow-up of ≥7 years \[[@CR17]\]. A hypothesis that has been corroborated in a number of studies is that early LRs are true recurrences and that late LRs are in fact new primary breast cancers; the latter group probably accounts for the majority of LR \[[@CR9], [@CR13]\]. We point out that our cohort consists of mutation carriers ascertained through a cancer genetic center, and a majority of them belong to multiple-case families. Under a model where the risk of late LR is modified by genetic and environmental factors, this risk of LR is higher for these carriers than for carriers ascertained through population-based programs. The decreased risk of LR conferred by adjuvant therapy could be mediated not only through killing of breast cancer cells from the primary tumor, but also through an effect on breast cancer precursors and changes in the microenvironment of the breast, making new tumor formation less likely. The difference in the risk of LR between carriers and sporadic cases is less clear in studies where more adjuvant treatment has been used and a majority of the patients have done an oophorectomy, supporting the notion that these measures reduce the risk for new primary breast cancers in the ipsilateral, as well as the contralateral, breast. In our study, the survival comparisons between BCT and M should be interpreted with caution. M was more common than BCT in the time period 1975--1989. Patients who died before they were known mutation carriers could be included in the study, but patients that were still alive when *BRCA1/2* testing was introduced in the mid-90s were probably more likely to be included, resulting in survivorship bias. M was also more common in the time period 2000--2011, when adjuvant treatment was more common and modern than before. Neither our cohort nor any of the other cohorts in published studies is large enough to properly adjust for all possible biases and confounders. Furthermore, in observational studies of surgical decisions, bias can never be fully accounted for. In the general population, large randomized trials of breast cancer patients have not shown a difference in survival between BCT and M. Still, in the general population, patients that are diagnosed with a LR have an inferior survival compared to patients without a LR \[[@CR22]\]. Despite the fact that some studies of *BRCA1/2* mutation carriers have shown a very high risk of LR, no study to date has shown a difference in survival. This contradiction could possibly be explained by a larger proportion of new primary breast cancers among carriers, which are less aggressive than early true recurrences, and more often curable \[[@CR13]\]. However, given large enough sample sizes and a cohort with a high rate of LR following BCT, a difference in long-term survival between BCT and M would be expected for carriers, since not all new primary breast cancers are curable. Apart from the inability to make reliable survival comparisons, there are other limitations to our study. First, the follow-up is too short to estimate cumulative lifetime risks of LR, which has not been done in previous studies either. Second, a small number of patients result in point estimates with wide confidence intervals and the inability to include some potentially important variables in the multivariable models. Still, our study confirms some of the findings from the larger collaborative study by Pierce et al. Third, by including breast cancer patients from 1975 and onwards, the proportion of patients receiving adjuvant treatment is lower than what it is today, which can overestimate the risk of LR after BCT. Fourth, we were not able to separate true local recurrences from new primary breast cancers by means of pathology or localization in the breast. The number of mutation carriers opting for M and BM has increased over the last decade, and the number opting for BCT has decreased \[[@CR23]\]; the trend is expected to continue further with evidence of a survival benefit with BM now starting to emerge \[[@CR5], [@CR24], [@CR25]\]. In the future, studies on the risk of LR after BCT in mutation carriers will therefore be harder to carry out and randomized trials are very unlikely. Still, there is a need for more studies, since BCT is considered an acceptable option for mutation carriers today. Data from retrospective studies can give relatively unbiased estimates of LR, but cannot properly measure psychosocial endpoints. In future studies, for an optimal generalizability to current standards, a very high proportion of patients treated with chemotherapy and prophylactic oophorectomy is needful. Furthermore, a long-term follow-up is demanded for estimation of cumulative lifetime risks of LR, which are pivotal for carriers with newly diagnosed breast cancer to know in order to make informed decisions about type of surgery. In conclusion, *BRCA1/2* mutation carriers treated with BCT and who resemble our cohort regarding ascertainment, age at diagnosis, adjuvant treatment, uptake of oophorectomy etc., have a high risk of LR, many of which are probably new primary breast cancers. This must be thoroughly discussed with the patient and is an example of how rapid treatment-focused genetic testing could influence choice of treatment. The study was approved by the Regional Ethical Review Board in Lund, and complies with the current laws of Sweden. The research was funded by grants from Skåne County Counsil's Research and Development Foundation, The Swedish Breast Cancer Association (BRO), The Swedish Cancer Society, and BioCARE. Conflict of interest {#d30e1828} ==================== The authors declare that they have no conflict of interest.

Introduction ============ Nano-sized materials have been increasingly used in the medical field to improve the target efficiency of drugs.[@b1-ijn-9-051]--[@b3-ijn-9-051] In order to successfully apply nanoparticles in drug delivery, their physical and chemical properties must first be understood, thereby assisting in controlling the biological responses to their use. Because drug delivery nanosystems transport pharmaceutical compounds in the body, it is important to understand their physiochemical properties to safely achieve a desired therapeutic effect. However, these drug delivery nanosystems have shown some limitations regarding the toxicity of the nanoscale materials in the body.[@b4-ijn-9-051],[@b5-ijn-9-051] In order to reduce their toxicity, it is crucial to study endocytosis, exocytosis, and clearance mechanisms for nanoparticles released from the nanoparticle--drug conjugates. Nanoparticles exposed to the bloodstream interact with opsonin proteins. When opsonin proteins attach to the surface of nanoparticles, they allow macrophages of the mononuclear phagocytic system (MPS) to easily recognize the nanoparticles and hence the nanoparticles eventually accumulate in the MPS organs, such as liver and spleen. These phenomena cause low targeting efficiency and severe systemic toxicity of the drug-delivery nanosystems. Therefore, this review focuses on endocytosis and exocytosis patterns of nanoparticles in mammalian cells with respect to their size, shape, and surface chemistry ([Figure 1](#f1-ijn-9-051){ref-type="fig"}). Nanoparticle stability ====================== Nanoparticles have been widely used in the fields of drug delivery and bioimaging because their size, shape, and surface properties can be precisely engineered for specific diseases.[@b6-ijn-9-051],[@b7-ijn-9-051] The nanoparticle surface can be modified with various targeting molecules (eg, antibody, peptide, aptamer, etc) in order to achieve efficient targeting to disease sites. Recently, many scientists have begun to investigate the effects of different sizes, shapes, and surface chemistries on endocytosis, toxicity, and gene regulation.[@b8-ijn-9-051]--[@b10-ijn-9-051] However, aggregates (strongly bonded between nanoparticles) and agglomerates (loosely bonded between nanoparticles or aggregates acting under weak forces, eg, van der Waals force) formed by forces between nanoparticles and components in biological media have not been fully considered to optimize their physicochemical properties for biological applications. It was recently suggested that the aggregates or agglomerates occur when the van der Waals attractive forces between nanoparticles are larger than the electrostatic repulsive forces ([Figure 2](#f2-ijn-9-051){ref-type="fig"}).[@b11-ijn-9-051] Aggregated or agglomerated forms of nanoparticles would behave differently within biological systems than would nanoparticles in their single form. Thus, size uniformity of nanoparticles should be considered when the effect of physical and chemical properties of nanoparticles on their interactions with biological systems is examined. In biological solutions, such as blood, saliva, and cell culture media, the surface chemistry of nanoparticles plays a crucial role in determining their behavior because they are directly related to types and compositions of biomolecules attached to the nanoparticle surface. The surface chemistries on the nanoparticle surface are dynamically changed because various biomolecules are attached and detached based on their binding affinity to the surface. Pre-coating of the nanoparticle surface with stabilizing molecules such as polyethylene glycol (PEG), deoxyribonucleic acid (DNA), and albumin has been utilized to reduce ionic strength and prevent nanoparticles from aggregation or agglomeration in the biological solutions.[@b12-ijn-9-051]--[@b14-ijn-9-051] Additionally, individual nanoparticles can be naturally coated with various biomolecules, forming the nanoparticle--protein complex, when solubilized in biological solutions. The stability lifetimes of the nanoparticle--protein complexes range from hours to days in the biological solutions. The proteins covering the nanoparticle surface further prevent the individual nanoparticles from aggregation or agglomeration. Because the formation of nanoparticle--protein complexes is mainly determined by surface chemistries of the nanoparticles, it is important to investigate which surface chemistry is the most favorable to form the nanoparticle--protein complex. Therefore, natural nanoparticle--protein complexes formed in biological environments would allow us to study how individual nanoparticles interact with various types of cells. When we study the endocytosis and exocytosis of nanoparticles, cells are treated with the nanoparticles in the culture medium containing various serum proteins. Most of the nanoparticles are first coated with the serum proteins and then met with the plasma membrane of cells. If the nanoparticles are already aggregated or agglomerated prior to binding to the membrane, their endocytosis patterns would differ from the endocytosis patterns of individual nanoparticles. The degree of aggregation or agglomeration of the nanoparticles can be determined by measuring time-dependent change of size and surface charge of the nanoparticles in the culture medium. The (hydrodynamic) size has been mainly analyzed using transmission electron microscopy (TEM) and dynamic light scattering (DLS) while the surface charge is determined by zeta potential measurements. In particular, the ultraviolet--visible (UV/Vis) spectrophotometry has also been used to monitor the size of gold nanoparticles because their localized surface plasmon resonance peaks can be shifted to a longer wavelength by increasing their size.[@b15-ijn-9-051] The DLS technique has been the most widely used to monitor size change because it directly measures hydrodynamic sizes of protein-coated nanoparticles in the biological solution with nanometer precision. Furthermore, the zeta potentials of protein-coated nanoparticles mostly appeared as a negative surface charge although the nanoparticles had different original surface chemistries. It suggests that most proteins attached on the nanoparticle surface seem to be negatively charged, regardless of their content and composition, although they may alter depending on the surface chemistries of the nanoparticles. Thus, the content and composition of proteins preferentially attached on the nanoparticle surfaces should be studied for the cellular uptake and immune response of nanoparticles.[@b16-ijn-9-051],[@b17-ijn-9-051] Nanoparticle endocytosis ======================== Endocytosis mechanism --------------------- All types of cells in the body use the endocytosis process to communicate with the biological environments. This process is an energy-dependent process through which cells internalize ions and biomolecules.[@b18-ijn-9-051] In particular, the cells internalize nutrients and signaling molecules to obtain energy and interact with other cells, respectively. The endocytosis pathways are typically classified into clathrin- and caveolae-mediated endocytosis, phagocytosis, macropinocytosis, and pinocytosis ([Table 1](#t1-ijn-9-051){ref-type="table"}). Clathrin- and caveolae-mediated endocytosis indicates receptor-mediated endocytosis. Many types of cells use the clathrin- and caveolae-mediated endocytosis pathways to internalize nanoscale materials, including viruses and nanoparticles.[@b19-ijn-9-051]--[@b21-ijn-9-051] These endocytosis pathways are the most important pathways for the internalization of nanoparticles into cells because the nanoparticles are directly coated with the plasma proteins when exposed to physiological solutions. The phagocytosis pathway is used when phagocytic cells internalize foreign materials with sizes larger than 0.5 μm.[@b16-ijn-9-051] The phagocytosis pathway is actin-dependent and restricted to professional phagocytes, such as macrophages, dendritic cells, and neutrophils. The macropinocytosis pathway is a non-specific process to internalize fluids and particles together into the cell, whereas the pinocytosis pathway absorbs biological fluids from the external environment of a cell.[@b22-ijn-9-051] These pathways are very important to translocate single nanoparticles with sizes below 10 nm into the cell. When nanoparticles are systemically administered into the body, they are confronted with many types of cells. Since nanoparticles have emerged as effective drug carriers to treat complex diseases, it has become crucial to understand nanoparticle endocytosis mechanisms. It is believed that the endocytosis efficiency of nanoparticles is dependent on the physicochemical properties, such as size, shape, and surface chemistry, as well as cell type. Thus, interaction of nanoparticles with cells depending on their physicochemical properties is discussed in the following sections. Factors affecting nanoparticle endocytosis ------------------------------------------ Nanoparticles circulating in the bloodstream happen to meet and internalize into many types of cells through the plasma membrane. The plasma membrane is a selectively permeable membrane that transfers materials that are essential for sustaining the cell's life. Naturally, materials necessary for the cell's life, such as ions and nano-sized proteins, can pass through the lipid bilayer using specialized membrane-transport protein channels.[@b23-ijn-9-051] Thus, the plasma membrane of cells would select the endocytosis pathways of nanoparticles depending on their size, shape, and surface chemistry ([Table 2](#t2-ijn-9-051){ref-type="table"}). ### Size Size-dependent cellular uptake of nanoparticles has been extensively investigated in various cell lines because the nanoparticle size has been known to be a key determinant of the uptake pathways. Many critical in vivo functions of nanoparticles, such as circulation time, targeting, internalization, and clearance, depend on their size. Thus, the cellular uptake of nanoparticles with various sizes is reviewed in this subsection. Much interest has focused on understanding size-dependent internalization of nanoparticles in cancer cells and fibroblasts. The cellular uptake of gold nanoparticles of various sizes was studied in human cervical cancer cells ([Figure 3](#f3-ijn-9-051){ref-type="fig"}).[@b8-ijn-9-051],[@b9-ijn-9-051],[@b24-ijn-9-051] Researchers at the University of Toronto, Canada, demonstrated that uptake mechanism and saturation concentration of nanoparticles was dependent on their size. The 50 nm gold nanoparticles showed the most efficient cellular uptake compared with other sizes. The cellular uptake of polystyrene (PS) nanoparticles of various sizes was also tested on a human colon adenocarcinoma cell line.[@b25-ijn-9-051] The PS nanoparticles with a size of 100 nm were taken up into the cells more efficiently than those with sizes of 50, 200, 500, and 1,000 nm. The internalization efficiency of the PS nanoparticles with a size of 50 nm was the lowest of all sizes. These two studies suggest that the patterns of cellular uptake could also vary according to nanoparticle material type. Although different cancer cell lines were used for each experiment, the stiffness of nanoparticles could affect their cellular uptake. Stiffer nanoparticles would interact tightly with the plasma membrane of cell, thereby causing rapid endocytosis. Single-walled carbon nanotubes (SWNTs) coated with DNA molecules were used to investigate length-dependent cellular uptake.[@b26-ijn-9-051] The results demonstrated that long (660 ± 40 nm) and short (130 ± 18 nm) SWNTs have lower uptake efficiencies in the fibroblasts than SWNTs with average lengths of 430 ± 35 nm and 320 ± 30 nm. The SWNTs with an average length of 320 ± 30 nm had the greatest uptake pattern. In addition to endocytosis in non-phagocytic cells, much attention has recently been paid to understanding interactions between nanoparticles and phagocytic cells such as macrophages, because it could be relevant to the design of nanoparticles to avoid the immune system, thus increasing their target efficiency. Nanoparticles with sizes larger than 0.5 μm have been known to enter phagocytic cells via phagocytosis pathways. Polymeric microspheres with diameters of 2--3 μm exhibited the maximal phagocytosis rate.[@b27-ijn-9-051] Interestingly, this size range coincides with the general size of the bacteria that are the most common targets of the MPS. On the other hand, there have been some efforts to examine size-dependent phagocytosis of nanoparticles smaller than 0.5 μm. Among many types of nanoparticles, lipid-based nanoparticles, including US Food and Drug Administration (FDA)-approved liposomes have been of particular interest in the drug-delivery field due to their drug-loading capability and biocompatibility. Lipid nanoparticles with sizes of 20, 50, and 100 nm were taken up into the macrophages by complement receptor-mediated phagocytosis.[@b28-ijn-9-051] In addition, liposomes with sizes ranging from 100 to 2,000 nm were also tested for their intracellular uptake in the macrophages.[@b29-ijn-9-051] The amount of liposomes taken up by the macrophages increased with size over the range 100--1,000 nm, but the uptake rate became constant with sizes over 1,000 nm. Size-dependent phagocytosis of gold nanoparticles was also studied in many research groups. It was reported that gold nanoparticles with sizes below 100 nm were phagocytosed via scavenger receptor-mediated phagocytosis.[@b30-ijn-9-051] Tsai et al[@b31-ijn-9-051] also demonstrated that gold nanoparticles with a size of 4 nm showed the highest uptake in the macrophages based on the number of nanoparticles taken up per cell, compared with those sized 11, 19, 35, and 45 nm. The 4 nm gold nanoparticles exhibited the highest potency in inhibiting tumor necrosis factor (TNF)-α production related to TNR9-mediated innate immune systems. Although the experimental results introduced here showed size-dependent cellular uptake of nanoparticles in many types of cells, the physical size would not be fully reflected when they meet the plasma membrane of the cell. Most nanoparticles tend to aggregate in biological solutions, increasing their overall size. The results regarding the size effect would be influenced by nanoparticle aggregation before entering the cell. Thus, it should be tested whether nanoparticles prepared to study their size-dependent endocytosis retain their singularity in the biological media before they enter the cell. ### Surface chemistry Surface chemistry (or surface charge) of nanoparticles can be determined by the chemical composition on the nanoparticle surface. The surface charge of nanoparticles can affect their efficiency and the pathway of cellular uptake, because biological systems consist of numerous biomolecules with various charges. Therefore, the charge of biomolecules covering the nanoparticle surface can influence the endocytosis patterns of nanoparticles. In this subsection, the effect of nanoparticle surface chemistries on the endocytosis pattern is discussed. For polymeric nanoparticles, carboxymethyl chitosan-grafted nanoparticles as negatively charged nanoparticles, and chitosan hydrochloride-grafted nanoparticles as positively charged nanoparticles, were used to test their cellular uptake efficiency.[@b32-ijn-9-051] The different surface charges significantly affected their uptake by macrophages. The positively charged nanoparticles exhibited a higher phagocytic uptake than did the negatively or neutrally charged nanoparticles. Moreover, when the uptake efficiency of the positively charged nanoparticles was compared with that of the negatively charged, neutrally charged, and PEGylated nanoparticles, the positively and negatively charged nanoparticles were internalized more rapidly than the neutrally and PEGylated charged nanoparticles. It was also demonstrated that negatively charged polymeric nanoparticles with diameters of around 100 nm were more efficiently phagocytized by macrophages than positively charged nanoparticles ([Figure 4](#f4-ijn-9-051){ref-type="fig"}).[@b33-ijn-9-051] Cellular uptake was also greater in the macrophages than in the monocytes. Furthermore, the surface functionalization with PEG, poloxamer, and poloxamine polymers prevented phagocytosis because these polymers protect the nanoparticles from ionic strength, promote particle dispersion, and reduce absorption of proteins in blood on their surface.[@b34-ijn-9-051] In addition, the uptake efficiency of PEGylated nanoparticles is closely related to PEG grafting density, which can determine the protein absorption.[@b17-ijn-9-051] That is, high PEG-grafting density inhibits protein adsorption on the nanoparticle surface in the biological solution. Interaction between nanoparticles and biological media can further lead to surface modification that eventually affects their phagocytosis through the attachment of complementary proteins and immunoglobulins.[@b35-ijn-9-051] Recently, there has been much effort to utilize biological nanomaterials in drug delivery applications, due to their biocompatibility and natural cell-binding ability. Apoferritin, a demineralized form of ferritin, was suggested as a drug carrier because it has been known to enter the cell via ferritin receptor-mediated endocytosis.[@b36-ijn-9-051],[@b37-ijn-9-051] Apoferritin can be further utilized for a switchable delivery system because the endocytosis can be reversibly inhibited in various ways. Additionally, the internal cavity of apoferritin can be used to load therapeutic molecules through channels on the protein shell. Other virus- and protein-based biomaterials also have great potential to serve as biocompatible nano-platforms in the drug-delivery system.[@b38-ijn-9-051] ### Shape As mentioned in previous subsections, size and surface properties of nanoparticles play crucial roles in controlling the interaction between nanoparticles and biological systems. The nanoparticle shape might also be important in determining biological behaviors of nanoparticles. For example, it has been reported that bacteria with various shapes, such as rods, spirals, and ellipsoids, have implications for macrophage recognition.[@b16-ijn-9-051] These shapes can directly affect the endocytosis pattern of bacteria. Recent experiments have demonstrated a shape effect of the nanoparticle on cellular uptake.[@b8-ijn-9-051],[@b39-ijn-9-051] Nanoscale rods exhibit the highest uptake in human cervical cancer cells, followed by spheres, cylinders, and cubes, and the cellular uptake of cylindrical particles depends strongly on their aspect ratio. However, the receptor-mediated endocytosis of gold nanorods was vastly decreased with increases in their aspect ratio. The comparison of intracellular uptake efficiency between rod-shaped and spherical nanoparticles has also been investigated in many types of cells.[@b40-ijn-9-051] Interestingly, the results demonstrated that the uptake of rod-shaped nanoparticles by macrophages was more efficient than that of the spherical nanoparticles, while the spherical nanoparticles were taken up by cervical cancer cells and human lung epithelial cells more efficiently than were rod-shaped nanoparticles. Nanoparticle exocytosis ======================= An understanding of the cellular uptake and organ distribution of nanoparticles is important when examining their targeting and therapeutic efficiency in drug-delivery applications. Nanoparticles administered into the body are eventually cleared by organs in the MPS, such as the liver and spleen. Nanoparticles remain in these organs for a long time after being taken up by the macrophages, which increases the likelihood of unintended acute or chronic toxicity. Thus, it is also crucial to study exocytosis of the internalized nanoparticles from many types of cells, particularly macrophages, to evaluate their biosafety. However, compared with investigations of nanoparticle endocytosis, relatively little effort has been made to investigate the exocytosis of nanoparticles that may be responsible for their systemic elimination and toxicity. Thus, the exocytosis of functionalized nanoparticles is reviewed in this section ([Table 3](#t3-ijn-9-051){ref-type="table"}). Many studies have compared the exocytosis of rod-shaped nanoparticles with that of spherical nanoparticles. Chithrani and Chan[@b9-ijn-9-051] examined differences in exocytosis phenomena between spherical and rod-shaped gold nanoparticles using various cell types ([Figure 5](#f5-ijn-9-051){ref-type="fig"}). The surface of gold nanoparticles was coated with transferrin proteins for their receptor-mediated endocytosis. Importantly, this work described that the cellular uptake could be considered as a result of competition between the thermodynamic driving force for wrapping and the receptor diffusion kinetics. In that respect, the 50 nm gold nanoparticles showed the fastest wrapping time, and therefore the receptor-ligand interaction could produce sufficient free energy to drive the nanoparticles into the cell. On the other hand, smaller nanoparticles with a slower wrapping time exhibited a faster rate of exocytosis. The exocytosis rate of the 14 nm nanoparticles was much faster than that of the 74 nm nanoparticles. In addition, the fraction of the rod-shaped nanoparticles released outside the cells was generally higher than that of the spherical nanoparticles. The exocytosis of peptide-coated gold nanoparticles was also investigated in endothelial cells.[@b41-ijn-9-051] Nanoparticles functionalized with KATWLPPR peptides have been known to bind to plasma membrane receptors on endothelial cells for endocytosis, while nanoparticles coated with KPRQPSLP peptides did not interact with the receptors for endocytosis. KATWLPPR peptide-coated nanoparticles taken up by cells were progressively exocytosed up until 6 hours. On the other hand, KPRQPSLP peptide-coated nanoparticles showed a more complex exocytosis profile. Interestingly, it was found that the exocytosed KPRQPSLP peptide-coated nanoparticles were re-taken up by the cells after 4 hours. The exocytosis of poly(D,L-lactide-*co*-glycolide) (PLGA) nanoparticles was also examined in vascular smooth muscle cells.[@b42-ijn-9-051] The size and zeta potential of PLGA nanoparticles coated with bovine serum albumin (BSA) were around 97 nm and --20 mV, respectively. The cellular uptake of nanoparticles increased with incubation time. The exocytosis of nanoparticles increased up to 65% of the internalized fraction within 30 minutes when nanoparticles in the culture media were removed. In addition, the exocytosis of nanoparticles was found to be energy-dependent because it was significantly inhibited with sodium azide and deoxyglucose. Furthermore, authors demonstrated that the exocytosis pattern of nanoparticles was dependent on the proteins in the medium, because the proteins were carried into the cells along with the nanoparticles and interacted with biological systems inside the cells. Exocytosis of polysaccharide cationic nanoparticles was also studied in airway epithelium cells.[@b43-ijn-9-051] The cationic polymer hydroxycholine was used to coat nanoparticles. After the surface modification, the nanoparticles appeared to be an approximate size of 60 nm. For exocytosis experiments, human bronchial epithelial cells were treated with the nanoparticles for 30 minutes. The amount of exocytosed nanoparticles increased significantly after 1 hour. In addition, cholesterol depletion completely blocked the exocytosis of nanoparticles, indicating that their exocytosis is cholesterol-dependent. The exocytosis patterns of superparamagnetic iron oxide nanoparticles and quantum dots were also studied in many research groups. Serda et al[@b44-ijn-9-051] investigated the intracellular trafficking of iron oxide nanoparticles delivered into macrophage cells using porous silicon microcarriers ([Figure 6](#f6-ijn-9-051){ref-type="fig"}). In the exocytosis process, the amine-functionalized nanoparticles enriched in the multivesicular bodies were incorporated into the membrane vesicles in the intracellular region and efficiently secreted from the cells 6 days after intracellular delivery. The exocytosis rate of 15 nm nanoparticles was faster than that of 30 nm nanoparticles, thus indicating that smaller nanoparticles are more favorable for exocytosis. Exocytosis of zwitterionic quantum dots was also examined in human cervical cancer cells ([Figure 7](#f7-ijn-9-051){ref-type="fig"}).[@b45-ijn-9-051] Quantum dots were coated with D-penicillamine to improve colloidal stability in biological solutions. The size of D-penicillamine-coated quantum dots was around 4 nm, which is much smaller than conventional nanoparticles studied for exocytosis. In the endocytosis process, most of these smaller nanoparticles were observed on the plasma membrane prior to internalization. In addition, the quantum dots were found to enter the cells via a clathrin-mediated endocytosis pathway and macropinocytosis. In the exocytosis process, some of the quantum dots trapped in endosomes were actively transported to the cell periphery and exocytosed to the media within 21 minutes after internalization. Summary and future prospects ============================ This review article summarizes the endocytosis and exocytosis of nanoparticles of different sizes, shapes, and surface chemistries in many types of cells. Generally, smaller nanoparticles seem to enter and exit the cell more efficiently. Spherical nanoparticles are more favorable to be internalized into the cell than cylindrical nanoparticles. Positively charged nanoparticles exhibited much higher rates of endocytosis than negatively or neutrally charged nanoparticles, while they seem to remain in the cell longer. Although nanoparticle-based drug delivery has been actively developed to treat complex diseases, concerns regarding nanoparticle biosafety have still been raised, limiting their clinical translations. Understanding the endocytosis of nanoparticles enables us to develop more efficient intracellular drug-delivery nanosystems, while understating their exocytosis allow us to develop more clearable delivery nanosystems after drug delivery. However, the exocytosis patterns of nanoparticles remain not yet fully understood, although the cellular uptake of nanoparticles has been studied by many research groups. Thus, a considerable amount of effort to study the endocytosis and exocytosis of nanoparticles should be made in order to develop clinically translatable targeted nanoparticles in the drug-delivery field. This work was supported by the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (grant no 2012M3A9C6050125), the National R&D Program for Cancer Control, Ministry for Health and Welfare (grant no 1220070), and the World Class University program funded by the Ministry of Education, Science and Technology, Republic of Korea (grant no R32-2008-000-10218-0). **Disclosure** The authors report no conflicts of interest in this work. {#f1-ijn-9-051} ![Scheme of aggregation or agglomeration mechanism. The stabilizing electrostatic forces (E~ES~) on the surface of bare nanoparticles are neutralized by NaCl ions in the biological solution, causing the van der Waals forces (E~vdW~) to drive formation of aggregation or agglomeration. The protein coating of nanoparticles can reduce the aggregation or agglomeration.\ **Note:** Reproduced with permission from Albanese A, Chan WC. Effect of gold nanoparticle aggregation on cell uptake and toxicity. *ACS Nano*. 2011;5:5478--5489.[@b11-ijn-9-051] Copyright © 2011 American Chemical Society.](ijn-9-051Fig2){#f2-ijn-9-051} ![(**A**) Schematic representation of gold nanoparticles. (**B**) Transmission electron microscope images of citrate-coated gold nanoparticles with various sizes. (**C**) Transmission electron microscope images of the gold nanoparticles entrapped in cellular vesicles. Graph showing the number of the gold nanoparticles per vesicle diameter.\ **Note:** Reproduced with permission from Chithrani BD, Ghazani AA, Chan WC. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. *Nano Lett*. 2006;6:662--668.[@b8-ijn-9-051] Copyright © 2007 American Chemical Society.](ijn-9-051Fig3){#f3-ijn-9-051} ![Kinetics of cellular uptake of negatively (--COOH) (**A**) and positively (--NH2) (**B**) charged polymeric nanoparticles in macrophages and monocytes (THP-1). Confocal fluorescence images (right) were taken after 2 hours' incubation with the indicated nanoparticles. Cell membrane was stained with red dyes and the nanoparticles were tagged with green dyes.\ **Note:** Reproduced with permission from Lunov O, Syrovets T, Loos C, et al. Differential uptake of functionalized polystyrene nanoparticles by human macrophages and a monocytic cell line. *ACS Nano*. 2011;5:1657--1669.[@b33-ijn-9-051] Copyright © 2011 American Chemical Society.\ **Abbreviation:** MFI, mean fluorescence index.](ijn-9-051Fig4){#f4-ijn-9-051} ![(**A**) Schematic depicting of transferrin-coated gold nanoparticles. (**B**) Kinetics of exocytosis patterns of the nanoparticles with different sizes. (**C**) Different stages of exocytosis patterns: (**a**) Movement of the vesicles containing nanoparticles toward the cell membrane; (**b**) Docking of one of the vesicles at the cell membrane; (**c**) Excretion of nanoparticles; (**d**) Cluster of nanoparticles after exocytosis.\ **Note:** Reproduced with permission from Chithrani BD, Chan WC. Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. *Nano Lett*. 2007;7:1542--1550.[@b9-ijn-9-051] Copyright © 2007 American Chemical Society.](ijn-9-051Fig5){#f5-ijn-9-051} ![(**A**) A scanning electron microscopy image of a macrophage showing endocytosis of porous silicon particles incorporated with iron oxide nanoparticles, intracellular partitioning of the particles, endosomal escape of the particles, and exocytosis of the incorporated iron oxide nanoparticles. (**B**) Exocytosis of iron oxide nanoparticles. Upper column shows transmission electron microscopy images of iron oxide nanoparticles released from the porous silicon carrier in a macrophage. Middle column shows transmission electron microscopy images of the released iron oxide nanoparticles located in the intracellular region of the macrophage 6 days after uptake of the porous silicon particles. Bottom column shows that graph displayed time-dependent iron content in the supernatant and transmission electron microscopy images showed the internalized iron oxide nanoparticles were exocytosed by membrane vesicles.\ **Note:** Reproduced with permission from Serda RE, Mack A, van de Ven AL, et al. Logic-embedded vectors for intracellular partitioning, endosomal escape, and exocytosis of nanoparticles. *Small*. 2010;6:2691--2700.[@b44-ijn-9-051] Copyright © 2010 John Wiley & Sons, Inc.](ijn-9-051Fig6){#f6-ijn-9-051} ![(**A**) Schematic diagram showing the endocytosis and exocytosis processes of D-penicillamine-coated quantum dots; a: Clathrin-mediated endocytosis; b: Macropinocytosis. (**B**) Interaction of D-penicillamine-coated quantum dots (green) with plasma membrane of a HeLa cell before internalization. The plasma membrane was stained with the red membrane dye. Scale bar: 10 μm. (**C**) Kinetics of exocytosis of D-penicillamine-coated quantum dots after removing the nanoparticles in the media.\ **Note:** Reproduced with permission from Jiang X, Röcker C, Hafner M, Brandholt S, Dörlich RM, Nienhaus GU. Endo- and exocytosis of zwitterionic quantum dot nanoparticles by live HeLa cells. *ACS Nano*. 2010;4:6787--6797.[@b45-ijn-9-051] Copyright © 2010 American Chemical Society.\ **Abbreviations:** QDs, quantum dots; min, minutes.](ijn-9-051Fig7){#f7-ijn-9-051} ###### Classification of endocytosis pathways Pathway Definition ---------------------- ----------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------ Specific pathway  Endocytosis Clathrin- and caveolin-mediated Energy-dependent process by which cells internalize biomolecules  Phagocytosis Mannose receptor-, complement receptor-, Fcγ receptor-, and scavenger receptor-mediated Actin-dependent endocytic process by which professional phagocytes (macrophages, dendritic cells and neutrophils) engulf particles with sizes larger than 0.5 μm Non-specific pathway  Macro-pinocytosis -- Endocytic process by which cells internalize fluids and particles together, and large vesicles (0.2--5 μm) are formed  Pinocytosis -- Endocytic process by which cells absorb extracellular fluids, small molecules and small vesicles (\~100 nm) are formed ###### Endocytosis of nanoparticles Nanoparticle Size (dm) Coating material Surface charge Cell types Summary Reference ------------------------------- ---------------------------- ---------------------------------------------------- ---------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------- Spherical AuNP 14, 50, 74, and 100 nm Citric acid Negative Human cervical cancer cell 50 nm AuNPs had the highest cellular uptake [@b8-ijn-9-051],[@b9-ijn-9-051],[@b24-ijn-9-051] 16 nm PEG, TAT, and NLS peptides -- Human cervical cancer cell PEG-coated AuNPs were not taken up at all by cells. TAT peptides facilitated the direct transfer of nanoparticles into cells. NLS peptide-modified AuNPs were found in the nucleus [@b46-ijn-9-051] Rod-shaped AuNP 40 × 14 nm and 74 × 14 nm Citric acid Negative Human cervical cancer cell Rod-shaped nanoparticles with a higher aspect ratio showed lower cellular uptake than those with a lower aspect ratio [@b8-ijn-9-051] Spherical and rod-shaped AuNP 15, 50, and 15 × 50 nm PEO with either hydroxyl, carboxyl, or amine group Negative or positive Human macrophage Nanorods were more efficiently taken up by macrophages than were spherical NPs [@b40-ijn-9-051] SPION 6 nm Dextran, aminodextran, heparin, or DMSA Negative or positive Human cervical cancer cell Dextran-coated NPs were not taken up by cells. DMSA-coated NPs displayed low cellular uptake. Heparin and aminodextran-coated NPs showed the highest cellular uptake [@b47-ijn-9-051] SWNT 130--660 nm DNA Negative Embryonic fibroblast 660 nm and 130 nm SWNTs showed lower cellular uptake than 430 nm and 320 nm SWNTs [@b26-ijn-9-051] Polymeric NP 150--500 nm CMC or CH Negative or positive Murine macrophage CH-coated NPs showed higher cellular uptake than CMC-coated NPs [@b32-ijn-9-051] PLGA NP 260--300 nm PVA or vitamin E TPGS Negative Human colon adeno-carcinoma cell Cellular uptake of vitamin E TPGS-coated NPs was higher than that of PVA-coated NPs [@b25-ijn-9-051] Apoferritin 12 nm -- -- Human intestinal epithelial cell Apoferrin enters the cell via ferritin receptor-mediated endocytosis [@b36-ijn-9-051] **Abbreviations:** AuNP, gold nanoparticles; CH, chitosan hydrochloride; CMC, carboxymethyl chitosan; dm, diameter; DMSA, dimercaptosuccinic acid; DNA, deoxyribonucleic acid; NLS, nuclear localization sequence; PEG, polyethylene glycol; PEO, poly(ethylene oxide); PLGA, poly(D,L-lactide-*co*-glycolide); PVA, polyvinyl alcohol; SPION, superparamagnetic iron oxide nanoparticles; SWNT, single-walled carbon nanotubes; TAT, HIV tat protein transduction domain; E TPGS, Vitamin E succinated polyethylene glycol 1000. ###### Exocytosis of nanoparticles ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- NP Size (dm) Coating material Surface charge Cell types Summary Reference --------------------- --------------------------------- ---------------------- ------------------------ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------ ------------------ Spherical AuNP 14, 50, and 74 nm Transferrin Negative Human fibroblast, cervical cancer, brain tumor cells Smaller NPs show a faster rate of exocytosis [@b9-ijn-9-051] 17 nm KATWLPPR and KPRQPSLP peptide Negative Human endothelial cell In KATWLPPR peptide, exocytosis of NPs taken up by cells progressively increase until 6 hours. In KPRQPSLP peptides, some exocytosed particles are re-taken up by the cells after 4 hours [@b41-ijn-9-051] Rod-shaped AuNP 20 × 30, 14 × 50, and 7 × 42 nm Transferrin Negative Human fibroblast, cervical cancer, brain tumor cells Nanorods with lower aspect ratios showed slower exocytosis.\ [@b9-ijn-9-051] The exocytosis rate of rod-shaped NPs was higher than that of spherical NPs Quantum dot 8 nm D-penicillamine Negative Human cervical cancer cells Half-life of exocytosed fraction was 21 minutes and reached saturation after 2 hours [@b45-ijn-9-051] PLGA NP 97 nm Bovine serum albumin Negative Human arterial vascular smooth muscle cells Half-life of exocytosed fraction was 30 minutes [@b42-ijn-9-051] Polysacch arides NP 60 nm Hydroxycholine Positive Human bronchial epithelial cell Cholesterol depletion blocked exocytosis of NPs [@b43-ijn-9-051] SPION 10 nm PEGylated amine Positive Murine macrophages SPIONs released from carriers in the intracellular region were exocytosed by membrane vesicles [@b44-ijn-9-051] ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Abbreviations:** AuNP, gold nanoparticle; dm, diameter; PEG, polyethylene glycol; PLGA, poly(D,L-lactide-*co*-glycolide); SPION, superparamagnetic iron oxide nanoparticles; NPs, nanoparticles.

Background {#Sec1} ========== The treatment of end-stage chronic kidney disease includes hemodialysis, peritoneal dialysis and kidney transplantation (KTx), while today KTx is the treatment of choice due to better survival, quality of life and cost effectiveness in comparison to maintenance dialysis \[[@CR1], [@CR2]\]. Short- and long-term graft survival after KTx have significantly improved within the last decades but there is still a substantial number of patients with declining transplant function and graft loss in the long term. Currently, the mean 20-year graft survival is only 30--40% \[[@CR3]\]. Consequently, there is a clear need to develop and test for new therapies that may help to preserve long-term graft function. In addition, identification of reliable candidate biomarkers would allow predicting long-term allograft function or select specific transplant patients that may benefit from new strategies. Metabolic acidosis (MA) is a well-known complication of CKD and associated with a variety of complications such as uremic bone disease, protein-energy wasting, chronic inflammation, insulin resistance, impairment of myocardial function and disturbances in mineral metabolism, growth hormone and thyroid gland function \[[@CR4]--[@CR6]\]. In addition, observational studies have shown that MA is associated with increased mortality in both, dialysis patients and patients with non-dialysis dependent CKD \[[@CR7]\]. Importantly, MA is highly prevalent in renal transplant patients with prevalences reported ranging from 12 to 58% and seems to be consistently more frequent and more severe when compared to non-transplant CKD cohorts with similar degrees of renal function \[[@CR8]--[@CR10]\]. In our clinic, a cross-sectional analysis among 823 unselected kidney transplant patients revealed metabolic acidosis in 58.1% of the patients defined by a serum bicarbonate of \< 24 mmol/l indicating a high prevalence of MA in this patient cohort \[[@CR8]\]. Notably, eGFR correlated best with serum bicarbonate levels at time of examination. These data could be confirmed by other authors \[[@CR9]\] and similar to CKD patients creatinine clearance was markedly lower in the group of transplant patients with MA compared to the non-acidotic patients. Over the past few years several studies have shown that MA plays an important role in the progression of CKD and that low serum bicarbonate levels are associated with a higher risk of progressing to end-stage renal disease \[[@CR5], [@CR7], [@CR11]--[@CR15]\]. Amelioration of MA in CKD patients with alkalinizing therapies delayed progression of GFR loss \[[@CR16]--[@CR18]\]. Furthermore, a recent study has shown for the first time that MA after KTx is associated with increased risk of graft loss and patient death as mentioned before for CKD patients. In this multicenter retrospective cohort study of 2318 patients, a low TCO~2~ level (\< 22 mmol/L) 3 months after transplantation was associated with increased risk of graft loss and death-censored graft failure even after adjusting for eGFR \[[@CR19]\]. However, to the best of our knowledge, no prospective trial has been initiated yet to test the role of alkali treatment in the prevention of progressive loss of renal allograft function. Apart from the crucial question if alkali treatment may have a beneficial effect on graft function there are at least two other important aspects of metabolic acidosis after kidney transplantation that are not fully understood yet, namely first the underlying pathomechanisms why MA occurs in KTx patients and second how metabolic acidosis may promote disease progression. It has been reported that MA is more common in patients who received a cadaveric organ compared to a transplant from a living donor \[[@CR20]\] but as of today, the influence of donor age, donor renal function and further pre-transplant factors on the occurrence of MA after KTx is still unclear \[[@CR10]\]. Other factors such as the intake of calcineurin inhibitors have been shown to be associated with the development of MA by specific changes in tubular epithelial cells which play a role in acid-base regulation \[[@CR8], [@CR20]--[@CR24]\]. In CKD, Wesson et al. investigated intensively the pathogenic effects of endothelin, aldosterone and angiotensin-II on the kidney and its impact on GFR \[[@CR13], [@CR14], [@CR17], [@CR25]--[@CR28]\]. Likewise, a role of the complement system and accumulation of interstitial ammonium had been early proposed to contribute to progression of CKD \[[@CR29]\]. Thus, it is essential to investigate the role and in particular the pathomechanisms of MA on graft function and outcome. These findings may help to develop new potential therapeutic targets, such as alkali therapy, that may help to preserve long-term graft survival in this population. To date, more than 3000 patients with a functioning kidney transplant are living in Switzerland and over 200,000 in the United States \[[@CR30]\]. Many of these patients already suffer from decreased graft function which favours the development of MA. Given the expanding pool of CKD patients - including former kidney transplant recipients - an alkali treatment study in kidney transplant patients is of prime importance and has the potential to show that such a treatment may slow or reduce the progression towards graft failure and significantly decrease the rate of end stage renal disease, and therefore prolonging long-term graft survival in KTRs. Methods {#Sec2} ======= Trial design {#Sec3} ------------ The Preserve-Transplant Study (PTS) is an investigator-initiated, prospective, patient-blinded, multi-center, randomized, controlled phase-IV trial with two parallel-groups comparing sodium bicarbonate to placebo. The study encompasses 300 patients, ≥ 18 years of age, at least 12 months after renal transplantation and with a serum bicarbonate level ≤ 22 mmol/l. A placebo was chosen as a comparator to the intervention because no equivalent active comparator is existing to date that would qualify as an appropriate comparator. Participants will be randomly allocated to receive sodium bicarbonate or placebo in a 1:1 distribution and will be followed up for 2 years (see Fig. [1](#Fig1){ref-type="fig"}). The trial is performed at three transplant centers in Switzerland, in Zurich, Berne and Geneva.Fig. 1Study Flowchart of the Preserve-Transplant Study. Numbers in circles define each study visit. Study visits 2, 7, and 11 include in addition to blood and spot urine tests 24 h-ambulatory blood pressure monitoring and 24 h urine collection Objectives and hypothesis {#Sec4} ------------------------- The primary objective of PTS is to test if alkali treatment will preserve kidney graft function and diminish the progression of chronic kidney disease in renal transplant patients by assesing the change in eGFR over 2 years from baseline compared to placebo. Additionally we want to investigate underlying pathomechanisms of metabolic acidosis and how metabolic acidosis may promote disease progression. A further objective is to identify reliable candidate biomarkers that would allow selecting specific transplant patients that may benefit from alkali therapy. This study assesses long-term safety of the study drug with a particular focus on new onset and worsening of hypertension, volume overload, and gastrointestinal side effects. Selected adverse events (AEs) and all serious adverse events (SAEs) will be documented regularly and compared between the treatment and placebo group. Trial outcomes {#Sec5} -------------- The primary outcome of this study is the change in renal function by assessing eGFR within 2 years. eGFR will be determined based on the CKD-EPI creatinine equation. By now, eGFR is the best parameter to assess kidney function and to predict graft and patient survival in kidney transplant recipients \[[@CR1]\]. Recent recommendations from the NKF-FDA Scientific Workshop and other publications show that eGFR decline over 2--3 years is an acceptable endpoint for CKD progression trials and clinical transplant research \[[@CR1], [@CR31]--[@CR33]\]. Additionally, several recent studies have used eGFR successfully to investigate the effect of alkali treatment on progression of chronic kidney disease \[[@CR13], [@CR14], [@CR28]\]. Moreover, creatinine is a well-established, standardized, and reliable parameter ubiquitously measured in routine clinical practice and compared to inulin or radiolabeled isotopes such as ^125^I Iothalamate or ^51^Cr EDTA very cost-effective and easy available. Important secondary outcomes include changes in serum bicarbonate levels, pH, sodium, potassium, and proteinuria measured as protein/creatinine or albumin/creatinine ratio in spot urine. As a safety outcome, 24-h ambulatory blood pressure monitoring will be performed at the beginning of the study and after 1 and 2 years to evaluate the effect of sodium bicarbonate on blood pressure. All outcomes of interest are shown in Table [1](#Tab1){ref-type="table"}**.**Table 1Study OutcomesPrimary OutcomeChange in renal function by assessing eGFR (CKD-EPI) within 2 yearsImportant Secondary OutcomesChanges in serum bicarbonate levels, pH, sodium, potassium, and proteinuria measured as protein/creatinine or albumin/creatinine ratio in spot urineSafety OutcomeTwenty-four-hour ambulatory blood pressure monitoring will be performed at the beginning of the study and after 1 and 2 yearsOther Outcomes of Interest• Changes in measurement of specific acid base transport proteins by urinary exosome collection\ • Changes in urinary ammonium excretion, inflammatory markers such as complement factors, and hormones involved in tubulo-interstitial nephritis/fibrosis such as endothelin and aldosterone\ • Histopathological grade of tubulo-interstitial fibrosis according to BANFF classification in kidney biopsies (where available) will be analyzed in both arms\ • Systemic calcification propensity in blood will be determined by the *T*~*50*~-Test (A test measuring the transformation time (*T*~*50*~) of primary to secondary calciprotein particles, where a long delay of *T*~*50*~ indicates a high residual capacity of the patients' serum to prevent the formation of secondary calciprotein particles and is therefore a sign of an intact endogenous defense against calcification) \[[@CR34]\]\ • Change of bone density and rejection rate Study population {#Sec6} ---------------- Kidney transplant patients will be recruited from all study centers according to the inclusion and exclusion criteria (Table [2](#Tab2){ref-type="table"}).Table 2Inclusion and Exclusion Criteria of the Preserve-Transplant StudyInclusion Criteria • Informed consent as documented by signature • Age ≥ 18 years and able to give informed consent • ≥ 12 months after renal transplantation • Stable clinical condition • Stable graft function over the last 6 months (creatinine changes ±15%) • eGFR between 15 and 89 ml/min/1.73 m^2^ • At least one serum bicarbonate measured ≤22 mmol/l within the last 6 monthsExclusion Criteria • Uncontrolled hypertension or use of \> 4 antihypertensive agents • Uncontrolled heart failure • Serum potassium \< 3.0 mmol/l • Serum sodium \> 150 mmol/l • Use of alkali in the preceding 4 weeks • Use of mineralocorticoid antagonists, topiramate, carbo anhydrase inhibitors or any drugs with similar effects • History of noncompliance with clinic visits • Hereditary fructose intolerance • Known hypersensitivity or allergy to the drug used in this study or to peanut, sorbitol, and soy • Pregnancy or breastfeeding • Intention to become pregnant during the course of the study • Lack of safe contraception, defined as: Female participants of childbearing potential, not using and not willing to continue using a medically reliable method of contraception for the entire study duration, such as oral, injectable, or implantable contraceptives, or intrauterine contraceptive devices, or who are not using any other method considered sufficiently reliable by the investigator in individual cases. Please note that female participants who are surgically sterilised / hysterectomised or post-menopausal for longer than 2 years are not considered as being of child bearing potential. • Suspected drug or alcohol abuse • Inability to follow the procedures of the study, e.g. due to language problems, psychological disorders, dementia, etc. of the participant • Enrolment of the investigator, his/her family members, employees and other dependent persons Study enrolment, randomization and blinding {#Sec7} ------------------------------------------- Potential candidates will be identified from the electronic hospital databases. After signature of the informed consent and successful screening patients will be randomized in a 1:1 ratio stratified by each study center and gender using a permuted block design. The block size will be concealed until the primary endpoint will be analyzed. The randomization lists remain within the electronic data management tool for the whole duration of the study. Thus, randomization will be conducted without any influence of the principal investigators or other study personnel. The study is single-blind on the patient side. Biobank {#Sec8} ------- Biobank samples are obtained at every study visit in all study centers and stored at − 80 °C for 5 years after termination of the study according to Swiss legal requirements. All samples or genetic data will be stored and used for future research purposes with the participant's consent. Investigational medicinal product {#Sec9} --------------------------------- Nephrotrans® (sodium hydrogen carbonate, ATC-Code: A02AH) has been approved and used since 1986 for the treatment of metabolic acidosis. Nephrotrans® is a safe, well-tolerated drug with very few and well-studied side effects. The study drug will be provided by SALMON Pharma GmbH in its commercially available formulation, used orally in this study and will be given at an initial dose of 500--1000 mg sodium hydrogen carbonate thrice daily. The dosage is chosen according to the body weight of the patient at baseline visit (\< 70 kg or ≥ 70 kg). If serum bicarbonate remains ≤22 mmol/l the dosage of sodium hydrogen bicarbonate will be titrated once to 1000--1500 mg thrice daily as for placebo too at visit 3 after 2 weeks. All patients will be blinded to the identity of the study drug. The study product will be administered for a total of 2 years. The placebo is also provided by SALMON Pharma GmbH and used as a comparator to the intervention in this study. All patients will have the best standard care for kidney transplant patients. Informed consent {#Sec10} ---------------- Written informed consent for participation will be obtained from all participants before any study related actions are taken. Patients will be informed that withdrawal from the study is possible at any time without giving reasons. Approvals {#Sec11} --------- The study has been approved by the Cantonal Ethics Committee of Zürich, Berne and Geneva and Swissmedic as the competent authority. The study has been registered on [ClinicalTrials.gov](http://clinicaltrials.gov) (NCT03102996). The project will be conducted in line with the Declaration of Helsinki as well as all national legal and regulatory requirements. In addition all researchers will follow the GCP guidelines. All non-substantial and substantial amendments of the protocol will be reported to the ethics committees and the competent authorities in Switzerland. Criteria for discontinuation {#Sec12} ---------------------------- Patients will be withdrawn from participation in the trial if they withdraw their consent or the investigator or sponsor withdraws the patient because of malcompliance with the study intervention. Additionally, if serum bicarbonate exceeds 35 mmol/l or falls below 16 mmol/l the patient will have to be excluded from the study for safety reasons. Pregnancy in women or a significant violation of the protocol as judged by the coordinating investigator also will lead to withdrawal of the participant. Patients will be withdrawn from study treatment permanently if a severe allergic or hypersensitivity reaction to the drug occurs or if the investigator feels that the treatment is harmful to the subject. In case of a treatment-related SAE the study drug will also be withdrawn from the patient. A study patient who discontinues study participation prematurely for any reason is defined as dropout if the subject has already been randomized. A study patient who terminates the study before randomization is regarded as a screening failure. Monitoring {#Sec13} ---------- Quality assurance will be based on a central and on-site monitoring. The central monitoring encompasses monitoring of data entry progress. Trial sites will also be regularly visited by independent monitors. Visits will focus on controlling regulatory compliance, monitoring of processes, and verification of data that cannot be monitored centrally. Data management {#Sec14} --------------- In the Preserve-Transplant study, all data will be entered electronically using an internet-based secure data base (secuTrial®) developed in agreement with the Good Clinical Practice (GCP) guidelines. Data integrity is enforced through a variety of mechanisms. Regular backup of the data is assured. Statistical analysis {#Sec15} -------------------- The analysis population consists of all patients who were randomized and have valid data for baseline and at least one follow-up. The primary and secondary endpoints will be analyzed with linear mixed models. Moreover, exploratory subgroup analyses are planned with respect to baseline eGFR to evaluate whether subgroups with lower baseline eGFR will show a better response to alkali treatment compared to subgroups with higher baseline eGFR. Any further details will be specified in the statistical analysis plan. Sample size {#Sec16} ----------- Based on a retrospective analysis of our own cohort, it was assumed that the mean annual eGFR decline in the placebo group will be 1.5 ml/min/1.73 m^2^ and that this decline can be reduced to 0 ml/min/1.73 m^2^ in the treatment group as a result of alkali therapy based on previously published data for CKD patients with metabolic acidosis. Power was determined on the basis of a two-sided, two-sample t-test of the slopes over 2 years. Group sizes of 150 will achieve 82% power to find a minimum detectable difference in the mean slope over 2 years of − 3.0 with estimated group standard deviations of 9.0 and with a significance level of 0.05 using a two-sided two-sample t-test. Therefore, a total number of 300 patients for the entire study is planned. Discussion {#Sec17} ========== Metabolic acidosis is a serious complication in patients with CKD with deleterious effects on kidney function and patient survival. A recent retrospective study has evidenced that MA is also associated with increased risk of graft loss and patient mortality in kidney transplant recipients \[[@CR19]\]. Although several studies have demonstrated a beneficial role for alkali therapy on the progression of CKD \[[@CR5], [@CR7], [@CR11]--[@CR15]\], no data is available if alkali treatment may also allow to better preserve renal allograft function in kidney transplant recipients with metabolic acidosis. The Preserve-Transplant Study is the first investigator-initiated prospective clinical trial that aims to test whether alkali treatment will help preserving kidney graft function and diminish the progression of chronic kidney disease in renal transplant patients. Thereby, alkali therapy may have the potential to become the first evidence-based adjuvant therapy for the preservation of graft function in kidney transplant patients. Consequently, the results of the PTS may provide evidence to adapt current recommendations and guidelines for the routine treatment of KTRs suffering from metabolic acidosis. The increasing demand for donor organs worldwide calls for solutions to help to prolong graft survival. The implementation of alkali therapy into the drug regimen of kidney transplant recipients would have a favorable risk-benefit ratio since alkali supplements are routinely used in CKD patients and represent a well-tolerated, safe and cost-effective treatment. Trial status and dissemination {#Sec18} ============================== The Preserve-Transplant randomized controlled clinical study has received governance approval and is registered at [ClinicalTrials.gov](http://clinicaltrials.gov) (NCT03102996). The trial started recruitment in June, 2017. The study will end in June 2021. First results from this trial are expected in the first quarter of 2022. Publications will be submitted to peer-reviewed journals and the results will be presented at national and international scientific conferences. AE : Adverse event CKD : Chronic kidney disease eGFR : Estimated Glomerular Filtration Rate ESRD : End stage renal disease GCP : Good clinical practice KTRs : Kidney transplant recipients KTx : Kidney transplantation MA : Metabolic acidosis PTS : Preserve-Transplant Study SAE : Serious adverse event We are grateful to all study nurses and study physicians participating in this study. We would like to thank SALMON Pharma GmbH for providing the study medication. Funding {#FPar1} ======= The trial is funded by the Swiss National Science Foundation (SNSF) Grant number 33IC30_166811. The study protocol has undergone an extensive peer-review process by the Swiss National Science Foundation. NM, CAW, NG and RPW participated in the design of the study. AW, NM, SA, KH, TFM, AR, DS and RPW participate in the execution and coordination of the study. AW and NM drafted the manuscript. All co-authors read and approved the final version of the manuscript. Ethics approval and consent to participate {#FPar2} ========================================== The study was approved by the lead cantonal ethics committee in Zurich (Switzerland) and the cantonal ethics committee in Berne (Switzerland) and Geneva (Switzerland), Registration number: 2016--02012. Consent for publication {#FPar3} ======================= Not applicable. Competing interests {#FPar4} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar5} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Correction to: Fraslin et al. Genet Sel Evol (2018) 50:60 10.1186/s12711-018-0431-9 {#Sec1} =================================================================================== After publication of this work \[[@CR1]\], we noted that there was an error in Table 3 Line 4: ^b^Omy2~**Omy3**~ should be ^b^Omy21~**Omy3**~. The correct Table [3](#Tab3){ref-type="table"} is included here.Table 3Results of QTL analysis using the model *M2* for resistance trait following injection or immersion challengesInfection routeQTLLRTmaxPosition (cM)CI (95%)Increase in survival rateResistance origin*P* value fixed effect*P* value interactionFixed_R (%)Fixed_S (%)Fixed_RFixed_SIMMERSIONOmy17~**Omy3**~13.97\*610--92387AP2AP2\*\*\*NSOmy25a~**Omy3**~10.41\*40--351018B57B57\*\*\*NS*Type 1 interaction*INJECTION^a^Omy3~**Omy29**~15.27\*\*8946--1051647AP2AP2\*\*\*\*\*\*IMMERSION^b^Omy21~**Omy3**~15.35\*\*9763--104439B57B57\*\*\*\*\*\*^b^Omy3~**Omy21**~40.73\*\*\*8782--932055AP2AP2\*\*\*\*\*\*^c^Omy3~**Omy2**~35.66\*\*\*8781--941744AP2AP2\*\*\*\*\*\*INJECTION^a^Omy29.2~**Omy3**~14.85\*238--49548B57AP2\*\*\*\*Omy17~**Omy25a**~15.85\*\*7353--791153AP2B57\*\*\*\*\*\*IMMERSIONOmy7.2~**Omy21**~11.48\*70--103531AP2B57\*\*\*\*\*\**Type 2 interaction*INJECTION^d^Omy25a~**Omy3**~25.49\*\*\*1410--185316B57B57\*\*\*\*^d^Omy3~**Omy25a**~35.35\*\*\*8986--925922AP2AP2\*\*\*\*\*\*Omy26~**Omy29**~11.75\*180--343026AP2AP2\*\*\*\*\*\*INJECTIONOmy17~**Omy29**~18.29\*\*\*7458--924711AP2B57\*\*\*\*\*\*IMMERSIONOmy24~**Omy2**~12.71\*40--19201B57AP2\*\*\*\*\*\**Type 3 interaction*IMMERSIONOmy7.1~**Omy2**~16.42\*\*6132--871919B57AP2\*\*\*\*\*\*The table presents chromosome-wide or genome-wide significant QTL detected for STATUS using model *M*2; Reciprocal interactions could be tested only for QTL detected in the first STATUS analysis (model *M*1); LRTmax = maximum of likelihood ratio test; Position in the genetic map in centimorgans (cM); CI = confidence interval; Chromosome-wide significant = \**P* ≤ 0.01; Genome-wide significant = \*\**P* ≤ 0.05 or \*\*\**P* ≤ 0.01; *P* values for fixed effect and interaction corrected with Benjamini--Hochberg method: Non-significant = NS; \**P* value ≤ 0.05; \*\*\**P* value ≤ 0.001^a^The reciprocal interaction could not be tested as a new QTL (Omy29.2~**Omy3**~-QTL) was detected with the reciprocal model^b,d^Reciprocal models for QTL pairs^c^The QTL in the reciprocal model (Omy2~**Omy3**~-QTL) was only suggestive (*P* ≤ 0.05) at the chromosome-wide level Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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INTRODUCTION {#s1} ============ Current treatment options for breast cancer are moving toward potent targeted therapies in general well tolerated and that can be tailored to an individual patient's tumor. There are now targeted therapeutic options available for nearly all breast cancer subtypes, exploiting the differing drivers of carcinogenesis within these individual tumors \[[@R1]\]. Indeed, a better understanding of the biology of breast cancer and the recent advances in the application of genomic technologies led to the identification of a number of molecular targets. Breast cancer is as a complex disease in which each tumor presents several genomic alterations and activated pathways \[[@R2], [@R3]\]. Therefore, there is a strong rationale to combine drugs based on the presence of multiple genomic alterations and/or pathway activation \[[@R4]\]. Among them are tyrosine kinase inhibitors directed at a number of targets (HER1, HER2, HER3, IGF receptor \[IGFR\], C-MET, FGF receptor \[FGFR\]), inhibitors of intracellular signaling pathways (PI3K, AKT, mammalian target of rapamycin \[mTOR\]), angiogenesis inhibitors and agents that interfere with DNA repair \[[@R5]\]. Some of these agents have shown remarkable activity and have become part of the standard of care in patients with breast cancer (exemplified by the anti-HER2 agents trastuzumab and lapatinib). Others have been recently approved for the treatment of specific breast cancer subtypes, such as the mTOR inhibitor everolimus in advanced luminal breast cancer and the poly(ADP-ribose)polymerase (PARP) inhibitor olaparib in metastatic breast cancer with germline BRCA1 or BRCA2 mutations \[[@R6]--[@R9]\]. One of the challenges that physicians are confronted with, is the ability to match each patient with the right therapy. Given the complexity of the cancer cell signal transduction networks, it may be more rational to inhibit more than one target or pathway at a time. Choices on drug combinations in clinical studies should be based on biological rationales and preclinical evidence of additive or synergistic effects. DNA repair deficiencies and activation of PI3K pathway are relatively common events in breast cancer. BRCA1/2 mutations have been associated with sensitivity to PARP1 inhibitors (synthetic lethality) and DNA alkylating agents (genotoxic) \[[@R9]\], while alterations in component of the PI3K pathway might confer sensitivity to PI3K and mTOR inhibitors. The mTOR inhibitor everolimus has been recently approved for the treatment of advanced ER+ breast cancer in a context of endocrine resistance \[[@R6]\]. Although everolimus combined with an aromatase inhibitor improved progression-free survival, not all patients respond to everolimus and even those who respond eventually develop resistance \[[@R10]\]. Patient stratification and predictive biomarker are still lacking \[[@R11]\]. Here we explored the therapeutic benefit of combining everolimus to a PARP inhibitor in two different patient-derived xenografts of BRCA2-mutated breast cancer with genomic alterations in the PI3K pathway. We show that everolimus combined to olaparib lead to unrepaired DNA damage and tumor regression *in vivo*, through a cross-talk between DNA repair and mTOR pathways. RESULTS {#s2} ======= Treatment of a BRCA2 mutated luminal breast cancer PDX by everolimus and olaparib results in tumor regression {#s2_1} ------------------------------------------------------------------------------------------------------------- To test whether the combination of DNA repair and mTOR inhibitors could be a relevant therapeutic strategy in tumors with genomic alterations in DNA repair and PI3K/AKT/mTOR pathway, we treat a PDX model established from a BRCA2 mutated breast cancer (HBCx-22 TamR) with the Parp inhibitor olaparib combined to the mTOR inhibitor everolimus. This PDX model has been established from an ER+ primary breast cancer \[[@R12]\] and has been rendered resistant to tamoxifen in mice, through long-term *in vivo* treatment and re-engraftment of xenograft that showed acquired resistance to tamoxifen, as previously described \[[@R13]\]. This tumor carries an in frame deletion in the *PIK3R1* gene and a frameshift inactivating mutation of *PTEN* associated to its protein loss (Table [1](#T1){ref-type="table"}). ###### Breast cancer subtypes and genomic characteristics of BRCA2-mutated PDX models ---------------------------------------------------------------------------------------------------------------------------------- HBCx22 TamR HBCx-17 ------------------------- ---------------------------------------------------- --------------------------------------------------- Subtype Luminal B Basal-like Germline BRCA2 mutation c.6405_6409del5, p.Asn2135Lysfs^\*^3 (NM_000059.3) c.6033_6034del, p.Ser2012GlnFs^\*^5 (NM_000059.3) *PIK3R1* c.1704_1727del, p.Arg569_Thr576del (NM_181523.1) Wild-type *PI3KCA* Wild-type Wild-type *PTEN* c.314G\>T, p.Cys105Phe (NM_000314.4)\ Intragenic deletion\ LOH and protein loss LOH and protein loss Reference Cottu et al. (2014) De Plater et al. (2010) ---------------------------------------------------------------------------------------------------------------------------------- The effect of everolimus, olaparib and evero-limus+olaparib treatments on the tumor growth of HBCx-22 TamR are shown in Figure [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}. In the monotherapy setting, everolimus and olaparib inhibited tumor growth with a TGI of 86% with 4 and 3 mice showing tumor regression, respectively (Figure [1B](#F1){ref-type="fig"}). The combination of the two treatments resulted in strong tumor growth inhibition (TGI of 96%) with 9/9 mice in tumor regression and 4 complete responses. Tumor growth inhibition was higher in the combination group as compared to the monotherapy groups (p\<0.0001 when compared to olaparib and p=0.0002 when compared to everolimus, Mann-Whitney *t*-test). {#F1} To analyze the effect of treatments on tumor, a second experiment was performed where treatments were started in larger tumors (tumor volume between 150 and 400 mm3), were administered during 3 weeks and samples were harvested before complete response occurred (3 hours after the last treatment). We then analyzed the expression of phospho-histone H3 (P-HistH3) and Ki67 by western blot and immunohistochemistry analysis, respectively. Expression of P-HistH3 and Ki67 were inhibited in both monotherapy groups and strongly reduced in the combination arm (Figure [1C](#F1){ref-type="fig"} and [1D](#F1){ref-type="fig"}). In summary, these results show that treatment by everolimus combined to olaparib results in strong tumor growth inhibition with 100% of animals showing tumor regression in a PDX of ER+ BRCA2 mutated breast cancer. Treatment by everolimus results in increased DNA damage and decreased expression of several proteins involved in DNA repair {#s2_2} --------------------------------------------------------------------------------------------------------------------------- The presence of double-strand break (DSB) in chromatin induces the phosphorylation of the histone H2AX, and this modification can be used as a marker for mechanistic studies of DSB induction and repair (44--46). To test the effect of olaparib and everolimus on DSB induction, we measured phosphorylation of H2AX (P-H2AX) by immunohistochemistry in tumors treated during 3 weeks and harvested 3 h after the last dose. Tumors treated with everolimus and olaparib alone showed an increased faction of P-H2AX positive cells (16% and 21% respectively, compared to 8% in the control group), indicating that not only the DNA repair inhibitor olaparib could increase DNA damage (as it is expected) but also the mTOR inhibitor everolimus (Figure [2A](#F2){ref-type="fig"}). In the combination arm, the fraction of P-H2AX was significantly higher than in both monotherapy groups, with 29% of P-H2AX positive cells. The formation of RAD51 foci in HBCx-22 TamR xenografts was impaired (Figure [2B](#F2){ref-type="fig"}), confirming that this BRCA2 deficient PDX is unable to repair DNA double strands breaks by homologous recombination. {#F2} To identify putative crosstalk events between mTOR and DNA repair, a Reverse Phase Protein Array (RPPA) analysis of multiple signaling pathways and DNA repair processes was performed on untreated and treated xenografts. The list of antibodies is provided in [Supplementary Table 1](#SD2){ref-type="supplementary-material"} and covered different signaling pathways (DNA repair, cell-cyle, PI3K/AKT/mTOR and MAPK pathways, apoptosis and chromatin remodeling pathways). Raw data of RPPA results are showed in [Supplementary Table 2](#SD3){ref-type="supplementary-material"}. Results of RPPA analysis indicate that treatment by everolimus and olaparib, both in the monotherapy and combination settings, decreased expression of several proteins involved in homologous recombination (RAD50, FANCD2, P-p53BP1), checkpoint control (P-CHK1 and P-CHK2), Toposimerase II alpha and chromatin remodeling (MST1 and SUV39H1) (Figure [3A](#F3){ref-type="fig"}). FANCD2 protein belongs to the Fanconi Anemia Pathway and is required for repair of DSB and intra-S-phase checkpoint activation \[[@R14]\], while SUV39H1 is a methyltransferase that directs H3K9 methylation on large chromatin domains adjacent to the DSB to promote activation of DSB-signaling proteins \[[@R15]\]. Inhibition of FANCD2 and SUV39H1 protein expression in HBCx22 TamR xenografts was confirmed by Western Blot analysis (Figure [3B](#F3){ref-type="fig"}). {#F3} RPPA analysis of proteins involved in PI3K and MAPK pathways showed strong inhibition of P-S6 in the everolimus treated xenografts (monotherapy and combination groups) and inhibition of AKT phosphorylation (Thr308) by everolimus and olaparib (Figure [4A](#F4){ref-type="fig"}). Phospho-MEK was also significantly decreased in all groups as compared to control. Western blot analysis of P-S6 and P-AKT confirmed RPPA results (Figure [4B](#F4){ref-type="fig"}). {#F4} In summary these results show that treatment by everolimus and olaparib, given as single agents and in combination, results in decreased expression of several proteins involved in DNA repair, chromatin remodeling and checkpoint control. The combination of mTOR and PARP inhibitors is also efficient in a basal-like BRCA2 breast cancer {#s2_3} ------------------------------------------------------------------------------------------------- Although BRCA2 mutations occur more frequently in ER+ breast tumors, several studies reported BRCA2 mutations in triple-negative breast cancer \[[@R16]--[@R18]\]. To test whether targeting mTOR and DNA repair could be a relevant therapeutic strategy in triple-negative breast cancer, we tested the combination of everolimus and olaparib in PDX model established from a BRCA2-mutated basal-like breast cancer \[[@R19]\]. This model carries a large deletion around the PTEN locus on chromosome 10q23 and shows loss of PTEN protein in IHC \[[@R20]\]. Treatment by everolimus and olaparib in the monotherapy setting resulted in TGI of 80% and 78%, calculated at day 40 (when mice of the control group were sacrificed) (Figure [5A](#F5){ref-type="fig"}). Tumor growth inhibition were similar to those obtained in the HBCx22 TamR xenograft, however no tumor regressions were observed in the monotherapy setting. In the combination setting, tumor growth inhibition was of 96% at day 40 and at day 55, all mice showed tumor regression and 2 of them complete response (Figure [5A](#F5){ref-type="fig"}). To measure the extent of unrepaired DNA damage, we analyzed P-H2AX expression in formalin-fixed tumors harvested at the end of the experiment. DNA damage was increase in xenografts treated by everolimus and olaparib given in the monotherapy setting, with a marked increase in the combination arm where the fraction of P-H2AX positive cells was of 50% (Figure [5B](#F5){ref-type="fig"}). To confirm results found in the HBCx22-TamR model, we analyzed the expression of PI3K markers, DNA repair proteins (RAD50 and FANCD2) and SUV39H1 by western blot. Results, shown in Figure [5C](#F5){ref-type="fig"} and [5D](#F5){ref-type="fig"}, show a decreased expression of RAD50, FANCD2 and SUV39H1 expression in treated tumors. Inhibition of RAD50 was evident in tumors treated with the drug combination, while SUV39H1 was also inhibited in olaparib-treated tumors and FANCD2 in everolimus-treated xenografts. As for HBCx-22 TamR xenografts, formation of RAD51 foci was not detected in this xenograft, indicating homologous recombination deficiency ([Supplementary Figure 1](#SD1){ref-type="supplementary-material"}). {#F5} In summary, these results indicate a strong anti-tumor effect of the everolimus+olaparib combination in the BRCA2 basal-like breast cancer PDX HBCx-17, with evidence of increased DNA damage in all treatment arms and inhibition of key component of the homologous recombination pathway. Everolimus-induced inhibition of FANCD2 was confirmed in this PDX model. The combination of mTOR and PARP inhibitors is synergistic *in vitro* in a BRCA2 mutated cell line {#s2_4} -------------------------------------------------------------------------------------------------- To determine whether combination of olaparib and everolimus is synergistic in BRCA2 mutated cells, we performed drug combination cell viability assays in two triple-negative breast cancer cell lines, HCC1395 (BRCA2 mutated) \[[@R21], [@R22]\] and BT20 (PI3KCA mutated) \[[@R22]\]. HCC1395 cell line, but not BT20, has a BRCAness phenotype, as determined by the number of genomic large-scale transitions (LST) \[[@R23]\]. Each combination was tested at multiple concentrations using a five-dose matrix. Olaparib and everolimus were tested from 0 to 10 μM. To quantify the combination strength, synergy scores and isobolograms were generated using the Loewe algorithm \[[@R24]\]. The matrix representing growth inhibition percentages and Loewe excess results are shown in Figure [6A](#F6){ref-type="fig"}. We observed that the combination of everolimus and olaparib were more synergistic in the HBC1395 cell line as compared to the BT20 cell line. The synergy scores were 8.6 and 3.81, respectively. Western blot analysis of cell lines showed inhibition of FANCD2 and SUV39H1 in the BRCA2-mutated HCC1395 cell line associated to increased expression of P-H2AX. P-S6 was equally inhibited by everolimus in BT20 and HCC1395. {#F6} DISCUSSION {#s3} ========== Hereditary breast cancer with germline mutations in *BRCA1* and *BRCA2* genes are characterized by a deficiency in DNA repair mechanisms that renders these tumors sensitive to platinum agents. In addition to platins, there has been increasing amount of information on the utility of the defects in DNA repair as targets for cancer therapy in BRCA-associated cancer \[[@R25]\]. Novel therapies like poly (ADP-ribose) polymerase (PARP) inhibitors are one of such example where the induction of double stranded breaks in DNA leads to cell death in tumor cell with homologous DNA repair deficiency \[[@R26]\]. So far combination treatment strategies have evaluated the efficacy and tolerability of olaparib with conventional chemotherapy, but recently several phase I trials have been launched to evaluate the tolerability of olaparib in combination with inhibitors targeting the PI3K/AKT/mTOR pathway. While PARP inhibitors and PI3K/AKT/mTOR inhibitors are effective when tested as single agents, cancers almost invariably develop resistance to these drugs through multiple mechanisms and find alternate ways to grow. This has prompted researchers to explore rational combinations of drugs that can help circumvent drug resistance. Breast cancers with germline BRCA mutations and activated PI3K pathway could be exceptional responder to a combination of DNA repair and PI3K pathway inhibitors. In this study we tested the combination of two FDA-approved drugs, olaparib and everolimus, in PDX models established from BRCA2-mutated breast cancer carrying molecular alteration in the PI3K pathway. This association was first tested in the HBCx-22 TamR PDX, established from a ER+ breast cancer with a *PIK3R1* mutation and PTEN protein loss, and rendered resistant to tamoxifen *in vivo* \[[@R12], [@R13]\]. Remarkably, the drug combination given for 60 days resulted in 100 % of tumor regressions with 40% of complete responses, while in the monotherapy setting only 40% of animals showed tumor regression and no complete responses were observed. In a previous work, the everolimus efficacy was tested in this PDX combined to different endocrine therapies (tamoxifen, fulvestrant and estrogen deprivation) resulting at best in tumor stabilization \[[@R13]\]. Analysis of P-H2AX after treatment showed increased DNA damage not only in the olaparib-treated tumors, but also after everolimus treatment, suggesting a link between mTOR and DNA repair. To further explore the pathways and cross-talks potentially affected by everolimus and olarparib treatments, an RPPA analysis on several proteins involved in DNA repair, cell death and signaling pathways was performed on the HBCx22 TamR PDX treated with the different drugs. Interestingly, the expression of different DNA repair proteins involved in homologous recombination such as FANCD2, RAD50, P-P53BP1, was decreased by everolimus and strongly decreased in the combination arm, confirming that inhibiting mTOR is sufficient to affect DNA repair proteins. FANCD2 belongs to the Fanconi anemia pathway and is involved in the DNA damage response by cooperating with BRCA1/2 proteins in homologous-recombination (HR)-mediated repair \[[@R27]\]. Recently, it has been showed that BRCA-deficient tumors have a compensatory increase in FANCD2 activity that maintains fork stability and promote alternative end-joining DNA repair \[[@R28]\]. Loss of FANCD2 in BRCA1/2-deficient tumors enhances cell death revealing a synthetic lethal relationship between FANCD2 and BRCA1/2, and identifying FANCD2 as a central player orchestrating the choice of DNA repair pathway at the replication fork. The regulation of FANCD2 by mTOR has been also shown in rhabdomyosarcoma xenografts, where treatment by the dual mTOR inhibitor AZD8055 significantly enhanced radiosensitivity and melphalan through a downregulation of FANCD2 expression, suggesting that mTOR may promote the repair of DNA double-strand breaks by sustaining FANCD2 \[[@R29]\]. Additional evidence of the cross-talk between mTOR and FANC2 was found Guo et al., who showed that mTOR inhibitors sensitizes T-cell lymphoblastic leukemia for chemotherapy-induced DNA damage via suppressing FANCD2 expression \[[@R30]\]. The inhibition of FANCD2 in BRCA2-mutated xenografts might result in impaired end-joining and synthetic lethality in a context of homologous recombination deficiency. Additional experiments will be necessary to properly demonstrate impaired end-joining DNA repair in everolimus-treated tumors and to validate FANCD2 as a key component of the cross-talk between mTOR and DNA repair in breast cancer. In the HBCx22 TamR xenografts, we also found inhibition of SUV39H1 in everolimus-treated tumors and complete expression loss in tumors treated by everolimus and olaparib. SUV39H1 is a methyltransferase that is rapidly loaded into the chromatin at DSBs and methylates H3K9, resulting in ATM-dependent phosphorylation of DSB repair proteins \[[@R15]\]. It was recently shown that mTOR inhibitors impair homologous recombination repair and synergize with PARP inhibitors through a negative regulation of SUV39H1 in BRCA-proficient triple-negative breast cancer cell lines \[[@R31]\]. *In vitro* experiments performed in two breast cancer cell lines, one showing a BRCAness phenotype due to a BRCA2 mutation, show an increased synergy between olaparib and everolimus in the BRCA2 mutated cell line as compared to the BRCA wild-type (Figure [6](#F6){ref-type="fig"}). Inhibition of FANCD2 and SUV39H1 proteins were found only in the BRCA2-mutated cell line. Other DNA repair proteins down-regulated in HBCx22 TamR by everolimus and olaparib treatments and strongly inhibited in the combination group, were RAD50, P-53BP1 and NBS1. Taken together these data suggest that inhibition of mTOR alone is sufficient to impair DNA repair and increase DNA damage, resulting in tumor regression and high percentage of complete response when combined to a PARP inhibitor. The *in vivo* activity of olaparib has been also tested in combination with a PI3K inhibitor in PDX models of BRCA-proficient triple-negative breast cancer, where it resulted in increased tumor growth inhibition, but not in tumor regression, through a mechanism based on increased phosphorylation of ERK and BRCA expression down-regulation \[[@R32]\]. In our BRCA2 --mutated xenograft, we did not observe an increased phosphorylation of ERK nor a decreased in BRCA1 gene expression after mTOR inhibition, indicating that this mechanism is not involved in the anti-tumor effect of olaparib and everolimus combination. The HBCx22 PDX model is the unique ER+ PDX of our cohort established from a BRCA2-mutated patient. As a consequence we could not validate our result in a second ER+ PDX model and tested the drug combination in a basal-like BRCA2 mutated PDX. BRCA2 mutated breast cancers are mostly luminal B (73%), while luminal A and basal-like breast cancer account for only 14% and 9%, respectively \[[@R33]\]. Although everolimus is approved only in ER+ breast cancer, it could be of potential interest to validate this drug combination in BRCA2 basal-like tumors. In the monotherapy setting, olaparib and everolimus treatment inhibited tumor growth, but no tumor regressions were observed. However, the association was as efficient as in the HBCx-22 TamR xenograft with 100% of animals showing tumor regression and 2 complete responses. Analysis of P-H2AX showed a marked increase of DNA damage in the combination setting as well as in both monotherapy arms and western blot analysis confirmed inhibition of FANCD2, RAD50 and SUV39H1 expression. In conclusion, our results indicate that combining everolimus and olaparib in BRCA2 mutated breast cancer strongly inhibits expression of key proteins involved in DNA repair and results in massive DNA damage and tumor regression *in vivo*. The present findings suggest that the combination of mTOR and PARP inhibitors could represent a promising therapeutic approach for the treatment of BRCA2 mutated breast cancers. Additional experiments will be necessary to evaluate this combination strategy in other tumors such as BRCA1-mutated cancer and somatic breast cancer showing a BRCAness phenotype. MATERIALS AND METHODS {#s4} ===================== PDX establishment and *in vivo* studies {#s4_1} --------------------------------------- Establishment of the PDX models HBCx-22 (ER+) and HBCx-17 (triple-negative) from primary BRCA2 mutated breast cancer was performed as previously described \[[@R12], [@R19]\]. Informed consent was obtained from patients before xenograft establishment. The HBCx22 TamR (tamoxifen-resistant) model has been established after tamoxifen treatment and tumor escape in an HBCx-22 xenograft \[[@R13]\]. Histology and IHC analysis of HBCx-17 and HBCx-22 PDX as compared to the original primary tumors have been previously published \[[@R12], [@R13], [@R19], [@R34], [@R35]\]. When tumors reached a volume of 60 to 200 mm3, mice were randomly assigned to the control or treated groups. Each group of treatment consisted of 7 or 8 mice. Everolimus and olaparib were purchased from Novartis and AstraZeneca, respectively. Everolimus and olaparib were administered orally at a dose of 15 mg/kg 3x/week and 100mg/kg 5x/week, respectively. Experiments complied with the current laws of France and were approved by Institut Curie ethical committee. Tumor volumes, tumor growth inhibition (TGI) and statistical significance of TGI were calculated as previously published \[[@R36], [@R37]\]. Percent change in tumor volume was calculated for each tumor using the following formula; \[(Vf-V0)/V0\]^\*^100; where V0=Initial volume (at the beginning of treatment) and Vf= Final volume (at the end of treatment). Classification of tumor response in waterfall plots: tumor regression, stabilization and progression corresponded to a percent of volume change lower, equal or greater than 0, respectively. Tumor sampling was performed 3 h after the last treatment. No specific toxicity was reported in the experiments. Breast cancer cell lines {#s4_2} ------------------------ The breast cancer cell lines BT20 and HCC1395 were purchased from the American Type Culture Collection cell lines (ATCC, LGC Promochem, Molsheim, France), authenticated by short tandem repeat profiling (data not shown) and cultured as previously described \[[@R38], [@R39]\]. *In vitro* cell viability assay {#s4_3} ------------------------------- *In vitro* cell viability assays were performed as described by Carita et al. \[[@R40]\]. Briefly, cells were seeded in three 96-well plates following a 6x6 matrix design. The day after, each drug was added following a matrix dilution format. 1:3 serial dilutions were tested to result in a total of six serial dilutions, including the DMSO control. Cell viability was measured after five days of drug treatment using the MTT assay (Sigma). Results were read using a spectrophotometer, and expressed as relative percentages of metabolically inactive cells compared with DMSO treated controls (percentage of growth inhibition). Combination effects were calculated with the Combination Analysis Module software, as previously described \[[@R24], [@R40]\]. A weighted "Synergy Score" was calculated across the dose matrix that adjusts for dose sampling and coverage and weights to favor combination effects at high inhibition levels. Immunohistochemistry {#s4_4} -------------------- Xenografted tumors were fixed in 10% neutral buffered formalin, paraffin embedded, and hematoxylin--eosin (H&E) stained to differentiate the human tumor components from the murine stroma. Tumor tissues were analyzed by immunohistochemistry (IHC) for expression of P-H2AXSer139 (Mouse Monoclonal Antibody, clone JBW301, Merck Millipore, Billerica, MA) and Ki67 (rabbit monoclonal antibody, clone E18-E, Clinisciences, Nanterre -- France). Slides immunostained with mouse and rabbit IgG were used as negative controls. Slides were incubated with anti-rabbit/mouse secondary antibodies (horseradish peroxidase complex) and DAB (3,3′-diaminobenzidine tetrahydrochloride) as the substrate for color development (ChromoMap Kit with Anti rabbit OmniMap, Ventana Medical System). P-H2AX scoring: homogenous nuclear staining and nuclei with 4 or more stained foci were considered positive for P-H2AX expression. For each tumor, the percentage of P-H2AX staining was evaluated in 7 different areas. Detection of RAD51 foci by immunofluorescence {#s4_5} --------------------------------------------- Slides were incubated with a rabbit primary antibody anti-RAD51 (Ab-1, Merck Millipore, Billerica, MA). A HRP-conjugated goat anti-rabbit IgG (H+L) (1/500) and Alexa Fluor^®^ 594-conjugated Streptavidin (1/500) were used as the secondary antibodies. As positive control for RAD51 foci we used a BRCA wt breast cancer PDX that is resistant to cisplatin treatment and shows intrinsic high levels of RAD51 foci. Western blotting {#s4_6} ---------------- Proteins were extracted from tumors using RIPA buffer (50 mM Tris HCL pH 8, 150 mM NaCl, 0.5% deoxycholic acid, 0.5% Triton), supplemented with protease and phosphatase inhibitors. Lysates were resolved on 10% agarose gels, transferred into nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) and immunoblotted with rabbit antibodies against GAPDH, SUV39H1, FANCD2, RAD50, P-HIST. H3, AKT, P-AKT, S6 or P-S6 (Cell Signaling). After washes, membranes were incubated with the appropriate secondary antibodies horseradish peroxidase-conjugated affinity-purified goat anti--rabbit (Jackson ImmunoResearch Laboratories, Inc., Interchim). Quantification of P-AKT, FANCD2, RAD50 and SUV39H1 was performed by the Multi Gauge software and normalized on GAPDH expression. Reverse phase protein array (RPPA) {#s4_7} ---------------------------------- Samples were prepared as described in \[[@R41]\] and printed onto nitrocellulose covered slides (Supernova, Grace Biolabs) using a dedicated arrayer (2470 arrayer, Aushon Biosystems) in five serial dilutions (2000 to 125 μg/ml) and two replicates per dilution. Arrays were labeled with 64 specific antibodies (see [Supplementary Table 1](#SD2){ref-type="supplementary-material"}) as described in \[[@R41]\]. Read-out was done using IRDye 800CW (LiCOR) on an Innoscan 710-AL scanner (Innopsys). For staining of total protein, arrays were incubated 30 min in Super G blocking buffer (Grace Biolabs), rinsed in water, incubated 5 min in 0,000005% Fast green FCF (Sigma) and rinsed again in water. Raw data were normalized using Normacurve \[[@R42]\]. SUPPLEMENTARY MATERIALS FIGURE AND TABLES {#s5} ========================================= **CONFLICTS OF INTEREST** The authors declare that they have no competing interests. PDX : patient-derived xenografts DSB : double-strand breaks TGI : tumor growth inhibition IHC : Immunohistochemistry RPPA : Reverse Phase Protein Array ER+ : Estrogen receptor positive TamR : tamoxifen-resistant

1. Introduction {#sec1-molecules-23-00346} =============== Suramin is a water-soluble polysulfonated naphtylurea. It was first synthesized in 1916 as a colorless derivate of trypan dyes for the treatment of African trypanosomiasis (reviewed by \[[@B1-molecules-23-00346]\]). Later on it was found that suramin had an effect against the Human-Immunodeficiency Virus \[[@B2-molecules-23-00346]\] and that non-Hodgkin lymphomas associated with this virus were sensitive to suramin treatment \[[@B3-molecules-23-00346]\]. Further investigation revealed broad antitumor activity of suramin and a wide variety of mechanisms of action were described. Among others, it was shown that suramin inhibits several different enzymes and growth factors, which may account for the antiretroviral, antiprotozoal and antitumor activity. In different tumor entities suramin was proposed to bind to a range of growth factors such as the vascular endothelial growth factor or the epidermal growth factor \[[@B1-molecules-23-00346]\]. Subsequently, suramin was tested in clinical trials of different metastatic tumor entities with emphasis on metastatic prostate cancer \[[@B4-molecules-23-00346],[@B5-molecules-23-00346],[@B6-molecules-23-00346],[@B7-molecules-23-00346]\]. In line with the seemingly unspecific mode of action of suramin, the clinical application revealed several side effects such as coagulopathy, adrenal insufficiency, nephrotoxicity or hepatotoxicity \[[@B1-molecules-23-00346],[@B6-molecules-23-00346]\]. One of the most frequently observed dose limiting side effects, however, is a toxic peripheral polyneuropathy, which occurs in 40--79% of suramin treated patients \[[@B6-molecules-23-00346],[@B8-molecules-23-00346],[@B9-molecules-23-00346]\]. As suramin is a charged molecule and does not cross the blood brain barrier it exerts neurotoxic effects in the peripheral nervous system only \[[@B10-molecules-23-00346]\]. Due to the potentially severe side effects, clinical use of suramin at the time of writing is limited to treatment of African trypanosomiasis and as an orphan drug for (metastatic) hormone refractory prostate cancer. Suramin-induced peripheral neuropathy most frequently shows as sensory-motor axonal neuropathy with sensory symptoms such as hypoesthesia, paresthesia, dysesthesia, hypoalgesia or pallhypesthesia \[[@B9-molecules-23-00346],[@B11-molecules-23-00346]\]. The incidence of a demyelinating Guillian-Barré like syndrome under suramin treatment was reported less frequently \[[@B12-molecules-23-00346]\]. Incidence and severity of suramin-induced neuropathy are influenced by plasma levels exceeding 350 µg/mL, the cumulative suramin dose and the duration of chemotherapy \[[@B12-molecules-23-00346],[@B13-molecules-23-00346]\]. A variety of different pathomechanisms was described: The inhibition of lysosomal enzymes by suramin was proposed to cause an experimental form of gangliosidosis with intracellular lamellar inclusion bodies in neurons and Schwann cells \[[@B14-molecules-23-00346],[@B15-molecules-23-00346]\]. Different experimental studies suggested the involvement of nerve growth factor and a binding affinity of suramin to the nerve growth factor receptor to account for the neurotoxic effect \[[@B16-molecules-23-00346],[@B17-molecules-23-00346]\]. Furthermore, Sun et al. described that suramin leads to a disturbance of the intracellular calcium (Ca^2+^) homoeostasis in dorsal root ganglia neurons (DRGN) \[[@B18-molecules-23-00346]\]. Intriguingly, other chemotherapeutic agents such as paclitaxel and salinomycin were also found to cause a peripheral neuropathy via a disturbance of intracellular Ca^2+^ levels \[[@B19-molecules-23-00346],[@B20-molecules-23-00346]\]. Thus, it appears that the disruption of the intracellular Ca^2+^ homeostasis might be a common pathomechanism in chemotherapy-induced polyneuropathies. The aim of this study was thus to establish preclinical models of suramin-induced neurotoxicity in order to further elucidate the underlying pathomechanism with an emphasis on Ca^2+^ signaling and to identify potential molecular targets suitable for neuroprotection. We observed that suramin induces neurotoxicity in vitro and in vivo. Calcium influx into DRGN from the extracellular space seems to partially mediate suramin-induced neurotoxicity, as Nimodipine, an L-Type voltage-gated calcium channel (VGCC) inhibitor, showed limited neuroprotective effects. 2. Results {#sec2-molecules-23-00346} ========== 2.1. Suramin Induces a Sensory Axonal-Demyelinating Polyneuropathy in C57Bl/6 Mice {#sec2dot1-molecules-23-00346} ---------------------------------------------------------------------------------- We previously characterized the behavioral and histological phenotype of chemotherapy-induced polyneuropathies in C57Bl/6 mice after application of paclitaxel, vincristine, cisplatin and bortezomib, respectively \[[@B21-molecules-23-00346]\]. In order to establish a mouse model of a suramin-induced polyneuropathy, we adapted a previously published protocol. Russell et al. had shown that a single intraperitoneal injection of 500 mg/kg bodyweight suramin can induce chemotherapy-induced polyneuropathy in rats \[[@B15-molecules-23-00346]\]. In mice, the published LD~50~ of intraperitoneal suramin application is 750 mg/kg bodyweight \[[@B22-molecules-23-00346]\]. To avoid unspecific general side effects, we lowered the dose to a single intraperitoneal injection of 250 mg/kg bodyweight suramin. Mice were tested for sensory and motor deficits on days 3, 8 and 13 after suramin application ([Figure 1](#molecules-23-00346-f001){ref-type="fig"}A). Suramin treatment resulted in a steady decline in bodyweight, which reached its maximum on day 6 with −1.44 g ± 0.65 g compared to baseline (not significant, Kruskal-Wallis test; [Figure 1](#molecules-23-00346-f001){ref-type="fig"}B). Afterwards, mice regained bodyweight and reached control levels by the end of the experiment. One animal, which had lost \>20% bodyweight compared to baseline, had to be sacrificed on day 6 according to predefined endpoints. As allodynia is a frequent symptom of chemotherapy-induced polyneuropathy, the mechanical withdrawal threshold was measured at various time points after suramin application. Suramin treated mice showed a significant decrease in the mechanical withdrawal threshold compared to vehicle-injected mice (Kruskal-Wallis test, *p* \< 0.0001 and *p* = 0.0296 on day 8 and 13, respectively; [Figure 1](#molecules-23-00346-f001){ref-type="fig"}C), indicative of increased sensitivity to mechanical stimuli due to suramin treatment. In the rotarod test we also observed a mild deficit in locomotor function in suramin treated mice compared to control animals, which reached significance on day 8 (repeated measures 2-way ANOVA, *p* = 0.0022; [Figure 1](#molecules-23-00346-f001){ref-type="fig"}D). As the rotarod test measures a complex behavior which is influenced by motor performance but also coordination and sensory function, the observed decline in performance could be mediated by sensory neuropathy as well as motor neuropathy. Given the normal behavior of suramin treated animals in their home cage, the small effect size and our observations regarding the sensory nervous system, the effect of suramin on rotarod performance is likely dominated by sensory neuropathy. Electrophysiological assessment showed that suramin application led to a significant decline in the sensory nerve action potential amplitude (repeated measures 2-way ANOVA, *p* = 0.0158; [Figure 1](#molecules-23-00346-f001){ref-type="fig"}E) as well as the sensory nerve conduction velocity (Kruskal-Wallis, *p* = 0.0329 and *p* = 0.0008 on days 8 and 13, respectively; [Figure 1](#molecules-23-00346-f001){ref-type="fig"}F). In conclusion, our data provide evidence that a single injection of 250 mg/kg bodyweight suramin in C57Bl/6 mice is sufficient to induce a predominantly sensory axonal-demyelinating polyneuropathy. 2.2. Effects of Suramin on Cell Viability and Calcium Homeostasis in Dorsal Root Ganglia Neurons {#sec2dot2-molecules-23-00346} ------------------------------------------------------------------------------------------------ We have previously shown that some cytostatic agents cause alterations in intracellular calcium (Ca^2+^) homeostasis in dorsal root ganglia neurons (DRGN) and thus contribute to the development of chemotherapy-induced polyneuropathy \[[@B19-molecules-23-00346],[@B20-molecules-23-00346],[@B23-molecules-23-00346],[@B24-molecules-23-00346]\]. In a first step, we were interested in the toxicity of suramin towards DRGN. We measured cell viability of DRGN in response to increasing concentrations of suramin and observed marked toxicity above 300 µM suramin concentrations. The calculated half maximal inhibitory concentration (IC~50~) of 22--24 h (hour) suramin treatment was 283 µM (non-linear regression analysis, 95% confidence interval 226--355 µM) in DRGN ([Figure 2](#molecules-23-00346-f002){ref-type="fig"}A). For the following experiments studying cell viability we used a dose of 400 µM. Next, we were interested in the effects suramin might exhibit on intracellular Ca^2+^ homeostasis. Sun and Windebank could previously show a calcium (Ca^2+^) influx into DRGN under "acute" suramin treatment \[[@B18-molecules-23-00346]\]. In order to quantify and localize the impact of suramin on intraneuronal Ca^2+^ homeostasis, Ca^2+^ imaging experiments were performed using the fluorescent Ca^2+^ indicator dye fura-2. A higher dose of 1 mM suramin was chosen in order to ensure robust activation of potential receptors in the plasma membrane of DRGN despite of the short incubation time. First, we monitored Ca^2+^ levels in DRGN cultured in Ca^2+^ free medium, which did not result in any changes of intracellular Ca^2+^ levels (unpaired two-sided *t*-test; [Figure 2](#molecules-23-00346-f002){ref-type="fig"}B). However, when DRGN were cultured in Ca^2+^ containing medium, we observed a strong increase of intracellular Ca^2+^ levels (SUR 65.2 nM ± 7.4 nM, VEH 3.2 nM ± 0.6 nM), which is in line with previous findings (Mann-Whitney-U test, *p* \< 0.0001; [Figure 2](#molecules-23-00346-f002){ref-type="fig"}C). These findings suggest that suramin exposure leads to an influx of Ca^2+^ from the extracellular space into the cytosol of DRGN via the plasma membrane. Intriguingly, the addition of suramin to DRGN cultures in Ca^2+^ containing imaging buffer caused different profiles of Ca^2+^ influx ([Figure 2](#molecules-23-00346-f002){ref-type="fig"}D): Most DRGN showed a rapid but transient Ca^2+^ influx followed by a slow Ca^2+^ increase (profile 1, 54%), others reacted with a slow but steady Ca^2+^ increase (profile 2, 23%) whereas some cells showed a short but transient Ca^2+^ increase only (profile 3, 18%). A smaller group of cells did not show any rise in intracellular Ca^2+^ levels after suramin treatment (profile 4, 7%) ([Figure 2](#molecules-23-00346-f002){ref-type="fig"}E). 2.3. Effects of Various Inhibitors of Plasmamembrane Channels on Suramin-Induced Neurotoxicity {#sec2dot3-molecules-23-00346} ---------------------------------------------------------------------------------------------- Our data obtained from Ca^2+^ imaging indicated, that suramin treatment leads to an increase of intracellular Ca^2+^ levels via a Ca^2+^ influx across the plasma membrane. In a next step, we aimed to identify specific Ca^2+^ channels in the plasma membrane which are modulated by suramin. It was previously shown that nimodipine, an inhibitor of L-Type VGCC, has a protective effect on axonal outgrowth in suramin treated DRGN \[[@B18-molecules-23-00346]\]. Based on our dose-response curve, we used suramin concentrations of 400 µM as a clinically relevant concentration that reproducibly led to a decrease of cell viability down to 52% ± 4% of vehicle treatment. We measured cell viability of suramin treated cells with and without co-incubation with various inhibitors of VGCC. Because of the complex chemical structure of suramin, we hypothesized that suramin could also induce Ca^2+^ influx into DRGN via transient-receptor-potential- (TRP-) channels. These are cation-conducting channels, which play an important role in sensory perception. A variety of TRP-channels subgroups are found in the plasma membrane of DRGN. At first, we screened for potential neuroprotective effects of different VGCC and TRP-channel inhibitors after co-incubation with 400 µM suramin using the MTT-assay. In case of neuroprotective effects on cell viability, calcium imaging was performed with those previously identified inhibitors. [Table 1](#molecules-23-00346-t001){ref-type="table"} summarizes the results of cell viability measurements of DRGN treated with 400 µM suramin in the presence of different VGCC- and TRP-inhibitors. We observed a dose-dependent increase of cell viability, when DRGN exposed to 400 µM suramin were co-incubated with nimodipine ([Figure 3](#molecules-23-00346-f003){ref-type="fig"}A). This effect reached statistical significance for nimodipine concentrations of 100 µM and 150 µM, but could not restore impaired cell viability to control levels (1-way ANOVA, *p* \< 0.0001; [Figure 3](#molecules-23-00346-f003){ref-type="fig"}B). We additionally measured intracellular Ca^2+^ levels of suramin treated DRGN under co-incubation with nimodipine. In line with the findings of the cell viability assays, we observed a significantly reduced Ca^2+^ influx in DRGN under co-incubation with suramin and nimodipine, compared to DRGN treated with suramin and the corresponding vehicle (Mann-Whitney-*U* test, *p* = 0.0229; *n* = 35 SUR/VEH, *n* = 34 SUR/Nimo; [Figure 3](#molecules-23-00346-f003){ref-type="fig"}C). Again, nimodipine was not able to prevent suramin-induced Ca^2+^ influx completely. Efonidipine, another L-Type channel antagonist, did not exert protective effects on suramin-induced cell death. However, data on efonidipine is hard to interpret as increasing doses of efonidipine in the absence of suramin per se exert toxic effects on cultured DRGN with a significantly lower cell viability (1-way ANOVA, *p* = 0.0388 (10 µM) and *p* \< 0.0001 (50 µM), respectively). The same problem was observed for the unselective VGCC and TRP inhibitor Ruthenium Red (1-way ANOVA, *p* = 0.0005; [Supplementary Materials Table S1](#app1-molecules-23-00346){ref-type="app"} and [Supplementary Materials Figure S1](#app1-molecules-23-00346){ref-type="app"}). Additional VGCC antagonists did not show any effects on cell viability of DRGN after suramin treatment ([Table 1](#molecules-23-00346-t001){ref-type="table"}). Next, we investigated various TRP channel inhibitors regarding their neuroprotective effects under suramin exposure. We only observed a small but significant protective effect on cell viability when DRGN were co-incubated with concentrations of 10--100 nM of the selective TRPV4 antagonist HC067047 (1-way ANOVA, *p* = 0.0294 (10 nM) and *p* = 0.0403 (100 nM), respectively; [Figure 3](#molecules-23-00346-f003){ref-type="fig"}D,E). However, HC067047 did not change Ca^2+^ influx due to suramin treatment (Mann-Whitney-*U* test; [Figure 3](#molecules-23-00346-f003){ref-type="fig"}F). Other TRP antagonists had no effects on cell viability after suramin exposure and measurements of intracellular Ca^2+^ levels of suramin treated DRGN did not show any alterations when DRGN were co-incubated with TRP antagonists. 2.4. Downstream Apoptotic Pathways and Experimental Limitations {#sec2dot4-molecules-23-00346} --------------------------------------------------------------- In order to investigate apoptotic pathways further downstream of Ca^2+^ influx, we measured caspase activity after suramin treatment of DRGN. Surprisingly, we did not detect any protective effects of caspase-3/7 or calpain inhibition on cell viability when DRGN were co-incubated with suramin and the respective inhibitors ([Supplementary Materials Table S2](#app1-molecules-23-00346){ref-type="app"}). We also observed a significant decrease of caspase-3/7 activity after 0, 6 and 12 h suramin treatment ([Supplementary Materials Figure S2A](#app1-molecules-23-00346){ref-type="app"}). It was previously described in the Jurkat cell line that suramin itself can inhibit caspase-3/7 activity \[[@B25-molecules-23-00346]\]. However, we also observed a significant increase in caspase-3/7 activity after 24 h suramin incubation. This observation is difficult to reconcile with the initially observed decrease of the luminescenct signal and we hypothesized that the luminescent assay signal might be quenched in the presence of suramin. Indeed, when we added suramin directly before luminescent signal detection, we also observed a significant decrease in measured caspase activity ([Supplementary Materials Figure S2B](#app1-molecules-23-00346){ref-type="app"}). We therefore suggest that suramin either binds under a chemical reaction directly to the luminophore and thereby alters photon emission or it potentially absorbs photons emitted by the luminophore. Nevertheless, the significantly increased caspase activity after 24 h of suramin incubation suggests caspase activation by suramin, even considering the experimental limitations of the assay. We additionally performed immunhistochemical staining in order to verify our hypothesis. During those experiments, suramin interaction with cell culture coated dishes was observed. DRGN did not adhere to cell culture dishes under suramin treatment when washed according to the staining protocol. All cell culture surfaces were coated with a well-established protocol for DRGN cultures using poly-[l]{.smallcaps}-lysin and laminin. Prighozinha et al. described Matrigel dissolvement by suramin, which is of interest as Matrigel consists of 61% laminin. It was hypothesized that negatively charged sulfate groups would account for laminin binding and Matrigel dissolvement \[[@B26-molecules-23-00346]\]. In line with these findings suramin was previously described to act as a heparan-sulfate analogue and antimetastic tumor activity of suramin was linked to this mode of action. Furthermore, suramin inhibition of binding of extracellular tumorcell-glycocalyx to the extracellular matrixprotein laminin was proposed to prevent tumor cell migration \[[@B27-molecules-23-00346],[@B28-molecules-23-00346],[@B29-molecules-23-00346]\]. The same mechanism could be responsible for reduced binding activity of DRGN to laminin-coated surfaces. This means that suramin could inhibit binding of extracellular glycocalyx of DRGN to laminin molecules of the coated cell culture dish. 3. Discussion {#sec3-molecules-23-00346} ============= The objective of this study was to elucidate the pathomechanism underlying suramin-induced neurotoxicity and its potential mechanistic link to neuronal Ca^2+^ dyshomeostasis. Intriguingly, different chemotherapeutic agents were found to disturb intracellular Ca^2+^ levels in neurons resulting in neuronal apoptosis. One example is paclitaxel, a chemotherapeutic agent causing a peripheral sensory neuropathy, which induces a dyshomeostasis of intracellular Ca^2+^ in DRGN \[[@B20-molecules-23-00346],[@B23-molecules-23-00346]\]. It was proposed that the phosphoinositide pathway is involved and causes Ca^2+^ release from the endoplasmic reticulum under treatment with paclitaxel. Salinomycin, another experimental chemotherapeutic agent, was found to cause increased intracellular Ca^2+^ levels by its ionophoric capacity \[[@B19-molecules-23-00346]\]. Furthermore, caspases and Ca^2+^ activated proteases such as calpains were shown to beinvolved in Ca^2+^-induced apoptosis. Caspase 12, a cytosolic caspase bound to the endoplasmic reticulum, also plays a role in Ca^2+^ triggered apoptosis \[[@B30-molecules-23-00346]\]. In the context of suramin, Sun and Windebank proposed that Ca^2+^ influx into the cytosol would be mediated by channels in the plasma membrane \[[@B18-molecules-23-00346]\]. Our Ca^2+^ imaging experiments confirm this hypothesis. In our study, we observed that suramin induces an increase of intracellular Ca^2+^ in DRGN only in Ca^2+^ containing buffer, whereas intracellular Ca^2+^ levels were not affected when DRGN were cultured in Ca^2+^ free buffer. This implies that suramin activates Ca^2+^ conducting channels in the plasma membrane causing a Ca^2+^ influx via the plasma membrane into the cytosol. Furthermore, it was shown that the [L]{.smallcaps}-type VGCC-inhibitor nimodipine can restore neuronal survival and axonal outgrowth after suramin treatment \[[@B18-molecules-23-00346]\]. In our experiments, we also observed that nimodipine improves cell viability compared to suramin/vehicle co-treatment. Our data however also suggest that nimodipine cannot fully reverse suramin's neurotoxic effects. In line with these findings, suramin-induced Ca^2+^ influx was significantly reduced when DRGN were co-incubated with nimodipine, but intracellular Ca^2+^ levels still remained elevated compared to control levels. Although these observations suggest that, the effect of nimodipine depends at least partially on inhibition of [L]{.smallcaps}-type VGCC, we observed robust neuroprotective effects only at comparatively high nimodipine concentrations above 100 µM. This concentration is at least an order of magnitude higher compared to the concentration of 1--10 µM typically used in in vitro studies to block [L]{.smallcaps}-type VGCC (e.g., \[[@B31-molecules-23-00346],[@B32-molecules-23-00346]\]), suggesting the possibility of additional molecular mechanisms. Among others, nimodipine was shown to interact with multidrug resistance transporters such as P-glycoprotein \[[@B33-molecules-23-00346]\] and at higher concentrations with voltage dependent potassium channels \[[@B34-molecules-23-00346]\]. To further investigate this aspect we used efonidipine as another specific inhibitor of [L]{.smallcaps}-type VGCC and ruthenium red as a broad range inhibitor of Ca^2+^ channels in the plasma membrane (including VGCC and TRP). Unfortunately, these substances were themselves toxic, making the results hard to interpret. Another possible source for the Ca^2+^ influx across the plasma membrane are TRP channels. We tested a number of TRP channel inhibitors targeting TRP channel 3 (TRPC3), TRP channel subfamily A (TRPA1), subfamily V (TRPV4) and subfamily M inhibitors, of which only the TRPV4 inhibitor yielded a slightly positive effect on suramin-induced cell death. This is an interesting finding, as TRPV4 has been shown to play an important role in neuropathic pain caused by paclitaxel \[[@B35-molecules-23-00346]\]. Given the diversity of TRP channels alone however, one limitation of the present work is that only a small subset of potentially relevant ion channels was studied. A logical next step for future experiments would be to repeat our experimental paradigm combined with whole cell patch clamp experiments to further characterize the mechanisms underlying Ca^2+^ influx. Subsequently additional inhibitors should be evaluated. DRGN exhibited different responses to suramin in terms of their intracellular Ca^2+^ levels. We hypothesize that suramin might stimulate different ion channels or receptors in the plasma membrane by its unspecific binding properties. It should also be noted that DRGN differ in receptor and ion channel status. VGCC expression patterns vary especially between small, medium and large diameter neurons \[[@B36-molecules-23-00346],[@B37-molecules-23-00346],[@B38-molecules-23-00346]\]. Also TRP-channel subtype expression was found to be variable in different DRGN \[[@B39-molecules-23-00346]\]. Therefore, the unspecific binding properties of suramin on the one hand and varying receptor/ion channel status on DRGN on the other hand could lead to different reaction patterns of Ca^2+^ influx in DRGN after suramin treatment. This observation might explain the small effect size and heterogenous results observed in case of TRPV4 inhibition and the lack of changes observed in Ca^2+^ imaging experiments. This experimental hurdle could be overcome by studying for instance only TRPV4 positive DRGN. Increasing intracellular concentrations of Ca^2+^ in the cytosol can trigger apoptotic processes by initiating different signaling pathways. Most frequently caspases and calpains are involved. Unexpectedly, we did not see any positive effects of caspase or calpain inhibition regarding cell viability after suramin treatment, which is in contrast to previous results of Sun and Windebank, who observed protective effects of calpain inhibition. Future studies should expand on this aspect as it suggests a more complex mechanism of cell death. Taken together, our data suggest a link between suramin-induced neurotoxicity and Ca^2+^ dyshomeostasis in DRGN and we could demonstrate that suramin treatment leads to a predominantly sensory axonal-demyelinating neuropathy in mice. However, it has also become clear in our experiments that suramin is a complex molecule and a whole array of cellular effects induced by suramin were described in the past (reviewed by \[[@B1-molecules-23-00346]\]) making it a "dirty" drug. A number of mechanisms may thus contribute to suramin-induced neurotoxicity in the peripheral nervous system including but not limited to Ca^2+^ dyshomeostasis, interference with nerve growth factor signaling \[[@B16-molecules-23-00346],[@B17-molecules-23-00346]\], glycolipid homeostasis \[[@B40-molecules-23-00346]\], inhibition of P2X/P2Y purinoreceptors (reviewed by \[[@B41-molecules-23-00346]\]) and mitochondrial toxicity \[[@B42-molecules-23-00346]\]. As suramin acts as inhibitor of calcium conducting P2X purinoreceptors it seems that this target does not play a role in suramin-induced calcium influx in DRGN \[[@B43-molecules-23-00346]\]. L-type Ca^2+^ channels seem to account only for a partial increase in intracellular Ca^2+^ levels after suramin treatment. Nevertheless, they present a potentially interesting molecular target as VGCC inhibitors such as nimodipine have already been approved for clinical use and could theoretically be easily translated to clinical application. Additional animal studies are however needed to prove a beneficial effect of VGCC inhibition in chemotherapy-induced polyneuropathy. Further ion channels in the plasma membrane also seem to mediate suramin-induced Ca^2+^ influx. One of those targets could be TRP channels, which have already been linked to chemotherapy-induced polyneuropathy in other antineoplastic substances such as paclitaxel. In summary, given suramin's complex biological and chemical characteristics and proven neurotoxicity, developing a neuroprotective co-treatment may help to overcome suramin's translational challenge and enable a broader clinical use of this drug. 4. Materials and Methods {#sec4-molecules-23-00346} ======================== 4.1. In Vivo {#sec4dot1-molecules-23-00346} ------------ ### 4.1.1. Animal Housing, Sample Sizes and Methods of Randomization and Blinding {#sec4dot1dot1-molecules-23-00346} We used 20 nine-week old male C57Bl/6 mice from Charles River (Sulzfeld, Germany) for the experiment. All procedures had been previously approved by the State Office for Health and Social Affairs (Landesamt für Gesundheit und Soziales (LaGeSo), Berlin, Germany, Reg.-Nr: G0092-10) and conformed to animal welfare guidelines. Mice were housed in groups of five in an enriched environment and a 12-hour light/dark cycle (7 a.m.--7 p.m.) with unlimited access to food and water. The a priori calculated sample size was 10 mice/group, based on previously observed effect sizes of electrophysiological parameters \[[@B21-molecules-23-00346]\], a power of 0.8 and an alpha-error of 0.05. Sample size calculation was done using G\*Power statistical software (Heinrich Heine Universität Düsseldorf, Düsseldorf, Germany) \[[@B44-molecules-23-00346]\]. Mice were randomized to one of two groups (SUR vs. VEH) using an online randomization tool (GraphPad Software, La Jolla, CA, USA; <https://www.graphpad.com/quickcalcs/randomize1.cfm>). Behavior experiments were conducted by a blind investigator. ### 4.1.2. Drug Preparation and Injection {#sec4dot1dot2-molecules-23-00346} Suramin (Tocris, Bioscience, Bristol, UK) was dissolved in Aqua dest. to a concentration of 50 mg/mL and diluted in sterile 0.9% saline to a final concentration of 25 mg/mL and injected once intraperitoneally at 10 µL/g bodyweight. As vehicle treatment, Aqua dest. was diluted 1:1 in 0.9% sterile saline and injected once intraperitoneally at 10 µL/g bodyweight. ### 4.1.3. Behavior Analysis {#sec4dot1dot3-molecules-23-00346} Mice were handled for five consecutive days before behavior experiments to ensure familiarization to the investigator and to minimize stress and anxiety. All behavior experiments were done in a dedicated laboratory with soundproof chambers during 10 a.m. and 4 p.m. The general wellbeing of the mice was checked daily and their weight recorded. The mechanical withdrawal threshold was obtained using the von Frey filament test as previously described \[[@B21-molecules-23-00346],[@B45-molecules-23-00346]\]. In brief: a hand-held force transducer was fitted with a 0.5 mm^2^ polypropylene tip. Mice were placed in a clear inverted plastic cage on a wire-mesh floor. Trained investigators applied increasing pressure to the center of the hind paws until a clear withdrawal response was evoked. If the animal did not exhibit a clear withdrawal response within 5 s the trial was deemed invalid and was repeated. The withdrawal threshold in grams (g) was automatically measured by the device (IITC, Woodland Hills, CA, USA). The maximum applied force was 10 g. For each time point, 5 trials were averaged. Locomotor function was measured with the rotarod test as previously described \[[@B21-molecules-23-00346],[@B45-molecules-23-00346]\]. Mice were placed on a rotating rod (TSE Systems, Bad Homburg, Germany), which gradually increased in speed from 4 to 40 rpm over the course of 300 s. The latency for the mice to fall off the rod was automatically measured by a floor sensor. Mice were trained in the task for four consecutive days prior to baseline measurement. For each time point, three trials for each animal were averaged. ### 4.1.4. Nerve Conduction Studies {#sec4dot1dot4-molecules-23-00346} Mice were anaesthetized with isoflurane 1.5%/50% O~2~ (volume/volume) and placed on a heating pad with a constant temperature of 37.5 °C (Harvard Apparatus, Holliston, MA, USA). Stimulating needle electrodes were placed at the base of the tail with the recording electrodes approx. 5 cm distal. A ground electrode was place in the middle. Single stimuli were applied to determine supramaximal level and afterwards 50 serial supramaximal stimuli were averaged to obtain the sensory nerve action potential (SNAP) amplitudes and NCV \[[@B21-molecules-23-00346],[@B45-molecules-23-00346]\]. 4.2. In Vitro {#sec4dot2-molecules-23-00346} ------------- ### 4.2.1. DRGN Cell Culture {#sec4dot2dot1-molecules-23-00346} DRGN cultures were obtained from P 0--3 Wistar rat neonates as described before \[[@B23-molecules-23-00346]\]. Cells were plated on poly-[l]{.smallcaps}-lysin and laminin coated 96-well plates for cell viability measurements and on cover slips with the same coating for Ca^2+^ imaging experiments. DRGN cultures were incubated overnight at 37 °C in a 95% air/5% CO~2~ humidified atmosphere before experimental use. Neurobasal A medium supplemented with B-27 (Life Technologies GmbH, Darmstadt, Germany), 0.5 mM glutamine, fresh nerve growth factor (10 ng/mL) was used for cell maintenance. ### 4.2.2. Calcium Imaging {#sec4dot2dot2-molecules-23-00346} Cells were mounted with Fura-2 AM 5 µM (Life Technologies GmbH, Darmstadt, Germany) for 30 min at 37 °C in a standard HEPES buffered solution: 130 mM NaCl/4.7 mM KCl/1 mM MgSO~4~/1.2 mM KH~2~PO~4~/1.3 mM CaCl~2~/20 mM Hepes/5 mM glucose (pH 7.4)/0.02% pluronic F-127 (Life Technologies GmbH, Darmstadt, Deutschland). After incubation cell cultures were washed with standard HEPES buffered solution and coverslips were placed in a Olympus IX 81 microscope equipped with a Uplan FLN oil objective 40×/1.13 (Olympus Corporation, Tokyo, Japan). For calcium imaging experiments in Ca^2+^ free extracellular medium a standard HEPES solution without Ca^2+^ was used: 130 mM NaCl/4.7 mM KCl/2.3 mM MgSO~4~/1.2 mM KH~2~PO~4~/10 mM EDTA/20 mM Hepes/5 mM glucose, pH 7.4. For experiments with ion channel inhibitors DRGN cultures were preincubated in standard HEPES buffered solution with the respective channel blocker for 5 min before Ca^2+^ levels were measured. Fluorescence signals were detected by a cooled CCD-camera and data was processed on a computer using Olympus xcellence imaging software. Intracellular Ca^2+^ concentrations \[(Ca^2+^) int\] (nM) were calculated using ratios of F340/F380 after background subtraction with the following equation: \[Ca^2+^\]int (nM) = *K*d\*Q\*(*R* − *R*~min~)/(*R*~max~ − *R*), where *K*d is the dissociation constant of Fura-2 for Ca^2+^ at room temperature (225 nM); Q is the fluorescence ratio of the emission intensity excited by 380 nm in the absence of Ca^2+^ to that during the presence of saturating Ca^2+^; and *R*~min~ and *R*~max~ are the minimal or maximal fluorescence ratios, respectively. Values for *R*~min~ and *R*~max~ were derived from in situ calibration using Ca^2+^ free HEPES buffered standard solution with 10 µM ionomycin for *R*~min~ values and HEPES buffered standard solution with CaCl~2~ for *R*~max~ values. Intracellular free Ca^2+^ concentrations were measured over 4.37 min adding 1 µM suramin or VEH (Aq. dest.) after 20 s and 10 µM ionomycin after 4.15 min (internal control). Cells with an increase of \<200 nM \[Ca^2+^ int.\] (Δ) in HEPES buffered solution with Ca^2+^ or \<50 nM \[Ca^2+^ int.\] (Δ) in HEPES buffered solution without Ca^2+^ were excluded from the data analysis. ### 4.2.3. MTT-Assay {#sec4dot2dot3-molecules-23-00346} DRGN cultures plated in 96 well plates were incubated with suramin and ion channel inhibitors (VGCC and TRP-channels) and respective controls for 24 h at 37 °C in a 95% air/5% CO~2~ humidified atmosphere. MTT was added to each well in a ratio of 1:10 and cell cultures were reincubated for 30 min at 37 °C in a 95% air/5% CO~2~ humidified atmosphere. Reaction was stopped using SDS in a ratio of 1:1. After cell dissolution overnight, absorption values were gained at a wavelength of 550 nm, lamp energy of 10,000 during 0.2 s in a microplate reader (Tristar LB941 Multimode Microplate Reader Berthold Technologies GmbH and Co. KG, Bad Wildbad, Germany). Values were background subtracted and standardized to percentage of control. For coincubation with suramin and ion channel inhibitors a second standardization was used and the mean difference suramin plus inhibitor to suramin plus vehicle treatment (ΔSUR/VEH) was calculated. ### 4.2.4. Caspase Assay {#sec4dot2dot4-molecules-23-00346} Caspase activity was assessed using the Promega *Caspase-Glo*^®^ *3/7 Assay kit* (Promega, Mannheim, Germany) according to the manufacturer's protocol as described \[[@B46-molecules-23-00346]\]. 4.3. Statistical Analysis and Exclusion Criteria {#sec4dot3-molecules-23-00346} ------------------------------------------------ The manuscript was prepared in accordance with ARRIVE guidelines \[[@B47-molecules-23-00346]\]. Data is presented as mean ± SEM with individual values depicted for in vivo studies. For MTT assay experiments, data was obtained from *n* = 3--6 individual experiments (biological replicates) with *n* = 3 technical replicates. For Ca^2+^ imaging experiments (*n*/*N*) describes the number of cells studied (*n*) in (*N*) independent cultures. Statistical analysis was performed using Prism v6.0 (GraphPad Software, La Jolla, CA, USA). Gaussian distribution of data was checked prior to statistical analysis using Shapiro-Wilk normality test. Normally distributed data was analyzed using unpaired two-sided *t*-tests (two groups), one-way ANOVA with Sidak post hoc test (≥three groups) or (repeated measures) 2-way ANOVA (two variables). Not normally distributed data was analyzed with Mann-Whitney-*U* test (2 groups) or Kruskal-Wallis test with Dunn's method (≥3 groups). *p* \< 0.05 was considered statistically significant and is depicted by asterisk. Only statistical outliers that conformed with Peirce criterion were excluded from the dataset \[[@B48-molecules-23-00346],[@B49-molecules-23-00346]\]. We would like to thank Petra Loge for excellent technical assistance. The researchers have received funding from the Federal Ministry of Education and Research via the grant Center for Stroke Research Berlin (01 EO 0801) and from the Deutsche Forschungsgemeinschaft (EXC 257 NeuroCure). PH receives funding from the Charité Clinician Scientist program funded by the Charité Universitätsmedizin Berlin and the Berlin Institute of Health. **Sample Availability:** Samples of the compounds are available from commercial sources. The Supplementary Materials are available online. ###### Click here for additional data file. W.B. and M.E. conceived and designed the experiments. D.v.d.A. performed the in vitro experiments. P.H., M.B. and S.P. performed the in vivo experiments. D.v.d.A., P.H. and W.B. analyzed the data and compiled the figures. D.v.d.A., P.H. and W.B. wrote the manuscript. All authors reviewed the manuscript. M.E. provided funding for the study. The authors declare no conflict of interest. Ca 2+ calcium DRGN dorsal root ganglia neurons VGCC voltage-gated calcium channels TRP transient-receptor-potential ###### Mouse model of suramin-induced sensory-motor polyneuropathy. (**A**) Schematic outline of the experiment: Adult C57Bl/6 mice received a single intraperitoneal injection of 250 mg/kg bodyweight suramin (SUR) or vehicle (VEH) on day 0. Mice were tested on the rotarod (RR) and with the von Frey (vF) test for locomotor deficits and alterations of mechanical withdrawal threshold four times. Electropysiological (Ephys) measurements were obtained on day 0, 8 and 13; (**B**) SUR treated mice showed a decline in body weight, but recovered by day 13. One animal had to be sacrificed due to weight loss \>20%; (**C**) SUR application resulted in a decrease of the mechanical withdrawal threshold and (**D**) locomotor function, which was most pronounced on day 8; (**E**) SUR treated mice showed a decline of the sensory nerve action potential (SNAP) amplitude as well as the (**F**) SNAP nerve conduction velocity (NCV), indicative of an axonal-demyelinating neuropathy. Group sizes: *n* = 10 (vehicle), *n* = 9--10 (suramin), \* *p* \< 0.05.   {#molecules-23-00346-f002} {#molecules-23-00346-f003} molecules-23-00346-t001_Table 1 ###### Summary of observed cell viability of DRGN after suramin treatment in the presence of different inhibitors. ----------------------------------------------------- ------------------------------------------------------------------- --------------------------------- ----------- --------------- --------------- -------------- -------------- -------------- ----------- ----------- **Substance** **Nimodipine (L-Type VGCC Inhibitor)** **A 967079 (TRPA1-Inhibitor)** SUR 400 µM + 0.1 µM 1 µM 10 µM 100 µM 150 µM 1 nM 10 nM 100 nM 1 µM 10 µM Change attributable to intervention (Δ% of SUR/VEH) +3.5 ±2.6 +2.7 ±2.0 +3.9 ±1.9 +15.1 \* ±2.9 +18.7 \* ±2.1 +0.2 ±2.3 +7.1 ±3.5 +3.0 ±2.4 +2.6 ±1.2 +2.8 ±4.8 **Substance** **Efonidipine (L-Type VGCC Inhibitor)** **HC 067047 (TRPV4 Inhibitor)** SUR 400 µM + 0.1 µM 1 µM 10 µM 50 µM 1 nM 10 nM 100 nM 1 µM 10 µM Change attributable to intervention (Δ% of SUR/VEH) +3.5 ±2.7 +0.5 ±2.6 +0.6 ±2.7 −1.6 ±3.2 −7.5 \* ±3.0 +7.2 \* ±2.9 +7.1 \* ±2.0 +1.1 ±1.2 +2.9 ±2.3 **Substance** **Ruthenium Red (unselective including VGCC and TRP Inhibition)** **Pyr 3 (TRPC3 Inhibitor)** SUR 400 µM + 10 nM 100 nM 1 µM 10 µM 1 nM 10 nM 100 nM 1 µM 10 µM Change attributable to intervention (Δ% of SUR/VEH) +8.1 ±4.2 +4.4 ±1.6 +7.6 ±3.9 −1.5 ±2.8 +0.2 ±2.5 −3.4 ±4.2 −1.3 ±3.8 −2.8 ±3.0 −2.6 ±2.5 **Substance** **SNX 482 (R-Type VGCC Inhibitor)** **Ononetin (TRPM3 Inhibitor)** SUR 400 µM + 2 nM 20 nM 200 nM 3 nM 30 nM 300 nM 3 µM 30 µM Change attributable to intervention (Δ% of SUR/VEH) −3.2 ±1.8 −2.2 ±3.0 +2.1 ±2.3 −5.9 ±2.6 +2.5 ±2.1 −2.2 ±2.4 −2.7 ±3.3 −1.9 ±2.7 **Substance** **Ω-Conotoxin MVIIC (N-, P-, Q-Type VGCC Inh)** SUR 400 µM + 1 nM 10 nM 100 nM 1 µM Change attributable to intervention (Δ% of SUR/VEH) −2.2 ±1.8 −2.0 ±2.6 +1.9 ±1.6 +1.9 ±3.3 ----------------------------------------------------- ------------------------------------------------------------------- --------------------------------- ----------- --------------- --------------- -------------- -------------- -------------- ----------- ----------- **\*** Statistically significant (*p* \< 0.05). [^1]: These authors contributed equally to this work.

Background ========== Cardiovascular diseases (CVD) are a major public health concern in the world, accounting for half of all non-communicable disease deaths worldwide \[[@B1]\]. Similar to findings from western countries \[[@B2],[@B3]\], risk factors for CVD in many developing countries have been well recognized \[[@B4],[@B5]\]. Diabetes, hypertension, cigarette smoking, alcohol drinking and overweight have been found to be major risk factors for CVD in China \[[@B6]-[@B8]\]. There is also growing evidence that the prevalence of CVD risk factors has been increasing, and clustering of CVD risk factors is common in China \[[@B9],[@B10]\]. Traditionally, epidemiological studies have focused on identifying individual-level risk factors for diseases. Recently in epidemiology, there is increasing interest in exploring the effect of population or group (contextual) variables on disease risks. People residing in the same community or context would share the same contextual or environmental exposure and statistical assumption of independence is usually not true. Without proper adjustment, simplistic analysis of contextual independent variables as if they vary independently across individual subjects would bias the result toward overestimation of association. Multilevel modelling \[[@B11]\] provides a useful solution to simultaneously examine the effects of individual-level and contextual-level variables. Discrimination of effects of contextual from individual independent variables is also important for public health. A good example of contextual concept is externality. While individual socioeconomic status (SES) variables such as income, unemployment and educational level \[[@B12]-[@B16]\] have been recognized to be important determinants of CVD risk factors, little is known about the influence of socioeconomic status of the neighbourhood. If such effects exist and are stronger than individual SES, all the residents would have a better health if they cooperate to raise the community SES than just compete to get to the most without making a contribution to local society. Another example would be independent effects of individual versus contextual ethnicity. CVD risk factors are associated with ethnicity \[[@B17]\] either through culture or genetics or both. As culture is contextual, one would expect strong effect of contextual ethnic effect if it is the main mechanisms. On the other hand, independent influence of genetic can be expressed as an individual ethnic effect. This is independent from the ethnic of the community where the subject resides. Yunnan province of China is a multi-ethnic area and has 52 ethnic groups. The terrain is mainly mountainous with high variability in level of socioeconomic development and ratio of ethnicities among communities. The risk for CVD among the population is high and also varies by geography \[[@B18]\]. Under such circumstance, our purpose in this study was to test the independent effects of contextual socioeconomic variables with adjustment for individual socioeconomic variables on risk factors for CVD in this study area. Understanding characteristics of village associated with CVD risk factors can assist health planning to allocate appropriate resources to the target area. Methods ======= Study design ------------ This was a cross-sectional community survey combined with investigation of contextual variables from existing official data sources. Study area and population ------------------------- Shi Lin County, a rural area of Kunming, the capital of Yunnan province (one of the poorest provinces in south-west China), was chosen as the study community. In 2004, it had a population of 205,186 and contained 10 townships, 90 villages and 65,135 families. The county was a typical minority rural county in Kunming, predominated by Yi ethnic group, and is one of the poor counties in Kunming, with a per capita income among peasants of US\$313 \[[@B19]\] in 2004. The total area was 1717 square kilometers, and mostly mountainous. Villages were scattered with a maximum distance of 80 kilometers from the main city, Kunming. Transportation by car was available throughout the year. Data source ----------- Socioeconomic characteristics of villages with regard to population size, adult literacy rate, proportion of Yi ethnic minority and distance from city were based on the 2000 census in China obtained from the local statistics office. Individual characteristics, village average income and CVD risk factor parameters were obtained from a cross-sectional community survey. Community survey ---------------- ### Sampling technique In order to obtain information on each township for subsequent spatial study, reported separately, standard cluster sampling technique \[[@B20]\] was modified to select three clusters or villages from each township. Altogether there were 30 study villages. In each selected village, from a name list of individuals aged ≥ 45 years obtained from the village committee, 200 subjects were chosen based on simple random sampling method. Data Collection and measurement ------------------------------- The questionnaire was adapted from that used by Inter-ASIA collaborative group \[[@B4]\]. The instrument was modified to suit the local situation. Twenty fifth-grade medical students from Kunming Medical College were trained to be data collectors under the supervision of two senior medical staff. The training included introduction to cardiovascular diseases, use of the questionnaire, conducting fasting blood sugar test and taking anthropometric measurements. In the morning of data collection, each participant was given full explanation of the research purpose, invited to participate and sign informed consent, and interviewed by one of the interviewers. Information on demographic characteristics, status of current cigarette smoking and alcohol drinking, blood pressure and fasting blood sugar test was obtained. Anthropometric measurements included height, weight, and waist and hip girth. Data collection was started in May 2005 and completed within 45 days. Three blood pressure (BP) measurements were made according to the American Heart Association recommendations \[[@B21]\]. After at least 5 minutes of rest in a sitting position, systolic and diastolic pressures were taken from the participant\'s right arm, using a mercury sphygmomanometer. In our study, BP measures were based on the average of three BP readings. Our method is slightly different from Health Survey for England/Scotland standard and MONICA standards \[[@B22]\], which keep the average of the second and third measurements and reject the first. Weight was measured using a balance beam scale. The measurement of height and weight was done with the participants standing on the scale wearing indoor clothes and barefoot. To ensure sufficient precision, height was measured to the nearest 0.2 centimeter, and weight was measured to the nearest 0.2 kilogram. Body mass index (BMI) was calculated as weight in kg divided by height in meters squared \[[@B23]\]. Waist girth was measured around the narrowest point between ribs and hips when viewed from the front after exhaling. Hip girth was measured at the point where the buttocks extended the maximum when viewed from the side. Waist-hip ratio was subsequently calculated. A random 10% of participants were selected from each village for fasting blood sugar study. The subjects were instructed to fast overnight for at least 10 hours. A small drop of fingerprick blood was obtained and put onto a special strip of paper, which was then inserted into the blood glucose monitor machine (Braun, Beijing NecKar Healthcare Company). The monitor measured and displayed the result within 15 seconds. This strip glucose technique had been validated against a standard test in Kunming Hospital, and found to give a 10% lower value. Values obtained from the participants\' blood stick test were therefore adjusted upward as appropriate. Ethical approval ---------------- This study was approved by the Ethics Committee of Faculty of Medicine, Prince of Songkla University, before carrying out the research. Definitions ----------- Cigarette smokers were defined as persons who had smoked at least 100 cigarettes in their lifetime, and those who smoked tobacco products during the survey time were classified as current smokers. Current drinkers were defined as persons who drank alcohol regularly during the previous 12 months (i.e. on 12 or more different days during the year). Hypertension was defined as a mean systolic blood pressure ≥ 140 mm Hg, diastolic blood pressure ≥ 90 mm Hg, and/or use of antihypertensive medications. Overweight was defined as a BMI of 25 kg/m^2^or greater \[[@B23]\]. Diabetes mellitus was defined as a fasting plasma glucose ≥ 7.0 mmol/l (126 mg/dl) \[[@B24]\] or the use of antidiabetic medications. Adult literacy rate was defined as the percentage of population aged 15 years and over who could both read and write with understanding a short simple statement on his/her everyday life. Outcome variables ----------------- The outcome variables included systolic blood pressure (mmHg), diastolic blood pressure (mmHg), fasting blood sugar (mmol/l), BMI (kg/m^2^), waist-hip ratio, current smoker and current drinker. Independent variables --------------------- Individual-level independent variables were age, sex, ethnicity, household income and education, which are known socio-economic risk factors. The contextual variables included population size, adult literacy rate, proportion of Yi ethnic minority, mean income and distance from city. Statistical analysis -------------------- Mean income of participants from each village was computed for using as a contextual variable since this information was not available in any report. Descriptive statistics were used for data summary. Age-sex-adjusted prevalence of each CVD risk factor was computed by indirectly standardizing to the overall sample \[[@B25]\]. The data were further analyzed using multilevel regression. The method of estimation was by constructing a Generalized Linear Model using Penalized Quasi-Likelihood, with individual characteristics at the first level and village socioeconomic status at the second. We examined all individual variables and fitted models for each of the village variables separately. Both individual and village characteristics were treated as fixed effects. Multilevel logistic regression was used to analyze the association between independent variables and binary individual outcome such as current smokers and drinkers, whereas multilevel linear regression was used to analyze CVD risk factors which had a continuous distribution such as blood pressure and fasting blood sugar. The levels of association were expressed as standardized beta coefficients and standard errors to allow easy comparison across different independent variables. All data analyses were done with R software, version 2.1.1 \[[@B26]\]. Results ======= A total of 6050 individuals aged ≥ 45 years was selected by the sampling process. Of these, 6006 participated (response rate = 99.3%). Among the participants, 611 randomly selected people received fasting blood sugar test. Their demographic characteristics are summarized in Table [1](#T1){ref-type="table"}. ###### Demographic characteristic of the study participants Variables n \% -------------------------------------------- ------- ------ Sex  Male 2905 48.4  Female 3101 51.6 Age  40--49 years 1102 18.3  50--54 years 1106 18.4  55--59 years 879 14.6  60--64 years 909 15.1  ≥ 65 years 2010 33.5 Ethnicity  Han 3664 61.0  Yi ethnic minority 2272 39.0 Educational level  Illiterate 3328 55.4  Primary (grade 1--6) or higher 2678 45.6 Approximate yearly household income (Yuan)  Max 20000  P~75~ 7000  P~50~ 4000  P~25~ 2000  Min 1100 Individual mean values of height, weight, waist girth, hip girth, BMI, waist-hip ratio, blood pressure and fasting blood sugar by sex are demonstrated in Table [2](#T2){ref-type="table"}. In general, men had slightly higher mean values of BMI, waist-hip ratio, systolic pressure (SBP), diastolic pressure (DBP) and fasting blood sugar than women. ###### CVD risk factor parameters by sex (mean ± SD\*) Variables Male Female All ------------------------------ ------------- ------------- ------------- Height (cm) 162.3 ± 7.4 153.7 ± 6.6 157.9 ± 8.2 Weight (kg) 58.7 ± 8.8 50.6 ± 7.8 54.5 ± 9.2 BMI\*\* (kg/m^2^) 22.3 ± 3.1 21.4 ± 3.0 21.8 ± 3.0 Waist girth (cm) 76.7 ± 8.2 66.8 ± 8.5 71.6 ± 9.7 Hip girth (cm) 87.0 ± 7.8 85.5 ± 7.9 86.2 ± 7.9 Waist-hip ratio 0.88 ± 0.07 0.78 ± 0.06 0.83± 0.08 Systolic pressure (mmHg) 120 ± 16 117 ± 17 118 ± 16 Diastolic pressure (mmHg) 79 ± 11 76 ± 11 77 ± 11 Fasting blood sugar (mmol/l) 5.5 ± 1.1 5.4 ± 1.0 5.4 ± 1.1 \* Standard deviation \*\* Body mass index, which has been promulgated by the World Health Organization as the most useful epidemiological measure of obesity. Table [3](#T3){ref-type="table"} summarizes the village contextual variables. Variations in adult literacy rate and percentage of minority ethnic were high. ###### Distribution of socioeconomic status among 30 villages Variables Max P~75~ P~50~ P~25~ Min -------------------------------------- ------ ------- ------- ------- ------ Population size 7217 3343 2410 1790 1120 Adult literacy rate\* (%) 73.0 54.5 47.0 32.5 29.0 Proportion of Yi ethnic minority (%) 97.0 64.8 33.5 4.8 1.5 Average yearly income (Yuan) 9200 7500 6200 5725 4900 Distance from city (km) 49.0 33.2 16.3 8.0 0.9 \* The percentage of population aged 15 years and over who could both read and write with understanding a short simple statement on his/her everyday life. Table [4](#T4){ref-type="table"} shows the distribution of age-sex-adjusted prevalence of CVD risk factors among the 30 villages. Males had somewhat higher prevalences of hypertension, diabetes and overweight and remarkably higher prevalences of current smokers and current drinkers than females. ###### Distribution of age -- and sex-adjusted prevalence (%) of CVD risk factors among 30 villages Variables Male Female ------------------ ------ -------- ------ ------ ------ ------ ------ ------ ------ ------ Hypertension 33.8 27.3 20.5 12.8 10.3 32.4 25.3 17.0 11.2 10.2 Diabetes 9.8 9.1 7.7 4.4 4.0 9.1 8.3 5.7 4.8 3.8 Overweight 24.5 21.4 14.7 10.0 9.5 20.6 15.8 11.8 9.7 8.9 Current smokers 72.0 65.3 52.0 33.5 31.0 3.0 2.0 1.0 0.2 0.2 Current drinkers 68.3 58.2 50.0 41.1 35.0 6.7 3.6 1.4 0.3 0.2 From results of multi-level analysis are shown in Table [5](#T5){ref-type="table"}. Residents of villages with large population size had increased mean individual SBP and waist-hip ratio. Those in communities with low literacy rate had increased individual waist-hip ratio and probability of smoking habit. Living in a village dominated by Yi ethnic minority increased individual probability to be a drinker. Subject in low income villages had increased SBP and BMI, but had decreased fasting blood sugar. Individuals in remote villages had increased mean SBP, higher fasting blood sugar and increased probability of being a current smoker. ###### Contextual effect of village characteristics on the CVD risk factors after adjustment for individual characteristics Predictors Systolic pressure (mmHg) Diastolic pressure (mmHg) Fasting blood sugar (mmol/l) BMI Waist-hip ratio Current smokers^†^ Current Drinkers^††^ ------------------------------------------- -------------------------- --------------------------- ------------------------------ ------------------ ------------------ -------------------- ---------------------- **Contextual variables:** Population size (X1000) 0.08\* (0.04) 0.02 (0.07) -0.08 (0.07) 0.09 (0.05) 0.10\* (0.05) 0.08 (0.28) 0.14 (0.10) Adult literacy rate (%) 0.006 (0.04) 0.01 (0.05) 0.11 (0.06) 0.02 (0.04) -0.12\*\*(0.04) -0.57\* (0.24) -0.03 (0.09) Proportion of Yi ethnic minority (%) -0.09 (0.05) -0.12 (0.08) 0.08 (0.08) -0.03 (0.06) -0.09 (0.05) 0.50 (0.33) 0.43\*\*\* (0.12) Income (× 1000 Yuan) -0.09\* (0.04) -0.07 (0.07) 0.34\*\*\* (0.07) -0.09\* (0.04) 0.02 (0.04) 0.57 (0.29) 0.11 (0.10) Distance from city (km) 0.11\*(0.04) 0.04 (0.07) 0.16\* (0.07) 0.03 (0.05) -0.02 (0.04) 0.75\*\* (0.30) 0.08 (0.11) **Individual variables:** Age 0.11\*\*\* (0.01) 0.06\*\* (0.01) 0.07\* (0.03) -0.09\*\*(0.01) 0.02\* (0.01) -0.12\*\* (0.04) -0.17\*\*\*(0.03) Sex (reference: male) -0.20\*\*\* (0.03) -0.19\*\* (0.03) -0.02 (0.08) -0.28\*\*(0.03) -1.24\*\* (0.02) -4.91\*\*\*(0.23) -3.86\*\*\*(0.14) Educational level (reference: illiterate) -0.02 (0.02) -0.02 (0.02) 0.09 (0.06) -0.003 (0.02) 0.01 (0.02) 0.06 (0.07) 0.04 (0.05) Ethnic (reference: Han) 0.11\*\* (0.03) 0.01 (0.04) 0.21 (0.11) -0.13\*\* (0.04) 0.04 (0.03) 0.04 (0.14) 0.29\*\* (0.11) Income (× 1000 Yuan) -0.01\*\* (0.002) -0.007\*\* (0.002) 0.001 (0.001) -0.01\*\*(0.002) 0.002 (0.002) 0.007 (0.007) 0.01 (0.008) \* p \< 0.05, \*\* p \< 0.01, \*\*\* p \< 0.001, † reference: non-current smokers, † † reference: non-current drinkers, † † † regression coefficient. ‡ standard error. Discussion ========== From this study, several geographical and socio-economic contextual variables were shown to exert an independent influence on CVD risk factors. While a previous study found high prevalence of CVD risk factors in poor rural villagers who were rarely exposed to the modern world \[[@B27]\], our findings that distance from the city is positively associated with SBP, fasting blood sugar and smoking do emphasize the need for more attention to the CVD problem in remote areas. Remoteness of the village in the study area is somewhat correlated with high altitude, which has been shown to cause an increase in blood pressure (BP) in other studies \[[@B28],[@B29]\]. A study from Taiwan indicated that people living in mountainous areas have the highest prevalence of diabetes compared to those living in metropolitan cities \[[@B30]\]. The mechanism of association therefore could be through physiological and/or lifestyle aetiology, and needs further investigation. While an inverse relationship between individual educational level and CVD risk factors has been reported in a number of studies \[[@B31]-[@B33]\], only a few have explored the effect of education at the contextual level and reported associations with various CVD risk factors, most of them in the direction not favourable for those living in poor education areas, such as an increased risk for smoking and increases in SBP \[[@B34]\] and diastolic pressure \[[@B35]\]. Our study yielded no evidence supporting any association between low individual educational level and CVD risk factors but a strong preventive effect on smoking and waist-hip ratio of increased literacy rate. In these rural communities, collective social life style is very strong. Influence of peers and neighbourhood on smoking and social activity, such as poor diet and lack of exercise, leading to obesity may overwhelm the effect of individual education. The inverse association of literacy rate with these two factors suggests that the educated communities rather than the uneducated ones have until now been the target for intervention programmes. Yi ethnic minority has increased risk for alcohol drinking in this study. Alcohol drinking is an integral part of Chinese culture. Distilled spirit is the primary beverage of choice for men, accounting for more than one-third of all drinks consumed \[[@B36]\]. A survey conducted in three centers of China indicated reasons for drinking were alcohol\'s positive social effects, relief of tension and worry, and relief of craving and withdrawal symptoms \[[@B37]\]. The phenomenon that ethnic minorities have more frequent reported alcohol intake than Han majority has been demonstrated in other studies \[[@B38],[@B39]\]. In our study, while individual Yi ethnicity has a positive relationship with alcohol drinking and blood pressure and negative relationship with BMI, community ethnicity has an effect only on drinking and not on other risk factor. These results thus link culture only with alcohol and not with other CVD risk factors. While western studies have demonstrated that deprivation in low income communities increases the level of various CVD risk factors such as SBP \[[@B34]\], overweight \[[@B40]\] and diabetes \[[@B41]\], our results agree with them on the protective effect of income against SBP and BMI but by contrast demonstrated an adverse effect on fasting blood sugar. This adversity is from village income and not individual income. This suggests that contextual lifestyle of the relatively rich communities must be investigated and appropriate intervention applied. Public environmental factors demanding more physical exercise such as transportation by bicycles and walking and predominant manual occupation such as farming may be more common in low income communities. The strength of our study lies in the large sample size and high response rate fulfilling the requirement for multilevel modelling \[[@B9]\]. The limitation of this study is that fasting blood sugar test was not done for all participants and none of the lipid profiles was available due to financial difficulties. Capillary blood glucose has slightly lower reading than plasma glucose level obtained from venous blood. Nevertheless, the relationship of blood sugar with contextual SES is illustrated, although further studies are needed to confirm these findings. Furthermore, social equity index such as Gini\'s coefficient for each community was not available. Thus we were unable to test the effect of local social equality on CVD risk factors. Conclusion ========== The study area shares the global trend of increased CVD risk factors, which are contributed to by contextual setting in addition to the known personal lifestyles. Unlike findings from the western countries, where most unfavourable outcome are more common among the low SES, local directions of association are inconsistent. While contextual SES development is associated with a few CVD risk factors, villages with high level of income are worse off in fasting blood sugar. Strategies of economic development should be reviewed to avoid their adverse effects on health. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= Cai Le carried out the study and drafted and revised the manuscript. Virasakdi Chongsuvivatwong conceptualized the research idea, participated in the design of the study, laid out the framework for data analysis, interpreted the results and helped to draft the manuscript and revised the discussion. Alan Geater participated in the design of the study and helped to draft and revised the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/7/72/prepub> Acknowledgements ================ This study is a part of the first author\'s thesis to fulfill the requirements for a PhD in Epidemiology at Prince of Songkla University. This research was greatly supported by Shi Lin Health Bureau. We would like to thank all the staff of this sector and the households surveyed for their valuable help and co-operation. Special thanks go to Bi Weihong, the head of Shi Lin Health Bureau, who helped us with the epidemiological data. The study was supported by a grant from Yunnan Provincial Natural Science Funds (Grant number: 2003C0022Q).

Introduction ============ Biliary tract cancer (BTC) is a rare malignancy comprising a heterogeneous group of diseases including intrahepatic/extrahepatic cholangiocarcinoma and gallbladder cancer. Up to 10,000 new BTC patients are diagnosed annually in the United States and Europe \[[@b1-crt-2017-526]\]. In Korea, BTC is one of leading causes of cancer-related deaths (sixth in males and seventh in females) \[[@b2-crt-2017-526]\]. Surgical resection is the only curative therapeutic modality for localized BTC; however, recurrence after curative resection is common, and most patients present with unresectable or metastatic disease at the time of diagnosis. These clinical characteristics in advanced BTC are associated with poor prognosis, with up to 10% 5-year overall survival (OS) rates \[[@b3-crt-2017-526]\]. After the success of the pivotal phase III ABC-02 study, gemcitabine plus cisplatin (GemCis) has been established as the standard first-line chemotherapy for patients with unresectable or metastatic BTC \[[@b4-crt-2017-526]\]. In that study, GemCis demonstrated significantly improved OS compared with gemcitabine alone (11.7 months vs. 8.1 months). The superiority of GemCis to gemcitabine alone was confirmed in a subsequent Japanese randomized phase II study (11.2 months vs. 7.7 months) \[[@b5-crt-2017-526]\]. Despite this improvement, the median survival remains \< 1 year for patients with advanced BTC, highlighting the large unmet need for improving the efficacy of systemic chemotherapy. Investigation of combination therapy including three cytotoxic chemotherapeutic agents has revealed a significant survival benefit in multiple cancer types \[[@b6-crt-2017-526],[@b7-crt-2017-526]\]. Particularly in pancreatic cancer, FOLFIRINOX, the combination of fluorouracil, irinotecan, and oxaliplatin, leads to significantly improved survival outcomes compared with gemcitabine monotherapy; this has been globally accepted as one of standard first-line regimens for patients with locally advanced or metastatic pancreatic cancer \[[@b7-crt-2017-526]\]. Considering these success, gemcitabine-free triplets including irinotecan, oxaliplatin, and fluorouracil might also be effective in treating advanced BTC. Furthermore, in a previous phase I study, biweekly triplet combination of oxaliplatin, irinotecan, and S-1 (oral fluoropyrimidine) (OIS) for multiple cancer types was associated with remarkable preliminary efficacy outcomes in advanced BTC, with four of nine BTC patients achieving partial response (PR) \[[@b8-crt-2017-526]\]. Based on these findings, we herein report the results of a single-arm, phase II study that aimed to evaluate the efficacy and safety of OIS in patients with unresectable or metastatic BTC. Materials and Methods ===================== 1. Eligibility -------------- Patients with histologically confirmed BTC were eligible if they were chemotherapy-naive and had inoperable locally advanced or metastatic disease. Other inclusion criteria included age of ≥ 19 years; measurable lesion according to the Response Evaluation Criteria in Solid Tumors (RECIST) ver. 1.1 \[[@b9-crt-2017-526]\]; Eastern Cooperative Oncology Group performance status of 0-2; adequate bone marrow, renal, and hepatic function; life expectancy of ≥ 3 months; and written informed consent. Patients were excluded if they had received chemotherapy for BTC. However, previous adjuvant chemotherapy without platinum was allowed if the interval between the completion of adjuvant chemotherapy and enrollment in the study was \> 6 months. 2. Treatment ------------ Patients received intravenous 65-mg/m^2^ oxaliplatin and 135-mg/m^2^ irinotecan on day 1 and 40-mg/m^2^ oral S-1 twice daily on days 1-7, every 2 weeks. This dosing schedule was based on a previous phase I study \[[@b8-crt-2017-526]\]. Treatment continued until disease progression, intolerable toxicity, or patient's withdrawal of consent. Doses were interrupted or modified for grade 3-4 hematological toxicities and grade 2-4 non-hematological toxicities according to the protocol. Primary prophylactic granulocyte-colony stimulating factor support was not allowed. In patients who did not exhibit disease progression during completion of the 12th cycle of OIS, continuation of S-1 monotherapy was allowed at the discretion of the attending physician. 3. Assessment ------------- Baseline assessment included medical history, physical examination, laboratory tests, and computed tomography (CT) of the chest, abdomen, and pelvis. Physical examination and laboratory tests were performed at each treatment cycle. For response evaluation, CT was performed every three cycles or in the presence of signs or symptoms indicating disease progression. Tumor responses were determined by local investigators according to the RECIST ver. 1.1. Toxicities were assessed every cycle and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events ver. 4.03. 4. Biomarker analysis --------------------- Somatic mutations and copy number variation analyses were performed on archival tumor tissues using targeted exome sequencing. Details of methods for next generation sequencing (NGS) experiments and bioinformatics analyses are described in the [Supplementary Methods](#SD3-crt-2017-526){ref-type="supplementary-material"}. Somatic mutations were manually reviewed using Integrative Gemonic Viewers, and correlative analyses were performed between these results and clinical outcomes. 5. Statistical analysis ----------------------- Primary endpoint was overall response rate (ORR) according to the local investigator's assessment. Secondary endpoints were progression-free survival (PFS), OS, and safety. Simon's optimal two-stage design was used to estimate sample size \[[@b10-crt-2017-526]\]. Estimated ORR of patients who received standard GemCis was approximately 15% according to our large retrospective analysis (P0) \[[@b11-crt-2017-526]\], and investigational OIS was expected to improve to 35% (P1) in ORR with two-sided α of 0.05 and power of 80%. We assumed that 20% improvement in ORR is acceptable for further investigation of OIS in randomized phase II or III trials. After expecting a 10% drop-out rate, target enrollment was 31 patients. In the first stage, during which 15 patients were enrolled, two patients with objective responses (complete response \[CR\] or PR) were required to proceed to the second stage of patient enrollment. At least 10 patients with objective responses were needed to declare OIS as effective. PFS was defined as the time from the initiation of study treatment to disease progression or death, whichever occurred first. OS was defined as the time from the initiation of study treatment to death from any cause. The Kaplan-Meier method was used to estimate time-to-event variables. All efficacy parameters were analyzed based on the full analysis set, which included all patients who met the eligibility criteria. A two-sided p-value of \< 0.05 was considered statistically significant. SPSS ver. 22.0 (IBM Corp., Armonk, NY) was used to perform all statistical analyses. 6. Ethical statement -------------------- This multicenter, open-label, single-arm, phase II study was conducted at two tertiary referral hospitals in Korea. The study protocol was approved by the Institutional Review Board of Hallym University Sacred Heart Hospital (2015-1020) and Asan Medical Center (2015-1070), and all patients provided written informed consent prior to enrollment. This study was conducted in accordance with the Declaration of Helsinki and the guidelines of Good Clinical Practice (ClinicalTrial. gov identifier: NCT02527824). Results ======= 1. Patients ----------- Between 22 October 2015, and 16 June 2016, 32 patients with histologically documented advanced BTC were enrolled. Baseline characteristics of the patients are summarized in [Table 1](#t1-crt-2017-526){ref-type="table"}. Median age was 64 years (range, 40 to 76 years); 24 patients (75%) were males. The most common condition for which study treatment was provided was initially metastatic disease (n=24, 75%); this was followed by recurrent disease after curative surgery (n=7, 22%) and locally advanced unresectable disease (n=1, 3%). Among seven patients who underwent surgery, there was no patient who received adjuvant chemotherapy and adjuvant radiotherapy was given in two patients. The most common location of tumors was intrahepatic (n=13, 41%), followed by gallbladder (n=11, 34%) and extrahepatic (n=8, 25%). Liver (n=23, 72%), lymph nodes (n=21, 66%), peritoneum (n=10, 31%), and lungs (n=7, 22%) were frequent metastatic sites. At the time of analysis, study treatment was ongoing in seven patients without disease progression and was discontinued in 25 patients because of disease progression (n=14), patient refusal (n=4), adverse events (n=4), and death without evidence of disease progression (n=3). 2. Efficacy ----------- All patients enrolled in this study had measurable disease. The waterfall plot for maximal percent changes in target lesions is presented in [Fig. 1](#f1-crt-2017-526){ref-type="fig"}. ORR was 50% (95% confidence interval \[CI\], 34 to 66) according to RECIST ver. 1.1. No patient achieved CR, whereas 16 patients (50%) achieved PR. Disease control rate, defined as the proportion of patients demonstrating CR, PR, or stable disease, was 88% (95% CI, 71 to 96). Progressive disease was the best response in three patients (9%). Tumor response could not be assessed in one patient (3%) because of early death prior to the first assessment. Response rates were highest in patients with gallbladder cancer (73%), followed by those in patients with extrahepatic (63%) and intrahepatic cholangiocarcinoma (23%, p=0.04). After a median follow-up period of 12.1 months (range, 0.3 to 16.2), median PFS was 6.8 months (95% CI, 4.8 to 8.8) ([Fig. 2](#f2-crt-2017-526){ref-type="fig"}). PFS rates at 6 and 12 months were 53% and 27%, respectively. Median OS was 12.5 months (95% CI, 7.0 to 18.0); OS rates at 6 and 12 months were 75% and 53%, respectively. Patients with extrahepatic cholangiocarcinoma had superior PFS (median, 15.3 months) compared to those with gallbladder cancer (5.9 months) and intrahepatic cholangiocarcinoma (3.0 months) ([Fig. 3](#f3-crt-2017-526){ref-type="fig"}); this difference was marginally significant (p=0.051). A similar trend was also observed in OS as median OS was better in patients with extrahepatic cholangiocarcinoma (median value not reached) than in those with intrahepatic cholangiocarcinoma (12.5 months) and gallbladder cancer (10.1 months); however, this was not statistically significant (p=0.21). Following progression on OIS, 50% patients (12/24) received second-line chemotherapy: 10 patients (83%) received GemCis and two patients who received flouropy-rimidine monotherapy (17%). One patient underwent concurrent chemoradiation as salvage therapy. 3. Safety profile ----------------- Safety analysis was available in 31 patients (97%), excluding one patient who was lost to follow-up after one cycle. A median of 12 cycles (range, 1 to 21) were administered. Study treatment was discontinued because of adverse events in four patients, and doses were interrupted and reduced in 17 (55%) and 13 (41%) patients, respectively. The mean relative dose intensity (the total delivered dose as a percentage of the targeted dose per unit time) was 87.0% (standard deviation \[SD\], 18.7%) for oxaliplatin, 86.4% (SD, 19.2%) for irinotecan, and 86.0% (SD, 19.3%) for S-1 during sixth cycles ([S1 Fig.](#SD1-crt-2017-526){ref-type="supplementary-material"}). Adverse events developed in ≥10% patients are listed in [Table 2](#t2-crt-2017-526){ref-type="table"}. The most common grade 3-4 adverse event was neutropenia (n=10, 32%), followed by diarrhea (n=2, 6%), and peripheral neuropathy (n=2, 6%). Despite the relatively high frequency of severe neutropenia, no patient developed febrile neutropenia. 4. Biomarker analysis --------------------- Eighteen of 32 patients (56%) were available for targeted exome sequencing. *TP53* (n=7, 42%), *KRAS* (n=5, 32%, including G12D in two, G13D in two, and G12A in one), *IDH1* (n=3, 16%), and *ARID1A* (n=2, 11%) were the most frequent genomic alterations in this study population. Although *TP53*mutations were not associated with PFS (p=0.43) or OS (p=0.76), *KRAS* mutations showed a marginal relationship with worse OS (p=0.07) ([S2 Fig.](#SD2-crt-2017-526){ref-type="supplementary-material"}); however, PFS was not associated with *KRAS* mutations (p=0.90). Although no statistical significance was observed (p=0.60), there was a trend toward greater tumor shrinkage (\> 50%) in patients without *KRAS* mutations (6/13, 46%) compared with that in patients harboring *KRAS* mutations (1/5, 20%). Discussion ========== In this single-arm, phase II study, we investigated the efficacy and safety of the triplet OIS regimen in chemotherapynaive patients with advanced BTC. Our findings suggested that OIS was well-tolerated and effective in patients with unresectable or metastatic BTC as first-line chemotherapy and that the prespecified primary endpoint was attained. OIS demonstrated an ORR of 50%, with median OS of 12.5 months, which were better than those in the pivotal phase III ABC-02 study (26% and 11.7 months, respectively) \[[@b4-crt-2017-526]\]. Meanwhile, median PFS of 6.8 months with OIS in the current study seems to be inferior to 8.1 months in the ABC-02 study. However, this comparison should be interpreted with caution because PFS outcomes might be affected by the presence of measurable lesions or the time interval of response assessment. In contrast to the ABC-02 study that imaging studies were conducted every 12 weeks, the current study performed response evaluation every 6 weeks. Additional support for this interpretation is provided by the Japanese phase II study as well as our previous retrospective analysis, which included response evaluation every 6 weeks and demonstrated shorter PFS (median, 5.2 to 5.8 months) despite comparable OS (10.4-11.2 months) \[[@b5-crt-2017-526],[@b11-crt-2017-526]\]. Compared with these Asian studies investigating GemCis \[[@b5-crt-2017-526],[@b11-crt-2017-526]\], median PFS was better with OIS (6.8 months vs. 5.2-5.8 months). Despite the dramatic improvement in response rates with OIS compared with those achieved with GemCis in previous studies \[[@b4-crt-2017-526],[@b5-crt-2017-526],[@b11-crt-2017-526]\], differences in survival outcomes between the two regimens were not remarkable. This finding might be because of the increased rate of unfavorable clinical features in our patient population compared with that in patient population included in the ABC-02 study \[[@b4-crt-2017-526]\]. Only 3% of patients in the current study had locally advanced non-metastatic disease (vs 27% in the ABC-02 study), and all patients had measurable lesions according to the RECIST criteria (vs. 79% in the ABC-02 study). In a previous retrospective analysis involving 740 patients treated with first-line GemCis, metastatic disease and the presence of measurable lesion according to the RECIST criteria were independent poor prognostic factors \[[@b11-crt-2017-526]\]. OIS was generally well-tolerated, with a toxicity profile consistent with that in the previous phase I study \[[@b8-crt-2017-526]\]. The dose intensities of oxaliplatin, irinotecan, and S-1 were well-maintained. Neutropenia, diarrhea, and peripheral neuropathy were the most frequent grade 3-4 toxicities associated with OIS and were manageable with dose interruption/modification and appropriate supportive care. Compared with GemCis in the ABC-02 study, OIS in our study showed increased incidence rates of grade 3-4 neutropenia (25% vs. 32%) and neuropathy (not reported vs. 6%). Although increases in the rate of certain toxicities with the use of triplet combination regimens rather than doublets might be inevitable, the current study showed that the safety profile of OIS was clinically acceptable. In the biomarker analysis using NGS, *KRAS* mutations were revealed as potential poor prognostic factors for patients with advanced BTC. Although the number of patients included in the biomarker analysis was too small to be conclusive, our results were consistent with those of previous studies \[[@b12-crt-2017-526],[@b13-crt-2017-526]\]. In terms of the potential impact of the primary tumor location to the efficacy of chemotherapy in BTC, the current study showed that survival outcomes might be better in patients with extrahepatic cholangiocarcinoma than in those with gallbladder cancer or intrahepatic cholangiocarcinoma. In previous prognostic factor analyses in patients with advanced BTC, primary tumor location was not associated with survival outcomes in patients treated with first-line GemCis \[[@b11-crt-2017-526]\]; however, intrahepatic cholangiocarcinoma was revealed as an independent poor prognostic factor for PFS and OS in patients treated with second-line fluorouracil-based chemotherapy \[[@b14-crt-2017-526]\]. These findings, together with those of the current study, suggest that the chemotherapeutic activity might differ among primary tumor sites, particularly with the use of fluorouracil-based regimens. Distinct anti-cancer activities between gemcitabine- and fluorouracilbased treatments in different primary tumor locations should therefore be investigated in future studies. The current study had an inherent limitation as a singlearm study based on small number of patients. Considering the high heterogeneity in clinical and genetic features of patients with BTC \[[@b15-crt-2017-526]\], cross-study comparisons using data from single-arm studies are difficult as heterogeneity might independently impact the clinical outcomes. Another drawback of this study is that sample size calculation was based on the results of our previous retrospective analysis, not on those of the pivotal randomized trial, considering the potential ethnic discrepancies on the activity of chemotherapy. To assess the clinical relevance and future development strategy of OIS in patients with advanced BTC, therefore, a randomized trial with a control arm including GemCis is necessary. In conclusion, triplet OIS regimen comprising oxaliplatin, irinotecan, and S-1 was feasible and effective as first-line chemotherapy in patients with unresectable or metastatic BTC. Further evaluation of OIS in randomized studies is warranted. S-1, irinotecan, and oxaliplatin were generously provided by Jeil Pharmaceuticals. This study was supported in part by Hallym University Research Fund (HURF-2015-38; HURF-2016-41), Anyang, Korea and grants from the Asan Institute for Life Sciences at Asan Medical Center in Seoul, Korea (2015-0753, 2017-728). Electronic Supplementary Material ================================= Supplementary materials are available at Cancer Research and Treatment website (<http://www.e-crt.org>). {#f1-crt-2017-526} {#f2-crt-2017-526} {#f3-crt-2017-526} ###### Patient characteristics Characteristic No. (%) (n=32) -------------------------------- ---------------- **Sex**  Male 24 (75)  Female 8 (25) **Age, median (range, yr)** 64 (40-76) **Primary tumor site**  Intrahepatic 13 (41)  Gallbladder 11 (34)  Extrahepatic 8 (25) **Disease status**  Initially metastatic 24 (75)  Recurrent 7 (22)  Locally advanced unresectable 1 (3) **ECOG performance status**  0 7 (22)  1 25 (78) **Previous surgery** 8 (25) **No. of metastatic sites**  0-2 18 (56)  3-5 14 (44) **Metastatic site**  Liver 23 (72)  Lymph nodes 21 (66)  Peritoneum 10 (31)  Lung 7 (22)  Bone 5 (16) ECOG, Eastern Cooperative Oncology Group. ###### Adverse events occurring in ≥ 10% of the patients Adverse event No. (%) (n=31) ----------------------- ---------------- --------- --------- Neutropenia 17 (55) 10 (32) 27 (87) Anemia 24 (77) 0 24 (77) Thrombocytopenia 22 (71) 0 22 (71) Elevated AST 11 (35) 0 11 (36) Elevated ALT 8 (26) 1 (3) 9 (29) Hyperbilirubinemia 5 (16) 0 5 (16) Anorexia 16 (52) 1 (3) 17 (55) Nausea 13 (42) 1 (3) 14 (45) Vomiting 12 (39) 1 (3) 13 (42) Diarrhea 16 (52) 2 (6) 18 (58) Constipation 10 (32) 0 10 (32) Fatigue 8 (26) 1 (3) 9 (29) Alopecia 11 (35) 0 11 (35) Skin pigmentation 3 (10) 0 3 (10) Peripheral neuropathy 14 (45) 2 (6) 16 (52) Stomatitis 6 (19) 0 6 (19) Edema 7 (23) 0 7 (23) AST, aspartate aminotransferase; ALT, alanine aminotransferase. [^1]: Changhoon Yoo and Boram Han contributed equally to this work.

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ *Bartonella* bacteria are new emerging pathogens causing diseases in humans and animals \[[@pone.0140856.ref001], [@pone.0140856.ref002]\]. The members of genus *Bartonella* are rod-shaped gram negative facultative intracellular bacteria that are fastidious and slow growing at aerobic conditions. They infect human and other mammalian hosts via infected-vectors such as fleas, ticks, and lice or the bite/scratch of an infected-animal \[[@pone.0140856.ref003]--[@pone.0140856.ref005]\]. Moreover, the infected arthropods could transmit *Bartonella* bacteria to human and other mammalian hosts via feces through superficial scratches in skin \[[@pone.0140856.ref006]\]; for example, *B*. *henselae* and *B*. *quintana* were transmitted to hosts via contaminated feces of infected cat fleas (*Ctenocephalides felis*) and human body lice (*Pediculus humanus*), respectively \[[@pone.0140856.ref007]\]. Pathogenesis involves the invasion of host's erythrocytes, endothelial cells, and dendritic cells which play an important role in the first line immune response to fight against pathogens \[[@pone.0140856.ref008], [@pone.0140856.ref009]\]. As a result of the immune system failure, a bacteremia persistent infection might occur \[[@pone.0140856.ref008], [@pone.0140856.ref010]\]. *Bartonella* genus comprises over 30 species and subspecies \[[@pone.0140856.ref011]\]. At least thirteen known or suspected species are thought to contribute to blood-borne infections in human \[[@pone.0140856.ref012]\]. Moreover, several studies suggested the role of *Bartonella* species as a potential causative agent for cases of unknown febrile illness as well as endocarditis in patients in Thailand \[[@pone.0140856.ref013]\]. The diversity of *Bartonella* species in several countries in Southeast Asia (Lao PDR, Cambodia, and Thailand) has been reported and the findings revealed that *Bartonella* species in rodents are much more diverse than in other animals, except bats. The species found in rodents included *B*. *elizabethae*, *B*. *coopersplainsensis*, *B*. *phoceensis*, *B*. *queenslandensis*, *B*. *rattimassiliensis*, *B*. *tribocorum* and three genotypes presumably representing new *Bartonella* species \[[@pone.0140856.ref014]\]. *Bartonella* transmission occurs mainly via horizontal transmission when arthropod vectors acquire *Bartonella* bacteria during the feeding of infected host and later they become infected and the infected vectors then transfer the bacteria to another host \[[@pone.0140856.ref005], [@pone.0140856.ref015]\]. Interestingly, some studies suggested that vertical and transstadial transmissions of *Bartonella* species in *Ixodes* ticks \[[@pone.0140856.ref016]\], deer ked \[[@pone.0140856.ref017]\], and transplacental in rodent populations \[[@pone.0140856.ref018]\]. High prevalence of *Bartonella* DNA and genotype diversity have been detected in arthropod vectors around the world. For example, ticks collected from dogs and donkeys in Peru were found to carry several *Bartonella* species, such as *B*. *rochalimae*, *B*. *quintana* and *B*. *elizabethae* \[[@pone.0140856.ref019]\]. In Taiwan, *B*. *tribocorum*, *B*. *elizabethae*, *B*. *queenslandensis*, *B*. *rochalimae*-like bacteria, *B*. *phoceensis*, and *B*. *rattimassiliensis* were detected in fleas and louse pools \[[@pone.0140856.ref020]\]. Several studies in Thailand have reported the detection of *B*. *henselae*, *B*. *clarridgeiae*, and *B*. *koehlerae* from cats and flea pools collected from the Thai-Myanmar border \[[@pone.0140856.ref021]\] and in the Bangkok area \[[@pone.0140856.ref022]\]. Moreover, novel species such as *B*. *tamiae* was recently isolated from whole blood of febrile patients from Thailand \[[@pone.0140856.ref023]\] and DNA belonging to this species was also detected from the pools of ticks and mites collected from rats in Thailand \[[@pone.0140856.ref015]\]. Though a number of papers on *Bartonella* in rodents from Thailand have been published, the comparative analysis of bartonellae between rodent hosts and ectoparasites has not been done. Our aim was to investigate the prevalence and diversity of *Bartonella* species in rodents and their ectoparasites, and to estimate the importance of this host-vector relationship for the transmission of *Bartonella* species in natural habitats of Thailand. Our results indicated a significant difference between bacterial communities recognized in mammals and arthropods. Materials and Methods {#sec002} ===================== Study sites and samples processing {#sec003} ---------------------------------- The study sites were located in different regions of Thailand. Rodents and their associated ectoparasites (ticks, fleas, mites, and lice) were collected from eight provinces within four regions of Thailand during the period of December 2012 to November 2013. The regions included the Northern region (Chiang Rai and Phayao provinces), the Southern region (Chumphon and Surat Thani provinces), the Eastern region (Rayong and Trat provinces) and the Northeastern region (Loei and Nong Bua Lam Phu provinces) ([Table 1](#pone.0140856.t001){ref-type="table"}). This study was carried out on private lands and the owners of the lands gave permission to conduct the study on their sites and the field studies did not involve endangered or protected species. Rodents were captured by live traps baited with bananas or dried fish. Rodents were collected from orchards, cultivated rice-fields, grassland areas, edges of dense forest, stream margins, and around houses. Traps were set for 3--5 nights and were checked early in the morning. Then, rodents were removed from the traps and later identified to species \[[@pone.0140856.ref024]\]. Captured rodents were killed by carbon dioxide and processed on the same day and at the site of capture. Blood and serum samples and rodent tissue samples (liver, spleen, kidney and lung) were collected and stored on dry ice, and transported to the AFRIMS laboratory. Rodent's ears were cut and stored in 70% ethanol for mite collections and the other ectoparasites (ticks, fleas, and lice) were collected from individual rodents by combing and stored in 70% ethanol for transportation to the laboratory. Mites in their larval stage (chigger) were collected from rodent's ears by paintbrush under the stereomicroscope and pooled by host. Three to five mites were selected from each pool and mounted on glass slides for morphological identification to genera and species if possible using taxonomic key \[[@pone.0140856.ref025]\]. Ectoparasites of each type (fleas, ticks, and lice) were identified morphologically \[[@pone.0140856.ref026], [@pone.0140856.ref027]\] and pooled by host, type, stage, and gender in 1.5 ml microcentrifuge tube. Pools of ectoparasites were subjected to DNA extraction procedures as described below. Louse species identification was performed following the previously published protocol \[[@pone.0140856.ref028]\]. Details of the ectoparasites collected from rodents in this study are provided in Table A in [S1 File](#pone.0140856.s002){ref-type="supplementary-material"}. 10.1371/journal.pone.0140856.t001 ###### Location coordinates of rodents and ectoparasites collection sites in Thailand (2012--2013). {#pone.0140856.t001g} Regions Provinces Districts Sub-districts Villages Latitude Longitude --------------- --------------------- --------------- ---------------- ------------------------- ---------------------- ----------------------- **North** **Chiangrai** Mae Chan Chanchawatai Ban Pagook 20°15\' 16.042\'\' N 99°56\' 2.144\'\' E Mae Chan Pa Sang Rong Khi 20°10\' 47.543\'\' N 99°50\' 48.505\'\' E **Phayao** Dok Khamtai Ban Tham Ban Sansai 19°6\' 54.234\'\' N 100°3\' 44.319\'\' E ** ** ** ** Dok Khamtai Ban Tham Ban Tham Mongkol 19°6\' 2.606\'\' N 100°2\' 25.71\'\' E **Northeast** **Loei** Dan Sai Na Di Ban Na Mue Muen 17°19\' 15.859\'\' N 101°8\' 58.304\'\' E Dan Sai Na Di Ban Na Ho 17°19\' 30.922\'\' N 101°8\' 51.766\'\' E Dan Sai Pak Man Ban Pak Man 17°29\' 42.137\'\' N 101°10\' 48.572\'\' E **NongBua Lam Phu** Si Bun Rueang Non Sa-at Ban Wang Khaen 16°55\' 9.883\'\' N 102°8\' 58.138\'\' E ** ** ** ** Si Bun Rueang Na Kok Ban Non Ngam 16°53\' 42.23\'\' N 102°13\' 5.375\'\' E **East** **Rayong** Pluak Daeng Map Yang Phon Sapan Seeyakmahanakhorn 13°0\' 59.404\'\' N 101°8\' 16.778\'\' E Pluak Daeng Map Yang Phon Sasithorn16 13°0\' 48.629\'\' N 101°7\' 5.664\'\' E Pluak Daeng Map Yang Phon Bo Win 13°1\' 23.012\'\' N 101°6\' 38.523\'\' E **Trat** Khlong Yai Khlong Yai Ban Suan Maprao 11°46\' 51.946\'\' N 102°52\' 36.364\'\' E ** ** ** ** Khlong Yai Hat Lek Ban Khlong Son 11°43\' 52.561\'\' N 102°54\' 1.699\'\' E **South** **Chumphon** Tha Sae Tha Kham Ban Dinkong 10°39\' 34.463\'\' N 99°6\' 11.62\'\' E Mueang Bangluek Ban Salaloy 10°39\' 34.650\'\' N 99°6\' 12.592\'\' E Mueang Bangluek Ban Nongnean 10°34\' 23.675\'\' N 99°12\' 56.937\'\' E **Surat Thani** Mueang Wat Pradu Wat Ma Pring 9°6\' 59.065\'\' N 99°16\' 34.42\'\' E   ** ** Phunphin Khao Hua Khwai Bang Or 9°5\' 16.771\'\' N 99°13\' 14.005\'\' E Surveillance activities were conducted in 4 regions and 8 provinces in Thailand. The Northern region (Chiangrai and Phayao provinces), the Northeastern region (Loei and Nong Bua Lam Phu provinces), and the Southern region (Chumphon and Surat Thani provinces). Genomic DNA extraction from rodent tissue and ectoparasites {#sec004} ----------------------------------------------------------- Genomic DNA was extracted from rodent livers using the Wizard® Genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer's instructions with some modifications. Briefly, the liver tissue was cut into pieces of approximately 3 millimeters in diameter and added to 600 μl of Nuclei Lysis Solution (Promega, Madison, WI). The mixture was homogenized with beads using a TissueLyser II machine (Qiagen, Hilden, Germany) at 25 Hz for 5 min twice. Subsequently, the mixture was incubated with 20 μl of Proteinase K solution (20 mg/ml) at 55°C for 1 hr, and then with 3 μl of RNase A (10 mg/ml) at 37°C for 15 min. Then 200 μl of protein precipitation solution (Promega, Madison, WI) was added and mixed vigorously by vortex. The mixture was kept on ice for 5 min. Insoluble materials were removed by centrifugation at 13,000 rpm for 4 min and the supernatant was transferred to a new tube. DNA was precipitated by adding 600 μl of isopropanol and then centrifuged at 13,000 rpm for 1 min. DNA pellet was washed using 70% ethanol and dried by SpeedVac™ concentrator (Thermo Scientific, Waltham, MA). Two hundred microliters of EB buffer (10 mM Tris Cl, pH 8.5) were used to resuspend dried DNA and stored at -20°C until further analysis. DNA extraction from the ectoparasites was performed according to the tissue extraction protocol from QIAamp^®^ DNA Mini Kit (Qiagen, Hilden, Germany) with some modification. Briefly, pools of ticks, fleas and lice were puncture in the presence of liquid nitrogen in a 1.5 ml microcentrifuge tube. Mites were punctured with a fine needle under microscopy. Next, ninety microliters of ATL lysis buffer were added to each sample and mixed thoroughly. Then, ten microliters of Proteinase K solution (20 mg/ml) was added and incubated at 56°C for 3 hr. One hundred microliters of AL buffer was added to the samples and mixed by pulse-vortexing for 15 sec then incubated at 70°C for 10 min. After that, 100 μl of absolute ethanol was added and the mixture was mixed by pulse-vortexing for 15 sec. Finally, the mixture was transferred to a QIAamp spin column and DNA was eluted in 50 l AE buffer. DNA solution was stored at -20°C until further analysis. *Bartonella* detection in rodent tissues and ectoparasites {#sec005} ---------------------------------------------------------- DNA extracts obtained from rodent tissues and ectoparasites were screened for the presence of *Bartonella* species using real-time PCR assay (qPCR) with TaqMan probe. A genus-specific assay targeting a transfer-mRNA gene (*ssr*A) of *Bartonella* species was used in this study following previously published protocol \[[@pone.0140856.ref029]\]. The primer pair, *ssr*A-F/ *ssr*A-R, and *ssr*A-probe were used to amplify 301 bp fragment of *ssr*A gene. The qPCR reaction (25 μl) consisted of 12.5 μl Platinum® Quantitative PCR SuperMix-UDG (Invitrogen, Grand Island, NY), 0.5 μM of each primer, and 0.1 μM of *ssr*A-Probe and 2 μl of DNA template or nuclease free water as non-template control. The qPCR conditions were performed as follows: UDG incubation at 50°C for 2 min and then initial denaturation at 95°C for 2 min followed by 45 cycles of 95°C for 15 sec and 60°C for 30 sec using the Chromo4™ Real-Time Detector (Bio-Rad, Hercules, CA). Every sample with a positive signal from the screening assay was subjected to confirmatory test. A different target gene of NADH Dehydrogenase Gamma Subunit gene (*nuo*G) of *Bartonella* species was used to confirm the positivity by conventional PCR assay. A 346 bp fragment of *nuo*G gene was amplified with *nuo*G-F and *nuo*G-R primer pair according to previously published protocol \[[@pone.0140856.ref030]\]. The PCR reaction mixture (25 μl) consisted of 1X of PCR buffer, 0.2 μM of each primer, 0.2 mM of dNTP, 1.25 U of Taq DNA Polymerase (Invitrogen, Grand Island, NY) and 5 μl of DNA template or nuclease free water as non-template control. PCR amplification was carried out using the Veriti® 96-well Thermal Cycler (Applied Biosystems, Foster City, CA) with the initial denaturation at 94°C for 3 min followed by 45 cycles of denaturation at 94°C for 45 sec, annealing at 55°C for 1 min and extension at 72°C for 1 min 30 sec, then incubated at 72°C for 10 min for the final extension step. The amplification product of 346 bp was observed with 1.5% agarose gel electrophoresis under the UV visualization. *Bartonella* culture from rodent blood {#sec006} -------------------------------------- Selected rodents, which were *Bartonella*-positive by molecular assays, were subjected to *Bartonella* culture following previously published protocol \[[@pone.0140856.ref031]\]. At the field sample site, whole blood was collected from each rodent by cardiac puncture and preserved in EDTA. Rodent whole blood was kept in -70°C until use. Briefly, whole blood was retrieved from the -70°C freezer and thawed at 4°C. Then, homogeneous whole blood was diluted 1:4 in 1X Dulbecco's Phosphate Buffered Saline (GIBCO, Grand Island, NY) containing 5--10% Fungizone (GIBCO, Grand Island, NY). Diluted blood sample (0.1 ml) was pipetted onto Brain Heart Infusion agar plates containing 5% rabbit blood (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD). Four to five agar plates were kept in a plastic bag and incubated at 37°C, 5% CO~2~ for up to 4 weeks. The agar plates were monitored once a week after the initial inoculation and once per three days after sub-culturing. Sub-culturing was continued until a pure culture was obtained. *Bartonella*-like colonies were recognized by colony morphology and then harvested into 10% sterile glycerol and kept in -70°C freezer for further confirmation and characterization. A portion of each *Bartonella*-like colony was subjected to DNA extraction using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following manufacturer's instruction and confirmed to be *Bartonella* species by citrate synthase gene (*glt*A) sequence identity. Citrate synthase gene (*glt*A) amplification {#sec007} -------------------------------------------- Amplification of *Bartonella glt*A gene was done using published primers, BhCS.781p and BhCS.1137n \[[@pone.0140856.ref032]\]. The PCR reaction (50 μL) consisted of 1X of PCR buffer, 2.5 mM of MgCl~2~, 0.2 μM of each primer, 0.2 mM of dNTP, 2.5 U of AmpliTaq Gold® DNA Polymerase (Applied Biosystems, Foster City, CA) and 2.5 μl of DNA template or nuclease free water as non-template control. The amplification conditions were as follows: the initial denaturation at 95°C for 3 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 1 min, then the final extension step at 72°C for 10 min. The amplification product (379 bp) was observed on 1.5% agarose gel electrophoresis. DNA sequencing {#sec008} -------------- Amplification products of *ssr*A or *glt*A genes were purified using QIAquick PCR Purification Kit (QIAGEN Inc., Valencia, CA) following the manufacturer's instruction and sent for sequencing at AITbiotech Pte. Ltd. (Singapore) Statistical analyses {#sec009} -------------------- The difference in the prevalence of *Bartonella* DNA in ectoparasites collected from *Bartonella*-positive and *Bartonella*-negative rodents was confirmed by Chi-Square Test and the critical range (*P* \< 0.005) was used. The statistical calculations were performed with IBM® SPSS® Statisic (version 22) software (Chicago, IL). Sequence and Phylogenetic analysis {#sec010} ---------------------------------- Sequence data were assembled using the Sequencher 5.1 software (Gene Code Corporation, Ann Arbor, MI) and the consensus sequences were used for analyses. Sequences were aligned and constructed a similarity matrix with the reference sequences of *Bartonella* species using Muscle algorithm implemented in MEGA 6.0 software \[[@pone.0140856.ref033]\]. Maximum likelihood (ML) trees based on Kimura's 2-parameter model (K2+G+I) were constructed using molecular evolutionary genetics analysis (MEGA) 6.0 software \[[@pone.0140856.ref033]\] and bootstrap analyses with 1,000 resamplings performed to test the robustness of the branching. Ethics Statement {#sec011} ---------------- Rodents trapping were carried out in the different locations of the provinces according to the institutional animal collection protocol entitled "Field Sampling of Small Mammal (Orders: Erinaceomorpha; Soricomorpha; Scandentia; Macroscelidea and Rodentia) Populations to Support Zoonotic Diseases Surveillance and Ectoparasite Collection" (PN\# 12--06) reviewed and approved by the USAMC-AFRIMS Institutional Animal Care and Use Committee (IACUC). All sampling procedures and experimental manipulations were reviewed and approved as part of obtaining the animal collection protocol (PN\# 12--06). Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition. Results {#sec012} ======= Prevalence rate of *Bartonella* species among rodent and their associated ectoparasites in Thailand {#sec013} --------------------------------------------------------------------------------------------------- A total of 619 rodents, 287 mite pools (3--153 mites/pool), 62 flea pools (1--14 fleas/pool), 35 louse pools (1--20 lice/pool), and 170 tick pools (1--24 ticks/pool) were collected from eight provinces within four regions of Thailand. Overall prevalence of *Bartonella* species in rodents was 17.6% (109/619); and prevalence in their associated ectoparasites varied substantially depending on ectoparasite type ([Table 2](#pone.0140856.t002){ref-type="table"}). The highest prevalence was found in lice (57.1%, 20/35 pools) followed by fleas \[25.8%, 16/62 pools (15 pools of females and 1 pool of males, Table B in [S1 File](#pone.0140856.s002){ref-type="supplementary-material"})\], with evident decline in ticks (3.5%, 6/170 pools of female ticks, Table C in [S1 File](#pone.0140856.s002){ref-type="supplementary-material"}) and mites (1.7%, 5/287 pools). A high *Bartonella* prevalence was found in rats collected from the North (22.9% in Phayao), the East (25.6% in Rayong), and the South (26.6% in Chumphon and 28.3% in Surat Thani), and a high prevalence in their ectoparasites was found in Eastern regions (23.3% in Rayong and 30.8% in Trat) as shown in [Table 2](#pone.0140856.t002){ref-type="table"}. 10.1371/journal.pone.0140856.t002 ###### Prevalence of *Bartonella* DNA among wild-caught rodents and their associated ectoparasites collected from different regions and provinces in Thailand, 2012--2013. {#pone.0140856.t002g}     No. of *Bartonella* DNA positive/total collected (% positive)[^a^](#t002fn001){ref-type="table-fn"}   --------------- ----------------- ----------------------------------------------------------------------------------------------------- ----------------- -------------------------------------------------- ------------------ ---------------------------------------------------- ------------------ **North** Chiangrai 19/138 (13.8) 0/57 (0) \- 1/2 (50.0) \- **1/59 (1.7)** **North** Phayao 14/61 (22.9) 0/15 (0) 0/2 (0) \- 5/157 (3.2)[^\#^](#t002fn002){ref-type="table-fn"} **5/174 (2.9)** **Northeast** Loei 6/53 (11.3) 0/25 (0) 3/11 (27.3) \- 0/7 (0) **3/43 (7.0)** **Northeast** NongBua Lam Phu 3/42 (7.1) 0/8 (0) 2/26 (7.7)[^£^](#t002fn003){ref-type="table-fn"} 0/1 (0) \- **2/35 (5.7)** **East** Rayong 10/39 (25.6) 0/14 (0) 1/2 (50.0) 5/8 (62.5) 1/6 (16.7) **7/30 (23.3)** **East** Trat 6/101 (5.9) 0/15 (0) 9/20 (45.0) 3/4 (75.0) \- **12/39 (30.8)** **South** Chumphon 21/79 (26.6) 5/73 (6.8) \- 9/16 (56.3) \- **14/89 (15.7)** **South** Surat Thani 30/106 (28.3) 0/80 (0) 1/1 (100.0) 2/4 (50.0) \- **3/85 (3.5)**   **Total** **109/619 (17.6)** **5/287 (1.7)** **16/62 (25.8)** **20/35 (57.1)** **6/170 (3.5)** **47/554 (8.5)** ^a^ *Bartonella* DNA was detected by *ssr*A and *nuo*G genes. The positivity for each sample was recorded only when 2 assays produced the concordant results. ^\#^ Two *B*. *indica* rats had 2 positive tick pools. ^£^ One *R*. *exulans* had 2 positive flea pools. Prevalence of *Bartonella* DNA in rodent species and their associated ectoparasites (mite, louse, flea, and tick) {#sec014} ----------------------------------------------------------------------------------------------------------------- *Bandicota indica* (45.1%, 279/619), *Rattus rattus* (26.3%, 163/619) and *R*. *exulans* (15.8%, 98/619) were the rodent species with highest rates of *Bartonella* prevalence ([Table 3](#pone.0140856.t003){ref-type="table"}). A high prevalence of *Bartonella* DNA was also found in *B*. *savilei* (35.7%) and *R*. *rattus* (32.5%) followed by *R*. *sabanus* (16.7%), *B*. *indica* (15.1%), *R*. *norvegicus* (12.5%), and *R*. *exulans* (3.1%). Rats of *B*. *indica* and *R*. *rattus* were heavily infested with a variety of ectoparasites (mite, tick, and louse) accounting for 84.3% of all ectoparasites collected from rodents in this study ([Table 4](#pone.0140856.t004){ref-type="table"}). 10.1371/journal.pone.0140856.t003 ###### Distribution of *Bartonella* DNA among rodent species in different regions and provinces of Thailand, 2012--2013. {#pone.0140856.t003g}   No. of *Bartonella* DNA positive/total collected (% positive)[^a^](#t003fn001){ref-type="table-fn"}   ----------------------- ----------------------------------------------------------------------------------------------------- ----------------- ---------------- --------------- ----------------- ---------------- ----------------- ------------------ ------------------- ***B*. *indica*** 17/119(14.3) 11/56(19.6) 1/4(25.0) 1/1(100) 0/2(0) 0/13(0) 0/20(0) 12/64(18.8) **42/279(15.1)** ***B*. *savilei*** 2/9(22.2)  -  - 2/4(50.0)  -  - 1/1(100)  - **5/14(35.7)** ***Mus caroli***  -  - 0/1(0)  -  -  -  -  - **0/1(0)** ***M*. *cervicolor*** 0/1(0)  -  -  - 0/2(0)  -  -  - **0/3(0)** ***R*. *berdmorei***  -  -  -  -  -  - 0/1(0)  - **0/1(0)** ***R*. *bukit***  -  - 0/3(0)  -  -  -  -  - **0/3(0)** ***R*.*exulans*** 0/3(0) 2/2(100) 1/26 (3.8) 0/33(0) 0/1(0) 0/28(0) 0/1(0) 0/2(0) **3/96(3.1)** ***R*. *losea*** 0/3(0) \- \- \- \- \- \- \- **0/3(0)** ***R*. *norvegicus*** \- \- \- \- 3/17(17.6) 2/23(8.7) \- \- **5/40(12.5)** ***R*. *rattus*** 0/3(0) 1/3(33.3) 3/3(100) 0/4(0) 7/17(41.2) 4/37(10.8) 20/56(35.7) 18/40(45.0) **53/163(32.5)** ***R*. *sabanus*** \- \- 1/6(16.7) \- \- \- \- \- **1/6(16.7)** ***R*. *surifer*** \- \- 0/10(0) \- \- \- \- \- **0/10(0)** **Total** **19/138(13.8)** **14/61(22.9)** **6/53(11.3)** **3/42(7.1)** **10/39(25.6)** **6/101(5.9)** **21/79(26.6)** **30/106(28.3)** **109/619(17.6)** ^a^ *Bartonella* DNA was detected by *ssr*A and *nuo*G genes. The positivity for each sample was recorded only when 2 assays produced the concordant results. 10.1371/journal.pone.0140856.t004 ###### Prevalence of *Bartonella* DNA-positive ectoparasites by rodent species based on detection of the *ssr*A gene fragment. {#pone.0140856.t004g}   No. of *Bartonella* DNA-positive pools/total collected (% positive) ----------------------- --------------------------------------------------------------------- ------------------ ------------------ ----------------- ------------------ ***B*. *indica*** 0/153 (0) 0/5 (0) 1/3 (33.3) 5/153 (3.3) 6/314 (1.9) ***B*. *savilei*** 0/9 (0) \- 1/1 (100) \- 1/10 (10.0) ***Mus*. *caroli*** \- \- \- \- \- ***M*. *cervicolor*** \- \- \- \- \- ***R*. *berdmorei*** 0/1 (0) \- \- \- 0/1 (0) ***R*. *bukit*** 0/2 (0) \- \- 0/1 (0) 0/3 (0) ***R*. *exulans*** \- 13/45 (28.9) 0/1 (0) 0/1 (0) 13/47 (27.7) ***R*. *losea*** 0/1 (0) \- \- \- 0/1 (0) ***R*. *norvegicus*** 0/1 (0) 1/1 (100) 1/2 (50.0) 0/2 (0) 2/6 (33.3) ***R*. *rattus*** 5/104 (4.8) 2/11 (18.2) 17/28 (60.7) 1/10 (10.0) 25/153 (16.3) ***R*. *sabanus*** 0/6 (0) \- \- 0/2 (0) 0/8 (0) ***R*. *surifer*** 0/10 (0) \- \- 0/1 (0) 0/11 (0) **Total** **5/287 (1.7)** **16/62 (25.8)** **20/35 (57.1)** **6/170 (3.5)** **47/554 (8.5)** The lice were identified as *Polyplax* spp. (61.1%) and *Hoplopleura* spp. (38.9%), fleas were identified as *Xenopsylla cheopis*, and ticks were identified as *Haemaphysalis* spp. Mites collected from rats were more diverse than other types of ectoparasites. Three to five mites were selected from each pool and morphologically identified to genus (subgenus), and species if possible. They were all identified as trombiculid mites (Trombiculidae family, Trombiculinae subfamily). The most predominant genera were as follows: *Gahrliepia* (39.9%), *Leptotrombidium* (34.3%), *Ascoschoengastia* (14.6%), *Blankaartia* (5.4%), *Schoengastia* (4.0%), *Helenicula* (1.5%) and *Lorillatum* (0.3%). Two major genera collected from rats were further identified to subgenus. The results showed that within the genus *Gahrliepia*, subgenus *Walchia* was the most prevalent followed by *Schoengastiella*, and then *Gahrliepia*. Almost all mites in *Leptotrombidium* genus belonged to subgenus *Leptotrombidium*. Among the tested ectoparasites, a high prevalence of *Bartonella* DNA was detected in lice (57.1%, 20/35) and fleas (25.8%, 16/62). High prevalence of *Bartonella* DNA was detected in 17/28 louse pools (60.7%) collected from *R*. *rattus* and 13/45 flea pools collected from *R*. *exulans*. Mites and ticks were mostly collected from *B*. *indica* and *R*. *rattus* and only 1.7% (5/287) of mite pools and 3.5% (6/170) of tick pools were positive for *Bartonella* DNA. Among *Bartonella*-positive trombiculid mites, 3 pools were *Leptotrombidium* genus, 1 pool was *Ascoschoengastia* genus, and the last pool did not have slide for morphological identification. Identification of *Bartonella* species in rodent hosts and their associated ectoparasites based on *glt*A sequence variations {#sec015} ----------------------------------------------------------------------------------------------------------------------------- A total of 16 *Bartonella* isolates were successfully cultured from 26 individual rodent blood samples. *Bartonella* species detected from rats and ectoparasites based on sequences and phylogenetic analyses of 318 bp *glt*A amplicon are presented in [Table 5](#pone.0140856.t005){ref-type="table"} and [Fig 1](#pone.0140856.g001){ref-type="fig"}. Percent identity to the reference *Bartonella glt*A sequences of *Bartonella* species detected in this study are summarized in [Table 6](#pone.0140856.t006){ref-type="table"}. Maximum-likelihood (ML) tree ([Fig 1](#pone.0140856.g001){ref-type="fig"}) shows the relationship between sequences generated from *Bartonella*-positive samples clustered into 8 different cladograms as described below. The majority of identified sequences (25/41 sequences) fell within *B*. *elizabethae* species complex. Within this *B*. *elizabethae* complex group (25 sequences), 15 *B*. *rattimassiliensis* were detected from 9 rats, 5 louse pools, and 1 mite pool sharing 96.5--100% identity with strain 16115 (AY515125) and strain THNA5-R09 (JX158360). Eight sequences of *B*. *tribocorum* were detected from 2 rats and 6 flea pools sharing 99.3--100% identity with strain IBS506 (AJ005494) and strain THSKR-020 (JX158363). One sequence of *B*. *queenslandensis* was detected from a flea pool sharing 99.6% identity with strain AUST/NH5 (EU111799). One sequence of undescribed *Bartonella* species within *B*. *elizabethae* complex was detected from a flea pool (\#F1644) with a percent identity ranging from 96.8 to 97.1% to *B*. *tribocorum* strain IBS506 (AJ005494) and strain THSKR-020 (JX158363). The rest of the detected *Bartonella* sequences (16 sequences) were identified into eight *B*. *coopersplainsensis* which were detected from 5 rats, 2 tick pools, and 1 louse pool sharing 99.0--100% identity with strain AUST/NH20 (EU111803). Seven *B*. *phoceensis* were detected solely from ectoparasites: 5 in louse pools, 1 in a mite pool, and 1 in a tick pool sharing 98.4--100% identity with *B*. *phoceensis* strain 16120 (AY515126) and *B*. *phoceensis* strain BR07 (GU056197). Interestingly, one sequence obtained from *Bartonella*-positive louse pool (\#L1026) was distantly related to the rest of the *Bartonella* species (66.3--68.2% identity) detected in this study, however, it was similar to some new strains of *Bartonella* species strain BCF03 (GU056190) and strain BR10 (GU056200) obtained from Taiwan with 99.3% identity ([Table 6](#pone.0140856.t006){ref-type="table"}). {#pone.0140856.g001} 10.1371/journal.pone.0140856.t005 ###### *Bartonella* species identified in rodents and their associated ectoparasites based on *glt*A gene sequence similarities {#pone.0140856.t005g} *Bartonella* species (*glt*A) Rodents Mites Fleas Lice [^c^](#t005fn003){ref-type="table-fn"} Ticks Total -------------------------------------------------------------- ---------------- ----------------------------------------- --------------- --------------------------------------------- -------------- ---------------- ***B*. *rattimassiliensis*** 9 1[^a^](#t005fn001){ref-type="table-fn"} \- 5 \- **15 (36.6%)** ***B*. *tribocorum*** 2 \- 6 \- \- **8 (19.5%)** ***B*. *elizabethae*** \- \- \- \- \- **-** ***B*. *queenslandensis*** \- \- 1 \- \- **1 (2.4%)** **Other *Bartonella* of *B*. *elizabethae* species complex** \- \- 1 \- \- **1 (2.4%)** ***B*. *phoceensis*** \- 1[^b^](#t005fn002){ref-type="table-fn"} 5 1 **7 (17.1%)** ***B*. *coopersplainsensis*** 5 \- \- 1 2 **8 (19.5%)** **New *Bartonella* species (GU056190)** \- \- \- 1[^d^](#t005fn004){ref-type="table-fn"} \- **1 (2.4%)** **Total** **16 (39.0%)** **2 (4.9%)** **8 (19.5%)** **12 (29.3%)** **3 (7.3%)** **41 (100%)** ^a^ *L*. *deliense*. ^b^ Mite species was not available since there were only 3 mites collected from this host and no slide was made for species identification. ^**c**^ *Bartonella* DNA was equally detected in *Polyplax* and *Hoplopleura* lice. ^d^ *Polyplax* spp. 10.1371/journal.pone.0140856.t006 ###### Percent similarity of *glt*A sequence for *Bartonella* identification detected from rodents (R) and their associated ectoparasites; Mite (M), flea (F), tick (T), and louse (L). {#pone.0140856.t006g}   \% Similarity ----------------------------------------------- --------------- ---------- ---------- ---------- ---------- --------- ---------- ---------- -------- ------ ---------- ---------- F1644 95.9 93.4 94 95.6 **97.1** 96.8 88.4 88.4 85.9 96.5 67.2 67.2 R1135 94 **97.1** **97.1** 93.1 94.7 94.3 87.8 87.8 85.3 95.3 67.2 67.2 R1188 92.8 **96.5** **96.5** 92.2 92.8 92.5 87.8 87.8 85 94.3 67.2 67.2 Group 1[^a^](#t006fn001){ref-type="table-fn"} 93.1 97.5 **100** 92.2 93.4 93.1 87.2 87.2 84.1 94.3 66.6 66.6 L1644 93.4 97.8 **99.6** 92.5 93.7 93.4 87.5 87.5 84.4 94.7 66.3 66.3 F1018 93.7 92.2 92.5 **99.6** 94 93.7 87.5 87.5 83.4 95.3 67.9 67.9 F2540 94.7 92.2 92.8 94.3 **99.3** 99 88.1 88.1 85.6 95.3 67.2 67.2 Group 2[^b^](#t006fn002){ref-type="table-fn"} 94.3 92.5 93.1 93.4 99.6 **100** 89 89 85.9 95.6 67.2 67.2 Group 3[^c^](#t006fn003){ref-type="table-fn"} 88.1 87.2 87.2 87.8 88.7 89 **100** **100** 88.7 90.3 67.6 67.6 L1181 87.5 86.2 86.2 87.2 87.8 88.1 **98.4** **98.4** 87.8 89.4 67.6 67.6 Group 4[^d^](#t006fn004){ref-type="table-fn"} 85.6 85.6 85 84.4 86.9 86.6 89.7 89.7 **99** 87.5 66.6 66.6 L1026 66.6 65.4 66.3 67.6 67.2 66.9 67.2 67.2 66.3 68.2 **99.3** **99.3** ^a^ Group 1 consists of sample no. R1023, R1025, R1026, R1028, R1181, R1195, R1197, L1028, M1200, L1206, L1354, and L1355. ^b^ Group 2 consists of sample no. R1012, R1033, F1413, F1437, F2386, F2495, and F2532. ^c^ Group 3 consists of sample no. T1005, L1025, M1189, L1194, L1227, and L1596. ^d^ Group 4 consists of sample no. R1072, R1073, R1136, R1144, R1168, L1080, T1513, and T1521. The distribution of *Bartonella* species found in the North and northeast seems to be a region-specific distribution. Four samples of *B*. *tribocorum* were found in the Northeast and 8 samples of *B*. *coopersplainsensis* were detected in the North, even though one *B*. *rattimassiliensis* was also detected in the North ([Fig 1](#pone.0140856.g001){ref-type="fig"} and [Table 5](#pone.0140856.t005){ref-type="table"}). In contrast, *Bartonella* species found in samples collected from the East and the South are quite diverse consisting of six different species; *B*. *rattimassiliensis*, *B*. *queenslandensis*, *B*. *tribocorum*, *Bartonella* species within *B*. *elizabethae* species complex, and a presumably new strain of *Bartonella* species. In this study, we found that the distribution of *Bartonella* species among rodents supports a host-specific pattern. Thus, five *B*. *coopersplainsensis* were solely detected from *B*. *indica*. Almost all *B*. *rattimassiliensis* (8/9) were detected from *R*. *rattus*, while two *B*. *tribocorum* were detected from *R*. *norvegicus* only. However, no specific pattern has been observed among ectoparasites. For example, two different *Bartonella* species (*B*. *rattimassiliensis* and *B*. *phoceensis*) were detected in mite pools ([Table 5](#pone.0140856.t005){ref-type="table"}), 3 species (*B*. *tribocorum*, *B*. *queenslandensis* and other *Bartonella* of *B*. *elizabethae* species complex) were detected in flea pools (*Xenopsylla cheopis)*, 4 species (*B*. *rattimassiliensis*, *B*. *phoceensis*, *B*. *coopersplainsensis* and new *Bartonella* species (GU056190)) were detected in louse pools (*Polyplax* spp. and *Hoplopleura* spp.), and 2 species (*B*. *phoceensis* and *B*. *coopersplainsensis*) were detected in tick pools (*Haemaphysalis* spp.). Prevalence and distribution of *Bartonella* species detected in ectoparasites collected from *Bartonella*-positive and *Bartonella*-negative rats {#sec016} ------------------------------------------------------------------------------------------------------------------------------------------------- The difference of *Bartonella* DNA prevalence in ectoparasites collected from *Bartonella*-positive and *Bartonella*-negative rats was investigated and presented in [Table 7](#pone.0140856.t007){ref-type="table"}. Among *Bartonella*-positive rats, 20/103 (19.4%) ectoparasite pools were positive for *Bartonella* species. In contrast, only 27/309 (8.7%) ectoparasite pools were positive from *Bartonella*-negative rats. Of these *Bartonella*-positive ectoparasites, louse pools possessed the highest prevalence of 65% (13/20) and 46.7% (7/15) for positive and negative rats, respectively ([Table 7](#pone.0140856.t007){ref-type="table"}). In general, *Bartonella* DNA prevalence in ectoparasites collected from positive rats (19.4%) were higher significantly (Chi-Square Tests, *P* = 0.003) comparing to ectoparasites from negative rats (8.7%). 10.1371/journal.pone.0140856.t007 ###### Comparison *of Bartonella* prevalence in ectoparasites collected from *Bartonella*-positive and *Bartonella*-negative rodents. {#pone.0140856.t007g}   Prevalence of *Bartonella* species in vectors among *Bartonella*-positive rodent Prevalence of *Bartonella* species in vectors among *Bartonella*-negative rodents --------------------------------------------- ---------------------------------------------------------------------------------- ----------------------------------------------------------------------------------- ----------------------------------------------------- -------- --------- ---------------------------------------------------- Mites 4 69 5.8 1 218 0.5 Fleas[^a^](#t007fn001){ref-type="table-fn"} 1 4 25 15 50 30 Ticks[^a^](#t007fn001){ref-type="table-fn"} 2 10 20 4 26 15.4 Lice 13 20 65 7 15 46.7 **Total** **20** **103** **19.4** [^**b**^](#t007fn002){ref-type="table-fn"} **27** **309** **8.7** [^**b**^](#t007fn002){ref-type="table-fn"} ^a^ There are 2 samples where *Bartonella* was detected in host (*R*. *rattus*), mite, and lice; 1--4 pools of ticks and fleas can be collected from one rodent. ^b^ *Bartonella* DNA prevalence in ectoparasites collected from positive rats (19.4%) were higher significantly (Chi-Square Tests, *P* = 0.003) comparing to ectoparasites from negative rats (8.7%). Discussion {#sec017} ========== Our study highlights the surveillance of *Bartonella* species among rodents and their associated ectoparasites (ticks, fleas, lice, and mites) in several regions across Thailand. The data demonstrated the high prevalence of *Bartonella* DNA in rats and their associated ectoparasites, especially in lice and fleas, as well as the finding of a diverse range of *Bartonella* species circulated among them. Previous studies have shown the high prevalence of *Bartonella* species among rats captured from Chiang Rai province (8.7%) \[[@pone.0140856.ref034]\] and from several regions across the country (41.5%) \[[@pone.0140856.ref035]\]. Although the prevalence in the latter study by Bai *et al*. was higher (41.5%, 137/330) than what we found (17.6%, 109/619), the proportion of rodent species are relatively the same except that *B*. *indica* was the highest number trapped in our study (45.1%, 279/619), while Bai *et al*. reported *R*. *rattus* as the most prevalent species investigated in their study (65.2%, 88/135). Given the higher rate of *Bartonella*-positive detected in *R*. *rattus* comparing to *B*. *indica* rat, the high *Bartonella* prevalence reported in Bai's study can be explain by rodent species composition. Similarly, high prevalence (41.3%) of *Bartonella* was reported in rodents and shrews in Taiwan with the highest prevalence was found in *R*. *norvegicus* (52.7%) \[[@pone.0140856.ref036]\]. Although *Bartonella* DNA was detected in all types of ectoparasites (ticks, lice, fleas, and mites) collected from rats in this study, the prevalence varied substantially between ectoparasite types. The highest rate was found in lice and fleas that were in agreement with the study conducted in Taiwan \[[@pone.0140856.ref020]\]. Though *Bartonella* DNA was found in lower prevalence in ticks and mite pools in both studies, Kabeya *et al*. reported high prevalence in mites (82.9%) collected from rats in Thailand and all *Bartonella* species detected from mites were identified into *B*. *tamiae* based on their DNA sequences \[[@pone.0140856.ref015]\]. Although we do not believe that rat-associated lice can transmit pathogens to humans because of their high specificity to the host, for example, *Polyplax* and *Hoplopleura* lice are more likely specific to *Rattus* rats or to some other relative rat species \[[@pone.0140856.ref037], [@pone.0140856.ref038]\], the circulation of bartonellae via different ectoparasites, including lice, can influence the diversity of a rat-associated *Bartonella* community. A diverse range of *Bartonella* species were isolated from whole blood of rats and shrews, including *B*. *rattimassiliensis*, *B*. *grahamii*, *B*. *elizabethae*, *B*. *tribocorum*, *B*. *coopersplainensis*, *B*. *phoceensis*, *B*. *queenslandensis*, and unknown genogroup \[[@pone.0140856.ref020], [@pone.0140856.ref032], [@pone.0140856.ref035], [@pone.0140856.ref036]\]. While only three of these species, *B*. *rattimassiliensis*, *B*. *tribocorum*, *B*. *coopersplainensis*, were isolated from rats in our study. *Bartonella* species found in rodent-associated ectoparasites were more diverse with seven different species found, including species isolated from their rat hosts. In this study, *glt*A gene sequence was used to identify *Bartonella* species, although ss*r*A gene was also sequenced and analyzed. We found that both genes were effective to identify *Bartonella* at the genus level; however, *glt*A gene sequence was found to be more suitable for species identification than *ssr*A gene sequence since the *glt*A sequence has expressed a higher range of variation comparing to the *ssrA*. Moreover, the availability of *glt*A reference sequences for *Bartonella* species in available databases is much higher than for *ssr*A genes that makes it a reliable and accurate tool for *Bartonella* species identification. Additionally, phylogenetic trees constructed from *Bartonella* culture isolates using both *ssr*A and *glt*A genes created quite similar phylogenetic tree topologies as shown in [S1 Fig](#pone.0140856.s001){ref-type="supplementary-material"}. Kabeya *et al*. \[[@pone.0140856.ref015]\] reported the detection of *B*. *tamiae*, previously isolated from Thai febrile illness patients, in mites and tick pools collected from rats in Thailand. Two dominant mite genus most infected with *B*. *tamiae* were *Leptotrombidium* (66.7%) and *Schoengastia* (78.6%), therefore, the author suggested the role of mite as potential vector for *B*. *tamiae* transmission to human patients. In this study, *Bartonella* DNA was also detected from mites of *Leptotrombidium* and *Ascoschoengastia* genera, although *B*. *tamiae* was not found in our study. Our finding supports the role of trombiculid mite as a vector for *Bartonella* transmission. Because of the difficulty of mite species identification, the mite species in each pool was determined based on 3--5 mites selected from each pool and mounted on a glass slide as mentioned in materials and methods. From our study, we found that *Bartonella* positive ectoparasites from *Bartonella*-positive rats was higher than in *Bartonella*-negative rats suggesting that this environment promotes the occurrence of horizontal transmission of *Bartonella* bacteria during the bite/feeding of vectors on rats. However, in order to prove infection route, transmission studies from an infected vector to naïve hosts/rats should be done in a controlled laboratory environment. Seasonal dynamics of *Bartonella* infection in natural populations of rats can also obscure a correlation between prevalence in rats and ectoparasites. *Bandicota* and *Rattus* rats are the most common reservoir hosts for *Bartonella* infection in Southeast Asia which raises a question about the potentially important role of these rodent-borne agents as sources of febrile illness in human populations in Thailand. Detection of *Bartonella* DNA similar to *B*. *tamiae*, isolated from three febrile patients \[[@pone.0140856.ref023]\], and from mites collected from rodents in Thailand \[[@pone.0140856.ref015]\] imply a potential connection role for transmission of the disease to humans. Data from Kosoy et al. 2010, support this potential transmission route came from isolation of *Bartonella* species, previously identified from rodent hosts, from Thai patients' blood and supported by analysis of history of the patients having an exposure to rats during the 2 weeks before the illness \[[@pone.0140856.ref035]\]. *Bartonella* species identified in these human cases included *B*. *elizabethae*, *B*. *rattimassiliensis*, and *B*. *tribocorum*, and the last two of these species were also detected in rodents, mites, fleas, and lice reported in this study. Disclaimer {#sec018} ========== Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Supporting Information {#sec019} ====================== ###### Phylogenetic relationships between *Bartonella* species isolated from rodent blood and some reference species according to the *ssr*A and *glt*A phylogenies. The GenBank accession numbers are shown for each reference sequences. Trees constructed from *ssr*A and *glt*A genes were able to discriminate all samples into two major clusters (C1 and C2), although some different branching patterns of sequences in C1 group (R1012, R1033, R1135, and R1188) were noticed. (TIF) ###### Click here for additional data file. ###### Additional data of ectoparasites collected from rodents and *Bartonella* DNA prevalence in the developmental stages of tick and flea. Detail information of ectoparasites collected from rodents in this study (Table A).*Bartonella* DNA detection in flea pools classified by gender (Table B) and in tick pools classified by stage and gender (Table C) (DOCX) ###### Click here for additional data file. We wish to thank Taweesak Monkanna and Opas Thachin (AFRIMS) for their assistances in the field work, Ms. Achareeya Korkusol for her technical support and analysis on louse identification by DNA sequencing and analysis. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: RT KK. Performed the experiments: KK SP SL. Analyzed the data: RT KK MK. Wrote the paper: RT KK ALS PWM.

Introduction ============ Cancer is among the most common causes of morbidity and mortality worldwide, with an estimated 14 million new cases and 8 million deaths in 2012, projected to rise by at least 70% by 2030.[@R1] Timely and accurate cancer statistics are crucial to identify priorities for cancer control strategies at the national level. Yet, only 34 of 194 World Health Organization (WHO) Member States presently report high-quality national mortality data,[@R2] while 68 countries provided high-quality incidence data for the last volume of *Cancer incidence in five continents*.[@R3] As a result, many policy-makers rely on national cancer incidence and mortality estimates of variable precision to inform cancer control priorities. GLOBOCAN, a project of the International Agency for Research on Cancer (IARC) provides estimates by cancer site and sex using the best available data in each country and several methods of estimation.[@R1] Producing high-quality estimates therefore requires a dual approach of improving the reported data (developing cancer registries and civil/vital registration systems) and a continual assessment of the validity of the estimation procedures to improve the methods used. This study focuses on the validity of the methods used in GLOBOCAN to derive national cancer incidence estimates, based on a retrospective comparison of these estimates to the observed national data in a setting with high quality cancer registry data. Although we focused on the methods most commonly used in high-income countries, we also aimed at providing insights into the validity of the methods more broadly, including methods used more predominantly in low- and middle-income countries. Methods ======= Recorded data ------------- To validate the nine methods used in GLOBOCAN to estimate national incidence in 2012 (GLOBOCAN 2012), long-term national and regional incidence and mortality data as well as 5-year relative survival estimates are required. Of the few countries with such data available, we selected Norway because of the consistently high quality of its cancer registry data, available nationally and by region. Cancer reporting is a legal requirement in Norway and data linkage procedures with the cause of death registry further increase the completeness of the information. For the period 2001--2005, data completeness was estimated at 98.8%, while 93.8% of the cases had been verified by examining biopsy samples under a microscope.[@R4] From the Nordic cancer database NORDCAN, we extracted Norwegian incidence and mortality data by region, year of diagnosis, cancer site, sex and 5-years age group (starting at 0--4 and ending at 85+) for the period 1983--2012.[@R5] We also extracted Norwegian 5-year relative survival proportions for each cancer site as well as incidence and mortality data from neighbouring countries Denmark, Finland, Iceland and Sweden.[@R5] As with GLOBOCAN 2012, national population data were obtained from the United Nations[@R6] while regional population data were extracted from NORDCAN.[@R5] Cancer sites of the recorded cases and deaths were grouped by the codes in the *International statistical classification of diseases and related health problems, 10th revision* (ICD-10) to correspond to the sites used in GLOBOCAN. Unspecified neoplasms of the uterus (ICD-10 code C55) were reallocated to the cervix (C53) and corpus uteri (C54) according to the respective proportions of these two sites in the different datasets.[@R7] We computed the number of cases by sex and cancer site in Norway in 2010 as the average of the recorded cancer cases between 2009 and 2011 to define a gold standard for comparisons. We then applied each of the nine methods used in GLOBOCAN 2012 to estimate the number of cancer cases in Norway in 2010, by sex and cancer site, and compared these estimates with the gold standard. Estimation methods ------------------ The GLOBOCAN methods are summarized in [Fig. 1](#F1){ref-type="fig"}, together with the algorithm used to select them in GLOBOCAN based on the availability of data in each country. More details can be found elsewhere.[@R1]^,^[@R8] [Fig. 2](#F2){ref-type="fig"} illustrates which method was used for each country within the GLOBOCAN 2012 project. {#F1} {#F2} The data required for each of the nine methods are summarized in [Table 1](#T1){ref-type="table"}.The methods used may produce under- or overestimates at different cancer sites. Therefore, presenting an overall number of cases based on the sum of site-specific numbers could be misleading, if aggregated overestimates and underestimates cancel each other out. We thus report separately the total number of cases underestimated and overestimated for each method. These were then aggregated to assess the differences between the results and the Norwegian recorded data. ###### Required conditions for reliable estimations for each of the nine methods used to estimate cancer incidence in GLOBOCAN 2012 Method Data required to use the method Conditions required for reliable estimations --------------------------------------------------------------------------------- ------------------------------------------------------------------------------------ --------------------------------------------------------- 1 Historical national incidence data -- Availability of robust data on cases/population size -- Recent incidence trends continue into near future 2 Recent national incidence data -- Availability of robust data on cases/population size -- Stable incidence rates in near future 3 National mortality data and M:I ratios from regional registries within the country -- Availability of robust data on cases/deaths -- Trends in incidence, mortality and survival are relatively stable over time -- Case fatality in combined regions representative nationally 4 National mortality data and M:I ratios from registries in neighbouring countries -- Availability of robust data on cases/deaths -- Trends in incidence, mortality and survival are relatively stable over time -- Case fatality in combined neighbouring countries representative nationally 5 National mortality and 5-year relative survival data -- Availability of robust data on deaths and survival -- Trends in incidence, mortality and survival are relatively stable over time -- Five-year survival proportion a reasonable proxy for clinical cure 6 Rates from one regional registry within the country -- Availability of robust data on cases/population size -- Incidence rates in single region representative nationally 7 Rates from multiple regional registries within the country -- Availability of robust data on cases/population size -- Incidence rates in combined regions representative nationally 8 Data from all sites by age and sex and frequency data by cancer site -- Availability of robust data on total cancer cases -- Total cases and cancer-specific frequencies representative nationally 9 Data from neighbouring countries -- Availability of robust data on cases/population size -- Incidence rates in combined neighbouring countries representative nationally M:I: mortality:incidence. All analyses were performed using the R software package (The R Project for Statistical Computing, Vienna, Austria). ### Method 1 Method 1 is based on projections of incidence rates. We performed two projections: (i) for 1A we used the computer program NORDPRED[@R9] and applied long-term data (1983--2007) and; (ii) for 1B we used the computer program DEPPRED[@R10] and applied medium-term data (1998--2007). ### Methods 2 to 7 For methods 2 to 7, we used incidence and/or mortality data from 2003--2007 to simulate a real-life situation where data from the latest volume of *Cancer incidence in five continents (Vol. X)* would be used.[@R3] The 2010 Norwegian mortality data used in methods 3 to 5 were estimated as in GLOBOCAN 2012 by projecting rates for the period 1988--2007 to 2008--2012. In method 3, mortality:incidence (M:I) ratios from regional registries are used as a proxy for national case-fatality rates. National incidence rates can then be inferred from national mortality data along with the M:I ratio. This is useful where regional registries are numerous but not necessarily nationally representative, as in Italy[@R11] or Japan.[@R12] Where no such regional population-based data are available, data from neighbouring countries can be used (method 4). To generate the M:I ratios used in method 3, we included recorded cancer cases and deaths from all regions of Norway except for the south-eastern region (that includes Oslo). In some high-income countries (e.g. France or Japan) national estimates are derived from regional cancer registry data that do not cover the capital city which is usually highly populated. We also included recorded cases and deaths from other Nordic countries for cancer sites with less than a hundred deaths in Norway (e.g. cancers of the larynx, testis and thyroid and Hodgkin lymphoma). Method 5 estimates national cancer incidence by using national mortality and 5-year relative cancer survival data, using the equation:where *M* is the mortality rate, *I* is incidence rate and *S* is the 5-year relative survival proportion. Method 6 was based on incidence data from the northern and western regions of Norway, while we selected the south-eastern region (including Oslo) for method 7. For GLOBOCAN estimations, regional incidence data are often only available from large cities, particularly in low- and middle-income countries (e.g. Uganda, Zimbabwe). ### Methods 8 and 9 The incidence rates from neighbouring countries used in methods 8 and 9 were computed using data from Nordic countries for the period 2009--2011. Results ======= In 2010, 14 507 new cancer cases were recorded in Norwegian men and 12 466 in women. Our corresponding estimates, based on GLOBOCAN methods, differed by 5.7--18.8% (834 to 2341 cases) from the observed data (excluding method 5). [Fig. 3](#F3){ref-type="fig"} summarizes the sex-specific numerical differences according to each method, with under- and overestimates reported separately, as well as the overall difference as a percentage with observed data. {#F3} Comparing incidence estimates to observed data across cancer sites by sex, estimates based on data from one regional cancer registry (method 7) performed best in men (mean of 5.7%, or 834 difference between estimated and observed cases), while projection of medium-term historical rates (method 1B) performed best in women (mean: 6.1% difference; 763 cases). When considering both sexes together, and among the methods usually used in high-income countries (methods 1 to 4), the most recent recorded rates applied to 2010 population (method 2) performed well with a 6.4% (1726 cases) difference between observed and estimated cases. However, when prostate and breast cancers were excluded, projection of rates (methods 1A and 1B) produced very similar overall estimates to those from method 2 (at most a 5.0% (723 cases, 1B) and 7.7% (958 cases, 1A) difference; [Fig. 3](#F3){ref-type="fig"}). Apart from methods 1A, 1B and 5, all methods tended to underestimate the total number of cases. Our estimates by cancer sites show variability in the performance of the different methods ([Fig. 4](#F4){ref-type="fig"}). Overall, methods commonly used in high-income countries performed quite well in estimating recent cancer incidence in Norway. Method 1A produced the closest estimates to observed data for lung cancer in both men and women (−0.5%; −7 cases and −1.1%; −13 cases, respectively). It also performed well for colorectal cancer in men (−0.1%; −1 case) and women (+2.4%; +45 cases). On the other hand, prostate cancer cases were overestimated by this method (+19.4%; +881 cases). Method 2 performed better than method 1A for breast (+2.3%; +67 cases) and prostate (−5.1%; −231 cases; [Fig. 5](#F5){ref-type="fig"}) cancers. Method 2 estimates for lung cancer were satisfactory in men (+1.2%; +18 cases) but less so in women (−13.9%; −168 cases; [Fig. 6](#F6){ref-type="fig"}). Methods 3 and 4 generally produced underestimations at major cancer sites except for melanoma of skin in women (+17.4%; +139 cases and +34.0%; +271 cases using methods 3 and 4, respectively). These two methods performed less well for rare cancers (e.g. gallbladder cancer or Hodgkin lymphoma) or those with a good prognosis (e.g. testis or thyroid cancers; [Table 2](#T2){ref-type="table"} and [Table 3](#T3){ref-type="table"}). {#F4} {#F5} {#F6} ###### Cancer incidence in males (Norwegian Cancer Registry, 2010) and estimates using GLOBOCAN methods Site (ICD-10 code) No. of observed cases No. of estimated cases (% difference from observed cases) ------------------------------------- ----------------------- ----------------------------------------------------------- ------------- ------------- ---------------- -------------- ------------- ------------- ------------- -------------- -------------- Head and neck (C00--14) 324 279 (−13.9) 288 (−11.1) 287 (−11.4) 306 (−5.6) 246 (−24.1) 221 (−31.8) 292 (−9.9) 298 (−8.0) 291 (−10.2) 335 (3.4) Oesophagus (C15) 168 159 (−5.4) 160 (−4.8) 154 (−8.3) 181 (7.7) 170 (1.2) 182 (8.3) 146 (−13.1) 166 (−1.2) 155 (−7.7) 217 (29.2) Stomach (C16) 293 296 (1.0) 294 (0.3) 355 (21.2) 281 (−4.1) 269 (−8.2) 276 (−5.8) 431 (47.1) 296 (1.0) 238 (−18.8) 297 (1.4) Colon-rectum (C18--21) 1948 1947 (0.0) 1882 (−3.4) 1895 (−2.7) 1834 (−5.8) 1668 (−14.4) 1994 (2.4) 2083 (6.9) 1797 (−7.8) 1617 (−17.0) 1428 (−26.7) Liver (C22) 120 105 (−12.5) 98 (−18.3) 94 (−21.7) 100 (−16.7) 99 (−17.5) 112 (−6.7) 91 (−24.2) 94 (−21.7) 113 (−5.8) 193 (60.8) Gallbladder (C23--24) 73 76 (4.1) 64 (−12.3) 67 (−8.2) 43 (−41.1) 25 (−65.8) 28 (−61.6) 68 (−6.8) 71 (−2.7) 60 (−17.8) 89 (21.9) Pancreas (C25) 332 377 (13.6) 385 (16.0) 352 (6.0) 376 (13.3) 309 (−6.9) 367 (10.5) 343 (3.3) 355 (6.9) 281 (−15.4) 329 (−0.9) Larynx (C32) 97 89 (−8.3) 82 (−15.5) 107 (10.3) 113 (16.5)^a^ 112 (15.5) 119 (22.7) 110 (13.4) 113 (16.5) 83 (−14.4) 104 (7.2) Lung (C33--34) 1561 1554 (−0.5) 1617 (3.6) 1579 (1.2) 1494 (−4.3) 1338 (−14.3) 1439 (−7.8) 1654 (6.0) 1549 (−0.8) 1357 (−13.1) 1357 (−13.1) Melanoma of skin (C43) 773 616 (−20.3) 672 (−13.1) 592 (−23.4) 669 (−13.5) 726 (−6.1) 815 (5.4) 503 (−34.9) 646 (−16.4) 741 (−4.1) 578 (−25.2) Prostate (C61) 4533 5414 (19.4) 5487 (21.1) 4302 (−5.1) 4165 (−8.1) 3897 (−14.0) 8741 (92.8) 4397 (−3.0) 4286 (−5.5) 3805 (−16.1) 3815 (−15.8) Testis (C62) 288 299 (3.8) 303 (5.2) 272 (−5.6) 229 (−20.5)^a^ 230 (−20.1) 172 (−40.3) 282 (−2.1) 256 (−11.1) 232 (−19.4) 185 (−35.8) Kidney (C64--66) 484 463 (−4.3) 452 (−6.6) 407 (−15.9) 404 (−16.5) 296 (−38.8) 418 (−13.6) 405 (−16.3) 406 (−16.1) 428 (−11.6) 400 (−17.4) Bladder (C67) 938 1045 (11.4) 1050 (11.9) 1000 (6.6) 1071 (14.2) 843 (−10.1) 1078 (14.9) 1060 (13.0) 956 (1.9) 809 (−13.8) 924 (−1.5) Brain (C70--72) 518 628 (21.2) 625 (20.7) 530 (2.3) 562 (8.5) 368 (−29.0) 563 (8.7) 527 (1.7) 515 (−0.6) 452 (−12.7) 412 (−20.5) Thyroid (C73) 77 80 (3.9) 87 (13.0) 67 (−13.0) 38 (−50.7)^a^ 38 (−50.7) 70 (−9.1) 61 (−20.8) 71 (−7.8) 65 (−15.6) 87 (13.0) Hodgkin lymphoma (C81) 73 94 (28.8) 73 (0.0) 76 (4.1) 54 (−26.0)^a^ 47 (−35.6) 45 (−38.4) 63 (−13.7) 80 (9.6) 62 (−15.1) 63 (−13.7) Non-Hodgkin lymphoma (C82--85, C96) 499 483 (−3.2) 485 (−2.8) 451 (−9.6) 426 (−14.6) 391 (−21.6) 444 (−11.0) 420 (−15.8) 473 (−5.2) 441 (−11.6) 455 (−8.8) Multiple myeloma (C88, C90) 218 210 (−3.7) 211 (−3.2) 202 (−7.3) 186 (−14.7) 169 (−22.5) 216 (−0.9) 187 (−14.2) 209 (−4.1) 194 (−11.0) 162 (−25.7) Leukaemia (C91--95) 363 339 (−6.6) 344 (−5.2) 336 (−7.4) 310 (−14.6) 292 (−19.6) 403 (11.0) 290 (−20.1) 361 (−0.5) 368 (1.4) 313 (−13.8) Other and unspecified 827 858 (3.8) 867 (4.8) 829 (0.2) 662 (−20.0) 569 (−31.2) 759 (−8.2) 826 (−0.1) 845 (2.2) 752 (−9.1) 839 (1.5) ICD-10: International statistical classification of diseases and related health problems, 10th revision; M:I mortality:incidence. ^a^ Cases/deaths from neighbouring countries were added to compute M:I ratios. ###### Cancer incidence in females (Norwegian Cancer Registry, 2010) and estimates using GLOBOCAN methods Site (ICD-10 code) No. of observed cases No. of estimated cases (% difference from observed cases) ------------------------------------- ----------------------- ----------------------------------------------------------- ------------- -------------- --------------- -------------- ------------- ------------- ------------- -------------- -------------- Head and neck (C00--14) 188 174 (−7.5) 187 (−0.5) 163 (−3.3) 165 (−12.2) 136 (−27.7) 157 (−16.5) 155 (−17.6) 174 (−7.5) 192 (2.1) 185 (−1.6) Oesophagus (C15) 58 62 (6.9) 56 (−3.5) 56 (−3.5) 56 (−3.5) 56 (−3.5) 61 (5.2) 53 (−8.6) 64 (10.3) 54 (−6.9) 85 (46.6) Stomach (C16) 196 201 (2.6) 218 (11.2) 230 (17.4) 210 (7.1) 188 (−4.1) 209 (6.6) 280 (42.9) 200 (2.0) 151 (−23.0) 197 (0.5) Colon-rectum (C18--21) 1894 1939 (2.4) 1947 (2.8) 1856 (−2.0) 1732 (−8.6) 1647 (−13.0) 2217 (17.1) 1961 (3.5) 1781 (−6.0) 1671 (−11.8) 1309 (−30.9) Liver (C22) 73 49 (−32.9) 59 (−19.2) 49 (−32.9) 74 (1.4) 65 (−11.0) 86 (17.8) 54 (−26.0) 48 (−34.3) 63 (−13.7) 94 (28.8) Gallbladder (C23--24) 84 83 (−1.2) 85 (1.2) 80 (−4.8) 44 (−47.6) 23 (−72.6) 34 (−59.5) 72 (−14.3) 79 (−6.0) 75 (−10.7) 96 (14.3) Pancreas (C25) 358 363 (1.4) 369 (3.1) 355 (−0.8) 361 (0.8) 286 (−20.1) 367 (2.5) 342 (−4.5) 348 (−2.8) 303 (−15.4) 331 (−7.5) Larynx (C32) 18 10 (−44.4) 14 (−22.2) 15 (−16.7) 17 (−5.6)^a^ 18 (0.0) 19 (5.6) 14 (−22.2) 18 (0.0) 19 (5.6) 21 (16.7) Lung (C33--34) 1210 1197 (−1.1) 1328 (9.8) 1042 (−13.9) 1172 (−3.1) 1026 (−15.2) 1118 (−7.6) 965 (−20.3) 1095 (−9.5) 1173 (−3.1) 1171 (−3.2) Melanoma of skin (C43) 797 698 (−12.4) 707 (−11.3) 630 (−21.0) 936 (17.4) 1068 (34.0) 1591 (99.6) 557 (−30.1) 675 (−15.3) 793 (−0.5) 646 (−19.0) Breast (C50) 2891 3059 (5.8) 2896 (0.2) 2958 (2.3) 2672 (−7.6) 2550 (−11.8) 4540 (57.0) 2818 (−2.5) 3062 (5.9) 2772 (−4.1) 3651 (26.3) Cervix (C53) 307 285 (−7.2) 296 (−3.6) 310 (1.0) 282 (−8.1) 234 (−23.8) 338 (10.1) 330 (7.5) 312 (1.6) 286 (−6.8) 250 (−18.6) Corpus uteri (C54) 744 801 (7.7) 763 (2.6) 715 (−3.9) 639 (−14.1) 632 (−15.1) 691 (−7.1) 721 (−3.1) 740 (−0.5) 721 (−3.1) 599 (−19.5) Ovary (C56) 479 432 (−9.8) 462 (−3.6) 489 (2.1) 451 (−5.8) 445 (−7.1) 559 (16.7) 477 (−0.4) 509 (6.3) 457 (−4.6) 392 (−18.2) Kidney (C64--66) 246 269 (9.3) 272 (10.6) 237 (−3.7) 215 (−12.6) 140 (−43.1) 263 (6.9) 232 (−5.7) 229 (−6.9) 216 (−12.2) 243 (−1.2) Bladder (C67) 359 378 (5.3) 326 (−9.2) 349 (−2.8) 351 (−2.2) 321 (−10.6) 438 (22.0) 353 (−1.7) 347 (−3.3) 329 (−8.4) 306 (−14.8) Brain (C70--72) 629 903 (43.6) 858 (36.4) 688 (9.4) 663 (5.4) 407 (−35.3) 788 (25.3) 672 (6.8) 672 (6.8) 542 (−13.8) 459 (−27.0) Thyroid (C73) 206 184 (−10.7) 176 (−14.6) 163 (−20.9) 189 (−8.3)^a^ 181 (−12.1) 236 (14.6) 177 (−14.1) 152 (−26.2) 206 (0.0) 242 (17.5) Hodgkin lymphoma (C81) 57 56 (−1.8) 54 (−5.3) 51 (−10.5) 25 (−56.1)^a^ 23 (−59.7) 39 (−31.6) 56 (−1.8) 50 (−12.3) 54 (−5.3) 54 (−5.3) Non-Hodgkin lymphoma (C82--85, C96) 412 398 (−3.4) 390 (−5.3) 374 (−9.2) 292 (−29.1) 274 (−33.5) 436 (5.8) 371 (−10.0) 369 (−10.4) 381 (−7.5) 385 (−6.6) Multiple myeloma (C88, C90) 159 164 (3.1) 164 (3.1) 162 (1.9) 126 (−20.8) 130 (−18.2) 207 (30.2) 151 (−5.0) 171 (7.6) 151 (−5.0) 129 (−18.9) Leukaemia (C91--95) 272 260 (−4.4) 253 (−7.0) 248 (−8.8) 216 (−20.6) 197 (−27.6) 307 (12.9) 223 (−18.0) 265 (−2.6) 276 (1.5) 218 (−19.9) Other and unspecified 829 910 (9.8) 857 (3.4) 855 (3.1) 762 (−8.1) 726 (−12.4) 921 (11.1) 807 (−2.7) 886 (6.9) 767 (−7.5) 876 (5.7) ICD-10: International statistical classification of diseases and related health problems, 10th revision; M:I mortality:incidence. ^a^ Cases/deaths from neighbouring countries were added to compute M:I ratios. Among the methods commonly used in low- and middle-income countries (methods 5 to 9), the method using mortality combined with 5-year relative survival proportion (method 5) produced quite large overestimates for cancers associated with good survival including melanoma of skin in women (+99.6%; +794 cases), prostate (+92.8%; +4208 cases) and breast (+57.0%; +1649 cases) and underestimates for cancers with small numbers of deaths, including testicular (−40.3%; −116 cases) or gallbladder (−61.6%; −45 cases in men, −59.5%; −50 cases in women) cancers. Estimates for lung and pancreatic cancers were similar to, or more accurate than, those obtained from method 3 and 4 ([Table 2](#T2){ref-type="table"} and [Table 3](#T3){ref-type="table"}). The performance of methods using data from one or more regional registries (methods 6 and 7) varied greatly by cancer site. Estimates for prostate, colorectal, lung and breast cancers were reasonable (less than 8% difference between estimates and observed data); method 6, however, underestimated female lung cancer estimates in our study (−20.3%; −245 cases). Despite the use of observed data (instead of GLOBOCAN estimates), results from methods 8 and 9 were also almost exclusively underestimates and their accuracy varied greatly by cancer site and sex ([Table 2](#T2){ref-type="table"} and [Table 3](#T3){ref-type="table"}). Discussion ========== Our results, validated against the high-quality data available from the Norwegian Cancer Registry, confirm that projections of historical national data are among the best methods to predict recent cancer incidence. They also suggest that, in selected populations, a site-specific approach is warranted for cancers where the level of incidence is driven by changes in diagnosis patterns (e.g. thyroid) or screening (e.g. breast, prostate). They also illustrate how the accuracy of national estimates based on geographic proxies -- including data from regional registries or neighbouring countries -- is highly dependent on the extent to which these datasets are representative of the scale and profile of the country of interest. In Norway, where long-term national cancer incidence data series are available, the projection of historical rates[@R9] (method 1A) resulted in a relatively good estimation of recent incidence statistics. Projections-based methods captured medium- to long-term trends reasonably but did not perform as well when there were recent changes in the trends. For example, prostate cancer rates increased by 4.3% annually in Norway between 1985 and 2008[@R13] but plateaued in recent years.[@R14] Thus method 2, which simply applies the most recent cancer incidence rates available to recent population data, performed better than a projection of historical rates in this context ([Fig. 5](#F5){ref-type="fig"}). On the other hand, lung cancer rates have uniformly increased in Norwegian women[@R15] in recent years, explaining the good quality of estimates based on trends for this cancer ([Fig. 6](#F6){ref-type="fig"}). Applied to the Norwegian data, methods 3 and 4 were less accurate than the first two methods and underestimated the overall number of cases. They were notably less reliable for cancer sites with small numbers of deaths such as thyroid (males) or testicular cancers. Although the incidence of testicular cancer has uniformly increased in Norway over recent decades, mortality from this cancer has declined since the late-1970s, leading to low numbers of annual deaths (13 deaths nationally in 2010).[@R14] In this context, methods 3 and 4 failed to accurately estimate incident cases in age groups where deaths are rare and tend to underestimate the overall cancer burden. Furthermore, these methods also depend on the representativeness of the proxy datasets used to compute the M:I ratios on which they rely. In GLOBOCAN 2012, method 5 was mainly used in the Caribbean, Latin America and some Asian countries. Applied to Norway, it performed equivalently or better than methods 3 and 4 in cancers with a poor prognosis such as lung or pancreatic cancer, for which the 5-year relative survival proportion in Norway is 15% and 6%, respectively, for male diagnoses 2009--2012.[@R14] However, the method was inadequate for cancers with good prognosis such as melanoma, breast or prostate cancers, where the 5-year relative survival rate was above 80%.[@R14] For the latter two cancers, cure is not apparent at 5 years and survival proportions continue to decline in further years of follow-up,[@R16] thus invalidating the equation used to calculate incidence ([Equation 1](#E1){ref-type="disp-formula"}). It is likely that method 5 combined with longer-term relative survival estimates would produce better incidence estimates for cancers with a good prognosis. In Norway, 10-year relative survival proportions for prostate and breast cancers are available and reduced to 58% and 71%, respectively.[@R17]^,^[@R18] However, such data are less frequently available than 5-year relative survival proportions, particularly in countries were method 5 would be applied. In many low- and middle-income countries, where curative treatments may not be available and hence the M:I ratio is higher, the 5-year survival proportion may be a better proxy of case-fatality. For example, 5-year relative survival proportions for breast cancer in Costa Rica was 68% for diagnoses 1995--2000[@R19] while the M:I ratio was 31.8% based on data from 1998--2002,[@R20] indicating that method 5 would produce reliable estimates in this setting. Because of the paucity of cancer data, national incidence in low- and middle-income countries is often estimated using datasets from regional registries or neighbouring countries. Most of the GLOBOCAN 2012 estimates for Africa and south-east Asia were based on such data (methods 6 to 9). Applying these methods to the Norwegian data illustrated the problem of a lack of representativeness of proxy datasets used to derive national cancer incidence. For example, method 7, where data from the country's capital city were used, provided relatively good overall estimates for Norway. In many low-income countries, the differences are likely to be considerably greater where there are marked differences in the profile of cancer in rural and urban settings. As an example, the breast cancer rate in Mumbai, India (a major urban area) was 31.0 per 100 000 person-years in 2008--2009, more than 2.5 times the rate observed in Barshi (12.3 per 100 000), a rural area, in 2009--2010.[@R21] Producing accurate national cancer incidence estimates is a difficult task that depends on multiple factors: the availability of high-quality cancer registry data, the use of valid and reproducible estimation methods and the representativeness of proxy datasets used for calculations. Because this study was performed using high-quality cancer registry data from a high-income country, the impact of data quality issues and regional variations of the cancer burden on our results are likely to be minimal. The findings should mainly reflect the intrinsic characteristics of the different methods of estimation. On the other hand, it also means that our site- and method-specific results cannot be generalized to other countries and may not be valid in different settings. However, our study provides general conclusions regarding the context in which the different methods are likely to produce reliable estimates, provided that the required data are available. The study provides a comparative assessment of the different methods of estimation of national incidence used in GLOBOCAN as well as some general guidance on the caveats associated with certain methods of estimation for specific cancer types. In particular, they indicate that in countries such as Norway with longstanding high quality population-based cancer registries, regional-based or trends-based estimates perform reasonably well in comparison with recorded incidence. However, such an evaluation of the validity of the estimates themselves is only possible in a few countries with high-quality national data. Elsewhere, data quality issues or a lack of national representativeness of regional datasets could potentially undermine the validity of the estimates and the evidence-based evaluation process. Assessment of uncertainty would also require additional adjustment for the completeness, accuracy and representativeness of the source information. Along with the continuous assessment and improvements of estimation methods, efforts should be targeted at supporting the development of cancer registration worldwide. The Global Initiative for Cancer Registry Development[@R22] is a global partnership launched in 2011 with a goal to increase the coverage and quality of registries in low- and middle-income countries. The partnership plays a critical role in capacity-building, to attain more robust data for national and global cancer estimation purposes and aid countries in the prioritization and evaluation of national cancer control plans. We thank the staff of population-based cancer registries worldwide, particularly those of Denmark, Finland, Iceland, Norway and Sweden. We also thank the NORDCAN Secretariat and the members of the Section of Cancer Surveillance at IARC. None declared.

1. Introduction {#sec1} =============== Convulsive interventions have been used to treat mental disorders since the 16th century up to the moment in the form of electroconvulsive therapy (ECT) \[[@B1]\]. The most common therapeutic indication of ECT is major depression disorder (MDD) and its effectiveness in reducing depressive symptoms has been confirmed in several studies \[[@B2]\]. However, the cognitive complications of ECT have been reported as the main limitation for its use. These side effects occur more in patients with depression. Cognitive side effects and memory deficits are considered as a major limitation to the use of ECT, with 12.4% prevalence for permanent anterograde amnesia in a community setting \[[@B3], [@B4]\]. Despite this, almost all patients return to their previous cognitive status within six months. However, some patients seriously complain of permanent drawbacks in their memory \[[@B3]\]. Disorientation, destruction of processing speed, anterograde and retrograde amnesia, impaired visual and spatial function, and word finding difficulty usually occur immediately after an ECT session. Except for anterograde memory impairment, other cognitive effects of ECT return to the baseline. Anterograde memory improvement occurs gradually, but point defects may still remain. Fraser et al. \[[@B5]\] have also shown that memory may be affected by ECT for a short-term (less than six months). Falconer et al. \[[@B6]\] showed that memory problems can be resolved after a one-month intervention. This study used Cambridge Neuropsychological Test Automated Battery (CANTAB) for cognitive assessments and showed that impairment in spatial recognition may be observed two weeks after ECT. Another study reported short-term and long-term deficits in autobiographical memory occurring shortly and two months after ECT, respectively, in patients with MDD. These patients had a poor performance in stating the exact details of stories and most had difficulty in remembering stories of others compared with their personal biography. Retrograde amnesia was reduced after 2 months of follow-up, but impairment in remembering the recent public events in detail continued still \[[@B7]\]. Although various medical interventions are suggested to reduce the cognitive deficits after ECT, no specific medicine has been found for improving them. Numerous studies investigated the preventive effects of galantamine \[[@B8]\], physostigmine \[[@B9]\], naloxone \[[@B10]\], dexamethasone \[[@B11]\], and piracetam \[[@B12]\] on ECT-associated memory deficit. Some have shown the positive effect of thyroid hormones on cognitive side effects of ECT \[[@B13]\]. However, there is a high tendency to use N-methyl-D-aspartate antagonists such as anesthetic ketamine and thyroid hormone. There also is some information about the use of physostigmine, thyroid hormone, and naloxone, indicating that they can reduce the psychological effects of ECT, but their effects are not well studied and identified yet. Liothyronine is used to increase the response to ECT and if administered at the beginning of an ECT course, response time and the incidence of cognitive problems are reduced \[[@B4]\]. In a study evaluating effects of liothyronine, piracetam, and placebo, biographical memory and mental control did not decrease in the liothyronine group, but orientation declined gradually until the last session of ECT and then significantly increased in a one-month follow-up \[[@B12]\]. Despite the effectiveness of ECT in treatment of MDD patients have concerns about the cognitive effects of ECT and memory deficits in particular. The main incentives of this research are the increasing use of ECT in treatment of MDD, scientific need for more precise information about it, and suggestion of previous researchers for further complementary studies. Accordingly, the present study investigates the effectiveness of liothyronine on ECT-induced memory deficit in patients with MDD. 2. Methodology {#sec2} ============== The study protocol was approved by the Institutional Review Board of Tabriz University of Medical Sciences in accordance with the principles of the Declaration of Helsinki. This trial is registered with the Iranian Clinical Trials Registry (IRCT: [IRCT201401122660N2](http://www.irct.ir/searchresult.php?keyword=&id=2660&number=2&prt=6002&total=10&m=1)). This is a double-blind clinical trial, in which the evaluator and the patients were unaware of the medication received by the patients (liothyronine or placebo). Sixty inpatients and outpatients with MDD who were referred to the ECT Ward, Razi Psychiatric Hospital, Tabriz, Northwest of Iran, were enrolled in this study. The diagnosis was based on the criteria of DSM-IV-TR, using the structured clinical interview for DSM-IV (SCID-IV). The inclusion criteria were the diagnosis of MDD, giving written informed consent, age between 20 to 50 years, Being candidate for receiving ECT based on diagnosis of a psychiatrist, and no history of ECT during the last 6 months. The exclusion criteria were any contraindications of liothyronine, drug and alcohol abuse, organic brain disorders, mental retardation (based on the patients\' history, physical examination, and medical records). Patients who experienced ECT-induced delirium or completed less than 6 sessions for any reasons were also excluded. Selected patients were randomly assigned to experimental and control groups and assessed by the Wechsler Memory Scale-Revised (WMS-R) test as described later. The two groups were matched in terms of depression severity. Then the experimental and control groups received liothyronine and placebo, respectively. Two months after the end of ECT sessions, WMS-R posttest was performed for both groups. For the intervention group, liothyronine (two tablets of 25 mcg) was administered orally from the day before beginning of ECT every morning until the last session. Lactose tablets which were similar to liothyronine in appearance were used as placebo in this study with the same directions as liothyronine for the intervention group. Patients were asked to stop any other medications during the study. ECT was administered by Thymatron DGX device (Somatics, ILC, lake Bluff, USA) through bilateral technique following a dose titration method. A bilateral dosage at 50--100% above seizure threshold of the patient (the first stimulus was set at 125 millicoulombs; the mean threshold was 100) was ensured to induce an acceptable convulsion duration between 15 seconds to 3 minutes. Thus in all sessions, ECT would have deemed ineffective if the convulsion had lasted less than 15 seconds and hence was not accounted as an effective ECT. In this state, the electric shock was repeated only once at the same session by 5--10% increase at repeated one. If convulsion had lasted longer than 3 minutes, patients would have been introduced to a neurologist for examination; however, this did not occur in this study. 6--12 ECT sessions were held for recruited patients, three times per week. 3. Measurements {#sec3} =============== 3.1. Wechsler Memory Scale-Revised {#sec3.1} ---------------------------------- Wechsler Memory Scale (WMS) was designed in 1970 by Wexler. The Revised WMS (WMS-R) consists of 5 subscales (general memory, attention/concentration, verbal memory, visual memory, and delayed recall) and evaluates different aspects of memory. Psychometric properties of Farsi version of WMS-R have been evaluated in Iran, in people aged 16 to 64 years. In this study, test-retest reliability coefficients were reported as 0.28 to 0.98 for the subscales and compositions. The average raw scores of the two groups of clinical and normal subjects were compared to evaluate the validity of WMS-R, indicating that the mean raw scores of the clinical group were significantly lower than that of the normal subjects \[[@B14]\]. 3.2. Hamilton Rating Scale for Depression {#sec3.2} ----------------------------------------- The Hamilton Depression Rating Scale (HRSD) is a 21-item multiple-choice scale which assesses various dimensions of depression: behavioral, physical, emotional, guilt, hypochondriasis, sex issues, job, suicide, and sleep disorders. This scale was used in this study to assess the severity of depression and to match the groups. The reliability and validity of the scale have been confirmed in several studies \[[@B15]\]. 3.3. Structured Clinical Interview for DSM-IV (SCID-IV) {#sec3.3} ------------------------------------------------------- This is a structured clinical interview based on DSM-IV criteria, for diagnosing MDD and other psychiatric disorders associated with Axis I and Axis II. SCID-IV has been used more than any other forms of psychiatric diagnostic interviews in psychological studies and has a global credibility. The reliability and validity of SCID-IV have been assessed in Iran, showing an acceptable reliability and validity \[[@B16]\]. 3.4. Statistical Methods {#sec3.4} ------------------------ All statistical analyses were performed using SPSS version 21. The data are reported by means of descriptive statistics (i.e., mean, SD, frequency, and percentage). Independent *t*-test was used to compare the significance of difference between the control and test groups. In addition, paired *t*-test was employed to determine differences in pretest and posttest mean scores from WMS-R in each group. Analysis of covariance (MANCOVA) was used to compare the difference between the posttest scores of both groups in WMS-R, with controlling the pretest. *P* values less than 0.05 were considered significant. 4. Findings {#sec4} =========== Sixty patients were enrolled in the liothyronine (*n* = 30) and placebo groups (*n* = 30). The minimum and maximum ages of the patients were 20 and 50 years. Most patients were female (76.7% of target group and 70% of controls), with an education level of diploma or higher (academic), married, housewife, and from urban area. All patients were diagnosed with major depressive disorder, without comorbidities or history of ECT in the recent six months. The majority of patients underwent 7 sessions of ECT. Demographic characteristics of these patients are presented in [Table 1](#tab1){ref-type="table"}. [Table 2](#tab2){ref-type="table"} depicts the scores of WMS-R in the liothyronine and placebo groups. A paired *t*-test was applied to scores of patients receiving placebo and no significant change was observed in verbal memory and general memory, while visual memory, attention/concentration, and delayed recall scores were significantly reduced after ECT (*P* \< 0.05). In patients receiving liothyronine, visual memory and attention/concentration had no significant change after ECT, but verbal memory and general memory scores were significantly increased in posttest measuring (*P* \< 0.01). The univariate analysis of covariance (with pretest control) was used to evaluate the effect of each group on all research\'s variables. Before using multivariate analysis of covariance (MANCOVA), normal distribution of the dependent variables and the associated variables, variance homogeneity and covariance homogeneity of the groups, and the weakness of correlation between the variables were ensured. [Table 3](#tab3){ref-type="table"} shows that difference in the mean scores of verbal memory between the two groups in posttest is significant (*F*(1,25) = 13.95, MS = 1149.05, *P* = 0.001, and Eta^2^ = 0.21). Difference in the mean scores of visual memory between the two groups in posttest is significant in both groups (*F*(1,25) = 5.03, MS = 785.70, *P* = 0.02, and Eta^2^ = 0.09). Difference in the mean scores of general memory between the two groups in posttest is significant (*F*(1,25) = 13.45, MS = 1744.04, *P* = 0.001, and Eta^2^ = 0.20). Difference in the mean scores of attention/concentration between the two groups in posttest is significant (*F*(1,25) = 5.87, MS = 795.05, *P* = 0.05, and Eta^2^ = 0.10). Difference in the mean scores of delayed recall between the two groups in posttest is significant (*F*(1,25) = 19.27, MS = 1662.12, *P* = 0.001, and Eta^2^ = 0.27). Therefore, it can be concluded that liothyronine effectively increases memory functioning (verbal memory, visual memory, general memory, attention/concentration, and delayed recall). Based on Eta square index, 9%--27% of improvement in verbal memory, visual memory, general memory, attention/concentration, and delayed recall was due to liothyronine. 5. Discussion {#sec5} ============= The results of this study showed that liothyronine can prevent memory deficit after ECT in patients with MDD. Despite clinical indication, patients may disagree to start or discontinue the ongoing treatment course with ECT \[[@B17]\] because of its well-known cognitive side effects \[[@B18]\]. Several efforts have been made to find a practical and effective preventive treatment for memory deficits after ECT. Add-on strategies seem to be the most popular \[[@B19]\]. Promising outcome has been reported by previous studies using thyroid hormone as an add-on strategy to ECT. These studies, not many, have not influenced guidelines yet, because of diversity in methodology and their limitations. The current study matches up with previous reports in terms of effect of liothyronine. Stern et al. \[[@B20]\] randomized 20 patients with MDD and reported that patients receiving liothyronine (*n* = 11) performed better in measure of remote personal memory, but not learning or recall. Hamidia et al. \[[@B21]\] also reported that liothyronine could prevent memory deficits after ECT in patients with MDD. Masoudzadeh et al. \[[@B22]\] stated that patients who received liothyronine achieved better scores on the depression and memory scales, beginning after 6 sessions of ECT. Both of latter studies also had a small sample size (40 and 30 in that order) but showed improvement on both the Hamilton Rating Scale for Depression and the Revised Wechsler Memory Scale. Stern et al. \[[@B23]\] repeated the same trial with 30 patients later and liothyronine could improve verbal learning performance and remote memory. The present study replicated these results with a larger sample size. Most of the previous studies did not examine memory subscales. Our findings show that the mean scores of the experimental group in terms of verbal memory and general memory had an improvement attributable to the use of liothyronine, though this improvement was small. Different mechanisms have been proposed to explain the discussed effect of liothyronine. A neuroprotective effect has been confirmed for triiodothyronine (T3) when electroconvulsive shock is administered to rats \[[@B24]\]. Additionally, anticonvulsive effect of thyrotropin releasing hormone will diminish when it is suppressed by exogene liothyronine. This may lower the required dose of electroshock and consequently decrease the damage and adverse cognitive effects \[[@B22]\]. Additionally, Siegrist and Kaiser \[[@B25]\] believe that the number of ECT sessions has no impact on memory and that decreased thyroxine (T4) may be the reason for the reduced rate of neural actin polymerization. Thus, when the level of T3 declines in patients receiving liothyronine, this leads to a temporary disruption in actin cytoskeleton and could protect cells from destructive effects of convulsion in amygdala and hippocampus neurons. These two constructs are very sensitive to convulsion and play an important role in memory and learning. This study had some limitations. In this study, the interactive effects of individual variables and variables such as the severity of depression and thyroid hormone alterations were not studied. We also suggest to conduct a study on patients with MDD who receive liothyronine for preventing cognitive effects of ECT but still suffer from symptoms of memory deficit after ECT to find predictive characteristics of effectiveness of such treatment. Selecting patients with MDD provided a homogenous sample; however, other studies with different samples are required to ensure that the beneficial effect is not only limited to depressed patients. We were also unable to take the seizure threshold into account or the stimulus charges of the effective electric shock. 6. Conclusion {#sec6} ============= The results showed that liothyronine improves the performance of verbal memory, visual memory, attention/concentration, and delayed recall in patients with MDD who receive ECT. This study, adds to the evidence that liothyronine may have a positive impact in preventing memory impairment caused by ECT in depressed patients. This study was supported by Research Center of Psychiatry and Behavioral Sciences, Tabriz University of Medical Sciences. Conflict of Interests ===================== The authors declare that they have no conflict of interests. ###### Demographic and individual characteristics of the patients undergoing ECT; each group included thirty patients.   Liothyronine Placebo --------------------------- ------------------ ------------------ Age 34.90 ± 8.06^\*^ 33.60 ± 7.99^\*^ Hamilton depression score 62.56 ± 5.82^\*^ 62.63 ± 6.96^\*^ Gender      Male 7 (23.3) 9 (30)  Female 23 (76.7) 21 (70) Education level      Primary school 7 (23.3) 8 (26.7)  Middle school 7 (23.3) 8 (26.7)  Graduate/postgraduate 16 (53.4) 14 (46.6) Marital status      Single 9 (30) 7 (23.3)  Married 20 (66.7) 17 (56.7)  Divorced/widowed 1 (3.3) 6 (20) Occupation      Employee 9 (30) 12 (40)  Housewife 21 (70) 18 (60) Location      City 24 (80) 24 (80)  Village 6 (20) 6 (20) Number of ECT sessions      Six 6 (20) 4 (13.3)  Seven 14 (46.7) 19 (63.9)  Eight 1 (3.3) 0  Ten 4 (13.3) 5 (16.7)  Twelve 5 (16.7) 2 (6.7) ^\*^As mean ± standard deviation. ###### Mean and standard deviation of the WMS-R and the results of paired *t*-test in both groups. Variables Liothyronine Placebo ------------------------- -------------- --------- ------- ------- ------- ------- Verbal memory 67.53 75.63 0.001 65.43 63.83 0.15 Visual memory 80.70 81.66 0.74 73.10 67.36 0.005 General memory 68.46 74.20 0.005 61.73 57.76 0.21 Attention/concentration 92.06 94.90 0.33 86.73 82.56 0.01 Delayed recall 72.03 76.06 0.066 64.43 60.40 0.02 ###### Results of univariate analysis of covariance of liothyronine effect on memory posttest with pretest control. Variables Sum of squares df Mean square *F* *P* value Eta^2^ Power ------------------------- ---------------- ---- ------------- ------- ----------- -------- ------- Verbal memory 1149.05 1 1149.05 13.95 \<0.001 0.21 0.95 Visual memory 785.70 1 785.70 5.03 0.025 0.09 0.61 General memory 1744.04 1 1744.04 13.45 0.001 0.20 0.94 Attention concentration 795.05 1 795.05 5.87 0.019 0.10 0.66 Delayed recall 1662.12 1 1662.12 19.27 \<0.001 0.27 0.99 [^1]: Academic Editor: Cristiano Capurso

Background {#Sec1} ========== Facioscapulohumeral muscular dystrophy 1 (FSHD1) has an autosomal dominant pattern of inheritance \[[@CR1]\] and manifests as a consequence of both genetic \[[@CR2]--[@CR4]\] and epigenetic disease mechanisms \[[@CR5]\]. FSHD is most commonly present in the second decade of life as asymmetric weakness of specific skeletal or facial muscle groups \[[@CR6]\]. Regardless of the genetic mechanism, FSHD results from abnormal expression of double homeobox protein 4 (DUX4) in skeletal muscle \[[@CR7]\]. The DUX4 gene is encoded in each of the 3.3 kb D4Z4 repeat units arrayed at chromosome 4q35. DUX4 expression is important early in development when it activates ZSCAN4 as part of a chromatin remodeling phase that occurs in 4 cell embryos \[[@CR8]\] and in most adult tissues DUX4 transcription is strongly repressed. A recent proteomics-based study identified proteins that specifically bind to the D4Z4 array and showed that nucleosome remodeling deacetylase (NuRD) and CAF-1 complexes repress DUX4 transcription in control myoblasts and induced pluripotent stem cells \[[@CR9]\]. The D4Z4 array length appears to be critical for DUX4 repression because FSHD-causing array contractions result in toxic DUX4 expression when they occur on a common 4q haplotype that includes a polyadenylation signal at the end of the last D4Z4 unit \[[@CR7], [@CR10], [@CR11]\]. Array length is not the only mediator of transcriptional repression because even in the context of an array contraction, DUX4 remains under significant although incomplete repression with cultured myoblasts showing stochastic bursts of transcription in a small fraction of myonuclei \[[@CR12]--[@CR14]\]. Differences in histone modifications associated with FSHD-causing contracted and normal length D4Z4 arrays have been difficult to identify because of the presence of multiple D4Z4 units near the telomere of chromosome 10, the normal D4Z4 array on the other allele of chromosome 4 and D4Z4-like sequences at multiple other genomic locations \[[@CR15]\]. As minor sequence differences of these arrays have been catalogued, primers with some specificity for D4Z4 units originating from chromosomes 4 and 10 have been developed \[[@CR16]\] and despite averaging PCR signals originating from each repeat on chromosomes 4 and 10 and from D4Z4 units within both pathogenic and non-pathogenic arrays, a decrease in the levels of H3K9me3 and 5-meC D4Z4 modifications \[[@CR16]\] and a decrease in cohesion and HP1γ association with D4Z4 have been observed in cells derived from FSHD-affected individuals \[[@CR17]\]. More recently, a bisulfite DNA sequencing approach has allowed methylation levels to be attributed to specific alleles identified by single nucleotide polymorphisms present in the sequenced regions and has convincingly demonstrated that CpG hypomethylation is localized to contracted arrays in cells from FSHD1-affected individuals \[[@CR18]\]. The ability to specifically characterize differences in histone modifications present on DUX4-expressing and non-expressing contracted D4Z4 arrays on chromosome 4 should reveal new and important epigenetic differences associated with FSHD. To determine the epigenetic changes that result in DUX4 transcription, we took advantage of the observation that FSHD-permissive 4qA alleles are polymorphic with two common haplotypes that differ in the length of the terminal D4Z4 unit \[[@CR3]\]. We also constructed and validated a DUX4 reporter that allows the detection and collection of DUX4 expressing cells using GFP fluorescence \[[@CR13]\]. This approach allows pathogenic (contracted) arrays to be specifically analyzed in the presence of other arrays in the same cells and allows comparisons of epigenetic differences present at D4Z4 in DUX4 expressing and non-expressing cells from the same myoblast population. By measuring the frequency of association of chromatin remodeling proteins and the abundance of epigenetic marks present on DNA and histones at D4Z4 arrays, we were able to follow the epigenetic state of normal and pathogenic D4Z4 arrays in stem cells and terminally differentiated human myocytes. Importantly, we were able to distinguish epigenetic differences present at DUX4 expressing and DUX4 non-expressing pathogenic arrays within the same myocyte population. We show that all D4Z4 arrays are bivalent with respect to repressive and activating histone modifications in stem cells and thus poised for DUX4 expression, consistent with the observation of DUX4 expression at the 4 cell embryonic stage where DUX4 appears to be instrumental in establishing the epigenetic profile of the early embryo \[[@CR8]\]. As stem cells commit to terminally differentiated states, long arrays adopt a repressive chromatin confirmation while histones on short arrays are left vulnerable to further modifications including the removal of H3K27me3 and acetylation of H3K9 leading to the activation of DUX4 transcription. Results {#Sec2} ======= Exclusive detection of pathogenic alleles produces an enhanced signal for analysis of FSHD-associated D4Z4 chromatin structure {#Sec3} ------------------------------------------------------------------------------------------------------------------------------ The contraction of the D4Z4 array associated with the allele variant 4qA is required to cause FSHD1 \[[@CR7], [@CR19]\]. There are two sub-haplotypes of 4qA that contain a permissive SSLP of 161 length associated with a poly A signal sequence at the distal end of the array \[[@CR7]\]. The sub-haplotypes differ at the distal most D4Z4 unit where the haplotype A161-L (Fig. [1](#Fig1){ref-type="fig"}a) contains an additional \~2 kb of non-translated D4Z4 sequence. Since this represents a different breakpoint in the most telomeric D4Z4 unit which is always partially present, we call this variant the long last partial (LLP) to distinguish it from the more common last partial D4Z4 unit that is shorter due to a breakpoint earlier in the last D4Z4 unit (SLP). Individuals with FSHD1 can have either a LLP or SLP sub-haplotype \[[@CR3]\] as both are permissive and associated with the poly A signal sequence. We utilized custom PCR primers that specifically amplify the A161-L but not the A161-S on 4qA alleles (Fig. [1](#Fig1){ref-type="fig"}b). The control and FSHD1 myoblasts utilized in this study were PCR amplified with these LLP primers and identified to have the 4q A161-L haplotype (Fig. [1](#Fig1){ref-type="fig"}c). The DUX4-interacting region 1 (DIR1) a region proximal to the LLP and common to all the genotypes studied \[[@CR20]\] was amplified with a separate set of primers as a control. We screened several myoblast cell lines to identify those that have a single permissive A-type array with a LLP sub-haplotype allowing us to specifically probe the chromatin structure of the DUX4 expressing array in cells from FSHD-affected individuals.Fig. 1PCR amplification of permissive alleles in FSHD cells. **a** Diagram of two unique terminal D4Z4 junctions of permissive alleles in FSHD. Both haplotypes contain a permissive SSLP of 161 bp length and a polyadenylation signal (ATTAAA). The junction of the distal end of the D4Z4 array is different resulting in a slightly longer 3′ untranslated region in the A161-L version \[also called long last partial (LLP)\]. Introns are noted by dashed lines between splice sites and exons are noted as black boxes with the thicker portion corresponding to the ORF of the DUX4 protein. PCR primers that uniquely identify the A161-L form are shown as arrows in the distal region of the array (LLP). **b** Table showing genotypes of the cell lines used to characterize the LLP primers. DIR1 primers are homologous to a common region present in all arrays just centromeric to the start. Note that FSHD1 and non-FSHD control myoblast lines both have permissive A161-L arrays with disease causing length of 12 kb in the FSHD line (2 repeats) and disease protective length in the control line (74 repeats). **c** DNA fragments amplified from genomic DNA purified from the cell lines shown in (**b**) and the expected result shown below. The locations of the LLP primers are shown in (**a**) DUX4 expressing and non-expressing FSHD myocytes are similarly hypomethylated on the pathogenic D4Z4 array {#Sec4} ---------------------------------------------------------------------------------------------------------- To determine whether DUX4 transcription is repressed by DNA methylation in FSHD myoblasts that don't express DUX4, we investigated basal CpG methylation levels at the permissive, contracted array using bisulfite conversion and PCR primers that uniquely amplify the LLP allele \[[@CR21]\]. D4Z4 in normal myocytes containing a LLP allele was 80% methylated, while the same region was only 8.8% methylated in myocytes from FSHD-affected individuals supporting previously published data (Fig. [2](#Fig2){ref-type="fig"}b, c) \[[@CR5], [@CR22]\]. Utilizing a fluorescing DUX4 reporter, we purified myocytes that expressed DUX4 from those that efficiently silenced DUX4 and show that myoblasts express 0.26--1.09% DUX4 and when differentiated the myocytes express 1.23--3.75% DUX4 (Additional file [1](#MOESM1){ref-type="media"}: Figure S1). These results suggest that even under optimal culture conditions, only a fraction of cells are expressing DUX4, suggesting that DUX4 repression is robust even in the disease state \[[@CR13]\]. We quantified LLP methylation levels by bisulfite conversion of genomic DNA from DUX4 expressing and DUX4 non-expressing cell populations. CpG methylation levels were similar whether the cells contained arrays that expressed DUX4 (12% methylation) or contained transcriptionally silent arrays (10% methylation) (Fig. [2](#Fig2){ref-type="fig"}d, e). Thus, even though the pathogenic array is hypomethylated in all FSHD myocytes, CpG methylation levels are not significantly different in DUX4-expressing and DUX4 non-expressing myocyte populations suggesting that additional epigenetic signals are responsible for the sporadic DUX4 expression from pathogenic arrays in FSHD.Fig. 2CpG methylation density from DUX4 expressing and DUX4 non-expressing arrays in FSHD and non-FSHD control myocytes. CpG methylation events are shown from sorted populations of bisulfite-treated non-FSHD control or FSHD-affected differentiated myocytes. Primers that uniquely amplify the 4qA-161-L haplotype were used so only methylation events from the 4q A161-L haplotype are shown. **a** Diagram of a full D4Z4 unit and terminal D4Z4 partial unit (LLP) with portions of full length (DUX4) or partial (DU) DUX4 genes shown as black rectangles. The position of LLP PCR primers are shown as converging arrows. **b** CpG methylation pattern in non-FSHD control myocytes containing a single LLP D4Z4 array (Fig. [1](#Fig1){ref-type="fig"}, 2081). **c** CpG methylation pattern of the LLP region in unsorted FSHD-affected differentiated myocytes (Fig. [1](#Fig1){ref-type="fig"}, 2349). **d** CpG methylation pattern of the LLP region in the DUX4 expressing population of cells from FSHD-affected differentiated myocytes (Fig. [1](#Fig1){ref-type="fig"}, 2349). **e** CpG methylation pattern of the LLP region in the DUX4 non-expressing population of cells from FSHD-affected differentiated myocytes (Fig. [1](#Fig1){ref-type="fig"}, 2349). The percentage of CpG methylation are indicated to the right-hand side for each group. The location of methylated cytosines is shown as red squares, and the location of unmethylated cytosines are shown as blue squares. DNA variants which result in a sequence but are no longer a CpG are colored white The D4Z4 array has a bivalent chromatin signature in stem cells that becomes univalent and associated with H3K27me3 labeled histones in FSHD myoblasts {#Sec5} ------------------------------------------------------------------------------------------------------------------------------------------------------ DUX4 has sequence homology to several homeotic transcription factors that determine cell fate early in development and most closely resembles homeotic transcription factors of the Paired class \[[@CR23]\]. A recurring regulatory theme among these developmentally important genes is the observation that early in development the chromatin structure is composed of a mixture of histones containing repressive modifications (H3K27me3) and activating modifications (H3K4me3) \[[@CR24]\]. These bivalent loci are thought to be poised to commit to silencing or expression depending on the cell fate choice. We used antibodies specific for H3K4me3 or H3K27me3 to immunoprecipitate chromatin from isogenic human iPS cells cloned from the normal and contracted D4Z4 populations present in an individual with FSHD who was mosaic for D4Z4 array size and contained a 4qA-L sub-haplotype. Single ChIP pull down with either H3K4me3 or H3K27me3 revealed that the isogenic human iPS mosaic clones had both marks present individually (Additional file [1](#MOESM1){ref-type="media"}: Figure S2). Further analysis showed that like the known bivalent locus POU4F3, the D4Z4 contracted and non-contracted stem cell clones contained both H3K4me3 and H3K27me3 labeled histones at D4Z4 and sequential ChIP experiments \[[@CR24]\] showed the marks were present at the same locus in the same cells confirming that D4Z4 is bivalent in stem cell populations (Fig. [3](#Fig3){ref-type="fig"}a). Re-examination of previously published ChIP-seq data \[[@CR25]\] revealed bivalent histone modifications in several human ES cell lines as well (Fig. [3](#Fig3){ref-type="fig"}b). However, fully committed primary myoblasts from FSHD muscle biopsies contained univalent D4Z4 loci yielding almost undetectable signal after sequential immunoprecipitation similar to the known univalent locus HOXA3 (Fig. [3](#Fig3){ref-type="fig"}a). We also confirmed previous observations \[[@CR17]\] that the D4Z4 array in myoblasts is labeled with H3K27me3 in affected and unaffected individuals using conventional immunoprecipitation (Fig. [3](#Fig3){ref-type="fig"}c). Myoblasts from FSHD-affected individuals did have higher H3K4me3 modification levels when compared to non-FSHD primary myoblasts from controls consistent with the presence of transcriptionally active arrays in some cells of the FSHD-affected population (Fig. [3](#Fig3){ref-type="fig"}c).Fig. 3The D4Z4 locus is bivalent in human stem cells and turns univalent in myoblast. **a** Sequential chromatin immunoprecipitation beginning with antibodies to H3K27me3 and followed by antibodies to H3K4me3 was utilized to investigate bivalency at the DUX4 locus. The reverse sequential ChIP with antibodies recognizing H3K4me3 first and antibodies recognizing H3K27me3s was also performed with similar results (data not shown). Isogenic human iPS cell clones isolated from individuals with mosaic distributions of D4Z4 lengths were used for this study. Chromatin purified from iPS clones with non-contracted (mosaic long clone) and contracted (mosaic short clone) iPS cell clones was utilized in ChIP and quantitative PCR amplification. The stem cell bivalent locus (POU4F3) \[[@CR25]\] and the stem cell univalent locus (HOXA3) \[[@CR24], [@CR25]\] were utilized as controls for comparison to the LLP region of D4Z4 in the iPS cell clones. Similarly, sequential ChIP was performed on myoblasts from non-FSHD control and FSHD myoblasts again uniquely amplifying the 4qA161-L allele in these cells. The arrow indicates signal was not detected. The pull down for the POU4F3 locus in the mosaic long clone was statistically significant using the Student's *t* test when compared to the HOXA3 locus in the mosaic long clone and the DUX4 locus in the normal primary myoblast (\**p* value ≤ 0.05). Similarly, the pull down for POU4F3 locus in mosaic short clone was significantly different when compared to the HOXA3 locus in the mosaic short clone (\**p* value ≤ 0.05). **b** ChIP-seq data \[[@CR25]\] were reprocessed and aligned to the human genome for DUX4 analysis. Similar levels of H3K27me3 and H3K4me3 at the DUX4 locus supports a bivalent chromatin structure and is consistent with our ChIP results in (**a**). **c** Chromatin immunoprecipitation of D4Z4 DNA with antibodies recognizing H3K4me3, H3K27me3, an EZH2 and selective amplification of the permissive allele in non-FSHD control and FSHD1 myoblasts. Comparison by *t* test of the mean percent input normalized to H3 between non-FSHD control to FSHD for H3K4me3 and H3K27me3 revealed statistical significance (\**p* value ≤ 0.05). The error bars show standard deviations The pathogenic D4Z4 array contains H3K9 acetylated histones in DUX4 expressing cells {#Sec6} ------------------------------------------------------------------------------------ CpG methylation analysis (Fig. [2](#Fig2){ref-type="fig"}) suggested that there are other epigenetic marks that distinguish DUX4 expressing and non-expressing D4Z4 arrays. We sorted DUX4 expressing and non-expressing myocytes from the same individual and screened D4Z4 associated histones for epigenetic modifications that might distinguish DUX4 expressing and non-expressing cells. Analysis of myogenic differentiation markers MYH1, MYH2 and MYOG revealed similar gene expression levels between the DUX4 expressing and non-expressing myocytes (Additional file [1](#MOESM1){ref-type="media"}: Figure S3) demonstrating that DUX4 expressing and non-expressing cells have similar differentiation profiles. Surprisingly levels of H3K4me3 and H3K9me2 modifications were similar between DUX4 expressing and non-expressing cells (Fig. [4](#Fig4){ref-type="fig"}a). However, histones containing H3K27me3 were fourfold more abundant on D4Z4 arrays that were not expressing DUX4 (Fig. [4](#Fig4){ref-type="fig"}a). In addition, histones containing H3K9 acetylation modifications were \~ fourfold more abundant at D4Z4 in DUX4 expressing cells (Fig. [4](#Fig4){ref-type="fig"}a). These findings demonstrate that even short D4Z4 arrays are under repressive epigenetic pressure despite the aberrant D4Z4 array length and suggest that repressive mechanisms and signals are intact in these cells but sporadically fail in a small population of cells.Fig. 4DUX4 expressing cells have increased H3K9 acetylation and decreased H3K27me3. **a** Differentiated myocytes from a FSHD1-affected individual were sorted into DUX4 expressing and non-expressing populations using a fluorescent DUX4-target reporter. Antibodies against inhibitory (H3K9me2, H3K27me3) and activating (H3K4me3, H3K9Ac) histone modifications were used to compare differences in modification levels between DUX4 expressing and non-expressing cell populations from the same culture. Percent input normalized to H3 is shown on the Y axis. **b** The levels of chromatin modifiers, EZH2 (member of the PRC2 complex that methylates H3K27), SUV39H1 (involved in H3K9 methylation) and structural protein CTCF were measured at the permissive contracted D4Z4 array and compared in DUX4 expressing and non-expressing FSHD-affected differentiated myocyte populations. Values shown are the percentage of signal obtained from input chromatin normalized to β-actin. Error bars show standard deviations of 6--12 replicates. Signal was determined by specific PCR amplification of the permissive allele using the LLP primers (see Fig. [1](#Fig1){ref-type="fig"}). The presence or absence of RNA polymerase 2 (Pol2) was used as a positive control. Statistical comparison was performed using *t* test with \**p* value ≤ 0.05 as being significant Chromatin modifications and mediators that suppress DUX4 transcription {#Sec7} ---------------------------------------------------------------------- Lysines at position 27 on the histone 3 tail are methylated by polycomb repressive complex 2 (PRC2), a complex of proteins that contains 4 main subunits (EZH2, SUZ12, EED and RbAp46/48) (for review see \[[@CR26]\]). We found similar levels of EZH2 association with D4Z4 in non-FSHD control and FSHD-affected myoblasts (Fig. [3](#Fig3){ref-type="fig"}c). However, we found an increase in EZH2 association at D4Z4 in DUX4 non-expressing cells again correlating with an increase in H3K27me3 modifications in DUX4 non-expressing cells and suggesting that PRC2 recruitment is a component of the repressive machinery at contracted pathogenic D4Z4 arrays (Fig. [4](#Fig4){ref-type="fig"}b). Antibody to RNA polymerase 2 was used as a positive control to indicate RNA transcription at the DUX4 expressing LLP locus. These results suggest that PRC2 is important for repression of DUX4 expression in the absence of H3K9me3 and CpG methylation and likely does not depend on H3K9me3 or CpG methylation for recruitment. A separate group of proteins including SUV39H1 mediates the methylation of H3K9 which in turn recruits HP1γ and the cohesion complex \[[@CR27]--[@CR29]\]. This complex is primarily active at repetitive DNA such as pericentromeric and telomeric heterochromatin, the latter being the location of FSHD-causing D4Z4 arrays. Although previous studies have shown that association of this complex with D4Z4 is highly reduced in the context of both FSHD1 and FSHD2 \[[@CR16], [@CR17]\], immunoprecipitation of SUV39H1 demonstrated that these proteins are preferentially associated with D4Z4 in DUX4 non-expressing cells, and therefore may also participate in the maintenance of DUX4 suppression in the context of FSHD (Fig. [4](#Fig4){ref-type="fig"}b). CTCF binding has been shown to be regulated by histone methylation and nucleosome occupancy over CTCF binding sites. CTCF can mediate transcriptional silencing or activation by creating accessible or inaccessible loops of chromatin at specific sites \[[@CR30]\]. We found CTCF to be more readily associated with transcriptionally silent arrays (Fig. [4](#Fig4){ref-type="fig"}b) suggesting CTCF also plays a role in repressing DUX4 transcription. Likewise, another structural component of chromatin and obvious candidate for transcriptional regulation of DUX4 is SMCHD1 \[[@CR31]\]. Mutations in SMCHD1 result in D4Z4 CpG hypomethylation and set up cellular conditions that result in sporadic expression of DUX4 without the need for very short D4Z4 array contractions \[[@CR32]\]. While SMCHD1 activity is central for DUX4 silencing, SMCHD1 levels were similar when comparing transcriptionally active and silenced arrays in FSHD1 myoblasts (data not shown). This observation suggests a role distinct from CTCF where SMCHD1, like "array length," is involved in stabilization of silent arrays so that sporadic DUX4 expression does not occur, but once destabilized, SMCHD1 appears to be minimally involved in the decision of whether DUX4 transcription is activated or repressed. Validation of ChIP findings by chemical inhibition of chromatin modifiers {#Sec8} ------------------------------------------------------------------------- We screened several chemicals known to inhibit enzymes involved in histone modification to validate our ChIP results and determine whether any could be used to reduce DUX4 expression. Our finding that histone acetylation promotes DUX4 expression suggests that histone deacetylase (HDAC) inhibitors should augment DUX4 expression and histone acetyltransferase (HAT) inhibitors may suppress DUX4 expression. We selected HDAC inhibitor RG2833 (RGFP109) \[[@CR33]\], and multiple HAT inhibitors (CPTH2 \[[@CR34]\], garcinol \[[@CR35]\], and C646 \[[@CR36]\]) and treated FSHD myocytes containing a DUX4 Luciferase reporter with increasing doses while measuring DUX4 activity and cell viability. As expected, HDAC inhibition with RG2833 resulted in increased DUX4 expression as well as expression of DUX4 target genes CCNA1 and MBD3L2 in a dose-dependent manner (Fig. [5](#Fig5){ref-type="fig"}a, b).  Higher doses (\> 10 µM) inhibited fusion and reduced the natural amplification of DUX4 target expression so apparent reductions of luciferase activity at higher doses are an artifact of this fusion dependent assay. DUX4 expression was reduced by treatment of cells with chemicals that inhibit HAT, however, cell viability and/or differentiation was concomitantly reduced so we were unable to conclude whether HAT inhibition directly affects DUX4 transcription.Fig. 5DUX4 and DUX4-target gene expression in response to chemical inhibition of chromatin modifiers. Dose escalations of the HDAC inhibitor RG2833 (**a**) or the EZH2 inhibitor GSK-126 (**c**). Cultured myoblasts were treated with the vehicle or the indicated drug at the time differentiation is initiated. Luciferase output from a DUX4-reporter is measured 48 h later, and cell numbers are estimated using the fluorescence signal from CellTiterFluor. Shown is the fold change in luciferase output normalized to cell number when the treatment is compared to vehicle alone. Analyses of all samples were performed in quadruplicate. Reductions in luciferase signal seen at higher drug concentrations (RG2833 25--50 µM and GSK-126 20--50 µM were a consequence of decreased myoblast fusion). Gene expression of DUX4 and its secondary targets CCNA1 and MBD3L2 for RG2833 at 10 µM concentration (**b**) and GSK-126 at 10 µM concentration (**d**) are shown. RNA expression was normalized to GAPDH (for DUX4) and RnaseP (for CCNA1 and MBD3L2) as endogenous controls. Analyses of all samples were performed in triplicate. *p* values were calculated using the Student's *t* test to compare vehicle and the treated group with \**p* ≤ 0.05 as being significant The finding that H3K27me3 modifications are a distinguishing epigenetic mark for DUX4 expressing and non-expressing arrays suggests that chemicals that inhibit H3K27 methylation should increase DUX4 expression in an FSHD1 context. PRC2 facilitates methylation of H3K27 and GSK-126 \[[@CR37]\] is a specific chemical inhibitor of the EZH2 component of PRC2. Consistent with these findings, previous studies have shown an increase in DUX4 expression from FSHD2 myoblasts treated with GSK-126 \[[@CR38]\] and here we treated FSHD1 myoblasts with GSK-126 and observed a dose-dependent increase in DUX4 expression and DUX4 targets from differentiated myoblasts further validating our ChIP findings (Fig. [5](#Fig5){ref-type="fig"}c, d). Higher doses (\>10 µM) inhibited fusion and reduced the natural amplification of DUX4 target expression so apparent reductions of luciferase activity at higher doses are an artifact of this fusion dependent assay.  Discussion {#Sec9} ========== Other groups have compared epigenetic marks at D4Z4 in FSHD myoblasts with D4Z4 epigenetic modifications in non-FSHD control myoblasts. These studies revealed a clear reduction in H3K9me3 at D4Z4 in people with FSHD and also show differences in H3K9me3-dependent D4Z4 association of HP1γ, cohesion, and SMCHD1 \[[@CR16], [@CR17]\]. Despite these established differences, most FSHD myoblasts are indistinguishable in their transcription profile when compared to non-FSHD control myoblasts including the lack of differences in expression of DUX4 and its downstream targets \[[@CR13]\]. Therefore, while D4Z4 chromatin changes clearly poise a muscle cell for deleterious expression of DUX4, additional events are required for the initiation of DUX4 expression. Using fluorescence from a DUX4 reporter, we previously measured the frequency of DUX4 expression in cultured primary myoblasts and differentiated myocytes from 5 different individuals and repeated this experiment with differentiated myocytes from 2 individuals in this study (Additional file [1](#MOESM1){ref-type="media"}: Figure S1). We found that the fraction of DUX4 expressing cells varied significantly from patient to patient and ranged from 0.26% in one individual to as high as 4.28% in another. Regardless of the absolute levels, this survey demonstrated that significant transcriptional repression is occurring in \> 95% of cells despite the clear genetic and epigenetic differences between FSHD and control cells \[[@CR13]\]. By carefully selecting myoblast cells from people with unique genotypes, we have characterized several epigenetic differences between DUX4 expressing and DUX4 non-expressing pathogenic D4Z4 arrays present in FSHD myocytes. This approach exhibits some important differences from previous epigenetic studies. Here we were able to measure transcription from a single allele, and perform ChIP studies on the same allele without contaminating signal from other very similar D4Z4 sequences present on the non-pathogenic D4Z4 array on chromosome 4 and the non-pathogenic D4Z4 arrays on chromosome 10 and elsewhere in the human genome. The enzymatic mediators of repressive chromatin modifications can be divided into two classes. Members of the first class (PRC2, SUV39H1) place H3K27me3 and H3K9me3 modifications at D4Z4, the latter facilitating binding of HP1γ and CTCF and are involved in silencing DUX4 expression from arrays that are hypomethylated at CpG residues due to contractions in length (FSHD1) or SMCHD1 deficiency (FSHD2). While H3K9me3 at D4Z4 has been shown to be reduced in FSHD \[[@CR16]\], H3K27me3 levels are not significantly different when comparing D4Z4-associated histone modifications in FSHD and control cells \[[@CR17], [@CR38], [@CR39]\]. Our results show differential labeling of histones with H3K27me3 when comparing DUX4 expressing and non-expressing cells from the same individual indicating that this histone modification may be important for transcriptional repression in the absence of H3K9me3 modification and associated HP1γ and cohesion complexes. Removal of the H3K27me3 repressive marks, along with H3K9 acetylation, results in DUX4 expression (Fig. [6](#Fig6){ref-type="fig"}). These observations are consistent with newly reported results showing the nucleosome remodeling deacetylase (NuRD) complex is important for DUX4 repression in unaffected cells and H3K9 acetylation could possibly be antagonized by HDAC1 and HDAC2 components of the NuRD complex \[[@CR9]\]. The observation that the majority of short pathogenic chromosome 4 arrays are silenced suggests that augmenting PRC2 activity may reduce DUX4 expression further and be a worthwhile therapeutic strategy for disease treatment.Fig. 6Increase in activating H3K9Ac mark leads to DUX4 expression in a small fraction of cultured FSHD myoblasts. The epigenetic landscape in human iPS and ES cells in the non-contracted (**a**) and contracted (**b**) D4Z4 locus start out as bivalent and become univalent upon differentiation into myoblasts. Even in the euchromatin state majority of the cells with the permissive D4Z4 arrays do not express DUX4 but a small fraction of cells express DUX4 due to specific activating histone mark Conclusions {#Sec10} =========== Our findings both extend and clarify the findings of others. DUX4 transcription in FSHD myoblasts appears to be fundamentally due to a defect in the association of a group of chromatin modifiers whose function is to stabilize repressive chromatin structure and make repressed but poised D4Z4 arrays no longer easily activated through covalent and structural modifications of the DNA. Members of this group of epigenetic modifiers require epigenetic modifications initiated by SUV39H1 (H3K9me3) and HP1γ/cohesion association and include DNMT3b \[[@CR40]\] an enzyme involved in methylating CpG residues, and SMCHD1 \[[@CR32]\] a structural molecule involved in organizing higher-order chromatin structure \[[@CR41], [@CR42]\]. A previous study also suggests that LRIF1 may facilitate the interaction of SMCHD1 with HP1γ \[[@CR41], [@CR43]\] The activities of these molecules and perhaps others create structural chromatin changes that prevent the "leaky" sporadic DUX4 expression seen in FSHD due to incomplete repression by the PRC2 complex. Methods {#Sec11} ======= Ethics statement {#Sec12} ---------------- Myoblast and fibroblast from individuals with FSHD and non-FSHD control myoblast from de-identified human biopsies were provided by the Fields Center for FSHD Research biorepository (<https://www.urmc.rochester.edu/neurology/fields-center/research-info/sharingbiologicalresources.aspx>) and utilized in this study. The non-FSHD control fibroblast and A9chr10 (hybrid) cells were obtained from the Coriell Institute for Medical Research with repository numbers GM05387 and GM11688, respectively. This study was performed in accordance and approval of the University of Washington Institutional Review Board. Generation of isogenic iPS cell clones and identification of array sizes {#Sec13} ------------------------------------------------------------------------ Fibroblasts from an individual with FSHD were genetically tested and were identified to have somatic mosaicism for the D4Z4 array size on chromosome 4. Human iPS cell clones were generated from this individual's fibroblasts using the protocol of Takahashi et al. \[[@CR44]\]. Individual clones were isolated and screened for their D4Z4 array sizes by hybridizing the p13E11 probe to Southern blots of DNA fragments separated by pulse field gel electrophoresis as described previously \[[@CR45]\] and according to the protocol posted on the website for the Fields Center for FSHD Research (<https://www.urmc.rochester.edu/fields-center/research-info/protocols.aspx>). Cell culture {#Sec14} ------------ Myoblasts were cultured in F10 medium (Life Technologies, Carlsbad, CA) supplemented with 20% fetal bovine serum (Thermo Scientific, Waltham, MA), 10 ng bFGF (Life Technologies), 1 µM dexamethasone (Sigma) and 1 × penicillin/streptomycin (Life Technologies). For differentiation the proliferating myoblasts were plated at a seeding density of 70,000 cells/cm^2^ and cultured with or without 1 mM EGTA in DMEM: F12 (1:1, Life Technologies) supplemented with 20% KOSR (Life Technologies) and 1 × penicillin/streptomycin (Life Technologies) for 48--72 h. Human stem cells were cultured on irradiated mouse embryonic fibroblasts in media containing F12/DMEM (Life Technologies), 20% knockout serum replacer (Life Technologies), 1 × penicillin/streptomycin (Life Technologies), 1% 100 mM sodium pyruvate (Life Technologies), 1 × non-essential amino acid (Life Technologies), 0.1% 0.1 M β-mercaptoethanol (Sigma) and 2 ng/ml FGF-2 (Life Technologies). Stem cell colonies were separated from irradiated mouse embryonic fibroblast layers using dispase (Life Technologies) and seeded to matrigel-coated dishes in mTESR1 media (Stemcell Technologies) and cultured at 37 °C in 5% oxygen and 5% CO~2~. Flow cytometry {#Sec15} -------------- FSHD myoblasts (see "[Cell Culture](#Sec14){ref-type="sec"}" section) expressing CDK4 and transduced with the reporter vector \[[@CR13]\] were differentiated in the presence of 1 mM EGTA to inhibit myoblast fusion and enzymatically detached from culture dishes using 0.05% trypsin-EDTA (Life Technologies). The cells were centrifuged, and the pellet was resuspended in PBS containing 1% fetal bovine serum (Thermo Scientific). The cells were sorted on a four-laser Aria II cell sorter, and data were analyzed using FlowJo at the University of Washington Cell Analysis Facility. GFP+ and GFP− cell populations were collected. These sorted cells were utilized for the bisulfite and Matrix ChIP assays. DNA CpG methylation analysis of LLP {#Sec16} ----------------------------------- DNA methylation was analyzed by bisulfite conversion (EpiTect Bisulfite Kit, Qiagen) of genomic DNA from differentiated myocytes. To perform the bisulfite analysis of the 4qA-L allele, previously published \[[@CR21]\] PCR oligonucleotide primers (BSS4qALF for forward 5′-TTATTTATGAAGGGGTGGAGTTTGTT and BSS3626R for reverse 5′-AACAAAAATATACTTTTAACCRCCAAAAA) were utilized. All PCRs were performed using the hotstart Taq (JumpStart REDTaq from Sigma) at 95 °C for 5 min, 95 °C for 30 s, 55 °C for 30 s, 40 cycles at 72 °C for 1 min and 72 °C for 1 min. The amplified 354-bp PCR product was then gel purified and cloned into pGEM-T-Easy vector system II (Promega) and transformed into DH10B electrocompetent cells. The DH10B cells that carried the ligated vectors were selected by color change on agar plates containing the ampicillin/X-gal/IPTG. The white colonies represented the vectors with the PCR product, while the blue colonies represented empty vectors. The white colonies were picked and bulked in LB medium. The plasmids containing the target DNA were extracted by using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced to reveal methylation distribution at a single molecule level. Chromatin immunoprecipitation {#Sec17} ----------------------------- ### Sequential and single ChIP {#Sec18} Adherent human iPS cells and myoblasts were enzymatically detached, centrifuged, washed in PBS and cross-linked with 1% formaldehyde for 20 min at room temperature. The cells were then incubated in 125 mM glycine at room temperature for 5 min to inhibit cross-linking. The resultant chromatin was washed in PBS and suspended in a low-detergent shearing buffer containing 150 mM NaCl, 50 mM Tris, pH = 7.5, 5 mM EDTA, 0.005% NP-40, 0.01% Triton X-100 and a cocktail of protease inhibitors. The chromatin was sheared for 2 cycles of 15 min each in a bioruptor to achieve DNA fragments of \< 500 bp. A total of 2 × 10^6^ cells were utilized per sequential ChIP or for input DNA. The sheared chromatin was incubated with the first antibody in an ultrasonic bath for 60 min at 4 °C. The chromatin-antibody mixture was then centrifuged, and the top 90% of the supernatant was pipetted and incubated in a protein A-agarose (Sigma) slurry at 4 °C for 45 min. The slurry was washed 4 times with cold immunoprecipitation buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, NP-40 (0.5% vol/vol), Triton X-100 (1.0% vol/vol) and lastly with TE buffer (10 mM Tris pH 8, 1 mM EDTA). The slurry was eluted in 30 mM DTT, 500 mM NaCl, 0.1% SDS by incubating at 37 °C for 20 min \[[@CR24]\]. The second antibody was added to the eluate and incubated in an ultrasonic bath for 60 min at 4 °C, followed by incubation with protein A-agarose and washed 4--5 times with cold immunoprecipitation buffer. Chromatin + antibodies and ethanol-precipitated input DNA were boiled in a slurry containing 10% Chelex-100 resin. The chromatin was further incubated with proteinase k at 55 °C for 30 min and then boiled for 10 min to remove protein contaminants. The chromatin was centrifuged, and the resulting supernatant was used for real-time PCR. Single ChIP experiments were performed with the above protocol without the addition of the second antibody. This protocol was adapted and modified according to previously published method for fast ChIP \[[@CR46]\]. The antibodies used were H3K27me3 (Abcam: ab8580) and H3K4me3 (Abcam: ab6002). Analyses of all samples were performed in triplicate. ### Matrix ChIP {#Sec19} ChIP was performed on differentiated myocytes utilizing a previously published microplate-based chromatin immunoprecipitation method (Matrix ChIP) \[[@CR47]\]. Briefly, 96-well microplates with reactin-bind protein A (Pierce) were incubated with protein A on a low-speed shaker at room temperature overnight. The next day, the wells were blocked with blocking buffer containing 5% BSA and immunoprecipitation buffer on a shaker at 40 °C for 60 min. Simultaneously, chromatin samples (see sequential ChIP to obtain chromatin) with blocking buffer and antibody were added to a new UV-modified polypropylene 96-well microplates (Genemate) and incubated in ultrasonic bath for 60 min at 4 °C. The blocking buffer was aspirated from the protein A-coated plate, and the chromatin + antibody mix was added to the wells and incubated in the ultrasonic bath for 60 min at 4 °C. The chromatin samples were washed 3 times with immunoprecipitation buffer and then TE buffer. Finally, elution buffer containing 25 mM Tris base, 1 mM EDTA (pH10) with proteinase K 200 µg/ml was added to the wells, then shaken for 30 s at 1400 rpms and incubated for 45 min at 55 °C and then 10 min at 95 °C. The 96-well plates were then briefly agitated and centrifuged for 3 min at \~ 500 g at 4 °C and were used for PCR. The antibodies utilized for Matrix ChIP were H3K4me3 (Abcam: ab12209), H3K9me2 (Abcam: ab1220), H3K27me3 (Aviva: OOAAH00064), H3K9Ac (ab 4441), Pol2 (Santa Cruz: SC47701), EZH2 (Active Motif: 39103), SUV39H1 (Aviva: 32471), CTCF (Cell Signaling: 28995), and H3 (Abcam: ab1791). Matrix ChIP experiments were performed in triplicate followed by qPCR in 6--12 replicates. The primer sets utilized for the ChIP assays were POU4F3: Forward-TGCTGCAAGAACCCAAATTC and Reverse-GTTCTGGGCGACATGAAAAA, DUX4 LLP: Forward-CGACGGAGACTCGTTTGG and Reverse-GCTTTTGACCGCCAGGAG, HOXA3: Forward-ATGTGGCTCTTGGCTTCTCA and Reverse-GCGCATTTTTGACCCAGTTA, DIR1: Forward-CTGGGAGAATGTGCTCAGGT and Reverse-GCCAGGATTGAACAGAGGAA, β-actin: Forward-AGCACAGCCTGGATAGCAAC and Reverse-TCTGAACAGACTCCCCATCC, Forward-TCTCCCTCCTCCTCTTCCTC and Reverse-TCGAGCCATAAAAGGCAACT for Matrix ChIP. Drug screening and generation of the NanoLuc reporter and delivery {#Sec20} ------------------------------------------------------------------ PCR amplified reporter cassette containing the 6 individual sequence motifs of the DUX4 DNA binding site sequence \[[@CR48]\] AGATAATTGAATCATGGGGT AATCCAATCATGGAGTAATTTAATCAGCCGTTAATTGAATCATGG GGTAATCCAATCATGGAGTAATTTAATCAGCCG followed by a minimal TATA box promoter upstream of the NanoLuc (Promega pNL1.2\[NlucP\] Vector) was cloned into the pRRLsincPPT-wpre third-generation lentivirus backbone \[[@CR49]\]. A neomycin resistance gene under the control of pTK promoter was cloned downstream of NanoLuc to establish G418 selection independent of reporter activation by the DUX4 protein. HEK-293T cells were utilized to package the lentiviral vector by polyethylenimine-mediated (PEI) co-transfection. FSHD myoblasts expressing CDK4 were then transduced with the lentivirus reporter similar to our previously published study \[[@CR13]\]. Cell viability assay {#Sec21} -------------------- The FSHD myoblasts transduced with the NanoLuc reporter were seeded in 384-well plates at a cell density of 4000--7000 cells per well in proliferating media along with the drug or DMSO (vehicle). After 24 h, the media were switched to differentiating media containing the drug or DMSO. After 48 h CellTiterFluor assay (Promega) was used to measure cell viability through fluorescent signal output on a BioTek multi-detection plate reader. Luciferase assay {#Sec22} ---------------- Luminescence was detected in the same cells through the Nano-Glo Luciferase assay (Promega) to measure Luciferase output, a measure of DUX4 expression. The cell-based assays were performed in quadruplicate. RNA preparation and qRT-PCR analysis {#Sec23} ------------------------------------ Adherent cells from the vehicle and drug-treated groups were lysed using 1 ml of TRIzol Cell Lysis Reagent (Life Technologies) at room temperature for 10 min. Chloroform was added at one-fifth volume of TRIzol reagent to separate the RNA containing aqueous phase from the TRIzol. The RNA was precipitated with 0.5 ml isopropyl alcohol from the aqueous phase. The RNA pellet was washed with 75% ethanol and allowed to air dry for 5--10 min and resuspended in RNAase-free water. The RNA sample was then incubated with DNAse I (NEB, Ipswich, MA) at 37 °C for 15 min followed by a cleanup step using the RNeasy column Qiagen kit. One microgram of DNAsed RNA was first primed with oligo dT primers and reverse-transcribed to cDNA using Superscript III First-Strand Synthesis System incubated at 65 °C for 5 min, followed by 50 °C for 50 min and 85 °C for 5 min. The cDNA was further diluted 1:4 to perform qRT-PCR using Roche Fast Start Universal SYBR Mastermix with ROX (Roche, Basel, Switzerland). The primer sets utilized for gene expression analysis were as follows DUX4: Forward-CTCCCGACACCCTCGGACAGCAC and Reverse-TCCAGGTTTGCCTAGACAGCGTC and GAPDH: Forward-GTGAAGGTCGGAGTCAAC and Reverse-TGAGGTCAATGAAGGGGTC. Pre-validated Taqman probes were utilized to analyze gene transcription of DUX4-activated secondary targets CCNA1 (Hs00171105_m1) and MBD3L2 (Hs00544743_m1) and RNAseP (4403326). Previously published cycling parameters were used (12) to perform the qRT-PCR using the ABI-7900HT machine. The gene expression analysis was performed in triplicate utilizing the 2^−ΔΔCt^ method. Statistical analyses {#Sec24} -------------------- Student's *t* test was utilized to calculate the statistical differences between the groups. A *p* value ≤ 0.05 was considered to be statistically significant and was denoted by asterisks. The error bars denote standard deviation. Additional file =============== {#Sec25} **Additional file 1: Figure S1.** Quantification of DUX4 expression in FSHD cell cultures. Flow cytometry analysis of GFP fluorescence intensity on x-axis and autofluorescence on the y-axis of non-FSHD control and 2 FSHD cell lines transduced with DUX4-activated GFP reporter. **a** Proliferating myoblasts and **b** Myocytes differentiated for 48 h in differentiating media containing 1mM EGTA. **Figure S2.** H3K27me3 and H3K4me3 marks on the D4Z4 LLP locus of the isogenic iPSC clones with non-contracted and contracted D4Z4 array. Single ChIP pull down with H3K27me3 alone or H3K4me3 alone revealed the presence of both marks in the iPS cells. **Figure S3.** Similar gene expression levels of myogenic differentiation markers in DUX4 expressing and DUX4 non-expressing FSHD myocytes. Previously published and unpublished RNA-seq data obtained from Rickard et al. \[[@CR13]\] were plotted above. Counts normalized gene expression levels were similar for MYH1, MYH2 and MYOG (markers for myogenic differentiation) with p values \> 0.05 using *t*-test between DUX4 expressing and non-expressing myocytes from **a** 2349 FSHD line and **b** 2084 FSHD line. FSHD : facioscapulohumeral muscular dystrophy LLP : long last partial iPS cells : induced pluripotent stem cell ChIP : chromatin immunoprecipitation PH performed ChIP analysis, flow cytometry, drug screening and wrote parts of the manuscript. KB developed the matrix ChIP assay platform. DGM made the isogenic cell lines and assisted in manuscript writing. All authors read and approved the final manuscript. Acknowledgements {#FPar1} ================ We thank Amanda Rickard and Rebecca Mellema for their technical assistance. We thank friends of FSH Research for their funding support to the Miller Lab. We also thank Dr. Rabi Tawil, director of the Fields Center for FSHD Research for providing fibroblasts and myoblasts from FSHD-affected individuals and volunteers who donated tissue to generate these cell lines. Competing interests {#FPar2} =================== The authors declare that they have no competing interests. Availability of data and materials {#FPar3} ================================== The datasets analyzed in the current study are available from the corresponding author upon request. Consent for publication {#FPar4} ======================= Written informed consent was obtained from study participants for participation in the study and for publication of this report. Ethics approval and consent to participate {#FPar5} ========================================== This study was done in accordance and approval of the University of Washington Institutional Review Board. Funding {#FPar6} ======= This study was supported by NIH-NIAMS R56 AR070778 and R01 AR06419701A1 to DGM and NIH R01 DK094934, R21 GM111439 and R33 CA191135 to KB. Publisher's Note {#FPar7} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Introduction ============ Essential fatty acids (EFAs): linoleic acid (LA, n-6, 18:2) and α-linolenic acid (ALA, n-3, 18:3) form precursors to their long chain metabolites γ-linolenic acid (GLA, n-6, 18:3), dihomo-GLA (DGLA, n-6, 20:3) and arachidonic acid (AA, n-6, 20:4); and eicosapentaenoic acid (EPA, n-3, 20:5) and docosahexaenoic acid (DHA, n-3, 22:6) respectively \[[@B1]-[@B3]\]. Our previous studies showed that polyunsaturated fatty acids (PUFAs) selectively induced tumor cells apoptosis though the sensitivity of various cancer cells to different fatty acids were found to be variable depending on the type of cancer cell being tested and the type and concentration of the fatty acid used \[[@B3]-[@B7]\]. Previously, it was reported that essential fatty acids and their metabolites suppress tumor cells growth both *in vitro*and *in vivo*. This tumoricidal action of fatty acids could be correlated to an increase in generation of free radicals in the tumor cells \[[@B8]\]. Subsequent studies showed that most polyunsaturated fatty acids were functional, and the inhibitory action of different types of n-3, n-6 and n-9 fatty acids does not depend on their unsaturation \[[@B9]\]. Among all the fatty acids tested, GLA, AA, EPA and DHA were found to be the most effective in inhibiting tumor cells growth, while LA and ALA were also effective but at much higher concentrations \[[@B3]-[@B5]\]. It was opined that n-6 fatty acids enhance tumor cell growth whereas n-3 fatty acids are beneficial since they arrest cancer growth. This differential action of n-3 and n-6 PUFAs in cancer has been attributed to the formation of pro-inflammatory eicosanoids from n-6 PUFAs whereas products formed from n-3 PUFAs are much less pro-inflammatory in nature \[[@B1]-[@B7],[@B10]-[@B13]\], though Trombetta A, *etc*. reported that AA, an n-6 PUFA, decreased human lung-tumor cell growth in a concentration-dependent manner, induction of cell death mainly evident at 100 mM concentration \[[@B14]\]. In the majority of previous investigations, n-3 and n-6 PUFAs were added to the tumor cell medium *in vitro*without a simultaneous study of these fatty acids on normal cells. Hence, it is not clear whether the concentrations of fatty acids used in these studies are non-toxic to normal cells at which they were found to be cytotoxic to tumor cells. In addition, little attention was paid to the ratio between n-6 and n-3 fatty acids as they exist in the body while performing these studies. It is important to note that in the plasma, n-6 PUFAs are present in large amounts compared to n-3 fatty acids (the ratio between n-6 PUFAs compared to n-3 PUFAs is \~7:1 in the serum) \[[@B15]\]. Furthermore, PUFAs are widely distributed in our food and hence, there could be a wide variation in the daily intake of these fatty acids among different populations and individuals depending on the type of diet and the quality of the food ingested. In general, the level of total fatty acids in the plasma/serum is \~200 mg/dl, and of which the percentage of LA is \~20% regardless of the differences in dietary pattern \[[@B15]\]. Previously, we observed that the action of LA on cancer cell growth depended on the type of cancer cells being tested and the concentration of fatty acids supplemented \[[@B3]-[@B7]\]. LA \~40 μg/ml/1 × 10^4^cells inhibited the growth of cancer cells whereas lower concentrations \~5-10 μg/ml/1 × 10^4^cells enhanced growth of some, if not all, types of cancer cells that were being tested \[[@B11]\]. In the present study, we evaluated the effect of the fatty acid on three cell lines, two of which were colorectal cancer cell lines, LOVO (undifferentiated) and RKO (semi-differentiated), and the human umbilical vein endothelial cells (HUVEC) taken as normal cell control in order to clarify the role of LA in the promotion and inhibition of the growth of cancer cells *in vitro*. In order to know the sensitivity of tumor cells to LA, in one set of studies we pre-incubated the cells for 24 hours with 100 μM of LA and then were subsequently exposed to various doses of LA to know whether the initial exposure to LA affects the survival of tumor cells. Materials and methods ===================== Materials --------- Linoleic acid (LA, 18:2 n-6) was obtained from Sigma (St. Louis, MO, USA). The colorectal cancer cell lines, LOVO (undifferentiated) and RKO (semi-differentiated), and normal cell line HUVEC (human umbilical vein endothelial cells) were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. PRMI medium 1640 and high-glucose DMEM Nutrient Mixture medium were purchased from GIBCO (Grand Island, NY, USA). MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) was provided by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. All other chemicals were of extra-pure grade or analytical grade. Cell culture and treatment -------------------------- Colorectal cancer cells (LOVO and RKO) and human normal cells (HUVEC) were cultured in PRMI Medium 1640 and high-glucose DMEM Nutrient Mixture medium separately, supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin in a humidified 37°C, 5% CO~2~incubator (Shellab, USA). LA was dissolved in 0.1 N NaOH and diluted to give a final concentration of 20 mM with the final concentration of NaOH was no more than 0.005 N, a concentration of NaOH at which it had little affect on the cells. Stock solutions were filter-sterilized and diluted with cell culture media for use in the study \[[@B16]\]. Both the colon cancer cells (LOVO and RKO) and normal cell (HUVEC) were treated with LA in two different ways: in the first group, the cells were cultured in the cell culture medium alone for 24 h prior to treatment with different doses of LA; whereas in the second group, cells were pre-incubated with 100 μM LA for 24 hr followed by treatment with different doses of LA as was done in the first group. Cell growth and viability assay ------------------------------- Cell proliferation was assessed using MTT assay (Roche, Mannheim, Germany). At different time intervals after incubation with LA, the number of viable cells grown in a 96-well plate was estimated by adding 20 μl of MTT solution (5 mg/ml in PBS). After 4 hr of incubation at 37°C, the stain was diluted with 150 μl of DMSO. The absorbance in each well was then measured with a microplate reader (Thermal Lab system, Finland) at 492 nm, and viability of cells was presented as percentage of the control \[[@B16]\]. Each treatment was replicated at least five times. Mitochondrial membrane potential detection ------------------------------------------ Cells (1 × 10^6^cells) obtained from control and various LA treatments were washed thrice with PBS, and resuspended at a final protein concentration of 0.75 mg/ml in PBS, 100 μl was taken for the following detection. 100 ng Rh123 (Rhodamine 123) were added to each sample. Upon incubation in the dark (15 min, at room temperature or 30 min at 4°C), the samples were washed with 300 μl PBS twice. The cells were resuspended in 200 μl PBS for analysis. Fluorescence intensity was carried out on a multifunctional micro-plate reader (SpectraMax M5, Molecular Devices) with a 96-well plate (side-opaque, clear bottom). The excitation and emission wavelengths for Rh123 were selected with monochromators set to 488 nm (5 nm slit width) and 530 nm (5 nm slit width), respectively. Each treatment was replicated thrice, and the final data was calculated as follows: corresponding fluorescence intensity (%) = F~sample~/F~CK~ Where: F~sample~and F~CK~are the intensities measured with microplate reader. \[[@B17]\] **ROS generationstudies** ------------------------- Cells were incubated with the cell permeant dye H~2~DCF-DA, which intracellularly de-esterifies to dichlorodihydrofluorescein (H~2~DCF). ROS oxidize H~2~DCF to the brightly fluorescent compound 2-, 7-dichlorofluorescein (DCF), which was monitored by flow cytometry following a previously described method \[[@B18]\] with modifications. Briefly, cells treated with LA or vehicle were incubated with 5 μM H~2~DCF-DA for 15 min at 37°C, detached from the plate with trypsin/EDTA, washed with PBS, resuspended in ice-cold PBS, and tested immediately. Triplicate samples were run in each experiment, and at least 5000 cells per sample were analyzed (excitation at 488 nm, emission at 500-530 nm) by *SpectraMax M5, Molecular Devices*. Mean fluorescence was calculated by using the program *SoftMax Pro Software Version 5 for Mac^®^and Windows^®^*. The final date was calculated as given below: Corresponding fluorescence intensity (%) = F~sample~/F~CK~ Where: F~sample~and F~CK~are the intensities measured with microplate reader. Estimation of cell MDA content and SOD activity ----------------------------------------------- The levels of MDA and the activity of SOD, the biomarkers of oxidative stress, were measured as described previously with commercial reagent kits purchased from Nanjing Kaiji Bioengineering Institute (Nanjing, China) \[[@B19]\]. The cell MDA content and SOD activity were also represented as corresponding value: Corresponding MDA (or SOD) concentration (%) = C~sample~/C~CK~ Where: C~sample~and C~CK~are the concentration of MDA (or SOD) measured with regent kits. Isolation and purification of mitochondria ------------------------------------------ The cells were harvested by centrifuging at 1,500 rpm for 5 min at 4°C, and washed twice with cold PBS and finally re-suspended in PBS. Cells were lysed by Ultrasonic Cell Disruption System (JY92-II, Chongqing, China) and centrifuged at 3,000 rpm for 10 min at 4°C. The supernatant thus obtained was centrifuged at 9,000 rpm for 10 min to obtain mitochondrial pellets that were washed twice with cold PBS. The final mitochondrial pellets were suspended in test medium (220 mmol/L Mannitol, 70 mmol/L Sucrose, 5 mmol/L HEPES, pH 7.2) for the following studies. The final protein concentrations of the mitochondrial suspension were all adjusted to 0.3 mg/mL (detected and corrected with Bradford protein analysis kit, Sangon, Shanghai, China) \[[@B20]\]. Measurement of cytochrome C content ----------------------------------- Isolated mitochondrial content of cytochrome C was measured at 520 nm in UV-vis spectroscopy (HP 8453, Hewlett-Packard, USA) by suspending them in a reaction medium containing 10 mg sodium dithionite and 0.5 mL of 0.3 mg/mL mitochondrial protein. Sample concentrations were determined based on a standard curve. Each of the experiments was replicated three times \[[@B20]\]. Measurement of the activities of caspases 9 and 3 ------------------------------------------------- The activities of caspases 9 and 3 were assayed using caspase-9 and caspase-3 activity assay kits separately, according to the manufacture\'s instructions (Keygen, Nanjing, China). After different treatments, 5 × 10^6^cells were harvested by centrifuging at 1,500 rpm for 5 min at 4°C, and washing twice with cold PBS, then resuspended in a cell lysis buffer. After incubation on ice for 60 min, the lysates were centrifuged for 20 min at 12,000 rpm, the supernatants were collected and protein concentrations were determined. Cell lysates (100 μg) were mixed with reaction buffer and incubated for 4 h at 37°C. The absorbance of the reaction mixture was measured in the wells at 405 nm using an ELISA reader (Thermal Labsystem, Helsinki, Finland) \[[@B20]\]. Statistical analysis -------------------- All results are expressed as means ± SD and significance of differences between various results was determined by Origin 7.0. Each treatment was carried out at least triple, and statistic analysis was shown as: \* meaning p \< 0.05, and for \*\* meaning p \< 0.01. Results ======= Cell growth and viability ------------------------- All the three cell lines studied showed increased cell proliferation at lower concentrations of LA (100-200 μM), while higher concentrations of LA (above 300 μM) suppressed the growth of the cells as determined by MTT assay (Fig. [1](#F1){ref-type="fig"}). {#F1} It is interesting to note that different cell lines showed different sensitivity to LA depending on the type of cell line being tested and the dose of LA supplemented (Fig. [1](#F1){ref-type="fig"}). Of three types of cells tested, HUVEC were the most resistant to the cytotoxic action of LA compared to the two cancer cell lines used in the present study. The colorectal cancer cell lines showed increased or no change in proliferation when exposed to 100 μM LA whereas HUVEC cells remained non-responsive even when supplemented with 400 μM LA. LA suppressed the proliferation of LOVO and RKO cells to a significant degree when supplemented with 300 μM of LA, whereas HUVEC cells showed decreased proliferation only when incubated with 600 μM of LA. Based on these results, we chose 200 μM and 400 μM for treatment of HUVEC, while 100 μM and 300 μM LA was used to study the effects of LA on colorectal cancer cells for subsequent studies. On the other hand, when we studied the effect of pre-incubation of cells with 100 μM of LA for 24 hours followed by further exposure to various doses of LA, it was noted that the sensitivity of HUVEC to the growth suppressive action of LA was enhanced. For instance, it was noted that almost 50% of HUVEC were still surviving even when treated with 400 μM LA after pre-treatment. Of the two cancer cell lines studied, LOVO cells were found to be resistant to the growth suppressive actions of LA even when exposed to 300 μM LA after pre-treatment, while, RKO cells showed increased sensitivity to the growth suppressive action of the fatty acid. Hence, we performed all subsequent mechanistic studies with HUVEC, LOVO and RKO cells by pre-incubating them with 100 μM for 24 hours followed by subsequent supplementation with LA as described. Cell mitochondrial membrane potential detection ----------------------------------------------- Cell mitochondrial membrane potential was evaluated as a measure of the degree of injury to the mitochondrial membrane which is reflected in the amount of permeability exhibited to Rh123 (Fig. [2](#F2){ref-type="fig"}): the higher the permeability the higher fluorescence of Rh123 could pass-through mitochondrial membrane indicating statically significant damage to the membrane. {#F2} HUVEC did not show any significant change in the cell mitochondrial membrane potential following the addition of LA, while the two cancer cell lines LOVO and RKO showed significant yet similar changes in the mitochondrial membrane potential in response to incubation with various treatments with LA. Higher concentration of LA (300 μM) with and without pre-treatment enhanced mitochondrial uptake of Rh123 by LOVO and RKO cells compared with their respective controls, while the low concentration treatments (100 μM of LA) did not show any significant change in mitochondrial membrane fluorescence permeability of Rh123 (Fig. [2](#F2){ref-type="fig"}). It is noteworthy that both LOVO and RKO cells showed similar increase in the mitochondrial membrane fluorescence permeability of Rh123 in response to incubation with 300 μM of LA with or without pre-incubation (Fig. [2](#F2){ref-type="fig"}). Pre-incubation with LA increased fluorescence intensity of LOVO and RKO cells, but not of HUVEC, suggesting that HUVEC are able to maintain their mitochondrial function while the colon cancer cells could not. In addition, pre-incubation of RKO cells with LA (100 μM) and subsequent exposure to 300 μM of LA led to a much higher fluorescence intensity of Rh123 in comparison to LOVO cells. The higher fluorescence intensity of Rh123 by RKO cells, the semi-differentiated cells, in comparison to the fluorescence intensity by LOVO cells, the undifferentiated cells, in response to the same dose of LA could be related to the cell differentiation state. The higher fluorescence intensity of Rh123 by LA-treated RKO cells in comparison to fluorescence intensity shown by LOVO cells indicates that LA is able to induce much higher damage to the mitochondrial membrane of RKO cells compared to the LOVO cells. These results indicate that RKO cells are much more sensitive to the toxic actions of LA compared to LOVO cells. Cell oxidative stress analysis ------------------------------ In order to know the degree of oxidative stress that occurred as a result of incubation with LA, we studied generation of ROS, MDA accumulation, and SOD activity in the cells. The amount of ROS generated by HUVEC when incubated with 200 μM LA with and without pre-incubation was at least 2 to 2.5 times higher compared to the untreated control while exposure to 400 μM of LA produced much less (only about 50% rise) increase (Fig. [3](#F3){ref-type="fig"}). In contrast, both the colorectal cancer cell lines RKO and LOVO studied showed much less significant change in the generation of ROS in response at both doses of LA (100 and 300 μM) added. There was no significant difference in the amount of ROS generated with or without pre-incubation with 100 μM of LA in colorectal cancer cell lines. {#F3} In contrast to the results obtained with regard to ROS generation, the accumulation of MDA in the cells showed distinctly different results. There were no significant changes in the levels of MDA accumulation in HUVEC following incubation with different doses of LA (Fig. [4](#F4){ref-type="fig"}), while colorectal cancer cells LOVO and RKO showed about 2 \~2.5 times increase compared to respective controls. Pre-incubation with LA (100 μM) significantly enhanced MDA accumulation in LOVO, while RKO cells showed enhancement in the MDA levels upon treatment with LA with and without pre-incubation with 100 μM of LA compared to the respective controls (Fig. [4](#F4){ref-type="fig"}). {#F4} LA treatment raised SOD activity in RKO cells to a significant degree in a time and dose dependent fashion (Fig. [5](#F5){ref-type="fig"}), while the there were no significant changes in SOD activity in HUVEC and LOVO cells. {#F5} Cell mitochondrial apoptosis pathway analysis --------------------------------------------- Cytochrome C release induces mitochondrial dependent apoptosis. An enhanced release of cytochrome C was noted in HUVEC treated with LA with and without pre-incubation with LA (100 μM) (Fig. [6](#F6){ref-type="fig"}). An increased release of cytochrome C was noted in both LOVO and RKO cells treated with LA with or without pre-treatment. It is noteworthy that the release of cytochrome C was much higher in cells that were treated with higher dose of LA (300 μM) with or without pre-incubation with LA (Fig. [6](#F6){ref-type="fig"}). {#F6} Enhanced release of cytochrome C activates caspase family that induces cell apoptosis. As expected, the changes in the activation of caspases 9 and 3 paralleled the changes seen with cytochrome C release in HUVEC, LOVO and RKO cells (Fig. [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"}). RKO cells showed much higher increases in the activation of caspase 3 compared to caspase 9, while LOVO cells showed much less activation of caspase 3 compared to caspase 9. {#F7} {#F8} Discussion ========== N-6 PUFAs are known for their critical role in many physiological functions but their role in cancer is still under debate. Both tumor promoting and inhibiting properties of n-6 fatty acids have been reported. It has been shown that n-6 fatty acids decrease human lung-tumor cell growth in a concentration-dependent manner \[[@B21],[@B14]\]. Our results suggested that low concentrations (≤ 200 μM) of LA promote colorectal cancer cell growth, while high levels (≥ 200 μM) induce apoptosis of the colorectal cancer cells *in vitro*. On the other hand, low concentrations of LA (≤ 200 μM) did not promote normal (HUVEC) cell proliferation while high concentrations (≥ 200 μM), which were cytotoxic to tumor cells, induced only 10\~20% decrease in the number of HUVEC. These results suggest that LA is toxic to tumor cells with little or no cytotoxic action on normal cells. Mitochondria are the main organelles where fatty acids and other energy donors are metabolized/oxygenized to support cell survival. In the present study, it was noted that the mitochondrial membrane potential is affected only when cells were exposed to ≥ 200 μM of LA, while low LA concentrations (≥ 200 μM) were without any affective. It was also observed that pre-treatment with LA followed by supplementation with high concentrations of LA induced significant increase in fluorescence suggesting that mitochondrial membrane loses its ability to function as an electron transporter and aerobic respirator (Fig. [2](#F2){ref-type="fig"}) at the dose tested. PUFAs enhance free radical generation \[[@B22]\] that may be related to the loss of mitochondrial function. Previously, it was reported that tumoricidal action of PUFAs could be correlated to an increase in generation of free radicals \[[@B8]\]. We also noted that GLA induced apoptosis of tumor cells by augmenting free radical generation only in the tumor cells but not normal cells \[[@B23]\]. Based on the results of the present study and previous results \[[@B24]\], we suggest that free radical generation is the main mediator of mitochondrial damage and apoptosis of tumor cells. Despite similar treatment schedules, the formation of ROS, lipid peroxides and the degree of apoptosis were found to be dissimilar between LOVO and RKO cells. It is noteworthy that the levels of SOD were significantly elevated in RKO compared with LOVO cells suggesting that oxidant stress is present to a significant degree in the former but not the latter. The increase in SOD levels in RKO could be secondary to the accumulation of significant amounts of MDA in these cells. The formation of ROS and lipid peroxides were not significantly increased in HUVEC suggesting that normal cells resist the formation of lipid peroxides and generation of ROS and thus are able to with stand oxidant stress induced by exposure to LA (Figs. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, &[5](#F5){ref-type="fig"}). MDA formulation is mainly due to (a) non-enzymatic free radical catalyzed lipid peroxidation and (b) AA derived cyclooxygenase pathway that can be enhanced by the addition of LA. Pre-treatment with LA (100 μM) followed by supplementation of 100 and 300 μM of LA enhanced the formation of lipid peroxides (MDA formation) in RKO cells compared with HUVEC and LOVO cells suggesting that accumulation or formation of lipid peroxides in cells may vary depending on the cell type. These results imply that accumulation of lipid peroxides may trigger apoptosis and generation of ROS and the levels of SOD could be determined by the amount and degree of lipid peroxides formed in the cells. Mitochondrial dysfunction leads to apoptosis due to the release of cytochrome C that, in turn, activates caspase-9 and caspase-3. Oxidant stress can induce mitochondrial dysfunction, alter its membrane potential, interrupt electron transport chain and induce apoptosis. LA-treated tumor cells showed significantly enhanced cytochrome C release, and activities of caspase-9 and caspase-3 (Figs. [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"}). Enhanced activity of caspase-9 and cytochrome c release was observed in LA-pre-treated RKO tumor cells that support the proposal that apoptosis in these cells are mediated by mitochondrial pathway (Figure [9](#F9){ref-type="fig"}). These results suggest that accumulation of LA in the tumor cells could facilitate apoptotic process since LA-pre-treated tumor cells were found to be much more vulnerable to apoptosis compared to untreated tumor and normal cells. {#F9} Cell differentiation status could affect their sensitivity to the cytotoxic actions of LA. This is supported by the observation that the differentiated cell line RKO was more sensitive to the cytotoxic action of LA in comparison to the undifferentiated cell line LOVO, especially after pre-incubation with LA. These results imply that LOVO cells may metabolize LA more efficiently and are able to detoxify the toxic metabolites of LA in comparison to RKO cells. Hence, it will be interesting to study the differential metabolic pathway of LA in RKO, LOVO cells and HUVEC as related to its cytotoxic action. Although the results of the present study indicated that colorectal cancer cell lines are more susceptible to the cytotoxic effects of LA compared with human umbilical endothelial cells, the inherent cell type differences may also have influenced the differential respones observed rather than tumor specific differences. Hence, more studies need to be performed to understand and elucidate the molecular mechanism(s) of the cytotoxic action of LA. In summary, it was observed that LA induces the formation of lipid peroxides that, in turn, could induce apoptosis of tumor cells. The differential cytotoxicity observed in two different colorectal cancer cell lines at different stages of differentiation suggests that the handling of LA by these cells is different and understanding its (LA) metabolism in these cells may lead to the identification toxic metabolites formed that drive its cytotoxic action. Abbreviations used ================== AA: arachidonic acid; ALA: α-linolenic acid; DGLA: dihomo-γ-linolenic acid; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; GLA: γ-linolenic acid; LA: linoleic acid; MDA: malondialdehyde; mPTP: mitochondrial permeability transition pore; MTT: 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; Rh123: Rhodamine 123; ROS: reactive oxygen species; SOD: Superoxide Dismutase. Declaration of competing interests ================================== The authors declare that they have no competing interests. Authors\' contributions ======================= SS and UND conceived the idea and designed the experiments. XL, HY and QM performed the experiments. SS participated in the design of the study and performed the statistical analysis. SS and UND drafted the manuscript and interpretation of the data. All authors read and approved the final manuscript. Acknowledgements ================ Dr. Das is in receipt of Ramalingaswami fellowship of the Department of Biotechnology, India during the tenure of this study.

Introduction {#s1} ============ The epidemiology of methicillin-resistant *Staphylococcus aureus* (MRSA) infections has changed dramatically during the last 15 years. While traditionally MRSA was a typical example of a nosocomial pathogen, it is now frequent found as causative agent of community-associated infections among patients without known risk factors for hospital-acquired (HA)-MRSA [@pone.0016419-Adam1], [@pone.0016419-Bukhari1], [@pone.0016419-Coombs1], [@pone.0016419-Elston1], [@pone.0016419-Fontanilla1], [@pone.0016419-Klein1], [@pone.0016419-Reyes1]. The molecular epidemiology of community-associated MRSA (CA-MRSA) is diverse, although certain clones appear to dominate on every continent. These predominant CA-MRSA clones cluster in different lineages: ST8/USA300, ST1/USA400, ST30/USA1100, ST93, ST59, ST80 and ST398 clone [@pone.0016419-Otter1]. In the USA, the first widely recognized CA-MRSA clone was USA400 (ST1). After the turn of the century, USA300 (ST8) emerged rapidly across the USA and replaced USA400 as the dominant clone in the USA responsible for the majority of skin and soft tissue infections. Subsequently, USA300 has been increasingly isolated outside the USA indicating pandemic spread. Within USA300 several subtypes exist of which PFGE-type USA300-0114 predominates [@pone.0016419-Diep1], [@pone.0016419-Witte1]. Comparative whole genome sequencing of 10 USA300 CA-MRSA and HA-MRSA isolates collected nationwide in the USA in 2002, 2003, and 2005 showed a limited number of single nucleotide polymorphisms and regions of differences among USA300 isolates, which suggests that USA300 has undergone rapid clonal expansion without great genomic diversification [@pone.0016419-Kennedy1]. So far whole genome comparisons of CA-MRSA are limited to isolates belonging to USA300. The aim of this study was to compare the genetic repertoire of different CA-MRSA clones with that of HA-MRSA from the USA and Europe through comparative genomic hybridization (CGH) to identify genetic clues that may explain the successful and rapid emergence of CA-MRSA [@pone.0016419-Witney1]. Materials and Methods {#s2} ===================== Bacterial isolates and nucleic acid extraction {#s2a} ---------------------------------------------- Thirty nine consecutive MRSA isolates collected in 2004, originating from hospitalized patients with serious invasive infections admitted at Cook County Hospital (Chicago IL, USA) were taken from a database. The isolates were phenotypically classified with MicroScan (West Sacramento, California) as methicillin-resistant. Twenty isolates were recovered \<48 h after admittance from patients who did not have prior health care exposure and were considered CA-MRSA. These isolates were collected between the 1^st^ and 28^th^ of July. Nineteen isolates were recovered (between July and October of 2004) from patients \>48 h after admission and these were classified as HA-MRSA ([Table S1](#pone.0016419.s001){ref-type="supplementary-material"}). Ten HA-MRSA isolates from The Netherlands, Germany, Belgium, and France were selected from a collection of 118 HA-MRSA isolates present at the University Medical Center Utrecht (UMCU) on the basis of their geographic origin, infections caused or nasal carriage and multilocus sequence type (ST) ([Table S1](#pone.0016419.s001){ref-type="supplementary-material"}). In addition, one CA-MRSA isolate, obtained in 2001 from a child that succumbed due to necrotizing pneumonia within 48 h of hospitalization (and without prior health care exposure) in the UMCU [@pone.0016419-vanderFlier1]. All isolates were cultured overnight on tryptic soy agar with sheep blood at 37°C. DNA was extracted with a Nucleospin Tissue kit according to manufacturer\'s protocol (Bioké, Leiden, The Netherlands). Plasmid DNA content was determined using S1 nuclease treatment (Takara Bio Europe, Saint-Germain-en-Laye, France), gel electrophoreses and southern blot with a 193 bp probe from the resolvase gene SAR719. We do not have an ethics approval and an informed consent, because the data we have used are anonymous and were not specifically collected for our study. The patients were admitted to the hospital, the samples were taken for diagnostic purposes by the treating physicians in order to appropriately treat the patients. These samples were anonymously put in a database from which they were recovered. In the Netherlands the Medical Ethical Committee does not require an approval for this kind of research and sampling. Typing of the MRSA isolates {#s2b} --------------------------- Colony morphology and standard techniques, like multiplex PCR for the 16S rRNA gene, *mecA* gene, and *nuc* gene were used to confirm whether the isolates were MRSA. All MRSA isolates from the USA were typed by MLST [@pone.0016419-Enright1]. The CA-MRSA isolates were further characterized by PFGE, *spa*, SCC*mec* and antimicrobial susceptibility patterns by MicroScan (Dade Behring Inc., West Sacramento, CA, USA) [@pone.0016419-Harmsen1], [@pone.0016419-Ito1], [@pone.0016419-McDougal1]. The antimicrobial agents tested were oxacillin, erythromycin, rifampicin, fluoroquinolones, clindamycin, gentamicin and tetracycline. The MIC values were interpreted according to the Clinical and Laboratory Standards Institute recommendations (2004) [@pone.0016419-NCCLS1]. Comparative genomic hybridization {#s2c} --------------------------------- Gene content of the CA- and HA-MRSA isolates was determined via CGH using a previously described multistrain (n = 7) PCR product *S. aureus* microarray [@pone.0016419-Lindsay1], [@pone.0016419-Witney1]. The array design is available in BµG\@Sbase (Accession No. A-BUGS-17; <http://bugs.sgul.ac.uk/A-BUGS-17>) and also ArrayExpress (Accession No. A-BUGS-17). Gene presence and divergence was determined using the algorithm described previously [@pone.0016419-Carpaij1]. Complete linkage hierarchical clustering with Euclidian distance of all the HA-MRSA and CA-MRSA isolates was used to visualize the genetic relatedness. Confirmation of genetic differences {#s2d} ----------------------------------- Differences in gene content between the USA300 CA-MRSA isolates from Chicago and USA300FPR3757 and USA300TCH1516 (for which the whole genome sequences are available) as identified by CGH were confirmed by PCR and sequencing. The primers were generated, if possible, using the USA300FPR3757 sequence and otherwise on the USA300TCH1516 or MRSA252 sequence. Amplification was carried out for 35 cycles with denaturation at 95°C for 30 seconds, annealing at the primer specific annealing temperature for 30 seconds, extension at 72°C for 45 seconds and a final extension at 72°C for 7 minutes. The presence of *arcA* considered specific for SCC~ACME~, *lukS-PV* and *lukF-PV,* encoding Panton-Valentine leukocidin, *sek* and *seq* encoding enterotoxin K and Q, respectively, in the USA300 strains from Chicago were determined by PCR as previously described [@pone.0016419-Diep1], [@pone.0016419-Lina1]. The primers and conditions used for confirmation are shown in the [Table S2](#pone.0016419.s002){ref-type="supplementary-material"}. Results {#s3} ======= Molecular epidemiology of CA- and HA-MRSA {#s3a} ----------------------------------------- The majority of the 19 HA-MRSA isolates from the USA belonged to ST5 (n = 14, 74%). From the five other isolates, four were classified as ST8 and one as ST45 ([Table S1](#pone.0016419.s001){ref-type="supplementary-material"}). Two of the 20 CA-MRSA isolates from the USA were *mecA* negative in the PCR, and were no longer considered MRSA. From the remaining 18 CA-MRSA isolates from the USA, 14 were typed by PFGE as USA300 and 13 represented the USA300-0114 subtype. One isolate (S16) had a slightly different PFGE subtype and differed by two bands ([Figure 1](#pone-0016419-g001){ref-type="fig"}). MLST was in agreement with these data, since 13 USA300 strains were ST8. One isolate (S08) represented ST858, a single locus variant of ST8 ([Table 1](#pone-0016419-t001){ref-type="table"}). All isolates, except one, belonged to *spa*-type t008. That isolate (S06) had a new *spa*-type, t4913 (11-19-36-21-17-34-24-34-22-25), one repeat difference with t008 ([Table 1](#pone-0016419-t001){ref-type="table"}). {#pone-0016419-g001} 10.1371/journal.pone.0016419.t001 ###### Demographic and clinical characteristics of the 18 CA-MRSA isolates from the USA based on MicroScan. {#pone-0016419-t001-1} Isolate Isolation date PFGE type ST (CC) *spa*-type SCC*mec* Isolation site Resistance[a](#nt101){ref-type="table-fn"} --------- ---------------- -------------------- --------- ------------ ---------- ------------------ -------------------------------------------- S01 07-02-2004 USA100 (subtype b) 5 t002 II Blood β,CD, L S02 07-02-2004 USA100 (subtype a) 5 t002 II Skin/soft tissue β,CD, L S03 07-04-2004 USA300-0114 8 t008 IV Skin/soft tissue β,E S04 07-04-2004 USA300-0114 8 t008 IV Blood β S05 07-03-2004 USA400 (subtype c) 1 t008 IV Skin/soft tissue β S06 07-07-2004 USA300-0114 8 t4913 IV Skin/soft tissue β,E S07 07-06-20 04 USA300-0114 8 t008 IV Skin/soft tissue β,E S08 07-09-2004 USA300-0114 858 (8) t008 IV Skin/soft tissue β,E S10 07-12-2004 USA300-0114 8 t008 IV Skin/soft tissue β,E S11 07-16-2004 USA300-0114 8 t008 IV Skin/soft tissue β,E S12 07-17-2004 USA100 (subtype b) 5 t002 II Cathether β,E,L S13 07-16-2004 USA300-0114 8 t008 IV Skin/soft tissue β S15 07-20-2004 USA300-0114 8 t008 IV Skin/soft tissue β,E S16 07-22-2004 USA300 8 t008 IV Blood β,E,R S17 07-24-2004 USA300-0114 8 t008 IV Skin/soft tissue β,E,T S18 07-26-2004 USA300-0114 8 t008 IV Skin/soft tissue β,E S19 07-28-2004 USA300-0114 8 t008 IV Skin/soft tissue β,E S20 07-28-2004 USA300-0114 8 t008 IV Skin/soft tissue β,E,T Resistance patterns were determined with MicroScan (Dade Behring INC. West Sacramento, CA, USA). β = β-lactam antbiotics, E =  erythromycin, R =  rifampicin, L =  quinolones, CD =  clindamycin, G =  gentamicin and T =  tetracycline. The other four CA-MRSA from the USA belonged to PFGE type USA400 (ST1) (n = 1) and USA100 (ST5) (n = 3) ([Table 1](#pone-0016419-t001){ref-type="table"}). The CA-MRSA isolate from The Netherlands belonged to ST80. The infections caused by the HA-MRSA isolates were more diverse than the infections caused by the CA-MRSA strains ([Table S1](#pone.0016419.s001){ref-type="supplementary-material"}). The patients with HA-MRSA suffered from pneumonia (n = 7), skin and soft tissue infections (n = 7), urinary tract infections (n = 3), sepsis (n = 8) and a bone infection (n = 1). Three isolates came from colonized patients and were collected with nasal swabs ([Table S1](#pone.0016419.s001){ref-type="supplementary-material"}). The majority of the CA-MRSA isolates from the USA (n = 14, 78%) caused skin and soft tissue infections. In three patients (S01, S04 and S16), the bacteria were isolated from blood. In one other (S12) case the bacteria were isolated from a catheter ([Table 1](#pone-0016419-t001){ref-type="table"}). Two of the 14 USA300 CA-MRSA isolates (S04 and S13) were only resistant to β-lactam antibiotics ([Table 1](#pone-0016419-t001){ref-type="table"}). The other 12 were also resistant to erythromycin. One isolate (S16) was also resistant to rifampicin. Differences in gene content between CA- and HA-MRSA {#s3b} --------------------------------------------------- Fully annotated microarray data have been deposited in BµG\@Sbase (accession number E-BUGS-108; <http://bugs.sgul.ac.uk/E-BUGS-108>). Hierarchical clustering based on the CGH-data showed no grouping of isolates based on community or hospital origin but on the basis of their ST type, which is in concordance with previous observations. Isolates with ST5 and -8 are split in different groups ([Figure 2](#pone-0016419-g002){ref-type="fig"}) [@pone.0016419-Lindsay1]. The 19 CA-MRSA isolates were dispersed among all isolates represented on the dendogram ([Figure 2](#pone-0016419-g002){ref-type="fig"}). This finding indicates that the CA-MRSA do not share a common and unique genetic repertoire. The PVL genes were present in all CA-MRSA isolates and absent in all HA-MRSA isolates based on the microarray data and confirmed by PCR. In addition, PCR demonstrated that *arcA, sek* and *seq* were present in all analyzed USA300 CA-MRSA. {#pone-0016419-g002} Genomic variation among USA300 CA-MRSA {#s3c} -------------------------------------- There were remarkable genetic differences between the 14 USA300 CA-MRSA isolates. A difference in gene content was not confined to one isolate but was found in nine of the 14 isolates ([Table 2](#pone-0016419-t002){ref-type="table"}), although isolate S04 had most variation compared to the other isolates. Furthermore, differences in plasmids were found among the 14 isolates from the USA. Although most isolates (n = 9, 65%) contained two plasmids of similar size (data not shown), four isolates did not contain any plasmids (S3, S6, S15 and S18) and one isolate (S20) had three plasmids, which were of the same size as the plasmids of USA300FPR3757. 10.1371/journal.pone.0016419.t002 ###### Genetic differences between the 14 USA300 isolates based on comparative genomic hybridization data and PCR. {#pone-0016419-t002-2} Gene MRSA252 (ORF) Location Results for --------------------------------------------------------------- --------------- ------------ ------------------------------------ ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- DNA repair protein SAR0617 MGE \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \- \+ \+ \+ \+ \+ Resolvase SAR0719 MGE \+ \- \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \- \+ Serine protease-like C SAR1906 Chromosome \- \+ \- \+ \+ \+ \- \+ \+ \+ \+ \+ \+ \+ \+ \+ Serine protease-like E SAR1902 Chromosome \- \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ Membrane protein SAR2132 MGE \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \- \+ \+ \+ \+ Hypothetical protein SAR1682 MGE \+ \- \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \- \- Phage protein SAR1554 MGE \- \+ \- \- \- \- \- \- \- \- \- \- \- \- \+ \+ Phage protein SAR2066 MGE \+ \+ \+ \+ \+ \+ \+ \- \+ \+ \+ \+ \+ \+ \+ \+ Exported protein (PCR)[a](#nt102){ref-type="table-fn"} SAR2565 Chromosome \+ \- \+ \+ \+ \+ \+ \+ \- \+ \+ \+ \+ \+ \- \- Exported protein (microarray)[b](#nt103){ref-type="table-fn"} SAR2565 Chromosome NR[c](#nt104){ref-type="table-fn"} \- NR NR NR \+ NR NR NR NR NR \- \+ \- \- \- Hypothetical protein SAR0056 MGE \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \- \+ \+ The results of the Exported gene (SAR2565) in the confirmation PCR, of which the primers are shown in [Table S2](#pone.0016419.s002){ref-type="supplementary-material"}. The results of the Exported gene (SAR2565) gained by the hybridization of the 14 USA300 isolates on the microarray earlier described. NR: not reliable. CGH (with PCR confirmation) indicated that nine genes were variably present among the 14 USA300 strains ([Table 2](#pone-0016419-t002){ref-type="table"}). In addition, one gene present in the sequenced USA300FPR3575 and USA300TCH1516 strains was absent in all 14 analyzed USA300 CA-MRSA isolates. This gene (SAR2565) encodes an exported protein ([Table 2](#pone-0016419-t002){ref-type="table"}). Seven of the in total 10 genes that were variably present or absent in USA300 isolates, were located on known mobile genetic elements (MGE) in the published genomes ([Table 2](#pone-0016419-t002){ref-type="table"}), whereas the three other genes were located in regions that are not considered to be mobile. This indicates that the genes that are variably present in USA300 are not located adjacent to each other, but dispersed across the genome (data not shown). The 10 genes encode for serine protease-like E protein (*splE*) (SAR1902), serine protease-like C protein (*splC*) (SAR1906); a resolvase (*tnpR*) (SAR0719), a putative DNA repair protein (SAR0617), the exported protein gene SAR2565, a gene encoding a membrane protein (SAR2132), two genes encoding phage proteins (SAR1554 and SAR2066) and two genes encoding hypothetical proteins (SAR1682 and SAR0056). The differentially present genes *splE* and *splC* are part of a 5 kb *spl* operon, encoded on genomic island νSaβ which is present in most, but not in all *S. aureus* strains [@pone.0016419-Reed1]. The other four genes of the *spl-*operon (*splA, splB, splD,* and *splF*) were present in all USA300 isolates analyzed by CGH. The resolvase gene (SAR719), which is absent in isolate S04, is located on a 26 kb plasmid (pUSA300HOUMR) in USA300TCH1516. This was confirmed by Southern blot (data not shown). The membrane protein gene (SAR2132), absent in isolate S18 has 58.62% (amino acid) similarity with the hypothetical protein YeeE in *E. coli* which is a putative transport system permease protein. This gene is present in the majority of the sequenced *S. aureus* strains (data not shown). There were two genes that were differentially present and that tentatively encode phage proteins. One of the hypothetical phage proteins (SAR2066) is similar to the C-terminal region of *Streptococcus thermophilus* bacteriophage Sfi21 hypothetical protein and is only present in the sequenced USA300 isolates, MRSA252, Mu3 and Newman strains. The second phage protein (SAR1554) is 97.96% (amino acid) similar to a *S. aureus* prophage phiPV83 protein which is differently present in the sequenced strains MRSA252, JH9, JH1 and S0385**.** Discussion {#s4} ========== The rapid emergence of specific MRSA clones, like USA300, first in the community followed by dispersal in hospitals is still largely unexplained. Our study confirmed previous findings that all CA-MRSA clones do not share a common genetic repertoire that is unique to these clones other than PVL toxin genes, which are present in most USA300 isolates [@pone.0016419-Ho1], [@pone.0016419-Lina1], [@pone.0016419-Nimmo1]. Despite being ubiquitously present in our set of USA300 isolates, USA300 isolates without the PVL and SCC~ACME~ genes have been described recently [@pone.0016419-Larsen1], [@pone.0016419-Monecke1]. This could mean that PVL and probably SCC~ACME~ have a less significant role in CA-MRSA virulence than previously assumed. In contrast to previously published studies by Tenover et al. and Kennedy et al., we found an unexpected high number of genetic differences among 13 PFGE subtype USA300-0114 clones isolated within the time-frame of one month from a single location [@pone.0016419-Kennedy1], [@pone.0016419-Tenover1]. Of the genes that were differentially present among the USA300 isolates from Chicago, or even completely absent, compared to the sequenced USA300 isolates, eight were identified for the first time as being differentially present among CA-MRSA, thus representing non-core CA-MRSA genes. However, it should be noted that divergent gene orthologues that are not represented on the array, may be present among the 14 USA300 isolates because USA300 was not used to create the microarray. Next to that the majority of the sequenced isolates used to generate the microarray described in this study were laboratory strains, which were collected years before the clinical USA300 specimens were accumulated, making it even more likely that USA300 strains and other CA-MRSA clones may contain novel genetic elements absent in the older strains. Non-core genes contribute significantly to the overall diversity of gene repertoires in a species. The vertically transmitted core genome encodes fundamental cellular processes, and the horizontally transmissible accessory genome encodes for a variety of secondary metabolites, resistance to specific toxins, virulence factors or antibiotics [@pone.0016419-Kazazian1]. Seven of the in total 10 genes that were variably present or absent in USA300 isolates, were located on mobile genetic elements (MGE) ([Table 2](#pone-0016419-t002){ref-type="table"}). This was as expected, because mobile genetic elements easily account for significant gene diversity among strains that are closely related with respect to the core genome. However, 3 of the 10 genes were located in regions that are not considered to be MGEs. In addition, the genetic differences, which are located on the MGEs involve only single genes and the isolates did not lose or acquire a complete genetic element. But we also found variation in the plasmid content of the 14 USA300 isolates. From literature (Kennedy et al. and Tenover et al.) it seems that the USA300 isolates used in these studies [@pone.0016419-Kennedy1], [@pone.0016419-Tenover1] were less diverse. The majority of these isolates are collected from different geographic regions and before 2004. This could suggest the USA300 isolates are less genetically diverse before 2004. However, in order to be certain about this, it will be necessary to analyze isolates from the same hospital in Chicago before 2004. Unfortunately we do not have these data. Two of the variably present genes are located on the *spl*-operon. The *spl* genes are positively controlled by *agr,* one of the regulators responsible for the global regulation of virulence factors in *S. aureus,* which is conserved in all staphylococcal species. The genes are most similar to a V8 protease, which can cleave the heavy chain of human immunoglobulin classes in vitro. In 64% of the isolates the s*pl* operon was present and no obvious role in virulence was demonstrated in intraperitoneally injected rats [@pone.0016419-Reed1], [@pone.0016419-Arvidsson1]. The observed genetic differences between the 14 USA300 isolates, recovered within the time-frame of one month and from a single location, suggests continuous evolution of this clone. In fact one may argue that following a period of intense and rapid clonal dispersion, USA300 has now entered a period of development of a polyclonal lineage rather than a single dispersing clone, which was also suggested by Kennedy et al [@pone.0016419-Kennedy1]. Whether the observed differences in gene content indicate random loss or acquisition of genes or reflect local adaptation, and whether these differences affect virulence remains to be investigated. Supporting Information {#s5} ====================== ###### **Characteristics of HA-MRSA isolates used in this study.** ^a^ Sequence type (ST) with clonal complex between brackets. (DOC) ###### Click here for additional data file. ###### **PCR primers used to validate the 10 genetic differences.** ^a^ MRSA252 ORF: MRSA252 open reading frame is the MRSA252 gene-number, which corresponds with the specific gene. (DOC) ###### Click here for additional data file. We acknowledge BuG\@s (the Bacterial Microarray Group at St George\'s, University of London) for supply of the microarrays and advice. From the BµG\@s group we would like to thank particularly K. Gould for array training and advice. **Competing Interests:**The authors have declared that no competing interests exist. **Funding:**M.J.M.B. was supported by the Netherlands Organization for Scientific Research (VICI NWO Grant 918.76.611). We acknowledge The Wellcome Trust for funding BuG\@S (the Bacterial Microarray Group at St George\'s University of London) for supply of the microarray and advice. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [^1]: Conceived and designed the experiments: RJLW AF MB NC. Performed the experiments: NC TR. Analyzed the data: NC JL AW JH. Contributed reagents/materials/analysis tools: MB RAW JL. Wrote the paper: NC RJLW AF MB. Corrected the manuscript: JL TR JH RAW AW.

Background and Significance =========================== Drug therapy in pediatric patients is a complex process. Children are not simply small adults, but are subject to continuous growth and variation in drug-metabolizing enzyme activity, which requires continuous adaption of dosages. Extrapolation of adult data to guide drug dosing in children requires great diligence and cannot replace clinical trials on efficacy, safety, and pharmacokinetics in pediatric patients. [@JR190022ra-1] [@JR190022ra-2] For most drugs, the costs of specific clinical trials and the development of an age-appropriate formulation (e.g., a liquid with good palatability, dosing accuracy for low doses, and without inappropriate preservatives) exceed the possible revenue, especially for conditions that seldom occur in minors. Both the U.S. and the European Union have introduced legislation for the development of pediatric medicines. Pharmaceutical companies are granted an additional 6 months\' patent extension as an incentive for pediatric trials on newly developed drugs. According to the pediatric regulation, since 2007 for all new drugs early in the developmental stage, a pediatric investigation plan has to be delivered, that will be assessed and agreed by the pediatric committee of the European Medicine Agency. [@JR190022ra-3] This is mandatory for drug licensing. Nonetheless, there is still a substantial number of medicines on the market that are not licensed for pediatric populations. Thus, off-label use is common in both ambulatory and hospital settings. [@JR190022ra-4] [@JR190022ra-5] [@JR190022ra-6] The Study of Health of Childs and Youth in Germany (KiGGS study) found that nearly one-third of medicines used in pediatric outpatients were not licensed for their age. [@JR190022ra-4] The younger the patient, the higher the prevalence of off-label uses. [@JR190022ra-4] [@JR190022ra-7] This prevalence is higher in the inpatient setting---and especially in intensive care units. [@JR190022ra-8] In Germany, there is currently no database that provides publicly accessible evidence-based information on drug dosages in children and adolescents. However, worldwide there are very few formularies providing evidence-based dosing recommendations and transparency on sources used. [@JR190022ra-9] Therefore, the information provided may not correspond to the best available evidence. Especially for the widespread off-label use, these details are essential to find a rational, best evidence-based therapy for a patient and at the same time minimize the risks during drug application. In other countries, such as the United Kingdom, the Netherlands, and Switzerland, a national database for pediatric medicines has brought significant progress in pediatric pharmacotherapy. [@JR190022ra-9] With the implementation of an electronic prescribing system at the Department of Pediatrics and Adolescent Medicine at the University Hospital Erlangen, Germany, in 2012, dosing recommendations based on a systematic research of clinical pharmacological evidence were established for internal use. [@JR190022ra-10] To expand access to this comprehensive information to a wider audience of pediatricians in Germany, initial funding was granted from the German Ministry of Health for the development of a Web-based online platform, termed Pediatric Dosing Recommendations (PaedDos). PaedDos is intended to serve as a German reference database for evidence-based dosing recommendations as well as pharmacological and pharmaceutical drug information in pediatrics. A separate journal article focusing on the pharmaceutical content of the platform is currently being finalized. To achieve a high level of acceptance among medical experts, the development of PaedDos was based on a user-centered design (UCD) approach with its iterative steps. [@OR190022ra-11] In a UCD approach, the end user---in this case the resident doctors, clinicians, and pharmacists---is highly involved in all steps of the development process and the user interface design. Functional scope and operation are tailored to the feedback of the test users. Through cyclic evaluations using various methods (e.g., pluralistic walkthroughs, user tests, thinking-aloud tests [@JR190022ra-12] [@JR190022ra-13] [@BR190022ra-14] [@BR190022ra-15] ), the system is constantly evaluated in terms of its usability and acceptability by the end users. Standardized scoring systems, for example, System Usability Scale (SUS), [@JR190022ra-16] [@JR190022ra-17] [@BR190022ra-18] can be used for objective usability assessment. Research shows that a UCD process increases the usability of applications in health care and thus can improve the productivity of systems, their accessibility, and reduces the risk of harm. [@JR190022ra-19] [@JR190022ra-20] [@JR190022ra-21] This article describes for the first time the development of Web-based online platform for pediatric dosing recommendation in Germany. The specific purpose of this article is: (1) to survey the demand of physicians for an evidence-based knowledge base, to check to what extend the requirements are fulfilled, and which new requirements arise, and (2) to assess a prototype of an online platform with end users in terms of usability and functionality. Methods ======= Initial Prototype Development ----------------------------- Pediatricians from the Erlangen Children\'s Hospital and the project management created a list of initial requirements for the online platform. One of these requirements was to tailor the online platform to usage by three main user groups according to the content, the presentation of the prepared information, as well as the functionality and the design. The first group consists of pediatricians in private practice, including general practitioners with a large proportion of pediatric patients, which later would be the largest and most important user group; the second group comprises clinicians from pediatric hospitals; and the third group are pharmacists, who also need information on dosages of drugs for children and adolescents in their daily work. The initial requirements led to paper mock-ups having a visual basis for discussions around the platform, its design, and its functionalities. After an internal feedback round, the platform and its content were divided into six modules: 1. Information (general information about the active substances). 2. Dosage (dosing recommendations). 3. Special dosage information (information about liver and kidney insufficiency). 4. Side effects and warnings (including overdosing and contraindications). 5. Drug--drug interactions. 6. Preparations (information about the available formulations like flavor). Subsequently, a first interactive prototype was developed, based on common Web technologies (HTML, CSS, JavaScript, PHP, and SQL) and frameworks (W3.CSS, Bootstrap, JQuery, AngularJS). This prototype was evaluated in two rounds with external users previously not involved in the project to analyze to what extent user requirements have already been considered and to derive further requirements. First Evaluation Round---Pluralistic Walkthrough ------------------------------------------------ During a pluralistic walkthrough in July 2017, experts and future end users of the system were invited to evaluate the initial prototype to draw attention to errors and provide information on its acceptance at an early stage of development. A pluralistic walkthrough is a collaborative usability evaluation method that involves both users and members of the product team. In this method, the test moderator presents the participants the system and invites discussion related to usability issues. At this development state, the system contained information in all modules for seven active ingredients. The active substances were selected from different groups (on-demand medication, antibiotics, antiepileptics, etc.). About 100 active substances were listed with dosing recommendations but not yet fully developed with all information in all modules. ### Participants In total, three participants, one representative of each of the main user groups, and the development team took part in the pluralistic walkthrough. ### Procedure The pluralistic walkthrough was divided into two parts. For the first part, three exemplary drug ordering scenarios with clindamycin, ibuprofen, and acetylsalicylic acid were defined. Based on these scenarios, the online platform was presented to the walkthrough participants. Next, the scope of search functions (search for active substances, trade name, indication, and Anatomical Therapeutic Chemical \[ATC\] code) and the live search functionality (search while typing) were demonstrated to the participants. Finally, all participants had the opportunity to test the system themselves with open scenarios and ask questions about the platform\'s user interface. In the second part of the pluralistic walkthrough, the participants were asked to write down both positive and negative aspects of the platform on cards. All cards were collected by the moderator on the flipchart, categorized, and prioritized in an open discussion with the participants. Participants were further encouraged to evaluate the user interface in terms of color, terminology, redundancy, content, and features. In addition, they were motivated to express wishes concerning the functionalities which the current prototype version did not yet meet. The complete walkthrough was recorded with the software Camtasia Studio ( <https://www.techsmith.de/camtasia.html> ) and later transcribed verbatim. The main statements, as well as the results of the categorization and prioritization of recommendations and wishes, were summarized to create an enhanced version of the requirements specification. The joint categorization process led to three requirement priority levels, from "high need for action," via "medium need for action," to "low need for action." Feedback on issues that required only minor adjustments was implemented into the platform immediately after the walkthrough. Second Evaluation Round---Online User Test ------------------------------------------ Prior to the second evaluation round, some requirements defined and analyzed during the pluralistic walkthrough were integrated into the development process to tailor the online platform more to the end users\' needs. The requirements defined under high and medium need for action could not be integrated due to various problems explained in the "Discussion" section. All other requirements were implemented. For the second evaluation round, the number and content of active substances in the database was expanded. For the online test, approximately 50 active substances with information in all modules were available to the users, as well as a total of approximately 150 listed active substances with dosage recommendations. ### Participants For the user test, pediatricians, pediatric clinicians, and pharmacists from Bavaria, Germany, were contacted by the authors. A total of 12 persons could be recruited who were willing to participate in the online test: 6 resident doctors, 3 clinicians, and 3 pharmacists. The chosen distribution reflects the intended distribution of end users after the release of the online platform. None of the participants had previously participated in the pluralistic walkthrough. The participants had no economic interest and were not part of the project team. ### Scenarios For the online user test, two different drug ordering scenarios were defined by the development team for each of the three end-user groups ( [Supplementary Appendix A](#SM190022ra-1){ref-type="supplementary-material"} , available in the online version). During the online user test, the participants got access to the revised prototype and were asked to evaluate the prototype based on their respective two scenarios. All scenarios were developed in collaboration with a pediatrician and pharmacists. ### Online Questionnaire After testing the prototype, the participants were instructed to answer questions about the content, functionality, and user-friendliness. The questions were based on the SUS [@JR190022ra-16] [@JR190022ra-17] [@BR190022ra-18] and a set of self-developed questions ( [Supplementary Appendix B](#SM190022ra-1){ref-type="supplementary-material"} \[available in the online version\]: questions about the design and functionality), focusing on the particular drug dosing information to capture an overall level of usability. All self-developed questions were designed by a usability expert at the Department of Medical Informatics in Erlangen and pretested in terms of understandability. A 5-point Likert scale (ranging from "does not apply at all" \[1\] to "completely applies" \[5\]) was used for numerical evaluation. If a question was answered in negative (\[1\], \[2\], or \[3\]), an additional question was displayed to ask the participant for details in free text. In total, the online questionnaire comprised 90 questions, categorized as follow: - Demographic questions ( *n*  = 11). - SUS questionnaire ( *n*  = 10). - Questions about the technical devices used ( *n*  = 5). - Questions about the scenarios ( *n*  = 12). - Questions about the design and functionality of the online platform ( *n*  = 49). - Positive and negative aspects of the online platform ( *n*  = 2). - Free-text field for notes and wishes of any kind ( *n*  = 1). The following demographic information was collected (with different answer options): user group (physician in private practice, clinician, pharmacist), professional experience in years (little: ≤ 10, medium: \> 10 and ≤ 20, much: \> 20 years), number of prescriptions per day (less: ≤ 10, medium: \> 10 and ≤ 25, many: \> 25 medications per day), and computer knowledge (subjective/own assessment: low, medium, high) to examine the dependencies of the results according to these aspects. ### System Usability Score To ensure global comparability of the system\'s usability with similar international developments and systems, the SUS was calculated. The SUS is a standardized questionnaire with a 5-point scale (ranging from "does not apply at all" to "completely applies"), whereby questions are alternately formulated positively and negatively. The statements relate to a range of aspects of system use, such as complexity, ease-of-use, and learnability. Overall SUS results range from 0 (poor) to 100 (very good). [@JR190022ra-17] [@BR190022ra-18] As described by Brooke, [@JR190022ra-16] the threshold values are worst imaginable (25), poor (38.5), ok (52), good (73), excellent (85), and best imaginable (100). To summarize SUS responses from participants, mean, median, minimum, and maximum were calculated. ### Procedure The online questionnaire was created with the online tool SoSci Survey ( <https://www.soscisurvey.de/> ). The participants had 2 weeks in December 2017 to complete the questionnaire for the two scenarios. In addition, each test user had the opportunity to explore the knowledge base independently, to search for dosing recommendations and information on active substances and to familiarize oneself with the functions, even during the processing of the questionnaire. The test users had full access to the online platform and its contents. ### Data Analysis Through the structure of the questions in 5-point Likert scales, a numerical evaluation of the questions was possible; frequencies were analyzed and means were calculated. With the exception of the SUS questionnaire, a rating of 5 was interpreted as positive and a rating of 1 as negative. Mean differences were compared between the three user groups and concerning a possible influence of information technology knowledge, years of work experience, and the number of daily prescriptions. The text-based answers were examined and scanned for significant statements, clustered, and also checked for dependencies. Results ======= Results of the Pluralistic Walkthrough---First Evaluation Round --------------------------------------------------------------- ### Positive Aspects Especially the clarity of the user interface and the structure were appreciated, so that the users were able to quickly comprehend the user interface layout and system navigation. The different search functions (by list of active substances, dropdown list of live search, search options) turned out to be very user-friendly and were well received by the participants. ### Negative Aspects Negative feedback (wishes, missing functions for a good workflow, and optimization potential of the system regarding design and structure) was categorized as high need for action, medium need for action, and low need for action. High need for action: - Interaction check of the active substance with other active substances (e.g., interactions with isoniazid). - Smart prioritization of active substances for the treatment of children (e.g., display ibuprofen before acetylsalicylic acid in search results for indication fever) based on the experts\' definitions. - A calculator for dosages (age, weight based on active substance), glomerular filtration rate according to Schwartz et al, [@JR190022ra-22] [@JR190022ra-23] and body surface. Medium need for action: - Compressing and standardization of the structure of the dosing recommendation table (previously 6 subdivisions, recommended only 2, maximum 4) ( [Fig. 1](#FI190022ra-1){ref-type="fig"} ). Low need for action: - Add application time (e.g., before/after a meal) to some active substances. - Applying a uniform font style and font size. - Addition of search function by trade name. The following optimization suggestions were implemented immediately after the walkthrough: - Links to the referenced sources in the individual dosing recommendation for quick and easy verification. - Prioritization of the drug monographs and dosing recommendations when the page is opened (not showing disclaimer). - Disclaimer as menu item, but not as main page. - Link to official form for spontaneous reporting of adverse drug reactions. {#FI190022ra-1} Results of the Online User Test---Second Evaluation Round --------------------------------------------------------- The results of the first evaluation round were implemented and a next prototype version was created, which was evaluated by an online test with end users. In the following, the results according to the different question sections are presented. ### Technical Devices Used Most participants ( *n*  = 10) responded that they would prefer to use PaedDos on a desktop personal computer. Nevertheless, they also expressed demand to use the system on mobile devices (smartphones: *n*  = 8, tablets: *n*  = 5; multiple answer selections were allowed). Therefore, a responsive design of the user interface for future PaedDos versions is recommended. Problems with the use of different browser types were not recorded. ### Design and Functionality of the Online Platform Almost all questions about the functions and design of the online platform received positive or very positive feedback. Except for one question (GP07_04), all averages and medians were above 3 and thus on the positive side of the answer scale ( [Fig. 2](#FI190022ra-2){ref-type="fig"} ). All answers to questions about design and functionality can be seen in [Table 1](#TB190022ra-1){ref-type="table"} . {#FI190022ra-2} ###### Excerpt of questionnaire and answers with mean, median, maximum, and minimum about the design and functions of the online platform ID Question Mean Median Maximum Minimum --------- ------------------------------------------------------------------------------------------------------------------------------------------------- ------ -------- --------- --------- GP02 If I had an automatic dosage calculator on the platform, I would use it 4.58 5 5 2 GP05 The possibility of searching according to the criteria "active substance," "trade name," "indication," and "ATC code" is sufficient for my work 4.64 5 5 3 GP15 The user interface of the online platform is visually attractive (colors, fonts, contrast) 3.83 4 5 2 GP16 The font size was easy for me to read 4.50 5 5 2 GP19 I believe that the online platform has the potential to improve the quality of the medication prescription for children and adolescents 4.67 5 5 3 GP23 If I had access to this online platform, I would most likely use it in the next months 4.58 5 5 4 GP24 Please specify for each module how often you would use it in your clinical routine GP24_01 Module 1: Information 3.33 3 5 2 GP24_02 Module 2: Dosage 4.50 4.5 5 4 GP24_03 Module 3: Special dosage information 3.92 4 5 3 GP24_04 Module 4: Side effects and warnings 4 4 5 3 GP24_05 Module 5: Interactions 3.83 4 5 2 GP24_06 Module 6: Preparations 4.08 4 5 3 Abbreviation: ATC, Anatomical Therapeutic Chemical. Note: Full questionnaire available in [Supplementary Appendix B](#SM190022ra-1){ref-type="supplementary-material"} (available in the online version). The function for searching via ATC codes was perceived as unimportant. As already expressed in the pluralistic walkthrough, there is a great demand for an automatic online dosage calculator. The free-text responses confirmed this pattern, insofar as four users explicitly mentioned a dosage calculator. ### Strengths and Weaknesses of the Online Platform The free-text answers to the pros and cons of the online platform were clustered. The classified answers to the strengths of the online platform are shown in [Table 2](#TB190022ra-2){ref-type="table"} . Multiple answers of a participant that were categorized in the same category were summed. The user-friendliness was perceived as positive by 8 out of 12 participants (e.g., "Fast, clear, easy"). ###### Clustered free-text answers (with examples) to the strengths of the online platform ------------------------------------------------------------------------------------------- Category Frequency ------------------------------------------------------------------------------- ----------- **Usability**\ **14** "Easy, fast", "Quick overview \[...\]", "\[...\] simple availability \[...\]" **Dosing recommendations**\ **6** "Therapy duration visible", "Dosage available for various indications" **Indication**\ **5** "Disease specific listing", "Linking drugs with indications" Preparations 4 Special dosage and application information 3 Search function 3 Active substance information 2 Approval status 1 Reference (transparency) 1 ------------------------------------------------------------------------------------------- The presentation, the information content, and the quick availability of the dosing recommendations have been explicitly named a total of 6 times as one of the great strengths of the system (e.g., "Fast dosage finding"). Particular emphasis was placed on the combination of indications and diseases for active substances. This was also named as one of the strengths by the participants (e.g., "Dosage can be viewed for different indications"). Participants\' free-text responses to existing weaknesses, suggestions for improvement, and wishes were classified into four categories ( [Table 3](#TB190022ra-3){ref-type="table"} ). Multiple answers of one participant that were categorized in the same category were summed. ###### Clustered free-text answers (with examples) to the weaknesses of the online platform ------------------------------------------------------------------------------------- Answer Frequency ------------------------------------------------------------------------- ----------- **Content and further information**\ **17** "Further drugs", "\[...\] side effects according to clinical relevance" **Design**\ **10** "Graphical representation", "standardized dose display" **Functions**\ **9** "List therapy alternatives", "Alternative drugs references \[...\]" **(Dosage-) Online-Calculator**\ **5** "Automatic calculation \[...\] after entering the weight" ------------------------------------------------------------------------------------- SUS Results ----------- Of the total of 12 participants, 2 had to be dropped from the SUS calculation because of partly missing answers, thus invalidating their results for the SUS. With an average value of 79.5 and a median of 81.25, the prototype of the online platform is classified between "good" and "excellent" and is thus regarded as an "acceptable system." [@JR190022ra-16] Every participant rated the system as "accepted" (minimum: 52.5; maximum: 97.5). Physicians in private practice tend to rate the system slightly lower, while pharmacists rate it slightly higher. Further, SUS results did not show any significant deviations according to the respective parameters ( [Table 4](#TB190022ra-4){ref-type="table"} ). ###### SUS mean and median according to the dependencies of the analyzed parameters Dependency on... Resident doctors Clinicians Pharmacists --------------------------------- ------------------ ----------------- ---------------- User group Average: 74 Average: 81.25 Average: 87.5 Median: 75 Median: 81.25 Median: 85 **Less** **Medium** **Much/Many** Experience in the profession Average: 80 Average: 78.5 Average: 81.25 Median: 82.5 Median: 80 Median: 81.25 Number of prescriptions per day Average: 78.75 Average: 85.625 Average: 80.83 Median: 78.75 Median: 86.25 Median: 80 **Low** **Medium** **High** Computer knowledge Average: - Average: 76.68 Average: 83.75 Median: - Median: 77.5 Median: 83.75 Abbreviation: SUS, System Usability Scale. In summary, the possible end users have expressed themselves very positively about the online platform and are already describing it as practical. Discussion ========== Principal Findings ------------------ The results of the study clearly show the added value that such a platform would provide for pediatric and adolescent medicine. Nine participants agreed completely that the platform has the potential to increase the quality of the medication prescription for children and adolescents. All participants stated that they would most likely use the online platform if they had access to this page. This confirms that the health care professionals in pediatrics and adolescent medicine want and need such an online platform and consider it to be useful. A serious problem in pediatrics is the extensive off-label use, where the summary of product characteristics rarely provides sufficient information for dosing of drugs in children and adolescents. Health care professionals, such as physicians and pharmacists, need to retrieve information from various sources, which often do not provide the evidence required to the intended uses. This is time-consuming, incompatible with the stressful daily routine, and entail an increased risk for the patients. PaedDos aims to provide children and adolescents with comprehensive, evidence-based dosing information and thus significantly supports pediatricians in the prescribing and pharmacists in the dispensing and verification process. During the pluralistic walkthrough, the use of varying fonts was criticized, leading to redesign and font standardization prior to the online user test. With the exception of 3 participants, everyone was completely satisfied with the font size of the system. This illustrates that the close user involvement and early system adaption were positively accepted. The positive feedback from the participants of the user test indicates that the UCD approach provides an effective way of structuring and simplifying the development process. Moreover, it might make the development comprehensible to end users. UCD facilitates the identification of requirements for design, function, and content, both on a general level and with respect to specific details. The implemented functionality related to those requirements that resulted from the pluralistic walkthrough in the first round (e.g., to link the source information) received a positive feedback when they were evaluated in the second round. This might serve as another example for the advantages of a UCD approach. The results of the user test indicate that the implemented search functions are sufficient for users to obtain information. The division of the contents into 6 modules made it possible to individually assess the added value of each module. The participants stated that the extent of use of dosing information (module 2) in clinical routine is expected to be "often to always." On average, users would often use information on specific dosage information (module 3), side effects and interactions (module 4 and module 5), and information on existing formulations in everyday clinical practice (module 6). Such a knowledge base should therefore not only contain information on the dosing recommendations and make them available to users, but should also provide further knowledge and evidence. General information on active substances, as offered in module 1 on the online platform, would be used occasionally by participants. Even if this demand is lower than for the other modules, it is nonetheless present, and it is therefore reasonable to integrate this information into the online platform. Two main points of criticism were expressed by the participants with the scope of the evaluations, which need to be discussed in detail. First, the presentation of the dosing recommendations was criticized, in particular the table width. Second, the demand for an online calculator was stated several times. The variety of information (single dose, daily dose, maximum daily dose, frequency, and standard dose) increases the width of the table of dosing recommendations, thereby forcing the user to scroll sideways, especially on screens of normal or small size. Nevertheless, it is necessary to present each of the different contents, since for some drugs the description of a single dose is important, while for others the description of the standard dose and frequency is important. As a result, some table cells are empty ( [Fig. 1](#FI190022ra-1){ref-type="fig"} ). It would be possible to hide them, but at the cost of fast information extraction, since the user would be forced to check the information twice. Therefore, it is important to always provide the information in the same place, or alternatively to apply an unambiguous label. Labeling options could be colors or symbols that would need to be checked in a further evaluation round. The pluralistic walkthrough and the online test have shown a strong demand for an online dosage calculator to facilitate daily work with pediatric medication and to improve the quality of care. The implementation of a dosage calculator would be especially helpful if PaedDos would be integrated in a providers\' electronic health record (EHR) system and input parameters for such formula (e.g., patient age, weight, and body surface) would be directly drawn from the patients\' medical record. Actually such a feature would be a core functionality of a pediatric EHR [@BR190022ra-24] and would be a future extension of our platform. However, such an integrated decision support functionality could fall under the regulations of the Medical Device Act (German: Medizinproduktegesetz [@OR190022ra-25] ) as well as Directive 95/46/EC, [@OR190022ra-26] and was thus not yet in the focus of our current research. To the question "What would be your reasons for nonuse" of an integrated calculator, a physician in private practice answered "False safety," which is an important point to consider. Doctors and others are using devices and systems that are not transparent from the user\'s point of view, which means that they have to rely on the system if they intend to use it. In a proven system, doctors could blindly accept dosing recommendations, which in the worst case could have serious consequences for the patient. It should therefore be noted that neither a dosage calculator nor an online platform for dosing recommendations can replace doctors\' decisions and knowledge, but can only support them. The doctor is still responsible for the medication, which makes the transparency of dosage calculations so important. The results showed a demand for advanced functions related to drug safety, such as an interaction check, or information about potential therapeutic alternatives. Such functions require that the necessary knowledge is made available to the platform. However, such extended functions make the validation procedure of the system and its contents more complex. The current PaedDos prototype can be seen as a starting point and might be extended in the future. Comparison with Other Online Platforms -------------------------------------- In the search for comparable systems, two systems were examined in more detail. The "kinderformularium" from the Netherlands [@OR190022ra-27] is a freely accessible platform for dosing recommendations in pediatrics published in 2008. Like PaedDos, this platform contains information on the level of evidence of dosage recommendations, systematic primary literature research, license status, national trade names, side effects and warning for use in children, contraindications, interactions, and adjustments in renal insufficiency. The information on dosages is not displayed in tabular form, but as a bullet list of text elements. The "kinderformularium" provides an implemented and CE certified calculator, which supports to combine the dosing recommendations with patient--individual variables, thus enabling the calculation of a patient--individual recommendation. [@JR190022ra-9] The University Children\'s Hospital Zurich has a Web site "kinderdosierung.ch" with information on dosing recommendations for pediatrics [@OR190022ra-28] only, but without warnings, side effects, interactions, etc. This platform focuses on inpatient drugs and is in German language like PaedDos. The system provides a calculator, which highlights the appropriate age group in the information list, copies it to the top of the site for easier reading, and calculates the dosing to the manually inserted patient parameters. The page is only accessible to qualified personnel and requires a login. All three platforms are Web-based, evidence-based, independent of the pharmaceutical industry, and structured according to drug, indication, route of application, age, and weight. However, no scientific publications were found describing technical details and evaluating the development process for "kinderformularium" and "kinderdosierung.ch." No studies could be found which address the usability aspects with the SUS of online platforms for dosing recommendations in pediatrics. Limitations ----------- Three limitations of the study have to be considered. First, the pluralistic walkthrough was conducted with only one representative from every end-user group. Actually, according to Bias, 6 to 10 participants are recommended, [@BR190022ra-29] but due to immense recruiting difficulties in this project phase (nonavailability of the physicians for the pluralistic walkthrough in addition to their professional activity) and time constraints of the project, this was not possible. For this reason, a sample size of three users representatives of the target audience was chosen, which according to Riihiaho [@JR190022ra-30] is also sufficient for this method. However, this limitation was compensated by subsequent system iterations and evaluations. Second, a total of 12 participants of the online test does not represent the entire PaedDos end-user group. Nevertheless, it is assumed that approximately 5 participants are sufficient to identify at least 80% of the major usability problems. [@OR190022ra-31] [@JR190022ra-32] [@OR190022ra-33] [@OR190022ra-34] In Brooke\'s retrospective study on SUS, [@JR190022ra-16] Tullis and Stetson [@OR190022ra-35] are referenced as having shown that it is possible to get reliable results with samples of 8 to 12 users. In addition, further iterations and evaluations are planned within the UCD, which will later include a larger sample. Third, the user test has been performed in a test setting and not in a real-world environment. Thus, the extent to which the actual end users are willing or able to use the system in their daily work cannot be anticipated with certainty. The need for an Internet connection or problems with the interoperability of the respective information system of the doctor\'s practice, pharmacy, or clinic could lead to nonuse of the online platform. This has to be examined in future field tests. Conclusion ========== There is a strong demand for an evidence-based pediatric dosage information system among resident doctors, clinicians, and pharmacists, which motivated the development of the PaedDos prototype. Applying an UCD approach during system development resulted in a rating between "good" and "excellent" (mean: 79.5, median: 81.25). Thus, the PaedDos prototype is regarded as an "acceptable system." The implemented search variants, such as live search, to get quickly to the desired information in different ways, received excellent feedback. The UCD approach proved to be a beneficial tool for optimizing usability. There is a strong demand for patient--individual support functions, in particular a dosage calculator. It has to be investigated in a field trial, similar to the studies described in Ateya et al [@JR190022ra-36] and Kim et al, [@JR190022ra-37] whether and to what extent the developed platform prevents dosing errors and in which ways it can support physicians. Future work will focus on the integration of additional drug information for pediatrics, the development of the responsive design, the creation of an app version for the user interface, and the integration of an online calculator. Clinical Relevance Statement ============================ As of now, no system providing evidence-based dosing information for children in Germany is publicly available for pediatricians in private practice, clinicians from pediatric hospitals, and pharmacists, who need information on dosages of drugs for children and adolescents. The aim of the PaedDos project was to establish a national Web platform for pediatric drug information. The platform presented in this study is a first prototype that was iteratively evaluated by real end users to provide such information for doctors and pharmacists in their daily work. Multiple Choice Questions ========================= 1. Which is the most explicitly mentioned additional feature for an online platform about dosing recommendations in pediatrics? A. Interaction check. B. Dosage calculator. C. List of alternative therapies. D. Interoperability to EPRs. **Correct Answer:** The correct answer is option b. In the scope of this study, interaction check and listing of alternative therapies was less named than a dosage calculator. A possible connection between the platform and an internal system or the electronic patient records (EPRs) was mentioned in a conversation with only one participant after the survey. 2. What is the problem with such a state-certified recommendation system? A. Parents of patients could complain in case of complications. B. If the system crashes, prescribing is not possible. C. Doctors could trust the implemented system too much. D. The doctor will be replaced by an electronic system. **Correct Answer:** The correct answer is option c. Information on this platform is aligned for health care professionals. To avoid misunderstandings of parents with regard to the dosing, the database should be restricted to health care professionals only. In case of a system crash, the doctor can still prescribe medication. A technical tool will never be able to replace a doctor, but is always there to support him. Like a stethoscope, an online reference book could also become a tool in the daily work of a doctor. Doctors still need to keep themselves informed. The present work was performed in (partial) fulfillment of the requirements for obtaining the degree "Dr. rer. biol. hum." from the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) (WR). **Conflict of Interest** None declared. Ethical approval was not required. Supplementary Material ====================== ###### Supplementary Material ###### Supplementary Material

I.. Introduction {#sec1} ================ Eyes and their movements are important in expressing a person\'s desires, needs and emotional states [@ref1]. The significance of eye movements with regards to the perception of and attention to the visual world is certainly acknowledged since it is the means by which the information needed to identify the characteristics of the visual world is gathered for processing in the human brain. Hence, robust eye detection and tracking are considered to play a crucial role in the development of human-computer interaction, creating attentive user interfaces and analyzing human affective states. Head movement is also found to be a natural, simple and effective way of pointing to objects, interaction and communication. Thus, head movement detection has received significant attention in recent research. One of the various purposes for head movement detection and tracking is to allow the user to interact with a computer. It also provides the ability to control many devices by mapping the position of the head into control signals. Eye tracking and head movement detection are widely investigated as alternative interface methods. They are considered to be easier to use than other methods such as voice recognition or EEG/ECG signals. They also have achieved higher accuracy and performance. In addition, using eye tracking or head movement detection as alternative interface, control or communication methods is beneficial for a wide range of severely disabled people who are left with minimal ability to perform voluntary motion. Eye and head movements are the least affected by disabilities because, for example, spinal cord injuries do not affect the ability to control them, as they are directly controlled by the brain. Combining eye tracking and head movement detection can provide a larger number for possible control commands to be used with assistive technologies such as a wheelchair. There are many approaches introduced in literature focusing on eye tracking. They can be used as a base to develop an eye tracking system which achieves the highest accuracy, best performance and lowest cost. Head movement detection has been receiving growing interest as well. There are many proposed approaches. Some approaches may be implemented using low computational hardware such as a microcontroller due to the simplicity of the used algorithm. This paper presents a survey of different eye tracking and head movement detection techniques reported in the literature along with examples of various applications employing these technologies. The rest of the paper is outlined as follows. Different methods of eye tracking are investigated in [Section II](#sec2){ref-type="sec"}. Fields of applications for eye tracking are described briefly in [Section III](#sec3){ref-type="sec"}. [Section IV](#sec4){ref-type="sec"} discusses several head movement detection algorithms and the applications of this technology are presented in [Section V](#sec5){ref-type="sec"}. Systems which use a combination of eye tracking and head movement detection are described in [Section VI](#sec6){ref-type="sec"}. Some of the commercial products of eye tracking and head movement detection are presented in VII. The results of using the investigated eye tracking and head movement detection algorithms are reported in [Section VIII](#sec8){ref-type="sec"}. Finally, [Section IX](#sec9){ref-type="sec"} draws the conclusions. II.. Eye Tracking {#sec2} ================= The geometric and motion characteristics of the eyes are unique which makes gaze estimation and tracking important for many applications such as human attention analysis, human emotional state analysis, interactive user interfaces and human factors. There are many different approaches for implementing eye detection and tracking systems [@ref2]. Many eye tracking methods were presented in the literature. However, the research is still on-going to find robust eye detection and tracking methods to be used in a wide range of applications. A.. Sensor-Based Eye Tracking (EOG) {#sec2a} ----------------------------------- Some eye tracking systems detect and analyze eye movements based on electric potentials measured with electrodes placed in the region around the eyes. This electric signal detected using two pairs of electrodes placed around one eye is known as electrooculogram (EOG). When the eyes are in their origin state, the electrodes measure a steady electric potential field. If the eyes move towards the periphery, the retina approaches one electrode and the cornea approaches the other. This changes the orientation of the dipole and results in a change in the measured EOG signal. Eye movement can be tracked by analyzing the changes in the EOG signal [@ref3]. B.. Computer-Vision-Based Eye Tracking {#sec2b} -------------------------------------- Most eye tracking methods presented in the literature use computer vision based techniques. In these methods, a camera is set to focus on one or both eyes and record the eye movement. The main focus of this paper is on computer vision based eye detection and gaze tracking. There are two main areas investigated in the field of computer vision based eye tracking. The first area considered is eye detection in the image, also known as eye localization. The second area is eye tracking, which is the process of eye gaze direction estimation. Based on the data obtained from processing and analyzing the detected eye region, the direction of eye gaze can be estimated then it is either used directly in the application or tracked over subsequent video frames in the case of real-time eye tracking systems. Eye detection and tracking is still a challenging task, as there are many issues associated with such systems. These issues include degree of eye openness, variability in eye size, head pose, etc. Different applications that use eye tracking are affected by these issues at different levels. Several computer-vision-based eye tracking approaches have been introduced. ### 1.. Pattern Recognition for Eye Tracking {#sec2b1} Different pattern recognition techniques, such as template matching and classification, have proved effective in the field of eye tracking. Raudonis et al. [@ref4] used principal component analysis (PCA) to find the first six principal components of the eye image to reduce dimensionality problems, which arise when using all image pixels to compare images. Then, Artificial Neural Network (ANN) is used to classify the pupil position. The training data for ANN is gathered during calibration where the user is required to observe five points indicating five different pupil positions. The system requires special hardware which consists of glasses and a single head-mounted camera and thus might be disturbing to the patient as it is in their field of view. The use of classification slows the system and hence it requires some enhancements to be applicable. In addition, the system is not considered a real-time eye tracking system. The proposed algorithm was not tested on a known database which means the quality of the system might be affected by changes in lighting conditions, shadows, distance of the camera, the exact position in which the camera is mounted, etc. The algorithm requires processing which cannot be performed by low computational hardware such as a microcontroller. Tang and Zhang [@ref5] suggested a method that uses the detection algorithm combined with gray prediction to serve eye tracking purposes. The GM(1,1) model is used in the prediction of the location of an eye in the next video frame. The predicted location is used as the reference for the region of eye to be searched. The method uses low-level data in the image in order to be fast but there are no experimental results evaluating the performance of the method. Kuo et al. [@ref6] proposed an eye tracking system that uses the particle filter which estimates a sequence of hidden parameters depending on the data observed. After detecting possible eyes positions, the process of eye tracking starts. For effective and reliable eye tracking, the gray level histogram is selected as the characteristics of the particle filter. Using low-level features in the image makes it a fast algorithm. High accuracy is obtained from the system; however, the real-time performance was not evaluated, the algorithm was tested on images not videos and the images were not taken from a known database and, thus, the accuracy and performance of the algorithm may decrease when utilized in a real-world application. Lui et al. [@ref7] suggested a fast and robust eye detection and tracking method which can be used with rotated facial images. The camera used by the system is not head mounted. A Viola-Jones face detector, which is based on Haar features, is used to locate the face in the whole image. Then, Template Matching (TM) is applied to detect eyes. Zernike Moments (ZM) is used to extract rotation invariant eye characteristics. Support Vector Machine (SVM) is used to classify the images to eye/non-eye patterns. The exact positions of the left and right eyes are determined by selecting the two positions having the highest values among the found local maximums in the eye probability map. Detecting the eye region is helpful as a pre-processing stage before iris/pupil tracking. Especially, it allows for eye detection in rotated facial images. This work presented a simple eye tracking algorithm but the results of the method evaluation were not reported and thus the proposed eye tracking method is weak and not usable. Hotrakool et al. [@ref8] introduced an eye tracking method based on gradient orientation pattern matching along with automatic template updates. The method detects the iris based on low level features and motion detection between subsequent video frames. The method can be used in applications that require real-time eye tracking with high robustness against change in lighting conditions during operation. The computational time is reduced by applying down-sampling on the video frames. The method achieves high accuracy. However, the experiments were performed on videos of a single eye, which eliminates all surrounding noise, and a known database was not used. The method detects the iris but does not classify its position. The method requires minimal CPU processing time among other real-time eye tracking methods investigated in this survey. The motion detection approach discussed in this paper is worth being considered in new algorithms to obtain the minimal required CPU processing time in eye tracking applications. Yuan and Kebin [@ref9] presented Local and Scale Integrated Feature (LoSIF) as a new descriptor for extracting the features of eye movement based on a non-intrusive system, which gives some tolerance to head movements. The feature descriptor uses two-level Haar wavelet transform, multi-resolution characteristics and effective dimension reduction algorithm, to find the local and scale eye movement features. Support Vector Regression is used in mapping between the appearance of the eyes and the gaze direction, which correspond to each eye\'s appearance. The focus of this method is to locate the eye without classifying its position or gaze direction. The method was found to achieve high accuracy in iris detection, which makes this method useful in iris detection and segmentation which is important for eye tracking. However, the real-time performance was not evaluated. Fu and Yang [@ref10] proposed a high-performance eye tracking algorithm in which two eye templates, one for each eye, are manually extracted from the first video frame for system calibration. The face region in a captured frame is detected and a normalized 2-D cross-correlation is performed for matching the template with the image. Eye gaze direction is estimated by iris detection using edge detection and Hough circle detection. They used their algorithm to implement a display control application. However, it has an inflexible calibration process. The algorithm was not tested on a variety of test subjects and the results were not clearly reported which requires the algorithm to be investigated carefully before choosing to implement it. Mehrubeoglu et al. [@ref11] introduced an eye detection and tracking system that detects the eyes using template matching. The system uses a special customized smart camera which is programmed to continuously track the user\'s eye movements until the user stops it. Once the eye is detected, the region of interest (ROI) containing only the eye is extracted with the aim of reducing the processed region. From their work, it can be concluded that it is a fast eye tracking algorithm with acceptable performance. The algorithm could be a nice feature to be added to modern cameras. A drawback is that the experiments were not performed using a database containing different test subjects and conditions, which reduces the reliability of the results. In addition, the algorithm locates the coordinates but does not classify the eye gaze direction to be left, right, up or down. ### 2.. Eye Tracking Based on Corneal Reflection Points {#sec2b2} Many computer vision based eye trackers use light reflection points on the cornea to estimate the gaze direction. [Fig. 1](#fig1){ref-type="fig"} shows the corneal reflection points in an eye image [@ref12]. Another name for eye images containing corneal reflection points is Purkinje Image. When using this approach, the vector between the center of the pupil and the corneal reflections is used to compute the gaze direction. A simple calibration procedure of the individual is usually needed before using the eye tracker [@ref2]--[@ref3][@ref4][@ref5][@ref6][@ref7][@ref8][@ref9][@ref10][@ref11][@ref12]. Fig. 1.Corneal reflection points [@ref12]. Yang et al. [@ref13] proposed a scheme which employs gray difference between the face, pupils and corneal reflection points for eye detection. The proposed scheme was tested under a cross-ratio-invariant-based eye tracking system. The test included users wearing glasses and other accessories and the results showed the ability of the system to eliminate the optical reflective effect of accessories and glasses. The scheme first prepares for gaze tracking by a preprocessing stage applied on cropped faces. This is particularly useful in applications which use a very close camera. The results are not detailed and not performed on a database containing various test subjects under different conditions which makes the algorithm weak when considered for use in real-world applications. In addition, the required CPU time was not addressed and thus the algorithm needs optimization to determine whether it works in real-time applications. Yang et al. [@ref14] presented an algorithm based on the difference in the gray level of the pupil region and the iris region. The image of the eye region is binarized and an estimate position of the pupil is detected. The exact position of the pupil is found by the vertical integral projection and the horizontal integral projection. The projection area contains the corneal glints and the pupil. The gray level of the pixels representing reflection points is the highest among all pixels. To apply their presented gaze tracking method, the points of corneal reflection must have known coordinates. The system uses hardware which adds limitations and inflexibility for pupil detection. The algorithm was tested on images containing a part of the face. It needs to be applied on different test subjects to prove the accuracy and performance of the algorithm. The required CPU time was not reported which does not make the algorithm feasible for adoption in real-time applications. However, the experiments showed that the algorithm achieves reasonably high accuracy. ### 3.. Eye Tracking Based on Shape {#sec2b3} Another approach for eye detection and tracking is to find the location of the iris or the pupil based on their circular shape or using edge detection. Chen and Kubo [@ref15] proposed a technique where a sequence of face detection and Gabor filters is used. The potential face regions in the image are detected based on skin color. Then, the eye candidate region is determined automatically using the geometric structure of the face. Four Gabor filters with different directions (0, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\pi/4$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\pi/2$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$3\pi/4$\end{document}$) are applied to the eye candidate region. The pupil of the eye does not have directions and thus, it can be easily detected by combining the four responses of the four Gabor filters with a logical product. The system uses a camera which is not head-mounted. The accuracy of the algorithm is not investigated and the required CPU time is not mentioned which does not make the algorithm preferable for real-world applications compared to other algorithms. Kocejko et al. [@ref16] proposed the Longest Line Detection (LLD) algorithm to detect the pupil position. This algorithm is based on the assumption that the pupil is arbitrary circular. The longest vertical and horizontal lines of the pupil are found. The center of the longest line among the vertical and horizontal lines is the pupil center. The proposed eye tracking system requires inflexible hardware which requires relatively difficult installation. The accuracy is not discussed and the performance of the system might be affected by changes in illumination, shadows, noise and other effects because the experiments were performed under special conditions and did not use a variety of test samples in different conditions. Khairosfaizal and Nor\'aini [@ref17] presented a straightforward eye tracking system based on mathematical Circular Hough transform for eye detection applied to facial images. The first step is detecting the face region which is performed by an existing face detection method. Then the search for the eye is based on the circular shape of the eye in a two-dimensional image. Their work added value to academic research but not to real-world applications. Pranith and Srikanth [@ref18] presented a method which detects the inner pupil boundary by using Circular Hough transformation whereas the outer iris boundary is detected by circular summation of intensity from the detected pupil center and radius. This algorithm can be used in an iris recognition system for iris localization because it is applied to cropped eye images. However, it needs further analysis to obtain its accuracy and real-time performance by applying it on a database containing variant images. Sundaram et al. [@ref19] proposed an iris localization method that identifies the outer and inner boundaries of the iris. The procedure includes two basic steps: detection of edge points and Circular Hough transform. Before these steps are applied, bounding boxes for iris region and pupil area are defined to reduce the complexity of the Hough transform as it uses a voting scheme that considers all edge points $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$(x_{i}, y_{i})$\end{document}$ over the image for all possible combinations of center coordinates $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$(x_{0}, y_{0})$\end{document}$ and different possible values of the radius $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$r$\end{document}$. The algorithm is applicable in applications where a camera close to the eye is used such as in iris recognition systems. The algorithm was tested on the UBIRIS database, which contains images with different characteristics like illumination change. The algorithm is relatively fast when compared to non real-time applications but it is not suitable for real-time eye tracking due to its complexity. Alioua et al. [@ref20] presented an algorithm that handles an important part of eye tracking, which is analyzing eye state (open/closed) using iris detection. Gradient image is used to mark the edge of the eye. Horizontal projection is then computed for the purpose of detecting upper and lower boundaries of the eye region and the Circular Hough transform is used for iris detection. This algorithm can be useful in eye blinking detection, especially since the possible output classes are limited: open, closed or not an eye. When used in a control application, it can increase the number of possible commands. Due to its importance, the algorithm is required to be fast but no required CPU time was mentioned, although indicating the required CPU time could be useful. ### 4.. Eye Tracking Using Dark and Bright Pupil Effect {#sec2b4} There are two illumination methods used in the literature for pupil detection: the dark pupil and the bright pupil method. In the dark pupil method, the location of a black pupil is determined in the eye image captured by the camera. This causes some issues when the user has dark brown eyes because of the low contrast between the brown iris and the black pupil. The bright pupil method uses the reflection of infrared light from the retina which makes the pupil appear white in the eye image. [Fig. 2](#fig2){ref-type="fig"} shows the dark and bright pupil effect. Fig. 2.(a) Dark and (b) Bright pupil effects [@ref2]. Yoo et al. [@ref21] proposed using cross-ratio-invariance for a gaze tracking algorithm. The proposed algorithm has been found to achieve very low accuracy. To enhance it, a virtual plane tangent to the cornea is added. The enhancement did not solve all issues as the system remains complex due to using two cameras to obtain the difference between dark and bright pupil images for pupil position detection [@ref22]. ### 5.. Eye Tracking Using Eye Models {#sec2b5} Eye models can be used for eye tracking. Zhu and Ji [@ref23] proposed two new schemes to allow natural head movement for eye tracking and minimize the calibration of the eye tracking system to only once for each new user. The first scheme estimates the 3-D eye gaze directly. The cornea of the eye is modeled as a convex mirror. Based on the characteristics of a convex mirror, the eye\'s 3-D optic axis is estimated. The visual axis represents the actual 3-D eye gaze direction of the user. It is determined after knowing the angle deviation between the visual axis and optic axis in calibration. The second scheme does not require estimating the 3-D eye gaze, and the gaze point can be determined implicitly using a gaze mapping function. The gaze mapping function is updated automatically upon head movement using a dynamic computational head compensation model. The proposed schemes require medical and physical background as well as image processing techniques. The schemes are complex and hence they are not applicable for real-time eye tracking. It would be beneficial if the used algorithms, equations and phases were optimized. In addition, the system uses inflexible hardware which is not preferred in real applications. The schemes were not tested on a database containing various test subjects and conditions. ### 6.. Hybrid Eye Tracking Techniques {#sec2b6} A mix of different techniques can be used for eye tracking. Huang et al. [@ref24] suggested an algorithm to detect eye pupil based on intensity, shape, and size. Special Infrared (IR) illumination is used and thus, eye pupils appear brighter than the rest of the face. The intensity of the eye pupil is used as the primary feature in pupil detection. However, some other bright objects might exist in the image. To separate the pupil from bright objects existing in the image, other pupil properties can be used, such as pupil size and shape. Support Vector Machine is used to locate the eye location from the detected candidates. The used hardware, including the IR LEDs and the IR camera, is not expensive. The algorithm has been used in a driver fatigue detection application. The algorithm can be considered a new beginning for real-time eye tracking systems if it is tested further with different test subjects and different classification functions in order to reach the most optimized eye algorithm. The required CPU time was not mentioned although it is important in driver fatigue detection applications as they are real-time applications. Using a corneal reflection and energy controlled iterative curve fitting method for efficient pupil detection was proposed by Li and Wee [@ref12]. Ellipse fitting is needed to acquire the boundary of the pupil based on a learning algorithm developed to perform iterative ellipse fitting controlled by a gradient energy function. This method uses special hardware which has been implemented specifically for this algorithm. It has been used in a Field-of-View estimation application and can be used in other applications. Coetzer and Hancke [@ref25] proposed a system for eye tracking which uses an IR camera and IR LEDs. It captures the bright and dark pupil images subsequently such that they are effectively the same image but each has been taken in different illumination conditions. Two groups of infrared LEDs are synchronized with the IR camera. The first is placed close to the camera\'s lens to obtain the bright pupil effect and the second about 19.5 cm away from the lens, to produce the dark pupil effect. The images are then subtracted from each other and a binary image is produced by thresholding the difference. This technique has been presented by Hutchinson [@ref26]. The binary image contains white blobs that are mapped to the original dark pupil image. The sub-images that result from the mapping are potential eye candidates. The possible eye candidate sub-images are classified into either eyes or non-eyes. Artificial neural networks (ANN), support vector machines (SVM) and adaptive boosting (AdaBoost) have been considered as classification techniques in this work. The system is ready for further improvements and enhancements. It was utilized in a driver fatigue monitoring system. It does not require calibration for each user because it uses a dataset for training and feature extraction. The best features to be used and flexible hardware implementation can be investigated further in order to make the algorithm a part of a bigger eye tracking system or eye location classification system. The images used in experiments were eye images after the background and noise were eliminated. This reduces the expectations of this algorithm performance in real applications. III.. Eye Tracking Applications {#sec3} =============================== The field of research in eye tracking has been very active due to the significant number of applications that can benefit from robust eye tracking methods. Many application areas employ eye tracking techniques. In the following, some of these areas are described. A.. Eye Control for Accessibility and Assistive Technology {#sec3a} ---------------------------------------------------------- People who have lost the control over all their muscles and are no longer able to perform voluntary movements as a result of diseases or accidents can benefit widely from eye tracking systems to interact and communicate with the world in daily life. Eye tracker systems provide many options for these individuals such as an eye-typing interface that could have text-to-speech output. They also enable eye-control, including directing electric wheelchairs or switching on the TV or other devices. Human computer interaction with graphical user interface actions or events may be classified into two main categories [@ref27]: •Pointing: moving the pointer over an object on the screen such as text or icon on the screen;•Selection: action interpreted as a selection of the object pointed to. Kocejko et al. [@ref16] introduced the "Eye Mouse" which people with severe disabilities can use. The mouse cursor position is determined based on the acquired information about the eye position towards the screen which provides the ability to operate a personal computer by people with severe disabilities. Raudonis et al. [@ref4] dedicated their eye tracking system to the assistance of people with disabilities. The system was used with three on-line applications. The first controls a mobile robot in a maze. The second application was "Eye Writer" which is a text-writing program. A computer game was the third application. Fu and Yang [@ref10] suggested employing information obtained from tracking the eye gaze to control a display based on video. Eye gaze is estimated and the display is controlled accordingly. Lupu et al. [@ref27] proposed "Asistsys" which is a communication system for patients suffering from neuro-locomotor disabilities. This system assists patients in expressing their needs or desires. B.. E-Learning {#sec3b} -------------- E-learning systems are computer-based teaching systems and are now very common. However, despite the fact that users are usually accustomed to machine interactions, the learning experience can be quite different. In particular, the "emotional" part is significant in the interaction between teacher and learner and it is missing in computer based learning processes. Calvi et al. [@ref28] presented "e5Learning" which is an e-learning environment that exploits eye data to track user activities, behaviors and emotional or "affective" states. Two main user states were considered: "high workload or non-understanding" and "tiredness." The author/teacher of the course is able to decide the duration the user should look at certain parts of the course content, whether this content was textual or non-textual. Porta et al. [@ref29] sought to build an e-learning platform which determines whether a student is having difficulty understanding some content or is tired or stressed based on the interpreted eye behavior. C.. Car Assistant Systems {#sec3c} ------------------------- Research is done on applying eye tracking methods in the vehicle industry with the aim of developing monitoring and assisting systems used in cars. For example, an eye tracker could be used in cars to warn drivers when they start getting tired or fall asleep while driving. Driver fatigue can be detected by analyzing blink threshold, eye state (open/closed) and for how long the driver\'s gaze stays in the same direction. Many eye tracking methods were used in this area of application [@ref24], [@ref25], [@ref30]. D.. Iris Recognition {#sec3d} -------------------- Iris recognition is being widely used for biometric authentication. Iris localization is an important and critical step upon which the performance of an iris recognition system depends [@ref18]--[@ref19]. E.. Field of View Estimation {#sec3e} ---------------------------- Another interesting application of eye tracking systems is that these systems can serve as an effective tool in optometry to assist in identifying the visual field of any individual, especially identifying blind spots of vision. Li and Wee [@ref12] used eye tracking to estimate the field of view to be used for augmented video/image/graphics display. IV.. Head Movement Detection {#sec4} ============================ The increased popularity of the wide range of applications of which head movement detection is a part, such as assistive technology, teleconferencing and virtual reality, have increased the size of research aiming to provide robust and effective techniques of real-time head movement detection and tracking. During the past decade, the field of real-time head movement detection has received much attention from researchers. There are many different approaches for head pose estimation. All investigated methods require high computational hardware and cannot be implemented using low computational hardware such as a microcontroller. A.. Computer-Vision-Based Head Movement Detection {#sec4a} ------------------------------------------------- One approach for head movement detection is computer vision-based. Liu et al. [@ref31] introduced a video-based technique for estimating the head pose and used it in a good image processing application for a real-world problem; attention recognition for drivers. It estimates the relative pose between adjacent views in subsequent video frames. Scale-Invariant Feature Transform (SIFT) descriptors are used in matching the corresponding feature points between two adjacent views. After matching the corresponding feature points, the relative pose angle is found using two-view geometry. With this mathematical solution, which can be applied in the image-processing field in general, the $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$x$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$y$\end{document}$, and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$z$\end{document}$ coordinates of the head position are determined. The accuracy and performance of the algorithm were not highlighted in the work and thus more work is needed to prove this algorithm to be applicable in real applications. Murphy-Chutorian and Trivedi [@ref32] presented a static head-pose estimation algorithm and a visual 3-D tracking algorithm based on image processing and pattern recognition. The two algorithms are used in a real-time system which estimates the position and orientation of the user\'s head. This system includes three modules to detect the head; it provides initial estimates of the pose and tracks the head position and orientation continuously in six degrees of freedom. The head detection module uses Haar-wavelet Adaboost cascades. The initial pose estimation module uses support vector regression (SVR) with localized gradient orientation (LGO) histograms as its input. The tracking module estimates the 3-D movement of the head based on an appearance-based particle filter. The algorithm was used to implement a very good driver awareness monitoring application based on head pose estimation. Using this algorithm in real-world applications will be effective for applications similar to a driver awareness monitoring system. It needs more software solution optimization before implementing in real applications. This algorithm can be considered to be a good addition to head tracking systems. Kupetz et al. [@ref33] implemented a head movement tracking system using an IR camera and IR LEDs. It tracks a 2×2 infrared LED array attached to the back of the head. LED motion is processed using light tracking based on a video analysis technique in which each frame is segmented into regions of interest and the movement of key feature points is tracked between frames [@ref34]. The system was used to control a power wheelchair. The system needs a power supply for the LEDs which could be wired or use batteries. The system can be improved to detect the maximum possible unique head movements to be used in different applications. It needs more experiments to prove its accuracy in addition to more theoretical proofs. Siriteerakul et al. [@ref35] proposed an effective method that tracks changes in head direction with texture detection in orientation space using low resolution video. The head direction is determined using a Local Binary Pattern (LBP) to compare between the texture in the current video frame representing the head image and several textures estimated by rotating the head in the previous video frame by some known angles. The method can be used in real-time applications. It can only find the rotation with respect to one axis while the others are fixed. It can determine the rotation with respect to the yaw axis (left and right rotations) but not neck flexion. Song et al. [@ref36] introduced a head and mouth tracking method using image processing techniques where the face is first detected using an Adaboost algorithm. Then, head movements are detected by analyzing the location of the face. Five head motions were defined as the basis of head movements. The geometric center of the detected face area is calculated and considered to be the head\'s central coordinates. These coordinates can be analyzed over time to trace the motion of the head. The used camera is not head-mounted. The method was found to be fast, which makes it applicable in simple applications for people with disabilities. However, the accuracy and performance of the method were not reported. Jian-zheng and Zheng [@ref37] presented a mathematical approach using image processing techniques to trace head movements. They suggested that the pattern of the head movements can be determined by tracing face feature points, such as nostrils, because the movements of the feature point and the movements of the head do not vary widely. Tracing the feature point with the Lucas-Kanade (LK) algorithm is used to find the pattern of head movements. GentleBoost classifiers are trained to use the coordinates of the nostrils in a video frame to identify the head direction. This technique is very appropriate for academic research as it is theoretically proved but it requires more testing and experiments on different test subjects and a known database containing variant samples in order to prove its reliability in applications. Zhao and Yan [@ref38] proposed a head orientation estimation scheme based on pattern recognition and image processing techniques. It uses artificial neural networks to classify head orientation. The face is first detected by adopting the YCbCr skin detection method. Then k key points that represent different head orientations are extracted and labeled. The coordinates and local textures of the labeled key points are used as the input feature vector of the neural networks. Head orientation estimation systems must have high real-time performance to be applicable in real-time applications. This scheme needs more optimization to be closer to real-time applications. Berjón et al. [@ref39] introduced a system which combines alternative Human-Computer Interfaces including head movements, voice recognition and mobile devices. The head movement detection part uses an RGB camera and image processing techniques. It employs a combination of Haar-like features and an optical flow algorithm. Haar-like features are used in detection of the position of the face. The optical flow detects the changes occurring in the position of the face detected within the image. The analysis for the system is shallow and did not provide any mathematical proofs or experimental tests to survive in real-world application fields. Zhao et al. [@ref40] presented another head movement detection method based on image processing which also used the Lucas-Kanade algorithm. In order to identify head movements accurately, the face is first detected, then the nostrils are located and the Lucas-Kanade algorithm is used to track the optical flow of the nostrils. However, the face position in the video may be approximate and, thus, the coordinates of the feature points might not reflect the head movement accurately. Therefore, they used inter-frame difference in the coordinate of feature points to reflect the head movement. This approach is applicable in head motion recognition systems and it is considered to be fast. More experiments are required using datasets which contain different subjects and cases in order to be considered as an option for real applications, especially since it ignored the challenges of different camera angles of view and normal noise challenges. Xu et al. [@ref41] presented a method which recovers 3-D head pose video with a 3-D cross model in order to track continuous head movement. The model is projected to an initial template to approximate the head and is used as a reference. Full head motion can then be detected in video frames using the optical flow method. It uses a camera that is not head-mounted. It can be considered a high complexity algorithm which is useful in academic research fields and further research can be done based on the findings of this work and possible applications may be suggested. B.. Acoustic-Signal-Based Methods {#sec4b} --------------------------------- Some head direction estimation systems localize the user\'s voice source (i.e., their mouth) to estimate head direction. Sasou [@ref42] proposed an acoustic-based head direction detection method. The method uses a microphone array that can localize the source of the sound. In each head orientation, the localized positions of the sounds generated by the user are distributed around unique areas that can be distinguished. Hence, if the boundaries between these unique areas are defined, the head direction can be estimated by determining which area corresponds to the generated sound. The work introduced a novel power wheelchair control system based on head orientation estimation. The system was enhanced with noise removal using back microphones. In real-world applications, this algorithm is very limited and can detect only three movements. Thus, it needs to be combined with other alternative control methods to increase the number of commands available to control the chair. In addition, it needs more tests and experimental analysis to prove the reliability and usability of the system, especially with the noise in the real world. C.. Accelerometer and Gyro-Sensor Based Methods {#sec4c} ----------------------------------------------- Many sensors such as gyroscopes and accelerometers can be used in head movement detection systems to obtain information on head movements. King et al. [@ref43] implemented a hands-free head movement classification system which uses pattern recognition techniques with mathematical solutions for enhancement. A Neural Network with the Magnified Gradient Function (MGF) is used. The MGF magnifies the first order derivative of the activation function to increase the rate of convergence and still guarantee convergence. A dual axis accelerometer mounted inside a hat was used to collect head movement data. The final data sample is obtained by concatenating the two channels of the dual accelerometer into a single stream to be used in the network with no additional pre-processing of the data. The proposed system needs more experiments to move it from being theoretically proved to being used in real world applications in different scenarios. No application based on the proposed method was suggested. A similar method was presented by Nguyen et al. [@ref44]. The method detects the movement of a user\'s head by analyzing data collected from a dual-axis accelerometer and pattern recognition techniques. A trained Bayesian Neural Network, which is an optimized version of a Neural Network, is used in the classification process where the head movement can be classified into one of four gestures. The algorithm still needs more experiments to make it usable in real world applications, as there was no mention of any possible applications. The use of Bayesian Neural Networks for optimization enhanced the performance compared with other head movement detection techniques which are based on pattern recognition. Manogna et al. [@ref45] used an accelerometer device fixed on the user\'s forehead. The accelerometer senses the tilt resulting from the user\'s head movement. This tilt corresponds to an analog voltage value that can be used to generate control signals. The implementation of this detection method is completely a simulation which represents a good proof of concept. However, it is missing actual accuracy analysis or real-time applicability investigation. The system is very flexible and can be applied in various applications Kim et al. [@ref46] presented a head tracking system in which the head pose is estimated based on data acquired using the gyro sensor. By applying an integral operation, the angular velocity obtained from the sensor is converted to angles. The system uses relative coordinates in head pose estimation instead of absolute coordinates. The proposal included a practical solution which is a computer mouse based on simple hardware implementation. This makes this system applicable in the real world. However, it needs more theoretical proofs and more experiments and accuracy analysis. The hardware implemented for the system could be enhanced and modified as it is not very comfortable for the user. D.. Hybrid Head Tracking Techniques {#sec4d} ----------------------------------- A combination of different techniques can be used in head tracking systems. Satoh et al. [@ref47] proposed a head tracking method that uses a gyroscope mounted on a head-mounted-device (HMD) and a fixed bird\'s-eye view camera responsible for observing the HMD from a third-person viewpoint. The gyroscope measures the orientation of the view camera in order to reduce the number of the parameters used in pose estimation. The bird\'s-eye view camera captures images of the HDM and, in order to find the remaining parameters that are needed for head pose estimation, feature key points are detected and processed to obtain their coordinates. Using a fixed camera, customized marker, gyroscope sensor and calibration process makes this proposal impractical for head tracking tasks. The time complexity of the algorithm has not been investigated which makes it a little far from being used in real-world applications, especially in that the paper mentions no suggested application. V.. Head Movement Detection Applications {#sec5} ======================================== A.. Accessibility and Assistive Technology {#sec5a} ------------------------------------------ Head movement has been found to be a natural way of interaction. It can be used as an alternative control method and provides accessibility for users when used in human computer interface solutions. Sasou [@ref42] introduced a wheelchair controlled by an acoustic-based head direction estimation scheme. The user is required to make sounds only by breathing. The direction of the head pose controls the direction of wheelchair. King et al. [@ref43] also presented a head-movement detection system to provide hands-free control of a power wheelchair. Song et al. [@ref36] mapped head movement-combined mouth movements to different mouse events, such as move, click, click and drag, etc. People with disabilities who cannot use a traditional keyboard or mouse can benefit from this system. B.. Video Surveillance {#sec5b} ---------------------- Video surveillance systems are now an essential part of daily life. Xie et al. [@ref48] presented a video-based system that captures the face or the head by using a single surveillance camera and performs head tracking methods to detect humans in the video scene. C.. Car Assistant Systems {#sec5c} ------------------------- Driver\'s attention recognition plays a significant role in driver assistance systems. It recognizes the state of the driver to prevent driver distraction. The driver\'s head direction can be an indication of his attention. Liu et al. [@ref31] used head pose detection to recognize driver\'s attention. Lee et al. [@ref49] introduced a system that detects when the driver gets drowsy based on his head movement. VI.. Combining Eye Tracking and Head Movement Detection {#sec6} ======================================================= Some research has been done on using a combination of eye tracking and head movements. Kim et al. [@ref50] presented a head and eye tracking system which uses the epipolar method along with feature point matching to estimate the position of the head and its rotational degree. The feature points are high brightness LEDs on a helmet. For eye tracking, it uses an LED for constant illumination and a Kalman filter is used for pupil tracking. Their presented work needs more theoretical proof. In addition, there is no mentioned optimization for the algorithm and the required CPU time analysis is not reported. There are a lot of hardware requirements for the system which makes it relatively expensive to implement. Iwata and Ebisawa [@ref51] introduced a good flexible eye mouse interface which is the pupil mouse system combined with head pose detection. The system detects pupil motion in the video frames by finding the difference between the bright and dark pupil images. Head direction is detected by tracing key feature points (nostrils). The nostrils were detected as the darker areas in the bright and dark images. The information obtained is mapped into cursor motion on a display. Using head pose detection to support the pupil mouse is a good idea which improved the overall performance. However, the head pose detection part increases the complexity of the pupil mouse algorithm which causes a need for a lot of optimization techniques, which may not be suitable for real-world applications. VII.. Eye/Head Tracking in Commercial Products {#sec7} ============================================== In the last decade, the advancing applicability of eye tracking technologies led to a significant interest in producing commercial eye tracking products. However, the research on head movement detection is not yet mature enough to be considered for commercial applications. Commercial eye tracking products have been used in several applications. In this section, some eye tracking products are discussed. A.. EagleEyes {#sec7a} ------------- EagleEyes eye tracking system was first developed by Professor Jim Gips at Boston College [@ref52]. It is primarily used for severely disabled children and adults. The eye movement signals are used to control the cursor on the computer screen. The computer then can be used to assist in communication and to help in education. It can also provide entertainment for the disabled and their families. There are different commercial and customized software programs that can be used in the system to provide the user with many options and functionalities. B.. BLiNK {#sec7b} --------- Another product is BLiNK, which is a simplified eye tracking system suitable for everyday use [@ref53]. There are many applications for BLiNK, such as computer input, gaming, taking orders, and learning. Another important application field is health care. It is used as an alternative communication method with patients who have lost the ability to write and speak. BLiNK is also useful for severely disabled people who have no or very limited movement capabilities. C.. Smi {#sec7c} ------- SMI also provides eye trackers which support many application fields [@ref54]. One of these applications is ophthalmology. The products provided by SMI are used for medical research in addition to related eye diseases investigation where the research results allow understanding the human eye and diagnosis of the disease. D.. Tobii {#sec7d} --------- Tobii eye trackers, along with the Tobii Studio eye tracking software, allow reliable eye tracking results to be obtained [@ref55]. There are many products provided by Tobii. One of these devices is the Tobii C12 AAC device which is by a wide range of disabled people to perform independent functionalities using optional eye or head control [@ref56]. The Tobii C12 AAC device is suitable to be used by people who suffer limitations or loss of functionality in their hands and other body parts due to stroke, diseases of the nervous system, etc. The device provides various control options. The flexible AAC software allows the user to choose either text or symbols for generating speech to communicate with the device. This provides the ability to ask and answer questions or use e-mail and text messages. There are multiple mounting systems for the device making it compatible with assistive technology including wheelchairs, walkers and bed frames. Tobii also has an academic program which supports university classrooms and research labs with easy and cost-effective eye tracking. Tobii products are also used by web designers for usability testing where the designers can get direct feedback on their work by analyzing what a user is looking at. VIII.. Experimental Results of Existing Eye/Head Tracking Techniques {#sec8} ==================================================================== The performance of eye tracking and head movement detection systems is evaluated in terms of accuracy and required CPU processing time. This section compares the results of the methods described earlier in the survey. A.. Performance of Eye Tracking Methods {#sec8a} --------------------------------------- [Table I](#table1){ref-type="table"} compares the described eye tracking methods in terms of eye detection accuracy, gaze angle accuracy and required CPU time. Table IComparison of Eye Tracking MethodsMethodDetection Accuracy (%)Angle Accuracy (degree)CPU time (ms)**Eye tracking using Pattern Recognition Raudonis *et al.*[@ref4]**100%N/AN/A**Kuo *et al.*[@ref6]**90%N/A N/A**Yuan and Kebin [@ref9]**N/A1N/A**Lui and Lui [@ref7]**94.1%N/A N/A**Khairosfaizal and Nor\'aini [@ref17]**86%N/AN/A**Hotrakool *et al.*[@ref8]**100%N/A12.92**Shape-based eye tracking Yang *et al.*[@ref13]**N/A0.5N/A**Yang *et al.*[@ref14]**N/AHorizontal: 0.327 Vertical: 0.3N/A**Mehrubeoglu *et al.*** [@ref11] *90%*N/A*49.7***Eye tracking using eye models Zhu and Ji [@ref23] (First scheme)**N/AHorizontal: 1.14 Vertical: 1.58N/A**Zhu and Ji [@ref23] (Second scheme)**N/AHorizontal: 0.68 Vertical: 0.83N/A**Eye tracking using hybrid techniques Li and Wee [@ref12]**N/A0.5N/A**Huang *et al.*[@ref24]**95.63%N/A N/A**Coetzer and Hancke [@ref25]**98.1%N/AN/A B.. Performance of Head Movement Detection Methods {#sec8b} -------------------------------------------------- The head detection accuracy, angle accuracy and required CPU time of described head movement detection methods are summarized in [Table II](#table2){ref-type="table"}. Table IIComparison of Head Tracking MethodsMethodDetection Accuracy (%)Angle Accuracy (degree)**Computer-vision-based head tracking Siriteerakul *et al*[@ref35]**N/A3.32**Zhao *et al.*[@ref40]***92.86%*N/A**Zhao, and H. Yan [@ref38]**90%N/A **Accelerometer and Gyro-sensor based methodsKing *et al.*[@ref43]**99.05%N/A**Nguyen *et al.*[@ref44]**93.75%N/A To use eye tracking and head movement detection methods in real-time systems, their time performance must be taken into consideration. The CPU time required for processing and analyzing eye and head movements must be minimized. This has only rarely been investigated in the experimental work of the reported methods. IX.. Conclusion {#sec9} =============== Eye tracking and head movement detection are considered effective and reliable human-computer interaction and communication alternative methods. Hence, they have been the subject of many research works. Many approaches for implementing these technologies have been reported in the literature. This paper investigated existing methods and presented a state-of-art survey on eye tracking and head movement detection. Many applications can benefit from utilizing effective eye tracking and/or head movement detection methods. However, the research is still facing challenges in presenting robust methods which can be used in applications to detect and track eye or head movements accurately. Eye tracking methods rarely investigate the required CPU time. However, real-time application requires investigating and optimizing the performance requirements. In addition, most studies do not test eye tracking using a known image database that contains variant images of different subjects in different conditions such as lighting conditions, noise, distances, etc. This makes the reported accuracy of a method less reliable because it may be affected by different test conditions. Head movement detection requires high computational hardware. A microcontroller, which is considered low computational hardware, cannot be used for implementing head movement detection algorithms reported in literature. More work and research is needed to provide eye tracking and head movement detection methods that are reliable and useful for real-world applications.  [Amer Al-Rahayfeh](#contrib1){ref-type="contrib"} is currently pursuing the Ph.D. degree in computer science and engineering with the University of Bridgeport, Bridgeport, CT, USA. He received the B.S. degree in computer science from Mutah University in 2000 and the M.S. degree in computer information systems from The Arab Academy for Banking and Financial Sciences in 2004. He was an Instructor at The Arab Academy for Banking and Financial from 2006 to 2008. He is currently a Research Assistant with the University of Bridgeport. His research interests include eye tracking and head movement detection for super assistive technology, wireless multimedia sensor networks, and multimedia database systems.  [Miad Faezipour](#contrib2){ref-type="contrib"} (S\'06--M\'10) is an Assistant Professor in the Computer Science and Engineering and Biomedical Engineering programs, University of Bridgeport, CT, and has been the Director of the Digital/Biomedical Embedded Systems and Technology Lab since July 2011. Prior to joining UB, she has been a Post-Doctoral Research Associate at the University of Texas at Dallas collaborating with the Center for Integrated Circuits and Systems and the Quality of Life Technology Laboratories. She received the B.Sc. degree in electrical engineering from the University of Tehran, Tehran, Iran, and the M.Sc. and Ph.D. degree in electrical engineering from the University of Texas at Dallas. Her research interests lie in the broad area of biomedical signal processing and behavior analysis techniques, high-speed packet processing architectures, and digital/embedded systems. She is a member of the IEEE EMBS and the IEEE Women in Engineering.

1. Introduction {#sec1-nutrients-12-01640} =============== Fatigue is a normal sensation that serves to prompt bodily rest following physical or mental exhaustion from daily activities. However, often fatigue is not resolved adequately by rest but is aggravated owing to quality of life (QOL) deterioration. It has been reported that approximately 30% of the Japanese population experience regular subjective fatigue \[[@B1-nutrients-12-01640],[@B2-nutrients-12-01640]\]. Chronic fatigue syndrome is a disorder diagnosed by profound disabling fatigue that persists for at least 6 months without relief and is not lessened by ordinary rest \[[@B3-nutrients-12-01640]\]. It is common in 20 to 50 years old during the prime period of their working life. It has been estimated that it affects some 80,000 to 240,000 individuals in Japan, and approximately one-third of these individuals are bedridden. Prevention of fatigue aggravation is thus of some importance. In the present study, we focused on an intervention study of anti-fatigue effects in healthy individuals experiencing fatigue in daily life that had continued for no longer than the 6 months standpoint from prevention of chronic fatigue. Fatigue is a condition in which cells and tissues are damaged by reactive oxygen species generated during excessive activity \[[@B4-nutrients-12-01640]\]. Recovery from fatigue occurs when this damage is adequately repaired; however, if the damage continues with inadequate repair due to the lack of necessary energy for recovery, an inflammatory response is elicited; this leads to conditions such as malaise and fever, consequently resulting in chronic fatigue \[[@B4-nutrients-12-01640]\]. Accordingly, antioxidants that inhibit damage from oxidative stress, and substances that can stimulate energy production are effective against fatigue. Ubiquinol, a reduced form of coenzyme Q~10~, is present in food and has been reported to have anti-fatigue activity \[[@B5-nutrients-12-01640]\]. This substance is a vitamin-like compound used in many countries as an ingredient in foods and supplements. As antioxidants increase ATP production by stimulating mitochondrial electron transport, their bioactivity consists of two elements---antioxidant activity and energy production that both generate an anti-fatigue effect. These two mechanisms likely contributed to many other effects that have been reported in human clinical studies, including enhanced cardiac function, anti-inflammatory effect on hematopoietic cells, sialagogic effects in middle- or advance-aged healthy individuals with dry-mouth condition, and improved conditions of patients with Parkinson's disease when used in combination with L-dopa \[[@B6-nutrients-12-01640]\]. Although ubiquinol is biosynthesized in humans, it is also present in food. Its level in various organs has been reported to reduce with age, suggesting reduction of biosynthetic activity with aging \[[@B7-nutrients-12-01640]\]. The biosynthesis pathway of ubiquinol, like that of cholesterol, involves metabolism by hydroxymethylglutaryl-CoA (HMG-CoA) reductase. This is likely the reason why its level in the blood has been reported to decrease by statins that lower the blood-cholesterol level \[[@B8-nutrients-12-01640]\]. It has been estimated that 3−5 mg of ubiquinol is ingested daily through diet; this amount is contributed largely by meats and seafood. It has been suggested that the amount of meat intake influences the amount of ubiquinol in the body \[[@B9-nutrients-12-01640]\]. According to research in patients with complete nutritional independence, approximately half of the blood ubiquinol is believed to originate from the diet \[[@B10-nutrients-12-01640]\]. After absorption, ubiquinol stays in blood as a lipoprotein component. It has been reported that the ubiquinol amount in blood is significantly higher in healthy individuals of advanced age than in elderly bedridden individuals, and that the risk of dementia is reduced approximately by half in individuals having high blood levels of ubiquinol \[[@B11-nutrients-12-01640]\]. In an open-label study on ubiquinol intake in patients with chronic fatigue syndrome, cognitive performance indicator of fatigue as well as the questionnaire score of depression symptoms improved along with an increased blood level of ubiquinol \[[@B5-nutrients-12-01640]\]. A double-blind study found that the blood ubiquinol level in patients with chronic fatigue syndrome was low prior to ubiquinol intake. Fatigue improvement, reduction in the episodes of wakefulness during sleep, and inhibition of decline in autonomic nerve functions were demonstrated following ubiquinol intake \[[@B5-nutrients-12-01640]\]. Another double-blind study in healthy individuals aged approximately 60 years found improvement in the mental QOL including subjective fatigue and improved spontaneous activity \[[@B12-nutrients-12-01640]\]. An interventional prospective cohort study among elderly residents suggested beneficial effects on QOL, including subjective fatigue, and improved maintenance of cognitive function \[[@B13-nutrients-12-01640]\]. This study also demonstrated that long-term ubiquinol intake over 5 years posed no safety issues. Thus, the beneficial effects of ubiquinol on fatigue have been shown not only in patients with chronic fatigue syndrome but also in healthy individuals; however, data in young individuals are scarce, being limited to a report of beneficial psychological effects and improved athletic performance in female university athletes \[[@B14-nutrients-12-01640]\]. Therefore, we targeted a wide range of age in young and old adults in the present study. The present double-blind study examined the anti-fatigue effect of ubiquinol in healthy participants aged between 20 and 64 years, experiencing mild fatigue in daily life. 2. Materials and Methods {#sec2-nutrients-12-01640} ======================== 2.1. Clinical Study {#sec2dot1-nutrients-12-01640} ------------------- The clinical study employed a written questionnaire on parameters such as fatigue, sleep, and lifestyle habits as well as the autonomic nerve function screening to select participants for evaluation of the anti-fatigue effects of a soft-capsule formulation containing ubiquinol. Seventy-one participants were selected out of 104 healthy individuals experiencing fatigue in daily life that had continued for more than 1 and less than 6 months and who were judged by the principal investigator as not having chronic fatigue syndrome with the checklist of diagnostic criteria \[[@B3-nutrients-12-01640]\]. This was a randomized, comparative, placebo-controlled, double-blind, parallel-group study designed in accordance with the recommendations of the Consolidated Standards of Reporting Trials guidelines. Written consent was obtained from participants after they were informed in advance about the study's objectives, methods, and expected clinical advantages and disadvantages of the treatment. This study was performed in compliance with the principles of the Declaration of Helsinki and was approved by the ethical-review board of the Ethics Committee of Osaka City University Center for Health Science Innovation (OCU-CHSI-IRB no. 16). The study was registered with the UMIN Clinical Trials Registry. The inclusion criterion required that participants were (1) healthy individuals at least 20 years old and (2) experiencing fatigue in daily life that had continued for more than 1 and less than 6 months. The study exclusion criteria included participants (1) receiving continuous treatment for disease or on medications; (2) intaking coenzyme Q~10~ continuously; (3) with a history of cardiovascular or neurological diseases; (4) with allergies, sleep disorders, or organic diseases that clearly contributed to fatigue; (5) who were pregnant, planning pregnancy, or nursing; (6) who had psychiatric diseases or history thereof; (7) who consumed alcohol in excess or could not avoid the use of medications that could potential affect the outcomes; (8) who had one or more severe diseases, such as diabetes mellitus, liver disease, kidney disease, heart disease, or other diseases; (9) who were participating in another clinical study within 1 month prior to giving consent or were participating in any other clinical trial; and (10) who were judged unsuitable by the principal investigator for any other reasons. Participants selected through preliminary screening were randomized by age and gender into three groups to receive 100 mg of ubiquinol, 150 mg of ubiquinol, or placebo (capsule without ubiquinol). Four participants who dropped out after the initiation of the study were excluded from analysis as well as five participants who were affected during the course of the study by bone fracture, initiation of treatment for hyperlipidemia, medication use for headache, strenuous exercise (long-distance running), and low rate of trial dietary product intake (≤80%). These screenings were performed based on the result of a 4-weeks daily journal recorded by each participant. Questions about dietary intake and alcohol intake were also included in this daily journal. To judge the subjects for analysis, we carefully checked the daily journal every four weeks, on the 2nd, and 3rd experimental days. The final analysis included 20 participants in the placebo group (age 41.3 ± 13.4 years; 13 females and 7 males), 20 in the 100-mg group (age 44.0 ± 9.8 years; 14 females and 6 males), and 22 in the 150-mg group (age 40.4 ± 11.8 years; 14 females and 8 males). The trial dietary product was a soft-capsule formula containing 50 mg of ubiquinol. The additional ingredients in capsules were rapeseed oil, diglycerol monooleate, beeswax, soy lecithin, and caramel as a coloring agent. Rapeseed oil was added to the placebo capsule instead of ubiquinol. Participants took three capsules once daily after breakfast continuously for 12 weeks (the placebo group took three placebo capsules; the 100-mg group took two active capsules and one placebo capsule; and the 150-mg group took three active capsules). The main outcome measures that were evaluated every 4 weeks included the autonomic nerve function, oxidative stress/antioxidative activity of blood samples, reaction time and rate of correct response in cognitive function tasks, subjective fatigue sensation, and the serum ubiquinol level. 2.2. Questionnaires {#sec2dot2-nutrients-12-01640} ------------------- The Chalder Fatigue Scale \[[@B15-nutrients-12-01640]\] and a modified version of the Osaka City University Hospital Fatigue Scale \[[@B16-nutrients-12-01640]\] were used for evaluating the severity of fatigue. The K6 scale \[[@B17-nutrients-12-01640]\] can measure mood and anxiety and the Center for Epidemiological Studies Depression Scale \[[@B18-nutrients-12-01640]\] can evaluate the symptoms of depression. The Pittsburgh Sleep Quality Index \[[@B19-nutrients-12-01640]\] and the Epworth Sleepiness Scale \[[@B20-nutrients-12-01640]\] were used to measure general sleepiness and daytime sleepiness, respectively. Previous studies have confirmed the reliability and validity of the Japanese versions of these questionnaires \[[@B16-nutrients-12-01640],[@B21-nutrients-12-01640],[@B22-nutrients-12-01640],[@B23-nutrients-12-01640],[@B24-nutrients-12-01640],[@B25-nutrients-12-01640]\]. We evaluated the level of subjective fatigue sensation using a visual analogue scale (VAS) from 0 representing "no fatigue," to 100 representing "total exhaustion" immediately prior to and after cognitive function testing. A VAS for sleepiness, depression, relaxation, and motivation was also used at the same measurement points. 2.3. Autonomic Nerve Function {#sec2dot3-nutrients-12-01640} ----------------------------- Autonomic nerve function is an objective marker for evaluating fatigue \[[@B4-nutrients-12-01640]\]. Data were generated by simultaneous electrocardiography and photoplethysmography using a Vital Monitor 302 system (Fatigue Science Laboratory, Osaka, Japan) in participants sitting quietly with their eyes closed for 3 min. Frequency analyses for R-R interval variation from electrocardiography and a-a interval variation as the second derivative of photoplethysmography were measured. The maximum entropy method that is capable of estimating the power-spectrum density from short time-series data and is adequate for examining short-duration changes in heart-rate variability under different conditions was used \[[@B26-nutrients-12-01640],[@B27-nutrients-12-01640]\]. Low frequency (LF) was taken as the power within a frequency range of 0.04--0.15 Hz and high frequency (HF) was as being within a frequency range of 0.15--0.4 Hz. HF is vagally mediated \[[@B28-nutrients-12-01640],[@B29-nutrients-12-01640]\], whereas LF originates from a variety of sympathetic and vagal mechanisms \[[@B30-nutrients-12-01640],[@B31-nutrients-12-01640]\]. We used log-transformed (ln) LF, ln HF, and ln LF/HF ratio \[[@B32-nutrients-12-01640]\] in the present study. Prior to 3-min autonomic nerve function testing, a practice test was conducted for a period of 1 min in accordance with previous studies \[[@B32-nutrients-12-01640],[@B33-nutrients-12-01640],[@B34-nutrients-12-01640],[@B35-nutrients-12-01640]\]. 2.4. Analyses of Oxidative Stress and Antioxidant Activity {#sec2dot4-nutrients-12-01640} ---------------------------------------------------------- The oxidative activity of serum samples was determined using a test for reactive oxygen metabolites-derived compounds (d-ROMs; Diacron International, Grosseto, Italy). The antioxidative activity was determined by measuring the biological antioxidant potential (BAP) (Diacron International) using a JCABM1650 automated analyzer (JEOL, Tokyo, Japan) \[[@B36-nutrients-12-01640]\]. The d-ROMs value was expressed in Carratelli Units (1 CARR U = 0.08 mg hydrogen peroxide/dL) \[[@B37-nutrients-12-01640]\]. The oxidative stress index (OSI) was calculated using the following formula: OSI = C × (d-ROMs/BAP) where C denotes a coefficient for standardization to set the mean OSI in healthy individuals at 1.0 (C = 8.85) \[[@B36-nutrients-12-01640]\]. All serum samples were stored at −80 °C until analysis. Assays for serum d-ROMs and BAP were performed at Yamaguchi University Graduate School of Medicine. 2.5. Analysis of Serum Ubiquinol {#sec2dot5-nutrients-12-01640} -------------------------------- Serum concentrations of ubiquinol were measured using LC/MS/MS (commissioned at Kaneka Techno Research Corporation). Specifically, 0.1 mL of serum was fractionated immediately after collection, mixed with 0.7 mL of isopropanol to prevent oxidation of ubiquinol and stored at −80°C until measurement. At the time of measurement, samples were centrifuged at 12,000 rpm for 5 min; supernatants were filtered through a membrane filter and subjected to LC/MS/MS analysis using the AB SCIEX Triple Quad5500 instrument. 2.6. Cognitive Functions {#sec2dot6-nutrients-12-01640} ------------------------ ### 2.6.1. Modified Advanced Trail Making Test {#sec2dot6dot1-nutrients-12-01640} To assess the cognitive function and volition of subjects, Task E in the modified Advanced Trail Making Test (mATMT) was implemented \[[@B38-nutrients-12-01640],[@B39-nutrients-12-01640],[@B40-nutrients-12-01640]\]. The mATMT is designed to assess cognitive function in terms of the response time and number of correct responses to tasks displayed on a computer screen; it further determines the task-to-task shifting speed as an indicator of volition. It is also used to assess the performance of fatigued humans \[[@B41-nutrients-12-01640],[@B42-nutrients-12-01640]\]. The participants were asked to respond to a total of 25 visual stimuli (numerals 1--13 and 12 different hiragana characters) displayed on the screen in random positions as follows: participants moved the cursor and clicked on the numerals and hiragana alternatively in the specified order, using the mouse, as quickly as possible for 5 consecutive minutes. During this task, cognitive function was evaluated by the number of correct/erroneous responses. Motivational response was evaluated in terms of response time from the end of one task to the start of the next task, with motivation rated higher as response time shortened. As in our previous studies \[[@B35-nutrients-12-01640],[@B42-nutrients-12-01640]\], participants practiced for a period of 1 min before performing Task E of the mATMT on each experimental day. ### 2.6.2. Modified Stroop Color-Word Test {#sec2dot6dot2-nutrients-12-01640} For the second assessment of cognitive function, the modified Stroop Color-Word Test (mSCWT) was used. In this test, the ability of the subject correctly to judge colors and characters in a pedestrian signal displayed on a computer screen was assessed \[[@B43-nutrients-12-01640]\]. Participants were asked to perform Task 1 (3 min) and Task 2 (6 min) as quickly and accurately as possible. In Task 1, the subject right-clicked the mouse upon receiving a flickering blue signal and left-clicked upon receiving a flickering red signal. In Task 2, the subject observed and judged characters appearing on the screen. Specifically, the subject right-clicked the mouse only when the word (in Japanese) appeared in blue. In other cases, the mouse was left-clicked. During these tasks, the time elapsed since clicking was displayed in 100-ms increments. Although the colors and characters were displayed randomly in each task, their frequencies were always constant. The frequencies in the Stroop trial (colors and characters mismatched) and the non-Stroop trial (colors and characters matched) were also constant. The result of each cognitive task in terms of it being a correct or error response was continuously presented on the personal computer display. Participants practiced for a total of 3 min (Task 1 for 1 min and Task 2 for 2 min) before performing the cognitive function task \[[@B43-nutrients-12-01640]\]. ### 2.6.3. Digit Symbol Substitution Test {#sec2dot6dot3-nutrients-12-01640} The Digit Symbol Substitution Test (DSST) is used to evaluate the visual processing speed; it was implemented as a measure of cognitive function \[[@B44-nutrients-12-01640],[@B45-nutrients-12-01640],[@B46-nutrients-12-01640]\]. Participants performed the task of entering as many numbers corresponding to the signals as possible within the 2-min time limit; the number of correct responses was used as the score. 2.7. Actigraphy (Sleep Parameters) {#sec2dot7-nutrients-12-01640} ---------------------------------- Actigraphy has been established as a valid and objective method of assessing sleep--wake parameters in natural settings \[[@B47-nutrients-12-01640]\]. Actigraphy devices are typically worn on the wrist and record movements that can be used to estimate sleep parameters \[[@B48-nutrients-12-01640]\]. Actigraphy data were obtained using the Actiwatch Spectrum (Philips Respironics, Murrysville, PA, USA) and processed using Actiware software (version 6.0.6, Philips Respironics, Murrysville, PA, USA); default settings (wake threshold medium, 10 min immobile for both sleep onset and offset criteria) were used. Averaged parameters of sleep time and sleep efficiency were calculated during the 12-week study period. 2.8. Statistical Analysis {#sec2dot8-nutrients-12-01640} ------------------------- Each measured value was represented as the mean ± standard deviation. Data analyses were performed by two-way analysis of variance with post-hoc Dunnet's multiple comparison test. *p*-values were corrected for number of post-hoc tests. Pearson's correlation analyses were also performed. All *p*-values \< 0.05 were considered statistically significant. Statistical analyses were performed using IBM SPSS Statistical Package version 25.0 (IBM, Armonk, NY, USA). 3. Results {#sec3-nutrients-12-01640} ========== [Table 1](#nutrients-12-01640-t001){ref-type="table"} shows the demographic characteristics of the study participants. The participants were randomized into three groups and had similar male to female ratio, mean age, and age range. The 62 analyzed participants were of 20--64 years of age, consisting of 10 participants in their 20s, 16 in their 30s, 15 in their 40s, 18 in their 50s, and 3 in their 60s. The effect of change in temperature was minimized by conducting the study during a relatively warm climate between March and June in Japan. The days and times for tests were fixed as much as possible to prevent the lifestyle pattern of the participants influencing the outcome. However, since the participants who were evaluated on Sunday had a day of rest, we focused the analyses mainly on the degree of change in each outcome measure (∆value). Before the first dosing, no statistically significant difference was observed among the three groups in any of the parameters. Because we recognized that the standard deviations tended to be large overall, we also analyzed the degree of change from before supplementation considering variation between individuals. Items in which a statistically significant improvement was observed are summarized in [Table 2](#nutrients-12-01640-t002){ref-type="table"}. In VAS for subjective sensation, the 150-mg group differed significantly from the placebo group in scores of VAS for fatigue, sleepiness and relaxation after the cognitive function task, and score of VAS for difference in sleepiness before and after the cognitive function task. Further, motivation in regard to cognitive function task and d-ROMs, serum oxidative stress markers, and ∆ln(LF/HF) balance of autonomic nerve function improved significantly compared with that in the placebo group. In the 100-mg group, scores of VAS for fatigue and sleepiness after the cognitive function task were significantly different compared with those in the placebo group. The serum ubiquinol level prior to supplementation was 0.96 ± 0.23 µg/mL (range, 0.50--1.46 µg/mL) and no correlation with age was observed at a level of 1.04 ± 0.22 µg/mL in males and 0.92 ± 0.24 µg/mL in females, although slightly lower levels in females were found (*p* = 0.07). At 4 weeks after the first dose, the groups receiving the active capsule showed a significant increase in serum ubiquinol level both compared with the group receiving placebo and that before first dosing (*p* \< 0.001). Although the 150-mg group showed higher levels than the 100-mg group, the difference was not statistically significant. Correlation analysis of the degree of increase in blood ubiquinol level and scores on evaluated items suggested the positive correlation with ∆VAS score for relaxation after cognitive function task ([Figure 1](#nutrients-12-01640-f001){ref-type="fig"}a) and ∆lnHF, indicating parasympathetic nerve activity ([Figure 1](#nutrients-12-01640-f001){ref-type="fig"}b), and a negative correlation with decline in ∆motivational response of the mATMT ([Figure 1](#nutrients-12-01640-f001){ref-type="fig"}c). The DSST score is inversely related to age. The rise in the DSST score was greater in the ubiquinol-intake groups than in the placebo group; however, the difference was not statistically significant. In the placebo group, a significant increase in the scores was observed at 8 weeks and 12 weeks compared with that before first dosing, suggesting that habituation due to testing every 4 weeks had an influence on learning effect ([Table 2](#nutrients-12-01640-t002){ref-type="table"}). The biochemical parameters did not reveal any changes indicative of safety issues. 4. Discussion {#sec4-nutrients-12-01640} ============= In the present double-blind study, we evaluated the anti-fatigue effects of ubiquinol in healthy individuals having mild subjective fatigue. Participants took three capsules of ubiquinol once daily for 12 weeks (the placebo group; 100-mg group or 150-mg ubiquinol groups). Our results indicate a certain level of improvement effects in the oxidative stress, cognitive function, and subjective fatigue sensation and sleepiness. Fatigue is a condition in which cells and tissues are damaged by reactive oxygen species generated during excessive activity \[[@B4-nutrients-12-01640]\]. In our previous study using a complex fatigue animal model where rats are exposed to relatively long-lasting stress and partial sleep deprivation, which humans often experience in their daily lives, plasma levels of total nitric oxide metabolite species were increased, indicating enhancement of systemic oxidative stress \[[@B49-nutrients-12-01640]\]. An increase in lipid peroxidation and a decrease in levels of antioxidant enzymes in the brain in the mouse model of chronic fatigue syndrome were also reported \[[@B50-nutrients-12-01640]\]. In human studies, the impact of oxidative stress at different levels of fatigue in healthy individuals and patients with chronic fatigue syndrome has been reported \[[@B51-nutrients-12-01640]\], suggesting that antioxidants including ubiquinol are effective against fatigue \[[@B4-nutrients-12-01640],[@B5-nutrients-12-01640]\]. Ubiquinol is an essential electron carrier and proton translocator in the mitochondrial respiratory chain. Ubiquinol is also an obligatory cofactor of the dihydroorotate dehydrogenase \[[@B52-nutrients-12-01640]\] and serves as a potent antioxidant in membranes by directly scavenging radicals and regenerating α-tocopherol \[[@B53-nutrients-12-01640],[@B54-nutrients-12-01640]\]. In the d-ROMs test, oxidative stress in the blood was evaluated by measuring hydroperoxides serum metabolites resulting from oxidative stress. In the serum, ubiquinol is primarily incorporated in lipoproteins, such as low-density lipoprotein (LDL), and its reducing activity on LDL-lipid peroxides and α-tocopherol radicals has been reported \[[@B55-nutrients-12-01640]\]. The significant reduction in d-ROMs level after ubiquinol intake in the 150-mg group is thought to be a result of an increased antioxidant ubiquinol level in the blood. In the 100-mg group, the significant reduction in d-ROMs level by ubiquinol administration was not observed influenced by some outliers of d-ROMs, which is above the second positive standard deviation (+2 SD) of mean value in the placebo group. The participants were assessed every 4 weeks on pre-fixed Saturday or Sunday mornings. The subjective fatigue sensation was evaluated with VAS before and after cognitive function tasks, which was also related to fatigue load. Although fatigue load includes both exercise load, such as bicycle pedaling, and mental load that imitate work on a computer, mental fatigue tasks were implemented in this study. Ubiquinol intake led to significant improvements in subjective fatigue sensation and sleepiness after the cognitive-fatigue load in both the 100-mg and 150-mg groups compared with that in the placebo group. These results appear to support previous reports of beneficial effects of ubiquinol on fatigue in the daily life of individuals of middle and advanced ages \[[@B12-nutrients-12-01640]\]. After 4 weeks of 150-mg ubiquinol intake, the motivated response to cognitive function tasks became faster and the cognitive function improved. No significant difference from the placebo group was observed after 8 and 12 weeks of intake; this suggests that habituation to the task had an impact on the placebo group whose response times gradually shortened, decreasing every to −0.03 s after 4 weeks, −0.08 s after 8 weeks, and −0.10 s after 12 weeks compared with the values before the first dose. The 150-mg group showed consistency at −0.14 s after 4 weeks, −0.13 s after 8 weeks, and −0.14 s after 12 weeks, suggesting improvement and maintenance of cognitive functions. Previous studies have also reported improvements in cognitive function following ubiquinol intake in patients with chronic fatigue syndrome \[[@B5-nutrients-12-01640]\] as well as in individuals of an advanced age \[[@B56-nutrients-12-01640]\]. The motivated response to cognitive function tasks (mATMT) and antioxidative activity also improved in a 4-week study in healthy individuals following the intake of hydrogen-rich water having antioxidative functions \[[@B35-nutrients-12-01640]\]. We believe that the correction of mitochondrial function and oxidative stress due to ubiquinol intake also took place in our study and was responsible for improved cognitive function performance; cognitive performance improvement correlated blood ubiquinol level. Conversely, no significant improvement due to ubiquinol intake was observed in terms of the DSST scores. DSST is a cognitive challenge in which participants quickly and accurately enter combinations of figures and numbers, and scores are reported to decrease significantly in patients with dementia or depression \[[@B44-nutrients-12-01640]\]. An interventional prospective cohort study with ubiquinol showed that the DSST scores correlated inversely with age and that the scores improved following ubiquinol intake for half a year or longer, suggesting beneficial effects in terms of improvement and maintenance of cognitive functions \[[@B13-nutrients-12-01640]\]. In the present study, the placebo group showed a gradual increase in the score from before intake, and scores at 8 and 12 weeks were significantly higher than that before the first dose. This is thought to have resulted from increased DSST proficiency from performing the test every 4 weeks. In the ubiquinol groups, compared with the scores before the first dose, scores increased significantly starting at 4 weeks of intake, and in the 150-mg group, it was also significant compared with the placebo group. However, no significant difference was observed between the degrees of change in each group. Although the effect of ubiquinol on DSST score improvement was not clear in this study, this could be owing to the relatively young age of the participants and the short intake period of 3 months. In the present study, an improvement in autonomic nerve function was observed in the 150-mg group only after 4 weeks of intake, and no significant improvement was observed after 8 or 12 weeks. However, the parasympathetic indicator lnHF and VAS score for relaxation after the cognitive function task correlated positively with the blood ubiquinol level. It was clear that continuous intake of ubiquinol also results in increased parasympathetic activity in patients with chronic fatigue syndrome \[[@B5-nutrients-12-01640]\]. Furthermore, symptoms of depression have been reported to improve along with the blood ubiquinol level in patients with chronic fatigue syndrome \[[@B5-nutrients-12-01640]\]. It may be inferred from the results of the present study that antioxidative activity due to ubiquinol intake modulated negative emotions and not only provided a subjective sense of relaxation but also stimulated the parasympathetic activity. The improvements in fatigue, relaxation, cognitive function, and autonomic nerve function obtained in this study from ubiquinol intake suggest a relationship with improved central nervous system functions. Molecular brain imaging and brain-activity-imaging studies based on positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) have shown the specific regions of the brain that are associated with central management of these functions and sensations. The orbitofrontal area is responsible for managing information related to subjective fatigue sensation \[[@B57-nutrients-12-01640]\]; the corpus striatum is involved in management of motivated response during the performance of cognitive tasks \[[@B58-nutrients-12-01640]\]; and the anterior cingulate gyrus is associated with control of the autonomic nerve activity \[[@B59-nutrients-12-01640]\]. Since it has been considered that fatigue could potentially accelerate the oxidation of the central nervous system including these regions of the brain \[[@B60-nutrients-12-01640]\], the present results may suggest that ubiquinol intake may have inhibited intracerebral oxidation. Interestingly, when intracerebral uptake of ubiquinol and ubiquinone were verified using PET, the uptake level of ubiquinol was considerably higher than that of ubiquinone \[[@B61-nutrients-12-01640]\]. Therefore, improvements in fatigue, relaxation, cognitive function, and autonomic nerve function seen in the present study might also be greater with ubiquinol than with ubiquinone. This point could be clarified by conducting comparative clinical trials of ubiquinone versus ubiquinol. There are limitations in the present study. In order to generalize our results, further study involving a larger number of participants is essential. We did not evaluate the effect of energy production by ubiquinol administration in the present study. Recovery from fatigue occurs when cells and tissues damaged by reactive oxygen species have been adequately repaired. However, the damage continues with inadequate repair in the case of the lack of necessary energy for recovery \[[@B4-nutrients-12-01640]\]. We previously demonstrated not only enhancement of oxidative stress but also decreases in amount of ATP and metabolites involved in the tricarboxylic acid (TCA) cycle, such as cis-aconitate and isocitrate, in the complex fatigue animal model \[[@B49-nutrients-12-01640]\]. Likewise, in patients with chronic fatigue syndrome, we revealed a profile of abnormal energy metabolism resulting from deficiencies in aconitase activity in the TCA cycle \[[@B62-nutrients-12-01640]\]. Accordingly, ubiquinol that avoids damage from oxidative stress and that can stimulate energy production may be effective against fatigue. Further study using metabolome analysis is needed to confirm the effect of energy production by ubiquinol administration on healthy individuals with mild fatigue. In the present study, although we mainly examined the effects of ubiquinol on the central nervous system, we did not directly evaluate the dynamics of ubiquinol in the brain. Human neuroimaging studies using PET and fMRI are thus underway in our laboratory to identify the mechanisms underlying the effects of ubiquinol intake on the central nervous system that can attenuate fatigue. 5. Conclusions {#sec5-nutrients-12-01640} ============== In the present study, results of evaluation of the effects of ubiquinol in healthy individuals with mild fatigue suggested that ubiquinol contributes to the improvement of QOL in individuals with mild fatigue by reducing fatigue and sleepiness following cognitive function load, promoting motivated engagement with cognitive function tasks while providing a relaxing effect, and reducing oxidative stress. We would like to thank Mika Furusawa and Machiko Nakatani for their excellent technical assistances. K.M. and Y.W. conceived and designed the experiments; K.M., A.T.S., and K.W. performed the experiments; K.M., A.T.S., and K.W. analyzed the data; and K.M., A.T.S., K.W., and Y.W. wrote the paper. All authors have read and agreed to the published version of the manuscript. This work was supported by KANEKA Corporation and partly supported by the Japan Science and Technology Agency (JST)/RIKEN Compass to Healthy Life Research Complex Program. Y.W. received the fund for the present study from KANEKA Corporation. The other authors declare that there are no conflicts of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. {#nutrients-12-01640-f001} nutrients-12-01640-t001_Table 1 ###### Participant characteristics. Variables Placebo 100 mg/day 150 mg/day -------------------------- ------------- ------------ ------------- Number 20 20 22 Sex (male:female) 7:13 6:14 8:14 Age (years; mean ± S.D.) 41.3 ± 13.4 44.0 ± 9.8 40.4 ± 11.8 Age (range in years) 20--64 23--58 20--61 S.D.: standard deviation. nutrients-12-01640-t002_Table 2 ###### Effects of ubiquinol by dosage group. -------------------------------------------------------------------------------------------------- Item Week Placebo 100 mg 150 mg --------------------------- -------------- -------------------- -------------------- ------------- Serum ubiquinol\ 0 0.96 ± 0.19 1.00 ± 0.29 0.93 ± 0.20 (µg/mL) 4 0.82 ± 0.19 2.96 ± 1.23 \*\*\* 3.53 ± 1.01 \*\*\* 8 1.15 ± 0.44 3.29 ± 1.40 \*\*\* 3.93 ± 1.40 \*\*\* 12 0.76 ± 0.17 2.61 ± 1.07 \*\*\* 3.27 ± 1.20 \*\*\* Pre-task fatigue\ 0 \- \- \- (score) 4 4.7 ± 22.2 −5.1 ± 16.5 −4.1 ± 16.9 8 7.0 ± 29.3 −4.5 ± 22.5 −3.4 ± 22.6 12 0.9 ± 22.0 −5.4 ± 20.7 −1.9 ± 22.1 Post-task fatigue\ 0 \- \- \- (score) 4 5.4 ± 28.1 −14.1 ± 11.9 \*\* −9.5 ± 21.8 \* 8 3.7 ± 23.0 −7.3 ± 19.5 −12.3 ± 21.4 \* 12 −2.1 ± 25.9 −10.9 ± 17.7 −3.6 ± 20.8 Post-task sleepiness\ 0 \- \- \- (score) 4 11.9 ± 27.0 −0.8 ± 13.5 \* −7.6 ± 18.7 \*\* 8 4.8 ± 25.1 −3.6 ± 20.4 −7.2 ± 25.4 12 3.8 ± 28.2 −2.2 ± 19.4 −3.3 ± 24.7 Post-task mood reduction\ 0 \- \- \- (score) 4 5.8 ± 14.0 −8.5 ± 16.2 \* 0 ± 26.4 8 2.2 ± 13.6 −4.4 ± 23.5 −1.6 ± 23.9 12 2.8 ± 15.6 −10.8 ± 16.8 \* 4.1 ± 24.4 ESS: sleepiness\ 0 \- \- \- (score) 4 0.65 ± 3.98 −1.10 ± 4.95 −0.59 ± 3.94 8 1.00 ± 4.95 −1.47 ± 2.74 \* −0.18 ± 3.80 12 0.50 ± 3.81 −0.32 ± 3.25 −0.86 ± 4.61 Post-task relaxation\ 0 \- \- \- (score) 4 4.5 ± 16.9 3.0 ± 16.7 5.3 ± 18.0 8 −0.3 ± 14.1 4.2 ± 18.2 11.7 ± 20.3 \* 12 0.6 ± 17.2 4.6 ± 13.2 7.0 ± 17.6 Sleepiness during task\ 0 \- \- \- (score) 4 6.1 ± 16.1 1.6 ± 18.5 −8.5 ± 18.3 \* 8 −5.5 ± 14.7 −1.7 ± 22.2 −7.0 ± 20.2 12 0.9 ± 15.7 −0.2 ± 22.1 −9.3 ± 27.3 Motivational response\ 0 \- \- \- (sec) 4 −0.03 ± 0.11 −0.03 ± 0.11 −0.14 ± 0.21 \* 8 −0.08 ± 0.11 −0.08 ± 0.11 −0.13 ± 0.17 12 −0.10 ± 0.11 −0.04 ± 0.11 −0.14 ± 0.20 d-ROMs\ 0 \- \- \- (U.CARR) 4 −17.3 ± 23.8 −1.0 ± 34.9 −26.4 ± 35.8 8 −7.3 ± 30.4 −6.1 ± 34.9 −31.0 ± 42.3 \* 12 −19.6 ± 33.9 −8.0 ± 47.9 −29.4 ± 41.5 DSST\ 0 63.6 ± 9.37 60.7 ± 9.07 68.4 ± 11.6 (score: absolute value) 4 65.4 ± 9.86 63.9 ± 8.54 72.1 ± 13.3 \* 8 67.1 ± 8.56 65.0 ± 9.24 73.9 ± 13.8 \* 12 69.9 ± 9.22 66.0 ± 11.5 76.0 ± 13.8 ln(LF/HF)\ 0 \- \- \- (ratio) 4 0.26 ± 0.72 0.13 ± 0.72 0.28 ± 1.04 8 0.47 ± 0.82 0.03 ± 0.95 −0.02 ± 0.84 \* 12 0.41 ± 0.81 0.36 ± 0.85 0.32 ± 1.15 -------------------------------------------------------------------------------------------------- Data show means ± standard deviations. \* *p* \< 0.05, \*\* *p* \< 0.01, \*\*\* *p* \< 0.001. Dunnett's multiple comparison vs. placebo. ESS: Epworth Sleepiness Scale, d-ROMs: reactive oxygen metabolites-derived compounds, DSST: Digit Symbol Substitution Test, ln(LF/HF): log-transformed low-frequency component power/high-frequency component power.

{#sp1 .448}

Introduction {#s1} ============ In many organisms, early embryonic development is directed exclusively by maternal products that are deposited into the female gamete during oogenesis. Following the clearance of a subset of these products ([@bib95]), transcription is initiated and the zygotic genome acquires developmental control ([@bib9]; [@bib25]; [@bib46]; [@bib86]). This handover is referred to as the maternal-to-zygotic transition and the onset of transcription is called zygotic genome activation (ZGA). The absolute time and number of cell cycles required before the first transcripts can be detected is species specific ([@bib86]). Additionally, from one gene to another the timing of transcriptional activation varies ([@bib1]; [@bib16]; [@bib27]; [@bib29]; [@bib53]; [@bib68]; [@bib69]; [@bib77]; [@bib87]). In fact, for some genes the first zygotic transcripts can be detected several cell cycles before the stage that is traditionally defined as the time point of ZGA ([@bib18]; [@bib29]; [@bib81]; [@bib94]). In spite of the progress made, it remains unclear how the onset of transcription in embryos is temporally regulated. Several lines of evidence suggest that the absence of transcription during early embryonic development could be due to limited levels of transcription factors ([@bib5]; [@bib90]). In this scenario, transcriptional activation would occur once a threshold level of these factors is reached. For example, experiments that used the transcriptional activity of injected plasmids as a read-out revealed that an increase in the amount of the potent, heterologous, transcriptional activator GAL4-VP16 can overcome transcriptional repression of its target gene in the early embryo ([@bib5]). However, it remained unclear whether limited levels of transcription factors contribute to the absence of endogenous transcription in early embryos. Additional support for the limited machinery model came from work showing that an increase in the concentration of the general transcription factor TBP can cause premature transcription from an injected -- and incompletely chromatinized -- DNA template in *Xenopus* embryos. This effect was maintained only when non-specific DNA was added to titrate chromatin assembly ([@bib5]; [@bib90]). These results suggested that low TBP levels may play a role in the absence of transcription during the early stages of *Xenopus* development, but that increasing TBP alone is not sufficient to cause sustained premature transcription. During the cleavage stages of *Xenopus* development, TBP levels increase due to translation, which suggests that TBP levels might contribute to the timely activation of transcription during ZGA ([@bib90]). Transcription factors have recently been identified that are required for the activation of the first zygotically expressed genes in *Drosophila* (Zelda) and zebrafish (Pou5f3, Sox19b, Nanog) ([@bib26]; [@bib47]; [@bib48]; [@bib50]; [@bib61]). RNA for these factors is maternally provided and their levels increase due to translation during the early cell cycles. This suggests the possibility that an increase in the concentration of these transcription factors might contribute to the shift from transcriptional repression to transcriptional activity. Although transcription factors levels clearly influence transcriptional activity during early embryogenesis, there is evidence to show that the transcriptional machinery is operational prior to ZGA ([@bib19]; [@bib54]; [@bib59], [@bib60]; [@bib73]) (see below). Thus, the timing of ZGA cannot be solely explained by a requirement to reach a threshold level of transcriptional activators. The finding that a premature increase in the number of nuclei or the amount of DNA resulted in premature transcription of injected plasmids in *Xenopus* embryos suggested that the transcriptional machinery is fully functional prior to genome activation and led to the excess repressor model ([@bib59]). This model postulates that a transcriptional repressor is titrated by binding to the exponentially increasing amount of genomic DNA, until it is depleted first from the soluble fraction, and then from DNA, to allow for the onset of transcription. Related studies in zebrafish and *Drosophila* have provided further evidence for this model. Endogenous transcription is initiated earlier in zebrafish embryos that accumulate DNA due to a defect in chromosome segregation ([@bib19]), and transcription is delayed in haploid *Drosophila* embryos compared to diploid embryos, albeit not for all genes ([@bib54]). The excess repressor model predicts that the repressor is present in large excess, at relatively stable levels while the genome is inactive, and can bind DNA with high affinity. Core histones fulfill these criteria ([@bib2]; [@bib92]). Moreover, when bound to DNA in the form of nucleosomes, histones can affect DNA accessibility for DNA-binding proteins. To date, two key studies have investigated the role of core histones in the temporal regulation of zygotic transcription in *Xenopus* embryos ([@bib5]; [@bib6]). Experiments that used the transcriptional activity of injected plasmids as a read-out revealed that premature transcription caused by an excess of non-specific DNA can be negated by the addition of histones ([@bib5]). More recently, the level of histones H3/H4 was shown to regulate the level of transcription in *Xenopus* egg extract and H3 was suggested to play a similar role in the embryo ([@bib6]). Taken together, these results support the idea that histones play a role in regulating the timing of zygotic transcription. If histones function as repressors according to the original excess repressor model, it would be predicted that a substantial reduction of the histone-density on DNA would cause the onset of transcription ([@bib6]; [@bib59]). However, while such a scenario might be possible for typical sequence-specific repressors of transcription, it is unlikely for histones. Histones assemble into histone octamers on DNA to form nucleosomes, the basic building blocks of chromatin. Thus, random depletion of nucleosomes from DNA would severely compromise the integrity of chromatin structure. Taken together, there is support for the idea that histone levels play a role in regulating the timing of zygotic transcription, but it remains unclear how this would mechanistically work. Furthermore, the observation that both activator and histone levels play a role in shifting the balance between repression and activation at genome activation remains to be clarified. Here, we analyze the onset of zygotic transcription in zebrafish embryos. With a quantitative approach, we show that the concentration of non-DNA-bound histones determines the timing of zygotic transcription and that all four core histones are required for this effect. The reduction in nuclear histone concentration that coincides with genome activation does not result in a significant change in nucleosome density, but rather allows transcription factors to successfully compete for DNA binding. In agreement with this, the association of transcription factors with the genome is sensitive to histone levels, and changing the concentration of transcription factors also affects the time of transcription. Our results show that transcription is regulated by a dynamic competition for DNA binding between histones and transcription factors. Transcription begins when the concentration of non-DNA-bound histones in the nucleus has sufficiently dropped so that the transcriptional machinery can outcompete histones for binding to DNA. Results {#s2} ======= In zebrafish, zygotic transcription starts \~3 hr post-fertilization, around the tenth cell division ([@bib1]; [@bib27]; [@bib29]; [@bib35]; [@bib69]) ([Figure 1A](#fig1){ref-type="fig"}). To analyze the onset of transcription in the embryo in detail, we identified six genes that are not maternally provided and that have previously been shown to be activated at the onset of genome activation ([@bib1]; [@bib69]) ([Figure 1---figure supplement 1A](#fig1s1){ref-type="fig"}). At the 1000-cell (1K) stage, transcripts can be detected, especially when choosing late stage embryos ([Figure 1B](#fig1){ref-type="fig"}). Thus, to clearly distinguish between transcription being off and on, we analyzed early 1K and (mid) high stage embryos. Using this approach, analysis by qPCR allowed us to detect consistent induction of these genes at high stage ([Figure 1B](#fig1){ref-type="fig"} and [Figure 1---figure supplement 1B](#fig1s1){ref-type="fig"}). We will refer to the stages before and after induction as before and following genome activation. To relate the onset of transcription to the number of cells present in the embryo, we next counted the number of cells in embryos ranging from 1K to dome stage. We imaged DAPI-stained nuclei on a two-photon microscope and counted them using the software Imaris ([Figure 1C](#fig1){ref-type="fig"}). Using nuclei as a proxy for cell number, we obtained counts that agreed with numbers of cells per embryo obtained by others for 1K ([@bib37]). In contrast, our cell count for high stage, for example, was slightly higher (1900 vs 1800) ([@bib37]), which is consistent with the later high stage which we analyzed ([Figure 1D](#fig1){ref-type="fig"} and [Figure 1---source data 1](#SD1-data){ref-type="supplementary-material"}). We conclude that the set of genes we selected is robustly induced at high stage, when embryos contain \~1900 cells, and thus represent a reliable system to analyze the onset of zygotic transcription during embryogenesis.10.7554/eLife.23326.003Figure 1.Assay to analyze the onset of transcription in zebrafish.(**A**) In zebrafish, transcription begins \~3 hr post-fertilization. Stage-specific drawings of representative embryos are adapted from [@bib41]) with permission. (**B**) Expression of six genes was analyzed by qPCR at 512-cell, early 1K, mid 1K, high and oblong stage in wild-type embryos. Inset shows the same data, focusing on 1K and high stage. Data were taken from [Figures 2B](#fig2){ref-type="fig"} and [3D](#fig3){ref-type="fig"}. Error bars represent SEM (n ≥ 4). (**C**) Cell counting of DAPI-stained nuclei imaged with a two-photon microscope. Multiple z-slices and four tiles were stitched to allow rendering in imaging software Imaris and the calculation of cell number. Image shown is a representative example of high stage. Scale bar, 100 μm. (**D**) Quantification of the number of cells at 1K, high, oblong, sphere and dome stage. Each data point represents a biological replicate consisting of three embryos. Error bars represent SEM (n = 3). (**E**) Western blots showing the protein level of histone H3, H4, H2A, and H2B in embryos at 8-cell, 64-cell, 256-cell and 1K stages. Tubulin was used to control for equal loading in each experiment. Blots shown are representative examples (n \> 3).**DOI:** [http://dx.doi.org/10.7554/eLife.23326.003](10.7554/eLife.23326.003)10.7554/eLife.23326.004Figure 1---source data 1.Cell numbers for wild-type zebrafish embryos.At every stage, cells were counted for three biological replicates, each consisting of three embryos.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.004](10.7554/eLife.23326.004)10.7554/eLife.23326.005Figure 1---figure supplement 1.Selection of genes and *eif4g2α* as a normalizer gene.(**A**) Schematic representation of gene-selection procedures. (**B**) Maternally loaded *eif4g2α* mRNA was used as a control in qPCR analyses. To test whether the level of *eif4g2α* mRNA was stable during the stages of interest (not degraded and independent of zygotic transcription), the level of *eif4g2α* mRNA was analyzed in wild-type embryos and in embryos that were injected with the transcription inhibitor α-amanitin. As a control, *sox19a* mRNA was analyzed. Indicated are the Ct's (threshold cycles in quantitative PCR analysis) for *eif4g2α* and *sox19a* as obtained at 512-cell, early 1K, high, oblong and late oblong in the presence (minus α-amanitin) and absence (plus α-amanitin) of zygotic transcription. Whereas the Ct's for *sox19a* decrease starting at high stage (green), the Ct's for *eif4g2α* do not (red). Moreover, α-amanitin inhibits the transcription of *sox19a* (black) but does not affect the levels of *eif4g2α* (blue). Together, this shows that *eif4g2α* is neither degraded nor zygotically transcribed during the stages we analyzed. Shown is a representative example of two replicates.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.005](10.7554/eLife.23326.005)© 1995 John Wiley and Sons. All Rights Reserved1995John Wiley and Sons[Figure 1](#fig1){ref-type="fig"} part A, lower panel sketches of embryos were reproduced from [@bib41] with permission. Increasing the levels of all core histones delays onset transcription and gastrulation {#s2-1} -------------------------------------------------------------------------------------- Experiments in *Xenopus* embryos led to the hypothesis that histone levels regulate the onset of zygotic transcription ([@bib5]; [@bib6]). To analyze whether in zebrafish, histones are potential candidates to be excess repressors, we analyzed the relative levels of the core histones---H3, H4, H2A and H2B---by Western blot. We found that they are present at relatively stable amounts from 8-cell to 1K stage ([Figure 1E](#fig1){ref-type="fig"}). Assuming that at 1K stage there are sufficient histones to wrap all genomes into chromatin, this suggests that histones are in excess relative to the amount of DNA during the earlier stages. Thus, histones could function as excess repressors of the zygotic genome in zebrafish. If histones function as excess repressors in zebrafish embryos, it would be predicted that their level would affect the onset of transcription. To test this, we analyzed the effect of increasing the amount of histones in the embryo on the timing of transcriptional activation. We injected a stoichiometric mixture of the four core histones (from here on referred to as histone cocktail, HC; see Materials and methods for more details) into embryos at the 1-cell stage and then analyzed the onset of transcription for the previously characterized set of genes ([Figures 2A](#fig2){ref-type="fig"} and [1B](#fig1){ref-type="fig"}). An increase in the amount of histones delayed the onset of transcriptional activation: transcripts were detected at high stage in uninjected embryos, whereas in embryos injected with histone cocktail, transcription was only induced at oblong stage, a complete developmental stage later ([Figure 2B](#fig2){ref-type="fig"} and [Figure 2---figure supplement 1A](#fig2s1){ref-type="fig"}). Comparison of gene expression levels in uninjected and injected embryos at high stage (when transcripts can consistently be detected in uninjected embryos) revealed that the level of induction is reduced significantly upon injection of the histone cocktail but not upon injection of BSA as a control ([Figure 2B](#fig2){ref-type="fig"} and [Figure 2---figure supplement 1A](#fig2s1){ref-type="fig"}, bar graphs). Extending the analysis further, a large set of genes in Nanostring analysis confirmed that the effect we observed is general, and not limited to six genes ([Figure 2C](#fig2){ref-type="fig"}, [Figure 2---figure supplement 2](#fig2s2){ref-type="fig"}, [Figure 2---source data 1](#SD2-data){ref-type="supplementary-material"}). Staging by morphology was corroborated by cell counting, with absolute time between the analyzed stages being constant, confirming that changes in the timing of transcription were not due to effects on cell cycle length or developmental progression ([Figure 2---figure supplement 1B](#fig2s1){ref-type="fig"}). Moreover, the injected histones can be incorporated into chromatin, as indicated by labeling one of them with Cy5 and detecting this label in chromatin when imaging embryos after injection ([Figure 2---figure supplement 1C](#fig2s1){ref-type="fig"}), confirming that they are functional. Together, these data show that an increase in the excess amount of histones in the embryo delays the onset of transcription.10.7554/eLife.23326.006Figure 2.Increasing the levels of all core histones delays onset of transcription and gastrulation.(**A**) Schematic representation of experimental procedure. Histone cocktail (HC) containing \~5800 genomes worth of histones, or BSA was injected into the yolk of 1-cell embryos and qPCR and NanoString analysis was carried out at stages around genome activation. Orange crosses represent the timing of stages used for the analysis. (**B**) Expression of *mxtx2* and *fam212aa* was analyzed by qPCR at early 1K, high, and oblong stage in uninjected, BSA-injected and HC-injected embryos. Bar graphs show the same data, focusing on high stage. Error bars represent SEM (n ≥ 13). \*\*\*p\<0.001 (two-tailed Student's t test, compared to BSA control). (**C**) Expression of 53 zygotically expressed genes was analyzed by NanoString analysis at high stage in uninjected, BSA-injected and HC-injected embryos. Mean counts of three independent biological replicates are shown. Location of *mxtx2* and *fam212aa* counts is indicated (See [Figure 2---figure supplement 2](#fig2s2){ref-type="fig"} for more details). (**D**) Relative expression level of *mxtx2* and *fam212aa* at high stage, for embryos injected with BSA, HC, and HC minus H3, H4, H2A, or H2B. Error bars represent SEM (n = 7). \*\*\*p\<0.001 (ordinary one-way ANOVA). (**E**) Brightfield images of embryos that were not injected, injected with BSA, or injected with HC. Boxed images represent the onset of gastrulation. Scale bar shown for the uninjected 2-cell embryo applies to all treatments except for 24 hpf embryos which have a different scale bar. All scale bars represent 250 μm. hpf, hours post-fertilization. (**F**) Bar graph shows the quantification of the extra time it takes embryos to start gastrulation upon injecting BSA, HC, or HC minus one histone, compared to uninjected embryos. Error bars represent SEM (n = 27 for BSA, n = 25 for HC, n = 7 for HC minus one histone experiments). \*\*\*p\<0.001 (ordinary one-way ANOVA with Tukey's multiple comparison test). In B and D, mRNA levels are normalized to the expression of *eif4g2α*.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.006](10.7554/eLife.23326.006)10.7554/eLife.23326.007Figure 2---source data 1.NanoString probe set.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.007](10.7554/eLife.23326.007)10.7554/eLife.23326.008Figure 2---figure supplement 1.Increasing the levels of all core histones delays onset of transcription.(**A**) Expression of *nnr*, *vox*, *sox19a*, and *grhl3* was analyzed by qPCR at early 1K, high, and oblong stage in uninjected, BSA-injected and HC-injected embryos. Bar graphs show the same data, focusing on high stage. Error bars represent SEM (n ≥ 13). \*\*\*p\<0.001 (two-tailed Student's t-test, compared to BSA control). (**B**) Staging by morphology was verified by cell counting. Each data point represents a single embryo. Error bars represent SEM. (**C**) Confocal microscope images of transgenic fish line Tg(h2afz:h2afz-GFP) injected with Cy5 conjugated to H4. Arrow points at chromatin in dividing cell. Scale bar, 20 μm. (**D**) Relative expression level of *nnr*, *vox*, *sox19a*, and *grhl3* at high stage, for embryos injected with BSA, HC, and HC minus H3, H4, H2A, or H2B. Error bars represent SEM (n = 7). \*p\<0.05; \*\*p\<0.01; \*\*\*p\<0.001 (ordinary one-way ANOVA). In A and D, expression is normalized to the expression of *eif4g2α*.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.008](10.7554/eLife.23326.008)10.7554/eLife.23326.009Figure 2---figure supplement 2.Increasing the levels of all core histones delays onset of transcription for a large number of genes.NanoString's nCounter technology was used to analyze changes in gene expression upon HC injection for a large number of genes (see Materials and methods for more details). From a custom probe set with 84 zygotically expressed genes and 12 controls genes (see [Figure 2---source data 1](#SD2-data){ref-type="supplementary-material"}), 53 genes that are induced at high stage were used for analysis. mRNA was collected from high stage embryos that were uninjected, BSA-injected and HC-injected (n = 3). (**A**) Fold difference in expression of twelve control genes, for embryos injected with BSA or HC compared to uninjected. Control genes are maternally provided and were previously shown to be stable from 512 cell to dome stage in NanoString analysis (data not shown). Error bars represent SEM (n = 3). No significant difference was detected between BSA or HC-injected in control genes (Ordinary one-way ANOVA with Tukey's multiple comparison test), showing that there are no differences in total RNA amount between samples. (**B**) Proportion of genes affected by BSA or HC injection at high stage in NanoString analysis. Error bars represent SEM (n = 3). A large proportion of genes are down-regulated in HC-injected embryos (86%) compared to BSA-injected embryos (4%). (**C**) Mean counts of three independent biological replicates in NanoString analysis for uninjected, BSA-injected and HC-injected embryos compared to a high (left) and 1K (right) standard from uninjected embryos. While uninjected and BSA-injected are statistically similar to the high standard, HC-injected is statistically similar to the 1K standard. Thus, HC-injected embryos that are developmentally at high stage, are transcriptionally delayed by one developmental stage. Error bars represent SEM (n = 3). \*\*\*p\<0.001 (Ordinary one-way ANOVA with Tukey's multiple comparison test). (**D**) Fold difference in mRNA counts for HC-injected and BSA-injected compared to uninjected embryos at high stage for all genes that were analyzed by qPCR in this study. Error bars represent SEM (n = 3). \*p\<0.05; \*\*p\<0.01; \*\*\*p\<0.001 (two-tailed Student's t-test ratio paired, compared to BSA-injected).**DOI:** [http://dx.doi.org/10.7554/eLife.23326.009](10.7554/eLife.23326.009) To test whether the effect we observe upon injecting the histone cocktail required an increase in the level of all four core histones, we next removed one histone at a time from the cocktail. The total protein content was kept constant by raising the level of the other three histones. Removing any histone from the histone cocktail impaired the ability of the histone cocktail to delay the onset of transcription ([Figure 2D](#fig2){ref-type="fig"} and [Figure 2---figure supplement 1D](#fig2s1){ref-type="fig"}). These results show that the injection of basic proteins into the embryo per se does not affect the onset of transcription. Moreover, these results argue that the effect of the histone cocktail relies on increasing the amount of all four histones and suggest that histones exert their repressive effect together. Since the onset of zygotic transcription is known to be required for gastrulation ([@bib34]; [@bib47]; [@bib96]), we analyzed the effect of injecting the histone cocktail on the onset of gastrulation. Embryos injected with the histone cocktail initiated gastrulation later than uninjected embryos ([Figure 2E](#fig2){ref-type="fig"}). Although there was a delay following injection of BSA, it was significantly shorter than that observed with the histone cocktail and appeared to be a non-specific effect of injection ([Figure 2F](#fig2){ref-type="fig"}). Following the onset of gastrulation, embryos appeared to develop normally ([Figure 2E](#fig2){ref-type="fig"}, 24 hpf). Removing any histone from the histone cocktail reduced the developmental delay we observed upon injecting the histone cocktail ([Figure 2F](#fig2){ref-type="fig"}). We note that the developmental delay in the minus-one histone experiments was not reduced to the level observed for BSA injections. We therefore expect that injecting histones has an additional effect on developmental progression that is independent of the delay in transcription. We conclude that the delay in transcription as a consequence of increased histone levels causes a delay in the onset of gastrulation. Decreasing the pool of available histones causes premature transcription {#s2-2} ------------------------------------------------------------------------ If the level of histones regulates zygotic genome activation, it can also be predicted that a reduction in histone levels would result in the premature induction of transcription. A large fraction of the histones that is present at the onset of transcription is loaded in the egg already as protein ([Figure 1E](#fig1){ref-type="fig"}). Pentraxin three is a soluble pattern recognition molecule that has been shown to rapidly and irreversibly bind to the core histones H3 and H4 ([@bib11]; [@bib17]). We injected mRNA encoding PTX3 fused to RFP, to reduce the pool of available histones H3 and H4 in the zebrafish embryo ([Figure 3A](#fig3){ref-type="fig"}). As expected, total levels of H3 and H4 were not affected upon injection of this fusion construct ([Figure 3B](#fig3){ref-type="fig"}). Next, we examined if H4 co-precipitated with RFP-tagged PTX3 ([Figure 3C](#fig3){ref-type="fig"}). Indeed, this histone associates with PTX3 in vivo, suggesting that the injection of PTX3 results in a reduction of the soluble amount of histones H3 and H4 in zebrafish cells. A decrease in the soluble amount of histones caused premature transcription activation: transcripts were detected at early 1K stage, while in the uninjected embryos, transcripts were only detected at mid 1K ([Figure 3D](#fig3){ref-type="fig"} and [Figure 3---figure supplement 1A](#fig3s1){ref-type="fig"}). We included embryos at mid 1K in this experiment, in order to increase our resolution for detecting changes in transcription. Comparison of gene expression levels in uninjected and *ptx3*-injected embryos at early 1K stage (one time-point prior to when genes are first induced in uninjected embryos) revealed that the level of expression is increased upon injection of *ptx3* mRNA ([Figure 3D](#fig3){ref-type="fig"} and [Figure 3---figure supplement 1A](#fig3s1){ref-type="fig"}, bar graphs). A control injection with *rfp* mRNA did not result in co-precipitation with H4 ([Figure 3C](#fig3){ref-type="fig"}), nor did it affect the onset of transcription ([Figure 3D](#fig3){ref-type="fig"} and [Figure 3---figure supplement 1A](#fig3s1){ref-type="fig"}). Staging by morphology was corroborated by cell counting ([Figure 3---figure supplement 1B](#fig3s1){ref-type="fig"}), with absolute time between the analyzed stages being constant. Taken together, our results provide evidence that the level of core histones in the embryo dictates the timing of transcriptional activation.10.7554/eLife.23326.010Figure 3.Decreasing the level of histones causes premature transcription.(**A**) Schematic representation of experimental procedure. *ptx3-rfp* or *rfp* (control) mRNA was injected into the cell of 1-cell embryos and qPCR analysis was carried out at stages around genome activation. Orange crosses represent the timing of stages used for the analysis. (**B**) Western blot analysis of PTX3, histone H3 and H4 levels at 512-cell, 1K and high stage in uninjected embryos, *rfp* and *ptx3-rfp* mRNA-injected embryos. Tubulin was used to control for equal loading. Blots shown are representative examples (n = 3). (**C**) Western blot analysis for histone H4 after a pull-down using an RFP antibody at 1K stage. Uncoupled beads were used as a negative control. Blot shown is a representative example (n = 3). (**D**) Expression of *mxtx2* and *fam212aa* was analyzed by qPCR at 512-cell, early 1K, and mid 1K stage in uninjected, *rfp* mRNA-injected and *ptx3-rfp* mRNA-injected embryos. Bar graphs focus on early 1K stage. Error bars represent SEM (n ≥ 4). \*p\<0.05; \*\*p\<0.01 (two-tailed Student's t-test, compared to *rfp* mRNA control). mRNA levels are normalized to the expression of *eif4g2α*.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.010](10.7554/eLife.23326.010)10.7554/eLife.23326.011Figure 3---figure supplement 1.Decreasing the level of histones causes premature transcription.(**A**) Expression of *nnr*, *vox*, *sox19a*, and *grhl3* was analyzed by qPCR at 512 cell, early 1K and mid 1K in uninjected embryos, embryos injected with *rfp* (control) mRNA and embryos injected with *ptx3-rfp* mRNA. Bar graphs show the same data, focusing on early 1K stage. Error bars represent SEM (n ≥ 4). n.s. p\>0.05; \*p\<0.05; \*\*p\<0.01 (two-tailed Student's t-test, compared to *rfp* mRNA control). Expression is normalized to the expression of *eif4g2α*. (**B**) Staging by morphology was verified by cell counting at the stages used for the analysis. Each data point represents a single embryo. Error bars represent SEM.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.011](10.7554/eLife.23326.011) Onset of transcription coincides with a reduction in nuclear histone concentration {#s2-3} ---------------------------------------------------------------------------------- If histones were to function as excess repressors according to the original excess repressor model, the concentration of non-DNA-bound histones would be predicted to decrease during the cleavage stages of development. To test this prediction, we determined the absolute (molar) content and, correspondingly, the number of molecules of core histones in embryos using a quantitative mass spectrometry approach we recently developed (Kumar et al., unpublished) ([Figure 4A](#fig4){ref-type="fig"} and Materials and methods for more details). We analyzed embryos ranging from 1-cell to shield stage, when gastrulation is well underway ([Figure 4B](#fig4){ref-type="fig"} and [Figure 4---source data 1](#SD3-data){ref-type="supplementary-material"}). We observed an increase in the levels of histone protein until 1K stage, after which levels remained reasonably stable until sphere stage. Then, a rapid increase was observed, which is most likely the result of translation of zygotically produced histone mRNAs. Knowing the absolute numbers of histones per embryo as well as the calculated number of histones required to wrap a genome ([Figure 4B](#fig4){ref-type="fig"}, [Figre 4---source data 1](#SD3-data){ref-type="supplementary-material"}), allowed us to derive the number of genomes worth of histones per embryo. From that, we derived the number of excess (non-DNA bound) histones per cell in genomes worth of histones, for embryos ranging from 1-cell to dome stage ([Figure 4C](#fig4){ref-type="fig"} and Materials and methods for more details). For example, at the 1-cell stage, there are 3098 times more histones per cell than are required to wrap the genome into chromatin. Due to the exponential increase in cell number during cleavage divisions, this number has dropped dramatically at 1K stage ([Figure 4C](#fig4){ref-type="fig"}). However, due to the large number of histones that is loaded in the oocyte, as well as the increase in histone level due to translation ([Figure 4B](#fig4){ref-type="fig"}), there are still nine genomes worth of non-DNA-bound histones per cell. Moreover, because what matters for protein-binding kinetics is the concentration, we next calculated the concentration of non-DNA-bound histones. Because the cleavage divisions are not accompanied by significant growth (the total animal cap volume increases by 29% from 128-cell to 1K, [Figure 4---figure supplement 1A](#fig4s1){ref-type="fig"}), the decreasing number of histones per cell is accompanied by a decreasing cellular volume, and the concentration of non-DNA bound histones in the cell does not change substantially ([Figure 4D](#fig4){ref-type="fig"}). Taken together, this shows that during transcription activation there is still a significant amount of non-DNA-bound histone and that the overall concentration of non-DNA-bound histones in the cell has not decreased by much.10.7554/eLife.23326.012Figure 4.Onset of transcription coincides with a reduction in nuclear histone concentration.(**A**) Our quantitative mass spectrometry approach. Zebrafish histones were quantified by comparing the abundances of native histone peptides with corresponding isotopically labeled peptides from the chimeric protein; chimeric protein was quantified by comparing the abundance of labeled (from chimera) and native (from standard) BSA peptides (see Materials and methods for more details). (**B**) Quantification of the number of histone H3, H4, H2A, and H2B per embryo at indicated stages by quantitative mass spectrometry. Error bars represent SEM (n = 3). (**C**) The excess number of histones per cell (in genomes worth) was calculated using H2B levels ([Figure 4---source data 1](#SD3-data){ref-type="supplementary-material"}) and cell numbers ([Figure 1---source data 1](#SD1-data){ref-type="supplementary-material"}), and by assuming an average of 1.5 genomes per cell (see Materials and methods for more details). For better visualization of the data at later developmental stages the values for 1-cell and 8-cell are not shown in the graph but are 3098 and 518, respectively. Error bars represent SEM (n = 3). GW, genomes worth of histones. (**D**) The total concentration of non-DNA-bound histones was calculated by dividing the excess genomes worth of histone H2B per embryo by the volume of the animal cap at the respective stages ([Figure 4---figure supplement 1A](#fig4s1){ref-type="fig"}). Error bars represent SEM of animal cap volumes (n = 3). GW, genomes worth of histones. (**E**) The nuclear concentration of non-DNA-bound histones was calculated from immunofluorescence (from left to right n = 12, 12, 14, 15) combined with live imaging and mass spectrometry data (see Materials and methods for more details). Error bars represent SEM of animal cap volumes (n = 3). (**F**) Relative differences in H2B intensity between chromatin fractions of 256-cell, 512-cell, 1K, and high stage embryos. Sphere stage embryos were used to determine the linear range of H2B detection (see also [Figure 4---figure supplement 1C](#fig4s1){ref-type="fig"}). Blots shown are representative examples (n ≥ 3). Plots show observed fold differences in H2B intensity in chromatin fractions comparing indicated stages compared to the differences that would be expected if the intensity were to scale with the amount of DNA (E, embryo). (**G**) Competition model. See text for more details (TFBS, transcription-factor-binding site).**DOI:** [http://dx.doi.org/10.7554/eLife.23326.012](10.7554/eLife.23326.012)10.7554/eLife.23326.013Figure 4---source data 1.Quantification of histone number by mass spectrometry.See Materials and methods for details on calculations.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.013](10.7554/eLife.23326.013)10.7554/eLife.23326.014Figure 4---source data 2.Two channel recording of H4-sfGFP and PCNA-RFP distributions from 8-cell to oblong stage.H4-sfGFP (left, green channel) intensities are transformed to logarithmic scale to compensate for intensity increase due to ongoing translation of mRNA into fluorescent fusion protein. PCNA-RFP (right, magenta channel) intensities are linear. Both channels are maximum z-projections, with a view upon the animal cap, time stamps are given in hour:minute format, starting with the first acquired frame.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.014](10.7554/eLife.23326.014)10.7554/eLife.23326.015Figure 4---figure supplement 1.Onset of transcription coincides with a reduction in nuclear histone concentration.(**A**) Changes in total animal cap volume, the fraction of the animal cap volume occupied by nuclei, and the size of individual nuclei for indicated stages. Volumes were measured by lightsheet microscopy of embryos injected with mRNA encoding H4-sfGFP and subsequent automated image analysis (error bars represent SEM, n = 3 embryos; offspring of transgenic PCNA-RFP was used to monitor integrity of imaged nuclei via a second color channel). (**B**) Image sequences showing nuclear import of H4-sfGFP fusion protein at 32- to 64- and 128-cell stages. Color scaling was kept constant and linear within each stage. Images show a representative maximum z-projection of a subset of a 3D microscopy stack of one of the embryos used in A. (**C**) The linear range of Western blots was determined using chromatin of sphere-stage embryos. Plotted are observed versus expected fold differences in H2B intensity using different numbers of embryos (n ≥ 3). Band intensities of test stages ([Figure 4F](#fig4){ref-type="fig"}) were only used for the analysis when they fell within the linear range.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.015](10.7554/eLife.23326.015) Because transcription takes place in the nucleus, we next wanted to investigate the concentration of non-DNA-bound histones in this compartment of the cell. First, we analyzed the dynamics of histone localization by lightsheet microscopy of living embryos ([Figure 4---figure supplement 1B](#fig4s1){ref-type="fig"} and [Figure 4---source data 2](#SD4-data){ref-type="supplementary-material"}). As expected, we found a close coordination between the formation of nuclei after cell division and the import of histones from the cytoplasm into the nucleus: during each cell cycle, non-DNA-bound histones are concentrated in the nucleus. A direct quantification of non-DNA-bound, endogenous histones in the nucleus is difficult, but by combining lightsheet microscopy measurements of both the nuclear volume fraction and the relative fluorescence intensity of histone H4 in cytoplasm and nucleus with the absolute amount of histone H4 as quantified by mass spectrometry, we were able to calculate the nuclear concentration of non-DNA-bound histones from 256-cell to oblong stage ([Figure 4E](#fig4){ref-type="fig"}, [Figure 4---figure supplement 1A](#fig4s1){ref-type="fig"} and see Materials and methods for more details). Importantly, our calculations indicate a decrease in the nuclear concentration of non-DNA-bound histones at the onset of transcription. In combination with our finding that histone levels determine the timing of transcription, this suggests that a decrease in the concentration of non-DNA-bound histones in the nucleus causes the onset of transcription during embryogenesis. Nucleosome density on DNA is unchanged during genome activation {#s2-4} --------------------------------------------------------------- We next analyzed whether the decreased concentration of non-DNA bound histones in the nucleus is accompanied by a reduced density of nucleosomes on chromatin. We quantified the amount of histone H2B in the chromatin fraction of embryos ranging from 256-cell to high stage. Comparing the amount of histone H2B between stages revealed that the level of H2B scales with the amount of DNA ([Figure 4F](#fig4){ref-type="fig"} and [Figure 4---figure supplement 1C](#fig4s1){ref-type="fig"}). This is in agreement with a previous study in which we found that the density of nucleosomes does not significantly change during genome activation ([@bib98]). Our results reveal that global nucleosome density on DNA does not change during genome activation. Taken together, this suggests that the concentration of non-DNA-bound histones in the nucleus determines the timing of transcription without the need for a significant change in global nucleosome density. A competition model for the onset of transcription {#s2-5} -------------------------------------------------- Our finding that the concentration of histones in the nucleus determines the onset of transcription without a significant change in global nucleosome density on DNA suggests that a simple depletion model cannot explain a role for histone levels in the timing of zygotic transcription. To explain the effect of histone levels on zygotic genome activation, we hypothesized that the transcriptional machinery (for simplicity referred to as transcription factors) competes with nucleosome-forming histones for binding to only a minimal fraction of the total DNA, corresponding to transcription-factor-binding sites ([Figure 4G](#fig4){ref-type="fig"}). In such a model, local substitution of nucleosomes by transcription factors allows for transcription to be activated, but will cause only localized changes in nucleosome positioning, and will barely affect the average nucleosome density. Transcription factors would lose the competition for DNA binding in the presence of an excess of histones (pre-ZGA), whereas a reduction of the concentration of non-DNA-bound histones in the nucleus would allow transcription factors to gain access to DNA (approaching ZGA) and initiate transcription (ZGA). Decreasing transcription factor levels delays the onset of transcription {#s2-6} ------------------------------------------------------------------------ If competition between nucleosome-forming histones and the transcriptional machinery determines the onset of transcription, it would be predicted that the levels at which transcription factors are present could also affect the timing of zygotic transcription. To test this, we changed the level of Pou5f3, a transcription factor that has been identified as being required for the activation of a large set of genes during genome activation ([@bib47]; [@bib48]; [@bib66]). To analyze the effect on the onset of transcription ([Figure 5A](#fig5){ref-type="fig"}), we selected five genes that are activated at the onset of genome activation and that have been identified as Pou5f3 targets ([Figure 1---figure supplement 1A](#fig1s1){ref-type="fig"}) ([@bib66]).10.7554/eLife.23326.016Figure 5.Direct experimental evidence for the competition model using endogenous genes and transcription factors.(**A**) Schematic representation of experimental procedure. Pou5f3 levels were decreased by injecting a morpholino, or increased by injecting *pou5f3* mRNA (in combination with *sox19b* mRNA) into the cell of 1-cell embryos. Controls used were a dead-end morpholino and *gfp* mRNA, respectively. qPCR and ChIP-qPCR analysis was carried out at stages around genome activation. Orange crosses represent the timing of stages used for the analysis. (**B**) Expression of *apoeb* and *dusp6* was analyzed by qPCR at 512-cell, early 1K, mid 1K and high stage in control and Pou5f3 morpholino-injected embryos. The data in the bar graphs focus on the mid 1K stage. Error bars represent SEM (n ≥ 4). \*p\<0.05 (two-tailed Student's t-test, compared to control MO). (**C**) Expression of *apoeb* and *dusp6* was analyzed by qPCR at 512-cell, early 1K and high stage in uninjected embryos, embryos injected with control mRNA and embryos injected with *pou5f3* and *sox19b* mRNA. Bar graphs focus on the early 1K stage. Error bars represent SEM (n ≥ 4). \*\*p\<0.01 (two-tailed Student's t-test, compared to control mRNA). (**D**) Binding of Pou5f3 to its respective binding sites for *apoeb* and *dusp6* ([@bib48]) and control region was analyzed by ChIP-qPCR at the early 1K stage in embryos injected with *pou5f3* + *sox19b* mRNA or *pou5f3* + *sox19b* mRNA plus histone cocktail. Enrichment of pulled-down fragments was normalized to input. Location of primer sets in respect to the transcription start-site used for ChIP-qPCR analysis are indicated by arrows. A genome control region on chromosome 23 was also used. Error bars represent SEM (n = 5). \*\*p\<0.01 (two-tailed Student's t-test ratio paired, compared to *pou5f3* + *sox19b* mRNA-injected embryos). In B and C, mRNA levels are normalized to the expression of *eif4g2α*.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.016](10.7554/eLife.23326.016)10.7554/eLife.23326.017Figure 5---figure supplement 1.Reducing transcription factor levels delays the onset of transcription.(**A**) Pou5f3 morpholino validation. Brightfield images of embryos that were injected with control or Pou5f3 morpholino. Embryos injected with Pou5f3 morpholino arrested at sphere or dome stage, as reported previously ([@bib12]). Scale bar, 250 μm. Western blot of embryos injected with 50 ng *pou5f3*-2xHA mRNA alone or in combination with 6 ng Pou5f3 moprholino. As expected, the morpholino reduces the expression of Pou5f3. (**B**) We injected embryos with Pou5f3 morpholino and analyzed the effect on transcription. Shown are the fold changes in relative expression levels of Pou5f3-morpholino-injected embryos compared to control morpholino-injected embryos at high stage for Pou5f3 targets *apoeb*, *dusp6*, *klf17*, *irx7*, and *klf2b*, and non-Pou5f3 targets *sox19a*, *grhl3* and *gadd45bb*. Reduction of Pou5f3 results in decreased transcription for all Pou5f3 target genes analyzed at high stage, confirming that they are regulated by Pou5f3. Non-targets were not affected. Error bars represent SEM (n = 4). n.s. p\>0.05; \*p\<0.05; \*\*p\<0.01; \*\*\*p\<0.001 (two-tailed Student's t-test, compared to control morpholino). (**C**) Expression of *klf17*, *irx7* and *klf2b* was analyzed by qPCR at 512-cell, early 1K, mid 1K and high stage in control and Pou5f3 morpholino-injected embryos. Bar graphs focus on mid 1K stage. Error bars represent SEM (n ≥ 4). \*\*p\<0.01; \*\*p\<0.001 (two-tailed Student's t-test, compared to control morpholino). (**D**) Western blot analysis of embryos injected with *sox19b*-2xHA mRNA alone and in combination with 2 ng of Sox19b morpholino validate the effect of the Sox19b morpholino. (**E**) Expression of *dusp6* was analyzed by qPCR at 512-cell, early 1K, mid 1K and high stage in control and Sox19b morpholino-injected embryos. *dusp6* was selected for analysis because it is the only gene from our selected Pou5f3-target genes that is also regulated by SoxB1 ([@bib47]). Error bars represent SEM (n ≥ 4). (**F**) Validation of FoxH1 target genes. To verify that the genes we selected require FoxH1 for their expression, we injected embryos with FoxH1 morpholino and analyzed the effect on transcription. Shown are the fold changes in relative expression levels of FoxH1 morpholino-injected embryos compared to control morpholino-injected embryos at high stage for FoxH1 targets *dusp6*, *flh*, *wnt11* and *gadd45bb*. Reduction of FoxH1 results in decreased transcription for all FoxH1 target genes. (**G**) Expression of *dusp6, flh, wnt11* and *gadd45bb* was analyzed by qPCR at 512-cell, early 1K, mid 1K and high stage in control and FoxH1 morpholino-injected embryos. All four target genes show a delay in the time when they are first transcribed in the FoxH1 morphants compared to control morpholino-injected embryos. Error bars represent SEM (n ≥ 4). (**H**) Staging by morphology was verified by cell counting at the stages used for the Pou5f3 morpholino analysis. Each data point represents a single embryo. Error bars represent SEM. Gene expression is normalized to the expression of *eif4g2α*.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.017](10.7554/eLife.23326.017)10.7554/eLife.23326.018Figure 5---figure supplement 2.Increasing transcription factor levels causes premature transcription.(**A**) Pou5f3 overexpression validation. Brightfield images of embryos that were injected with control or *pou5f3* mRNA. Developmental defects at 24 hpf resembled the ventralized phenotypes described upon *pou5f3-VP16* overexpression ([@bib7]). Scale bar, 250 μm. Western blot using an HA antibody shows the protein level of Pou5f3-2xHA and Sox19b-2xHA in embryos at 1K stage after injection at the 1 cell stage. Blot shown is a representative example (n = 2). (**B**) To test whether Pou5f3 and Sox19b are sufficient to drive expression of the genes we selected, we increased the level of both transcription factors and analyzed the effect on transcription. Shown are the expression levels of *apoeb*, *dusp6*, *klf17*, *irx7*, and *klf2b* at high stage for embryos injected with *pou5f3* + *sox19b* mRNA relative to control mRNA-injected. Error bars represent SEM (n ≥ 4). Increasing the levels of Pou5f3 and Sox19b only increased the relative expression level of *apoeb* and *dusp6*, suggesting that Pou5f3 and Sox19b are not sufficient to drive expression of the other genes. Error bars represent SEM (n ≥ 4). n.s p\>0.05; \*p\<0.05 (two-tailed Student's t-test, compared to control mRNA). (**C**) Expression of *klf17*, *irx7* and *klf2b* and was analyzed by qPCR at 512-cell, early 1K, and high stage in uninjected, control and *pou5f3* + *sox19b* mRNA-injected embryos. Bar graphs focus on the early 1K stage. Error bars represent SEM (n ≥ 4). n.s p\>0.05 (two-tailed Student's t-test, compared to control mRNA). (**D**) Staging by morphology was verified by cell counting at the stages used for the analysis. Each data point represents a single embryo. Error bars represent SEM. In B and C, expression is normalized to the expression of *eif4g2α*.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.018](10.7554/eLife.23326.018) To reduce the level of Pou5f3, we used a previously characterized morpholino ([@bib12]) and confirmed its effect by analyzing the morphology of injected embryos and the effect of the morpholino on the translation of injected RNA encoding Pou5f3 ([Figure 5---figure supplement 1A](#fig5s1){ref-type="fig"}). We verified that the selected Pou5f3-target genes require Pou5f3 for their expression and that other genes do not ([Figure 5---figure supplement 1B](#fig5s1){ref-type="fig"}), and analyzed the effect of a reduction in Pou5f3 levels on the timing of transcription of target genes ([Figure 5A](#fig5){ref-type="fig"}). Consistent with our model, a reduction in the amount of Pou5f3 delayed the onset of transcriptional activation: transcripts were detected in the middle of 1K stage in embryos injected with control morpholino, while in the embryos injected with Pou5f3 morpholino, the genes started to be transcribed at high stage ([Figure 5B](#fig5){ref-type="fig"} and [Figure 5---figure supplement 1C](#fig5s1){ref-type="fig"}). We analyzed embryos at mid 1K in this experiment, because the delay that we observe is weaker than with the histone cocktail. Comparison of gene expression levels in control morpholino and Pou5f3 morpholino-injected embryos at mid 1K (when transcripts can first be detected in control morpholino-injected embryos) revealed that the level of induction is significantly reduced upon injection of Pou5f3 morpholino ([Figure 5B](#fig5){ref-type="fig"} and [Figure 5---figure supplement 1C](#fig5s1){ref-type="fig"}, bar graphs). Performing similar experiments for two additional transcription factors (Sox19b and FoxH1) revealed that this effect is general, and not specific to Pou5f3 ([Figure 5---figure supplement 1D--G](#fig5s1){ref-type="fig"}). Staging by morphology was corroborated by cell counting with absolute time between the analyzed stages being constant ([Figure 5---figure supplement 1H](#fig5s1){ref-type="fig"}). Together, these data show that a decrease in the level of transcription factors in the embryo delays the onset of transcription of target genes. Increasing transcription factor levels causes premature transcription {#s2-7} --------------------------------------------------------------------- Next, we analyzed the effect of increasing the level of Pou5f3 on the transcription of the selected Pou5f3 target genes ([Figure 5A](#fig5){ref-type="fig"}). We co-injected mRNA coding for Sox19b because it has been shown that Pou5f3 and Sox19b often co-occupy their target genes ([@bib14]; [@bib48]; [@bib66]). Injecting mRNA encoding these transcription factors resulted in overexpression of both proteins and the expected phenotypes for Pou5f3 overexpression ([Figure 5---figure supplement 2A](#fig5s2){ref-type="fig"}) ([@bib7]). Although Pou5f3 was required for the expression of all genes we selected ([Figure 5---figure supplement 1B](#fig5s1){ref-type="fig"}), Pou5f3 and Sox19b were only sufficient to increase the expression level of *apoeb and dusp6* at high stage ([Figure 5---figure supplement 2B](#fig5s2){ref-type="fig"}). In agreement with this observation, overexpression of Pou5f3 and Sox19b resulted in premature expression of *apoeb and dusp6*: transcripts were detected at early 1K stage in embryos injected with *pouf53* and *sox19b*, whereas in the embryos injected with control mRNA, transcripts could be detected only at high stage ([Figure 5C](#fig5){ref-type="fig"}). Comparison of gene expression levels in control, and *pouf53* and *sox19b* mRNA-injected embryos at early 1K stage (one time-point prior to when genes are first induced in uninjected embryos) revealed that the level of expression is increased significantly upon injection of *pou5f3* and *sox19b* mRNA for *apoeb and dusp6* ([Figure 5C](#fig5){ref-type="fig"}, bar graphs). Such an effect on the timing of transcription was not observed for the other genes ([Figure 5---figure supplement 2C](#fig5s2){ref-type="fig"}). Staging by morphology was corroborated by cell counting, with absolute time between the analyzed stages being constant ([Figure 5---figure supplement 2D](#fig5s2){ref-type="fig"}). These experiments show that an increase in the level of Pou5f3 and Sox19b in the embryo can cause premature transcription. Taken together, our results show that changing the concentration of endogenous transcription factors can affect the onset of transcription. This is in agreement with our model in which the relative levels of histones and transcription factors determine the onset of transcription. Transcription factor binding is sensitive to histone levels {#s2-8} ----------------------------------------------------------- If transcription factors and histones compete for DNA binding, it would be predicted that transcription factor binding is sensitive to histone levels. To directly test competition at the chromatin level, we determined whether the binding of the transcriptional machinery is affected by histone levels. We analyzed the binding of Pou5f3 to its predicted target sites upstream of *apoeb* and *dusp6* by chromatin immunoprecipitation (ChIP) and identified co-precipitated DNA fragments by qPCR ([Figure 5D](#fig5){ref-type="fig"}). In embryos that were injected with mRNA encoding both Pou5f3 and Sox19b, we found that the binding of Pou5f3 was readily detected at early 1K stage ([Figure 5D](#fig5){ref-type="fig"}, white bars). When the HC was co-injected, binding of the transcription factor was reduced ([Figure 5D](#fig5){ref-type="fig"}, gray bars). We expect, but did not test, that nucleosome density is concordantly increased at these binding sites. A control region in genomic DNA did not show any binding of Pou5f3 ([Figure 5D](#fig5){ref-type="fig"}). Taken together, this shows that the binding of an endogenous transcription factor is sensitive to the amount of histones present in the embryo. Experimental evidence for competition using a heterologous transgene {#s2-9} -------------------------------------------------------------------- Our results support a model in which transcription is regulated by the relative levels of histones and transcription factors. Endogenous gene regulation, however, is intrinsically complex, with multiple transcription factors providing input on the same gene, and often there is limited information on the number and strength of activator-binding sites. Because this might have affected the results we obtained with endogenous transcription factors and genes ([Figure 5](#fig5){ref-type="fig"}), we decided to take advantage of a heterologous system to confirm our results. The integrated inducible transgene TRE:GFP ([Figure 6A](#fig6){ref-type="fig"}) contains seven binding sites for tTA--VP16 as well as a CMV promoter and is strictly dependent on tTA--VP16 for its expression (data not shown). tTA-VP16 was tagged with HA and a protein product was detected at the 1K stage following injection of mRNA ([Figure 6---figure supplement 1A](#fig6s1){ref-type="fig"}). Injection of 5 pg of mRNA encoding the heterologous transcription factor tTA-VP16 resulted in the detection of transcripts at high stage, in accordance with the onset of zygotic transcription of endogenous genes ([Figure 6B](#fig6){ref-type="fig"}). Next, we analyzed the transcriptional activity of this transgene upon injection of 300 pg of mRNA encoding tTA-VP16 and we observed that transcripts could be detected at early 1K ([Figure 6B](#fig6){ref-type="fig"}). Comparison of gene expression levels at early 1K stage (one stage prior to when genes are first induced in embryos injected with 5 pg of mRNA) in embryos injected with 5 and 300 pg of mRNA revealed that the number of transcripts is increased significantly upon increasing the level of transcription factor ([Figure 6B](#fig6){ref-type="fig"}, bar graph). Next, we tested whether an increase in histone levels would negate the effect of high levels of transcription factor. As predicted by the competition model, the increase in transcriptional activity that is observed upon the injection of 300 pg of tTA-VP16 mRNA is lost when the histone cocktail is co-injected ([Figure 6C](#fig6){ref-type="fig"}). Finally, we determined whether the binding of tTA is affected by histone levels. We analyzed the binding of tTA-VP16 to the TRE sites in the transgene by ChIP-qPCR ([Figure 6A](#fig6){ref-type="fig"}). We found that upon injecting 300 pg of tTA-VP16 mRNA, the binding of the transcription factor was readily detected at early 1K stage ([Figure 6D](#fig6){ref-type="fig"} and [Figure 6---figure supplement 1B](#fig6s1){ref-type="fig"}). As expected, binding of the transcription factor was significantly reduced when the HC was co-injected. Control regions within the transgene and in genomic DNA did not show any binding of tTA-VP16 ([Figure 6D](#fig6){ref-type="fig"} and [Figure 6---figure supplement 1B](#fig6s1){ref-type="fig"}). This shows that the binding of a heterologous transcription factor is sensitive to the amount of histones present in the embryo. These results are in agreement with the results obtained with the endogenous transcription factors ([Figure 5](#fig5){ref-type="fig"}). Taken together, our data provide direct evidence for a model in which competition between histones and transcription factors determines the onset of transcription.10.7554/eLife.23326.019Figure 6.Direct experimental evidence for the competition model using a heterologous transgene.(**A**) Schematic representation of the experimental procedure and TRE:GFP transgene. tTA-VP16 and/or histone levels were increased by injecting mRNA or HC into the cell or yolk, respectively, of 1-cell transgenic embryos. The TRE element contains seven binding sites for tTA-VP16 and is joined to a CMV promoter. qPCR and ChIP-qPCR analysis was carried out at stages around genome activation. Orange crosses represent the timing of stages used for the analysis. (**B**) Expression of *gfp* was analyzed by qPCR at 512-cell, early 1K and high stage in embryos injected with 5 or 300 pg *tTA-VP16* mRNA. Bar graphs focus on the early 1K stage. Error bars represent SEM (n ≥ 4). \*p\<0.05 (two-tailed Student's t-test, compared to 5 pg *tTA-VP16* mRNA). (**C**) Expression of *gfp* was analyzed by qPCR at early 1K stage in embryos injected with 5 pg, 300 pg *tTA-VP16* mRNA and 300 pg *tTA-VP16* mRNA plus histone cocktail. Error bars represent SEM (n = 4). \*p\<0.05 (Ordinary one-way ANOVA). (**D**) Binding of tTA-VP16 to the TRE element and control regions was analyzed by ChIP-qPCR at the early 1K stage in embryos injected with 300 pg *tTA-VP16* mRNA or 300 pg *tTA-VP16* mRNA plus histone cocktail. Enrichment of pulled-down fragments was normalized to input. Primer sets used for ChIP-qPCR analysis are indicated by arrows in panel A. A control region on the transgene was used in addition to a genome control region on chromosome 23. Error bars represent SEM (n = 3). \*p\<0.05 (two-tailed Student's t-test ratio paired, compared to 300 pg *tTA-VP16* mRNA-injected embryos). In B and C, mRNA levels are normalized to the expression of *eif4g2α*.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.019](10.7554/eLife.23326.019)10.7554/eLife.23326.020Figure 6---figure supplement 1.Direct experimental evidence for the competition model using a heterologous transgene.(**A**) Western blot using an HA antibody shows the protein level at 1K stage after injection of either 5 pg or 300 pg *tTA-VP16* mRNA. Tubulin was used to control for equal loading. Blot shown is a representative example (n = 2). (**B**) Binding of tTA-VP16 to the TRE element was analyzed by ChIP-qPCR at the early 1K stage in embryos injected with 300 pg *tTA-VP16* or 300 pg *tTA-VP16* mRNA plus histone cocktail. Enrichment of pulled-down fragments was normalized to input. All primers used for ChIP-qPCR are indicated using arrows. Error bars represent SEM (n = 3). \*p\<0.05 (two-tailed Student's t-test ratio paired, compared to 300 pg *tTA-VP16* mRNA plus histone cocktail).**DOI:** [http://dx.doi.org/10.7554/eLife.23326.020](10.7554/eLife.23326.020) Discussion {#s3} ========== In this study, we have shown that the concentration of all four core histones determines the onset of transcription in zebrafish embryos by competing with transcription factors for binding to DNA. Upon fertilization, there is a large excess of histones stockpiled in the embryo and transcription starts when the concentration of non-DNA-bound histones in the nucleus drops, and the transcriptional machinery gains access to DNA. Thus, the relative concentrations of both histones and transcription factors determine the timing of zygotic genome activation ([Figure 4G](#fig4){ref-type="fig"}). Our observations provide, to our knowledge, the first example of a developmental transition in which competition for DNA binding between histones and transcription factors plays an important role in transcriptional regulation. All core histones are important for timing of transcription {#s3-1} ----------------------------------------------------------- Our observation that histone levels affect the time of transcription onset in zebrafish embryos is in agreement with previous studies that showed a role of histones in the regulation of transcription in early *Xenopus* embryos and extracts ([@bib5]; [@bib6]). However, our finding that histones are neither completely depleted from the soluble fraction, nor generally depleted from chromatin, argues against a model in which a global loss of nucleosome density on chromatin causes the onset of transcription ([@bib6]). Our work does not exclude the importance of other factors, such as the linker histone H1 ([@bib74]), the embryonic form of which is stably present during zebrafish genome activation, but it establishes that core histones themselves function as actual repressors of transcription. Our discovery that all core histones are required to regulate the onset of transcription suggests that the nucleosome is important for the repressor function of histones. In a previous study, premature transcription of injected plasmids caused by an excess of non-specific DNA was negated by the addition of the four core histones, but histones were not tested separately and it was not clear whether one or more histones were required for the observed effect on transcription ([@bib5]). This left open the possibility that single histones could repress transcriptional activity in the embryo, for example by binding to a transcription factor and preventing it from binding to DNA. However, our observation that in the embryo all core histones are important for the regulation of transcription would then require all histones to independently take part in this mode of repression. Because this is a very unlikely scenario, we propose that repression takes place close to DNA, where histones are assembled into a histone octamer to form the nucleosome. Changes in nuclear histone concentration during genome activation {#s3-2} ----------------------------------------------------------------- We propose that genome activation follows a decrease in the concentration of non-DNA bound histones in the nucleus. One possible way to explain the reduction in nuclear histone concentration is the exponential increase in DNA content during the cleavage stages of zebrafish development. Because histones have a high affinity for DNA, an increase in the amount of DNA would titrate out non-DNA-bound histones. Several experiments have indeed shown that changes in DNA content can affect the time of transcription in *Drosophila*, zebrafish and *Xenopus* ([@bib19]; [@bib54]; [@bib59]; [@bib73]). Our data suggest that these effects were the result of reducing the concentration of non-DNA-bound histones. In zebrafish embryos, the amount of histones is so large that the increase in DNA content leading up to genome activation may contribute only moderately to a decrease in the concentration of non-DNA-bound histones in the nucleus. Another possible explanation for the decrease in nuclear histone concentration is the marked increase in the ratio of nuclear over cytoplasmic volume during the cleavage stages ([Figure 4---figure supplement 1A](#fig4s1){ref-type="fig"}). We suggest that this may limit the capacity of the nucleus to concentrate histones. The process of nuclear transport has been investigated in great detail ([@bib38], [@bib39]; [@bib42], [@bib43]), and we can assume that the nuclear envelope can create a certain fold difference in concentrations between the nucleus and cytoplasm. During the initial stages of zebrafish development, the nucleus occupies only a very small fraction of the cell volume (1.1% at 128-cell stage, [Figure 4---figure supplement 1A](#fig4s1){ref-type="fig"}). As a result, when the nucleus concentrates histones up to the maximum fold difference between cytoplasm and nucleus, the cytoplasmic histone concentration is hardly altered. Later in development, when approaching the onset of zygotic transcription, the nucleus takes up a notably larger part of the total cell volume (7.1% at high stage, [Figure 4---figure supplement 1A](#fig4s1){ref-type="fig"}). Now, when histones are imported into the nucleus, the concentration of histones in the cytoplasm noticeably decreases. The nuclear envelope is still able to create approximately the same fold difference of concentrations, but due to the reduced cytoplasmic concentration, the achieved nuclear concentration is not as high as during the initial stages. Thus, in this scenario, the nuclear histone concentration decreases due to the increasing relative nuclear size, which alters the distribution of histones among cellular compartments. It may be expected that the relative increase in nuclear size affects the nuclear concentration of transcription factors as well, and it remains to be seen how the concentration of histones and transcription factors change with respect to each other in order to activate transcription. Experiments recently performed in *Xenopus* showed that changing the size of the nucleus affects the timing of transcription ([@bib33]), providing further evidence for a role of nuclear size in regulating the onset of transcription. Competition for DNA binding determines transcription onset {#s3-3} ---------------------------------------------------------- Our finding that the onset of transcription depends on the concentration of histones, but also on the concentration of transcription factors, is consistent with previous studies that suggested an important role for transcriptional activators in the temporal regulation of zygotic transcription ([@bib5]; [@bib73]; [@bib90]). Because transcription can be induced prior to the onset of genome activation, both by adding DNA ([@bib19]; [@bib54]; [@bib59]) or removing histones (this study), we suggest that transcription factors required for the onset of transcription are in principle present prior to genome activation. To shift the balance from repression to activation, the relative concentrations of histones and transcription factors need to be changed in favor of transcription factors. This would explain previous observations in *Xenopus*, where the addition of TBP (in combination with adding DNA) or GAL4-VP16 resulted in premature transcription ([@bib5]; [@bib90]). Based on our findings, those experiments would have shifted the balance in favor of transcriptional activity, similar to the effect observed when we increased transcription factor levels or decreased histone levels. Our model in which histones and transcription factors dynamically compete for DNA binding to regulate transcription in the embryo is consistent with the notion that most transcription factors cannot bind DNA when it is wrapped around a nucleosome and thus compete with nucleosomes for DNA access ([@bib4]; [@bib3]; [@bib28]; [@bib56]; [@bib57]; [@bib75]; [@bib76]; [@bib78]; [@bib85]). Initially, there is a large excess of histones stockpiled in the embryo and transcription starts when the concentration of non-DNA-bound histones in the nucleus drops and the transcriptional machinery gains access to DNA. In contrast to a competition model that was previously proposed ([@bib73], [@bib72]), transcription factors do not need to be pre-bound to DNA in order to compete with histones, but rather, they dynamically compete with histones for DNA binding. Our experiments did not address when competition takes place during the cell cycle. Competition for DNA binding might either occur immediately following replication, on temporarily naked DNA, or following chromatin assembly. Recent studies assessing the nucleosome landscape following replication have revealed that replication-coupled nucleosome assembly initially outcompetes transcription factors for binding to DNA but that chromatin remodeling and phasing of nucleosomes by remodelers and transcription factors occurs rapidly thereafter ([@bib21]; [@bib75]; [@bib88]). Future experiments will determine the details of competition in the early embryo, with its rapid cell cycles and large pool of soluble histones. To gain further insight in the molecular details of competition that lead up to genome activation, it will be important to determine which factors compete with histones for DNA binding. In theory, the binding of all factors that require access to DNA could be affected by histone levels, suggesting that competition might take place at many levels of transcription regulation: the formation of higher order chromatin structure, chromatin remodeling, the binding of transcription factors, and the assembly of the basal transcription complex. Our results show that the transcription factors that have been identified to regulate many genes during genome activation in zebrafish (Pou5f3 and Sox19b) ([@bib47]; [@bib48]), as well as FoxH1 and the heterologous transcription factor tTA-VP16, compete with histones for binding to DNA ([Figures 5](#fig5){ref-type="fig"} and [6](#fig6){ref-type="fig"}). In this context, it is interesting to note that the transcription factors that have been identified to play a role in genome activation have either been suggested to be pioneer factors ([@bib47]; [@bib48]), or there is indication for such a role because of their homology with mammalian pioneer factors ([@bib47]; [@bib48]; [@bib82]). Pioneer factors are able to interact with DNA that is nucleosome bound ([@bib97]). In the context of the competition model, it will be interesting to see whether these factors also have pioneering activity in the early embryo, and how this affects their role in activating transcription in the embryo. General relevance of competition in development {#s3-4} ----------------------------------------------- The applicability of the competition model might extend well beyond the onset of zygotic transcription in zebrafish. First, given the excess of histones in a large number of species including *Drosophila*, *Xenopus*, and zebrafish ([@bib2]; [@bib49]; [@bib55]; [@bib67]; [@bib89]; [@bib92]), it is likely that histone levels play a role in the timing of zygotic transcription across these species. As discussed, in *Xenopus* embryos there is indeed evidence for a role of histone levels in regulating transcriptional activity ([@bib5]; [@bib6]). Additional experiments will be required to determine whether the competition model we propose applies to these and other species. Second, the onset of zygotic transcription in the embryo takes place in the context of the mid-blastula transition and is accompanied by a lengthening of the cell cycle and changes in chromatin structure. Although it had previously been suggested that the rapid cell cycles lacking G1 and G2 phases might interfere with productive transcription during early developmental stages ([@bib15]; [@bib20]; [@bib40]), it was recently shown that the lengthening of the cell cycle might be a direct consequence of the onset of transcription in *Drosophila* embryos ([@bib10]). This would suggest that what regulates the onset of zygotic transcription might also dictate the lengthening of the cell cycle. Finally, post-translational modification of histones often requires a chromatin-modifying enzyme to bind to DNA, much like transcription factors. Thus, competition is likely to affect the de novo modification of histones as well, explaining why many histone modifications are only observed around the onset of zygotic transcription in their temporal profile ([@bib51]; [@bib89]). Importantly, we observe an effect on the timing of transcription by adding unmodified histones. This suggests that post-translational modifications of histones are either downstream of the timing of transcriptional activation, or the enzymes that modify histones are not limiting in the embryo. The competition model can explain why genome activation is gene specific ([@bib1]; [@bib16]; [@bib27]; [@bib29]; [@bib53]; [@bib68]; [@bib69]; [@bib77]; [@bib87]), and even why the first zygotic transcripts can be detected several cell cycles before the stage that is traditionally defined as the time point of ZGA ([@bib18]; [@bib29]; [@bib81]; [@bib94]). The sensitivity of genes for a given histone concentration logically depends on their enhancers and the affinity and concentration of the transcription factors that bind to them. In this context, it is interesting to note that many genes that are activated during zebrafish genome activation respond to the same set of transcription factors, which are also the most highly translated transcription factors before genome activation ([@bib47]; [@bib48]). Conversely, the affinity of transcription factors and the number of transcription-factor-binding sites might provide a mechanism to explain why some genes overcome repression earlier than others ([@bib29]). Indeed, in *Drosophila*, it has been shown that the number of transcription-factor-binding sites as well as the level of transcription factors can affect the timing of gene expression ([@bib23]; [@bib24]). Recent literature suggests that histone levels might play a role in the regulation of transcription during developmental transitions other than genome activation. In contrast to the situation in early embryos, where histone and DNA levels do not scale, it was generally believed that in somatic cells, histone production is tightly coupled to replication ([@bib62]). Recently, however, histone levels have been shown to change during ageing and differentiation ([@bib22]; [@bib30]; [@bib36]; [@bib63]) suggesting that histone levels might not be as tightly coupled to DNA replication as previously thought. Moreover, histone chaperones were identified as inhibitors of reprogramming ([@bib13]; [@bib32]) and it was proposed that the lack of histone chaperones facilitates transcription factor binding ([@bib13]). Taken together, these studies might suggest that the availability of histones could play a role in the regulation of transcription during differentiation and reprogramming. We have shown that the onset of transcription is regulated by a dynamic competition for DNA binding between histones and transcription factors. This suggests that the relative levels of histones and transcription factors in the nucleus determine the time at which transcription begins in the embryo. Future studies will be required to improve our understanding of the molecular mechanism of competition, the regulation of repressor and activator concentrations in the nucleus, and the role of competition during other developmental transitions. M**aterials** and methods {#s4} ========================= Zebrafish husbandry and manipulation {#s4-1} ------------------------------------ Zebrafish were maintained and raised under standard conditions. Wild-type (TLAB) (WT-TL RRID:[ZIRC_ZL86](https://scicrunch.org/resolver/ZIRC_ZL86), WT-AB RRID:[ZIRC_ZL1](https://scicrunch.org/resolver/ZIRC_ZL1)) and transgenic embryos were dechorionated immediately upon fertilization, synchronized and allowed to develop to the desired stage at 28°C. Stage was determined by morphology and corroborated by cell counting. In terms of absolute time, the time between collected stages around ZGA was consistent between all conditions within an experiment and for all experiments. Histone cocktail and BSA (A9418; Sigma, St. Louis, MO) were injected into the yolk at the 1-cell stage at 22 ng per embryo. Pou5f3 anti-sense morpholino was injected at 6 ng per embryo, together with 1 ng of p53 morpholino ([@bib45]). Sox19b anti-sense morpholino ([@bib64]) was injected at 2 ng per embryo and FoxH1 anti-sense morpholino ([@bib71]) was injected at 4 ng per embryo, together with 1 ng of p53 morpholino. Dead-end ([@bib91]) or control morpholino were injected as a control at the same concentration. Morpholino sequences can be found in [Table 1](#tbl1){ref-type="table"}. α amanitin (A2263; Sigma) was injected at the 1-cell stage at a concentration of 0.2 ng per embryo. 2.8 mg/ml rhodamine-dextran (D3307; Molecular Probes, Eugene, OR) was used as an injection marker for the HC and BSA experiments. For all other injections, 0.1% Phenol red (P0290; Sigma) was injected. Bright-field images of whole embryos were acquired on a Leica M165 C dissecting scope equipped with a Leica MC170 HD camera (Leica, Wetzlar, Germany).10.7554/eLife.23326.021Table 1.List of morpholinos used.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.021](10.7554/eLife.23326.021)MorpholinosTargetSequenceCompanyReferencep535'-GCGCCATTGCTTTGCAAGAATTGGeneTools[@bib45]Pou5f35′-CGCTCTCTCCGTCATCTTTCCGCTAGeneTools[@bib12]Sox19b5\'-ACGAGCGAGCCTAATCAGGTCAAACGeneTools[@bib64]Foxh15\'-TGCTTTGTCATGCTGATGTAGTGGGGeneTools[@bib71]Dead-end5\'-GCTGGGCATCCATGTCTCCGACCATGeneTools[@bib91]Ctrl MO5\'-CCTCTTACCTCAGTTACAATTTATAGeneToolsGeneTools, LLC mRNA production and injection {#s4-2} ----------------------------- mRNA was synthesized using the Ambion mMESSAGE mMACHINE SP6 Transcription Kit (AM1430; ThermoFisher Scientific, Waltham, MA). Human PTX3 cDNA was cloned into a pCS2+ vector with a C-terminal RFP. *ptx3-rfp* and *rfp* mRNA were injected into the cell at the 1-cell stage at a concentration of 300 pg per embryo. Zebrafish Pou5f3 and Sox19b cDNA were cloned into a pCS2+ vector containing 2xHA sequences. For gene expression experiments, *pou5f3-2xHA* and *sox19b-2xHA* mRNA were each injected into the cell of 1-cell embryos at 300 pg per embryo. mRNA encoding cytoplasmic *gfp* was injected as a control at 600 pg per embryo. For ChIP-qPCR experiments, *pou5f3-2xHA* and *sox19b-mEos2* mRNA were each injected into the cell of 1 cell embryos at 150 pg per embryo. A subsequent injection of either histone cocktail or mock (histone buffer) into the yolk was carried out. Human H4 cDNA was cloned into a pCS2+ vector with C-terminal sfGFP (50550; addgene, Cambridge, MA) ([@bib65]). mRNA encoding H4-sfGFP was injected into the cell of 1-cell embryos at 240 pg per embryo. tTA-VP16 DNA was cloned into a pCS2+ vector containing 2xHA sequences. mRNA encoding tTA-VP16-2xHA was injected into the cell of 1-cell Tg(TRE:GFP) embryos either at 5 pg or 300 pg per embryo. The combination injection of 300 pg *tTA-VP16-2xHA* mRNA and histone cocktail, involved two subsequent injections into the cell of 1-cell embryos and yolk, respectively. The 300 pg *tTA-VP16-2xHA* mRNA only injections also received a secondary mock injection into the yolk. Quantitative PCR {#s4-3} ---------------- Twenty-five embryos per developmental stage were snap frozen in liquid nitrogen. RNeasy Mini Kit (74104; Qiagen, Venlo, the Netherlands) was used to extract RNA. For Tg(TRE:GFP) embryos, contaminating DNA was removed from RNA preparations using the DNA-free Kit (AM1906; ThermoFisher Scientific). mRNA was converted to cDNA using the iScript cDNA Synthesis Kit (1708891; Bio-Rad Laboratories, Hercules, CA). SYBR green (AB-1158.; ThermoFisher Scientific) with Rox (R1371; ThermoFisher Scientific; 100 nM) was used as the qPCR master mix. Primers were used at a final concentration of 500 nM and sequences can be found in [Table 2](#tbl2){ref-type="table"}. Two or three technical replicates were performed for each sample. Ct values were normalized to the maternally loaded gene *eif4g2a* or input in ChIP-qPCR analysis. Relative mRNA expression levels were calculated via 1/(2^∧^(gene-*eif4g2a*)). Fold difference was calculated by dividing the relative mRNA expression level value of the test sample over control.10.7554/eLife.23326.022Table 2.List of primers used. Location of primer sets with respect to transcription start-sites are indicated in brackets.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.022](10.7554/eLife.23326.022)Primer listGenePrimers*eif4g2α*5'-GAGATGTATGCCACTGATGAT5'-GCGCAGTAACATTCCTTTAG*mxtx2*5'-ACTGACTGCATTGCTCAA5'-ACCATACCTGAATACGTGATT*fam212aa*5'-GCAAATGAGTATCTAAAACTGCT5'-CATCATATAGCGCATCTGGT*nnr*5'-GAGACATACCACAGGTGAAGC5'-CCGCTCTGGTCTGTTGC*vox*5'-TTATTCGTCGGGTTATGAGAG5'-AACCAAGTTCTGATCTGTGT*sox19a*5'-GAGGATGGACAGCTACGG5'-CTATAGGACATGGGGTTGTAG*grhl3*5'-AGACGAGCAGAGAGTCCT5'-TTGCTGTAATGCTCGATGATG*apoeb*5'-GCAGAGAGCTTGACACACTAA5'-TGCATTCTGCTCCATCATGG*dusp6*5\'-AGCCATCAGCTTTATTGATGAG5\'-CAAAGTCCAAGAGTTGACCC*klf17*5'-ATAGTTCGGGACTGGAAAGTTG 5'- TGAGGTGTTGTCGTTGTCAG*irx7*5'-TGGCACACATTAGCAATTCC5'-GCATGATCTTCTCGCCTTTG*klf2b*5'-GCTCTGGGAGGATAGATGGA5'-CTCGGAGTGGGAGATGAAC*flh*5\'-CACTGAAGCTCAGGTTAAAGTC5\'-ACAATCTGGGGAAAATCATGG*wnt11*5\'-CAGACAGGTGCTTATGGACT5\'-CATCTCTCGGGGCACAAG*gadd45bb*5\'-CAACTCATGAATGTGGATCCAG5\'-ATGCAGTGAAGGTCTCTTGG*GFP*5'-GCACCATCTTCTTCAAGGAC5'-TTGTCGGCCATGATATAGACPou5f3 binding site (−2270 *apoeb*)5\'-TAAAGTGAGCAAATGTATGGCC5\'-TTTGTTGATTAAATCGCTTGTGAPou5f3 binding site (−3095 *dusp6*)5\'-CATATGTTAAGCGGGGTGAAAC5\'-ATCCTGTCTCCTGTGTCATTTGTRE binding site (−222)5\'-TCTTGATAGAGAGGCTGCAAAT5\'-TCGAGATGGGCCCTTGATATRE binding site (13)5\'-TCGTATAGGGATAACAGGGTAATG5\'-TACACGCCTACCTCGACCTRE binding site (217)5\'-GTACGGTGGGAGGCCTATAT5\'-CTTCTATGGAGGTCAAAACAGCTransgene control5\'-CTCTACAAATGTGGTATGGCTG 5\'-ATTACCCTGTTATCCCTAAGGCGenomic control5\'-CCATCATATTCACATCTTGCAAG5\'-GTTCGTATGAACCGGAAGC Staging embryos by cell counting {#s4-4} -------------------------------- Embryos were fixed in 4% formaldehyde in Danieau's solution at 4°C overnight. The next day, embryos were washed with Danieau's solution and then permeabilized with 0.2% Triton X in Danieau's solution for 30 min. Subsequently, embryos were incubated for 10 min in DAPI (1 µg/ml) and then washed several times with Danieau's solution. Embryos were placed in an inverted agarose holder and covered with Danieau's solution for imaging. An upright Zeiss LSM 780 NLO microscope equipped with a coherent Chameleon Vision II infrared laser was used for two-photon excitation (Carl Zeiss AG, Oberkochen, Germany). DAPI was excited with 780 nm and detected using a non-descanned GaAsP detector (BIG-Module) with BP450/60 or SP485. Samples were imaged with either a Zeiss W Plan-Apochromat 20 × 1.0 or 40 × 1.0 dipping objective. Images were acquired using a four tile scan of multiple z-sections (3--3.5 µm steps). Tiles were stitched with the ZEN software (RRID:[SCR_013672](https://scicrunch.org/resolver/SCR_013672), Zeiss). Images were imported into the Imaris software (RRID:[SCR_007370](https://scicrunch.org/resolver/SCR_007370), Bitplane, Belfast, Northern Ireland) and the spot tool was used to calculate cell number. Western blotting {#s4-5} ---------------- Embryos were manually deyolked at the desired stage and snap frozen in liquid nitrogen. For all proteins, equal numbers of embryos were analyzed for each developmental stage (H4 and H2A \[*n* = 10\], all other proteins \[*n* = 5\]). Samples were boiled with SDS loading buffer at 98°C, run on 4--12% polyacrylamide NuPAGE Bis-Tris gels (NP0321BOX; ThermoFisher Scientific) and blotted onto a nitrocellulose membrane (10600002; GE Life Sciences, Chicago, IL). Primary antibodies were incubated at RT for 1 hr or overnight at 4°C and secondary antibodies were incubated at RT for 45 min. Primary and secondary antibodies used are listed in [Tables 3](#tbl3){ref-type="table"} and [4](#tbl4){ref-type="table"}, respectively. Membranes were analyzed on an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) or via chemiluminescent detection (GE Life Sciences) and X-ray film (GE Life Sciences). Tubulin was examined visually on all blots as a loading control.10.7554/eLife.23326.023Table 3.List of primary antibodies used.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.023](10.7554/eLife.23326.023)Primary antibodiesTarget/NameCompanyCompany codeRRID\[WB\]\[IF\]\[IP\]H3Abcamab1791[AB_302613](https://scicrunch.org/resolver/AB_302613)1:10,000H4Abcamab10158[AB_296888](https://scicrunch.org/resolver/AB_296888)1:10001:300H2AAbcamab18255[AB_470265](https://scicrunch.org/resolver/AB_470265)1:1000H2BAbcamab1790[AB_302612](https://scicrunch.org/resolver/AB_302612)1:3000α-tubulinSigmaT6074[AB_477582](https://scicrunch.org/resolver/AB_477582)1:20,000RFPAbcamab152123[AB_2637080](https://scicrunch.org/resolver/AB_2637080)ExcessPTX3Cosmo BioPPZ1724[AB_1962280](https://scicrunch.org/resolver/AB_1962280)1:15,000RNA Pol IIBioLegendMMS-126R[AB_10013665](https://scicrunch.org/resolver/AB_10013665)1:1000HAAbcamab9110[AB_307019](https://scicrunch.org/resolver/AB_307019)1:5000ExcessIgG from rabbit serumSigmaI5006[AB_1163659](https://scicrunch.org/resolver/AB_1163659)Excess[^1]10.7554/eLife.23326.024Table 4.List of secondary antibodies used.**DOI:** [http://dx.doi.org/10.7554/eLife.23326.024](10.7554/eLife.23326.024)Secondary antibodiesNameCompanyCompany codeRRID\[WB\]\[IF\]Alexa 488 goat anti-mouse IgG H&LThermoFisherA-11029[AB_138404](https://scicrunch.org/resolver/AB_138404)1:1000Alexa 594 goat anti-rabbit IgG H&LThermoFisherA-11037[AB_2534095](https://scicrunch.org/resolver/AB_2534095)1:500IRDye 800CW donkey anti-rabbit IgG H&LLI-CORP/N 926--32213[AB_621848](https://scicrunch.org/resolver/AB_621848)1:20,000IRDye 800CW donkey anti-mouse IgG H&LLI-CORP/N 926--32212[AB_621847](https://scicrunch.org/resolver/AB_621847)1:20,000Peroxidase AffiniPure goat anti-rabbit IgG H&LJackson ImmunoResearch111-035-144[AB_2307391](https://scicrunch.org/resolver/AB_2307391)1:20,000Peroxidase AffiniPure rabbit anti-mouse IgG H&LJackson ImmunoResearch315-035-003[AB_2340061](https://scicrunch.org/resolver/AB_2340061)1:20,000[^2] Quantitative mass spectrometry {#s4-6} ------------------------------ We selected five proteotypic peptides ([@bib93]) for each of the four histones: H3, H4, H2A and H2B. The peptides do not discriminate between known histone variants for H3 and H2A. A chimeric gene encoding these peptides ([@bib8]) in addition to reference peptides from BSA and Glycjogen Phosphorylase B (PhosB) (five each), and flanked by Strep- and His-tags, was chemically synthesized (Gene Art, ThermoFisher Scientific). This gene was expressed in a Lys, Arg dual-auxotroph *E. coli* strain (BL21DE3pRARE) that was grown in media complemented with ^13^C^15^N-Arg and ^13^C-Lys (Silantes, Munich, Germany). In a separate LC-MS/MS experiment, we established that the full-length chimeric protein was correctly expressed and the rate of incorporation of isotopically labeled amino acids was ca. 99%. The gel band corresponding to the chimeric protein was co-digested with the gel slab containing the histones from the samples of interest ([@bib80]) and with the band containing the exactly known amount of the reference protein (BSA). The recovered tryptic peptides were analysed by nanoLC-MS/MS on a LTQ Orbitrap Velos coupled with Dionex Ultimate 3000 nano-HPLC system (ThermoFisher Scientific). Three biological replicates were analyzed for each sample, with two technical replicates for each sample. The peptides were separated using C18 reversed phase column (Acclaim PepMap 100) over a linear gradient from 0 to 55% solvent B in a mixture of solvents A and B, delivered in 120 min (Solvent A 0.1% FA, Solvent B 60% ACN + 0.1% FA). The identification of peptides was performed using Mascot v2.2.04 (Matrix Science, London, United Kingdom) against a custom-made database composed of sequences from all histones, BSA, PhosB, affinity tag and the sequences of common contaminants such as human keratins and porcine trypsin. The raw abundances of extracted ion chromatograms (XIC) peaks of peptide precursors were reported by Progenesis LC-MS v4.1 software (Nonlinear Dynamics, Newcastle, United Kingdom). First the chimeric protein was quantified by comparing the abundances of BSA peptides comprised in its sequence with the corresponding peptides obtained by co-digestion of a known amount of BSA protein standard. In turn, the molar content of target zebrafish histones was inferred from the content of the chimera protein and the ratio of relative abundances of XIC peaks of precursor ions of matching pairs of labeled (originating from chimera) and unlabeled (originating from histones) peptides. Note that all peptides were recovered from the same in-gel digest and quantified at the same LC-MS/MS run. Histone calculations {#s4-7} -------------------- Absolute histone amounts were measured using mass spectrometry. The number of histones bound to a diploid zebrafish genome was calculated as 31,324,994 per genome for each histone. To arrive at this calculation ([Figure 4---source data 1](#SD3-data){ref-type="supplementary-material"}), we have previously shown that the average distance between the centers of neighboring nucleosomes in the zebrafish embryo around genome activation is 187 base pairs ([@bib98]). The size of a zebrafish genome is 1.46 Gb (GRCz10). This was multiplied by two to reach the diploid genome size which was then divided by the nucleosome repeat length to arrive at the number of nucleosomes per genome. As each histone is represented twice in a nucleosome, this number was multiplied by two to arrive at 3.13 × 10^7^ copies of each histone that are required to wrap one zebrafish genome into chromatin (see [Figure 4---source data 1](#SD3-data){ref-type="supplementary-material"}). To arrive at numbers of histones in 'genomes worth of histones', the actual number of histones was divided by the amount of histones required to wrap one diploid genome. For the excess of histones per cell calculation ([Figure 4D](#fig4){ref-type="fig"}), the level of H2B ([Figure 4---source data 1](#SD3-data){ref-type="supplementary-material"}) and the cell numbers in [Figure 1---source data 1](#SD1-data){ref-type="supplementary-material"} were used. Because we have shown that all four core histones contribute to the repressor effect, we used the level of H2B for these calculations, as H2B is the lowest abundant histone and therefore may be limiting for the formation of nucleosomes. We subtracted the number of histones that are assumed to be bound to DNA, which amounts to one to two genomes worth of histones assuming replication (we used the average). The total concentration of non-DNA bound histones was calculated by dividing the total amount of non-DNA-bound histones by the volume of the animal cap ([Figure 4---figure supplement 1A](#fig4s1){ref-type="fig"}). Histone cocktail {#s4-8} ---------------- Recombinant histones were of human origin and produced in *E. Coli* (NEB, Ipswich, MA; 1 mg/ml: H3.1 M2503S, H4 M2504S, H2A M2502S, H2B M2505S). We used human histones because histones are highly conserved between species and these are readily available. To remove DTT, H3.1 was dialyzed in histone buffer (300 mM NaCl, 1 mM EDTA, 20 mM NaPO~4~, pH 7.0 at 25°C) using a Slide-A-Lyzer MINI dialysis device, 7K MWCO (ThermoFisher Scientific) at RT for 30 min or at 4°C overnight. Stoichiometric amounts of all four core histones were combined, spun for 5 min at 6600 rcf on a bench top centrifuge, supernatant was removed, and recovery of histones was measured via quantitative Western blot analysis and calculated using a standard. On average 5756 genomes worth of histone were injected with an error of ±388 (n = 3). NanoString analysis {#s4-9} ------------------- This method involves assigning a unique color-coded barcode to transcripts of interest for single-molecule imaging and counting. The number of times the unique barcode is detected, is used as a readout of the expression level or number of 'counts' for the gene of interest. We developed a custom-made probe set of zygotically expressed genes as well as control genes ([Figure 2](#fig2){ref-type="fig"}, [Figure 2---source data 1](#SD2-data){ref-type="supplementary-material"}). Probe-sets were hybridized to 100 ng of mRNA extracted from a batch of 25 embryos using the RNeasy Mini Kit and processed following the manufacturer's recommendations (NanoString Technologies, Seattle, WA) ([@bib44]). More information about the analysis can be found in the legend of [Figure 2---figure supplement 2](#fig2s2){ref-type="fig"}. Chromatin fractionation {#s4-10} ----------------------- At the desired stage, 65--100 embryos were manually deyolked and snap frozen in a cell lysis buffer (CLB: 10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl~2~, 0.34 M sucrose, 10% glycerol, 0.1% NP-40, 1x protease inhibitor (Roche, Basel, Switzerland)) ([@bib58]). Thawed embryos were shaken at 4°C for 5 min, then placed on ice and flicked intermittently for 5 min. Samples were spun in a bench top centrifuge at 1700 rcf for 5 min. Supernatant was removed and the pellet was washed with CLB. After another spin, the pellet was washed with a nuclear lysis buffer (3 mM EDTA, 0.2 mM EGTA). The sample was spun down and resuspended with high-salt solubilization buffer (50 mM Tris-HCL pH 8.0, 2.5 M NaCl, 0.05% NP-40, 1x protease inhibitor) ([@bib79]). The sample was vortexed for 2 min and placed on a rotator at RT for 10 min. The complete sample was then used in Western blot analysis. Co-immunoprecipitation {#s4-11} ---------------------- Per IP, 500 staged embryos were deyolked as previously described ([@bib52]). Cells were immediately resuspended in cell lysis buffer (10 mM Tris-HCl at pH 7.5, 10 mM NaCl, 0.5% NP-40, 1x protease inhibitor (Roche)), and lysed for 15 min on ice. Nuclei were pelleted by centrifugation and the supernatant was collected and rotated overnight at 4°C with 25 mL of protein G magnetic Dynabeads (Invitrogen, Carlsbad, CA) that had been pre-bound to an excess amount of antibody. Bound complexes were washed six times with RIPA buffer (50 mM HEPES at pH 7.6, 1 mM EDTA, 0.7% DOC, 1% Igepal, 0.5 M LiCl, 1x protease inhibitor) followed by 10 min of boiling in SDS loading buffer. Beads from the sample were subsequently removed by centrifugation and Western blotting was used for further analysis. Cy5 labeling {#s4-12} ------------ Histone H4 was incubated with Cyanine5 NHS ester (10:1 molar ratio) (Lumiprobe, Hannover, Germany) rotating overnight at 4°C. The next day, the solution was dialyzed in histone buffer for 30 min at RT. \~1 ng was injected into embryos of the Tg(h2afz:h2afz-GFP) transgenic fish line ([@bib70]) and embryos were imaged live on an upright LSM 510 META microscope equipped with a Zeiss W Plan-Apochromat 40 × 1.0 dipping objective. GFP was excited at 488 nm, detected with a PMT using BP527.5/545 and a pinhole size of 72 µm. Cy5 was excited at 633 nm, detected with the META detector using BP649-756 and a pinhole size of 96 µm. Images are 512\*512 pixels, pixel size is 0.22 µm and were acquired with eight-bit mode. Quantification of nuclear concentration of non-DNA-bound histones {#s4-13} ----------------------------------------------------------------- We determined the concentration of non-DNA bound histones in the nucleus as follows. First, we obtained volumetric data from live embryos, in which H4-sfGFP fusion protein was translated from injected mRNA to label animal cap and cell nuclei, at low- and high-intensity levels, respectively (see 'Live embryo tracking of nuclei and animal cap volumes' and 'Automated image analysis' below). Imaging live embryos prevented volume alterations due to fixation, permeabilization, and wash steps in immunofluorescence. Next, we determined relative histone distributions in cytoplasm and nucleus by immunofluorescence detection of endogenous histone H4, thus avoiding potential offsets or sub-cellular redistribution of the endogenous histone pool due to the addition of labeled fusion protein (see 'Immunofluorescence' and 'Automated image analysis' below). Then, we combined volumetric and nuclear-over-cytoplasmic intensity ratio data to allocate the total amount of histone H4 per embryo, as measured by mass spectrometry ([Figure 4---source data 1](#SD3-data){ref-type="supplementary-material"}), to the cytoplasmic and the nuclear sub-compartment (see 'Calculation of non-DNA-bound nuclear histone concentration' below). Lastly, aiming to calculate the concentration of only non-DNA-bound histones, the histones bound on chromatin in a given nucleus were subtracted from the total nuclear concentration of histones. Live embryo tracking of nuclei and animal cap volumes {#s4-14} ----------------------------------------------------- To monitor the volumes of the animal cap and individual nuclei as well as nuclear import dynamics, histone H4 and PCNA were imaged in whole live embryos at a time resolution of 2 min or faster (see [Figure 4---source data 2](#SD4-data){ref-type="supplementary-material"}). H4 was introduced as a fusion with sfGFP by mRNA injection. PCNA was monitored using offspring of transgenic fish with PCNA-RFP (Tg(bactin:RFP-pcna) \[[@bib84]\]). Embryos were mounted in glass capillaries with 1% low-melting agarose (Invitrogen) dissolved in 0.3x Danieau's solution and imaged with a Zeiss Z.1 lightsheet microscope using a 10x water dipping objective (NA 0.5) for acquisition, a lightsheet thickness below 5 µm, and dual side illumination ([@bib31]). The microscopy chamber was filled with 0.3x Danieau's and kept at 28.5°C. Optical sectioning was 1 or 1.5 µm, time resolution was 2 min or faster for the acquisition of a whole 3D stack. Immunofluorescence {#s4-15} ------------------ A time series of wild-type TLAB embryos covering 64-cell to sphere stages was collected, immunostained following a protocol optimized for full transparency and penetration of antibody, and imaged using a Zeiss Z.1 lightsheet microscope. Wild-type TLAB embryos were transferred at a given stage by transfer from 0.3x Danieau's into 2% formaldehyde in 0.3x Danieau's with 0.2% Tween-20 and left to fix overnight at 4°C. On the next day, embryos were washed three times for 10 min in PBST (Dulbecco's PBS with 0.1% Tween-20), then further permeabilized by washing twice in double-distilled water followed by 5 min waiting at room temperature, and then blocked with 4% BSA in PBST with 1% DMSO for at least 30 min. Primary antibodies against histone H4 and RNA polymerase II were diluted in 2% BSA in PBST with 1% DMSO and applied for incubation at 4°C for at least 48 hr. Embryos were washed three times for 10 min in PBST. Secondary antibodies were diluted in 2% BSA in PBST with 1% DMSO and applied overnight or longer at 4°C. Embryos were then washed three times for at least 10 min in PBST and stored at 4°C until imaging. Mounting for imaging was done in glass capillaries using 2% low-melting agarose dissolved in Dulbecco's PBS. 3D stacks were acquired using a 20x water dipping objective (NA 1.0) for acquisition, a lightsheet thickness below 5 µm, and dual side illumination. The microscopy chamber was filled with Dulbecco's PBS. Optical sectioning was 1 µm or less. Automated image analysis {#s4-16} ------------------------ Microscopy data were analyzed with a custom MatLab code using the Open Microscopy Environment bioformats plugin for stack reading. Nuclei were segmented using iterative thresholding for individual nuclei to compensate for differing intensities across the sample. 3D segments representing nuclei were dilated in two steps, giving a once- and a twice-extended shell around any given nucleus ([@bib83]). The once-extended shell was removed from the twice-extended shell, along with any other nuclei that happened to be covered by the twice-extended shell. The resulting 3D segment thus covered cytoplasm in the vicinity of a given nucleus. The nucleus and the cytoplasm 3D segments were then used as masks to extract the mean intensity of a given cell's nucleus and cytoplasm. The animal cap was segmented in 3D based a single, global threshold determined from maximum intensity z-projections using Otsu's method. (For code, see Hilbert L. 2016 GitHub. <https://github.com/lhilbert/NCRatio_Analysis>. a7a5849). For live-imaging data, individual nuclei were tracked across consecutive time frames based on minimal centroid distances. The volume fraction of the animal cap taken up by nuclei was calculated from the sum of volumes of all nuclei detected in a given stage, at their individual times of maximal extension in the respective cell cycle. (For code, see Hilbert L. 2016 GitHub. <https://github.com/lhilbert/NucCyto_Ratio_TimeLapse>. 55ed0fc). For immunofluorescence data, nuclei were segmented based on the Pol II signal, which exhibited strong nuclear localization during interphase for all stages. Nuclear and cytoplasmic intensities for both Pol II and H4 were then extracted based on the Pol II segmentation as described above. To remove nuclei that were not in interphase or suffered signal degradation due to excessive spherical aberration or out-of-focus light, only nuclei with a nuclear-over-cytoplasmic intensity ration of greater than two were included in the analysis. Intensity ratios were strongly affected by background staining, so that H4 intensity values were corrected by subtraction of background levels before calculating ratios. Background levels were obtained from control embryos incubated with secondary, but not primary antibodies, which were imaged in the same session and with the same settings as the fully stained samples. Calculation of non-DNA-bound nuclear histone concentration {#s4-17} ---------------------------------------------------------- To obtain nuclear histone concentration values, one considers that the total number of histones must correspond to the contributions from all cells' cytoplasm and nuclei, $H_{total} = \left\lbrack H_{nuclear} \right\rbrack \times V_{nucleus}^{sum} + \left\lbrack H_{cytoplasm} \right\rbrack \times V_{cytoplasm}^{sum}$, where $H_{total}$, $H_{nuclear}$, $H_{cytoplasm}$ are the total, the nuclear, and the cytoplasmic concentration of endogenous histone H4, respectively. $V_{nucleus}^{sum}$ and $V_{cytoplasm}^{sum}$ are the summed volumes of all cells' nuclei and cytoplasm, respectively. Dividing both sides by the total animal cap volume, $V_{total}$, one finds$$\frac{H_{total}}{V_{total}} = \left\lbrack H_{nuclear} \right\rbrack \times v + \left\lbrack H_{cytoplasm} \right\rbrack \times \left( {1 - v} \right),$$ where $v = V_{nucleus}^{sum}/V_{total}$ is the fraction of the total animal cap volume taken up by nuclei (also corresponds to the average fraction of cell volume occupied by the cell nucleus). Considering the N/C intensity ratio, *R*, to represent the concentration ratio, $R \approx \left\lbrack H_{nuclear} \right\rbrack/\left\lbrack H_{cytoplasm} \right\rbrack,$ one can solve for $\left\lbrack H_{nuclear} \right\rbrack$,$$\left\lbrack H_{nuclear} \right\rbrack = \frac{H_{total}}{V_{total}} \times \frac{R}{1 + v\left( {R - 1} \right)}.$$ Realizing that this measured nuclear concentration results from non-DNA-bound histones as much as chromatin bound histones, one needs to subtract the concentration of chromatin bound histones to arrive at the non-DNA-bound histone H4 concentration,$$\left\lbrack H_{free} \right\rbrack = \left\lbrack H_{nucleus} \right\rbrack - \left\lbrack H_{bound} \right\rbrack = \left\lbrack H_{nucleus} \right\rbrack - \frac{g}{V_{nucleus}^{single}}.$$ *g* quantifies the number of complete, histone wrapped genomes (in units of genomes worth) being present in the volume of an individual nucleus, $V_{nucleus}^{single}$. Dropping the *single* superscript for ease of notation, the final expression is$$\left\lbrack H_{free} \right\rbrack = \frac{H_{total}}{V_{total}} \times \frac{R}{1 + v\left( {R - 1} \right)} - \frac{g}{V_{nucleus}}.$$ We measured all variables except *g* on the right hand side using mass spectrometry ($\left\lbrack H_{total} \right\rbrack$), lightsheet imaging of whole live embryos injected with mRNA for H4-sfGFP ($V_{total},~v,~V_{nucleus}$, see above), or immunofluorescence of endogenous histone H4 (*R*) (see [Figure 4---figure supplement 1B](#fig4s1){ref-type="fig"}). *g* was assigned a value of 1.5 genomes worth, to fall between 1 (before replication of the genome) and 2 (complete replication of the genome), under the assumption of full occupation of the DNA by histones. Chromatin immunoprecipitation {#s4-18} ----------------------------- Per IP, \~550 staged embryos were fixed at RT for 15 min in 1.85% formaldehyde. The fixative was quenched with 125 mM glycine and rotation at RT for 5 min. Embryos were then rinsed 3x in ice cold PBS (Accugene, Willowbrook, IL), resuspended in cell lysis buffer (same as co-IP) and lysed for 15 min on ice. Nuclei were pelleted by centrifugation, resuspended in nuclear lysis buffer (50 mM Tris-HCl at pH 7.5, 10 mM EDTA, 1% SDS, 1x protease inhibitor) and lysed for 10 min on ice. Two volumes of IP dilution buffer (16.7 mM Tris-HCl at pH 7.5, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1x protease inhibitor) was added and the sample was sonicated to produce DNA fragments of between 200 and 300 bp (for Pou5f3-2xHA ChIP) or 400 and 500 bp (for tTA-VP16-2xHA ChIP) as determined using a bioanalyzer. 0.8% Triton X was added to the chromatin, which was then centrifuged to remove residual cellular debris. A sample was saved for input and the rest was divided over 25 mL of protein G magnetic Dynabeads that had been pre-bound to an excess amount of either HA antibody or IgG control antibody ([Table 3](#tbl3){ref-type="table"}). These were rotated overnight at 4°C. Bound complexes were washed six times with RIPA buffer followed by TBS. Elution buffer (50 mM NaHCO3, 1% SDS) was added to the beads, which were then vortexed and incubated for 15 min at RT on a rotator. Elutant was collected after centrifugation at 13,200 rcf, and beads were subjected to a repetition of the elution step. The same volume of elution buffer was added to the input sample, and 300 mM NaCl was added to all samples to reverse crosslink at 65°C overnight. Three volumes of 100% ethanol was added and samples were incubated for 1 hr at −80°C. Samples were spun at 13,200 rcf at 4°C for 10 min followed by supernatant removal and air drying. 100 µL water was added and samples were shaken at RT for 5 hr. A PCR purification kit (Qiagen) was used before qPCR analysis. Sample-size determination {#s4-19} ------------------------- A minimum of three biological replicates were used for each experiment, with most experiments having four or more biological replicates (see figure legends for sample size). Funding Information =================== This paper was supported by the following grants: - http://dx.doi.org/10.13039/501100001659Deutsche Forschungsgemeinschaft to Shai R Joseph. - http://dx.doi.org/10.13039/501100004189Max-Planck-Gesellschaft to Shai R Joseph, Máté Pálfy, Lennart Hilbert, Jens Karschau, Vasily Zaburdaev, Andrej Shevchenko, Nadine L Vastenhouw, Mukesh Kumar. - http://dx.doi.org/10.13039/501100000854Human Frontier Science Program CDA00060/2012 to Nadine L Vastenhouw, Máté Pálfy. This work was supported by MPI-CBG core funding, a Human Frontier Science Program Career Development Award (CDA00060/2012) to NLV, a fellowship of the Dresden International Graduate School for Biomedicine and Bioengineering (DIGS-BB) granted by the German Research Foundation (DFG) to SRJ, and an ELBE Postdoctoral Fellowship granted by the Center for Systems Biology Dresden to LH. We thank members of the Vastenhouw laboratory, F Buchholz, D Drechsel and K Steinberg-Bains for help and advice, J Brugues, M Krause, YT Lin, IK Patten, P Tomancak, A Tóth, and J Rink for discussions and critical reading of the manuscript, the Norden lab for kindly providing the Tg(bactin:RFP-pcna) fish and the following MPI-CBG Services and Facilities for their support: the Biomedical Services (Fish unit), the Light Microscopy Facility, and Scientific Computing. Additional information {#s5} ====================== The authors declare that no competing interests exist. SRJ, Conceptualization, Resources, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing---review and editing. MP, Conceptualization, Data curation, Investigation, Visualization, Writing---review and editing. LH, Conceptualization, Software, Investigation, Visualization, Methodology, Writing---review and editing. MK, Conceptualization, Investigation, Methodology, Writing---review and editing. JK, Conceptualization, Investigation, Writing---review and editing. VZ, Conceptualization, Supervision, Funding acquisition, Investigation, Writing---review and editing. AS, Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing---review and editing. NLV, Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Methodology, Writing---original draft, Project administration. 10.7554/eLife.23326.025 Decision letter Adelman Karen Reviewing editor Harvard University , United States In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. \[Editors' note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.\] Thank you for choosing to send your work entitled \"Histone level determines the timing of zygotic genome activation in zebrafish\" for consideration at *eLife*. Your full submission has been evaluated by Janet Rossant (Senior Editor) and three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in *eLife*. As you will see, the reviewers broadly agreed on several key weaknesses in this work: 1\) The extensive overlap with previous work; 2\) The absence of a clear demonstration that adding excess histones alters nucleosome levels on DNA (or that depletion of H2B reduces nucleosome occupancy); 3\) Model that perhaps over-simplifies the scenario. Nothing in this manuscript or previous work pinpoints nucleosomes as the repressors. It remains entirely possible that it is a factor that binds nucleosomes -- and not the octamer itself -- that inhibits transcription. However, all reviewers also saw the potential in this system and encourage the authors to delve deeper into this interesting question to shed new light on zygotic genome activation. *Reviewer \#1:* Work described in this manuscript tests the idea that histone levels define the timing of zygotic genome activation, using zebrafish as a model system. Background on the question: The idea that core histones repress zygotic transcription in the early embryo dates back to the early 90s (Prioleau et al., Cell 1994) and was explored in detail in *Xenopus oocytes* and embryos (e.g. Almouzni and Wolffe, EMBO, 1995). These studies involved injection of excess DNA, core histones, TBP and the activator VP16, leading to the conclusion that transcriptional quiescence before zygotic genome activation was due to a general repressive effect of histones and a defect in transcription activation. Work from the Wolffe lab also noted that they had not established that core histones were directly responsible for repression, but that nucleosome-binding proteins such as linker histones could functionally carry out repression. This possibility was recently supported by work in *Drosophila* (Perez-Montero et al. Dev. Cell, 2013) which showed that a specific variant of linker histone H1 affected the timing of zygotic genome activation (see also nice preview entitled \"A histone timer for zygotic genome activation, by Tamkun and colleague, Dev Cell, 2013). The current manuscript explores the role of histones in zebrafish genome activation. Quantitative mass spec nicely measures the histone levels across early development, confirming their gross excess over DNA. Injection of a cocktail of core histones is found to delay gastrulation and the onset of zygotic transcription (as measured by qPCR of 6 transcripts). All core histones were required for a maximal delay in genome activation, indicating that the formation of an entire nucleosome is essential for full repression. The reciprocal experiments were also performed, wherein the level of histone H2B is reduced to decrease the potential for nucleosome formation. These experiments show a modest premature activation of zygotic transcription. The model proposed is well in line with that from the Wolffe lab, involving both the titration of inhibitory nucleosomes and the involvement of an activator that can overcome such repression at selected sites. Overall, the manuscript is well written and the hypothesis being tested is clear. The experiments seem to be carefully performed and the results are generally convincing. I like the quantitative mass spec and the focus on activation of endogenous genes (previous work largely looked at activation of genes located on exogenous DNA). One caveat is that the effects of histone H2B depletion on gene activation appears small, and I would encourage the authors to consider trying to reduce more than one core histone to achieve stronger effects. My primary concern is that the conclusions do not go much beyond the model established in the mid-90s in *Xenopus*, reducing the impact of this work. Further, the authors should acknowledge that their work does not define core histones as the repressors, only that repression requires nucleosome formation (as in earlier work). I see this as a missed opportunity, given the very nice system developed. Why not target linker histones, specific transcription activators or repressors? That would allow them to go beyond what has been shown in *Xenopus* and *Drosophila* to define direct repressors and activators in their system. *Reviewer \#2:* In the manuscript \"Histone level determines the timing of zygotic genome activation in the zebrafish\", the authors demonstrate that excessive quantities of histone proteins repress transcription during the early stages of zebrafish development. When injected with even more excess quantities of histone proteins, development and onset of transcription is delayed. These findings show are exciting and suggest a clever mechanism for regulating the timing of transition from maternal to zygotic transcription. If the authors could address the following issues, the manuscript would be of interest to the readers of *eLife*. 1\) The authors give a compelling argument for the role of excess nucleosomes in preventing zygotic transition, however some direct evidence of the excess nucleosomes actually interacting with DNA is necessary to support their data. Showing maintenance of nucleosomes on the tested loci upon overexpression of histones and loss of nucleosome occupancy when the H2B morpholino is used would provide some evidence of the effect of the excess nucleosomes. As well, using ChIP to assay binding of transcriptional machinery to show differences with excess or loss of histone proteins. 2\) While the authors show a role for nucleosomes in the timing of transition, they do not address the idea that a repressive chromatin remodeling complex or some other factor may still contribute to the timing of transition as well. Hdac1 is a likely candidate for this role, but there are many repressors that could contribute. It would be helpful for the authors to test Hdac1 or other repressors or discuss the possibility that other repressors may exist. 3\) Authors estimate that the entire genome is fully bound once every 170bp with histones, with no mention of possible variations. While this is obviously an estimate, it would be helpful to at least note that this is an estimate based on the expectation of full occupancy of the genome with histones during the entire course of experiments. 4\) The histone calculations are a bit confusing, particularly with the phrase \"2x1011 copies of each per embryo\". It would be helpful to state histone proteins rather than copies. Is this actually per embryo or per cell? If it is per embryo, then the calculations for excess amounts of histones added for later experiments are confusing, since the measurement is taken at the 8 cell stage, so 8 there would be genomes worth of histones. It seems like the 1.8x1011 histone proteins should be divided by 8x the number of histones in one genome rather than one single genome, giving an excess of 636 rather than 5049. 5\) Calculations of the fold excess are slightly different using the numbers in the paper. Using the numbers from the paper, the fold excess at the 1000 cell stage calculates to 705 rather than 715. 1.8x1011 histone proteins divided by 35,425,427 comes out to 5081 rather than 5049. *Reviewer \#3:* The question of how a developing embryo controls the transition from reliance on maternally loaded mRNA to activation of zygotic expression is a central question in developmental biology. The current manuscript examines this phenomenon in the context of early zebrafish development. The authors hypothesize that maternally loaded histones compete with the transcriptional machinery and that zygotic transcription ensues upon reaching a critical ratio of DNA to histones. They take the general approach of manipulation of the histone:DNA ratio by (1) microinjection of exogenous recombinant human histones or (2) depletion of endogenous H2B using morpholino technology, assessing (1) transcriptional states via RT-PCR of selected protein-coding genes as well as (2) developmental progression. These experiments are highly reminiscent of papers published about 20 years ago by the Mechali (Prioleau et al., (1994) Cell 77: 439-449) and Wolffe (Almouzni and Wolffe, (1995) EMBO J 14: 1752-1765) labs in which the histone:DNA ratio was manipulated in early *Xenopus* development via microinjection of DNA. Regardless of the difference in technique, the current manuscript and the Mechali/Wolffe studies come to an identical conclusion -- that the ratio of histones to DNA in early development is a critical determinant of the onset of zygotic transcription. Mechali and colleagues state \"Our data suggest that the large excess of histones represses gene activation during early development...\". Given this precedent in the literature, it is unclear to me why the current manuscript states repeatedly that the importance of the histone:DNA ratio in zygotic gene activation has never been established (i.e. in Abstract -- \"the titration of a transcriptional repressor by exponentially increasing amounts of DNA has been suggested as a possible mechanism..., but the repressor has never been identified.\"). Mechanistically, I do not understand what exactly the current manuscript is measuring. The authors purchase recombinant human histones (NEB). These are supplied as purified, single polypeptides -- not reconstituted into octamers or any other physiologically relevant complex. They then mix the histones together and inject the cocktail into embryos. It is not clear to me what the fate of the injected histones would be in this scenario. Recombinant histones at low salt concentration will be largely unstructured, certainly not present as octamers (and likely not as H3/H4 tetramers or H2A/H2B heterodimers). I encourage the authors to provide biochemical evidence that injected histones can associate in biochemical species similar to existing histone pools in the embryo -- which are largely associated with histone chaperones. The conclusion by the authors that nucleosomes are the relevant species in their experiments competing with transcription factors for DNA (as opposed to non-specific interactions of unstructured basic proteins) seems poorly supported by the data. \[Editors' note: what now follows is the decision letter after the authors submitted for further consideration.\] Thank you for resubmitting your work entitled \"Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos\" for further consideration at *eLife*. Your revised article has been favorably evaluated by Robb Krumlauf (Senior Editor), a Reviewing Editor, and two reviewers. The manuscript has been considerably improved, and would now be suitable for publication once several major concerns below are addressed. Summary: By utilizing efficient quantitative techniques, Joseph et al. present a very interesting study that supports a competitive interaction between histones and transcription factors to set the time for ZGA. They conclude that histones and critical transcription factors compete in binding to DNA, and eventually, the concentration of non-DNA bound core histone decides the timing of ZGA during zebrafish gastrulation. By injecting stoichiometric mixture of all four core histones they showed that increasing their levels delays the onset of zygotic transcription. On the other hand, decreasing levels of histones, by sequestering H4 with PTX3, shows an opposite effect. Using a quantitative mass spectrometry approach, the authors calculated the number of non-DNA bound histones per cell in genomes worth of histone, and showed that this parameter drops dramatically as embryos approach gastrulation. This provides opportunity for critical transcription factors (such as Pou5f3) to bind to DNA and initiate transcription. Following this argument, increasing and decreasing the level of Pou5f3 predates and delays the onset of ZGA, respectively. Finally, in the context of a TRE-GFP reporter construct, by injecting either a heterologous transcription factor (rTA-VP16) alone, or by co-injecting it with histone cocktails, a ChIP-PCR analysis provided further evidence for the competitive interaction between histones and transcription factors to bind to TRE elements in the GFP reporter. Altogether, this study provides a detailed mechanistic analysis to answer a few important issues related to a long-standing concept in the field. The quantitative techniques followed in this study highlights the usefulness of zebrafish to answer critical questions which could be otherwise difficult in other model systems. Such analyses would be beneficial for the zebrafish community as a whole. Taken together, this would be suitable for publication in \"*eLife*\" if the following concerns could be addressed or explained properly in an updated manuscript. Major points that must be addressed in a revised manuscript: 1\) In [Figure 5](#fig5){ref-type="fig"}, the authors only tested Pou5f3 as an example transcription factor that is important for ZGA. The authors posit that \'specific\' effects needed for ZGA are the result of competition with transcription factors, and suggest that only specific TFs could compete with histones (e.g. only those TF needed for activation of the first ZGA genes). It would be nice to see this idea better fleshed out. Did the authors try to knockdown other important developmental factors (like Pou5f1, *Sox2*, Nanog) to see the effect on ZGA? What about non-developmental factors? According to their model, the competition between ZGA-related transcription factors and histones should not be specific to Pou5f3 only. Rather, knockdown of any other pluripotency factor should have the same ZGA-related phenotype. The specificity/ generality of this phenomenon should be tested by knockdown of at least one additional TF. 2\) The authors should clarify the nature of their competition model, through additional experiments, or clarifications in the text. At present (and as detailed below) it is not clear how the authors envision that excess histones are functioning, and the text and data are not consistent in this regard. For this work to have the desired impact, we encourage the authors to work towards clearly articulating and substantiating their model. A\) The reviewers were concerned about the absence of proof for a competition model in the data presented, and alternative interpretations for the data presented are plausible: for example, histone \'excess\' may affect replication timing, thereby affecting timing of transcriptional activation at ZGA. While the integrated transgene and VP16 experiments is strong, one would like to see it \'working\' in an endogenous target: at the very least, the same ChIP experiment performed in excess of histone should be done for the Apoeb gene for Pouf3t and Sox19 transcription factors. Otherwise, it is difficult to reconcile it with the specificity in competition program discussed by the authors in the subsection "Competition for DNA binding between histones and transcription factors regulates the onset of transcription". B\) Perhaps an additional possibility to address whether the competition model is indeed occurring, would be to reduce histone chaperone activity at the \'early\' 1K stage. This should increase non-DNA bound histones, and therefore should have the same effect as the injection cocktail. C\) If the effect on transcriptional activation is assumed to be due to non-DNA bound histones (subsection "Onset of transcription coincides with a reduction in nuclear histone concentration" and Summary) -- why is the addition of Histone cocktail effective considering that the authors conclude that \'this led to efficient chromatin incorporation of the histones injected as determined using Cy5 labelling\' (subsection "Increasing the levels of all core histones delays onset of transcription and gastrulation")? D\) Along the same lines, in the Discussion, the authors state that, because the 4 core histones are necessary to see the effects of the injected cocktail on transcriptional activation, this suggests a role for the nucleosome for the repressor function of histones. This statement is not consistent with the interpretation that it is the \'non-DNA bound histones\' that is effecting ZGA timing (e.g. nucleosome is composed by the histone octamer plus the DNA). Given the biochemical properties of the 4 histones, it is also unlikely that the histones injected remain as octamers in the cells. E\) Similarly, related to the PTX3 experiments ([Figure 3](#fig3){ref-type="fig"}, subsection "Decreasing the level of histones causes premature transcription"), the authors conclude that \'total levels of H3 and H4 are not affected upon PTX3 expression\', but three lines below, they conclude that effective levels of histone H3 and H4 are decreased. While \'effective\' may indicate that they are not necessarily free to be incorporated into chromatin, this experiment is also at odds with the hypothesis that \'non-DNA-bound histones\' regulate ZGA timing. F\) The authors conclude that histones are effectively incorporated by doing immunofluorescence with Cy5-labelled H4. However, nuclear localisation is not a proof for incorporation -- did the authors try the same immunofluorescence with e.g. either pre-extraction with tritron or salt? 3\) In [Figure 6](#fig6){ref-type="fig"}, the authors check the occupancy of rTA-VP16 on TRE elements under histone cocktail (HC) overexpression. This experiment shows a decrease in rTA-VP16 level at TRE upon HC injection, but they did not comment if this leads to a concomitant increase in histone binding at TRE. It would be good to include a comment related to this issue in the respective Results section. 10.7554/eLife.23326.026 Author response \[Editors' note: the author responses to the first round of peer review follow.\] *As you will see, the reviewers broadly agreed on several key weaknesses in this work:* *1) The extensive overlap with previous work;* We agree with the reviewers that previous work using injected species of exogenous DNA had suggested that histones might play a role in transcription regulation in the embryo. We apologize for not doing enough justice to this work and now clarified this part of the manuscript. Questions that remained after previous studies, however, include: - Whether histones regulate the timing of zygotic transcription (transcription of endogenous genes in the embryo). - If so, which histones are involved (important for understanding the mechanism). - How this all should work (the mechanism). In our revised manuscript, we address these questions. We first set up in an in vivo assay to analyze the onset of zygotic transcription ([Figure 1](#fig1){ref-type="fig"}) and show that all core histones are required to determine the timing of transcription in the embryo ([Figures 2](#fig2){ref-type="fig"}, [3](#fig3){ref-type="fig"}). Then we directly analyze the mechanism. We first quantified the levels of histones in the embryo, nucleus and on chromatin. We found a reduction in nuclear histone concentration that coincides with genome activation but this is not accompanied by a loss of nucleosome density on DNA. This led us to propose a model in which a competition for DNA binding between transcription factors and non-DNA bound histones regulates the onset of transcription ([Figure 4](#fig4){ref-type="fig"}). To test this model, we changed the levels of endogenous transcription factors and analyzed its effect on the timing of transcription. In agreement with our model, the levels of transcription factors also affect the timing of transcription ([Figure 5](#fig5){ref-type="fig"}). We further test the competition model using a heterologous transgene ([Figure 6](#fig6){ref-type="fig"}). Using this strategy, we could confirm the importance of transcription factor levels for the timing of transcription and we were able to show that histone levels affect the binding of a transcription factor. Taken together, we show that the onset of transcription in the zebrafish embryo is regulated by a competition for DNA binding between histones and transcription factors. Our study provides, to our knowledge, the first example of a developmental transition in which competition for DNA binding between histones and transcription factors plays an important role in transcriptional regulation. *2) The absence of a clear demonstration that adding excess histones alters nucleosome levels on DNA (or that depletion of H2B reduces nucleosome occupancy);* We show that histone levels regulate the time of zygotic transcription. Transcription starts when the nuclear concentration of histones drops, but this is not accompanied by a loss of nucleosome density on DNA. This suggests that the onset of transcription is regulated by the concentration of non-DNA bound histones in the nucleus. Based on this data, we propose that the onset of transcription in the embryo is regulated by a competition for DNA binding between non-DNA bound histones and transcription factors and we provide direct experimental evidence for this model. We therefore propose that the effect of changing histone levels experimentally is exerted through changes in the non-DNA bound concentration of histones and not by changes in global nucleosome density. *3) Model that perhaps over-simplifies the scenario. Nothing in this manuscript or previous work pinpoints nucleosomes as the repressors. It remains entirely possible that it is a factor that binds nucleosomes -- and not the octamer itself -- that inhibits transcription.* This is where we have really expanded the manuscript. Our experiments show that the four core histones mediate the repressor effect. Moreover, our finding that histones are neither completely depleted from the soluble fraction, nor generally depleted from chromatin, argues against a model in which a global loss of nucleosome density on chromatin causes the onset of transcription. This strongly suggests that histones themselves function as the actual repressors of transcription and argues against a role for the depletion of proteins associated with core histones. Furthermore, the model presented in the earlier submission did indeed simplify the scenario. We now present data that the levels of activators are also important. We provide direct evidence for a model in which competition for DNA binding between histones and activators regulates the onset of transcription in the embryo. *However, all reviewers also saw the potential in this system and encourage the authors to delve deeper into this interesting question to shed new light on zygotic genome activation.* *Reviewer \#1:* Work described in this manuscript tests the idea that histone levels define the timing of zygotic genome activation, using zebrafish as a model system. *Background on the question: The idea that core histones repress zygotic transcription in the early embryo dates back to the early 90s (Prioleau et al., Cell 1994) and was explored in detail in Xenopus oocytes and embryos (e.g. Almouzni and Wolffe, EMBO, 1995). These studies involved injection of excess DNA, core histones, TBP and the activator VP16, leading to the conclusion that transcriptional quiescence before zygotic genome activation was due to a general repressive effect of histones and a defect in transcription activation. Work from the Wolffe lab also noted that they had not established that core histones were directly responsible for repression, but that nucleosome-binding proteins such as linker histones could functionally carry out repression. This possibility was recently supported by work in Drosophila (Perez-Montero et al. Dev. Cell, 2013) which showed that a specific variant of linker histone H1 affected the timing of zygotic genome activation (see also nice preview entitled \"A histone timer for zygotic genome activation, by Tamkun and colleague, Dev Cell, 2013).* We agree with the reviewer that in our previous manuscript, we did not do enough justice to the work that has previously shown a role for histones in repressing transcription in the early *Xenopus* embryo. We apologize and now discuss existing work more clearly and identify the aspects which needed further work. *The current manuscript explores the role of histones in zebrafish genome activation. Quantitative mass spec nicely measures the histone levels across early development, confirming their gross excess over DNA. Injection of a cocktail of core histones is found to delay gastrulation and the onset of zygotic transcription (as measured by qPCR of 6 transcripts). All core histones were required for a maximal delay in genome activation, indicating that the formation of an entire nucleosome is essential for full repression. The reciprocal experiments were also performed, wherein the level of histone H2B is reduced to decrease the potential for nucleosome formation. These experiments show a modest premature activation of zygotic transcription. The model proposed is well in line with that from the Wolffe lab, involving both the titration of inhibitory nucleosomes and the involvement of an activator that can overcome such repression at selected sites.* *Overall, the manuscript is well written and the hypothesis being tested is clear. The experiments seem to be carefully performed and the results are generally convincing. I like the quantitative mass spec and the focus on activation of endogenous genes (previous work largely looked at activation of genes located on exogenous DNA). One caveat is that the effects of histone H2B depletion on gene activation appears small, and I would encourage the authors to consider trying to reduce more than one core histone to achieve stronger effects.* As suggested, we have changed our approach to reduce histones. Since we see by mass spectrometry that a large amount of histones is present as protein from fertilization on ([Figure 4B](#fig4){ref-type="fig"}), we decided to change our approach and target protein levels. Now we use PTX3 to target histone proteins. We show in vivo that PTX3 interacts with H4 protein, and the effect of PTX3 is greater than with the morpholino. We also expand our Discussion, and indicate that due to a role of activator levels in our competition model, pushing genome activation back will always be harder than forward (with more histones). Activators might be limiting because they need to be translated (Pou5f3, Nanog, *Sox2* are highly translated during development) and because many of these activators might function cooperatively, they all need to be present at the right concentration at the right time. *My primary concern is that the conclusions do not go much beyond the model established in the mid-90s in Xenopus, reducing the impact of this work. Further, the authors should acknowledge that their work does not define core histones as the repressors, only that repression requires nucleosome formation (as in earlier work). I see this as a missed opportunity, given the very nice system developed. Why not target linker histones, specific transcription activators or repressors? That would allow them to go beyond what has been shown in Xenopus and Drosophila to define direct repressors and activators in their system.* With respect to the novelty of our work, and the identity of the repressor, we refer to the key points. *Reviewer \#2:* *In the manuscript \"Histone level determines the timing of zygotic genome activation in the zebrafish\", the authors demonstrate that excessive quantities of histone proteins repress transcription during the early stages of zebrafish development. When injected with even more excess quantities of histone proteins, development and onset of transcription is delayed. These findings show are exciting and suggest a clever mechanism for regulating the timing of transition from maternal to zygotic transcription. If the authors could address the following issues, the manuscript would be of interest to the readers of eLife.* *1) The authors give a compelling argument for the role of excess nucleosomes in preventing zygotic transition, however some direct evidence of the excess nucleosomes actually interacting with DNA is necessary to support their data. Showing maintenance of nucleosomes on the tested loci upon overexpression of histones and loss of nucleosome occupancy when the H2B morpholino is used would provide some evidence of the effect of the excess nucleosomes. As well, using ChIP to assay binding of transcriptional machinery to show differences with excess or loss of histone proteins.* To address these points, we did the following: We have chemically tagged H4 and show that it incorporates into chromatin ([Figure 2---figure supplement 1C](#fig2s1){ref-type="fig"}). We have done chromatin fractionation and show that nucleosome density does not change globally at the time of genome activation ([Figure 4F](#fig4){ref-type="fig"}). In our competition model, local changes in nucleosome density (where RNA pol II or TF bind) result in transcription. Exactly as suggested, we used ChIP-qPCR to assess the binding of a transcriptional activator in response to changing histone levels. Increasing histone levels causes a reduction of binding of the activator to its target site ([Figure 6](#fig6){ref-type="fig"}). In agreement with this, increasing activator levels leads to premature transcription ([Figures 5](#fig5){ref-type="fig"}, [6](#fig6){ref-type="fig"}). *2) While the authors show a role for nucleosomes in the timing of transition, they do not address the idea that a repressive chromatin remodeling complex or some other factor may still contribute to the timing of transition as well. Hdac1 is a likely candidate for this role, but there are many repressors that could contribute. It would be helpful for the authors to test Hdac1 or other repressors or discuss the possibility that other repressors may exist.* Here, we show that histone levels in combination with transcription factor levels are sufficient to change the timing of transcription. We agree with the reviewer, however, that chromatin remodeling complexes might also contribute to genome activation. We have now included a discussion on how the activity of chromatin remodelers might also be mediated by histone levels. *3) Authors estimate that the entire genome is fully bound once every 170bp with histones, with no mention of possible variations. While this is obviously an estimate, it would be helpful to at least note that this is an estimate based on the expectation of full occupancy of the genome with histones during the entire course of experiments.* The number we used is based on MNase-Seq experiments and the average nucleosome repeat length derived from that (Zhang et al., 2014). We have clarified this in the text. Of note is that the nucleosome repeat length that we use is not critical for our model. Even variations of nucleosome occupancy from 160 to 200 do not substantially affect our calculations of the total nor nuclear histone concentration. *4) The histone calculations are a bit confusing, particularly with the phrase \"2x1011 copies of each per embryo\". It would be helpful to state histone proteins rather than copies. Is this actually per embryo or per cell? If it is per embryo, then the calculations for excess amounts of histones added for later experiments are confusing, since the measurement is taken at the 8 cell stage, so 8 there would be genomes worth of histones. It seems like the 1.8x1011 histone proteins should be divided by 8x the number of histones in one genome rather than one single genome, giving an excess of 636 rather than 5049.* We apologize for the confusion and provide more clarity with our calculations in the new manuscript (Materials and methods and [Table 2](#tbl2){ref-type="table"}). *5) Calculations of the fold excess are slightly different using the numbers in the paper. Using the numbers from the paper, the fold excess at the 1000 cell stage calculates to 705 rather than 715. 1.8x1011 histone proteins divided by 35,425,427 comes out to 5081 rather than 5049.* This discrepancy was due to rounding our numbers. There is more clarity in our new [Table 2](#tbl2){ref-type="table"}. *Reviewer \#3:* *The question of how a developing embryo controls the transition from reliance on maternally loaded mRNA to activation of zygotic expression is a central question in developmental biology. The current manuscript examines this phenomenon in the context of early zebrafish development. The authors hypothesize that maternally loaded histones compete with the transcriptional machinery and that zygotic transcription ensues upon reaching a critical ratio of DNA to histones. They take the general approach of manipulation of the histone:DNA ratio by (1) microinjection of exogenous recombinant human histones or (2) depletion of endogenous H2B using morpholino technology, assessing (1) transcriptional states via RT-PCR of selected protein-coding genes as well as (2) developmental progression.* For a summary of the data in the revised manuscript we refer to the key points. *These experiments are highly reminiscent of papers published about 20 years ago by the Mechali (Prioleau et al., (1994) Cell 77: 439-449) and Wolffe (Almouzni and Wolffe, (1995) EMBO J 14: 1752-1765) labs in which the histone:DNA ratio was manipulated in early Xenopus development via microinjection of DNA. Regardless of the difference in technique, the current manuscript and the Mechali/Wolffe studies come to an identical conclusion -- that the ratio of histones to DNA in early development is a critical determinant of the onset of zygotic transcription. Mechali and colleagues state \"Our data suggest that the large excess of histones represses gene activation during early development...\". Given this precedent in the literature, it is unclear to me why the current manuscript states repeatedly that the importance of the histone:DNA ratio in zygotic gene activation has never been established (i.e. in Abstract -- \"the titration of a transcriptional repressor by exponentially increasing amounts of DNA has been suggested as a possible mechanism..., but the repressor has never been identified.\").* We apologize for not clearly reviewing existing data previously, and included a thorough discussion of existing work in our revised manuscript. With respect to the novelty of our work, we refer to the key points. Mechanistically, I do not understand what exactly the current manuscript is measuring. The authors purchase recombinant human histones (NEB). These are supplied as purified, single polypeptides -- not reconstituted into octamers or any other physiologically relevant complex. They then mix the histones together and inject the cocktail into embryos. It is not clear to me what the fate of the injected histones would be in this scenario. Recombinant histones at low salt concentration will be largely unstructured, certainly not present as octamers (and likely not as H3/H4 tetramers or H2A/H2B heterodimers). I encourage the authors to provide biochemical evidence that injected histones can associate in biochemical species similar to existing histone pools in the embryo -- which are largely associated with histone chaperones. The conclusion by the authors that nucleosomes are the relevant species in their experiments competing with transcription factors for DNA (as opposed to non-specific interactions of unstructured basic proteins) seems poorly supported by the data. To address these points, we did the following: We provide proof that the human NEB histones are functional. We have chemically tagged H4 that shows that it localizes to the nucleus and incorporates into chromatin ([Figure 2---figure supplement 1C](#fig2s1){ref-type="fig"}). Additionally, the experiments that remove one histone from the cocktail and elevate the levels of the other three, argue against a non-specific effect of an unstructured basic protein. If the delay in transcription was a result of randomly injecting unstructured protein, then the histone minus one experiment should also have the same effect. Which they don't. \[Editors\' note: the author responses to the re-review follow.\] *Major points that must be addressed in a revised manuscript:* *1) In [Figure 5](#fig5){ref-type="fig"}, the authors only tested Pou5f3 as an example transcription factor that is important for ZGA. The authors posit that \'specific\' effects needed for ZGA are the result of competition with transcription factors, and suggest that only specific TFs could compete with histones (e.g. only those TF needed for activation of the first ZGA genes). It would be nice to see this idea better fleshed out. Did the authors try to knockdown other important developmental factors (like Pou5f1, Sox2, Nanog) to see the effect on ZGA? What about non-developmental factors? According to their model, the competition between ZGA-related transcription factors and histones should not be specific to Pou5f3 only. Rather, knockdown of any other pluripotency factor should have the same ZGA-related phenotype. The specificity/ generality of this phenomenon should be tested by knockdown of at least one additional TF.* We apologize for the confusion. We agree with the reviewers that, according to our model, the competition between ZGA-related transcription factors and histones should not be specific to Pou5f3. We have clarified this in the text. Following the reviewers' suggestion, we now show that knockdown of Sox19b and FoxH1 also results in a delay in the transcription of target genes (subsection "Decreasing transcription factor levels delays the onset of transcription", last paragraph, [Figure 5---figure supplement 1D-G](#fig5s1){ref-type="fig"}). To further substantiate the generality of the role of histone levels in genome activation, we have also included NanoString analysis of embryos injected with the histone cocktail (subsection "Increasing the levels of all core histones delays onset transcription and gastrulation", second paragraph, [Figure 2C](#fig2){ref-type="fig"} and [Figure 2---figure supplement 2](#fig2s2){ref-type="fig"}). This shows that transcription of an important number of genes is delayed as a consequence of increased histone levels. Given that these genes are likely regulated by a variety of transcription factors, this supports the notion that histone levels affect the binding of many transcription factors. *2) The authors should clarify the nature of their competition model, through additional experiments, or clarifications in the text. At present (and as detailed below) it is not clear how the authors envision that excess histones are functioning, and the text and data are not consistent in this regard. For this work to have the desired impact, we encourage the authors to work towards clearly articulating and substantiating their model.* Thank you for raising this point. We have detailed below what experiments we have done and what changes we have made to the text to address it. *A) The reviewers were concerned about the absence of proof for a competition model in the data presented, and alternative interpretations for the data presented are plausible: for example, histone \'excess\' may affect replication timing, thereby affecting timing of transcriptional activation at ZGA.* That is a good point. Replication timing is not affected in our experiments, and we have now clarified this in the text (For example, subsection "Increasing the levels of all core histones delays onset transcription and gastrulation", second paragraph and subsection "Decreasing the pool of available histones causes premature transcription", just to mention a few). *While the integrated transgene and VP16 experiments is strong, one would like to see it \'working\' in an endogenous target: at the very least, the same ChIP experiment performed in excess of histone should be done for the Apoeb gene for Pouf3t and Sox19 transcription factors. Otherwise, it is difficult to reconcile it with the specificity in competition program discussed by the authors in the subsection "Competition for DNA binding between histones and transcription factors regulates the onset of transcription".* This is an excellent point. We used the transgene because the regulation of endogenous genes is complex and the data is therefore harder to interpret. However, we now show that the binding of Pou5f3 on apoeb (and dusp6, an additional gene for which we saw premature transcription upon Pou5f3/Sox19b overexpression), is also reduced in the presence of more histones (subsection "Transcription factor binding is sensitive to histone levels", [Figure 5D](#fig5){ref-type="fig"}). *B) Perhaps an additional possibility to address whether the competition model is indeed occurring, would be to reduce histone chaperone activity at the \'early\' 1K stage. This should increase non-DNA bound histones, and therefore should have the same effect as the injection cocktail.* This is in principle a good idea. However, the lack of information on chaperones in zebrafish, as well as the -- related -- lack of specific drugs make this experiment difficult to perform and interpret. Especially in combination with the fact that in the best-case scenario this experiment would recapitulate the histone cocktail effect without providing more (quantitative) insight in the competition model, we decided not to try these experiments. *C) If the effect on transcriptional activation is assumed to be due to non-DNA bound histones (subsection "Onset of transcription coincides with a reduction in nuclear histone concentration" and Summary) -- why is the addition of Histone cocktail effective considering that the authors conclude that \'this led to efficient chromatin incorporation of the histones injected as determined using Cy5 labelling\' (subsection "Increasing the levels of all core histones delays onset of transcription and gastrulation")?* This experiment was designed to show that the histones we inject can incorporate in chromatin, and thus become part of, and behave like, the endogenous pool of histones available for competition. Like endogenous histones in the early embryo, not all histones will incorporate into chromatin due to their excess levels. We have now clarified this in text (subsection "Increasing the levels of all core histones delays onset transcription and gastrulation", second paragraph). *D) Along the same lines, in the Discussion, the authors state that, because the 4 core histones are necessary to see the effects of the injected cocktail on transcriptional activation, this suggests a role for the nucleosome for the repressor function of histones. This statement is not consistent with the interpretation that it is the \'non-DNA bound histones\' that is effecting ZGA timing (e.g. nucleosome is composed by the histone octamer plus the DNA). Given the biochemical properties of the 4 histones, it is also unlikely that the histones injected remain as octamers in the cells.* We apologize for the confusion. Our model is based on the knowledge that the four core histones are required to form a histone octamer (and nucleosome). Of course, the histones only form a histone octamer when associating with DNA and it is therefore expected that the concentration of soluble histones dictates how likely a nucleosome is formed on an available piece of DNA. Thus, we propose that the soluble histones compete with transcription factors for binding, but ultimately it is either the histone octamer or transcription factor that occupies DNA. We have clarified this in the text (throughout). *E) Similarly, related to the PTX3 experiments ([Figure 3](#fig3){ref-type="fig"}, subsection "Decreasing the level of histones causes premature transcription"), the authors conclude that \'total levels of H3 and H4 are not affected upon PTX3 expression\', but three lines below, they conclude that effective levels of histone H3 and H4 are decreased. While \'effective\' may indicate that they are not necessarily free to be incorporated into chromatin, this experiment is also at odds with the hypothesis that \'non-DNA-bound histones\' regulate ZGA timing.* In order to compete for DNA binding, histones need to be available (see also above). Therefore, when histones are not available to bind to DNA (because they are bound by PTX3), this lowers their effective concentration. We have clarified this in the text (subsection "Decreasing the pool of available histones causes premature transcription"). *F) The authors conclude that histones are effectively incorporated by doing immunofluorescence with Cy5-labelled H4. However, nuclear localisation is not a proof for incorporation -- did the authors try the same immunofluorescence with e.g. either pre-extraction with tritron or salt?* We apologize for the confusion. The conclusion that histones are incorporated was not based on the nuclear localization of the labeled histone but rather on the fact that we see the label in chromatin during metaphase. We have now clarified this in the text (subsection "Increasing the levels of all core histones delays onset transcription and gastrulation", second paragraph) and in [Figure 2---figure supplement 1C](#fig2s1){ref-type="fig"}. *3) In [Figure 6](#fig6){ref-type="fig"}, the authors check the occupancy of rTA-VP16 on TRE elements under histone cocktail (HC) overexpression. This experiment shows a decrease in rTA-VP16 level at TRE upon HC injection, but they did not comment if this leads to a concomitant increase in histone binding at TRE. It would be good to include a comment related to this issue in the respective Results section.* We assume that there is an increase in histone binding but we have not looked at this. We have now clarified this in the text (subsection "Transcription factor binding is sensitive to histone levels") in the section on Pou5f3 binding ([Figure 5D](#fig5){ref-type="fig"}), which is the first time we test the effect of histone levels on transcription factor binding in the revised version of the manuscript. [^1]: WB, Western blotting; IF, immunofluorescence; IP, immunoprecipitation. [^2]: WB, Western blotting; IF, immunofluorescence.

{#sp1 .540}

Introduction ============ Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer-related death worldwide.[@B1], [@B2] CRC is caused by mutations that target oncogenes, tumor suppressor genes and genes related to DNA repair mechanisms. Interestingly, noncoding RNAs account for 90% of total transcribed RNAs in the human genome.[@B3] Long noncoding RNAs (lncRNAs) are functionally defined as transcripts \>200 nucleotides in length with no protein coding potential. They also number in the tens of thousands, many of which are uniquely expressed in differentiated tissues or specific cancer types.[@B4] LncRNAs regulate cellular processes depending on their cellular localization: nuclear lncRNAs are enriched for functionality involving chromatin interactions, transcriptional regulation, and RNA processing, while cytoplasmic lncRNAs can modulate mRNA stability or translation and influence cellular signaling cascades.[@B5] Since the lncRNA CCAT1 was identified in CRC, numerous lncRNAs have been characterized along with their oncogenic or tumor suppressor functions in CRC.[@B6]-[@B9]. SNHG6 is a housekeeping gene from the 5\'TOP family that encodes two non-coding RNAs (ncRNAs): U87 C/D box snoRNA (SNORD87),[@B10] and lncRNA SNHG6,[@B11] which has been demonstrated to be as a potential oncogene in various human cancers.[@B12]-[@B14] In this study, we investigated SNGH6 expression in different human cancers using a TCGA dataset, and found that SNHG6 was highly expressed in CRC with a poor prognosis. Our study demonstrated that SNHG6 may act as an oncogene in CRC by activating the TGF-β/Smad signaling pathway via binding UPF1 and inducing epithelial-mesenchymal transition (EMT) through regulating ZEB1. Materials and methods ===================== The Cancer Genome Atlas (TCGA) database, GEO database, StarBase and bioinformatics analysis ------------------------------------------------------------------------------------------- TCGA and GEO data of different cancers was selected by GEPIA and UALCAN, so examine whether any significant differences in SNHG6 expression existed between paired normal and tumor tissues. Fold change \> 1.5 and *P*-value \< 0.01 between the tumor and normal tissues were considered as significant. The starBase v2.0[@B15] was used to selected downstream interacting protein. Clinical specimens ------------------ Clinical CRC specimens and paired normal tissues were collected from 77 patients who underwent surgical treatment for CRC at Nanfang Hospital of Southern Medical University after obtaining informed consent. A diagnosis of CRC was histopathologically confirmed for each patient sample. Cancer tissues and matched normal tissues were stored at -80℃ until use. The protocols used in this study were approved by our hospital\'s Protection of Human Subjects Committee. Cell culture, plasmid construction, lentiviral construction and cell transfections ---------------------------------------------------------------------------------- Human normal colon epithelial cell line (FHC) and human colorectal cancer cell lines (HT29, CaCO~2~, SW480, SW620, RKO, HCT116 and LoVo) were purchased from the Cell Bank of Type Culture Collection (CBTCC, Chinese Academy of Sciences, Shanghai, China) and were cultured in DMEM (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA). Cells were maintained at 37℃ in a water-saturated atmosphere with 5% CO~2~. In order to overexpress SNHG6, full-length SNHG6 was cloned into the expression vector pCMV (Vigene, Shandong, China) and transfected into RKO cells by using Lipofectamin^TM^ 3000 (Invitrogen, Carlsbad, CA) according to the manufacturer\'s instructions. Knockdown of SNHG6 was accomplished using three different designed shRNAs (Cyagen, Guangzhou, China) that were transfected into RKO cells according to the manufacturer\'s instructions. RNA isolation, cDNA synthesis, and quantitative real-time PCR ------------------------------------------------------------- Total RNAs were extracted from cells or tissues with Trizol solution (TaKaRa, Dalian, China). Quantitative real-time polymerase chain reaction (qRT-PCT) was performed using the PrimeScript RT Reagent Kit and SYBR Premix Ex Taq (TaKaRa, Dalian, China) following the manufacturer\'s instructions. Our results were normalized to the expression of glyeraldehyde-3-phosphate dehydrogenase (GAPDH) or U6. The specific primers used are listed in Table [1](#T1){ref-type="table"}. qRT-PCR results were analyzed to obtain Ct values of amplified products, and data was analyzed by the 2^-ΔΔCt^ method. Cell proliferation assay ------------------------ Cell proliferation was estimated using a Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). Overexpression transfected RKO cells and HCT116 cells as well as RKO knockdown cells were seeded on the 96-well plates and each were cultured for 0h, 24h, 48h, 72h, 96h respectively. At the different time point, 10μL CCK-8 was added to the well and incubated for 2 hours. An absorbance value (OD) of 450nm was determined on the microplate reader. Transwell assay --------------- Cell migration and invasion assays were measured by trawnswell chamber (8μm pore size, Corning), and for cell invasion, the transwell chambers were also matrigel-coated. The lower chamber was filled with 500μL of 20% FBS medium. Transfected RKO cells (6×10^4^) in 200μL of serum-free medium were gently loaded onto each filter insert (upper chamber) and then incubated at 37℃ for 48h. The filter inserts were removed from the chambers, fixed with methanol for 10min and stained with hematoxylin for 20 min. The samples were subsequently washed, dried and mounted onto slides. The migratory cells were stained blue, visualized under and inverted microscope and then counted in five random fields for statistical analysis. Wound healing assay ------------------- Transfected overexpression and knockdown RKO cells were cultured in DMEM with 2% fetal bovine serum. Wounds were made in the cell monolayer using a 10-μl plastic pipette tip. The size of the wound was imaged and measured after 48h of wound formation. The cell migration area was measured with dashed areas and normalized to control cells. *In vivo* experiments --------------------- 4-week-old male nude mice were purchased from the Central Laboratory of Animal Science, Wuhan University (Wuhan, China) and were maintained in a specific pathogen-free facility. RKO cells stably transfected with SNHG6-shRNA or scramble-shRNA were harvested from 60mm plates and suspended at 5×10^6^ cells/ml. The suspended cells (200μl) were subcutaneously injected into the left hip of 4 mice (4 weeks old) each group, and the mice were sacrificed 4 weeks after injection. The tumor volume (V) was obtained by measuring the length (L) and width (W) of the tumor with vernier calipers, and which was calculated using the formula V = (L×W^2^) × 0.5. Western blot analysis --------------------- Total protein was extracted from cells using RIPA lysis buffer. Extracted proteins were mixed with loading buffer, separated by SDS-PAGE and transferred to PVDF membranes, which were subsequently blocked with a 5% solution of non-fat milk for 1h. Membranes were then incubated with primary antibody \[GAPDH, UPF1, 1:5000, Proteintech; smad2, p-smad2, smad3, p-smad3, E-cadherin, N-cadherin, Vimentin, ZEB1, Slug, Snail, MMP9, MMP2, 1:1000, Cell Signaling Technology\] according to the manufacturer\'s instructions. Then the membranes were washed three times with TBST and incubated with appropriate secondary antibodies for 1h at room temperature. The ECL chemiluminescence system was used to detect the signal. Statistical analysis -------------------- The SPSS 17.0 statistical analysis software was used for statistical analysis of experimental data. The significance of differences between groups was estimated by Student\'s t-test. Additionally, multiple group comparisons were analyzed with one-way ANOVA. Statistically significant correlation between SNHG6 and UPF1 expression levels in CRC tissues and cell lines was analyzed by Pearson\'s correlation analysis. The overall survival probability was analyzed using Kaplan-Meier method and calculated using the log-rank test. \* *P*\<0.05, \*\**P*\<0.01, and \*\*\**P*\<0.001 were considered significant. Results ======= SNHG6 is differentially expressed in CRC tumor and normal tissues and associated with CRC progression ----------------------------------------------------------------------------------------------------- According to TCGA, SNHG6 is significantly upregulated in colorectal cancer tissues in comparison with the normal counterparts (Fig. [1](#F1){ref-type="fig"}a-c,*P* \< 0.01). Additionally, we used the Kaplan-Meier method analysis (log-rank test) to explore the relationship between SNHG6 expression and patient prognosis from GEO dataset (GSE17538). We found that patients with high levels of SNHG6 had a significantly shorter overall survival than those with low levels of SNHG6 (Fig. [1](#F1){ref-type="fig"}d, *P* = 0.0162). SNHG6 is upregulated in colorectal cancer tissues and cell lines ---------------------------------------------------------------- We used qRT-PCR to observe that SNHG6 was significantly upregulated in CRC tissues based on samples from 77 colorectal cancer patients (Fig. [2](#F2){ref-type="fig"}a, *P* \< 0.001). High levels of SNHG6 was also confirmed in CRC cell lines (Fig. [2](#F2){ref-type="fig"}b). Furthermore, we detected SNHG6 localization because the activities of lncRNAs depended on their subcellular distribution. By analyzing cytoplasmic and nuclear RNA fractions from CRC cells, we found that SNHG6 was localized preferentially in the cytoplasm (Fig. [2](#F2){ref-type="fig"}e-f). SNHG6 promotes CRC cell invasion and migration *in vitro* --------------------------------------------------------- The biological function of SNHG6 in CRC cells was determined by constructing plasmid vectors harboring SNHG6 or an empty vector. SNHG6 was examined in RKO cells with overexpression of SNHG6, and was then transfected with SNHG6-specific shRNAs to knockdown SNHG6 (Fig. [2](#F2){ref-type="fig"}c-d, *P* \< 0.01). According to the knockdown efficiency of SNHG6, we chose shSNHG6\#2 as functional shRNA. Both transwell assays and wound healing assays showed that SNHG6 upregulation significantly promoted the invasion and migration of RKO cells compared with the control, and SNHG6 knockdown also reduced the metastasis ability in RKO cells (Fig. [3](#F3){ref-type="fig"}a-b). Finally, we also found that when SNHG6 knockdown in RKO cells, the levels of MMPs which are directly involved in the invasiveness of cells were downregulated (Fig. [5](#F5){ref-type="fig"}e). SNHG6 promotes CRC cell proliferation *in vitro* and knockdown of SNHG6 represses tumor growth *in vivo* -------------------------------------------------------------------------------------------------------- CCK-8 assays demonstrated that overexpression of SNHG6 resulted in a higher proliferative capacity in RKO cells and HCT116 cells compared with that of parallel stable cell lines containing the empty vector; SNHG6 knockdown significantly decreased RKO cells growth (Fig.[4](#F4){ref-type="fig"}a-c). In order to investigate the roles of SNHG6 in tumorigenesis *in vivo*, RKO cells were stably transfected with SNHG6-shRNA \# 2 and control cells were injected into the left hips of male nude mice. We found that after 25 days, SNHG6-shRNA \# 2 inhibited tumor growth compared to the control group (Fig.[4](#F4){ref-type="fig"}c, *P* \< 0.05). SNHG6 regulates TGF-β/Smad by targeting UPF1 and inducing EMT by ZEB1 --------------------------------------------------------------------- In order to understand the mechanism by which SNHG6 contributed to CRC, we performed bioinformatic analysis using StarBase v2.0 (Table [2](#T2){ref-type="table"}) and found that Up-frameshift Protein 1 (UPF1) may be a target gene of SNHG6; a function which has been demonstrated in HCC. [@B11] Then we used qRT-PCR and western blot to determine SNHG6 and UPF1 expression. We found that UPF1 was upregulated in RKO cells when SNHG6 was knocked down, and it has an inverse correlation with SNHG6 in CRC tissues (Fig.[5](#F5){ref-type="fig"}a-b). UPF1 was already reported as a tumor suppressor gene for HCC by targeting Smad7 and affecting the TGF-β pathway.[@B16] Thus, we hypothesized that SNHG6 promoted CRC cells tumorigenesis by regulating the ability of UPF1 to mediate the TGF-β/Smad pathway. We further used western blot to investigate the relationship between SNHG6 and UPF1. Our results demonstrated that the expression of UPF1 protein, and the Smad7 downstream TGF-β pathway proteins, such as p-Smad2 and p-Smad3, were decreased with SNHG6 knockdown whereas total Smad2 and Smad3 expression level was not significantly altered (Fig. [5](#F5){ref-type="fig"}c). We were able to conclude that SNHG6 regulated the expression of UPF1 and affected the TGF-β pathway. Furthermore, a previous study demonstrated that SNHG6 could affect ZEB1 through sponging miR-101-3p.[@B11] As previously reported, ZEB1 is a crucial transcription factor of EMT which can regulate the expression of many EMT markers.[@B17] We speculated that SNHG6 could also induce EMT by upregulating ZEB1 expression in CRC via miR-101-3p. We searched TargetScan database finding that miR-101-3p has two predicted binding sites with ZEB1 (Fig.[5](#F5){ref-type="fig"}f). We detected miR-101-3p and ZEB1 expression by qRT-PCR, finding that miR-101-3p was upregulated and ZEB1 was downregulated while SNHG6 knockdown in RKO cells (Fig.[5](#F5){ref-type="fig"}g-h). To further explore the effect of targeting SNHG6 on EMT, we found that knockdown of SNHG6 in RKO cells resulted in increased expression of E-cadherin but decreased expression of ZEB1, N-cadherin, Vimentin, Slug and Snail compared to control cells by western blot(Fig.[5](#F5){ref-type="fig"}i). The above data indicated that SNHG6 may induce EMT by regulating ZEB1 via sponging miR-101-3p. Discussion ========== LncRNAs are involved in numerous biological and cellular pathways by interacting with various macromolecules such as DNA, chromatin, proteins, and various RNA species; including mRNAs, microRNAs, and other lncRNAs.[@B18] Recent studies have implied that lncRNAs are widely involved in proliferation, invasion, and metastasis and thus represent potential prognostic biomarkers in colorectal cancer, such as CCAT,[@B19] DANCR,[@B20] CRNDE.[@B21] Small nucleolar RNAs (snoRNAs) are another class of small non-coding RNA molecules, which are concentrated in the nucleoli and have a stable metabolism.[@B22] Their main function is to participate in the post-transcriptional modification of rRNA and other RNAs in the cytoplasm.[@B23] Most snoRNAs are encoded by host genes and are processed from the introns of pre-mRNAs. However, recent studies have also indicated that snoRNA host genes could affect cell proliferation, transformation and tumorigenesis in a variety of human cancers, such as SNGH1 in HCC[@B24], [@B25] and CRC,[@B26], [@B27] SNGH5 in GC[@B28], [@B29] and CRC.[@B30]. SNHG6 has been reported to have an oncogenic role in tumors such as HCC,[@B11], [@B12], [@B31] glioma,[@B32], esophageal squamous cell carcinoma[@B33] and osteosarcoma.[@B14] The current study showed that SNHG6 was significantly overexpressed in human CRC tissues as well, with poor prognosis. Moreover, we applied *in vitro* and *in vivo* methods to reveal the involvement of SNHG6 in CRC tumorigenesis, such as CRC cellular growth and metastasis. The subcellular localization of lncRNAs is also a critical factor to determine their functions by providing them different opportunities to interact with different molecules.[@B34]. For instance, lncRNAs localized in nucleus tend to be involved in transcriptional and epigenetic regulations by interacting with genomic DNA, chromatin, transcription factors, chromatin regulators, spliceosomes and other nuclear proteins.[@B18] Meanwhile, cytosolic lncRNAs are frequently implicated in post-transcriptional, translational, and posttranslational regulatory processes through interactions with various key factors in epigenetic and signaling pathways.[@B35] Based on the findings of previous study suggested that SNHG6 was mostly located in cytoplasm, where it could bind to proteins and microRNAs. UPF1, a part of the human nonsense-mediated mRNA decay (NMD) substrate, could mediate RNA decay processes and destabilize the encoding TGF-β inhibitor, Smad7, stimulating TGF-β signaling.[@B36] This gene was reported to be a tumor suppressor gene regulating cell proliferation and differentiation in HCC.[@B16] Moreover, UPF1 gene was found to be commonly mutated in pancreatic adenosquamous carcinoma (ASC), which represents the first known example of genetic alterations in a NMD gene in human tumors.[@B37] In this study, we used StarBase v2.0 finding that UPF1 may be a target gene of SNGH6. Our results confirmed that knockdown SNHG6 by shRNA increased UPF1 and overexpression of SNHG6 decreasing UPF1, while regulating TGF-β/Smad signaling pathway in CRC cells. Meanwhile, SNHG6 has been reported to regulate ZEB1 by sponging miR-101-3p in gastric cancer.[@B13] We used qRT-PCR and western blot confirmed that SNHG6 could regulate ZEB1 and induce EMT in CRC cells. Conclusion ========== In summary, our study revealed that SNHG6 could play an oncogenic role in CRC. SNHG6 promoted tumor cell proliferation and metastasis by activating the TGF-β/Smad pathway via binding UPF1. Meanwhile SNHG6 could regulate ZEB1 by inducing EMT via miR-101-3p (Fig.[6](#F6){ref-type="fig"}). These findings suggest that SNHG6 is an important prognostic factor and therapeutic target for CRC. This study was supported by Guangzhou Pilot Project of Clinical and Translational Research Center (early gastrointestinal cancer, No. 7415696196402), Guangdong gastrointestinal disease research center (No.2017B02029003). lncRNA : long noncoding RNA SNHG6 : small nucleolar RNA host gene 6 CRC : colorectal cancer TCGA : The Cancer Genome Atlas qRT-PCR : quantitative real-time PCR UPF1 : Up-frameshift Protein 1 HCC : Hepatocellular carcinoma EMT : epithelial-mesenchymal transition ZEB1 : zinc finger E-box binding homeobox 1 CCK-8 : Cell Counting Kit-8. {#F1} {#F2} {#F3} {#F4} {#F5} {#F6} ###### List of qRT-PCR primers Gene Forward primer (5\'-3\') Reverse primer(5\'-3\') ------------ ------------------------------ ------------------------- SNHG6 TTGAGGTGAAGGTGTATG GGTAACGAAGCAGAAGTA UPF1 TCACGGCACAGCAGATCAACAAG CGGCTTCTCCAGGTCCTCCAG miR-101-3p CGCGCGTACAGTACTGTGATAA-CTGAA ZEB1 ACTGTTTGTAGCGACTGGATT TAAAGTGGCGGTAGATGGTA GAPDH CGAGCCACATCGCTCAGACA GTGGTGAAGACGCCAGTGGA U6 CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT ###### Partial Human RBP-LncRNA interactions of SNHG6 from StarBase v2.0 (target sites ≥ 5) Name LncRNA Name Target Sites Clip-seq Read Number ---------- ------------- -------------- ---------------------- UPF1 SNHG6 48 218 U2AF65 SNHG6 20 1128 eIF4AIII SNHG6 11 413 IGF2BP1 SNHG6 5 41 FMRP SNHG6 5 5000 LIN28 SNHG6 5 148 [^1]: \*These two authors contributed equally to this work. [^2]: Competing Interests: The authors have declared that no competing interest exists.

Introduction {#s1} ============ Fruit ripening involves the well-orchestrated coordination of several regulatory steps in both climacteric and non-climacteric fruit, together with strong metabolic and physiological changes ([@CIT0025]). Fig fruit (*Ficus carica*) are categorized as climacteric; they show a rise in respiration rate and ethylene production at the onset of their ripening phase ([@CIT0033]; [@CIT0005]), similar to that of the climacteric tomato, apple, and mango fruit ([@CIT0012]; [@CIT0001]; [@CIT0060]). However, unlike climacteric fruit, figs harvested before they are fully ripe never complete their ripening process to reach commercially desirable parameters of size, color, flavor, and texture ([@CIT0008]). This challenges the idea that fig fruit are climacteric. In addition,1-methylcyclopropene (1-MCP), which blocks the effects of ethylene during ripening, increases ripening-related ethylene production in fig fruit following pre- or post-harvest application in an unexpected auto-inhibitory manner ([@CIT0049]; [@CIT0038]; [@CIT0010]). A molecular study of ethylene-related genes in figs ([@CIT0011]) showed that *FcERF12185*, an ethylene signal-transduction gene, might be responsible for the non-climacteric auto-inhibition of ethylene production in the fruit. Unlike most fig ethylene-response factor (ERF) genes, *FcERF12185* expression does not increase during ripening, and is induced upon 1-MCP treatment ([@CIT0011]). It has been suggested that the plant hormone abscisic acid (ABA) regulates fruit ripening and senescence in both climacteric and non-climacteric fruit ([@CIT0061], [@CIT0062]; [@CIT0052]; [@CIT0027]). In climacteric fruit, endogenous ABA levels increase before the onset of ripening and subsequently decrease until the fruit is fully ripe. However, in non-climacteric fruit, ABA levels increase from maturation to harvest ([@CIT0044]; [@CIT0027]). The fig fruit bears a unique closed inflorescence structure, the syconium. This closed inflorescence produces an aggregate fruit, which is composed of small individual drupelets that develop from the ovaries enclosed in the receptacle ([@CIT0051]). Development of female fruit in the common fig consists of three phases: phase I is characterized by rapid growth in fruit size; in phase II, the fruit remains nearly the same size, color, and firmness; in phase III, ripening occurs, with fruit growth, color change, softening, and alteration of the pulp texture to an edible state ([@CIT0008]). Endogenous ABA is reported to increase as the ripening phase progresses ([@CIT0040]). The endogenous ABA content in the fruit is determined by the dynamic balance between its biosynthesis and catabolism. Zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), short-chain alcohol dehydrogenase (ABA2), and abscisic aldehyde oxidase participate in ABA biosynthesis, while ABA-8'-hydroxylase (ABA8OX) and ABA-glucosyltransferase participate in its catabolism ([@CIT0042]; [@CIT0045]; [@CIT0055]; [@CIT0019]; [@CIT0043]; [@CIT0027]; [@CIT0040]). The effect of ABA on ethylene biosynthesis, fruit ripening, and senescence has been extensively studied in the model plant tomato. Exogenous ABA treatment increases ABA content in tomato fruit, and induces the expression of ethylene-biosynthesis genes, namely 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (*ACS*) and ACC oxidase (*ACO*). On the other hand, treatment of tomato fruit with the ABA inhibitors fluridone or nordihydroguaiaretic acid (NDGA) inhibits *ACS* and *ACO* and delays ripening and softening ([@CIT0062]; [@CIT0036]). In particular, a significant reduction in the activity of *SlNCED1*, a key gene in tomato ABA biosynthesis, by RNAi, reduces the expression of genes encoding major cell wall-catabolic enzymes, and increases the accumulation of pectin during ripening, which leads to a significant extension of the shelf-life of climacteric tomato fruit ([@CIT0052]). Similarly, exogenous ABA application significantly promotes the ripening process of the non-climacteric strawberry fruit, whereas fluridone inhibits it. Down-regulation of the ABA-biosynthesis gene *FaNCED1* in strawberry significantly decreases ABA levels and produces non-colored/unripe fruit ([@CIT0018]). Moreover, studies in apple, banana, grape, and sweet cherry have shown that exogenous application of ABA enhances fruit ripening by up-regulating ethylene production, as well as anthocyanin and sugar accumulation ([@CIT0021]; [@CIT0026]; [@CIT0002]; [@CIT0020]; [@CIT0013]; [@CIT0031]). In fig fruit, genes of the ABA biosynthesis and catabolism pathways have been isolated and their expression characterized during ripening ([@CIT0040]).In addition, [@CIT0011] performed a comprehensive study focusing on ethylene-biosynthesis, MADS-box, and ethylene signal-transduction genes during natural ripening and their interactions with 1-MCP. However, the relationship between ABA and ethylene during the onset of fruit ripening in fig remains to be fully elucidated. The plant-specific NAC family of proteins containing the NAC domain is one of the largest transcription factor families in plants. An increasing number of NAC genes are being identified and studied in both monocotyledonous and dicotyledonous plants . In tomato, 74 NAC or NAC-like genes belonging to 12 subfamilies have been identified, with high expression of *SlNAC4--9* being found during fruit development and ripening ([@CIT0024]). *SlNAC1* has a broad influence on tomato fruit ripening, and its over-expression during ripening has been found to be regulated through both ethylene-dependent and ABA-dependent pathways ([@CIT0032]). Reduced expression of *SlNAC4* by RNAi in tomato results in delayed fruit ripening, with suppressed chlorophyll breakdown and decreased ethylene synthesis being mediated mainly through reduced expression of system-2 ethylene-biosynthesis genes, and with carotenoids being reduced via alteration of the flux of the carotenoid pathway ([@CIT0063]). In addition, *SlNAC4* is down-regulated by ABA treatment while *SlNAC5*, *6*, *7*, and *9* are up-regulated ([@CIT0024]). Involvement of NAC genes in banana fruit ripening was found via interactions with ethylene-signaling components ([@CIT0047]). In figs, 27 NAC genes have been identified during fruit ripening ([@CIT0009]); however, their roles during this process are still unknown. In the present work, we used exogenous ABA, ethephon, and the ABA inhibitors fluridone and NDGA to characterize the effects of ABA on fig fruit ripening. The expression levels of ABA- and ethylene-biosynthesis genes were determined, together with those of ripening-associated transcription factors and signal-transduction genes. The levels of endogenous ABA and ethylene were also quantified to determine how ABA triggers ethylene production to start the ripening process. This study also examined the genes that likely to be induced during on-tree fruit ripening. The non-climacteric ripening behavior of fig fruit associated with ABA is also discussed. Materials and methods {#s2} ===================== Plant material and sample preparation {#s3} ------------------------------------- Fruit of common fig (*Ficus carica* L.) cv. Brown Turkey, 37 mm in diameter with a greenish-yellow ostiole color (just before the rapid increase in endogenous ABA production and the start of ethylene production), were selected for on-tree treatment with ABA, ethephon, fluridone, and NDGA in a fig orchard located at the Agricultural Research Organization -- Volcani Center, Israel. Treatments were applied by injecting 1 ml of the following into the fruit through the ostiole with a plastic syringe: 1.89 mM ABA (Valent Bioscience Corporation, USA), 0.7 mM ethephon (Ishihara Sangyo Kaisha, Ltd., Japan), 0.15 mM NDGA, or 0.2 mM fluridone (both Sigma-Aldrich). ABA and ethephon were injected once at time 0, whilst NDGA and fluridone were injected three times at 12-h intervals starting at time 0. The size and ostiole color of the fruit used in the experiments and the treatment concentrations were selected on the basis of several preliminary trials (data not shown). Fruit treated with ddH~2~O served as the control group and ethephon-treated fruit served as a positive control. Three separate experiments were performed: (i) ABA and ethephon treatment; (ii) fluridone treatment; and (iii) NDGA treatment. These trials were conducted during the summers of 2015 and 2016 (July--August), with average day and night temperatures of 32 °C and 24 °C, respectively. For fruit treated with ABA and ethephon, samples were collected at 0, 12, 24, 48, 72, and 96 h after treatment (HAT). Samples for the NDGA and fluridone treatments were collected at 0, 24, 32, 48, 72, and 96 HAT. In total, 600 fruit were treated in each experiment and physiological changes were examined. Nine fruit were harvested at each time interval for molecular characterization, divided into three biological replicates consisting of three fruit each. Fruit receptacle and inflorescence tissues were collected separately and stored at --80ºC until further analysis. Fruit diameter and firmness {#s4} --------------------------- Fruit fresh weight was determined immediately on sampling. Width was measured using a standard caliber, and was recorded as the maximal horizontal diameter. Fruit firmness was measured using an Inspekt Table Blue universal testing machine with 5 kN capacity (Hegewald & Peschke MTP, Nossen, Germany), and expressed in Newtons as F(N), the energy used to deform the fruit to 5% of its diameter, as in [@CIT0041]). Measurement of ethylene production {#s5} ---------------------------------- Ethylene production was determined according to [@CIT0010] in a total of seven fruit at each sampling time. Individual fruit were enclosed in a 0.75-l airtight glass jar for 2 h at room temperature (20 °C)and ethylene was quantified using a Varian 3300 GC instrument with a flame-ionization detector (FID) (Varian Inc., CA, USA), a stainless-steel column (length 1.5 m, outside diameter 3.17 mm, internal diameter 2.16 mm) packed with HayeSep T, particle size 0.125--0.149 mm (Alltech Associates Inc., IL, USA), and helium as the carrier gas (5 ml min^--1^). ABA extraction and quantification {#s6} --------------------------------- Frozen receptacles or inflorescences were ground into powder and 0.5-g samples were dissolved in 3 ml of 80% acidified methanol and 1% acetic acid in 15-ml tubes. d4-ABA (7 µl of a 10× dilution) was added as an internal standard and, after vortexing, the mixture was stored at 4 °C for 2 h. Samples were centrifuged at 1510 *g* for 30 min at 4 °C and the upper phase was transferred to a new tube. The pellet was extracted again by adding 3 ml of 80% acidified methanol and storing overnight at 4 °C, before further centrifuging. The separated upper phases were combined and then evaporated at 40 °C down to 200 µl and stored at --20 °C for further analysis. LC--MS analyses were conducted using a UPLC-Triple Quadrupole-MS (Waters Xevo TQ, MS, USA). Samples were centrifuged for 15 min at 13 362 *g* and 1 µl of supernatant was injected into the LC--MS instrument. Separation was performed on a 2.1 × 100 mm^2^, 1.7-µm ACQUITY UPLC BEH C18 column with a VanGuard pre-column (BEH C18 1.7 µm, 2.1 × 5 mm^2^). The chromatographic and MS parameters were as follows: the mobile phase consisted of water (phase A) and acetonitrile (phase B), both containing 0.1% formic acid, in gradient-elution mode. The solvent gradient program was as follows: 5% to 95% A over 0.1 min, 25% to 75% A over 2 min, 35% to 65% A over 2.5 min, 40% to 60% A over 3 min, held at 95% to 5% A for 4 min. At the end of the gradient, the column was washed with 95% B (3 min) and re-equilibrated to initial conditions for 3 min. The flow rate was 0.3 ml min^--1^, and the column temperature was kept at 35 °C. All of the analyses were performed using the ESI source in positive ion mode with the following settings: capillary voltage 3.5 kV, cone voltage 26 V, desolvation temperature 350 °C, desolvation gas flow 650 l h^--1^, source temperature 150 °C. Quantitation was performed using multiple reaction monitoring (MRM) acquisition by monitoring 247/187, 247/173 (RT=3.93, dwell time of 78 ms for each transition) for ABA and 251/191, 251/177 (RT=3.93, dwell time of 78 ms) for d4-ABA (used as an internal standard). The LC--MS data were acquired using the MassLynx V4.1 software (Waters). RNA extraction and cDNA synthesis {#s7} --------------------------------- Total RNA was extracted according to [@CIT0016]. The RNA concentration was determined in a Nanodrop ND-1000 spectrophotometer, and its integrity was checked by running 1 μl in a 1% (w/v) agarose gel stained with Bromophenol Blue. Total RNA was digested with RQ-DNase (Promega). Complementary DNA was synthesized, using Oligo-dT primers, using a VERSO cDNA kit (Thermo Scientific). High-throughput real-time quantitative PCR {#s8} ------------------------------------------ High-throughput real-time qPCR was performed on a BioMark 96.96 Dynamic Array with TaqMan Gene Expression Assays (Applied Biosystems) at the Weizmann Institute of Science (Israel). Three biological replicates were used for each treatment. Primers were designed with the Primer3 software, and synthesized by Metabion (Germany) and Hylabs (Israel) (see [Supplementary Table S1](#sup1){ref-type="supplementary-material"} at *JXB* online). The expression levels of the target genes were normalized to the control gene *actin*, as in [@CIT0011], and analysed using the Δ*C*~t~ method ([@CIT0030]). Results {#s9} ======= Effects of exogenous ABA application on physiological and metabolic changes during fig fruit ripening {#s10} ----------------------------------------------------------------------------------------------------- Fig fruit of 37 mm diameter with a greenish-yellow ostiole color were identified as being just before the rapid increase in endogenous ABA and initiation of ethylene production ([Fig. 1A](#F1){ref-type="fig"}). Application of exogenous ABA or ethephon resulted in fruit with significantly larger diameters at 12, 24, 48, and 72 HAT compared to controls ([Fig. 1B](#F1){ref-type="fig"}). ABA- and ethephon-treated fruit were significantly softer at 24 and 48 HAT compared to controls ([Fig. 1C](#F1){ref-type="fig"}), and the change in fruit color associated with ripening started earlier than in controls ([Fig. 1D](#F1){ref-type="fig"}). {#F1} Effects of exogenous ABA application on expression levels of genes of the ABA metabolism and ethylene biosynthesis pathways {#s11} --------------------------------------------------------------------------------------------------------------------------- To understand the role of exogenous ABA in fig fruit ripening, the expression levels of six previously characterized ([@CIT0040]) ABA-biosynthesis and catabolism genes were determined ([Fig. 2](#F2){ref-type="fig"}, [Supplementary Fig. S1](#sup1){ref-type="supplementary-material"}). Exogenous ABA application during the onset of ripening increased the expression levels of these genes. In particular, expression of *FcNCED2*, the key ABA-biosynthesis gene in fig fruit, was increased by ABA and ethephon treatments in both the inflorescences (4.2- and 7.9-fold change, respectively) and the receptacles (1.8- and 2.1-fold change, respectively) at 12 HAT, as compared to untreated controls ([Fig. 2](#F2){ref-type="fig"}). *FcNCED2* continued to be expressed at moderately high levels in ABA- and ethephon-treated fruit in the inflorescences (2.4- and 1.9-fold change, respectively) and the receptacles (2.2- and 1.9-fold change, respectively) at 24 HAT. In addition, moderately high expression of *FcNCED2* was maintained at 72 HAT in inflorescences, but not in receptacles. ABA treatment also increased the expression of the ABA-biosynthesis gene *FcABA2*, to 5.6-fold that of controls in the inflorescence at 12 HAT ([Fig. 2](#F2){ref-type="fig"}). Unexpectedly, ethephon down-regulated *FcABA2* expression at 24 HAT in the inflorescence ([Fig. 2](#F2){ref-type="fig"}). {#F2} We then determined the effect of exogenous ABA application on ethylene-biosynthesis genes in the fruit ([Fig. 2](#F2){ref-type="fig"}, [Supplementary Fig. S1](#sup1){ref-type="supplementary-material"}). *FcACS4* was highly up-regulated by ABA and ethephon in the inflorescences (85.4- and 55.3-fold change, respectively) and in the receptacles (22.2- and 26.8-fold change, respectively) at 12 HAT, as compared to untreated controls. In addition, it was moderately up-regulated by ABA treatment in the inflorescences at 24, 48, and 72 HAT (4.5-, 3.4-, and 4.1-fold change, respectively). In ethephon-treated inflorescences, *FcACS4* was moderately up-regulated at 24 HAT (3.1-fold change), whilst in the receptacles it was moderately up-regulated by ABA treatment at 24 and 48 HAT (3.7- and 4.4-fold change, respectively). Similarly, *FcACS4* was moderately up-regulated at 48 HAT (4.5-fold change) in ethephon-treated receptacles ([Fig. 2](#F2){ref-type="fig"}). The ethylene-biosynthesis gene *FcACOL* was highly up-regulated at 12 HAT by ABA and ethephon application in both the inflorescences (10.8- and 11.5-fold change, respectively) and the receptacles (6.6- and 6.5-fold change, respectively) compared to controls. At 24 HAT, it was moderately up-regulated by ABA in the inflorescences (2.8-fold change) and the receptacles (2.8-fold change). Similarly, compared to controls, *FcACO2* was up-regulated by ABA at 12 and 24 HAT in both the inflorescences (5.3- and4.5-fold change, respectively) and the receptacles (2.8- and5.2-fold change, respectively), and it was also up-regulated by ethephon at 12 and 24 HAT in the inflorescences (13.9- and 2.2-fold change, respectively) and the receptacles (3.5- and 2.4-fold change, respectively) ([Fig. 2](#F2){ref-type="fig"}). Interestingly, in the ABA-treated receptacles, *FcSAM2* and *FcSAM3* were moderately up-regulated (2.4- and 2.9-fold change, respectively) at 12 HAT ([Supplementary Fig. S1](#sup1){ref-type="supplementary-material"}). Effects of fluridone and NDGA application on physiological and metabolic alterations during fig fruit ripening {#s12} -------------------------------------------------------------------------------------------------------------- Fluridone treatment delayed the ripening of treated fig fruit; the fruit diameter was significantly smaller at 48 HAT and fruit remained significantly firmer at 32, 48, and 72 HAT compared to controls ([Fig. 3A](#F3){ref-type="fig"}). The effect of fluridone on fruit firmness appeared to be transient as control and treated fruit showed no significant difference at 96 HAT. In addition, by 96 HAT ABA levels had increased 6- and 4-fold in the inflorescences and receptacles, respectively, of untreated fruit. Notably, we found that fluridone prevented ABA accumulation in both the inflorescences and receptacles, and delayed ethylene production. ###### Effects of (A) fluridone and (B) NDGA on diameter, firmness, ABA production in inflorescences and receptacles, and changes in color of fig fruit. Fluridone and NDGA were applied before the onset of ripening. Data are means (±SE) of 9 fruit. Significant differences compared with the control were determined using Student's *t*-test: \**P*\<0.05.   Similarly, NDGA also delayed ripening, with treated fruit having significantly smaller diameters and remaining firmer at 32, 48, and 72 HAT compared to controls ([Fig. 3B](#F3){ref-type="fig"}). NDGA significantly delayed ABA accumulation as well as ethylene production in the inflorescences and receptacles. Effects of fluridone and NDGA on expression levels of ABA metabolic pathway and ethylene-biosynthesis genes {#s13} ----------------------------------------------------------------------------------------------------------- To investigate the changes in expression patterns of ripening-related genes caused by fluridone and NDGA, the expression of genes of the ABA biosynthetic and catabolic pathways and ethylene biosynthesis were examined ([Fig. 4](#F4){ref-type="fig"}, [Supplementary Fig. S2](#sup1){ref-type="supplementary-material"}). Fluridone did not significantly down-regulate *FcNCED2* ([Fig. 4A](#F4){ref-type="fig"}); however, compared to controls, it inhibited the expression of *FcABA2* in inflorescences at 32, 48, and 72 HAT (2.5-, 8.4-, and 22.6-fold change, respectively) and in receptacles at 32 and 72 HAT (3.4- and 4.7-fold change, respectively). The ethylene-biosynthesis gene *FcACS2* was down-regulated at 32, 48, and 72 HAT in both inflorescences (4.6-, 4-, and 4.3-fold change, respectively) and receptacles (5.1-, 2.7-, and 4.8-fold change, respectively) compared to controls. In addition, *FcACS4* was down-regulated at 48 and 72 HAT in inflorescences (3.2- and 2.7-fold change, respectively) and at 32 and 72 HAT in receptacles (3.3- and 3.7-fold change, respectively). Similarly, expression levels of *FcACOL* and *FcACO2* were slightly reduced at 48 HAT in inflorescences as compared to controls. ###### Expression patterns of ABA-biosynthesis genes (*FcNCED2* and *FcAB*A*2*) and ethylene-biosynthesis genes (*FcACS2*, *FcACS4*, *FcACOL*, and *FcACO2*) in (A) fluridone-treated and (B) NDGA-treated fig fruit. I, inflorescence; R, receptacle. Expression is relative to the *actin* gene. Treatment was applied before the onset of ripening. Data are means (±SE) of three biological replicates.   Interestingly, NDGA treatment caused down-regulation of *FcNCED1* at 32, 48, and 72 HAT in the receptacles (3.6-, 7.1-, and 9.8-fold change, respectively; [Supplementary Fig. S2B](#sup1){ref-type="supplementary-material"}). In addition, *FcNCED2* was significantly inhibited at 24, 32, and 48 HAT in inflorescences (18.4-, 3.8-, and 20-fold change, respectively) and in receptacles at 24 and 48 HAT (4.3- and 2.9-fold change, respectively; [Fig. 4B](#F4){ref-type="fig"}). At 32 and 72 HAT, *FcABA2* was inhibited in both inflorescences and receptacles. Expression of the ABA-catabolism gene *FcABA8OX* was elevated at 48, 72, and 96 HAT in both NDGA-treated inflorescences (13-, 5-, and 2.4-fold change, respectively) and receptacles (4.3-, 3.5-, and 3.2-fold change, respectively) compared to controls ([Supplementary Fig. S2B](#sup1){ref-type="supplementary-material"}). The genes responsible for the first steps of ethylene biosynthesis, *FcSAM2* and *FcSAM3*, were moderately down-regulated in inflorescences (2.8- and 2.4-fold change, respectively) and receptacles (2.9- and 2.2-fold change, respectively) at 12 HAT compared to controls ([Supplementary Fig. S2B](#sup1){ref-type="supplementary-material"}). In addition, *FcACS2* and *FcACS4* were down-regulated in inflorescences and receptacles of NDGA-treated fruit up to 72 HAT. In particular, *FcACS2* was strongly down-regulated at 32 HAT (47.2-fold change) in inflorescences ([Fig. 4B](#F4){ref-type="fig"}). Similarly, *FcACS4* was strongly down-regulated by NDGA treatment at 24 and 32 HAT (24.7- and 16.5-fold change, respectively) in inflorescences. NDGA-treated fruit also showed strong down-regulation of *FcACOL* at 48 HAT (9.9-fold change) and *FcACO2* at 32 HAT (8.6-fold change) in the inflorescences. Effects of exogenous ABA application on expression levels of MADS-box, NAC, and ERF genes {#s14} ----------------------------------------------------------------------------------------- To investigate the effects of ABA application on expression of potential ripening-regulator and ethylene signal-transduction genes, 15 MADS-box, 10 NAC, and 12 ERF genes were analysed ([Fig. 5](#F5){ref-type="fig"}, [Supplementary Figs. S3, S5, S7](#sup1){ref-type="supplementary-material"}). *FcMADS8*, *FcMADS14*, and *FcMADS15* were moderately up-regulated relative to controls by treatment with ABA and ethephon in inflorescences at 24 HAT ([Fig. 5](#F5){ref-type="fig"}). Similarly, *FcMADS8* and *FcMADS14* were up-regulated in receptacles at 24 and 48 HAT, while *FcMADS15* was up-regulated at 48 and 72 HAT. *FcNAC1*, *FcNAC2*, and *FcNAC5* were up-regulated by ABA and ethephon treatment at 12 and 24 HAT in both inflorescences and receptacles, and *FcERF9006* was up-regulated at 12, 24, and 48 HAT in both inflorescences and receptacles. {#F5} Effects of fluridone and NDGA on expression levels of MADS-box, NAC, and ERF genes {#s15} ---------------------------------------------------------------------------------- To determine the effects of fluridone and NDGA on the expression of potential ripening-regulator and ethylene signal-transduction genes, MADS-box, NAC and ERF genes were analysed ([Fig. 6](#F6){ref-type="fig"}, [Supplementary Figs. S4, S6, S8](#sup1){ref-type="supplementary-material"}). Compared to controls, *FcMADS8*, *FcMADS14*, and *FcMADS15* were moderately down-regulated by fluridone and NDGA treatments at 32 HAT in inflorescences and receptacles ([Fig. 6](#F6){ref-type="fig"}). Similarly, *FcNAC1* was slightly down-regulated by NDGA at 72 HAT in inflorescences, and also at 48 and 72 HAT in receptacles ([Fig. 6B](#F6){ref-type="fig"}). There was no effect of fluridone on *FcNAC1* in inflorescences, although this gene was down-regulated in receptacles at 72 HAT ([Fig. 6A](#F6){ref-type="fig"}). Interestingly, *FcNAC2* and *FcNAC5* were down-regulated by NDGA at 32 HAT in both inflorescences and receptacles ([Fig. 6B](#F6){ref-type="fig"}). In addition, *FcNAC5* was down-regulated by fluridone at 32 HAT in inflorescences and receptacles ([Fig. 6A](#F6){ref-type="fig"}). *FcERF9006* was down-regulated by both fluridone and NDGA up to 72 HAT in both inflorescences and receptacles ([Fig. 6](#F6){ref-type="fig"}). {#F6} Correlation between ripening-related genes and ABA in fluridone- and NDGA-treated fruit {#s16} --------------------------------------------------------------------------------------- Potential coordination between expression of ripening-related genes and ABA during fig fruit ripening was determined by Pearson's correlation analysis. The analysis reflected the degree of coordination between gene expression and hormonal changes in the reproductive (inflorescence) and non-reproductive (receptacle) tissues of treated and non-treated fruit ([Fig. 7](#F7){ref-type="fig"}). Fluridone treatment strongly altered the correlation between ABA and ripening-related genes in both tissues. In particular, *FcSAM2* and *FcSAM3* were negatively correlated with ABA in untreated fruit, whereas they were positively correlated in inflorescences and receptacles of fluridone-treated fruit ([Fig. 7A](#F7){ref-type="fig"}). Fluridone negatively altered the correlation of *FcABA2* with ABA in the inflorescence but not in the receptacle. In addition, *FcZEP*, *FcMADS14*, and *FcMADS15* were negatively correlated with ABA in the inflorescence, whereas there was no correlation in the receptacle ([Fig. 7A](#F7){ref-type="fig"}). {#F7} Interestingly, NDGA treatment resulted in a positive correlation of *FcACS2* and *FcNCED1* with ABA in the inflorescence; however, *FcABA2* was negatively correlated with ABA in the NDGA-treated inflorescence ([Fig. 7B](#F7){ref-type="fig"}). *FcNCED3* was negatively correlated with ABA in inflorescences but positively correlated with ABA in the receptacles of all treated and untreated fruit. Furthermore, highly positive correlations of the key ABA-biosynthesis gene *FcNCED2* and of the ethylene-biosynthesis genes *FcACS4*, *FcACOL*, and *FcACO2* with ABA were detected in the inflorescences of treated and untreated fruit ([Fig. 7](#F7){ref-type="fig"}). Discussion {#s17} ========== Fig fruit has traditionally been perceived as climacteric because its ripening process follows an increase in respiration rate and ethylene production ([@CIT0005]). However, ripening-related ethylene production has been found to increase in an unexpected auto-inhibitory manner after application of 1-MCP ([@CIT0049]; [@CIT0038]; [@CIT0010]). This paradox was explained by [@CIT0011] in a molecular study of potential ripening-regulatory and ethylene-related genes in the receptacles (vegetative tissue) and inflorescences (reproductive tissue). More recently, endogenous ABA production has been observed to increase as the result of an increase in expression levels of ABA-biosynthesis genes at the onset of fruit ripening ([@CIT0040]). These data suggest that both ABA and ethylene are crucial players in the ripening process of figs, and we examined their potential interplay in the current study. Exogenous ABA enhances fruit ripening by inducing the expression of genes in the ABA metabolism and ethylene biosynthesis pathways {#s18} ---------------------------------------------------------------------------------------------------------------------------------- Exogenous ABA treatment induced earlier onset of ripening, followed by a rapid increase in fruit diameter, softening, and purple coloration of the outer peel. Ethephon had the same effect and was used as a positive control ([Fig. 1](#F1){ref-type="fig"}). Concomitant with the early onset of ripening, both ABA-biosynthesis genes (particularly *FcNCED2*) and ethylene-biosynthesis genes (particularly *FcACS4*, *FcACOL*, and *FcACO2*) were up-regulated as a result of exogenous ABA treatment in inflorescences and receptacles at 12 and 24 HAT ([Fig. 2](#F2){ref-type="fig"}). These results are in general agreement with previous work on tomato where exogenous ABA was found to promote ripening by enhancing ethylene biosynthesis ([@CIT0062]; [@CIT0036]). An increase in endogenous autocatalytic ethylene production as a result of application of exogenous ABA is well known in several other fruits such as banana ([@CIT0021]), melon ([@CIT0053]), peach, and grapes ([@CIT0061]). Exogenous ABA also enhances the ripening process of non-climacteric strawberry fruit ([@CIT0018]). In citrus, exogenous ABA accelerates ripening and induces expression of the ethylene-biosynthesis gene *CsACO1*. It also enhances fruit color, significantly decreases organic acid content, and increases sugar accumulation ([@CIT0058]). The key ABA-biosynthesis gene *NCED* has been cloned and characterized from various climacteric fruit species, such as apple ([@CIT0026]), peach ([@CIT0048]), tomato ([@CIT0003]), and melon ([@CIT0053]), as well as from non-climacteric fruit such as orange ([@CIT0039]), grape ([@CIT0059]), and strawberry ([@CIT0018]). In particular, in climacteric tomato fruit, *LeNCED1* initiates ABA biosynthesis at the onset of fruit ripening, and may act as a ripening inducer ([@CIT0062]). Similarly, *FaNCED1* and *FaNCED2* have been reported to regulate ABA biosynthesis during the onset of ripening in non-climacteric strawberry fruit ([@CIT0018]; [@CIT0017]). To date, three genes encoding the NCED enzyme have been isolated from the inflorescence and receptacle tissues of both pollinated and parthenocarpic fig fruit during ripening ([@CIT0040]). Our current study showed that expression of *FcNCED2* was up-regulated as early as 12 HAT, while expression of the ethylene-biosynthesis gene *FcACS4* was only up-regulated at 24 HAT in control fruit. In addition, another ABA-biosynthesis gene, *FcABA2*, showed a similar temporal pattern of up-regulation, but only in control fruit inflorescences ([Fig. 2](#F2){ref-type="fig"}). The simultaneous up-regulation of key ABA- and ethylene-biosynthesis genes, especially in the inflorescence, suggested that there was major up-regulation of ethylene-biosynthesis genes after the up-regulation of ABA-biosynthesis genes, as seen in tomato ([@CIT0062]; [@CIT0036]), and confirmed that the ethylene-dependent ripening process in fig is coordinated by the reproductive part of the fruit---the inflorescence---as stated by [@CIT0011] and [@CIT0040]). Fluridone and NDGA reduce expression of ABA-biosynthesis genes, thereby altering fruit ripening {#s19} ----------------------------------------------------------------------------------------------- Fruit treated with fluridone showed delayed onset of ripening, followed by a slower increase in diameter and a slower decrease in firmness of the fruit compared with controls ([Fig. 3A](#F3){ref-type="fig"}). Endogenous ABA accumulation and ethylene production were strongly suppressed as a result of the down-regulation of ABA- and ethylene-biosynthesis genes. Similarly, application of NDGA reduced the expression of ABA-biosynthesis genes. Specifically, NDGA reduced the expression of both *FcNCED2* and *FcABA2*, whereas fluridone only lowered the expression level of *FcABA2* ([Fig. 4](#F4){ref-type="fig"}). Although fluridone and NDGA act in different ways to suppress ABA biosynthesis, the ethylene-biosynthesis genes *FcACS2*, *4*, *FcACOL*, and *FcACO2* were strongly down-regulated by both inhibitors, as also occurs in the fruit of tomato ([@CIT0062]) and strawberry ([@CIT0018]). In tomato, down-regulation of *SlNCED1* by RNAi leads to down-regulation of genes encoding major cell wall-catabolic enzymes during ripening, resulting in firmer fruit ([@CIT0052]), whilst in strawberry, down-regulation of *FaNCED1* results in a significant decrease in ABA levels and in fruit that lack color ([@CIT0018]). As with *SlNCED1* in tomato ([@CIT0052]), *FcNCED2* in fig was down-regulated by NDGA (*FcNCED2* shares 71.72% identity with *SlNCED1*, [Supplementary Fig. S9](#sup1){ref-type="supplementary-material"}); it is therefore plausible that NDGA treatment altered the expression of genes related to regulation of cell wall modification in fig: further experiments are needed to clarify this point. Application of NDGA to fig up-regulated the ABA-catabolism pathway gene *FcABA8OX*, which could have been responsible for the decrease in total active ABA accumulation. This would be in agreement with work in tomato by [@CIT0036], where *ABA8OX* was slightly up-regulated after NDGA treatment; however, no effect of fluridone was observed. Responses of potential ripening regulators to exogenous ABA, fluridone, and NDGA treatments {#s20} ------------------------------------------------------------------------------------------- The ripening process in fleshy fruits is regulated by plant hormones and involves numerous transcription factors ([@CIT0001]; [@CIT0022]). Members of the MADS-box gene family have been found to regulate ripening in several climacteric species: *RIN* and *TAGL1* in tomato, *PLENA* in *Prunus persica* (peach), *MADS1--5* in banana, and *MADS8* and *MADS9* in apple ([@CIT0057], [@CIT0056]; [@CIT0015]; [@CIT0054]; [@CIT0007]; [@CIT0014]). In addition, genes from the MADS-box gene family are involved in non-climacteric fruit ripening, such as *MADS9* in strawberry ([@CIT0046]). In the unique climacteric fig fruit, eight MADS-box transcripts have been identified and partially isolated from fruit developing on the tree ([@CIT0009]). An in-depth study of the transcriptome data of fig fruit during different ripening stages revealed seven new MADS-box transcripts ([@CIT0040]). In addition, *FcMADS8*, which is highly similar to *SlRIN*, shows an increase in expression during the ripening of both fig inflorescences and receptacles ([@CIT0011]). Here, we found that *FcMADS8*, *14*, and *15* were moderately up-regulated by ABA application in both inflorescences and receptacles at 24 HAT, whereas they were inhibited by fluridone and NDGA ([Figs 5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}). This was similar to tomato fruit, where expression of *MADS-RIN* is elevated by exogenous ABA, and suppressed by NDGA when endogenous ABA is inhibited ([@CIT0036]). Among the 27 NAC transcripts of fig fruit described by [@CIT0009], *FcNAC1*, *2*, and *5* showed moderate up-regulation by ABA treatment ([Fig. 5](#F5){ref-type="fig"}). On the other hand, *FcNAC2* and *5* were down-regulated by both fluridone and NDGA treatments while *FcNAC1* was down-regulated by NDGA only in the receptacle ([Fig. 6](#F6){ref-type="fig"}). A similar effect has been noted in tomato fruit where *NOR*, a member of the NAC domain family that functions upstream of ethylene in the tomato fruit ripening cascade, is elevated by exogenous ABA and suppressed by NDGA ([@CIT0036]). In this context, our results suggested that *FcNAC1*, *2*, and *5* interacted in their role in fig fruit ripening, similar to *MaNAC1* and *MaNAC2* in banana fruit ripening via interaction with the ethylene-signaling pathway ([@CIT0047]). Furthermore, overexpression of *SlNAC1* has been shown to regulate tomato fruit ripening through both ethylene-dependent and ABA-dependent pathways ([@CIT0032]). Reduced expression of *SlNAC4* by RNAi in tomato results in delayed fruit ripening, suppression of chlorophyll breakdown, a decrease in ethylene biosynthesis by a reduction in the expression of ethylene-biosynthesis genes, and a reduction in carotenoids by alterations in fluxes in the carotenoid pathway ([@CIT0063]). Surprisingly, [@CIT0024] showed that *SlNAC4* is down-regulated by ABA treatment whereas *SlNAC5*, *6*, *7*, and *9* are up-regulated in tomato fruit. The ERFs are a large family of transcription factors that includes gene repressors and activators, with a degree of functional redundancy among its members ([@CIT0023]; [@CIT0029]). In fig fruit, ERFs are positive ripening regulators, except for *FcERF12185* that is only highly expressed at the fully ripe stage and is probably not responsible for the climacteric rise in ethylene or the metabolic ripening processes in the earlier stages ([@CIT0011]). Among the ERF transcripts isolated by [@CIT0011], we demonstrated here that *FcERF9006* expression is up-regulated by exogenous ABA and ethephon treatment, whereas it is down-regulated by fluridone and NDGA in both inflorescences and receptacles ([Figs 5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}). In climacteric tomato, *LeERF2*, *LeERF3*, and *LeERF4* are down-regulated by ABA treatment and *LeERF4* is more highly expressed after NDGA treatment ([@CIT0036]). Indeed, temporary down-regulation of most *FcERF*s was observed in the unique climacteric fig fruit following pre-harvest treatment with 1-MCP ([@CIT0011]). Non-climacteric characteristics of fig fruit exclude them from the conventional climacteric category {#s21} ---------------------------------------------------------------------------------------------------- Despite the classification of fig fruit as climacteric, when harvested prior to complete ripening they never reach the commercially desirable parameters of size, color, flavor, and texture ([@CIT0008]). Surprisingly, 1-MCP markedly enhances ethylene production in figs ([@CIT0049]; [@CIT0038]; [@CIT0010]), similar to its effect in non-climacteric fruit such as citrus ([@CIT0034]). This confirms the non-climacteric, auto-inhibitory regulation of ethylene synthesis, rather than the autocatalytic pattern typical of climacteric fruit ([@CIT0010]). Apart from ethylene, several recent studies have shown that ABA plays an important role in the regulation of fruit development and ripening in both climacteric and non-climacteric fruit ([@CIT0061], [@CIT0062]; [@CIT0052]; [@CIT0027]). In the mature fig fruit, endogenous ABA is present before ethylene is produced, and is supposed to trigger the ripening process ([Fig. 1A](#F1){ref-type="fig"}). The increase in accumulation of endogenous ABA during the onset of fig ripening is similar to that in other climacteric fruit such as tomato, avocado, and apple ([@CIT0004]; [@CIT0026]; [@CIT0050]; [@CIT0062]; [@CIT0027]). However, instead of tapering off after the onset of ripening, as in climacteric fruit, the endogenous ABA content in control plants continued to rise until the fig fruit was fully ripe, as in non-climacteric strawberry fruit ([Fig. 1A](#F1){ref-type="fig"}; [@CIT0018]). Interestingly, this phenomenon was observed in both the reproductive inflorescence tissue and the vegetative receptacle tissue, with high expression of major ABA-biosynthesis pathway genes ([Figs 2](#F2){ref-type="fig"}, [4](#F4){ref-type="fig"}). On the other hand, expression of ethylene-related genes was different for the two tissues, with only the inflorescence showing expression similar to that in climacteric fruit ([@CIT0011]). In agreement with previous studies, our results showed that ethylene was produced at high levels at the 30--50% ripening stage, with significantly high expression of the ethylene-biosynthesis gene *FcACS4* in the inflorescence, although expression levels rose in both tissues ([@CIT0010], [@CIT0011]; [Fig. 1A](#F1){ref-type="fig"}). Increased expression of ethylene-biosynthesis genes was also observed in petioles (vegetative tissues) of fruit at different ripening stages---from green to fully ripe ([Supplementary Fig. S10](#sup1){ref-type="supplementary-material"}). These expression levels were very low compared to those in the inflorescences and receptacles, and they played no part in fruit ripening. Similarly, very low expression of ethylene-biosynthesis genes in receptacles relative to inflorescences would lead to the production of minor amounts of ethylene, which might not be enough for the ripening process, but a high amount of endogenous ABA production until full ripeness, as in strawberry, could lead to non-climacteric ripening by promoting sugar accumulation, fruit pigmentation, and softening, as described in a non-climacteric fruit-ripening model ([@CIT0028]). This distinct mechanism in the receptacle could be synchronized by the ABA-biosynthesis genes *FcZEP*, *FcNCED1*, and *FcNCED3*, the expression of which was positively correlated with ABA in the receptacle but negatively correlated in the inflorescence during natural ripening of untreated fruit ([Fig. 7](#F7){ref-type="fig"}). Different ripening mechanisms for specific organs have also been reported in strawberry, where ethylene is involved in the ripening of achenes (reproductive organ) but not the receptacles (vegetative organ) ([@CIT0035]). Indeed, strawberry is considered a non-climactic fruit where ABA promotes ripening by increasing its endogenous levels until full ripeness ([@CIT0018]). This unique characteristic of ripening, showing both climacteric and non-climacteric behavior, has also been reported in 'Elizabeth' melon (*Cucumis melo*), although it is regarded as a climacteric fruit ([@CIT0053]). Non-climacteric fruit are considered to be a separate group that does not follow the typical climacteric ripening pattern. However, the identification of MADS-box genes in both climacteric and non-climacteric fruit suggests that at least some aspects of ripening are shared between these two categories ([@CIT0006]). In strawberry, a MADS-box *SEPALLATA* gene (*SEP1/2*) is needed for normal fruit development and ripening ([@CIT0046]). Similarly, in banana, which is classified as a climacteric fruit, the MADS-box *SEP3* gene also displays ripening-related expression ([@CIT0007]). In the fig fruit, *FcMADS8* expression increases during ripening on the tree and is inhibited by 1-MCP treatment ([@CIT0011]). Interestingly, *FcMADS8* was inhibited by fluridone and NDGA in the inflorescence at 32 and 48 HAT ([Fig. 6](#F6){ref-type="fig"}). During the ripening of pepper fruit, genes involved in ethylene biosynthesis are not induced; however, genes downstream of ethylene perception, such as cell wall-related genes, *ERF3*, and carotenoid-biosynthesis genes, are up-regulated ([@CIT0037]). In fig fruit, *FcERF12185* is up-regulated by 1-MCP treatment during ripening and may play a role in regulating ethylene-synthesis system 1 and in causing the non-climacteric behavior of ethylene production ([@CIT0011]). However, expression of *FcERF12185* was not affected by application of ABA, fluridone, or NDGA in our current study ([Supplementary Figs S7, S8](#sup1){ref-type="supplementary-material"}). In contrast, the expression level of *FcERF9006* was altered by application of ABA, fluridone, and NDGA, which suggests a role for this gene in non-climacteric and/or ABA-dependent ripening of fig fruit ([Figs 5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}). In conclusion, involvement of ABA during fig fruit ripening was confirmed in our study by observed alterations in ripening as the result of the application of exogenous ABA, ethephon, fluridone, and NDGA. The unique ripening nature of fig fruit, in which two tissues act separately, is illustrated in a proposed model ([Fig. 8A](#F8){ref-type="fig"}). The alterations in the expression levels of ABA-metabolic pathway, ethylene-biosynthesis, MADS-box, NAC, and ERF genes in the fruit by on-tree application of ABA, ethephon, fluridone, NDGA, and 1-MCP ([@CIT0011]) are summarized in [Fig. 8B](#F8){ref-type="fig"}, C. A highly positive correlation of the ABA-biosynthesis gene *FcNCED2* and the ethylene-biosynthesis gene *FcACS4* with endogenous ABA in the inflorescence demonstrates a key role in fig fruit ripening. We plan to further utilize these as candidate genes for genome editing to slow down the ripening process. ![Proposed model of ABA-regulatory and -biosynthesis genes in fig fruit. (A) ABA interplay with ethylene in fig fruit during ripening. (B) Changes in activity of ABA metabolic pathway genes in the fruit following on-tree exogenous application of ABA, ethephon, NDGA and fluridone. (C) Changes in activity of ethylene-biosynthesis, MADS-box, NAC, and ERF gene in the fruit following on-tree exogenous application of ABA, ethephon, NDGA, fluridone, and 1-MCP ([@CIT0011]). Downward arrows indicate down-regulated genes in treated fruit compared to untreated controls; upward arrows indicate up-regulated genes. The arrows are color-coded as follows: red, ABA; purple, ethephon; green, NDGA; blue, fluridone; black, 1MCP.](ery33308){#F8} Supplementary data {#s22} ================== Supplementary data are available at *JXB* online. Table S1. Primers used for high-throughput real-time qPCR. Fig. S1. Expression pattern of ABA-metabolism and ethylene-biosynthesis genes in ABA- and ethephon-treated fig fruit. Fig. S2. Expression pattern of ABA-metabolism and ethylene-biosynthesis in fluridone- and NDGA-treated fig fruit. Fig. S3. Expression pattern of MADS-box genes in inflorescence and receptacle tissues following ABA and ethephon treatment. Fig. S4. Expression pattern of MADS-box genes in inflorescence and receptacle tissues following fluridone and NDGA application. Fig. S5. Expression pattern of NAC genes in inflorescence and receptacle tissues following ABA and ethephon application. Fig. S6. Expression pattern of NAC genes in inflorescence and receptacle tissues following fluridone and NDGA application. Fig. S7. Expression pattern of ERF genes in inflorescence and receptacle tissues following ABA and ethephon application. Fig. S8. Expression pattern of ERF genes in inflorescence and receptacle tissues following fluridone and NDGA application. Fig. S9. Alignment of *FcNCED2* amino acid sequence and *SlNCED1* protein. Fig. S10. Expression of ethylene-biosynthesis genes in the petiole of fig fruits at different developmental stages during ripening. ###### Click here for additional data file. The authors wish to thank Dr Adi Faigenboim for help in data analysis. Funding was provided by the Ministry of Agriculture, Bet Dagan, Israel.

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Background {#Sec1} ========== Over the past 20--30 years, suicide rates have declined overall in European countries with previously high rates of suicide, such as Denmark, Estonia, Germany, Hungary, and Sweden \[[@CR1]\]. Ever since the adoption of a national suicide prevention strategy in 2008, \[[@CR2]\] the suicide rate among males in Sweden has declined further \[[@CR3]\]. The prevention strategy involves nine strategic action areas, with two of these areas, 'medical, psychological and psychosocial improvements' and 'lethal means restriction', being specifically applicable within health care. The prevention strategy recommends early interventions with a focus on treating depression, restricted prescriptions of sleeping pills, and increased use of new less-toxic antidepressants \[[@CR2]\]. However, suicide still accounts for the deaths of around 1500 individuals per year in Sweden, with rates of 15.75 suicides per 100,000 males and 7.09 per 100,000 females having been reported in 2016 \[[@CR3]\]. The suicide rate of ≥15 per 100,000 is among the highest rates reported worldwide \[[@CR4]\]. Suicide rates and suicide attempts are often associated with a diagnosis of severe psychiatric or somatic illness \[[@CR4], [@CR5]\], and vary across lifetime and gender \[[@CR4]\]. It is important to note that around 90% of individuals who commit suicide have a documented history of a psychiatric disease \[[@CR6]--[@CR8]\]. The psychiatric disorders with a high lifetime risk of suicide are the affective disorders \[[@CR9]\], particularly bipolar disorder \[[@CR10]\], alcohol or substance use disorders \[[@CR11], [@CR12]\], schizophrenia \[[@CR13], [@CR14]\], and personality disorders \[[@CR15], [@CR16]\]. Indeed, previous research has emphasized treatment interventions for psychiatric illness to reduce suicidal ideation and suicidal behavior. Psychopharmacological treatment \[[@CR17]\], cognitive therapy for suicide prevention (CT-SP) \[[@CR18]\], cognitive behavioral therapy (CBT) \[[@CR1], [@CR19]\], dialectical behavior therapy (DBT) \[[@CR20]\], and electroconvulsive therapy (ECT) \[[@CR21], [@CR22]\] have all been found to be effective for preventing suicide. In addition, research also suggests that repeated suicide attempts can be significantly reduced or prevented by rather short psychotherapeutic interventions \[[@CR23]\]. Despite promising results \[[@CR19], [@CR24]\], further research into suicide as an outcome is warranted to draw firm conclusions concerning the impact of different interventions on the suicide rate \[[@CR1], [@CR5], [@CR25]\]. In particular, more studies are needed on treatments that combine psychotropic medication and psychotherapy to refine treatment recommendations for suicidal behavior \[[@CR26]\]. Hence, the necessary requirements for successful interventions for patients with suicidal behavior or suicidal ideation are not yet fully understood \[[@CR27]\]. Furthermore, lifetime suicide risk has been suggested to be hierarchical, meaning that psychiatric inpatients have the highest risk of suicide, while the risk is lower in psychiatric outpatients, and even lower in individuals with no history of contact with psychiatric services \[[@CR9]\]. Suicide risk is also higher shortly after the onset of psychiatric illness and among recently hospitalized psychiatric patients with suicidal ideation or a history of suicide attempts \[[@CR9], [@CR28]\]. Additionally, patients admitted to psychiatric inpatient care are at higher risk of suicide shortly after admission, during hospitalization, during periods of authorized hospital leave, and at discharge, with the suicide risk remaining elevated up to 12 months post-discharge \[[@CR29]--[@CR31]\]. In relation to this, a study by Appleby et al. showed that a reduction in care efforts during the 12 months prior to suicide was observed significantly more often for suicide victims than for controls \[[@CR32]\]. The suicide cases more often had reduced outpatient appointment frequencies, less supervision, and lower drug doses than the controls, with the authors finding strong associations between each of these three factors and completed suicide. These findings have also been confirmed in more recent research \[[@CR33]\]. Hence, maintained and regular contact with psychiatric services and the avoidance of abrupt cessation of mental health care appear to lower the risk of suicide \[[@CR5]\]. In addition, frequent follow-ups or outreach, especially after missed mental health visits, have been shown to reduce repeated suicide attempts \[[@CR24]\]. In summary, research shows that suicidal behavior can be prevented through the application of appropriate medical, psychotherapeutic, or psychosocial interventions, and that reduced health care consumption is associated with suicide. However, important limitations of the previous research are that the above-mentioned factors were largely examined separately, rather than in combination, and the studies did not use control groups drawn from patients with the same diagnoses. To determine whether these different factors are independently associated with suicide, investigation of multiple associated factors is needed using data from suicide cases and adequately matched control subjects. The overarching aim of the current study was to examine whether completed suicide in psychiatric patients in a Swedish population was associated with the quantity and nature of previous medical and psychosocial treatment interventions, with this being accomplished by comparing cases of suicide with matched control psychiatric patients. The study tested the following two hypotheses: *Controls have a higher frequency of psychiatric outpatient visits than suicide cases.Controls have a higher occurrence of psychosocial treatment interventions than suicide cases.* Method {#Sec2} ====== Study design {#Sec3} ------------ This study used a retrospective psychiatry-based case-control design. Data from the period 1 January 2007 to 31 December 2013 were obtained from the Swedish National Cause of Death Registry \[[@CR3]\], psychiatric and medical records, and the statistics of the Swedish total population \[[@CR34]\]. Study setting {#Sec4} ------------- The study catchment area (Örebro County) covers a population of 285,395, with a mix of urban and rural areas. In 2013, the region had suicide rates of 21.7 suicides per 100,000 male inhabitants and nine suicides per 100,000 female inhabitants. These rates were higher than the Swedish national rates for both males (16.2 per 100,000) and females (7.5 per 100,000) during the same period \[[@CR35]\]. The specialist psychiatric unit belongs to a general university hospital with 918 beds, of which 136 are part of the psychiatric care unit. In 2013, the psychiatric units offered 40,020 days of inpatient psychiatric care and 131,137 outpatient department visits. Persons outside specialist psychiatry with visits only to community services outside of health care, or those at private clinics, are not included in this study. Sample and participant selection {#Sec5} -------------------------------- ### Suicide cases {#Sec51} Suicide cases were identified using the Swedish National Cause of Death Registry. According to the registry, during the period 1 January 2007 to 31 December 2013, a total of 339 individuals (69.3% men) from Örebro County died secondary to suicide (codes X60--X84) or undetermined intent (codes Y10--Y34) classified in accordance with the International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10) \[[@CR35]\]. The national identification numbers of these individuals were used to search the electronic psychiatric medical records of the University Hospital of Örebro. This revealed that 154 (45.4%) of these 339 individuals had received psychiatric care at this regional centre during the 2 years prior to their death. These 154 individuals (65.6% men) were therefore included in the present study as suicide cases. Cases classified as uncertain suicides were also included, as their exclusion may have led to an underestimation of the suicide rate. The national statistics on causes of death include cases of uncertain cause of death as possible suicides, as analyses indicate that the majority of uncertain cases are probable suicides \[[@CR3], [@CR36]\]. In the subsequent text, the term suicide refers to both definite suicide and uncertain suicide. #### Controls {#FPar2} The 154 control subjects were identified through the electronic psychiatric medical records of the University Hospital of Örebro during the matching procedure (see next section). #### Case-control matching procedure {#FPar3} A hospital statistician outside the research team personnel matched the suicide cases with control subjects on the basis of the following: 1) a history of contact with psychiatric services in the year that the suicide case died, 2) age, 3) sex, and 4) primary psychiatric diagnosis. For the primary psychiatric diagnosis, consistency was required in terms of the first two or three digits of the respective ICD-10 code (e.g., F32.2 or F32). When applicable, efforts were made to control for a comorbid diagnosis of psychoactive substance use disorder (F10-F19), as comorbid substance use disorders increase the risk for suicide \[[@CR37], [@CR38]\]. For suicide cases below the age of 25 years, a control of similar age (± 2 years) was sought. In older suicide cases, controls with a maximum age difference of 2 years were primarily sought; however, a maximum age difference of 10 years was accepted. A total of 113 (73.4%) suicide case-matched control pairs met the stringent primary diagnosis matching criteria (e.g., F32.2). Twenty-five (16.2%) pairs were matched on the basis of two digits (e.g., F32), and two (1.3%) pairs were matched on the basis of a diagnostic cluster (e.g., Mood disorders F30--F39). Thirteen suicide cases (8.5%) lacked a primary psychiatric diagnosis. Of these, 12 were matched with controls without psychiatric diagnoses, while a control with a primary diagnosis of depression was selected as the optimal match for the remaining case (0.6%). A total of 15 suicide cases had a comorbid diagnosis of psychoactive substance use (F10--F19). Of these, nine were matched to a control with a similar comorbidity. For suicide cases younger than 25 years, 16 out of 18 pairs (88.9%) were matched according to the study criteria. For the remaining two pairs, the age-interval was extended to ±3 years and ± 7 years. The majority (87.5%) of the 136 case-control pairs in the age group 25 years or older were matched according to the study criteria (i.e., a maximum age-interval of ±2 years); however, the age-interval was extended to ±3 years in seven pairs, ± 4 years in three pairs, and ± 5 years in five pairs. In the remaining pair, a 96-year-old suicide case was matched to an 85-year-old control. The mean follow-up time in days was calculated from the date of the first psychiatric care contact included in the study to the date of suicide (for suicide cases), or to the date of last contact included in the study (for controls). The mean follow-up time was 446 days (SD 256.4) for suicide cases and 479 days (SD 254.1) for control subjects, with no significant difference being found between suicide cases and controls according to an independent *t*-test analysis (*t* = 1.13, df 306, *p* = 0.259). Measures {#Sec6} -------- ### The Swedish National Cause of death registry {#Sec52} The suicide cases in this study were identified from the National Cause of Death registry. This contains information on the deaths of all Swedish citizens, including deaths occurring outside Sweden. The registry does not include information concerning the deaths of people seeking asylum, undocumented migrants, or visitors to Sweden. Information concerning suicide is based on death certificates. These are completed by a physician following a clinical or forensic autopsy, with the cause of death being classified in accordance with the ICD-10 codes \[[@CR35]\]. The annual rate of missing data on causes of death for Swedish citizens is less than 2% \[[@CR3]\]. For the purposes of the present study, the following data were collected: national identification number, municipality, date of death, underlying cause of death, and medical evaluation of whether death by self-harm was intentional or of uncertain intent. #### Data from medical records for suicide cases and controls {#FPar5} Information concerning psychiatric outpatient visits and psychiatric and somatic admissions in the 2 year period prior to each suicide was retrospectively collected from electronic medical records. The data concerned all care received from the 1st January 2005 until the 31st December 2013. For example, for a suicide case with a date of death of 14th March 2007, data were collected from 14th March 2005 to 14th March 2007 inclusive. Care consumption for controls was included from the same year as the death of the case, and the data were collected for 2 years back in time. For each suicide case and control subject, data-files were created from electronic psychiatric medical records, and if applicable, from the relevant somatic medical records. From these files, data were retrieved concerning the number of psychiatric outpatient visits, the number of psychiatric admissions, the total number of days of hospitalization (from admission to discharge) for each psychiatric admission, the total number of days spent as a psychiatric inpatient, the occurrence of somatic hospitalization, and the ICD-10 diagnoses within the 2 year study period. #### Psychiatric interventions {#FPar6} Information concerning the interventions implemented during outpatient visits and/or inpatient care in psychiatry (e.g., psychological treatment, prescriptions of psychotropic drugs, and ECT) was gathered from the electronic medical records. #### Somatic comorbidity and serious suicide attempts {#FPar7} Data on somatic hospitalization at a specialist medical unit together with diagnoses according to ICD-10 codes (see Appendix for ICD-codes) were used as indicators of somatic comorbidity, and somatic comorbidity was treated as a possible covariate in the analyses, as it is assumed to increase suicide risk. Somatic comorbidity did not include hospitalization secondary to injuries or suicide attempts. A serious suicide attempt was defined as any intentional attempt to end life that led to hospitalization at a somatic specialist medical unit (e.g., due to intoxication). The ICD-10 codes for previous suicide attempts are provided in the Appendix. Statistical analysis {#Sec7} -------------------- Descriptive statistics were used to describe the distribution of psychiatric diagnoses, sex, and age in the suicide cases and control subjects. The chi-square test and Fisher's exact test were used to test for significant case-control differences in the occurrence (yes/no) of outpatient or inpatient psychiatric care, somatic hospitalization due to comorbidity, somatic hospitalization due to previous serious suicide attempts, and specific psychiatric interventions. The Mann Whitney U-test was used for case-control comparisons involving non-normally distributed variables (e.g., the frequency of health care provision). Treatment interventions/covariates for which case-control differences (*p* \< 0.1) were found (see Table [2](#Tab2){ref-type="table"}) were evaluated as predictors or covariates using stepwise forward logistic regression models (i.e., a selection method using the likelihood ratio) to examine their association with suicide. This analysis was used to compute odds ratios (ORs) and confidence intervals (CIs) with suicide (yes = 1, no = 0) as a binary dependent outcome variable. Correlations between the included interventions and covariates were examined with Spearman correlation analyses. All statistical analyses were performed using SPSS for windows version 22 (IBM, New York). A power calculation was performed on the basis of assumptions of 25% exposure to treatment interventions in controls, an OR of 2, and a confidence level of 95%. This showed that to obtain a power of 80% for the detection of significant differences, a sample size of 154 patients per group was required. If the exposure to treatment among controls was only 5%, an OR \> 3.2 would be required to detect significant differences with this sample size. Results {#Sec8} ======= The majority of cases died within 2 weeks from their last care contact (median times of 12 days in men and 11 days in women). The distributions of age, sex, and primary psychiatric diagnoses according to ICD-10 \[[@CR35]\] among the suicide cases and controls are summarized in Table [1](#Tab1){ref-type="table"}. The distribution of comorbidities was similar for both groups, with a total of 15 suicide cases and 14 controls having a comorbid substance use disorder (F10--F19). In terms of socio-demographics, the suicide cases and controls did not differ significantly in education, living situation, or occupation (see Table [1](#Tab1){ref-type="table"}). Table 1Characteristics of the study population after matching for age, sex, and psychiatric diagnosis, and results of chi-square-tests/*t*-tests (suicide cases versus controls)Suicide cases *n* = 101 Men^4^Controls n = 101\ Men^4^Suicide cases *n* = 53 Women^4^Controls n = 53\ Women^4^All suicide cases *n* = 154All controls\ n = 154Test statistic, pMean age: years (SD^5^)Range45.9 (17.6)18--8445.9 (17.4)17--8449.6 (16.4)13--9649.4 (15.5)13--8547.1 (17.2)13--9647.1 (16.8)13--85*t*: −0.040, *p* = .97^6^Educational level^*1,8*^ n (%)χ^2^: 4.085, df 2, *p* = .13^7^ Low28 (27.7)44 (43.6)15 (28.3)16 (30.8)43 (27.9)60 (39.0) Medium54 (53.5)42 (41.6)25 (47.2)25 (48.1)79 (51.3)67 (43.5) High18 (17.8)14 (13.9)11 (0.8)11 (21.2)29 (18.8)25 (16.2)Occupational status^8^ n (%)χ^2^: 0.14, df 1, *p* = .71^7^ Employed37 (36.6)29 (28.7)12 (22.6)17 (32.1)49 (32.0)46 (30.1) Unemployed64 (63.4)72 (71.3)40 (75.5)35 (66.0)104 (67.5)107 (69.5)Family status^8^ n (%)χ^2^: 3.23, df 3, *p* = .36^7^ Cohabiting with partner (no children)12 (11.9)12 (11.9)8 (15.1)11 (20.8)20 (13.0)23 (14.9) Cohabiting with partner and children14 (13.9)20 (19.8)4 (7.5)8 (15.1)18 (11.7)28 (18.2) Single parent with children11 (10.9)5 (5.0)5 (9.4)8 (15.1)16 (10.4)13 (8.4) Living alone64 (63.4)64 (63.4)35 (66.0)25 (47.2)99 (64.3)89 (57.8)Diagnoses used for matching^2^ n (%)Depressive disorders(F32--F33, F34.1)27 (26.7)28 (27.7)24^2^ (45.3)24 (45.3)51 (33.1)52 (33.8)χ^2^: 0.047, df 7, *p* = 1.00^7^Mental and behavioural disorders due to psychoactive substance use (F10--F19)28 (27.7)28 (27.7)5 (9.4)5 (9.4)33 (21.4)33 (21.4)Neurotic, stress-related, and somatoform disorders (F40--F48)13 (12.9)13 (12.9)8 (15.1)8 (15.1)21 (13.6)21 (13.6)Schizophrenia, schizotypal, and delusional disorder (F20--29)9 (8.9)9 (8.9)5 (9.4)5 (9.4)14 (9.1)14 (9.1)Disorder of adult personality and behaviour (F60--F69)7 (6.9)7 (6.9)4 (7.5)4^2^ (7.5)11 (7.1)11 (7.1)Bipolar disorder (F31)3^2^ (3)3 (3)4 (7.5)4 (7.5)7 (4.5)7 (4.5)Disorder of psychological development / behavioral and emotional disorders (F80--89, F90--98)3 (3)3 (3)003 (1.9)3 (1.9)Psychiatric examination^3^11 (10.9)10 (9.9)3 (5.7)3 (5.7)14 (9.1)13 (8.4)^1^ Low: elementary school or grade 1--9; Medium: high school or other education below university level; High: university or university college^2^ Diagnoses were mental and behavioural disorders referring to chapter V in the International Statistical Classification of Diseases, 10th Revision classifications (ICD-10)The diagnoses were registered as primary diagnoses in all but two cases and one control, where the secondary diagnoses were used (see ^2^ in the suicide case and control columns above)^3^ Persons who received psychiatric care without being assigned a primary psychiatric diagnosis^4^ A comorbid diagnosis of psychoactive substance use (F10-F19) was present in 22 patients (five male and seven female suicide cases; five male and five female controls)^5^ SD = standard deviation^6^ Results from Independent Samples *t*-test^7^ Results from Pearson Chi-Square Tests^8^ Number of patients with missing data: educational level: 5, occupational status: 2, family status: 2 Provision of psychiatric care {#Sec9} ----------------------------- Medical and psychological assessments, supportive conversation, and efforts to improve everyday functioning were regular contents of both psychiatric inpatient and outpatient care. During the 2 years before the suicide, outpatient psychiatric care was received by 305 patients (305/308, 99%) in the total cohort, and inpatient care was received by 155 patients (155/308, 50.3%) in psychiatric specialist units. Three of the suicide cases received inpatient care only, while all controls had at least one outpatient visit. Similar proportions of the occurrence (yes/no) of outpatient care were observed in the suicide case and control groups; however, during the 2 years before death, suicide cases made a significantly lower number of visits to outpatient psychiatry than controls. On average, the control subjects attended five times as many outpatient visits as suicide cases. No significant case-control differences were found in the occurrence of inpatient psychiatric care, involuntary inpatient admissions, the number of psychiatric inpatient admissions, or the total days of hospitalization (for details see Table [2](#Tab2){ref-type="table"}). Table 2Case-control comparisons of clinical variables and somatic inpatient care because of somatic comorbidity or serious suicide attemptsSuicide cases\ n = 154Controls\ *n* = 154n (%)^11^n (%)^11^Test statistic, pProvision of psychiatric care Outpatient visits (yes)151 (98.1)154 (100)F, *p* = .248^9^ Inpatient admissions (yes)80 (51.9)75 (48.7)χ^2^: 0.32, df 1, *p* = .569^10^ of which involuntary admissions (yes)15 (9.7)9 (5.8)χ^2^: 1.63, df 1, *p* = .202^10^ Number of outpatient visits, Md^1^4 (1--217)19.5 (1--209)U, *p* = .001^9^ Number of admissions, Md^1^2 (1--11)2 (1--22)U, *p* = .954^11^ Hospitalization days, Md^1^20.5 (1--693)16 (1--191)U, *p* = .443^11^Psychiatric interventions Intervention forms  Combination treatment (yes) ^2^17 (11.0)41 (26.6)χ^2^: 12.27, df 3, *p* = .007^10^  Psychotherapy (yes)^3^2 (0.9)2 (0.9)  Psychotropics (yes)^4^119 (77.2)98 (63.6)  Other (yes)^5^16 (10.4)13 (8.4)  Number of psychotherapy sessions, Md^1^5 (1--47)13 (1--142)U, *p* = .504^11^  CBT (including DBT)8 (5.2)17 (11)χ^2^: 3.53, df 1, *p* = .060^10^  PDT12 (7.8)14 (9.1)χ^2^: 0.17, df 1, *p* = .838^10^  Other structured psychotherapy6 (3.9)18 (11.7)χ^2^: 6.51, df 1, *p* = .011^10^  Electroconvulsive therapy^6^13 (8.4)13 (8.4)χ^2^: 0.00, df 1, *p* = 1.00^10^Provision of somatic inpatient care Somatic comorbidity (yes)^7^50 (32.5)30 (19.5)χ^2^: 6.75, df 1, *p* = .009^10^ Serious suicide attempts^8^30 (19.5)14 (9.1)χ^2^: 6.79, df 1, p = .009^101^ Md: Median (minimum to maximum)^2^ Combination treatment: psychotropics and psychotherapy (CBT, DBT, PDT or other structured psychotherapy)^3^ Mono treatment: psychotherapy without psychotropics^4^ Mono treatment: psychotropics without psychotherapy^5^ Other supportive interventions^6^ All patients with ECT were prescribed psychotropic drugs. Two ECT cases (controls) also received psychotherapy^7^ Somatic specialist medical inpatient treatment because of somatic comorbidity. Diagnostic ICD-10 codes provided in the Appendix^8^ Somatic specialist medical inpatient treatment because of serious suicide attempts. Diagnostic ICD-10 codes provided in the Appendix^9^ Results from Fisher's Exact Test (F)^9^ Results from Pearson Chi-Square Tests (χ^2^)^10^ Results from Mann Whitney U-test (U)^11^ n (%) = number (percent) of patients in each group Psychiatric interventions {#Sec10} ------------------------- Psychiatric interventions included prescriptions of psychotropic drugs, ECT, and psychotherapeutic interventions such as CBT, DBT, psychodynamic therapy (PDT), or other structured psychological treatment approaches (e.g., integrated psychotherapy using a mix of several psychotherapeutic approaches to meet the needs of the patient). These different interventions were received by 279 (90.6%) patients in the total sample (suicide cases: 89.6%, controls: 91.6%). Case-control comparisons revealed no differences in the occurrence of ECT or psychotherapy without combined use of psychotropic drugs. By contrast, significant case-control differences were found in the provision of combination therapy (i.e., both psychotherapy and prescription of psychotropic drugs), which was significantly more frequent in controls, while mono-therapy with psychotropic drugs without psychotherapy was significantly more frequent in the suicide cases. Between group comparisons in the type of psychotherapy received showed 'other structured psychotherapy' to be significantly more frequent in controls, while the number of psychotherapy sessions received did not differ significantly between the groups (for details see Table [2](#Tab2){ref-type="table"}). Provision of somatic inpatient care {#Sec11} ----------------------------------- A significant case-control difference was found for somatic hospitalization. Around one-third of suicide cases and a fifth of controls had a somatic comorbidity that required specialist inpatient medical treatment (Table [2](#Tab2){ref-type="table"}). In the suicide cases, the most common somatic comorbid diagnoses were diseases of the musculoskeletal system (*n* = 10, 6.5%, ICD-10: chapter M) and diseases of the circulatory system (*n* = 8, 5.2%, ICD-10: chapter I). In the controls, the most common somatic comorbid diagnoses were diseases of the musculoskeletal system (*n* = 5, 3.2%, ICD-10: chapter M) and diseases of the digestive system (n = 5, 3.2%, ICD-10: chapter K). The somatic diagnoses in suicide cases and controls are specified in the Appendix. Serious suicide attempts that required somatic inpatient treatment were significantly more prevalent among the suicide cases than the controls during the 2 years before death. The suicide attempt diagnoses are specified in the Appendix. The distribution between groups is shown in Table [2](#Tab2){ref-type="table"}. Factors and treatment interventions associated with suicide {#Sec12} ----------------------------------------------------------- The psychiatric interventions and covariates showing associations (*p* \< 0.1) with suicide (see Table [2](#Tab2){ref-type="table"}) in the present cohort were analyzed using a stepwise forward selection method (likelihood ratio) in a logistic regression analysis. The full model included the following six variables: 'number of visits in outpatient psychiatry', 'intervention form' (i.e., psychotropic prescription without psychotherapy, combination therapy, psychotherapy without psychotropics, other supportive interventions), 'CBT' (including DBT), 'other structured psychotherapy', 'somatic hospitalization due to serious suicide attempt', and 'somatic hospitalization due to somatic comorbidity'. The results from the full stepwise logistic regression model (Table [3](#Tab3){ref-type="table"}) indicated that a high frequency of outpatient visits was significantly negatively associated with suicide, while other structured psychotherapy approaches did not reach significance (*p* = .056). The occurrence of serious suicide attempts was significantly associated with successful suicide. This full model explained 9.9% of the variance (Table [3](#Tab3){ref-type="table"}). Table 3Results from a binominal logistic regression (full model) with a stepwise forward selection method (likelihood ratio). Suicide cases, *n* = 154; Controls, n = 154Full model\*B (SE)WaldSig.OR (CI)95% CI for ORLowerUpperNumber of outpatient visits−0.012 (0.004)7.908.0050.9880.9800.996Serious suicide attempt (yes)^1^1.043 (0.364)8.233.0042.8391.3925.790Other structured psychotherapy−0.970 (0.509)3.641.0560.3790.1401.027Note: The dependent outcome variable *'Suicide'* was dichotomized (Yes = 1, No = 0)Abbreviations: B: beta coefficient, SE: standard error, OR: odds ratio / expected beta coefficient, CI: confidence interval for OR. For each step, the entry testing was based on the significance of the score statistic, and removal testing was based on the probability of a likelihood-ratio statistic. Degrees of freedom (df): 1 in all steps, except for 'Intervention form' (df = 3)^1^ Somatic specialist medical inpatient treatment due to serious suicide attempt; for diagnostic ICD-10 codes, see Appendix^\*^Full model: The six independent predictor variables/covariates entered in the stepwise forward analysis were: 'Number of visits' (continuous variable), 'Intervention form' (categorical variable: Combination therapy, Psychotherapy, Psychotropics (*reference*), Other), 'CBT' (incl. DBT), and 'Other structured psychotherapy', 'Serious suicide attempt', and 'Somatic comorbidity' (dummy coded \[Yes = 1, No = 0\] variablesFull model fit: R^2^ = 0.099 (Nagelkerke). Model χ2 = 23.818; *p* = .001 Spearman correlation analyses revealed significant (*p* \< .01) correlations between the 'number of outpatient visits' and the various psychiatric interventions (*r* = .27 to .46). To further investigate the impact of other variables on suicide, we excluded the strongest variable 'number of outpatient visits' from an additional stepwise logistic regression model, but included all the other variables used in the full model. The results from this model indicated that the intervention consisting of combined pharmacotherapy and psychotherapy treatment was the only intervention that was significantly negatively associated with suicide, while previous severe suicide attempts remained significantly associated with suicide. This additional model explained 8.4% of the variance (Table [4](#Tab4){ref-type="table"}). Table 4Results from a binominal logistic regression (additional model) with a stepwise forward selection method (likelihood ratio). Suicide cases, n = 154; Controls, n = 154Additional model\*\*B (SE)WaldSig.OR (CI)95% CI for ORLowerUpperIntervention form:--12.153.007------ Combination treatment ^2^−1.113 (0.325)11.746.0010.3290.1740.621 Psychotherapy^3^−0.065 (1.010)0.004.9490.9370.1296.788 Psychotropics^4^*reference* Other^5^0.053 (0.401)0.017.8951.0540.4802.316Serious suicide attempt (yes)^1^0.950 (0.357)7.074.0082.5851.2845.206Note: The dependent outcome variable *'Suicide'* was dichotomized (Yes = 1, No = 0)Abbreviations: B: beta coefficient, SE: standard error, OR: odds ratio / expected beta coefficient, CI: confidence interval for OR. For each step, the entry testing was based on the significance of the score statistic, and removal testing was based on the probability of a likelihood-ratio statistic. Degrees of freedom (df): 1 in all steps, except for 'Intervention form' (df = 3)^1^ Somatic specialist medical inpatient treatment due to serious suicide attempt; for diagnostic ICD-10 codes, see Appendix^2^ Combination treatment: psychotropics and psychotherapy (CBT, DBT, PDT, or other structured psychotherapy)^3^ Mono treatment: psychotherapy without psychotropics^4^ Mono treatment: psychotropics without psychotherapy^5^ Other interventions (e.g., supportive conversation)\*\*Additional model: Five predictor variables/covariates entered in the stepwise forward analysis: 'Intervention form', 'CBT' (incl. DBT), 'Other structured psychotherapy', 'Serious suicide attempt', and 'Somatic comorbidity'. The variable 'Number of outpatient visits' was excluded from this model. Additional model fit: R^2^ = 0.084 (Nagelkerke). Model χ2 = 20.155; p = .001 Discussion {#Sec13} ========== The results of the present case-control comparisons indicate that a higher frequency of outpatient visits and provision of combination therapy were significantly negatively associated with suicide, and a history of serious suicide attempts and somatic comorbidity were significantly more frequent in the suicide cases. The results will be further discussed below. Over the 7-year inclusion period, about 45% of total suicide cases in the county had treatment contact with specialist psychiatric care before suicide; this is the same amount as reported by Ahmedani et al. \[[@CR33]\], but somewhat higher than the proportion of around a third reported by Luoma et al. \[[@CR39]\]. The suicide cases received fewer psychiatric treatment interventions and attended fewer outpatient visits than the control group. No case-control differences were found in the number of psychiatric inpatient admissions or the total number of days of psychiatric inpatient care. Compared with suicide cases, a higher proportion of controls received intervention in the form of systematic psychological treatment in combination with psychotropic medication. One possible explanation for this finding is that the suicide cases died during the early course of their disease, i.e., before they had commenced or completed treatment. However, this finding could also indicate that, for various reasons, the suicidal patients received fewer interventions demonstrated to be effective in reducing suicidal behavior. These data support our first hypothesis that controls have a higher frequency of psychiatric outpatient visits than suicide cases. The results also partly support our second hypothesis that controls have a higher occurrence of medical and psychosocial treatment interventions than suicide cases, with the exception of medical treatment as mono-therapy, which was significantly more frequent in the suicide cases, and the occurrence of ECT, which was equally prevalent in both groups. The number of psychotherapy sessions did not differ between the groups, and hence did not seem to protect from suicide as a single factor. However, as part of the combination treatment (psychotherapy and medication), the presence (rather than quantity) of psychotherapy seemed to lower the risk of suicide. In addition, we found somatic comorbidity that required somatic hospitalization to be significantly more prevalent among suicide cases. This is in line with other studies \[[@CR8], [@CR29], [@CR40]--[@CR43]\] reporting an elevated risk of suicidal behavior or completed suicide with the occurrence of a somatic disease. To reduce the risk of suicide in patients with somatic comorbidity, it is important that psychiatric interventions (for example, appropriate pain management) are offered close in time to critical events in the course of the somatic illness, i.e., at initial diagnosis, times of deterioration or relapse, and the transition to palliative care \[[@CR42], [@CR44], [@CR45]\]. Somatic comorbidity, used as a covariate in our study, did not independently contribute to increasing suicide, nor was it identified as a unique factor associated with suicide in the multivariate analysis. By contrast, a severe suicide attempt during the last 2 years before suicide was independently associated with suicide, which is in accord with several other studies \[[@CR5], [@CR9], [@CR28], [@CR42], [@CR46]\]. The suicides in this study occurred in a median time of 11 (females) or 12 days (males) from the patients' last time of contact with psychiatric outpatient services. This might indicate that suicidal patients require frequent outpatient visits or close follow-up. This points in the same direction as earlier research \[[@CR5]\] and the global suicide prevention strategy of the World Health Organization (WHO) \[[@CR47]\], which emphasizes follow-up as one of the most important methods of suicide prevention. Continuation of treatment beyond the stage of clinical recovery has also been found to be protective in patients with a high risk of suicide \[[@CR32]\]. Strength and limitations {#Sec14} ------------------------ The present study has several strengths. First, suicide data were extracted from the National Causes of Death Registry, thus ensuring that all suicide cases within Örebro County were included. Second, suicide cases and controls were matched according to factors previously associated with suicide risk, i.e., age, sex, and primary psychiatric diagnosis, and both suicide cases and controls were current or past patients within psychiatry \[[@CR5], [@CR48]\]. The successful matching using stringent diagnostic criteria (three digits) on primary ICD-10 F-diagnoses for the majority (73.4%) of the sample is a strength. This means that in the majority of the suicide cases, the levels of severity were also taken into account through matching with a control with a similar severity of symptoms (for example, the third diagnosis digit specifies if a depression**/**recurring depression was mild, moderate, or severe). Other facts that indicate that important aspects of the suicide case and control groups were similar are that the groups did not differ significantly in the distribution of comorbid substance use, educational level, living situation, employment, or follow-up time. A third strength was that the retrospective design made it possible to collect data for all patients who died from suicide, regardless of the level of suicide risk before death. In randomized controlled trials, patients with an immediate suicide risk are often excluded for patient safety reasons \[[@CR49]\]. A fourth strength was the use of data from electronic psychiatric medical records. The WHO \[[@CR47]\] has emphasized the need to use data from hospital-based systems to develop strategies to prevent both suicide attempts and completed suicides. The electronic psychiatric medical records contain important data on treatment interventions implemented not only by psychiatrists, but also by psychologists, psychotherapists, psychiatric nurses, and other medical personnel. The use of such data (so far not collected in the Swedish national patient register) enabled us to examine whether the patients benefited from a combination treatment. Fifth, two major risk factors for suicide were controlled for as covariates in the forward logistic regression analysis. The present study also has limitations. First, the associations between suicide and fewer visits to outpatient psychiatry or less combination treatment might be explained by factors not possible to control or match for. For example, patients who are suicidal may be offered more frequent visits, but may choose to decline further visits or interventions for unknown reasons. A poor adherence to treatment is identified as another factor that increases the risk of suicide \[[@CR13], [@CR32]\]. Alternatively, fewer visits or less combination treatments could also be a result of a clinical decision where a psychologist may be reluctant to offer psychotherapy to severely symptomatic and functionally impaired patients because of their potentially high suicide risk. Psychotherapy is one of the drivers of frequent outpatient visits in psychiatry. Severely functionally impaired patients may be offered support by community services outside of psychiatric care, and such data are not included in the psychiatric records. We found no way to retrospectively collect data on the level of function in suicide cases, and were therefore unable to compare the groups in this respect. Moreover, help-seeking behavior among patients might influence both the frequency of psychiatric outpatient visits and the risk of suicide. Therefore, selection effects may have influenced the negative associations found between suicide and more frequent outpatient visits and interventions. In addition, the occurrence or number of psychiatric inpatient admissions, involuntary treatment periods, or differences in follow-up time between suicide cases and controls may have affected the possible number of outpatient visits. However, the present study found no significant differences between the groups in this regard, and it does not therefore explain the limited numbers of outpatient visits among the suicide cases. Another limitation of our study is that it was not possible to match for all comorbidities or other factors that are assumed to increase the suicide risk; for example, levels of comorbid anxiety. A recent large prospective study of comorbid anxiety disorders in mood disorder patients found no significant differences in survival curves for patients with or without anxiety comorbidity \[[@CR50]\]. However, other well-known risk factors for suicide, such as previous suicide attempts and somatic comorbidity, which differed significantly between our suicide cases and controls, were controlled for in the multivariate analysis. Furthermore, we lacked information on treatments and outpatient visits in clinics outside of the Region Örebro County, including private psychotherapy. However, all outpatient visits within the county health care system are likely to have been recorded in the electronic medical records, although some data regarding the content of some visits may have been missing. We also lack information about referrals. Nevertheless, the quality of the medical record content is unlikely to have differed between cases and controls. Another limitation was the relatively small number of patients included in the study, which precludes firm conclusions regarding the effects of certain interventions, including ECT. Participants were not matched for time passed since first contact with psychiatry, which may limit the comparability between the groups. Finally, suicide risk may have been influenced by non-health care system factors, such as negative life stressors shortly before death \[[@CR51]--[@CR54]\]. Although such factors were beyond the scope of the present analyses, they could add valuable knowledge if they were to be included in future research. Conclusions {#Sec15} =========== In the studied sample, completed suicide was significantly negatively associated with the quantity of psychiatric outpatient visits and the presence of psychotropic medication combined with psychotherapy, but not with the number of inpatient treatments or the quantity of psychotherapy sessions. Underlying causes for these differences need to be further studied before any firm conclusion can be drawn on whether the studied interventions may have a protective effect against suicide, or whether the associations may have been attributable to other factors that were not possible to control for in this study. Appendix {#Sec16} ======== ICD-10 codes used in the study {#Sec17} ------------------------------ *ICD-10 codes in suicide attempts that required somatic inpatient care (cases).* **T060.** **T148.** **T213, T292.** **T312.** **T424, T427, T432, T438, T439.** **T509, T519, T559,** **T659.** **T719.** ***ICD-10 codes in suicide attempts that required somatic inpatient care (controls).*** **T424, T425, T427, T432, T433, T436,** **T509, T510, T519.** **Somatic comorbidity, ICD-10 codes during somatic hospitalization (cases).** A: A414. B: B182. C: C549, C569, C649. E: E871. G: G122 (2 patients), G250, G442, G931. H: H811. I: I109, I210, I269, I479, I489, I495. J: J441, J690. K: K250, K400, K550, K859, K860. L: L890. M: M059, M161, M170, M245, M329, M511, M544, M545, M549, M628. N: N409. O: O049. R: R074, R119, R509, R559, R568, R568. **Somatic comorbidity, ICD-10 codes during somatic inpatient hospitalization (controls).** A: A045. B: B182. C: C649. D: D259. G: G403, G431, G459. H: H811 I: I200, I269, I639J: J159, J441, J939. K: K359, K501, K567, K579, K829. M: M059, M171, M191, M243, M462. R: R074, R104, R401, R568. CBT : Cognitive behavioral therapy CT-SP : Cognitive therapy for suicide prevention DBT : Dialectical behavior therapy ECT : Electroconvulsive therapy ICD-10 : International Statistical Classification of Diseases and Related Health Problems,10th Revision PDT : Psychodynamic therapy **Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. We would like to acknowledge the efforts of Hedda Linderstam, Emilia Gustad, Anna Wadefjord, Anna Karlsson, Ann-Kesti Strandell, and Ville Cato during the data collection, the matching process and analyses. TS made substantive contributions to the design of the study, collection of data, matching process, analysis and interpretation of data, and drafting and revising the manuscript. FH, AN and MT were involved in designing the study, interpretation of data, and drafting and revising the manuscript. All authors have read and approved the final manuscript. Financial support for this study was provided by the Foundation for Medical Research 'Nyckelfonden', which is based at the University Hospital of Örebro, Sweden \[OLL-621651, 2016\], and by the Region Örebro County Research Committee \[OLL-483691\]. None of the funding bodies had any part in designing the study, the data collection, the analyses, the interpretation of data, or in writing the manuscript. Open access funding provided by Örebro University. The dataset analyzed during the current study is available from the corresponding author on request. This study was approved by the Regional Ethics Review Board in Uppsala, Sweden (D. no 2015/138). The requirement for informed consent from control subjects was waived due to the retrospective nature of the analyses. All other study procedures were performed in accordance with the Declaration of Helsinki. Not applicable. The authors declare that they have no competing interests.

By 2030, 72.1 million people in the USA will be 65 years or older and will represent 20% of the US population, expanding the need for speech-language pathology services while increasing costs ([@b1-ijt-09-25]). Not only are demographics changing, but people are also experiencing extended work days reducing the capacity to commit to in-person services ([@b5-ijt-09-25]; [@b12-ijt-09-25]). Equitable access to services continues to challenge current service delivery models as evidenced by ongoing difficulties with recruitment and retention of speech-language pathologists in rural and remote areas and by servicing bilingual populations with qualified speech-language pathologists ([@b5-ijt-09-25]; [@b12-ijt-09-25]). Furthermore, many individuals with communication disorders also have co-occurring physical disabilities that prohibit access to in-person services. Telepractice may offer a solution by providing convenient and cost effective access to speech-language pathology (SLP) services at a distance. While the advantages of telepractice are obvious in terms of reducing costs and improving access, another benefit of telepractice is found with the provision of services to clients in their functional environments, which is considered best practices in many areas of rehabilitation ([@b11-ijt-09-25]) and is supported by the World Health Organization (WHO) intervention framework ([@b13-ijt-09-25]). Telepractice has the potential to improve client outcomes by targeting the functional environment, sustaining services, facilitating self-management, and reducing costs. A 2002 survey conducted by the America Speech-Language-Hearing Association (ASHA) revealed that fewer than 21% of respondents had received training in telepractice methods ([@b2-ijt-09-25]). Of those who were trained, 47% reported receiving on-the-job training, 44% completed continuing education courses, and 19% were trained during graduate school. According to a national survey that evaluated the current state of telepractice training in graduate programs, only 26% of the reporting universities were providing academic and clinical training in telepractice ([@b9-ijt-09-25]). A more recent 2016 ASHA telepractice survey indicated that 58.5% of respondents received telepractice training by an employer, while only 6.9% of respondents had received telepractice training in graduate school ([@b3-ijt-09-25]). When comparing the results of all three surveys over a 14-year period from 2002 to 2016, there appear to be differing results related to receiving telepractice training in graduate school. Comparing the 2002 and 2016 ASHA surveys, there were more clinicians trained in telepractice methods in graduate school in 2002 than in 2016 ([@b2-ijt-09-25]; [@b3-ijt-09-25]). That goes against what is expected considering that telepractice is more prevalent now and widely accepted, then it was 14 years ago. Also, the 2015 graduate school survey ([@b9-ijt-09-25]), reported that 26% of graduate programs were providing telepractice training; however, only 6.9% of respondents reported receiving training in graduate school in the 2016 ASHA survey ([@b3-ijt-09-25]). It is anticipated that the number of graduate programs offering telepractice training will only increase as time advances. The differing results could have several explanations. One explanation may be that while 26% of graduate programs provided telepractice training, perhaps not all students received the training. A second explanation may be that the participants of the survey may have graduated from programs before telepractice training was offered. Regardless of the difference in results, it is clear that most clinicians do not receive telepractice training in graduate school. The majority of the training is occurring with employers. The previous surveys provided information about demographics of clinicians and clients being served by telepractice, areas of service delivery, sources of training, and preparation for telepractice ([@b2-ijt-09-25], [@b3-ijt-09-25]; [@b9-ijt-09-25]). To design effective telepractice models with clients and to train SLP graduate students in telepractice methods, further information about telepractice was needed regarding costs, methodology differences between in-person and telepractice, types of learning opportunities offered, and manipulation of the client's environment from clinicians who are currently using telepractice as a service delivery model. A survey was created and administered targeting the need for further information. This article will describe the results of that survey. METHODS ======= DESCRIPTION OF THE SURVEY AND SAMPLING PROCEDURES ------------------------------------------------- The survey consisted of seven sections, 66 questions, and was approved by West Chester University's Institutional Review Board. The seven sections were (1) demographics, (2) licensing and licensure regulations, (3) costs and equipment, (4) synchronous and asynchronous learning opportunities, (5) use of the client's environment and caregiver/e-helper interactions, (6) method adaptations, and (7) overall impressions of telepractice. A majority of the questions required a response from a selection of multiple choice options. Some of the questions were answered with either Yes or No. There were some open-ended response options where participants could provide comments. For the multiple choice questions, participants could select more than one response when it was appropriate to do so. To ensure that experienced telepractice practitioners participated in the survey, the survey was sent to ASHA Special Interest Group (SIG) 18 affiliates through the Community Discussion Board as a web-based Qualtrics survey link. In addition, the same Qualtrics survey link was emailed to participants who attended the Waldo County General Hospital Speech-Language Pathology Telepractice Training program in Maine, USA. The survey was open and accepting responses over the summer of 2016. After respondents offered their informed consent to participate in the survey, they were directed to the first question of the survey. If respondents did not offer their informed consent to participate, then the survey ended. There were 67 participants; 59 SLPs and four audiologists. Sixty-two of the 67 respondents were providing telepractice services at the time of survey. The following results section will be organized by the seven overall sections within the survey. RESULTS ======= PROFILE OF THE PARTICIPANTS BY DEMOGRAPHICS ------------------------------------------- The majority of respondents were servicing clients via telepractice from the ages of 6--17 years. Interestingly, all ages were represented from under six months of age to above 75 years. Treatment was the most common service offered across 93% of the respondents. Supervision of graduate student clinicians and clinical fellows were reported by 15% and 12% of the respondents, respectively. Half of the respondents reported consulting with other professionals about clients without the client or caregiver present and half of the respondents indicated that telepractice was being used for follow up or monitoring of previously learned skills. Half of the respondents reported using a hybrid approach (i.e., both in-person and telepractice sessions) to service clients. Fifty-six percent of respondents reported using only telepractice to service clients. Reasons given for using a hybrid approach versus telepractice only were: requirements of the state, professional judgment based on initial interview and assessment, distance, computer skills, and parent/caregiver involvement. Only 22% of the respondents have denied a client from participating in telepractice services and 37% of the respondents had recommended a switch from telepractice sessions to in-person only sessions. Reasons given were: client skills were better served via in-person, comorbidity (i.e., blindness, deafness, limited mental capacity, and severe dysphagia, etc.) or behaviors which significantly compromised the ability to participate in the virtual environment, bias of some team members, poor support at home, bad internet connection, and feeding therapy. Forty-three percent of respondents indicated that clients were charged a cancellation rate if the client ended telepractice prematurely or missed a planned session. The amount varied from \$20 to half the original rate to the full rate. Forty-three percent of the respondents were self-employed and 49% were employees of governmental agencies, public, private, and non-profit organizations. Sixty-nine percent of the participants indicated that they were using telepractice for their primary employment. For the respondents who were not using telepractice as part of their primary employment, 59% were self-employed. Schools (i.e., preschool, elementary, and secondary) were the most common facilities for serving clients via telepractice as indicated by 91% of respondents. The second most common was in the client's home as indicated by 56% of the respondents. Thirteen percent for international and 11% for special day/residential schools were third and fourth, respectively. Related to Health Insurance Portability and Accountability Act ([@b10-ijt-09-25]) compliance, 84% reported using a platform that was promoted as HIPAA compliant, 48% indicated that the client signs a permission form to allow telepractice, 56% had written policies and procedures related to HIPAA, and 58% used HIPAA policies established by the employer. Participants indicated the security measures that were in place to ensure no breaches in confidentiality. Sixty-five percent used unique passwords, 62% used encryption, 60% used a secure connection via virtual private network, 53% used unique meeting numbers, and 50% used hardware/software firewalls. PROFILE OF THE PARTICIPANTS BY LICENSING AND LICENSURE REGULATIONS ------------------------------------------------------------------ The respondents reported on the number of state licenses that they maintained: one state (39%), two states (28%), three states (17%), and four or more (15%). Thirty-four percent of respondents reported that they were restricted from doing telepractice due to state licensure regulations, whereas 46% indicated that they were not restricted. One restriction that varies across states may involve providing assessment by telepractice. Eighty-eight percent of participants indicated that the states in which they are licensed allow assessment via telepractice. PROFILE OF PARTICIPANTS BY COSTS AND EQUIPMENT ---------------------------------------------- Forty-nine percent of participants indicated that the money needed to begin/implement telepractice was from \$500--\$2000 and 22% indicated that over \$2000 was needed. The participants included the following in the estimate: computer, headphones, microphone, software, marketing materials, telepractice training, licenses in various states, and high speed internet. The large range of costs may be explained by whether or not the respondents were employed by a government agency (49%) or self-employed (43%). Practitioners who were self-employed probably had increased costs to implement telepractice. Fifty-five percent of respondents indicated that costs including training were not reimbursed by an employer. The necessary equipment needed to begin/implement telepractice was determined by a telepractice continuing education course (56%), personal trial and error (45%), internet search (43%), and consulting with a clinician already involved in telepractice (43%). The type of web camera and microphone used varied slightly across client and clinician. According to the respondents, the client was more likely to use the internal microphone (50%) and the internal web camera (63%) on the device. The respondents indicated that the use of internal versus external microphones by clinicians was essentially the same at 41% and 40%, respectively. According to the respondents, clients preferred laptops (59%) over desktops (35%) with only 5% of clients preferring tablets. Clinicians recommended laptops to the clients (57%) and desktops (42%) with no recommendations for tablets or smartphones. In a majority of responses, the equipment needs were supplied by both the clinicians and the clients (46%) and in some cases by the employer (25%). According to the respondents' judgment, technical difficulties may interfere with telepractice occasionally (53%), rarely (36%), and frequently (8%). PROFILE OF PARTICIPANTS BY SYNCHRONOUS AND ASYNCHRONOUS LEARNING ---------------------------------------------------------------- ### OPPORTUNITIES Synchronous learning opportunities are conducted live and in real-time with both the client and clinician present, whereas asynchronous learning opportunities are completed by the client only outside of the live sessions. Synchronous interactions are typically held through videoconferencing. Respondents indicated the type of platform used for synchronous exchanges: WebEx (42%), Zoom (35%), and other (28%) were most common. The other platforms included VSee, Vidyo, GoToMeeting, custom employer platform, and WiZIQ. Reasons for choosing the various platforms were cost, familiarity, ease of use, encryption, employer-provided, quality, security, reliability, recommendation from the Waldo County General Hospital Speech-Language Pathology Telepractice Training program, and consistency with HIPAA standards. Asynchronous experiences were typically offered through three main forms of delivery: email (73%), recorded videos (38%), and custom programs (20%). The three most common types of asynchronous opportunities involved the following: homework exercises (81%), recording speech samples (31%), and recording communication interaction samples (27%). Clients typically completed the asynchronous activities less than once a week (29%), once a week (20%), once a day (18%), and other times (29%) varied based on client need and involvement of caregiver. PROFILE OF PARTICIPANTS BY USE OF CLIENT'S ENVIRONMENT AND CAREGIVER/E-HELPER ----------------------------------------------------------------------------- Seventy-two percent of respondents indicated using the client's environment (i.e., the setting in which the client lives, works, and plays) during telepractice sessions. Sixty percent reported using the environment synchronously and 29% reported using the environment asynchronously. For assessment, the client's environment was never used (33%), rarely used (17%), sometimes used (26%), frequently used (12%), and always used (10%). For treatment, the client's environment was never used (9%), rarely used (10%), sometimes used (40%), frequently used (17%), and always used (12%). Eighty-nine percent of respondents reported using communicative partners in the client's environment to utilize telepractice methods. The most common communicative partners used were: caregiver (59%), e-helper (48%), other (30%), children (19%), spouse (17%), and grandparents (15%). Other communicative partners included: parent, teacher, coworkers, instructional aide, and classmates. Based on the judgment of the respondents, 59% indicated that the use of caregivers was used to its full potential in telepractice methods. Forty-five percent reported using caregiver intervention differently for in-person sessions as compared to telepractice sessions, whereas 55% of respondents indicated that caregiver interactions were not used differently. The domains of caregiver practice in telepractice included: assisting with technology (85%), generalization of newly learning behaviors (73%), practice newly learning behaviors (67%), homework (58%), direct intervention (30%), and assist with assessment (26%). Fifty percent of respondents reported that use of caregivers in telepractice was sometimes needed and 29% reported that use of caregivers in telepractice was frequently needed. PROFILE OF PARTICIPANTS BY METHOD ADAPTATIONS IN TELEPRACTICE ------------------------------------------------------------- The length of typical telepractice sessions reported by the respondents varied from 15--30 minutes (26%), 30--45 minutes (42%), 45--60 minutes (28%), and over 60 minutes (3%). Respondents indicated that the session length between in-person and telepractice was essentially equal (80%). The respondents indicated that the following domains needed to be adjusted for effective telepractice sessions: technology (83%), therapy materials (64%), cueing (verbal/visual/physical/tactile, 57%), caregiver/e-helper interaction (55%), environment (46%), reinforcement (39%), SLPs communication (37%), time (26%), and frequency of sessions (8%). Seventy-three percent of respondents indicated that telepractice requires different skills from traditional in-person sessions. The following methods need to be adjusted for telepractice sessions: therapy targets to match technology (69%), motivation (67%), administering standardized tests to a child (63%), reinforcement (61%), cueing (55%), home program/exercises (36%), administering standardized tests to an adult (30%), helping parent administer test to child (13%), and other (5%). Eighty-four percent agreed that additional training was required for telepractice. PROFILE OF PARTICIPANTS BY OVERALL IMPRESSIONS OF TELEPRACTICE -------------------------------------------------------------- Respondents indicated that telepractice improved their ability to work with clients (36% agree and 35% strongly agree). Seventeen percent were neutral as to whether or not telepractice improved their ability to work with clients and only 10% strongly disagreed with the statement. Ninety-six percent of the respondents reported that, in their opinion, clients were satisfied with telepractice, while 92% of the respondents indicated that clinicians were satisfied with therapy delivered via telepractice. The respondents indicated their client's main complaints about telepractice were using equipment (33%), other (21%), and prefer in-person interaction (14%). Respondents indicated that other complaints included: auditory issues with the microphones, not paid for by insurance/Medicare, and slow internet. DISCUSSION ========== As telepractice models are designed and SLP graduate programs facilitate training in telepractice, some key issues need to be addressed that were highlighted from the results of the current survey. One, either a hybrid approach (i.e., in-person and telepractice) or a telepractice only approach can be used with clients. Deciding which one is best for the client is a major consideration. As we train graduate students in telepractice, we need to help them develop the ability to determine the best approach for each client. Two, licensure regulations by state need to be targeted in the training of future practitioners; for example, investigating state laws on providing assessment via telepractice is one type of regulation that future practitioners must know when implementing telepractice. Three, costs of telepractice may extend beyond equipment and include additional training, marketing materials, and maintaining multiple licenses. Clinicians need to consider all that is needed for costs and equipment to be successful with telepractice. Four, choosing a software program for synchronous exchanges is a major consideration. Clinicians need to uphold HIPAA standards and client confidentiality by creating HIPAA compliant procedures and methods. Future practitioners need to be trained on how to develop such procedures and methods. Five, as we train future clinicians and design new telepractice models, asynchronous learning opportunities need to be explored and become more prevalent in both in-person and telepractice sessions. Clients and clinicians work together synchronously for a finite amount of time during each week. Asynchronous opportunities extend that finite time to the client's functional environment. Using caregivers or e-helpers offers an advantage by integrating increased use of asynchronous opportunities. Six, skills of clinicians need to be developed on how to use the client's environment and the caregiver or e-helper with activities beyond help with technology. Both the environment and caregiver/e-helper need to be more involved with direct intervention of newly learning skills. The use of the environment in improving client outcomes is supported by the [@b13-ijt-09-25]. Seven, the session length may be equal between in-person and telepractice, but methods must be adapted to be effective in the telepractice environment. Such methods include: communication style and timing, motivation, therapy targets, cueing, reinforcement, etc. In conclusion, the results of the survey have provided additional information from practicing clinicians using telepractice that extend the work of previous surveys focused on demographics, areas of service delivery, and preparation and training for telepractice ([@b2-ijt-09-25], [@b3-ijt-09-25]; [@b9-ijt-09-25]). Such information is needed to design new telepractice models and to facilitate telepractice training in SLP graduate programs. The survey described in this article was supported by the National Institute on Deafness and Other Communication Disorders, R15DC014566, of the National Institutes of Health. The author would like to thank Dr. Ellen Cohn for reviewing the survey and providing feedback on the content. In addition, the author would like to thank Mr. Michael Towey for emailing the Qualtrics survey link to attendees of the Waldo County General Hospital Speech-Language Pathology Telepractice Training program in Maine, USA. Finally, the author would like to thank Ms. Elizabeth Fedak and Ms. Kaeli MacArthur for helping to prepare the survey, organize the results, and present the findings at the Pennsylvania Speech-Language-Hearing Association Conference in 2017.

Introduction {#sec1} ============ Autoinflammatory syndromes are an incompletely understood spectrum of diseases that involve spontaneous inflammation caused by genetic variants of the innate immune system. Unlike autoimmune diseases, no high-titer autoantibodies are associated with the disease process. We report a case of autoinflammatory skin and bone disease that flared within 1 month of replacing alendronate with teriparatide therapy. Teriparatide, a recombinant form of parathyroid hormone (PTH), is used for the treatment of osteoporosis and may be prescribed for dermatology patients requiring long-term prednisone therapy. Bisphosphonates such as alendronate are used to treat osteoporosis and autoinflammatory disease; they possess anti-inflammatory properties and the ability to remodel bone. We hypothesize that replacing alendronate with teriparatide triggered this disease flare through stimulatory effects on inflammatory cytokines, specifically interleukin (IL)-1 and recommend caution when choosing a drug to treat osteoporosis in patients with autoinflammatory skin and bone disorders. Case report {#sec2} =========== A man in his 60s with a 10-year history of pustulosis, pyoderma gangrenosum, dissecting cellulitis, and erosive arthropathy initially presented to our clinic with widespread exudative ulcerations, pustules, vegetative crusted plaques, and keloidal scarring primarily on the back, upper chest, face, head, and neck. Pertinent laboratory test results included a normal white blood cell (WBC) count, normal calcium level, and a negative rheumatoid factor. Erythrocyte sedimentation rate was elevated at 52 mm/h (normal range \[NR\], 0--19 mm/h), and alkaline phosphatase level was elevated at 197 U/L (NR, 45--129 U/L). Wound culture grew 1+ methicillin-susceptible *Staphylococcus aureus*, sensitive to trimethoprim-sulfamethoxazole, which offered little improvement. Adalimumab was initiated, and after mild improvement, dapsone and later cyclosporine were added. His condition continued to worsen; thus, high-dose prednisone along with alendronate therapy for osteoporosis was initiated. After marked improvement of skin and joints, adalimumab and trimethoprim-sulfamethoxazole were continued, prednisone was tapered to 5 mg daily, and cyclosporine and dapsone were gradually discontinued. The patient\'s condition remained well controlled for more than 1 year. On follow-up bone densitometry, baseline osteopenia of the proximal femur and early osteoporosis of the lumbar spine and femoral neck remained unchanged. Based on these findings and endocrinology consultation, alendronate was replaced with teriparatide. Within 1 month of starting teriparatide, the patient\'s joint complaints abruptly flared, and widespread ulcerations and crusted plaques recurred on the scalp, neck, and trunk ([Fig 1](#fig1){ref-type="fig"}). Complaints of eye, knee, and hand pain ensued. Conjunctival redness, knee swelling, and bilateral finger swelling ([Fig 2](#fig2){ref-type="fig"}) were noted on physical examination. Although the patient remained afebrile, his WBC count increased to 24,500 (NR, 4.5--11) with 87% neutrophils (NR, 40%--75%). Calcium level increased to 10.8 mg/dL (NR, 8.7--10.4 mg/dL), and alkaline phosphatase level remained elevated at 209 U/L. No previous WBCs, including those drawn during high-dose prednisone therapy, had been this elevated, and all previous calcium levels had been in the normal range. Wound cultures grew rare *Proteus*, rare β-hemolytic group G *Streptococcus*, and 1+ methicillin-susceptible *S aureus*, again sensitive to trimethoprim-sulfamethoxazole. Teriparatide was promptly discontinued, alendronate restarted, and prednisone increased. Adalimumab and trimethoprim-sulfamethoxazole regimens were continued. After minimal improvement and based on its success in treating other autoinflammatory conditions, anakinra, 100 mg subcutaneous daily, was substituted for the adalimumab. After 30 days of anakinra therapy, the patient\'s skin lesions showed marked improvement with no new ulcerations or pustules. Despite insurance denial of further anakinra and adalimumab, the patient\'s condition steadily improved allowing for tapering of prednisone. At 9-month follow-up his disease was well controlled on prednisone, 5 mg daily. Discussion {#sec3} ========== Autoinflammatory disorders involving skin and joints are a spectrum of disease. They include the following syndromes: pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA); synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO); aseptic multifocal osteomyelitis, periostitis, and pustulosis, seen in deficiency of interleukin-1 receptor antagonist (DIRA); and pyoderma gangrenosum, acne, and suppurative hidradenitis (PASH).[@bib1], [@bib2], [@bib3] Our patient\'s widespread pustulosis, pyoderma gangrenosum, dissecting cellulitis, erosive arthropathy, and response to anti-inflammatory agents indicate his condition exists on the spectrum of these related syndromes. Overproduction of IL-1β was found to occur in PAPA syndrome and is a major therapeutic target of the disorder.[@bib1] Anakinra, an IL-1 receptor antagonist, has proven successful in controlling flares of PAPA and deficiency of interleukin-1 receptor antagonist and improving symptoms in pyoderma gangrenosum, acne, and suppurative hidradenitis and SAPHO,[@bib1], [@bib2], [@bib3] implying a major etiologic role of IL-1β in these disorders. Nitrogenous bisphosphonates have been effective in treating bone involvement in the autoinflammatory syndromes of SAPHO, chronic recurrent multifocal osteomyelitis, and diffuse sclerosing osteomyelitis of the mandible.[@bib4], [@bib5], [@bib6] Patients treated with bisphosphonates for these diseases have shown improvements ranging from decreased pain levels to reduction of lesions on bone imaging.[@bib4], [@bib5], [@bib6] The beneficial effects of bisphosphonates may be attributed to bone remodeling and anti-inflammatory properties, specifically lowering secretions of IL-1β, IL-6, and tumor necrosis factor-α.[@bib4] Teriparatide, an anabolic agent, is a recombinant form of PTH given intermittently to increase number and activity of osteoblasts, ultimately improving bone mass and skeletal architecture.[@bib7] In contrast to bisphosphonates, which lower inflammatory cytokine production, studies have found that PTH increases IL-1 and IL-6 production and acts synergistically with IL-1 to promote bone resorption.[@bib8], [@bib9] We postulate that the initiation of teriparatide concurrent with discontinuation of alendronate flared our patient\'s autoinflammatory disease possibly owing to net stimulatory effects on cytokine production, specifically IL-1. The patient\'s hypercalcemia concurrent with this flare provides evidence of a teriparatide effect, as transiently elevated calcium levels are commonly reported during treatment.[@bib10] Anakinra, an IL-1 receptor antagonist, was successful in improving and inducing remission of our patient\'s disorder, supporting a key role of IL-1 in our patient\'s condition. Although infection might precipitate flares of autoinflammatory syndromes, no new significant pathogens grew on skin culture during our patient\'s flare. He remained afebrile on chronic trimethoprim-sulfamethoxazole therapy. Here we report a change in osteoporosis management contemporaneously linked to an acute flare of an autoinflammatory disorder. Teriparatide is known to promote and work synergistically with IL-1; thus, it is reasonable to believe its use combined with the removal of alendronate\'s anti-inflammatory properties may have led to an increased inflammatory state; we thus recommend caution when choosing a drug to treat osteoporosis in patients with autoinflammatory skin and bone disorders. Funding sources: None. Conflicts of interest: None declared. {#fig1} {#fig2}

Introduction {#Sec1} ============ In paediatric palliative care (PPC), most seriously ill children are predominantly cared for at home \[[@CR18], [@CR31], [@CR43]\]. Therefore, parents of a child with a life-limiting disease (LLD) are confronted with increased caregiving demands, and also have to cope with the inevitability of a premature death of their child \[[@CR12]\]. The spectrum of LLDs requiring palliative care during childhood is broad and heterogeneous. LLDs are generally divided into four categories (Table [1](#Tab1){ref-type="table"}) \[[@CR1]\]. The duration of PPC and the needs of these children vary widely among the categories.Table 1Categories of life-limiting diseases \[[@CR1]\]CategoryDescriptionExamplesCategory 1Life-threatening conditions for which curative treatment may be feasible but can fail. Where access to palliative care services may be necessary when treatment fails or during acute crisis, irrespective of the duration of that threat to lifeCancer; irreversible organ failure of heart, liver or kidneyCategory 2Conditions where premature death is inevitable, where there may be long periods of intensive treatment aimed at prolonging life and allowing participation in normal childhood activitiesCystic fibrosis; muscular dystrophyCategory 3Progressive conditions without curative treatment options, in which treatment is exclusively palliative and may commonly extend over many yearsBatten's disease; mucopolysaccharidosesCategory 4Irreversible but non-progressive conditions causing severe disability, leading to susceptibility to health complications, and likelihood of premature deathSevere cerebral palsy; multiple disabilities such as following brain or spinal cord injury Because PPC is a relatively young specialty, current knowledge on parental caregiving mainly relies on studies in chronically ill children, not facing life-limiting issues of their disease and in children treated for cancer. It shows that the parenting role intensifies and expands beyond routine physical care \[[@CR21], [@CR33], [@CR38], [@CR44], [@CR48]\]. This expanded parenting role includes nursing, technical and emotional tasks, such as providing childcare, learning about the disease and its treatment options, managing their child's disease, organising all aspects of their child's daily life and care and managing their own particular situation \[[@CR4], [@CR11], [@CR12], [@CR21], [@CR39], [@CR44], [@CR47], [@CR48]\]. Studies on parental caregiving in PPC are mainly performed in paediatric oncology and focus on the end-of-life (EOL). Besides the expansion of caregiving tasks, these studies show that parents have to deal with uncertainty and to adapt to an accumulation of losses related to their child's physical and functional decline \[[@CR6], [@CR14], [@CR26]\]. Although parents intend to act in their child's best interest, including a good death, many of them struggle with facing reality and the timely transition from preserving their child at all costs towards being prepared to let their child die \[[@CR2], [@CR10], [@CR14], [@CR16], [@CR23]\]. Moreover, parents emphasise they have to 'navigate uncharted territory' and lack professional guidance, resulting in feelings of isolation and abandonment \[[@CR15], [@CR37]\]. A recent review on chronic care situations in children showed the discrepancy between the parental learning needs and the information provided by healthcare professionals (HCPs), stressing the necessity to elicit the parents' perspectives and to take the families' complete situation into account \[[@CR30]\]. Paediatric illnesses or injuries affect many children and parents because they are often brought into healthcare settings under adverse and often life-threatening circumstances \[[@CR20], [@CR32]\]. These circumstances concern potentially traumatic medical experiences that might lead to stress responses \[[@CR20]\]. As such, parenting a seriously ill child is often approached from the perspective of stress \[[@CR38], [@CR48]\], as is represented in the paediatric medical traumatic stress model \[[@CR20], [@CR32]\]. In PPC, parental caregiving is also considered distressing and potentially traumatic for parents \[[@CR25], [@CR32], [@CR36]\], since living towards a child's death understandably causes disruption and grief \[[@CR12]\]. Studies indicate that parents of children with a LLD, who perceive a high risk of life threat and complications, are at increased risk of post-traumatic stress disorder (PTSD) \[[@CR19], [@CR32]\]. While HCPs cannot protect parents from such risks, they should however try to strengthen the parents' resilience and prevent distress as much as possible \[[@CR12], [@CR34], [@CR35]\]. This starts with an understanding of parental caregiving from the parents' perspective. In addition, since driven by technical and medical improvements PPC may last over many years, a clear understanding of the content of parental caregiving in PPC from the parents' perspective becomes increasingly important. Therefore, the aim of this article is to provide a generic and comprehensive overview of parental caregiving, based on the lived experience of parents caring for a child with a LLD. Methods {#Sec2} ======= To elucidate the parents' perspective, we conducted an interpretative qualitative interview study using an inductive thematic analysis \[[@CR3], [@CR8], [@CR40]\]. This study was part of a larger study to evaluate a pilot with a paediatric palliative care team (PPCT; Box 1). The focus of this article is to provide insight into parental caregiving in PPC from the parents' perspective. The role of the PPCT from the parents' perspective will be described in a separate article.Box 1 Description of the paediatric palliative care team (PPCT)In June 2012, the first Dutch PPCT was initiated as a 3-year pilot project at the Emma Children's Hospital in Amsterdam. The multidisciplinary PPCT consists of five specialised paediatric nurses trained and experienced in PPC, two child life specialists, a psychologist, a social worker and a chaplain. Additionally, two paediatric oncologists and two paediatricians are committed for regular consultation. The PPCT is responsible for the coordination, continuity and quality of PPC, irrespective of the child's place of residence. They strive to avoid acute demands for support by a proactive attitude. The support provided by the PPCT is continuous throughout the disease trajectory, including a 24-h availability and bereavement care. The PPCT bridges the gaps between home and hospital and navigates parents through the complex care processes by regular contact through phone calls, e-mails, and personal visits at home and during hospitalisations. In addition, the PPCT strengthens regular care at home by educating and coaching the other healthcare professionals involved. If regular care fails, the PPCT is competent and qualified to take over the care by providing temporary nursing care at home. For the possibility to discuss patients, maximum exchange of palliative care knowledge and optimal deployment and collaboration between team members, the PPCT has weekly multidisciplinary conferences. Sample {#Sec3} ------ A purposive sample of Dutch-speaking parents of children with a LLD primarily residing at home who were referred to the PPCT from a Dutch university children's hospital (Emma Children's Hospital, Amsterdam) was included. Referral to the PPCT ensured a general agreement among HCPs that PPC was indicated and thus provided access to families of children with a variety of diseases, who could maximally inform us about parental caregiving in PPC \[[@CR28]\]. To capture a wide range of perspectives, variation in selected children was sought with respect to malignant (MD) and non-malignant diagnoses (NMD) and phase of the disease trajectory that increases the need for PPC: the palliative trajectory. Based on literature, four phases of the palliative trajectory were distinguished: diagnostic phase, phase of loss of normality (adjusting to new normality), phase of decline and the dying phase \[[@CR15], [@CR45]\]. Parents of 35 cases were identified as eligible. A member of the PPCT or the treating physician introduced this study to the parents and asked permission for the researchers to contact them. In six identified cases, the introducing HCP considered the parents' situation too vulnerable to inform them about the study. Parents of 29 children were invited by telephone to participate by the researchers. In five cases, parents refused participation. Reasons for refusal were as follows: no time (*n* = 2), too burdensome (*n* = 2) and unknown reason (*n* = 1). In total, 24 mothers and 18 fathers of 24 children were interviewed. For patient characteristics, see Table [2](#Tab2){ref-type="table"}. In three cases, parents (*n* = 6) were intentionally approached to participate after the child's death, and in three cases, a second interview after the child's death was performed with five parents to gain deeper insight into parental caregiving during the end-of-life and dying phase.Table 2Characteristics of the parents (*n* = 42) and their ill child (*n* = 24)CharacteristicsNumber (*N*)Percentages (%)Gender parent Male1843 Female2457Age of parent^a^ \<3025 30--402973 \>40923Marital stage Married/cohabiting3890 Divorced/not cohabiting410Education Low^b^512 Middle^c^1536 High^d^2252Age of child at first interview 0--11^e^4^e^ 1--513^f^54^f^ 5--12729 12--1628 ≥1614Child gender Male1250 Female1250Child diagnosis Non-malignant disease (total)1563  Congenital anomalies1146  Neurodegenerative disease28  Metabolic disease28 Malignant disease (total)938  Central nervous system tumour521  Bone/soft tissue sarcoma28  Neuroblastoma14  Leukaemia14Time since diagnosis 0--6 months28 6--12 months313 1--2 years729 2--5 years833  \> 5 years417Palliative phase at first interview Diagnostic phase00 Phase of loss of normality1563 Phase of decline625 Dying phase313Siblings per case 0521 11146 2729 314Percentages may not equal 100 due to rounding^a^Age of two parents is missing^b^Low: primary school, lower secondary general education, lower vocational education^c^Middle: higher secondary general education, intermediate vocational education^d^High: higher vocational education, university^e^In one case, the interview took place after the child's death^f^In two cases, the interview took place after the child's death Data collection {#Sec4} --------------- In total, 47 individual open interviews took place at home. The interviews were held between August 2013 and November 2015 and lasted from 30 min to 2 h. The interviewers (LV, MK, MB) were independent researchers from a different university from where the PPCT is seated. A topic list (Supplement [1](#MOESM1){ref-type="media"}) based on literature and experts' knowledge was used to guide the interviews. Topics relevant for this study were parenting, parental caregiving, care facilities, parents' life, self-efficacy and family life. Interviews were audio recorded and transcribed verbatim. The study was approved by the research ethics committee of the Academic Medical Centre Amsterdam (June 12, 2013; Reference number: W13_120 no. 13.17.0153). Written informed consent was obtained from all participating parents. Data analysis {#Sec5} ------------- An inductive thematic analysis was used \[[@CR3], [@CR8], [@CR40]\] in accordance with methods that optimise validity and rigour \[[@CR29]\]. During the entire process, three researchers (LV, MK, JvD) supported by a research assistant were involved. They used joint meetings to reach agreement on interpretation of the data and findings and worked towards consensus. Therefore, researcher triangulation was ensured to improve reliability and validity of the analysis. The thematic analysis consisted of three phases. Firstly, the researchers (re)read the transcripts of eight interviews individually to get familiar with common aspects and phrases in the context of the interview \[[@CR3], [@CR8], [@CR40]\]. At least two researchers analysed and initially coded with paper and pencil the eight transcripts individually and compared interpretations together. The meaning of the separate text fragments was determined by interpreting them in light of the whole interview \[[@CR8], [@CR22]\]. The initial codes were recoded, resulting in an adapted code list with themes and concepts at a more abstract and conceptual level \[[@CR8]\]. During the second phase, every new interview was read and discussed by at least two researchers. One researcher (LV) and a trained research assistant coded all transcripts, supported by the software program NVivo10. After coding each transcript, the code tree was evaluated and, if necessary, revised. The different codes were sorted into potential themes, which were defined and refined \[[@CR3]\]. To guide constant comparison, the research team went back and forth between the different steps and the entire dataset to capture the key aspects of the themes in the raw transcripts. Thirdly, the researchers identified the relationship between the themes \[[@CR3]\] and integrated the themes into a descriptive model \[[@CR40]\]. To ensure validity and provide transparency of the results, an audit trail was made by the core members of the research team (LV, MK, JvD), in consultation with the other researchers involved in this study, to record methodological choices and substantive ideas and concepts related to the interpretation of the data. Saturation was reached on a conceptual level. The results were externally validated by two parent association representatives, respectively for children with malignant and non-malignant diseases. In addition, we checked our findings in an expert meeting among nine HCPs experienced in paediatrics, PPC and/or homecare. This has not led to adjustments of the results. Results {#Sec6} ======= Being a 'good parent' {#Sec7} --------------------- All parents expressed their ambition to be a good parent for their child in the extraordinary situation of knowing that their child's life is limited and within an unknown time span where they will have to direct their child's EOL. In response to the perceived vulnerability, the disease-related suffering and the efforts their child had to make due to his/her illness, parents desired to be the best parents their child could wish for. The wish to be a good parent became manifest in three aims parents consciously or unconsciously strived for, as identified from their narratives. In addition, four groups of tasks connected to the aims were identified (Fig. [1](#Fig1){ref-type="fig"}). First, the aims and the way parents struggled to achieve the aims are described. Then, the related parental tasks are presented. Representative quotations were chosen to illustrate the identified aims and tasks (Tables [3](#Tab3){ref-type="table"} and [4](#Tab4){ref-type="table"}).Fig. 1Aims and tasks of parents caring for a child with a LLD Table 3Quotes that illustrate the parents' wish to be a 'good parent' and the parents' aims in the care for their child with a LLD, chosen from eight interviews with parentsThemeQuoteBeing a good parentCase 7: boy, 9 years, NMD. Father: *When he dies, if he could think, this is the way it had to be, it sucks that I had this disease but I could not have had better parents with this disease, to go this route with me. That should be his conclusion on the last day.*\ Case 23: boy, 4 years, MD. Father: *It takes a lot of energy to have such a child, besides that the care takes a lot of energy. You are going to think about many things, whether you like it or not. Do I mind it? No, I do not mind it because he deserves everything, he deserves the best, so I do everything for him. Yes, we give him whatever we can give him. It just takes a lot of energy, that's all. So we are exhausted when we go to sleep in the evening, you are exhausted. But I do not mind.*Controlled symptoms and controlled diseaseCase 21: girl, 3 years, NMD. Mother: *You also want her not to be sick too often, that she has energy, because that was a problem for a long time. For that reason she started the night ventilation. And you see she has more energy and feels good and she has less pneumonia and lower respiratory tract infection, and that was absolutely the main goal. And that remains the goal, to have her as well as possible and that she can enjoy life as much as possible.*Case 20: girl, 6 years, MD. Mother: *Last week I contacted the doctor,* via *a telephone consultation, because I found that she was deteriorating, she started choking, drooled a lot. And her speech was worse. So since last week, in consultation with the doctor, we started the Dexamethasone again. And again today a telephone consultation with her about how it is now and whether that is enough.*A life worth livingCase 2: girl, 4 years, NMD. Father: *My only concern is that she can have fun and enjoy herself and whether that is with a clothes pin or with a ball, having fun is the most important to me. Then this is feasible for her and then it is ok.*Case 23: boy, 4 years, MD. Mother: *Yes, that he has time for as long as possible, and can be light-hearted and have fun, enjoy himself, enjoy being together. That he can enjoy the little things. That we can do the things that give him energy. And yeah, that he is having a good time.*Family balanceCase 14: girl, 1 year, NMD. Father: *Since the birth of Maaike, life has been like riding a large high-speed train going 400 km an hour. And it is very difficult to relax and be there for each other. Look, the most important thing is Maaike and her well-being, but we also have to be there for each other, so Machteld (mother) and I have to work to maintain our relationship, otherwise it will go wrong. But right now being there for each other is proving very difficult. And we both know that we have to do that... but well, Maaike is currently recovering from an illness, and we currently do not dare to leave her in other people's care. So the decision is very simple, you choose Maaike. But we do know that it's very important to go out for an evening, to go out to dinner together, to be with each other. But yeah, right now that's not possible.*Case 13: boy, 5 years, MD. Mother: talking about the time that her son received chemotherapy: *It was just very tough for everyone, also for Jayden (sibling, 6 years), because he had to miss us very often because he was with his grandparents. Yeah and we do not want that anymore. \[...\] Jayden, he has to be ok, he has to come out as unscathed as possible, to come through it. To some extent this is not possible, but you need to support him in the best possible way.*Some quotes are slightly modified to improve readability. Names are fictitious*MD* malignant disease, *NMD* non-malignant disease Table 4Quotes that illustrate the parents' tasks in the care for their child with a LLD, chosen from eight interviews with parentsThemeQuoteProviding basic and complex careCase 21: girl, 3 years, NMD. Mother: *We are busy with everyday things, because she cannot do them herself. You have to make sure she sits one moment and lies down the next. PEG-tube care, make sure her lungs do not fill up, suctioning. Oxygen. And on and on. And also with her bowel movements, that that all goes well. But also that she develops in her own way, so you are also occupied with the standing-table or working her muscles, but also doing something creative with her. It's is always a lot. But it is something that you are already very used to. And, of course, providing medication, that's what you start with, and tube feeding. That kind of stuff I'm actually doing now as part of her daily care.*Case 17: girl, 9 years, MD. Father: *The only kind of care that we have now is the home care; they will replace the tube if Tessa's tube has fallen out, otherwise we do everything ourselves. Administering the chemotherapy, which is easy, and the medication; that's all easy, otherwise she doesn't really have any facilitated care, no.*Organising good quality care and treatmentCase 6: boy, 2 years, NMD. Mother: *There are so many things you have to think about, and that takes a lot of energy. You have to give direction to all the caregivers. And 'PGB' (resources) and the home care organisation, you're also busy with them and then there is a hearing and what not. There is always something. And usually such bureaucratic matters are not settled at once. So it takes you a great deal of time to arrange everything, to make clear to people what you mean and to fill out a lot of forms or call a lot of people again and again to say please hurry up... It really is a kind of full-time job to arrange it all successfully.*Case 22: girl, 6 years, MD. Mother: *Then we were gathering information, exploring this and that... and we discovered that the irradiation method with protons for children with brain tumours did not yet exist in the Netherlands. However, it did exist in Germany, France, Spain, Switzerland. \[..\] So we talked about it with the hospital. After much humming and hawing we went to Germany and did the irradiation there. We were so happy that we chose to do it there, and it didn't bother her at all.*Making sound decisions while managing risksCase 8: boy, 6 years, NMD. Father: *We don't use day care or other child care. And that I think has to do with two reasons, firstly because Linda (mother) is very happy to be with him. And she likes to keep him close to her. And that when I am at work I have peace of mind because he is with Linda when I'm not there. And secondly because we are afraid that if he, for example, were picked up by a taxi and went to school every day, the chance of his getting a respiratory infection would absolutely be increased and that is the second reason not to do that at this time.*Case 23: boy, 4 years, MD. Father: *You have to make a number of decisions very soon. Which road are you taking, which one you're not. \[\...\] It goes very quickly. You get very little time to think calmly, you don't have time to think, you have to make choices. Yes, I found it difficult. But there is no other way. We were told there are three choices, you do nothing, you take the treatment in one particular university hospital or in this university hospital. And we were told all the pros and cons. And we finally said, ok, tomorrow we will call and let you know our choice. And we chose quality of life and quality for our son, that he had the least pain, so we opted for the short-term treatment, and we did not chose science.*Organising a good family balanceCase 6: boy, 2 years, NMD. Father: *That is really divided. At home, we do everything together, but if he gets sick, I go to the hospital with him. My wife stays with the children and takes care of everything at home and comes to the hospital every other day with one of the children or alone. And if he is really doing poorly, then she comes every day. But we need to create a bit of peace and quiet at home, with the other children.*Case 17: girl, 9 years old, MD. Mother: *And the difficulty with Tessa is, you have to explain everything, why do I have to take this and why do I get that? But on the other hand she pulls us through it each time, like okay...we have to... And also the other two, the twins, have to go to school, so luckily we still have our structure to some extent... meals on time, on time to bed. So there is a rhythm. We try to keep to that as much as possible. At half past six it should be quiet here. Then they have to sleep. Then mom and dad also have some time together.*Some quotes are slightly modified to improve readability. Names are fictitious*MD* malignant disease, *NMD* non-malignant disease Aims {#Sec8} ---- The parents' aims were (1) controlled symptoms and controlled disease, (2) a life worth living and (3) family balance. ### Controlled symptoms and controlled disease {#Sec9} All parents described that it was of major importance for them to reach controlled symptoms and controlled disease of their ill child. They mentioned that controlling symptoms was of major relevance for maximal comfort for their child or at least that inconveniences were minimised as much as possible. Additionally, they mentioned that controlling the disease was a matter of preventing loss of their child or maximally prolonging their child's life. At some point during the disease process, parents were informed by HCPs that cure from the disease or preserving life of their child was no longer possible. Despite this information, many parents, both from the MD and NMD group, still had the ambition to find possible treatments to control their child's disease. Therefore, parents constantly worked to reduce, relieve or prevent symptoms of the disease and side effects of the treatment and also searched for optimal treatment to prevent further progression of the disease. When parents believed that controlling the disease was no longer a realistic aim, their focus shifted towards merely comfort care, whereby symptom control remained important. ### A life worth living {#Sec10} Despite their focus on controlled symptoms and controlled disease, parents emphasised seeing their child as a beloved person who deserves a life worth living for the time that is left. They wanted their child to have fun, enjoy his/her life and make as much out of his/her life as possible. Most parents felt challenged to create a life worth living for their child. Especially when their child had limited abilities or when their child deteriorated and lost his/her abilities for life fulfilment, parents put even more efforts towards creating a life worth living. ### Family balance {#Sec11} Parents aimed a variety of aspects that could be categorised as family balance. Family balance is a situation in which all individual family members can keep going, experience well-being and are able to develop within their full potential. It also contains established parental responsibilities, such as earning an income or organising a holiday. Parents felt challenged in achieving this aim because they continuously had to adapt to the demanding care situation and to rearrange family life, while meeting the needs and interests of all family members involved. They mentioned that they had to deal with the limited flexibility in daily life, the disruption of the self-evident togetherness of their family and the siblings' need for attention. Most parents could not immediately leave home for spontaneous or unexpected activities because they needed more time in advance to schedule and prepare activities than 'healthy' families. Planned activities often needed cancelling, for instance when symptoms of their child suddenly worsened. Due to the extensive involvement in childcare and the intensive use of healthcare facilities, including hospitalisations, the opportunity to be together as a family was limited. Parents felt forced to give priority to the needs of their ill child, thus the siblings often came in second place. Many parents noticed that siblings received less attention and often had to wait until they had finished the care, which they could not postpone, for their ill child. Parents felt uncomfortable about this situation but often lacked the opportunity to act otherwise given the urgency of the needs of their ill child. Struggling to achieve all aims {#Sec12} ------------------------------ From the parents' stories, it could be concluded that they became aware of the three aims over time. In addition, they experienced difficulties in balancing the aims because they tried to achieve the three aims at the same time. ### Becoming aware of the aims {#Sec13} Parents mentioned how, initially, they were fully occupied by the aim of controlled symptoms and controlled disease as first priority. Parents emphasised that they, in their perspective, had learnt how to control the symptoms and to limit the burden and progression of the disease and how to respond to their child's care needs. During this learning process, many parents had felt thrown back on their own. However, they became familiar with the disease, the treatment options, their child's special needs and preferences, the healthcare system and what the new world of their child entailed. Most parents had developed their ability to assess, decide on and perform all disease-related tasks; however, some parents continued to struggle with the complexity of achieving controlled symptoms and controlled disease and felt uncertain, particularly when the disease progressed or when complications occurred. For parents, the focus on creating a life worth living for their child was boosted once the LLD was diagnosed or, in MD, when it became clear for parents that treating cancer was no longer considered to be effective. From the start of their child's disease, most parents intuitively felt the importance of a family balance. However, they accepted the disrupted family balance because their first and second aim of controlling symptoms and controlling disease and living a meaningful life had priority. Family balance obtained a clearer focus when the disease trajectory lasted longer and when the disease and symptom management and the child's well-being were at a manageable level. ### Balancing the aims {#Sec14} In the context of their child's inevitable death, parents wanted to do everything as well as possible and tried to maximise all separate aims. However, they experienced that the efforts for creating a life worth living for their ill child and achieving a family balance were easily overruled by the efforts for controlling symptoms and, if possible, controlling disease, because the child's symptoms or disease always intruded to the foreground. Consequently, controlled symptoms and controlled disease appeared to stay the predominant aim for parents. A life worth living for their ill child was the second dominant aim. Parents mainly succeeded herein when they, in their perspective, had controlled the symptoms and, if possible, the disease. Only when their child's death was near, some parents ignored their first aim in order to create a life worth living. For example, while their child had pain and wanted to play with friends, parents decided to delay the start of pain medication in order to enable their child to experience life fulfilment instead of being asleep as a side effect of the medication. Achieving the first and second aim was a prerequisite to work towards a family balance. Therefore, many parents described their family balance as fragile, as it was rapidly disturbed by an increase of the symptoms, progression of the disease or a decrease of the child's well-being. In these situations, the aim for a family balance was easily overruled by the parents' need to control the symptoms and, if still realistic, to control the disease and by their ideal of a meaningful life. Because parents tried to achieve all three aims, they had to keep several balls in the air at the same time. Some parents became aware of the necessity to balance between the aims, were able to develop themselves herein and increasingly took direction to achieve all three aims. For example, some parents realised that they also needed to give attention to their partner, other children and/or friends; otherwise, all these relations would be lost after their child's death. Other parents felt overwhelmed by the multiplicity and complexity of the first aim and were not able to look beyond controlling their child's symptoms and disease. Tasks {#Sec15} ----- With maximal commitment, parents performed many intertwined tasks, originating from the child's disease and the abovementioned aims. Four groups of tasks were identified: (1) providing basic and complex care, (2) organising good quality care and treatment, (3) making sound decisions while managing risks and (4) organising a good family life. The accomplishment of the tasks by parents determined the level of achievement of their aims, varying per family and child. ### Providing basic and complex care {#Sec16} For many parents, the caregiving tasks to achieve controlled symptoms and controlled disease and to create a life worth living were unavoidable and numerous. The caregiving tasks consisted of assisting in the child's activities of daily living (ADL), symptom management, medical technical procedures, offering sleep support, supporting well-being and creating life fulfilment for their child. Many parents described how they learnt to provide complex medical care, such as preparing and providing medication, suctioning, giving tube feeding or fixing a prosthesis. These procedures needed to be attuned to their child's needs, abilities, coping strategies and learned routines and rituals to help him/her to accept undergoing the procedures of which some were life sustaining. All parents felt the need and took their responsibility to monitor the child's physical condition and well-being and to intervene adequately when needed. Consequently, parents had limited control over their efforts for controlled symptoms and controlled disease, including how these efforts influenced their daily life. Besides this responsibility, parents emphasised that they considered themselves as being the best carers because they cared with parental love attuned to their child's needs. Therefore, they found it difficult to leave (complex) care to others, such as HCPs or informal carers. Many parents mentioned that even when they were supported by homecare nurses or during hospitalisations, they still provided many components of care. Additionally, when HCPs or informal carers became involved, parents had to guide them towards providing the care for their child attuned to his/her needs. The intensity of providing basic and complex care largely varied per family and child and throughout the disease trajectory. Some parents could not lose their child out of sight to prevent the risk of severe life-threatening situations, which resulted in 24-h per day caring for their child, whereas others provided mainly routine childcare and facilitated their child to attend school. ### Organising good quality care and treatment {#Sec17} Many parents emphasised how they had searched for the best treatment and professional support. They coordinated care and care facilities and arranged many practical things, such as equipment, reimbursements and medication at home. Some parents also arranged things, such as a special computer or toys, to enable their child to communicate and to develop, which potentially improved the child's well-being. Over time, some parents arranged (more) homecare to have some respite in favour of having time for the siblings or themselves (family balance). Because parents felt final responsible for their child and family, they primarily performed this task. Many parents described this task as time-consuming and difficult because it was an ongoing process, even when their child was stable, and it never succeeded at once. Also, many actors and organisations were involved in creating well-organised childcare, making this task even more complex. Moreover, parents mentioned that this task required many efforts and valuable time, which they preferred to spend with their child. Especially when their child's death was near, parents wanted to enjoy the time left together with their child and not being busy with arranging care and practical things. ### Making sound decisions while managing risks {#Sec18} Parents described that they made numerous, both minor and major, decisions in daily life aimed at maximising accomplishment of the three aims. For example, "is it safe enough to let my 15-year-old son take a shower by himself despite the risk of falling due to his amputated leg?" Because some parents, for instance, decided to accept day care to gain time for the siblings or to let their child attend school because he/she did enjoy this, their ability to control the disease and symptoms according to their own standards could decrease. Therefore, many parents often felt forced to weigh arguments, consider alternatives and make decisions to achieve a justifiable balance between their aims. Especially, making decisions entailed weighing the major risks of losing their child. Parents explained how they decided to maximally protect their child by reducing the risk of worsening the symptoms and/or the disease or provoking a life-threatening situation. This often resulted in protective behaviour that increased their workload. For example, some parents avoided day care to minimise the risk of infection. At other occasions, parents consciously chose not to emphasise the control of symptoms and control of disease and accepted a possible risk of deterioration in favour of a life worth living or a good family balance. Because parents felt responsible for making the right decisions, they often felt tensions, particularly when they had to make decisions that allowed EOL to come or when they had to choose the least bad option. For instance, when new symptoms appeared, they had to choose between accepting the increase of the symptom load of their child or adapting medication running the risk of new or increased side effects. Many parents told that by moments, they felt overwhelmed by the large amount of decisions they had to make and felt overruled by the type and the impact of decisions and the short period of time they had available for making these far-reaching decisions. Some parents mentioned they had to negotiate for sufficient time until they felt comfortable to make a sound decision. ### Organising a good family life {#Sec19} Most parents described that having a child with a LLD affected all family members. In favour of a family balance, they tried to integrate their extraordinary situation in daily life. They supported their child to attach to normal life or embedded their situation in a life as normal as possible. To relieve their workload, many parents chose to divide the caregiving tasks and the responsibility of earning an income. This could result in feelings of shortcoming and having lost relevant aspects of personal development, for one or both parents. Many parents described they discovered in particular that rhythm and routines were helping in integrating caregiving in daily life. Rhythm took form of a daily pattern, providing guidance in the things that had to be done, such as wake up in time, providing childcare, cooking, housekeeping and bringing the children to school. Routines were tasks where they no longer had to think about and which became part of their pattern, for example how to prepare medication or when to give tube feeding. Parents told that by applying family rhythm and childcare routines, they gained time and energy for other activities. When parents were aware of their aim for a family balance, they continuously had to weigh the needs of the individual family members. Sometimes, this meant that they had to reorganise care in favour of the family balance. For example, parents decided that when their child would be hospitalised again, one of them stayed with their child and one of them stayed at home with the siblings to pay them proper parental attention and stay connected. This choice came instead of staying in the hospital of both parents and letting the siblings stay at their grandparents as they did before. Discussion {#Sec20} ========== We identified that, based on their ambition to be a good parent in a situation where parents tried to prevent child loss and had to direct child loss in the end, parents strived for three main aims in caring for their child with a LLD. Parents primarily aimed for optimal controlled symptoms and controlled disease. Over time, the aims of a life worth living and a family balance gained importance. Since the time with their child is finite, parents developed a major need to concurrently achieve each separate aim and felt under pressure because everything had to be as good as possible. However, they could not always succeed herein, resulting in considerable distress for parents. To achieve the three aims, parents performed four interconnected tasks: providing basic and complex care, organising good quality care and treatment, making sound decisions while managing risks and organising a good family life. These tasks were relentless for parents because the accomplishment of the tasks determined the level of achievement of the aims. Although MD and NMD and their disease trajectories differed, the aims and tasks as experienced by parents in both groups were quite similar. From this study, it follows that being a good parent included not only maintaining their child's health and ensuring that their child had a good life, which is earlier described \[[@CR10], [@CR24], [@CR46], [@CR47]\] but also achieving a situation in which all family members could keep going and experienced well-being. This study adds that the parents' aims not only guided parental caregiving but also encouraged them to leave no stone unturned to achieve all these aims, particularly controlled symptoms and controlled disease and a life worth living. As such, many parents were absolutely involved in childcare and ran the risk of a suppressed family life. In addition, the pressure for parents accumulated by doing the utmost in a limited time while also creating a rewarding time. These aspects contributed, together with the threat of future child loss, to the parents' distress and could be an explanation for the increased risk of a PTSD \[[@CR19], [@CR32]\]. Some of the identified parental tasks are earlier reported but not yet in a comprehensive overview within the context of PPC as was done in this study. Like others, we found that parents felt largely responsible to provide childcare by themselves no matter how complex it is \[[@CR4], [@CR6], [@CR11], [@CR12], [@CR44], [@CR47], [@CR48]\] and did everything to organise the best quality of care and treatment for their child. Parents also organised a good family life, which was creating time for themselves and their partner and, consistent with earlier research, maintaining any sense of normal life \[[@CR6]\] and emotionally supporting their child and the siblings \[[@CR4], [@CR21], [@CR27], [@CR39]\]. Parents felt that succeeding in their tasks largely depended on their own efforts, even when they were supported by HCPs. However, their wish to be a good parent was a powerful internal motivator to conduct all tasks, as was till now only indicated in paediatric oncology \[[@CR10], [@CR13]\]. Many parents had felt abandoned and had difficulty in searching for the diagnosis and the best treatment, and in developing parental caregiving, which is in line with studies addressing parents' role to 'navigate uncharted territory' \[[@CR15], [@CR37], [@CR47]\]. Parents mentioned this as exhausting and stressful at times, as was also described by Woodgate et al. \[[@CR47]\]. It was seen that parents became experts in their child's care attuned to his/her needs; however, many of them (had) felt disrupted and sometimes powerless to improve their child's and own situation. Parents felt tensions in searching for support on the one hand and the burden of this support on the other hand, due to the limited time left with their child and the risk of a rapid disruption of their situation. Parents wanted to be there for their child and strived for preserving their parenting role and relationship with their child. Consequently, consistent with previous studies, parents found it difficult to entrust the care to informal caregivers or HCPs who, in their perception, may lack the ability to provide care at the same level as they do \[[@CR6], [@CR45], [@CR47]\]. By refusing to withdraw from their caregiving tasks, some parents sacrificed their emotional and physical well-being, as previously described \[[@CR6], [@CR13]\]. This study showed that it is not only the complex palliative and EOL decisions but also the numerous minor decisions related to daily hassles that required attention and efforts from parents. Many parents felt inexperienced and overwhelmed to make complex and difficult EOL decisions in a limited period of time, as was also seen by Hinds et al. and Carnevale et al. \[[@CR5], [@CR10]\]. This study adds that parents also made many smaller decisions, whereas PPC research mainly focuses on EOL decisions \[[@CR5], [@CR7], [@CR9], [@CR10], [@CR41], [@CR42]\]. While making minor decisions, parents weighed the risks and the aims in daily life because every minor decision could have a major impact on controlling the symptoms and/or disease and the quality of life of their child and family. A further exploration of parental decision-making during the palliative phase could be helpful. This study had some strengths and limitations. It was noticed that some HCPs prevented or delayed participation of eligible parents because they considered them too vulnerable or burdened, which is known as gate keeping and often seen in palliative care research \[[@CR17]\]. This might have resulted in an underestimation of the parents' difficulties and efforts to achieve the aims and to perform all tasks. The sample mainly consisted of native Dutch parents of one university hospital. Differing cultural and ethnic backgrounds were not captured. Nevertheless, we included both mothers and fathers and our sample showed a wide variation in diagnosis and phase of the palliative trajectory. In addition, we were able to provide the perspective of parents who currently cared for their child with a LLD. These aspects enabled us to give a realistic and comprehensive overview of parental caregiving in PPC. Our sample included a relatively large amount of children with NMD. This might have resulted in an overestimation of providing basic and complex care throughout the entire palliative trajectory because these children appeared to be more dependent on parental care for ADL than children with MD. Although Dutch people are relatively highly educated, in this study, they were overrepresented. Highly educated parents might be more capable in searching for and organising the best care for their child and might be more able to take over homecare tasks because their professional positions provided them the flexibility to do so. Therefore, in reality, the parental distress following from the aims and tasks might be even higher than seen in this study. On the other hand, it was noticed that in many families (irrespective of their education level), one parent and during the EOL phase often both parents quitted their job, which in most cases is partially financially supported by the Dutch government, enabling parents of seriously ill children to provide childcare at home. Implications for practice {#Sec21} ------------------------- This study shows more in-depth what parents face and how they combine parenting and caregiving. In addition to the PPC that professionals currently provide to the child and family, it could be helpful to discuss at times on a meta-perspective with parents the content of parental aims, the related tasks and the bottlenecks from both the parents' and professionals' perspective. This is preferably done from the start of the disease trajectory to decrease parents' distress and to strengthen their resilience, since the awareness of their child's LLD often overwhelms them. Based on the meta-perspective, an exploration of what is needed for parents to succeed at home can be made and organised by the parents and their homecare team together with a PPCT. Therefore, they have to define and organise the help and support in a way that is acceptable for and provides relief to parents. For example, help in the household or for other daily tasks or someone to bring a sibling to school or sports. The support should be provided by a (healthcare) professional whom parents trust and who gives active direction to the parents while at the same time not taking over their parenting role. By doing so, PPC becomes tailored to the individual needs of families in PPC and better accepted. Electronic supplementary material ================================= {#Sec22} Below is the link to the electronic supplementary material.ESM(PDF 18.5 kb) ADL : Activities of daily living EOL : End-of-life HCP : Healthcare professional LLD : Life-limiting disease MD : Malignant disease NMD : Non-malignant disease PPC : Paediatric palliative care PPCT : Paediatric palliative care team PTSD : Post-traumatic stress disorder Revisions received: 04 November 2016; 07 Dec 2016; (2) Provided Funding information has to be tagged. We thank all the parents who participated in this study. We also thank Madelief Buijs for conducting the first interviews and Doris Verwijmeren, who supported us as a research assistant, for coding the transcripts. Authors' contributions {#FPar1} ====================== Conception or design of the work: all authorsData collection: LV, MKData analysis and interpretation: LV, MK, JvDDrafting the article: LV, MKCritical revision of the article: MK, ASM, DB, DC, MG, JvDFinal approval of the version to be published: all authors Conflict of interest {#FPar2} ==================== The authors declare that they have no conflict of interest. Funding {#FPar3} ======= The study was sponsored by the Netherlands Organisation for Health Research and Development (ZonMw), Grant Number 82-82100-98-208. The authors declare that they have no financial relationship with this organisation that sponsored the research. Ethical approval {#FPar4} ================ All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent {#FPar5} ================ Informed consent was obtained from all individual participants included in the study. [^1]: Communicated by David Nadal

Many cancer cells are addicted to driver oncogenes or to cancer-selective survival factors, and their proliferation and survival is highly dependent on oncogenic signaling pathways.[@b1],[@b2] Therefore, molecularly targeted drugs that selectively inhibit these pathways are critically important for the pharmacological treatment of advanced cancer.[@b3] Presently, various inhibitors of oncogenic kinase pathways are available for the clinical treatment of cancer, such as inhibitors of oncogenic tyrosine kinases (for example, EGFR, HER2, BCR-ABL, and ALK), the RAF/MEK/ERK pathway, the PI3K/AKT/mTOR pathway, and multikinases.[@b4] However, after treatment with each agent, cancer cells soon acquire drug-resistant phenotypes by several mechanisms including gatekeeper mutations in the target kinases and bypassing of signaling pathways.[@b5],[@b6] To improve treatment outcomes, additional next-generation inhibitors that possess better activity or overcome drug resistance to the primary agent should be further developed. Target validation of agents is critically important for the development of new compounds as clinical antitumor agents. In the initial stages of drug development, high-throughput screens are usually carried out based on enzyme inhibition assays. As a result, candidate agents that have the potential to inhibit target enzymes are screened out. In some cases, however, the agents are found to affect additional target molecules in cancer cells and cause unexpected cytotoxicity during drug development or in clinical trials,[@b7],[@b8] which may mislead the selection of proper cancer subtypes for the agents and cause delay or failure in clinical trials. To ensure rational targeted therapy, target validation of compounds should be carried out with multiple reliable and unbiased methods. Genome-wide gene expression analysis is an unbiased method to evaluate the mode of action of chemical compounds.[@b9] We previously analyzed gene expression data of cancer cells that were mainly treated with classical antitumor agents, including DNA topoisomerase inhibitors, anti-metabolites, and tubulin-binding agents. We showed that the gene signature data reflected the modes of action of the respective agents.[@b10] However, it is still not clear whether this signature-based analysis could widely be applied to classify the target pathways of molecularly targeted agents in cancer. To address these questions, in this study, we comprehensively obtained and analyzed gene expression data of cancer cells treated with 83 anticancer drugs or related agents covering most clinical (small molecule) anticancer drugs, such as oncogenic receptor tyrosine kinase inhibitors and other kinase inhibitors as well as inhibitors of promising molecular cancer targets. Our data indicated that this gene expression-based analysis efficiently classified the oncogenic kinase inhibitors as well as other classes of agents in a target pathway-dependent manner. Our data provide a platform to evaluate molecular pathways or primary cellular targets of compounds for further development of antitumor agents. Materials and Methods ===================== Cell lines and compounds ------------------------ Human colon cancer HT-29 cells, ovarian cancer SKOV3 cells, leukemia K562 cells, and prostate cancer PC3 cells were obtained and cultured as described previously.[@b10]--[@b12] Human lung cancer H2228 cells were obtained from ATCC (Manassas, VA, USA). Human lung cancer PC-9 cells were a kind gift from Dr. Kazuto Nishio (Department of Genome Biology, Kinki University Faculty of Medicine, Osaka, Japan).[@b13] These cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 100 μg/mL kanamycin. The anticancer drugs or compounds used in our analysis are listed in Table[1](#tbl1){ref-type="table"}. The agents were obtained as described in [Table S1](#sd2){ref-type="supplementary-material"}. Stock solutions of the compounds were prepared using dimethyl sulfoxide as a solvent or as described previously.[@b10] We examined the growth inhibitory effect of each agent ([Fig. S1](#sd1){ref-type="supplementary-material"}) and determined the GI~50~ values ([Table S1](#sd2){ref-type="supplementary-material"}). Growth inhibition assays were carried out and the GI~50~ values for each agent was determined as described previously.[@b10] ###### Cancer cell line--anticancer drug combinations used in this study Cell Compound Criteria Target/Mode of action -------------------------------------------------- ------------------------------------------------- ----------------------------------------------- ----------------------- K562 Imatinib BCR-ABL inhibitor BCR-ABL/KIT Dasatinib BCR-ABL inhibitor BCR-ABL/Src Nilotinib BCR-ABL inhibitor BCR-ABL Bosutinib BCR-ABL inhibitor BCR-ABL/Src Ponatinib BCR-ABL inhibitor BCR-ABL (T315I) SN-38 DNA damaging agent Topoisomerase I Doxorubicin DNA damaging agent DNA intercalator/Topoisomoerase II PC-9 Gefitinib EGFR/HER2 inhibitor EGFR Erlotinib EGFR/HER2 inhibitor EGFR Afatinib EGFR/HER2 inhibitor EGFR/HER2 Trametinib RAF/MEK/ERK inhibitor MEK SN-38 DNA damaging agent Topoisomerase I Doxorubicin DNA damaging agent DNA intercalator/Topoisomerase II H2228 Crizotinib ALK inhibitor ALK Alectinib ALK inhibitor ALK SN38 DNA damaging agent Topoisomerase I Doxorubicin DNA damaging agent DNA intercalator/Topoisomerase II SKOV3 Lapatinib EGFR/HER2 inhibtor EGFR/HER2 SN-38 DNA damaging agent Topoisomerase I Doxorubicin DNA damaging agent DNA intercalator/Topoisomerase II HT-29 Vemurafenib RAF/MEK/ERK inhibitor BRAF (V600E) Dabrafenib RAF/MEK/ERK inhibitor BRAF (V600E) Trametinib RAF/MEK/ERK inhibitor MEK U-0126 RAF/MEK/ERK inhibitor MEK Everolimus[†](#tf1-1){ref-type="table-fn"} PI3K/AKT/mTOR inhibitor mTOR Temsirolimus[†](#tf1-1){ref-type="table-fn"} PI3K/AKT/mTOR inhibitor mTOR PP242[†](#tf1-1){ref-type="table-fn"} PI3K/AKT/mTOR inhibitor mTOR BKM120 PI3K/AKT/mTOR inhibitor PI3K BEZ235 PI3K/AKT/mTOR inhibitor PI3K/mTOR AKT Inhibitor VIII PI3K/AKT/mTOR inhibitor AKT 1/2 Regorafenib Multikinase inhibitor VEGFR, RAF, KIT, RET etc Sorafenib Multikinase inhibitor VEGFR, RAF etc Pazopanib Multikinase inhibitor VEGFR, PDGFR,, KIT, FGFR etc Sunitinib Multikinase inhibitor VEGFR, PDGFR, KIT etc Cabozantinib Multikinase inhibitor VEGFR, MET,RET,KIT,FLT1/3/4 etc Vandetanib Multikinase inhibitor VEGFR, EGFR etc Axitinib Multikinase inhibitor VEGFR, KIT, PDGFR etc Gefitinib EGFR/HER2 inhibitor EGFR Erlotinib EGFR/HER2 inhibitor EGFR Afatinib EGFR/HER2 inhibitor EGFR/HER2 Lapatinib EGFR/HER2 inhibitor EGFR/HER2 Crizotinib ALK inhibitor ALK Alectinib ALK inhibitor ALK SU11274 MET inhibitor MET AG1024 IGFR inhibitor IGF1R PDGFR inhibitor V PDGFR inhibitor PDGFR Dasatinib BCR-ABL/Src inhibitor BCR-ABL/Src CDK4 inhibitor Cell cycle inhibitor CDK4 NU6102 Cell cycle inhibitor CDK1/Cyclin B ATM/ATR kinase inhibitor DNA damage check point inhibitor ATM,ATR SB218078 DNA damage check point inhibitor CHK1 CHK2 inhibitor II DNA damage check point inhibitor CHK2 GSK-3 inhibitor IX GSK-3 inhibitor GSK-3 FH535 β-catenin/TCF inhibitor β-catenin/TCF Celecoxib COX2 inhibitor COX2 BI 2536 Mitosis inhibitor Polo-like kinase Aurora kinase inhibitor III Mitosis inhibitor Aurora kinase Docetaxel[†](#tf1-1){ref-type="table-fn"} Mitosis inhibitor Tubulin Paclitaxel[†](#tf1-1){ref-type="table-fn"} Mitosis inhibitor Tubulin Vincristine[†](#tf1-1){ref-type="table-fn"} Mitosis inhibitor Tubulin Trichostatin A[†](#tf1-1){ref-type="table-fn"} HDAC inhibitor HDAC Vorinostat[†](#tf1-1){ref-type="table-fn"} HDAC inhibitor HDAC Romidepsin HDAC inhibitor HDAC HT-29 5-Aza-2′-deoxycytidine DNA methyltransferase inhibitor DNA methyltransferase Decitabine DNA methyltransferase inhibitor DNA methyltransferase Bortezomib[†](#tf1-1){ref-type="table-fn"} Proteasome inhibitor Proteasome Carfilzomib Proteasome inhibitor Proteasome MG-132[†](#tf1-1){ref-type="table-fn"} Proteasome inhibitor Proteasome MLN-4924 Nedd8 conjugation inhibitor Nedd8 activating enzyme 17-AAG[†](#tf1-1){ref-type="table-fn"} Hsp90 inhibitor Hsp90 Geldanamycin[†](#tf1-1){ref-type="table-fn"} Hsp90 inhibitor Hsp90 PKR inhibitor RNA-dependent protein kinase inhibitor RNA-dependent protein kinase (PKR) Ruxolitinib JAK inhibitor JAK TX-1918 Eukaryotic elongation factor-2 kinase inhibitor Eukaryotic elongation factor-2 kinase (eEF2K) Vismodegib Hedgehog pathway inhibitor SMO SN-38[†](#tf1-1){ref-type="table-fn"} DNA damaging agent Topoisomerase I Doxorubicin[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA intercalator/Topoisomerase II Camptothecin[†](#tf1-1){ref-type="table-fn"} DNA damaging agent Topoisomerase I inhibitor Topotecan[†](#tf1-1){ref-type="table-fn"} DNA damaging agent Topoisomerase I inhibitor Mitoxantrone[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA intercalator/Topoisomerase II Etoposide[†](#tf1-1){ref-type="table-fn"} DNA damaging agent Topoisomerase II inhibitor Amrubicin DNA damaging agent Topoisomerase II inhibitor Cisplatin[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA cross-linker Melphalan[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA cross-linker Oxaliplatin[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA cross-linker Neocarzinostatin[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA cleavage Bleomycin[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA cleavage Nimustine[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA alkylator Mitomycin C[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA alkylator 5-FU[†](#tf1-1){ref-type="table-fn"} DNA damaging agent Pyrimidine Gemicitabine[†](#tf1-1){ref-type="table-fn"} DNA damaging agent Pyrimidine Methotrexate[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DHFR 6-Mercaptopurine[†](#tf1-1){ref-type="table-fn"} DNA damaging agent Purine Actinomycin D[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA replication/RNA synthesis Pemetrexed[†](#tf1-1){ref-type="table-fn"} DNA damaging agent DNA/RNA synthesis 2-Deoxyglucose[†](#tf1-1){ref-type="table-fn"} ER stress inducer Glycolysis Tunicamycin[†](#tf1-1){ref-type="table-fn"} ER stress inducer N-glycosylation Thapsigargin[†](#tf1-1){ref-type="table-fn"} ER stress inducer SERCA A23187[†](#tf1-1){ref-type="table-fn"} ER stress inducer Ca2+ ionophore Gene expression data of these compounds were reported previously.[@b10] 17-AAG, 17-*N*-allylamino-17-demethoxygeldanamycin; AKT, protein kinase B; ALK: anaplastic lymphoma kinase; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; BCR-ABL, fusion gene of breakpoint cluster region protein (BCR) and Abelson murine leukemia viral oncogene homolog (ABL); CDK4, cyclin-dependent kinase 4; CHK, checkpoint kinase; DHFR, dihydrofolate reductase; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; FGFR, fibroblast growth factor receptor; 5-FU, 5-fluorouracil; GSK3, glycogen synthase kinase 3; HDAC, histone deacetylase; HER2, human EGFR-related 2; Hsp90, heat shock protein 90; IGF1R, insulin-like growth factor 1 receptor; KIT, mast/stem cell growth factor receptor; MET, hepatocyte growth factor receptor; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; PI3K, phosphoinositide 3-kinase; PKR, protein kinase RNA-activated; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; SMO, smoothened; T-cell factor (TCF); VEGFR, vascular endothelial growth factor receptor. Drug treatment and GeneChip analysis ------------------------------------ For gene expression analysis, we chose a concentration of drugs that were 3- to 10-fold greater than the GI~50~ value and caused \>80% growth inhibition after 48 h of treatment, and gene expression data were obtained after 6 h of treatment[@b10] Drug treatment concentrations and treatment duration for each agent are summarized in [Table S1](#sd2){ref-type="supplementary-material"}. Total RNA was extracted using an RNeasy Mini kit (Qiagen, Hilden, Germany). Microarray analysis was carried out as described previously with the GeneChip Human Genome U133 Plus 2.0 array (Affymetrix, Santa Clara, CA, USA).[@b10] The signature data will be released on our website (<http://scads.jfcr.or.jp/db/cs/>). Statistical analysis -------------------- All analyses were carried out using the statistical programming language R version 2.15.0 (<http://www.r-project.org/>) and Bioconductor version 2.10 (<http://bioconductor.org/>). Data preprocessing ------------------ The R package software of Affymetrix Microarray Suite 5.0 was used to generate signal intensities for each of the HG-U133 Plus 2.0 arrays in the study. Expression values were normalized to a mean target level of 100. Identifying gene signatures --------------------------- Gene sets were extracted and classified as up- or downregulated after exposure to the drug. For each treatment sample, we calculated treatment-to-control ratio statistics, where, if any intensity value was \<50, the value was replaced as 50. For the hierarchical clustering and the principal component analyses, we selected probe sets if the treatment-to-control ratio was \>3 for upregulated genes or less than one-third for downregulated genes and the intensity of at least the treatment or control was \>300 ([Table S2](#sd3){ref-type="supplementary-material"}). To identify the signature gene sets characteristic of some drug subsets, we extracted the probe sets whose expression changes after drug treatment were statistically significantly different between the drug subsets and other agents. Statistical evaluations were carried out using Student's *t*-test. Probes with more than a twofold differential expression and a *P*-value of \<0.05 were extracted. Hierarchical clustering ----------------------- Probe sets for hierarchical clustering comprised the collection of all gene signatures. We carried out hierarchical clustering using the logarithm of the sample and probe set ratio statistics. We used Ward's method for linkage and Pearson's correlation for distance metric. Principal component analysis ---------------------------- We carried out a principal component analysis based on the cancer cell gene expression data to examine 3-D clustering patterns of subclasses of anticancer drugs (oncogenic kinase inhibitors, HDAC inhibitors, proteasome inhibitors, mitosis inhibitors, and DNA damaging agents). We plotted cancer cells treated with anticancer drugs in a 3-D space consisting of three principal components. We used the same probe sets used for hierarchical clustering in this analysis. Gene ontology analysis ---------------------- To interpret the extracted gene signatures, we used gene ontology analyses using the DAVID analytical tool;[@b14],[@b15] this analysis is a method of highlighting relevant gene ontology terms associated with a given gene signature. Analysis with the C-map algorithm (connectivity scoring analysis) ----------------------------------------------------------------- To investigate the relationship between gene signature and compound, we adopted the connectivity score based on the Kolmogorov--Smirnov statistic as developed by Lamb *et al*.[@b9] For each treatment sample, all probe sets were ranked based on the treatment-to-control ratio and the rank matrix was configured using a similar method to Lamb *et al*.[@b9] We modified our program and calculated the connectivity scores for all compounds, as described previously.[@b10] Western blot analysis ===================== Cells were lysed in TNE buffer (150 mM NaCl, 1.0% NP-40, 1 mM EDTA, and 10 mM Tris--HCl, pH 8.0) supplemented with 1× protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and PhosSTOP phosphatase inhibitor cocktail (Roche, Mannheim, Germany). Western blot analysis was carried out as described previously,[@b16] using the following primary antibodies: anti-phospho-p70S6 kinase (p70S6K), anti-p70S6K, anti-phospho-AKT, anti-AKT, anti-phospho-ERK (Cell Signaling Technology, Danvers, MA, USA), anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (Sigma, St. Louis, MO, USA). Results ======= Comprehensive collection of gene expression data related to molecularly targeted, anticancer drug effects --------------------------------------------------------------------------------------------------------- In our previous analysis, we obtained gene expression data from human colon cancer HT-29 cells treated with 35 compounds mainly consisting of classical antitumor agents.[@b10] For comprehensive transcriptomic analysis, we further obtained gene expression data from cancer cells treated with the most commonly used clinical molecularly targeted anticancer drugs, such as inhibitors of driver oncogenes (EGFR, HER2, BCR-ABL, ALK), RAF/MEK/ERK pathway inhibitors, PI3K/AKT/mTOR pathway inhibitors, multikinase inhibitors, HDAC inhibitors, and proteasome inhibitors (Table[1](#tbl1){ref-type="table"}). Alongside the anticancer compounds that are presently in clinical trials, we also included "promising" next-generation targeted inhibitors in our analysis, such as inhibitors of several receptor tyrosine kinases (MET, IGF1R, PDGFR), regulators of the cell cycle/check point (CDK4, ATM/ATR, CHK1, CHK2, Aurora kinase, and Polo-like kinase), β-catenin/TCF, COX2, and NAE.[@b17]--[@b20] We used HT-29 cells because it is a commonly used, solid tumor cell line and we have used it in our previous analyses.[@b10] We obtained transcriptomic data for all the agents in HT-29 cells with the exception of the BCR-ABL inhibitors that did not suppress HT-29 cell proliferation. Moreover, in the cases of drugs whose primary targets preferentially exist in specific types of cancer cell lines, we also used additional cell lines such as BCR-ABL-positive K562 cells[@b21] for the BCR-ABL inhibitors, mutant EGFR-expressing PC-9 cells[@b13] for the EGFR inhibitors, EML4-ALK fusion-positive H2228 cells for the ALK inhibitors,[@b22] and HER2-overexpressing SKOV3 cells[@b23] for the HER2 inhibitor. To estimate the effect of cell type difference on the gene expression analysis, we treated the cell lines with SN38 and doxorubicin, and obtained gene expression data as reference data (the gene expression data will be released on our website, <http://scads.jfcr.or.jp/db/cs/>). Gene signatures reflect the target pathways of molecularly targeted drugs ------------------------------------------------------------------------- As summarized in [Table S2](#sd3){ref-type="supplementary-material"}, we extracted genes whose expression was up- or downregulated by the analyzed agents. To compare the gene expression data of the compounds, we carried out a hierarchical clustering analysis with the acquired 129 gene expression datasets for cancer cells treated with 83 agents (4869 probe sets whose expression was up- or downregulated more than threefold in at least one of the datasets). As shown in Figure[1](#fig01){ref-type="fig"}, we observed that the compounds targeting similar molecules or molecular pathways were clustered together, such as DNA damaging agents, HDAC inhibitors, proteasome inhibitors, and inhibitors of mitosis-related molecules. These results indicate that the gene expression signatures reflect the primary target pathways of the drugs, as shown in our previous study.[@b10] Moreover, in this study, we found that most of the inhibitors of oncogenic kinase pathways formed a large cluster distinct from classical antitumor agents or from other classes of agents. The data for the oncogenic kinase inhibitors in K562, PC-9, and SKOV3 cells were also clustered together with those of the oncogenic kinase inhibitors in HT-29 cells, whereas the data for the DNA damaging agents, SN-38 and doxorubicin, in multiple cancer cell lines were clustered together. Principal component analysis confirmed that the kinase inhibitors were clustered together and that this cluster was distinct from those of other classes of agents (Fig.[2](#fig02){ref-type="fig"}). These data indicate that the kinase inhibitors affect a shared molecular pathway in cancer cells distinct from other classes of antitumor agents. We further extracted signature genes whose expression was commonly modified by oncogenic kinase inhibitors ([Table S3](#sd4){ref-type="supplementary-material"}). Subsequent gene ontology analysis with the DAVID bioinformatics database revealed that several categories of genes, such as those involved in transcriptional regulation or apoptosis, were enriched in the signature genes (Table[2](#tbl2){ref-type="table"}). ###### Gene ontology (GO) analysis of oncogenic kinase inhibitor signature genes GO term *P*-value FDR --------------------------------------------------------------------------------- ----------- -------- GO:0009952 anterior/posterior pattern formation 0.0004 0.0052 GO:0003002 regionalization 0.0013 0.0185 GO:0048806 genitalia development 0.0014 0.0204 GO:0045944 positive regulation of transcription from RNA polymerase II promoter 0.0019 0.0274 GO:0006355 regulation of transcription, DNA-dependent 0.0023 0.0332 GO:0007242 intracellular signaling cascade 0.0025 0.0353 GO:0042127 regulation of cell proliferation 0.0025 0.0355 GO:0051252 regulation of RNA metabolic process 0.0028 0.0397 GO:0042981 regulation of apoptosis 0.0028 0.0400 GO:0043067 regulation of programmed cell death 0.0030 0.0422 GO:0010941 regulation of cell death 0.0030 0.0431 GO:0043065 positive regulation of apoptosis 0.0036 0.0513 GO:0043068 positive regulation of programmed cell death 0.0037 0.0528 GO:0010942 positive regulation of cell death 0.0038 0.0538 GO:0007389 pattern specification process 0.0039 0.0549 GO:0010557 positive regulation of macromolecule biosynthetic process 0.0045 0.0638 GO:0045893 positive regulation of transcription, DNA-dependent 0.0056 0.0785 GO:0031328 positive regulation of cellular biosynthetic process 0.0057 0.0793 GO:0051254 positive regulation of RNA metabolic process 0.0058 0.0812 GO:0007548 sex differentiation 0.0058 0.0815 GO:0009891 positive regulation of biosynthetic process 0.0061 0.0848 Signature probe sets whose expression changes after drug treatment were significantly different between the oncogenic kinase inhibitors and other agents were extracted based on the Student's t-test (fold-change values of more than 2 and the *P*-value of less than 0.05). We carried out GO analyses using the DAVID analytical tool to extract relevant GO terms associated with the gene signature. FDR, false discovery rate. {#fig01} {#fig02} Classification of oncogenic kinase inhibitors based on gene expression signature -------------------------------------------------------------------------------- To examine whether the gene signature analysis could further distinguish the kinase inhibitors depending on their modes of action, we next focused on the gene signatures in HT-29 cells. As shown in Figure[3](#fig03){ref-type="fig"}, within the kinome-targeted agents, drugs with similar target pathways were clustered together, such as: (i) RAF/MEK/ERK pathway inhibitors; (ii) PI3K/AKT/mTOR pathway inhibitors; (iii) EGFR/HER2 inhibitors; (iv) multikinase inhibitors targeting VEGFR and PDGFR (shown as "multikinase inhibitors (1)" in Fig.[3](#fig03){ref-type="fig"}); and (v) multikinase inhibitors targeting VEGFR and RAF (shown as "multikinase inhibitors (2)" in Fig.[3](#fig03){ref-type="fig"}). Analyses of the gene expression signatures of BEZ235, vemurafenib, and gefitinib with the C-map algorithms further confirmed that the signatures of these agents were significantly similar to those of other drugs targeting the same or similar pathways in HT-29 cells (Table[3](#tbl3){ref-type="table"}A--C). These results indicated that the gene signature analysis could classify the kinome-targeted agents in a target pathway-dependent manner. ###### Compounds similar to (A) BEZ235, (B) vemurafenib, (C) gefitinib (10 μM in HT29 cells) and (D) gefitinib (0.6 μM in PC-9 cells) with regards to gene expression changes after treatment Rank Cell Compound Concentration Unit Score Up_score Down_score ------- ------- ----------------------------------- --------------- ------ --------- ---------- ------------ \(A\) 1 HT-29 BEZ235 1.00E-06 M 1.00000 0.9979 −0.99954 2 HT-29 BKM120 3.00E-06 M 0.97648 0.97167 −0.97878 3 HT-29 AKT Inhibitor VIII 1.00E-05 M 0.89995 0.96704 −0.83055 4 HT-29 Temsirolimus 1.00E-05 M 0.87040 0.80446 −0.93412 5 HT-29 PP242 1.00E-05 M 0.85915 0.82501 −0.89109 6 HT-29 6-Mercaptopurine 1.00E-04 M 0.84061 0.70322 −0.97586 7 HT-29 Cabozantinib 3.00E-05 M 0.81859 0.77389 −0.86118 8 HT-29 Crizotinib 1.00E-05 M 0.80053 0.80713 −0.79189 9 HT-29 Lapatinib (10 μM) 1.00E-05 M 0.79923 0.64398 −0.95245 10 HT-29 ATM&\# x002F;ATR kinase inhibitor 1.00E-05 M 0.79442 0.7299 −0.85690 11 HT-29 Methotrexate 1.00E-06 M 0.78992 0.70036 −0.87746 12 HT-29 Sorafenib 1.00E-05 M 0.77319 0.56009 −0.98431 13 HT-29 Everolimus 1.00E-05 M 0.75731 0.78196 −0.73073 14 HT-29 Vandetanib 1.00E-05 M 0.74860 0.72206 −0.77323 15 PC-9 Gefitinib (30 μM) 3.00E-05 M 0.74129 0.63593 −0.84476 \(B\) 1 HT-29 Vemurafenib 3.00E-05 M 1.00000 0.99762 −0.99770 2 HT-29 Cabozantinib 3.00E-05 M 0.95797 0.96799 −0.94347 3 HT-29 U-0126 3.00E-05 M 0.93570 0.89185 −0.97516 4 HT-29 Dabrafenib 1.00E-05 M 0.87493 0.80785 −0.93791 5 HT-29 Vandetanib 1.00E-05 M 0.86775 0.86776 −0.86367 6 HT-29 Sunitinib 1.00E-05 M 0.85795 0.82050 −0.89138 7 HT-29 Sorafenib 1.00E-05 M 0.84555 0.83440 −0.85274 8 HT-29 Regorafenib 3.00E-05 M 0.81791 0.74200 −0.89000 9 HT-29 PDGF inhibitor V 1.00E-05 M 0.77796 0.83640 −0.71588 10 HT-29 Gefitinib (30 μM) 3.00E-05 M 0.77393 0.75086 −0.79339 11 HT-29 Pazopanib 3.00E-05 M 0.74890 0.69189 −0.80240 12 HT-29 Gefitinib (10 μM) 1.00E-05 M 0.74553 0.75449 −0.73308 13 HT-29 PP242 1.00E-05 M 0.73347 0.65308 −0.81042 14 HT-29 AKT inhibitor VIII 1.00E-05 M 0.72928 0.76986 −0.68529 15 HT-29 Erlotinib 3.00E-05 M 0.72384 0.70626 −0.73802 \(C\) 1 HT-29 Gefitinib (10 μM) 1.00E-05 M 1.00000 0.99927 −0.99945 2 HT-29 Gefitinib (30 μM) 3.00E-05 M 0.96045 0.96838 −0.95129 3 HT-29 Erlotinib 3.00E-05 M 0.94112 0.99669 −0.88435 4 HT-29 Sunitinib 1.00E-05 M 0.93169 0.99170 −0.87050 5 HT-29 Sorafenib 1.00E-05 M 0.91256 0.94111 −0.88283 6 HT-29 Pazopanib 3.00E-05 M 0.90385 0.8882 −0.91834 7 HT-29 Lapatinib (10 μM) 1.00E-05 M 0.89179 0.95223 −0.83022 8 HT-29 PDGF inhibitor V 1.00E-05 M 0.80332 0.83498 −0.77063 9 HT-29 Dasatinib 1.00E-07 M 0.76608 0.58031 −0.95086 10 HT-29 Thapsigargin 1.00E-08 M 0.74753 0.95102 −0.54308 11 HT-29 Vandetanib 1.00E-05 M 0.74082 0.89791 −0.58278 12 HT-29 AG1024 3.00E-05 M 0.73856 0.93070 −0.54548 13 HT-29 Vemurafenib 3.00E-05 M 0.72601 0.89795 −0.55314 14 PC-9 Erlotinib (30 μM) 3.00E-05 M 0.70436 0.75877 −0.64905 15 HT-29 Tunicamycin 3.00E-06 g/mL 0.68796 0.88138 −0.49367 \(D\) 1 PC-9 Gefitinib (0.6 μM) 6.00E-07 M 1.00000 0.99652 −0.99634 2 PC-9 Erlotinib (0.6 μM) 6.00E-07 M 0.98035 0.96886 −0.98486 3 PC-9 Erlotinib (30 μM) 3.00E-05 M 0.93176 0.93554 −0.92133 4 PC-9 Gefitinib (30 μM) 3.00E-05 M 0.92112 0.92387 −0.91180 5 PC-9 Afatinib 3.00E-08 M 0.86916 0.82167 −0.91045 6 PC-9 Trametinib 1.00E-06 M 0.60445 0.45342 −0.75116 7 HT-29 U-0126 3.00E-05 M 0.60392 0.58254 −0.62100 8 HT-29 Cabozantinib 3.00E-05 M 0.59445 0.56836 −0.61630 9 HT-29 Vemurafenib 3.00E-05 M 0.58051 0.53379 −0.62308 10 HT-29 PP242 1.00E-05 M 0.54759 0.55305 −0.53822 11 HT-29 Vandetanib 1.00E-05 M 0.52336 0.54008 −0.50290 12 HT-29 Dabrafenib 1.00E-05 M 0.51811 0.40252 −0.63000 13 HT-29 Sunitinib 1.00E-05 M 0.51786 0.45561 −0.57641 14 HT-29 Gefitinib (30 μM) 3.00E-05 M 0.50888 0.5259 −0.48823 15 HT-29 PP242 1.00E-05 M 0.50778 0.5421 −0.46985 AKT, protein kinase B; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein; PDGF, platelet-derived growth factor. Compounds in our data that showed high similarity in their gene signatures to the given compounds were extracted using C-map algorithms. Top 15 data among the acquired 129 datasets are shown. {#fig03} Moreover, we also evaluated the signature of gefitinib obtained in the mutant EGFR-expressing PC-9 cells using the C-map algorithms. The gefitinib signature of PC-9 cells showed significant similarity to those of oncogenic kinase inhibitors of HT-29 cells, including the gefitinib signature in HT-29 cells, while the top hits were other EGFR inhibitors of PC-9 cells (Table[3](#tbl3){ref-type="table"}D). These data indicated that, for the agents whose targets are selectively expressed in certain subtypes of cancer, use of data obtained in specific cancer cell lines could aid accurate evaluation of the drug target pathways based on the signature analysis. To observe biological differences in the signature genes between subclasses of the kinase inhibitors, we further extracted genes that showed significantly selective expression in cells treated with RAF/MEK/ERK and PI3K/AKT/mTOR pathway inhibitors ([Table S4](#sd5){ref-type="supplementary-material"}). Gene ontology analysis revealed characteristic features of each gene set (Table[4](#tbl4){ref-type="table"}). Namely, the gene set specific for the RAF/MEK/ERK pathway inhibitors not only contained genes related to cell proliferation, protein kinase cascades, and cell death, but also genes involved in phosphate metabolic processes. In contrast, the gene set specific for the PI3K/AKT/mTOR pathway inhibitors was characteristically related to erythrocyte homeostasis, response to hypoxia, and angiogenesis, as well as cell proliferation and protein kinase cascades. ###### Gene ontology (GO) analysis of signature genes of (A) RAF/MEK/ERK inhibitors and (B) phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) inhibitors GO term *P*-value FDR ----------------------------------------------------------------------------- ----------- ---------- \(A\) GO:0042127 regulation of cell proliferation \<0.0001 \<0.0001 GO:0008285 negative regulation of cell proliferation \<0.0001 0.0002 GO:0006469 negative regulation of protein kinase activity 0.0001 0.0011 GO:0033673 negative regulation of kinase activity 0.0001 0.0013 GO:0007243 protein kinase cascade 0.0001 0.0013 GO:0051348 negative regulation of transferase activity 0.0001 0.0017 GO:0043407 negative regulation of MAP kinase activity 0.0007 0.0115 GO:0008219 cell death 0.0007 0.0116 GO:0016265 death 0.0008 0.0122 **GO:0006793 phosphorus metabolic process** 0.0008 0.0131 GO:0006796 phosphate metabolic process 0.0008 0.0131 GO:0007242 intracellular signaling cascade 0.0010 0.0158 GO:0045321 leukocyte activation 0.0012 0.0191 GO:0044092 negative regulation of molecular function 0.0013 0.0198 GO:0010557 positive regulation of macromolecule biosynthetic process 0.0013 0.0206 GO:0045859 regulation of protein kinase activity 0.0015 0.0238 GO:0043549 regulation of kinase activity 0.0019 0.0289 GO:0031328 positive regulation of cellular biosynthetic process 0.0019 0.0290 GO:0009891 positive regulation of biosynthetic process 0.0021 0.0322 GO:0051338 regulation of transferase activity 0.0024 0.0363 GO:0040012 regulation of locomotion 0.0026 0.0397 **GO:0019220 regulation of phosphate metabolic process** 0.0026 0.0402 **GO:0051174 regulation of phosphorus metabolic process** 0.0026 0.0402 GO:0051270 regulation of cell motion 0.0026 0.0406 GO:0001775 cell activation 0.0029 0.0446 GO:0002521 leukocyte differentiation 0.0040 0.0609 GO:0000188 inactivation of MAPK activity 0.0043 0.0655 GO:0043405 regulation of MAP kinase activity 0.0052 0.0784 GO:0045449 regulation of transcription 0.0057 0.0849 GO:0006366 transcription from RNA polymerase II promoter 0.0060 0.0896 GO:0051252 regulation of RNA metabolic process 0.0061 0.0913 GO:0030097 hemopoiesis 0.0062 0.0927 GO:0042113 B cell activation 0.0063 0.0940 GO:0045941 positive regulation of transcription 0.0065 0.0968 \(B\) GO:0042127 regulation of cell proliferation 0.0001 0.0015 **GO:0034101 erythrocyte homeostasis** 0.0001 0.0015 GO:0007169 transmembrane receptor protein tyrosine kinase signaling pathway 0.0008 0.0122 GO:0048872 homeostasis of number of cells 0.0014 0.0227 GO:0007243 protein kinase cascade 0.0021 0.0333 GO:0048514 blood vessel morphogenesis 0.0036 0.0569 GO:0008284 positive regulation of cell proliferation 0.0039 0.0614 **GO:0001666 response to hypoxia** 0.0041 0.0643 **GO:0070482 response to oxygen levels** 0.0049 0.0766 **GO:0001525 angiogenesis** 0.0059 0.0902 GO:0007167 enzyme-linked receptor protein signaling pathway 0.0063 0.0973 Signature probe sets whose expression changes after drug treatment were significantly different between the RAF/MEK/ERK inhibitors (or PI3K/AKT/mTOR inhibitors) and other agents in HT29 cells were extracted using the Student's *t*-test (fold-change values of more than 2 and the *P*-value of less than 0.05). We carried out GO analyses using the DAVID analytical tool. FDR, false discovery rate. Characteristic GOs for each signature were indicated as bold letters (phosphate metabolic process-related GOs for the RAF?MEK?ERK inhibitors and the GOs related to erythrocyte homeostasis,response to hypoxia, and angiogenesis for the PI3K ?AKT?mTOR inhibitors). Gene signature analyses revealed potential new target pathways of some anticancer drugs --------------------------------------------------------------------------------------- As described above, the anticancer drugs were basically clustered in a target pathway-dependent manner. However, we also found several agents that were clustered in unexpected positions. As shown in Figure[1](#fig01){ref-type="fig"}, CDK4 inhibitor, AG1024 (IGF1R inhibitor), and FH535 (β-catenin/TCF inhibitor) unexpectedly showed similar gene expression signatures with the ER stress inducers. Amrubicin is an anthracycline drug that is supposed to target DNA topoisomerase II.[@b24] However, the agent was not clustered together with other topoisomerase II inhibitors but instead with the proteasome inhibitors (Fig.[1](#fig01){ref-type="fig"}). These data suggest potential novel modes of action for these agents. Among these drugs with unexpected gene signatures, we focused on vismodegib, a Hedgehog pathway inhibitor,[@b25] because our clustering analysis suggested its possible similarity with the oncogenic kinase inhibitors (Fig.[1](#fig01){ref-type="fig"}). To validate whether vismodegib could affect kinase signaling pathways, we examined its effect on the phosphorylation of components in the MEK/ERK and AKT/mTOR pathways. As shown in Figure[4](#fig04){ref-type="fig"}, vismodegib clearly suppressed the phosphorylation of p70S6K, a molecule downstream of mTOR, in HT-29 cells as well as in PC3 cells in which the AKT/mTOR pathways are strongly activated. As a positive control, we also observed inhibition of p70S6K phosphorylation by temsirolimus, a clinically used mTOR inhibitor. In contrast, ERK and AKT phosphorylation was not significantly affected by vismodegib treatment, although we observed a marginal inhibition of ERK phosphorylation in PC3 cells (Fig.[4](#fig04){ref-type="fig"}). These data indicated that our gene signature analysis successfully revealed a novel action of vismodegib on the mTOR pathway. {#fig04} Discussion ========== During anticancer drug development, the molecular target of each candidate compound should be strictly determined with reliable methods. In the present study, we showed that gene signature-based analysis can classify oncogenic pathway inhibitors in a target pathway-dependent manner and is a powerful tool to evaluate the molecular targets of compounds. We prepared subsets of genes for the signature analysis and showed that the signature reflected the modes of action of the agents (Figs[3](#fig03){ref-type="fig"}). These data indicated that the analysis worked well to validate target pathways of the agents. Overall, most inhibitors of oncogenic kinase pathways formed a unique cluster in the hierarchical clustering analysis as well as in the principal component analysis. These data suggested that this signature-based analysis could predict the potential of compounds to affect oncogenic signaling pathways. Our analysis further revealed that, of the kinome-targeted drugs, agents with similar molecular targets showed similar gene expression signatures. These data indicate the gene signature analysis is effective in validating target molecules or pathways of kinome-targeted compounds. In addition to the kinase inhibitors, other compounds that target similar molecular pathways were also clustered together. For instance, NAE is a component of the NEDD8 conjugation pathway that regulates protein turnover upstream of the proteasome.[@b20] MLN4924, a specific inhibitor of NAE, was clustered with proteasome inhibitors (Fig.[1](#fig01){ref-type="fig"}). Aurora kinase and Polo-like kinase are involved in the process of mitosis.[@b18] Inhibitors of these molecules (Aurora kinase inhibitor III and BI2536) were clustered with tubulin-binding agents, the classical inhibitors of mitosis. These results clearly indicate that the gene expression signatures reflect the primary target pathways of the agents. However, we observed some kinase inhibitors that were clustered in an unexpected way in our signature analysis, such as ALK inhibitors that were not clustered with the majority of the oncogenic kinase inhibitors (Fig.[1](#fig01){ref-type="fig"}). These data potentially suggest that these agents could affect unique downstream pathways; however, we should also take into account off-target effects, because in this study we used these agents at higher doses than the clinically relevant concentrations. Our gene expression analysis also assigned some antitumor drugs to unexpected modes of action. One agent was vismodegib, an inhibitor of the Hedgehog pathway,[@b25] whose gene expression pattern showed significant similarity with those of the oncogenic kinase inhibitors. Our "wet" experiments confirmed that vismodegib actually inhibits the mTOR pathway. These data indicate that the signature-based analysis was effective in identifying novel target pathways of the drugs. Endoplasmic reticulum stress is involved in the mode of action of some anticancer drugs.[@b26],[@b27] In this study, celecoxib, a selective inhibitor of COX2, showed a similar gene signature to that of ER stress inducers (Fig.[1](#fig01){ref-type="fig"}). This result is consistent with previous reports showing that the cytotoxic effect of celecoxib correlates with ER stress.[@b7],[@b28] Additionally, we also found several agents that were clustered together with ER stress inducers, such as a CDK4 inhibitor, AG1024 (IGF1R inhibitor), and FH535 (β-catenin/TCF inhibitor). These results suggest that these agents could affect ER stress pathways. In the signature-based analysis, careful interpretation of results was required. First, we needed to administer relatively high doses of agents that were less cytotoxic to cancer cells. As for the EGFR inhibitors, gefitinib and erlotinib, we tested how the drug concentration would affect the result of the signature analysis and found that high-dose treatment (30 μM) still showed significant similarity in gene signature to that of low-dose treatment (0.6 μM) (Table[3](#tbl3){ref-type="table"}D). Nevertheless, for such high-dose treatment data, we should be careful to confirm whether the signature reflects the physiological mode of action of the agents. Second, as we mentioned above, gene signatures of the agents could depend on cell context in some cases. As we have shown, the signatures of oncogenic kinase inhibitors in different cancer cell lines showed significant similarity (Fig.[1](#fig01){ref-type="fig"}). However, the target pathway-based classification was more accurately achieved using the data of a single cancer cell line (Fig.[3](#fig03){ref-type="fig"}). These data would be valuable to examine the cell context effect on the signature analysis. We further showed that, for the agents whose targets are selectively expressed in a certain subtype of cancer, use of data obtained in specific cancer cells could help accurate evaluation of drug target pathways (Table[3](#tbl3){ref-type="table"}D). In this aspect, our data would be valuable because we obtained the gene signature data using multiple specific cancer cell lines. Finally, the signature analysis could reveal target "pathways" of each agent, but the analysis would not be enough to completely define target "molecules" of the agent (for example, inhibitors of mitotic pathways showed similar gene signatures despite the direct target of each agent being different). Considering these points, integrated approaches with signature analysis and other methods would be important for accurate evaluation of the molecular targets of antitumor compounds. There are several other publicly available databases related to compounds' transcriptomic data. Connectivity map (C-map) (<https://www.broadinstitute.org/cmap/>) is a pioneering database that contains genome-wide transcriptome data for more than 1000 compounds.[@b9] In addition, several other databases containing drug-related gene expression data have recently been established, such as the Library of Integrated Cellular Signatures (<http://www.lincsproject.org/>) and the Cancer Cell Line Encyclopedia (<https://www.broadinstitute.org/ccle/home>). These are huge databases, but they do not focus on anticancer drugs, nor do they cover all antitumor agents. Our database is unique in that it is a compact database focusing on anticancer drugs and it covers genome-wide gene expression data of most clinically available anticancer compounds as well as promising inhibitors of molecular cancer targets. Moreover, we are updating the database by adding newly approved agents' data. Our website (<http://scads.jfcr.or.jp/db/cs/>) also provides an online analysis tool for users to easily compare the gene signature of query compounds to those in our database. These aspects make our database more updated and user-friendly, particularly for oncologists, than other public databases providing gene expression data. It should also be noted that our data were obtained using HT-29 cells as well as the specific driver oncogene-expressing cell lines, whereas the C-map and the other databases used different types of cells. Therefore, we believe that the combination of our database and others would provide more robust information to estimate modes of action of anticancer compounds. In conclusion, we obtained and analyzed gene expression data for a wide variety of molecularly targeted agents. This is a unique, comprehensive analysis of gene expression related to the pathways of molecularly targeted anticancer drugs. Our data will not only be beneficial in classifying antitumor agents but could also be valuable as a reference database to evaluate the modes of action of new candidate compounds in drug development. The present study was supported by the Scientific Support Programs for Cancer Research Project/Screening Committee of Anticancer Drugs, Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors thank Ms. H Shibaya and Ms. N. Kawata for their helpful support with data analysis and manuscript preparation. Disclosure Statement ==================== The authors have no conflict of interest. AKT : protein kinase B ALK : anaplastic lymphoma kinase ATM : ataxia telangiectasia mutated ATR : ataxia telangiectasia and Rad3-related protein BCR-ABL : fusion gene of breakpoint cluster region protein (BCR) and Abelson murine leukemia viral oncogene homolog (ABL) CDK4 : cyclin-dependent kinase 4 CHK : checkpoint kinase C-map : connectivity map DAVID : Database for Annotation, Visualization and Integrated Discovery EGFR : epidermal growth factor receptor EMK4-ALK : fusion gene of echinoderm microtubule-associated protein-like 4 (EMK4) and ALK ER : endoplasmic reticulum GI~50~ : concentration that causes 50% growth inhibition HDAC : histone deacetylase HER2 : human EGFR-related 2 IGF1R : insulin-like growth factor 1 receptor MET : hepatocyte growth factor (HGF) receptor NAE : NEDD8-activating enzyme NEDD8 : neural precursor cell expressed, developmentally down-regulated 8 mTOR : mammalian target of rapamycin PDGFR : platelet-derived growth factor (PDGF) receptor PI3K : phosphoinositide 3-kinase TCF : T-cell factor VEGFR : vascular endothelial growth factor receptor Supporting Information ====================== Additional supporting information may be found in the online version of this article: **Fig. S1.** Growth inhibition data of anticancer drugs and related agents used in this analysis. **Table S1.** Compounds used in the analysis. **Table S2.** Number of upregulated and downregulated gene signatures for anticancer drugs and related agents used in this analysis. **Table S3.** Signature gene set characteristic of oncogenic kinase inhibitors. **Table S4.** Signature gene sets characteristic of RAF/MEK/ERK and PI3K/AKT/mTOR inhibitors. [^1]: These authors contributed equally to this work. [^2]: **Funding Information**Ministry of Education, Culture, Sports, Science and Technology, Japan

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ The cellular and molecular mechanisms that underlie protein misfolding are a matter of major concern for studies conducted in several scientific centers all over the world. Under denaturing conditions, a growing number of proteins and peptides that fail to fold properly into their native structure, are led to the formation of highly ordered, insoluble aggregates, the so-called amyloid fibrils \[[@pone.0173163.ref001]\]. Amyloidogenicity, the ability of proteins to self-assemble into these well-defined fibrillar structures, was initially associated with a group of functionally unrelated proteins \[[@pone.0173163.ref002]\]. Meanwhile, targeted *in vitro* experiments revealed that amyloid formation is a universal phenomenon for polypeptide chains \[[@pone.0173163.ref003]\] and thus, this concept was the onset of a new era in protein misfolding, since a great number of novel amyloidogenic proteins and peptides were uncovered \[[@pone.0173163.ref004]\]. Noteworthy, proteins, ranging from bacteria to humans, have been also found to adopt the same amyloid architecture, as part of their nature \[[@pone.0173163.ref005], [@pone.0173163.ref006]\]. A vast amount of data, regarding amyloid fibril formation, present both in pathological and physiological conditions, is currently organized into freely available databases \[[@pone.0173163.ref007]--[@pone.0173163.ref011]\]. Amyloid fibril formation is widely observed and directly linked to the pathology of a range of widespread human diseases, known as amyloidoses \[[@pone.0173163.ref002]\]. Amyloidoses are a group of aggregation-disorders, where full-length amyloidogenic proteins or fragments of larger amyloidogenic protein precursors, precipitate and deposit, forming amyloid plaques and resulting in organ or tissue dysfunction \[[@pone.0173163.ref012], [@pone.0173163.ref013]\]. Literature data indicate the implication of more than one amyloidogenic proteins in the evolution of different amyloidoses. In the case of Senile Systemic Amyloidosis, co-operation of several Apolipoproteins and ATTR is recorded \[[@pone.0173163.ref014], [@pone.0173163.ref015]\], whereas in Alzheimer's disease, apart from Aβ, proteinaceous components such as ACys, ATTR and AGel were found \[[@pone.0173163.ref016]\]. To date, the extent to which co-deposition in amyloid plaques has impacted the development of amyloidoses between putative unrelated amyloidogenic proteins, remains unclear. Experimental work over the past ten years has revealed an intriguing, synergistic phenomenon between amyloidogenic proteins \[[@pone.0173163.ref017]\]. *In vitro* experiments highlighted the capacity of Aβ peptide under specific conditions to seed the polymerization process for α-synuclein \[[@pone.0173163.ref018]\], Tau \[[@pone.0173163.ref019]\] or APrp protein \[[@pone.0173163.ref020]\]. Similar experiments were performed on several well-characterized amyloidogenic proteins \[[@pone.0173163.ref021]--[@pone.0173163.ref023]\]. Further to *in vitro* assays, animal models demonstrated the co-deposition of Aβ and Tau proteins \[[@pone.0173163.ref024]\] or APrp protein \[[@pone.0173163.ref020]\] in transgenic models. However, a hidden perspective emerges from this molecular association; amyloid "cross-seeding" could explain mechanistically the way by which misfolded proteins co-deposite, and propose possible, attractive candidates for the development of novel therapeutic strategies of aggregation-related diseases. An apt example towards this direction is the protective role of the amyloidogenic ACys in neurodegenerative diseases \[[@pone.0173163.ref025]\]. The interactomes \[[@pone.0173163.ref026], [@pone.0173163.ref027]\], a systems biology approach, were viable complements to proteomics, in an attempt to look at "the big picture" of protein-protein interactions (PPIs). Gaining a proper understanding of PPIs contributed to several problems in the field of biological and medical research \[[@pone.0173163.ref028]--[@pone.0173163.ref030]\] and served as a reference for further targeted experimentation \[[@pone.0173163.ref031]\]. Systematic PPI studies are essential, in order to fully comprehend the molecular mechanisms that trigger human diseases \[[@pone.0173163.ref032], [@pone.0173163.ref033]\]. However, a subject poorly explored so far is deviating PPIs associated with amyloidogenic/amyloid forming proteins. To date, only a few studies utilized a protein interaction network framework, to obtain information regarding the Alzheimer's \[[@pone.0173163.ref034]--[@pone.0173163.ref036]\] or Huntington's disease \[[@pone.0173163.ref037]\] and to construct the Amyloid precursor protein interactome \[[@pone.0173163.ref038]--[@pone.0173163.ref040]\]. Incomplete knowledge on direct and/or indirect interactions of proteins "prone-to-misfold", emphasizes the need to focus on the amyloid protein-protein interaction network. Here we introduce the amyloid interactome, a systematic approach to study "macroscopically" interactions between previously unrelated human amyloidogenic proteins, associated with distinct pathologies. Our ultimate goal was to find a common denominator for amyloid formation, unveil the relationships that govern amyloidogenicity and, subsequently, guide further experimental studies on protein misfolding. Materials and methods {#sec002} ===================== Amyloid classification {#sec003} ---------------------- In order to classify amyloidogenic proteins, all protein-precursors were sorted into three categories: - *in vivo* amyloid forming protein: the precursor protein, or a peptide segment--derived from the precursor protein--, self assembles into typical amyloid fibrils, affecting one or more tissues or organs in human. These proteins, from a clinical perspective, give rise to distinct amyloidoses or play a pathological role in neurodegenerative or endocrine diseases \[[@pone.0173163.ref002]\]. - *in vitro* amyloid forming protein: the precursor protein, or commonly a peptide segment--derived from the precursor protein--, that was reported to self assemble into amyloid-like fibrils, at experimental level. The amyloidogenicity of proteins comprising this list may be speculative. Only human precursor proteins are mentioned in this category. - protein related to amyloid fibril formation: the protein is associated with other *in vivo* amyloid forming proteins, but has no amyloid properties recorded. Amyloid interactome datasets {#sec004} ---------------------------- Amyloidogenic proteins were firstly obtained from a literature-curated dataset, peer-reviewed in 2014, by the International Society of Amyloidosis (<http://www.amyloidosis.nl/>). This list included human proteins known to self-assemble into typical amyloid fibrils *in vivo*, along with intracellular inclusions with known biochemical composition \[[@pone.0173163.ref002]\]. In addition to this, the set was enriched with proteins that form amyloid fibrils *in vitro* \[[@pone.0173163.ref041]\]. To expand this dataset, AmyLoad \[[@pone.0173163.ref007]\] was used as a source of supplementary proteins, characterized to form amyloid-like fibrils *in vitro* at experimental level. A final addition included several UniProtKB \[[@pone.0173163.ref042]\] entries, gathered elaborately to incorporate reviewed proteins related to amyloid fibrils. Overall, the dataset contained 145 non-redundant amyloidogenic protein precursors. [S1 Table](#pone.0173163.s005){ref-type="supplementary-material"} provides a detailed catalogue of the aforementioned proteins, mapped to a UniProtKB Accession Number (AC). The subsequent construction of the network incorporated only well-characterized *in vivo* amyloidogenic proteins, published by Sipe et al. \[[@pone.0173163.ref002]\], excluding Enfurvitide an anti-retroviral peptide drug \[[@pone.0173163.ref043]\], as well as Immunoglobulin Light and Heavy Chains. In the case of Immunoglobulin chains (e.g. Bence Jones proteins \[[@pone.0173163.ref044]\]), their variety in human population did not allow the identification of a unique protein precursor, related to amyloid fibril formation. The final seed-dataset included 28 proteins, related to *in vivo* amyloid fibril formation ([Table 1](#pone.0173163.t001){ref-type="table"}), which were subsequently used for the collection of protein-protein interactions. Protein nomenclature follows abbreviations established by Sipe et al. \[[@pone.0173163.ref002]\]. 10.1371/journal.pone.0173163.t001 ###### The dataset of 28 proteins related to in vivo amyloid fibril formation. {#pone.0173163.t001g} Protein Precursor Name[\*](#t001fn001){ref-type="table-fn"} Abbreviation UniProtKB AC ------------------------------------------------------------- -------------- -------------- Amyloid beta A4 protein Aβ P05067 Apolipoprotein A-I AApoAI P02647 Apolipoprotein A-II AApoAII P02652 Apolipoprotein A-IV AApoAIV P06727 Beta-2-microglobulin Aβ2M P61769 Calcitonin ACal P01258 Corneodesmosin ACor Q15517 Cystatin-C ACys P01034 Fibrinogen alpha chain Afib P02671 Galectin 7 AGal P47929 Gelsolin AGel P06396 Insulin AIns P01308 Integral membrane protein 2B ABri/ ADan Q9Y287 Islet Amyloid Polypeptide AIAPP P10997 Kerato-epithelin Aker Q15582 Lactadherin AMed Q08431 Lactoferrin ALac P02788 Leukocyte cell-derived chemotaxin-2 ALECT2 O14960 Lysozyme C Alys P61626 Major prion protein APrP P04156 Natriuretic peptides A AANF P01160 Odontogenic Ameloblast-Associated Protein AOAAP A1E959 Prolactin APro P01236 Pulmonary surfactant associated protein C APSP P11686 Semenogelin-1 ASem1 P04279 Serum amyloid A-1 AA1 P0DJI8 Serum amyloid A-2 AA2 P0DJI9 Transthyretin ATTR P02766 \*Protein nomenclature follows abbreviations published by Sipe *et al*. Assembling the protein-protein interaction dataset {#sec005} -------------------------------------------------- UniProtKB ACs were used to query IntAct \[[@pone.0173163.ref045]\], BioGRID \[[@pone.0173163.ref046]\] and STRING \[[@pone.0173163.ref047]\] databases, in order to extract experimentally verified PPIs for the 28 proteins related to *in vivo* amyloid fibril formation ([Table 1](#pone.0173163.t001){ref-type="table"}). This process resulted in three independent PPI datasets, derived from each database (data not shown). In general protein-protein interaction data contain experimentally verified interactions, along with data derived from prediction methods. These last data do not have the high reliability often attributed to them and thus, in order to avoid extracting automatic text-mining results from the plethora of scientific articles related to amyloid fibril formation, BioGRID and STRING datasets were excluded from any further analysis. IntAct PPIs, gather highly curated experimental data, which ensured the quality and consistency of information of our dataset \[[@pone.0173163.ref048]\]. The interaction data from IntAct (05--2016) were retrieved in a MITAB 2.5 format file \[[@pone.0173163.ref049]\], which is appropriate for Perl parsing, without the loss of information regarding PPIs. An editing process of the file allowed the removal of all the non-human interactions, and additional screening was performed to dismiss interactions with chemical compounds. The resulting set included 355 protein nodes with 762 edges. In order to create a more robust network, the interactions deposited in IntAct between all 355 proteins were retrieved, whilst an extra processing allowed for the removal of self-loops and duplicated edges. Thus, a final dataset of 1178 PPIs between 353 human proteins was obtained, after the exclusion of two protein nodes that had only self interactions (See [Results and discussion](#sec007){ref-type="sec"}). Visualization and analysis of the network {#sec006} ----------------------------------------- For the visualization and the analysis of the network we followed the protocol introduced by Nastou et al. \[[@pone.0173163.ref050]\]. Cytoscape 3.2.1 \[[@pone.0173163.ref051]\] was used to manipulate, analyze and visualize our data, since it ideally provides all the necessary and improved applications for the analysis of biological networks \[[@pone.0173163.ref052]\]. The analysis of simple and complex network topology parameters, was performed by NetworkAnalyzer \[[@pone.0173163.ref053]\]. The Cytoscape.js JavaScript library \[[@pone.0173163.ref054]\] was used to create interactive networks, is available at this link: <http://83.212.109.111/amyloid_interactome>. Clustering analysis was performed with clusterMaker \[[@pone.0173163.ref055]\], utilizing the Markov Clustering algorithm (MCL), an optimal choice for biological interaction networks assembled from high-throughput experiments. Different inflation values, between 1.8 and 3.0, were used during the clustering process (data not shown). The inflation value was finally set to 1.8, as it has been proved to be the most suitable for biological networks \[[@pone.0173163.ref050], [@pone.0173163.ref056]\], in consistency with the observation that values above 1.8, result in extreme network fragmentation and the creation of clusters with minor biological significance (See [Results and discussion](#sec007){ref-type="sec"}). BiNGO \[[@pone.0173163.ref057]\], an application for Cytoscape and WebGestalt \[[@pone.0173163.ref058]\], an online server, were both used to perform a functional enrichment analysis of the network. BiNGO determines Gene Ontology (GO) \[[@pone.0173163.ref059]\] categories that are statistically overrepresented in a set of proteins in a biological network (e.g. a cluster), and thus, aids in the detection of functional modules. WebGestalt was used to further supplement this analysis, since, besides GO term it can perform Disease Association and KEGG pathway analysis. The hypergeometric method was used, and significance was set at an adjusted P-value of \<0.05 for BiNGO and \<0.01 for WebGestalt (Benjamini and Hochberg method). Significant categories, driven by only two or less proteins, were discarded due to the high potential for false signals in such cases. [S1 Fig](#pone.0173163.s001){ref-type="supplementary-material"} outlines the overall study design of the amyloid interactome ([S1 Fig](#pone.0173163.s001){ref-type="supplementary-material"}). Results and discussion {#sec007} ====================== The study of the dynamics, structure and function of protein-protein interaction networks (PPINs) has proven crucial for the understanding of many biological phenomena \[[@pone.0173163.ref060]--[@pone.0173163.ref063]\]. Hence, network theory is a sophisticated approach to study the puzzling phenomenon of amyloidogenicity. The amyloid interactome displays the interacting partners of *in vivo* amyloid forming proteins in a flat and detailed protein map ([Fig 1](#pone.0173163.g001){ref-type="fig"}). The results presented in this work, combine interactions from specialized networks of protein aggregation \[[@pone.0173163.ref034], [@pone.0173163.ref035], [@pone.0173163.ref037]--[@pone.0173163.ref040], [@pone.0173163.ref064]\] and eventually, assemble a new set of functionally unconnected proteins into a network that would possibly fill the missing pieces of protein aggregation and shed light towards the exploitation of novel disease protein-targets. ![The amyloid interactome.\ Interaction data for the creation of this network were gathered from the publicly available database IntAct \[[@pone.0173163.ref045]\] and Cytoscape \[[@pone.0173163.ref051]\] was used as a visualization tool (Interactive network available at <http://83.212.109.111/amyloid_interactome>). The network consists of 353 nodes and 1178 edges. Proteins are depicted as nodes and interactions as edges. Red-coloured nodes represent known *in vivo* amyloidogenic proteins, whereas yellow-coloured nodes represent *in vitro* amyloid-forming proteins or proteins related to amyloid fibril formation (see also Tables [1](#pone.0173163.t001){ref-type="table"} and [2](#pone.0173163.t002){ref-type="table"}). Green-coloured nodes are proteins, listed as other interaction partners. Hubs and bottlenecks are depicted as triangles (▲) and squares (■), respectively. Protein-nodes, which are both hubs and bottlenecks are shown as diamonds (◆). Important molecular chaperones are highlighted with a blue outline.](pone.0173163.g001){#pone.0173163.g001} Construction of the amyloid interactome {#sec008} --------------------------------------- In an effort to build the human amyloid interactome, *in vivo* amyloid-forming proteins were obtained from a peer-review library, published by Sipe et al. \[[@pone.0173163.ref002]\] ([Table 1](#pone.0173163.t001){ref-type="table"}). Overall the protein-protein interaction network contains 353 protein nodes and 1178 protein-protein interaction edges, between them ([Fig 1](#pone.0173163.g001){ref-type="fig"}). Surprisingly, among the proteins listed in the network, 23 amyloidogenic proteins construct a giant connected component ([Fig 1](#pone.0173163.g001){ref-type="fig"}, red nodes), whereas 13 proteins ([Fig 1](#pone.0173163.g001){ref-type="fig"}, yellow nodes), recorded as *in vitro* amyloid-forming proteins and proteins related to amyloid fibril formation ([S1 Table](#pone.0173163.s005){ref-type="supplementary-material"}), are also identified. A list of these significant proteins is available in [Table 2](#pone.0173163.t002){ref-type="table"}. Among them, proteins forming intracellular inclusions bodies were reported (Tau, Actin, NACP, HD) \[[@pone.0173163.ref002]\], while proteins, found as co-deposits in the Alzheimer's disease, emerged (Apo-E, PS-1, PS-2, Tau) \[[@pone.0173163.ref065]\]. 10.1371/journal.pone.0173163.t002 ###### The dataset of 13 proteins related to amyloid fibril formation. {#pone.0173163.t002g} Protein Precursor Name[\*](#t002fn001){ref-type="table-fn"} Abbreviation UniProt AC ------------------------------------------------------------- -------------- ------------ ***In vitro amyloid-forming protein precursors*** alpha beta Crystallin (ABC) HspB5 P02511 alpha-Synuclein NACP P37840 Apolipoprotein C-II Apo-CII P02655 Caspase-3 precursor CASP-3 P42574 Cystic fibrosis transmembrane conductance regulator CFTR P13569 Huntingtin (Polyq expanded) HD P42858 Presenilin 1 PS-1 P49768 Presenilin 2 PS-2 P49810 Spectrin SH3 Spectrin Q13813 Tau Tau P10636 ***Proteins related to amyloid fibril formation*** Actin, cytoplasmic 1 Actin P60709 Apolipoprotein E Apo-E P02649 Ataxin 1 Ataxin-1 P54253 \*Protein nomenclature follows the most cited abbreviations in literature. Notably, APro, an anterior pituitary hormone known to self-assemble into amyloid fibrils \[[@pone.0173163.ref066]\], did not manage to join the giant connected component created by the other 23 amyloidogenic proteins in the network, whilst the only interactor identified was the prolactin receptor ([Fig 1](#pone.0173163.g001){ref-type="fig"}, right). This "detachment" of an important hormone together with the absence of AIAPP should receive a renewed emphasis \[[@pone.0173163.ref067]\]. Namely, for AIAPP no experimental verified partners were recorded, since only speculative approaches attempted to describe possible interaction partners \[[@pone.0173163.ref068]\]. AA1 and AA2 proteins, which can form amyloid fibrils after partial proteolysis \[[@pone.0173163.ref069]\], are important components of the High Density Lipoprotein (HDL) complex \[[@pone.0173163.ref070]\]. However, direct interactions of AA1 and AA2 are not recorded in IntAct. Therefore, at first glance, the network which has emerged designates a correlation among the majority of otherwise unrelated *in vivo* and also *in vitro* amyloidogenic proteins, along with proteins related to amyloid fibril formation. Network analysis based on graph theory {#sec009} -------------------------------------- One of the most fruitful approaches to extract relative biological conclusions from the structure of the amyloid interactome is to computationally calculate its topological parameters. ### Simple topology parameters {#sec010} An assessment of simple topology parameters revealed intriguing information. A random graph with the same number of nodes and edges has been used as a "null model" to draw conclusions regarding certain topological traits of our network. In general, small-world graphs describe networks, where nodes can be reached from each other by traversing a small number of edges, and, thus their average path length is small and their clustering coefficient (transitivity) is high, compared to a random graph \[[@pone.0173163.ref071]\]. For the amyloid interactome, both the clustering coefficient (CC = 0.187) and the characteristic path length (CPL = 3.083) are higher and lower, respectively, than those of the corresponding random graph (CC = 0.003, CPL = 3.290), signifying that a small number of steps is needed, for one amyloidogenic protein to reach another. For example, Aβ can "reach" ATTR, by crossing AApoAI in only two (2) steps, verifying experimental data which pinpoint ATTR as a promising biomarker of the Alzheimer's disease \[[@pone.0173163.ref072]\] ([Fig 1](#pone.0173163.g001){ref-type="fig"}, top). Furthermore, measuring the density of our network (0.019), a value lower than 0.1 was recorded, a result in accordance with other sparsely connected biological networks \[[@pone.0173163.ref073]\]. Since our network complies with all these criteria, we can safely conclude that it has small-world properties \[[@pone.0173163.ref074]\]. ### Complex topology parameters {#sec011} In addition to having small-world features, biological networks are commonly scale-free \[[@pone.0173163.ref075]\]. The most important parameter to gain an insight on the scale-free nature of a biological network is the node degree distribution \[[@pone.0173163.ref076]\]. In our case, the distribution is of the following form: $$P\left( k \right) = 117.86k^{- 1.236}$$ decaying as a power law (P(k) \~k^-γ^). According to this finding, the network has scale-free properties \[[@pone.0173163.ref076]\] and specifically it consists of a few hubs ([Fig 1](#pone.0173163.g001){ref-type="fig"}, triangles and diamonds) connected with multiple nodes ([S2A Fig](#pone.0173163.s002){ref-type="supplementary-material"}). More importantly, hubs ([S2 Table](#pone.0173163.s006){ref-type="supplementary-material"}) seem to play a crucial role in our network, since the degree exponent (γ) is lower than 2 \[[@pone.0173163.ref077]\]. Generally, networks enriched with hubs, are robust against random node deletions \[[@pone.0173163.ref078], [@pone.0173163.ref079]\] as these disturbances do not affect the average path length severely ([S2D Fig](#pone.0173163.s002){ref-type="supplementary-material"}). Nevertheless, the removal of particular hubs, such as the Aβ or APrP ([Fig 1](#pone.0173163.g001){ref-type="fig"}), can drastically alter the average path length and so, our network can be generally addressed as 'robust yet fragile' \[[@pone.0173163.ref080]\]. As shown in [Fig 1](#pone.0173163.g001){ref-type="fig"}, several proteins act as "bridges", immediately connecting many, otherwise distantly or unconnected proteins in the network, thus, increasing the network's interconnectivity ([Fig 1](#pone.0173163.g001){ref-type="fig"}, squares and diamonds). The influence of these "bridging" proteins is expressed with high betweeness centrality values, indicating their role as bottlenecks, key connectors for the communication of other important proteins in the interactome, like hubs \[[@pone.0173163.ref081], [@pone.0173163.ref082]\] ([S2B Fig](#pone.0173163.s002){ref-type="supplementary-material"}). [S3 Table](#pone.0173163.s007){ref-type="supplementary-material"} introduces the top 20 bottlenecks of the interactome. Among the topological parameters mentioned above, node degree, and betweenness centrality distributions were carefully studied. Fifteen protein-nodes were identified as both hubs and bottlenecks, whereas 5 proteins have high degree (hubs) and 5 proteins have high betweenness centrality (bottlenecks). Impressively, 6 out of the 23 *in vivo* amyloidogenic proteins have a major contribution on the interactome, demonstrating their crucial role in the network ([S2](#pone.0173163.s006){ref-type="supplementary-material"} and [S3](#pone.0173163.s007){ref-type="supplementary-material"} Tables). Additionally, the amyloid interactome has an average clustering coefficient distribution, that follows approximately the scaling law *C(k) \~ k*^*-1*^ ([S2C Fig](#pone.0173163.s002){ref-type="supplementary-material"}), indicating the ability of this network to form functional modules (clusters) with biological significance (See Clustering Analysis) \[[@pone.0173163.ref083]\]. Overall, the network analysis based on graph theory revealed that the amyloid interactome appears to be enriched with interactions between amyloidogenic proteins. Finally, in order to further examine the role of selected and random perturbations in the stability of the amyloid interactome, we performed a "lethality" test \[[@pone.0173163.ref078]\]. A multistep procedure included the gradual removal of proteins, randomly ("failure") and in descending order of node degree and betweeness centrality ("attacks"). The rapid increase in the network's characteristic path length (CPL) during the targeted "attacks", in contrast to the slow increase during its "failure", puts emphasis on the significance of the removed proteins ([S2D Fig](#pone.0173163.s002){ref-type="supplementary-material"}). The above analysis gave us valuable information regarding central components of the network (hubs and bottlenecks), ranging from single proteins to entire modules ([S2](#pone.0173163.s006){ref-type="supplementary-material"} and [S3](#pone.0173163.s007){ref-type="supplementary-material"} Tables). However, due to inevitable technical biases present in interaction data \[[@pone.0173163.ref084]\], all results produced from such analyses should be carefully examined. Aβ publication biases, for example, may lead to the overestimation of the role of certain constituents of the network, in expense of others. Graph theory based analysis combined with other validation approaches were utilized, in our case, to further address the aforementioned issues. The amyloid interactome unravels interconnections between amyloidogenic proteins {#sec012} -------------------------------------------------------------------------------- In general, it is believed that disease-related proteins in a protein-protein interaction network are more interconnected than non-disease proteins \[[@pone.0173163.ref085]\], a claim in accordance with our findings. [Fig 1](#pone.0173163.g001){ref-type="fig"} deciphers the complex interactions governing amyloidogenicity, by interconnecting well-characterized amyloidogenic proteins (red nodes) with a heterogeneous collection of proteins related to protein aggregation (yellow nodes). Nevertheless, it is possible to understand that not all proteins on the interactome were directly related, meaning that indirect links may occur. Consequently, amyloid forming proteins, such us Tau, NACP and HD, which were excluded from our initial seed-dataset (See [Materials and methods](#sec007){ref-type="sec"}), were ultimately retrieved during the interactome construction process ([S3C Fig](#pone.0173163.s003){ref-type="supplementary-material"}). Impressively, the interaction network consolidates a number of human proteins, which have been shown to form amyloids *in vitro*. Frequently, *in vitro* aggregation assays are oriented towards protein segments, responsible to drive proteins from their native structure to the amyloid state, in place of full-length proteins \[[@pone.0173163.ref086]\]. Evidence at experimental level prove that "aggregation-prone" segments are indeed sufficient to lure a protein precursor into forming typical amyloid fibrils, and thus, these full-length protein precursors are characterized as "amyloidogenic" \[[@pone.0173163.ref087]\]. Interconnections between *in vivo* (red nodes) and *in vitro* amyloid forming proteins or protein segments (HspB5, Apo-CII, PS-1, PS-2, Spectrin, See [Table 2](#pone.0173163.t002){ref-type="table"}) may extend biological expectations, related to protein-aggregation ([S3A Fig](#pone.0173163.s003){ref-type="supplementary-material"}). Looking deeper into the crowded topography of the amyloid interactome, a great variety of "amyloid-binding proteins" is included. These amyloid specific molecules are basically a list of divergent proteins, capable of interacting with assemblies, derived from amyloidogenic proteins \[[@pone.0173163.ref088]--[@pone.0173163.ref090]\]. A broad range of Aβ contributors, for example, includes molecular chaperones or co-chaperones, apolipoproteins and other amyloid-forming proteins, enhanced with various functional characteristics. Since cells have adapted a mechanism to avoid the accumulation of incorrectly folded proteins, the Gene Ontology (GO) term \[[@pone.0173163.ref059]\] enrichment analysis in the entire interactome revealed the overrepresentation of GO terms regarding regulatory mechanisms (positive or negative regulation), while the most important GO term recorded is "response to stress" (GO ID: 6950) ([S4 Table and S4A Fig](#pone.0173163.s004){ref-type="supplementary-material"}). In particular, knowledge of the biological role of non-amyloidogenic hubs and bottlenecks in our interactome ([S3B Fig](#pone.0173163.s003){ref-type="supplementary-material"}), is gathered and shown in [S5 Table](#pone.0173163.s009){ref-type="supplementary-material"}. As expected, proteins highly interconnected are involved in signal transduction and in several metabolic processes. Impressively, though, a vast amount of topologically important proteins is related with stress pathways, highlighting possible novel disease protein targets, mediating amyloidogenicity (See Rational Design of Protein Inhibitors). The most abundant interaction detected is the one between amyloidogenic proteins and well-known regulatory proteins, the so-called chaperones ([Fig 1](#pone.0173163.g001){ref-type="fig"}). As [Fig 1](#pone.0173163.g001){ref-type="fig"} illustrates, chaperones together with co-chaperones dynamically participate in the interactome (nodes with blue border). This finding was, more or less, an expected phenomenon, since molecular chaperones are molecules dedicated to suppress amyloid formation \[[@pone.0173163.ref091], [@pone.0173163.ref092]\] and usually have many interactors \[[@pone.0173163.ref093]\]. The expert review, by Yerbury & Kumita, presents an extended group of amyloid-specific chaperones and discusses their implications \[[@pone.0173163.ref094]\]. Our interaction network, apart from validating existing data, demonstrates that chaperones exhibit high connectivity and at the same time high betweeness centrality, meaning that a sudden removal of such a node would result to the elimination of many important interactions in the network ([Fig 1](#pone.0173163.g001){ref-type="fig"} and [S2D Fig](#pone.0173163.s002){ref-type="supplementary-material"}). These findings are in accordance with previously published interaction networks, associated with aging \[[@pone.0173163.ref095]\] or stress \[[@pone.0173163.ref096]\], where chaperones participate as special constituents. Clustering and functional enrichment analysis {#sec013} --------------------------------------------- ### Clustering analysis {#sec014} The core of our study is the Amyloid Interactome, an interaction network represented as a large interconnected network with embedded functional sub-networks. Consequently, in order to further evaluate functional modules, a network clustering analysis was performed, utilizing the MCL algorithm \[[@pone.0173163.ref097]\]. The network was divided in 20 clusters, 11 of which composed of three or more nodes ([Fig 2](#pone.0173163.g002){ref-type="fig"}), while 9 contained only two proteins and were not further analyzed. The inflation value of 1.8 allowed the creation of compact clusters, preventing the network's fragmentation. ![Clustering analysis of the amyloid interactome.\ The 11 clusters with 3 or more nodes of the amyloid interaction network, derived utilizing the MCL algorithm \[[@pone.0173163.ref097]\]. Cytoscape \[[@pone.0173163.ref051]\] was used as a visualization tool. The visual legend summarizes the shortcuts of node colour and node shape (See also [Fig 1](#pone.0173163.g001){ref-type="fig"}). The highly connected subnetwork of the first cluster within the amyloid interactome reveals the strong affinity between 7 amyloidogenic proteins (cluster 1---red nodes) and the integral representation of the proteins presented in [Table 2](#pone.0173163.t002){ref-type="table"} (cluster 1---yellow nodes) (Interactive cluster subnetworks available at <http://83.212.109.111/amyloid_interactome>).](pone.0173163.g002){#pone.0173163.g002} The most important cluster, retrieved after the MCL implementation, consists of 186 protein-nodes ([Fig 2](#pone.0173163.g002){ref-type="fig"}, cluster 1). The results revealed the strong association between 7 *in vivo* amyloidogenic proteins ([Fig 2](#pone.0173163.g002){ref-type="fig"}, cluster 1---red nodes) and 13 proteins related to amyloid fibril formation ([Fig 2](#pone.0173163.g002){ref-type="fig"}, cluster 1---yellow nodes). Impressively, the full list of proteins included in the amyloid interactome and recorded as *in vitro* amyloid forming proteins or proteins related to amyloid fibril formation ([Table 2](#pone.0173163.t002){ref-type="table"}), is solidly represented in this first cluster. This finding further validates the strong interconnection observed in our initial interaction network ([Fig 1](#pone.0173163.g001){ref-type="fig"}), implying that all these 13 proteins are likely important determinants of protein aggregation. Notably, the most important subgroup of our interaction network ([Fig 2](#pone.0173163.g002){ref-type="fig"}, cluster 1) includes direct or indirect well-known amyloidogenic interaction partners of Aβ. Namely, amyloid forming Apolipoproteins (AApoAI, AApoAI*I*) are catalytic binding partners \[[@pone.0173163.ref098]\], whereas ACys \[[@pone.0173163.ref016], [@pone.0173163.ref099]\] and AGel \[[@pone.0173163.ref100], [@pone.0173163.ref101]\] prevent Aβ from accumulation. Moreover, Aβ cooperated with 9 (HspB5, Spectrin, Actin, PS-1, PS-2, ApoE, CASP-3, Tau, NACP) out of the 13 proteins of [Table 2](#pone.0173163.t002){ref-type="table"}, both in the entire network and the fragmented network, a finding that might stimulate new ideas about the nature of Aβ interactions. The amyloidogenic proteins, APrP and AKer are located in the second cluster, together with Clusterin ([Fig 2](#pone.0173163.g002){ref-type="fig"}, cluster 2---node with blue border), an extracellular chaperone, present in disease-associated extracellular amyloid deposits \[[@pone.0173163.ref102]\] and a proteasome functional subunit (Proteasome subunit alpha type-3). The absence of Clusterin from cluster 1, though, has prompted increasing interest, since the well-studied molecular chaperone is an existing protein target for the Alzheimer's disease and so, we would expect a strong correlation with Aβ \[[@pone.0173163.ref103]\]. Nevertheless, MCL algorithm results point toward a possible relationship between Clusterin and Aker or APrp, a finding that remains to be elucidated. Additional proteins of this cluster are associated with transcriptional regulation and pre-mRNA splicing, since the over-expression of prions influences normal cellular proteins, participating in apoptosis or cell signaling \[[@pone.0173163.ref104]\]. Transthyretin, a potent inhibitor of Aβ \[[@pone.0173163.ref072]\], created a separate cluster together with Small ubiquitin-related modifier 3. The remaining complexes, consist of less than 7 nodes, where, with the exception of cluster 4, each one contains only one amyloidogenic protein ([Fig 2](#pone.0173163.g002){ref-type="fig"}, clusters 4--11). ### Functional enrichment analysis {#sec015} Functional interpretation of the data, derived from each cluster, was performed using BiNGO \[[@pone.0173163.ref057]\] and thus, statistically significant GO terms \[[@pone.0173163.ref059]\] were obtained for three functional categories (biological process, molecular function and cellular component). Due to the excess of information derived from this analysis, terms with great statistical and biological significance were manually selected to functionally characterize each cluster. Importantly, as mentioned before, similar subcategories with the entire network analysis resulted from the cluster functional analysis, and "response to stress" was the most significant function in the majority of the clusters (details of cluster 1 GO enrichment are shown in [S6 Table](#pone.0173163.s010){ref-type="supplementary-material"}). Therefore, it is apparent from all the above results that the amyloid interaction map locates in "spatial proximity" proteins related to stress (chaperones, co-chaperones and amyloidogenic proteins), which arise as a response to pathological conditions \[[@pone.0173163.ref088]\] ([S4B Fig](#pone.0173163.s004){ref-type="supplementary-material"}). Nevertheless, biological systems are dynamic, meaning that a complex succession of events may occur over the course of time, in contrast with a protein-protein interaction network. Therefore, certain events described on the amyloid interactome are based on a static system and thus, this analysis could produce certain artificial results that should be addressed carefully to draw biologically significant conclusions. ### Pathway analysis and disease association {#sec016} KEGG pathway analysis was performed, in an attempt to detect common metabolic pathways, in which the network's proteins participate. A complex series of signaling pathways including the MAPK signaling pathway, B-cell and T-cell signaling pathways and the insulin signaling pathway are associated with the network's proteins. Additional disease association analysis, conducted with WebGestalt \[[@pone.0173163.ref105]\], revealed significant associated disorders for every cluster. In the first cluster ([Fig 2](#pone.0173163.g002){ref-type="fig"}), for example, Tauopathies, Dementia and Alzheimer's disease constitute the most significant group of pathologies. Pinpointing the components of such disease pathways is a promising perspective and thus, a detailed analysis and a novel joined network of diseases related to amyloidoses is currently being under construction (research article in preparation). It is important to note that a delicate feature of our interactome, and its subsequent fragmentation, is the vast amount of experimental data on Aβ peptide polymerization, owing to the worldwide prevalence of the Alzheimer's disease and the shortage of data on other less studied amyloidogenic proteins. Rational design of protein inhibitors {#sec017} ------------------------------------- Moving a step forward it seems interesting to investigate the correlation between biological factors, participating in the amyloid interactome that could influence amyloidogenicity. To determine whether significant elements of the amyloid interactome might indicate common properties of good candidates to be targeted by therapy, hubs and bottlenecks were thoroughly examined ([Fig 3](#pone.0173163.g003){ref-type="fig"}). {#pone.0173163.g003} An important group of proteins, dynamically participating in the interactome, is the group of molecular chaperones ([S3D Fig](#pone.0173163.s003){ref-type="supplementary-material"}). Their active role in controlling protein aggregation and their close relation to amyloidogenic proteins is the "hidden weapon\" of the protein machinery and the reason why 4 out of 9 chaperones were recorded as hubs and bottlenecks in the amyloid interactome ([Fig 1](#pone.0173163.g001){ref-type="fig"}). The transient nature of real-time interactions between chaperones and their partners, though, results in unrealistic low connectivity, in signaling and mitochondrial protein--protein interaction networks \[[@pone.0173163.ref106]\]. Contradictory theories on whether chaperones are "guilty or innocent" during the protein aggregation process provoke an intense debate. In particular, several experimental studies revealed the co-localization of chaperones with various amyloidogenic proteins \[[@pone.0173163.ref107], [@pone.0173163.ref108]\], whereas other experimental work reported on the inhibitory properties of chaperones, when added during fibrillation \[[@pone.0173163.ref102], [@pone.0173163.ref109], [@pone.0173163.ref110]\]. Impressively, molecular chaperones have been shown to inhibit the formation of amyloid fibrils even when present at extremely sub-stoichiometric ratios, comparing to amyloid forming protein \[[@pone.0173163.ref111]\]. [Fig 3](#pone.0173163.g003){ref-type="fig"} illustrates the interaction partners of selected molecular chaperones, prospect for the rational design of aggregation inhibitors. All characteristic path lengths for the subnetworks shown in [Fig 3](#pone.0173163.g003){ref-type="fig"} are reduced approximately by one degree, in comparison with those of the entire amyloid interactome (CPL \~ 2.4). This result quantifies the great importance of chaperones as mediators of communication between amyloidogenic proteins. Plasma proteins, on the other hand, is another promising group of protein-targets, considering that it includes proteins with many interaction partners, which have a wide range of physiological functions. Noteworthy, this group of proteins contains significant amyloidogenic components of the amyloid interactome, such as ATTR and Aβ2M. Among them, Serum albumin is a co-chaperone with significant topological features in the amyloid interactome, since it acts both as a hub and a bottleneck ([Fig 3A and 3B](#pone.0173163.g003){ref-type="fig"}). As [Fig 3](#pone.0173163.g003){ref-type="fig"} illustrates, Serum albumin interacts directly or indirectly with *in vivo* amyloidogenic proteins and proteins related to amyloid fibril formation (See [Fig 3](#pone.0173163.g003){ref-type="fig"} legend for details). Normally, Albumin is a prevalent transporter of human plasma, known to carry a wide range of molecules, but under *in vitro* conditions was found to self-assemble into typical amyloid fibrils \[[@pone.0173163.ref112]\]. Nevertheless, it is worth mentioning that Serum Albumin was absent from our initial non-redundant list of amyloidogenic proteins ([S1 Table](#pone.0173163.s005){ref-type="supplementary-material"}), since such an entry was not found recorded neither in our literature sources nor deposited in AmyLoad or UniProtKB. Despite the "aggregation-prone" nature of Albumin, the presence of this plasma protein promotes neuronal survival \[[@pone.0173163.ref113]\] or inhibits amyloid fibrillation in *in vitro* designed experiments \[[@pone.0173163.ref114]\], while according to the proteomic analysis, conducted by Hye et al., is an eligible biomarker for the Alzheimer's Disease \[[@pone.0173163.ref115]\]. Therefore, because of its central role in the interactome ([Fig 3A and 3B](#pone.0173163.g003){ref-type="fig"}) and the previously recorded features as a potent inhibitor of fibrillation, it seems that human plasma Albumin is a challenging molecule, which might stimulate new ideas about the design of anti-amyloid drugs. From the above discussion, we pinpointed hubs as the most competent candidates regarding the therapeutic intervention of amyloidogenicity. Opposing studies, though, suggest that proteins with low connectivity would be more efficient therapeutic targets for neurodegenerative diseases, since hubs are generally considered as "elegant features" for the robustness of an interactome \[[@pone.0173163.ref035]\]. Therefore, our computational approach should be followed by a variety of *in vitro*, cellular and *in vivo* experiments, in order to verify our speculations. Conclusions {#sec018} =========== Given the complexity of the molecular mechanisms driving amyloid fibrillation, a frequently used strategy is directed towards studying amyloidogenicity at molecular level, although, this approach is not always feasible, due to the dynamics of protein misfolding. In this study, we attempted to answer the crucial question of amyloidogenicity, following the principles of systems biology, by assembling a group of "miscellaneous" proteins into a common biological interaction network. The amyloid interactome illustrates a united interaction network of critical hypotheses, regarding the irregular protein aggregation, since it represents an integrated protein map of *in vivo* amyloidogenic proteins, together with *in vitro* amyloid forming proteins or proteins related to amyloid fibril formation. The amyloidogenic and non-amyloidogenic elements of the amyloid interactome eventually come together to form a complex "tapestry" of protein-protein interactions. Based on the complex network theory analysis, this network exhibits topological properties that are similar to other significant interaction networks. Surprisingly, our approach, apart from validating all previously experimentally verified direct or indirect protein interactions, allowed us to stress the importance of novel protein targets. Our integrated project has raised plentiful questions and could serve as the driving force to guide the experimental process in the challenging field of protein aggregation, even at the molecular level. Nevertheless, it should be addressed that the amyloid interactome was built based on the current knowledge of protein-protein interactions, meaning that there is a "publication bias" between over and understudied amyloidogenic proteins. Individual proteins should be carefully validated, utilizing the appropriate methodology, in order to enhance the significance of our observations. Therefore, the strength of the amyloid interactome lies in the perspective to identify key mediators of amyloidogenicity that could be targeted therapeutically. Supporting information {#sec019} ====================== ###### Study design workflow of the amyloid interactome. An overview of the basic protocol, used to create and analyze the amyloid interactome. (TIF) ###### Click here for additional data file. ###### Distributions for three complex topological parameters of the amyloid interactome and results from "Lethality Testing". **(A) Node degree distribution** in log-log plot. The red line shows that the distribution decays as a power law (*P(k) = 117*.*86k*^*-1*.*236*^). Nodes on the upper left corner of the chart (high node degree) are hubs in the amyloid interactome. **(B) Betweenness centrality distribution** with the horizontal axis in a logarithmic scale. Nodes on the right quarter of the chart (high betweeness centrality) are bottlenecks in the network. **(C) Average clustering coefficient distribution.** The red line shows that it follows approximately the scaling law (*C(k) = 0*.*816k*^*-0*.*647*^), designating the network's ability to form clusters. **(D) Lethality testing.** This chart shows the effect of the gradual removal of random nodes (blue circles) and the gradual removal of hubs (black triangles) and bottlenecks (grey squares), on the Characteristic Path Length (CPL) of the network (For detailed discussion please refer to [Results and discussion](#sec007){ref-type="sec"} section). (TIF) ###### Click here for additional data file. ###### Detailed features of the amyloid interactome. **(A)** Interactions between *in vivo* amyloidogenic proteins (red-coloured nodes) and *in vitro* amyloid forming proteins or proteins related to amyloid fibril formation (yellow-coloured nodes). **(B)** Yellow-coloured nodes represent *in vitro* amyloid forming proteins or proteins related to amyloid fibril formation and are a delicate feature of the amyloid interactome. **(C)** Representation of the key role of non-amyloidogenic hubs and bottlenecks in the amyloid interactome. Triangles are proteins acting as hubs, squares are proteins acting as bottlenecks and diamonds are proteins acting as both. **(D)** Nodes with blue borders represent proteins characterized as chaperones or co-chaperones (Interactive network available at <http://83.212.109.111/amyloid_interactome>). (TIF) ###### Click here for additional data file. ###### GO functional analysis of enriched terms in the biological process ontology for the entire amyloid interactome and the first cluster. Functionally grouped networks of enriched categories were generated both for the amyloid interactome (A) and cluster 1 (B). GO terms are represented as nodes. The colour gradient of each circle corresponds to the p-value of the associated GO term. White-coloured nodes are not statistically significant nodes, but are parent nodes of statistically significant GO terms. Different node sizes are indicative of varying frequencies of the proteins correlated with each GO term (See [S4](#pone.0173163.s008){ref-type="supplementary-material"} and [S6](#pone.0173163.s010){ref-type="supplementary-material"} Tables). (TIF) ###### Click here for additional data file. ###### The non-redundant, detailed catalogue of *in vivo* and *in vitro* amyloidogenic proteins or peptide fragments or proteins related to amyloid fibril formation. This extended list of proteins includes proteins known to self-assemble into typical amyloid fibrils *in vivo*, along with intracellular inclusions with known biochemical composition, published by Sipe *et al*. in 2014 \[[@pone.0173163.ref002]\]. In addition to this, the list includes proteins which form amyloid fibrils *in vitro* \[[@pone.0173163.ref045]\], protein segments obtained from AmyLoad \[[@pone.0173163.ref005]\] and finally, UniProtKB entries \[[@pone.0173163.ref046]\] of proteins related to amyloid fibril formation. A UniProtKB Accession Number is provided for each protein. The original source library of each protein is tagged with a cross (+). The Digital Object Identifier (DOI) code is provided, when it is available (See [Materials and methods](#sec007){ref-type="sec"}). (PDF) ###### Click here for additional data file. ###### The top 20 hubs of the amyloid interactome. The 20 proteins with the highest node degrees are considered as hubs in the amyloid interactome. 6 of these proteins belong to the dataset of the amyloidogenic proteins, described in [S1 Table](#pone.0173163.s005){ref-type="supplementary-material"}, whilst the rest of the hubs exhibit numerous functions, acting mainly as chaperones, signal transducers or structural constituent of the cell (See [Results and discussion](#sec007){ref-type="sec"}). (PDF) ###### Click here for additional data file. ###### The top 20 bottlenecks of the amyloid interactome. The 20 proteins with the highest betweeness centralities are considered bottlenecks in the amyloid interactome. 6 of these proteins belong to the dataset of the amyloidogenic proteins, described in [S1 Table](#pone.0173163.s005){ref-type="supplementary-material"}. 15 bottlenecks exhibit high node degree values and are also considered as hubs in this network ([S2 Table](#pone.0173163.s006){ref-type="supplementary-material"}). (PDF) ###### Click here for additional data file. ###### Amyloid interactome GO term enrichment. A p-value of 10E-14 was set as a gathering threshold for Biological Process and Cellular Component, whereas a value of 10E-8 was set as threshold for Molecular Function. Proteins of the entire amyloid interactome were subjected to a GO term enrichment analysis using BiNGO \[[@pone.0173163.ref056]\]. The UniProtKB ACs of proteins that are characterized by overrepresented GO terms in the entire amyloid interactome are given in this table, along with their number and their frequency in the network. The adjusted p-value suggests the importance of these GO terms in the proteins of the amyloid interactome. (PDF) ###### Click here for additional data file. ###### GO Terms enrichment of the 18 important, non-amyloidogenic hubs and bottlenecks identified on the amyloid interactome. The majority of proteins, which are characterised as hubs and bottlenecks are involved in signal transduction and in several metabolic processes. The most abundant GO term, though, is response to stress, in accordance with the most represented group of the amyloid interactome (See [S4 Table](#pone.0173163.s008){ref-type="supplementary-material"}). (PDF) ###### Click here for additional data file. ###### Enriched GO categories of Cluster 1, derived from the amyloid interactome. Enriched categories for Biological Process are those with p\<10E-12, for Cellular Component with p\<10E-14 and for Molecular Function with p\<10E-8. Proteins of the first cluster of the amyloid interactome were subjected to a GO term enrichment analysis using BiNGO \[[@pone.0173163.ref056]\]. The UniProtKB ACs of proteins that are characterized by overrepresented GO terms in this cluster are given in this table, along with their number and their frequency in the network. The adjusted p-value suggests the importance of these GO terms in the proteins of cluster 1. (PDF) ###### Click here for additional data file. ###### Web application user guide. Detailed description of the structure of the interactive Amyloid Interactome--Web application is available at <http://83.212.109.111/amyloid_interactome>. (PDF) ###### Click here for additional data file. This project was financially supported by the Greek State Scholarships Foundation, through the Program: ''RESEARCH PROJECTS FOR EXCELLENCE IKY/SIEMENS" (2015--2017). [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: **Conceptualization:** PLT KCN SJH VAI.**Data curation:** PLT KCN KVB.**Formal analysis:** KCN KVB.**Funding acquisition:** VAI.**Investigation:** PLT KCN KVB CVM.**Methodology:** PLT KCN.**Project administration:** SJH VAI.**Resources:** VAI.**Software:** KCN.**Supervision:** VAI.**Visualization:** PLT KCN KVB.**Writing -- original draft:** PLT KCN KVB SJH VAI.

Introduction ============ Aerosol delivery of therapeutic gene is made directly to the respiratory tract without first delivery to other organs and tissues. Due to these advantages, many studies have been applied to gene therapy for lung disease, such as lung cancer ([@B1]), asthma ([@B2]) and chronic obstructive pulmonary disease ([@B3]) using aerosol delivery. Aerosol therapeutic delivery system consists of aerosol generator, delivery-chamber, drug-carrier and air flow controller. The gene carriers are broadly classified into two groups: viral and non-viral vector systems. Non-viral gene carriers were attracted increasingly because of their many advantages such as low immunogenicity, large-scale production and safety ([@B4]). Chitosan, a natural polysaccharide, is safe, biocompatible and mucoadhesive materials with high cationic charge potential ([@B5]). However, this material has low transfection efficiency as a result of weak release from endosomes into the cytoplasm ([@B6]). In our previous study, we have overcome this problem by grafting with the polyethylenimine (PEI) (CHI-*g*-PEI) ([@B7]). However, pulmonary toxicity and gene expression by *in-vivo*aerosol gene delivery have not been investigated yet. Therefore, in the current study, we present information from both gene expression and pulmonary toxicity in the lung through our aerosol gene delivery system and CHI-*g*-PEI as an aerosol gene carrier. Experimental ============ *Materials* Chitosan (molecular weight, 100 kDa; deacetylation degree, 87.7%) was kindly supplied from Jakwang (Ansung, Korea). Branched PEI 25K was obtained from Sigma-Aldrich (St. Louis, MO, USA). Branched PEI 1800 Da was purchased from Wako (Osaka, Japan). pcDNA3.1-GFP was purchased from Invitrogen (Carlsbad, CA, USA). The plasmids were propagated in *E. coli*, extracted by the alkalilysis technique, and purified by QIAGEN kit (Chatsworth, CA, USA). *Aerosol gene delivery system* The nose-only exposure chamber (NOEC) system consists of dual cylindrical box and 4 small tubes ([Figure 1](#F1){ref-type="fig"}). {#F1} Conical acrylic tubes were connected to the main chamber and mice (male C57BL/6, 8\~10 wks old, SLC, Hamaguchi, Japan) were placed in the tubes. All animal experiments were performed in accordance with the guidelines of Seoul National University for the care and use of animals. Nebulizer (Dusturbo, Seoul, Korea)-generated aerosols were entered into the NOEC. Operation of the aerosol generation process by nebulizer was as follows: air flow rate was 2 L/min (*lpm*) by a mass flow controller (MFC, Brooks Instruments, Hatfield, PA, USA) and the complex was composed of plasmid DNA; 200 μg and polymer (PEI 25K; 260 μg, N/P ratio of 10 and CHI-g-PEI; 1,400 μg, N/P ratio of 35) was added to 20 mL deionized water. Aerosol size distributions were measured with a dust monitor (Grimm Aerosol Technik, Germany). Size distribution measurements were taken in the NOEC. *Transfection efficiency* To determine the efficiency of gene transfection, animals were exposed to aerosol containing GFP-PEI and GFP-Chi-*g*-PEI complex for 30 min in NOEC system. Animals were sacrificed 48 h after the inhalation and routinely fixed lungs were cryosectioned for confocal laser scanning microscopy (CLSM, Carl Zeiss-LSM510, Germany). CLSM images were quantified and analyzed by a computerized system (Media Cyber-netics, Silver Spring, MD, USA). *Pulmonary toxicity analysis* For the assessment of pulmonary toxicity of CHI-*g*-PEI and PEI, mice were exposed to aerosols, two times in a week for a total of 4 weeks. The control group was exposed to air filtered by a high-efficiency particulate air (HEPA) filter and polymer groups were exposed to aerosol containing PEI; 260 μg and CHI-g-PEI; 1,400 μg without DNA in distilled water, respectively. At the end of exposure, bronchioalveolar lavage (BAL) fluid from mice was obtained by whole-lung lavage. As a marker of cellular damage, lactate acid dehydrogenase (LDH) activity in BAL fluid was measured using an automated biochemical analyzer (VITALAB, Merck, the Netherlands). *Histopathological examination* For histopathological analysis, lungs were removed from each animal. The organs were immersion-fixed in 10% neutral buffered formalin. After routine tissue processing, the tissues were embedded in paraffin and the tissue sections (5 μm) were then prepared for hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) stain. The slides were evaluated under light microscopy. *Statistical analysis* All results are expressed as mean ± standard error. A multiple variance of a Student's t-test (Graphpad Software, San Diego, CA, USA) was used to compare the test groups with those obtained from unexposed control group. The level of significance was set at p \< 0.05 and p \< 0.01. Results and Discussion ====================== In this study, we investigated the gene expression and pulmonary toxicity of CHI-*g*-PEI as aerosol gene carrier. We found that CHI-*g*-PEI is safe to use and shows higher transfection than PEI in aerosol gene delivery to animals, and enhanced efficiency was achieved using our aerosol gene delivery system. *Aerosols characterization* Geometric Mean Diameter (GMD) of nebulized aerosols in N-OEC measurements was as follows: GFP-PEI; 0.39 and GFP-CHI-*g*-PEI complex; 0.42 μm, respectively. Geometric Standard Deviation (GSD) was 2.46 and 2.26, respectively. Our study demonstrated that GMD results obtained from GFP-PEI were not different from those of GFP-CHI-*g*-PEI. However, the horizontal chamber is easy to handle and has a space saving in the aerosol delivery system. The mean diameters of generated aerosols over 1 μm are known to be increased with solute concentration of the solution ([@B8]); however, the solute concentration of generated aerosols under 1 μm size can be different from that of solution in the nebulizer reservoir ([@B9]). Measured size distributions showed that large portion of generated droplets are smaller than 1 μm, so the efficiency of trasfections might be different for each case. *Transfection efficiency* As shown in [Figure 2(a)](#F2){ref-type="fig"}, compared with the control group, the green intensity of GFP was dominant in the polymer group (PEI and CHI-*g*-PEI). In addition, the CHI-g-PEI group showed 15.65% enhancement of the fluorescence intensity as compared to the PEI group ([Figure 2b](#F2){ref-type="fig"}). It was already reported that polyethylenimine-graft-chitosan (PEI-g-chitosan) showed higher gene expression than PEI (25 K) in liver due to the higher level of amine content in the DNA-carrier complex ([@B10]). Furthermore, gene expressions by PEI are affected by the serum ([@B11], [@B12]), whereas, chitosan increased the transfection efficiency with serum ([@B13]). The gene expression of low molecular weight polyethylenimine grafted N-maleated chitosan (NMC-g-PEI) was not affected in the presence of serum ([@B12]). In a previous study, we also observed higher transfection efficiencies with CHI-*g*-PEI compared to PEI in the presence of serum ([@B7]). Taken together, these results indicate that CHI-*g*-PEI with serum in the lung enhances the efficiency of the aerosol gene delivery. {#F2} *Pulmonary toxicity* Our results showed that LDH levels in BAL were higher in the PEI group compared to the CHI-*g*-PEI group ([Figure 3](#F3){ref-type="fig"}). H and E analysis of the CHI-*g*-PEI and PEI groups showed no detectable change compared to the control group ([Figure 4](#F4){ref-type="fig"}). However, the results of our present experiment stained PAS showed that CHI-g-PEI was secreted small amount of mucin compared to the PEI ([Figure 5](#F5){ref-type="fig"}). Mucin has high molecular weight and is produced by epithelial tissues and they protect our body from toxicants by forming the mucosal barrier ([@B14]). Moreover, mucin secretion indicated that repeated inhalation of PEI could induce cellular immune responses in the lung. Complexes of PEI with mucin break the PEI/DNA interactions and it probably reduces the gene expression in the lung ([@B15]). {#F3} {#F4} {#F5} We showed that PEI has high cytotoxicity in diverse cell line types ([@B7]). However, some studies of aerosol delivery to animals using PEI investigated the difficulties with toxicity ([@B16]). Apparent discrepancies between our results for PEI toxicity and those of earlier studies may be due to differences in design. In our study, we used the N-OEC system with repeated inhalation of polymer, whereas previous studies applied a whole-body exposure chamber (W-BEC) system with single exposure. W-BEC is frequently used in long-term inhalation studies, as it is suitable for efficient delivery of a large number of animals without restraint. However, W-BEC requires a large amount of test materials; thus, this method limits practical application. On the other hand, our N-OEC has various advantages, including simple control, low cost, and suitability for repeated aerosol delivery. In summary, CHI-*g*-PEI was safe to use and showed higher transfection than PEI in aerosol gene delivery to animals, and enhanced efficiency was achieved by use of our aerosol gene delivery system. Therefore, CHI-*g*-PEI has the potential to be safe and efficient gene carrier and this system would be applicable for future studies. This work was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2011-0002169). This research was a part of the Technology Development Program for Agriculture, the Ministry for Food, Agriculture, Forestry and Fisheries, Korea. MHC was also partially supported by the Research Institute for Veterinary Science. JTK is grateful for the financial support from BK21 program.

Introduction {#s1} ============ It was reported that fetal mortality rate at 20 weeks of gestation or more was 6.22 deaths per 1,000 in United States, in which the fetal mortality rate for twins was 2.7 times higher compared to singletons [@pone.0065050-MacDorman1]. The higher risk of twin pregnancies may due to several reasons, for instance, twin--twin transfusion syndrome (TTTS) [@pone.0065050-Simpson1]. There are more than 4,500 TTTS cases per year in the U.S. [@pone.0065050-1]. Moreover, a significantly increasing risk has been observed in monozygotic (MZ) twins in previous studies [@pone.0065050-Glinianaia1]. Therefore, zygosity is an important parameter in prenatal diagnosis for twin pregnancies. The diagnosis of zygosity for twin pregnancies relies on the determination of chorionicity by ultrasound scanning within 14 gestational weeks, with 89.8% sensitivity and 99.5% specificity [@pone.0065050-Stenhouse1]--[@pone.0065050-NationalGuideline1]. However, the accuracy of ultrasound detection declines dramatically due to thinner chorionicity in the second trimester [@pone.0065050-Lee1]. Invasive approaches such as amniocentesis or cord blood sampling combined with microsatellite DNA markers could also detect zygosity with high accuracies, but it presents a potential miscarriage at a risk of 0.5--1% [@pone.0065050-Kan1]. Thus there is a huge demand for a noninvasive method to accurately determine the zygosity type without the limitation of gestational age. The discovery of cell-free fetal DNA (cff-DNA) in maternal plasma opened a new direction for noninvasive prenatal diagnosis [@pone.0065050-Lo1]. Combined with the rapidly developing massively parallel sequencing(MPS) technology, Qu *et al.* recently observed the fluctuation of cff-DNA concentration among autosomes between dizygotic (DZ) and MZ twin pregnancies. The SD variation of the fluctuation from 8 samples was regarded as the indication to determine the zygosity [@pone.0065050-Qu1]. However, the method lacked evaluation of sensitivity and specificity. Herein, we developed a noninvasive method based on maternal plasma target region sequencing to determine zygosity of twin pregnancies. We successfully determined two DZ, MZ twin pregnancies and two simulated MZ twin pregnancies through our mathematical model and obtained satisfactory sensitivity and specificity *in silico*. Our study provides a practical alternative approach for zygosity determination in clinical practice. Results {#s2} ======= Bioinformatic Pipeline Establishment {#s2a} ------------------------------------ In order to determine the zygosity, we employed a bioinformatic method using a conditional probability model. We defined ***L~i~*** to measure the zygosity tendency of each available paternal-only heterozygous SNP locus (where maternal genotype was homozygous), and ***L*** value which was the geometric mean of ***L~i~*** to represent the global tendency. The zygosity could be determined if its ***L*** value passed its corresponding cut-off. In order to get the cut-offs, we generated simulated samples with different gradients of cff-DNA concentration from 10.00% to 30.00% and sequence depth from 300× to 1300×, and got a series of real cut-offs (***L~R~***) with the boundaries of \>95% confidence interval (CI) (**[Table S1](#pone.0065050.s003){ref-type="supplementary-material"}**). Based on these scattered ***L~R~***, we used least squared method (LSM) to obtain two approximate mathematical expressions of DZ and MZ dynamic cut-offs respectively (**Materials and Methods**). After getting the fitting expressions, we established a comprehensive pipeline, which included sequence reads alignment, parental genotype detection, total cff-DNA concentration estimation, calculation of ***L*** among clinical samples and zygosity determination by comparing ***L*** to its corresponding dynamic cut-off. Six clinical samples were recruited to assess the accuracy of our methodology. Finally, we used more simulated samples to depict the sensitivity and specificity of our methodology under various detecting conditions *in silico*. Clinical Samples and Data Productions {#s2b} ------------------------------------- Four twin pregnancies named Sample1, 2, 5 and 6 were enrolled from Women's Hospital School of Medicine Zhejiang University and Peking University Third Hospital, whose gestational ages were 20^+2^ and 19^+4^, 20 and 20^+4^ weeks, respectively. We also enrolled two singletons named Sample3 and Sample4 with gestational age of 19 and 8 weeks from Women's Hospital School of Medicine Zhejiang University and BGI-Shenzhen. Sample1 and Sample2 had already been diagnosed as DZ by invasive procedure aminocyte karyotyping suggesting mixed-gender twin pregnancies. Sample5 and Sample6 were diagnosed as MZ by ultrasound scanning. 4.43 Gbp and 11.47 Gbp clean data were extracted from maternal plasma Sample1 and Sample2, corresponding with 930.87× and 1363.25×sequence depth. 95.81% and 97.51% of target region was covered by at least one read. For maternal plasma Sample3 and Sample4, 4.53 Gbp and 2.55 Gbp clean data were extracted respectively. The sequence depth was 519.34× and 446.89×, and the corresponding coverage of target region was 95.36% and 96.88%. For Sample5 and Sample6, we obtained 16.26 Gbp and 15.59 Gbp clean data, corresponding to 492.3× and 271.2× sequence depth, 99.85% and 98.68% of target region depth ([**Table 1**](#pone-0065050-t001){ref-type="table"}). 10.1371/journal.pone.0065050.t001 ###### Data production of 6 clinical samples. {#pone-0065050-t001-1} Sample Production(Gbp) Coverage(%)[\*](#nt101){ref-type="table-fn"} Depth(×)[\*](#nt101){ref-type="table-fn"} ---------------- ----------------- ---------------------------------------------- ------------------------------------------- Father Sample1 0.41 98.18 219.63 Mother Sample1 0.37 96.01 192.54 Father Sample2 0.46 98.22 228.93 Mother Sample2 0.51 96.29 268.76 Plasma Sample1 4.43 95.81 930.87 Plasma Sample2 11.47 97.51 1363.25 Father Sample3 0.33 95.06 130.30 Mother Sample3 0.38 95.37 146.03 Father Sample4 0.10 92.59 51.47 Mother Sample4 0.37 94.18 185.33 Plasma Sample3 3.16 95.36 519.34 Plasma Sample4 2.55 96.88 446.89 Father Sample5 2.05 99.64 97.36 Mother Sample5 1.51 98.12 76.80 Father Sample6 2.25 99.60 106.58 Mother Sample6 1.26 97.98 64.27 Plasma Sample5 16.26 99.85 492.30 Plasma Sample6 15.59 98.68 271.20 "Coverage (%)" and "Depth (×)" mean the coverage and average sequencing depth in the target region. Estimation of Total cff-DNA Concentration and Zygosity Determination {#s2c} -------------------------------------------------------------------- Genotypes of parental genomes were analyzed by SOAPsnp [@pone.0065050-Li1], and only parental-specific homozygous loci in the form of ♀AA♂BB were selected. Then the sequence reads from those loci were used to estimate the total cff-DNA concentration. We obtained 1,209, 1,057, 1,090 and 986, 1,150 and 1,241 parental-specific homozygous loci from Sample1-6 respectively. And the total cff-DNA concentrations of Sample1-6 were estimated at 27.04%, 22.12%, 23.35%, 9.36%, 18.83% and 25.16%, respectively. According to our mathematical model, paternal-only heterozygous loci in the form of ♀AA♂AB were used to calculate ***L*** values. 708 and 603 loci were available for Sample1 and Sample2 to obtain 1.891 and 1.554 of ***L*** values, which were both above their corresponding DZ cut-offs (\>1.162 and \>1.172 for DZ, while \<0.938 and \<0.928 for MZ), indicating both samples were DZ ([**Figure 1a**](#pone-0065050-g001){ref-type="fig"}). ***L*** values of Sample3 and Sample4 were calculated as 0.639 and 0.757 through 564 and 610 available loci respectively, which were both lower than the MZ respective cut-offs (\<0.921 and \<0.799 for MZ, while \>1.179 and \>1.301 for DZ)([**Figure 1b**](#pone-0065050-g001){ref-type="fig"}). Additionally, by using 554 and 558 available loci of Sample5 and Sample6 respectively, ***L*** of this two samples were 0.763 and 0.784, both below their MZ cut-offs (\<0.903 and \<0.918 for MZ, while \>1.197 and \>1.182 for DZ) **(** [**Figure 1c**](#pone-0065050-g001){ref-type="fig"} **)**.The results for these six samples showed the zygosity of twin pregnancies could be determined using our bioinformatic method through maternal plasma target region sequencing. {ref-type="fig"}, the two points were both above the corresponding DZ cut-off, indicating both samples were correctly determined as DZ. In [Figure 1b and 1c](#pone-0065050-g001){ref-type="fig"}, the four points were all under the corresponding MZ cut-off, meaning simulated and real MZ twin pregnancies were all correctly determined.](pone.0065050.g001){#pone-0065050-g001} Estimation of Sensitivity and Specificity *in Silico* {#s2d} ----------------------------------------------------- To further understand the performance of our method, we simulated sequence data with different gradients of cff-DNA concentration and sequence depth (**Materials and Methods**). Overall, the sensitivity, which was defined as MZ accuracy, increased with the enhancement of cff-DNA concentration and sequence depth. It could achieve 99.90% on the condition of 10.00% total cff-DNA concentration and 300× target region sequence depth ([**Figure 2a**](#pone-0065050-g002){ref-type="fig"}). Also, the specificity (DZ accuracy) of 15.00% total cff-DNA concentration and 500× target region depth was as high as 97.00% ([**Figure 2b**](#pone-0065050-g002){ref-type="fig"}). It was notable that the results from *in silico* showed a relatively high accuracy to determine MZ twins than DZ twins, which might be partially related to the systematic loss of paternal-specific alleles in the maternal plasma sequence data. Meanwhile, we also found that the total cff-DNA concentration plays a more decisive effect than the sequence depth in the zygosity determination. (**[Table S2](#pone.0065050.s004){ref-type="supplementary-material"}**). {ref-type="fig"} indicated the specificity (DZ) and sensitivity (MZ) with different total cff-DNA concentration and sequence depth respectively. The detailed grey level of each square represented the related accuracy according to the legend on the right, ranging from 0% to 100%.](pone.0065050.g002){#pone-0065050-g002} Discussion {#s3} ========== In this study, we developed a practical method to noninvasively determine the zygosity of twin pregnancies by using target region sequencing for maternal plasma. The method consists of determination of empirical dynamic thresholds, cff-DNA concentration estimation and likelihood ratios calculation. The zygosity types of 4 clinical twin pregnancies samples were determined successfully as well as those of 2 singletons. The simulation data also showed that more than 99.90% of the MZ simulated samples with total cff-DNA concentration as much as 10.00% were correctly determined by using about 2.00 Gbp sequence data. Moreover, the sensitivity was improved apparently with the increment of cff-DNA concentration. Parental genotypes were necessary information in our current method. Since the judgments of fetal genotypes mostly relied on the prior probabilities ensured by parental genotypes in the Bayesian model, parental genomes helped filtering useless and disruptive SNP loci, e.g. loci in the form of ♂AA♀AA and ♂AB♀AB. Therefore we could use only the paternal-specific heterozygous loci (♂AB♀AA) to calculate the likelihood ratio and estimate the percentage cff-DNA through parental-specific homozygous loci (♂AA♀BB). Comparing with conventional approaches, this sequencing-based approach has several advantages. Firstly, cff-DNA detection has been reported to as early as four weeks [@pone.0065050-Collins1], which has much less limitation of sampling time restriction than ultrasound scanning. Secondly, maternal blood sampling avoids the risk of miscarriage carried by invasive procedure. Lastly, we used an empirical dynamic threshold for DZ and MZ twin pregnancies to improve the accuracy of zygosity detection, which could significantly minimize the fluctuation of cff-DNA between different loci. However, our bioinformatic model still needs to be improved in the following studies. Firstly, we constructed this model on the hypothesis of the same contribution to cff-DNA concentration in twin pregnancies, but previous studies have reported a variable combination of cff-DNA concentration for each fetus. False signal might be obtained in MZ detection if there is a significant bias in the distribution of cff-DNA concentration. Referring to some clinical information such as crown-rump length (CRL), the fractional cff-DNA concentration of MZ twins may be preliminarily ensured. Secondly, this high throughput sequencing approach could be only used to distinguish MZ and DZ twin pregnancies. The detailed physiological structure of the placenta for MZ twins, such as monochorionic-monoamniotic (MCMA) twins and monochorionic-diamniotic (MCDA) twins, could be determined by only the combination of ultrasound scanning with sequencing test. Besides ultrasound scanning and invasive prenatal test, few prenatal detection approaches could be provided for twin pregnancies due to limited accuracy. Here we demonstrated a sequencing-based noninvasively approach to detect zygosity, which could give clues for twins specific diseases, such as TTTS, as well as gender determination and sex-linked monogenetic diseases [@pone.0065050-Prior1], [@pone.0065050-Emery1]. Our study also encourages the application of sequencing technology using maternal plasma to meet rigorous clinical needs, especially on twin pregnancies. Materials and Methods {#s4} ===================== Sample Recruitment and Library Construction {#s4a} ------------------------------------------- Six pregnant women, including four of which with twin pregnancies and the rest two with singleton pregnancies, were recruited for this study. Written informed content was obtained from each participant and approval was obtained from the Institutional Review Board of BGI-Shenzhen. 5 ml maternal blood was drawn into EDTA-anticoagulated tubes, and plasma samples were isolated using two-steps centrifugation. Cell-free DNA was extracted from 600 µl maternal plasma following the instruction of QIAamp DNeasy Blood & Tissue Kit (Qiagen). DNA libraries were prepared in accordance with previous study [@pone.0065050-Dan1], [@pone.0065050-Lau1]. We also collected 5 ml paternal peripheral blood to construct the model. Genomic DNA (gDNA) for whole blood were extracted and used to construct pre-capture libraries with 200 bp insert size. Targets Regions Capture and Sequencing {#s4b} -------------------------------------- We designed two versions of probes, both covering 4,524 SNPs from 22 autosome chromosomes (**[Table S3](#pone.0065050.s005){ref-type="supplementary-material"}**). The SNPs were selected from dbSNP build 131 with at least of 0.3 of MAF. DNA libraries were hybridized with the capture probes at 65°C for 22--24 hours, in accordance with the manufacturer's instructions. After hybridization, the captured targets were selected by pulling down the biotinylated probe/target hybrids with M-280 streptavidin Dynabeads (Invitrogen). Then, the targeted-DNA libraries were enriched by PCR amplification. And the PCR products were purified by QIAquick PCR Purification Kit. These libraries were subjected to target enrichment and then precede paired-end (PE) 90 cycles sequencing on Illumina HiSeq2000 Analyzers (following the manufacturer's standard cluster generation and sequencing protocols). The PE reads were mapped to the human reference genome (Hg19, Build37.3) using SOAP2 [@pone.0065050-Li2] with maximally five mismatches. PCR duplication and non-unique alignments reads were also removed before following analysis. The genotypes of 4,524 SNPs for parents and fetus were detected using SOAPsnp. All the raw sequencing data had submitted to NCBI SRA (<http://www.ncbi.nlm.nih.gov/sra>) and the Submission ID is SRA071774. Bioinformatic Model for Zygosity Determination {#s4c} ---------------------------------------------- To noninvasively determine zygosity using maternal plasma sequencing, we constructed a comprehensive bioinformatic model based on paternal-specific heterozygous SNP loci. Those loci provide applicable information to determine the zygosity in the massive background of the maternal homozygotes on these SNPs. We defined ***L~i~*** as a likelihood ratio to measure the tendency of zygosity of a single locus. Through the simulation of 10,000 loci with the same fetal genotype (Type I) and 10,000 with different fetal genotypes (Type II), we discovered the natural logarithm of ***L~i~*** (ln ***L~i~***) of most loci of Type I was lower than 0 while ln ***L~i~*** of most loci of Type II was higher than 0 (**[Figure S1](#pone.0065050.s001){ref-type="supplementary-material"}**). As most loci could increase the signal-noise ratio in our zygosity determination, we used the cumulative difference ***L*** brought by all ***L~i~*** to enrich the signal and regarded it as the effective evidence to determine the zygosity. We firstly calculated cff-DNA concentration based on parental-specific homozygous SNP loci. For each available biparental homozygous SNP locus (♂AA♀BB), where the fetal genotypes of both twins are definite to be AB, we calculated the ratio as the percentage cff-DNA from this locus, where ***d*** meant the depth of the allele A or B. Then the percentage cff-DNA was estimated by calculating the average value of all the ratios. The total calculating formula is: As for the detailed calculation, for each available paternal-specific heterozygous SNP locus (♂AB♀AA), the conditional probability of DZ twins was calculated as: Conditional probability of MZ twins was calculated as: In the equation, ***F~0~*** and ***F~1~*** stood for DZ fetuses and ***F*** for MZ fetuses; ***G~F0,\ F1~*** and ***G~F~*** mean genotype for fetuses; ***B~i~*** mean the observation of base distribution at a typical locus in maternal plasma; ***j*** stood for the number of fetal genotypes. Theoretically, genotypes in paternal-specific heterozygous loci should be the same in MZ twins, while probably different in DZ twins. Here we used ***L~i~*** as an odd ratio between the conditional probability of DZ and MZ twins pregnancies to quantify the tendency of zygosity: ***L~i~*** value should be larger than 1 if there was a DZ twins pregnancies. We employed ***L*** as the numerically cumulative difference of ***L~i~*** to describe the global tendency of zygosity. The total likelihood ratio ***L*** value would be calculated by at least hundreds of paternal-specific heterozygous loci as a geometrical mean: Considering the fluctuation of sequencing depths and cff-DNA concentration, we set a dynamic threshold for ***L*** values to determine zygosity. Of 4,524 autosomal SNP loci, we randomly generated maternal plasma sequence results of 500 paternal-specific heterozygous loci for DZ and MZ twin pregnancies to calculate the likelihood ratio ***L*** value. In order to obtain a series of real cut-offs (***L~R~***) as the boundaries of \>95% CI, we simulated six different sequence depths from 300× with 200× of gradient increasing and five different cff-DNA concentrations from 10.00% with 5.00% of gradient increasing, for 500 DZ and 500 MZ samples. Hereinto we used least squared method (LSM) to get two approximate mathematical expressions of DZ and MZ dynamic cut-offs respectively by using ***L~R~***. It was expressed as:where ***f*** means cff-DNA concentration and ***D*** represents sequence depth. The reliability of the fitting expressions was validated by calculating the multiple correlation coefficients ***R^2^***. The results for DZ and MZ expressions were 0.98 and 0.95 respectively, indicating the reasonability of the expressions. For better understanding of our methodology, we illustrated a three-dimensional figure through our fitting expressions of ***L*** to exhibit the broader feasible region with the enhancement of percentage cff-DNA and sequence depth (**[Figure S2](#pone.0065050.s002){ref-type="supplementary-material"}**). The figure showed that DZ twins' feasible region was upon the upper surface, while MZ twins' feasible region was below the inferior surface. Lastly, additional 1,000 DZ and 1,000 MZ simulated samples were generated to estimate the sensitivity and specificity for different cff-DNA concentration and sequence depth *in silico*. Supporting Information {#s5} ====================== ###### ***L~i~* distribution of two types of loci.** 10,000 loci of Type I, which were represented by using red pillars, meant those with fetal genotypes in concordance. While 10,000 loci of Type II, which were represented by using green pillars, meant those without fetal genotypes in concordance. (TIF) ###### Click here for additional data file. ###### **The three-dimensional feasible region of zygosity determination.** The zone beyond the surface above meant the feasible region for DZ twins, while the zone under the surface below meant the feasible region for MZ twins. (TIF) ###### Click here for additional data file. ###### **Real cut-offs with different total cff-DNA concentration and sequence depth.** (DOC) ###### Click here for additional data file. ###### **Evaluation results of sensitivity and specificity *in silico*.** (DOC) ###### Click here for additional data file. ###### **Information about the 4524 autosomal SNP loci.** (XLS) ###### Click here for additional data file. We sincerely thank our colleagues at the BGI-Shenzhen for sequencing. We thank Ruoyu Zhang for excellent advice and revise of the manuscript. [^1]: **Competing Interests:**Jing Zheng, Jing Guo, Huijuan Ge, Xuchao Li, Chunlei Zhang, Haojun Jiang, Weiping Tang, Weiwei Xie, Hongyun Zhang, Fuman Jiang, Shengpei Chen, Wei Wang, Xun Xu, Fang Chen, Hui Jiang are employees of BGI-Shenzhen and none of the other authors have any financial relationship with BGI-Shenzhen. Also Huijuan Ge, Jing Zheng and Xuchao Li have filed patent applications on the effective noninvasive zygosity determination by maternal plasma target region sequencing. The patent number is PCT/CN2012/075969 and the date is 2012-5-23. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. [^2]: Conceived and designed the experiments: JZ JG HG. Performed the experiments: JZ HG Haojun Jiang WX. Analyzed the data: JG XL HZ FJ. Contributed reagents/materials/analysis tools: CX YW YZ HH LP WT. Wrote the paper: JZ JG CZ SC FC Hui Jiang. Supplied the direction for this study: FC Hui Jiang WW XX.

All relevant data are within the paper. Introduction {#sec001} ============ Acetaminophen-induced toxicity, like many other drugs, may have a great deal of variations at the molecular, cellular, tissue, organ and organism levels \[[@pone.0145965.ref001]--[@pone.0145965.ref002]\]. Metabolic alterations and increased oxidative stress is considered to be the key aspects of hepatotoxicity and apoptotic as well as necrotic cell death by acetaminophen (APAP) \[[@pone.0145965.ref003]--[@pone.0145965.ref005]\]. The initial events in APAP-induced toxic injury lead to the activation of a secondary innate immune response by up regulation of proinflammatory cytokines and inflammasome \[[@pone.0145965.ref006]--[@pone.0145965.ref007]\]. Thus, alterations in the microenvironment by macrophages and their chemical communication and coordination with tissues play a major role in the progression and prevention of drug-induced toxicities and tissue repair. The precise molecular mechanism of APAP cytotoxicity however, is still controversial \[[@pone.0145965.ref008]--[@pone.0145965.ref009]\]. Reports suggest glutathione (GSH) depletion, oxidative stress and mitochondrial dysfunction in APAP-induced toxicity \[[@pone.0145965.ref003],[@pone.0145965.ref010]--[@pone.0145965.ref011]\].The general consensus in APAP-induced toxicity is that the drug is mainly metabolized by various cytochrome P450s such as CYP2E1, CYP3A4, CYP1A2 and CYP1A1 to its active metabolite, mainly N-acetyl-p-benzoquinone imine (NAPQI) \[[@pone.0145965.ref012]--[@pone.0145965.ref013]\] which conjugates with GSH causing depletion of cellular GSH pools and increase in oxidative stress. Studies have suggested that APAP toxicity exhibited a biphasic response in which the metabolism of APAP is responsible for initial toxicity followed by mitochondrial dysfunctions \[[@pone.0145965.ref009],[@pone.0145965.ref014]--[@pone.0145965.ref019]\]. The selective inhibition of proinflammatory signaling and induction of autophagy which removes damaged mitochondria, attenuates APAP-induced liver toxicity \[[@pone.0145965.ref007], [@pone.0145965.ref018], [@pone.0145965.ref020]--[@pone.0145965.ref021]\]. Both cytotoxic and cytoprotective effects of macrophages have been reported in APAP-induced toxicity \[[@pone.0145965.ref019], [@pone.0145965.ref022]--[@pone.0145965.ref023]\]. Our previous study on J774.2 macrophages demonstrated that APAP induces cytotoxicity and apoptosis by increasing ROS production, depletion of GSH pool, increase in oxidative stress and mitochondrial dysfunction \[[@pone.0145965.ref024]--[@pone.0145965.ref025]\]. Using macrophages and HepG2 cells as in vitro models, we have recently reported that aspirin treatment also induces oxidative stress and mitochondrial dysfunction, albeit at different levels \[[@pone.0145965.ref026]--[@pone.0145965.ref029]\]. Induction of cellular resistance has also been reported after APAP treatment. Several studies suggest that APAP treatment may also develop resistance towards drug toxicity by altering multidrug resistance protein, JNK-dependent signaling, autophagy in cells under in vitro conditions and in vivo in mice \[[@pone.0145965.ref020], [@pone.0145965.ref030]--[@pone.0145965.ref031]\]. There are multiple metabolic factors which determine the APAP-induced initial or long term cytotoxicity, which may or may not be dependent upon GSH depletion, metabolic activation and detoxification of the drug by the enzymes. However, APAP toxicity in vivo and in vitro has been correlated with CYP450s enzymes, particularly with CYP2E1 and CYP3A4 which metabolizes APAP to its toxic metabolites \[[@pone.0145965.ref013], [@pone.0145965.ref032]\] and could be blocked by CYP2E1 modulators such diallyl sulfide (DAS), a major garlic constituent, and antioxidants such as N-acetylcysteine \[[@pone.0145965.ref017],[@pone.0145965.ref033]--[@pone.0145965.ref034]\].The objective of the present study was to elucidate the molecular mechanisms of differential toxicity of APAP in HepG2 cells and macrophages and the protection of cytotoxicity by DAS. The main focus of our study is to highlight the role of drug metabolizing enzymes, glutathione metabolism, oxidative stress and mitochondrial function in APAP induced cytotoxicity and cytoprotection by DAS. Materials and Methods {#sec002} ===================== Reagents {#sec003} -------- N-Acetyl-p-aminophenol (acetaminophen, APAP), diallyl sulfide (DAS), 5,5'-dithio bis-2-nitrobenzoic acid (DTNB), NADPH, glutathione reductase, 1-chloro 2,4-dinitrobenzene (CDNB), oxidized glutathione (GSSG) and reduced glutathione (GSH), N-ethyl malleimide (NEM), ethacrynic acid, 4-hydroxynonenal (4-HNE), cumene hydroperoxide, dimethylnitrosamine (DMNA), erythromycin, Coenzyme Q2, succinate, cytochrome c, ethoxyresorufin and methoxyresorufin and ATP bioluminescent assay kit were purchased from Sigma-Aldrich Fine Chemicals (St Louis, MO, USA). Apoptosis detection kit for flow cytometry was from BD Pharmingen (BD Biosciences, San Jose, USA) and comet assay kits were procured from Cell Biolabs, Inc. (San Diego, CA, USA). Kits for mitochondrial membrane potential were purchased from R&D Systems, (MN, USA). 2', 7'-dichlorofluorescein diacetate (DCFDA) was purchased from Molecular Probe (Eugene,OR,USA). Aconitase assay kit was procured from Oxis International Inc. (Portland, OR, USA). HepG2 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and murine macrophage J774.2 cells were purchased from European Collection of cell cultures (Health Protection Agency Culture Collections, Salisbury, UK). Antibodies against microsomal GST and GST A4-4 were generous gifts from Prof. Ralf Morgenstern, Karolina Institute, Stockholm, Sweden and Prof. Bengt Mannervic, Uppsala University, Uppsala, Sweden, respectively. Polyclonal antibodies against CYP1A1, CYP1A2, CYP2E1 and CYP3A4 were purchased from Amersham Int. Plc. (Amersham, UK) and GSTpi specific isoenzyme from Biotrin (Dublin, Ireland). MDR1and β-actin antibodies were procured from Santa Cruz Biotechnology, Inc, (CA, USA). Reagents for cell culture and for SDS-PAGE and Western blot analyses were purchased from Gibco BRL (Grand Island, NY, USA) and from Bio Rad Laboratories (Richmond, CA, USA) respectively. Cell Culture, Treatment and Fractionation {#sec004} ----------------------------------------- Macrophage J774.2 cells and HepG2 cells were grown in poly-L-lysine coated 75 cm^2^ flasks (\~2.0--2.5 x10^6^ cells/ml) in DMEM medium supplemented with 10% heat inactivated fetal bovine serum in the presence of 5% CO~2~-95% air at 37°C. Cells were treated with APAP (10 μmol/ml) for 18 hours after treatment with or without 200 μM DAS for 24 h. After the desired time of treatment, cells were harvested, washed with PBS (pH 7.4) and homogenized in H-medium buffer (70 mM sucrose, 220 mM mannitol, 2.5 mM HEPES, 2 mM EDTA, and 0.1 mM phenylmethylsulfonylfluoride, pH7.4) at 4°C. Mitochondria and postmitochondrial (PMS) fractions were prepared by centrifugation and the purity of the isolated fractions for cross contamination was checked as described before \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\]. Control cells were treated with vehicle alone. The choice of time and doses were based on our previous publications and literatures using acetaminophen in these cell lines as well as MTT viability test \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\]. DNA Fragmentation, Apoptosis, ROS Assays {#sec005} ---------------------------------------- ### DNA fragmentation {#sec006} DNA fragmentation was assayed by UV transillumination after staining the electrophoretically (by 1.5% agarose gel) separated fragments with 0.5 μg/ml ethidium bromide as described before \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\]. ### Comet assay {#sec007} Cellular DNA damage was assessed using the single cell gel electrophoresis or comet assay according to the vendor's protocol. Briefly, following treatment, cells were washed in cold PBS, centrifuged and resuspended at 1x10^5^ cells/ml in cold PBS. An aliquot of the resuspended cells was combined with low melting point agarose at 1:10 ratio, mixed and this cell suspension transferred to 3-well glass slides, ensuring complete well coverage and kept in the dark for 15 min at 4°C. Slides were then incubated for 1h at 4°C in lysis buffer. Following lysis, the slides were placed in 1.2% NaOH solution for 30 min at 4°C. The slides were then transferred to a horizontal electrophoresis chamber containing electrophoresis buffer (300mM NaOH, 1mM EDTA, pH\>13) and run for 15--30 min at 1volt/cm. The slides were then washed with distilled water, followed by 70% ethanol and then stained using the Vista Green DNA dye at room temperature for 15 min. The slides were then analyzed using an Olympus fluorescence microscope. Typical representation from 3 such experiments has been shown. ### Apoptosis {#sec008} The apoptosis assay by flow cytometry using Annexin V conjugated FITC and propidium iodide was performed according to the vendor's protocol as described in the previous study \[[@pone.0145965.ref024]--[@pone.0145965.ref026]\]. The apoptotic cells were estimated by the percentage of cells that stained positive for Annexin V-FITC. ### ROS assay {#sec009} The intracellular production of ROS was measured by using lucigenin-coupled chemiluminiscence assay which preferentially measures superoxides as described before \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\]. Measurement of GSH Metabolism {#sec010} ----------------------------- Total cellular GSH concentration was measured by enzymatic recycling of oxidized glutathione by Griffith's method using NADPH and GSH-reductase as described before \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\].Total GST activity was measured in APAP treated cells by using CDNB as substrate \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\]. Substrate specific GST pi and GST A4-4 activities were measured using ethacrynic acid and 4-hydroxynonenal (4-HNE) as substrates. Microsomal GST activity was measured in the presence of NEM using CDNB as substrate. GSH-peroxidase activity was measured using cumene hdroperoxide as substrate as described before \[[@pone.0145965.ref027]--[@pone.0145965.ref028]\]. Measurement of CYP450 Activities {#sec011} -------------------------------- Post-mitochondrial supernatant was used to measure the activities of the enzymes,CYP1A1,CYP1A2, CYP2E1 and CYP3A4, using ethoxyresorufin, methoxyresorufin, DMNA and erythromycin, respectively, as substrates as described before \[[@pone.0145965.ref025],[@pone.0145965.ref028], [@pone.0145965.ref035]\]. Measurement of ATP Level {#sec012} ------------------------ The ATP content in the cell lysate was determined using an ATP Bioluminescent cell assay kit according to the manufacturer's suggestion and samples were read using the TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA). A standard curve for different concentrations of ATP (5--500 nM) was used to calculate the concentration of ATP in the control and treated cells. Measurement of Mitochondrial Membrane Potential (Δψm) {#sec013} ----------------------------------------------------- The mitochondrial membrane potential (Δψm) was measured by flow cytometry using a fluorescent cationic dye according to the vendor's protocol (DePsipher^TM^, R &D System Inc.). DePsipher has the property of aggregating upon membrane polarization forming an orange-red fluorescent (absorption/emission 585/590nm) compound. If the membrane potential is reduced, the dye cannot access the transmembrane space and remains in its green fluorescent (510/527nm) monomeric form. Measurement of Enzymes of Krebs Cycle and Mitochondrial Respiratory Complexes {#sec014} ----------------------------------------------------------------------------- The freshly isolated mitochondria (5μg protein) from control and treated macrophage and HepG2 cells were suspended in 1.0 ml of 20 mM potassium phosphate buffer, pH 7.4, in the presence of the detergent, lauryl maltoside (0.2%). Mitochondrial matrix enzyme aconitase activity was measured by NADPH coupled conversion of citrate to isocitrate in the presence of isocitrate dehydrogenase using Bioxytech Aconitase-340 assay kit as described before \[[@pone.0145965.ref026]\]. NADH-ubiquinone oxidoreductase (Complex I), and cytochrome c oxidase (complex IV) were measured using the substrates ubiquinone and reduced cytochrome c, respectively, as described before \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\]. SDS-PAGE and Western Blot Analysis {#sec015} ---------------------------------- Protein (50 μg/well), from the sub-cellular fractions prepared as described above, was separated on 12% SDS-PAGE according to the method of Laemmli (1970) \[[@pone.0145965.ref036]\]. Electrophoresed proteins were transferred to nitrocellulose membrane and subjected to Western blotting \[[@pone.0145965.ref037]\]. Transferred proteins were then probed with rabbit antibodies against CYP2E1, CYP3A4, CYP1A1 and CYP1A2 (1:1000 dilution), GST A4-4, pi, microsomal GST1-1 (1:1000 dilution), and subsequently detected with peroxidase conjugated species specific secondary antibodies (1:5000 dilution). The signals were visualized and further densitometric analysis performed using the Typhoon FLA 9500 system (GE Healthcare, Uppsala, Sweden) and expressed as relative intensity (R.I) compared to the untreated control. β-Actin was used as a loading control. \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\]. Statistical Analysis {#sec016} -------------------- Statistical comparison of control and drug treated groups was analyzed using SPSS software (version 21) by ANOVA followed by Dunnett's post-hoc analysis. The values are expressed as mean ± SEM and p values≤0.05 were considered significant. Results {#sec017} ======= Effect of APAP on DNA Fragmentation, Apoptosis, and ROS Production {#sec018} ------------------------------------------------------------------ [Fig 1](#pone.0145965.g001){ref-type="fig"} shows that the macrophages are much more sensitive to APAP treatment than HepG2 cells as indicated by the fragmentation and laddering of DNA in these cells. Comet single cell gel electrophoresis assay showed enhanced DNA fragmentation in macrophages after treatment with APAP (10 μmol/ml) for 18 h). No significant DNA fragmentation or laddering was however observed in HepG2 cells under these conditions. {#pone.0145965.g001} Similarly, more cell death was observed in macrophages than in HepG2 cells after APAP treatment ([Fig 2](#pone.0145965.g002){ref-type="fig"} right upper panel, 56.8% vs 21%) when compared with control untreated cells. Interestingly, HepG2 cells exhibited a higher level of early apoptosis than that seen in macrophages ([Fig 2](#pone.0145965.g002){ref-type="fig"} right lower panel, 48% vs 19.8%). These results suggest more late apoptosis (necrotic) cell death in macrophages by APAP than in HepG2 cells. These results confirm our earlier electron microscopic studies on macrophages treated with different doses of APAP at different time points \[[@pone.0145965.ref025]\]. Our results also show that DAS treatment resulted in a significant reduction of apoptosis in both the cell systems. However, the protective effect of DAS treatment on necrotic cell death induced by APAP was more apparent in HepG2 cells than in macrophages. These results suggest that macrophages are more sensitive to APAP induced toxicity than HepG2 cells. {#pone.0145965.g002} APAP treatment in macrophages and HepG2 cells has significantly increased ROS production ([Fig 3](#pone.0145965.g003){ref-type="fig"}). Pretreatment with DAS significantly reduced the level of ROS production in APAP treated HepG2 cells but not in macrophages. DAS alone does not cause any appreciable alterations in ROS production in these cell lines. ![Effect of APAP on ROS production.\ Intracellular production of ROS was measured using lucigenin coupled method and chemiluminescence was measured using Turner's luminometer as described in Materials and Methods \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\]. The values expressed are mean ±SEM for at least three determinations. Asterisks (\*) indicate significant difference (p ≤0.05) from control values, \# indicate significant difference (P ≤0.05) from APAP-treated group.](pone.0145965.g003){#pone.0145965.g003} Effect of APAP on GSH Metabolism {#sec019} -------------------------------- A marked decrease in total GSH level in the mitochondrial compartment and cytosolic fraction was observed in macrophages after APAP treatment ([Fig 4A](#pone.0145965.g004){ref-type="fig"}). GSH concentration was reduced significantly only in the mitochondrial compartment of HepG2 cells ([Fig 4A](#pone.0145965.g004){ref-type="fig"}). DAS treatment exhibited a significant recovery in the concentration of GSH. However the concentration of mitochondrial GSH was still below the control untreated cells. These results again suggest that the macrophages are more sensitive to APAP- induced cytotoxicity than HepG2 cells. ![Effect of APAP on GSH metabolism.\ J774.2 macrophages andHepG2 cells were treated with APAP and DAS alone or in combination and GSH levels in the mitochondria and post-mitochondrial supernatant (PMS) were measured by enzymatic method as described in the Materials and Methods (4A). Total GST-conjugating activity was measured using CDNB as a substrate (4B). Microsomal GST activity was measured using CDNB as a substrate in the presence of NEM as an activator of membrane-bound microsomal GST (4C). Ethacrynic acid was used as substrate to measure GSTpi isoenzyme (4D) and 4-HNE was used to measure GSTA4-4 isoenzyme (4E) as described before \[[@pone.0145965.ref024]--[@pone.0145965.ref028]\]. GSH-Px activity was measured using cumene hydroperoxide as a substrate (4F). Results are expressed as mean ± SEM of three determinations. Asterisks (\*) indicate significant difference (p ≤0.05) from control values, \# indicate significant difference (p ≤0.05) from APAP-treated group.](pone.0145965.g004){#pone.0145965.g004} Interestingly, differential GSH-CDNB conjugating activity of glutathione S-transferase enzyme was observed in the macrophages and HepG2 cells treated with APAP ([Fig 4B](#pone.0145965.g004){ref-type="fig"}). While a significant decrease in enzyme activity was observed in the cytosolic and mitochondrial compartments in macrophages, a significant increase in total GSH-CDNB conjugating activity was observed in HepG2 cells. Pretreatment with DAS brought the enzyme activity close to that of control untreated cells. On the other hand, membrane bound microsomal GST was only significantly increased in HepG2 cells after APAP treatment but not in macrophages ([Fig 4C](#pone.0145965.g004){ref-type="fig"}). In order to further investigate the role of specific GST isoenzymes in GSH-conjugation in these cell lines, we further studied the enzyme activity using isoenzyme specific substrates, ethacrynic acid for GST pi and 4-hydroxynonenal (4-HNE) for GST A4-4. GST pi activity was increased in APAP- treated macrophages as well as HepG2 cells ([Fig 4D](#pone.0145965.g004){ref-type="fig"}). On the other hand, 4-HNE-conjugating GST A4-4 activity was slightly increased in HepG2 cells, while a marked reduction in enzyme activity was observed in the macrophage cells ([Fig 4E](#pone.0145965.g004){ref-type="fig"}). This was also confirmed by SDS-PAGE/Western blot analysis of enzyme protein expression using isoenzyme-specific antibodies (described later). DAS treatment alone had little effect on GST pi activity in these cells. However, GST A4-4 activity was enhanced in both cell lines. These results suggest the differential responses of macrophages and HepG2 cells towards APAP- induced cytotoxicity. GSH-peroxidase (GSH-Px) activity, on the other hand, was significantly inhibited in the mitochondrial and post-mitochondrial compartments after APAP treatment both in the macrophages as well as in HepG2 cells ([Fig 4F](#pone.0145965.g004){ref-type="fig"}). DAS treatment resulted in partial recovery of enzyme activity only in the post-mitochondrial compartment of HepG2 cells. Effects of APAP on CYP450 Activities {#sec020} ------------------------------------ We used isoenzymes-specific substrates to measure the microsomal activities of CYP2E1, CYP3A4, CYP1A1 and CYP1A2 in macrophages and HepG2 cells treated with APAP and DAS. While CYP2E1 activity was significantly lower in the macrophages, the enzyme activity was markedly (56%) increased in HepG2 cells ([Fig 5A](#pone.0145965.g005){ref-type="fig"}). As expected, DAS treatment in the macrophages resulted in a 2--3 fold decrease in activity. However, unlike in the macrophages, the enzyme activity remained higher than that seen in the control untreated HepG2 cells ([Fig 5A](#pone.0145965.g005){ref-type="fig"}). {#pone.0145965.g005} Interestingly, alterations in CYP3A4 activity exhibited a different pattern. A marked increase (about 2-fold) in enzyme activity was only observed in the macrophages while a significant decrease (30%) was observed in HepG2 cells ([Fig 5B](#pone.0145965.g005){ref-type="fig"}). These results have further confirmed our earlier observation on the alterations of different CYPs in macrophages and HepG2 cells \[[@pone.0145965.ref025]\]. CYP1A1 and CYP1A2 activities in both the cell lines have demonstrated a more or less similar pattern of alterations ([Fig 5C and 5D](#pone.0145965.g005){ref-type="fig"}). A 2--3 fold increase in enzyme activities was observed after APAP treatment in both the cell lines. DAS treatment, either alone or in the presence of APAP, had no significant effects on CYP1A1enzyme activity. CYP1A2 activity was, however, significantly reduced after DAS treatment in APAP-induced macrophages and HepG2 cells ([Fig 5D](#pone.0145965.g005){ref-type="fig"}). Effect of APAP on Mitochondrial Functions: ATP Production, Membrane Potential and Respiratory Enzymes Complexes {#sec021} --------------------------------------------------------------------------------------------------------------- APAP treatment reduced the ATP levels drastically (40--80%) in HepG2 cells and macrophages ([Fig 6A](#pone.0145965.g006){ref-type="fig"}). Interestingly, DAS treatment recovered the ATP levels close to the control cells. The mitochondrial membrane potential, as determined by cationic dye membrane permeability, demonstrated an increased loss of membrane potential and DAS treatment has brought the potential close to that of the control untreated cells ([Fig 6B](#pone.0145965.g006){ref-type="fig"}). These results suggest that alterations in ATP production after APAP treatment are correlated with the disturbance in mitochondrial potential gradient. The mitochondrial matrix enzyme, aconitase, which is a sensitive marker for mitochondrial oxidative stress related dysfunction, was also found to be profoundly inhibited after APAP treatment which confirms our earlier observation \[[@pone.0145965.ref024]--[@pone.0145965.ref025]\]. DAS treatment recovered the enzyme activity close to the control untreated level ([Fig 6C](#pone.0145965.g006){ref-type="fig"}). However, a differential effect of APAP on the mitochondrial membrane bound respiratory enzyme complexes was observed in the macrophages and HepG2 cells. While activities of Complex I (NADH-Ubiquinone oxidoreductase) and terminal oxygen utilizing enzyme, Complex IV (cytochrome c oxidase) were negligibly disturbed in the HepG2 cells, there was marked inhibition in the activities of both enzymes in the APAP-treated macrophages ([Fig 6D](#pone.0145965.g006){ref-type="fig"}). DAS treatment alone had no appreciable effects on these enzyme activities. Only a marginal recovery of Complex I enzyme activity was observed after DAS treatment in APAP-treated macrophages. {#pone.0145965.g006} Effect of APAP on the Expression of Proteins {#sec022} -------------------------------------------- [Fig 7A](#pone.0145965.g007){ref-type="fig"} shows the expression of cytosolic and microsomal GST proteins' expression using isoenzyme-specific antibodies. APAP treatment resulted in almost 2-fold induction of GST pi protein in the macrophages as well as HepG2 cells. DAS pre-treatment brought the levels close to normal, more so in the macrophages. GSTA4-4, a member of the alpha GST family which conjugates lipid peroxidation product, 4-HNE, was slightly increased after APAP treatment in HepG2 cells. However, a significant reduction in the expression of GSTA4-4 was observed in the macrophages. This differential expression may be associated with the level of GSH metabolism and 4-HNE conjugation in these cell lines. On the other hand, the microsomal GST (MGST1-1) expression after APAP treatment was not significantly altered in macrophages while it was increased in the HepG2 cells. DAS treatment had minimal effects on the expression of microsomal GST. {#pone.0145965.g007} [Fig 7B](#pone.0145965.g007){ref-type="fig"} shows the differential expression of various CYP450s in the macrophages and HepG2 cells after APAP and DAS treatment. While CYP2E1 protein expression was inhibited by APAP treatment, it was induced about 2 fold in HepG2 cells. DAS, a known inhibitor of CYP2E1 activity and expression, as expected, has markedly inhibited the enzyme expression in these cell lines. The pattern of expression of CYP3A4, on the other hand, was reversed. While a 2-fold increase after APAP treatment was observed in the macrophages, a marginal decrease in the expression was observed in HepG2 cells. The differential expression of CYP2E1 and CYP3A4 may be implicated with the preferential isoenzyme involved in the metabolism of APAP in macrophages and HepG2 cells and are in agreement of our previous reports \[[@pone.0145965.ref024]--[@pone.0145965.ref025]\]. CYP1A1 and CYP1A2 expression, on the other hand, was markedly increased in these cells and DAS treatment has brought the expression level close to the controls. [Fig 7C](#pone.0145965.g007){ref-type="fig"} shows the expression of multidrug resistance glycoprotein, MDR1 in macrophages and HepG2 cells treated with APAP and DAS. Both cell lines exhibited an increased expression of MDR1 protein. However, the increased level of expression was more significant in HepG2 cells compared to macrophages. DAS treatment reversed the expression of the protein close to the controls. These results may suggest differential drug-conjugate exclusion which may be detrimental for drug-induced cytotoxicity and resistance in these cell lines. Discussion {#sec023} ========== Metabolism of APAP, a hepatotoxin, by a family of CYP450s, depletion of GSH, which conjugates the toxic metabolite of APAP and a burst of oxidative damages have all been implicated in APAP-induced toxicity \[[@pone.0145965.ref003],[@pone.0145965.ref008],[@pone.0145965.ref009],[@pone.0145965.ref021]\]. Several antioxidants and activators of GSH pool, such as N-acetylcysteine, dithiothreitol, tea polyphenols, diallyl sulfide (DAS), taurine, melatonin, ascorbic acid, vitamin E, etc. have been shown to be beneficial in preventing APAP-induced toxicity \[[@pone.0145965.ref033],[@pone.0145965.ref038]--[@pone.0145965.ref039]\]. However, the precise mechanism of cytotoxicity in different tissues and cellular systems is not clear. Macrophages have been implicated both in the attenuation as well as augmentation of APAP-induced responses \[[@pone.0145965.ref022], [@pone.0145965.ref023]\]. We have previously shown that APAP induced cytotoxicity and cell death in macrophages is associated with increased oxidative stress, alterations in GSH pool, oxidative protein carbonylation and activation of mitochondrial apoptotic signals \[[@pone.0145965.ref024], [@pone.0145965.ref025]\]. Our present study, using both macrophages and HepG2 cells has further confirmed that the cytotoxicity induced by APAP is indeed associated with increased oxidative stress and mitochondrial dysfunction. However, our results have also demonstrated that the macrophages appear to be more sensitive to APAP toxicity compared to HepG2 cells at the same dose and time point. This was demonstrated by increased DNA breakdown and apoptosis and depletion of GSH in the macrophages compared to HepG2 cells. These differential effects of APAP cytotoxicity in macrophages and HepG2 cells appears to be associated with the differential metabolism of APAP by CYP2E1 and CYP3A4 in these cells lines as well as differences in GSH metabolism by different isoenzymes of GST. As shown, macrophages exhibited a higher induction of CYP3A4 activity while a lower activation of CYP2E1 and GST A4-4 activities were observed. On the other hand, HepG2 cells exhibited higher activity of CYP2E1 and GSH-conjugating activities of CDNB by GSTpi and 4-HNE by GSTA4-4. CYP3A4 activity however, was, lower in HepG2 cells compared to the macrophages. On the other hand, the microsomal membrane bound GSH conjugating isoenzyme, was activated only in HepG2 cells but not in the macrophages. SDS-PAGE and Western blot analysis also confirmed the differential expression of CYP450 and GST isoenzymes in these cell lines. These results clearly suggest a differential mechanism of APAP activation by CYPs and conjugation by GST isoenzymes in the macrophages and HepG2 cells. Treatment with DAS, a garlic constituent with known CYP2E1 inhibitory activity, recovered the changes observed in CYP2E1 activity in HepG2 cells but not in the macrophages. On the other hand, DAS treatment alone had very little effect on the alterations in CYP3A4, CYP1A1 and CYP1A2 activities. GSH-Px activity in macrophages as well as in HepG2 cells was significantly lower in both cell systems suggesting increased oxidative stress and confirming our previously published results. DAS treatment marginally recovered the altered GSH-Px activity in both cell systems. Thus DAS acts not only via the inhibition of CYP2E1 activity but also as an antioxidant in these cell systems. There are numerous reports suggesting the involvement of DAS in protection against APAP toxicity due to the inhibition of CYP2E1, and increasing GSH metabolism, which is suggestive of an antioxidant effect for DAS \[[@pone.0145965.ref033], [@pone.0145965.ref040]\]. Interestingly, the expression of multi drug resistance protein, MDR1, which plays a role in drug exclusion and detoxification, including APAP \[[@pone.0145965.ref030]--[@pone.0145965.ref031], [@pone.0145965.ref041]\], was also markedly higher in HepG2 cells compared to macrophages which might be associated with increased efflux or detoxification of APAP from the HepG2 cells resulting in the development of some resistance in these cells, as seen by the low level of apoptosis and DNA damage in comparison to macrophages. Increased expression and activities of GST in APAP-treated HepG2 cells also support this observation. We have further demonstrated that mitochondrial respiratory function and ATP synthesis was markedly affected by APAP treatment in macrophages as well as in HepG2 cells. The increased ROS production and apoptosis observed in APAP-treated macrophage J774.2 and HepG2 cells might be implicated with mitochondrial dysfunction in these cell systems. This was further confirmed by drastic inhibition of mitochondrial membrane potential, decreased activity of aconitase, a mitochondrial matrix enzyme and marker for oxidative stress, and by inhibition of the inner membrane respiratory enzymes complexes. This has been reported earlier using APAP and other NSAIDs. \[[@pone.0145965.ref024]--[@pone.0145965.ref029], [@pone.0145965.ref042]\]. Increased ROS production and mitochondrial oxidative stress also results in increased oxidative glutathionylation of respiratory complex I resulting in the inhibition of enzyme activity and disruption of energy homeostasis in APAP toxicity \[[@pone.0145965.ref043]--[@pone.0145965.ref046]\]. In summary, our results suggest that the differential cytotoxicity of APAP in macrophage J774.2 and HepG2 cells might be associated with their ability to metabolize and detoxify APAP differently in these cell systems. Also, there is some indication that MDR1 gene may also play a role in determining the sensitivity of macrophages and HepG2 cells towards APAP toxicity. These results may have long term implications to better understand the role of macrophages and HepG2 cells in the toxicology and pharmacology of APAP. Authors wish to acknowledge the support from the Research Committee Grant, CMHS, UAE University, UAE. Supports from Sheikh Hamdan bin Rashid Al Maktoum Medical Research Award and Terry Fox Cancer Research Fund (UAE) are also acknowledged. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: HR. Performed the experiments: AJ HR. Analyzed the data: AJ HR. Contributed reagents/materials/analysis tools: AJ HR. Wrote the paper: HR AJ.

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ Illegal, unreported, and unregulated (IUU) fishing undermine efforts to sustainably manage fish stocks and threaten fish populations worldwide \[[@pone.0143960.ref001]\]. Managers must know as much as possible about the extent, character (e.g., gear types, target/bycatch species, timing, location), and motivations of illegal fishing to effectively develop and implement regulations. However, quantifying illegal fishing is inherently difficult: it is generally covert and significant incentives exist for informants to withhold information \[[@pone.0143960.ref002]\]. Furthermore, budget and human resource constraints often restrict efforts to monitor illegal resource use, especially in developing countries \[[@pone.0143960.ref003]\]. There is a need to develop inexpensive yet informative methods for quantifying illegal fishing and its impacts. Indirect observation, the use of signs of illegal activity as an indicator of non-compliance, has been commonly used to characterize illegal resource use in terrestrial systems \[[@pone.0143960.ref004]\], but has been infrequently used in marine systems \[[@pone.0143960.ref005]\], and to our knowledge, has never been used in freshwater systems. In marine systems, dynamite blast craters \[[@pone.0143960.ref006],[@pone.0143960.ref007]\] and derelict fishing gear \[[@pone.0143960.ref008]\] have been used as indicators of illegal fishing, but have generally failed to quantitatively measure non-compliance \[[@pone.0143960.ref005]\]. Most successful quantifications of illegal fishing compare the amount of derelict fishing gear inside and outside reserve boundaries \[[@pone.0143960.ref009]--[@pone.0143960.ref012]\], but such comparisons are of little use in places without reserves or where the areas outside reserves are undesirable to fishers. The full capacity for indirect observation to reveal rich and quantitative information about illegal fishing remains unexplored. Indirect observation offers several advantages over other approaches for assessing illegal fishing. It does not require large amounts of labor, specialized equipment, or training and can be recorded during routine enforcement patrols or biological surveys \[[@pone.0143960.ref013]\]. Repeated surveys can reveal spatial and temporal patterns of non-compliance \[[@pone.0143960.ref008]--[@pone.0143960.ref010],[@pone.0143960.ref014]\] that can be compared to changes in fish communities to examine the effects of illegal fishing \[[@pone.0143960.ref015]\]. Although indirect observation generally cannot identify specific violators or motivations for non-compliance, they can contribute to a comprehensive understanding of non-compliance when combined with other methods, such as direct questioning \[[@pone.0143960.ref009]--[@pone.0143960.ref010]\]. In this study, we used a mixed-method approach to evaluate the extent, character, and motivations of illegal gillnet fishing in Lake Hovsgol National Park (LHNP), Mongolia and its impact on the lake's fish populations, especially that of the endangered endemic Hovsgol grayling (*Thymallus nigrescens*). Despite the closure of the park to gillnet fishing in 1992, illegal fishing is known to persist \[[@pone.0143960.ref016]--[@pone.0143960.ref017]\]. We used four complementary methods to describe this fishery and evaluate its impacts: (1) surveys for derelict fishing gear, an indirect indicator of fishing activity, to evaluate how much illegal fishing is occurring, where illegal fishing is occurring, and what gear is being used; (2) interviews with herders living within the park and park rangers to validate and contextualize the results of the surveys for derelict fishing gear; (3) biological monitoring to identify fish species vulnerable to gillnet fishing and evaluate changes in population abundance potentially caused by fishing; and (4) data-poor stock assessment methods to estimate the effort required to overexploit the Hovsgol grayling population. Overall, we demonstrate the ability for a mixed-method approach to describe an illegal gillnet fishery and suggest that these methods could be used to effectively and inexpensively assess illegal fishing and its impacts in other protected areas. Methods {#sec002} ======= Study site {#sec003} ---------- Lake Hovsgol (51°05'50"N, 100°30'E) is located in the mountains of northern Mongolia at the southern edge of the Siberian taiga forest. It is the 19^th^ largest lake in the world by volume (480 km^3^) and has a maximum depth of 262 m and surface area of 2,760 km^2^ \[[@pone.0143960.ref018]\]. The lake was established as a National Park in 1992 and is mostly undeveloped. The majority of the resident population lives in two towns on the lakeshore: Hatgal (pop. 2,980) and Hankh (pop. 2,460; \[[@pone.0143960.ref019]\]). Tourist camps line the southwestern shore and herding families live intermittently along the lakeshore (**[Fig 1](#pone.0143960.g001){ref-type="fig"}**). Most of the park's \~35,000 annual visitors enter and remain in the southern portion of the park \[[@pone.0143960.ref020]\]. {#pone.0143960.g001} Lake Hovsgol has ten fish species, the most abundant of which, the Hovsgol grayling (*Thymallus nigrescens*), is endemic to the lake and is listed as endangered on the Mongolian Red List due to climate change and illegal fishing \[[@pone.0143960.ref016]\]. Hovsgol grayling are more common in littoral areas than pelagic areas and are most abundant along the western shore \[[@pone.0143960.ref021]\]. A portion of the grayling population spawns in tributary streams in late spring while another portion spawns in the littoral in late summer \[[@pone.0143960.ref022]\]. The prevalence, fidelity, and success of these spawning strategies are unknown. The sparse literature on Mongolian fisheries suggests that commercial fishing for Hovsgol grayling, lenok (*Brachymystax lenok*), roach (*Rutilus rutilus*), perch (*Perca fluviatilis*), and burbot (*Lota lota*) removed as much as 200--400 tons annually before the park was established (\[[@pone.0143960.ref023]\]; **[S1 Table](#pone.0143960.s001){ref-type="supplementary-material"}**). Despite the ban on gillnet fishing, active gillnets are often observed and grayling and lenok are frequently sold in Hatgal and along the southwestern shore road. Recreational hook-and-line fishing is legal within the park and is regulated through permits and season and bag limits. Subsistence fishing during the spring spawning migration, though officially illegal, is generally tolerated. Surveys for derelict fishing gear {#sec004} --------------------------------- We surveyed and collected derelict fishing gear at ten sites on the Lake Hovsgol shoreline in July 2013 and resurveyed six of these sites in July 2014 (**[Fig 1](#pone.0143960.g001){ref-type="fig"}**). Although fishing gear found in the 2013 surveys could represent several years of accumulation and even pre-date the ban on gillnet fishing, gear found in the 2014 resurveys must represent accumulation over the preceding year, since all gear was removed from these sites during the 2013 surveys. Sites were selected as part of a long-term fish monitoring study \[[@pone.0143960.ref021]\]; though non-random, they provide excellent spatial coverage and access to points and bays on all sides of the lake. In 2013, we censused 54.9 km of shoreline (10 sites, 13 transects, 0.4--8.5 km each, \~13% of total shoreline) for all anthropogenic debris, including derelict fishing gear, between the water and wrack lines \[[@pone.0143960.ref024]\]. In 2014, we recensused 31.9 km of the original transects (7 sites/transects, 1.3--8.3 km each) for derelict fishing gear only. Because transect widths were variable, we report linear (km^-1^) rather than areal (km^-2^) debris density. Derelict fishing gear was classified into the following gillnet categories: whole net, net fragment, float line, lead line, foam float, or bottle float (**[S1 Fig](#pone.0143960.s006){ref-type="supplementary-material"}**); and hook-and-line categories: rod, monofilament, lure, or bobber. Bottles, string/rope, and stakes without mesh, floats, weights, or lines were not considered fishing gear. We weighed each item and measured the mesh size (knot to knot distance) of every whole gillnet or gillnet fragment. Interviews with herders and rangers {#sec005} ----------------------------------- The Rutgers University Internal Review Board (IRB) approved our interview protocol (Protocol E14-675) and all respondents gave informed verbal consent (written consent is problematic in former Soviet regions) as approved by the IRB. We used a semi-structured questionnaire to interview ten herding families from three sites (**[Fig 1](#pone.0143960.g001){ref-type="fig"}**) about their fishing habits, fishing activity they observe, and status and conservation of fish in the lake (**[S1 Appendix](#pone.0143960.s013){ref-type="supplementary-material"}**). The first household at each site was selected opportunistically and additional households were recommended by this family based on proximity and availability. This "snowball sampling" method is commonly used to find respondents in isolated or hard-to-access groups \[[@pone.0143960.ref025]\]. There was no indication that recommendations were biased towards fishing or non-fishing households. We interviewed seven male and three female heads of household. Family and herd sizes ranged from 3--7 people and 4--630 animals, respectively. We used a different semi-structured questionnaire to interview five park rangers, including the head ranger, from 5 of 17 ranger districts (**[Fig 1](#pone.0143960.g001){ref-type="fig"}**) about the frequency and character of illegal fishing, actions taken against illegal fishers, and status and conservation of fish in the lake (**[S2 Appendix](#pone.0143960.s014){ref-type="supplementary-material"}**). The interviewed rangers were male and had worked as rangers for 3--15 years. They were responsible for districts that varied in area (22--398 ha) and number of families (32--1,264 families). Biological sampling, gillnet catch efficiency, and population trends {#sec006} -------------------------------------------------------------------- We used fish monitoring data to estimate catch rates for gillnet mesh sizes used by fishers and to evaluate changes in fish population abundance and body size. The Rutgers University Animal Care and Facilities Committee approved our fish sampling protocol (Protocol 11--005). Permission to conduct field research (Permit 6/445) was granted by the Mongolian Ministry of Environment and Green Development (MEGD). In July 2009 and 2011--13, we set two monofilament horizontal gillnets at seven of the ten surveyed sites (**[Fig 1](#pone.0143960.g001){ref-type="fig"}**). Both gillnets were 2 m deep and 20 m long with 4 m panels of 2.54, 3.81, 5.08, 6.35, and 7.62 cm bar mesh. They were set at least 100 m apart, perpendicular to shore, using a stationary bottom set in water \< 10 m deep, and were fished overnight (8.5--10.5 hr) at each location. Captured fish were identified and measured to the nearest millimeter in total length. Weights for fish without weight measurements were estimated using length-weight parameters derived from our data (**[S2 Fig](#pone.0143960.s007){ref-type="supplementary-material"}**). Vulnerability of fish to gillnets can vary depending on species, body size, and mesh size. We calculated catch-per-unit-of-effort (CPUE) for each gillnet panel in terms of count and biomass (\#/kg 10 m^-1^ night^-1^) to determine species-specific and overall catch rates for each mesh size. We also calculated the species-specific CPUE of each gillnet set in terms of count and biomass (\#/kg night^-1^) and used linear mixed effects models to examine changes in species-specific abundance from 2009--13 while accounting for sampling site as a random effect on the model intercepts. Decreases in body size can be a useful indicator of fishing impacts when changes in abundance cannot be accurately assessed \[[@pone.0143960.ref026]\]. Therefore, we also used linear mixed effects models to examine changes in species-specific body size (length/weight) from 2009--13. P-values were generated through likelihood ratio tests of the full models and null 'intercept only' models. All analyses were performed in R version 3.2.0 \[[@pone.0143960.ref027]\] and mixed effects models were fit using the *lme4* package \[[@pone.0143960.ref028]\]. Potential population level impacts on Hovsgol grayling {#sec007} ------------------------------------------------------ We used methods commonly used in data-poor fisheries management to estimate the maximum sustainable yield (MSY) for Hovsgol grayling and evaluate the likelihood that illegal gillnet fishing could approach or exceed this threshold. Fishing at a rate greater than that which results in MSY is a common definition of overfishing \[[@pone.0143960.ref029]\]. Meta-analyses have shown that fish life history traits can be used to estimate natural mortality rates \[[@pone.0143960.ref030]\], which can in turn be used to estimate F~MSY~ \[[@pone.0143960.ref031]\], the fishing mortality rate resulting in MSY. We estimated the Hovsgol grayling natural mortality rate (*M*) using three separate life history invariant approaches (**[Table 1](#pone.0143960.t001){ref-type="table"}**) and applied the Zhou et al. \[[@pone.0143960.ref031]\] method to estimate F~MSY~ as *0*.*87\*M*. We used a length-converted catch curve analysis \[[@pone.0143960.ref032]\] to calculate total mortality (total mortality = fishing mortality + natural mortality) to place an upper limit on possible natural mortality rates and estimate current fishing mortality rates. More details on the mortality estimation methods are provided in **[S3 Appendix](#pone.0143960.s015){ref-type="supplementary-material"}**. 10.1371/journal.pone.0143960.t001 ###### Natural mortality rates estimated by life history invariant methods and estimates of the effort required to exceed the sustainable harvest associated with each mortality rate. {#pone.0143960.t001g}             \# nights \# fishers \% families ---------------------------------------------------------- --------------------------------- ------ ------ ------ --------- ----------- ------------ ------------- Hoenig~nls~ from Then et al. \[[@pone.0143960.ref056]\] 4.899 \* t~max~ ^-0.916^ 0.37 0.06 0.32 330,869 22,058 220.6 14.6% Pauly~nls-T~ from Then et al. \[[@pone.0143960.ref056]\] 4.118 \* K^0.73^ \* Linf^-0.33^ 0.27 0.15 0.24 255,285 17,019 170.2 11.3% Gunderson \[[@pone.0143960.ref057]\] 1.79 \* GSI 0.30 0.12 0.26 279,557 18,637 186.4 12.3% ^1^ See **[S6 Fig](#pone.0143960.s011){ref-type="supplementary-material"}** for life history traits used in analysis. ^2^ *F = Z--M*, where Z is 0.42 from the length-converted catch curve analysis (**[S7 Fig](#pone.0143960.s012){ref-type="supplementary-material"}**). ^3^ *F* ~*MSY*~ *= 0*.*87 \* M*, from Zhou et al. \[[@pone.0143960.ref031]\]. ^4^ *MSY = (1- exp(-F* ~*MSY*~ *)) \* BIOMASS*, where Hovsgol grayling biomass is 1,214,400 kg based on Ahrenstorff et al. \[[@pone.0143960.ref021]\]. ^5^ Number of nights required to reach MSY assuming fishers use 50-m of optimal mesh gillnet each night (15 kg grayling night^-1^). ^6^ Number of fishers required to reach MSY assuming each fisher uses 50-m of optimal mesh gillnet 100 nights per year. ^7^ Percentage of families participating in the fishery assuming a resident population of 5,440 and average family size of 3.6 people per household (1,511 families; NSOM \[[@pone.0143960.ref019]\]). We then calculated MSY for each F~MSY~ estimate using the Ahrenstorff et al. \[[@pone.0143960.ref021]\] hydroacoustic biomass estimate for Hovsgol grayling (4.4 ± 0.9 kg ha^-1^) and estimated the number of nights of gillnet fishing required to reach each MSY assuming fishers use 50-m gillnets with 2.54-cm mesh, the optimal mesh size for targeting grayling (\~15 kg grayling night^-1^; see *[Gillnet catch efficiency](#sec012){ref-type="sec"}*results). Finally, we estimated the number of fishers required to achieve each MSY assuming fishers use 50-m of gillnet 100 nights year^-1^. These assumptions seem reasonable given the number of nets used by observed and self-reported fishers and reports that fishing continues throughout the winter (see *[Interviews with herders and rangers](#sec005){ref-type="sec"}*results). Results {#sec008} ======= Surveys for derelict fishing gear {#sec009} --------------------------------- A total of 220 (5.78 kg) and 281 (3.82 kg) pieces of derelict fishing gear were collected in the 2013 and 2014 surveys, respectively. Fishing gear comprised 25% of the total weight of plastic debris observed during the 2013 surveys \[[@pone.0143960.ref024]\]. Derelict gillnet material, the majority of fishing gear found in both years (**[Fig 2](#pone.0143960.g002){ref-type="fig"}**), was found in all but two 2013 transects and all 2014 transects (**[Fig 1](#pone.0143960.g001){ref-type="fig"}**). Foam floats were the most abundant gillnet debris items by count, likely due to their ability to separate from nets and disperse widely; gillnet fragments were the most abundant gillnet debris items by weight, likely due to their large size and heavy lead lines. Gillnet fragments ranged from 2--8 cm in mesh size with 3--4 cm mesh being the most common by both count and weight (**[Fig 2](#pone.0143960.g002){ref-type="fig"}**). All six active gillnets observed had 3.0 cm mesh. The density of derelict gillnet material varied among transects, but in both years, Site 7, the most remote and difficult to access site, had the lowest density of gillnet material and Site 10 (Har Us), the primary location of the spring spawning migration fishery, had the highest density of gillnet material. The density of derelict gillnet material in resurveyed sites was higher in 2014 than 2013 at all but Site 7 suggesting that illegal fishing may be increasing (**[Fig 1](#pone.0143960.g001){ref-type="fig"}**). {#pone.0143960.g002} Interviews with herders {#sec010} ----------------------- All of the interviewed herding families (n = 10) reported fishing and observing others fishing (**[S4 Table](#pone.0143960.s004){ref-type="supplementary-material"}**). Families on the eastern shore reported fishing with gillnets repeatedly throughout the year and during the spring grayling spawning migration. They also reported observing commercial gillnet fishers from Hatgal during the winter and during the spring spawning migration, and they reported finding enforcement ineffective. In contrast, families on the northwestern shore reported fishing with rods or by hand only once per spring spawning migration. They reported no commercial fishing activity and found enforcement effective. All of the families reported that Russian visitors fish recreationally year-round but especially in winter with ice fishing rods and gillnets (**[S4 Table](#pone.0143960.s004){ref-type="supplementary-material"}**). All of the families reported fishing primarily for Hovsgol grayling and primarily for household consumption; only one family from the eastern shore reported selling fish (**[S4 Table](#pone.0143960.s004){ref-type="supplementary-material"}**). Families reported fishing primarily during the spawning migration because (1) grayling soup is healthy after the long winter; (2) fish are more abundant and easier to catch than any other time; (3) herders are too busy to fish, or they live away from the lake, the rest of the year; (4) cooking grayling soup interferes with milk production, their principal food source; and (5) eating grayling allows them to delay the slaughtering of herd animals until they have had time to fatten. Nearly all of the interviewed herders stated that fish population sizes have decreased dramatically (**[S4 Table](#pone.0143960.s004){ref-type="supplementary-material"}**). Many recalled that migrating fish were once so numerous that the rivers appeared to "be only fish and no water." Most of the herders also asserted that fish body sizes have decreased and that large lenok and burbot have become especially rare (**[S4 Table](#pone.0143960.s004){ref-type="supplementary-material"}**). The herders stated that "local people should protect the lake and fish" but offered few concrete ideas for achieving this objective (**[S4 Table](#pone.0143960.s004){ref-type="supplementary-material"}**). Interviews with rangers {#sec011} ----------------------- The rangers reported that recreational, commercial, and subsistence fishing all occur in LHNP (**[S5 Table](#pone.0143960.s005){ref-type="supplementary-material"}**). The rangers agreed that the majority of recreational fishers are non-local Mongolians or foreigners who fish with rods primarily in summer but also through the ice in winter. The rangers reported that recreational fishers are generally permitted and compliant with the law. All but one ranger reported that local Mongolians use gillnets to target Hovsgol grayling and lenok for subsistence or commercial purposes (**[S5 Table](#pone.0143960.s005){ref-type="supplementary-material"}**). The rangers reported that subsistence fishers fish almost exclusively at river mouths during the spring spawning migration and that commercial fishers come predominantly from Hatgal due to that town's proximity to the developed southwestern shore and the city of Mörön. The rangers asserted that the town of Hankh is too remote and undeveloped for commercial fishing to be viable. The rangers reported that commercial gillnet fishing occurs year-round and that fishing when the lake is freezing, thawing, or entirely frozen may even be preferred (**[S5 Table](#pone.0143960.s005){ref-type="supplementary-material"}**). The rangers were divided on the status of fish in the lake: three rangers reported that fish population sizes are decreasing and two rangers reported that they are increasing (**[S5 Table](#pone.0143960.s005){ref-type="supplementary-material"}**). The rangers who reported fish population sizes to be decreasing reported that lenok have become especially rare. The majority of rangers reported that fish body sizes have not changed (**[S5 Table](#pone.0143960.s005){ref-type="supplementary-material"}**). The rangers were also divided on the best approach to conservation. The head ranger asserted that the native Great Cormorant (*Phalacrocorax carbo*) population is the primary threat to fish and that their population must be controlled. Another ranger suggested that grayling die naturally after the spring spawning migration (an assertion that is not supported by the scientific literature) and that these migrations must therefore be prevented. The remaining rangers emphasized the importance of improved enforcement during the spawning migration (**[S5 Table](#pone.0143960.s005){ref-type="supplementary-material"}**). The rangers offered a detailed description of fishing at Har Us mineral spring (Site 10), the primary location of the spring grayling spawning migration fishery. Mineral springs are culturally important to Mongolians and visiting this spring in May-June is a longstanding social tradition. Rangers are instructed not to enforce the gillnet ban on fishers at Har Us during this time. The rangers reported that over 570 people visited the spring in 2013 and set a total of 60--100 nets per day with an average catch of 50--70 grayling per net. They estimated that 3,600 grayling were caught per day during peak migration (Jun 7--12) and 1,000--1,500 grayling per day from May 30-Jun 6 and Jun 13--24. Based on this report, we estimate that the Har Us fishery removes \~33,000 fish annually. Gillnet catch efficiency and population trends {#sec012} ---------------------------------------------- The 2.54-cm mesh in our survey gillnets maximized total nightly catch by numbers because it maximized the catch of the abundant Hovsgol grayling (**[S3 Fig](#pone.0143960.s008){ref-type="supplementary-material"}**). The 3.81- and 5.08-cm mesh sizes showed similar catch rates and maximized total nightly catch by biomass because they maximized the catch of larger-bodied lenok and burbot (**[Fig 3](#pone.0143960.g003){ref-type="fig"}**); however, the median nightly catch biomass of the 2.54-cm mesh was comparable to those of the 3.08- and 5.81-cm mesh and the 2.54-cm mesh captured fish during every gillnet set, while the larger mesh sizes were often observed empty. {#pone.0143960.g003} Analysis of the biological monitoring data identified significant reductions in body size for three species over the sampling period (2009--13), but a significant change in CPUE for only one species. Linear mixed effects regression on species-specific CPUE indicates that only burbot population abundance decreased significantly from 2009--13 (**[Fig 4](#pone.0143960.g004){ref-type="fig"}; [S4 Fig](#pone.0143960.s009){ref-type="supplementary-material"}**). Linear mixed effects regression on body size indicates that grayling, roach, and burbot body size decreased significantly from 2009--13 (**[Fig 5](#pone.0143960.g005){ref-type="fig"}; [S5 Fig](#pone.0143960.s010){ref-type="supplementary-material"}**). The abundance and body size of other species remained constant. {#pone.0143960.g004} {#pone.0143960.g005} Potential population level impacts on Hovsgol grayling {#sec013} ------------------------------------------------------ Estimates of Hovsgol grayling natural mortality (*M*) ranged 0.25--0.37 (**[Table 1](#pone.0143960.t001){ref-type="table"}**). A total mortality estimate of 0.42 (**[S7 Fig](#pone.0143960.s012){ref-type="supplementary-material"}**) implies fishing moralities of 0.06--0.15, all of which are less than their associated F~MSY~ estimates (**[Table 1](#pone.0143960.t001){ref-type="table"}**). The F~MSY~ estimates imply MSY values of \~255--331 metric tons yr^-1^, which could be achieved in \~17,000--22,000 nights of fishing with 50-m optimal mesh gillnets (**[Table 1](#pone.0143960.t001){ref-type="table"}**). Although these estimates seem large for a low-density resident population, they could be achieved by 170--220 fishers using 50-m of optimal mesh gillnet 100 nights year^-1^ (roughly twice per week). With an estimated permanent population of 5,440 in LHNP and average family size of 3.6 people \[[@pone.0143960.ref019]\], this effort could be attained if 11.3--14.6% of families participated in the fishery (**[Table 1](#pone.0143960.t001){ref-type="table"}**). Alternatively, this effort could be attained if every family living in the park fished with 50-m of optimal mesh gillnet 11.3--14.6 nights per year. Discussion {#sec014} ========== Knowledge of illegal fishing in Lake Hovsgol National Park (LHNP) has been anecdotal and limited in its usefulness to managers, but with a mixed-method approach, we have empirically described the extent, character, and motivations of illegal fishing and its potential impact on the lake's fish populations. Our mixed-method approach reveals a fuller understanding of illegal fishing in LHNP than using a single method alone. Each method validates, contextualizes, and builds upon the others to construct a consistent story for a complex fishery: **(1) surveys for derelict fishing gear** quantitatively describe the extent, location, and methods of fishing: gillnet fishing is widespread and increasing and fishers generally use 3--4 cm mesh gillnet; **(2) interviews with herders and park rangers** contextualize these results by qualitatively describing the motivations of fishers, character of fishing, and status of fish in the lake: many residents gillnet fish for subsistence during the spring grayling spawning migration, some residents gillnet fish commercially year-round, and fish population sizes are decreasing; **(3) biological monitoring** documents the vulnerability of fish to gillnets as well as population-level trends in fish abundance and body size: the gillnet mesh sizes used by fishers efficiently target Hovsgol grayling and grayling, burbot, and roach exhibit negative population-level trends; and **(4) data-poor stock assessment analyses** demonstrate that plausible levels of fishing effort by Lake Hovsgol residents using gillnets have the capacity to result in overexploitation of the Hovsgol grayling population. Though seemingly intuitive, the use of multiple methods to quantify and characterize illegal resource use has been rare and should be more widely used by conservation scientists and resource managers \[[@pone.0143960.ref004],[@pone.0143960.ref005]\]. Our surveys for derelict fishing gear are an improvement to previous studies because we use repeated surveys to measure re-accumulation rates and biological monitoring data to evaluate the vulnerability of fish to the gear observed in surveys. The majority of studies have focused on comparing the density of derelict gear inside and outside marine reserves for quantifying non-compliance and fail to measure or report accumulation rates (e.g., \[[@pone.0143960.ref009]--[@pone.0143960.ref011]\]). A few studies have measured the accumulation rates of derelict gear among habitat types to inform cleanup efforts but have not used the results to understand non-compliance (e.g., \[[@pone.0143960.ref008],[@pone.0143960.ref033],[@pone.0143960.ref034]\]). Only Williamson et al. \[[@pone.0143960.ref014]\] and the present study have linked these objectives and used both the density and re-accumulation rate of derelict fishing gear to evaluate temporal and spatial trends in non-compliance. By measuring re-accumulation, we show not only that the observed gillnet was used recently and does not pre-date the ban on gillnet fishing, but also that gillnet fishing is becoming increasingly common. Neither Williamson et al. \[[@pone.0143960.ref014]\] or our study properly control for the influence of habitat characteristics (e.g., shore/bottom cover or wind/wave exposure) on accumulation and future studies must consider these covariates when identifying hotspots of illegal fishing. Although our interview method likely underestimates the rate of non-compliance \[[@pone.0143960.ref035],[@pone.0143960.ref036]\], it provides a relative description of the frequency of illegal fishing and important information about the motivations for non-compliance, which cannot be gained using other respondent-based approaches \[[@pone.0143960.ref004]\]. The biases and limitations of direct questioning (DQ) can be reduced when researchers have long-standing relationships with the community \[[@pone.0143960.ref037],[@pone.0143960.ref038]\] or by interviewing multiple stakeholders \[[@pone.0143960.ref037],[@pone.0143960.ref039]\]. In our study, this likely contributes to the discrepancy in personal fishing habits reported by herders on the eastern and western shores. Whereas eastern shore herders, with whom we have long partnerships, reported frequent gillnet use, western shore herders reported fishing by hook and line or by hand only. Although this may reflect real geographic differences, it may also reflect social desirability bias \[[@pone.0143960.ref040]\], as western shore herders might be less comfortable revealing sensitive information to us. In our study, this bias is partially corrected by interviewing multiple stakeholders and by inquiring about observed illegal behavior \[[@pone.0143960.ref037],[@pone.0143960.ref039]\]. For example, herders were more likely than park rangers to characterize enforcement as ineffective and park rangers were more likely than herders to describe illegal fishing. Similarly, although some respondents were likely to underreport personal fishing, they may not be as likely to underreport observed fishing by others. Because of these biases, recent papers promote the randomized response (RRT; \[[@pone.0143960.ref041]\]) and item count techniques (ICT; \[[@pone.0143960.ref042]\]) over DQ for quantifying non-compliance \[[@pone.0143960.ref036],[@pone.0143960.ref043]--[@pone.0143960.ref045]\], but we argue that DQ more easily and fully reveals the motivations for non-compliance \[[@pone.0143960.ref004]\], which is essential information for successful management \[[@pone.0143960.ref046]\]. RRT and ICT incentivize honest responses about illegal behavior by protecting anonymity and generally generate more accurate estimates of the proportion of the sample population engaging in illegal behavior \[[@pone.0143960.ref035],[@pone.0143960.ref036]\]; however, these approaches require large sample sizes and prevent researchers from implicitly discerning motivations for non-compliance by linking behaviors with covariates or from explicitly inquiring about the motivations for non-compliance \[[@pone.0143960.ref045]\]. DQ, on the other hand, allows researchers to inquire about the motivations for non-compliance, importance of natural resources to culture or livelihood, and desire for changes to management rules. Managers must consider the socioeconomic functions of resource use and DQ should remain in the conservation science toolbox. Although the population-level impacts observed in our biological monitoring data cannot necessarily be attributed to illegal fishing, they indicate the importance of improving fisheries management in LHNP, especially given the feasibility for gillnet fishers to overexploit the Hovsgol grayling population, as indicated by the data-poor stock assessment analysis. These calculations represent a simplification of population dynamics made necessary by the lack of time series of fishery removals or estimates of biological parameters needed for more complex data-poor assessment methods \[[@pone.0143960.ref047]\]. However, our indirect estimates of *M* for Hovsgol grayling are similar to direct estimates of *M* for Arctic grayling (*T*. *arcticus*), a close relative (0.29 average; **[S3 Table](#pone.0143960.s003){ref-type="supplementary-material"}**). Furthermore, all of our MSY estimates indicate that overexploitation is possible even with only a small percentage of the population participating in the fishery using gillnets, an inexpensive and widely available fishing gear. The threat of overexploitation is not unrealistic given that grayling, as a taxonomic group, can be susceptible to anthropogenic influences as has been seen with the extirpation of many North American Arctic grayling populations in Montana and Wyoming \[[@pone.0143960.ref048]\]. Salmonids are vulnerable to exploitation and other disruptions during their spring spawning migrations \[[@pone.0143960.ref049]\] and managers must carefully consider the value and impact of the spring spawning migration fishery. The results of our mixed-method approach indicate that illegal fishing is a problem in Lake Hovsgol but that fish also serve an important socioeconomic function. An effective management system will need to incorporate the needs of local people as well as address the synergistic pressures of climate change, water pollution, increasing tourism, and invasive species on LHNP's fish populations. In the last 40 years, regional air temperatures have increased 2.1°C \[[@pone.0143960.ref050]\], a rate of warming more than three times faster than the global average \[[@pone.0143960.ref051]\], which has prompted the drying of many of Lake Hovsgol's previously reliable streams and loss of grayling spawning habitat \[[@pone.0143960.ref016],[@pone.0143960.ref017]\]. Increasing tourism may result in increased fishing pressure, habitat destruction, water pollution, and invasive species introductions without proper management. Lake Hovsgol is already heavily polluted with household trash and will only become more polluted with additional strains on its inadequate waste management system \[[@pone.0143960.ref024]\]. Although no invasive species have established to date, the successful introduction of a new fish or aquatic plant species could alter this otherwise intact ecosystem \[[@pone.0143960.ref052]\]. Fishing, historically uncommon in Mongolia's pastoralist culture, may be gaining prevalence as a new source of food, income, or recreation, especially as climate change makes herding more difficult \[[@pone.0143960.ref053]\] and urban Mongolians acquire more globalized tastes in food and leisure \[[@pone.0143960.ref054]\]. At the same time, Mongolia aims to protect 30% of the country by 2030, more than doubling the area currently under protection \[[@pone.0143960.ref055]\]. These trends forecast continued conflicts between economic and conservation objectives and the way in which these conflicts are resolved or ignored in the iconic LHNP could shape future protected area management in the country. Supporting Information {#sec015} ====================== ###### Large-bodied fish species in Lake Hovsgol, Mongolia and their historic catch\*, market price^†^, and fine per illegally caught fish^‡^. (DOCX) ###### Click here for additional data file. ###### Life history invariant methods selected for estimating Hovsgol grayling natural mortality rate. (DOCX) ###### Click here for additional data file. ###### Arctic grayling (*Thymallus arcticus*) natural mortality rates reported in the literature. (DOCX) ###### Click here for additional data file. ###### Responses of ten herding families interviewed about their personal fishing habits, fishing activity they observe, and status and conservation of fish in the lake. (DOCX) ###### Click here for additional data file. ###### Responses of five park rangers interviewed about the frequency and character of illegal fishing, actions taken against illegal fishers, and status and conservation of fish in the lake. (DOCX) ###### Click here for additional data file. ###### Diagram of a typical Mongolian horizontal gillnet and its components. (PNG) ###### Click here for additional data file. ###### Length-weight relationships for the five most abundant fish species in gillnet catches in Lake Hovsgol. Note variable y-axis scales. (PNG) ###### Click here for additional data file. ###### Catch-per-unit-of-effort (CPUE; \# 10 m^-1^ night^-1^) by mesh size for the five most abundant species in gillnet catches and the sum of their catch. Data from the two 5-panel sequential mesh gillnets used at seven sites in 2009 and 2011--2013 (14 sets yr^-1^ x 4 yr = 56 sets total). Boxplots indicate median (heavy black line), interquartile range (IQR; box), 1.5 times the IQR (whiskers), and extreme values (open circles). Note variable y-axis scales. (PNG) ###### Click here for additional data file. ###### Trends in the abundance of the five most abundant fish species in gillnet catches from 2009--2013. Points indicate the CPUE (\# net^-1^ night^-1^) of each 5-panel sequential mesh gillnet set (2 nets site^-1^ x 7 sites yr^-1^ = 14 sets yr^-1^). Dark lines indicate linear mixed effects regressions fit to the catch data, gray shading indicates the confidence interval for each regression, and dashed lines indicate the prediction interval for the data. Points are jittered around year for display. P-values are indicated in the upper right corner of each panel. Note variable y-axis scales. (PNG) ###### Click here for additional data file. ###### Trends in the body size of the five most abundant fish species in gillnet catches from 2009--2013. Points indicate the total length (mm) of every fish caught in gillnet sets that year (2 nets site^-1^ x 7 sites yr^-1^ = 14 sets yr^-1^). Dark lines indicate linear mixed effects regressions fit to the catch data, gray shading indicates the confidence interval for each regression, and dashed lines indicate the prediction interval for the data. P-values are indicated in the upper right corner of each panel. Points are jittered around year for display. Note variable y-axis scales. (PNG) ###### Click here for additional data file. ###### Estimates of the life history characteristics used to calculate natural mortality (*M*) for Hovsgol grayling. See **[Table 1](#pone.0143960.t001){ref-type="table"}**for *M* estimation methods and results. In **(A),** *L* ~*inf*~, *K*, and *t* ~*max*~ were estimated from aged otoliths and a von Bertalanffy growth model (black line) fit through the observed age-size relationship and origin (Tsogotsaikhan et al. in review). In **(B),** *GSI* was estimated as the mean gonadosomatic index (GSI) for all observed grayling (Jensen, unpublished data). In **(B),** the black line indicates a linear regression fit and the grey shading indicates the confidence interval for the regression. Life history characteristics are marked and labeled in both panels. (PNG) ###### Click here for additional data file. ###### The (A) length and (B) length-converted age structure of the Hovsgol grayling population. The length strucutre was observed in the Ahrenstorff et al. (2012) hydroacoustic surveys. In **(B)**, the solid black line indicates a linear regression fit to the log-transformed trailing arm of the age structure. The dashed black lines indicate the confidence interval for the regression. Z is equal to the negative slope of the regression. (PNG) ###### Click here for additional data file. ###### Herder interview questionnaire. (DOCX) ###### Click here for additional data file. ###### Park ranger interview questionnaire. (DOCX) ###### Click here for additional data file. ###### Total and natural mortality estimation. (DOCX) ###### Click here for additional data file. ###### Supplemental references. (DOCX) ###### Click here for additional data file. We are grateful for the devoted work of our many field assistants. We also thank Steven Alexander, Talia Young, and the members of the Jensen lab for their helpful feedback on the manuscript. The Taimen Conservation Fund, Mongol Ecology Center, Sweetwater Travel, and Hovsgol Travel provided field support and assistance. This work was supported by a NSF-IRES grant (OISE 1064843) to OPJ, a USAID-PEER grant (Project 98) to BM, and American Center for Mongolian Studies and Grayling Research Trust grants to CMF. CMF is funded by a NMFS-Sea Grant Population Dynamics Fellowship. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: BM CMF OPJ. Performed the experiments: BM CMF OPJ. Analyzed the data: CMF OPJ. Wrote the paper: CMF OPJ. Conducted and translated interviews: BM.

Background ========== The pendulum of scientific opinion often swings back and forth in the light of new data and hypotheses. 150 years ago Darwin\'s observations pushed opinion towards believing the universal tree of life (TOL) existed for the first time \[[@B1]\]. This view was pushed to an extreme 30 years ago as Woese pioneered the use of sequence data to build universal trees \[[@B2]\]. But the pendulum has begun to swing back the other way in the past decade, as a wealth of prokaryotic genomic data has demonstrated a higher than expected frequency of horizontal gene transfer (HGT). Ford Doolittle and Eric Bapteste\'s arguments against the TOL hypothesis are quite compelling \[[@B3]\], and this view seems to be gaining support \[[@B4]\]. These authors argue that HGT is so rampant that tree-like representations of prokaryotic species contain too little information to capture evolutionary histories. Their work questions whether the metaphor of the TOL is inspired from a historical bias from the taxonomy of eukaryotes, and therefore should not be applied to prokaryotes. This is an important and worthwhile question to ask. Resolving the eukaryotic tree is a distinct problem because there is much less horizontal transfer and a much better preserved fossil record. The conclusion of Doolittle and Bapteste is not so much that the inability to build the tree is the problem, rather it is forcing the data into a tree that needs to be questioned, and in a pluralistic framework, avoided, since this model does not allow a precise description of the evolutionary processes. The TOL and tree of cells (TOC) should be one and the same. However, the meaning of the former has become the trees we can build, and the latter has become the hypothetical tree we cannot build. This difference was recently discussed in \[[@B5]\]. The reason the TOC is truly a tree is simple and has been stated by many before us. Every extant cell on this planet is the daughter of a cell that came before it \[[@B6]\]. Prokaryotic cells divide by binary fission. Therefore there must truly be a TOC in the prokaryotic superkingdoms. Nobody seems to dispute this. If every daughter cell\'s membrane kept track of who its parent was, reconstructing the evolution of cell divisions would be a trivial task. But since there is no selective pressure for cells to do that, so we are left with a more difficult task. Since the membranes do not keep track of heredity we chose a different representation of ancestry, the genome. The genetic material of the cell does keep track of its parents in some sense as there is selective pressure to ensure fidelity of replication. All of the issues the community is currently having with the TOL hypothesis stem from the simple fact that genomes are not a perfect representation of membrane history. Membrane heredity is a tree-like structure, but all of the recent work on the pervasiveness of HGT has shown that genome heredity is often more of a network than a tree. We are beginning to have enough technology to reconstruct genomic evolution, but we are only beginning to realize how vastly different that is from cellular evolution. However, even genomic evolution makes little sense without the light of cellular evolution. Ernest Rutherford said, \"Physics is the only real science. The rest are just stamp collecting\". Some biologists have taken this as a challenge to create universal laws in biology on par with those in physics. This is a noble endeavor, and has produced many interesting results, but the goal should to be to collect stamps in a way that is justified by the laws. The promise of the TOL did just that. It was a collection of every living thing as well as the laws that organized that collection. Instead of a consensus TOL emerging from the vast amount of genomic data available, the community was faced with the disappointment that very few genes are universally conserved. The universal sequence tree created from 31 concatenated proteins \[[@B7]\] has been criticized as \"the tree of 1%\" because the average prokaryote genome has about 3000 genes \[[@B8]\]. They argue that even if this gene set did produce a reliable tree it would only reflect a small portion of evolution, since this is such a tiny portion of the genome. The assumption that genomic histories were congruent with cellular histories hid the fact that much of the collection could not be explained by the TOL hypothesis. The community was lost in the woods without knowing it under tree monism. We worry that the sound arguments against the TOL hypothesis will shift focus away from evolutionary histories. For instance Dagan *et al.*\[[@B9]\] have quantified rates of horizontal transfer for every gene family. This is a tour de force of quantifying a law in biology. However, they do not give examples of the rates for any specific family, or cite any example they found where a horizontal transfer played a role in speciation. They also say their results are independent of the vertical tree used as input, which we find worrying. The overall rates of HGT may not change, but we assume the rates for each family almost certainly would. Not worrying about that difference is getting lost in the woods to us because the real history of the stamp collection is lost in search of a concise law. Here we argue for the need to be cautious about how far away from the TOL hypothesis we swing, as novel sources of data already bring into question the conclusions supported by genomics. The arguments against the TOL are centered on the idea that the modern synthesis of biology from 50 years ago was too eukaryote-centric. We hope to offer a perspective that will spare this current synthesis from being labeled too genome-centric 50 years from now. We are not arguing for tree monism. Instead we are attempting to demonstrate that the TOC becomes even more important under a pluralistic approach. Not all genes contribute equally to the cell, and we will demonstrate that vertical inheritance of function has a more pronounced signal than vertical inheritance of genetic material. Using only universal sequences to attempt to resolve the TOC is a narrowing strategy, and we will discuss alternative sources of data that may still shed light on this problem. Results and Discussion ====================== The Great Tree of Cellular Function ----------------------------------- The attempts at resolving the TOL using universal sequences only represent a small part of the history of genomes, but what portion of the history of cells does it represent? Genomic methods represent all genes equally. Subsequently, a gene that is only expressed under specific conditions can be just as useful as a housekeeping gene for building a species phylogeny if both are present across the same set of genomes. If we created a concatenated protein sequence, both proteins would be counted as equals proportional to their length despite vast differences in their actual abundance as proteins in the cell. Here lies the fundamental shortcoming of genomics; confusing the genome and the cell. If we wish to measure a gene\'s contribution to the cell there are many different metrics: essentiality, abundance of proteins, number of transcripts, and portion of total weight are just a few. Any of these will give dramatically different proportions than simply counting the copy number within the genome. The abundance of many proteins present in *Escherichia coli\'s*cytoplasm has recently been calculated experimentally \[[@B10]\] as well as for the entire cell of *Leptospira interrogans*\[[@B11]\]. For the first time data are available to measure what portion of a prokaryotic cell each protein comprises. All of these numbers should be taken with a grain of salt due to experimental noise, but the trend is clear; the core proteins make up a larger portion of the cell than the genome. The data used to calculate these values are available as Additional files [1](#S1){ref-type="supplementary-material"} and [2](#S2){ref-type="supplementary-material"}. We argue abundance is a good proxy for evolutionary importance because there is a correlation between the abundance of a protein and the energy a cell invests into producing it. It has been demonstrated that highly expressed proteins have been optimized to use less energetically costly amino acids \[[@B12]\], and that highly abundant proteins are shorter on average \[[@B10]\]. The abundant proteins justify the use of a large portion of the cell\'s energy despite these optimizations, so they must be important. Proteins perform most of the functions in the cell. Comparing how many of the same functions two cells are doing at the same time is a good measure of similarity. The downside of abundance is it is dynamic during a single generation, while genomes are static. This makes direct comparison more difficult, but it still gives insight into the evolution of genomes. Our point is not that this data magically fixes all problems with the TOL hypothesis, but rather that many important details are left unresolved in our understanding of the big picture that still may come into focus. Let us consider the so called \"tree of 1%\". The authors list 36 genes that are universal but claim that only 31 have not been horizontally transferred \[[@B7]\], although later analysis claims the number is actually 22 \[[@B13]\]. However, there are arguments that a TOL is still meaningful despite a large incongruence between individual gene trees \[[@B14]\], but a detailed argument against that view is presented in \[[@B4]\]. 34 of these genes are present in the *E. coli*data set that measures abundance for 1103 proteins. For this argument let us consider the universal set because in this case the HGTs appear to be displacements of genes that were already present. That is to say the function of these genes was vertically inherited despite HGTs of the genetic material. This brings up the point that there are two distinct forms of HGT that we need to consider that are currently not distinguished enough; functional innovations (relative to the recipient genome) and displacements. For example, the histories of the 20 aminoacyl-tRNA synthetases (AARSs) contain many inconsistencies, the result of HGTs \[[@B15]\]. However, it still appears that most of these enzymes date back to the last universal common ancestor (LUCA), with the possible exceptions of AsnRS and GlnRS. HGT makes many of these proteins unusable for reconstructing a universal TOC as their sequence trees would be inconsistent with the cellular history. However, the HGTs of this family would displace a copy of the gene that was already present. In that sense there is no functional innovation caused by the transfer. Even though the genetic material was inherited horizontally the functional content was still transmitted vertically, and would still be consistent with the TOC. We argue events like this are far more deleterious to tree reconstruction algorithms than they are to the recipient cells. Current methods for estimating HGT rely on measuring inconsistencies between sequence trees or looking for unusual compositional features \[[@B16]\], so there is no way for them to distinguish between innovations and displacements. We must also consider the role functional redundancy plays in prokaryotes. There are nearly two hundred known cases of non homologues enzymes performing analogous reactions \[[@B17]\]. HGT of such enzymes should be relatively easy since they can plug into existing metabolic pathways. Therefore any current measure of the vertical inheritance of genetic material is a lower bound on functional vertical inheritance. If one wishes to measure the size of the vertical component of evolution it must be done in terms of function as well as genetic material. The universal genes are about 3% of the E. coli dataset if we count all genes equally. However, it we count proteins by abundance, the 34 from the universal core make up 6.6% of the data set (Table [1](#T1){ref-type="table"}). That would double the thickness of the vertical component! A tree of 2% may not seem dramatically better than a tree of 1% but the point is that the universal proteins make up a larger portion of the cell than the genome. This gets quite dramatic when one takes into consideration that 84.5% of the abundance in this dataset is made up of ribosomal protein L33. L33 is universal across the Bacteria, but absent in the Archaea. Although it is not universal it still must be very ancient. The fact that a large portion of the cytosolic proteome of an extant cell would be present in the cytosol of LUCA truly speaks to the fact that vertical inheritance can be a major force along long evolutionary time scales. This is consistent with previous work that showed older genes are expressed at higher levels than younger genes \[[@B18]\]. However, the authors did not quantify the contribution of the universal proteins in this manner. ###### Coverage in terms of cellular versus genomic abundance in *E. coli*\'s cytosol. Gene Set Protein Abundance Coverage Genomic Abundance Coverage --------------------------------------- ---------------------------- ---------------------------- Universal 34 6.57% 3.08% Core *Enterobacteriaceae* 99.96% 79.22% All Enterobacteriaceae 99.80% 61.34% Non-Ribosomal Core Enterobacteriaceae 85.14% 78.10% Non-ribosomal All Enterobacteriaceae 81.70% 59.33% The protein abundance from *L. interrogans*\[[@B11]\] provides us an opportunity to test the trends we see in *E. coli*. This data set was not dominated by a single protein like L33 in *E. coli*. The 36 universal proteins make up only about 1% of this genome, but 5.4% of the entire cell\'s abundance (Table [2](#T2){ref-type="table"}). A tree of 5% starts to sound significant! This shows the vertical component of the TOL is five times thicker than genomics alone would lead us to believe. Again, regardless of the nature of LUCA, extant cells still have a significant amount of function in common with her. ###### Coverage in terms of cellular versus genomic abundance in a *L. interrogans cell*. Gene Set Protein Abundance coverage Protein Abundance Coverage ----------------- ---------------------------- ---------------------------- Universal 36 5.09% 0.96% Spirochete Core 30.99% 11.28% The TOL hypothesis has also been challenged on the grounds that currently defined taxa may have a very small genomic core (the intersection of their gene sets) and very large pan genomes (the union of their gene sets) \[[@B3],[@B4]\]. We analyzed the genomic cores\' abundances to explore their contribution to the cell. A subset of the proteins in any Enterobacteriaceae genome are conserved in enough genomes to be considered part of their genomic core, and even fewer are conserved across all nine genomes studied in \[[@B19]\]. This is a very diverse set of bacteria ranging from endosymbionts to free-living species. In each case the coverage in terms of genes is much smaller than the coverage in terms of protein abundance (Table [1](#T1){ref-type="table"}). The most dramatic example is that the genes conserved in all nine genomes only account for about 61% of the genes in this dataset, but they account for 99.8% of the protein abundance! Since the ribosomal proteins are so dominant in the data set, we repeated the same measures excluding ribosomal proteins as was done in the initial study. The results are not as impressive, but in each case the core proteins are up to 20% more abundant in the cell than they are in the genome. Measuring the similarity of species based on shared gene content greatly underestimates their functional conservation. We also considered a set of genes conserved across four Spirochete genomes \[[@B20]\] that includes both obligate and non-obligate parasites. 412 proteins that were conserved across four Spirochete species were mapped to the abundance data set. This accounts for only 11.3% of the *L. interrogans*genome, but makes up 31% of the cell when counted by abundance (Table [2](#T2){ref-type="table"}). There is stronger conservation between these species than genomic data implies. Again, even though these species have vastly different lifestyles a large cellular core has remained conserved between them. We assigned COGs (clusters of orthologous genes) to every gene in the *L. interrogans*dataset using the STRING database \[[@B21]\]. This allowed us to compare the relative age of a COG (the percentage of bacterial genomes that have a particular COG) to the genomic or cellular portion of this dataset that COG composes. A cumulative plot of genomic and cellular abundance reveals that at every level genomic abundance underestimates cellular abundance (Figure [1](#F1){ref-type="fig"}). In some cases the difference can be as high as 20%. A similar plot for the *E. coli*dataset was not informative because the dominating feature is ribosomal protein L33 (data not shown). {#F1} We mapped the tree inconsistency scores (ISs) for all trees from the forest of life in \[[@B22]\] to these datasets to test our proposal that abundance is a barrier to HGT. IS is defined as the frequency the splits in a specific tree are found in all single gene phylogenies in the forest of life. Therefore it is an estimate of the horizontal transfer rate for each family. The authors of that work found that ISs have a bimodal distribution, with many families having very low IS (below .6 of the average IS) or very high IS (above 1.4 of the average IS). They noticed many ribosomal proteins had very low IS and proposed this is due to their numerous physical interactions. If highly abundant proteins are less likely to be transferred their trees should not have high ISs. However, there may still be proteins that are not abundant with very low ISs for reasons besides abundance, so we do not think a comparison of average ISs is informative. Instead we counted how many high ISs were found in the 100 most abundant proteins that had an IS. We repeated this measure excluding ribosomal proteins to ensure this result is due to abundance and not just physical constraints on protein complexes. P-values were estimated by taking 10,000 permutations of 100 ISs taken out of all possible ISs. In each case they were significantly less high ISs among the most abundant proteins than would be expected by chance (Table [3](#T3){ref-type="table"}). Abundance does not mean it is impossible for a transfer to occur, but it certainly seems to limit it. Even though the backbone of vertical inheritance may appear to be dwarfed by HGT on the genomic scale, it is clear that vertical inheritance plays a larger role in the cell than the genome. ###### Inconsistency scores (ISs) for the most abundant proteins in each data set. Proteins with high IS P-value ---------------------------------------------------------------------- ----------------------- --------- 100 most abundant proteins with IS in *L. interrogans* 7 .0067 100 most abundant non-ribosomal proteins with IS in *L. interrogans* 9 .033 100 most abundant non-ribosomal proteins with IS in *E. coli* 6 .0013 100 most abundant non-ribosomal proteins with IS in *E. coli* 9 .033 The most abundant proteins have fewer high ISs which implies abundance is a barrier to HGT. One of the major criticisms of tree monism is the arbitrary methods used to deal with incongruent data and ambiguities in a single tree of life model \[[@B4]\]. Bapteste *et al.*cite the ambiguous placement of *Aquifex aeolicus*in a supertree as an example of conflicting signals; sometimes they are placed near the Proteobacteria and sometimes near *Thermotoga maritima*\[[@B23]\]. We propose that abundance could be used to consider the relative weights of two incongruent signals such as these. We predict the vertical signal would become clear if we could measure the abundance of proteins shared between *A. aeolicus, T. maritima*, and the Proteobacteria. It is not just the number of genes that are shared that matters; one needs to consider what their contribution to the cell is. Placing sequence trees in their cellular context ------------------------------------------------ So far we have argued the large role vertical inheritance of function has played in evolution is larger than the network of genomes implies. But how can we proceed forward if that tree is not the one reconstructed by gene sequence trees? We must first discuss the very reasons the TOL metaphor is appealing in the first place. Trees are the natural representation of replicating processes \[[@B24]\]. As discussed in \[[@B4]\] the tree representation has three strengths: 1) it provides a hierarchy for classification; 2) shared traits are implied by that hierarchy; and 3) ancestral traits are inferred from the branch order of the tree. We believe the strength of the hierarchy rests on the last two points so we will address those first. The branching order of a true species tree can be read like a timeline. It implies the ancestral state for each group, as well as the transition that defined the groupings. Without a timeline much of evolution becomes gibberish. There are many transitions that can be polarized. The biggest transition in evolution is between the prokaryotes and eukaryotes. It is truly impossible to represent endosymbiosis in a tree representation, but an undirected network would just say that the eukaryotes are related to both the archaea and bacteria. However, it is not just the relationships we need to know to understand this event; the key point is the directionality of the transition. The eukaryotes came from the prokaryotes. That is to say there was a time where prokaryotes existed but eukaryotes did not. Trees have many shortcomings for representing prokaryotic evolution, but any data structure that lacks temporality is even worse. Since there is no real branch order on any of the current universal sequence trees or networks, some take this as evidence that the origin of all the major prokaryotic taxa are contemporaneous (the condensed cladogenesis model) \[[@B25]\] or are the result of intense periods of HGT (the biological big bang model) \[[@B26]\]. If the major events in prokaryotic evolution all happen near the root of the tree, then it might be really be impossible to reconstruct life\'s history. However, there are no fossils that unambiguously mark the origin of any major prokaryotic taxa (reviewed in \[[@B27]\]). Therefore the justification for both of these models comes from a lack of resolution in both the fossil and sequence data. It is critical we make a distinction between the big picture of evolutionary history being hard to resolve and it not existing. We argue against the expectation that every major prokaryotic taxa showed up at the same moment in evolutionary history. Not all innovations are possible immediately in evolution; they need the right push in selective pressure from the environment. The most important examples are processes that require an oxygenic atmosphere such as sterol synthesis \[[@B28]\]. Oxygen dependent metabolism could not thrive before the great oxidation event that occurred about 2.3 billion years ago \[[@B29]\]. This can be used to constrain the ages of several branches of the TOL \[[@B30]\]. This begs the question of whether the major prokaryotic taxa are contemporaneous in origin or not. Despite the disagreement between the current macrophylogenies \[[@B31]-[@B33]\], they imply the major prokaryotic taxa did not appear at the same time. The disagreement between these phylogenies is not in terms of how to define the major taxa but rather in the proper way to polarize the data, especially the indels (insertion deletions) which we have discussed \[[@B34]\]. However, the distribution of these traits themselves implies specific taxa evolved before another, regardless of the direction of each polarization. For example, there is a large insert in HSP70 (heat shock protein 70) that is present across the Gram-negative bacteria, but absent in the Gram-positives. There is no reason to assume the insertion deletion event occurred early in evolution. That event would be contemporaneous to the change in membrane structure. There must be a branch order between the Gram-positives and Gram-negatives; even if it is not resolved in sequence trees. One would be very hard pressed to draw a detailed scenario of transfers that explains the distribution of fixed indels better than a more timeline like structure. There are numerous similar divides that can be drawn across the prokaryotes. Relatively stable traits like these must used as guides to reconstruct the TOC. If the origins of the major clades really are very close to contemporaneous we should not expect it to be possible to reconstruct a macrophylogeny at all using such traits. Despite their disagreement, we take the macrophylogenies as evidence that the origins of major prokaryotic taxa could not all be contemporaneous. If the clades arose out of intense periods of HGT the indels and other data points should have largely independent histories. Instead many indels appear on the same points on the tree, which act to independently verify each other. Why would there be more signal left in the rare events instead of the sequence alone? We propose an alternative model where cladogenesis is primarily caused by revolutions in the \"abundome.\" Some proteins that were highly abundant in LUCA may be entirely absent in an extant cell. This would be a result of major events that led to dramatic changes in gene expression of even the most conserved genes. Such a change in the abundome could make some HGTs more deleterious since the protein would be plugged into a modified core. It is possible these events could actually be periods of reduced HGT. Abundance data may make it possible to quantify what Simpson coined \"quantum evolution\" when referring to the metazoan fossil record \[[@B35]\], the idea that changes in one part of an organism can trigger a domino effect of rapid evolution, on a molecular level in prokaryotes. There is an inverse relationship between population size and evolutionary rate \[[@B36]\]. Initially members of a novel niche could evolve rapidly. It is also possible that could lead to a population size that is large enough to cause extreme purifying selection, essentially freezing ribosomal sequences. It seems impossible to predict the effects cladogenesis will have on sequences without taking into account other sources of data. This view that revolutions in abundances play a role in cladogenesis is supported by the observation that there are major changes in gene regulation between the major prokaryotic taxa \[[@B37]\]. Since gene expression is a major driver of evolutionary rates \[[@B38]\], there is no reason to expect protein sequence to be well behaved across these events even if cellular population size remained constant. That, taken with the new observation that evolutionary rates vary greatly between prokaryotic groups \[[@B39]\] implies our null hypothesis should be that sequence trees would not resolve the branch order of the major prokaryotic groups even in the complete absence of HGT. Therefore we are in complete agreement that a tree created by concatenating protein sequences together is not the TOL or the TOC. But we do not take that as evidence the TOC does not exist, is not resolvable, or is not useful as a concept for understanding prokaryotic evolution. It just means the community needs to move beyond primary sequence analysis. It will be possible to look at the coevolution between the cellular and genomic cores as abundance data becomes available from more species. This will allow us to divide extant species into groups that have maintained a cellular backbone. Evolution within these groups should be well suited for study using sequence since there will be fewer confounding factors. This view is supported by the growing list of prokaryotic clades that form well defined sequence trees discussed in \[[@B5]\]. If we can identify the innovation between groups that leads to the differences in abundances it may be possible to polarize these transitions in the manner pioneered in \[[@B31]\]. This approach creates a timeline that is appropriate for classification purposes, and thus we approach the advantages of the TOL while reconstructing the TOC. Of course there will be traits that do not fit that timeline and we must consider them in a pluralistic fashion. However, the timeline will allow us to polarize many of the HGTs and place true innovations in their proper context. Current sequence-based methods could be made much less arbitrary by comparing them against these other lines of data. Combining the TOC with genomic histories would capture all the positive aspects of the TOL hypothesis, while accommodating HGT. It takes a universal sequence that has not been horizontally transferred and has evolved at a steady rate to build a universal tree. As discussed above that does not leave us with very much data. It takes two widely distributed paralogous proteins to polarize an indel \[[@B40]\], which leaves us with even less. Therefore we are very interested in non-ubiquitous traits that may be useful in resolving the branch order of the prokaryotic taxa. We have found protein structure to be a highly useful tool for studying evolution, but hopefully there are others as well. A transition within quaternary structure only requires a protein to be universal within a taxon of interest. We have presented two transitions in quaternary structure that exclude the root from the Archaea: Anbu evolving into the 20s proteasome \[[@B41]\] and PyrD 1A evolving into PyrD 1B \[[@B34]\]. Neither of these proteins is in Ciccarelli *et al*.\'s dataset because they are not universal. But they are derived structures that are universal enough in the Archaea to provide compelling independent arguments that exclude the root of the TOL from within the Archaea. Even if the proteasome sequences have been horizontally transferred all over the Archaea it does not take away from the fact all Archaea have a proteasome (those would be horizontal displacements). Therefore a protein might be useful for resolving a branch order in the tree even if there is major incongruence between the cellular and genetic history. It is currently possible to predict the tertiary structure for about half of a prokaryotic proteome \[[@B42]\]. However, quaternary interactions are not being predicted from sequence in the same way. This gives us hope that there are still untapped sources of data that might resolve the branch order of the major prokaryotic taxa. Conclusion ========== We must keep in mind the humor of calling the central metaphor for evolution \"the tree of life\". The phrase first appears in Genesis 2:9: And the LORD God made all kinds of trees grow out of the ground\--trees that were pleasing to the eye and good for food. In the middle of the garden were the tree of life and the tree of the knowledge of good and evil. There is irony in using the name of a tree central to the creation story to argue against that very myth. Therefore we doubt that any phrase will ever pack as much punch as the \"tree of life\", even if the pattern of common descent is more of a web. It is very important that the community stops labeling any tree derived from a single data source the TOL. The recent attempts to resolve the TOC using primary sequence should be labeled \"universal sequence trees\", a name that is grounded in the limitations of the data. The title TOC should be reserved for branch orders that are supported by several lines of independent evidence, and the TOL should be the synthesis of those branch orders and horizontal process. Perhaps the most important line of reasoning that the TOL exists is the fact that HGT is so rampant. Why is HGT possible at all? The answer is obviously common descent. If it was not for common descent the genetic code would not be universal, and most HGTs would not even be translatable in their new host. Many biological parts are interchangeable because they have evolved in conjunction with the same systems. Therefore, we argue the very reason the TOC is so hard to reconstruct is because it exists!! It has now become clear that many expectations about prokaryotic evolution were based too heavily on observations of eukaryotes. There is truly a fundamental divide in the way these two groups use the communal gene pool as a genetic memory \[[@B43]\]. However if we give up on the eukaryotes as a model, it is not clear what our expectation of prokaryotes should be. It does not make sense to us to criticize a tree as \"the tree of 1%\" without providing a justified cutoff of n% that would be enough for the vertical component of genomic evolution to be meaningful. We think the TOL crisis would be worse if it was the \"tree of 99%\", as it would be quite difficult to explain the phenotypic differences between humans and *E. coli*. It is remarkable any genes are conserved since LUCA, and therefore the TOL still rings true to us. Likewise, it is not clear what level of genome conservation between strains of prokaryotes would be satisfying enough to consider them evolving in a tree-like manner. It is true that two strains of the same species may have relatively few genes in common, but we have argued above this is probably an exaggeration of functional distance. Abundance data from different strains under similar growth condition will shed light on their true functional differences. We predict this gap will be much smaller than it appears from counting genes. Of course one reaches a point where two species live under different enough conditions that comparing their abundances is like comparing apples to oranges. But since expression is highly correlated with evolutionary rates \[[@B38]\] these are probably cases where primary sequence analysis would fail too. More abundance data may shed light on why some branches of the tree are so much harder to resolve than others. Fortunately species most likely to exchange genes horizontally live in similar conditions \[[@B23]\]. This means it will be possible to compare the relative contribution of horizontal and vertical inheritance to the cell when protein abundance data are available from different taxa living in the same environment. The landscape of genomes is rapidly being filled, and many higher level taxa are now well sampled. Despite this, there is no consensus on the TOL and many are ready to abandon the notion that we will ever reconstruct it. There is still plenty of data that needs to be generated to elucidate the history of cells. More information on protein abundance will shed light on the true revolutions in the history of cells and help prioritize conflicting signals in the genetic material. Protein and cellular structure will help us polarize the major events in evolution. It seems to us that genomes simply are not enough to study genomics. Of course it would be naïve to expect that some new data source will be a magic bullet that will resolve the TOC. Instead we must realize each data source has its shortcomings, many of which cannot be illuminated except in the context of other data. It is not just that we need more data, we need more details. Automated methods fall prey to numerous confounding factors but can still be highly informative. They must always be supplemented by experts whose intuitions have been tempered by careful examination of details from multiple data sources. Therefore the best way to move forward is to take sequence data off center stage and supplement it with these other data sources. This view can be summarized by several dualities, best exemplified by the classic symbol of yin and yang (Figure [2](#F2){ref-type="fig"}). The basic lesson of this symbol is that one finds the darkest point in the center of the light, and the brightest light in the center of the darkness. It is only through understanding the interplay between the light and dark that one gains insight into their true nature. Neither can exist without the other. A new understanding of evolution comes from the study of the interplay between a series of dualities. It is now clear that there is large distinction between heredity of genetic material and of membranes. There is clearly a duality in Darwin\'s theory of descent with modification; the history of variation is well described by a network and the history of selection is well described by a tree. A web of life (WOL) may be more factual than a single TOC, but we argue it is a less accurate depiction of life\'s history. It is possible to precisely represent the relationship between most extant genetic material on this planet using a network. But without a tree (or time line) of life this undirected graph is mostly functional displacements and shifts in redundancy in our opinion. The \"light\" in the confounding \"darkness\" of horizontal transfer must be the TOC. Of course the \"darkest\" points are in the center of the \"light\" too. Endosymbiosis is clearly a non-vertical event that has profoundly influenced the structure of the tree of function. Likewise there are many horizontal innovations that were important for shaping the prokaryotic tree of function. We feel the most productive way to move forward is create a duality between the horizontal transfers that shape evolution and those that confound our tree building algorithms. The point is that neither of these extremes invalidates the other; they complement each other. Darwin wrote, \"Thus, from the war of Nature, from famine and death, the most exalted object which we are capable of conceiving, namely, the production of the higher animals, directly follows.\" His understanding that death comes from life, and life comes from death fits perfectly with the symbol of yin and yang (the link between the quote and symbol of yin and yang was noted in \[[@B44]\]). {#F2} The increase in the size of the functional tree argued here may not be enough to persuade everyone saving the TOL is possible. Some prefer to be cautious and label this the \"central trend in the forest of life\" \[[@B22]\]. The fact that a large chunk of universal cellular function has remained conserved and its sequence behaves in a mostly tree-like manner after billions of years makes the reconstruction of the TOC seem possible despite the issues of HGT. Resolving and rooting this tree are meaningful problems that are worth pursuing. That is not to say that the tree is resolved simply by taking a consensus or average of universal gene sequences. However, those who rather look at the forest of life need to keep in mind that there are some trees in that forest that are much older and larger than the others. The central tree(s) must be the landmarks used to navigate the rest of the forest. HGT has clearly shaped the prokaryotic world, but if we do not keep in mind the histories of both genomes and cells we will end up lost in the woods. Methods ======= Abundance data were taken from \[[@B10],[@B11]\]. The universal core proteins are defined in \[[@B7]\]. The Enterobacteriaceae genomic core was defined in \[[@B19]\]. The Spirochete genomic core was defined in \[[@B20]\]. All COG annotations were taken from the STRING database \[[@B21]\]. All inconsistency scores were taken from \[[@B22]\]. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= REV conceived the study and analyzed the data. PEB assisted in writing the manuscript. All authors read and approved the final manuscript. Reviewers\' Comments ==================== Reviewer\'s report 1 -------------------- Eric Bapteste, Université Pierre et Marie Curie, UMR CNRS 7138, 75005 Paris, France This paper, in many respects well-balanced, proposes a strategy to reconstruct a tree of cells, and discusses questions regarding the TOL in ways, that in my opinion, should be significantly improved to be convincing. The authors begin by acknowledging that genetic evolution is largely reticulated in prokaryotes, but that genetic and cellular evolution should be distinguished, since, importantly, cellular evolution could be accurately described by a tree. They argue that the tree of cells could be reconstructed by (i) considering the distribution of functions (e.g. the repertoires of functions present in genomes) rather than the repertoires of genes; and by (ii) giving a greater weight to genes that are abundantly expressed in cells (rather than giving similar weigth to all genes) to define the branching pattern of cellular evolution. Genes with a greater abundance, they propose, would be less easy to transfer. In that regard, they introduce a novel (and possibly quite sound) \'complexity hypothesis\' based on abundance. In the complexity hypothesis, genes with more interactions are expected to be less transferred, in the \'abundant hypothesis\', genes with the most abundant expression are expected to be less transferred. Whether the evolution of functions is tree-like as the authors repeatedly claim could be tested by reconstructing a phylogenetic network based on the functional content of genomes. The authors should do it in a revised version of the MS and add that analysis and a figure to their paper. This test of the tree-like evolution of functions would improve the paper, since in some parts of their manuscript, the authors mention the problematic possibility that even functions evolution could be to some extent affected by HGT (e.g. p.19). In particular, the extent to which such repertoires of functions could be convergent to adapt to some environments (i.e. animal guts, or hypersaline environments) is probably partly an open question, that could complicate the interpretation of the branching pattern in such trees. Likewise, the authors mention that indels (another type of slowly evolving characters, in their view less affected by HGT) appear on the same points on a tree. It would be tempting as well to see how a phylogenetic network of these data on indels look (e.g. how tree-like the distribution of indels is), and how it matches with the tree of functions. *Author\'s response:* *We are arguing that the tree of function requires a well developed tree of cells to define when lines of cells gained or lost function. Our other work focuses on reconstructing a large portion of the tree of cells, and hints of the tree of function can be seen in it (in preparation). In the tree of functions the source of the function does not matter. The ancestral line did not have the function and the derived group does, which is well described by a branch order on a tree. Whether other branches on the tree have the same function is irrelevant to those cells from a functional point of view unless they are in competition. To really build the tree of functions one would also need to represent the relationships between different functions. We think the history of cells, genomes, and functions are long term goals that cannot begin to be reconstructed in a single figure, but we hope we have argued they are goals worth pursuing still*. *We agree it would be interesting to map characters such as indels onto networks*. Gene abundance, especially in extinct cells, may be quite difficult to quantify. The data are currently limited to decide which genes are abundant and which ones are not. Nonetheless, this limitation could be a chance, since it should be possible to test whether these abundant genes evolve vertically or not, by aligning these genes (and corresponding controls) and by searching possible traces of recombination in them, or evidence of inconsistent or odd branchings in their trees. Knowing whether these genes appear to recombine/transfer in proportions comparable with that of non abundant (control) genes would help evaluating the authors\' claim that abundantly expressed genes are less affected by LGT. Such an analysis should also be added to a revised version of this paper. Indeed if molecular changes accumulate in these sequences largely due to non vertical processes, at some evolutionary depth, the proposition made by the authors that such genes would better describe vertical evolution than other markers (and thus should be preferred in case of conflict between markers) would simply be wrong. *Author\'s response:* *We have added an analysis of the inconsistency scores for the highly abundant proteins. This data supports our proposal that abundance is a barrier to horizontal transfer*. The authors\' conclusions that the tree of functions (should it be consistent with the data, once reconstructed) is a good proxy of the tree of cells, itself a perfect match of the Tree of Life, is very arguable. The tree of functions, the tree of cells and the Tree of Life can hardly be one same thing. They can hardly be considered isomorphic for a simple reason: they do not have the same explanatory powers, nor the same explanatory scopes. Evolution in general is much more than the evolution of cells, or the evolution of functions (even if these two aspects are very important to understand evolution). The problem is that biological diversity resulting from evolution by far exceeds these two aspects: many evolutionary units (recombined genes, operons, transferred genes, mosaic genomes, consortia, communities, \'acellular\' and \'intercellular evolution\' mediated by mobile elements such as phages and plasmids) cannot be exactly mapped onto a tree of functions or onto a tree of cells. The evolutionary fates of these objects are partly (and sometimes largely or totally) uncoupled with the ones described by the tree of functions or by the tree of cells. The tree of functions - if it can be reconstructed- would certainly be informative about the evolution of functions; the tree of cells divisions - if it can be reconstructed- will be informative about a part of cellular evolution. However such a tree of cell divisions won\'t inform us about most of what cannot be considered as mere details in evolution: the lifestyle, adaptation, processes creating and sustaining the genetic diversity, the selection pressures at play and the evolution of species (that is how remarkable groups of organisms emerged (or failed to emerge) from the interplay of evolutionary processes). *Author\'s response:* *You are absolutely right that each of these trees has different explanatory scopes. The tree of functions is certainly not the tree of cells. Our argument is that tree-like thinking is more useful when abstracting beyond the level of genetic material. One would need to combine the tree of cells, tree of functions, and networks of genomes to get the explanatory power of the dreamt TOL. We are not pattern monists, so we have no problem with that*. *We completely disagree that the trees of cells would not be informative to studying adaptation. It might not provide much explanatory power on its own, but how can one study adaptation without a history of the cell? The tree of cells provides the snapshots of before and after the adaptive process. Mapping any other evolutionary data source back onto the tree of cells makes it more informative. Without the history you cannot say what any of the processes you listed actually changed in a cell. The bottom line is the contribution of these other factors is always on the tree of cells. That is why we argue the tree of cells becomes more important under a pluralistic model*. This limited explanatory power of such a tree is even clearly demonstrated in this manuscript: the \'cellular core\' of four spirochetes is already uninformative about the Spirochetes lifestyles. Using these \'abundant genes\' would not allow explaining much of the spirochetes biology, and thus of these species origin (how some become obligate parasites for instance). The duality between the tree of cells and the network of genes, well acknowledged by the authors, seems irreducible, because real and relevant to our understanding of evolution. In other words, while the authors rigthly argue that the genome and the cell should not be confused with one another, they seem to be tempted to approximate the entire biology and the entire evolution by the history of cell divisions. This confusion too should be discouraged. *Author\'s response:* *The tree of function is not just the abundant genes; it would include the losses and gains that define the adaptations to these different environments. We are not arguing cell divisions are all that matter, but rather their history is necessary for a true understanding of these other processes*. It would certainly be interesting to polarize in time many evolutionary scenarios, but it does not follow that, based on the history of some genes with a slower dynamic and based on some \'frozen\' features, we will be able to infer the independent histories of the other genes and of the other organismal properties evolving with a distinct (faster) pace. In that respect, knowing the tree of cell division might not help much in understanding precise gene histories (for instance). A tree of cells will have some useful explanatory power but not as much as a dreamt TOL. *Author\'s response:* *We agree that some genes histories will still not make sense even in the framework of something that resembles the TOL. Our point is that without some branch order for the major prokaryotic groups it becomes difficult to come up a meaningful history for ANY gene. The tree would let us differentiate the slow and fast properties, which would give great insight to evolutionary processes that are either tree-like or network-like*. The authors\' choice to keep using the phrase \'tree of life\' when referring to the pattern of common descent even if it is more of a web, because this would somehow annoy creationists, is in my opinion not a good idea. I do not think creationists should dictate us any of our scientific agenda, or influence our wording, as they have no scientific competence to evaluate evolutionary studies. When phenomena are not tree-like, we should not call them a tree. When they are tree-like and are supported by several lines of independent evidence, we should call them the \'most corroborated evolutionary tree\' or the \'best evolutionary tree\' but not the tree of life, because maybe features did not evolve in a tree like fashion, and thus cannot be reduced to that scheme to be fully understood. *Author\'s response:* *If this was our only reason for this title you would be absolutely correct. We have presented many reasons besides this why we think \'tree of life\' is a worthy title for the combination of the histories of cells and genomes. It is not about annoying creationists, which comes quite naturally to us. We agree that they should not dictate our agenda, but clearly they have already shaped our wording. The point is 150 years ago the phrase \'tree of life\' invoked a vision of a talking snake and a magic apple. Now it is a story that involves genomes, viruses, and algorithms. The meaning of the \'tree of life\' will continue to evolve, but it will continue to provide an explanation of where life came from. We don\'t think any other title could ever have quite the same aesthetic value, but beauty is in the eye of the beholder*. There is then a cost to do as if the Tree of Life existed (but not testing this scientific hypothesis): it reifies parts of the tree, like the nodes and the branches. Lawrence and Rechtless have masterfully shown that nodes, when conceived as points of speciations, are not \'real\'. When prokaryotic species do not evolve by a series of dichotomies, it is a delusion to impose a dichotomy to describe a speciation. *Author\'s response:* *Lawrence and Retchless*\[[@B45]\]*have demonstrated that nodes are fuzzy in terms of genetic material due to varying levels of recombination during the divergence process. If we consider a tree of functions then the nodes are real. Consider an ancestral state that lacks a function. The function is gained (through HGT or innovation). There is now a derived and ancestral node that can be described on a tree, but the history of their genetic material is no longer so well behaved. Their may be a fuzzy functional intermediate, but that would not be a stable state due to selective pressures. Again trees appear a better data structure if we abstract past the genome*. The root poses a comparable issue. The authors keep referring to LUCA, as if there were one one last universal common ancestor of all life that was a cell. The literature on early life is, to say the least, divided about this notion. Invoking LUCA to prove that there is a tree, and the tree to prove that there is a LUCA, without any principled way (or any test) to refute that there is a LUCA or that there is a tree is unfortunately a circular argument. That all cells share a given gene/function does not mean that all cells evolved vertically. If LUCA ever existed (which I doubt, and most importantly which in my view could explain more than a part of early evolution), how big was its pangenome? What kind of mobile elements drove its evolution? We need to make sure that a prioris about the tree and about LUCA being real do not already bias our conclusions, if these a prioris cannot be tested. Otherwise, we might reinforce our habits of tree-thinkers, but not necessarily our knowledge of evolution per se. *Author\'s response:* *The issue of LUCA is certainly muddled in the literature. We direct the readers to a recent empirical argument that there must have been a LUCA, even if it was a community*\[[@B46]\]. *You are right in pointing out we are biased in our view of LUCA. Our other work has led us to support Cavalier-Smith\'s assertion the Chloroflexi are the earliest branching extant group. Therefore, we assume LUCA had a relatively small pan genome. If one accepts the canonical rooting between the Archaea and Bacteria the idea of a large pan genomed LUCA is certainly more appealing because of the large differences between the prokaryotic superkingdoms. If LUCA was a large pan genome our focus should be on what genes could NOT have been in that community and must be younger than LUCA*. In that regards, I have a few detailed suggestions where some simplifications could maybe be corrected in the text. p.3: The authors wrote: If every daughter cell\'s membrane kept track of who its parent was, reconstructing evolution would be a trivial task. I feel that this is a bit misleading, it would inform us on a part of evolution: If every daughter cell\'s membrane kept track of who its parent was, reconstructing evolution **of cell divisions**would be a trivial task. It would not tell us anything on how species taxa, and genes, and phages, and communities, etc. evolved. *Author\'s response:* *A more precise statement, which we have adopted*. p.3: It is very arguable that even genomic evolution makes little sense without the light of cellular evolution. There is certainly lots of knowledge to be gained from metagenomic analyses, from the study of mobile elements, from the study of gene evolution, lots of patterns and processes to explain, even without the light of cellular evolution. This is not to say that we would not benefit from that particular light. But this light will mostly make a \'genealogical\' sense on evolution, and evolution is more than genealogy. *Author\'s response:* *Studying evolution without genealogy makes little sense to us. These are all important processes, and we certainly can learn a lot about them without the TOC. However, we argue the TOC gives a deeper understanding of each of them. Without considering genealogy in metagenomics one basically has a laundry list of genes, and it might not even be clear which of them are from the same cell. If one has knowledge of how the cells in the community are related, they can reconstruct the history of the mobile elements and examine what their impact on the community was. Likewise studying gene evolution without genealogy seems limiting, as the impact the gene has is ultimately on the fitness of the cell. The genealogy is necessary to integrate these processes into a bigger picture and to see what they actually changed*. p.4: The TOL does not become even more important under a pluralistic approach, quite the opposite: it is regionalized under a pluralistic approach, as no single model can explain everything about evolution under that perspective. Finding the tree of cells for instance remains an important and ambitious goal, but not the alpha and omega of evolutionary research. The importance of the TOL thus decreases while the importance of additional interesting evolutionary questions increases. *Author\'s response:* *We have changed this to TOC in the text. We are arguing building it is the alpha, but not the omega. The fact that the TOC will be used to formulate more questions than initially expected makes it more important*. p.5. It seems to me that proteins abundancy and core cellular features might be a basal make-up of cellular lineages on which further adaptations are adjusted. If there is some ratchet, abundant proteins can not be easily gotten rid of, but that does not mean that most of the evolutionary dynamic concerns these proteins and their coding genes. *Author\'s response:* *Evolution is certainly not just about the abundant genes. We have included abundance data to add a dimension beyond the genome to study the big picture in evolution*. p.5. Comparing how many of the same functions two cells are doing at the same time is a good measure of similarity, but is it a good proxy of the genealogy ? This depends on the amount of convergence and selective pressures on functions induced by the environment. Is not it possible that bacteria of the gut microbiomes (or of a salty environment) will perform the same functions at the same time even if they are not directly related ? *Author\'s response:* *This is certainly a vital question to answer. Abundance data from similar environments will allow us to test this in the future, but for now we are left to speculate*. p\. 6. For a detailed argument that a TOL is not as meaningful as claimed in \[[@B13]\], when there is a large incongruence between individual gene trees, see Bapteste et al. Biol Direct. 2009 Sep 29;4:34. Prokaryotic evolution and the tree of life are two different things. *Author\'s response:* *We lean towards your arguments in this case unless one can deal with the incongruence in a non-arbitrarily manner as we have proposed here*. p.11: I absolutely disagree with the following statement: Trees have many shortcomings for representing prokaryotic evolution, but any data structure that lacks temporality is even worse. Reconstructing a wrong tree (imposing an irrelevant structure to the data because of our a prioris) is the worst thing one can do. We can learn a lot from unrooted gene trees, on which by definition there is no temporality. *Author\'s response:* *We mean that we need the benefits of the trees we have listed, while trying to accommodate the shortcomings of that representation. We are not for forcing data to fit the tree. We are saying the data that does not fit the tree can only be noticed and studied once you have the tree. There are certainly other representations that are useful, but the TOC is necessary for weaving them into a coherent story*. p\. 18 and p. 19: When commenting on dualities, the text becomes pretty metaphysical in places. I do not see why \'the\' light must be \'the TOL\': a light can be a tree of cells, another light a tree of functions, and so on. Why do you need only one light to explore the darkness of evolution anyway, while so many processes are occuring, creating a diversity of phenomena that calls for more than one explanation ? *Author\'s response:* *We think a discussion of the metaphor used to represent the process of life should have a healthy dose of metaphysics. The nice thing about yin and yang is that you can switch them and it still tells the same story. We have chosen light because in this case the answer the TOL provides (if it exists) is more directly readable than the WOL. There should be multiple lights, but we cannot see how anything could be more useful to understanding the history of genes than understanding the history of cells. These are the two primary replicative processes in evolution. The assumption they were the same process is the source of the problem. As we begin to separate them we must keep them connected. Therefore we feel the yin and yang is fitting. One could argue the confusion caused by forcing data to fit a tree is the darkness and the light is the realization of processes like HGT. The point is that HGT and the TOC are inseparable and cannot exist without the other, regardless of how we label each one*. A thinking about evolution in terms of yin and yang is possibly not entailed by the quote on (I believe) Darwin\'s malthusianism. I doubt that historians of sciences and/or philosophers of sciences would be convinced that this is a proper use of that particular quote. I do not think it is needed in the paper. *Author\'s response:* *We probably did go a bit too far in our use of this quote. We have softened the implications, but have kept the quote. We see the yin and yang in that quote, as well as in the data, regardless of what Darwin was thinking when he wrote it*. p.18: I disagree with this sentence: A web of life may be more factual than a single TOL, but we argue it is a less accurate depiction of life\'s history.. The authors possibly have in mind a fairly simple web of shared genes. But even these graphs can be further studied to gain knowledge on history. Dagan and Martin for instance have shown how such networks can be exploited to learn about life\'s history. And what about phylogenetic networks for taxa with a limited amount of HGT: are they worse than a tree to describe life\'s history? To me this kind of claim is counterproductive, as it fails to acknowledge that it might just be time to change our habits and our thinking about how evolution should be described. *Author\'s response:* *We are making a similar point to the one you made above about explanatory power. The WOL does explain as much as the dreamt TOL. Therefore we would be settling for too little if we thought it was enough*. I also would like to make some further precisions: p.2. The conclusion of Doolittle and Bapteste is not so much that the inability to build the tree is the problem, rather it is forcing the data into a tree that needs to be questionned, and in a pluralistic framework, avoided, since this model does not allow a precise description of the evolutionary processes. *Author\'s response:* *Changed*. p.17: The authors write that it is not clear what level of genome conservation between strains of prokaryotes would be satisfying enough to consider them evolving in a tree-like manner. It is true that two strains of the same species may have relatively few genes in common, but we have argued above this is probably an exaggeration of functional distance. Even if the second sentence might be correct, its association with the first one suggest that the authors tend to overlook the importance of recombination in prokaryotic genomes, a major process that is not tree-like. This non-tree like phenomenon can in part be masked by zooming out at a higher taxonomical level, still the real processes responsible for evolution are not tree-like. In that respect, a tree of cells or a tree of functions will fall short in explaining major evolutionary processes at play on genomes. *Author\'s response:* *We are trying to emphasize the many tree-like patterns that could be masked by recombination of genetic material. Certainly a network is needed to understand the history of the genome. Our key point is that does not mean a network necessarily describes the evolution of cellular function better even in the presence of recombination*. p.17: Just like genomes simply are not enough to study genomics, cells (or functions) are not enough to study evolution: you need to include phages, plasmids, etc. *Author\'s response:* *True, but in reality phages and plasmids only really affect evolution when they enter cells*. p.19: The duality that the authors propose between HGT that shape evolution and HGT that confound our tree building algorithms seems a distinction between good and bad HGTs. This distinction (if it can be achieved, how ?) could help them building an evolutionary tree, but it would not make the processes of evolution and prokaryotic genetic evolution more tree-like in nature. Both good and bad HGTs are non strictly vertical processes. *Author\'s response:* *This distinction can only be made by having a hypothetical ancestral genome before the transfer occurred, which is not a trivial task is given the amount of transfers that have occurred. If the function is novel to the recipient cells lets call it a good HGT. In both cases there is non-tree like evolution, but we argue that bad HGTs are just red herrings leading us away from the tree that does exist. Our point is that HGTs are not strictly non vertical from a functional perspective*. Overall, in many places of the MS the authors could advantageously replace TOL by Tree of Cells, which would address (simply) most of my concerns. *Author\'s response:* *Calling the TOL a universal sequence tree in much of the current literature would have addressed many of our concerns as well, but your arguments have convinced us TOC is more precise in several places. We think a compromise is to use the term TOL to refer to the combination of the network of genomes and tree of cells. It would not be entirely a tree, but it would have nearly same explanatory power as the original TOL hypothesis. We hope this work adds precision to these terms instead of just muddying the waters*. To sum up, I feel that the current title of the manuscript is misleading, unnecessarily dramatic, and should be modified. *The title is meant to be dramatic. We have explained what mean by \'lost in the woods\' a little better in the introduction. Now that we have changed TOL to TOC in many places we explain the title as rescuing the explanatory power of the TOL by remembering the WOL needs to be grounded in the TOC. We feel the need to be dramatic because many appear ready to abandon the TOC because it is confused with the TOL*. What this MS proposes is how a tree of cellular functions, equated with the tree of cells, could possibly be reconstructed by taking into account additional (novel) sources of data (such as the functional repertoire of genomes and the abundance of expressed genes in the cells) rather than by focusing on the mere gene content of genomes, and by giving comparable weights to the phylogenetic signal(s) of each individual gene. The Tree of Life and the tree of cells are however two different things: in particular they do not offer similar explanations of evolution. The tree of cells is by definition more limited in its scope than the legendary Tree of Life. It is then important to stress that the tree that could be saved if the author\'s proposition hold is (and that is already quite good) either the tree of functions or the tree of cells. The title also suggests that evolutionary biologists would be lost without this one tree. I think this claim is unduly pessimistic, and stems from our acquired habits to explain evolution with a tree model. Evolutionary biologists won\'t be lost without the tree of life: they will be challenged. They will need to reconsider their practices, their goals, and their explanatory toolkits to make sense of an evolution that is not just tree-like. There are lots of fascinating researches to be done to learn about the evolutionary processes and mechanisms, that do not require the inference of a unique tree of life, i.e. to harvest the phylogenetic forest of unrooted trees (see Lapointe et al. Trends in Micro, in press), or to exploit genome networks (see Dagan and Martin\'s works; Fani, Fondi et al.\'s works, Lima-Mendez, Leplae et al.\'s or Halary et al.\'s works). Our explanations will be different, but evolutionary biologists won\'t be out of job or hopeless. Such a possibility could/should have been explored more by the authors, as they reckon that it is not clear what our expectation of prokaryote should be. Precisely, clarifying this expectation, with the least possible a prioris, is an exciting prospect for evolutionary science. *Author\'s response:* *Processes and mechanisms are certainly important, but evolution is about history to us. We only care about the mechanisms and processes because they caused the history. You cannot understand the mechanisms or processes without the history. We certainly believe that networks have a lot to teach us, but they only are meaningful when grounded in a TOC. Therefore the TOC becomes more important as we try to understand the processes that do not fit into that scheme*. This is why I finally beg all authors who might be tempted to send me some more papers to review on themes such as \'rescuing the TOL\' or \'saving the TOL\' in a near future not to: I have definitely said all I had to say on that issue for a little while, and it is time for me to move on more exciting research topics ྶ. *Author\'s response:* *We sincerely thank you for one last round on this subject. Unfortunately for you, your insightful review of this manuscript will probably make others want to continue this discussion with you. But we understand the need to move on from this topic*. Additional specific comments ---------------------------- ### Further questions p.8: Do large pangenomes have larger repertoires of functions ? If so, won\'t that affect the reconstruction of a tree of functions ? *Author\'s response:* *A large pangenome may have a large amount of functional redundancy. This will not be a problem if one has a good functional outgroup, but that requires a well defined TOC*. p\. 9: Why should we assume that COGs that are the most widely distributed in extant taxa are the most ancient ones? Why can not they be highly transferred ? *Author\'s response:* *You are right that some of these could be the result of frequent transfers. To the best of our knowledge there is no case of a young protein being transferred to the majority of a superkingdom, but there are many proteins clearly in the ancestor of a superkingdom that have been retained. Therefore in general the most widely distributed proteins are the oldest. It seems we would need the tree of cells, tree of functions, and network of genomes to be certain though, so for now this is a reasonable estimate*. p\. 19: The fact that a large chunk of universal cellular function has remained conserved and its sequence behaves in a mostly tree-like manner after billions of years earns it the title of TOL in our opinion. How is this a fact ? How has this been tested ? *Author\'s response:* *We have changed this one to TOCs to soften it. We are calling the 5% of the \'abundome\' represented by the universal proteins a large chunk, which is certainly arguable. However, this number increase if one considers the functional content of the last common ancestor of each superkingdom instead. The nearly universal trees in*\[[@B22]\]*have a high level of consistency. Therefore, we think this statement is justified in its current form*. ### Unclear sentences p.1. \'results\': What do you mean by ***proper***selective context ? *Author\'s response:* *Differentiating between whether it is a good or bad horizontal transfer*. p.2. Why should the woods be \'woods of ***neutral***evolution\' ? *Author\'s response:* *Because we believe most of the noise coming from HGT are actually just displacements*. p.5. What do you mean by: The downside of abundance is it is dynamic, while genomes are static. ? What time-line/evolutionary scale do you have in mind ? At the TOL level, genomes are very dynamic. *Author\'s response:* *It is true on evolutionary time scales genomes are dynamic. We mean within a single cell*. p\. 12: This entire section: The disagreement between these phylogenies is not in terms of how to define the major taxa but rather in the proper way to polarize the data, especially the indels (insertion deletions) which we have discussed \[[@B34]\]. However, the distribution of these traits themselves implies specific taxa evolved before another, regardless of the direction of each polarization. For example, there is a large insert in HSP70 (heat shock protein 70) that is present across the Gram-negative bacteria, but absent in the Gram-positives. One form of the protein must have predated the other. There is no reason to assume all the informative indels were fixed early in evolution, and one would be very hard pressed to draw a detailed scenario of transfers that explains their distribution better than a more timeline like structure. is unclear, and should be somehow rewritten. If this is a philosophical point (rather than an empirical comment on the data distribution), I would say that the best explanations are not necessarily the ones that match a tree, these latter are only the simplest explanations. When irrelevant, they do not help much. *Author\'s response:* *This is an empirical comment. We arguing if HGT was truly so rampant as to annihilate any trace of the TOC it should not be possible to find independent traits that support these phylogenies. We have rewritten it to try to make our point clearer. We see no need to invoke a more complicated explanation if the simple one works*. p.13: Abundance data may make it possible to quantify what Simpson coined \"quantum evolution\" when referring to the metazoan fossil record \[[@B35]\] on a molecular level in prokaryotes. This sentence needs to be developped or better explained (as it is I do not recognize Simpson\'s theory - that gives a main role to the environmental selection in quantum evolution- if I recall correctly, as a particularly valid analogy here). *Author\'s response:* *We have inherited our use of this term from Cavalier-Smith. To be precise we mean events where there is a domino effect across numerous proteins that results in rapid evolution. We are arguing that if some major change in abundance was tolerated by rapidly shifting the abundance of other proteins it would very difficult to resolve with sequence data regardless of HGT*, p. 16: This highly finctional sentence makes no sense whatsoever to me: We think the TOL crisis would be worse if it was the \"tree of 99%\", as it would be quite difficult to explain the phenotypic differences between humans and *E. coli*. It is remarkable any genes are conserved since LUCA, and therefore the TOL still rings true to us. *Author\'s response:* We are saying there tree of 1% argument makes no sense without a null hypothesis. This purely fictional sentence is an example of another tree we could be dealing with that would cause a different set of problems. Put another way, what% did the community expect to be conserved before genomic sequences were available and why? ### Typos p\. 2: Bapteste is spelled strangely. p.17: But since expression is highly correlated without evolutionary rates \[[@B38]\] Do you mean \'with\' not without, don\'t you ? p.19, first line: a word is missing before \'has\'. *Author\'s response:* *We have corrected all of these*. Reviewer\'s report 2 -------------------- *Arcady Mushegian, Department of Binformatics, Stowers Institute for Medical Research, Kansas City, Missouri, USA*. I have read the manuscript by Valas and Bourne with considerable interest, wholeheartedly agreeing with several ideas in it and disagreeing with some. The best home for this study is probably in the *Opinion*category within *Biology Direct*- this is not really a research paper. *Author\'s response:* *We feel the paper is both research and opinion, and hopefully it will fit fine in either category*. There are two main themes, one of which is more of a research proposal, the other more of a philosophy-of-science talking point. The research proposal is essentially to enhance the utility of genic traits by assigning weights to them - the weights which, directly or indirectly, estimate relative contribution of each gene to the phenotype; if I understand the proposal correctly, the significance of the phylogenetics signal can therefore be reordered by the \"functional rank\" of the sequence from which this signal was obtained. I think this is a good proposal, and Adami/Wilke and Koonin\'s groups, among others, have already said a lot about gene \"relative importance\"; important technical details of all that have not been worked out, however. The more methodological question, of what to make out of the purported lack of tree-like signal, or of the Doolittle and Bapteste\'s \"pattern pluralism\" and other related proposals, is also of interest, and my intuition runs close to the author\'s, but I still think that he is led astray by the setups of the problem in the literature. In more detail, much of the \"conceptual\" literature on the HGT is hand waving about \"rampant\", \"massive\" etc. aspects of horizontal transfer. This usually refers to the large number of events observed in a particular dataset, but generally fails to acknowledge that this high *number*of events usually accounts for a small *proportion*of the genes in the dataset and correspondingly relatively *low average ratio*of horizontal to vertical branches in the trees. (Ninety-nine percent of the trees, for example, may show some evidence of HGT, but in the vast majority of these trees, there may be just one or very few HGT events, and so on; see, e.g., Pubmed 19077245, 18062816 and 15799709). Thus, instead of talking about the applicability of the TOL \"metaphor\", perhaps we should be talking about TOL quantitative model, the alternatives to it, and which model or mixed model is best compatible with the data. *Author\'s response:* *We agree with your sentiment towards these results. We are all for a mixed model, but it needs to be a true duality where cells and genomes are treated as such, instead of just a reticulated network of genomes*. The author states about Doolittle and Bapteste\'s proposal: \"A key point of their work is that any data can be forced to fit a tree, even if that representation of the data makes no sense\" - in fact, this has been known for a long time; the same can be said about any representation of the evolutionary process (e.g., alignment algorithm will align even unrelated sequences, and network algorithm will build a network even on a hierarchical set of OTUs); and finally, so what? *Author\'s response:* *We have changed this sentence to one suggested by Eric Bapteste in his review. The other two examples you bring up are valid. It is easy to forget the results of high powered computing tools we have still are prone to \'garbage in garbage out\'. Some alignments are probably forced to fit, but the authors are saying ALL universal trees are forced to fit. It took their persuasive argument to demonstrate how forced many of the genome trees are. It is the scale of the problem that makes their work worthwhile*. In other words, the author should stop fighting the windmills: the goal of phylogenetics should not be to build a tree, nor to build any graph with another kind of predetermined topology, but to improve our understanding of which evolutionary events actually happened and led to the observed data. I think this accommodates \"pattern pluralism\" naturally. *Author\'s response:* *We agree, but one must have some data structure in mind when designing algorithms and strategies to reconstruct these histories. We feel trees capture the history of the events better, and that is needed to supplement the networks to get anywhere*. I would advise to the author to get all this out of the way early and to focus on the relatively independent proposal of including protein abundance and other information (such as perhaps correlated essentiality and degree of conservation) into the judgement of importance or relevance of any particular tree topology for phylogenetics. I would like to see the discussion of several points in more detail. *Author\'s response:* *We\'d rather let the reader see our wild speculation and eastern symbolism after a little well grounded research*. 1\. \"A cumulative plot of genomic and cellular abundance reveals that at every level genomic abundance underestimates cellular abundance\" - so what does this tell us about phylogeny? Also, the datasets that are available to us are full of parasitic microorganisms whose genomes may be experiencing net gene loss, which contributes to the reduction of the \"genomic abundance\" of almost all categories of genes. Would the picture change if we focus on free-living/saprotrophic organisms? *Author\'s response:* *This implies that when a large group of trees is in agreement about phylogeny that forest is a large portion of those cells. That makes the phylogeny more historically real to us. It would certainly be interesting to focus on abundance in parasites and their free living relatives. We assume that most of genes they retain would be highly abundant proteins in free living cells, and they mostly streamline what is usually necessary to power that core. That would be consistent with higher levels of conservation when measured by protein versus genomic abundance in the spirochete data set, but more data here would certainly be informative*. 2\. The authors want to rescue the tree by bringing in the functional importance/protein abundance (phenotype), as discussed before. In this case, would not the change of function be equivalent to a HGT, and would this be less or more often than a true genetic HGT? *Author\'s response:* *Functional changes are never neutral, and we argue HGTs are neutral most of the time so they are not equivalent. It is hard to speculate on the frequency of such events because so there are so many ORFans in sequenced genomes, and so many proteins beyond that which have not been functionally characterized. If that portion of genomic space represents nuanced novel functions they could be more frequent than HGT. We think this is exactly the sort of question that requires both a tree and a network to answer properly*. Reviewer\'s report 3 -------------------- Celine Brochier, Laboratoire de Chimie Bactérienne, CNRS-Aix Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France In this paper Valas and Bourne propose an original approach to reconstruct the tree of life. To my point of view this contribution is more an opinion than an experimental paper. This is at odds with the organisation of the paper that includes a large \"results\" section (11 pages), whereas the real experimental part of the work is represented by a single figure and one paragraph (1 page). By contrast, the discussion\" section is rather short (3 pages). I think it would be more appropriate to combine the results and the discussion sections into a single section, with subdivisions corresponding to the different points that are discussed. Finally, I think it is important to clearly classify this manuscript as an opinion and not as an experimental paper. *Author\'s response:* *We have combined the results and discussion as suggested. We agree that this paper is not a traditional research paper, but we still feels it belongs in that category as it is a combination of novel research, opinion, and review*. The contribution of Valas and Bourne comes within the scope of the hot debate around the Tree Of Life (TOL). Indeed, based on genomic data the suitability of tree-like structures to represent the evolutionary history of all organisms has been highly debated \[[@B4],[@B8],[@B47]\]. The two main arguments are that in prokaryotes (1) the evolutionary history of genes is different from the evolutionary history of organisms because of horizontal gene transfers (HGT) \[[@B4]\], and (2) HGT may be so frequent that a substantial part of the genes in a genome have been affected by HGT. Then, jumping from genomes to organisms (perhaps because we have entered in a \"*too genomic-centric*\" area, as stated by the authors), this has led to the conclusion that, at least for prokaryotes, a tree-like structure does not reflect the evolution of genomes, which will be better represented by a network (NOG, Network Of Genes). However, if nobody can deny that HGT have played an important role in evolution (and not only in prokaryotes), it is also undisputable that cell division in prokaryotes occurs by the division of a mother cell in two daughter cells. It is therefore theoretically possible to trace-back the history of cell lineages and to represent it with a tree-like structure, the TOL. However, the TOL and the NOG are often confounded, maybe because genes are the only informational entities that are transmitted from one generation to another, whereas TOL and NOG represent two different things that are equally interesting and highly complementary to understand the evolution of living organisms \[[@B5],[@B14]\]. The authors state these points well by writing: \"*All of the issues the community is currently having with the TOL hypothesis stem from the simple fact that genomes are not perfect representation of membrane history*. \[\...\] *even genomic evolution makes little sense without the light of cellular evolution*\". The challenge is now to reconstruct the TOL in a NOG context. Classical approaches consist to identify (and to analyse) the sets of genes that may be used to reconstruct the different parts of TOL. This step is important because it appears a utopia to think that it is possible to fully resolve the TOL (from the root to the leaves) based on the analysis of a few universal genes. This would be the tree of 1%. In fact, it would be cleaver to divide the problem by looking at the set of genes suitable to reconstruct different parts of the TOL. For example, the set of genes suitable to resolve the phylogeny of animals will probably be different to the set of genes that may be used to trace back the relationships within Methanococcales (Archaea). This is well known by botanists and zoologists who used different sets of characters for different levels of their classifications. Ideally, the TOL should be a synthetic drawing showing the relationships between organisms (not species, which are artificial entities, or genomes) by combining the results obtained by the phylogenetic analyses of different sets of genes. In this case, the TOL will not be the tree of 1% but the tree of dozens of percents, each gene contributing to resolve some parts of the TOL. Here the authors propose a radically different approach based on the vertical inheritance of functions rather than on the vertical inheritance of genetic material. The approach is based on the assumption that all genes do not contribute equally to the cell: some are more important than others. The authors underline that the gene contribution to the cell should be an important criterion to take into account when reconstructing the TOL. As the authors point out there are different metrics to measure the gene contribution to a cell: \"*essentiality, abundance of proteins, number of transcripts, portion of total weight, etc*\". 1\) My first question is how to organize these factors in a hierarchy, i.e. which criterion is the more suitable to represent protein importance in a cell? And what do these factors exactly represent from an evolutionary point of view? Is it possible to develop evolutionary models for such data (that are mainly quantitative and not discrete characters)? Unfortunately the authors do not propose methodological approaches to analyse such data. I think this is important to discuss about their suitability to reconstruct the TOL. Among these factors, the authors chose to study the abundance of proteins in cells (the \"*adundome*\"). Based on recently published data on the abundance of proteins present in the cytoplasm of *Escherichia coli*cells (GammaProteobacteria) and of the complete proteome of *Leptospira interrogans*(Spirochaetes), the authors argue that \"*abundance is a good proxy for evolutionary importance because there is a correlation between the abundance of a protein and the energy the cell invests into producing it*\". *Author\'s response:* *We do not have precise answers for these questions, but they are certainly going to be important to answer. Our demonstration that abundance is a barrier to transfer supports the notion it is evolutionary important. But there are abundant genes that have been transferred. It would be naïve to say those are less important. It might not be possible to precisely quantify how important each gene is to the cell, but we have demonstrated that in general the important genes evolve in a more tree-like manner. We are hopeful it will be possible to develop evolutionary models for the evolution of \"abundomes\", but we doubt they will behave well enough to resolve the TOL or TOC on their own. Rather we think they will be tools to help us understand how the TOC was shaped. In either case it would certainly be premature to begin developing these methods from the two datasets currently available as they are not directly comparable. A sampling of many strains of E. coli seems like a good place to start addressing these ideas*. 2\) This raises my second question: what does the \"*evolutionary importance*\" of a protein mean from an evolutionary point of view? The word \"*importance*\" is a subjective and indefinite criterion. The abundance is one side of the importance, essentiality is another. Indeed, a protein may be important even if it is not abundant in a cell (e.g. transcription regulators). The authors should discuss more this point. *Author\'s response:* *Importance is certainly a subjective term. We are not arguing that abundance is a perfect representation of importance but it is seems to be a straightforward and objective measure. We argue that as we measure the importance of function in some meaningful way the importance of HGT will begin to shrink, and the vertical component will grow in size*. 3\) More problematic, and this is partially raised by the authors, the abundance of a protein is a dynamic parameter that may vary across cells depending for example of their lifestyle. More importantly, for a given cell the relative abundance of its proteins may vary in time, depending for example on the developmental state reached (e.g. cells in exponential growth or in stationary phase, etc) or environmental conditions. How to take this difficulty into account? *Author\'s response:* *There are certainly going to be many difficulties in using abundance data. We argue the proteins that remain abundant under a variety of conditions are probably the most important, but surely there will be many interesting caveats to discover as more data become available. Again, we are not suggesting using this data to build phylogenetic trees, but rather as a tool to better understand the ones created from other data sources*. 4\) I think the large paragraph on indels and the timing of appearance of prokaryotic phyla should be removed because it is beyond the scope of the paper, and I am afraid that the reader will loose grasp on the logical succession of ideas. Same remark for the paragraph dealing with protein structures. On the contrary, I think the authors should rather focus on their proposal to use \"abundome\" to reconstruct the TOL and in particular on the methodological aspects. *Author\'s response:* *We are not arguing the \"abundome\" data can reconstruct the TOC, but we think the indels and quaternary structures can. In some sense we are using abundance data to show the phylogenies created using other data sources are meaningful despite the arguments against the TOL. Most of these arguments are against sequence based methods, so we think it is appropriate to include our other work as a demonstration the TOC is still evolutionary meaningful and can still be resolved*. 5\) Finally, I have a few comments regarding assumptions that are made on phylogenetic studies based on gene sequence analyses. The authors say that \"*current methods for estimating HGT rely on measuring inconsistencies between sequence trees or looking for unusual compositional features, so there is no way for them to distinguish between innovations and displacements*\". I think this statement should be toned down because, in the case of phylogenetic studies, most of the time a careful examination of trees allows discriminating between gene acquisition and gene replacement. This is for example the case for aminoacyl-tRNA synthetases (that are discussed in the paper), where clear cases of gene replacements can be identified. *Author\'s response:* *The aminoacyl-tRNA synthetases are a special case because the combination of their trees and knowledge of the essentiality of their function implies these are displacements. It is much more difficult to conclude that from the trees alone. It is not impossible to discriminate between these scenarios, but it seems like many do not seem to worry about the difference when looking at forests of phylogenetic trees*. Very minor points: I do not understand the sentence \"*We argue events like this are far more deleterious to tree reconstruction algorithms than they are to the recipient cells*.\" *Author\'s response:* *We feel that sequence has persisted as the primary tool to study evolution because of the relative ease algorithms can represent it, as opposed to these other sources of data. Again we are trying to emphasize the difference between displacement and innovation needs to be made by including other data sources*. The legend of Figure [1](#F1){ref-type="fig"} is poorly understandable. I did not understand the last sentence of the abstract. *Author\'s response:* See our reply to Eric Bapteste I disagree with the allusions to Darwin in the discussion section \"*There is clearly a duality in Darwin\'s theory of descent with modification; the history of variation is well described by a network and the history of selection is well described by a tree*\". First, this sentence is not clear. Second the history of variation may be represented by a tree: for example, the evolutionary history of a gene (irrespective to HGT) may be depicted by a tree and it is possible to indicate on each branch the mutations that occurred, and therefore to follow the history of variation of this gene. *Author\'s response:* *We are speaking in very general terms. Your example is correct, but the variation of organisms is in terms of their entire genomes. The gene cannot really be selected for independently of the rest of the cell and genome, so the variation is relative to them as well. It was assumed for a long time the history of these two processes is the same, and we think it has become time to explicitly separate them*. Supplementary Material ====================== ###### Additional file 1 ***E. coli*data Columns A-E of additional file**[1](#S1){ref-type="supplementary-material"}**are taken directly from supplemental material of**\[[@B10]\]. The universal core proteins are defined in \[[@B7]\]. The Enterobacteriaceae genomic core was defined in \[[@B19]\]. All COG annotations were taken from the STRING database \[[@B21]\]. All inconsistency scores were taken from \[[@B22]\]. ###### Click here for file ###### Additional file 2 ***L. interrogans*data**. Columns A-D of additional file [2](#S2){ref-type="supplementary-material"} are taken directly from supplemental material of \[[@B11]\]. The Spirochete genomic core was defined in \[[@B20]\]. All inconsistency scores were taken from \[[@B22]\]. ###### Click here for file Acknowledgements ================ We would like to thank Russell Doolittle, William Loomis, Stella Veretnik, and the entire Bourne laboratory for useful discussions. We would like to thank Pere Puigbo for providing the inconsistency score data.

Introduction ============ Neurosensory symptoms, such as tinnitus and dizziness, are frequently observed in elderly people and even more so in patients with dementia. Five-year and 10-year incidence rates of 18.0% and 12.7% were reported for tinnitus from the Blue Mountains Hearing Study and the Beaver Dam Epidemiology of Hearing Loss Study, respectively.[@b1-cia-13-1121],[@b2-cia-13-1121] Epidemiological studies have found an increase in the prevalence of tinnitus as a function of age.[@b3-cia-13-1121] Jahn et al reported 1-year prevalence rates for significant dizziness of 20% in persons older than 60 years, 30% in those older than 70 years, and 50% in those older than 80 years.[@b4-cia-13-1121] In elderly patients with dementia, we found prevalence rates between 13% and 52% for tinnitus and between 14.2% and 77.5% for dizziness in five clinical trials.[@b5-cia-13-1121]--[@b9-cia-13-1121] Age-related hearing loss is likely to account for higher rates of tinnitus in the elderly and may even contribute to cognitive decline and the development of Alzheimer's disease (AD) and other dementias.[@b10-cia-13-1121]--[@b13-cia-13-1121] In a case-control study of elderly patients with neurological disorders, those with dementia had a particularly high rate of falls (60% in 1 year), which may indicate a higher prevalence of dizziness and impaired equilibrium control.[@b14-cia-13-1121] Given the high comorbidity of tinnitus and dizziness in dementia and the findings from earlier studies in which *Ginkgo biloba* extract EGb 761^®^ (Dr Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany) alleviated tinnitus and dizziness or vertigo,[@b15-cia-13-1121],[@b16-cia-13-1121] measures of tinnitus and dizziness were included in recent clinical trials of EGb 761^®^ in patients with dementia. When considering why *G. biloba* extract EGb 761^®^ may alleviate tinnitus, dizziness, or vertigo in patients with dementia, the pathomechanisms underlying these symptoms should be taken into account. Neurons of the central vestibular and auditory systems, cochlear hair cells, and vestibular sensory cells have a high energy demand in order to maintain and continuously restore their transmembrane electrical potential. Impaired mitochondrial function and impaired perfusion are thought to contribute to both cochlear and vestibular dysfunction and sensory cell degeneration.[@b17-cia-13-1121] EGb 761^®^ improves inner ear and cerebral blood flow by decreasing blood viscosity; it also improves mitochondrial function and energy metabolism, which altogether may play a role in improving inner ear and brain function in elderly people with dementia who often have vascular disorders and compromised mitochondrial function.[@b18-cia-13-1121]--[@b20-cia-13-1121] The antiapoptotic and neuroprotective properties of EGb 761^®^ may inhibit aging-related loss of cochlear and vestibular sensory cells,[@b21-cia-13-1121]--[@b24-cia-13-1121] which may play a role in tinnitus and vertigo.[@b25-cia-13-1121],[@b26-cia-13-1121] Coping with the distress of tinnitus as well as compensating for vestibular dysfunction involves both learning and neuroplasticity. EGb 761^®^ enhances neuroplasticity, improves learning, and accelerates vestibular compensation.[@b18-cia-13-1121],[@b27-cia-13-1121],[@b28-cia-13-1121] Tinnitus is likely to cause distress and anxiety, while dizziness often causes unsteadiness and fear of falling. Due to anxiolytic effects and by attenuating the activation of the stress axis, EGb 761^®^ may decrease the distress in both conditions.[@b18-cia-13-1121],[@b29-cia-13-1121],[@b30-cia-13-1121] By improving the speed of information processing, it may improve gait and reduce unsteadiness.[@b18-cia-13-1121] Here, we present a meta-analysis of the trials that used rating scales for the assessment of presence and severity of tinnitus and dizziness. The question addressed by this meta-analysis was whether, taking into account all available evidence, EGb 761^®^ treatment was superior to placebo in alleviating tinnitus or dizziness or both in patients with dementia who had one or both of these neurosensory symptoms at pre-treatment examination. Materials and methods ===================== In 2014, Gauthier and Schlaefke published a systematic review and meta-analysis of randomized, placebo-controlled, double-blind clinical trials of *G. biloba* extract EGb 761^®^ in patients with mild to moderate dementia (AD, vascular dementia \[VaD\], mixed dementia, ie, AD with cerebrovascular disease \[CVD\]).[@b31-cia-13-1121] The search strategy is described in detail in their original paper.[@b31-cia-13-1121] Our aim was to provide an update on studies until October 2017. We did not identify any further relevant studies. Briefly, PubMed, including and excluding MedLine (from beginning to October 2017), EMBASE (from January 2006 to October 2017), and PASCAL (from beginning to end of 2015, no further update of PASCAL existed beyond this date) were searched using the following search terms (with \* characterizing a wild-card, and the items AND and OR being used as Boolean functions): (ginkg\* OR gingk\*) AND clinical trial\[pt\] for PubMed including MedLine, ((ginkg\* OR gingk\*) NOT medline\[sb\]) AND (clinical\* OR trial OR randomized) for PubMed excluding Medline, (GINKGO OR GINGKO) AND (HUMAN/CT OR HOMME/CTFR) for PASCAL, and (ginkgo or gingko) AND CT=(CLINICAL TRIAL; CLINICAL STUDY; DOUBLE BLIND PROCEDURE) AND py\>2005 for EMBASE. The papers retrieved were assessed for eligibility by two scientists independently and trials were selected for the review, if 1) the diagnoses were established in accordance with generally accepted diagnostic criteria, 2) the treatment periods were at least 20 weeks, and 3) outcome measures covered at least two of the three conventional domains (cognition, global judgment, activities of daily living). For the current meta-analysis, we applied two additional inclusion criteria, requiring that 4) the presence and severity of tinnitus or dizziness or both were assessed and 5) assessment was done before the start and after the end of randomized treatment. Five of the seven trials included in the meta-analysis by Gauthier and Schlaefke met these additional criteria.[@b5-cia-13-1121]--[@b9-cia-13-1121] All trials were sponsored by Dr Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany. The risk of bias was low for all five trials, with Jadad scores of 3 and 5 ([Table 1](#t1-cia-13-1121){ref-type="table"}).[@b32-cia-13-1121] In all five trials, 11-point box scales were used to assess the presence and severity of tinnitus and dizziness. The 11-point box scale is a type of visual analog scale (VAS) consisting of 11 adjacent boxes which contain ascending numbers from 0 to 10 and descriptors for the extremes only, thus providing a distinct number of possible responses in a single dimension. Such types of 11-point VASs are often used as measures for pain,[@b33-cia-13-1121],[@b34-cia-13-1121] and have also been found useful for the assessment of other unpleasant and distressing symptoms, such as dizziness[@b35-cia-13-1121] or tinnitus.[@b33-cia-13-1121],[@b36-cia-13-1121],[@b37-cia-13-1121] In the studies reviewed here, 11-point box scales for tinnitus and dizziness were administered with the extreme ends indicating "no tinnitus at all" (0) and "extremely severe tinnitus" (10) or "no dizziness at all" (0) and "extremely severe dizziness" (10). One study enrolled patients exclusively with AD,[@b5-cia-13-1121] while all other studies also accepted patients with VaD or AD with CVD.[@b6-cia-13-1121]--[@b9-cia-13-1121] In one study, two different doses of EGb 761^®^ (240 mg or 120 mg) and placebo were compared;[@b5-cia-13-1121] however, the patients treated with 120 mg of EGb 761^®^ were excluded from the present meta-analysis. In all other studies, patients received either 240 mg of EGb 761^®^ or placebo. For each study, EGb 761^®^ and placebo treatment were compared with regard to mean differences between baseline and end of treatment on both 11-point box scales for tinnitus and dizziness. These calculations, conducted with SAS version 9.3, were based on individual patient data, which were provided by Dr Willmar Schwabe GmbH & Co. KG. Based on the study-specific mean differences, weighted mean differences (and corresponding forest plots) were calculated using meta-analytical models to compare EGb 761^®^ and placebo treatments by Review Manager (version 5). Due to the somewhat different inclusion criteria and design of the trial conducted by Schneider et al[@b5-cia-13-1121] compared to the other four trials, which were similarly designed, a random effects model was chosen for the analysis and a fixed effects model as a sensitivity analysis. Only data from patients who had tinnitus or dizziness at baseline (baseline scores \>0 on the respective 11-point box scale) were included in the respective meta-analyses. Missing data were replaced by the last-observation-carried-forward method. Results ======= In the five studies, a total of 1,972 patients, aged 50--98 years, were randomly assigned to receive EGb 761^®^ at a daily dose of 240 mg or placebo. The treatment periods were 22--26 weeks. The full analysis comprised a total of 1,942 patients, of whom 904 were diagnosed with probable AD, 374 had probable VaD, and 664 had mixed dementia. Study characteristics and demographics are provided in [Table 1](#t1-cia-13-1121){ref-type="table"}. Dementia-related baseline scores and outcomes are provided in detail by Gauthier and Schlaefke.[@b31-cia-13-1121] Altogether, 773 patients had tinnitus at baseline (EGb 761^®^, 372; placebo, 401). In the individual studies, prevalence of tinnitus ranged between 13% and 52%; the average severity ratings varied between \~2.7 and 4.0. The baseline scores for the 11-point box scale related to tinnitus are provided in [Table 2](#t2-cia-13-1121){ref-type="table"}. A total of 1,040 patients reported dizziness at baseline (EGb 761^®^, 528; placebo, 512). Prevalence in the individual studies ranged from 13% to 77%; the average severity ratings varied between 2.5 and 4.3. The baseline scores for the 11-point box scale related to dizziness are shown in [Table 3](#t3-cia-13-1121){ref-type="table"}. A mean reduction in tinnitus severity for the EGb 761^®^-treated patients compared to placebo was observed in all five trials. The difference in favor of EGb 761^®^ was statistically meaningful in four trials. Overall, there was a weighted mean difference of −1.06 (95% CI: −1.77, −0.36) favoring EGb 761^®^ at *p* = 0.003 ([Figure 1](#f1-cia-13-1121){ref-type="fig"}). This was similar to the result of the sensitivity analysis by the fixed effects model: −0.97 (95% CI: −1.16, −0.78; *p* \< 0.001). Considering that the average severity of tinnitus was between 2.7 and 4.0 at baseline,[@b5-cia-13-1121],[@b6-cia-13-1121] the weighted mean difference corresponds to an improvement over placebo effects by 27%--40% of baseline severity in the single studies. A greater reduction in dizziness was observed for the EGb 761^®^-treated patients in four of the five trials comprising 96% of all patients included in the meta-analysis. The differences in favor of EGb 761^®^ were statistically meaningful in three trials comprising 80% of all patients. Overall, there was a weighted mean difference of −0.77 (95% CI: −1.44, −0.09) favoring EGb 761^®^ at *p* = 0.03 ([Figure 2](#f2-cia-13-1121){ref-type="fig"}) and similar to the fixed effects model calculated as a sensitivity analysis: −0.98 (95% CI: −1.15, −0.81; *p* \< 0.001). The average severity of dizziness at baseline was between 2.5 and 4.3.[@b5-cia-13-1121],[@b6-cia-13-1121] Therefore, the weighted mean difference corresponds to an improvement over placebo effects of between 18% and 31% of baseline severity in the four larger trials. Discussion ========== In our meta-analysis, we included five randomized, placebo-controlled clinical trials of *G. biloba* extract EGb 761^®^ in patients with mild to moderate dementia. Using 11-point box scales to assess the presence and severity of tinnitus and dizziness, we found that a considerable proportion of the patients enrolled for their diagnoses of dementia, and not for tinnitus or dizziness, had such neurosensory symptoms. On average, these symptoms were mild to moderate at baseline. Overall, we found EGb 761^®^ to be clearly superior to placebo in alleviating both tinnitus and dizziness. This is in line with the results of earlier trials in patients with tinnitus or vertigo,[@b15-cia-13-1121],[@b16-cia-13-1121] in whom these neurosensory symptoms were the main complaints. It is also in line with conclusions from systematic reviews that found EGb 761^®^, but not other *G*. *biloba* extracts, to be effective in the treatment of tinnitus and vertigo.[@b15-cia-13-1121],[@b16-cia-13-1121] EGb 761^®^ is a defined extract of *G. biloba* leaves that is obtained by a proprietary multi-step extraction procedure during which pharmacodynamically active molecules are enriched and harmful compounds are removed. EGb 761^®^ is adjusted to 22.0%--27.0% ginkgo flavonoids calculated as ginkgo flavone glycosides and 2.0%--7.0% terpene lactones consisting of 2.8%--3.4% ginkgolides A, B, C and 2.6%--3.2% bilobalide and contains less than 5 ppm ginkgolic acids. High batch-to-batch consistency, which is a prerequisite for the generalization of study results to daily clinical practice, has been demonstrated. Extracts that result from different productive processes are inherently different in composition, and in pharmacodynamic and clinical activity.[@b18-cia-13-1121] Our literature search did not identify any randomized, placebo-controlled clinical trials of any other Ginkgo extract in patients with dementia in which effects on tinnitus or dizziness were evaluated. The clinical relevance of the effects is difficult to assess. Reductions in tinnitus severity by 27%--40% over and above the placebo effect may represent clinically relevant effect sizes. The effects on dizziness are less pronounced, but may still be clinically relevant in the given population of elderly people with ischemic and neurodegenerative CNS changes, who are particularly prone to falling. When evaluating clinical relevance in this context, the fact that the patients had mild to moderate dementia has to be kept in mind. Tinnitus may contribute to cognitive decline in dementia.[@b12-cia-13-1121],[@b13-cia-13-1121] Both tinnitus and dizziness cause anxiety, fear (fear of enduring annoyance and fear of falling, respectively), and distress.[@b1-cia-13-1121],[@b4-cia-13-1121],[@b38-cia-13-1121],[@b39-cia-13-1121] Tinnitus often leads to sleep disturbances,[@b1-cia-13-1121],[@b39-cia-13-1121] thus adding to the burden of night-time disturbances frequently experienced by patients with dementia.[@b40-cia-13-1121] Stress-induced activation of the hypothalamic--pituitary--adrenal axis (the so-called stress axis) tends to further compromise cognitive functioning in patients with cognitive impairment.[@b41-cia-13-1121] Focusing on and being distracted by tinnitus may additionally impair attention and processing speed, ie, the cognitive abilities already compromised in patients with dementia.[@b39-cia-13-1121] Dizziness and fear of falling often lead to reduced physical activity, decreased participation in social life, disability, and frailty.[@b42-cia-13-1121] Physical activity is not only necessary to maintain muscle mass and postural control,[@b43-cia-13-1121],[@b44-cia-13-1121] it is also closely correlated with cognitive performance in elderly people with and without cognitive impairment.[@b45-cia-13-1121]--[@b47-cia-13-1121] Moreover, dizziness is not only associated with fear of falling, it also increases the risk of falling and the incidence of falling-related injuries, such as hip fractures.[@b4-cia-13-1121] However, hospitalization and major surgery increase the risk of confusion and delirium and may accelerate cognitive decline in patients with dementia.[@b48-cia-13-1121] Our meta-analysis has some limitations related to the studies included. First, the symptom ratings were done by patients with dementia, which may cast doubts on their reliability. However, patients with mild to moderate dementia are still able to understand questions about ringing in the ears and dizziness, when asked in simple language. They may have problems remembering the intensity of tinnitus or dizziness at an earlier point in time, but the 11-point box scales ask about the current severity, and not about changes. Rating errors due to cognitive problems may possibly increase scatter, but does not build a consistent pattern of treatment benefits across the studies as found in the present meta-analysis. Second, no examinations of auditory or vestibular functions were performed, and disabilities related to tinnitus and dizziness were not assessed within the studies. As a consequence, it is not known to what extent alleviation of tinnitus and/or dizziness may have contributed to the improvement in everyday functioning and quality of life. After all the examinations required by current guidelines for clinical trials in dementia have been performed, the patients are usually too exhausted to undergo further examinations during the same visit. Investigators were free to perform all examinations required for medical reasons, but any findings not related to the studies were not documented in the case record forms. As a result, to what extent dizziness was related to vestibular vertigo or to non-vestibular disorders remains unknown. Yet, the dizziness-related problems in patients with dementia are largely the same. It is possible, however, that different proportions of patients with vestibular vertigo and non-vestibular dizziness contributed to the heterogeneity of the outcomes related to dizziness. Third, average baseline scores for tinnitus and dizziness were around 3, ie, about one-third of the range of the scales, in most trials. As a consequence, the relative effect sizes may lead to an overestimation of their clinical relevance. On the other hand, floor effects cannot be excluded, so that the real therapeutic potential of the drug could be underestimated. In general, patients complaining of tinnitus or dizziness should be examined for causes and contributors that are amenable to specific treatments, irrespective of whether or not they have dementia. Assessment of auditory or vestibular function might provide additional information about the underlying causes. However, in many patients the causes of neurosensory symptoms remain unclear, and even if known, there are often no causal or cause-specific treatments. As outlined earlier, EGb 761^®^ interferes with a number of mechanisms that seem to contribute to dysfunction and degenerative processes in the inner ear and cerebral structures. Conclusion ========== *G. biloba* extract EGb 761^®^, at daily doses of 240 mg, alleviated both tinnitus and dizziness in patients with dementia, ie, in patients who are particularly vulnerable to such disturbances. This should be taken into account when choosing an appropriate treatment for patients with dementia and neurosensory symptoms. H Mueller and R Hoerr made their contribution during working hours paid by Dr Willmar Schwabe GmbH & Co. KG. There was no other funding for this work. Rainer Spiegel and Roger Kalla share first authorship. **Author contributions** R Spiegel and R Kalla contributed equally to the manuscript. All authors contributed to the interpretation of the data and the intellectual content of the manuscript and approved the final version to be published. H Mueller performed the meta-analyses. **Disclosure** R Maire received a speaker honorarium from Schwabe Pharma AG, Kuessnacht, Switzerland; R Kalla and G Mantokoudis were supported by the Swiss National Science Foundation (Grant \#320030-173081); R Ihl received speaker honoraria from Dr Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany; H Mueller and R Hoerr are full-time employees of Dr Willmar Schwabe GmbH & Co. KG receiving fixed salaries. R Spiegel reports no conflicts of interest in this work. ![Meta-analysis of changes in tinnitus severity across five clinical trials using the 11-point box scale (weighted mean differences \[95% CI\] from random effects model).](cia-13-1121Fig1){#f1-cia-13-1121} ![Meta-analysis of changes in dizziness severity across five clinical trials using the 11-point box scale (weighted mean differences \[95% CI\] from random effects model).](cia-13-1121Fig2){#f2-cia-13-1121} ###### Study characteristics and demographics (mean ± standard deviation for age) Study Jadad score Treatment duration (weeks) EGb 761^®^ Placebo ------------------------------------------ ------------- ---------------------------- ------------ --------- --------- ----- ---- -------- Schneider et al, 2005[@b5-cia-13-1121] 3 26 170 56 78 ± 7 174 52 77 ± 7 Napryeyenko et al, 2007[@b6-cia-13-1121] 5 22 198 72 65 ± 8 197 72 63 ± 8 Nikolova et al, 2013[@b9-cia-13-1121] 3 22 196 57 69 ± 8 201 60 69 ± 8 Ihl et al, 2011[@b7-cia-13-1121] 5 24 202 69 65 ± 10 202 66 65 ± 9 Herrschaft et al, 2012[@b8-cia-13-1121] 5 24 200 70 65 ± 9 202 69 65 ± 9 **Note:** Full analysis set. **Abbreviation:** N, number of patients. ###### Eleven-point box scale baseline scores for patients with tinnitus (mean, standard deviation) Study EGb 761^®^ Placebo ------------------------------------------ ------------ ------------- ----- ------------- Schneider et al, 2005[@b5-cia-13-1121] 19 2.84 ± 1.80 24 2.54 ± 1.82 Napryeyenko et al, 2007[@b6-cia-13-1121] 102 4.03 ± 1.61 104 3.89 ± 1.45 Nikolova et al, 2013[@b9-cia-13-1121] 64 3.53 ± 1.94 72 2.94 ± 1.81 Ihl et al, 2011[@b7-cia-13-1121] 90 2.92 ± 1.52 107 2.92 ± 1.54 Herrschaft et al, 2012[@b8-cia-13-1121] 97 2.90 ± 1.64 94 3.02 ± 1.41 **Abbreviation:** N, number of patients. ###### Eleven-point box scale baseline scores for patients with dizziness (mean, standard deviation) Study EGb 761^®^ Placebo ------------------------------------------ ------------ ------------- ----- ------------- Schneider et al, 2005[@b5-cia-13-1121] 24 2.29 ± 1.23 22 2.64 ± 1.97 Napryeyenko et al, 2007[@b6-cia-13-1121] 149 4.37 ± 1.42 157 4.22 ± 1.43 Nikolova et al, 2013[@b9-cia-13-1121] 88 3.52 ± 1.84 71 3.18 ± 2.04 Ihl et al, 2011[@b7-cia-13-1121] 136 3.04 ± 1.54 139 3.02 ± 1.33 Herrschaft et al, 2012[@b8-cia-13-1121] 131 3.10 ± 1.37 123 3.01 ± 1.39 **Abbreviation:** N, number of patients.

Background {#Sec1} ========== It was estimated that over 2.2 billion participants worldwide are at risk of iodine deficiency. Iodine deficiency remains a major public health problem \[[@CR1], [@CR2]\]. Understanding the role of risk factors is key to developing a clear and effective strategy for improving global health. In low-income countries relatively few risk factors such as micronutrient deficiency are responsible for a large percentage of and ill health \[[@CR3]\]. The mandatory use of iodized salt reduces or eliminates iodine deficiency \[[@CR3]\]. In South Sudan according to Gaffar and Mahafuz, in a 2005 survey, households consuming iodized salt had increased from 40 to 73% \[[@CR4]\]. Although the International Council for Control of iodine deficiency disorders (ICCID) indicates that South Sudan, Sudan and Ethiopia have only 35% or less households with access to iodized salt, neighboring Kenya and Uganda records 90% and more \[[@CR3], [@CR5]--[@CR7]\]. However, high goiters prevalence persist in these high coverage iodized salt intake regions. Severe iodine deficiency results in impaired thyroid hormone synthesis and or/thyroid enlargement (goiter). Population effects of severe iodine deficiency, termed iodine deficiency disorders (IDDs), include endemic goiter, hypothyroidism, cretinism, decreased fertility rate, increased infant mortality and mental retardation \[[@CR8]\]. There is no data from south Sudan describing the association of goiter and iodine deficiency. The purpose of this study was to determine the prevalence iodine deficiency among adult patients with goiter and associated factors in Unity state of south Sudan. Country context {#Sec2} --------------- The Health Care System in Sudan is currently described as critical, the country's most important industry oil, which is a source of 98% of the country's revenue suffers frequent shut downs due to insecurity and political upheaval. There is an enormous, largely unmet demand for health services including immediate humanitarian crises often involving internally displaced persons. There are inadequate human resources for health, infrastructure and other resources to meet current needs \[[@CR9]\]. Method {#Sec3} ====== Study design {#Sec4} ------------ A cross-sectional descriptive community based study to determine the prevalence of iodine deficiency. Study setting {#Sec5} ------------- South Sudan; involving three counties: Rubkona county with a population of 100,236 has 170 villages health facilities in an area of 3,368 km^2^, its major livelihood activities are farming, live stock and fishing. Koch county with a population of 74,863, Guit county with a population of 33,004 \[[@CR3], [@CR10]\]. These three counties were purposively selected because they have a stable population with no much people migration as a result of better security and civil order compared to the other 6 counties in the country. No established patient records exist in these counties to give a sense of the extent of the problem. The population in this area is socio economically homogenous, its rural and engage in subsistence activities and the majority reside along the river Nile where floods frequently occur. Study population {#Sec6} ---------------- All Adults (both males and females) aged 18 years and above residing in Rubkona, Guit and Koch counties of the Republic of south Sudan with a visibly enlarged thyroid gland were enrolled. Sampling procedure {#Sec7} ------------------ Each county had health centers to which study participants presented. Three health centers from Rubkona County, two from Guit, and one from Koch County were selected randomly out of the nine as study sites. The number of centers selected from each County was determined by probability proportionate to size of the population of the three counties. Data collection {#Sec8} --------------- Local radio announcements were made a week prior to starting the study to encourage and alert the communities about the availability of free neck medical examinations, and testing. The persons who responded to the invitation and were above 18 years and provided written informed consent, were examined. The physical examination for the presence of goiter was by inspection (visible goiter) and palpation. The simplified classification of goiter by World Health Organization (WHO) \[[@CR11]\] was used as Grade 0, 1 and 2. Grade 1: A goiter is palpable but not visible where the neck is in the normal position. A thyroid gland was considered goitrous when each lateral lobe had a volume greater than the terminal phalanx of the thumbs of the subjects examined. The examinations were performed by trained research assistants (clinical officers and medical officers). Grade 2: A swelling in the neck that is clearly visible when the neck is in a normal position and is consistent with an enlarged thyroid when the neck is palpable. Unique codes were assigned to each participant to ensure confidentiality. A questionnaire was completed and two fresh on-spot urine samples was collected from the participants as they came in and examined for iodine concentration and another sample was sent to another laboratory for quality control assurance. The Sandell-kolthoff method was used \[[@CR12]\] which depends on iodine's role as a catalyst in the reduction of cericammonium sulfate (yellow color) to the cerous form (colorless) in the presence of arsenious acid. Results were interpreted in accordance to WHO classification \[[@CR13]\]. Optimal levels ranges 100-199 mcg/l, mild deficiency ranges 50--99 mcg/l, Moderate ranges 20--24 mcg/l, severe ranges \<20 mcg/l, more than adequate rages 200--299 mcg/l, and excessive \>299 mcg/l. Study variables {#Sec9} --------------- Socio demographic data included; age, gender, family history of goiter, dietary intake of iodized salt, food with goitrogens, medications, and levels of urinary iodine concentration (UIC) measure and goiter. Data analysis {#Sec10} ------------- Continuous data were summarized into mean and standard deviations and medians. Other categorical data were summarized as frequencies and percentages. The prevalence of iodine deficiency was the number of patients with goiter who had urine iodine below 100 mcg/l to the total number of goiter patients sampled for urine iodine concentration. Ethical considerations {#Sec11} ---------------------- Ethical approval was obtained from Makerere College of Health Sciences, School of Medicine Research and Ethics Committee, and the Ugandan National Council of Science and Technology and the Ministry of Health, South Sudan. All participants provided informed written consent. Results {#Sec12} ======= The study was conducted in the three counties of the Unity State in South Sudan between April and June 2012. A total of 286 patients with endemic goiter were interviewed and urinary iodine excretion was assessed. The mean age was 38 years (SD 9) with median age of 38 years (see Table [1](#Tab1){ref-type="table"}).Table 1**Clinical features and socio- demographic profile of the respondents**VariableCategoryFrequencyPercentage (%)**County (Payam)**Rubkona17963Guit6824Koch3914**Gender**Male248Female26292**Occupation**\*Peasants25790^†^Salaried/wage workers3011^∞^Business10.4**Symptoms of presenting goiter**Neck swelling28599Palpitation31Anxiety41Difficulty in breathing20.7Voice change10.3Temperature intolerance20.7Profuse sweating31.0**Signs of goiter**Thyroidectomy scar10.4Tenderness of mass43Hoarseness of voice10.7Grade 1 goiter15654.5Grade 2 goiter13045.5\*Subsistence farmers ^†^Low income/salaried workers ^∞^Business. Of all the 286 participants, 262 (92%) were females, and 257 (90%) were peasants. (179) 63% were from Rubkona with 68 (24%) and 39 (14%) from Guit and Koch Payams respectively. The median duration of stay at the respective addresses was 36 years (25--42), 255 participants (90%) were indigenous (born within that area). Those who came from other places had stayed for median duration of 21 years (11--29). A handful (30 participants (11%) came from the Khartoum, Durfur, Bar el gazel and Malakal states. All respondents presented with a neck swelling, followed by anxiety 4 (2%), palpitations and profuse sweating each at 3 (1.0%). Voice change was seen in one respondent (0.3%). 156 (55%) had grade 1 goiter, 130 (46%) had grade 2 goiter, 1 (0.4%) had a thyroidectomy scar, 1 (0.7%) had hoarseness of voice, and tenderness of the swelling 4 (3%) see Table [2](#Tab2){ref-type="table"}. The median urinary iodine secretion of participants with goiter was 152 μg/ml (IQ 101, 197) with mean of 151 and a standard deviation of 72 μg/ml. The median was preferred because the data on the urinary iodine secretion was not normally distributed with the majority of the participants skewed to the higher levels. See Table [3](#Tab3){ref-type="table"}.Table 2**Factors associated with goiter in unity state Southern Sudan, 2012**VariableCategoryFrequencyPercent (%)Thyroid statusNodular27998Multinodular72Previous surgeryYes11No28599Present treatmentNone27496Anti thyroid drugs62Iodine31Sorghum/MaizeYes286100Iodized salt ingestionYes10838No17462\*Ingested iodized salt and had iodine deficiency-259^†^Did not ingest iodized salt and had iodine deficiency-4616Family history of goitreYes62No27697\*2 were moderate and 23 were severe.^†^38 were moderate and 8 were severe.Table 3**Factors associated with goiter (urinary iodine levels as a proxy variable)**VariableF testP valueAge0.4440.506Sex0.0460.830County0.2240.800Duration of stay0.9730.325Family history1.4250.234Community history of goiter1.8451.176Ingestion of iodized salt0.1310.878**Diet**Maize0.4740.492Sorghum1.4450.230Previous history of thyroidectomy0.1690.681Metabolic status of the thyroid0.3130.575 Table [4](#Tab4){ref-type="table"} indicates the magnitude of the problem 75% of the respondents had optimal to excess levels iodine excretion in their urine, deficiency was observed in 25% of the respondents while 7% had moderate to severe iodine deficiency levels. Of the 286 respondents, 279 (98%) had nodular goiter, 274 (96%) had never received any treatment, only one was had prior thyroidectomy done. All the respondents reported sorghum and maize as their predominant food, and 108 (38%) had been ingesting iodized salt. Family history of goiter was reported among 6(2%) of the respondents.Table 4**Comparison of the respondents' results with WHO classification of iodine deficiency in Unity State**WHO ClassificationCut offsFrequencyPercent (%)Severe deficiency\<20114Moderate deficiency20 - 4993Mild deficiency50 - 995118Optimal100 - 19914952More than adequate200 - 2995820Possibly excess\>29983 Table [5](#Tab5){ref-type="table"} shows the comparison of variables among those that consumed iodized salt and those that did not.Table 5**Comparing variables of those who consumed iodized salts vs those that did not consume iodized salt**VariableThose who consumed iodized saltsThose that did not consume iodized saltGenderMale1014Positive family history of goiter23Iodine deficiency-- Severe238Iodine deficiency-- Moderate238Iodine deficiency -- Mild00More than adequate1837Optimal iodine6286Excess iodine35The correlation between goiter size and urinary iodine levels Chi- square 4.461 p = 0.813. 274(96%) had never received any treatment for the goiter.In Figure [1](#Fig1){ref-type="fig"}, a participant with a goiter is shown.Figure 1**Illustration of a participant with a goiter in South Sudan, 2012.** Discussion {#Sec13} ========== We set out to investigate the prevalence and factors associated with iodine deficiency among goiter patients in rural south Sudan. We found that patients with goiters were predominantly female (ratio 11:1). They had this thyroid condition nearly all their lives (mean age of 38 years and duration of symptoms was 35 years). Only 9 out of 286 the participants had received any form of medical attention. This attests to the degree of limitation of access to health care. They were mostly the rural poor, only 38% had access to iodized salt, contrary to Gaffar findings in 2005 \[[@CR4]\] which suggested that households consuming iodized salt in south Sudan had increased from 50% to 70%. However the UIC data in this study indicates that 52% had optimum iodine levels and 25% clearly deficient. These data therefore suggests that nearly half of the study population had sub optimal iodine levels and therefore at risk of thyroid dysfunction. Some neighboring countries in the region such as Uganda and Kenya have over 90% of their populations consuming iodized salt \[[@CR14]\] and it is therefore anticipated that iodine deficiency associated disorders are minimized which would include cretinism among children, goiter formation and hypo or hyper thyroidism. What is also clear from these data is that, some individuals that ingested iodized salt were found to be iodine deficient. Only half of the individuals that did not ingest iodized salt were iodine deficient. Among the reasons for this apparent discrepancy is the fact that deficiency of iodine may be influenced by differences in diet. Assessment of progress in controlling iodine deficiency is shifting from reliance on physical assessment of goiter to biochemistry, using urinary iodine concentrations. While the latter is less subjective, it measures current iodine states (over the last few days before sampling) whereas a physical goiter reflects a considerably longer history. None the less the goiter prevalence does respond and reductions can be observed at least from year to year with increased iodine intakes \[[@CR15]--[@CR17]\]. Trends in goiter prevalence from available repeated national surveys from 31 countries show a consistent picture of almost universal substantial improvement often from 30% to 40% down to single digit percentages over 10 -- 20 years \[[@CR14]\]. Most respondents had stayed with goiters for over 35 years, giving a sense of endemicity of iodine deficiency and limited access to health care. Universal iodized salt program was only introduced in 2005 by the Unity State government \[[@CR18]\]. Over 75% had sufficient iodine using urinary iodine as a marker, the presence of goitres precedes the USI program. Perhaps what we are seeing is a cumulative effect of iodine deficiency of the pre 2005 era. Most participants were women and most likely bore children during the period of iodine deficiency. This is similar to other studies where the ratio is skewed to women \[[@CR14]\]. However enrollment of men was unexpectedly low, perhaps it could be explained by differences in health seeking behavior. The impact of micronutrient deficiency is immense and such communities carry a heavy burden of it. The consequences of iodine deficiency include but not limited to goiter formation, thyroiditis and cretinism in children \[[@CR19]--[@CR21]\]. It may be worthwhile investigating iodine disorders among children and pregnant women in such communities and the barriers to control or elimination of iodine deficiency \[[@CR21], [@CR22]\]. The staple foods consumed were sorghum and maize. In the absence of iodized salt, they are unlikely to offer sufficient iodine intake \[[@CR23]\]. The challenges of war and political upheaval cannot be under estimated in contributing to inadequate access to health care and lack of public health interventions including assessing micronutrient deficiency and the impact of interventions in place \[[@CR9]\]. Study limitations {#Sec14} ----------------- Even though it was community based, it was not a house to house survey. We depended on participants responding to radio announcements. It is possible that many may not have turned up. However this method of mobilization is not new in the area, it has successfully been used before for child immunization campaigns. Ultra-sonography has been used in epidemiological studies to assess thyroid size, leading to much higher estimates of goiter prevalence than in studies in which goiter size was assessed by physician examination \[[@CR24]\]. Therefore it is likely that this study under estimates it's prevalence in the population investigated. In this study, we used urinary iodine excretion as a proxy to body iodine. It is a limitation in a way that it may not reflect iodine status of a participant in the past. Conclusion {#Sec15} ========== In the population studied, the percentage of participants with median urinary iodine between 100-200 micro g/l indicating adequate intake and optimal nutrition was 52% leaving out nearly half of the population. Goiter was prevalent among the rural poor mostly women. Access to iodized salt for the rural poor in the underdeserved areas of south Sudan and sub Saharan Africa needs to be urgently improved. **Competing interests** The authors declare that they have no competing interests. **Authors' contributions** CCC originated the concept, collected data. GM wrote the first draft. GM, IC, FJO, KN performed critical reviews for intellectual content. All authors approved of the final manuscript. Prof. James Tumwine (Makerere University), General Taban Deng Gai, the Governor, Unity State authorities; Hon. Dr. Chenkel and Dr. Manong of the Ministry of Health, staff of Bentiu hospital, Rubkona military Hospital, Koch Hospital and Guit county PHCU staff, Mr.William Garjang of Rubkona County and James Pui of Guit county.