Arum maculatum is particularly well-known for its potent dung-like volatile organic compound emissions and specialized floral chamber, which traps pollinators—mainly Psychoda phalaenoides and Psychoda grisescens—overnight. However, little is known about the genes underlying the production of many Arum maculatum volatile organic compounds, and their influence on variation in pollinator attraction rates. Therefore, we performed de novo transcriptome sequencing of Arum maculatum appendix and male floret tissue collected during anthesis and postanthesis, from 10 natural populations across Europe. These RNA-seq data were paired with gas chromatography–mass spectrometry analyses of floral scent composition and pollinator data collected from the same inflorescences. Differential expression analyses revealed candidate transcripts in appendix tissue linked to malodourous volatile organic compounds including indole, p-cresol, and 2-heptanone. In addition, we found that terpene synthase expression in male floret tissue during anthesis significantly covaried with sex- and species-specific attraction of Psychoda phalaenoides and Psychoda grisescens. Taken together, our results provide the first insights into molecular mechanisms underlying pollinator attraction patterns in Arum maculatum and highlight floral chamber sesquiterpene (e.g. bicyclogermacrene) synthases as interesting candidate genes for further study.Deceptive pollination often involves volatile organic compound emissions that mislead insects into performing nonrewarding pollination. Among deceptively pollinated plants,  Angiosperms have evolved to produce a wide array of specialized metabolites to mediate their interactions with other organisms and their environment, including volatile organic compounds (VOCs) emitted from flowers, leaves, fruits, and roots. While VOCs represent a relatively small proportion of all plant metabolomic diversity , they plArum maculatum L. (Araceae).Pollinator-mediated selection is known to influence floral morphology  and coloA. maculatum is a common European woodland flower with a long history of study, due to its deceptive pollination strategy which involves olfactory deception through brood-site mimicry  during anthesis which are also present in cow manure  grisescens (Tonnoir) , which is surrounded by a leaf-like bract (the spathe). The spathe surrounds the fertile flowers, creating a basal trap chamber with only a narrow opening at the top of the chamber, which is surrounded by a ring of hair-like sterile flowers. The scent of the floral chamber during anthesis appears to be dominated by 1 sesquiterpene, bicyclogermacrene  and not terpenes, which form a proportionally larger part of the floral scent bouquet in A. maculatum  use these results to identify differentially expressed transcripts putatively related to VOC biosynthesis, and (3) to characterize and compare differential expression in these transcripts across Europe, with a focus on populations with divergent pollinator attraction patterns. We predicted that during anthesis, AAA metabolism would be highly expressed in appendix tissue, while sesquiterpene synthases would be highly expressed in male floret tissue. We further expected to observe differential expression of transcripts associated with AAA metabolism and TPSs in inflorescences with dung-like  vs. sesquiterpene-dominated floral bouquets, respectively. Finally, given that the A. maculatum population in Forêt du Gâvre, France, appears to attract almost exclusively P. grisescens  investigate variation in A. maculatum between April and May 2019 , and pumping air over them for 30 min at an air flow rate of 200 ml/min. All samples were analyzed using a gas chromatography (Agilent 7890a) mass spectrometry (Agilent 5975c) system. Desorption of the VOCs was carried out in a thermal desorption unit  with an initial temperature of 50°C, followed by a ramp of 60°C/min until 250°C (followed by a 3.5-min hold time). Before injection, the VOCs were cryo-focused  at −80°C, then released with a ramp of 12°C/min until 260°C (hold time 6 min). PTV solvent vent at 14 PSI and 50 ml/min flow was used for injection into a HP5-MS column in constant flow mode (0.9 ml/min) with helium as the carrier gas. The Mass Selective Detector (MSD) transfer line was set at 280°C. Scan mode was used for the MS acquisition with a solvent delay (1 min), over a mass range of 33–300 m/z; MS source and quadrupole temperatures were set at 230 and 150°C, respectively. Electron ionization (70 eV) was used. Major ions were recorded for each integrated peak using Agilent Chemstation, and compound identifications were derived from NIST 2.3 (library v.17) and published Kovats retention indices; all names used in our analyses should therefore be considered hypotheses. Appendix temperatures were recorded at the time of VOC sampling using FLIR thermographic imaging, and ambient air temperature was recorded using a thermistor.Before collecting tissue samples, we noninvasively sampled the dynamic headspace VOCs emitted by each Immediately following VOC sampling (i.e. during anthesis), we used sterile scalpel blades to collect 1-mm thick slices of appendix and/or male floret tissue. As a control, male floret tissue samples were also collected from inflorescences the following morning, after pollinator attraction had ended. All samples were individually preserved in RNAlater (Thermo Fischer Scientific) and stored at −80°C until extraction. After collecting each during-anthesis male floret tissue sample, we closed and sealed the small window we cut in floral chamber, so that pollinator trapping was not affected. During the morning following anthesis, we collected all insects trapped within inflorescences, preserved them in 70% ethanol and identified them to at least the suborder level. Psychodidae were further identified to species level (including sex) following taxonomic descriptions and illustrations .All samples and tissue types were extracted using identical methods; approximately 100 mg of tissue was rinsed in sterile RNase-free water, flash frozen in liquid nitrogen, powdered in a QIAGEN TissueLyser II, and immediately extracted using a QIAGEN RNeasy Plant Mini Kit, following the manufacturer’s protocol. Extraction quality and concentration were verified using an Agilent Fragment Analyzer. Three Illumina TruSeq Stranded mRNA polyA libraries were prepared from these samples, following the manufacturer’s recommended protocol. The first library contained the appendix RNA samples, and the second and third each contained a mix of during anthesis and postanthesis male floret tissue samples from all populations. Each library was then sequenced on its own lane  using an Illumina HiSeq 4000 (150-bp paired-end sequencing) at the Lausanne Genomic Technologies Facility (Switzerland).FilterUncorrectablePEfastq.py (https://github.com/harvardinformatics/TranscriptomeAssemblyTools).We performed an initial quality filtering of all raw read files using fastp  to removA. maculatum transcriptome  using Trinity v2.11.0  to identify candidate coding regions and searches the OrthoDB, Pfam-A, Rfam, and uniref90 protein databases for transcript annotations, with an E-value threshold of 1 × 10−5. Unannotated transcripts were not removed from the final A. maculatum reference transcriptome. We also uploaded the EvidentialGene output to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server  term analysis with the R package TopGO  and male floret (n = 16) tissue collected during anthesis . Second, we compared male floret tissue collected during anthesis (n = 14) against their paired control samples from the morning following anthesis (n = 14). Paired control samples were not collected for 2 inflorescences from Sokobanja, Serbia; these individuals were excluded from this analysis. Finally, we compared transcript expression in during-anthesis male floret tissue sampled in the Forêt du Gâvre population against all other populations, to identify transcripts putatively linked to this population’s exclusive attraction of P. grisescens. To account for some of the effects of isolation by distance, we further split this analysis along the 2 main neutral genetic clusters (n = 5) against during-anthesis male floret tissue from (1) France, Switzerland, and Italy (n = 7) and (2) Serbia (n = 4).We performed 3 sets of differential expression analyses, each addressing one of the main aims of our study. Our first comparison was between appendix  our complete reference transcriptome and (2) sets of significantly differentially expressed genes. Since the genes responsible for the production of 2-heptanone and p-cresol in A. maculatum are unclear and unknown respectively, we also used homology searches to identify additional candidate genes. Here, we performed BLASTp searches of genes known involved in the biosynthesis of the aforementioned VOCs in other plants and bacteria, against a database of all of our Transdecoder predicted peptide sequences using, with an e-value cutoff of 1 × 10−5. After all candidate transcripts were identified, we visualized variation in their average expression across all tissue types and populations using the R package pheatmap , performed a multiple sequence alignment of these sequences using Clustal Omega . Then, weMemoire , and adeBetween 27,933,352 and 66,825,898  paired-end reads from individual tissue samples passed all quality filtering steps . These wA total of 49,779 transcripts remained in our expression matrix following prefiltering in DESeq2 (i.e. >0.75 CPM in at least 7 samples). Principal components analysis of filtered transcript expression  revealedP-values <0.05; log2fold change ±1), after controlling for population effects. Furthermore, we found that VOC biosynthetic activity is significantly elevated in A. maculatum appendix tissue during anthesis. Many major biosynthetic pathways  were significantly enriched in the appendix tissue during anthesis, whereas transcripts related to pollen production were significantly enriched in male floret tissue  revealed 8,683 transcripts with significantly greater expression in appendix tissue and 6,581 transcripts with significantly greater expression in male floret tissue . While tryptophan (i.e. indole) biosynthesis was elevated in male florets during anthesis, we did not identify increased expression of other putative VOC biosynthetic pathways  and (2) 327 and 175 transcripts were, respectively, differentially expressed in Forêt du Gâvre vs. Serbian populations . However, no transcripts putatively linked to VOC biosynthesis were identified among these transcripts.Our third and final set of differential expression analyses, comparing male floret tissue during anthesis among populations, revealed that (1) 84 and 9 transcripts were, respectively, differentially expressed in Forêt du Gâvre vs. all other French, Swiss, and northern Italian populations  subunit, which catalyzes the conversion of indole-3-glycerolphosphate to indole.Consistent with recent large-scale surveys of al scent . We furtr = 0.988, P < 0.001). This enzyme may further catalyze the conversion of 4-hydroxyphenyllactate to p-coumaric acid. However, we did not identify any homologs of bacterial proteins linked to the production of 4-hydroxyphenylacetate and/or p-cresol  and hydroxyphenylpyruvate reductase (HPPR). Pearson correlation tests further identified several transcripts correlated with HPPR expression in appendix tissue during anthesis ; notablyp-coumaric acid—appear to be produced by A. maculatum, transcripts responsible for the production of benzenoid and phenylpropanoid VOCs generally appeared to be absent or weakly expressed in A. maculatum. Finally, we identified putative homologs of methylketone synthases (MKS1 and MKS2), which are known to be involved in 2-heptanone biosynthesis  compounds—particularly A. maculatum transcripts putatively encoding all proteins in both the mevalonate and MEP pathways. During anthesis, the MEP pathway appears to be more highly expressed than the mevalonate pathway, particularly in appendix tissue (d-xylulose-5-phosphate synthase (DXS), which was highly expressed in appendix tissue, also featured the greatest isoform diversity among all terpene backbone synthesis genes. Furthermore, we identified 108 putative TPS transcripts expressed in appendix and male floret tissue during anthesis. A phylogeny of all putative A. maculatum TPSs is given in We identified x tissue . The firA. maculatum VOCs (e.g. humulenes and germacrenes); others, such as 2-methylisoborneol (2-MIB) synthase, catalyze the production of terpenes not previously described in A. maculatum. However, in the case of the latter, the transcript we identified likely encodes a novel TPS, given that we identified an uncharacterized Colocasia esculenta (Araceae) gene, which shares 92.5% identity with our transcript, as opposed to approximately 22.8% identity with the bacterial 2-MIB synthase  in appendix tissue samples emitting high quantities of an unnamed sesquiterpene with a nonpolar Kovats Retention Index of 1681, which is known to be a strong predictor of P. grisescens attraction . However, we did identify significant concordance between putative TPS expression in male floret tissue and Psychodidae pollinator composition trapped within inflorescences ; the first coinertia axis split TPS expression based on species , while the second coinertia axis split TPS expression based on sex  via a pathway homologous to tyrosine degradation in bacteria  has also been reported in p-coumaric acid from phenylalanine, where also abundantly expressed in the appendix during anthesis. However, downstream proteins producing common FBP VOCs  would further modify p-coumaric acid into precursors for the aforementioned FBP VOCs, but this gene was expressed at relatively low levels in appendix tissue. Interestingly, experimental knock-outs of C3H in Petunia × hybrida have demonstrated that downregulation of this gene also leads to unexpected production of p-cresol  and cinnamate 4-hydroxylase (C4H), which produce A. maculatum VOC with a “fermented” scent. In this study, we were able to identify several methylketone synthase homologs (MKS1 and MKS2), which produce 2-heptanone and related compounds in tomato plants , which can further modify volatile terpenes through oxidation, methylation, or acylation  homolog appears to be correlated with the production of at least 1 unnamed A. maculatum sesquiterpene (Kovats RI 1681). This unnamed sesquiterpene was the single strongest predictor of P. grisescens attraction in a large-scale survey of A. maculatum pollinators  to attract females . Such terpene emissions in the floral chamber could stimulate the movement of trapped Psychodidae over the male and female florets, aiding in pollination and pollen dispersal; however, further experiments are needed to test these hypotheses. A recent gas chromatography–electroantennography , and the aforementioned trimethyltridecatetraene synthase homolog, which may produce an unnamed sesquiterpene with a Kovats Retention Index of 1681 (covarying with P. grisescens). Notably, these same compounds were highlighted as key predictors of pollinator species trapped by A. maculatum inflorescences in a random forest analysis with a larger sample size . Our results highlight a need for further focused study of VOCs within the trap chamber, given that specific sesquiterpenes appear to be an important aspect of this highly specialized lure-and-trap pollination system.Our data provide a deeper understanding of the relationships between transcript expression, floral scent, and pollinator attraction in https://www.ncbi.nlm.nih.gov/bioproject/PRJNA856436). Additional data, including the files and R code needed to reproduce all analyses and figures, have been archived in the Zenodo repository 6806366 (https://doi.org/10.5281/zenodo.6806365).G3 online.jkac175_Appendix_S1Click here for additional data file.jkac175_Appendix_S2Click here for additional data file.


The use of tumor markers aids in the early detection of cancer recurrence and prognosis. There is a hope that they might also be useful in screening tests for the early detection of cancer. Here, the question of finding ideal tumor markers, which should be sensitive, specific, and reliable, is an acute issue. Human plasma is one of the most popular samples as it is commonly collected in the clinic and provides noninvasive, rapid analysis for any type of disease including cancer. Many efforts have been applied in searching for “ideal” tumor markers, digging very deep into plasma proteomes. The situation in this area can be improved in two ways—by attempting to find an ideal single tumor marker or by generating panels of different markers. In both cases, proteomics certainly plays a major role. There is a line of evidence that the most abundant, so-called “classical plasma proteins”, may be used to generate a tumor biomarker profile. To be comprehensive these profiles should have information not only about protein levels but also proteoform distribution for each protein. Initially, the profile of these proteins in norm should be generated. In our work, we collected bibliographic information about the connection of cancers with levels of “classical plasma proteins”. Additionally, we presented the proteoform profiles (2DE patterns) of these proteins in norm generated by two-dimensional electrophoresis with mass spectrometry and immunodetection. As a next step, similar profiles representing protein perturbations in plasma produced in the case of different cancers will be generated. Additionally, based on this information, different test systems can be developed. In a broad sense, tumor biomarkers are components that are either produced directly or indirectly because of a tumor. Moreover, these biomarkers can be common cellular products that are overproduced by cancer cells or the products of genes that are expressed only during malignant transformation. Thus, a tumor marker that is present in significant quantities indicates the presence of cancer. The marker can be present inside the tumor or enter the bloodstream ,2. This https://www.hupo.org/plasma-proteome-project accessed on 10 September 2022). Now, this initiative has achieved great success in plasma protein analysis (http://plasmaproteomedatabase.org/index.html accessed on 10 September 2022)  th th265] ter 2022) . Accordihttps://glyconnect.expasy.org/browser/proteins/780 accessed on 10 September 2022). In SWISS-2DPAGE, alpha-1-B glycoprotein is represented as a chain of six spots  [The two-dimensional electrophoresis pattern of alpha-1-B glycoprotein represents a long chain of spots in the pI-range 4–6 and Mw ~ 54,000  that is https://world-2dpage.expasy.org/ accessed on 10 September 2022 [https://glygen.org/protein/P01023#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of alpha-2-M represents a chain of heavy Mw spots (mostly in the pI-range 5.8–6.3) . This pahttps://glygen.org/protein/P08697#glycosylation accessed on 10 September 2022). In SWISS-2DPAGE, alpha-2-antiplasmin is represented as a chain of seven spots .The two-dimensional electrophoresis pattern of α2AP represents a chain of spots in the pI-range 4–6 with Mw ~ 50,000  that is https://world-2dpage.expasy.org/ accessed on 10 September 2022 and has a characteristic for multiple glycosylation profiles, where acidic spots have higher Mw [https://www.uniprot.org/uniprot/P02750 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of LRG1 represents a chain of spots in the pI-range 3.5–5.0 with Mw ~ 40,000–50,000 . This pahttps://glygen.org/protein/P01011#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ACT represents a chain of spots in the pI-range 4.0–5.0 and Mw 50–60,000 . This pahttps://glygen.org/protein/Q15848#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of adiponectin represents a chain of spots in the pI-range 5.0–5.5 and Mw 26,000 . There aThe two-dimensional electrophoresis pattern of adiponectin represents a chain of spots in the pI-range 4.5–6.0 and Mw ~ 70,000 . There ahttps://glygen.org/protein/P02768#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of albumin represents a chain of spots in the pI-range 5.5–6.5 and Mw ~ 70,000 . Its pathttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of AMBP represents two groups of spots in the pI-range 4.0–6.5: alpha-1-microglobulin with Mw ~ 26,000 and inter-alpha-trypsin inhibitor light chain/bikunin that is assembled in a high Mw complex by a chondroitin-like glycosaminoglycan (GAG) cross-linking with Mw 120,000 . In the https://glygen.org/protein/P01019#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of angiotensinogen represents a chain of spots in the pI-range 4.0–6.4 and Mw ~ 50,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ATIII represents a chain of eight spots in the pI-range 4.5–6.0 and Mw ~ 50,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoA-I represents a chain of spots in the pI-range 4.5–6.5 and Mw ~ 26,000 . In the The two-dimensional electrophoresis pattern of apoA-II represents a chain of spots in the pI-range 4.5–6.0 and Mw ~ 9000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoA-IV represents a chain of spots in the pI-range 4.5–6.0 and Mw ~ 40,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo B-100 represents a long chain of spots in the pI-range 3.5–7.5 and heavy Mw > 120,000 . Apo B-1https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo-CI represents a chain of spots in the pI-range 7.8–8.5 and Mw ~ 6000 . This prhttps://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated (3 O-linked glycans at 4 sites) (https://www.glygen.org/protein/P02655 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo-CII represents a chain of spots in the pI-range 4.8–5.2 and Mw ~ 8000 . In the https://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated (4 O-linked glycans at 1 site) (https://www.glygen.org/protein/P02655 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoC-III represents a chain of spots in the pI-range 3.8–6.1 and Mw ~ 9000 . In the https://www.uniprot.org/uniprotkb/P05090/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoD represents an unusual set of spots in the pI-range 3.5–6.5 and Mw from ~15,000 to ~26,000 and 80,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoE represents a chain of spots in the pI-range 4.5–6.5 and Mw ~ 35,000 . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo-F represents a set of spots in the pI-range 3.5–4.2 and Mw from ~15,000 to ~32,000 . ApoF cahttps://glygen.org/protein/P02749#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apo-H represents a chain of spots in the pI-range 6.2–8.4 and Mw ~ 52,000 , which ihttps://glygen.org/protein/O95445#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of apoM represents a chain of spots in the pI-range 4.5–6.5 and Mw ~ 22,000 . There aThe results of several studies suggest that changes in the complement system can not only promote an antitumor response but can also influence tumor development through proliferation, survival, angiogenesis, and invasiveness ,268. Thehttps://glygen.org/protein/P00736#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern represents only a chain of spots of the complement C1r subcomponent in the pI-range 4.5–6.2 and Mw ~ 80,000  that corhttps://glygen.org/protein/P09871#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis-pattern represents a chain of five spots of C1s in the pI-range from 4.0 to 4.9 and Mw ~ 80,000 . The clehttps://glygen.org/protein/P02747#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of C1q represents a long horizontal chain of spots in the pI-range 3.0–9.5 with Mw ~ 23,000 and a vertical chain of heavy complexes  with pI ~ 9.0 . It was https://glygen.org/protein/P05156#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement factor I represents the chains of many spots in the pI-range 4.5–6.8 from Mw ~ 64,000 (complement factor I) to Mw ~ 30,000 (the complement factor I heavy and light chains) . In the https://glygen.org/protein/P00751#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement factor B represents the chains of spots in the pI-range 4.5–6.8 with Mw ~ 90,000 . The clehttps://www.phosphosite.org accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement factor D represents two spots  . The prohttps://glygen.org/protein/P08603#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement factor D represents a long chain of spots in the pI-range 5.5–7 with Mw ~ 140,000 . It was https://glygen.org/protein/P06681#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C2 represents a chain of spots in the pI-range 6–7 with Mw ~ 80,000 . It was https://glygen.org/protein/P01024#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C3 represents a cluster of spots in the pI-range 3.5–7.5 and Mw from ~30,000 to 180,000 . In the https://glygen.org/protein/P0C0L4#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C4-A represents a wide cluster of spots in the pI-range 3.0–10.0 and Mw from ~25,000 to 190,000 . There ahttps://glygen.org/protein/P0C0L5#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C4-B represents a wide cluster of spots in the pI-range 3.0–10.0 and Mw from ~35,000 to 19,0000 . In the https://glygen.org/protein/P01031#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C4-B represents a wide cluster of spots in the pI-range 5.0–6.8 and Mw ~ 70,000–190,000 . It was https://glygen.org/protein/P13671#glycosylation accessed on 10 September 2022), and 5 sites of phosphorylation (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C6 represents a chain of spots in the pI-range 4.0–6.5 and Mw ~ 100,000 . It was https://glygen.org/protein/P10643#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C7 represents a chain of spots in the pI-range 4.5–6.5 and Mw ~ 100,000 . It was https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of the complement C9 represents a chain of spots in the pI-range 4.5–5.5 and Mw ~ 60,000 . The prohttps://glygen.org/protein/P00915#glycosylation accessed on 10 September 2022), phosphorylation (11 sites), acetylation (5 sites), and ubiquitylation (2 sites) (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of CAB represents a chain of spots in the pI-range 5–7 and Mw ~ 28,000 . It was https://glygen.org/protein/P08185#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of CBG represents a chain of spots in the pI-range 3.7–5.1 and Mw ~ 50,000 . It was https://glygen.org/protein/P08185#glycosylation accessed on 10 September 2022.The two-dimensional electrophoresis pattern of CBPN represents a chain of spots in the pI-range 4.5–7 and Mw ~ 50,000 . It was https://glygen.org/protein/P08571#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of CD14 represents a chain of spots in the pI-range 4.5–5.8 and Mw ~ 40,000 . It was https://glygen.org/protein/P00450#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ceruloplasmin represents a chain of spots in the pI-range 4.0–6.2 and Mw ~ 120,000 . In the https://glygen.org/protein/P06276#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of cholinesterase represents a chain of five spots in the pI-range 4.5–5.2 and Mw ~ 65,000 . There ahttps://glygen.org/protein/P10909#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ceruloplasmin represents a chain of 18 spots in the pI-range 4.5–6.5 and Mw ~ 35,000 . In the https://glygen.org/protein/Q96KN2#glycosylation accessed on 10 September 2022).The two-dimensional electrophoresis pattern of beta-Ala-His dipeptidase represents two spots around pI 5.0 and Mw ~ 54,000 . It was https://www.glygen.org/protein/P22792 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of carboxypeptidase N subunit 2 represents nine spots with pI 3.5–5.5 and Mw ~ 65,000 . It is khttps://www.uniprot.org/uniprotkb/P02741/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of CRP represents a single spot  . In the https://www.glygen.org/protein/Q16610 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of ECM1 represents a spot  . It was https://www.glygen.org/protein/P23142 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of FIBL-1 represents a chain of four spots  . It was https://www.glygen.org/protein/O75636 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of ficolin-3 represents a chain of five spots  . It was https://www.glygen.org/protein/P02765 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of fetuin-A represents a set of proteoforms  . In the https://www.glygen.org/protein/Q9UGM5 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of fetuin-B represents a chain of proteoforms  . The prohttps://www.glygen.org/protein/P02671 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of FBA represents several sets of chains with pI 5.0–7.5  . In the https://www.glygen.org/protein/P02675 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of FBB represents a chain of spots  . In the https://www.glygen.org/protein/P02679 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of FGG represents a chain of spots with pI 4.5–7  . In the https://www.glygen.org/protein/P02751 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of fibronectin represents a chain of spots with pI 4.5–6.7  . The prohttps://www.phosphosite.org accessed on 10 September 2022).The two-dimensional electrophoresis pattern of gelsolin represents a chain of spots with pI 4.5–6.5  . The prohttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots with pI 4.9–6.9  . It is phttps://www.glygen.org/protein/P69905 accessed on 10 September 2022), phosphorylated (17 sites), acetylated (4 sites), and ubiquitinated (8 sites) (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots with pI 7.5–9  . In the https://www.glygen.org/protein/P68871 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of this protein represents a chain of spots with pI 6.5–6.9  . In the https://www.glygen.org/protein/P02790 accessed on 10 September 2022)).The two-dimensional electrophoresis pattern of this protein represents a chain of spots with pI 5–6.9  . In the https://www.glygen.org/protein/P05546 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of this protein represents a chain of spots with pI 4.9–6.5  . The prohttps://www.glygen.org/protein/P00738 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of Hp represents ~16 spots of beta chains with pI 4.8–6.0  and 3 spots of alpha 2 chain . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots with pI 4.6–6.5  . The prohttps://www.glygen.org/protein/P04196 accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of this protein represents a chain of spots with pI 4.5–7.8  and spots around pI/Mw: 5.5/53,000 . In the https://www.glygen.org/protein/P05155 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a long chain of spots with pI 3.2–5.2 . The protein is heavily glycosylated  (https://www.phosphosite.org/ accessed on 10 September 2022).In our experiments, 2DE patterns of these proteins are presented by the chains of the precursor proteoforms and the mature ITIH1 . The prohttps://www.glygen.org/protein/P29622 accessed on 10 September 2022, https://www.phosphosite.org/ accessed on 10 September 2022.In our experiments, the 2DE pattern of this protein is presented as a cluster of proteoforms around pI/Mw: 6–7/40–120,000 . The prohttps://www.glygen.org/protein/P01042 accessed on 10 September 2022), phosphorylated, acetylated, and ubiquitinated (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents multiple spots  . In the https://www.glygen.org/protein/P04180 accessed on 10 September 2022), phosphorylated, and ubiquitinated (https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents three spots  . The prohttps://www.uniprot.org/uniprotkb/P51884/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents chains of spots  . The proThe two-dimensional electrophoresis pattern of this protein represents just a single spot  .https://www.uniprot.org/uniprotkb/P36955/entry accessed on 10 September 2022).The two-dimensional electrophoresis-pattern of this protein represents a chain of spots  . The prohttps://www.uniprot.org/uniprotkb/Q96PD5/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . The prohttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . The prohttps://www.uniprot.org/uniprotkb/P00747/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents two chains of multiple spots  and  . In the https://www.uniprot.org/uniprotkb/P27169/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the The two-dimensional electrophoresis pattern of this protein represents two spots  . The prohttps://www.uniprot.org/uniprotkb/P07225/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . The prohttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents two spots  . This prhttps://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated  (https://www.glygen.org/protein/P02743 accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . This prhttps://www.phosphosite.org/ accessed on 10 September 2022) and glycosylated .The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . This prhttps://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated (1 O-linked glycan) (https://www.uniprot.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents one spot  . Howeverhttps://www.phosphosite.org/ accessed on 10 September 2022), and glycosylated (1 O-linked glycan) (https://www.uniprot.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of S100-A9 represents one spot  . This prhttps://www.uniprot.org/uniprotkb/P05452/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . This prhttps://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . This prhttps://www.uniprot.org/uniprotkb/P00734/entry accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a chain of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022). The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the https://www.phosphosite.org/ accessed on 10 September 2022).The two-dimensional electrophoresis pattern of this protein represents a cluster of spots  . In the Tumorigenesis leads to multiple variations in the human plasma proteome that can be dynamic and alterable during the progress of the disease. Practically all major, so-called “classical”, plasma proteins change abundances or PTMs. The majority of these proteins are secreted by the liver, so it could be anticipated to see these changes only in the case of liver cancer. However, they can be observed with other tumors as well as cancer induces disturbances in the blood homeostasis that is supported by “classical plasma proteins”. It follows that it is possible to search the specific/unspecific ways of tumor prediction not only through the detection of products of the tumor but also by analyzing the changes in “classic plasma proteins”. It is relevant to mention that plasma analysis by a very different approach, differential scanning calorimetry (DSC), can give us a hint. Typically, DSC is used to determine the partial heat capacity of macromolecules as a function of temperature, from which their structural stability during thermal denaturation can be assessed. The method is very sensitive and allows precise determination of thermally-induced conformational transitions of proteins present in plasma. There are already quite a few publications showing that DSC can be used to distinguish between normal and cancerous plasma samples ,274. Morhttps://vermillion.com/ova-products accessed on 10 September 2022). The observation of enhanced levels of clusterin, ITIH4, antithrombin-III, and C1RL in sera of endometrial cancer patients allowed a mathematical model to be built to detect cancer samples [It follows that there is a possibility of building test systems based on these major  proteins. What is important is that many examples of such systems have been introduced already. For example, the relationship between inflammation and clinical outcome is described using the Modified Glasgow Prognostic Scale (mGPS), which includes levels of C-reactive protein (CRP) and albumin . The com samples . Accordi samples . The dec samples , NSCLC [ samples , nasopha samples , esophag samples , and BC  samples , but it  samples . The lev samples ,87 but i samples ,89. A si samples .Another aspect that should be considered is the appearance of proteoforms produced by genetic polymorphisms, alternative splicing, PTMs, etc. These events change the charge (pI) and the weight (Mw) of the protein. Because of that, the experimental pI/Mw of the proteins can be different from the theoretical ones. This leads to the production of sets of proteoforms that in our case are detected as 2DE patterns. There is a belief that some 2DE patterns can be different between norm and cancer and could be used as specific biomarkers. Thus far, there are not many such examples, but progress in proteomics methods should improve the situation ,278. Prohttp://2de-pattern.pnpi.nrcki.ru/ accessed on 10 September 2022 [Our aim is to build a comprehensive proteoform database containing norm and cancer samples ber 2022 . GlioblaThe pooled human plasma was from healthy male donors (age 20–47 years) ,279. Depv/v) ampholytes, pH 3–10, protease inhibitor cocktail) and then with 100 μL of rehydrating buffer  buffer, pH 3–11 NL, 0.001% bromophenol blue). Immobiline DryStrip 3–11 NL (7 cm) was passively rehydrated by this solution for 4 h at 4 °C. IEF was run on Hoefer™ IEF100 . After IEF, strips were incubated 10 min in the equilibration solution  glycerol, 1% DTT), following in the same solution with 5% IAM instead of DTT. The strips were sealed with a hot solution of 0.5% agarose prepared in electrode buffer  on top of the polyacrylamide gel (14%), and run in the second direction [2) was shredded and treated with trypsin. Tryptic peptides were eluted from the gel by extraction solution (5% (v/v) ACN, 5% (v/v) formic acid) and dried in Speed Vac. In the case of a semi-virtual 2DE, the 18-cm Immobiline DryStrip 3–11 NL was cut into 36 equal sections after IEF. For complete reduction, 300 μL of 3 mM DTT and 100 mM ammonium bicarbonate were added to each section and incubated at 50 °C for 15 min. For alkylation, 20 μL of 100 mM IAM were added and samples were incubated in the dark at r.t. for 15 min. The peptides were eluted with 60% acetonitrile and 0.1% TFA and dried in Speed Vac.The detailed process was described previously . In shorirection . Gels stv/v) formic acid. Tandem mass spectrometry analysis was conducted in duplicate on an Orbitrap Q-Exactive mass spectrometer . The data were analyzed by Mascot “2.4.1”  or SearchGui [A detailed procedure was described previously ,280. PepearchGui  using thOnly 100% confident results of protein identification were selected. Two unique peptides per protein were required for all protein identifications. Exponentially modified PAI (emPAI), the exponential form of protein abundance index (PAI) defined as the number of identified peptides divided by the number of theoretically observable tryptic peptides for each protein, was used to estimate protein abundance .w/v) BSA) or rabbit polyclonal anti-Hp . Secondary goat anti-mouse immunoglobulins G labeled by horseradish peroxidase  were used in TBS containing 3% (w/v) nonfat dry milk (1/5000 dilution). The reaction was developed using ECL  and X-ray film (Amersham Hyper film ECL).Plasma proteins (0.5 mg) were run by 2DE . Proteins were transferred  from the gel onto PVDF membrane  using two sheets of thick paper , saturated with 48 mM Tris, 39 mM glycine, 0.037% SDS, 20% ethanol. The membrane was treated following a protocol of Blue Dry Western  and treahttp://plasmaproteomedatabase.org/index.html accessed on 10 September 2022 [For now, proteomics is collecting big data about the human plasma proteome ber 2022 . These dber 2022 ,285. In


Total hip arthroplasty is a widely performed surgical procedure, which enables patients to regain mobility, alleviates pain, and improves overall quality of life. Periarticular multimodal drug infiltration (PAI) is increasingly being used as an effective postoperative pain management, decreasing the systemic consumption of opioids. Extensive postoperative skin necrosis without a deep joint infection as a complication of total hip arthroplasty with PAI has not yet been described.A 71-year-old patient who underwent total hip arthroplasty of the right hip for primary osteoarthritis through the Direct Anterior Approach presented postoperatively a large area of necrotic skin at the incision. Joint infection was excluded. An extensive debridement was performed and the tissue defect was reconstructed by a pedicled anterolateral thigh flap. The skin maintained a satisfactory appearance at 1 year postoperatively, and the hip was pain-free with restored ranges of motion. The patient was able to walk with no support and without limitation.We address the possible risk factors, discuss the use of epinephrine in PAI and explore possible treatment options for such a complication.The online version contains supplementary material available at 10.1186/s12891-023-06643-z. Total hip arthroplasty (THA) is a common surgical treatment which provides reliable functional outcomes in patients suffering from hip osteoarthritis. Effective postoperative pain control is most important to enable prompt rehabilitation, reduce postoperative complications, and reduce the length of hospital stay. Pain control is achieved by several methods, including systemic treatments with Non-Steroidal Anti-inflammatory Drugs, opioids, and regional treatments such as epidural anaesthesia, peripheral nerve blocks, and periarticular multimodal drug infiltration (PAI). Up until recently, the efficacy of PAI in pain relief following total hip arthroplasty during rest and activity, and its impact on the length of hospital stay and total opioid consumption was not fully established. A recent meta-analysis concluded that PAI showed better pain relief and less opioid consumption and is safe and effective following total hip arthroplasty . HoweverThis case is presented in accordance with the 2013 CARE checklist.A 71-year-old male, non-smoker, with a previous history of cardiac ischemia was admitted for a THA. The patient was positioned in the supine position on a traction table. Surgery was performed under general anaesthesia through the Direct Anterior Approach. The patient received a fully hydroxyapatite-coated uncemented stem , an uncemented cup , and a ceramic-highly-crosslinked polyethylene bearing surface Fig. . The patConsidering the presence of a large skin necrosis and the high risk of infection of the underlying hip joint, we decided to perform a single-staged surgical debridement and reconstruction of the skin and soft tissue defect using a pedicled fasciocutaneous anterolateral thigh flap (ALT) of about 15 × 6 cm Fig. ; video. Wound complications after joint replacement can result in significant morbidity to the patient with increased risk on infections, further surgeries, increased recovery period and hospital stay . Wound cInterestingly, in our patient, the skin necrosis was first noted on day 8 postoperatively. The dressings had been changed at day 3 and day 6 and a clean wound with no oozing or redness was described. In other reported cases of skin necrosis following local anaesthetics infiltration with epinephrine, skin changes only appeared around day 5. This difference could be due to the site of injection, which was the penoscrotal junction in one case , and theBased on expertise from our department of infectious diseases, we excluded the possibility of acute periprosthetic joint infection as the bacterial culture of synovial fluid was negative with a WBC count of 4650 cells/µl (< 10 000 cells/µl) in the joint fluid . A broadMalnutrition compromises wound healing as decreased protein reserves affects collagen synthesis and fibroblast proliferation and decreased albumin levels promotes tissue oedema formation . It has PAI is a multimodal drug solution comprised of three active agents, long-acting local anaesthetics such as ropivacaine and bupivacaine, nonsteroidal anti-inflammatory drugs (NSAIDs) and epinephrine. Local anaesthetics block voltage-gated sodium channels in nociceptors, thereby decreasing pain transmission. Injection of NSAIDs such as ketorolac reduces pain by inhibiting the production of proinflammatory mediators such as prostaglandins. Epinephrine is a nonspecific alpha- and beta-adrenergic agonist, widely used in local anaesthesia with the rationale that through local vasoconstriction it decreases absorption thereby increasing the duration of analgesia. While some studies have reported that the inclusion of epinephrine in PAI prolongs the duration of local anaesthetics, other studies have reported no significant increase in the duration when epinephrine is added to ropivacaine due to the intrinsic vasoconstrictive effect of ropivacaine . A recenOther possible causes for wound complications include the use of retractors and their pressure effects on skin and soft tissues. However, we have routinely performed total hip arthroplasty through the Direct Anterior Approach (DAA) for more than 12 years (approx. 400 hips/year), and we have not observed this issue previously . Also, gThe DAA has dramatically gained in popularity over the last 15 years because it is a muscle sparing.approach, with a potential reduction of postoperative pain and length of stay. As the skin is thinner in the anterior aspect of the hip, the risks for wound complications might be increased. However, there have been several studies, reviews and meta-analyses which compared early outcomes of total hip replacement through the main different surgical approaches, and reported similar rates of wound complications  16]. Wi. Wi16]. In this case, we performed surgical debridement and reconstruction of the skin and soft tissue defect using a pedicled fasciocutaneous anterolateral thigh flap. This method is a reliable and versatile option for loco-regional reconstruction due to the large skin and soft tissue availability, reliable blood supply, long vascular pedicle and wide arc of rotation . FurtherTo the best of our knowledge, this case represents the first evidence of a non-infectious and non-warfarin related extensive skin necrosis as a complication following total hip arthroplasty. In such cases, further testing should be performed to rule out possible causes such as infection and malnutrition. We achieved a good outcome with extensive debridement and wound reconstruction using a pedicled fasciocutaenous anterolateral thigh flap. As the exact cause of skin necrosis cannot be determined from a single case report, larger studies are required. Additionally, further clinical trials are needed to establish the risk and benefits of using epinephrine in PAI. Meanwhile, we decided to change our practice and not to use epinephrine anymore.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Nature Communications 10.1038/s41467-023-38746-5, published online 25 May 2023Correction to: The Supplementary Information associated with this Article contained text highlights corresponding to tracked changes that were made during revision. The HTML has been updated to include the correct version of the Updated Supplementary InformationIncorrect Supplementary Information


Nature Communications 10.1038/s41467-023-38087-3, published online 22 April 2023Correction to: The original version of the Updated Supplementary Information


Nature Communications 10.1038/s41467-022-35768-3, published online 19 January 2023Correction to: Updated Supplementary Information


Proteinuria and glomerular endotheliosis are characteristics of glomerular injury in preeclampsia, a hypertensive disorder in human pregnancy. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are biomarkers of acute/chronic renal tubule injury. To determine if tubule injury occurs in preeclampsia, we determined maternal plasma and urine NGAL and KIM-1 levels and evaluated NGAL and KIM-1 expression in kidney biopsy specimens from women with preeclampsia.n = 100), preeclampsia (n = 83), and pregnancy complicated with chronic hypertension (n = 20). Plasma and urine levels of NGAL and KIM-1 were measured by ELISA. Kidney biopsy tissue sections from patients with preeclampsia (n = 5) were obtained from Pathology Archives and processed to determine NGAL and KIM-1 expression by immunostaining and high kidney solution images were assessed by electron microscopy (EM).Prenatal and postpartum maternal blood and urinary samples were collected from three groups of pregnant women: normal pregnancy (p < 0.01. In normal pregnancy, both plasma and urine levels of NGAL and KIM-1 at 24–48 h after delivery and 6–8 weeks postpartum were relatively comparable to that of antenatal levels. In preeclampsia, urine, but not plasma, NGAL levels were reduced at 6–8 weeks postpartum compared to the antenatal levels, p < 0.05. Although maternal and urine KIM-1 levels were reduced at 6–8 weeks postpartum compared to the antenatal levels in preeclampsia, the levels were still higher than those in normal pregnancy. Positive expression of NGAL and KIM-1 was detected in proximal tubule epithelial cells in kidney tissue specimens from preeclampsia but not in non-pregnancy controls. EM examination showed glomerular and tubular injury in preeclampsia.Prenatal plasma and urine levels of NGAL and KIM-1 were significantly higher in preeclamptic than in normal controls, Our findings of increased maternal levels and urine secretion of NGAL and KIM-1, along with the upregulation of NGAL and KIM-1 expression in tubular epithelial cells in preeclampsia, provide plausible evidence that tubular injury exists in preeclampsia. The higher postpartum NGAL and KIM-1 levels in preeclamptic pregnancies indicate that tubular injury would not resolve within 2–3 months after delivery and suggest that proper follow-up and management of kidney function in women with preeclampsia would be necessary to reduce chronic kidney diseases in those women later in life. Proteinuria and glomerular endotheliosis are the classic hallmarks of renal dysfunction/pathology in preeclampsia, a hypertensive disorder in human pregnancy. Recent studies also indicate that other than glomerular endotheliosis, podocyte injury is present in preeclampsia, as demonstrated by increased podocyte shedding and podocyte protein shedding in women with preeclampsia –4. IncreIn addition to glomerular and podocyte injury in preeclampsia, emerging evidence also suggests that this pregnancy disorder is a risk factor for kidney tubular injury in women during pregnancy . There an = 100), pregnant women complicated with chronic hypertension (n = 20), and normotensive pregnant controls (n = 83). Institutional Review Board's (IRB) approval was obtained and informed consent was obtained from all the study subjects. Plasma and urinary samples were aliquot and stored at −70° until assay. Normotensive pregnancy is defined as pregnancy with a maternal blood pressure of <140/90 mmHg, absence of proteinuria, and medical and obstetrical complications. Chronic hypertension (CHT) in pregnancy is defined as either a documented history of high blood pressure before pregnancy or with a blood pressure of ≥140/90 mmHg on two occasions more than 24 h apart before the 20th week of gestation. Diagnosis of preeclampsia is based on the American College Obstetricians and Gynecologists (ACOG) criteria: sustained systolic blood pressure of ≥140 mmHg or a sustained diastolic blood pressure of ≥90 mmHg on two separate readings; proteinuria measurement of 2+ or more on the dipstick, or 24 h urine protein collection with ≥300 mg or protein creatinine ratio > 0.3 in the specimen after 20 weeks of gestation. Smokers or patients with signs of infection were excluded. To avoid clinical phenotypic differences in preeclampsia, patients complicated with nephritic syndrome, diabetic mellitus, or gestational diabetes were also excluded from the study. Patient demographic and clinical information was obtained via medical record review and is presented in A total of 203 pregnant women were recruited for the study. Maternal venous blood and urine specimens were collected either in a clinic or Labor and Delivery in University Health hospital, Louisiana State University Health Sciences Center-Shreveport (LSUHSC-S), including patients diagnosed with preeclampsia  and human KIM-1 DuoSet ELISA kit (Cat# DY1750) were purchased from R&D Systems . The range of the standard curve for NGAL is 4.9–5,000 pg/ml and for KIM-1 is 0.95–2,000 pg/ml. For the NGAL assay, plasma was 1:20 diluted and urine was 1:10 diluted. For the KIM-1 assay, both plasma and urine specimens were 1:2 diluted. All assays were performed following the manufacturer's instructions. All specimens were measured in duplicate. Between-assay and within-assay variations were <8% for both assays.n = 5) and control cases (n = 5) were obtained retrospectively from the Department of Pathology Archives at LSUHSC-Shreveport. For the preeclamptic cases, a kidney biopsy was done for diagnostic purposes due to persistent hypertension, massive proteinuria, or other complications after clinical treatment. For the controls, kidney tissue was taken from a normal portion of kidney surgical cases that were performed for excluding neoplasia. Control cases (non-pregnant) were anonymous, without hypertension or renal disease. IRB approval was obtained. Clinical information on the five preeclamptic cases is presented in Kidney biopsy tissue sections from preeclamptic  for 2 h, tissue sections were incubated with anti-NGAL or anti-KIM-1 antibody overnight at 4°C and then followed by incubation of biotinylate-conjugated secondary antibody (anti-rabbit IgG) for 2 h. NGAL (rabbit mAb D4M8L) and KIM-1 (rabbit mAb E1R9N) antibodies were purchased from Cell Signalling Technology . Biotinylate-conjugated goat anti-rabbit secondary antibody was obtained from Vector Laboratories . Slides without primary antibody staining served as a negative control. Nuclei counterstain was done by hematoxylin. Stained slides were reviewed under an Olympus microscope . Images were captured in the area containing glomerular and/or tubules by a digital camera.post-hoc test. Unpaired t-test, Mann–Whitney test, or Chi-square test were used to compare results between preeclamptic and normotensive or pregnancy complicated with chronic hypertension groups. Paired t-test was used to compare biomarker concentrations within each group before delivery and 6–8 weeks postpartum. Pearson product–moment correlation coefficient (Pearson r) was used to analyze the correlation between (1) maternal KIM-1 and NGAL, (2) urine KIM-1 and NGAL, (3) urine and maternal NGAL, and (4) urine and maternal KIM-1. A probability level of <0.05 was considered statistically significant.Clinical data are presented as mean ± SD . Data foPatient demographic data and clinical characteristics including maternal age, body mass index (BMI), blood pressure, gestational age at specimen collection and delivery, and medical history with chronic hypertension or preeclampsia are presented in n = 100) than those in normotensive controls (n = 83), p < 0.01. While maternal NGAL, but not KIM-1, levels were also significantly higher in pregnant women complicated with CHT (n = 20) than in normotensive controls, p < 0.01.Among these subjects, urine specimens were also obtained from 27 normotensive pregnant women, 20 women complicated with CHT, and 53 women with preeclampsia. Urine NGAL and KIM-1 levels were also significantly higher in women with preeclampsia than in normotensive pregnant controls. Urine NGAL and KIM-1 levels were increased, but not statistically significant, in women complicated with CHT compared to those in normotensive controls .p < 0.05. In comparison, urine NGAL, but not urine KIM-1, levels were reduced at 24–48 h after delivery. Although urine NGAL and KIM-1 levels were significantly reduced at 6–8 weeks postpartum compared to the antenatal levels in preeclampsia (p < 0.05 for NGAL and p < 0.01 for KIM-1), both urine NGAL and KIM-1 levels were still higher in preeclampsia than in normotensive controls.Among these subjects, maternal blood from 18 normotensive and 39 preeclamptic pregnancies and urine specimens from 21 normotensive and 35 preeclamptic pregnancies were also obtained at 24–48 h after delivery and 6–8 weeks postpartum. Results of plasma and urine NGAL and KIM-1 levels are shown in r = 0.320, and in preeclampsia, r = 0.317, but not in normal pregnant women  uriwith CHT ; and (4)with CHT .Expression of NGAL and KIM-1 was determined in kidney biopsy tissue sections from five patients who were clinically diagnosed with preeclampsia in the most recent pregnancy. Kidney tissue sections from a normal portion of the kidney surgical cases served as control. Representative transmission electron microscopy micrographs of glomeruli and proximal tubule from a non-hypertensive control and a patient with preeclampsia are shown in This study highlights the kidney tubular injury in preeclampsia, by assessment of maternal and urinary levels of NGAL and KIM-1, and by evaluation of NGAL and KIM-1 expression in kidney biopsy specimens from women with preeclampsia. We found that both maternal and urine levels of NGAL and KIM-1 were significantly higher in women with preeclampsia than in normotensive pregnant controls. We further found that NGAL and KIM-1 expression was upregulated in proximal tubule epithelial cells in kidney biopsy specimens from women with preeclampsia but not in controls. Since NGAL and KIM-1 are considered biomarkers for acute and chronic kidney tubular injury , 9, 11, To determine if tubular injury was restored after delivery in preeclampsia, we measured maternal and urinary NGAL and KIM-1 levels at 24–48 h after delivery and 6–8 weeks postpartum. It is interesting to note that both maternal and urinary levels of NGAL and KIM-1 at 24–48 h after delivery and 6–8 weeks postpartum were comparable to those of antenatal levels in normotensive pregnant women. While in preeclampsia, although maternal plasma and urine levels of NGAL and KIM-1 reduced at 6–8 weeks postpartum compared to those of antenatal levels, NGAL and KIM-1 levels were still much higher than the levels in normotensive controls, which suggests that kidney injury may not be resolved within 2–3 months after delivery in preeclampsia.de novo synthesis of NGAL is markedly increased, levels of NGAL increase rapidly and can be easily detected in urine, blood, and other body fluids  who had a history of CHT in the preeclampsia group, only a few of them had 6–8 weeks postpartum specimens collected and measured. Therefore, it is not known if those CHT pregnancies impact the elevated NGAL and KIM-1 levels at 6–8 weeks postpartum in the preeclampsia group. Although kidney specimens in the control group were from non-pregnant patients, we believed that immunostaining and electron microscopic results provide convincing information and support the concept that kidney tubule injury is present in preeclampsia. In addition, >75% of subjects were African American in our study. Since the disproportionate population was evenly distributed among the three study groups which represent the demographic and ethnic disparities in the Shreveport community, we do not believe that racial differences would affect our results.In summary, we found that maternal and urinary levels of NGAL and KIM-1 were significantly increased in women with preeclampsia compared to normotensive pregnant controls. We also found upregulation of NGAL and KIM-1 expression in proximal tubule epithelial cells in kidney specimens from women who had preeclampsia. Electron microscopic study further revealed loss of integrity of proximal tubule epithelial cells, showing vacuolization, apical blebbing, and loss of brush borders. All these findings demonstrate that kidney tubular injury exists in preeclampsia. It is well-accepted that preeclampsia is a risk factor for cardiovascular diseases in women later in life. Since preeclampsia is also a risk factor for subsequent end-stage renal diseases , proper The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRB: H09-085, Kidney Podocyte Injury in Preeclampsia. The patients/participants provided their written informed consent to participate in this study.YW and DL contributed to the study design. YW, YG, XG, and DC contributed to the sampling process, assays, and data collection. YW and YG performed the statistical analysis. YW wrote the first draft of the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted version.


These losses are still underperceived by the academic community, and by a global society that is disconnected from biocultural diversity. To reconnect society with biocultural diversity, we integrate temporal and spatial dimensions of seasonal cycles, by combining two conceptual frameworks: ecological calendars and the “3Hs” model of the biocultural ethic . The latter values the vital links between human and other‐than‐human co‐inhabitants, their life habits , and the structure and processes of their shared habitats. This integration enhances an understanding of links between cultural practices and the life cycles of biocultural keystone species. As a synthesis, we use the term biocultural calendars to emphasize their co‐constitutive nature that result from interactions between dynamic biophysical and cultural processes embedded in specific ecosystems and cultures. These calendars link astronomical, biological, and cultural seasonal cycles that sustain life and enhance the integration of Indigenous and scientific knowledge to confront challenges of climate change faced from local to global scales. To illustrate this integration, we examine cultural practices and socio‐environmental changes across four contrasting ethnolinguistic communities in southwestern South America, from southern to northern Chile along a marked climatic gradient to show the broad application of the concept of biocultural calendars.Since the mid‐20th century, the so‐called Great Acceleration ( Biocultural calendars are co‐constitutively generated through interactions between dynamic biophysical and cultural processes3Hs model values the vital links among human and other‐than‐human co‐inhabitants, their habits, and shared habitatsThe biocultural ethic's Biocultural calendars are based on seasonal cycles of keystone species that are part of communities of co‐inhabitants This synchrony is particularly relevant in the context of climate change because it makes explicit the inextricable links between cultures and nature. Changes in cultures involve changes in nature, and vice versa. Communities that are locally attuned to biophysical and cultural habitats have an enhanced capacity to adapt their life habits to variabilities of temperature, rainfall, and other climatic events.In this article we complement ecological calendars with the biocultural ethic to develop the concept of biocultural calendars. By interrelating phenological events in the biophysical realm with worldviews and practices in the cultural realm, biocultural calendars help us to understand the links between different life habits in contrasting habitats and annual seasons. This attribute of biocultural calendars is crucial in the context of climate change. To develop this concept, we organize our article in two parts. First, we describe the biocultural calendars of Indigenous and other local communities from four climatically contrasting regions of Chile. Second, we concisely discuss the broader implications of this biocultural systemic, contextual, and co‐evolutionary understanding. We propose that biocultural calendars could help set social‐environmental priorities, policies, and actions to conserve critical habitats and biocultural diversity not only in Chile but also in other heterogeneous regions of the planet. We show that our biocultural outlook introduces an understanding that transcends the purely descriptive plane, since it implies an ethical responsibility.1.1In Latin America, the history of Indigenous peoples and local communities is one in which their life habits have fostered co‐inhabitation with high levels of biodiversity Toledo, . Globallbiocultural calendars, an expression recently used by Mariana Landwehr (Zea mays L.) in Zinacantán is part of the cultural identity and is present in the social, political, religious, ceremonial, ritual and gastronomic organization during a well‐defined biocultural calendar  Rozzi, . AdditioIn this section we use the concept of biocultural calendars to examine cultural practices and socio‐environmental changes in southwestern South America, specifically in Chile Figure . We orgaanalemmas (or lemniscates), showing the position of the sun in the sky as seen from a fixed location on Earth at a fixed time ), began to paint the leaves giving rise to autumn. The trees revived months later giving rise to spring. This was the beginning of the annual cycles ) and the exemplary sanction for hoarding this vital element was death  Oerst.) is collected for the construction of canoes  Hook.fil.). Different types of baskets are made with its fibers: steapa with a tight weave are used to collect berries, and keichi with a loose weave are used to collect shellfish  is the season that begins when the leaves of the high‐deciduous beech or hanis (Nothofagus pumilio (Poepp. & Endl.) Krasser) turn red (lush). Fall is also marked by the arrival of abundant sardines ) and caiquenes ) are fat, and can be hunted more easily ) inhabiting the intertidal zones are central for the Yahgan diet, because they exhibit low seasonal variation in abundance ), American mink ), and muskrat ), is having a major impact on forest and wetland habitats and is decimating bird populations, especially of ground nesting species  that is acquired from the high‐deciduous beech tree (hanis) on the slopes of the mountains (tulara) in the fall. Ina, winter, is marked by long nights and snow, associated with lampia (black) and yakua (white), respectively. Iahakisi (spring) the short (iaha) summer (kisi), is signaled by the shoots of leaves that color this season with arlampia (green). Kisi, the summer, arrives with its interminable days where kurlampia (blue) inundates the sea and sky.To illustrate the biocultural conservation initiative in Cape Horn  inhabited by ca. 12,000 people ) and other seaweeds are harvested during the pilcanes ) and snoeks or “sierra” ). During their spawning period, these large fish approach the coast chasing schools of sardines. Fishing weirs are used throughout the year to capture other species such as the Patagonian blennie or “róbalo” (Eleginops maclovinus). Finally, fishing weirs are a refuge for multiple intertidal species, such as rock fish, crustaceans, and seaweeds that support coastal families. Thus, fishing weirs generate relationships of human‐nature reciprocity since these anthropogenic habitats stimulate greater biodiversity at the beaches ) and carnivorous mollusks, such as the Chilean abalone or “loco” ). In 1989, these fishing habitats and habits were included in the Chilean legal decree 18,892 on Benthic Resources Management and Exploitation Areas.3.4Since mid‐twentieth century, neoliberal economic policies have instilled private property rights, economic efficiency, and deregulation  offer opportunities for adapting to current social‐environmental problems. For example, today local fishers recognize the high intelligence of sea lions and maintain a “mutual understanding” of co‐inhabitation. Fishers must vary their fishing practices to avoid interference by sea lions which quickly detect the new sites, baits, and techniques used by fishers FSP, . On the 44.14.1.1Lafkenche means people (che) of the sea (lafken)   land (mapu). For them, this territory has both material and symbolic cultural significance ):Millenary bird that works by the light of day, and this it divides into four parts: the daybreak, the morning, the noon‐time, and the afternoon; symbol of life, work, results and production, family, planting, harvest and animals ) possesses special powers to connect the human with the divine and nature  land, that is like a reflection of the Mapu; it is inhabited not only by planets and stars, but also by relatives and friends who have died and now are working there just like they did in the Mapu   the Wenumapu and the Mapu.Mapu, the land “of here” where human beings live, suffer, and love. Mapu, also called mother earth (ñukemapu), is delicate and must be loved. The land is worked freely and fraternally, like brothers (peñi). In the ñukemapu, birds and trees should be treated as equals. To achieve this, the ancestors consumed only what they needed.Minchemapu, the land of below (minche) that encompasses all that is underneath, the rivers and the soil, is like the subsoil , the Lloyka is a “full‐time healer bird” that enhances the growth of medicinal plants. In Aillapan's poem the bird says: “I have always healed my people with pure herbs. For this, my name is full‐time healer”  and lawen (remedy). Aillapan explains that this healer bird wishes to cure its own gaudy red colored chest that carries the blood of the Mapuche people  Hanst. (Gesneriaceae), Cissus striata Ruiz & Pav. (Vitaceae), Eccremocarpus scaber Ruiz & Pav. (Bignoniaceae), Elytropus chilensis (A.DC.) Müll.Arg. (Apocynaceae), Lapageria rosea Ruiz & Pav. (Philesiaceae), Tropaeolum speciosum Poepp. & Endl. (Tropaeolaceae), Lardizabala funaria (Molina) Looser, Boquila trifoliolata (DC.) Decne. , and Berberidopsis corallina Hook.fil. (Berberidopsidaceae)  Hariot, 1892), involves synchronizing harvesting and drying habits with the periods in which the seaweeds grow. In addition, the climate that allows them to be dried on the shore for storage and marketing is considered; otherwise, it would rot.Along the coasts, gathering activities also take place in intertidal and subtidal marine habitats that host a variety of seaweeds, fish, and shellfish  are immediately attended to because their precariousness causes social imbalance  a variety of fungi are gathered. Community activities are reduced giving place to conversations within each family, usually commenting on the experiences they had during the spring and summer. In winter (pükem), the season of greatest deprivation, community practices include times for collective thought and reflection  implies flowing with the vitality of plants and animals through community activities linked to respectful use of forests  inserted in a matrix of forests and shrublands , the sea urchin Loxechinus albus , the sea snail Tegula atra , and the crab Taliepus dentatus  are four keystone species for the Lafkenche diet  and plants (such as voqui) are salient (see above). Among human families, when faced with unforeseen events, such as periods of drought during spring or summer, Lafkenche and Williche families do not hesitate to exchange seeds that have been culturally modified for centuries and that they know will germinate and grow under stressful conditions  they continue exchanging seeds and begin harvesting fruits. Additionally, young lambs and calves are born, thereby generating a season of opulence. Hence, ecological seasonal patterns and the sequence of social activities among Lafkenche people are closely linked. These biocultural links reinforce knowledge and customs that nourish a sense community, which includes human and other‐than‐human co‐inhabitants  and Eucalyptus L'Hér. species , which did not acknowledge the presence of the Indigenous peoples on the coasts of Chile. This lack of recognition triggered a social movement by the Mapuche‐Lafkenche Indigenous people, producing a shift of the Indigenous demands from the interior lands to the sea. After over a decade of negotiations, in 2008, the government of Chile introduced the “Lafkenche Law” (Law 20.249) establishing a new conservation category in Chile: the coastal‐marine space of the original peoples   on the northeast border of Chile with Bolivia at 4,000 m altitude. This area has large wetlands supported by underground saline aquifers that are home to a great diversity of native fauna such as flamingos and camelids  Figure . Summer 5.1.2achachila) are ancestors and sacred sites. These sites make the reproduction of life possible and must be respected and remembered in rituals and offerings. Their seasonal changes are the changes of human time; this is how Aymara biocultural calendars have been forged . Manqha Pacha is the world below; it is the place under the Earth where what has already happened is kept, where the gaze is directed toward the past time (Nayra Pacha). It is a sacred space where spirits dwell, and where the waters are lost and the vegetation ends. Aka Pacha is at the center and harmonizes opposites; it is where the Aymara people live. It is where the present time (Jicha Pacha), in which we humans live, work, rest, and dream  and llama (L. glama). Indigenous Aymara communities have sustained models of life coupled with marked seasonal contrasts, which alternate rain and drought, heat and cold  and the Chilean flamingo (Phoenicopterus chilensis), have the world's largest populations in this high Andean region.Central co‐inhabitants in the high Andes are the domesticated camelids alpaca . IDAs are understood as territorial units where the Chilean state supports the protection of common resources and access to them by Indigenous communities with three central goals: to overcome poverty, to preserve Indigenous cultures, and to comply with their demands for land and water. The criteria for the designation of IDAs are based on the ancestral occupation of the territory, high density of Indigenous populations, ecological homogeneity, and dependence on natural resources such as land, water, flora, and fauna. IDAs have helped, but often for the defense of these habitats and water resources, Aymara communities have had to appeal to international conventions, such as the Convention 169 of the International Labour Organization (ILO) and/or the Ramsar Convention   S.C. Lindstrom, 2011) is the most eaten red algae . Tsimshian people watch the stalks of the stinging nettle mature and elongate; as they grow in the coastal habitats, so do the seaweed fronds grow in the intertidal zone. The red laver is harvested in late spring, generally in May, so much that in the Tsimshian language this month is called ‘‘the month of the seaweed’’ , but including today cattle of European origin (with higher water requirements). These nomadic habits are intertwined in a complex scenario with other uses of water, principally mining. Mining activities have degraded wetlands and their biodiversity, and triggered accelerated depopulation of Andean rural areas along with disruption of biocultural calendars.Fourth, a conceptual understanding of biocultural conservation approach contributes to balancing the interests and needs among different stakeholders. Just as the Chilean state has created Indigenous Development Areas (IDAs) to protect biological and cultural diversity, worldwide international programs have embraced the concept of including human communities and their habitats in conservation programs (e.g., Man and Biosphere Program, Guevara & Laborde, To address this type of conflicts, a biocultural calendars integrates concepts of ecological calendars and the biocultural ethic to cope with social and environmental dimensions of climate change. The drivers of climate change are of two types: direct and indirect (MEA, Fifth, the four former concluding remarks show how the concept of ect MEA, . Direct ect MEA, . In thisThe authors declare no conflicts of interest relevant to this study.