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10.1101/2022.05.06.490893 | A Saccharomyces cerevisiae Model for Overexpression of Ntg1 a Base Excision DNA Repair Protein Reveals Novel Genetic Interactions | The Base Excision Repair (BER) pathway repairs oxidative DNA damage, a common and detrimental form of damage to the genome. Although biochemical steps in BER have been well define, little is understood about how the pathway is regulated. Such regulation is critical, as cells must respond rapidly to DNA damage while avoiding aberrant activation of repair proteins that can produce DNA damage as intermediates in the repair pathway. Indeed, overexpression of the human BER protein, NTHL1, a DNA N-glycosylase, can cause genomic instability and early cellular hallmarks of cancer. We developed a Saccharomyces cerevisiae model to explore how overexpression of NTHL1 may impair cellular function. Overexpression of Ntg1, the budding yeast orthologue of NTHL1, impairs cell growth. To dissect mechanisms underlying this growth defect, we overexpressed either wild-type Ntg1 or a catalytically inactive variant of Ntg1 (ntg1catdead). Consistent with results obtained for NTHL1, both variants of Ntg1 impair cell growth, but only the wild-type protein causes accumulation of double-strand breaks and chromosome loss. We screened a panel of DNA repair mutants for resistance/sensitivity to overexpression of wild-type Ntg1 or ntg1catdead. This analysis identified several cellular pathways that protect cells from Ntg1-induced damage, providing insight into interplay between DNA repair pathways. Finally, we identified a link to sumoylation and probed how this post-translational modification could contribute to regulation of Ntg1 function. This study describes a budding yeast system to understand how cells regulate and respond to dysregulation of the BER pathway.
Take AwayO_LIOverexpression of a base excision DNA repair protein impairs cell growth
C_LIO_LIOverexpression of a base excision DNA repair protein can cause DNA damage
C_LIO_LIMultiple mechanisms cause DNA damage from overexpression of a repair protein
C_LIO_LIDNA repair pathways functionally interact to protect cells from DNA damage
C_LIO_LIPrecise regulation of the activity of DNA repair proteins is critical
C_LI | genetics |
10.1101/2022.05.06.490906 | Insulin Receptor Loss Impairs Mammary Tumorigenesis in Mice | Breast cancer (BC) prognosis and outcome are adversely affected by increased body weight, obesity and the obesity-associated type 2 diabetes. Hyperinsulinemia, which is commonly seen as part of metabolic reprograming in the obese state, has been associated with higher risk of death and recurrence in BC. Up to 80% of breast cancers overexpress the insulin receptor (INSR) de novo, which correlates with worse prognosis. To directly probe the role of insulin signaling in mammary tumorigenesis in the mouse, we generated the MMTV-driven polyoma middle T (PyMT) and ErbB2/Her2 BC models, respectively, with coordinate mammary epithelium-restricted deletion of the INSR. In both models, deletion of either one or both copies of the INSR in the mammary gland led to a marked delay in tumor onset and burden, including in mice fed to mimic conditions of human obesity. Longitudinal monitoring of generated mouse models revealed that tumor initiation, rather than progression and metastasis, were impacted by INSR deletion. The similarity of phenotypes elicited by the deletion of one or both copies of INSR raises the possibility that there is a dose-dependent threshold for the contribution of INSR to mammary tumorigenesis. | cancer biology |
10.1101/2022.05.04.490681 | Knockout of mitogen-activated protein kinase 3 causes barley root resistance against Fusarium graminearum. | The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are less understood in crops. Here, microscopic, phenotyping, proteomic and biochemical analyses revealed that independent TALEN-based knockout lines of Hordeum vulgare MITOGEN-ACTIVATED PROTEIN KINASE 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of hyphae was considerably deregulated, mycelia development was less efficient and number of appressoria and their penetration potential were significantly reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild type, but it was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with the elevated abundances of catalase and ascorbate peroxidase. Moreover, differential proteomic analysis revealed downregulation of defense-related proteins in wild type, and the upregulation of peroxidases, lipid transfer proteins, and cysteine proteases in HvMPK3 KO lines after 24h of F. graminearum inoculation. Consistently with proteomic analysis, microscopic observations showed an enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to the barley root resistance against Fusarium root rot. | plant biology |
10.1101/2022.05.06.490883 | Modelling the within-host spread of SARS-CoV-2 infection, and the subsequent immune response, using a hybrid, multiscale, individual-based model. Part I: Macrophages. | Individual responses to SARS-CoV-2 infection vary significantly, ranging from mild courses of infection that do not require hospitalisation to the development of disease which not only requires hospitalisation but can be fatal. Whilst many immunological studies have revealed fundamental insights into SARS-CoV-2 infection and COVID-19, mathematical and computational modelling can offer an additional perspective and enhance understanding. The majority of mathematical models for the within-host spread of SARS-CoV-2 infection are ordinary differential equations, which neglect spatial variation. In this article, we present a hybrid, multiscale, individual-based model to study the within-host spread of SARS-CoV-2 infection. The model incorporates epithelial cells (each containing a dynamical model for viral entry and replication), macrophages and a subset of cytokines. We investigate the role of increasing initial viral deposition, increasing delay in type I interferon secretion from epithelial cells (as well as the magnitude of secretion), increasing macrophage virus internalisation rate and macrophage activation, on the spread of infection. | systems biology |
10.1101/2022.05.06.490908 | Antigenicity is preserved with fixative solutions used in human gross anatomy: A mice brain immunohistochemistry study. | BackgroundHistology remains the gold standard to assess human brain biology, so ex vivo studies using tissue from brain banks are standard practice in neuroscientific research. However, a larger number of specimens could be obtained from gross anatomy laboratories. These specimens are fixed with solutions appropriate for dissections, but whether they also preserve brain tissue antigenicity is unclear. Therefore, we perfused mice brains with solutions used for human body preservation to assess and compare the tissue quality and antigenicity of the main cell populations.
Methods28 C57BL/6J mice were perfused with: 4% formaldehyde (FAS, N=9), salt-saturated solution (SSS, N=9), and alcohol-solution (AS, N=10). The brains were cut into 40m sections for antigenicity analysis and were assessed by immunohistochemistry of four antigens: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP-astrocytes), ionized calcium binding adaptor molecule1 (Iba1-microglia), and myelin proteolipid protein (PLP). We compared the fixatives according to multiple variables: perfusion quality, ease of manipulation, tissue quality, immunohistochemistry quality, and antigenicity preservation.
ResultsThe perfusion quality was better using FAS and worse using AS. The manipulation was very poor in SSS brains. FAS and AS fixed brains showed higher tissue and immunohistochemistry quality than the SSS brains. All antigens were readily observed in every specimen, regardless of the fixative solution.
ConclusionSolutions designed to preserve specimens for human gross anatomy dissections also preserve tissue antigenicity in different brain cells. This offers opportunities for the use of human brains fixed in gross anatomy laboratories to assess normal or pathological conditions.
SIGNIFICANCE STATEMENTNeuroscientists currently obtain tissue samples from brain banks. Alternatively, a much larger amount of tissue may be obtained from bodies donated to gross anatomy laboratories. However, they are preserved with different fixative solutions that are used for dissection purposes. We ignore if these solutions also preserve antigenicity of the main cell populations, essential in neuroscientific research. This work is the first to show that two solutions currently used in human gross anatomy laboratories preserve sufficient histological quality, in addition to preserve antigenicity of the main cell populations of the mice brain. This work opens the door to the use of human brain tissue obtained from anatomy laboratories in neuroscientific research. | neuroscience |
10.1101/2022.05.05.490815 | Immunological findings in a group of individuals who were non-responders to standard two-dose SARS-CoV-2 vaccines | Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic. The virus has infected more than 505 million people and caused more than 6 million deaths. However, data on non-responders to SARS-CoV-2 vaccines in the general population are limited. The objective of the study is to comprehensively compare the immunological characteristics of non-responders to SARS-CoV-2 vaccines in the 18-59 years with that in the 60 years and older using internationally recognized cutoff values. Participants included 627 individuals who received physical examinations and volunteered to participate in COVID-19 vaccination from the general population. The main outcome was an effective seroconversion characterized by anti-SARS-CoV-2 spike IgG level of at least 4-fold increase from baseline. Profiling of naive immune cells was analyzed prior to vaccination to demonstrate baseline immunity. Outcomes of effective seroconversion in the 18-59 years with that in the 60 years and older were compared. The quantitative level of the anti-spike IgG was significantly lower in the 60 years and older and in men among the 18-59 years. There were 7.5% of non-responders among the 18-59 years and 11.7% of non-responders in the 60 years and older using the 4-fold increase parameter. The effective seroconversion rate was significantly related to the level of certain immune cells before vaccination, such as CD4 cells, CD8 cells and B cells and the age. An individual with a titer of anti-SARS-CoV-2 spike IgG that is below 50 BAU/mL might be considered a non-responder between 14-90 days after the last vaccine dose. Booster vaccination or additional protective measures should be recommended for non-responders as soon as possible to reduce disease severity and mortality. | immunology |
10.1101/2022.05.05.490850 | ORF6 protein of SARS-CoV-2 inhibits TRIM25 mediated RIG-I ubiquitination to mitigate type I IFN induction. | Evasion and antagonism of host cellular immunity upon SARS-CoV-2 infection confers a profound replication advantage on the virus and contributes to COVID-19 pathogenesis. We explored the ability of different SARS-CoV-2 proteins to antagonize the host innate immune system and found that the ORF6 protein mitigated type-I IFN (interferon) induction and downstream IFN signaling. Our findings also corroborated previous reports that ORF6 blocks the nuclear import of IRF3 and STAT1 to inhibit IFN induction and signaling. Here we show that ORF6 directly interacts with RIG-I and blocks downstream type-I IFN induction and signaling by inhibiting K-63 linked ubiquitination of RIG-I by the E3 Ligase TRIM25. This involves ORF6-mediated targeting of TRIM25 for degradation, also observed during SARS-CoV-2 infection. The type-I IFN antagonistic activity of ORF6 was mapped to its C-terminal cytoplasmic tail, specifically to amino acid residues 52-61. Overall, we provide new insights into how the SARS-CoV-2 ORF6 protein inhibits type I-IFN induction and signaling through distinct mechanisms. | microbiology |
10.1101/2022.05.06.490915 | Reaction Conditions Promoting the Specific Detection of SARS-CoV-2 NendoU Enzymatic Activity | Methods that enable rapid detection of SARS-CoV-2 provide valuable tools for detecting and controlling Covid-19 outbreaks and also facilitate more effective treatment of infected individuals. The predominant approaches developed use PCR to detect viral nucleic acids or immunoassays to detect viral proteins. Each approach has distinct advantages and disadvantages, but alternatives that do not share the same limitations could enable substantial improvements in outbreak detection and management. For instance, methods that have comparable sensitivity to PCR, but that are not prone to the false-positive results that stem from the tendency of PCR to detect molecular degradation products could improve accurate identification of infected individuals. An alternative approach with potential to achieve this entails harnessing the unique enzymatic properties of SARS-CoV-2 enzymes to generate SARS-Cov-2-specific signals that indicate the presence of the virus. This route benefits from the high sensitivity provided by enzymatic signal amplification and also the fact that signal is generated only by intact viral enzymes, not degradation products. Here, we demonstrate enzymatic reaction conditions that enable the preferential detection of NendoU of SARS-CoV-2, versus several of its orthologues, with a fluorogenic oligonucleotide substrate. These compositions provide a possible technical foundation for a novel approach for detecting SARS-CoV-2 that has distinct advantages from current approaches. | microbiology |
10.1101/2022.05.06.490907 | GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility. | Coronavirus disease 19 (COVID-19) is a persistent global pandemic with a very heterogeneous disease presentation ranging from a mild disease to dismal prognosis. Early detection of sensitivity and severity of COVID-19 is essential for the development of new treatments. In the present study, we measured the levels of circulating growth differentiation factor 15 (GDF15) and angiotensin-converting enzyme 2 (ACE2) in plasma of severity-stratified COVID-19 patients and healthy control patients and characterized the in vitro effects and cohort frequency of ACE2 SNPs. Our results show that while circulating GDF15 and ACE2 stratify COVID-19 patients according to disease severity, ACE2 missense SNPs constitute a risk factor linked to infection susceptibility. | molecular biology |
10.1101/2022.05.06.490944 | Notch signaling functions in non-canonical juxtacrine manner in platelets to amplify thrombogenicity | BackgroundNotch signaling is an evolutionarily conserved pathway that dictates cell fate decisions in mammalian cells including megakaryocytes. Existence of functional Notch signaling in enucleate platelets that are generated as cytoplasmic buds from megakaryocytes still remains elusive.
MethodsPlatelets were isolated from human blood by differential centrifugation under informed consent. Expression of transcripts as well as peptides of Notch1 and DLL-4 in platelets was studied by employing RT-qPCR, Western analysis and flow cytometry. Platelet activation responses that include aggregation, secretion of granule contents and platelet-leucocyte interaction were analyzed by Borns aggregometry, flow cytometry, Western analysis and lumi- aggregometry. Shedding of extracellular vesicles from platelets was documented with Nanoparticle Tracking Analyzer. Platelet adhesion and thrombus growth on immobilized matrix was quantified by employing microfluidics platform. Intracellular free calcium in Fura-2-loaded platelets was monitored from ratiometric fluorescence spectrophotometry. Coagulation parameters in whole blood were studied by thromboelastography. Ferric chloride-induced mesenteric arteriolar thrombosis in murine model was imaged by intravital microscopy.
ResultsHere we demonstrate significant expression of Notch1 and its ligand, the Delta-like ligand (DLL)- 4, as well as their respective transcripts, in human platelets. Synthesis and surface translocation of Notch1 and DLL-4 were upregulated when cells were challenged with physiological agonists like thrombin. DLL-4, in turn, instigated neighbouring platelets to switch to activated phenotype, associated with cleavage of Notch receptor and generation of its intracellular domain (NICD). DLL-4-mediated pro-thrombotic attributes were averted by pharmacological inhibition of {gamma}-secretase and phosphatidylinositol 3-kinase. Inhibition of Notch signaling, too, restrained agonist-induced platelet activation, and significantly impaired arterial thrombosis in mice, suggestive of synergism between thrombin- and DLL-4-mediated pathways. Strikingly, prevention of DLL-4-Notch1 interaction by a blocking antibody abolished platelet aggregation and extracellular vesicle shedding induced by thrombin.
ConclusionsOur study presents compelling evidence in support of non-canonical Notch signaling that propagates in juxtacrine manner within platelet aggregates and synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, targeting Notch signaling can be investigated as a potential anti-platelet/anti-thrombotic therapeutic approach.
FundingThis research was supported by J. C. Bose National Fellowship (JCB/2017/000029) and grants received by D. Dash from the Indian Council of Medical Research (ICMR) under CAR (71/4/2018-BMS/CAR), Department of Biotechnology (DBT) (BT/PR-20645/BRB/10/1541/2016) and Science and Engineering Research Board (SERB) (EMR/2015/000583), Government of India. S.N. Chaurasia is a recipient of financial assistance from the ICMR. M. Ekhlak is a recipient of CSIR-SRF and V. Singh is a recipient of UGC-SRF. D. Dash acknowledges assistance from the Humboldt Foundation, Germany. Funders have no role in the design, analysis and reporting of the study. | cell biology |
10.1101/2022.05.05.490776 | Allelic gene polymorphisms suspected to diversify the individual early metabolic response upon influenza H3N2 and SARS-CoV-2 infections | In this brief report, we point to virus infection time-dependent transcript levels of polymorphic ASMTL genes in human nasal epithelial cells from seven cell origins. Our observations encourage focused top-down and hypothesis-driven studies in support of more efficient allelic genotyping to identify targets for early resilience prediction. | cell biology |
10.1101/2022.05.06.490640 | Interrogating the Precancerous Evolution of Pathway Dysfunction in Lung Squamous Cell Carcinoma Using XTABLE | Lung squamous cell carcinoma (LUSC) is a type of lung cancer with a dismal prognosis that lacks adequate therapies and actionable targets. This disease is characterized by a sequence of low and high-grade preinvasive stages with increasing probability of malignant progression. Increasing our knowledge about the biology of these premalignant lesions (PMLs) is necessary to design new methods of early detection and prevention, and to identify the molecular processes that are key for malignant progression. To facilitate this research, we have designed XTABLE, an open-source application that integrates the most extensive transcriptomic databases of PMLs published so far. With this tool, users can stratify samples using multiple parameters and interrogate PML biology in multiple manners, such as two and multiple group comparisons, interrogation of genes of interests and transcriptional signatures. Using XTABLE, we have carried out a comparative study of the potential role of chromosomal instability scores as biomarkers of PML progression and mapped the onset of the most relevant LUSC pathways to the sequence of LUSC developmental stages. XTABLE will critically facilitate new research for the identification of early detection biomarkers and acquire a better understanding of the LUSC precancerous stages. | bioinformatics |
10.1101/2022.05.06.490845 | Insulin receptors in pancreatic acinar cells contribute to KrasG12D-driven cancer initiation in the context of diet-induced obesity | The rising incidence of pancreatic cancer is largely driven by increased prevalence of obesity and type 2 diabetes (T2D). Hyperinsulinemia is a cardinal feature of obesity and T2D, and is associated with increased cancer incidence and mortality. Genetic reduction of insulin production suppresses formation of pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in mice with mutant Kras. However, it remained unclear whether hyperinsulinemia exerts its tumorigenic effects directly on the cells that give rise to PanINs or indirectly on the tumor microenvironment. Here, we tested whether diet-induced hyperinsulinemia contributes to pancreatic cancer directly through insulin receptor (Insr) signaling in KrasG12D-expressing pancreatic acinar cells. Loss of insulin receptors in wild-type or KrasG12D-expressing acinar cells did not significantly influence fasting glucose or insulin. Strikingly, mice lacking Insr in KrasG12D-expressing acinar cells and their progeny had a significant reduction in PanIN plus tumor area in males (2.7-fold) and females (5.3-fold) compared to control mice. Mechanistically, proteome analyses implicated a reduction in digestive enzymes among altered protein networks in mice protected from PanINs, and together with phospho-proteome analysis, linked the spliceosome, ribosome, and secretory pathway to insulin signaling in context of pancreatic cancer initiation. Collectively, these data demonstrate that insulin receptor signaling in acinar cells promotes PanIN initiation in the context of obesity. | cancer biology |
10.1101/2022.05.06.490942 | Heterologous expression of a lycophyte protein enhances angiosperm seedling vigor | Seedling vigor is a key agronomic trait that determines juvenile plant performance. Angiosperm seeds develop inside fruits and are connected to the mother plant through vascular tissues. Their formation requires plant-specific genes, such as BREVIS RADIX (BRX) in Arabidopsis thaliana roots. BRX family proteins are found throughout the angiosperms but also occur in non-vascular bryophytes and non-seed lycophytes. They consist of four conserved domains, including the tandem "BRX-domains". We found that bryophyte or lycophyte BRX homologs can only partially substitute for Arabidopsis BRX (AtBRX) because they miss key features in the linker between the BRX-domains. Intriguingly however, expression of a BRX homolog from the lycophyte Selaginella moellendorffii (SmBRX) in A. thaliana wildtype background confers robustly enhanced root growth vigor that persists throughout the life cycle. This effect can be traced back to a substantial increase in seed and embryo size, and can be reproduced with a modified, "SmBRX-like" variant of AtBRX. Our results thus suggest that BRX variants could serve as biotechnological tools to boost seedling vigor and shed light on the activity of ancient, non-angiosperm BRX family proteins. | plant biology |
10.1101/2022.05.06.490896 | Expansion of information in scientific research papers | Presenting information in papers allows readers to see the evidence for the research claims. The amount of information presented to readers is increasing in high impact factor scientific journals. The aim of the present study was to determine whether there was a similar expansion in the amount of information presented to readers in subject-specific journals. We examined 878 research papers that were published in the journals Biology of Reproduction and Reproduction during the first six months of 1989, 1999, 2009, and 2019. Although there were few differences between the journals, we found that between 1989 and 2019 the number of figures increased 1.5-fold, the number of figure panels increased 3.6-fold, and the number of display items increased 5.6-fold. Amongst the display items, the number of images per paper increased 10-fold, and the number of graphs per paper increased 3.7-fold. The median paper in 1989 was 8 pages long, contained 6 tables and/or figures, with 1 image and 4 graphs. In 2019 the median paper was 12 pages long, contained 7 tables and/or figures, with 13 images and 15 graphs. This expansion of information in subject-specific journals implies that authors, reviewers, and editors need to help readers digest complex biological messages without causing information overload.
Lay summaryWe are living in an age of science and information. The amount of information presented in research papers has increased over time in the top science journals. Our research examined whether there has been a similar expansion in information in two influential subject-specific journals. We counted how much information was presented in 878 research papers across a 30-year period in the journals Biology of Reproduction and Reproduction. There were few differences between the two journals. But there was a striking increase in the information presented to readers in 2019 compared with 1989. The typical paper in 1989 was 8 pages long and contained 1 picture and 4 graphs. In 2019 the typical paper was 12 pages long and contained 13 pictures and 15 graphs. This expansion of information means that subject-specific journals must balance the presentation of complex biological messages with the risk of causing information overload. | scientific communication and education |
10.1101/2022.05.06.490867 | SARS-CoV-2 evolution and patient immunological history shape the breadth and potency of antibody-mediated immunity | Since the emergence of SARS-CoV-2, humans have been exposed to distinct SARS-CoV-2 antigens, either by infection with different variants, and/or vaccination. Population immunity is thus highly heterogeneous, but the impact of such heterogeneity on the effectiveness and breadth of the antibody-mediated response is unclear. We measured antibody-mediated neutralisation responses against SARS-CoV-2Wuhan, SARS-CoV-2, SARS-CoV-2{delta} and SARS-CoV-2o pseudoviruses using sera from patients with distinct immunological histories, including naive, vaccinated, infected with SARS-CoV-2Wuhan, SARS-CoV-2 or SARS-CoV-2{delta}, and vaccinated/infected individuals. We show that the breadth and potency of the antibody-mediated response is influenced by the number, the variant, and the nature (infection or vaccination) of exposures, and that individuals with mixed immunity acquired by vaccination and natural exposure exhibit the broadest and most potent responses. Our results suggest that the interplay between host immunity and SARS-CoV-2 evolution will shape the antigenicity and subsequent transmission dynamics of SARS-CoV-2, with important implications for future vaccine design.
Author SummaryNeutralising antibodies provide protection against viruses and are generated because of vaccination or prior infections. The main target of anti-SARS-CoV-2 neutralising antibodies is a protein called Spike, which decorates the viral particle and mediates viral entry into cells. As SARS-CoV-2 evolves, mutations accumulate in the spike protein, allowing the virus to escape antibody-mediated immunity and decreasing vaccine effectiveness. Multiple SARS-CoV-2 variants have appeared since the start of the COVID-19 pandemic, causing various waves of infection through the population and infecting-in some cases-people that had been previously infected or vaccinated. Since the antibody response is highly specific, individuals infected with different variants are likely to have different repertoires of neutralising antibodies. We studied the breadth and potency of the antibody-mediated response against different SARS-CoV-2 variants using sera from vaccinated people as well as from people infected with different variants. We show that potency of the antibody response against different SARS-CoV-2 variants depends on the particular variant that infected each person, the exposure type (infection or vaccination) and the number and order of exposures. Our study provides insight into the interplay between virus evolution and immunity, as well as important information for the development of better vaccination strategies. | microbiology |
10.1101/2022.05.06.490953 | DNA methylation Dependent Restriction of Tyrosine Hydroxylase Contributes to Pancreatic β-cell Heterogeneity | The molecular and functional heterogeneity of pancreatic {beta}-cells is well recognized. Pancreatic islets harbor a small subset of {beta}-cells that co-express Tyrosine Hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of this sub-population within islets is essential for appropriate insulin secretion. However, the distinguishing characteristics of this subpopulation and the mechanisms that restrict TH expression in {beta}-cells are not known. Here, we define the specific molecular and metabolic characteristics of the TH+ {beta}-cells and show that TH expression in {beta}-cells is restricted by DNA methylation patterning during {beta}-cell lineage specification. Ablation of de novo DNA methyltransferase Dnmt3a in the pancreatic- and endocrine-progenitor lineages results in a dramatic increase in the proportion of TH+ {beta}-cells, while {beta}-cell specific ablation of Dnmt3a has no effect on this sub-population. We demonstrate that maintenance of Th promoter DNA methylation patterns is essential for its continued restriction in postnatal {beta}-cells, and that loss of DNA methylation dysregulates TH expression in {beta}-cells in response to chronic overnutrition, contributing to impairment of {beta}-cell identity. These data highlight the essential requirement of DNA methylation patterning in regulating endocrine cell fates, and reveal a novel role of DNA methylation in {beta}-cell heterogeneity. | developmental biology |
10.1101/2022.05.06.490966 | Draft genome of six Cuban Anolis lizards and insights into genetic changes during the diversification | The detection of various type of genomic variants and their accumulation processes during species diversification and adaptive radiation is important for understanding the molecular and genetic basis of evolution. Anolis lizards in the West Indies are good models for studying the mechanism of the evolution because of the repeated evolution of their morphology and the ecology. In this study, we performed de novo genome assembly of six Cuban Anolis lizards with different ecomorphs and thermal habitats (Anolis isolepis, Anolis allisoni, Anolis porcatus, Anolis allogus, Anolis homolechis, and Anolis sagrei). As a result, we obtained six novel draft genomes with relatively long and high gene completeness, with scaffold N50 ranging from 5.56-39.79 Mb, and vertebrate Benchmarking Universal Single-Copy Orthologs completeness ranging from 77.5% to 86.9%. Subsequently, we performed comparative analysis of genomic contents including those of mainland Anolis lizards to estimate genetic variations that had emerged and accumulated during the diversification of Anolis lizards. Comparing the repeat element compositions and repeat landscapes revealed differences in the accumulation process between Cuban trunk-crown and trunk-ground species, LTR accumulation observed only in A. carolinensis, and separate expansions of several families of LINE in each of Cuban trunk-ground species. The analysis of duplicated genes suggested that the proportional difference of duplicated gene number among Cuban Anolis lizards may be associated to the difference of their habitat range. Furthermore, Pairwise Sequentially Markovian Coalescent analysis proposed that the effective population sizes of each species might have been affected by Cubas geohistory. Hence, these six novel draft genome assemblies and detected genetic variations can be a springboard for the further genetic elucidation of the Anolis lizards diversification.
SignificanceAnolis lizard in the West Indies is excellent model for studying the mechanisms of speciation and adaptive evolution. Still, due to a lack of genome assemblies, genetic variations and accumulation process of them involved in the diversification remain largely unexplored. In this study, we reported the novel genome assemblies of six Cuban Anolis lizards and analyzed evolution of genome contents. From comparative genomic analysis and inferences of genetic variation accumulation process, we detected species- and lineage-specific transposon accumulation processes and gene copy number evolution, considered to be associated with the adaptation to their habitats. Additionally, we estimated past effective population sizes and the results suggested its relationship to Cubas geohistory. | genomics |
10.1101/2022.05.06.490842 | Mechanism of the cadherin-catenin F-actin catch bond interaction | Mechanotransduction at cell-cell adhesions is crucial for the structural integrity, organization, and morphogenesis of epithelia. At cell-cell junctions, ternary E-cadherin/{beta}-catenin/E-catenin complexes sense and transmit mechanical load by binding to F-actin. The interaction with F-actin, described as a two-state catch bond, is weak in solution but is strengthened by applied force due to force-dependent transitions between weak and strong actin-binding states. Here, we provide direct evidence from optical trapping experiments that the catch bond property principally resides in the E-catenin actin-binding domain (ABD). Consistent with our previously proposed model, deletion of the first helix of the five-helix ABD bundle enables stable interactions with F-actin under minimal load that are well-described by a single-state slip bond, even when E-catenin is complexed with {beta}-catenin and E-cadherin. Our data argue for a conserved catch bond mechanism for adhesion proteins with structurally similar ABDs. We also demonstrate that a stably bound ABD strengthens load-dependent binding interactions between a neighboring complex and F-actin, but the presence of the other E-catenin domains weakens this effect. These results provide mechanistic insight to the cooperative binding of the cadherin-catenin complex to F-actin, which regulate dynamic cytoskeletal linkages in epithelial tissues. | biophysics |
10.1101/2022.05.04.490552 | Ongoing Replication Stress Response and New Clonal T Cell Development Discriminate Between Liver and Lung Recurrence Sites and Patient Outcomes in Pancreatic Ductal Adenocarcinoma | Background and AimsMetastatic pancreatic adenocarcinoma (mPDAC) is lethal, yet a subset of patients who have metastatic disease that spreads only to the lung have better outcomes. We identified unique transcriptomic and immune features that distinguish patients who develop metastases in the liver (liver cohort) versus those with lung-avid but liver-averse mPDAC (lung cohort).
MethodsWe used clinical data from the Oregon Pancreas Tissue Registry to identify PDAC patients with liver and/or lung metastases. Gene expression and genomic alteration data from 290 PDAC tumors were used to identify features unique to patients from the liver and lung cohorts. In parallel, T cell receptor sequencing data from 289 patients were used to identify immune features unique to patients in the lung cohort.
ResultsLung cohort patients had better survival outcomes than liver cohort patients. Primary tumors from patients in the liver cohort expressed a novel gene signature associated with ongoing replication stress (RS) response predictive of poor patient outcome independent from known subtypes. In contrast, patients with tumors lacking the RS response signature survived longer, especially if their tumors had alterations in DNA damage repair genes. A subset of patients in the lung cohort demonstrated new T cell clonal development in their primary and metastatic tumors leading to diverse peripheral blood TCR repertoires.
ConclusionLiver-avid metastatic PDAC is associated with an ongoing RS response, whereas tumors lacking the RS response with ongoing T cell clonal responses may have unique vulnerabilities allowing long-term survival in patients with lung-avid, liver-averse metastatic PDAC. | cancer biology |
10.1101/2022.05.06.490600 | Chromosomal position of ribosomal protein genes impacts long term evolution of Vibrio cholerae. | It is unclear how gene order within the chromosome influences bacterial evolution. The genomic location of genes encoding the flow of genetic information is biased towards the replication origin (oriC) in fast-growing bacteria. To study the role of chromosomal location on cell physiology we relocated the S10-spec- locus (S10), harboring half of ribosomal protein genes, to different chromosomal positions in the fast-growing pathogen V. cholerae. We found that growth rate, fitness and infectivity inversely correlated the distance between S10 and oriC. To gain insight into the evolutionary effect of RP genomic position, we evolved strains bearing S10 at its current oriC-proximal location or derivatives harboring the locus far from it (i.e. 1.5Mbp). Populations deep sequencing on average 1 mutation fixed each 100 generations, mainly at genes linked to flagellum regulation, lipopolysaccharide synthesis, chemotaxis, biofilm and quorum sensing. Along the experiment, populations showed an increment in biofilm forming capacity. All populations increased their growth rate. However, growth rate advantage of populations bearing S10 at an oriC-proximal persisted along the experiment over those where the main ribosomal protein gene cluster locates at an oriC-distal position. This indicates that suppressor mutations cannot compensate S10 genomic location. We selected fast-growing clones displaying a [~]10% growth rate increment finding that they harbored inactivating mutations at, among other sites, flagellum master regulators flrAB regardless S10 genomic location. The introduction of these mutations on naive V. cholerae strains resulted in a [~]10% increase in growth rate. Our study therefore demonstrates that the location of ribosomal protein genes conditions the evolutionary trajectory of growth rate in the long term. While genomic content is highly plastic in prokaryotes, gene order is an underestimated factor that conditions cellular physiology and lineage evolution. The lack of suppression enables artificial gene relocation for genetic circuit reprogramming. | microbiology |
10.1101/2022.05.06.490877 | Characteristics of intestinal microflora and dysbiosis in relation to the disease duration in patients with Meniere's disease | Menieres disease is an intractable disease characterized by paroxysms of intense rotatory dizziness, hearing loss, and other auditory symptoms. It is believed to be caused by endolymphatic edema of the vestibule and cochlea. The mechanism underlying endolymphatic edema is believed to be stress-related. Endolymphatic edema identification using cochlear contrast-enhanced MRI (hybrid of reversed image of positive endolymph signal and native image of positive perilymph signal [HYDROPS]) is an objective test for Menieres disease, enabling reliable diagnosis of this disease. Since dysbiosis of the intestinal microflora has been reported in stress-induced depression and irritable colitis, a similar abnormality may also occur in stress-triggered Menieres disease. Therefore, we investigated the gut microbiota of 10 patients (6 males and 4 females; mean age, 49.6 {+/-} 8.1 years) diagnosed with Menieres disease and marked endolymphatic edema on HYDROPS. These 10 patients underwent intestinal microbiota examination, and the results of the examination were evaluated in relation to the duration of the disease, the results of auscultation of the affected side, and Dizziness Handicap Inventory (DHI) scores. A significant negative correlation was found between the disease duration and Shannon diversity index and Faiths phylogenetic diversity, which indicated dysbiosis of the intestinal microflora. No correlation was found between the indicators of microbial diversity and the results of audiometry or the DHI on the affected side. Dysbiosis of the intestinal microbiota worsened with increasing duration of Menieres disease. Moreover, Akkermansia muciniphila was not detected in any patient with Menieres disease. Thus, despite the small number of cases in this study (n = 10), the findings indicate the possibility of abnormalities of the intestinal microflora in Menieres disease. | microbiology |
10.1101/2022.05.06.490959 | Local adaptation of Aedes aegypti mosquitoes to Wolbachia-induced fitness costs | Aedes aegypti mosquito eggs can remain quiescent for many months before hatching, allowing populations to persist through unfavorable conditions. Aedes aegypti infected with the Wolbachia strain wMel have been released in tropical and subtropical regions for dengue control. wMel reduces the viability of quiescent eggs, but this physiological cost might be expected to evolve in natural mosquito populations that frequently experience stressful conditions. We therefore compared the costs of wMel infection for quiescent egg viability in field-derived and laboratory populations. Quiescent egg viability was highly variable in wMel-infected populations, with greater costs of wMel in field-derived populations. In contrast, there was little variation between matched field-derived and long-term laboratory populations lacking wMel, suggesting that laboratory adaptation does not influence this trait and that differences are due to wMel infection. Comparisons of populations collected a year apart show a decline in costs under laboratory rearing conditions involving a rapid turnover of mosquito generations; this pattern was consistent across populations despite their origin, suggesting adaptation of mosquitoes to the wMel infection under laboratory conditions. Reciprocal crossing experiments confirm that differences in quiescent egg viability were mainly due to the genetic background and not Wolbachia alone. wMel-infected mosquitoes hatching from long-term quiescent eggs showed partial loss of cytoplasmic incompatibility and female infertility, highlighting additional costs of long-term quiescence. Our study provides the first evidence for a shift in Wolbachia phenotypic effects following deliberate field release and establishment and it highlights interactions between Wolbachia infections and local adaptation. The unexpected changes in fitness costs observed here suggest potential tradeoffs with undescribed fitness benefits of the wMel infection. | evolutionary biology |
10.1101/2022.05.06.490478 | Structure of C. elegans TMC-1 complex illuminates auditory mechanosensory transduction | The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction (MT) channel. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding MT, the composition, structure and mechanism of the MT complex has remained elusive. Here we report the single particle cryo-EM structure of the native MT TMC-1 complex isolated from C. elegans. The 2-fold symmetric complex is composed of 2 copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits while the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of particles in addition harbors a single arrestin-like protein, ARRD-6, bound to a CALM-1 domain. Single- particle reconstructions and molecular dynamics simulations show how the MT complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating. | biochemistry |
10.1101/2022.05.06.490940 | A curated data resource of 214K metagenomes for characterization of the global resistome | The growing threat of antimicrobial resistance (AMR) calls for new epidemiological surveillance methods, as well as a deeper understanding of how antimicrobial resistance genes (ARGs) have transmitted around the world. The large pool of sequencing data available in public repositories provides an excellent resource for monitoring the temporal and spatial dissemination of AMR in different ecological settings. However, only a limited number of research groups globally have the computational resources allowing analyses of such data. We retrieved 442 Tbp of sequencing reads from 214,095 metagenomic samples from the European Nucleotide Archive (ENA) and aligned them using a uniform approach against ARGs and 16S/18S rRNA genes. Here, we present the results of this extensive computational analysis and share the counts of reads aligned. Over 6.76 {middle dot} 108 read fragments were assigned to ARGs and 3.21 {middle dot} 109 to rRNA genes, where we observed distinct differences in both the abundance of ARGs and the link between microbiome and resistome compositions across various sampling types. This collection is another step towards establishing a global surveillance of AMR and can serve as a resource for further research into the environmental spread and dynamic changes of ARGs. | bioinformatics |
10.1101/2022.05.06.490939 | Effects of anti-fibrotic standard of care drugs on senescent human lung fibroblasts | RationaleCellular senescence is crucial in the progression of idiopathic pulmonary fibrosis (IPF), but it is yet unclear whether the standard-of-care (SOC) drugs nintedanib and pirfenidone have senolytic properties.
ObjectivesWe attempted to illuminate the effects of SOC drugs on senescent normal and IPF lung fibroblasts in vitro.
MethodsColorimetric/fluorimetric assays, qRT-PCR, and western blotting were used to evaluate the effect of SOC drugs on senescent normal and IPF lung fibroblasts.
ResultsSOC drugs did not induce apoptosis in the absence of death ligands in either normal or IPF senescent cells. Nintedanib increased caspase-3 activity in the presence of Fas Ligand (FasL) in normal but not in IPF senescent fibroblasts. Conversely, nintedanib enhanced B cell lymphoma (Bcl)-2 expression in senescent IPF lung fibroblasts. Moreover, in senescent IPF cells, pirfenidone alone induced mixed lineage kinase domain-like pseudokinase (MLKL) phosphorylation, provoking necroptosis. However, fragmented gasdermin D, indicating pyroptosis, was not detected under any condition. In addition, SOC drugs increased transcript levels of fibrotic and senescence markers in senescent IPF fibroblasts, whereas D+Q inhibited all these markers. Finally, D+Q enhanced growth differentiation factor 15 (GDF15) transcript and protein levels in both normal and IPF senescent fibroblasts.
ConclusionsIn the presence and absence of the extrinsic pro-apoptotic ligands, SOC drugs failed to trigger apoptosis in senescent fibroblasts, possibly due to enhanced Bcl-2 levels and the activation of the necroptosis pathway. SOC drugs elevated fibrotic and senescence markers in IPF lung fibroblasts. Together, these data demonstrated the inefficacy of SOC in targeting senescent cells. Further investigation is required to fully elucidate the therapeutic implications of SOC drugs on other senescent cell types in IPF. | pharmacology and toxicology |
10.1101/2022.05.06.490929 | The peptide GOLVEN10 controls nodule and lateral root organogenesis and positioning along the longitudinal root axis | O_LIGLV/RGF peptide encoding genes can be identified in genomes of all plants that can form roots or root-like structures suggesting they were essential for transition of plants to land.
C_LIO_LIIn Medicago truncatula, five of fifteen GOLVEN(GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide coding genes were induced during nodule organogenesis and to a varying extent under nitrogen deficiency and auxin treatment. Expression of MtGLV9 and MtGLV10 at nodule initiation sites was dependent on the transcription factor NODULE INCEPTION.
C_LIO_LIOverexpression of all five nodule-induced GLV genes in M. truncatula hairy roots as well as application of the corresponding synthetic peptides resulted in a 25-50% reduction in nodule number indicating GOLVENs are negative regulators of nodule organogenesis.
C_LIO_LIThe peptide GOLVEN10 shifted the position of the first formed lateral root (rhizotaxis) as well as the first formed nodule along the longitudinal primary root axis, a phenomenon we term nodulotaxis, thereby reducing the absolute length of the zone of lateral organ formation on roots.
C_LIO_LIApplication of synthetic GOLVEN10 peptide caused an increase in cell number but not cell length in each root cortical cell layer causing an increase in root length and a consequent spatiotemporal delay in formation of the first lateral organ.
C_LI
Plain Language SummaryNodule positioning is an understudied trait, yet it determines the length of the root that can support nodule formation and consequently the total number of functional nodules formed. We identify for the first time, genetic factors called GOLVEN peptides that alter nodule and lateral root positioning on the primary root along with several other traits including nodule organ initiation and root architecture. | plant biology |
10.1101/2022.05.06.490928 | Insulin docking within the open hemichannel of connexin 43 may reduce risk of amyotrophic lateral sclerosis | BackgroundType 2 diabetes (T2D), characterized by hyperinsulinemia, protects motor neurons against amyotrophic lateral sclerosis (ALS). Type 1 diabetes and a total lack of insulin are associated with increased risk of ALS. Connexin 43 (Cx43), an astrocyte protein, operates as an open pore via which toxic substances from the astrocytes reach the motor neurons.
MethodsIn the current study we performed molecular docking of insulin with monomeric Cx31, monomeric Cx43, and hexameric Cx31 to assess whether insulin might affect the pore. Hexameric Cx31 and hexameric Cx43 have hemichannels composed of 6 subunits that work as transmembrane channels, binding together to form gap junction intercellular channels that open and close. We used the program AutoDock Vina Extended for the molecular docking study.
ResultsCx31 shares amino acid and structural similarity to Cx43 and insulin docks to the same position of the N-terminal domain of monomeric Cx31 and monomeric Cx43. Insulin docks within the open hemichannel of hexameric Cx31, potentially blocking it. The block may be responsible for the protective relationship of T2D to ALS.
ConclusionInsulin might be a treatment for ALS, especially since insulin enhances glucose-stimulated insulin secretion in healthy humans. An insulin secretogogue such as oral sulfonylurea or glinide might also be of value. | neuroscience |
10.1101/2022.05.06.489035 | Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 to drive osteoarthritis | ObjectivesSynovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/{beta}-catenin signaling, given its emerging importance in arthritis.
MethodsWe subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain-of-function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages, and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types.
ResultsWe uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/{beta}-catenin signaling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages, and chondrocytes.
ConclusionsSynovial fibroblasts assume distinct functional identities during PTOA, and Prg4hi lining fibroblasts secrete the Wnt agonist Rspo2 to drive pathological crosstalk in the joint after injury. | cell biology |
10.1101/2022.05.06.490948 | Messenger-RNA Modification Standards and Machine Learning Models Facilitate Absolute Site-Specific Pseudouridine Quantification | Enzyme-mediated chemical modifications to mRNA are important for fine-tuning gene expression, but they are challenging to quantify due to low copy number and limited tools for accurate detection. Existing studies have typically focused on the identification and impact of adenine modifications on mRNA (m6A and inosine) due to the availability of analytical methods. The pseudouridine ({Psi}) mRNA modification is also highly abundant but difficult to detect and quantify because there is no available antibody, it is mass silent, and maintains canonical basepairing with adenine. Nanopores may be used to directly identify {Psi} sites in RNAs using a systematically miscalled base, however, this approach is not quantitative and highly sequence dependent. In this work, we apply supervised machine learning models that are trained on sequence-specific, synthetic controls to endogenous transcriptome data and achieve the first quantitative {Psi} occupancy measurement in human mRNAs. Our supervised machine learning models reveal that for every site studied, different signal parameters are required to maximize {Psi} classification accuracy. We show that applying our model is critical for quantification, especially in low-abundance mRNAs. Our engine can be used to profile {Psi}-occupancy across cell types and cell states, thus providing critical insights about physiological relevance of {Psi} modification to mRNAs. | genomics |
10.1101/2022.05.06.490934 | Atomic protein structure refinement using all-atom graph representations and SE(3)-equivariant graph neural networks | Three-dimensional (3D) protein structures reveal the fundamental information about protein function. The state-of-art protein structure prediction methods such as Alphafold are being widely used to predict structures of uncharacterized proteins in biomedical research. There is a significant need to further improve the quality and nativeness of the predicted structures to enhance their usability. Current machine learning methods of refining protein structures focus mostly on improving the backbone quality of predicted structures without effectively leveraging and enhancing the conformation of all atoms including sidechains, while molecular simulation methods are computationally intensive and time-consuming.
SummaryIn this work, we develop ATOMRefine, a deep learning-based, end-to-end, all-atom protein structural model refinement method. It uses a SE(3)-equivariant graph transformer network that is equivariant to the rotation and translation of 3D structures in conjunction with a novel graph representation of all atoms to directly refine protein atomic coordinates of all the atoms in a predicted tertiary structure represented as a molecular graph. The method is first trained and tested on the structural models in AlphafoldDB whose experimental structures are known, and then blindly tested on 69 CASP14 regular targets and 7 CASP14 refinement targets. ATOMRefine improves the quality of both backbone atoms and all-atom conformation of the initial structural models generated by AlphaFold. It also performs better than the state-of-the-art refinement methods in multiple evaluation metrics including an all-atom model quality score - the MolProbity score based on the analysis of all-atom contacts, bond length, atom clashes, torsion angles, and sidechain rotamers. As ATOMRefine can refine a protein structure quickly, it provides a viable, fast solution for improving protein geometry and fixing structural errors of predicted structures through direct coordinate refinement. | bioinformatics |
10.1101/2022.05.06.490351 | Metagenomic analysis of coprolites from three Late Pleistocene megaherbivores from the Southwestern United States. | 1.BackgroundDetermining the life-history traits of extinct species is often difficult from skeletal remains alone, limiting the accuracy of studies modeling past ecosystems. However, the analysis of the degraded endogenous bacterial DNA present in paleontological fecal matter (coprolites) may enable the characterization of specific traits such as the hosts digestive physiology and diet. An issue when evaluating the microbial composition of coprolites is the degree to which the microbiome is representative of the hosts original gut community versus the changes that occur in the weeks following deposition due to desiccation. Analyses of paleontological microorganisms are also relevant in the light of recent studies linking the Late Pleistocene and Early Holocene extinctions with modern-day zoonotic pathogen outbreaks.
MethodsShotgun sequencing was performed on ancient DNA (aDNA) extracted from coprolites of the Columbian mammoth (Mammuthus Columbi), Shasta ground sloth (Nothrotheriops shastensis) and paleontological bison (Bison sp.) collected from caves on the Colorado Plateau, Southwestern USA. The novel metagenomic classifier MTSv, parameterized for studies of aDNA, was used to assign bacterial taxa to sequencing reads. The resulting bacterial community of coprolites was then compared to those from modern fecal specimens of the African savannah elephant (Loxodonta africana), the brown-throated sloth (Bradypus variegatus) and the modern bison (Bison bison). Both paleontological and modern bison fecal bacterial communities were also compared to those of progressively dried cattle feces to determine whether endogenous DNA from coprolites had a microbiome signal skewed towards aerobic microorganisms typical of desiccated fecal matter.
ResultsThe diversity of phyla identified from coprolites was lower than modern specimens. The relative abundance of Actinobacteria was increased in coprolites compared to modern specimens, with fewer Bacteroidetes and Euryarchaeota. Firmicutes had a reduced relative abundance in the mammoth and bison coprolites, compared to the African savanna elephants and modern bison. There was a significant separation of samples in NMDS plots based on their classification as either paleontological or modern, and to a lesser extent, based on the host species. Increasingly dried cattle feces formed a continuum between the modern and paleontological bison samples.
ConclusionOur results reveal that any coprolite metagenomes should always be compared to desiccated modern fecal samples from closely related hosts fed a comparable diet to determine the degree to which the coprolite metagenome is a result of desiccation versus true dissimilarities between the modern and paleontological hosts. Also, a large-scale desiccation study including a variety of modern species may shed light on life-history traits of extinct species without close extant relatives, by establishing the proximity of coprolite metagenomes with those from dried modern samples. | paleontology |
10.1101/2022.05.06.490932 | Highly efficient healing of critical sized articular cartilage defect in situ using a chemically nucleoside-modified mRNA-enhanced cell therapy | Critical sized cartilage defects heal poorly and MSC-based therapies holds promise functional cartilage regeneration either used alone or in combination with growth factors. However, Recombinant protein growth factors were proven to have minimal benefits while to have adverse side effects and high cost. Nonviral mRNA delivery provides a promising, alternative approach to delivering therapeutic proteins within defect lesion for an extended period of time. Despite successful therapeutic outcome in bone and other vascularized tissues, the therapeutic application of mRNA in poorly vascularized tissues such as cartilage is still facing many challenges and rarely studied. We report here using chemically modified messenger RNA encoding TGF-{beta}3(TGF-{beta}3 cmRNA) to enhance the therapeutic efficacy of BMSCs to efficient repair of cartilage defect. Local administration of TGF-{beta}3 cmRNA enhanced BMSCs therapy restored critical-sized cartilage defects in situ in a rat model within 6 weeks with structural and molecular markers similar to its nature counterparts. In addition, the development of osteoarthritis caused by cartilage damage was prevented by this mRNA-enhanced BMSCs therapy evidenced by minimal late-stage OA pharmacological presentations. This novel mRNA enhanced-MSC technology extend the development of new therapeutic approaches for treating functional cartilage repair. | synthetic biology |
10.1101/2022.05.06.490483 | Developmental Morphogens Direct Human Induced Pluripotent Stem Cells Towards an Annulus Fibrosus-like Cell Phenotype | Therapeutic interventions for intervertebral disc herniation remain scarce due to the inability of endogenous annulus fibrosus (AF) cells to respond to injury and drive tissue regeneration. Unlike other orthopaedic tissues, such as cartilage, delivery of exogenous cells to the site of annular injury remains underdeveloped, largely due to a lack of an ideal cell source and the invasive nature of cell isolation. Human induced pluripotent stem cells (iPSCs) can be differentiated to specific cell fates using biochemical factors and are, therefore, an invaluable tool for cell therapy approaches. While differentiation protocols have been developed for cartilage and fibrous connective tissues (e.g., tendon), the signals that regulate the induction and differentiation of human iPSCs towards the annulus fibrosus fate remain unknown. Here, we screened a number of candidate factors (and their combinations) and assessed the transcriptomic signatures of key signaling factors involved in embryonic AF development and differentiated function. The transcriptional signatures of treated cells were compared to those of mature human AF cells, and conditions that promoted expression of annulus fibrosus extracellular matrix genes and key transcription factors involved in embryonic AF development were identified. These findings represent an initial approach to guide human induced pluripotent stem cells towards an annulus fibrosus-like fate for cellular delivery strategies. | cell biology |
10.1101/2022.05.06.490937 | Modulation of Tau Tubulin Kinases (TTBK1 and TTBK2) Impacts Ciliogenesis | Tau tubulin kinase 1 and 2 (TTBK1 and TTBK2) are highly homologous kinases that are expressed and mediate disease-relevant pathways predominantly in the brain. Distinct roles for TTBK1 and TTBK2 have been delineated. While efforts have been devoted to characterizing the impact to TTBK1 inhibition in diseases like Alzheimers disease and amyotrophic lateral sclerosis, TTBK2 inhibition has been less explored. TTBK2 serves a critical function during cilia assembly. Given the biological importance of these kinases, we designed a targeted library from which we identified several chemical tools that engage TTBK1 and TTBK2 in cells and inhibit their downstream signaling. Indolyl pyrimidinamine 10 significantly reduced the expression of primary cilia on the surface of human induced pluripotent stem cells (iPSCs). Furthermore, analog 10 phenocopies TTBK2 KO in iPSCs, confirming an essential role for TTBK2 in ciliogenesis. | cell biology |
10.1101/2022.05.06.490951 | Morphogen Directed Coordination of GPCR Activity Promotes Primary Cilium Function for Downstream Signaling | Primary cilium dysfunction triggers catastrophic failure of signal transduction pathways that organize through cilia, thus conferring significant pressure on such signals to ensure ciliary homeostasis. Intraflagellar transport (IFT) of cargo that maintains the primary cilium is powered by high ciliary cAMP. Paradoxically, Sonic Hedgehog (SHH) signaling, for which ciliary function is crucial, triggers a reduction in ciliary cAMP that could blunt downstream signaling by slowing IFT. We investigated this paradox and mapped a novel signal relay driven by SHH-stimulated prostaglandin E2 that stabilizes ciliary cAMP flux through by activating Gs-coupled EP4 receptor. Chemical or genetic blockade of the SHH-EP4 relay cripples cAMP buffering, which leads to decreased intraciliary cAMP, short cilia, and attenuated SHH pathway induction. Accordingly, EP4-/- mice show pronounced ciliary defects and altered SHH-dependent neural tube patterning. Thus, SHH orchestrates a sophisticated ciliary GPCR-cAMP signaling network that ensures primary cilium fitness for a robust downstream signaling response. | cell biology |
10.1101/2022.05.06.490954 | Independent regulation of mtDNA quantity and quality resets the mitochondrial genome in C. elegans primordial germ cells | Mitochondria contain an independent genome, called mtDNA, which contains essential metabolic genes. Although mtDNA mutations occur at high frequency, they are inherited infrequently, indicating that germline mechanisms limit their accumulation. To determine how germline mtDNA is regulated, we examined the control of mtDNA quantity and quality in C. elegans primordial germ cells (PGCs). We show that PGCs generate a bottleneck in mtDNA number by segregating mitochondria into lobe-like protrusions that are cannibalized by adjacent cells, reducing mtDNA content two-fold. As PGCs exit quiescence and divide, mtDNAs replicate to maintain a set point of [~]200 mtDNAs per germline stem cell. Whereas PGC lobe cannibalism eliminates mtDNAs stochastically, we show that the kinase PINK1, operating independently of Parkin and autophagy, preferentially reduces the fraction of mutant mtDNAs. Thus, PGCs employ parallel mechanisms to control both the quantity and quality of the founding population of germline mtDNAs. | cell biology |
10.1101/2022.05.06.490935 | New hypotheses of cell type diversity and novelty from comparative single cell and nuclei transcriptomics in echinoderms | Cell types are the fundamental building blocks of metazoan biodiversity and offer a powerful perspective for inferring evolutionary phenomena. With the development of single-cell transcriptomic techniques, new definitions of cell types are emerging. This allows a conceptual reassessment of traditional definitions of novel cell types and their evolution. Research in echinoderms, particularly sea star and sea urchin embryos have contributed significantly to understanding the evolution of novel cell types, in particular the primary mesenchyme cells (PMCs) and pigment cells that are found in sea urchin but not sea star embryos. This paper outlines the development of a gene expression atlas for the bat star, Patiria miniata, using single nuclear RNA sequencing (snRNA-seq) of embryonic stages. The atlas revealed 22 cell clusters covering all expected cell types from the endoderm, mesoderm and ectoderm germ layers. In particular, four distinct neural clusters, an immune cluster, and distinct right and left coelom clusters were revealed as distinct cell states. A comparison with Strongylocentrotus purpuratus embryo single cell transcriptomes was performed using 1:1 orthologs to anchor and then compare gene expression patterns. S. purpuratus primordial germ cell equivalents were not detected in P. minata, while the left coelom of P. miniata has no equivalent cell cluster in S. purpuratus. Pigment cells of S. purpuratus map to clusters containing immune mesenchyme and neural cells of P. miniata, while the PMCs of S. purpuratus are revealed as orthologous to the right coelom cluster of P. miniata. These results suggest a new interpretation of the evolution of these well-studied cell types and a reflection on the definition of novel cell types. | evolutionary biology |
10.1101/2022.05.06.490957 | Reduced representation sequencing accurately quantifies relative abundance and reveals population-level variation in Pseudo-nitzschia spp. | Certain species within the genus Pseudo-nitzschia are able to produce the neurotoxin domoic acid (DA), which can cause illness in humans, mass-mortality of marine animals, and closure of commercial and recreational shellfisheries during toxic events. Understanding and forecasting blooms of these harmful species is a primary management goal. However, accurately predicting the onset and severity of bloom events remains difficult, in part because the underlying drivers of bloom formation have not been fully resolved. Furthermore, Pseudo-nitzschia species often co-occur, and recent work suggests that the genetic composition of a Pseudo-nitzschia bloom may be a better predictor of toxicity than prevailing environmental conditions. We developed a novel next-generation sequencing assay using restriction site-associated DNA (2b-RAD) genotyping and applied it to mock Pseudo-nitzschia communities generated by mixing cultures of different species in known abundances. On average, 94% of the variance in observed species abundance was explained by the expected abundance. In addition, the false positive rate was low (0.45% on average) and unrelated to read depth, and false negatives were never observed. Application of this method to environmental DNA samples collected during natural Pseudo-nitzschia spp. bloom events in Southern California revealed that increases in DA were associated with increases in the relative abundance of P. australis. Although the absolute correlation across time-points was weak, an independent species fingerprinting assay (Automated Ribosomal Intergenic Spacer Analysis) supported this and identified other potentially toxic species. Finally, we assessed population-level genomic variation by mining SNPs from the environmental 2bRAD dataset. Consistent shifts in allele frequencies in P. pungens and P. subpacifica were detected between high and low DA years, suggesting that different intraspecific variants may be associated with prevailing environmental conditions or the presence of DA. Taken together, this method presents a potentially cost-effective and high-throughput approach for studies aiming to evaluate both population and species dynamics in mixed samples.
HighlightsO_LI2bRAD method facilitates species- and population-level analysis of the same sample
C_LIO_LIMethod accurately quantifies species relative abundance with low false positives
C_LIO_LIConsistent shifts in allele frequencies were detected between high and low DA years
C_LIO_LICertain Pseudo-nitzschia spp. populations may be more associated with DA presence
C_LI | genomics |
10.1101/2022.05.06.490936 | Proteome Remodeling of the Eye Lens at 50 Years Identified with Data-Independent Acquisition | The eye lens is responsible for focusing and transmitting light to the retina. The lens does this in the absence of organelles yet maintains transparency for at least five decades before onset of age-related nuclear cataract (ARNC). It is hypothesized that oxidative stress contributes significantly to ARNC formation. It is additionally hypothesized that transparency is maintained by a microcirculation system (MCS) that delivers antioxidants to the lens nucleus and exports small molecule waste. Common data-ependent acquisition (DDA) methods are hindered by dynamic range of lens protein expression and provide limited context to age-related changes in the lens. In this study we utilized data-independent acquisition (DIA) mass spectrometry to analyze the urea insoluble, membrane protein fractions of 16 human lenses subdivided into three spatially distinct lens regions to characterize age-related changes, particularly concerning the lens MCS and oxidative stress response. In this pilot cohort, we measured 4,788 distinct protein groups, 46,681 peptides, and 7,592 deamidated sequences, more than in any previous human lens DDA approach. Our results reveal age-related changes previously known in lens biology and expand on these findings, taking advantage of the rich dataset afforded by DIA. Principally, we demonstrate that a significant proteome remodeling event occurs at approximately 50 years of age, resulting in metabolic preference for anaerobic glycolysis established with organelle degradation, decreased abundance of protein networks involved in calcium-dependent cell-cell contacts while retaining networks related to oxidative stress response. Further, we identified multiple antioxidant transporter proteins not previously detected in the human lens and describe their spatiotemporal and age-related abundance changes. Finally, we demonstrate that aquaporin-5, among other proteins, is modified with age by PTMs including deamidation and truncation. We suggest that the continued accumulation of each of these age-related outcomes in proteome remodeling contribute to decreased fiber cell permeability and result in ARNC formation. | biochemistry |
10.1101/2022.05.06.490945 | Replicative Instability Drives Cancer Progression | In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress have been shown to exhibit genomic instability and poor survival rates across tumor types. Here we utilize-omics data from two independent consortia to identify the genetic underpinnings of replication stress, therapy resistance, and primary carcinoma to brain metastasis in BRCA wildtype tumors. In doing so, we have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by genomic evolution secondary to replicative challenge. Our data supports a model whereby defective single-strand break repair, translesion synthesis, and non-homologous end joining effectors drive RIN. Collectively, we find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution.
Statement of SignificanceDefining the genetic basis of genomic instability with wildtype BRCA repair effectors is a significant unmet need in cancer research. Here we identify and characterize a pan-cancer cohort of tumors driven by replicative instability (RIN). We find that RIN drives therapy resistance and distant metastases across multiple tumor types. | cancer biology |
10.1101/2022.05.07.490557 | Single-cell atlas of mouse limb development reveals a complex spatiotemporal dynamics of skeleton formation | Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex nonconsecutive three-dimensional pattern.
Single-cell RNA sequencing of the developing mouse forelimb revealed three progenitor states: naive, proximal and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments.
Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest a new model for limb skeleton development. | developmental biology |
10.1101/2022.05.07.491018 | A synthetic tear protein resolves dry eye through promoting corneal nerve regeneration. | Corneal architecture is essential for vision and is greatly perturbed by the absence of tears due to the highly prevalent disorder dry eye. With no regenerative therapies available, pathological alterations of the ocular surface in response to dryness, including persistent epithelial defects and poor wound healing, result in lifelong morbidity. Here, using a mouse model of aqueous-deficient dry eye, we reveal that topical application of the synthetic tear protein lacripep reverses the pathological outcomes of dry eye through restoring the extensive network of corneal nerves that are essential for tear secretion, barrier function, epithelial homeostasis and wound healing. Intriguingly, the restorative effects of lacripep occur despite extensive immune cell infiltration, suggesting tissue reinnervation and regeneration can be achieved under chronic inflammatory conditions. In summary, our data highlight lacripep as a first-in-class regenerative therapy for returning the cornea to a near homeostatic state in individuals who suffer from dry eye.
TeaserTopical application of a synthetic tear protein repairs dry eye-mediated corneal damage through driving functional sensory reinnervation. | cell biology |
10.1101/2022.05.07.491023 | Crosstalk between age accumulated DNA-damage and the SIRT1-AKT-GSK3b axis in urine derived renal progenitor cells | The aging process is manifested by a multitude of interlinked biological processes. These processes contribute to genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. Together these are recognized as of the main risk factors of the worlds most prevalent diseases, such as neurodegenerative disorders, cancer, cardiovascular disease, and metabolic disease.
The mammalian ortholog of the yeast silent information regulator (Sir2) SIRT1 is a NAD+-dependent class III histone deacetylase and has been recognized to be involved in many of the forementioned processes. Therefore, its activity is connected to aging via the regulation of apoptosis, cell differentiation, development, stress response, metabolism, and tumorigenesis. Furthermore, the physiological activity of several sirtuin family members has been connected to the regulation of life span of lower organisms (Caenorhabditis elegans and Drosophila melanogaster) as well as mammals. Aging in somatic cells of mammals is accompanied by mutations and other forms of DNA damage. These might manifest in transient cell cycle arrest associated with DNA repair, apoptosis, senescence, or cell differentiation. The activity of SIRT1 has previously been reported to be regulated by the DNA damage response pathway. On the one hand, SIRT1 is recruited from ATM to DBS and is required for DNA damage repair, but on the other hand, SIRT1 activity was also found to be negatively regulated by genotoxic stress via the interaction of ATM with Deleted in Breast Cancer 1 (DBC1). Increased levels of DBS are associated with downregulation of ATM and lower phosphorylation levels of AKT and GSK3{beta}, with significant implications for mesenchymal stem cell (MSC) maintenance and differentiation. In this proposed "stem cell checkpoint," the ATM signalling pathway initiated by DBS maintains MSCs and blocks their differentiation. Based on this, it has already been established that in senescent mesenchymal stem cells, SIRT1 expression is decreased, while its overexpression delays the onset of senescence and loss of differentiation capacity/ability.
In the present study, we provide evidence that SIX2-positive urine derived renal progenitor cells-UdRPCs isolated directly from human urine show typical hallmarks of aging when obtained from elderly donors. This includes the transcriptional downregulation of SIRT1 and its downstream targets AKT and GSK3{beta}. This transcriptional downregulation is accompanied by an increase in DNA damage and transcriptional levels several cell cycle inhibitors such as P16, reflecting possibly the ATM induced "stemness checkpoint" to maintain UdRPC stemness and differentiation capacity. We provide evidence that the renal progenitor transcription factor SIX2 binds to the coding sequence of SIRT1 and both factors mutually influence the transcription of each other. Furthermore, we show that the SIRT1 promoter region is methylation sensitive and becomes subsequently methylated in UdRPCs derived from aged donors, dividing them into SIRT1 high and low expressing UdRPCs. This downregulation might render the cells more vulnerable to endogenous noxae accelerating the accumulation of DNA damage and ultimately the accumulation of aging associated hallmarks. | cell biology |
10.1101/2022.05.06.490961 | Protrusion growth driven by myosin-generated force | Actin-based protrusions are found on the surface of all eukaryotic cells, where they support diverse biological activities essential for life. Models of protrusion growth hypothesize that actin filament assembly provides the mechanical force for bending the plasma membrane outward. However, membrane-associated myosin motors are also abundant in protrusions, though their potential for contributing growth-promoting force remains unexplored. Using a novel inducible system that docks myosin motor domains to membrane binding modules with temporal control, we found that the application of myosin-generated force to the plasma membrane is sufficient for driving robust elongation of protrusions. Protrusion growth scaled with motor accumulation, required active, barbed end-directed force, and was independent of cargo delivery or the recruitment of canonical barbed end elongation factors. Application of growth-promoting force was also supported by structurally distinct myosin motor domains and membrane binding modules. We conclude that myosin-generated force can drive protrusion growth and this mechanism is likely active in diverse biological contexts. | cell biology |
10.1101/2022.05.07.490949 | MorphoFeatures: unsupervised exploration of cell types, tissues and organs in volume electron microscopy | Electron microscopy (EM) provides a uniquely detailed view of cellular morphology, including organelles and fine subcellular ultrastructure. While the acquisition and (semi-)automatic segmentation of multicellular EM volumes is now becoming routine, large-scale analysis remains severely limited by the lack of generally applicable pipelines for automatic extraction of comprehensive morphological descriptors. Here, we present a novel unsupervised method for learning cellular morphology features directly from 3D EM data: a convolutional neural network delivers a representation of cells by shape and ultrastructure. Applied to the full volume of an entire three-segmented worm of the annelid Platynereis dumerilii, it yields a visually consistent grouping of cells supported by specific gene expression profiles. Integration of features across spatial neighbours can retrieve tissues and organs, revealing, for example, a detailed organization of the animal foregut. We envision that the unbiased nature of the proposed morphological descriptors will enable rapid exploration of very different biological questions in large EM volumes, greatly increasing the impact of these invaluable, but costly resources. | developmental biology |
10.1101/2022.05.02.490263 | Musashi1 and its short C-terminal variants regulate pluripotency states in embryonic stem cells | Musashi1 (MSI1) is a marker for adult stem cells, but little is known for its expression and function in pluripotent stem cells (PSCs). Here we report that MSI1 is expressed in embryonic stem cells (ESCs) and is required for pluripotency maintenance. We found that there exit short c-terminal MSI1 variants (MSI1-C, hMSI1272-362 or mMSI1138-362) in naive but not primed ESCs. When overexpressed, MSI1 and MSI1-C variants facilitate primed-to-naive pluripotency transition by elevating the pluripotency of primed hESCs toward a formative-like state, enable better survival of hESCs in human-mouse interspecies cell completion, and enhance the ability of blastoid formation of hESCs after naive induction. Mechanistically, we show that the MSI1-C variants, though lacking RNA recognition motifs, bind to RNAs, enhance stress resistance and upregulate DNA damage repair genes. Thus, this study demonstrates that ESCs utilize MSI1 and the newly identified short MSI1-C proteins as double swords to regulate pluripotency states. | developmental biology |
10.1101/2022.05.05.490722 | The effect of dietary chitin on Atlantic salmon (Salmo salar) chitinase activity, gene expression, and microbial composition | BackgroundChitin is a common component in the natural diet of many fish, and a range of chitinases with the potential to down chitin have been identified. Yet whether chitin is metabolized in fish is still unclear. Here we used a combination of chitinase activity assay, transcriptomics, and 16S rRNA bacterial analysis to assess the effect of chitin supplementation on Atlantic salmon gene expression and microbial community.
ResultsAtlantic salmon express multiple genes associated with chitin metabolism, and we show that the expression and activity of Atlantic salmon chitinases are not affected by the addition of dietary chitin. We do, however, demonstrate an association between gut microbial composition, chitinase activity in the gut, and host chitinase expression.
ConclusionThe findings presented here support the idea that chitin metabolism genes are linked to the maintenance of a chitin-based barrier in the teleost gut. These results contribute to a greater understanding of chitin metabolism in fish. | genetics |
10.1101/2022.05.07.487787 | Evolution towards small colony variants of pandemic multidrug resistant ST131 Escherichia coli isolates from a 10-year bone infection | BackgroundChronic wounds are usually challenging to treat due to underlying medical conditions of the individual and as they readily become infected by microorganisms due to the failure of mechanical and physiological first line innate immune responses. We report here the characterization of host adaptation of five E. coli genomes including three E. coli ST131 genomes that occurred concomitantly with Enterococcus faecalis from a 10-year chronic wound infection after a foot fracture during the 2004 tsunami.
MethodsThe five E. coli strains were characterized by various microbiological and genomic approaches. Microbiological methods were antimicrobial resistance, growth in different media and biofilm formation. Genomic methods were determination of the genome sequence by PacBio RSII and Illumina sequencing. Phylogenetic analyses and genome alterations such as single nucleotide polymorphisms, deletion and rearrangements that led to pseudogenes and chromosomal inversions were documented. Relevant selected metabolic and physiological pathways were analyzed for integrity.
FindingStrains of two initially present sequence types, including the highly antimicrobial resistant ST405 clone, were subsequently replaced by isolates of the ubiquitous ST131 clone. The three E. coli ST131 strains showed a heavily host-adapted genome with a high number of pseudogenes and a large chromosomal inversion compared to ST131 reference strains. Furthermore, two of three E. coli ST131 isolates were small colony variants with its genetic basis in multiple genome alterations including pseudogenes and deletions in the pathway for heme biosynthesis. Pseudogene analysis indicated also the three ST131 strains to be mutator strains. Although enhanced capability of biofilm formation of the ST131 isolates was indicated by the agar plate assay, the a liquid culture biofilm assay did not display pronounced biofilm formation suggesting unconventional modes of biofilm formation.
InterpretationST131 clone members, which originally appeared as commensal strains can cause urinary tract and blood stream infections and are ubiquitously found in the environment including waste water and in animals. ST131 strains have presumably been already acquired from the environment on occurrence of the initial foot fracture and can persist in wounds showing an outmost genome plasticity and adaptability which might causing the chronic infection. Although co-infection with E. faecalis might have supported chronicity, these findings indicate that in individuals with underlying metabolic diseases wound infection by ST131 E. coli isolates can be a health risk.
FundingThis work was partially funded by ALF. | microbiology |
10.1101/2022.05.07.491039 | Multi-level force-dependent allosteric enhancement of αE-catenin binding to F-actin by vinculin | Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via {beta}-catenin, - catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in E-catenin conformation enables it to recruit the actin-binding protein vinculin to cell-cell junctions, where it contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell-cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (-) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. Further, load is distributed asymmetrically among complexes, such that only one bears the majority of applied load. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (-) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization. | biophysics |
10.1101/2022.05.07.490997 | A zebrafish embryo screen utilizing gastrulation for identification of anti-metastasis drugs | Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a phenotype-based chemical screen utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on our hypothesis that small molecules which interrupt zebrafish gastrulation might suppress metastasis progression of cancer cells, we designed a drug screening concept which uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables 100 chemicals to be tested in five hours through just observing epiboly progression of a test chemical-treated embryos. In the screen, embryos at two-cell stage are firstly corrected and then developed to sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to 90% epiboly stage. Finally, positive hit chemicals that interrupt epiboly progression are selected through comparing epiboly progression of a test chemical-treated embryos with that of vehicle-treated embryos under a stereoscopic microscope. Previous study subjected 1280 FDA-approved drugs into the screen and identified Adrenosterone and Pizotifen as epiboly-interrupting drugs. Mouse models of metastasis validated that the drugs could suppress metastasis of breast cancer cells. Furthermore, the screen can be diverted to a chemical genetic screening platform for identification of metastasis-promoting genes. Hydroxysteroid (11-Beta) Dehydrogenase 1 (HSD11b1) and serotonin receptor 2C (HTR2C), which are primary target of Adrenosterone and Pizotifen respectively, induces epithelial-mesenchymal transition (EMT) and promote metastasis of breast cancer cells. | cancer biology |
10.1101/2022.05.07.491029 | Herpes simplex virus 1 entry glycoproteins form stable complexes prior to and during membrane fusion | Herpesviruses - ubiquitous pathogens that cause persistent infections - have some of the most complex cell entry mechanisms. Entry of the prototypical herpes simplex virus 1 (HSV-1) requires coordinated efforts of 4 glycoproteins, gB, gD, gH, and gL. The current model posits that the glycoproteins do not interact prior to receptor engagement and that binding of gD to its receptor causes a "cascade" of sequential pairwise interactions, first activating the gH/gL complex and subsequently activating gB, the viral fusogen. But how these glycoproteins interact remains unresolved. Here, using a quantitative split-luciferase approach, we show that pairwise HSV-1 glycoprotein complexes form prior to fusion, remain stable throughout fusion, and do not depend on the presence of the cellular receptor. Based on our findings, we propose a revised "conformational cascade" model of HSV-1 entry. We hypothesize that all 4 glycoproteins assemble into a complex prior to fusion, with gH/gL positioned between gD and gB. Once gD binds to a cognate receptor, the proximity of the glycoproteins within this complex allows for efficient transmission of the activating signal from the receptor-activated gD to gH/gL to gB through sequential conformational changes, ultimately triggering the fusogenic refolding of gB. Our results also highlight previously unappreciated contributions of the transmembrane and cytoplasmic domains to glycoprotein interactions and fusion. Similar principles could be at play in other multicomponent viral entry systems, and the split luciferase approach used here is a powerful tool for investigating protein-protein interactions in these and a variety of other systems.
IMPORTANCEHerpes simplex virus 1 (HSV-1) infects the majority of humans for life and can cause diseases ranging from painful sores to deadly brain inflammation. No vaccines or curative treatments currently exist. HSV-1 infection of target cells requires coordinated efforts of four viral glycoproteins. But how these glycoproteins interact remains unclear. Using a quantitative protein interaction assay, we found that HSV-1 glycoproteins form stable, receptor-independent complexes. We propose that the 4 proteins form a complex, which could facilitate transmission of the entry-triggering signal from the receptor-binding component to the membrane fusogen component through sequential conformational changes. Similar principles could be applicable across other multicomponent protein systems. A revised model of HSV-1 entry could facilitate the development of therapeutics targeting this process. | microbiology |
10.1101/2022.05.07.491029 | Herpes simplex virus 1 entry glycoproteins form stable complexes prior to and during membrane fusion | Herpesviruses - ubiquitous pathogens that cause persistent infections - have some of the most complex cell entry mechanisms. Entry of the prototypical herpes simplex virus 1 (HSV-1) requires coordinated efforts of 4 glycoproteins, gB, gD, gH, and gL. The current model posits that the glycoproteins do not interact prior to receptor engagement and that binding of gD to its receptor causes a "cascade" of sequential pairwise interactions, first activating the gH/gL complex and subsequently activating gB, the viral fusogen. But how these glycoproteins interact remains unresolved. Here, using a quantitative split-luciferase approach, we show that pairwise HSV-1 glycoprotein complexes form prior to fusion, remain stable throughout fusion, and do not depend on the presence of the cellular receptor. Based on our findings, we propose a revised "conformational cascade" model of HSV-1 entry. We hypothesize that all 4 glycoproteins assemble into a complex prior to fusion, with gH/gL positioned between gD and gB. Once gD binds to a cognate receptor, the proximity of the glycoproteins within this complex allows for efficient transmission of the activating signal from the receptor-activated gD to gH/gL to gB through sequential conformational changes, ultimately triggering the fusogenic refolding of gB. Our results also highlight previously unappreciated contributions of the transmembrane and cytoplasmic domains to glycoprotein interactions and fusion. Similar principles could be at play in other multicomponent viral entry systems, and the split luciferase approach used here is a powerful tool for investigating protein-protein interactions in these and a variety of other systems.
IMPORTANCEHerpes simplex virus 1 (HSV-1) infects the majority of humans for life and can cause diseases ranging from painful sores to deadly brain inflammation. No vaccines or curative treatments currently exist. HSV-1 infection of target cells requires coordinated efforts of four viral glycoproteins. But how these glycoproteins interact remains unclear. Using a quantitative protein interaction assay, we found that HSV-1 glycoproteins form stable, receptor-independent complexes. We propose that the 4 proteins form a complex, which could facilitate transmission of the entry-triggering signal from the receptor-binding component to the membrane fusogen component through sequential conformational changes. Similar principles could be applicable across other multicomponent protein systems. A revised model of HSV-1 entry could facilitate the development of therapeutics targeting this process. | microbiology |
10.1101/2022.05.06.490989 | Conservation of energetic pathways for electroautotrophy in the uncultivated candidate order Tenderiales | Electromicrobiology can be used to understand extracellular electron uptake in previously undescribed chemolithotrophs. Enrichment and characterization of the uncultivated electroautotroph "Candidatus Tenderia electrophaga" using electromicrobiology led to the designation of the order Tenderiales. Representative Tenderiales metagenome assembled genomes (MAGs) have been identified in a number of environmental surveys, yet a comprehensive characterization of conserved genes for extracellular electron uptake has thus far not been conducted. Using comparative genomics we identified conserved orthologous genes within the Tenderiales and nearest neighbor orders important for extracellular electron uptake based on a previously proposed pathway from "Ca. Tenderia electrophaga". The Tenderiales contained a conserved cluster we designated uetABCDEFGHIJ, which encodes proteins containing features that would enable transport of extracellular electrons to cytoplasmic membrane bound energy transducing complexes such as two conserved cytochrome cbb3 oxidases. For example, UetJ is predicted to be an extracellular undecaheme c-type cytochrome that forms a heme wire. We also identified clusters of genes predicted to facilitate assembly and maturation of electron transport proteins, as well as cellular attachment to surfaces. Autotrophy among the Tenderiales is supported by the presence of carbon fixation and stress response pathways that could allow cellular growth by extracellular electron uptake. Key differences between the Tenderiales and other known neutrophilic iron oxidizers were revealed, including very few Cyc2 genes in the Tenderiales. Our results reveal a possible conserved pathway for extracellular electron uptake and suggests the Tenderiales have an distribution unlimited ecological role coupling metal or mineral redox chemistry and the carbon cycle in marine and brackish sediments.
ImportanceElectromicrobiology enables enrichment and identification of chemolithotrophic bacteria capable of extracellular electron uptake to drive energy metabolism and CO2 fixation. The recently described order Tenderiales contains the uncultivated electroautotroph "Candidatus Tenderia electrophaga". The "Ca. Tenderia electrophaga" genome contains genes proposed to make up a previously undescribed extracellular electron uptake pathway. Here we use comparative genomics to show that this pathway is well conserved among Tenderiales spp. recovered by metagenome assembled genomes. This conservation extends to near neighbors of the Tenderiales, but not to other well-studied chemolithotrophs including iron and sulfur oxidizers. Our findings suggest that extracellular electron uptake may be pervasive among the Tenderiales and the geographic location from which metagenome assembled genomes were recovered offers clues to their natural ecological niche. | microbiology |
10.1101/2022.05.06.490984 | The DNA replication protein Orc1 from the yeast Torulaspora delbrueckii is required for heterochromatin formation but not as a silencer-binding protein | To understand the process by which new protein functions emerge, we examined how the yeast heterochromatin protein Sir3 arose through gene duplication from the conserved DNA replication protein Orc1. Orc1 is a subunit of the origin recognition complex (ORC), which marks origins of DNA replication. In Saccharomyces cerevisiae, Orc1 also promotes heterochromatin assembly by recruiting the structural proteins Sir1-4 to silencer DNA. In contrast, the paralog of Orc1, Sir3, is a nucleosome-binding protein that spreads across heterochromatic loci in conjunction with other Sir proteins. We previously found that a non-duplicated Orc1 from the yeast Kluyveromyces lactis behaved like ScSir3 but did not have a silencer-binding function like ScOrc1. Moreover, K. lactis lacks Sir1, the protein that interacts directly with ScOrc1. Here, we searched for the presumed intermediate state in which non-duplicated Orc1 possesses both the silencer-binding and spreading functions. In the non-duplicated species Torulaspora delbrueckii, which has an ortholog of Sir1 (TdKos3), we found that TdOrc1 spreads across heterochromatic loci independently of ORC, as ScSir3 and KlOrc1 do. This spreading is dependent on the nucleosome binding BAH domain of Orc1 and on Sir2 and Kos3. However, TdOrc1 does not have a silencer-binding function: T. delbrueckii silencers do not require ORC binding sites to function, and Orc1 and Kos3 do not appear to interact. Instead, Orc1 and Kos3 both spread across heterochromatic loci with other Sir proteins. Thus, Orc1 and Sir1/Kos3 originally had different roles in heterochromatin formation than they do now in S. cerevisiae. | genetics |
10.1101/2022.05.06.490984 | The DNA replication protein Orc1 from the yeast Torulaspora delbrueckii is required for heterochromatin formation but not as a silencer-binding protein | To understand the process by which new protein functions emerge, we examined how the yeast heterochromatin protein Sir3 arose through gene duplication from the conserved DNA replication protein Orc1. Orc1 is a subunit of the origin recognition complex (ORC), which marks origins of DNA replication. In Saccharomyces cerevisiae, Orc1 also promotes heterochromatin assembly by recruiting the structural proteins Sir1-4 to silencer DNA. In contrast, the paralog of Orc1, Sir3, is a nucleosome-binding protein that spreads across heterochromatic loci in conjunction with other Sir proteins. We previously found that a non-duplicated Orc1 from the yeast Kluyveromyces lactis behaved like ScSir3 but did not have a silencer-binding function like ScOrc1. Moreover, K. lactis lacks Sir1, the protein that interacts directly with ScOrc1. Here, we searched for the presumed intermediate state in which non-duplicated Orc1 possesses both the silencer-binding and spreading functions. In the non-duplicated species Torulaspora delbrueckii, which has an ortholog of Sir1 (TdKos3), we found that TdOrc1 spreads across heterochromatic loci independently of ORC, as ScSir3 and KlOrc1 do. This spreading is dependent on the nucleosome binding BAH domain of Orc1 and on Sir2 and Kos3. However, TdOrc1 does not have a silencer-binding function: T. delbrueckii silencers do not require ORC binding sites to function, and Orc1 and Kos3 do not appear to interact. Instead, Orc1 and Kos3 both spread across heterochromatic loci with other Sir proteins. Thus, Orc1 and Sir1/Kos3 originally had different roles in heterochromatin formation than they do now in S. cerevisiae. | genetics |
10.1101/2022.05.07.491049 | Comparative and longitudinal analysis of axial and retinal biometry in prospective models of hyperopia | Structured AbstractO_ST_ABSPurposeC_ST_ABSTo quantify changes in axial and retinal biometry in aging hyperopic mouse models.
MethodsFundus photographs and ocular biometric measurements from Mfrprd6, Prss56glcr4, Adipor1tm1Dgen, C1qtnf5tm1.1(KOMP)Vlcg and Prss56em2(IMPC)J homozygotes and C57BL/6J control mice were ascertained longitudinally up to one year of age. Parameters including axial length (AL), central corneal thickness (CCT), anterior chamber depth (ACD), lens thickness (LT), outer nuclear layer thickness (ONLT), retinal thickness (RT), vitreous chamber depth (VCD) and posterior length (PL) were measured using Spectral Domain-Optical Coherence Tomography imaging. Mixed-model analysis of variance and factorial analysis of covariance, using body size as a covariate, followed by post-hoc analysis was performed to identify significant strain differences.
ResultsStrain specific changes in axial and retinal biometry along with significant effects of age, sex and body size on AL were noted. Mfrprd6, Prss56glcr4, Adipor1tm1Dgen and Prss56em2(IMPC)J homozygotes had significantly shorter AL than controls. While a comparable decrease in PL was observed in Mfrprd6, Prss56glcr4, and Adipor1tm1Dgen homozygotes, the decrease was attributed to changes in different posterior components from each mutant. Mfrprd6 and Adipor1tm1Dgen homozygotes developed regularly sized fundus spots across the ocular globe, which differed from the large bright spots seen in aged Prss56glcr4 and Prss56em2(IMPC)J homozygotes. While ONLT of C1qtnf5tm1.1(KOMP)Vlcg mice was less than controls, AL and fundus images appeared normal.
ConclusionsThis study highlights differences in contributions of ocular components to AL among hyperopic mouse models with decreased AL. Understanding the mechanisms through which these proteins function, will help to elucidate their role in controlling ocular growth. | genetics |
10.1101/2022.05.06.490972 | Comparative single-cell transcriptomes of dose and time dependent epithelial-mesenchymal spectrums | Epithelial-mesenchymal transition (EMT) is a key cellular process involved in development and disease progression. Single-cell transcriptomes can characterize intermediate EMT states observed in tumors and fibrotic tissues, but previous in vitro models focused on time-dependent responses after stimulation with single dose of EMT signals. It was therefore unclear whether single-cell transcriptomes support stable intermediate EMT phenotypes crucial for disease progression. We performed single-cell RNA-sequencing with human mammary epithelial cells treated with various concentrations of TGF-{beta}. We found that the dose-dependent EMT harbors multiple intermediate states at the single-cell level after two weeks of treatment, suggesting a stable continuum. After correcting batch effects from experiments, we performed comparative analyses of the dose- and time-dependent EMT. We found that the dose-dependent EMT shows a stronger anti-correlation between epithelial and mesenchymal transcriptional programs and a better resolution of transition stages compared to the time-dependent process. These properties enable higher sensitivity to detect genes whose expressions are associated with core EMT regulatory networks. Nonetheless, we found cell clusters unique to the time-dependent EMT, which correspond to en route cell populations that do not appear at steady states. Furthermore, combining dose- and time-dependent cell clusters gave rise to more accurate prognosis for cancer patients compared to individual EMT spectrum. Our new data and analyses reveal a stable EMT continuum at the single-cell resolution and the transcriptomic level. The dose-dependent experimental model can complement the widely used time-course experiments to reflect physiologically or pathologically relevant EMT phenotypes in a comprehensive manner. | genomics |
10.1101/2022.05.07.491048 | Discovery of diverse human BH3-only and non-native peptide binders of pro-apoptotic BAK indicate that activators and inhibitors use asimilar binding mode and are not distinguished by binding affinity or kinetics | Apoptosis is a programmed form of cell death important for the development and maintenance of tissue homeostasis. The BCL-2 protein family controls key steps in apoptosis, dysregulation of which can lead to a wide range of human diseases. BCL-2 proteins comprise three groups: anti-apoptotic proteins, pro-apoptotic proteins, and BH3-only proteins. BAK is one of two pro-apoptotic proteins, and previous work has shown that binding of certain BH3-only proteins such as truncated BID (tBID), BIM, or PUMA to BAK leads to mitochondrial outer membrane permeabilization, the release of cytochrome c, and ultimately cell death. This process, referred to as activation, involves the BH3-stimulated conversion of BAK from monomer to dimer and then to oligomers that promote membrane disruption. Crystal structures of putative intermediates in this pathway, crosslinking data, and in vitro functional tests have provided insights into the activation event, yet the sequence-function relationships that make some but not all BH3-only proteins function as activators remain largely unexamined. In this work, we used computational protein design, yeast surface-display screening of candidate BH3-like peptides, and structure-based energy scoring to identify ten new binders of BAK that span a large sequence space. Among the new binders are two peptides from human proteins BNIP5 and PXT1 that promote BAK activation in liposome assays and induce cytochrome-c release from mitochondria, expanding current views of how BAK-mediated cell death may be triggered in cells. High-resolution crystal structures and binding experiments revealed a high degree of similarity in binding geometry, affinity, and association kinetics between peptide activators and inhibitors, including peptides described previously and those identified in this work. We propose a model for BAK activation that is based on differential engagement of BAK monomers vs. the BAK activation transition state that integrates our observations with previous reports of BAK binders, activators, and inhibitors. | biochemistry |
10.1101/2022.05.07.491048 | Discovery of diverse human BH3-only and non-native peptide binders of pro-apoptotic BAK indicate that activators and inhibitors use asimilar binding mode and are not distinguished by binding affinity or kinetics | Apoptosis is a programmed form of cell death important for the development and maintenance of tissue homeostasis. The BCL-2 protein family controls key steps in apoptosis, dysregulation of which can lead to a wide range of human diseases. BCL-2 proteins comprise three groups: anti-apoptotic proteins, pro-apoptotic proteins, and BH3-only proteins. BAK is one of two pro-apoptotic proteins, and previous work has shown that binding of certain BH3-only proteins such as truncated BID (tBID), BIM, or PUMA to BAK leads to mitochondrial outer membrane permeabilization, the release of cytochrome c, and ultimately cell death. This process, referred to as activation, involves the BH3-stimulated conversion of BAK from monomer to dimer and then to oligomers that promote membrane disruption. Crystal structures of putative intermediates in this pathway, crosslinking data, and in vitro functional tests have provided insights into the activation event, yet the sequence-function relationships that make some but not all BH3-only proteins function as activators remain largely unexamined. In this work, we used computational protein design, yeast surface-display screening of candidate BH3-like peptides, and structure-based energy scoring to identify ten new binders of BAK that span a large sequence space. Among the new binders are two peptides from human proteins BNIP5 and PXT1 that promote BAK activation in liposome assays and induce cytochrome-c release from mitochondria, expanding current views of how BAK-mediated cell death may be triggered in cells. High-resolution crystal structures and binding experiments revealed a high degree of similarity in binding geometry, affinity, and association kinetics between peptide activators and inhibitors, including peptides described previously and those identified in this work. We propose a model for BAK activation that is based on differential engagement of BAK monomers vs. the BAK activation transition state that integrates our observations with previous reports of BAK binders, activators, and inhibitors. | biochemistry |
10.1101/2022.05.07.491030 | Comparative genomic and crystal structure analyses identify a collagen glucosyltransferase from Acanthamoeba Polyphaga Mimivirus | Humans and Acanthamoeba Polyphaga Mimivirus share numerous homologous genes, including collagens and collagen-modifying enzymes. To explore the homology, we performed a genome-wide comparison between human and mimivirus using DELTA-BLAST (Domain Enhanced Lookup Time Accelerated BLAST) and identified 52 new mimiviral proteins that are homologous with human proteins. To gain functional insights into mimiviral proteins, their human protein homologs were organized into Gene Ontology (GO) and REACTOME pathways to build a functional network. Collagen and collagen-modifying enzymes form the largest subnetwork with most nodes. Further analysis of this subnetwork identified a putative collagen glycosyltransferase R699. Protein expression test suggested that R699 is highly expressed in E coli, unlike the human collagen-modifying enzymes. Enzymatic activity assays showed that R699 catalyzes the conversion of unique galactosylhydroxylysine within the GXXXUG motif (U=galactosylhydroxylysine) to glucosylgalactosylhydroxylysine on collagen using uridine diphosphate glucose (UDP-Glc) as a sugar donor, suggesting R699 is a mimiviral collagen galactosylhydroxylysyl glucosyltransferase (GGT) with defined substrate specificity. Structural study of R699 produced the first crystal structure of a collagen GGT with a visible UDP-Glc. Sugar moiety of the UDP-Glc resides in a previously unrecognized pocket. Mn2+ coordination and nucleoside-diphosphate binding site are conserved among GGT family members and critical for R699s collagen GGT activity. To facilitate further analysis of human and mimiviral homologous proteins, we presented an interactive and searchable genome-wide comparison website for quickly browsing human and Acanthamoeba Polyphaga Mimivirus homologs, which is available at RRID Resource ID: SCR_022140 or https://guolab.shinyapps.io/app-mimivirus-publication/. | biochemistry |
10.1101/2022.05.07.491021 | The metastatic capacity of high-grade serous ovarian cancer cells changes along disease progression: inhibition by mifepristone | Background: Reductionist two-dimensional (2D) in vitro assays have long been the standard for studying the metastatic abilities of cancer cells. However, tri-dimensional (3D) organotypic models provide a more complex environment, closer to that seen in patients, and thereby provide a more accurate representation of their true capabilities. Our laboratory has previously shown that the antiprogestin and antiglucocorticoid mifepristone can reduce the growth, adhesion, migration, and invasion of various aggressive cancer cells assessed using 2D assays. In this study, we characterize the metastatic capabilities of high-grade serous ovarian cancer cells generated along disease progression, in both 2D and 3D assays, and the ability of cytostatic doses of mifepristone to inhibit them. Methods: High-grade serous ovarian cancer cells collected from two separate patients at different stages of their disease were used throughout the study. The 2D wound healing and Boyden chamber assays were used to study migration, while a layer of extracellular matrix was added to the Boyden chamber to study invasion. A 3D organotypic model, composed of fibroblasts embedded in collagen I and topped with a monolayer of mesothelial cells was used to further study cancer cell adhesion and mesothelial displacement. All assays were studied in cells representing different stages of disease progression in the absence or presence of cytostatic doses of mifepristone. Results: 2D in vitro assays demonstrated that the migration and invasive rates of the cells isolated from both patients decreased along disease progression. Conversely, in both patients, cells representing late-stage disease demonstrated a higher adhesion capacity to the 3D organotypic model than those representing an early-stage disease. This adhesive behaviour is associated with the in vivo tumor capacity of the cells. Regardless of these differences in adhesive, migratory, and invasive behaviour among the experimental protocols used, cytostatic doses of mifepristone were able to inhibit the adhesion, migration, and invasion rates of all cells studied, regardless of their basal capabilities over reductionist or organotypic metastatic in vitro model systems. Finally, we demonstrate that when cells acquire the capacity to grow spontaneously as spheroids, they do attach to a 3D organotypic model system when pre-incubated with conditioned media. Of relevance, mifepristone was able to cause dissociation of these multicellular structures. Conclusion: Differences in cellular behaviours were observed between reductionist 2D and 3D assays when studying the metastatic capabilities of high-grade serous ovarian cancer cells representing disease progression. Mifepristone inhibited these metastatic capabilities in all assays studied. | cancer biology |
10.1101/2022.05.06.487898 | NetAct: a computational platform to construct core transcription factor regulatory networks using gene activity | A major question in systems biology is how to identify the core gene regulatory circuit that governs the decision-making of a biological process. Here, we develop a computational platform for constructing core transcription-factor regulatory networks, named NetAct, using both transcriptomics data and literature-based transcription factor-target databases. NetAct robustly infers the activities of regulators using target expression, constructs networks based on transcriptional activity, and integrates mathematical modeling for validation. We show that NetAct outperforms existing algorithms in inferring transcriptional activity from our benchmark tests. We illustrate the application of NetAct to model networks driving TGF-beta induced Epithelial-Mesenchymal transition and macrophage polarization. | systems biology |
10.1101/2022.05.06.487898 | NetAct: a computational platform to construct core transcription factor regulatory networks using gene activity | A major question in systems biology is how to identify the core gene regulatory circuit that governs the decision-making of a biological process. Here, we develop a computational platform for constructing core transcription-factor regulatory networks, named NetAct, using both transcriptomics data and literature-based transcription factor-target databases. NetAct robustly infers the activities of regulators using target expression, constructs networks based on transcriptional activity, and integrates mathematical modeling for validation. We show that NetAct outperforms existing algorithms in inferring transcriptional activity from our benchmark tests. We illustrate the application of NetAct to model networks driving TGF-beta induced Epithelial-Mesenchymal transition and macrophage polarization. | systems biology |
10.1101/2022.05.06.490990 | The proteomic and transcriptomic landscapes altered by Rgg2/3 activity in Streptococcus pyogenes | Streptococcus pyogenes, otherwise known as Group A Streptococcus (GAS), is an important and highly adaptable human pathogen with the ability to cause both superficial and severe diseases. Understanding how S. pyogenes senses and responds to its environment will likely aid in determining how it causes a breadth of diseases. One regulatory network involved in GASs ability to sense and respond to the changing environment is the Rgg2/3 quorum sensing (QS) system, which responds to metal and carbohydrate availability and regulates changes to the bacterial surface. To better understand the impact of Rgg2/3 QS on S. pyogenes physiology, we performed RNA-seq and TMT-LC-MS/MS analysis on cells in which this system was induced or disrupted. Primary findings confirmed that pheromone stimulation in wildtype cultures is limited to the induction of operons whose promoters contain previously determined Rgg2/3 binding sequences. However, supplementing exogenous pheromone to a deletion mutant of rgg3, a strain that endogenously produces elevated amounts of pheromone, led to extended alterations of the transcriptome and proteome, ostensibly by stress-induced pathways. Under such exaggerated pheromone conditions (Drgg3+SHP), a connection was identified between Rgg2/3 and the stringent response. Mutation of relA, the bifunctional guanosine tetra- and penta-phosphate nucleoside synthetase/hydrolase, and alarmone synthase genes sasA and sasB, impacted culture doubling times and disabled induction of Rgg2/3 in response to mannose, while manipulation of Rgg2/3 signaling modestly altered nucleotide levels. Our findings indicate that excessive pheromone production or exposure places stress on GAS resulting in an indirect altered proteome and transcriptome beyond primary pheromone signaling. | microbiology |
10.1101/2022.05.06.490987 | Temporally integrated transcriptome analysis reveals ASFV pathology and host response dynamics | African swine fever virus (ASFV) causes a lethal swine hemorrhagic disease and is currently responsible for widespread damage to the pig industry. The molecular mechanisms of ASFV pathogenicity and its interaction with host responses remain poorly understood. In this study, we profiled the temporal viral and host transcriptomes in porcine alveolar macrophages (PAMs) infected at 6, 12, 24 and 48 hours with highly virulent (SY18) and low virulent (HuB20) ASFV strains. We first identified profound differences in the virus expression programs between SY18 and HuB20, while the transcriptome dynamics in host cells were dominated by infection time. Through integrated computational analysis and experimental validation, we identified differentially expressed genes and related biological processes, and elaborated differential usage of the NF-kappaB related pathways by the two virus strains. In addition, we observed that compared to the highly virulent SY18 strain, HuB20 infection quickly activates expression of receptors, sensors, regulators, as well as downstream effectors, including cGAS, STAT1/2, IRF9, MX1/2, suggesting rapid induction of a strong immune response. Lastly, we constructed a host-virus coexpression network, which shed light on pathogenic functions of several ASFV genes. Taken together, these results will provide a basis for further mechanistic studies on the functions of both viral and cellular genes that are involved in different responses.
Author SummarySince it was first described in Kenya in 1921, ASF has spread across sub-Saharan Africa, the Caribbean, the Western Europe, the Trans-Caucasus region, and the Russian Federation. Recent out-breaks have also been reported in Asia, which has devastated the pig industry, resulting in an approximately 40% reduction in pork worldwide. In the absence of effective vaccine or treatment, the mortality for infections with highly virulent strains approaches 100%, while low virulent strains causing less mortality spreads fast recently. Nevertheless, the mechanisms of ASFV pathogenicity, especially the differences between highly and low virulent strains remain poorly understood. Here, we used RNA-seq to analyze the viral and host transcriptome changes in PAMs infected with a virulent strain (SY18) or an attenuated strain (HuB20) at different stages. We found that the presence of ASFV significantly affected the cellular transcriptome profile. In addition, we did temporal and described the dynamic expression programs induced in the host cells by ASFV infection of different virulence strains. In particular, we identified differential gene expression patterns in host innate immune responses and expressed cytokines and chemokines between ASFV strains of different virulence. Our study provides new insights into ASFV pathogenicity research and novel drug or vaccine targets. | microbiology |
10.1101/2022.05.06.490987 | Temporally integrated transcriptome analysis reveals ASFV pathology and host response dynamics | African swine fever virus (ASFV) causes a lethal swine hemorrhagic disease and is currently responsible for widespread damage to the pig industry. The molecular mechanisms of ASFV pathogenicity and its interaction with host responses remain poorly understood. In this study, we profiled the temporal viral and host transcriptomes in porcine alveolar macrophages (PAMs) infected at 6, 12, 24 and 48 hours with highly virulent (SY18) and low virulent (HuB20) ASFV strains. We first identified profound differences in the virus expression programs between SY18 and HuB20, while the transcriptome dynamics in host cells were dominated by infection time. Through integrated computational analysis and experimental validation, we identified differentially expressed genes and related biological processes, and elaborated differential usage of the NF-kappaB related pathways by the two virus strains. In addition, we observed that compared to the highly virulent SY18 strain, HuB20 infection quickly activates expression of receptors, sensors, regulators, as well as downstream effectors, including cGAS, STAT1/2, IRF9, MX1/2, suggesting rapid induction of a strong immune response. Lastly, we constructed a host-virus coexpression network, which shed light on pathogenic functions of several ASFV genes. Taken together, these results will provide a basis for further mechanistic studies on the functions of both viral and cellular genes that are involved in different responses.
Author SummarySince it was first described in Kenya in 1921, ASF has spread across sub-Saharan Africa, the Caribbean, the Western Europe, the Trans-Caucasus region, and the Russian Federation. Recent out-breaks have also been reported in Asia, which has devastated the pig industry, resulting in an approximately 40% reduction in pork worldwide. In the absence of effective vaccine or treatment, the mortality for infections with highly virulent strains approaches 100%, while low virulent strains causing less mortality spreads fast recently. Nevertheless, the mechanisms of ASFV pathogenicity, especially the differences between highly and low virulent strains remain poorly understood. Here, we used RNA-seq to analyze the viral and host transcriptome changes in PAMs infected with a virulent strain (SY18) or an attenuated strain (HuB20) at different stages. We found that the presence of ASFV significantly affected the cellular transcriptome profile. In addition, we did temporal and described the dynamic expression programs induced in the host cells by ASFV infection of different virulence strains. In particular, we identified differential gene expression patterns in host innate immune responses and expressed cytokines and chemokines between ASFV strains of different virulence. Our study provides new insights into ASFV pathogenicity research and novel drug or vaccine targets. | microbiology |
10.1101/2022.05.06.490995 | Highly specialized carbohydrate metabolism capability in Bifidobacterium strain associated with intestinal barrier maturation in early preterm infants | "Leaky gut", or high intestinal barrier permeability, is common in preterm newborns. The role of microbiota in this process remains largely uncharacterized. We employed both short- and long-read sequencing of the 16S rRNA gene and metagenomes to characterize the intestinal microbiome of a longitudinal cohort of 113 preterm infants born between 240/7-326/7 weeks of gestation. Enabled by enhanced taxonomic resolution, we found significantly increased abundance of Bifidobacterium breve and a diet rich in mothers breastmilk to be associated with intestinal barrier maturation during the first week of life. We combined these factors using genome-resolved metagenomics and identified a highly specialized genetic capability of the Bifidobacterium strains to assimilate human milk oligosaccharides and host-derived glycoproteins. Our study proposed mechanistic roles of breastmilk feeding and intestinal microbial colonization in postnatal intestinal barrier maturation; these observations are critical towards advancing therapeutics to prevent and treat hyperpermeable gut-associated conditions, including necrotizing enterocolitis. | microbiology |
10.1101/2022.05.06.490981 | A comprehensive list of the replication promoters of Bunyavirales reveals a unique promoter structure in Nairoviridae differing from other virus families | Bunyaviruses belong to the order Bunyavirales, the largest group of RNA viruses. They infect a wide variety of host species around the world, including plants, animals and humans, and pose a major threat to public health. Major families in the order Bunyavirales have tri-segmented negative-sense RNA genomes, the 5 and 3 ends of which form complementary strands that serve as a replication promoter. Elucidation of the mechanisms by which viral RNA-dependent RNA polymerase recognizes the promoter to initiates RNA synthesis is important for understanding viral replication and pathogenesis, and for developing antivirals. A list of replication promoter configuration patterns may provide details on the differences in the replication mechanisms among bunyaviruses. Here, by using public sequence data of all known bunyavirus species, we constructed a comprehensive list of the replication promoters comprising 40 nucleotides in both the 5 and 3 ends of the genome that form a specific complementary strand. We showed that among tri-segmented bunyaviruses, viruses belonging to the family Nairoviridae, including the highly pathogenic Crimean-Congo hemorrhagic fever virus, have evolved a GC-rich promoter structure that differs from that of other bunyaviruses. The unique promoter structure might be related to the large genome size of the family Nairoviridae among bunyaviruses. It is possible that the large genome architecture confers a pathogenic advantage. The promoter list provided in this report is expected to be useful for predicting virus family-specific replication mechanisms of segmented negative-sense RNA viruses. | microbiology |
10.1101/2022.05.06.490991 | Quorum sensing regulation of a major facilitator superfamily transporter affects multiple streptococcal virulence factors | Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or Group A strep, one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show that the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells.
ImportanceThe production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds that expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple secreted virulence factors and accounts for a documented QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between the expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport. | microbiology |
10.1101/2022.05.07.490347 | Stem Cell Secretome Promotes Scarless Corneal Wound Healing and Rescues Corneal Sensory Nerves | Corneal scarring is a leading cause of blindness in the world. In this study, we explored the therapeutic potential of corneal stromal stem cell (CSSC)-derived secretome in a mechanical debridement mouse model of corneal scarring. CSSC secretome was able to promote scarless corneal wound healing. The mechanisms include 1) dampening inflammation with reduced CD45+, CD11b+/GR1+, and CD11b+/F4/80+ inflammatory cells in the wounded corneas; 2) reducing fibrotic extracellular matrix deposition such as collagen IV, collagen 3A1, SPARC, and alpha-SMA; 3) rescuing sensory nerves. The proteomic analysis shows upregulated proteins related to wound healing and cell adhesion which boost scarless wound healing. It also shows upregulated neuroprotective proteins in CSSC secretome related to axon guidance, neurogenesis, neuron projection development, and neuron differentiation. Four unique complement inhibitory proteins CD59, vitronectin, SERPING1, and C1QBP found in CSSC secretome contribute to reducing a complement cascade mediating cell death and membrane attacking complex autoantibodies after corneal injury. This study provides novel insights into mechanisms of stem cell secretome induced scarless corneal wound healing and neuroprotection and identifies regenerative proteins in the CSSC secretome. | cell biology |
10.1101/2022.05.07.491011 | The rescue of epigenomic abnormalities in ICF1 patient iPSCs following DNMT3B correction is incomplete at a residual fraction of H3K4me3-enriched regions | Background: Bi-allelic hypomorphic mutations in DNMT3B disrupt DNA methyltransferase activity and lead to Immunodeficiency, Centromeric instability, Facial anomalies syndrome, type 1 (ICF1). While several ICF1 phenotypes have been linked to abnormally hypomethylated repetitive regions, the unique genomic regions responsible for the remaining disease phenotypes remain largely uncharacterized. Here we explored two ICF1 patient-induced pluripotent stem cells (iPSCs) and their CRISPR/Cas9 corrected clones to determine whether gene correction can overcome DNA methylation defects and related/associated changes in the epigenome of non-repetitive regions. Results: Hypomethylated regions throughout the genome are highly comparable between ICF1 iPSCs carrying different DNMT3B variants, and significantly overlap with those in ICF1-peripheral blood and lymphoblastoid cell lines. These regions include large CpG island domains, as well as promoters and enhancers of several lineage-specific genes, in particular immune-related, suggesting that they are pre-marked during early development. The gene corrected ICF1 iPSCs reveal that the majority of phenotype-related hypomethylated regions re-acquire normal DNA methylation levels following editing. However, at the most severely hypomethylated regions in ICF1 iPSCs, which also display the highest increased H3K4me3 levels and enrichment of CTCF-binding motifs, the epigenetic memory persisted, and hypomethylation was uncorrected. Conclusions: Restoring the catalytic activity of DNMT3B rescues the majority of the aberrant ICF1 epigenome. However, a small fraction of the genome is resilient to this reversal, highlighting the challenge of reverting disease states that are due to genome-wide epigenetic perturbations. Uncovering the basis for the persistent epigenetic memory will promote the development of strategies to overcome this obstacle. | genomics |
10.1101/2022.05.07.491051 | Chemical structures of cyclic ADP ribose (cADPR) isomers and the molecular basis of their production and signaling | Cyclic ADP ribose (cADPR) isomers are important signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via NAD hydrolysis, yet their chemical structures are unknown. We show that v-cADPR (2cADPR) and v2-cADPR (3-cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of v-cADPR (2-cADPR)-producing TIR domains reveal that conformational changes are required for the formation of the active assembly that resembles those of Toll-like receptor adaptor TIR domains, and mutagenesis data demonstrate that a conserved tryptophan is essential for cyclization. We show that v2-cADPR (3-cADPR) is a potent activator of ThsA effector proteins from Thoeris anti-phage defence systems and is responsible for suppression of plant immunity by the effector HopAM1. Collectively, our results define new enzymatic activities of TIR domains, reveal the molecular basis of cADPR isomer production, and establish v2-cADPR (3-cADPR) as an antiviral signaling molecule and an effector-mediated signaling molecule for plant immunity suppression. | biochemistry |
10.1101/2022.05.06.490980 | Microfluidic chemostatic bioreactor for high-throughput screening and sustainable co-harvesting of biomass and biodiesel in microalgae | As a renewable and sustainable source for energy, environment, and biomedical applications, microalgae and microalgal biodiesel have attracted great attention. However, their applications are confined due to the cost-efficiency of microalgal mass production. One-step strategy and continuous culturing systems could be solutions. However, current studies for optimization throughout microalgae-based biofuel production pipelines are generally derived from the batch culture process. Better tools are needed to study algal growth kinetics in continuous systems. A microfluidics chemostatic bioreactor array was presented, providing low-adhesion cultivation for algae in the gas, nutrition, and temperature (GNT) well-controlled environment with high throughput. The chip wasused to mimic the continuous culture environment of bioreactors. It allowed simultaneously studying of 8x8 different chemostatic conditions on algal growth and oil production in parallel on a 7x7 cm2 footprint. On-chip experiments of batch and continuous cultures of Chlorella. sp. were performed to study growth and lipid accumulation under different nitrogen concentrations. The results demonstrated that microalgal cultures can be regulated to grow and accumulate lipids concurrently, thus enhancing lipid productivity in one step. The developed on-chip culturing condition screening, which was more suitable for continuous bioreactor, was achieved at a half shorter time, 64-times higher throughput, and less reagent consumption. It could be used to establish chemostat cultures in continuous bioreactors which can dramatically accelerate the development of renewable and sustainable algal for CO2 fixation and biosynthesis and related systems for advanced sustainable energy, food, pharmacy, and agriculture with enormous social and ecological benefits.
TEASERSustainable microfluidic bioreactor for 64 times higher-throughput screening CO2 fixation and biomass and biodiesel production in microalgae. | bioengineering |
10.1101/2022.05.06.490965 | A Standardized Ontology for Naming tRNA-derived RNAs Based on Molecular Origin | Small RNAs processed from transfer RNA are recognized to have regulatory functions distinct from protein synthesis. This rapidly advancing field has led to a constellation of transcript nomenclatures. Building upon the accepted tRNA naming system and linking tRNA-derived small RNAs to their molecular sources, we propose an improved nomenclature. Facilitated by the tDRnamer system, it will promote inter-study comparisons leveraging a biologically-rooted nomenclature for this emerging class of intriguing regulators. | bioinformatics |
10.1101/2022.05.07.491036 | Structural heterogeneity of amyloid aggregates identified by spatially resolved nanoscale infrared spectroscopy | Amyloid plaques, composed of aggregates of the amyloid beta (A{beta}) protein, are one of the central manifestations of Alzheimers disease pathology. Aggregation of A{beta} from amorphous oligomeric species to mature fibrils has been extensively studied. However, significantly less in known about early-stage aggregates compared to fibrils. In particular, structural heterogeneities in prefibrillar species, and how that affects the structure of later stage aggregates are not yet well understood. Conventional spectroscopies cannot attribute structural facets to specific aggregates due to lack of spatial resolution, and hence aggregates at any stage of aggregation must be viewed as having the same average structure. The integration of infrared spectroscopy with Atomic Force Microscopy (AFM-IR) allows for identifying the signatures of individual nanoscale aggregates by spatially resolving spectra. In this report, we use AFM-IR to demonstrate that amyloid oligomers exhibit significant structural variations as evidenced in their infrared spectra, ranging from ordered beta structure to disordered conformations with predominant random coil and beta turns. This heterogeneity is transmitted to and retained in protofibrils and fibrils. We show for the first time that amyloid fibrils do not always conform to their putative ordered structure and structurally different domains can exist in the same fibril. We further show the implications of these results in amyloid plaques in Alzheimers tissue using infrared imaging, where these structural heterogeneities manifest themselves as lack of expected beta sheet structure. | biophysics |
10.1101/2022.05.06.490933 | The trade-off function of photorespiration in a changing environment | The photorespiratory pathway in plants comprises metabolic reactions distributed across several cellular compartments. It emerges from the dual catalytic function of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which either carboxylates or oxygenates ribulose-1,5-bisphosphate (RuBP). Carboxylation reactions produce 3-phospho-glycerate (3PGA) molecules which are substrate for central carbohydrate metabolism while oxygenation forms 2-phosphoglycolate (2PG) molecules which are substrate for the multicompartmental recovery process of photorespiration. Further, 2PG is a strong inhibitor of several enzymes involved in the Calvin-Benson-Bassham cycle which challenges the experimental and theoretical study of carbon assimilation, photorespiration and metabolic regulation in vivo. Here, an approach of structural kinetic modeling (SKM) is presented to investigate the extend of stabilization of CBC and carbohydrate metabolism by photorespiration. Further, our approach highlights the importance of feedback regulation by 2-PG for alleviation of environmental perturbation. Our findings indicate that oxygenation of RuBP by Rubisco significantly stabilizes CBC activity and, thus, carbohydrate metabolism. Based on our findings, we suggest a trade-off function of photorespiration which reduces carbon assimilation rates but simultaneously stabilizes metabolism by increasing plasticity of metabolic regulation within the chloroplast. Furthermore, our analysis suggests a stabilizing effect of increasing the partition of newly assimilated carbon going towards sucrose biosynthesis. With this, our analysis sheds light on the role of a multicompartmental metabolic pathway in stabilizing plant metabolism within a changing environment. | plant biology |
10.1101/2022.05.07.490946 | A task-general connectivity model reveals variation in convergence of cortical inputs to functional regions of the cerebellum | While resting-state fMRI studies have provided a broad picture of the connectivity between human neocortex and cerebellum, the degree of convergence of cortical inputs onto cerebellar circuits remains unknown: Does each cerebellar region receive input from a single cortical area or convergent inputs from multiple cortical areas? Here we use task-based fMRI data to build a range of cortico-cerebellar connectivity models, each allowing for a different degree of convergence. We compared these models by their ability to predict cerebellar activity patterns for novel task sets. Models that allow some degree of convergence provided the best predictions, arguing for convergence of multiple cortical inputs onto single cerebellar voxels. Importantly, the degree of convergence varied across the cerebellum with the highest convergence observed in areas linked to language, working memory, and social cognition. These findings suggest important differences in the way that functional subdivisions of the cerebellum support motor and cognitive function. | neuroscience |
10.1101/2022.05.06.490982 | Splashed E-box and AP-1 motifs cooperatively drive regeneration-response and shape regeneration abilities | Injury triggers genetic program to induce gene expression for regeneration. Several studies have recently reported the identification of regeneration-response enhancers (RREs) in zebrafish; however, it remains unclear whether a common mechanism operates in RREs. Here, we show that E-box and activator protein 1 (AP-1) motifs cooperatively function as RREs. We identified three RREs from the fn1b promoter by a search of conserved sequences and an in vivo transgenic assay for regeneration-response in zebrafish. Two of them derived from transposons displayed RRE activity only when combined with the -0.7 kb fn1b promoter, while another non-transposable element functioned as a standalone enhancer. A search for transcription factor-binding motifs and validation by transgenic assay revealed that both of E-box and AP-1 motifs are necessary and sufficient for RREs. Such RREs responded to variety of tissue injuries including zebrafish heart and Xenopus limb bud regenerations. Our findings highlight that regeneration is regulated by merging two activating signals evoked by tissue injuries. It is speculated that a large pool of potential enhancers in the genome shaped regenerative capacities during evolution.
SUMMARY STATEMENTThe study revealed that regeneration-response enhancer is composed of two transcription factor-binding motifs. The fidelity of regeneration-dependent gene expression is ensured by merging two activating signals evoked by injuries. | developmental biology |
10.1101/2022.05.07.490958 | Climate and ant richness explain the global distribution of ant-plant mutualisms | Biotic interactions are known to play an important role in shaping species geographic distributions and diversity patterns. However, the role of mutualistic interactions in shaping global diversity patterns remains poorly quantified, particularly with respect to interactions with invertebrates. Moreover, it is unclear how the nature of different mutualisms interacts with abiotic drivers and affects diversity patterns of mutualistic organisms. Here, we present a global-scale biogeographic analysis of three different ant-plant mutualisms, differentiating between plants bearing domatia, extrafloral nectaries (EFNs), and elaiosomes, based on comprehensive geographic distributions of ~15,000 flowering plants and ~13,000 ant species. Domatia and extrafloral nectaries involve indirect plant defenses provided by ants, while elaiosomes attract ants to disperse seeds. Our results show distinct biogeographic patterns of different ant-plant mutualisms, with domatium- and EFN-bearing plant richness decreasing sharply from the equator towards the poles, while elaiosome-bearing plants prevail at mid-latitudes. Contemporary climate, especially mean annual temperature and precipitation, emerge as the most important predictor of ant-associated plant diversity. In hot and moist regions, typically the tropics, domatium- and EFN-bearing plant richness increases with related ant guild richness, while in warm regions plants with elaiosomes are strongly linked to interacting ants. Our results suggest that ant richness in combination with climate drives the spatial variation of plants bearing domatia, extrafloral nectaries, and elaiosomes, highlighting the importance of mutualistic interactions for understanding plant biogeography and its response to global change. | ecology |
10.1101/2022.05.07.490958 | Climate and ant richness explain the global distribution of ant-plant mutualisms | Biotic interactions are known to play an important role in shaping species geographic distributions and diversity patterns. However, the role of mutualistic interactions in shaping global diversity patterns remains poorly quantified, particularly with respect to interactions with invertebrates. Moreover, it is unclear how the nature of different mutualisms interacts with abiotic drivers and affects diversity patterns of mutualistic organisms. Here, we present a global-scale biogeographic analysis of three different ant-plant mutualisms, differentiating between plants bearing domatia, extrafloral nectaries (EFNs), and elaiosomes, based on comprehensive geographic distributions of ~15,000 flowering plants and ~13,000 ant species. Domatia and extrafloral nectaries involve indirect plant defenses provided by ants, while elaiosomes attract ants to disperse seeds. Our results show distinct biogeographic patterns of different ant-plant mutualisms, with domatium- and EFN-bearing plant richness decreasing sharply from the equator towards the poles, while elaiosome-bearing plants prevail at mid-latitudes. Contemporary climate, especially mean annual temperature and precipitation, emerge as the most important predictor of ant-associated plant diversity. In hot and moist regions, typically the tropics, domatium- and EFN-bearing plant richness increases with related ant guild richness, while in warm regions plants with elaiosomes are strongly linked to interacting ants. Our results suggest that ant richness in combination with climate drives the spatial variation of plants bearing domatia, extrafloral nectaries, and elaiosomes, highlighting the importance of mutualistic interactions for understanding plant biogeography and its response to global change. | ecology |
10.1101/2022.05.07.491037 | Analysis of CheW-like domains provides insights into organization of prokaryotic chemotaxis systems | The ability to control locomotion in a dynamic environment provides a competitive advantage for microorganisms, thus driving the evolution of sophisticated regulatory systems. Nineteen known categories of chemotaxis systems control motility mediated by flagella and Type IV pili, plus other cellular functions. A key feature that distinguishes chemotaxis systems from generic two-component regulatory systems is separation of receptor and kinase functions into distinct proteins, linked by CheW scaffold proteins. This arrangement allows for formation of varied arrays with remarkable signaling properties. We recently analyzed sequences of CheW-like domains found in CheA kinases and CheW and CheV scaffold proteins. Sixteen Architectures of CheA, CheW, and CheV proteins contain ~94% of all CheW-like domains, forming six Classes with likely functional specializations. We surveyed chemotaxis system categories and proteins containing CheW-like domains in ~1900 prokaryotic species, the most comprehensive analysis to date. The larger sample size revealed previously unknown insights. Co-occurrence analyses suggested that chemotaxis systems occur in non-random combinations within species, increasing our understanding of evolution of chemotaxis. Furthermore, many Types of CheW-like domains occurred predominantly with specific categories of chemotaxis systems, suggesting specialized functional interactions. For example, Class 2 (Type CheW.IC) domains exhibit properties spanning the primary Classes of CheW-like domains in CheA and CheW proteins. CheW.IC frequently co-occurred with methyl-accepting coiled coil (MAC) proteins, which contain both receptor and kinase functions. Although MAC proteins should not need CheW scaffolds to connect receptor and kinase functions, co-occurrence suggested that MAC systems may nevertheless benefit from array formation facilitated by CheW.IC domains. | bioinformatics |
10.1101/2022.05.06.490994 | PHi-C2: interpreting Hi-C data as the dynamic 3D genome state | Hi-C is a widely used assay for studying three-dimensional (3D) genome organization across the whole genome. Here, we present PHi-C2, a Python package supported by mathematical and biophysical polymer modeling, that converts an input Hi-C matrix data into the polymer models dynamics, structural conformations, and rheological features. The updated optimization algorithm to regenerate a highly similar Hi-C matrix provides a fast and accurate optimal solution compared to the previous version by eliminating a computational bottleneck in the iterative optimization process. Besides, we newly set up the availability on Google Colab workflow to run, easily change parameters and check the results in the notebook. Overall, PHi-C2 can be a valuable tool to mine the dynamic 3D genome state embedded in Hi-C data.
Availability and ImplementationPHi-C2 as the phic Python package is freely available under the GPL license and can be installed from the Python package index. The source code is available from GitHub at https://github.com/soyashinkai/PHi-C2. Without preparing a Python environment, PHi-C2 can run on Google Colab (https://bit.ly/3rlptGI).
Contactsoya.shinkai@riken.jp or sonami@riken.jp | bioinformatics |
10.1101/2022.05.06.490985 | Inhibition of Aromatase by Hops, Licorice Species, and their bioactive compounds in Postmenopausal Breast Tissue | Breast cancer risk continues to rise post menopause. Endocrine therapies are employed to prevent postmenopausal breast cancer in high-risk women. However, their adverse effects have reduced acceptability and overall success in cancer preventionatural products such as hops (Humulus lupulus) and pharmacopoeial licorice (Glycyrrhiza) species, which are used for managing menopausal symptoms, have demonstrated estrogenic and chemopreventive properties. Their beneficial effects on aromatase activity and expression as important factors in postmenopausal breast carcinogenesis are understudied. The presented data show that Gycyrrhiza inflata (GI) has the highest aromatase inhibition potency among these plants. Moreover, phytoestrogens such as 8-prenylnaringenin from hops as well as liquiritigenin and 8-prenylapigenin from licorice are shown to be potent bioactives, in line with computational docking studies. 8-Prenylnaringenin, GI extract, liquiritigenin, and licochalcone A all suppress aromatase expression in postmenopausal womens breast tissue. Collectively, these data suggest that these natural products may have breast cancer prevention potential for high-risk postmenopausal women. | pharmacology and toxicology |
10.1101/2022.05.07.491027 | Composition structures and biologically meaningful logics: plausibility and relevance in bipartite models of gene regulation | Boolean network models have widely been used to study the dynamics of gene regulatory networks. However, such models are coarse-grained to an extent that they abstract away molecular specificities of gene regulation. In contrast, bipartite Boolean network models of gene regulation explicitly distinguish genes from transcription factors (TFs). In such models, multiple TFs may simultaneously contribute to the regulation of a gene by forming heteromeric complexes. The formation of heteromeric complexes gives rise to composition structures in the corresponding bipartite network. Remarkably, composition structures can severely restrict the number of Boolean functions (BFs) that can be assigned to a gene. The introduction of bipartite Boolean network models is relatively recent, and so far an empirical investigation of their biological plausibility is lacking. Here, we estimate the prevalence of composition structures arising through heteromeric complexes in Homo sapiens. Moreover, we present an additional mechanism by which composition structures arise as a result of multiple TFs binding to the cis-regulatory regions of a gene and we provide empirical support for this mechanism. Next, we compare the restriction in BFs imposed by composition structures and by biologically meaningful properties. We find that two types of minimally complex BFs, namely nested canalyzing functions (NCFs) and read-once functions (RoFs), are more restrictive than composition structures. Finally, using a compiled dataset of 2687 BFs from published models, we find that composition structures are highly enriched in real biological networks, but that this enrichment is most likely driven by NCFs and RoFs. | systems biology |
10.1101/2022.05.06.490960 | Pattern dynamics and stochasticity of the brain rhythms | Our current understanding of brain rhythms is based on quantifying their instantaneous or time-averaged characteristics. What remains unexplored, is the actual structure of the waves--their shapes and patterns over finite timescales. To address this, we used two independent approaches to link wave forms to their physiological functions: the first is based on quantifying their consistency with the underlying mean behavior, and the second assesses "orderliness" of the waves features. The corresponding measures capture the waves characteristic and abnormal behaviors, such as atypical periodicity or excessive clustering, and demonstrate coupling between the patterns dynamics and the animals location, speed and acceleration. Specifically, we studied patterns of{theta} and{gamma} waves, and Sharp Wave Ripples, and observed speed-modulated changes of the waves cadence, an antiphase relationship between orderliness and acceleration, as well as spatial selectiveness of patterns. Further-more, we found an interdependence between orderliness and regularity: larger deviations from steady oscillatory behavior tend to accompany disarrayed temporal cluttering of peaks and troughs. Taken together, our results offer a complementary--mesoscale--perspective on brain wave structure, dynamics, and functionality. | neuroscience |
10.1101/2022.05.06.490952 | Reinstatement of Pavlovian responses to alcohol cues by stress | RationaleStress may contribute to relapse to alcohol use in part by enhancing reactivity to cues previously paired with alcohol. Yet, standard models of stress-induced reinstatement generally use contingent presentations of alcohol-paired cues to reinforce instrumental behaviors, making it difficult to isolate the ability of cues to invigorate alcohol-seeking.
ObjectiveHere we sought to test the impact of stress on behavioral responses to alcohol-paired cues, using a model of stress-induced reinstatement of Pavlovian conditioned approach, inspired by Nadia Chaudhris work on context-induced reinstatement.
MethodsLong Evans rats were trained to associate one auditory cue with delivery of alcohol or sucrose and an alternative auditory cue with no reward. Following extinction training, rats were exposed to a stressor prior to being re-exposed to the cues under extinction conditions. We assessed the effects of yohimbine, intermittent footshock and olfactory cues paired with social defeat on responses to alcohol-paired cues, and the effects of yohimbine on responses to sucrose-paired cues.
ResultsThe pharmacological stressor, yohimbine, enhanced Pavlovian responses to both alcohol and sucrose cues, but intermittent footshock and social defeat cues did not.
ConclusionsWhile yohimbine elicited reinstatement of Pavlovian conditioned responses, these effects may be unrelated to activation of stress systems. | neuroscience |
10.1101/2022.05.06.490926 | habCluster: Identifying Geographical Boundary among Intraspecific Units Using Community Detection Algorithm in R | Conservation management for a species generally rests on intraspecific units, while identification of their geographic boundaries is necessary for the implementation. Intraspecific units can be discriminated using population genetic methods, yet an analytical approach is still lacking for detecting their geographic boundaries. Here, based on landscape connectivity, we present a geographical boundary delineation method, habCluster, using community detection algorithm. Community detection is an algorithm in graph theory used to identify clusters of highly connected nodes within a network. We assume that the habitat raster cells with better connections tend to form a continuous habitat patch than the others, thus making the range of an intraspecific unit. The method is tested on grey wolf (Canis lupus) habitat in Europe and on giant panda (Ailuropoda melanoleuca) habitat in China. The habitat suitability for grey wolves and giant pandas were evaluated using species distribution modeling. Each cell in the habitat suitability index (HSI) raster is treated as a node and directly connected with its eight neighbor cells. The edge weight between nodes is the distance between the center of them weighted by the average of their HSI values. We implement habCluster using R programming language with inline C++ code to speed up the computing. The geographical clusters detected were compared with the HSI maps for both species and with the nature reserves for giant panda. We found the boundaries of the clusters delineated using habCluster could serve as a good indicator of habitat patches, and they match generally well with nature reserves in the giant panda case. habCluster can provide spatial analysis basis for conservation management plans such as monitoring, translocation and reintroduction, as well as for population structure research. | ecology |
10.1101/2022.05.07.491010 | Life history traits and a method for continuous mass rearing of the planthopper Pentastiridius leporinus, a vector of the causal agent of syndrome "basses richesses" in sugar beet | BACKGROUNDThe planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of the {gamma}-3 proteobacterium Candidatus Arsenophonus phytopathogenicus which causes the syndrome "basses richesses" (SBR) in sugar beet. SBR is a new and fast spreading disease in Central Europe that leads to high yield losses. To date the development of management strategies is hampered by insufficient knowledge about general life history traits of the planthopper and, most importantly, the year round availability of insects reared under controlled conditions. Rearing of P. leporinus has been considered challenging and to date no protocol exists.
RESULTSHere we describe a method for mass rearing P. leporinus on sugar beet from egg to adult, which has produced five generations and >20,000 individuals between June 2020 and March 2022. An alternative host such as wheat is not necessary for completing the life cycle. No-choice experiments showed that P. leporinus lays 139.1 {+/-} 132.9 eggs on sugar beet, whereas no oviposition was observed on its nymphal host wheat. Head capsule width was identified as a trait that unequivocally distinguished the five nymphal instars. Developmental time from first instar to adult was 193.6 {+/-} 35.8 days for males and 193.5 {+/-} 59.2 days for females. Infection rates of adults were tested with nested polymerase chain reaction (PCR). The results demonstrated that 70-80% of reared planthoppers across all generations carried the SBR proteobacterium.
CONCLUSIONThe mass rearing protocol and life history data will help overcome an important bottleneck in SBR research and enhance efforts in developing integrated pest management tools. | ecology |
10.1101/2022.05.06.490977 | DeepLUAD: An efficient approach for lung adenocarcinoma pattern classification | Histopathological analysis of whole-slide images is the gold standard technique for diagnosis of lung cancer and classifying it into types and subtypes by specialized pathologists. This labor-based approach is time and effort consuming, which led to development of automatic approaches to assist in reducing the time and effort. Deep learning is a supervised classification approach that is well adapted for automatic classification of histopathological images. We aimed to develop a deep learning-based approach for lung adenocarcinoma pattern classification and generalize the proposed approach to the classification of the major non-small cell lung cancer types. Three publicly available datasets were used in this study. A deep learning approach for histopathological image analysis using convolutional neural networks was developed and incorporated into automatic pipelines to accurately classify the predominant patterns on the whole-slide images level and non-small cell lung cancer types on patch-level. The models were evaluated using the confusion matrix to perform an error analysis and the classification report to compute F1-score, recall and precision. As results, the three models have shown an excellent performance with best combination of hyper-parameters for training models. First and second models predicted adenocarcinoma predominant patterns on two different datasets with an accuracy, respectively, of 96.15% and 89.51%. The third model has exceeded an accuracy of 99.72% in classifying major non-small cell lung cancer types. The proposed deep learning-based lung cancer classification approach can be used to assist pathologists in identifying of lung adenocarcinomas patterns. | bioinformatics |
10.1101/2022.05.07.491035 | Generalizable strategy to analyze domains in the context of parent protein architecture: A CheW case study | Domains are the three-dimensional building blocks of proteins. An individual domain can be found in a variety of protein architectures that perform unique functions and are subject to different evolutionary selective pressures. We describe an approach to evaluate the variability in amino acid sequences of a single domain across architectural contexts. The ability to distinguish the different evolutionary paths of one protein domain can help determine whether existing knowledge about a specific domain will apply to an uncharacterized protein. Such discrimination can lead to insights and hypotheses about function, as well as guide experimental priorities. We developed and tested our approach on CheW-like domains (PF01584), which mediate protein/protein interactions and are difficult to compare experimentally. CheW-like domains occur in CheW scaffolding proteins, CheA kinases, and CheV proteins that regulate bacterial chemotaxis. We chose 16 protein Architectures that included 94% of all CheW-like domains found in nature. Because some Architectures had more than one CheW-like domain, CheW-like domains were divided into 21 distinct Contexts. The CheW-like domain sequences were closely related within most Contexts; however, one Context was subdivided into three Types. The resulting 23 sequence Types coalesced into five or six Classes of CheW-like domains, which we described in detail. In addition, we created SimpLogo, an innovative method for visualizing amino acid composition across large sets of multiple sequence alignments of arbitrary length. SimpLogo offers substantial advantages over standard sequence logos for comparison and analysis of related protein sequences. The R package for SimpLogo is freely available. | bioinformatics |
10.1101/2022.05.07.490938 | The funny current If is essential for the fight-or-flight response in cardiac pacemaker cells | The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating {beta} adrenergic receptors ({beta}ARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs. If is critical for pacemaker activity, however, its role in the fight-or-flight response remains controversial. In this study, we used AP waveform analysis, machine learning, and dynamic clamp experiments in acutely-isolated SAMs from mice to quantitatively define the AP waveform changes and role of If in the fight-or-flight response. We found that while {beta}AR stimulation significantly altered nearly all AP waveform parameters, the increase in AP firing rate was only correlated with changes in a subset of parameters (diastolic duration, late AP duration, and diastolic depolarization rate). Dynamic clamp injection of the {beta}AR-sensitive component of If showed that it accounts for approximately 41% of the fight-or-flight increase in AP firing rate and 60% of the decrease in the interval between APs. Thus, If is an essential contributor to the fight-or-flight increase in heart rate. | physiology |
10.1101/2022.05.07.490900 | Learning consistent subcellular landmarks to quantify changes in multiplexed protein maps | Highly multiplexed quantitative subcellular imaging holds enormous promise for understanding how spatial context shapes the activity of our genome and its products at multiple scales. Yet unbiased analysis of subcellular organisation across experimental conditions remains challenging, because differences in molecular profiles between conditions confound differences in molecular profiles across space. Here, we introduce a deep-learning framework called CAMPA (Conditional Autoencoder for Multiplexed Pixel Analysis), which uses a variational autoencoder conditioned on cellular states and perturbations to learn consistent molecular signatures. Clustering the learned representations into subcellular landmarks allows quantitative comparisons of landmark sizes, shapes, molecular compositions and relative spatial organisation between conditions. By performing high-resolution multiplexed immunofluorescence on human cells, we use CAMPA to reveal how subnuclear organisation changes upon different perturbations of RNA production or processing, and how different membraneless organelles scale with cell size. Furthermore, by integrating information across the cellular and subcellular scales, we uncover new links between the molecular composition of membraneless organelles and bulk RNA synthesis rates of single cells. We anticipate that CAMPA will greatly accelerate the systematic mapping of multiscale atlases of biological organisation to identify the rules by which context shapes physiology and disease. | systems biology |
10.1101/2022.05.07.491046 | Single-cell RNA sequencing reveals molecular features of postnatal maturation in the murine retinal pigment epithelium | Transcriptomic analysis of the mammalian retinal pigment epithelium (RPE) aims to identify cellular networks that influence ocular development, maintenance, function, and disease. However, available evidence points to RPE cell heterogeneity in the native tissue, which adds complexity to transcriptomic analysis. Here, to assess cell heterogeneity, we performed single-cell RNA sequencing of RPE cells from two young adult male C57BL/6J mice. Following quality control to ensure robust transcript identification limited to cell singlets, we detected 13,858 transcripts among 2,667 and 2,846 RPE cells, respectively. Dimensional reduction by principal component analysis and uniform manifold approximation and projection revealed six distinct cell popu-lations. All clusters expressed transcripts typical of RPE cells; the smallest (C1, containing 1-2% of total cells) exhibited hallmarks of stem and/or progenitor cells. Placing C1-6 along a pseudotime axis suggested a relative decrease in melanogenesis and stem/progenitor gene expression, and a corresponding increase in visual cycle gene expression upon RPE maturation. K-means testing of all detected transcripts identified additional expression patterns that may advance understanding of RPE stem/pro-genitor cell maintenance and the evolution of cellular metabolic networks during development. This work provides new insights into the transcriptome of the mouse RPE and a baseline for identifying experimentally induced transcriptional changes in future studies of this tissue. | systems biology |
10.1101/2022.05.07.490875 | Allogeneic testes transplanted into castrated adult medaka (Oryzias latipes) are engrafted and can function over a long period for production of donor-derived offspring by natural mating | Germ cell transplantation protocols generally require induced immunosuppression in the recipient to avoid rejection of the transplanted cells; cytotoxic drugs may also be used to deprive the recipient germ cells and create a germ cell niche that allows donor-derived germ cells to thrive. In the present study, we demonstrate that in medaka, allogeneic adult testicular tissue will engraft in adult recipients immediately after the rough castration of the recipients and without use of immunosuppressive drugs. The allografted testes are retained in the recipient body for at least three months and are able to produce viable gametes that yield offspring after natural mating. Some recipients showed a high frequency (over 60%) of offspring derived from spermatozoa produced by the transplanted germ cell tissues. Histological analyses showed that allografted testicular tissues included both germ cells and somatic cells that had established within an immunocompetent recipient testis. The relative simplicity of this new approach will benefit investigations of the basic processes of reproductive immunology and will improve the technique of gonadal tissue transplantation. | zoology |
10.1101/2022.05.07.491003 | Validation of tWO novel primers for THE promising amplification of the mitogenomic Cytochrome c Oxidase subunit I (COI) barcoding region in Artemia aff. sinica (Branchiopoda, Anostraca) | Due to the lack of a taxonomic key for the identification of Artemia species, molecular markers have been increasingly used for phylogenetic studies. The mtCOI marker is a regularly considered marker for the molecular systematics of Artemia populations. The proposed universal and specific primers have mostly failed to amplify the Artemia aff. sinica mtCOI marker, and on the whole, the successfully amplified products of the PCR were inefficient, primarily through the representation of poly-peak or incomplete sequences. We presumed that if a forward primer could be developed regarding the joint regions of the last part of the previous gene (tRNATyr) and the beginning of the target gene mtCOI, the sequence could be relevant to the target-sequence of mtCOI. Thus, here, we describe a new set of primers, which could be used to amplify the mtCOI barcoding region of Artemia aff. sinica Cai, 1989, with a high performance of sequencing. The new primer set worked well also for other Artemia bisexual species, as well as for parthenogenetic populations. It is recommended that joint regions between the previous/next gene(s) and the target marker, could be aimed at when designing specific primers for other markers and taxa. | zoology |
10.1101/2022.05.07.491002 | Cyst size variability in invasive American Artemia franciscana Kellogg, 1906 (Branchiopoda: Anostraca) in Asia: a commercial approach | Artemia is the most common live food which is used in aquaculture worldwide. This study reports on biometrical variation of introduced American Artemia franciscana cyst from 24 non-native localities and two native habitats in Asia and USA, respectively. Results showed the largest diameter of untreated cyst, diameter of decapsulated cyst and thicker chorion ordinarily belong to invasive populations. Because of the small cysts, which have an effect on increasing quantity per unit weight and could be the cause of increased hatching efficiency, commercial productions of A. franciscana cyst from native sources should potentially be considered higher quality than productions from non-indigenous environments. Principal Component Analysis revealed that all cyst batches from San Francisco Bay were classified in one group and the most invasive populations could arrange in other separated group. Although, diameter of decapsulated cyst and chorion thickness showed a negative and significant correlation among invasive populations, there was no significant relationship within native populations. These observations contrast with biometrical patterns of parthenogenetic populations. | zoology |
10.1101/2022.05.07.491031 | Oxytocin attenuates microglial activation and restores social and non-social memory in the APP/PS1 mouse model of Alzheimer's disease | Alzheimers disease (AD) is the main cause of dementia in the elderly and is characterized by memory loss, social withdrawal and neurodegeneration, eventually leading to death. Brain inflammation has emerged as a key pathogenic mechanism in AD. We hypothesized that oxytocin, a pro-social hypothalamic neuropeptide with anti-inflammatory properties, could have therapeutic actions in AD. We investigated oxytocin production in mouse models of AD, and evaluated the therapeutic potential of intranasal oxytocin. We observed lower levels of hypothalamic oxytocin in wild-type mice following brain infusion of amyloid-{beta} oligomers (A{beta}Os), as well as in APP/PS1 AD model mice. Treatment of APP/PS1 mice with intranasal oxytocin reduced microglial activation and favored deposition of A{beta} in dense core plaques, a potentially neuroprotective mechanism. Oxytocin further alleviated social and non-social memory impairments in APP/PS1 mice. Our findings point to oxytocin as a potential therapeutic target to reduce brain inflammation and correct memory deficits in AD. | neuroscience |
10.1101/2022.05.07.491020 | ATXN2 is a target of N-terminal proteolysis | Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder caused by the expansion of the poly-glutamine (polyQ) tract of Ataxin-2 (ATXN2). Other polyQ-containing proteins such as ATXN7 and huntingtin are associated with the development of neurodegenerative diseases when their N-terminal polyQ domains are expanded. Furthermore, they undergo proteolytic processing events that produce N-terminal fragments that include the polyQ stretch, which are implicated in pathogenesis. Interestingly, N-terminal ATXN2 fragments were reported in a brain extract from a SCA2 patient, but it is currently unknown whether an expanded polyQ domain contributes to ATXN2 proteolytic susceptibility. Here, we used transient expression in HEK293 cells to determine whether ATXN2 is a target for specific N-terminal proteolysis. We found that ATXN2 proteins with either normal or expanded polyQ stretches undergo proteolytic cleavage releasing an N-terminal polyQ-containing fragment. We identified a short amino acid sequence downstream of the polyQ domain that is necessary for N-terminal cleavage of full-length ATXN2 and sufficient to induce proteolysis of a heterologous protein. However, this sequence is not required for cleavage of a short ATXN2 isoform produced from an alternative start codon located just upstream of the CAG repeats encoding the polyQ domain. Our study extends our understanding of ATXN2 posttranslational regulation by revealing that this protein can be the target of specific proteolytic cleavage events releasing polyQ-containing products that are modulated by the N-terminal domain of ATXN2. N-terminal ATXN2 proteolysis of expanded polyQ domains might contribute to SCA2 pathology, as observed in other neurodegenerative disorders caused by polyQ domain expansion. | neuroscience |
10.1101/2022.05.06.490781 | Alone in a crowd: Effect of a nonfunctional lateral line on expression of the social hormone parathyroid hormone 2. | Parathyroid hormone 2 (Pth2) is a vertebrate-specific neuropeptide whose thalamic expression is upregulated by social contact with conspecifics. However, social interactions fail to stimulate pth2 expression in isolated zebrafish whose lateral line hair cells have been chemically ablated. These results suggest that modulation of pth2 by social context is acutely dependent on mechanosensory information from the lateral line. However, it is unclear how a congenital loss of lateral line function influences the ability of zebrafish to interpret their social environment. In this study, we measure pth2 levels in zebrafish mutants lacking hair cell function in either the lateral line only, or in both the inner ear and lateral line. Socially-raised lateral line mutants express lower levels of pth2 relative to wild type siblings, but there is no further reduction when all sensory hair cells are nonfunctional. However, social isolation of hair cell mutants causes a further reduction in pth2 expression, pointing to additional unidentified sensory cues that influence pth2 production. Lastly, we report that social context modulates fluorescent transgenes driven by the pth2 promoter. Altogether, these data suggest that lateral line mutants experience a chronic sense of isolation, even when raised in a social environment.
SUMMARY STATEMENTExpression of the pro-social neuropeptide pth2 is downregulated in larval zebrafish with a congenital loss of lateral line function. Thus, even in social environments, fish with compromised lateral lines may experience chronic loneliness. | neuroscience |