Biology subjects

Chen, S.

Publications and source records attributed to Chen, S..

At least 883 records · Page 49Linked to original sources

bioRxiv2020

Simulation of Ca2+ oscillations in astrocytes mediated by amyloid beta in Alzheimer's disease

Disruptions of astrocyte Ca2+ signaling is important in Alzheimers disease (AD) with the unclear mechanism of amyloid beta peptide (A{beta}). We have modified our previous computational model of spontaneous Ca2+ oscillations in astrocytes to investigate the effects of A{beta} on intracellular Ca2+ dynamics. The simulation results have shown consistence with the previous experiments. A{beta} can increase the resting concentration of intracellular Ca2+ and change the regime of Ca2+ oscillations by activating L-type voltage-gated calcium channels and the metabolic glutamate receptors, or by increasing ryanodine receptors sensitivity and Ca2+ leakage, respectively. This work have provided a toolkit to study the influence of A{beta} on intracellular Ca2+ dynamics in AD. It is helpful for understanding the toxic role of A{beta} during the progression of AD. Statement of SignificanceAlzheimers disease (AD) is the most common neurodegenerative disease with the unclear mechanism of amyloid beta peptide (A{beta}). This work have implemented a computational model to address the Ca2+ dynamics of astrocyte mediated by A{beta} with the four different pathways: voltage-gated calcium channels, metabotropic glutamate receptors 5, ryanodine receptor channels and membrane leak. The Ca2+ oscillations and bifurcation diagram indicate that astrocytes exhibit ionic excitability mediated by A{beta} and become the potential targets of A{beta} neurotoxicity. We expect this shared computational model would advance the understanding of AD.

biophysics↗

bioRxiv2020

Differential responses of transplanted stem cells to the diseased environment unveiled by a single molecular NIR II cell tracker

Stem cell therapy holds high promises in regenerative medicine. The major challenge of clinical translation is to precisely and quantitatively evaluate the in vivo cell distribution, migration, and engraftment, which cannot be easily achieved by current techniques. To address this issue, for the first time, we have developed a single molecular cell tracker with a strong fluorescence signal in the second near-infrared (NIR-II) window (1000-1700 nm) for real-time monitoring of in vivo cell behaviors in both healthy and diseased animal models. The NIR-II tracker (CelTrac1000) has shown complete cell labeling with low cytotoxicity and profound long-term tracking ability for 30 days in high temporospatial resolution for semi-quantification of the biodistribution of primary mesenchymal stem cell and induced pluripotent stem cell-derived endothelial cells. Taking advantage of the unique merits of CelTrac1000, the responses of transplanted stem cells to different diseased environments have been discriminated and unveiled. Furthermore, we also demonstrate CelTrac1000 as a universal and effective technique for ultrafast real-time tracking of the cellular migration and distribution in a single cell cluster resolution, along with the lung contraction and heart beating. As such, this single molecular NIR-II tracker will shift the optical cell tracking into a single cell cluster and millisecond temporospatial resolution for better evaluating and understanding stem cell therapy, affording optimal doses and efficacy. Significance StatementFor the first time, we synthesized a NIR-II tracker (CelTrac1000) for ultrafast real-time tracking of the migration trajectory of transplanted mesenchymal stem cells in the circulatory system with a single cell cluster resolution. Taking advantage of the merits of CelTrac1000, the responses of transplanted stem cells to different diseased environments, including acute lung injury, myocardial infarction, and middle cerebral artery occlusion, have been discriminated and unveiled in mice models. As such, our approach can help correlate critical biomedical information in stem cell therapies, such as stem cell dosing and engraftment and their relationships with efficacy, providing more accurate therapeutic treatment and outcomes in certain diseases during a long evaluation period (>30 days) in comparison with the commercial Qtracker (7-10 days).

bioengineering↗

bioRxiv2020

Schistosoma Japonicum infection in Treg-specific USP21 knock-out mice

USP21, an E3 de-ubiquitin enzyme playing vital roles in physiological activities, is important for Treg cells to maintain immune homeostasis and control immune tolerance. To understand its diverse functions and potential mechanism is essential for disease development. We, using the USP21 gene-conditional knock-out mice model of Schistosoma Japonicum infection, found more cercariae developed into adults, and more eggs deposited in the liver in KO mice. However, immunohistochemistry showed the degree and the area of egg granuloma and liver fibrosis were both reduced. This suggested knock-out USP21 did affect the immunoregulation between schistosomes and the host. In KO mice the content of IFN-gamma and IL-4, and the expression of anti-SEA IgG and anti-SWAP IgG both increased in the liver, spleen and blood by flow cytometry, while the content of IL-10, lL-17A, IL-23, IL-9 and the expression of USP21 and anti-SEA IgM decreased. This indicated USP21-knockout-Tregs promoted both Th1-type and Th2-type immunity and inhibited other immunities during schistosomes infection, which disordered the host immunity. This study revealed the immunomodulatory of USP21 and preliminarily suggests it might be essential to regulate the complex immune network between the host and schistosomes. USP21 provides a new possible target for schistosomiasis treatment in the future. Author summarySchistosomiasis is a common neglected tropical disease that affects more than 230 million people worldwide. Therefore, the study on the mechanism of immune interaction between schistosomas and the host is not only helpful for the understanding of immune homeostasis, but also helpful for the further development of the treatment of schistosomiasis. Ubiquitin Specific Protease 21(USP21) has been shown to be involved in the regulation of many biological processes, such as maintaining immune homeostasis and regulating cell growth. Here, we observed that the specific deletion of USP21 led to the decrease of mices ability to resist schistosomes infection and promoted the survival of schistosomes. It was also proved that unstable regulatory T cells would produce polarization phenomenon and promote differentiation to helper T cells, which would lead to disorder of immune response in mice. However, this process reduced the serious immune pathological damage caused by egg granuloma. Our findings reveal that USP21 may be an important molecule regulating immune interaction between Schistosoma japonicum and the host.

immunology↗

bioRxiv2020

Global Landscape of Native Protein Complexes in Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 (hereafter: Synechocystis) is a model organism for studying photosynthesis, energy metabolism, and environmental stress. Though known as the first fully sequenced phototrophic organism, Synechocystis still has almost half of its proteome without functional annotations. In this study, we obtained 291 protein complexes, including 24,092 protein-protein interactions (PPIs) among 2062 proteins by using co-fractionation and LC/MS/MS. The additional level of PPIs information not only revealed the roles of photosynthesis in metabolism, cell motility, DNA repair, cell division, and other physiological processes, but also showed how protein functions vary from bacteria to higher plants due to the changed interaction partner. It also allows us to uncover functions of hypothetical proteins, such as Sll0445, Sll0446, S110447 participating in photosynthesis and cell motility, and Sll1334 regulating the expression of fatty acid. Here we presented the most extensive protein interaction data in Synechocystis so far, which might provide critical insights into the fundamental molecular mechanism in Cyanobacterium.

biochemistry↗

bioRxiv2020

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

The outbreak of coronavirus disease (COVID-19) in China caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. It is currently no specific viral protein targeted therapeutics yet. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein is yet to be clear. Herein, we have determined the 2.7 [A] crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although overall structure is similar with other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the {beta}-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

molecular biology↗

bioRxiv2020

Rapid & reversable mutations generate subclonal genetic diversity

Most genetic changes have negligible reversion rates. As most mutations that confer resistance to an adversary condition (e.g., drug treatment) also confer a growth defect in its absence, it is challenging for cells to genetically adapt to transient environmental changes. Here we identify a set of rapidly reversible drug resistance mutations in S. pombe that are caused by Microhomology mediated Tandem Duplication (MTD), and reversion back to the wild-type sequence. Using 10,000x coverage whole-genome sequencing we identify near 6000 subclonal MTDs in a single clonal population, and determine using machine learning how MTD frequency is encoded in genome. We find that sequences with the highest predicted MTD rates tend to generate insertions that maintain the correct reading frame suggesting that MTD formation has shaped the evolution of coding sequences. Our study reveals a common mechanism of reversible genetic variation that is beneficial for adaptation to environmental fluctuations and facilitates evolutionary divergence.

genetics↗

bioRxiv2020

Ageing compromises mouse thymus function and remodels epithelial cell differentiation

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

immunology↗

bioRxiv2020

Multi-epitope vaccine design using an immunoinformatics approach for 2019 novel coronavirus in China (SARS-CoV-2)

A new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. An in silico approach based on the available virus genome was applied to identify 19 high immunogenic B-cell epitopes and 499 human-leukocyte-antigen (HLA) restricted T-cell epitopes. Thirty multi-epitope peptide vaccines were designed by iNeo Suite, and manufactured by solid-phase synthesis. Docking analysis showed stable hydrogen bonds of epitopes with their corresponding HLA alleles. When four vaccine peptide candidates from the spike protein of SARS-CoV-2 were selected to immunize mice, a significantly larger amount of IgG in serum as well as an increase of CD19+ cells in ILNs was observed in peptide-immunized mice compared to the control mice. The ratio of IFN-{gamma}-secreting lymphocytes in CD4+ or CD8+ cells in the peptides-immunized mice were higher than that in the control mice. There were also a larger number of IFN-{gamma}-secreting T cells in spleen in the peptides-immunized mice. This study screened antigenic B-cell and T-cell epitopes in all encoded proteins of SARS-CoV-2, and further designed multi-epitope based peptide vaccine against viral structural proteins. The obtained vaccine peptides successfully elicited specific humoral and cellular immune responses in mice. Primate experiments and clinical trial are urgently required to validate the efficacy and safety of these vaccine peptides. ImportanceSo far, a new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. Different from the development approaches for traditional vaccines, the development of our peptide vaccine is faster and simpler. In this study, we performed an in silico approach to identify the antigenic B-cell epitopes and human-leukocyte-antigen (HLA) restricted T-cell epitopes, and designed a panel of multi-epitope peptide vaccines. The resulting SARS-CoV-2 multi-epitope peptide vaccine could elicit specific humoral and cellular immune responses in mice efficiently, displaying its great potential in our fight of COVID-19.

bioinformatics↗

bioRxiv2020

Notch1 regulates breast cancer stem cell function via a non-canonical cleavage-independent pathway

Current treatment of triple negative breast cancer patients is hindered by a high incidence of chemoresistance (30-50%). The prevailing theory is that resistance and subsequent recurrence is driven by cancer stem cells. Unfortunately, the functional characterization of cancer stem cells at the molecular level is still incomplete. We show here, that within the canonical breast cancer stem cell population, a subset of cells characterized by high Notch1 expression possesses the tumor-initiating property associated with cancer stem cells. Moreover, the tumor initiating property of these high Notch1-expressing breast cancer stem cells is mediated by a cleavage independent Notch signaling pathway culminating in the repression of SIRT1. Of note, the Notch1-mediated repression of SIRT1 is required not only for tumor initiation, but also for chemoresistance in breast cancer stem cells. Strikingly, inhibition of SIRT1 obviates the requirement for Notch1, marking the first example of conferring cancer stem cell function by inhibiting the activity of a single protein. We also demonstrate that progenitor-like mammary epithelial cells, which possess both luminal and basal properties, are also characterized by high Notch1 expression and repression of SIRT1 via the non-canonical pathway. These findings provide the first functional mechanistic requirements for tumor initiation by breast cancer stem cells and suggest that activation of the non-canonical Notch1 pathway is hardwired into tumor-initiating progenitor cells and thus a prerequisite for tumor initiation. Statement of SignificanceWe demonstrate that chemoresistant and tumor-initiating properties of breast cancer stem cells are driven by repression of SIRT1 via non-canonical Notch signaling, suggesting a novel therapeutic strategy for triple negative breast cancer.

cancer biology↗

bioRxiv2020

FL-QSAR: a federated learning based QSAR prototype for collaborative drug discovery

MotivationQuantitative structure-activity relationship (QSAR) analysis is commonly used in drug discovery. Collaborations among pharmaceutical institutions can lead to a better performance in QSAR prediction, however, intellectual property and related financial interests remain substantially hindering inter-institutional collaborations in QSAR modeling for drug discovery. ResultsFor the first time, we verified the feasibility of applying the horizontal federated learning (HFL), which is a recently developed collaborative and privacy-preserving learning framework to perform QSAR analysis. A prototype platform of federated-learning-based QSAR modeling for collaborative drug discovery, i.e, FL-QSAR, is presented accordingly. We first compared the HFL framework with a classic privacy-preserving computation framework, i.e., secure multiparty computation (MPC) to indicate its difference from various perspective. Then we compared FL-QSAR with the public collaboration in terms of QSAR modeling. Our extensive experiments demonstrated that (1) collaboration by FL-QSAR outperforms a single client using only its private data, and (2) collaboration by FL-QSAR achieves almost the same performance as that of collaboration via cleartext learning algorithms using all shared information. Taking together, our results indicate that FL-QSAR under the HFL framework provides an efficient solution to break the barriers between pharmaceutical institutions in QSAR modeling, therefore promote the development of collaborative and privacy-preserving drug discovery with extendable ability to other privacy-related biomedical areas. Availability and implementationThe source codes of the federated learning simulation and FL-QSAR are available on the GitHub: https://github.com/bm2-lab/FL-QSAR

bioinformatics↗

bioRxiv2020

CRISPR Screen in Regulatory T Cells Reveals Ubiquitination Modulators of Foxp3

Regulatory T cells (Tregs) are required to control immune responses and maintain homeostasis but are a significant barrier to anti-tumor immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of pro-inflammatory properties2, can promote autoimmunity and/or facilitate more effective tumor immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. Despite improved functional genetic tools that now allow for systematic interrogation, dissection of the gene regulatory programs that modulate Foxp3 expression has not yet been reported. In this study, we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Tregs and applied this technology to perform a targeted loss-of-function screen of [~]490 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We discovered several novel modulators including ubiquitin-specific peptidase 22 (Usp22), Ataxin 7 like 3 (Atxn7l3) and ring finger protein 20 (Rnf20). Members of the deubiquitination module of the SAGA chromatin modifying complex, Usp22 and Atxn7l3, were discovered to be positive regulators that stabilized Foxp3 expression; whereas the screen suggested Rnf20, an E3 ubiquitin ligase, is a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein and created defects in their suppressive function that led to spontaneous autoimmunity but protected against tumor growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Tregs could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Tregs. These results reveal novel modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

immunology↗

bioRxiv2020

Accurate localization of linear probe electrodes across multiple brains

Recently developed silicon probes have large numbers of recording electrodes on long linear shanks. Specifically, Neuropixels probes have 960 recording electrodes distributed over 9.6 mm shanks. Because of their length, Neuropixels probe recordings in rodents naturally span multiple brain areas. Typical studies collate recordings across several recording sessions and animals. Neurons recorded in different sessions and animals have to be aligned to each other and to a standardized brain coordinate system. Here we report a workflow for accurate localization of individual electrodes in standardized coordinates and aligned across individual brains. This workflow relies on imaging brains with fluorescent probe tracks and warping 3-dimensional image stacks to standardized brain atlases. Electrophysiological features are then used to anchor particular electrodes along the reconstructed tracks to specific locations in the brain atlas and therefore to specific brain structures. We performed ground-truth experiments, in which motor cortex outputs are labelled with ChR2 and a fluorescence protein. Recording from brain regions targeted by these outputs reveals better than 100 m accuracy for electrode localization.

neuroscience↗

bioRxiv2020

Ovarian Cancer Risk Variants are Enriched in Histotype-Specific Enhancers that Disrupt Transcription Factor Binding Sites

Quantifying the functional effects of complex disease risk variants can provide insights into mechanisms underlying disease biology. Genome wide association studies (GWAS) have identified 39 regions associated with risk of epithelial ovarian cancer (EOC). The vast majority of these variants lie in the non-coding genome, suggesting they mediate their function through the regulation of gene expression by their interaction with tissue specific regulatory elements (REs). In this study, by intersecting germline genetic risk data with regulatory landscapes of active chromatin in ovarian cancers and their precursor cell types, we first estimated the heritability explained by known common low penetrance risk alleles. The narrow sense heritability [Formula] of both EOC overall and high grade serous ovarian cancer (HGSOCs) was estimated to be 5-6%. Partitioned SNP-heritability across broad functional categories indicated a significant contribution of regulatory elements to EOC heritability. We collated epigenomic profiling data for 77 cell and tissue types from public resources (Roadmap Epigenomics and ENCODE), and H3K27Ac ChIP-Seq data generated in 26 ovarian cancer-relevant cell types. We identified significant enrichment of risk SNPs in active REs marked by H3K27Ac in HGSOCs. To further investigate how risk SNPs in active REs influence predisposition to ovarian cancer, we used motifbreakR to predict the disruption of transcription factor binding sites. We identified 469 candidate causal risk variants in H3K27Ac peaks that break TF motifs (enrichment P-Value < 1x10-5 compared to control variants). The most frequently broken motif was REST (P-Value = 0.0028), which has been reported as both a tumor suppressor and an oncogene. These systematic functional annotations with epigenomic data highlight the specificity of the regulatory landscape and demonstrate functional annotation of germline risk variants is most informative when performed in highly relevant cell types.

genetics↗

bioRxiv2020

The influence of peptide context on signalling and trafficking of glucagon-like peptide-1 receptor biased agonists

Signal bias and membrane trafficking have recently emerged as important considerations in the therapeutic targeting of the glucagon-like peptide-1 receptor (GLP-1R) in type 2 diabetes and obesity. In the present study, we have evaluated a peptide series with varying sequence homology between native GLP-1 and exendin-4, the archetypal ligands on which approved GLP-1R agonists are based. We find notable differences in agonist-mediated signalling, endocytosis and recycling, dependent both on the introduction of a His [->] Phe switch at position 1 and the specific mid-peptide helical regions and C-termini of the two agonists. These observations were linked to insulin secretion in a beta cell model and provide insights into how ligand factors influence GLP-1R function at the cellular level. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/961524v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@cb425borg.highwire.dtl.DTLVardef@719e37org.highwire.dtl.DTLVardef@14a45b9org.highwire.dtl.DTLVardef@127afd1_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2020

Clonal Evolution of Acute Myeloid Leukemia Revealed by High-Throughput Single-Cell Genomics

One of the pervasive features of cancer is the diversity of mutations found in malignant cells within the same tumor; a phenomenon called clonal diversity or intratumor heterogeneity. Clonal diversity allows tumors to adapt to the selective pressure of treatment and likely contributes to the development of treatment resistance and cancer recurrence. Thus, the ability to precisely delineate the clonal substructure of a tumor, including the evolutionary history of its development and the co-occurrence of its mutations, is necessary to understand and overcome treatment resistance. However, DNA sequencing of bulk tumor samples cannot accurately resolve complex clonal architectures. Here, we performed high-throughput single-cell DNA sequencing to quantitatively assess the clonal architecture of acute myeloid leukemia (AML). We sequenced a total of 556,951 cells from 77 patients with AML for 19 genes known to be recurrently mutated in AML. The data revealed clonal relationship among AML driver mutations and identified mutations that often co-occurred (e.g., NPM1/FLT3-ITD, DNMT3A/NPM1, SRSF2/IDH2, and WT1/FLT3-ITD) and those that were mutually exclusive (e.g., NRAS/KRAS, FLT3-D835/ITD, and IDH1/IDH2) at single-cell resolution. Reconstruction of the tumor phylogeny uncovered history of tumor development that is characterized by linear and branching clonal evolution patterns with latter involving functional convergence of separately evolved clones. Analysis of longitudinal samples revealed remodeling of clonal architecture in response to therapeutic pressure that is driven by clonal selection. Furthermore, in this AML cohort, higher clonal diversity ([≥]4 subclones) was associated with significantly worse overall survival. These data portray clonal relationship, architecture, and evolution of AML driver genes with unprecedented resolution, and illuminate the role of clonal diversity in therapeutic resistance, relapse and clinical outcome in AML.

cancer biology↗

bioRxiv2020

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract

PurposeTo characterize the effects of netarsudil on the aqueous humor outflow tract distal to the trabecular meshwork (TM). We hypothesized that netarsudil increases outflow facility in eyes with and without circumferential ab interno trabeculectomy (AIT) that removes the TM. Methods64 porcine anterior segment cultures were randomly assigned to groups with (n=32) and without circumferential AIT (n=32). Cultures were exposed to 0.1, 1, and 10 M netarsudil (N= 8 eyes per concentration). For each concentration, IOP and vessel diameters were compared to their respective pretreatment baselines. Outflow tract vessel diameters were assessed by spectral-domain optical coherence tomography (SDOCT) and rendered in 4D (XYZ time-series). ResultsNetarsudil at 1 M reduced IOP in both eyes with TM (-0.60{+/-}0.24 mmHg, p = 0.01) and in eyes without TM (-1.79{+/-}0.42 mmHg, p<0.01). At this concentration, vessels of the distal outflow tract dilated by 72%. However, at 0.1 M netarsudil elevated IOP in eyes with TM (1.59{+/-}0.36 mmHg, p<0.001) as well as in eyes without TM (0.23{+/-}0.32 mmHg, p<0.001). Vessels of the distal outflow tract constricted by 31%. Similarly, netarsudil at a concentration of 10 M elevated IOP both in eyes with TM (1.91{+/-}0.193, p<0.001) and in eyes without TM (3.65{+/-}0.86 mmHg, p<0.001). At this concentration, outflow tract vessels constricted by 27%. ConclusionIn the porcine anterior segment culture, the dose-dependent IOP changes caused by netarsudil matched the diameter changes of distal outflow tract vessels. Hyper- and hypotensive properties of netarsudil persisted after TM removal.

physiology↗

bioRxiv2020

3D-Reconstruction of the Human Conventional Outflow System by Ribbon Scanning Confocal Microscopy

PurposeRisk for glaucoma is driven by the microanatomy and function of the anterior segment. We performed a computation-intense, high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM) of the outflow tract of two human eyes. We hypothesized this would reveal important species differences when compared to existing data of porcine eyes, an animal that does not spontaneously develop glaucoma. MethodsAfter perfusing two human octogenarian eyes with lectin-fluorophore conjugate and optical clearance with benzyl alcohol benzyl benzoate (BABB), anterior segments were scanned by RSCM and reconstructed in 3D for whole-specimen rendering. Morphometric analyses of the outflow tract were performed for the trabecular meshwork (TM), limbal, and perilimbal outflow structures and compared to existing porcine data. ResultsRSCM provided high-resolution data for IMARIS-based surface reconstruction of outflow tract structures in 3D. Different from porcine eyes with an abundance of highly interconnected, narrow, and short collector channels (CCs), human eyes demonstrated fewer CCs which had a 1.5x greater cross-sectional area (CSA) and 2.6x greater length. Proximal CC openings at the level of Schlemms canal (SC) had a 1.3x larger CSA than distal openings into the scleral vascular plexus (SVP). CCs were 10.2x smaller in volume than the receiving SVP vessels. Axenfeld loops, projections of the long ciliary nerve, were also visualized. ConclusionIn this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes. Human CCs spanned several clock-hours and were larger than in porcine eyes. These species differences may point to factors downstream of the TM that increase our vulnerability to glaucoma. Grant informationNational Eye Institute K08EY022737 (NAL); Initiative to Cure Glaucoma of the Eye and Ear Foundation of Pittsburgh (NAL); Wiegand Fellowship of the Eye and Ear Foundation of Pittsburgh (YD); P30-EY08098 (NAL); Department grant by Research to Prevent Blindness (NAL); an unrestricted fellowship grant from the Xiangya Hospital of Central South University (SC).

physiology↗

bioRxiv2020

Regulation of olfactory-based sex behaviors in the silkworm by genes in the sex-determination cascade

Insect courtship and mating depend on integration of olfactory, visual, and tactile cues. Compared to other insects, Bombyx mori, the domesticated silkworm, has relatively simple sexual behaviors as it cannot fly. Here by using CRISPR/Cas and electrophysiological techniques we found that courtship and mating behaviors are regulated in male silk moths by mutating genes in the sex determination cascade belonging to two conserved pathways. Loss of Bmdsx gene expression significantly reduced the peripheral perception of the major pheromone component bombykol by reducing expression of the product of the BmOR1 gene which completely blocked courtship in adult males. Interestingly, we found that mating behavior was regulated independently by another sexual differentiation gene, Bmfru. Loss of Bmfru completely blocked mating, but males displayed normal courtship behavior. Lack of Bmfru expression significantly reduced the perception of the minor pheromone component bombykal due to the down regulation of BmOR3 expression; further, functional analysis revealed that loss of the product of BmOR3 played a key role in terminating male mating behavior. Our results suggest that Bmdsx and Bmfru are at the base of the two primary pathways that regulate olfactory-based sexual behavior. Author SummaryThe fundamental insect sexual behaviors, courtship and mating, result from successful integration of olfactory, vision, tactile and other complex innate behaviors. In the widely used insect model, Drosophila melanogaster, the sex determination cascade genes fruitless and doublesex are involved in the regulation of courtship and mating behaviors; however, little is known about the function of these sexual differentiation genes in regulating sex behaviors of Lepidoptera. Here we combine genetics and electrophysiology to investigate regulation pathway of sexual behaviors in the model lepidopteran insect, the domesticated silk moth, Bombyx mori. Our results support the presence of two genetic pathways in B. mori, named Bmdsx-BmOR1-bombykol and Bmfru-BmOR3-bombykal, which control distinct aspects of male sexual behavior that are modulated by olfaction. This is the first comprehensive report about the role of sex differentiation genes in the male sexual behavior in the silk moth.

genetics↗