Biology subjects

Chen, B.

Publications and source records attributed to Chen, B..

At least 307 records · Page 17Linked to original sources

bioRxiv2020

Glycans function as a Golgi export signal to promote the constitutive exocytic trafficking

Most proteins in the secretory pathway are glycosylated. However, the role of glycans in the membrane trafficking is still unclear. Here, we discovered that transmembrane secretory cargos, such as interleukin 2 receptor subunit or Tac, transferrin receptor and cluster of differentiation 8a, unexpectedly displayed substantial Golgi localization when their O-glycosylation was compromised. By quantitatively measuring their Golgi residence times, we found that the apparent Golgi localization of these O-glycan deficient cargos is due to their slow Golgi export. The super-resolution microscopy method that we previously developed revealed that O-glycan deficient Tac chimeras localize at the interior of the trans-Golgi cisternae. The O-glycan was observed to be both necessary and sufficient for the efficient Golgi export of Tac chimeras. By sequentially introducing O-glycosylation sites to {beta}-galactoside -2,6-sialyltransferase1, we demonstrated that the O-glycans effect on the Golgi export is probably additive. Finally, the finding that N-glycosylated GFP substantially reduces the Golgi residence time of Tac chimera suggests that the N-glycan might have a similar effect. Therefore, both O- and N-glycan might function as a generic Golgi export signal at the trans-Golgi to promote the constitutive exocytic trafficking.

cell biology↗

bioRxiv2020

Distinct conformational states of SARS-CoV-2 spike protein

The ongoing SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic has created urgent needs for intervention strategies to control the crisis. The spike (S) protein of the virus forms a trimer and catalyzes fusion between viral and target cell membranes - the first key step of viral infection. Here we report two cryo-EM structures, both derived from a single preparation of the full-length S protein, representing the prefusion (3.1[A] resolution) and postfusion (3.3[A] resolution) conformations, respectively. The spontaneous structural transition to the postfusion state under mild conditions is independent of target cells. The prefusion trimer forms a tightly packed structure with three receptor-binding domains clamped down by a segment adjacent to the fusion peptide, significantly different from recently published structures of a stabilized S ectodomain trimer. The postfusion conformation is a rigid tower-like trimer, but decorated by N-linked glycans along its long axis with almost even spacing, suggesting possible involvement in a mechanism protecting the virus from host immune responses and harsh external conditions. These findings advance our understanding of how SARS-CoV-2 enters a host cell and may guide development of vaccines and therapeutics.

biochemistry↗

bioRxiv2020

A putative long noncoding RNA-encoded micropeptide maintains cellular homeostasis in pancreatic β-cells

Micropeptides (microproteins) encoded by transcripts previously annotated as long noncoding RNA (IncRNAs) are emerging as important mediators of fundamental biological processes in health and disease. Here we applied two computational tools to identify putative micropeptides encoded by lncRNAs that are expressed in the human pancreas. We experimentally verified one such micropeptide encoded by a {beta}-cell- and neural cell-enriched lncRNA TUNAR (also known as TUNA, HI-LNC78 or LINC00617). We named this highly conserved 48-amino-acid micropeptide Beta cell- and Neural cell-regulin (BNLN). BNLN contains a single-pass transmembrane domain and localized at the endoplasmic reticulum in pancreatic {beta}-cells. Overexpression of BNLN lowered ER calcium levels, increased cytosolic calcium levels, and maintained ER homeostasis in response to high glucose challenge. To determine the physiological and pathological roles of BNLN, we assessed the BNLN expression in islets from mice fed with a high-fat diet and a regular diet, and found that BNLN is suppressed by diet-induced obesity (DIO). Conversely, overexpression of BNLN elevated glucose-stimulated insulin secretion in INS-1 cells. Lastly, BNLN overexpression enhanced insulin secretion in islets from lean and obese mice as well as from humans. Taken together, our study provides the first evidence that lncRNA-encoded micropeptides play a critical role in pancreatic {beta}-cell function and provides a foundation for future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes.

molecular biology↗

bioRxiv2020

Long Noncoding RNA Derived from LncRNA-mRNA Co-expression Networks Modulates the Locust Phase Change

Long noncoding RNAs (lncRNAs) regulate various biological processes from gene expression to animal behavior. Although protein-coding genes, microRNAs, and neuropeptides play important roles in the regulation of phenotypic plasticity in migratory locust, empirical studies on the function of lncRNAs in the process remain limited. Here, we applied high-throughput RNA-seq to characterize the expression patterns of lncRNAs and mRNAs in the time course of the locust phase change. LncRNAs displayed more rapid response at the early stages of the time-course treatments than mRNA expression. Functional annotations demonstrated that early changed lncRNAs employed different pathways in isolation and crowding processes to cope up with the changes in population density. Finally, two overlapping hub lncRNA loci in the crowding and isolation networks were screened to be functionally verified. Experimental validation indicated that LNC1010057 could act as a potential regulator to modulate the locust phase change. This work offers new insights into the mechanism underlying the locust phase change and expands the scope of lncRNA functions in animal behavior.

bioinformatics↗

bioRxiv2020

Reversal of Infected Host Gene Expression Identifies Repurposed Drug Candidates for COVID-19

The repurposing of existing drugs offers the potential to expedite therapeutic discovery against the current COVID-19 pandemic caused by the SARS-CoV-2 virus. We have developed an integrative approach to predict repurposed drug candidates that can reverse SARS-CoV-2-induced gene expression in host cells, and evaluate their efficacy against SARS-CoV-2 infection in vitro. We found that 13 virus-induced gene expression signatures computed from various viral preclinical models could be reversed by compounds previously identified to be effective against SARS- or MERS-CoV, as well as drug candidates recently reported to be efficacious against SARS-CoV-2. Based on the ability of candidate drugs to reverse these 13 infection signatures, as well as other clinical criteria, we identified 10 novel candidates. The four drugs bortezomib, dactolisib, alvocidib, and methotrexate inhibited SARS-CoV-2 infection-induced cytopathic effect in Vero E6 cells at < 1 {micro}M, but only methotrexate did not exhibit unfavorable cytotoxicity. Although further improvement of cytotoxicity prediction and bench testing is required, our computational approach has the potential to rapidly and rationally identify repurposed drug candidates against SARS-CoV-2. The analysis of signature genes induced by SARS-CoV-2 also revealed interesting time-dependent host response dynamics and critical pathways for therapeutic interventions (e.g. Rho GTPase activation and cytokine signaling suppression).

genomics↗

bioRxiv2020

Extended depth of focus multiphoton microscopy via incoherent pulse splitting

We present a phase mask that can be easily added to any multi-photon raster scanning microscope to extend the depth of focus five-fold at a small loss in lateral resolution. The method is designed for ultrafast laser pulses or other light-sources featuring a low coherence length. In contrast to other methods of focus extension, our approach uniquely combines low complexity, high light-throughput and multicolor capability. We characterize the point-spread function in a two-photon microscope and demonstrate fluorescence imaging of GFP labeled neurons in fixed brain samples as imaged with conventional and extended depth of focus two-photon microscopy.

biophysics↗

bioRxiv2020

Ibrutinib blocks YAP1 activation and reverses BRAFi resistance in melanoma cells

Most BRAF-mutant melanoma tumors respond initially to BRAFi/MEKi therapy, although few patients have durable long-term responses to these agents. The goal of this study was to utilize an unbiased computational approach to identify inhibitors which reverse an experimentally derived BRAFi resistance gene expression signature. Using this approach, we found that ibrutinib effectively reverses this signature and we demonstrate experimentally that ibrutinib re-sensitizes a subset of BRAFi-resistant melanoma cells to vemurafenib. Ibrutinib is used clinically as a BTK inhibitor; however, neither BTK deletion nor treatment with acalabrutinib, another BTK inhibitor with reduced off-target activity, re-sensitized cells to vemurafenib. These data suggest that ibrutinib acts through a BTK-independent mechanism in vemurafenib re-sensitization. To better understand this mechanism, we analyzed the transcriptional profile of ibrutinib-treated BRAFi-resistant melanoma cells and found that the transcriptional profile of ibrutinib was highly similar to that of multiple SRC kinase inhibitors. Since ibrutinib, but not acalabrutinib, has significant off-target activity against multiple SRC family kinases, it suggests that ibrutinib may be acting through this mechanism. Furthermore, genes either upregulated or downregulated by ibrutinib treatment are enriched in YAP1 target genes and we showed that ibrutinib, but not acalabrutinib, reduces YAP1 activity in BRAFi-resistant melanoma cells. Taken together, these data suggest that ibrutinib, or other SRC family kinase inhibitors, may be useful for treating some BRAFi/MEKi-refractory melanoma tumors.

cancer biology↗

bioRxiv2020

Iatrogenic Iron Promotes Neurodegeneration and Activates Self-protection of Neural Cells against Exogenous Iron Attacks

Metal implants are used worldwide, with millions of metal nails, plates and fixtures grafted during orthopaedic surgeries. Iron is the most common element of these metal implants. As time passes metal elements can be corroded and iron can be released from the implants in the form of ferric (Fe3+) or ferrous (Fe2+). These iron ions can permeate the surrounding tissues and enter circulation; importantly both Fe3+ and Fe2+ freely pass blood brain barrier (BBB). Can iron from implants represent a risk factor for neurological diseases? This remains an unanswered question. In this study, we discovered that the probability of metal implants delivered through orthopaedic surgeries was higher in patients of Parkinsons diseases (PD) or ischemic stroke than in healthy subjects. This finding instigated subsequent study of iron effects on neuronal cells. In experiments in vivo, we found that iron selectively decreased presence of divalent metal transporter 1 (DMT1) in neurones through increasing the expression of Ndfip1, which degrades DMT1 and rarely exists in glial cells. At the same time iron accumulation increased expression of DMT1 in astrocytes and microglial cells and triggered reactive astrogliosis and microglial activation. Facing the attack of excess iron, glial cells act as neuroprotectors to uptake more extracellular iron by up-regulating DMT1, whereas neurones limit iron uptake through decreasing DMT1 operation. Cerebral accumulation of iron was associated with impaired cognition, locomotion and mood. Excess iron thus affects neural cells and could increase the risk of neurodegeneration.

neuroscience↗

bioRxiv2020

Establishment of Etv5 gene knockout mice as a recipient model for spermatogonial stem cell transplantation

Transplantation of spermatogonial stem cells (SSCs) is an alternative reproductive method to achieve conservation and production of elite animals in livestock production. Creating a recipient animal without endogenous germ cells is important for effective SSC transplantation. However, natural mutants with depletion of SSCs are difficult to obtain, and drug ablation of endogenous germ cells is arduous to perform for practical use. In this study, we used mouse models to study the preparation of recipients with congenital germ cell ablation. We knocked out (KO) Ets-variant gene 5 (Etv5) in mice using the CRISPR/Cas9 system. The testicular weight of Etv5-/- mice was significantly lower than that of wild-type (WT) mice. The germ cell layer of the seminiferous tubules gradually receded with age in Etv5-/- mice. At 12 weeks of age, the tubules of Etv5-/- mice lacked germ cells (Sertoli cell-only syndrome), and sperm were completely absent in the epididymis. We subsequently transplanted allogeneic SSCs with enhanced green fluorescent protein (EGFP) into 3-(immature) or 7-week-old (mature) Etv5-/- mice. Restoration of germ cell layers in the seminiferous tubules and spermatogenesis was observed in all immature testes but not in mature adult testes at 2 months post-transplantation. The presence of heterologous genes Etv5 and EGFP in recipient testicular tissue and epididymal sperm by PCR indicated that sperm originated from the transplanted donor cells. Our study demonstrates that, although Etv5-/- mice could accommodate and support foreign germ cell transplantation, this process occurs in a quite low efficiency to support a full spermatogenesis of transplanted SSCs. However, using Etv5-/- mice as a recipient model for SSC transplantation is feasible, and still needs further investigation to establish an optimized transplantation process.

bioengineering↗

bioRxiv2020

Converting lateral scanning into axial focusing to speed up 3D microscopy

In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of 3D volumetric imaging. Recently, by conjugating either a movable-mirror to the image plane or an electrotuneable lens (ETL) to the back-focal plane respectively, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits axial scanning rate (usually only 10-100 Hz for piezoelectric or voice coil based actuators), while ETLs introduce spherical and higher order aberrations, thereby preventing high-resolution imaging. Here, we introduce a novel optical design that can transform a lateral-scan motion into a spherical-aberration-free, high-resolution, rapid axial scan. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in image space. We characterize the optical performance of this remote focusing technique and use it to accelerate axially swept light-sheet microscopy (ASLM) by one order of magnitude, allowing the quantification of rapid vesicular dynamics in 3D.

bioengineering↗

bioRxiv2020

Potential T-cell and B-cell Epitopes of 2019-nCoV

As of early March, 2019-nCoV has infected more than one hundred thousand people and claimed thousands of lives. 2019-nCoV is a novel form of coronavirus that causes COVID-19 and has high similarity with SARS-CoV. No approved vaccine yet exists for any form of coronavirus. Here we use computational tools from structural biology and machine learning to identify 2019-nCoV T-cell and B-cell epitopes based on viral protein antigen presentation and antibody binding properties. These epitopes can be used to develop more effective vaccines and identify neutralizing antibodies. We identified 405 viral peptides with good antigen presentation scores for both human MHC-I and MHC-II alleles, and two potential neutralizing B-cell epitopes near the 2019-nCoV spike protein receptor binding domain (440-460 and 494-506). Analyzing mutation profiles of 68 viral genomes from four continents, we identified 96 coding-change mutations. These mutations are more likely to occur in regions with good MHC-I presentation scores (p=0.02). No mutations are present near the spike protein receptor binding domain. Based on these findings, the spike protein is likely immunogenic and a potential vaccine candidate. We validated our computational pipeline with SARS-CoV experimental data. Significance StatementThe novel coronavirus 2019-nCoV has affected more than 100 countries and continues to spread. There is an immediate need for effective vaccines that contain antigens which trigger responses from human T-cells and B-cells (known as epitopes). Here we identify potential T-cell epitopes through an analysis of human antigen presentation, as well as B-cell epitopes through an analysis of protein structure. We identify a list of top candidates, including an epitope located on 2019-nCoV spike protein that potentially triggers both T-cell and B-cell responses. Analyzing 68 samples, we observe that viral mutations are more likely to happen in regions with strong antigen presentation, a potential form of immune evasion. Our computational pipeline is validated with experimental data from SARS-CoV.

microbiology↗

bioRxiv2020

O-GlcNAcylation and oxidation contribute to atrial fibrillation in diabetes by activating CaMKII

Diabetes mellitus and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF in patients. However, the mechanism(s) underlying this clinical association is unknown. Elevated protein O-GlcNAcylation (OGN) and reactive oxygen species (ROS) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by OGN (OGN-CaMKII) and ROS (ox-CaMKII). We induced type 1 (T1D) and type 2 diabetes (T2D) in a portfolio of genetic mouse models capable of dissecting the role of OGN and ROS at CaMKII and the type 2 ryanodine receptor (RyR2), an intracellular Ca2+ channel implicated as an important downstream mechanism of CaMKII- mediated arrhythmias. Here we show that T1D and T2D significantly increased AF, similar to observations in patients, and this increase required CaMKII. While T1D and T2D both require ox-CaMKII to increase AF, they respond differently to loss of OGN-CaMKII or OGN inhibition. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF, and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by diverse mechanisms and targets, including CaMKII and RyR2. The proarrhythmic consequences of OGN- and ox-CaMKII differ between T1D and T2D. These results provide new and unanticipated insights into the mechanisms for increased AF in diabetes mellitus, and suggest successful future therapies will need to be different for AF in T1D and T2D.

molecular biology↗

bioRxiv2020

A Refined View of Airway Microbiome in Chronic Obstructive Pulmonary Disease at Species and Strain-levels

Little is known about the species and strain-level diversity of the airway microbiome, and its implication in chronic obstructive pulmonary disease (COPD). Here we report the first comprehensive analysis of the COPD airway microbiome at species and strain-levels. The full-length 16S rRNA gene was sequenced from sputum in 98 stable COPD patients and 27 age-matched healthy controls, using the third-generation Pacific Biosciences sequencing platform. Individual species within the same genus exhibited reciprocal relationships with COPD and disease severity. Species dominant in health can be taken over by another species within the same genus in GOLD IV patients. Such turnover was also related to enhanced symptoms and exacerbation frequency. Ralstonia mannitolilytica, an opportunistic pathogen, was significantly increased in COPD frequent exacerbators. There were inflammatory phenotype-specific associations of microbiome at the species-level. One group of four pathogens including Haemophilus influenzae and Moraxella catarrhalis, were specifically associated with sputum mediators for neutrophilic inflammation. Another group of seven species, including Tropheryma whipplei, showed specific associations with mediators for eosinophilic inflammation. Strain-level detection uncovered three non-typeable H. influenzae strains PittEE, PittGG and 86-028NP in the airway microbiome, where PittGG and 86-028NP abundances may inversely predict eosinophilic inflammation. The full-length 16S data augmented the power of functional inference and led to the unique identification of butyrate-producing and nitrate reduction pathways as significantly depleted in COPD. Our analysis uncovered substantial intra-genus heterogeneity in the airway microbiome associated with inflammatory phenotypes and could be of clinical importance, thus enabled a refined view of the airway microbiome in COPD. "Take-home" messageThe species-level analysis using the third-generation sequencing enabled a refined view of the airway microbiome and its relationship with clinical outcome and inflammatory phenotype in COPD.

microbiology↗

bioRxiv2019

Slo2 potassium channel function depends on a SCYL1 protein

Slo2 potassium channels play important roles in neuronal function, and their mutations in humans cause epilepsies and cognitive defects. However, little is known how Slo2 function is regulated by other proteins. Here we found that the function of C. elegans Slo2 (SLO-2) depends on adr-1, a gene important to RNA editing. However, slo-2 transcripts have no detectable RNA editing events and exhibit similar expression levels in wild type and adr-1 mutants. In contrast, mRNA level of scyl-1, which encodes an orthologue of mammalian SCYL1, is greatly reduced in adr-1 mutants due to deficient RNA editing at a single adenosine in its 3-UTR. SCYL-1 physically interacts with SLO-2 in neurons. Single-channel open probability of SLO-2 in neurons is reduced by [~]50% in scyl-1 knockout whereas that of human Slo2.2/Slack is doubled by SCYL1 in a heterologous expression system. These results suggest that SCYL-1/SCYL1 is an evolutionarily conserved regulator of Slo2 channels.

neuroscience↗

bioRxiv2019

Dual indexed design of in-Drop single-cell RNA-seq libraries improves sequencing quality and throughput

The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. Here, we engineered a new dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. We overcame the index-hopping issue, demonstrated significant improvements in base-calling accuracy, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior library structures. Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

molecular biology↗

bioRxiv2019

Patient stratification of clear cell renal cell carcinoma using the global transcription factor activity landscape derived from RNA-seq data

Clear cell renal cell carcinoma represents the most common type of kidney cancer. Precision medicine approach to ccRCC requires an accurate stratification of patients that can predict prognosis and guide therapeutic decision. Transcription factors are implicated in the initiation and progression of human carcinogenesis. However, no comprehensive analysis of transcription factor activity has been proposed so far to realize patient stratification. Here we propose a novel approach to determine the subtypes of ccRCC patients based on global transcription factor activity landscape. Using the TCGA cohort dataset, we identified different subtypes that have distinct upregulated biomarkers and altered biological pathways. More important, this subtype information can be used to predict the overall survival of ccRCC patients. Our results suggest that transcription factor activity can be harnessed to perform patient stratification.

systems biology↗

bioRxiv2019

OCTAD: an open workplace for virtually screening therapeutics targeting precise cancer patient groups using gene expression features

One approach to precision medicine is to discover drugs that target molecularly defined diseases. Voluminous cancer patient gene expression profiles have been accumulated in public databases, enabling the creation of a cancer-specific expression signature. By matching this signature to perturbagen-induced gene expression profiles from large drug libraries, researchers can prioritize small molecules that present high potency to reverse expression of signature genes for further experimental testing of their efficacy. This approach has proven to be an efficient and cost-effective way to identify efficacious drug candidates. However, the success of this approach requires multiscale procedures, imposing significant challenges to many labs. Therefore, we present OCTAD: an open workplace for virtually screening compounds targeting precise cancer patient groups using gene expression features. We release OCTAD as a web portal and standalone R workflow to allow experimental and computational scientists to easily navigate the tool. In this work, we describe this tool and demonstrate its potential for precision medicine.

bioinformatics↗

bioRxiv2019

Developmental plasticity of hearing sensitivity in red-eared slider Trachemys scripta elegans

Developmental plasticity of hearing sensitivity (DPHS) has been verified in some groups of vertebrates. Turtles face a trade-off between terrestrial and aquatic hearing in different acoustic environments throughout ontogeny. However, how chelonian hearing sensitivity changes throughout ontogeny is still unclear. To verify DPHS in turtles, auditory brainstem responses (ABR) were compared using hearing thresholds and latencies in female red-eared slider (Trachemys scripta elegans) aged 1 week, 1 month, 1 year, and 5 years, and the results showed hearing sensitivity bandwidths of approximately 200-1100, 200-1100, 200-1300, and 200-1400 Hz, respectively. The lowest threshold sensitivity was approximately 600{square}Hz. Below 600 Hz, ABR threshold decreased rapidly with increasing age (1 week to 1 year), with significant differences between age groups, but no significant difference between the 1- and 5-year age groups (stimulus frequency, 200-600 Hz). Above 600 Hz, ABR threshold was the lowest in the 5-year age group. These findings show that aging was accompanied by hearing sensitivity changes, suggesting rapid, frequency-segmented development during ontogeny. This variability in hearing sensitivity differs from that reported in other vertebrates, and allows adaptation to acoustically distinct environments throughout ontogeny. Our findings further elucidate the developmental patterns of the vertebrate auditory system.

animal behavior and cognition↗