Biology subjects

Geng, J.

Publications and source records attributed to Geng, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A feedback-driven IoT microfluidic, electrophysiology, and imaging platform for brain organoid studies

The analysis of tissue cultures, particularly brain organoids, requires a sophisticated integration and coordination of multiple technologies for monitoring and measuring. We have developed an automated research platform enabling independent devices to achieve collaborative objectives for feedback-driven cell culture studies. Our approach enables continuous, communicative, non-invasive interactions within an Internet of Things (IoT) architecture among various sensing and actuation devices, achieving precisely timed control of in vitro biological experiments. The framework integrates microfluidics, electrophysiology, and imaging devices to maintain cerebral cortex organoids while measuring their neuronal activity. The organoids are cultured in custom, 3D-printed chambers affixed to commercial microelectrode arrays. Periodic feeding is achieved using programmable microfluidic pumps. We developed a computer vision fluid volume estimator used as feedback to rectify deviations in microfluidic perfusion during media feeding/aspiration cycles. We validated the system with a set of 7-day studies of mouse cerebral cortex organoids, comparing manual and automated protocols. The automated protocols were validated in maintaining robust neural activity throughout the experiment. The automated system enabled hourly electrophysiology recordings for the 7-day studies. Median neural unit firing rates increased for every sample and dynamic patterns of organoid firing rates were revealed by high-frequency recordings. Surprisingly, feeding did not affect firing rate. Furthermore, performing media exchange during a recording showed no acute effects on firing rate, enabling the use of this automated platform for reagent screening studies.

neuroscience↗

bioRxiv2023

Internet-connected cortical organoids for project-based stem cell and neuroscience education

The introduction of internet-connected technologies to the classroom has the potential to revolutionize STEM education by allowing students to perform experiments in complex models that are unattainable in traditional teaching laboratories. By connecting laboratory equipment to the cloud, we introduce students to experimentation in pluripotent stem cell-derived cortical organoids in two different settings: Using microscopy to monitor organoid growth in an introductory tissue culture course, and using high density multielectrode arrays to perform neuronal stimulation and recording in an advanced neuroscience mathematics course. We demonstrate that this approach develops interest in stem cell and neuroscience in the students of both courses. All together, we propose cloud technologies as an effective and scalable approach for complex project-based university training. HIGHLIGHTS- Development of cortical organoids as pedagogical tools for undergraduate education. - Organoids implemented in a tissue culture course through cloud-enabled microscopy. - Multielectrode arrays allow for live organoid manipulation in a mathematics course. - Students self-report increased interest in neuroscience and stem cells topics.

scientific communication and education↗

bioRxiv2023

Distinct mutational processes shape selection of MHC class I and class II mutations across primary and metastatic tumors

Disruption of antigen presentation via loss of MHC expression is a strategy whereby cancer cells escape immune surveillance and develop resistance to immunotherapy. We developed the personalized genomics algorithm Hapster and accurately called somatic mutations within the MHC genes of 10,001 primary and 2,199 metastatic tumors, creating a catalog of 1663 nonsynonymous mutations that provide key insights into MHC mutagenesis. We found that MHC-I genes are among the most frequently mutated genes in both primary and metastatic tumors, while MHC-II mutations are more restricted. Recurrent deleterious mutations are found within haplotype and cancer-type specific hotspots associated with distinct mutational processes. Functional classification of MHC residues revealed significant positive selection for mutations disruptive to the B2M, peptide, and T-cell binding interfaces, as well as MHC chaperones. At the cohort level, all cancers with positive selection for MHC mutations are responsive to immune checkpoint inhibitors, underscoring the translational relevance of our findings.

cancer biology↗

bioRxiv2022

Systems analysis of metabolic responses to a mixed meal test in an obese cohort reveals links between tissue metabolism and the gut microbiota

Individuals with prediabetes and type 2 diabetes mellitus (T2DM) have poor ability to adapt to diet-triggered perturbations. We investigated global metabolic responses to a mixed meal test (MMT) in morbidly obese individuals with different diabetic status by performing plasma metabolomic profiling. Abnormal metabolism of carbohydrates, (branched-chain) amino acids, fatty acids and acylcholines in individuals with (pre)diabetes was observed. Moreover, differences in metabolic responses were associated with altered fecal metagenomics and transcriptomes of liver, jejunum and adipose tissues, which revealed a modified gut microbiome and multi-tissue metabolism in individuals having insulin resistance. Finally, using integrative machine learning models, we built a predictive model based on metabolomics data after 2h MMT, and identified possible new biomarkers for glycemic control including N-acetylaspartate and phenylalanine-derived metabolites that may be useful for diagnosis, intervention and prevention of T2DM.

systems biology↗

bioRxiv2022

ZNF598 responds to mitochondrial stress to abort stalled translation on mitochondrial outer membrane and maintain tissue homeostasis

Translational control exerts immediate effect on the composition, abundance, and integrity of the proteome. Ribosome-associated quality control (RQC) handles ribosomes stalled at the elongation and termination steps of translation, with ZNF598 in mammals and Hel2 in yeast serving as key sensors of translation stalling and coordinators of downstream resolution of collided ribosomes, termination of stalled translation, and removal of faulty translation products. The physiological regulation of RQC in general and ZNF598 in particular in multicellular settings is underexplored. Here we show that ZNF598 undergoes regulatory K63-linked ubiquitination and its level is upregulated upon mitochondrial stress in mammalian cells and Drosophila. Overexpression of ZNF598 protects against mitochondrial stress. In Drosophila models of neurodegenerative diseases and patient cells, ZNF598 overexpression aborted stalled translation of mitochondrial outer membrane-associated mRNAs and removed faulty translation products causal of disease. These results shed lights on the regulation of ZNF598 and its important role in mitochondrial homeostasis.

cell biology↗

bioRxiv2022

Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X

Dynamic Ca2+ signals reflect acute changes in membrane excitability (e.g. sensory response), and also mediate intracellular signaling cascades normally of longer time scales (e.g., Ca2+-dependent neuritogenesis). In both cases, chronic Ca2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca2+ indicators. Here, we demonstrate that the recently developed GCaMP-X outperforms GCaMP in long-term probe expression and/or chronic Ca2+ imaging. GCaMP-X shows much improved compatibility with neurons and thus more reliable than GCaMP as demonstrated in vivo by acute Ca2+ responses to whisker deflection or spontaneous Ca2+ fluctuations over an extended time frame. Chronic Ca2+ imaging data ([≥]1 month) are acquired from the same set of cultured cortical neurons, unveiling that spontaneous/local Ca2+ activities would progressively develop into autonomous/global Ca2+ oscillations. Besides the morphological indices of neurite length or soma size, the major metrics of oscillatory Ca2+, including rate, amplitude, synchrony among different neurons or organelles have also been examined along with the developmental stages. Both neuritogenesis and Ca2+ signals are dysregulated by GCaMP in virus-infected or transgenic neurons, in direct contrast to GCaMP-X without any noticeable side-effect. Such in vitro data altogether consolidate the unique importance of oscillatory Ca2+ to activity-dependent neuritogenesis, as one major factor responsible for the distinctions between GCaMP vs GCaMP-X in vivo. For the first time with GCaMP-X of long-term expression in neurons, spontaneous and sensory-evoked Ca2+ activities are imaged and evaluated both in vitro and in vivo, providing new opportunities to monitor neural development or other chronic processes concurrently with Ca2+ dynamics.

neuroscience↗

bioRxiv2021

Internet of Things Architecture for High Throughput Biology

The Internet of Things (IoT) provides a simple framework to easily control online devices. IoT is now a commonplace tool used by technology companies, but it is rarely used in biology experiments. IoT can benefit research through alarm notifications, automation, and the real-time monitoring of experiments. We developed and implemented an IoT architecture to control biological devices used in experiments. We developed our own electrophysiology, microscopy, and microfluidic devices so that may be controlled through a unified IoT architecture. The system allows each device to be monitored and controlled through an online web tool. We present our IoT architecture so other labs may replicate it for their own experiments.

bioengineering↗

bioRxiv2021

Cryo-EM structures of human TMEM120A and TMEM120B

TMEM120A (Transmembrane protein 120A) was recently identified as a mechanical pain sensing ion channel named as TACAN, while its homologue TMEM120B has no mechanosensing property1. Here, we report the cryo-EM structures of both human TMEM120A and TMEM120B. The two structures share the same dimeric assembly, mediated by extensive interactions through the transmembrane domain (TMD) and the N-terminal coiled coil domain (CCD). However, the nearly identical structures cannot provide clues for the difference in mechanosensing between TMEM120A and TMEM120B. Although TMEM120A could mediate conducting currents in a bilayer system, it does not mediate mechanical-induced currents in a heterologous expression system, suggesting TMEM120A is unlikely a mechanosensing channel. Instead, the TMDs of TMEM120A and TMEM120B resemble the structure of a fatty acid elongase, ELOVL7, indicating their potential role of an enzyme in lipid metabolism.

biophysics↗

bioRxiv2021

Light-weight Electrophysiology Hardware and Software Platform for Cloud-Based Neural Recording Experiments

ObjectiveNeural activity represents a functional readout of neurons that is increasingly important to monitor in a wide range of experiments. Extracellular recordings have emerged as a powerful technique for measuring neural activity because these methods do not lead to the destruction or degradation of the cells being measured. Current approaches to electrophysiology have a low throughput of experiments due to manual supervision and expensive equipment. This bottleneck limits broader inferences that can be achieved with numerous long-term recorded samples. ApproachWe developed Piphys, an inexpensive open source neurophysiological recording platform that consists both hardware and software. It is easily accessed and controlled via a standard web interface through Internet of Things (IoT) protocols. Main ResultsWe used a Raspberry Pi as the primary processing device and Intan bioamplifier. We designed a hardware expansion circuit board and software to enable voltage sampling and user interaction. This standalone system was validated with primary human neurons, showing reliability in collecting real-time neural activity. SignificanceThe hardware modules and cloud software allow for remote control of neural recording experiments as well as horizontal scalability, enabling long-term observations of development, organization, and neural activity at scale.

bioengineering↗

bioRxiv2021

CD147 antibody specifically and effectively inhibits infection and cytokine storm of SARS-CoV-2 variants

SARS-CoV-2 and its variants are raging worldwide. Unfortunately, the global vaccination is not efficient enough to attain a vaccine-based herd-immunity and yet no special and effective drug is developed to contain the spread of the disease. Previously we have identified CD147 as a novel receptor for SARS-CoV-2 infection. Here, we demonstrated that CD147 antibody effectively inhibits infection and cytokine storm caused by SARS-CoV-2 variants. In CD147KO VeroE6 cells, infections of SARS-CoV-2, its variants (B.1.1.7, B.1.351) and pseudovirus mutants (B.1.1.7, B.1.351, B.1.525, B.1.526 (S477N), B.1.526 (E484K), P.1, P.2, B.1.617.1, B.1.617.2) were decreased. Meanwhile, CD147 antibody effectively blocked the entry of variants and pseudomutants in VeroE6 cells, and inhibited the expression of cytokines. A model of SARS-CoV-2-infected hCD147 transgenic mice was constructed, which recapitulated the features of exudative diffuse alveolar damage and dynamic immune responses of COVID-19. CD147 antibody could effectively clear the virus and alveolar exudation, resolving the pneumonia. We found the elevated level of cyclophilin A (CyPA) in plasma of severe/critical cases, and identified CyPA as the most important proinflammatory intermediate causing cytokine storm. Mechanistically, spike protein of SARS-CoV-2 bound to CD147 and initiated the JAK-STAT pathway, which induced expression of CyPA. CyPA reciprocally bound to CD147, triggered MAPK pathway and consequently mediated the expression of cytokine and chemokine. In conclusion, CD147 is a critical target for SARS-CoV-2 variants and CD147 antibody is a promising drug to control the new wave of COVID-19 epidemic.

immunology↗

bioRxiv2021

Total Biosynthesis of Triacsin Featuring an N-hydroxytriazene Pharmacophore

Triacsins are an intriguing class of specialized metabolites possessing a conserved N-hydroxytriazene moiety not found in any other known natural products. Triacsins are notable as potent acyl-CoA synthetase inhibitors in lipid metabolism, yet their biosynthesis has remained elusive. Through extensive mutagenesis and biochemical studies, we here report all enzymes required to construct and install the N-hydroxytriazene pharmacophore of triacsins. Two distinct ATP-dependent enzymes were revealed to catalyze the two consecutive N-N bond formation reactions, including a glycine-utilizing hydrazine-forming enzyme, Tri28, and a nitrous acid-utilizing N-nitrosating enzyme, Tri17. This study paves the way for future mechanistic interrogation and biocatalytic application of enzymes for N-N bond formation.

biochemistry↗

bioRxiv2020

BMSCs differentiated into neurons, astrocytes and oligodendrocytesalleviatedthe inflammation and demyelination of EAE mice models

Multiple sclerosis (MS) is a complex, progressive neuroinflammatory disease associated with autoimmunity. Currently, effective therapeutic strategy was poorly found in MS. Experimental autoimmune encephalomyelitis (EAE) is widely used to study the pathogenesis of MS. Previous studies have shown that bone marrow mesenchymal stem Cells (BMSCs) transplantation could treat EAE animal models, but the mechanism was divergent. Here, we systematically evaluated whether BMSCs can differentiate into neurons, astrocytes and oligodendrocytes to alleviate the symptoms of EAE mice. We used Immunofluorescence staining to detect MAP-2 neurons marker, GFAP astrocytes marker, and MBP oligodendrocytes marker expression to evaluate whether BMSCs can differentiate. The effect of BMSCs transplantation on inflammatory cell invasion and demyelination in EAE mice were detected by Hematoxylin-Eosin (H&E) and Luxol Fast Blue (LFB) staining. Inflammatory factors expression was detected by ELISA and RT-qPCR. Our results showed that BMSCs could be induced to differentiate into neuron cells, astrocytes and oligodendrocyte in vivo and in vitro. In addition, BMSCs transplant improved the survival rate and weight, and reduced neurological function scores and disease incidence of EAE mice. Moreover, BMSCs transplant alleviated the inflammation and demyelination of EAE mice. Finally, we found that BMSCs transplantation down-regulated the expression levels of pro-inflammatory factors TNF-, IL-1{beta} and IFN-{gamma}, and up-regulated the expression levels of anti-inflammatory factors IL-10 and TGF-{beta}. In conclusion, this study found that BMSCs could alleviate the inflammatory response and demyelination in EAE mice, which may be achieved by the differentiation of BMSCs into neurons, astrocytes and oligodendrocytes in EAE mice.

neuroscience↗

bioRxiv2020

Mpl is activated by dimers of MPN-linked calreticulin mutants stabilized by disulfide bonds and ionic interactions

Myeloproliferative neoplasms (MPNs) are frequently driven by insertions and deletions within the gene encoding calreticulin (CRT). CRTDel52 and CRTIns5 are recurrent mutations. Although oncogenic transformation requires both mutated CRT and the myeloproliferative leukemia protein (Mpl), the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. Our studies reveal that the novel C-domain of CRTDel52 encodes specificity both for Mpl binding and for disulfide-mediated CRT dimerization. Disulfide-stabilized CRTDel52 dimers and multimers are observed in MPN patient-derived platelet lysates and in transfected mammalian cells. Cysteine mutations within both the novel C-domain (C400A and C404A) and the conserved N-domain (C163A) of CRTDel52 are required to reduce disulfide-mediated dimers and multimers of CRTDel52. Based on these data and published structures of crystalized CRT oligomers, we tested the relevance of ionic interactions between charged residues proximal to C163 at the N-domain dimerization interface. Charge alteration at these residues affected dimerization and multimerization of both wild type and CRTDel52. Elimination of intermolecular disulfides and disruption of ionic interactions at both proposed dimerization interfaces was required to abrogate the ability of CRTDel52 to induce cytokine-independent cell proliferation via Mpl. Based on these findings, we propose a structural model of the Mpl-activating CRTDel52 unit as a covalently-linked dimer that is stabilized by disulfides and ionic interactions at both the C-domain and N-domain. MPNs exploit a natural dimerization interface of CRT combined with C-domain gain-of-functions to achieve cell transformation.

cancer biology↗

bioRxiv2020

SORBS2 is a genetic factor contributing to cardiac malformation of 4q deletion syndrome

Chromosome 4q deletion is one of the most frequently detected genomic imbalance events in congenital heart disease (CHD) patients. However, a portion of CHD-associated 4q deletions do not include known CHD genes. Alignment of those 4q deletions defined a minimal overlapping region including only one gene-SORBS2. Histological analysis of Sorbs2-/- heart revealed atrial septal hypoplasia/aplasia or double atrial septum. Mechanistically, SORBS2 had a dual role in maintaining sarcomeric integrity of cardiomyocytes and specifying the fate of second heart field (SHF) progenitors through c-ABL/NOTCH/SHH axis. In a targeted sequencing of a panel of known and candidate CHD genes on 300 CHD cases, we found that rare SORBS2 variants were significantly enriched in CHD patients. Our findings indicate that SORBS2 is a regulator of cardiac development and its haploinsufficiency may contribute to cardiac phenotype of 4q deletion syndrome. The presence of double atrial septum in Sorbs2-/- hearts reveals the first molecular etiology of this rare anomaly linked to paradoxical thromboembolism.

genetics↗

bioRxiv2020

Global Profiling of 2-hydroxyisobutyrylome in Common Wheat

As a novel post-translational modification (PTM), lysine 2-hydroxyisobutyrylation (Khib) has been found to play a role in active gene transcription in mammalian cells and yeast, but the function of Khib proteins in plants remains unknown. In this study, we used western blot to demonstrate that Khib is an evolutionarily-conserved PTM in wheat and its donators, with the highest Khib abundance occurring in hexaploidy wheat. Additionally, global profiling using affinity purification and mass spectroscopy of 2-hydroxyisobutyrylome revealed that there were 3348 lysine modification sites from 1074 proteins in common wheat (Triticum aestivum L.). Moreover, bioinformatic data indicated that Khib proteins participate in a wide variety of biological and metabolic pathways. Immunoprecipitation and western blot confirmed that Khib proteins had an in vivo origin. A comparison of Khib and other major PTMs revealed that Khib proteins were simultaneously modified by multiple PTMs. Using mutagenesis experiments and Co-IP, we demonstrated that Khib on K206 is a key regulatory modification of phosphoglycerate kinase enzymatic activity and found that de-Khib on K206 affects protein interactions. Furthermore, Khib production of low-molecular-weight proteins was a response to the deacetylase inhibitors nicotinamide and trichostatin A. This study provides evidence that enhances our current understanding of Khib in wheat plants, including the cooperation between this PTM and metabolic regulation.

plant biology↗

bioRxiv2019

An Array of 60,000 Antibodies for Proteome-Scale Antibody Generation and Target Discovery

Antibodies are essential for elucidating the roles of genes decoded by genome sequencing. However, affordable technology for proteome-scale antibody generation does not exist. To address this, we developed the Proteome Epitope Tag Antibody Library (PETAL) and its array. PETAL consists of 62,208 mAbs against 15,199 peptides from diverse proteomes. PETAL harbors binders for a great multitude of proteins in nature due to antibody multispecificity, an intrinsic feature of an antibody. Distinctive combinations of 10,000-20,000 mAbs were found to target specific proteomes by array screening. Phenotype-specific mAb-target pairs were discovered for maize and zebrafish samples. Immunofluorescence and flow cytometry mAbs for human membrane proteins and ChIP-seq mAbs for transcription factors were identified from respective proteome-binding PETAL mAbs. Differential screening of cell surface proteomes of tumor and normal tissues discovered internalizing tumor antigens for antibody-drug conjugates. By discovering high affinity mAbs at a fraction of current time and cost, PETAL enables proteome-scale antibody generation and target discovery.

systems biology↗

bioRxiv2018

Identifying the Biosynthetic Gene Cluster for Triacsins with an N-hydroxytriazene Moiety

Triacsins are a family of natural products containing an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, the biosynthesis of triacsins has remained unknown. Here, we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.

biochemistry↗

bioRxiv2018

The Impact of Reward on Attention in Schizophrenia

Traditionally, attention was thought to be directed by either top-down goals or bottom-up salience. Recent studies have shown that the reward history of a stimulus feature also acts as a powerful attentional cue. This is particularly relevant in schizophrenia, which is characterized by motivational and attentional deficits. Here, we examine the impact of reward on selective attention.\n\nForty-eight people with schizophrenia (PSZ) and 34 non-psychiatric control subject (NCS) discriminated the location of a target dot appearing inside a left circle or right circle. The circles were different colors, one of which was associated with reward via pre-training. In the first 2 blocks, targets were equally likely to appear in the left or right circle. In the last 4 blocks, the target was 75% likely on one side, thus allowing us to separately examine how attention was impacted by reward (color) and probability (location).\n\nPSZ had slower overall reaction times (RTs) than NCS. Both groups showed robust effects of spatial probability and reward history, with faster RTs for the rewarded color and for the more probable location. These effects were similar in PSZ and NCS. Negative symptom severity correlated with overall RT slowing, but there were no correlations between symptoms and reward-associated biasing of attention.\n\nPSZ demonstrated RT slowing but normal reward history and spatial probability-driven RT facilitation. These results are conceptually similar to prior findings showing intact implicit reward effects on response bias, and suggest that implicit processing of reward and probability is intact in PSZ.

neuroscience↗