Biology subjects

Zou, P.

Publications and source records attributed to Zou, P..

28 records · Page 2Linked to original sources

bioRxiv2022

Bright and sensitive red voltage indicators for imaging action potentials in brain slices and pancreatic islets

As fast developing tools for observing cellular membrane potential, red-emitting genetically encoded voltage indicators (GEVIs) reduce auto-fluorescence background, allow multiplexed recordings, and enable all-optical electrophysiology, but have been limited by either insensitivity or dimness. Here, we report a pair of red GEVIs, Cepheid1b/s, with improved sensitivity, brightness, and photostability. Cepheid1 indicators faithfully report cellular excitability in pancreatic islets and neural activity in acute brain slices.

neuroscience↗

bioRxiv2022

ame-miR-34 modulates the larval body weight and immune response of Apis mellifera workers to Ascosphara apis invasion

MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera ligustica larvae to A. apis invasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT-PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut of A. m. ligustica. RT-qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (P < 0.001) in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (P < 0.001), while that in the inhibi- tor-miR-34 group was significantly downregulated (P < 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibi- tor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. apis-infected 4-, 5- and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in the A. apis-infected 4-, 5- and 6-day-old larval guts were significantly downregu- lated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. m. ligustica larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.

molecular biology↗

bioRxiv2022

Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome

Mapping the subcellular organization of proteins is crucial for understanding their biological functions. Herein, we report a reactive oxygen species induced protein labeling and identification (RinID) method for profiling subcellular proteome in the context of living cells. Our method capitalizes on a genetically encoded photocatalyst, miniSOG, to locally generate singlet oxygen that reacts with proximal proteins. Labeled proteins are conjugated in situ with an exogenously supplied nucleophilic probe, which serves as a functional handle for subsequent affinity enrichment and mass spectrometry-based protein identification. From a panel of nucleophilic compounds, we identify biotin-conjugated aniline and propargyl amine as highly reactive probes. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we apply RinID in the mitochondrial matrix, capturing 394 mitochondrial proteins with 97% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum.

biochemistry↗

bioRxiv2022

First-in-patient dose prediction for adeno-associated virus-mediated hemophilia gene therapy using allometric scaling

Rational first-in-patient (FIP) dose selection is critical for successful clinical development of gene therapy. In this study, the author compared the performance of two allometric scaling approaches and body weight-based dose conversion approach for FIP dose prediction for adeno-associated virus (AAV)-mediated hemophilia gene therapy. The performance of the three approaches was examined using preclinical and clinical efficacy data of nine AAV vectors. In general, body weight-based direct conversion of effective doses in monkeys or dogs was more likely to underestimate FIP dose but worked better for one bioengineered vector with a high transduction efficiency specifically in humans. In contrast, allometric scaling between gene efficiency factor (logGEF) and body weight (logW) was likely to overestimate FIP dose but worked better than the other two approaches when vector capsid-specific T-cell responses were detected in patients. The third approach, allometric scaling between logGEF and W-0.25 was appropriate for FIP dose prediction in the absence of T-cell responses to AAV vectors or a dramatic difference in vector transduction efficiency between animals and humans. A decision tree was tentatively proposed to facilitate approach selection for FIP dose prediction in AAV-mediated hemophilia gene therapy.

pharmacology and toxicology↗

bioRxiv2019

Exponential Growth of Glioblastoma In Vivo Driven by Rapidly Dividing and Outwardly Migrating Cancer Stem Cells

How overall tumor growth emerges from the properties of functionally heterogeneous tumor cell subpopulations is a fundamental question of cancer biology. Here we combined lineage tracing, continuous monitoring of tumor mass, proliferation assays and transcriptomics with mathematical modeling and statistical inference to dissect the growth of glioblastoma in mice. We found that tumors grow exponentially at the rate of symmetric divisions of brain tumor stem cells (BTSCs). Spatial modeling predicts, and data show, that BTSCs accumulate at the tumor rim rather than in the core. The physiological differentiation hierarchy downstream of BTSCs is preserved in mice and humans: transit amplifying progenitors give rise to terminally differentiated cells. Consistent with our quantification of the mechanisms underlying tumor growth, molecular data show elevated expression of cell cycle-and migration-related genes in BTSCs. Our systematic approach reveals fundamental properties of glioblastoma and may be transferable to the study of other animal models of cancer.

cancer biology↗

bioRxiv2019

A Genetically Encoded Fluorescent Sensor Enables Real-time Detection of the Intracellular GTP:GDP Ratio

The interconversion of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) is integral to a wide variety of biological cellular activities. However, analytical methods which directly detect the ratio of intracellular GTP and GDP concentrations have not been available. Herein, we report GNEPS, a genetically encoded fluorescent sensor that enables real-time monitoring of the GTP:GDP ratio, which is a fusion protein comprising a eukaryotic G-protein and a circularly permuted yellow fluorescent protein. GNEPS has distinct fluorescence spectra between its GTP-bound and GDP-bound states. Its apparent fluorescence signal therefore depends upon the competitive binding of GTP and GDP. Live cell imaging experiments demonstrated that GNEPS can be used to monitor spatiotemporal changes in the intracellular GTP:GDP ratio in various cell types and organelles in response to metabolic perturbations. We anticipate that GNEPS will become a valuable tool for understanding the metabolic and regulatory contributions of guanosine nucleotides.

biochemistry↗

bioRxiv2018

Ex vivo dynamics of human glioblastoma cells in a microvasculature-on-a-chip system correlates with tumor heterogeneity and subtypes

The perivascular niche (PVN) plays an essential role in brain tumor stem-like cell (BTSC) fate control, tumor invasion, and therapeutic resistance. Herein we report on the use of a microvasculature-on-a-chip system as a PVN model to evaluate the dynamics of BTSCs ex vivo from 10 glioblastoma patients. We observed that BTSCs preferentially localize in the perivascular zone. Live cell tracking revealed that the cells residing in the vicinity of microvessels had the lowest motility, while a fraction of cells on the microvessels unexpectedly possessed the highest motility and migrated over the longest distance. These results indicate that the perivascular zone is a niche for BTSCs, while the microvascular tracks are also a path for long-distance tumor cell migration and invasion. Additionally, the degree of co-localization between tumor cells and microvessels varied significantly across patients. To validate the results from our microvasculature-on-a-chip system, we used single-cell transcriptome sequencing (10 patients and 21,750 single cells in total) to identify the subtype of each tumor cell. The co-localization coefficient was found to correlate positively with proneural (stem-like) or mesenchymal (invasive) but not classical (proliferative) tumor cells. Furthermore, we found that a gene signature profile including PDGFRA correlated strongly with the \"homing\" of brain tumor cells to the PVN. Our findings demonstrated that ex vivo dynamics of human brain tumor cells in a microvasculature-on-a-chip model can recapitulate in vivo tumor cell dynamics, heterogeneity, and subtypes, representing a new route to the study of human tumor cell biology and uncover patient-specific tumor cell functions.

bioengineering↗

bioRxiv2018

Transgenic Zebrafish Models Reveal Distinct Molecular Mechanisms for Cataract-linked alphaA-Crystallin Mutants

Mutations in the small heat shock proteins -crystallins have been linked to autosomal dominant cataracts in humans. Extensive studies in vitro have revealed a spectrum of alterations to the structure and function of these proteins including shifts in the size of the oligomer, modulation of subunit exchange and modification of their affinity to client proteins. Although mouse models of these mutants were instrumental in identifying changes in cellular proliferation and lens development, a direct comparative analysis of their effects on lens proteostasis has not been performed. Here, we have transgenically expressed cataract-linked mutants of A- and B-crystallin in the zebrafish lens to dissect the underlying molecular changes that contribute to the loss of lens optical properties. Zebrafish lines expressing these mutants displayed a range of morphological lens defects. Phenotype penetrance and severity were dependent on the mutation even in fish lines lacking endogenous -crystallin. The mechanistic origins of these differences were investigated by the transgenic co-expression of a destabilized human{gamma} D-crystallin mutant. We found that the R49C but not the R116C mutant of A-crystallin promoted aggregation of {gamma}D-crystallin, although both mutants have similar affinity to client proteins in vitro. Our working model attributes these differences to the propensity of R49C, located in the buried N-terminal domain of A-crystallin, to disulfide crosslinking as previously demonstrated in vitro. Our findings complement and extend previous work in mouse models and emphasize the need of investigating chaperone/client protein interactions in appropriate cellular context.

genetics↗

bioRxiv2018

All-optical electrophysiology for high-throughput functional characterization of human iPSC-derived motor neuron model of ALS

Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch clamp electrophysiology. Here we describe an optimized all-optical electrophysiology, \"Optopatch\", pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron model of ALS. In a comparison of neurons with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus, and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results to numerical simulations of simple conductance-based neuronal models and to literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.

neuroscience↗

bioRxiv2017

Autonomous Combinatorial Color Barcoding For Multiplexing Single Molecule RNA Visualization

Single molecular fluorescence in situ hybridization (smFISH) detects RNA transcripts with spatial information and digital molecular counting. However, the broad usage of smFISH is still hindered by the complex chemical probe conjugation or microscopy set-up, especially for investigating multiple gene expression. Here we present a multiple fluorophore enzymatic labeling method (termed HuluFISH) for smFISH probes to achieve flexible combinatorial color barcoding in single hybridization step. The multiplex capacity of HuluFISH follows an exponential growth with the increase of the number of fluorophore types. We demonstrate that this method can be used to detect cellular heterogeneity in embryonic mouse brain on single cell level.

molecular biology↗