Biology subjects

Zhou, A.

Publications and source records attributed to Zhou, A..

50 records · Page 3Linked to original sources

bioRxiv2021

An evaluation of resonant scanning as a high-speed imaging technique for two-photon imaging of cortical vasculature

We demonstrate a simple, low-cost two-photon microscope design with both galvo-galvo and resonant-galvo scanning capabilities. We quantify and compare the signal-to-noise ratios and imaging speeds of the galvo-galvo and resonant-galvo scanning modes when used for murine neurovascular imaging. The two scanning modes perform as expected under shot-noise limited detection and are found to achieve comparable signal-to-noise ratios. Resonant-galvo scanning is capable of reaching desired signal-to-noise ratios using less acquisition time when higher excitation power can be used. Given equal excitation power and total pixel dwell time between the two methods, galvo-galvo scanning outperforms resonant-galvo scanning in image quality when detection deviates from being shot-noise limited.

neuroscience↗

bioRxiv2021

Multi-site phosphorylation of yeast Mif2/CENP-C promotes inner kinetochore assembly

Kinetochores control eukaryotic chromosome segregation by connecting chromosomal centromeres to spindle microtubules. Duplication of centromeric DNA necessitates kinetochore disassembly and subsequent reassembly on the nascent sisters. To search for a regulatory mechanism that controls the earliest steps of kinetochore assembly, we studied Mif2/CENP-C, an essential basal component. We found that Polo-like kinase (Cdc5) and Dbf4-dependent kinase (DDK) phosphorylate the conserved PEST region of Mif2/CENP-C and that this phosphorylation directs inner kinetochore assembly. Mif2 phosphorylation promotes kinetochore assembly in a reconstituted biochemical system, and it strengthens Mif2 localization at centromeres in cells. Disrupting one or more phosphorylation sites in the Mif2-PEST region progressively impairs cellular fitness and sensitizes cells to microtubule poisons. The most severe Mif2-PEST mutations are lethal in cells lacking otherwise non-essential Ctf19 complex factors. These data suggest that multi-site phosphorylation of Mif2/CENP-C is a robust switch that controls inner kinetochore assembly, ensuring accurate chromosome segregation.

cell biology↗

bioRxiv2021

Diamond Raman Laser and Yb Fiber Amplifier for In Vivo Multiphoton Fluorescence Microscopy

Here we introduce a fiber amplifier and a diamond Raman laser that output high powers (6.5 W, 1.3 W) at valuable wavelengths (1060 nm, 1250 nm) for multiphoton excitation of red-shifted fluorophores. These custom excitation sources are both simple to construct and cost-efficient in comparison to similar custom and commercial alternatives. Furthermore, they operate at a repetition rate (80 MHz) that allows fast image acquisition using resonant scanners. We demonstrate our systems compatibility with fast resonant scanning, the ability to acquire neuronal images, and the capability to image vasculature at deep locations (>1 mm) within the mouse cerebral cortex.

neuroscience↗

bioRxiv2021

Atria: An Ultra-fast and Accurate Trimmer for Adapter and Quality Trimming

BackgroundAs Next Generation Sequencing takes a dominant role in terms of output capacity and sequence length, adapters attached to the reads and low-quality bases hinder the performance of downstream analysis directly and implicitly, such as producing false-positive single nucleotide polymorphisms (SNP), and generating fragmented assemblies. A fast trimming algorithm is in demand to remove adapters precisely, especially in read tails with relatively low quality. FindingsWe present a trimming program named Atria. Atria matches the adapters in paired reads and finds possible overlapped regions with a super-fast and carefully designed byte-based matching algorithm (O(n) time with O(1) space). Atria also implements multi-threading in both sequence processing and file compression and supports single-end reads. ConclusionsAtria performs favorably in various trimming and runtime benchmarks of both simulated and real data with other cutting-edge trimmers. We also provide an ultra-fast and lightweight byte-based matching algorithm. The algorithm can be used in a broad range of short-sequence matching applications, such as primer search and seed scanning before alignment. Availability & ImplementationThe Atria executables, source code, and benchmark scripts are available at https://github.com/cihga39871/Atria under the MIT license.

bioinformatics↗

bioRxiv2021

Establishment of Strigolactone-Producing Bacterium-Yeast Consortium

Strigolactones (SLs) are a class of phytohormones playing diverse roles in plant growth and development, yet the limited access to SLs is largely impeding SL-based foundational investigations and applications. Here, we developed Escherichia coli-Saccharomyces cerevisiae consortia to establish a microbial biosynthetic platform for the synthesis of various SLs, including carlactone, carlactonic acid, 5-deoxystrigol (5DS, 6.65{+/-}1.71 {micro}g/L), 4-deoxyorobanchol (4DO, 3.46{+/-}0.28 {micro}g/L), and orobanchol (OB, 19.36{+/-}5.20 {micro}g/L). The SL-producing platform enabled us to conduct functional identification of CYP722Cs from various plants as either OB or 5DS synthase. It also allowed us to quantitatively compare known variants of plant SL biosynthetic enzymes in the microbial system. The titer of 5DS was further enhanced through pathway engineering to 47.3 {micro}g/L. This work provides a unique platform for investigating SL biosynthesis and evolution and lays the foundation for developing SL microbial production process.

synthetic biology↗

bioRxiv2021

In vitro and in vivo preclinical studies predict REGEN-COV protection against emergence of viral escape in humans

Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. As rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of minor virus variants in SARS-COV-2 isolates found in COVID-19 patients or identified from preclinical in vitro and in vivo studies. This study demonstrates that a combination of non-competing antibodies, REGEN-COV, not only provides full coverage against current variants of concern/interest but also protects against emergence of new such variants and their potential seeding into the population in a clinical setting.

microbiology↗

bioRxiv2020

Chondrocytes in the resting zone of the growth plate are maintained in a Wnt-inhibitory environment

Chondrocytes in the resting zone of the postnatal growth plate are characterized by slow cell cycle progression, and encompass a population of parathyroid hormone-related protein (PTHrP)-expressing skeletal stem cells that contribute to the formation of columnar chondrocytes. However, how these chondrocytes are maintained in the resting zone remains undefined. We undertook a genetic pulse-chase approach to isolate slow cycling, label-retaining chondrocytes (LRCs) from the growth plate using a chondrocyte-specific doxycycline-controllable Tet-Off system regulating expression of histone 2B-linked GFP. Comparative RNA-seq analysis identified significant enrichment of inhibitors and activators for Wnt/{beta}-catenin signaling in LRCs and non-LRCs, respectively. Activation of Wnt/{beta}-catenin signaling in PTHrP+ resting chondrocytes using Pthrp-creER and Apc-floxed allele impaired their ability to form columnar chondrocytes. Therefore, slow-cycling chondrocytes are maintained in a canonical Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating maintenance and differentiation of PTHrP+ skeletal stem cells of the postnatal growth plate.

developmental biology↗

bioRxiv2020

REGN-COV2 antibody cocktail prevents and treats SARS-CoV-2 infection in rhesus macaques and hamsters

An urgent global quest for effective therapies to prevent and treat COVID-19 disease is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987+REGN10933) targeting non-overlapping epitopes on the SARS-CoV-2 spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques and golden hamsters and demonstrate that REGN-COV-2 can greatly reduce virus load in lower and upper airway and decrease virus induced pathological sequalae when administered prophylactically or therapeutically. Our results provide evidence of the therapeutic potential of this antibody cocktail.

microbiology↗

bioRxiv2020

Prohibitin protects against cigarette smoke extract-induced cell apoptosis in cultured human pulmonary microvascular endothelial cells

Prohibitin is an evolutionarily conserved and ubiquitously expressed protein in eukaryocyte. It mediate many important roles in cell survival, apoptosis, autophagy and senescence. In the present study, we aimed to explore the role of prohibitin in cigarette smoke extract (CSE)-induced apoptosis of human pulmonary microvascular endothelial cells (HPMECs). For this purpose, HPMECs were trasfected with prohibitin and challenged with CSE. Our results showed that CSE exposure inhibited prohibitin expression in a dose-dependent manner in HPMECs. Overexpression of prohibitin could protect cell from CSE-induced injury by inhibiting CSE-induced cell apoptosis, inhibiting reactive oxygen species (ROS) production, increase mitochondrial membrane potential, increase the content of mitochondrial transcription factor A (mtTFA), IKK/{beta} phosphorylation and I{kappa}B- degradation. CSE decreases prohibitin expression in endothelial cells and restoration of prohibitin expression in these cells can protect against the deleterious effects of CSE on mitochondrial and cells. We identified prohibitin is a novel regulator of endothelial cell apoptosis and survival in the context of cigarette smoke exposure.

pharmacology and toxicology↗

bioRxiv2020

Multiple environmental parameters impact core lipid cyclization in Sulfolobus acidocaldarius

Environmental reconstructions based on microbial lipids require understanding the coupling between environmental conditions and membrane physiology. The paleotemperature proxy TEX86 is built on the observation that archaea alter the number of five- and six-membered rings in the hydrophobic core of their glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipids when growing at different temperatures. However, recent work with these archaea also highlights a role for other factors, such as pH or energy availability in determining the degree of core lipid cyclization. To better understand the role of these variables we cultivated a model Crenarchaeon, Sulfolobus acidocaldarius, over a range in temperature, pH, oxygen flux, or agitation speed, and quantified the changes in growth rate, biomass yield, and core lipid compositions. The average degree of cyclization in core lipids correlated with growth rate under most conditions. When considered alongside other experimental findings from both the thermoacidophilic and mesoneutrophilic archaea, the results suggest the cyclization of archaeal lipids records a universal response to energy availability at the cellular level. Although we isolated the effects of individual parameters, there remains a need for multi-factor experiments (e.g., pH + temperature + redox) to establish a robust framework to interpret biomarker records of environmental change.

microbiology↗

bioRxiv2020

Lower Soil Carbon Loss Due to Persistent Microbial Adaptation to Climate Warming

Soil microbial respiration is an important source of uncertainty in projecting future climate and carbon (C) cycle feedbacks. Despite intensive studies for two decades, the magnitude, direction, and duration of such feedbacks are uncertain, and their underlying microbial mechanisms are still poorly understood. Here we examined the responses of soil respiration and microbial community structure to long-term experimental warming in a temperate grassland ecosystem. Our results indicated that the temperature sensitivity of soil microbial respiration (i.e., Q10) persistently decreased by 12.0{+/-}3.7% across 7 years of warming. Integrated metagenomic and functional analyses showed that microbial community adaptation played critical roles in regulating respiratory acclimation. Incorporating microbial functional gene abundance data into a microbially-enabled ecosystem model significantly improved the modeling performance of soil microbial respiration by 5-19%, compared to the traditional non-microbial model. Model parametric uncertainty was also reduced by 55-71% when gene abundances were used. In addition, our modeling analyses suggested that decreased temperature sensitivity could lead to considerably less heterotrophic respiration (11.6{+/-}7.5%), and hence less soil C loss. If such microbially mediated dampening effects occur generally across different spatial and temporal scales, the potential positive feedback of soil microbial respiration in response to climate warming may be less than previously predicted.

ecology↗

bioRxiv2019

Energy flux controls tetraether lipid cyclization in Sulfolobus acidocaldarius

Microorganisms regulate the composition of their membranes in response to environmental cues. Many archaea maintain the fluidity and permeability of their membranes by adjusting the number of cyclic moieties within the cores of their glycerol dibiphytanyl glycerol tetraether (GDGT) lipids. Cyclized GDGTs increase membrane packing and stability, which has been shown to help cells survive shifts in temperature and pH. However, the extent of this cyclization also varies with growth phase and electron acceptor or donor limitation. These observations indicate a relationship between energy metabolism and membrane composition. Here we show that the average degree of GDGT cyclization increases with doubling time in continuous cultures of the thermoacidophile Sulfolobus acidocaldarius (DSM 639). This is consistent with the behavior of a mesoneutrophile, Nitrosopumilus maritimus SCM1. Together, these results demonstrate that archaeal GDGT distributions can shift in response to electron donor flux and energy availability, independent of pH or temperature. Paleoenvironmental reconstructions based on GDGTs thus capture the energy available to microbes, which encompasses fluctuations in temperature and pH, as well as electron donor and acceptor availability. The ability of Archaea to adjust membrane composition and packing may be an important strategy that enables survival during episodes of energy stress.\n\nSignificance StatementMicrobial lipid membranes protect and isolate a cell from its environment while regulating the flow of energy and nutrients to metabolic reaction centers within. We demonstrate that membrane lipids change as a function of energy flux using a well-studied archaeon that thrives in acidic hot springs and observe an increase in membrane packing as energy becomes more limited. These observations are consistent with chemostat experiments utilizing a low temperature, neutral pH, marine archaeon. This strategy appears to regulate membrane homeostasis is common across GDGT-producing lineages, demonstrating that diverse taxa adjust membrane composition in response to chronic energy stress.

microbiology↗

bioRxiv2018

The Lineage-Specific Transcription Factor CDX2 Navigates Dynamic Chromatin to Control Distinct Stages of Intestine Development

Lineage-restricted transcription factors, such as the intestine-specifying factor CDX2, often have dual requirements across developmental time. Embryonic-loss of CDX2 triggers homeotic transformation of intestinal fate, while adult-onset Cdx2-loss compromises critical physiological functions but preserves intestinal identity. It is unclear how such diverse requirements are executed across the developmental continuum. Using primary and engineered human tissues, mouse genetics, and a multi-omics approach, we demonstrate that divergent CDX2 loss-of-function phenotypes in embryonic versus adult intestines correspond to divergent CDX2 chromatin-binding profiles in embryonic versus adult stages. CDX2 binds and activates distinct target genes in developing versus adult mouse and human intestinal cells. We find that temporal shifts in chromatin accessibility correspond to these context-specific CDX2 activities. Thus, CDX2 is not sufficient to activate a mature intestinal program, but rather, CDX2 responds to its environment, targeting stage-specific genes to contribute to either intestinal patterning or maturity. This study provides insights into the mechanisms through which lineage-specific regulatory factors achieve divergent functions over developmental time.

developmental biology↗

bioRxiv2018

MYC Dysregulates Mitotic Spindle Function Creating a Dependency on TPX2

The MYC oncogene promotes tumorigenesis in part by facilitating cell cycle entry thus driving cellular proliferation. Tumors that overexpress MYC frequently demonstrate aneuploidy, numerical chromosome alterations associated with highly aggressive cancers, rapid tumor evolution, and poor patient outcome. While the role of MYC in overcoming the G1/S checkpoint is well established, it remains poorly understood whether MYC induces chromosomal instability (CIN). Here, we identify a direct influence of MYC on mitotic progression. MYC overexpression induces defects in microtubule nucleation and spindle assembly promoting chromosome segregation defects, micronuclei and CIN. We examined which mitotic regulators are required for the survival of MYC-overexpressing cells and found a reliance on high TPX2 expression. TPX2, a master microtubule regulator, is overexpressed together with MYC in multiple cell lines, in mouse tumor models and in aggressive human breast cancers. High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in cells with deregulated MYC, whereas TPX2 depletion blocks mitotic progression, induces cell death and prevents tumor growth. Importantly, attenuation of MYC expression reverses the mitotic defects observed, even in established tumor cell lines, implicating an ongoing role for high MYC in the persistence of a CIN phenotype in tumors. Here, we implicate the MYC oncogene as a regulator of spindle assembly and dynamics and identify a new MYC-TPX2 synthetic-lethal interaction that could represent a future therapeutic strategy in MYC-overexpressing cancers. Our studies suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.

cell biology↗