Biology subjects

Wu, Z.

Publications and source records attributed to Wu, Z..

At least 19 recordsLinked to original sources

bioRxiv2018

A genetically encoded fluorescent sensor for rapid and specific in vivo detection ofnorepinephrine

Norepinephrine (NE) and epinephrine (Epi), two key biogenic monoamine neurotransmitters, are involved in a wide range of physiological processes. However, their precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring these molecules in vivo. Here, we developed a family of GPCR Activation-Based NE/Epi (GRABNE) sensors with a 230% peak {Delta}F/F0 response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second rapid kinetics, high specificity to NE vs. dopamine. Viral- or transgenic- mediated expression of GRABNE sensors were able to detect electrical-stimulation evoked NE release in the locus coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and hypothalamus of freely moving mice. Thus, GRABNE sensors are a robust tool for rapid and specific monitoring of in vivo NE/Epi transmission in both physiological and pathological processes.

neuroscience↗

bioRxiv2018

Construction of a germline-specific RNAi tool in C. elegans

Analysis of complex biological functions usually requires tissue-specific genetic manipulations in multicellular organisms. The C. elegans germline plays regulatory roles not only in reproduction, but also in metabolism, stress response and aging. Previous studies have used mutants of rrf-1, which encodes an RNA-directed RNA polymerase, as a germline-specific RNAi tool. However, the rrf-1 mutants showed RNAi efficiency in somatic tissues. Here we constructed a germline-specific RNAi strain by combining an indel mutation of rde-1, which encodes an Argonaute protein that functions cell autonomously to ensure RNAi efficiency, and a single-copy rde-1 transgene driven by the sun-1 germlinespecific promoter. The germline RNAi efficiency and specificity are confirmed by RNAi phenocopy of known mutations, knockdown of GFP reporter expression, as well as quantitative RT-PCR measurement of tissue-specific mRNAs upon RNAi knockdown. The germline-specific RNAi strain shows no obvious deficiencies in reproduction, lipid accumulation, thermo-tolerance and life span compared to wild-type animals. By screening an RNAi sub-library of phosphatase genes, we identified novel regulators of thermo-tolerance. Together, we have created a useful tool that can facilitate the genetic analysis of germline-specific functions in C. elegans.

genetics↗

bioRxiv2018

Removing Confounding Factors Associated Weights in Deep Neural Networks Improves the Prediction Accuracy for Healthcare Applications

The proliferation of healthcare data has brought the opportunities of applying data-driven approaches, such as machine learning methods, to assist diagnosis. Recently, many deep learning methods have been shown with impressive successes in predicting disease status with raw input data. However, the \"black-box\" nature of deep learning and the high-reliability requirement of biomedical applications have created new challenges regarding the existence of confounding factors. In this paper, with a brief argument that inappropriate handling of confounding factors will lead to models sub-optimal performance in real-world applications, we present an efficient method that can remove the influences of confounding factors such as age or gender to improve the across-cohort prediction accuracy of neural networks. One distinct advantage of our method is that it only requires minimal changes of the baseline models architecture so that it can be plugged into most of the existing neu-ral networks. We conduct experiments across CT-scan, MRA, and EEG brain wave with convolutional neural networks and LSTM to verify the efficiency of our method.

bioinformatics↗

bioRxiv2018

Virus infection might cause cells producing black swimming dots

Black swimming dots (BSDs) are nanoscale dot-like contaminants in the dishes of cultured cells. Until now, the identity of BSDs has not yet been determined. In our recent study, we proposed that BSDs per se are nonliving inorganic nanoparticles yet should derive from the cells infected with an unidentified airborne pathogen. We showed the pathogen possessed the characteristics including airborne transmitted, cell-dependent, insensitive to antibiotics, filterable through 0.1 m membrane. These properties prompt us to speculate that the pathogen of BSDs might be one kind of unidentified virus-like organism. However, the imperfection is that we have not isolated this putative pathogen from BSD+ samples [1]. Here, we report a further investigation of finding the virus-like pathogen in the serum and tissue extracts from BSD+ mice. Most importantly, this virus-like pathogen can be reisolated from the BSD+ extracts-inoculated, diseased BSD- cells.\n\nTo de ...

cell biology↗

bioRxiv2018

Gating TrkB switch by methylglyoxal enables GLO1 as a target for depression

The endogenous metabolite methylglyoxal (MGO) has recently emerged as a potential mediator of psychiatric disorders, such as anxiety and depression, but its precise mechanism of action remains poorly understood. Here, we find that MGO concentrations are decreased in the prefrontal cortex and hippocampus in rats subjected to chronic stress, and low-dose MGO treatment remarkedly enhances resilience to stress and alleviates depression-like symptoms. This effect is achieved by MGOs promotion on the synaptic plasticity in prefrontal cortex and hippocampus. Both in vitro and in vivo experiments show that MGO provokes the dimerization and autophosphorylation of TrkB and the subsequent activation of downstream Akt/CREB signaling, which leads to a rapid and sustained expression of brain-derived neurotrophic factor (BDNF). We further demonstrate that MGO directly binds to the extracellular domain of TrkB, but not its intracellular domain. In addition, we also identify a natural product luteolin and its derivative lutD as potent inhibitors of Glyoxalase 1 and validate their antidepressant effects in chronic stress rat models. The antidepressant role of endogenous MGO provides a new basis for the understanding and therapeutic intervention design for stress-associated mental disorders.

neuroscience↗

bioRxiv2018

Do mechanical strain magnitude and rate drive bone adaptation in adult women? A 12-month prospective study.

Although there is strong evidence that certain activities can increase bone density and structure in some individuals, it is unclear what specific mechanical factors govern the response. This is important because understanding the effect of mechanical signals on bone could contribute to more effective osteoporosis prevention methods and efficient clinical trial design. The degree to which strain rate and magnitude govern bone adaptation in humans has never been prospectively tested. Here, we studied the effects of a voluntary upper extremity compressive loading task in healthy adult women during a twelve month prospective period. One hundred and two women age 21-40 participated in one of two experiments. (1): low (n=21) and high (n=24) strain magnitude. (2): low (n=21) and high (n=20) strain rate. Control: (n=16): no intervention. Strains were assigned using subject-specific finite element models. Load cycles were recorded digitally. The primary outcome was change in ultradistal integral bone mineral content (iBMC), assessed with QCT. Interim timepoints and secondary outcomes were assessed with high resolution pQCT (HRpQCT). Sixty-six subjects completed the intervention, and interim data were analyzed for 77 subjects. Both the low and high strain rate groups had significant 12-month increases to ultradistal iBMC (change in control: -1.3{+/-}2.7%, low strain rate: 2.7{+/-}2.1%, high strain rate: 3.4{+/-}2.2%), total iBMC, and other measures. \"Loading dose\" was positively related to 12-month change in ultradistal iBMC, and interim changes to total BMD, cortical thickness and inner trabecular BMD. Subjects who gained the most bone completed, on average, 130 loading bouts of (mean strain) 550 {varepsilon} at 1805 {varepsilon}/s. Those with the greatest gains had the highest loading dose. We conclude that signals related to strain magnitude, rate, and number of loading bouts contribute to bone adaptation in healthy adult women, but only explain a small amount of variance in bone changes.

physiology↗

bioRxiv2018

Visualizing ATP-dependent substrate-processing dynamics of the human 26S proteasome at near-atomic resolution

The proteasome is an ATP-dependent 2.5-megadalton machine responsible for ubiquitylated protein degradation in all eukaryotic cells. Here we present cryo-EM structures of the substrate-engaged human 26S proteasome in seven conformational states at 2.8-3.6 [A] resolution, captured during polyubiquitylated protein degradation. These structures visualize a continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to processive substrate translocation, during which ATP hydrolysis sequentially navigate through all six ATPase subunits. Three principle modes of coordinated ATP hydrolysis are observed, featuring hydrolytic events in two oppositely positioned ATPases, in two consecutive ATPases, and in one ATPase at a time. They regulate deubiquitylation, translocation initiation and processive unfolding of substrates, respectively. A collective power stroke in the ATPase motor is generated by synchronized ATP binding and ADP release in the substrate-engaging and disengaging ATPases, respectively. It is amplified largely in the substrate-disengaging ATPase, and propagated unidirectionally by coordinated ATP hydrolysis in the third consecutive ATPase.

biochemistry↗

bioRxiv2018

Systematic characterization of genome editing in primary T cells reveals proximal genomic insertions and enables machine learning prediction of CRISPR-Cas9 DNA repair outcomes

The Streptococcus pyogenes Cas9 (SpCas9) nuclease has become a ubiquitous genome editing tool due to its ability to target almost any location in DNA and create a double-stranded break1,2. After DNA cleavage, the break is fixed with endogenous DNA repair machinery, either by non-templated mechanisms (e.g. non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ)), or homology directed repair (HDR) using a complementary template sequence3,4. Previous work has shown that the distribution of repair outcomes within a cell population is non-random and dependent on the targeted sequence, and only recent efforts have begun to investigate this further5-11. However, no systematic work to date has been validated in primary human cells5,7. Here, we report DNA repair outcomes from 1,521 unique genomic locations edited with SpCas9 ribonucleoprotein complexes (RNPs) in primary human CD4+ T cells isolated from multiple healthy blood donors. We used targeted deep sequencing to measure the frequency distribution of repair outcomes for each guide RNA and discovered distinct features that drive individual repair outcomes after SpCas9 cleavage. Predictive features were combined into a new machine learning model, CRISPR Repair OUTcome (SPROUT), that predicts the length and probability of nucleotide insertions and deletions with R2 greater than 0.5. Surprisingly, we also observed large insertions at more than 90% of targeted loci, albeit at a low frequency. The inserted sequences aligned to diverse regions in the genome, and are enriched for sequences that are physically proximal to the break site due to chromatin interactions. This suggests a new mechanism where sequences from three-dimensionally neighboring regions of the genome can be inserted during DNA repair after Cas9-induced DNA breaks. Together, these findings provide powerful new predictive tools for Cas9-dependent genome editing and reveal new outcomes that can result from genome editing in primary T cells.

cell biology↗

bioRxiv2018

Dissecting differential signals in high-throughput data from complex tissues

Samples from clinical practices are often mixtures of different cell types. The high-throughput data obtained from these samples are thus mixed signals. The cell mixture brings complications to data analysis, and will lead to biased results if not properly accounted for. We develop a method to model the high-throughput data from mixed, heterogeneous samples, and to detect differential signals. Our method allows flexible statistical inference for detecting a variety of cell-type specific changes. Extensive simulation studies and analyses of two real datasets demonstrate the favorable performance of our proposed method compared with existing ones serving similar purpose.

bioinformatics↗

bioRxiv2018

A Bayesian Approach to Restricted Latent Class Models for Scientifically-Structured Clustering of Multivariate Binary Outcomes

SO_SCPLOWUMMARYC_SCPLOWThis paper presents a model-based method for clustering multivariate binary observations that incorporates constraints consistent with the scientific context. The approach is motivated by the precision medicine problem of identifying autoimmune disease patient subsets or classes who may require different treatments. We start with a family of restricted latent class models or RLCMs (e.g., Xu and Shang, 2018). However, in the motivating example and many others like it, the unknown number of classes and the definition of classes using binary states are among the targets of inference. We use a Bayesian approach to RCLMs in order to use informative prior assumptions on the number and definitions of latent classes to be consistent with scientific knowledge so that the posterior distribution tends to concentrate on smaller numbers of clusters and sparser binary patterns. The paper derives a posterior sampling algorithm based on Markov chain Monte Carlo with split-merge updates to efficiently explore the space of clustering allocations. Through simulations under the assumed model and realistic deviations from it, we demonstrate greater interpretability of results and superior finite-sample clustering performance for our method compared to common alternatives. The methods are illustrated with an analysis of protein data to detect clusters representing autoantibody classes among scleroderma patients.

bioinformatics↗

bioRxiv2018

Black swimming dots in cell culture: the identity, detection method and judging criteria

Black swimming dots (BSDs) -- the biological UFO in cell culture -- have perplexed biologists for decades. BSDs are extremely tiny dots found in dishes of cultured cells. It is still controversial on their origin and identity. BSDs are very hard to be removed and bring adverse impact to cell experiments (Supplementary Table 1). Here we wish to answer three urgent questions about BSDs. First, is the identity of BSDs nonliving matter or living organism? Second, is there any reliable method to tell whether the donor cattle for FBS production or the animals for primary cells isolation carry BSDs? Third, what are the judging criteria for BSDs when \"tiny black dots\" were observed? In 2015, we happened to observe BSDs in the perivitelline space of mouse oocytes and embryos; and the cells derived from these mice exhibited typical properties of BSDs infected cells (described in Supplementary Information). With these BSDs infected (BSD+) mice, we demonstrate that BSDs per se are ...

cell biology↗

bioRxiv2018

Comparison of Generally Recognized as Safe Organic Acids for Disinfecting Fresh-cut Lettuce

In this study, we aimed to determine the organic acids (acetic, lactic, citric, malic, propionic, succinic, and tartaric acids; 1% and 0.5%, w/w or v/v) that were most effective for fresh-cut lettuce disinfection based on analysis of quality (i.e., color, electrolyte leakage, and sensory quality) and microbial examination. The results showed that these acids did not negatively affect the color quality (i.e., L*, a*, b*, whiteness index, and sensory color). Additionally, 0.5% lactic acid led to the lowest electrolyte leakage (0.83%), which was not significantly different (p > 0.05) from that of distilled water (0.46%). Lactic acid (1%) did not affect the sensory quality and led to the highest microbial reduction (1.45 log reduction in aerobic plate counts [APCs]; 2.31 log reduction in molds and yeasts [M&Y]) and was therefore recommended as the primary choice for lettuce disinfection. Malic acid (0.5%), with a 1.07% electrolyte leakage rate, 0.73 log reduction in APCs, and 1.40 log reduction in M&Y, was better than the other six acids (0.5%) and was recommended as a pH regulator and a potential synergistic agent for oxidizing sanitizers. Acetic acid (1%) negatively affected the sensory quality and led to the highest electrolyte leakage (2.90%). Microbial analysis showed that propionic acid (0.5% and 1%) was ineffective for disinfection of lettuce (p > 0.05); thus, acetic and propionic acids were not recommended. Our results provide insights into the choice of sanitizers and formula design in food safety.\n\nIMPORTANCESince chlorine is forbidden in several countries, generally recognized as safe organic acids are used in minimal processing industries and in household sanitizers. The disinfection efficacy of organic acids has been studied when used alone or with oxidizing sanitizers. However, since different antibacterial mechanisms, contact time, fresh produce, and concentration have been reported, the acids most effective for single fresh produce disinfection, especially that of lettuce, an important salad vegetable, are not known. Moreover, in developing countries, because of imperfections in field management, cold chain transportation, and minimal processing industry development, the demand for low-cost household sanitizers is greater than that for minimally processed fresh produce. In this work, microbial load in lettuce was determined after disinfecting with seven GRAS organic acids. The changes in quality were also determined. These results provide insights into the choice of minimal processing sanitizers and a formula design for household sanitizers.

microbiology↗

bioRxiv2018

Intraspecific Polymorphism for the Presence and Absence of Entire Mitochondrial Chromosomes

Although mitochondrial genomes are typically thought of as single circular molecules, these genomes are fragmented into multiple chromosomes in many eukaryotes, raising intriguing questions about inheritance and (in)stability of mtDNA in such systems. A previous comparison of mitochondrial genomes from two different individuals of the angiosperm species Silene noctiflora found variation in the presence of entire mitochondrial chromosomes. Here, we expand on this work with a geographically diverse sampling of 25 S. noctiflora populations. We also included the closely related species S. turkestanica and S. undulata, with the latter exhibiting a surprising phylogenetic placement nested within the diversity of S. noctiflora mitochondrial haplotypes. Using a combination of deep sequencing and PCR-based screening for the presence of 22 different mitochondrial chromosomes, we found extensive variation in the complement of chromosomes across individuals. Much of this variation could be attributed to recent chromosome loss events. Despite the fragmented structures of these mitochondrial genomes and the evidence for occasional biparental inheritance in other Silene species, we did not find any indication of recombination between distinct mitochondrial haplotypes either within or among mitochondrial chromosomes, which may reflect the extreme paucity of nucleotide sequence polymorphism and/or the high selfing rate in this species. These results suggest that the massively expanded and fragmented mitochondrial genomes of S. noctiflora may have entered a phase of genome reduction in which they are losing entire chromosomes at a rapid rate.

plant biology↗

bioRxiv2018

Cerebellar nuclei neurons dictate growth of the cortex through developmental scaling of presynaptic Purkinje cells

Efficient function of neural systems requires the production of specific cell types in the correct proportions. Here we report that reduction of the earliest born neurons of the cerebellum, excitatory cerebellar nuclei neurons (eCN), results in a subsequent reduction in growth of the cerebellar cortex due to an accompanying loss of their presynaptic target Purkinje cells. Conditional knockout of the homeobox genes En1 and En2 (En1/2) in the rhombic lip-derived eCN and granule cell precursors leads to embryonic loss of a subset of medial eCN and cell non-autonomous and location specific loss of Purkinje cells, with subsequent proportional scaling down of cortex growth. We propose that subsets of eCN dictate the survival of their specific Purkinje cell partners, and in turn sonic hedgehog secreted by Purkinje cells scales the expansion of granule cells and interneurons to produce functional local circuits and the proper folded morphology of the cerebellum.

developmental biology↗

bioRxiv2018

Functional repair after ischemic injury through high efficiency in situ astrocyte-to-neuron conversion

Mammalian brains have largely lost internal neural regeneration capability except for a few discrete neurogenic niches. After brain injury, the cerebral cortex is especially difficult to repair due to its extremely low rate of adult neurogenesis. Previous studies have converted glial cells into neurons, but the total number of neurons generated is rather limited, casting doubt about its therapeutic potential. Here, we demonstrate that high-efficiency neuroregeneration can be achieved in adult mammalian brains by making use of an engineered AAV Cre-FLEX system to convert a large number of reactive astrocytes into functional neurons. Specifically, using a combination of GFAP::Cre and FLEX-NeuroD1 AAV system, we were able to regenerate enough new neurons from astrocytes to cover about 40% of the neurons lost from an ischemic injury (400 NeuN+ new neurons/mm2), compared to previously reported an average of <1% of cortical neurons (2-8 NeuN+ neurons/mm2) in an ischemic-injured adult mammalian cortex. Importantly, this in situ astrocyte-to-neuron conversion process also improved survival of injured pre-existing neurons, (additional 400 neurons/mm2), leading to a repaired motor cortex with layered cortical structures. Moreover, NeuroD1-converted neurons not only form functional neural circuits but also rescue motor and memory deficits after ischemic injury. Our results establish the proof-of-principle that a highly efficient in situ astrocyte-to-neuron conversion approach provides a novel treatment for neurological disorders that are in need of new neurons.

neuroscience↗

bioRxiv2018

smCounter2: an accurate low-frequency variant caller for targeted sequencing data with unique molecular identifiers

MotivationLow-frequency DNA mutations are often confounded with technical artifacts from sample preparation and sequencing. With unique molecular identifiers (UMIs), most of the sequencing errors can be corrected. However, errors before UMI tagging, such as DNA polymerase errors during end-repair and the first PCR cycle, cannot be corrected with single-strand UMIs and impose fundamental limits to UMI-based variant calling.\n\nResultsWe developed smCounter2, a UMI-based variant caller for targeted sequencing data and an upgrade from the current version of smCounter. Compared to smCounter, smCounter2 features lower detection limit at 0.5%, better overall accuracy (particularly in non-coding regions), a consistent threshold that can be applied to both deep and shallow sequencing runs, and easier use via a Docker image and code for read pre-processing. We benchmarked smCounter2 against several state-of-the-art UMI-based variant calling methods using multiple datasets and demonstrated smCounter2s superior performance in detecting somatic variants. At the core of smCounter2 is a statistical test to determine whether the allele frequency of the putative variant is significantly above the background error rate, which was carefully modeled using an independent dataset. The improved accuracy in non-coding regions was mainly achieved using novel repetitive region filters that were specifically designed for UMI data.\n\nAvailabilityThe entire pipeline is available at https://github.com/qiaseq/qiaseq-dna under MIT license.

bioinformatics↗

bioRxiv2018

Rapid proteotyping reveals cancer biology and drug response determinants in the NCI-60 cells

We describe the rapid and reproducible acquisition of quantitative proteome maps for the NCI-60 cancer cell lines and their use to reveal cancer biology and drug response determinants. Proteome datasets for the 60 cell lines were acquired in duplicate within 30 working days using pressure cycling technology and SWATH mass spectrometry. We consistently quantified 3,171 proteotypic proteins annotated in the SwissProt database across all cell lines, generating a data matrix with 0.1% missing values, allowing analyses of protein complexes and pathway activities across all the cancer cells. Systematic and integrative analysis of the genetic variation, mRNA expression and proteomic data of the NCI-60 cancer cell lines uncovered complementarity between different types of molecular data in the prediction of the response to 240 drugs. We additionally identified novel proteomic drug response determinants for clinically relevant chemotherapeutic and targeted therapies. We anticipate that this study represents a significant advance toward the translational application of proteotypes, which reveal biological insights that are easily missed in the absence of proteomic data.

systems biology↗

bioRxiv2018

The plasma miR-122 basal levels respond to circulating catecholamine in rats

miR-122 in circulation is a promising non-invasive biomarker as a replacement or supplement of current serum biomarkers for liver injuries. But the concept was questioned by recent studies, mainly due to its release from hepatocytes in absence of overt cellular injuries. In this study, we reported that the hepatic metabolism of circulating catecholamines resulted in the release of hepatocyte-specific miR-122. Acute stress-induced hepatocellular deformation was histopathologically different from drug-induced liver injury with significant increases of plasma miR-122 levels. The basal levels of human plasma miR-122 could be significantly altered by emotional responses. Interday variances of plasma miR-122 measurements were reduced effectively by stress-relief measures. The metabolism of basal circulating norepinephrine and epinephrine in liver might contribute to the basal levels of plasma miRNAs expressed in hepatocytes.

molecular biology↗