Biology subjects

Jiang, H.

Publications and source records attributed to Jiang, H..

31 records · Page 2Linked to original sources

bioRxiv2018

Ratiometric assays of autophagic flux in zebrafish for analysis of familial Alzheimer’s disease-like mutations

Protein aggregates such as those formed in neurodegenerative diseases can be degraded via autophagy. To assess changes in autophagic flux in zebrafish models of familial Alzheimers disease (fAD) mutations, we first developed a transgene, polyQ80-GFP-v2A-GFP, expressing equimolar amounts of aggregating polyQ80-GFP and a free GFP internal control in zebrafish embryos and larvae. This assay detects changes in autophagic flux by comparing the relative strength of polyQ80-GFP and free GFP moiety signals on western immunoblots probed with an antibody detecting GFP. However, the assays application is limited by the toxicity of polyQ80-GFP, and because aggregation of this protein may, itself, induce autophagy. To overcome these issues, we subsequently developed a similar ratiometric assay where expression of a GFP-Lc3a-GFP transgene generates initially equimolar amounts of GFP-Lc3a (directed to autophagic degradation) and a free GFP internal control. The sensitivity of this latter assay is reduced by a cellular protease activity that separates Lc3a from GFP-Lc3a, thus contributing to the apparent free GFP signal and somewhat masking decreases in autophagic flux. Nevertheless, the assay demonstrates significantly decreased autophagic flux in zebrafish lacking presenilin2 gene activity supporting that the Presenilin2 protein, like human PRESENILIN1, plays a role(s) in autophagy. Zebrafish heterozygous for a typical fAD-like, reading-frame-preserving mutation in psen1 show decreased autophagic flux consistent with observations in mammalian systems. Unexpectedly, a zebrafish model of the only confirmed reading-frame-truncating fAD mutation in a human PRESENILIN gene, the K115Efs mutation of human PSEN2, shows possibly increased autophagic flux in young zebrafish (larvae).

molecular biology↗

bioRxiv2018

A Novel QconCAT-Based Proteomics Method for Determining Allele-Specific Protein Expression (ASPE): a New Approach to Identify Cis-acting Genetic Variants

Measuring allele-specific expression (ASE) is a powerful approach for identifying cis-regulatory genetic variants. Here we developed a novel targeted proteomics method for quantification of allele-specific protein expression (ASPE) based on scheduled high resolution multiple reaction monitoring (sMRM-HR) with a heavy stable isotope-labeled quantitative concatamer (QconCAT) internal protein standard. This strategy was applied to the determination of the ASPE of UGT2B15 in human livers using the common UGT2B15 nonsynonymous variant rs1902023 (i.e. Y85D) as the marker to differentiate expressions from the two alleles. The QconCAT standard contains both the wild type tryptic peptide and the Y85D mutant peptide at a ratio of 1:1 to ensure accurate measurement of the ASPE of UGT2B15. The results from 18 UGT2B15 Y85D heterozygotes revealed that the ratios between wild type Y allele and mutant D allele varied from 0.60 to 1.46, indicating the presence of cis-regulatory variants. In addition, we observed no significant correlations between the ASPE and mRNA ASE of UGT2B15, suggesting the involvement of different cis-acting variants in regulating the transcription and translation processes of the gene. This novel ASPE approach provides a powerful tool for capturing cis-genetic variants involved in post-transcription processes, an important yet understudied area of research.

molecular biology↗

bioRxiv2018

In situ administration of cytokine combinations induces tumor regression

Recent advances in cancer immunotherapy suggest a possibility of harnessing the immune system to defeat malignant tumors, but complex immunosuppressive microenvironment confined the therapeutic benefits to a minority of patients with solid tumors. Here we constructed a lentivector based inducible system to evaluate the therapeutic effect of cytokines in established tumors. Doxycycline (Dox) induced local expression of cytokine combinations exhibited strong synergistic effect, leading to complete regression of tumors. Notably, IL12+GMCSF+IL2 expression induced eradication of tumors in all mice tolerated with this treatment, including those bearing large tumors of ~15mm in diameter, and generated an intensive systemic antitumor immunity. Other combinations with similar immune regulatory roles also induced tumor elimination in a majority of mice. Moreover, intratumoral injection of chitosan/IL12+GMCSF+IL2 solution induced complete response in all tested syngeneic tumor models, regardless of various tumor immunograms. These results provide a versatile method for the immunotherapy of intractable malignant neoplasm.

cancer biology↗

bioRxiv2018

Artefactual formation of pyruvate from in-source conversion of lactate

RationaleLactate and pyruvate are high abundance products of glucose metabolism. Analysis of both molecules as part metabolomics studies in cellular metabolism and physiology have been aided by advances in liquid chromatography-mass spectrometry (LC-MS).\n\nMethodsWe used ion pairing-chromatography and negative ion mode ESI on an QExactive HF to perform stable isotope assisted metabolomics profiling of lactate and pyruvate metabolism.\n\nResultsUsing an LC-MS method for polar metabolite analysis we discovered an artefactual formation of pyruvate from in-source fragmentation of lactate. Surprisingly, this in-source fragmentation has not been previously described, thus we report this identification to warn other investigators. This artefact was detected by baseline chromatographic resolution of lactate and pyruvate by LC with confirmation of this artefact by stable isotope labeling of lactate and pyruvate.\n\nConclusionsThese findings have immediate implications for metabolomics studies by LC-MS and direct infusion MS, especially in negative ion mode, whereby users should resolve lactate from pyruvate or robustly quantify the potential formation of pyruvate from higher abundance lactate in their assays.

biochemistry↗

bioRxiv2018

Large-scale two-photon imaging revealed super-sparse population codes in V1 superficial layer of awake monkeys

Efficient coding has been proposed as a general principle for the sensory systems. The efficient coding hypothesis predicts that neuronal population responses should be sparse, but limited by the measurement techniques, the precise estimates of the population sparseness of visual cortical neurons are still uncertain. Here, we employed large-scale two-photon calcium imaging to examine the neuronal population activities in V1 superficial layers of awake macaques in response to a large set of natural images. We found that only 0.5% of these neurons on average responded strongly to any given natural image with response strength above half of their individual peak responses, which is more than tenfold sparse over those reported by early studies. We further showed that these sparse population activities contain sufficient information for discriminating images with high accuracy. This study provided the first accurate measure of sparseness in V1 neuronal population responses, which support super-sparse neural codes in primates.

neuroscience↗

bioRxiv2017

Terminal uridylyltransferases target RNA viruses as part of the innate immune system in animals

RNA viruses are a major threat to animals and plants. RNA interference (RNAi) and the interferon response provide innate antiviral defense against RNA viruses. Here we performed a large-scale screen using C. elegans and its natural pathogen, the Orsay virus (OrV), and identified cde-1 as important for antiviral defense. CDE-1 is a homologue of the mammalian TUT4/7 terminal uridylyltransferases; its catalytic activity is required for its antiviral function. CDE-1 uridylates the 3' end of the OrV RNA genome and promotes its degradation, independently of the RNAi pathway. Likewise, TUT4/7 uridylate influenza A virus (IAV) mRNAs in mammalian cells. Deletion of TUT4/7 leads to increased IAV mRNA and protein levels. We have defined 3' terminal uridylation of viral RNAs as a conserved antiviral defense mechanism.

immunology↗

bioRxiv2017

Dissection of purified LINE-1 reveals distinct nuclear and cytoplasmic intermediates

1.Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications and quantitative mass spectrometry, we have characterized the proteins associated with distinctive L1 macromolecular complexes. Our findings support the presence of multiple L1-derived retrotransposition intermediates in vivo. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in template-primed reverse transcription.

molecular biology↗

bioRxiv2017

Changing geographic patterns and risk factors for avian influenza A(H7N9) infection in China

The 5th epidemic wave in 2016-2017 of avian influenza A(H7N9) virus in China caused more human cases than any previous waves but the factors that may explain the recent range expansion and surge in incidence remain unknown. We investigated the effect of anthropogenic, poultry and wetland information and of market closures on all epidemic waves (1-5). Poultry predictor variables recently became much more important than before, supporting the assumption of much wider H7N9 transmission in the chicken reservoir, that could be linked to increases in pathogenicity. We show that the future range expansion of H7N9 to northern China may translate into a higher risk of coinciding peaks with those of seasonal influenza, leading to a higher risk of reassortments. Live-poultry market closures are showed to be effective in reducing the local incidence rates of H7N9 human cases, but should be paired with other prevention and control measures to prevent transmission.

epidemiology↗

bioRxiv2017

Accuracy, Reproducibility And Bias Of Next Generation Sequencing For Quantitative Small RNA Profiling: A Multiple Protocol Study Across Multiple Laboratories

Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.

genomics↗

bioRxiv2017

Simulating retinal encoding: factors influencing Vernier acuity

Humans resolve the spatial alignment between two visual stimuli at a resolution that is substantially finer than the spacing between the foveal cones. In this paper, we analyze the factors that limit the information at the cone photoreceptors that is available to make these acuity judgments (Vernier acuity). We use open-source software, ISETBIO1 to quantify the stimulus and encoding stages in the front-end of the human visual system, starting with a description of the stimulus spectral radiance and a computational model that includes the physiological optics, inert ocular pigments, eye movements, photoreceptor sampling and absorptions. The simulations suggest that the visual system extracts the information available within the spatiotemporal pattern of photoreceptor absorptions within a small spatial (0.12 deg) and temporal (200 ms) regime. At typical display luminance levels, the variance arising from the Poisson absorptions and small eye movements (tremors and microsaccades) both appear to be critical limiting factors for Vernier acuity.

neuroscience↗

bioRxiv2017

Research on Mitochondrial DNA Mutations in Patients with SCA3/MJD

Spinocerebellar ataxia type 3 (SCA3) is a degenerative neurological disorders caused by trinucleotide repeat expansion within the ataxin-3 gene. It is characterized by multi-system involvement and diverse clinical phenotypes, which cannot be fully explained the length of the CAG repeats. One possible explanation for the phenotypic heterogeneity could be the presence of mitochondrial DNA mutations that modify disease severity. To explore the role of Mitochondrial DNA(mtDNA) variations in SCA3 pathogenesis, we analyzed polymorphisms of six mitochondrial genes, MT-LT1, MT-ND1, MT-CO2, MT-TK, MT-ATP8 and MT-ATP6, in 102 unrelated SCA3/MJD patients and 100 healthy controls. The results showed that there were 24 variations of those mtDNA genes in the SCA3 patients and only 10 in the unrelated healthy controls. There was no difference of the relative mtDNA copy number variation between the SCA3 patients and healthy controls (93.20 vs. 89.66, P>0.05). In the group of SCA3 patients, the relative mtDNA copy number showed a negative correlation between the number of CAG repeats (r=-0.210, P < 0.05), but did not correlate with the age at diagnosis, the age of onset, disease duration, ICARS scores and SARA scores. Our research demonstrated that the frequency of mutated mtDNA in SCA3 patients was higher than that in the healthy group. The mtDNA relative copy number in SCA3 patients was not significantly different compared to the healthy group. Thus, the copy number might not be treated as a biomedical indicator when measuring the severity of illness in SCA3 patients.

genetics↗

bioRxiv2017

Biocuration as an undergraduate training experience: Improving the annotation of the insect vector of Citrus greening disease

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory, and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models. All data are available on https://citrusgreening.org/.

genomics↗

bioRxiv2016

Time dependent genetic analysis links field and controlled environment phenotypes in the model C4 grass Setaria

Vertical growth of plants is a dynamic process that is influenced by genetic and environmental factors and has a pronounced effect on overall plant architecture and biomass composition. We have performed twelve controlled growth trials of an interspecific Setaria italica x Setaria viridis recombinant inbred line population to assess how the genetic architecture of plant height is influenced by developmental queues, water availability and planting density. The nondestructive nature of plant height measurements has enabled us to monitor vertical growth throughout the plant life cycle in both field and controlled environments. We find that plant height is reduced under water limitation and high density planting and affected by growth environment (field vs. growth chamber). The results support a model where plant height is a heritable, polygenic trait and that the major genetic loci that influence plant height function independent of growth environment. The identity and contribution of loci that influence height changes dynamically throughout development and the reduction of growth observed in water limited environments is a consequence of delayed progression through the genetic program which establishes plant height in Setaria. In this population, alleles inherited from the weedy S. viridis parent act to increase plant height early, whereas a larger number of small effect alleles inherited from the domesticated S. italica parent collectively act to increase plant height later in development.

plant biology↗