Biology subjects

Xiao, H.

Publications and source records attributed to Xiao, H..

100 records · Page 6Linked to original sources

bioRxiv2020

Establishing Prokaryotic Expression System of Angiotensin- Converting Enzyme 2 (ACE2) gene in pigs

In this paper, ACE2 gene of pigs was cloned and the purified protein was obtained via the prokaryotic expression system. Polyclonal antibody of high titer and sensitivity was obtained using Wastar rats immunization method and is then used to determine of the expression of ACE2 using immunohistochemistry. The sequence of ACE2 in pigs covered 2418 nucleotides and coded 805 amino acid (aa) residues. Sequence homology analysis showed that the ACE2 sequence in pigs is highly conserved among species at the nucleotide and amino acid levels. Genetic evolution analysis revealed that ACE2 gene in pigs has the shortest genetic distance with that in goats while residing in a totally different branch from that in zebra fishes. Analysis of protein structure predicted that ACE2 protein is a transmembrane secreted protein with high hydrophilicity, containing a signal peptide sequences locating between 1aa to 17aa. The ACE2 fusion protein expressed (under the induction with 1.0 mmol/L IPTG for 10 h) was of approximately 100 kDa and mainly existed in inclusion body. Wastar rats immunization showed that the titer of the anti-ACE2 antiserum in rats was 1: 3200. Western blot showed that the antibody binds specifically. Immunohistochemistry showed that the ACE2 protein was expressed in all major tissues of pigs. It is the first time that polyclonal antibody of ACE2 in pigs was obtained and the expression of ACE2 was confirmed. These results will provide a basis for investigating on ACE2s biological activity in pigs.

biochemistry↗

bioRxiv2020

CRNKL1 is a highly selective regulator of intron-retaining HIV-1 and cellular mRNAs

The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely-spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knock-down. Depletion of DHX38, WDR70 and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of cellular mRNAs with selectively retained introns. Thus, CRNKL1 dependent nuclear retention seems to be a novel mechanism for the regulation of cytoplasmic levels of intron-retaining cellular mRNAs that is harnessed by HIV-1 to direct its complex splicing pattern.

molecular biology↗

bioRxiv2019

The rice PPR756 coordinates with MORFs for multiple RNA editing in mitochondria

In land plants, the pentatricopeptide repeat (PPR) proteins form a large family involved in post-transcriptional processing of RNA in mitochondria and chloroplasts, which is critical for plant development and evolutionary adaption. Although studies showed a number of PPR proteins generally influence the editing of organellar genes, few of them were characterized in detail in rice. Here, we report a PLS-E subclass PPR protein in rice, PPR756, loss of function of which led to the abolishment of RNA editing events among three mitochondrial genes including atp6, ccmC, and nad7. Their defective C-to-U transformation then resulted in improper amino acid retention which could cause abortive pollen development. Furthermore, PPR756 could bind to the three target genes directly and interact with three OsMORFs (multiple organellar RNA editing factors): OsMORF1, OsMORF8-1, and OsMORF8-2. The knock-out plants of PPR756 exhibited retarded growth and greener leaves during the early vegetative stages, along with sterile pollen and lower seed setting at the reproductive stage. These results established a role for PPR756 in rice development, participating in RNA editing of three various transcripts and cooperating with OsMORFs via an editosome manner in rice.

developmental biology↗

bioRxiv2019

Mutant Zp1 results in Zona Pellucida Lacking and Female Infertility in Rats and Humans

Zona pellucida (ZP) plays a vital role in reproductive processes including oogenesis, fertilization and preimplantation development of embryo. The ZP of humans is composed of four glycoproteins (ZP1-ZP4), same as rats ZP. Our previous research reported a first case of human infertility due to ZP1 mutation, but the mechanism was unclear. Here we developed a genome editing in vivo rat model and a co-transfected in vitro cell model to investigate the pathogenic effect. In rat homozygous for the homologous mutation, ZP were absent in all of collected eggs. Further the growing and fully grown oocytes in the mutant ovaries completely lack a ZP but with detectable intracellular ZP1 protein. After mating with male rats, none of the mutant female rats got pregnant. Moreover, the co-transfected cell experiments and the ovarian experiments showed that the truncated ZP1 sequestered intracellularly ZP3 and ZP4 to impede their release outside, resulting in an intracellular accumulation of ZP1, ZP3 and ZP4, leading to absence of ZP in mutant oocytes. Our results clearly establish the causal role of ZP1 mutation on ZP defects and female infertility.\n\nSummary statementRat model mirrored completely the phenotypes observed in humans, infertility and abnormal eggs that lack a zona pellucida, through the negative effects of ZP1 mutation.

genetics↗

bioRxiv2019

Quantitative multiplexed proteomics of mouse microglia by flow-cytometric sorting reveals a core set of highly-abundant microglial proteins

BackgroundProteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies. MethodsWe coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) to obtain a highly-pure microglial proteome and identified a core set of highly-abundant microglial proteins in adult mouse brain. We interrogated existing human proteomic data for Alzheimers disease (AD) relevance of highly-abundant microglial proteins and performed immuno-histochemical and in-vitro validation studies. ResultsQuantitative multiplexed proteomics by TMT-MS of CD11b+ MACS-enriched (N = 5 mice) and FACS-isolated (N = 5 mice), from adult wild-type mice, identified 1,791 proteins. A total of 203 proteins were highly abundant in both datasets, representing a core-set of highly abundant microglial proteins. In addition, we found 953 differentially enriched proteins comparing MACS and FACS-based approaches, indicating significant differences between both strategies. The FACS-isolated microglia proteome was enriched with cytosolic, endoplasmic reticulum, and ribosomal proteins involved in protein metabolism and immune system functions, as well as an abundance of canonical microglial proteins. Conversely, the MACS-enriched microglia proteome was enriched with mitochondrial and synaptic proteins and higher abundance of neuronal, oligodendrocytic and astrocytic proteins. From the 203 consensus microglial proteins with high abundance in both datasets, we confirmed microglial expression of moesin (Msn) in wild-type and 5xFAD mouse brains as well as in human AD brains. Msn expression is nearly exclusively found in microglia that surround A{beta} plaques in 5xFAD brains. In in-vitro primary microglial studies, Msn silencing by siRNA decreased A{beta} phagocytosis and increased lipopolysaccharide-induced production of the pro-inflammatory cytokine, tumor necrosis factor (TNF). In network analysis of human brain proteomic data, Msn was a hub protein of an inflammatory co-expression module positively associated with AD neuropathological features and cognitive dysfunction. ConclusionsUsing FACS coupled with TMT-MS as the method of choice for microglial proteomics, we define a core set of highly-abundant adult microglial proteins. Among these, we validate Msn as highly-abundant in plaque-associated microglia with relevance to human AD.

neuroscience↗

bioRxiv2019

Microglial ERK activation is a critical regulator of pro-inflammatory immune responses in Alzheimer’s disease

BackgroundThe mitogen-activated protein kinase (MAPK) pathway is a central regulator of gene expression, pro-survival signaling, and inflammation. However, the importance of MAPK pathway signaling in regulating microglia-mediated neuroinflammation in Alzheimers Disease (AD) remains unclear. Here we examined the role of MAPK signaling in microglia using pre-clinical in-vitro and in-vivo models of AD pathology integrated with quantitative proteomics studies of post-mortem human brains.\n\nMethodsWe performed multiplexed immunoassay analyses of MAPK phosphoproteins, particularly ERK1/2, in acutely-isolated microglia and brain tissue from wild-type and 5xFAD mice. Neuropathological studies of mouse and human brain tissues were performed to quantify total and phosphorylated ERK protein in AD. The importance of ERK signaling in unstimulated and interferon {gamma} (IFN{gamma})-stimulated primary microglia cultures was investigated using NanoString transcriptomic profiling, coupled with functional assays of amyloid {beta} (A{beta}) and neuronal phagocytosis. Receptor tyrosine kinases (RTKs) likely responsible for ERK signaling in homeostatic microglia and disease-associated-microglia (DAM) states and ERK-regulated human AD risk genes were identified using gene expression data. Total and phosphorylated MAPKs in human post-mortem brain tissues were measured in quantitative proteomic datasets.\n\nResultsPhosphorylated ERK was the most strongly up-regulated signaling protein within the MAPK pathway in microglia acutely isolated from 5xFAD brains. Neuroinflammatory transcriptomic and phagocytic profiling of mouse microglia confirmed that ERK is a critical regulator of IFN{gamma}-mediated pro-inflammatory activation of microglia, although it was also important for constitutive microglial functions. Phospho-ERK was an upstream regulator of disease-associated microglia (DAM) gene expression (Trem2, Tyrobp), as well as of several human AD risk genes (Bin1, Cd33, Trem2, Cnn2). Among RTKs that signal via ERK, CSF1R and MERTK were primarily expressed by homeostatic microglia while AXL and FLT1 were likely regulators of ERK signaling in DAM. Within DAM, FLT4 and IGF1R were specifically expressed by pro- and anti-inflammatory DAM sub-profiles respectively. In quantitative proteomic analyses of post-mortem human brains from non-disease, asymptomatic and cognitively-impaired AD cases, ERK1 and ERK2 were the only MAPK pathway signaling proteins with increased protein expression and positive associations with neuropathological grade. Moreover, in a phospho-proteomic study of post-mortem human brains from controls, asymptomatic and symptomatic AD cases, we found evidence for a progressive increased flux through the ERK signaling pathway.\n\nConclusionsOur integrated analyses using pre-clinical models and human proteomic data strongly suggest that ERK phosphorylation in microglia is a critical regulator of pro-inflammatory immune response in AD pathogenesis and that modulation of ERK via upstream RTKs may reveal novel avenues for immunomodulation.

neuroscience↗

bioRxiv2019

Computational analysis of transcriptome signature repurposes low dose trifluoperazine for the treatment of fragile X syndrome in mouse model

Fragile X syndrome (FXS), caused by mutations in fragile X mental retardation 1 gene (FMR1), is a prevailing genetic disorder of intellectual disability and autism. Currently, there is no efficacious medication for FXS. Here, we use transcriptome landscape as a holistic molecular phenotype/endpoint to identify potential therapeutic intervention. Through in silico screening with public gene signature database, computational analysis of transcriptome profile in Fmr1 knockout (KO) neurons predicts therapeutic value of an FDA-approved drug trifluoperazine. Through experimental validation, we find that systemic administration of low dose trifluoperazine at 0.05 mg/kg attenuates multiple FXS- and autism-related behavioral symptoms. Moreover, computational analysis of transcriptome alteration caused by trifluoperazine suggests a new mechanism of action against PI3K (Phosphatidylinositol-4,5-bisphosphate 3-kinase) activity. Consistently, trifluoperazine suppresses PI3K activity and its down-stream targets Akt (protein kinase B) and S6K1 (S6 kinase 1) in neurons. Further, trifluoperazine normalizes the aberrantly elevated activity of Akt and S6K1 and enhanced protein synthesis in FXS mouse. In conclusion, our data demonstrate promising value of gene signature-based computation in identification of therapeutic strategy and repurposing drugs for neurological disorders, and suggest trifluoperazine as a potential practical treatment for FXS.

neuroscience↗

bioRxiv2019

Quantitative analysis and medium components optimizing for culturing a fastidious bacterium Christensenella minuta

Christensenella minuta is a heritable bacterium with controversial physiologies associating with both obesity and potential pathogenicity. Since this bacterium is fastidious to culture, it is hardly to well understand its biological feature. We develop a strategy for statistical analysis of this low abundant strain and optimize culture condition to make a significant improvement on its biomass and facilitate the researches about the metabolism and function of this bacterium. Basing on the fluorogenic quantitative technology, a quantitative approach was successfully constructed for Christensenella minuta by plotting Ct value from fluorescence quantitative PCR against the logarithm of concentration gradient of plasmids containing 16S rDNA of the strain. This method exhibited to have specificity on analyzing the strain biomass statistically. For improving the strain biomass, \"komodo\" predicted to optimize medium components and metabonomics analysis explored the catabolites addition effects on culture improvement. With the aid of Plackett-Burman and Box-Behnken in Design-Expert 8.0.6, the PB and response surface experiment were designed and analyzed from the single factor results. On the modified GAM medium, the strain concentration was found increasing markedly by 10 times. The addition of some amino acids, vitamins and inorganic salts has contributions for the strain multiplication, especially L-cysteine, VB6 and NaCl. The addition of 55 mg/L of L-cysteine, 20.5 mg/L VB6 and 55 g/L NaCl into the modified GAM medium increased the biomass by 3.59 times compared to the biomass on only modified GAM medium according to the response surface experiment. Through the newly constructed method, we successfully analyzed the amount of Christensenella minuta and obtained a novel medium to increase biomass significantly.\n\nImportanceChristensenella minuta is a heritable bacterium with controversial physiologies associating with both obesity and potential pathogenicity. Since this bacterium is fastidious to culture, it is hard to well understand its biological feature. We develop a strategy for statistical analysis of this low abundant strain and optimize culture condition to make a significant improvement on its biomass and facilitate the researches about the metabolism and function of this bacterium. This work combined the prediction tools and experiments to improve the medium components of C. munita and successfully enhance the culturing and increase biomass by more than 10-fold. From this perspective, the project throws some new ideas and also enables access to new knowledge and information in uncultured microbial resources.

microbiology↗

bioRxiv2019

Improving diagnosis and monitoring of treatment response in pulmonary tuberculosis using the molecular bacterial load assay (MBLA)

ObjectivesBetter outcomes in tuberculosis require new diagnostic and treatment monitoring tools. In this paper we evaluated the utility of a marker of M. tuberculosis viable count, the Molecular Bacterial Load assay (MBLA) for diagnosis and treatment monitoring of tuberculosis in a high burden setting. MethodsPatients with smear positive pulmonary tuberculosis from two sites in Tanzania and one each in Malawi and Mozambique. Sputum samples were taken weekly for the first 12 weeks of treatment and evaluated by MBLA and mycobacterial growth indicator tube method (MGIT). ResultsThe results of high and low positive control samples confirmed inter site reproducibility. Over the 12 weeks of treatment there was a steady decline in the viable bacterial load as measured by the MBLA that corresponds to rise in time to a positive result (TTP) in the Mycobacterial Growth Indicator Tube. Both MBLA and MGIT provided similar time to test negativity. Importantly, as treatment progressed samples in MGIT were increasingly likely to be contaminated, which compromised the acquisition of results but did not affect MBLA samples. ConclusionsMBLA produces a reproducible measure of Mtb viable count comparable to that of MGIT that is not compromised by contamination in a real-world setting. As a molecular test, the results can be available in as little as four hours and could allow health care professionals to identify rapidly patients who are failing therapy.

microbiology↗

bioRxiv2017

A Rice Dual-localized Pentatricopeptide Repeat Protein is involved in Organellar RNA Editing with MORFs

Flowering plants engage in diverse RNA editing events in mitochondrion and chloroplast on post-transcriptional process. Although several PPRs and MORFs were identified as RNA editing factors, the underlying mechanism of PPRs and the cooperation among them are still obscure. Here, we identified a rice dual-localized PPR mutant Ospgl1. Loss-of-function of OsPGLl resulted in defect of chloroplast RNA editing at ndhD-878 and mitochondrial RNA editing at ccmFc-543, which can be restored via complementary validation. Despite the synonymous editing on ccmFc-543, loss of editing at ndhD-878 caused failure of conversion from serine to leucine, leading to the dysfunction of chloroplast and defective in photosynthetic complex, further studies demonstrated OsPGL1 directly bound to both two transcripts. The interaction between three MORFs (MORF2/8/9) and OsPGL1 were confirmed in vitro and in vivo, implied OsPGL1 functioned on RNA editing via an editosome. It also suggested MORFs assisted and contributed to the flexible PPR-RNA recognition model during RNA editing through the cooperation with PPRs. These results provide new insight into the relationship between RNA editing and plant development on chloroplast.\n\nHighlightWe firstly characterized a dual-localized PPR protein which is required for RNA editing in mitochondrion and chloroplast simultaneously. OsPGL1 binds to two distinguish target transcripts directly and cooperated with MORFs.

molecular biology↗