Biology subjects

He, J.

Publications and source records attributed to He, J..

At least 19 recordsLinked to original sources

bioRxiv2018

Multi-Center Study of Resectable Lung Lesions by Ultra-Deep Sequencing of Targeted Genes in Plasma Cell-Free DNA to Assess Nodule Malignancy and Detect Lung Cancers

BACKGROUNDEarly detection of lung cancer to allow curative treatment remains challenging. Cell-free circulating tumor DNA (ctDNA) analysis may aid in malignancy assessment and early cancer diagnosis of lung nodules found in screening imagery.\n\nMETHODSThe multi-center clinical study enrolled 192 patients with operable occupying lung diseases. Plasma ctDNA, white blood cell genomic DNA (gDNA) and tumor tissue gDNA of each patient were analyzed by ultra-deep sequencing to an average of 35,000X of the coding regions of 65 lung cancer-related genes.\n\nRESULTSThe cohort consists of a quarter of benign lung diseases and three quarters of cancer patients with all histopathology subtypes. 64% of the cancer patients is at Stage I. Gene mutations detection in tissue gDNA and plasma ctDNA results in a sensitivity of 91% and specificity of 88%. When ctDNA assay was used as the test, the sensitivity was 69% and specificity 96%. As for the lung cancer patients, the assay detected 63%, 83%, 94% and 100%, for Stage I, II, III and IV, respectively. In a linear discriminant analysis, combination of ctDNA, patient age and a panel of serum biomarkers boosted the overall sensitivity to 80% at a specificity of 99%. 29 out of the 65 genes harbored mutations in the lung cancer patients with the largest number found in TP53 (30% plasma and 62% tumor tissue samples) and EGFR (20% and 40%, respectively).\n\nCONCLUSIONPlasma ctDNA was analyzed in lung nodule assessment and early cancer detection while an algorithm combining clinical information enhanced the test performance.

clinical trials↗

bioRxiv2018

Comparison of tri-exponential decay vs. bi-exponential decay and full fitting vs. segmented fitting for modeling liver intravoxel incoherent motion diffusion MRI

PurposeTo determine whether bi- or tri-exponential models, and full or segmented fittings, better fit IVIM imaging signal of healthy livers.\n\nMaterials and methodsDiffusion-weighted images were acquired with a 3-T scanner using respiratory-triggered echo-planar sequence and 16 b-values (0[~]800 s/mm2). Eighteen healthy volunteers had liver scanned twice in the same session, and then once again in another session. Region of interest (ROI)-based measurements were processed with bi-exponential model full fitting and segmented fitting (threshold b-value = 80 s/mm2), as well as tri-exponential model full fitting and segmented fitting (threshold b-value = 200 s/mm2).\n\nResultsWith all scans signal averaged, bi-exponential model full fitting showed Dslow=1.14, Dfast=193.6x10-3 mm2/s, and PF=16.9%, and segmented fitting showed Dslow=1.03, Dfast=56.7x10-3 mm2/s, and PF=21.3%. IVIM parameters derived from tri-exponential model were similar for full fitting and segmented fitting, with a slow (Dslow=0.98x10-3 mm2/s; Fslow=76.4 or 76.6%), a fast (Dfast=15.1 or 15.4x10-3 mm2/s; Ffast=11.8 or 11.7%) and a very fast (DVfast=445.0 or 448.8x10-3 mm2/s; FVfast=11.8 or 11.7 %) diffusion compartments. Tri-exponential model provided an overall better fit than bi-exponential model. For bi-exponential model, full fitting provided better fit at very low and low b-values compared with segmented fitting with the later tended to underestimate Dfast, however, segmented method demonstrated lower error in signal prediction for high b-values. Compared with full fitting, tri-exponential segmented fitting offered better scan-rescan reproducibility.\n\nConclusionFor healthy liver, tri-exponential modelling is preferred than bi-exponential modelling. For bi-exponential model, segmented fitting underestimates Dfast, but offers more accurate estimation of Dslow.

biophysics↗

bioRxiv2018

Induction of Sertoli cells from human fibroblasts by NR5A1 and GATA4

Sertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). The induction of human Sertoli cells from fibroblasts could provide cellular sources for fertility and transplantation treatments. Here, we report the in vitro reprogramming of human fibroblasts to Sertoli cells and characterize these human induced Sertoli cells (hiSCs). Initially, five transcriptional factors (NR5A1, GATA4, WT1, SOX9 and DMRT1) and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, i.e., NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles that are similar to those of primary human Sertoli cells. Consistent with the known cellular properties of Sertoli cells, hiSCs attract endothelial cells and exhibit high number of lipid droplets in the cytoplasm. More importantly, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. In addition, hiSCs suppress the production of IL-2 and proliferation of human T lymphocytes. When hiSCs were cotransplanted with human embryonic kidney cells, these xenotransplanted human cells survived longer in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli-only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.

developmental biology↗

bioRxiv2018

Microbial Cells Harboring a Mitochondrial Gene Are Capable of CO2 Capture

Global warming is escalating with increased temperatures reported worldwide. Given the enormous land mass on the planet, biological capture of CO2 remains a viable approach to mitigate the crisis as it is economical and easy to implement. In this study, a gene capable of CO2 capture was identified via selection in minimal media. This mitochondrial gene named as OG1 encodes the OK/SW-CL.16 protein and shares homology with cytochrome oxidase subunit III of various species and PII uridylyl-transferase from Loktanella vestfoldensis SKA53. CO2 capture experiments indicate that {delta}13C was substantially higher in the cells harboring the gene OG1 than the control in the nutrition-poor media. This study suggests that CO2 capture using engineered microorganisms in barren land can be exploited to address the soaring CO2 level in the atmosphere, opening up vast land resources to cope with global warming.\n\nIMPORTANCEGlobal warming crisis is deteriorating with increased CO2 levels in the atmosphere each year. Action must be taken before catastrophic consequences occur in the not-so-distant future. Biological capture of CO2 is a feasible approach to alleviate the current crisis. We have identified a mitochondrial gene which demonstrated CO2 utilization capability. Data presented in this study suggest that CO2 capture using engineered microorganisms can be harnessed to address the ever-rising CO2 level in the atmosphere.

bioengineering↗

bioRxiv2018

Regulation by HSP70/90 in the different tissues and testis development of male cattle (Cattle-yak and Yak)

HSP70/90 play important role in testis develop and spermatozoa regulation, but the contact of HSP70/90 with infertility in cattle is unclear. Here, we focus on male cattle-yak and yak, which to investigate the expression and localization of HSP70/90 in different tissues, and explore the influence of HSP70/90 to infertility. In our study, a total of 54 cattle (24 cattle-yak and 30 yak) were examined. The HSP90 mRNA of cattle-yak was cloned first and found amino acid variation in HSP90, which led to difference at protein spatial structure compare with yak. To investigate whether the expression of HSP70/90 mRNA and protein are different in cattle-yak and yak, we used real-time quantitative PCR (qRT-PCR) and Western blot (WB) to examine them. We found that the expression level of HSP70/90 mRNA and protein are disparity in different tissues and testis development stages, and obviously high expression was observed in testicle during juvenile and adult, Moreover, it s interestingly in which the HSP70 expression is significant high in yak whereas HSP90 in cattle-yak (P<0.01). On this bases, we detect the location of HSP70/90 in testis by immunohistochemical (IHC) and immunofluorescence (IF), the results demonstrate that HSP70/90 were located in the epithelial cells, spermatogenic cells and mesenchymal cells. In summary, our study proved the expression of HSP70/90 are different in tissues, and the expression of HSP90 is obviously high in testis of cattle-yak, which propose that the infertility of cattle-yak may cause from up-regulating of HSP90.

biochemistry↗

bioRxiv2018

RNAi screening identifies a new Toll from shrimp that restricts WSSV infection through activating Dorsal to induce antimicrobial peptides

The function of Toll pathway defense against bacterial infection has been well established in shrimp, however how this pathway responds to viral infection is still largely unknown. In this study, we report the Toll4-Dorsal-AMPs cascade restricts the white spot syndrome virus (WSSV) infection of shrimp. A total of nine Tolls from Litopenaeus vannamei namely Toll1-9 are identified, and RNAi screening in vivo reveals the Toll4 is important for shrimp to oppose WSSV infection. Knockdown of Toll4 results in elevated viral loads and renders shrimp more susceptible to WSSV. Furthermore, Toll4 could be a one of upstream pattern recognition receptor (PRR) to detect WSSV, and thereby leading to nuclear translocation and phosphorylation of Dorsal, the known NF-{kappa}B transcription factor of the canonical Toll pathway. More importantly, silencing of Toll4 and Dorsal contributes to impaired expression of a specific set of antimicrobial peptides (AMPs) such as anti-LPS-factor (ALF) and lysozyme (LYZ) family, which exert potent anti-WSSV activity. Two AMPs of ALF1 and LYZ1 as representatives are demonstrated to have the ability to interact with several WSSV structural proteins. Taken together, we therefore identify the Toll4-Dorsal pathway mediates strong resistance to WSSV infection by inducing some specific AMPs.\n\nAuthor summaryThe TLR pathway mediated antiviral immune response is well identified in mammals, yet, Toll pathway governing this protection in invertebrates remains unknown. In the present study, we uncover that a shrimp Toll4 from a total of nine Tolls in L. vannamei confers resistance to WSSV thought inducing the NF-{kappa}B transcription factor Dorsal to inspiring the production of some antimicrobial peptides (AMPs) with antiviral activity. The anti-LPS-factor (ALF) and lysozyme (LYZ) family are identified as the Toll4-Dorsal pathway targeted genes with the ability to interact with viral structural proteins in response to WSSV infection. These results suggest that the Toll receptor induces the expression of AMPs with antiviral activity could be a general antiviral mechanism in invertebrates and Toll pathway established antiviral defense could be conserved during evolution.

immunology↗

bioRxiv2018

Cerebrovascular damages induce lymphatic invasion into brain parenchyma to guide vascular regeneration

Damage to regional cerebrovascular network and neuronal tissues occurs during acute cerebrovascular diseases, such as ischemic stroke. The promotion of vascular regeneration is the most promising therapeutic approach. To understand cellular and molecular mechanisms underlying brain vascular regeneration, we developed two zebrafish cerebrovascular injury models using genetic ablation and photochemical thrombosis. Although brain parenchyma is physiologically devoid of lymphatic vasculature, we found that cerebrovascular injuries induce rapid ingrowth of meningeal lymphatics into the injured parenchyma. The ingrown lymphatics on one hand become lumenized drain interstitial fluid to resolve brain edema, on the other hand act as \"growing tracks\" for nascent blood vessels. The ingrown lymphatic vessels undergo apoptosis and clearance after cerebrovascular regeneration. This study reveals a pathological function of meningeal lymphatics, through previously unexpected ingrowth into brain parenchyma and a newly identified lymphatic function as vascular \"growing tracks\".\n\nHIGHLIGHTSCerebrovascular injuries induce lymphatic ingrowth into the injured brain parenchyma The ingrown lymphatics drain interstitial fluid to resolve brain edema Nascent blood vessels use the ingrown lymphatic vessels as \"growing tracks\" The ingrown lymphatic vessels undergo apoptosis after vascular regeneration completes

developmental biology↗

bioRxiv2018

Improved prediction of chronological age from DNA methylation limits it as a biomarker of ageing

DNA methylation is associated with age. The deviation of age predicted from DNA methylation from actual age has been proposed as a biomarker for ageing. However, a better prediction of chronological age implies less opportunity for biological age. Here we used 13,661 samples (from blood and saliva) in the age range of 2 to 104 years from 14 cohorts measured on Illumina HumanMethylation450/EPIC arrays to perform prediction analyses. We show that increasing the sample size achieves a smaller prediction error and higher correlations in test datasets. We demonstrate that smaller prediction errors provide a limit to how much variation in biological ageing can be captured by methylation and provide evidence that age predictors from small samples are prone to confounding by cell composition. Our predictor shows a similar or better performance in non-blood tissues including saliva, endometrium, breast, liver, adipose and muscle, compared with Horvaths across-tissue age predictor.

bioinformatics↗

bioRxiv2018

Effect of Lactobacillus reuteri on intestinal microflora and immune parameters: involvement of sex differences

Probiotic candidate L. reuteri was screened out for in vivo experiments based on a relatively higher gastrointestinal tolerance and moderate adhesiveness. As results shown in in-vivo experiments, a significantly higher level of IL-12 at low-dose group was found both in females and males. Higher levels of T-lymphocytes were also observed in females compared to control group, however, males displayed a reduction expcept for CD8-positive cells in ileum. In comparison to the control group, the relative abundance of phylotypes in the phylum Bacteroidetes (genus of Bacteroides, Prevotella) and Firmicutes (genus of ClostridiumIV) exihibited a reserve shift between sexes after L. reuteri intervened. Meanwhile, the relative abundance of several taxa (Acetobacteroides, Lactobcaillus, bacillus) also differed markedly in sexes at low-dose group, together with microbiota diversity, as indicated by Shannon index.\n\nImportanceSexual dimorphism has triggered researchers attention. However, the relationship between immune parameters and gut microbiota caused by Lactobacillus at different dosage are not fully elucidated. In present research, the possible probiotic role of L. reuteri DMSZ 8533 on immunomodulation and effect on fecal microbiota composition were investigated. Our findings demonstrate the importance of L. reuteri DMSZ 8533 as a potential probiotic strain with an immunomodulatory effect, which also alters the microflora composition depending on the sex of the host.

microbiology↗

bioRxiv2018

Elucidating synergistic dependencies in lung adenocarcinoma by proteome-wide signaling-network analysis

Signaling pathway models are largely based on the compilation of literature data from heterogeneous cellular contexts. Indeed, de novo reconstruction of signaling interactions from large-scale molecular profiling is still lagging, compared to similar efforts in transcriptional and protein-protein interaction networks. To address this challenge, we introduce a novel algorithm for the systematic inference of protein kinase pathways, and applied it to published mass spectrometry-based phosphotyrosine profile data from 250 lung adenocarcinoma (LUAD) samples. The resulting network includes 43 TKs and 415 inferred, LUAD-specific substrates, which were validated at >60% accuracy by SILAC assays, including \"novel substrates of the EGFR and c-MET TKs, which play a critical oncogenic role in lung cancer. This systematic, data-driven model supported drug response prediction on an individual sample basis, including accurate prediction and validation of synergistic EGFR and c-MET inhibitor activity in cells lacking mutations in either gene, thus contributing to current precision oncology efforts.

systems biology↗

bioRxiv2018

Transcriptional deconvolution reveals consistent functional subtypes of pancreatic cancer epithelium and stroma

Bulk tumor tissues comprise intermixed populations of neoplastic cells and multiple lineages of stromal cells. We used laser capture microdissection and RNA sequencing to disentangle the transcriptional programs active in the malignant epithelium and stroma of pancreatic ductal adenocarcinoma (PDA). This led to the development of a new algorithm (ADVOCATE) that accurately predicts the compartment fractions of bulk tumor samples and can computationally purify bulk gene expression data from PDA. We also present novel stromal subtypes, derived from 110 microdissected PDA stroma samples, that were enriched in extracellular matrix- and immune-associated processes. Finally, we applied ADVOCATE to systematically evaluate cross-compartment subtypes spanning four patient cohorts, revealing consistent functional classes and survival associations despite substantial compositional differences.

systems biology↗

bioRxiv2017

Unconsciously Implanted Visuoauditory Memory in the Presence of Cholecystokinin Retrieved in Behavioral Contexts

We investigated whether visuoauditory association can be artificially implanted in rodents and then retrieved in a behaviorally relevant context. Rats were trained to approach the left or right hole of a behavioral apparatus to retrieve a reward depending on the side of electrical stimulation of the auditory cortex (EAC) they received and mice were fear-conditioned to EAC. Next, an irrelevant visual stimulus (VS) was repeatedly paired with EAC in the presence of cholecystokinin (CCK) or with activation of terminals of entorhinal CCK neurons in the auditory cortex. In subsequent behavioral testing with VS, rats approached the hole associated with reward availability and mice showed a freezing response to the VS. A CCK antagonist blocked the establishment of visuoauditory association, whereas a CCK agonist rescued the deficit of association. Our findings provide a scientific foundation for \"memory implantation\" and indicate that CCK is the switching chemical for formation of visuoauditory association.

neuroscience↗

bioRxiv2017

Single Molecule Sequencing of Cell-free DNA from Maternal Plasma for Noninvasive Trisomy Detection

The demand of non-invasive prenatal testing for autosomal aneuploidy using cell-free fetal DNA (cffDNA) in maternal plasma is a highly sought-after diagnostic, with a rapidly growing market. Current approaches developed by next generation sequencing (NGS) need PCR amplifcation during sample preparation, which results in amplification bias in GC-rich areas of the human genome. With these approaches, the minimum fetal fraction in maternal plasma is 4% for the small differences in circulating cfDNA between trisomic and disomic pregnancies to be detectable. In this paper, we performed single molecule sequencing of cell-free DNA from maternal plasma for noninvasive trisomy 13, 18 and 21 detections using the GenoCare platform. We found that single molecule sequencing is sensitive enough to detect these chromosome abnormalities when the fetal DNA fraction is as low as 2%. Compared to the Hiseq2500 platform, no significant GC bias was observed. The improved sensitivity and unbiased GC readout make GenoCare a promising platform for autosomal aneuploidy detections, even in the very early stage of pregnancy.

genomics↗

bioRxiv2017

The Early Diagnosis in Lung Cancer by the Detection of Circulating Tumor DNA

BackgroundRemarkable advances for clinical diagnosis and treatment in cancers including lung cancer involve cell-free circulating tumor DNA (ctDNA) detection through next generation sequencing. However, before the sensitivity and specificity of ctDNA detection can be widely recognized, the consistency of mutations in tumor tissue and ctDNA should be evaluated. The urgency of this consistency is extremely obvious in lung cancer to which great attention has been paid to in liquid biopsy field.\n\nMethodsWe have developed an approach named systematic error correction sequencing (Sec-Seq) to improve the evaluation of sequence alterations in circulating cell-free DNA. Averagely 10 ml preoperative blood samples were collected from 30 patients containing pulmonary space occupying pathological changes by traditional clinic diagnosis. cfDNA from plasma, genomic DNA from white blood cells, and genomic DNA from solid tumor of above patients were extracted and constructed as libraries for each sample before subjected to sequencing by a panel contains 50 cancer-associated genes encompassing 29 kb by custom probe hybridization capture with average depth >40000, 7000, or 6300 folds respectively.\n\nResultsDetection limit for mutant allele frequency in our study was 0.1%. The sequencing results were analyzed by bioinformatic expertise based on our previous studies on the baseline mutation profiling of circulating cell-free DNA and the clinicopathological data of these patients. Among all the lung cancer patients, 78% patients were predicted as positive by ctDNA sequencing when the shreshold was defined as at least one of the hotspot mutations detected in the blood (ctDNA) was also detected in tumor tissue. Pneumonia and pulmonary tuberculosis were detected as negative according to the above standard. When evaluating all hotspots in driver genes in the panel, 24% mutations detected in tumor tissue (tDNA) were also detected in patients blood (ctDNA). When evaluating all genetic variations in the panel, including all the driver genes and passenger genes, 28% detected in tumor tissue (tDNA) were also detected in patients blood (ctDNA). Positive detection rates of plasma ctDNA in stage I lung cancer patients is 85%, compared with 17% of tumor biomarkers.\n\nConclusionWe demonstrated the importance of sequencing both circulating cell-free DNA and genomic DNA in tumor tissue for ctDNA detection in lung cancer currently. We also determined and confirmed the consistency of ctDNA and tumor tissue through NGS according to the criteria explored in our studies. Our strategy can initially distinguish the lung cancer from benign lesions of lung. Our work shows that the consistency will be benefited from the optimization in sensitivity and specificity in ctDNA detection.

cancer biology↗

bioRxiv2017

Cholecystokinin release triggered by presynaptic NMDA receptors produces LTP and sound-sound associative memory formation

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA-dependent long-term potentiation (LTP). There is evidence that entorhinal cortex enables neocortical neuroplasticity through cholecystokinin (CCK)-containing neocortical projections. Here we show that a CCKB antagonist blocks high-frequency stimulation (HFS)-induced LTP in the auditory cortex, whereas local infusion of CCK induces LTP. CCK-/- mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; administration of CCK rescued associative learning. HFS of CCK-containing entorhino-neocortical projection neurons in anesthetized mice enabled cue-cue associative learning. Furthermore, when one cue was pre-conditioned to footshock, the mouse showed a freezing response to the other cue, indicating that the mice had formed an association. HFS-induced neocortical LTP was completely blocked by either NMDA antagonist or CCK-BR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation (LFS). Moreover, in the presence of CCK LTP was still induced, even after blockade of NMDA receptors. Local application of NMDA induced CCK release in the neocortex. To identify how NMDA receptor switches LTP, a stimulation protocol of 25 pulse-pairs was adopted to replace HFS; NMDA-dependent LTP was induced with the inter-pulse intervals between 10 and 100 ms, but not with those of 5 and 200 ms. LTP-mediated plasticity was linked to localization of the NMDA receptor subunit NR2a on cortical CCK terminals originating in the entorhinal cortex. These novel findings suggest that presynaptic NMDA receptors on CCK terminals control the release of CCK, which enables neocortical LTP and formation of cue-cue associative memory.\n\nOne Sentence SummaryPresynaptic NMDA receptors switches the release of CCK from entorhinal neurons, which enables neocortical LTP and formation of sound-sound associative memory.

neuroscience↗

bioRxiv2017

IVIM parameters have good scan-rescan reproducibility when evidential motion contaminated and poorly fitted image data are removed

BackgroundIntravoxel Incoherent Motion (IVIM) diffusion MRI is a promising technique for liver pathology evaluation, but this techniques scan-rescan reproducibility has been reported to be unsatisfactory.\n\nObjectiveTo understand whether IVIM MRI parameters for liver parenchyma can be good after removal of motion contaminated and/or poorly fitted image data.\n\nMaterial and MethodsEighteen healthy volunteers had liver scanned twice at the same session to assess scan-rescan repeatability, and again in another session after an average interval of 13 days to assess reproducibility. Diffusion weighted image were acquired with a 3T scanner using respiratory-triggered echo-planar sequence and 16 b-values (0 to 800 s/mm2). Measurement was performed on the right liver with segmented-unconstrained least square fitting. Image series with evidential anatomical mismatch, apparent artifacts, and poorly fitted signal intensity vs. b-value curve were excluded. A minimum of three slices was deemed necessary for IVIM parameter estimation of a liver.\n\nResultsWith total 54 examinations, 6 scans did not satisfy inclusion criteria, leading to a success rate of 89%; and 14 volunteers were finally included. With each scan a mean of 5.3 slices (range: 3-10 slices) were utilized for analysis. Using threshold b-value=80s/mm2, the coefficient of variation and within-subject coefficient of variation for repeatability and reproducibility were: 2.86% and 4.24% for Dslow, 3.81% and 4.24%, for PF, 18.16% and 24.88% for Dfast; and those for reproducibility were 2.48% and 3.24% for Dslow; 4.91% and 5.38% for PF; 21.18% and 30.89% for Dfast.\n\nConclusionIVIM parameter scan-rescan reproducibility can be potentially good.

biophysics↗

bioRxiv2017

The Novel Monocomponent FAD-dependent Monooxygenase HpaM Catalyzes the 2-Decarboxylative Hydroxylation of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135

5-hydroxypicolinic acid (5HPA) is a natural pyridine derivative that can be microbially degraded. However, the physiological, biochemical, and genetic foundation of the microbial catabolism of 5HPA remains unknown. In this study, a gene cluster hpa (which is involved in degradation of 5HPA in Alcaligenes faecalis JQ135) was cloned and HpaM was identified as a novel monocomponent FAD-dependent monooxygenase. HpaM shared a sequence only 31% similarity with the most related protein 6-hydroxynicotinate 3-monooxygenase (NicC) of Pseudomonas putida KT2440. hpaM was heterologously expressed in E. coli BL21(DE3), and the recombinant HpaM was purified via Ni-affinity chromatography. HpaM catalyzed the 2-decarboxylative hydroxylation of 5-HPA, thus generating 2,5-dihydroxypyridine (2,5-DPH). Monooxygenase activity was only detected in the presence of FAD and NADH, but not of FMN and NADPH. The apparent Km values of HpaM toward 5HPA and NADH were 45.4 and 37.8 , respectively. Results of gene deletion and complementation showed that hpaM was essential for 5HPA degradation in Alcaligenes faecalis JQ135.\n\nImportancePyridine derivatives are ubiquitous in nature and important chemical materials that are currently widely used in agriculture, pharmaceutical, and chemical industries. Thus, the microbial degradation and transformation mechanisms of pyridine derivatives received considerable attention. Decarboxylative hydroxylation was an important degradation process in pyridine derivatives, and previously reported decarboxylative hydroxylations happened in the C3 of the pyridine ring. In this study, we cloned the gene cluster hpa, which is responsible for 5HPA degradation in Alcaligenes faecalis JQ135, thus identifying a novel monocomponent FAD-dependent monooxygenase HpaM. Unlike 3-decarboxylative monooxygenases, HpaM catalyzed decarboxylative hydroxylation in the C2 of the pyridine ring in 5-hydroxypicolinic acid. These findings deepen our understanding of the molecular mechanism of microbial degradation of pyridine derivatives. Furthermore, HpaM offers potential for applications to transform useful pyridine derivatives.

microbiology↗

bioRxiv2017

Resequencing the Escherichia coli genome by GenoCare single molecule sequencing platform

Next generation sequencing (NGS) has revolutionized life sciences research. Recently, a new class of third-generation sequencing platforms has arrived to meet increasing demands in the clinic, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single molecule sequencing platform from Direct Genomics to resequence the E. coli genome and show comparable performance to the Illumina MiSeq system. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. With a consensus sequence of 99.71% nucleotide identity to that of the Illumina MiSeq systems, GenoCare was determined to be a reliable platform for single-molecule sequencing, with strong potential for clinical applications.

genomics↗