Transcriptome analysis of Δmig1Δmig2 mutant reveals their roles in methanol catabolism, peroxisome biogenesis and autophagy in methylotrophic yeast Pichia pastoris

Lei Shi,Xiaolong Wang,Jinjia Wang,Ping Zhang,Fei Qi,Menghao Cai,Yuanxing Zhang,Xiangshan Zhou 한국유전학회 2018 Genes & Genomics Vol.40 No.4

Two catabolite repressor genes (MIG1 and MIG2) were previously identified in Pichia pastoris, and the derepression of alcohol oxidase (AOX) expression was realized in Δmig1 or Δmig1Δmig2 mutants grown in glycerol, but not in glucose. In this study, genome-wide RNA-seq analysis of Δmig1Δmig2 and the wild-type strain grown in glycerol revealed that the expression of numerous genes was greatly altered. Nearly 7% (357 genes) of approximately 5276 genes annotated in P. pastoris were significantly upregulated, with at least a two-fold differential expression in Δmig1Δmig2; the genes were mainly related to cell metabolism. Approximately 23% (1197 genes) were significantly downregulated; these were mainly correlated with the physiological characteristics of the cell. The methanol catabolism and peroxisome biogenesis pathways were remarkably enhanced, and the genes AOX1 and AOX2 were upregulated higher than 30-fold, which was consistent with the experimental results of AOX expression. The Mig proteins had a slight effect on autophagy when cells were grown in glycerol. The expression analysis of transcription factors showed that deletion of MIG1 and MIG2 significantly upregulated the binding of an essential transcription activator, Mit1p, with the AOX1 promoter, which suggested that Mig proteins might regulate the AOX1 promoter through the regulation of Mit1p. This work provides a reference for the further exploration of the methanol induction and catabolite repression mechanisms of AOX expression in methylotrophic yeasts.

Construction of Recombinant Pichia pastoris Carrying a Constitutive AvBD9 Gene and Analysis of Its Activity

( Jian Tu ),( Kezong Qi ),( Ting Xue ),( Haiting Wei ),( Yongzheng Zhang ),( Yanli Wu ),( Xiuhong Zhou ),( Xiaolong Lv ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.12

Avian beta-defensin 9 (AvBD9) is a small cationic peptide consisting of 41 amino acids that plays a crucial rule in innate immunity and acquired immunity in chickens. Owing to its wide antibacterial spectrum, lack of a residue, and failure to induce bacterial drug resistance, AvBD9 is expected to become a substitute for conventional antibiotics in the livestock and poultry industries. Using the preferred codon of Pichia pastoris, the mature AvBD9 peptide was designed and synthesized, based on the sequence from GenBank. The P. pastoris constitutive expression vector pGHKα was used to construct a pGHKα-AvBD9 recombinant plasmid. Restriction enzyme digestion was performed using SacI and BglII to remove the ampicillin resistance gene, and the plasmid was electrotransformed into P. pastoris GS115. High-expression strains with G418 resistance were screened, and the culture supernatant was analyzed by Tricine-SDS-PAGE and western blot assay to identify target bands of about 6 kDa. A concentrate of the supernatant containing AvBD9 was used for determination of antimicrobial activity. The supernatant concentrate was effective against Escherichia coli, Salmonella paratyphi, Salmonella pullorum, Pseudomonas aeruginosa, Enterococcus faecalis, and Enterobacter cloacae. The fermentation product of P. pastoris carrying the recombinant AvBD9 plasmid was adjusted to 1.0 × 108 CFU/ml and added to the drinking water of white feather broilers at different concentrations. The daily average weight gain and immune organ indices in broilers older than 7 days were significantly improved by the AvBD9 treatment.

Iron metabolism protein transferrin receptor 1 involves in cervical cancer progression by affecting gene expression and alternative splicing in HeLa cells

Huang Nan,Wei Yaxun,Cheng Yi,Wang Xiaolong,Wang Qi,Chen Dong,Li Wenjing 한국유전학회 2022 Genes & Genomics Vol.44 No.6

Background: Transferrin receptor 1 (TfR1), encoded by TFRC, is a key regulator of iron homeostasis and plays important roles in many diseases, including cancers. Objective: To decipher the underlying molecular functions of TfR1 based on its influence on transcriptome profile in cancer cells. Methods: In this study, we first identified the expression pattern and prognostic influence of TFRC in cervical cancer patients from TCGA database. To explore the regulatory outcomes of TfR1 from the view of whole transcriptome profile, we generated TFRC knockdown (TFRC-KD) HeLa cells and negative control (NC) cells using short hairpin RNA (shRNA) method. Unbiased transcriptome sequencing (RNA-seq) experiment was used to analyze the global expression level and alternative splicing (AS) changes between TFRC-KD and NC cells. Results: We found TFRC was consistently elevated in cervical cancer samples and tightly associated with prognosis of patients. Differential expression analysis revealed that 629 differentially expressed genes (DEGs) were identified between TFRC-KD and NC. Functional enrichment analysis of these DEGs revealed that TFRC-KD extensively disturbed cell physiology related pathways, including immunity, cell metabolism and gene expression. Moreover, dysregulated AS profile also indicated that TfR1 has important roles in the AS regulation. Hundreds of TfR1-regulated AS genes were involved in DNA repair, cell death, transcription and viral reproduction pathways, which were tightly associated with cancer cell progression. Conclusions: In summary, we for the first time explored the molecular functions of TfR1 at transcriptional and post-transcriptional levels. These results demonstrate TfR1 participates in the progression of cervical cancer by affecting the expression and AS levels of genes in cancer associated pathways, which greatly extends our understanding of TfR1 functions besides iron homeostasis and provide novel options in cancer treatment by targeting TfR1.

Step-by-step identification of industrial robot dynamics model parameters and force-free control for robot teaching

Binrui Wang,Junwei Fang,Shunan Qi,Ling Wang,Xiaolong Liu,Haijun Ren 대한기계학회 2023 JOURNAL OF MECHANICAL SCIENCE AND TECHNOLOGY Vol.37 No.7

In order to solve the problem of lack of flexibility in direct teaching of industrial robots under complex working conditions, dynamic inertia term compensation is combined with the traditional force-free control algorithm to reduce the traction in the teaching process in this paper. Firstly, considering the multi-dimensional, strongly nonlinear and multi-parameter characteristics of the 6-DOF manipulator dynamic model, a step-by-step parameters identification method based on genetic algorithm is proposed. This method can effectively reduce the computational complexity and improve the optimization speed of industrial robot dynamic model identification. Secondly, based on the internal torque analysis of industrial robot joints, a forcefree compensation control algorithm based on the torque control is designed. Finally, the effectiveness of the step-by-step robot dynamic model parameter identification method is verified by a traction teaching experiment. In the verification experiment, the relative error rate between the predicted torque and the actual torque calculated from the identification results is 1.64 % to 5.99 %. After increasing the inertia term compensation, the traction force of the teaching process is reduced by 28.6 %. The experimental results show that the identification result is more accurate, and the proposed force-free control algorithm significantly improves the compliance of the teaching process.

SIRT5-related desuccinylation modification of AIFM1 protects against compression-induced intervertebral disc degeneration by regulating mitochondrial homeostasis

Mao Jianxin,Wang Di,Wang Dong,Wu Qi,Shang Qiliang,Gao Chu,Wang Huanbo,Wang Han,Du Mu,Peng Pandi,Jia Haoruo,Xu Xiaolong,Wang Jie,Yang Liu,Luo Zhuojing 생화학분자생물학회 2023 Experimental and molecular medicine Vol.55 No.-

Mitochondrial dysfunction plays a major role in the development of intervertebral disc degeneration (IDD). Sirtuin 5 (SIRT5) participates in the maintenance of mitochondrial homeostasis through its desuccinylase activity. However, it is still unclear whether succinylation or SIRT5 is involved in the impairment of mitochondria and development of IDD induced by excessive mechanical stress. Our 4D label-free quantitative proteomic results showed decreased expression of the desuccinylase SIRT5 in rat nucleus pulposus (NP) tissues under mechanical loading. Overexpression of Sirt5 effectively alleviated, whereas knockdown of Sirt5 aggravated, the apoptosis and dysfunction of NP cells under mechanical stress, consistent with the more severe IDD phenotype of Sirt5 KO mice than wild-type mice that underwent lumbar spine instability (LSI) surgery. Moreover, immunoprecipitation-coupled mass spectrometry (IP-MS) results suggested that AIFM1 was a downstream target of SIRT5, which was verified by a Co-IP assay. We further demonstrated that reduced SIRT5 expression resulted in the increased succinylation of AIFM1, which in turn abolished the interaction between AIFM1 and CHCHD4 and thus led to the reduced electron transfer chain (ETC) complex subunits in NP cells. Reduced ETC complex subunits resulted in mitochondrial dysfunction and the subsequent occurrence of IDD under mechanical stress. Finally, we validated the efficacy of treatments targeting disrupted mitochondrial protein importation by upregulating SIRT5 expression or methylene blue (MB) administration in the compression-induced rat IDD model. In conclusion, our study provides new insights into the occurrence and development of IDD and offers promising therapeutic approaches for IDD.

Correspondence on Editorial regarding “Baveno- VII criteria to predict decompensation and initiate non-selective beta-blocker in compensated advanced chronic liver disease patients”

Yu Jun Wong,Sanchit Sharma,Giulia Tosetti,Xiaolong Qi,Massimo Primignani 대한간학회 2023 Clinical and Molecular Hepatology(대한간학회지) Vol.29 No.1

Baveno-VII criteria to predict decompensation and initiate non-selective beta-blocker in compensated advanced chronic liver disease patients

Yu Jun Wong,Chen Zhaojin,Guilia Tosetti,Elisabetta Degasperi,Sanchit Sharma,Samagra Agarwal,Liu Chuan,Chan Yiong Huak,Li Jia,Qi Xiaolong,Anoop Saraya,Massimo Primignani 대한간학회 2023 Clinical and Molecular Hepatology(대한간학회지) Vol.29 No.1

Background/Aims: The utility of Baveno-VII criteria of clinically significant portal hypertension (CSPH) to predict decompensation in compensated advanced chronic liver disease (cACLD) patient needs validation. We aim to validate the performance of CSPH criteria to predict the risk of decompensation in an international real-world cohort of cACLD patients. Methods: cACLD patients were stratified into three categories (CSPH excluded, grey zone, and CSPH). The risks of decompensation across different CSPH categories were estimated using competing risk regression for clustered data, with death and hepatocellular carcinoma as competing events. The performance of “treating definite CSPH” strategy to prevent decompensation using non-selective beta-blocker (NSBB) was compared against other strategies in decision curve analysis. Results: One thousand one hundred fifty-nine cACLD patients (36.8% had CSPH) were included; 7.2% experienced decompensation over a median follow-up of 40 months. Non-invasive assessment of CSPH predicts a 5-fold higher risk of liver decompensation in cACLD patients (subdistribution hazard ratio, 5.5; 95% confidence interval, 4.0–7.4). “Probable CSPH” is suboptimal to predict decompensation risk in cACLD patients. CSPH exclusion criteria reliably exclude cACLD patients at risk of decompensation, regardless of etiology. Among the grey zone, the decompensation risk was negligible among viral-related cACLD, but was substantially higher among the non-viral cACLD group. Decision curve analysis showed that “treating definite CSPH” strategy is superior to “treating all varices” or “treating probable CSPH” strategy to prevent decompensation using NSBB. Conclusions: Non-invasive assessment of CSPH may stratify decompensation risk and the need for NSBB in cACLD patients.