Identification of SARS-CoV-2 m6A modification sites correlate with viral pathogenicity.

It has recently been found that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) m6A modifications can affect viral replication and function. However, no studies to date have shown a correlation between SARS-CoV-2 m6A modifications and viral pathogenicity. In this study, we analyzed m6A modification in 2,190,667 SARS-CoV-2 genomic RNAs. m6A modifications of SARS-CoV-2 from different lineages, causing mild or severe COVID-19 and showing breakthrough for different vaccines were analyzed to explore correlations with viral pathogenicity. The results suggested that the presence of more m6A modifications in the SARS-CoV-2 N region (positive strand) correlates with weaker pathogenicity. In addition, we identified three m6A modification sites correlating with weak pathogenicity (924 in ORF1ab, 15,659 in ORF1ab, 28,288 in N, 28,633 in N and 29,385 in N, 29,707 in 3'UTR) and one with strong pathogenicity (74 in 5'UTR). These results provide new information for understanding the prevalence of SARS-CoV-2 and controlling the virus.

Nonstructural N- and C-tails of Dbp2 confer the protein full helicase activities.

Human DDX5 and its yeast ortholog Dbp2 are ATP-dependent RNA helicases that play a key role in normal cell processes, cancer development, and viral infection. The crystal structure of the RecA1-like domain of DDX5 is available but the global structure of DDX5/Dbp2 subfamily proteins remains to be elucidated. Here, we report the first X-ray crystal structures of the Dbp2 helicase core alone and in complex with ADP at 3.22 Å and 3.05 Å resolutions, respectively. The structures of the ADP-bound post-hydrolysis state and apo-state demonstrate the conformational changes that occur when the nucleotides are released. Our results showed that the helicase core of Dbp2 shifted between open and closed conformation in solution but the unwinding activity was hindered when the helicase core was restricted to a single conformation. A small-angle X-ray scattering experiment showed that the disordered amino (N) tail and carboxy (C) tails are flexible in solution. Truncation mutations confirmed that the terminal tails were critical for the nucleic acid binding, ATPase, and unwinding activities, with the C-tail being exclusively responsible for the annealing activity. Furthermore, we labeled the terminal tails to observe the conformational changes between the disordered tails and the helicase core upon binding nucleic acid substrates. Specifically, we found that the nonstructural terminal tails bind to RNA substrates and tether them to the helicase core domain, thereby conferring full helicase activities to the Dbp2 protein. This distinct structural characteristic provides new insight into the mechanism of DEAD-box RNA helicases.

[Histone ubiquitylation and its roles in DNA damage response].

Ubiquitylation is an essential type of protein post-translational modifications (PTMs) in eukaryotes, which mediates various biological processes by regulating the subcellular localization, activity, and stability of proteins. Histones, as the main protein ingredients of chromatin, are closely coupled with DNA activities such as replication, transcription and repair, and therefore are the hotspots of PTMs. After DNA damage, histone ubiquitylations are involved in DNA damage response (DDR) by regulating nucleosome structure, activating cell cycle checkpoints, remodeling the nucleosome, and the recruitment and assembly of repair factors. Meanwhile, histone ubiquitylations can also crosstalk with other types of PTMs to regulate DDR processes. In this review, we summarize how the site-specific histone ubiquitylation forms signal network and contributes to DDR, which may shed light on the further study of how histone codes formed by histone PTMs affect the entire DDR processes.

Ginsenoside Rg1 Promotes the Migration of Olfactory Ensheathing Cells via the PI3K/Akt Pathway to Repair Rat Spinal Cord Injury.

The aim of this study was to determine the effects of ginsenoside Rg1 on the migration of olfactory ensheathing cells (OECs) in vitro, and its influence on the therapeutic efficacy of OECs transplanted in vivo for the treatment of spinal cord injury (SCI). Primary cultured and purified OECs (prepared from rats) were treated with ginsenoside Rg1. The wound healing test indicated that ginsenoside Rg1 promoted the migration of OECs. Real-time RT-PCR demonstrated that ginsenoside Rg1 upregulated the expression of migration-related factors of OECs, including matrix metalloproteinases-2 (MMP-2), MMP-9, and neural cell adhesion molecule 1 (NCAM1). Moreover, Western blot analysis indicated that ginsenoside Rg1 significantly promoted the migration of OECs via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. An SCI rat model was induced in vivo using a revised Allen's method. The Basso, Beattie, and Bresnahan (BBB) scores and histological analysis demonstrated that OECs, which were treated with ginsenoside Rg1, exhibited significant improvement in SCI compared with both the control group and the OEC group. Thus, ginsenoside Rg1 may represent a novel treatment target for SCI.

Urinary vimentin mRNA as a potential novel biomarker of renal fibrosis.

Renal fibrosis is a histological outcome of chronic kidney disease (CKD) progression. However, the noninvasive detection of renal fibrosis remains a challenge. Here we constructed a renal fibrosis target mRNA array and used it to detect urinary mRNAs of CKD patients for investigating potential noninvasive biomarkers of renal fibrosis. We collected urine samples from 39 biopsy-proven CKD patients and 11 healthy controls in the training set. Urinary mRNA profiles of 86 genes showed a total of 21 mRNAs that were differentially expressed between CKD patients and controls (P < 0.05), and vimentin (VIM) mRNA demonstrated the highest change fold of 9.99 in CKD vs. controls with robust correlations with decline of renal function and severity of tubulointerstitial fibrosis. Additionally, VIM mRNA further differentiated patients with moderate-to-severe fibrosis from none-to-mild fibrosis group with an area of the curve of 0.796 (P = 0.008). A verification of VIM mRNA in the urine of an additional 96 patients and 20 controls showed that VIM is not only well correlated with renal function parameters but also correlated with proteinuria and renal fibrosis scores. Multiple logistic regression and receiver-operating characteristics analysis further showed that urine VIM mRNA is the best predictive parameter of renal fibrosis compared with estimated glomerular filtration rate, serum creatinine, and blood urea nitrogen. In addition, there is no improved predictive performance for the composite biomarkers to predict renal fibrosis severity compared with a single gene of VIM. Overall, urinary VIM mRNA might serve as a novel independent noninvasive biomarker to monitor the progression of kidney fibrosis.

Engineering of Ralstonia eutropha for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose.

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with Ralstonia eutropha relies on the addition of propionate during fermentation, and propionate consumption is one of the major factors affecting the cost of PHBV production. In this study, 7 strains were obtained by genetic manipulating the methylcitric acid cycle and the methylmalonyl-CoA pathway in R. eutropha. Disruption of prpC1 and prpC2 genes did not affect cell growth and PHBV accumulation. All 7 strains were able to accumulation high amounts of PHBVs with 3HV fractions of 0.41-29.1 mol% during cultivation in flasks. Fermentation in 7.5-L fermenter showed that genetically engineered Rem-8 was able to yield biomass of 132.8 CDWg/L, of which 68.6% were PHBV with 3HV fraction of 26.0 mol% in the biopolymer, indicating promising potentials of commercialization in the future.

Elevation of B-type natriuretic peptide is a sensitive marker of left ventricular diastolic dysfunction in patients with maintenance haemodialysis.

OBJECTIVE: To determine the clinical value of B-type natriuretic peptide (BNP) in diagnosing left ventricular diastolic dysfunction (LVDD) associated with maintenance haemodialysis (MHD) population. METHODS: Plasma BNP was determined in 59 MHD patients with normal ejection fraction. The ratio of early to late annular velocity (E'/A') was determined by tissue Doppler imaging as a parameter of diastolic function. RESULTS: LVDD occurred in 66% of the patients. Receiver-operating characteristic curve analyses identified a cut-off of 353.6 pg ml(-1) as the one with the highest sensitivity and specificity for detecting LVDD. CONCLUSIONS: Plasma BNP may serve as a potential biomarker in diagnosing LVDD in MHD patients with normal systolic function.

[Construction of Corynebacterium glutamicum/E. coli shuttle promoter-probe vector].

Based on the replication origins of the C. glutamicum pXZ10145 and the Escherichia coli ColE1 plasmid, a novel Corynebacterium glutamicum/Escherichia coli shuttle vector pAK6 was constructed. This vector was able to replicate in C. glutamicum and E. coli. Plasmid pAK6 carried multiple cloning site useful for gene cloning, kanamysin- and ampicillin-resistance-encoding gene. Furtherly based on the shuttle vector pAK6, a promoter-probe vector was developed for the isolation of promoter elements from C. glutamicum . This vector carried the promoterless chloramphenicol acetyltranstersae (CAT) gene as a reporter downstream from useful cloning site. For testing this promoter-probe vector, C. glutamicum genomic DNA was digested to completion with Sau3AI and the fragments shot-gun cloned into its unique Bgl II. Two fragments exhibiting promoter activity were isolated. By measuring CAT activity, the strength of promoter fragments was assayed. After being sequenced, promoter sequences were predicted by using BDGP Neural Network Promoter Prediction V2.2 and the similarities to the regions of the consensus promoter sequence or the known promoters were confirmed.

[Cloning, sequence analysis and expression of anthranilate synthetase gene in Corynebacterium pekinense].

Anthranilate synthetase (EC4.1.3.27;AS) genes from wild-type Corynebacterium pekinense AS1.299 and its mutant PD-67 were cloned and sequenced. Analysis of PCR fragments revealed that three ORFs existed, which corresponded to trpL, trpE and trpG gene, respectively. Six bases changes that resulted in the changes of five amino acids were found in the trpE structural gene of C. pekinense PD-67 and a single-base change that resulted in an amino acid substitution was found in the trpG structural gene of C. pekinense PD-67.A homology comparison revealed that C. pekinense AS1.299 was closely related to Corynebacterim glutamicum ATCC 13032 and Brevibacterium lactofermentum. An internal promoter was found in the upstream of the trpL gene from C. pekinense and it functioned in E. coli, but a single-base exchange (A to G) existed in the-35 box of PD-67. The trpEG genes from the wild-type strain and its mutant were expressed both in C. pekinense AS1.299 and PD-67, and the specific enzyme activities of transformed C. pekinense were much higher than that of the parental strains. The amplification of the activity of AS yielded 22.39% increase of L-tryptophan production, but the cell growth became slower than PD-67.

[Cloning, sequence analysis and expression of alanine racemase gene in Pseudomonas putida].

Two distinct alanine racemase genes from Pseudomonas putida 200 were cloned and sequenced. DadX encodes a peptide of 357 amino acids with a calculated molecular weight of 38.82kDa. The putative product of alr gene is a peptide of 409 amino acids with molecular weight of 44.182kDa. A homology comparison revealed identities of 96.64%, 71.99%, 44.88% and 47.37% of the DadX alanine racemase to those from P. putida KT2440, Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli, respectively. The amino acids sequence deduced from alr gene showed the homologies of 94.38%, 22.89%, 25.72% and 26.44% to those from the microorganisms above, respectively. Two motifs believed essential to the enzyme activity are found both in DadX and Alr, such as pyridoxal-5'-phosphate binding site. Both dadX and alr were expressed in E. coli TG1. Neither alanine racemase activity or serine racemase activity was detected in the host strain. Only alanine racemase activity was found in E. coli TG1/pCTD. But both E. coli TG1/pCTA and TG1/pCBA exhibit activity toward L-alanine and L-serine. Transcription of alr gene in E. coli is independent from extraneous promoter, a result confirmed by the significant enzyme activity observed in the E. coli TG1/pCBA, which indicates the presence of a possible promoter upstream the structure gene.

[Cloning, sequence analysis and expression of N-acetylglutamate kinase gene in Corynebacterium crenatum].

N-Acetylglutamate kinase (EC 2.7.2.8;NAGK) genes from wild-type Corynebacterium crenatum AS 1.542 and a L-arginine-producing mutant C. crenatum 971.1 were cloned and sequenced. Analysis of argB sequences revealed that only one ORF existed, which used ATG as the initiation codon and coded a peptide of 317 amino acids with a calculated molecular weight of 33.6kDa. Only one nucleotide difference was found in the structure gene and the difference did not cause a change of amino acid by comparison of the gene sequences between the wild type C. crenatum AS 1.542 and the mutant 971.1. The ORF sequence of argB from C. crenatum AS 1.542 showed homologies of 99.89%, 76.62%, 37.94% to those from Corynebacterium glutamicum ATCC 13032, Corynebacterium efficient YS-314 and Escherichia coli k12. And the amino acid sequence deduced from ORF displayed homologies of 100%, 78.55%, 25.25% to those from microorganisms above, respectively. An internal promoter was found in the upstream of the argB gene from C. crenatum. The argB gene from C. crenatum AS 1.542 was expressed both in C. crenatum AS 1.542 and 971.1. The NAGK activity of transformed C. crenatum AS 1.542 was greatly increased by the induction of IPTG. The NAGK activity of transformed C. crenatum 971.1 was almost twice as much as that of C. crenatum 971.1 under the same induction. The amplification of the NAGK activity yielded 25% increase of L-arginine production in C. crenatum 971.1.

[Expression of feedback-resistant aspartate kinase gene in Corynebacterium crenatum].

The AEC-resistant aspartate kinase gene from C. crenatum CD945 was cloned into vector pJC1. Its expression was investigated both in the wild type C. crenatum AS1.542 and its mutant C. crenatum CD945. The result showed that C. crenatum AS1.542 harboring AK(fbr) gene could grow on the defined medium with the co-existence of 12 mg/mL both of AEC and L-threonine respectively. Overexpression of AK(fbr) gene in C. crenatum CD945 results in a 4-fold increase of specific enzyme activity than the parental strain. The amplification of the activity of aspartate kinase yields 22% increase of L-lysine production and 23% increase of L-lysine productivity without affecting the growth rate.