PEG modification increases thermostability and inhibitor resistance of Bst DNA polymerase.

Polyethylene glycol modification (PEGylation) is a widely used strategy to improve the physicochemical properties of various macromolecules, especially protein drugs. However, its application in enhancing the performance of enzymes for molecular biology remains underexplored. This study explored the PEGylation of Bst DNA polymerase, determining optimal modification reaction conditions. In comparison to the unmodified wild-type counterpart, the modified Bst DNA polymerase exhibited significantly improved activity, thermal stability, and inhibitor tolerance during loop-mediated isothermal amplification. When applied for the detection of Salmonella in crude samples, the modified enzyme demonstrated a notably accelerated reaction rate. Therefore, PEGylation emerges as a viable strategy for refining DNA polymerases, helping in the development of novel molecular diagnostic reagents.

Improvement of duplex-specific nuclease salt tolerance by fusing DNA-binding domain of DNase from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix.

Salt tolerance is an important property of duplex-specific nuclease (DSN). DSN with high salt tolerance can be more widely used in genetic engineering, especially in the production of nucleic acid drugs. To improve the salt tolerance of DSN, we selected five DNA-binding domains from extremophilic organisms, which have been shown the ability to improve salt tolerance of DNA polymerases and nucleases. The experimental results demonstrated that the fusion protein TK-DSN produced by fusing a N-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs domain from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix, has a significantly improved salt tolerance. TK-DSN can tolerate the concentration of NaCl up to 800 mM; in addition, the ability of digesting DNA was also enhanced during in vitro transcription and RNA purification. This strategy provides the method for the personalized customization of biological tool enzymes for different applications.

B-class MADS-box TM6 is a candidate gene for tomato male sterile-1526.

KEY MESSAGE: Tomato male sterile-1526 locus was fine-mapped to an interval of 44.6 kb, and a B-class MADS-box gene TM6 was identified as the candidate gene. Male sterile lines have been widely used for hybrid seed production in many crop plants. The tomato male sterile-1526 (ms-1526) mutant displays abnormal stamens and exerted stigmas and is suitable for practical use. In this study, the ms-1526 locus was fine-mapped to a 44.6 kb interval that contained four putative genes. Thereinto, Solyc02g084630 encodes tomato B-class MADS-box gene TM6 (syn. TDR6), which plays an important role in stamen development. Sequencing revealed that there was a 12.7 kb deletion in the ms-1526 region, where the promoter and first four exons of the TM6 gene were absent. ms-1547, an allele of ms-1526, also contained the same deletion in the TM6 gene. And the other allele ms-15 mutant contained a single-nucleotide polymorphism (SNP, C to A) in the coding region of the TM6 gene, which led to a missense mutation (G to W). The codominant insertion/deletion (InDel) marker MS26D and codominant derived cleaved amplified polymorphic sequence (dCAPS) marker MS15C were developed based on the deletion and SNP, respectively. A real-time quantitative reverse-transcription PCR showed that expression of the TM6 gene was barely detectable in the flowers of the ms-1526 and ms-1547 mutants. In addition, other floral organ identity genes, pollen development marker genes, and pistil marker genes were differentially expressed between wild type and mutant flowers. These findings may facilitate functional analysis of the TM6 gene and help in the marker-assisted selection of ms-15 and its alleles in tomato breeding.

[Effects of Wenhua Juanbi Recipe on the Expressions of DNA Methyltransferases in Peripheral Blood Mononuclear Cells of Collagen-Inducing Arthritis Rats].

Objective To observe the effect of Wenhua Juanbi Recipe (WJR) on the expres- sions of DNA methyltransferases (DNMTs) in peripheral blood mononuclear cells (PBMCs) of collagen- inducing arthritis (CIA) , and to study its mechanism for treating CIA. Methods Totally 90 Wistar rats were randomly divided into the model group (n =80) and the normal control group (n = 10). Rats of the model group were injected with type II collagen of bovine (BC II) emulsion from the tail to establish CIA model. Successfully modeled 50 CIA rats were randomly divided into five groups, i.e., the model group, the methotrexate (MTX) group, the low dose WJR group, the middle dose WJR group, the high dose WJR group, 10 in each group. Rats in the model group were administered with normal saline by gastrogavage, once per day. Rats in low, middle, and high dose WJR groups were administered with WJR by gas- trogavage at the daily dose of 22. 9, 45. 8, 68. 7 g/kg, respectively (once per day). Rats in the MTX group were administered with MTX suspension (0.78 mg/kg) by gastrogavage, once per week for 30 successive days. The paw swelling was evaluated using volume method (draining volume). PBMCs were extrac- ted from each group after intervention. mRNA expression levels of DNMTs (DNMT1 , DNMT3a, DNMT3b) were detected by real-time quantitative PCR. Results Compared with the normal group, the paws were obviously swollen in the model group (P <0. 01). Compared with the model group, swollen paws were obviously alleviated in low, middle, and high dose WJR groups, and the MTX group (P <0.01). Compared with before treatment in the same group, swollen paws were obviously alleviated in low, middle, and high dose WJR groups, and the MTX group (P <0. 01 ). Compared with the normal group, expression levels of DNMT1, DNMT3a, and DNMT3b in PBMCs were obviously lowered in the model group (P <0.01). Compared with the model group, expression levels of DNMT1 , DNMT3a, DNMT3b in PBMCs were obviously elevated in low, middle, and high dose WJR groups, and the MTX group (all P <0. 01). There was no sig- nificant difference in expression levels of DNMT1, DNMT3a, or DNMT3b in PBMCs among low, middle, and high dose WJR groups (P>0.05). Conclusions Expression levels of DNMTs in PBMCs of CIA rats decreased. WJR up-regulated the expression level of DNMTs in PBMCs of CIA rats in no obvious dose de- pendent way. One of WJR's mechanisms for treating CIA might be up-regulating expression levels of DN- MTs, and adjusting the state of DNA methylation.

DHX33 mediates p53 to regulate mevalonate pathway gene transcription in human cancers.

Tumor suppressor p53 is frequently null or mutated in human cancers. Here in this study, DHX33 protein was found to be induced in p53 null cells in vitro, and in p53 mutant lung tumorigenesis in vivo. Cholesterol metabolism through mevalonate pathway is pivotal for cell proliferation and is frequently altered in human cancers. Mice carrying mutant p53 and KrasG12D alleles showed upregulation of mevalonate pathway gene expression. However upon DHX33 loss, their upregulation was significantly debilitated. Additionally, in many human cancer cells, DHX33 knockdown caused inhibition of mavelonate pathway gene transcription. We propose DHX33 locates downstream of mutant p53 and Ras to regulate mevalonate pathway gene transcription and thereby supports cancer development in vivo.

Effects of the periodic fasting-mimicking diet on health, lifespan, and multiple diseases: a narrative review and clinical implications.

Dietary restriction and fasting have been recognized for their beneficial effects on health and lifespan and their potential application in managing chronic metabolic diseases. However, long-term adherence to strict dietary restrictions and prolonged fasting poses challenges for most individuals and may lead to unhealthy rebound eating habits, negatively affecting overall health. As a result, a periodic fasting-mimicking diet (PFMD), involving cycles of fasting for 2 or more days while ensuring basic nutritional needs are met within a restricted caloric intake, has gained widespread acceptance. Current research indicates that a PFMD can promote stem cell regeneration, suppress inflammation, extend the health span of rodents, and improve metabolic health, among other effects. In various disease populations such as patients with diabetes, cancer, multiple sclerosis, and Alzheimer's disease, a PFMD has shown efficacy in alleviating disease symptoms and improving relevant markers. After conducting an extensive analysis of available research on the PFMD, it is evident that its advantages and potential applications are comparable to other fasting methods. Consequently, it is proposed in this review that a PFMD has the potential to fully replace water-only or very-low-energy fasting regimens and holds promise for application across multiple diseases.

Fine Mapping of fw6.3, a Major-Effect Quantitative Trait Locus That Controls Fruit Weight in Tomato.

Tomato (Solanum lycopersicum) is a widely consumed vegetable, and the tomato fruit weight is a key yield component. Many quantitative trait loci (QTLs) controlling tomato fruit weight have been identified, and six of them have been fine-mapped and cloned. Here, four loci controlling tomato fruit weight were identified in an F2 population through QTL seq.; fruit weight 6.3 (fw6.3) was a major-effect QTL and its percentage of variation explanation (R2) was 0.118. This QTL was fine-mapped to a 62.6 kb interval on chromosome 6. According to the annotated tomato genome (version SL4.0, annotation ITAG4.0), this interval contained seven genes, including Solyc06g074350 (the SELF-PRUNING gene), which was likely the candidate gene underlying variation in fruit weight. The SELF-PRUNING gene contained a single-nucleotide polymorphism that resulted in an amino acid substitution in the protein sequence. The large-fruit allele of fw6.3 (fw6.3HG) was overdominant to the small-fruit allele fw6.3RG. The soluble solids content was also increased by fw6.3HG. These findings provide valuable information that will aid the cloning of the FW6.3 gene and ongoing efforts to breed tomato plants with higher yield and quality via molecular marker-assisted selection.

Analysis of the effect of NEKs on the prognosis of patients with non-small-cell lung carcinoma based on bioinformatics.

NEKs are proteins that are involved in various cell processes and play important roles in the formation and development of cancer. However, few studies have examined the role of NEKs in the development of non-small-cell lung carcinoma (NSCLC). To address this problem, the Oncomine, UALCAN, and the Human Protein Atlas databases were used to analyze differential NEK expression and its clinicopathological parameters, while the Kaplan-Meier, cBioPortal, GEPIA, and DAVID databases were used to analyze survival, gene mutations, similar genes, and biological enrichments. The rate of NEK family gene mutation was high (> 50%) in patients with NSCLC, in which NEK2/4/6/8/ was overexpressed and significantly correlated with tumor stage and nodal metastasis status. In addition, the high expression of NEK2/3mRNA was significantly associated with poor prognosis in patients with NSCLC, while high expression of NEK1/4/6/7/8/9/10/11mRNA was associated with good prognosis. In summary, these results suggest that NEK2/4/6/8 may be a potential prognostic biomarker for the survival of patients with NSCLC.

Berberine attenuates choline-induced atherosclerosis by inhibiting trimethylamine and trimethylamine-N-oxide production via manipulating the gut microbiome.

Trimethylamine-N-oxide (TMAO), a derivative from the gut microbiota metabolite trimethylamine (TMA), has been identified to be an independent risk factor for promoting atherosclerosis. Evidences suggest that berberine (BBR) could be used to treat obesity, diabetes and atherosclerosis, however, its mechanism is not clear mainly because of its poor oral bioavailability. Here, we show that BBR attenuated TMA/TMAO production in the C57BL/6J and ApoE KO mice fed with choline-supplemented chow diet, and mitigated atherosclerotic lesion areas in ApoE KO mice. Inhibition of TMA/TMAO production by BBR-modulated gut microbiota was proved by a single-dose administration of d9-choline in vivo. Metagenomic analysis of cecal contents demonstrated that BBR altered gut microbiota composition, microbiome functionality, and cutC/cntA gene abundance. Furthermore, BBR was shown to inhibit choline-to-TMA conversion in TMA-producing bacteria in vitro and in gut microbial consortium from fecal samples of choline-fed mice and human volunteers, and the result was confirmed by transplantation of TMA-producing bacteria in mice. These results offer new insights into the mechanisms responsible for the anti-atherosclerosis effects of BBR, which inhibits commensal microbial TMA production via gut microbiota remodeling.

A small-molecule inhibitor of PCSK9 transcription ameliorates atherosclerosis through the modulation of FoxO1/3 and HNF1α.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that down-regulates hepatic low-density lipoprotein receptor (LDLR) by binding and shuttling LDLR to lysosomes for degradation. The development of therapy that inhibits PCSK9 has attracted considerable attention for the management of cardiovascular disease risk. However, only monoclonal antibodies of PCSK9 have reached the clinic use. Oral administration of small-molecule transcriptional inhibitors has the potential to become a therapeutic option. METHODS: Here, we developed a cell-based small molecule screening platform to identify transcriptional inhibitors of PCSK9. Through high-throughput screening and a series of evaluation, we found several active compounds. After detailed investigation on the pharmacological effect and molecular mechanistic characterization, 7030B-C5 was identified as a potential small-molecule PCSK9 inhibitor. FINDINGS: Our data showed that 7030B-C5 down-regulated PCSK9 expression and increased the total cellular LDLR protein and its mediated LDL-C uptake by HepG2 cells. In both C57BL/6 J and ApoE KO mice, oral administration of 7030B-C5 reduced hepatic and plasma PCSK9 level and increased hepatic LDLR expression. Most importantly, 7030B-C5 inhibited lesions in en face aortas and aortic root in ApoE KO mice with a slight amelioration of lipid profiles. We further provide evidences suggesting that transcriptional regulation of PCSK9 by 7030B-C5 mostly depend on the transcriptional factor HNF1α and FoxO3. Furthermore, FoxO1 was found to play an important role in 7030B-C5 mediated integration of hepatic glucose and lipid metabolism. INTERPRETATION: 7030B-C5 with potential suppressive effect of PCSK9 expression may serve as a promising lead compound for drug development of cholesterol/glucose homeostasis and cardiovascular disease therapy. FUND: This work was supported by grants from the National Natural Science Foundation of China (81473214, 81402929, and 81621064), the Drug Innovation Major Project of China (2018ZX09711001-003-006, 2018ZX09711001-007 and 2018ZX09735001-002), CAMS Innovation Fund for Medical Sciences (2016-I2M-2-002, 2016-I2M-1-011 and 2017-I2M-1-008), Beijing Natural Science Foundation (7162129).

SlGID1a Is a Putative Candidate Gene for qtph1.1, a Major-Effect Quantitative Trait Locus Controlling Tomato Plant Height.

Plant height is an important agronomic trait in crops. Several genes underlying tomato (Solanum lycopersicum) plant height mutants have been cloned. However, few quantitative trait genes for plant height have been identified in tomato. In this study, seven quantitative trait loci (QTLs) controlling plant height were identified in tomato. Of which, qtph1.1 (QTL for tomato plant height 1.1), qtph3.1 and qtph12.1 were major QTLs and explained 15, 16, and 12% of phenotypic variation (R2), respectively. The qtph1.1 was further mapped to an 18.9-kb interval on chromosome 1. Based on the annotated tomato genome (version SL2.50, annotation ITAG2.40), Solyc01g098390 encoding GA receptor SlGID1a was the putative candidate gene. The SlGID1a gene underlying the qtph1.1 locus contained a single nucleotide polymorphism (SNP) that resulted in an amino acid alteration in protein sequence. The near-isogenic line containing the qtph1.1 locus (NIL-qtph1.1) exhibited shorter internode length and cell length than the wild type (NIL-WT). The dwarf phenotype of NIL-qtph1.1 could not be rescued by exogenous GA3 treatment. Transcriptome analysis and real-time quantitative reverse transcription PCR (qPCR) showed that several genes related to biosynthesis and signaling of GA and auxin were differentially expressed in stems between NIL-qtph1.1 and NIL-WT. These findings might pave the road for understanding the molecular regulation mechanism of tomato plant height.

A Physic Nut Stress-Responsive HD-Zip Transcription Factor, JcHDZ07, Confers Enhanced Sensitivity to Salinity Stress in Transgenic Arabidopsis.

Homeodomain-leucine zipper (HD-Zip) transcription factors are reported to play crucial roles in the growth, development, and stress responses of plants. However, there is little knowledge of the molecular mechanisms involved in physic nut's stress tolerance generally, or the functions of its HD-Zip genes. In the present study, a HD-Zip family transcription factor, designated JcHDZ07, was isolated from physic nut. Expression profile analysis showed that salinity stress inhibited the expression of JcHDZ07. Transient expression of JcHDZ07-YFP in Arabidopsis protoplast cells revealed that JcHDZ07 was a nuclear-localized protein. Additionally, no obvious difference in growth and development between wild-type and JcHDZ07-overexpressing plants was observed in the absence of stress. Our results further indicated that JcHDZ07 overexpressing transgenic plants had lower proline contents, lower survival rates, and activities of catalase and superoxide dismutase, but higher relative electrical leakage and malonaldehyde contents compared with wild-type plants under salinity stress conditions, suggesting that overexpression of JcHDZ07 confers enhanced sensitivity to salinity stress in transgenic Arabidopsis. Expression of salt stress-responsive genes were upregulated in leaves of transgenic plants under salinity stress, but less strongly than in wild-type plants. Collectively, our results suggest that JcHDZ07 functions as an important regulator during the process of plant responses to salinity stress.

A putative bHLH transcription factor is a candidate gene for male sterile 32, a locus affecting pollen and tapetum development in tomato.

The tomato (Solanum lycopersicum) male sterile 32 (ms32) mutant has been used in hybrid seed breeding programs largely because it produces no pollen and has exserted stigmas. In this study, histological examination of anthers revealed dysfunctional pollen and tapetum development in the ms32 mutant. The ms32 locus was fine mapped to a 28.5 kb interval that encoded four putative genes. Solyc01g081100, a homolog of Arabidopsis bHLH10/89/90 and rice EAT1, was proposed to be the candidate gene of MS32 because it contained a single nucleotide polymorphism (SNP) that led to the formation of a premature stop codon. A codominant derived cleaved amplified polymorphic sequence (dCAPS) marker, MS32D, was developed based on the SNP. Real-time quantitative reverse-transcription PCR showed that most of the genes, which were proposed to be involved in pollen and tapetum development in tomato, were downregulated in the ms32 mutant. These findings may aid in marker-assisted selection of ms32 in hybrid breeding programs and facilitate studies on the regulatory mechanisms of pollen and tapetum development in tomato.