Association of KRAS, NRAS, BRAF and PIK3CA gene mutations with clinicopathological features, prognosis and ring finger protein 215 expression in patients with colorectal cancer.

The relationships of KRAS, NRAS, BRAF and PIK3CA gene mutations with the clinicopathological features and prognosis of colorectal cancer (CRC) in patient are lacking. Furthermore, the role of ring finger protein 215 (RNF215) in CRC patients with KRAS, NRAS, BRAF and PIK3CA mutations remains unclear. In the present study, 182 surgical resection specimens from patients with primary CRC for retrospective analysis, were collected. KRAS/NRAS/BRAF/PIK3CA gene mutations were confirmed by an amplification-refractory mutation system. Immunohistochemistry (IHC) was conducted to confirm KRAS, NRAS, BRAF and PIK3CA protein expression. RNF215 expression in patients with CRC was evaluated using TIMER 2.0 database and IHC. The individual mutation rates of KRAS, NRAS, BRAF and PIK3CA were 40.7% (74/182), 4.4% (8/182), 4.4% (8/182) and 3.3% (6/182), respectively. The KRAS exon 2 mutation rate was the highest (61.5%, 64/104), and these mutations mainly occurred at codons 12 and 13. KRAS/NRAS/BRAF/PIK3CA wild-type CRC patients had significantly longer overall survival and disease-free survival than mutated KRAS/NRAS/BRAF/PIK3CA CRC patients (P<0.05). Overall, 45.4% (5/11) of patients with PIK3CA mutations had concomitant KRAS mutations. The KRAS/NRAS/BRAF/PIK3CA gene mutation rate in patients with lymph node metastasis (76.1%, 35/46) was significantly higher than that in patients without lymph node metastasis (50.8%, 69/136) (P=0.0027). There were no significant differences in IHC expression between patients with and without KRAS, NRAS, BRAF and PIK3CA mutations (P>0.05). The TIMER 2.0 analysis showed that RNF215 expression was significantly higher in the mutated BRAF group than in the wild-type BRAF group in CRC (P<0.05). In conclusion, KRAS is the most commonly mutated gene, and KRAS mutations may be a poor prognostic factor for patients with CRC. KRAS wild-type patient resistance may be related to PIK3CA gene mutations, although this needs further verification in larger cohorts. BRAF mutations may be associated with RNF215 expression in patients with CRC.

Molecular epidemiology and recombination of enterovirus D68 in China.

Enterovirus D68 (EV-D68), a member of Enterovirus genus of the Picornaviridae family, mainly causes respiratory system-related diseases as well as neurological complications in some patients. At present, there is no effective vaccine or treatment for the virus. The aim of this research was to systematically analyse the molecular epidemiology, recombination and changes in the epitope of EV-D68 in China from 2008 to 2022. Through phylogenetic analysis based on VP1 sequences, it was found that there was limited information about EV-D68 infection before 2011 and that EV-D68 infection was dominated by the A2 gene subtype from 2011 to 2013 and the B3 genotype from 2014 to 2018, during which A2 and B3 were coprevalent and alternately prevalent. We also constructed a phylogenetic tree using the EV-D68 full-length genome sequences, and the genotype of each sequence was consistent with that of the VP1 sequence evolutionary tree. Recombination analysis showed that MH341715 underwent intertypic recombination with the A2 genotype MH341729 at the 5' untranslated region (5'UTR) and that P1-P3 underwent recombination with the B3 genotype MH341712. The capsid protein VP1 is one of the most important structural proteins. In VP1, the BC-loop (89-105 amino acids) and DE-loop (140-152 amino acids) are the most variable domains on the surface of the virus and are associated with epitopes. In this study, it was found that the dominant amino acid composition of the BC-loop and DE-loop continued to change with the epidemic of the virus; the amino acid composition also differed in different regions of the same genotypes. The ongoing genomic and molecular epidemiology of EV-D68 remains important for predicting emergence of new viruses and preventing major outbreaks of respiratory diseases.

Molecular mechanism and research progress on pharmacology of ferulic acid in liver diseases.

Ferulic acid (FA) is a natural polyphenol, a derivative of cinnamic acid, widely found in Angelica, Chuanxiong and other fruits, vegetables and traditional Chinese medicine. FA contains methoxy, 4-hydroxy and carboxylic acid functional groups that bind covalently to neighbouring adjacent unsaturated Cationic C and play a key role in many diseases related to oxidative stress. Numerous studies have shown that ferulic acid protects liver cells and inhibits liver injury, liver fibrosis, hepatotoxicity and hepatocyte apoptosis caused by various factors. FA has protective effects on liver injury induced by acetaminophen, methotrexate, antituberculosis drugs, diosbulbin B and tripterygium wilfordii, mainly through the signal pathways related to TLR4/NF-κB and Keap1/Nrf2. FA also has protective effects on carbon tetrachloride, concanavalin A and septic liver injury. FA pretreatment can protect hepatocytes from radiation damage, protects the liver from damage caused by fluoride, cadmium and aflatoxin b1. At the same time, FA can inhibit liver fibrosis, inhibit liver steatosis and reduce lipid toxicity, improve insulin resistance in the liver and exert the effect of anti-liver cancer. In addition, signalling pathways such as Akt/FoxO1, AMPK, PPAR γ, Smad2/3 and Caspase-3 have been shown to be vital molecular targets for FA involvement in improving various liver diseases. Recent advances in the pharmacological effects of ferulic acid and its derivatives on liver diseases were reviewed. The results will provide guidance for the clinical application of ferulic acid and its derivatives in the treatment of liver diseases.

Interannual variations of Sargassum blooms in the Yellow Sea and East China Sea during 2017-2021.

Golden tide, caused by Sargassum horneri, is becoming another periodic and trans-regional harmful macroalgal bloom in the Yellow Sea (YS) and East China Sea (ECS) other than the green tide. In this study, we employed high-resolution remote sensing, field validations, and population genetics to investigate the spatiotemporal development pattern of Sargassum blooms during the years 2017 to 2021 and explore the potential environmental factors that influence them. Sporadic floating Sargassum rafts could be detected in the middle or northern YS during autumn and the distribution area then occurred sequentially along the Chinese and/or western Korean coastlines. The floating biomass amplified significantly in early spring, reached its maximum in two to three months with an evident northward expansion, and then declined rapidly in May or June. The scale of the spring bloom was much larger than the winter one in terms of coverage, suggesting an additional local source in ECS. The blooms were mostly confined to waters with a sea surface temperature range of 10-16℃, while the drifting pathways were consistent with the prevailing wind trajectory and surface currents. The floating S. horneri populations exhibited a homogenous and conservative genetic structure among years. Our findings underscore the year-round cycle of golden tides, the impact of physical hydrological environments on the drifting and blooming of pelagic S. horneri, and provide insights for monitoring and forecasting this emerging marine ecological disaster.

Exploring lemology teaching with "internet plus" flipped classroom pedagogy.

BACKGROUND: To investigate the use of flipped classroom pedagogy based on "Internet plus" in teaching viral hepatitis in the lemology course during the COVID-19 epidemic. METHODS: This study included students from the clinical medicine general practitioner class at Nanjing Medical University's Kangda College, with the observation group consisting of 67 students from the 2020-2021 school year and the control group consisting of 70 students from the 2019-2020 school year. The observation group used "Internet plus" flipped classroom pedagogy, while the control group used conventional offline instruction. The theory course and case analysis ability scores from the two groups were compared and analyzed, and questionnaire surveys were administered to the observation group. RESULT: After the flipped classroom, the observation group had significantly higher theoretical test scores (38.62 ± 4.52) and case analysis ability scores (21.08 ± 3.58) than the control group (37.37 ± 2.43) (t = 2.024, P = 0.045) and (19.16 ± 1.15) (t = 4.254, P < 0.001), respectively. The questionnaire survey in the observation group revealed that the "Internet plus" flipped classroom pedagogy approach can help enhance students' enthusiasm to learn, clinical thinking ability, practical application ability, and learning efficiency, with satisfaction rates of 81.7%, 85.0%, 83.3%, and 78.8%, respectively; 89.4% of students expressed hope that whenever physical classes resumed, the offline courses could be combined with this pedagogy approach. CONCLUSION: The use of the "Internet plus" flipped classroom pedagogy technique for teaching viral hepatitis in a lemology course boosted students' theory learning ability as well as their case analysis ability. The majority of students were pleased with this type of instruction and hoped that whenever physical classes resumed, the offline courses may be integrated with the "Internet plus" flipped classroom pedagogical approach.

Seroprevalence of coxsackievirus A6 and enterovirus A71 infection in humans: a systematic review and meta-analysis.

Hand, foot, and mouth disease (HFMD) is a common infectious disease in children. Enterovirus A71 (EV-A71) is one of the main pathogens, and coxsackievirus A6 (CVA6) has gradually become the dominant pathogen of HFMD in recent years. This study was conducted mainly to assess the serological prevalence of EV-A71 and CVA6 antibodies in people of different ages, sexes, and regions through a systematic review and meta-analysis. A comprehensive study was performed based on the EV-A71 and CVA6 serological literature published before May 2022. Heterogeneity analysis (Cochrane's Q test and the I2 statistic) and random effect models were adopted. Subgroup and meta-regression analyses were used to identify potential sources of heterogeneity in the data, and all analysis was performed using STATA version 16.0. This study included 71 studies involving 55,176 people from 13 countries that met the inclusion criteria. The serological prevalence of EV-A71 antibody in different studies was 4.31-88.8%, and that of CVA6 antibody was 40.8-80.9%. Meta-analysis results showed that the serum positive rate for EV-A71 antibody was 45.9% (95% CI: 37.6-54.1%). The rate in the Chinese population was 47.8% (95% CI: 42.4-53.2%), and in the other countries, it was 38% (95% CI: 23-55%). The serum positive rate for CVA6 antibody was 58.3% (95% CI: 46.5-70.2%). The rate in the Chinese population was 49.1% (95% CI: 38.3-59.9%), and in the other countries, it was 68% (95% CI: 51-83%). Subgroup analysis was also conducted. The seroprevalence of EV-A71 and CVA6 antibodies is related to age rather than gender or region. The rates of EV-A71 and CVA6 seropositivity are considerably lower in children younger than five years of age. However, the rates gradually increase with age. The findings of this study suggest that children under five years of age may be susceptible to EV-A71 and CVA6. Thus, safety education and vaccination should be strengthened accordingly. This study provides a basis for understanding the risk factors for EV-A71 and CVA6 infection in China and for deciding how to formulate standard preventive measures to prevent the spread of the virus.

A Novel Antihypertensive Pentapeptide Identified in Quinoa Bran Globulin Hydrolysates: Purification, In Silico Characterization, Molecular Docking with ACE and Stability against Different Food-Processing Conditions.

The addition of food derived antihypertensive peptides to the diet is considered a reasonable way to prevent and lower blood pressure. However, data about stability of antihypertensive peptides against different food-processing conditions are limited. In this study, through Sephadex G-15 gel chromatography and RP-HPLC separation, UPLC-ESI-MS/MS analysis and in silico screening, a novel ACE-inhibitory pentapeptide Ser-Ala-Pro-Pro-Pro (IC50: 915.03 µmol/L) was identified in quinoa bran globulin hydrolysate. The inhibition patterns on angiotensin-I-converting enzyme and safety of SAPPP were studied using molecular docking and in silico predication, respectively. Results demonstrated that SAPPP could noncompetitively bind to active sites PRO519 and SER461 of ACE through short hydrogen bonds. SAPPP was resistant to different pH values (2.0-10.0), pasteurization conditions, addition of Na+, Mg2+, Fe3+ or K+, and the simulated gastrointestinal digestion. In contrast, SAPPP was unstable against heating at 100 °C for more than 50 min and the treatment of Zn2+ (5 mmol/L). These results indicated that peptides derived from quinoa globulin hydrolysates can be added into foods for antihypertension.

Improved Production of ε-Poly-L-Lysine in Streptomyces albulus Using Genome Shuffling and Its High-Yield Mechanism Analysis.

ε-Poly-L-lysine (ε-PL), a natural food preservative, has recently gained interest and mainly produced by Streptomyces albulus. Lacking of efficient breeding methods limit ε-PL production improving, knockout byproducts and increase of main product flux strategies as a logical solution to increase yield. However, removing byproduct formation and improving main product synthesis has seen limited success due to the genetic background of ε-PL producing organism is not clear. To overcome this limitation, random mutagenesis continues to be the best way towards improving strains for ε-PL production. Recent advances in Illumina sequencing opened new avenues to understand improved strains. In this work, we used genome shuffling on strains obtained by ribosome engineering to generate a better ε-PL producing strain. The mutant strain SG-86 produced 144.7% more ε-PL than the parent strain M-Z18. Except that SG-86 displayed obvious differences in morphology and ATP compared to parent strain M-Z18. Using Illumina sequencing, we mapped the genomic changes leading to the improved phenotype. Sequencing two strains showed that the genome of the mutant strain was about 2.1 M less than that of the parent strain, including a large number of metabolic pathways, secondary metabolic gene clusters, and gene deletions. In addition, there are many SNPs (single nucleotide polymorphisms) and InDels (insertions and deletions) in the mutant strain. Based on the results of data analysis, a mechanism of ε-PL overproduction in S. albulus SG-86 was preliminarily proposed. This study is of great significance for improving the fermentation performance and providing theoretical guidance for the metabolic engineering construction of ε-PL producing strains.

Ferulic acid exhibits anti-inflammatory effects by inducing autophagy and blocking NLRP3 inflammasome activation.

Background: Inflammation is involved in the healing process; however, when inflammation is overactivated, multiple diseases can occur. The continued discovery of new anti-inflammatory drugs is crucial in the treatment of inflammation-linked diseases. Objectives: Ferulic acid (FA), a precursor necessary for lignan synthesis, is widely distributed in plant-based whole foods and is a strong antioxidant. However, the effect of FA on the expression level of inflammatory factors in macrophages has not been fully clarified. The current study aimed to explore the anti-inflammatory effect and mechanism of ferulic acid. Results: The results showed that THP-1 cells were induced to differentiate into macrophages by Phorbol-12-myristate-13-acetate (PMA), and THP-1-derived macrophages were stimulated by LPS to establish an inflammatory cell model. Compared with the control group, low (5 µmol·mL-1), medium (10 µmol·mL-1), and high (20 µmol·mL-1) concentration ferulic acid groups have decreased cell viability and increased apoptosis rate in a dose-dependent manner. FA reduced the transcriptional levels of Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). Importantly, FA-induced autophagy and inhibited NLRP3 inflammasome activation. 3-MA (a widely used autophagy inhibitor) enhanced the secretion of TNF-α, IL-6 and IL-1ß. Moreover, autophagy inhibition by 3-MA resulted in increased proteins expression associated with NLRP3 inflammasome signaling pathway. Besides, the inhibition of inflammasome activation by MCC950 reduced the expression of TNF-α, IL-6 and IL-1ß. Conclusion: It is concluded that FA enhanced autophagy, inhibited the activation of NLRP3 inflammasome and reduced the expression and release of inflammatory factors.

A novel subgenotype C6 Enterovirus A71 originating from the recombination between subgenotypes C4 and C2 strains in mainland China.

Recombination plays important roles in the genetic diversity and evolution of Enterovirus A71 (EV-A71). The phylogenetics of EV-A71 in mainland China found that one strain DL71 formed a new subgenotype C6 with unknown origin. This study investigated the detailed genetic characteristics of the new variant. DL71 formed a distinct cluster within genotype C based on the genome and individual genes (5'UTR, VP4, VP1, 2A, 2B, 2C, 3D, and 3'UTR). The average genetic distances of the genome and individual genes (VP3, 2A, 2B, 2C, 3A, 3C, and 3D) between DL71 and reference strains were greater than 0.1. Nine recombination events involving smaller fragments along DL71 genome were detected. The strains Fuyang-0805a (C4) and Tainan/5746/98 (C2) were identified as the parental strains of DL71. In the non-recombination regions, DL71 had higher identities with Fuyang-0805a than Tainan/5746/98, and located in the cluster with C4 strains. However, in the recombination regions, DL71 had higher identities with Tainan/5746/98 than Fuyang-0805a, and located in the cluster with C2 strains. Thus, DL71 was a novel multiple inter-subgenotype recombinant derived from the dominant subgenotype C4 and the sporadic subgenotype C2 strains. Monitoring the emergence of new variants by the whole-genome sequencing remains essential for preventing disease outbreaks and developing new vaccines.

Monitoring the therapeutic efficacy of CA4P in the rabbit VX2 liver tumor using dynamic contrast-enhanced MRI.

PURPOSE: The present work aims to evaluate whether dynamic contrast-enhanced magnetic resonance Imaging (DCE-MRI) can monitor non-invasively the blocking effect on microvessels of the Combretastatin-A4-phosphate (CA4P) and assess the therapeutic efficacy. METHODS: Forty rabbits were implanted the VX2 tumors specimens. Two weeks later, serial MRI (T1 weighted image, T2 weighted image and DCE) were performed at 0 h, 4 h, 24 h, 3 d and 7 d after CA4P (10 mg/kg) or saline treatment. The parameters of DCE (Ktrans, Kep, Ve and iAUC60) of enhancement tumor portions were measured. Then all the tumor samples were stained to count microvessel density (MVD). At last, two-way repeated measures ANOVA was used to analyze the difference between and within groups. The correlation between the Ktrans, Kep, Ve, iAUC60 and MVD was analyzed by using the Pearson correlation analysis and Spearman's rank correlation. RESULTS: The Ktrans and iAUC60 in the CA4P group were lower than the values of the control group at 4 h after treatment, which have significant differences (D-value: -0.133 min-1, 95%CI: -0.169~-0.097 min-1，F = 59.109, p < 0.001 for Ktrans; D-value: -10.533 mmol/sec, 95%CI: -17.147~-3.919 mmol/sec，F = 11.110, and p = 0.003 for iAUC60). In the CA4P group, the Ktrans and iAUC60 reached the minimum values at 4 h. There were significant differences between 4 h and other different time points of the Ktrans and iAUC60 in the treatment group (all p < 0.01). The parameters Ktrans (r = 0.532, P = 0.016 and r = 0.681, P = 0.001, respectively) and iAUC60 (r = 0.580, P = 0.007 and r = 0.568, P = 0.009, respectively) of 7 days showed correlation with MVD in both groups, while Kep and Ve did not show correlation with MVD (P > 0.05). CONCLUSION: The blocking effect of microvessels after CA4P treatment can be evaluated by DCE-MRI, and the parameters of quantitative Ktrans and semi- quantitative iAUC60 can assess the change of the tumor angiogenesis noninvasively.

Molecular epidemiology and recombination of Enterovirus A71 in mainland China from 1987 to 2017.

Enterovirus A71 (EV-A71) is an important pathogen of severe hand, foot, and mouth disease (HFMD) in young children. This study aimed to retrospectively analyze the molecular epidemiology and recombination of EV-A71 in mainland China during 1987-2017. Phylogenetic tree showed that besides the previously reported subgenotypes A, B5, C0, C2, C3, and C4, a new subgenotype C6 emerged in mainland China. Recombination analysis indicated that C4 EV-A71 was derived from a common ancestor as a "double-recombinant" virus by intertypic recombination between C EV-A71 and CVA4, CVA5, CVA14, and CVA16 strains in P3 region and intratypic recombination between C and B EV-A71 strains in P2 region. The B5 EV-A71 shared high similarity with C EV-A71 in P1 region while it contained an unidentified sequence in P2 and P3 regions with two possible recombination patterns: one occurred between C4 EV-A71 and CVA3, CVA5, CVA6, CVA10, and CVA12 stains with one breakpoint in 3C, and the other occurred between C1, C2, C3, and C5 EV-A71 and CVA4, CVA5, CVA14, and CVA16 strains with two breakpoints in the 2A/2B junction and 3C. The C2 EV-A71 was probably a recombinant virus between C4 EV-A71 and CVA8 strains with two breakpoints located in the 5'UTR and 2A/2B junction. Moreover, an incredible recombination of C6 EV-A71 occurred between C4 and C2 EV-A71 with multiple breakpoints. Thus, continuous studies on EV-A71 genome characteristics are still useful and essential for monitoring emergence of new viruses and preventing HFMD outbreaks.

Association between platelet count and the risk and progression of hand, foot, and mouth disease among children.

OBJECTIVE: We aimed to evaluate the association between platelet (PLT) count and the risk and progression of hand, foot, and mouth disease (HFMD). METHODS: In total, 122 HFMD patients and 40 healthy controls were enrolled in the study. The differences between variables among the different subgroups were compared. Logistic regression analyses were performed to assess the relationship between various parameters and HFMD risk/progression. Sensitivity analysis was conducted by detecting the trend of the association between PLT count quartiles and HFMD risk/progression. A generalized additive model was used to identify the nonlinear relationship between PLT count and HFMD risk/progression. The relationship between gender and PLT count as well as the risk/progression of HFMD was detected using a stratified logistic regression model. RESULTS: Significant differences were observed in terms of age, male/female ratio, white blood cell (WBC) count, and PLT count between patients with stage I-II, III-IV HFMD and healthy controls. Moreover, the alanine aminotransferase and magnesium levels between patients with stage I-II and III-IV HFMD significantly differed. Moreover, a significant difference was noted in the male/female ratio among the different PLT groups. The group with a low PLT count had a lower risk of HFMD progression than the group with a high PLT count (Q4) (p=0.039). Lower age, male gender, and WBC count were found to be associated with HFMD risk. Meanwhile, PLT count was correlated to HFMD progression. The sensitivity analysis yielded a similar result using the minimally adjusted model (p for trend=0.037), and minimal changes were observed using the crude and fully adjusted model (p for trend=0.054; 0.090). A significant nonlinear relationship was observed between PLT count and HFMD progression after adjusting for age, gender, and WBC (p=0.039). CONCLUSIONS: PLT was independently associated with HFMD progression in a nonlinear manner.

Development and validation of a novel immune-related prognostic model in hepatocellular carcinoma.

BACKGROUND: Growing evidence has suggested that immune-related genes play crucial roles in the development and progression of hepatocellular carcinoma (HCC). Nevertheless, the utility of immune-related genes for evaluating the prognosis of HCC patients are still lacking. The study aimed to explore gene signatures and prognostic values of immune-related genes in HCC. METHODS: We comprehensively integrated gene expression data acquired from 374 HCC and 50 normal tissues in The Cancer Genome Atlas (TCGA). Differentially expressed genes (DEGs) analysis and univariate Cox regression analysis were performed to identify DEGs that related to overall survival. An immune prognostic model was constructed using the Lasso and multivariate Cox regression analyses. Furthermore, Cox regression analysis was applied to identify independent prognostic factors in HCC. The correlation analysis between immune-related signature and immune cells infiltration were also investigated. Finally, the signature was validated in an external independent dataset. RESULTS: A total of 329 differentially expressed immune-related genes were detected. 64 immune-related genes were identified to be markedly related to overall survival in HCC patients using univariate Cox regression analysis. Then we established a TF-mediated network for exploring the regulatory mechanisms of these genes. Lasso and multivariate Cox regression analyses were applied to construct the immune-based prognostic model, which consisted of nine immune-related genes. Further analysis indicated that this immune-related prognostic model could be an independent prognostic indicator after adjusting to other clinical factors. The relationships between the risk score model and immune cell infiltration suggested that the nine-gene signature could reflect the status of tumor immune microenvironment. The prognostic value of this nine-gene prognostic model was further successfully validated in an independent database. CONCLUSIONS: Together, our study screened potential prognostic immune-related genes and established a novel immune-based prognostic model of HCC, which not only provides new potential prognostic biomarkers and therapeutic targets, but also deepens our understanding of tumor immune microenvironment status and lays a theoretical foundation for immunotherapy.

Association between platelet count and the risk and progression of hand, foot, and mouth disease among children

OBJECTIVE: We aimed to evaluate the association between platelet (PLT) count and the risk and progression of hand, foot, and mouth disease (HFMD). METHODS: In total, 122 HFMD patients and 40 healthy controls were enrolled in the study. The differences between variables among the different subgroups were compared. Logistic regression analyses were performed to assess the relationship between various parameters and HFMD risk/progression. Sensitivity analysis was conducted by detecting the trend of the association between PLT count quartiles and HFMD risk/progression. A generalized additive model was used to identify the nonlinear relationship between PLT count and HFMD risk/progression. The relationship between gender and PLT count as well as the risk/progression of HFMD was detected using a stratified logistic regression model. RESULTS: Significant differences were observed in terms of age, male/female ratio, white blood cell (WBC) count, and PLT count between patients with stage I-II, III-IV HFMD and healthy controls. Moreover, the alanine aminotransferase and magnesium levels between patients with stage I-II and III-IV HFMD significantly differed. Moreover, a significant difference was noted in the male/female ratio among the different PLT groups. The group with a low PLT count had a lower risk of HFMD progression than the group with a high PLT count (Q4) (p=0.039). Lower age, male gender, and WBC count were found to be associated with HFMD risk. Meanwhile, PLT count was correlated to HFMD progression. The sensitivity analysis yielded a similar result using the minimally adjusted model (p for trend=0.037), and minimal changes were observed using the crude and fully adjusted model (p for trend=0.054; 0.090). A significant nonlinear relationship was observed between PLT count and HFMD progression after adjusting for age, gender, and WBC (p=0.039). CONCLUSIONS: PLT was independently associated with HFMD progression in a nonlinear manner.

Efficiently activated ε-poly-L-lysine production by multiple antibiotic-resistance mutations and acidic pH shock optimization in Streptomyces albulus.

ε-Poly-L-lysine (ε-PL) is a food additive produced by Streptomyces and is widely used in many countries. Working with Streptomyces albulus FEEL-1, we established a method to activate ε-PL synthesis by successive introduction of multiple antibiotic-resistance mutations. Sextuple mutant R6 was finally developed by screening for resistance to six antibiotics and produced 4.41 g/L of ε-PL in a shake flask, which is 2.75-fold higher than the level produced by the parent strain. In a previous study, we constructed a double-resistance mutant, SG-31, with high ε-PL production of 3.83 g/L and 59.50 g/L in a shake flask and 5-L bioreactor, respectively. However, we found that R6 did not show obvious advantages in fed-batch fermentation when compared with SG-31. For further activation of ε-PL synthesis ability, we optimized the fermentation process by using an effective acidic pH shock strategy, by which R6 synthetized 70.3 g/L of ε-PL, 2.79-fold and 1.18-fold greater than that synthetized by FEEL-1 and SG-31, respectively. To the best of our knowledge, this is the highest reported ε-PL production to date. This ε-PL overproduction may be due to the result of R99P and Q856H mutations in ribosomal protein S12 and RNA polymerase, respectively, which may be responsible for the increased transcription of the ε-poly-lysine synthetase gene (pls) and key enzyme activities in the Lys synthesis metabolic pathway. Consequently, ε-PL synthetase activity, intracellular ATP, and Lys concentrations were improved and directly contributed to ε-PL overproduction. This study combined ribosome engineering, high-throughput screening, and targeted strategy optimization to accelerate ε-PL production and probe the fermentation characteristics of hyperyield mutants. The information presented here may be useful for other natural products produced by Streptomyces.

Differential protein expression of a streptomycin-resistant Streptomyces albulus mutant in high yield production of ε-poly-l-lysine: a proteomics study.

ε-Poly-l-lysine (ε-PL), produced by Streptomyces albulus, is an excellent antimicrobial agent which has been extensively used in the field of food and medicine. In our previous study, we have improved ε-PL production by S. albulus M-Z18 through iterative introduction of streptomycin resistance. To decipher the overproduction mechanism of high-yielding mutant S. albulus SS-62, we conducted a comparative proteomics analysis between S. albulus SS-62 and its parent strain S. albulus M-Z18. Approximately 11.5% of the predicted S. albulus proteome was detected and 401 known or putative regulatory proteins showed statistically differential expression levels. Expression levels of proteins involved in ε-PL precursor metabolism and energy metabolism, and proteins in the pathways related to transcriptional regulation and translation were up-regulated. It was indicated that mutant SS-62 could not only strengthen the ε-PL precursor metabolism and energy metabolism but also tune the pathways related to transcriptional regulation and translation, suggesting a better intracellular metabolic environment for the synthesis of ε-PL in mutant SS-62. To confirm these bioinformatics analyses, qRT-PCR was employed to investigate the transcriptional levels of pls, frr and hrdD and their transcription levels were found to have increased more than 4-fold. Further, overexpression of pls and frr resulted in an increase in ε-PL titer and the yield of ε-PL per unit cell. This report not only represents the first comprehensive study on comparative proteomics in S. albulus, but it would also guide strain engineering to further improve ε-PL production.

Development of Microtiter Plate Culture Method for Rapid Screening of ε-Poly-L-Lysine-Producing Strains.

ε-Poly-L-lysine (ε-PL) produced by Streptomyces albulus possesses a broad spectrum of antimicrobial activity and is widely used as a food preservative. To extensively screen ε-PL-overproducing strain, we developed an integrated high-throughput screening assay using ribosome engineering technology. The production protocol was scaled down to 24- and 48-deep-well microtiter plates (MTPs). The microplate reader assay was used to monitor ε-PL production. A good correlation was observed between the fermentation results obtained in both 24-(48)-deep-well MTPs and conventional Erlenmeyer flasks. Using this protocol, the production of ε-PL in an entire MTP was determined in <5 min without compromising on accuracy. The high-yielding strain selected through this protocol was also tested in Erlenmeyer flasks. The result showed that the ε-PL production of the high-yielding mutants was nearly 45% higher than that of the parent stain. Thus, development of this protocol is expected to accelerate the selection of ε-PL-overproducing strains.

Hsa-let-7c-5p augments enterovirus 71 replication through viral subversion of cell signaling in rhabdomyosarcoma cells.

BACKGROUND: Human enterovirus 71 (EV71) causes severe hand, foot and mouse disease, accompanied by neurological complications. During the interaction between EV71 and the host, the virus subverts host cell machinery for its own replication. However, the roles of microRNAs (miRNAs) in this process remain obscure. RESULTS: In this study, we found that the miRNA hsa-let-7c-5p was significantly upregulated in EV71-infected rhabdomyosarcoma cells. The overexpression of hsa-let-7c-5p promoted replication of the virus, and the hsa-let-7c-5p inhibitor suppressed viral replication. Furthermore, hsa-let-7c-5p targeted mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) and inhibited its expression. Interestingly, downregulation of MAP4K4 expression led to an increase in EV71 replication. In addition, MAP4K4 knockdown or transfection with the hsa-let-7c-5p mimic led to activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas the hsa-let-7c-5p inhibitor inhibited activation of this pathway. Moreover, EV71 infection promoted JNK pathway activation to facilitate viral replication. CONCLUSIONS: Our data suggested that hsa-let-7c-5p facilitated EV71 replication by inhibiting MAP4K4 expression, which might be related to subversion of the JNK pathway by the virus. These results may shed light on a novel mechanism underlying the defense of EV71 against cellular responses. In addition, these findings may facilitate the development of new antiviral strategies for use in future therapies.

Exogenous brain-derived neurotrophic factor relieves pain symptoms of diabetic rats by reducing excitability of dorsal root ganglion neurons.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes lacking of effective treatments. Enhanced excitability of dorsal root ganglion (DRG) neuron plays a crucial role in the progression of diabetic neuropathic hyperalgesia. Brain-derived neurotrophic factor (BDNF) is known as a neuromodulator of nociception, but whether and how BDNF modulates the excitability of DRG neurons in the development of DPN remain to be clarified. This study investigated the role of exogenous BDNF and its high-affinity tropomyosin receptor kinase B (TrkB) in rats with streptozotocin-induced diabetic neuropathic pain. The results showed that continued intrathecal administration of BDNF to diabetic rats dramatically alleviated mechanical and thermal hyperalgesia, as well as inhibited hyperexcitability of DRG neurons. These effects were blocked by pretreatment with TrkB Fc (a synthetic fusion protein consisting of the extracellular ligand-binding domain of the TrkB receptor). The expression of BDNF and TrkB was upregulated in the DRG of diabetic rats. Intrathecal administration of BDNF did not affect this upregulation. These data provide novel information that exogenous BDNF relieved pain symptoms of diabetic rats by reducing hyperexcitability of DRG neurons and might be the potential treatment of painful diabetic neuropathy.