Advances in experimental models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by persistent articular inflammation and joint damage. RA was first described over 200 years ago; however, its etiology and pathophysiology remain insufficiently understood. The current treatment of RA is mainly empirical or based on the current understanding of etiology with limited efficacy and/or substantial side effects. Thus, the development of safer and more potent therapeutics, validated and optimized in experimental models, is urgently required. To improve the transition from bench to bedside, researchers must carefully select the appropriate experimental models as well as draw the right conclusions. Here, we summarize the establishment, pathological features, potential mechanisms, advantages, and limitations of the currently available RA models. The aim of the review is to help researchers better understand available RA models; discuss future trends in RA model development, which can help highlight new translational and human-based avenues in RA research.

Secretory expression of amylosucrase in Bacillus licheniformis through twin-arginine translocation pathway.

Amylosucrase (EC 2.4.1.4) is a versatile enzyme with significant potential in biotechnology and food production. To facilitate its efficient preparation, a novel expression strategy was implemented in Bacillus licheniformis for the secretory expression of Neisseria polysaccharea amylosucrase (NpAS). The host strain B. licheniformis CBBD302 underwent genetic modification through the deletion of sacB, a gene responsible for encoding levansucrase that synthesizes extracellular levan from sucrose, resulting in a levan-deficient strain, B. licheniformis CBBD302B. Neisseria polysaccharea amylosucrase was successfully expressed in B. licheniformis CBBD302B using the highly efficient Sec-type signal peptide SamyL, but its extracellular translocation was unsuccessful. Consequently, the expression of NpAS via the twin-arginine translocation (TAT) pathway was investigated using the signal peptide SglmU. The study revealed that NpAS could be effectively translocated extracellularly through the TAT pathway, with the signal peptide SglmU facilitating the process. Remarkably, 62.81% of the total expressed activity was detected in the medium. This study marks the first successful secretory expression of NpAS in Bacillus species host cells, establishing a foundation for its future efficient production. ONE-SENTENCE SUMMARY: Amylosucrase was secreted in Bacillus licheniformis via the twin-arginine translocation pathway.

Change of sexual behavior among men who have sex with men before, during and after COVID-19 pandemic in China: a cross-sectional study.

INTRODUCTION: The COVID-19 epidemic control and prevention strategies affected people's sexual activities and behaviors. Little was known about long-term effects of COVID-19 prevention and control strategies on sexual behaviors among men who have sex with men (MSM). This study aimed to examine changes in risky sexual behaviors of MSM before and after the local epidemic. METHODS: An online survey was conducted nationwide from June 1 to June 10, 2022. MSM aged 16 years and above, residing in China were recruited through convenience sampling. A generalized estimating equation model with modified Poisson regression was used to analyze changes in multiple sexual partners, unprotected sex, mobility for sexual activity, and recreational substance use before and after the local epidemic. RESULTS: Compared to the pre-pandemic (36.5%), the prevalence of multiple sexual partners (11.5%) significantly decreased during the local epidemic and then increased after the local epidemic (25.2%) but remained lower than pre-pandemic, as did the prevalence of unprotected sex (31.1%, 19.4%, and 26.1%), mobility for sexual activity (7.5%, 2.8%, and 4.1%) and recreational substance use (47.7%, 27.2%, and 39.5%). Compared to the pre-pandemic, higher declines in the prevalence of risky sexual behaviors during the local epidemic existed among MSM living without a regular partner (44% decrease in unprotected sex and 46% in recreational substance use), with a bachelor's degree and above (70% decrease in multiple sex partners, 39% in unprotected sex, 67% in mobility for sexual activity and 44% in recreational substance use), higher incomes (70% decrease in multiple sex partners), self-identified gay or bisexual/unsure (38-71%), and HIV infection (49-83% decrease respectively in these four indicators). After the local epidemic, the declines in the above indicators compared to the pre-pandemic were correspondingly. And higher declines existed among MSM living without a regular partner (8% decrease in unprotected sex and 13% in recreational substance use), with a bachelor's degree and above (33% decrease in multiple sex partners), higher incomes (55% decrease in mobility for sexual activity), self-identified gay (51% decrease in mobility for sexual activity), and HIV infection (32%, 68%, 24% decrease respectively in unprotected sex, mobility for sexual activity and recreational substance use). CONCLUSIONS: Risky sexual behaviors reduced considerably during the local epidemic, then seemed rebounded after the outbreak but wouldn't return to pre-pandemic levels. More attention should be paid to vulnerable people with lower socio-economic status, HIV-positive, and sexual minorities for sustained HIV and COVID-19 prevention.

Excess mortality and associated factors among people living with HIV initiating highly active antiretroviral therapy in Luzhou, China 2006-2020.

BACKGROUND: To estimate crude mortality, excess mortality, and standardized mortality rates (SMR) among people living with HIV (PLHIV) initiating highly active antiretroviral therapy (HAART) in Luzhou, China 2006-2020, and assess associated factors. METHODS: PLHIV initiating HAART in the HIV/AIDS Comprehensive Response Information Management System (CRIMS) in Luzhou, China 2006-2020 were included in the retrospective cohort study. The crude mortality, excess mortality, and SMR were estimated. Multivariable Poisson regression model was used for analyzing risk factors associated with excess mortality rates. RESULTS: The median age among 11,468 PLHIV initiating HAART was 54.5 years (IQR:43.1-65.2). The excess mortality rate decreased from 1.8 deaths/100 person-years (95% confidence interval [CI]:1.4-2.4) in 2006-2011 to 0.8 deaths/100 person-years (95%CI:0.7-0.9) in 2016-2020. SMR decreased from 5.4 deaths/100 person-years (95%CI:4.3-6.8) to 1.7 deaths/100 person-years (95%CI:1.5-1.8). Males had greater excess mortality with the eHR of 1.6 (95%CI:1.2-2.1) than females. PLHIV with CD4 counts ≥ 500 cells/µL had the eHR of 0.3 (95%CI:0.2-0.5) in comparison to those with CD4 counts < 200 cells/µL. PLHIV with WHO clinical stages III/IV had greater excess mortality with the eHR of 1.4 (95%CI:1.1-1.8). PLHIV with time from diagnosis to HAART initiation ≤ 3 months had the eHR of 0.7 (95%CI:0.5-0.9) compared to those with time ≥ 12 months. PLHIV with initial HAART regimens unchanged and viral suppression had the eHR of 1.9 (95%CI:1.4-2.6) and 0.1 (95%CI:0.0-0.1), respectively. CONCLUSIONS: The excess mortality and SMR among PLHIV initiating HAART in Luzhou, China decreased substantially from 2006 to 2020, but the mortality rate among PLHIV was still higher than general population. PLHIV who were male, with baseline CD4 counts less than 200 cells/µL, WHO clinical stages III/IV, time from diagnosis to HAART initiation ≥ 12 months, initial HAART regimens unchanged, and virological failure had a greater risk of excess deaths. Early and efficient HAART would be significant in reducing excess mortality among PLHIV.

High-efficiency chromosomal integrative amplification strategy for overexpressing α-amylase in Bacillus licheniformis.

Bacillus licheniformis is a well-known platform strain for production of industrial enzymes. However, the development of genetically stable recombinant B. licheniformis for high-yield enzyme production is still laborious. Here, a pair of plasmids, pUB-MazF and pUB'-EX1, were firstly constructed. pUB-MazF is a thermosensitive, self-replicable plasmid. It was able to efficiently cure from the host cell through induced expression of an endoribonuclease MazF, which is lethal to the host cell. pUB'-EX1 is a nonreplicative and integrative plasmid. Its replication was dependent on the thermosensitive replicase produced by pUB-MazF. Transformation of pUB'-EX1 into the B. licheniformis BL-UBM harboring pUB-MazF resulted in both plasmids coexisting in the host cell. At an elevated temperature, and in the presence of isopropyl-1-thio-ß-d-galactopyranoside and kanamycin, curing of the pUB-MazF and multiple-copy integration of pUB'-EX1 occurred, simultaneously. Through this procedure, genetically stable recombinants integrated multiple copies of amyS, from Geobacillus stearothermophilus ATCC 31195 were facilely obtained. The genetic stability of the recombinants was verified by repeated subculturing and shaking flask fermentations. The production of α-amylase by recombinant BLiS-002, harboring five copies of amyS, in a 50-l bioreactor reached 50 753 U/ml after 72 hr fermentation. This strategy therefore has potential for production of other enzymes in B. licheniformis and for genetic modification of other Bacillus species.

Transmission Dynamics of the Rubella Virus Circulating in China During 2010-2019: 2 Lineage Switches Between Genotypes 1E and 2B.

BACKGROUND: To provide a better understanding of the progress on rubella control and elimination in China, a genetic analysis was conducted to examine the transmission pattern of the endemic rubella virus in China during 2010-2019. METHODS: A total of 4895 strains were obtained from 29 of the 31 provinces in mainland China during 2010-2019. The genotyping regions of the strains were amplified, determined, and assembled. Genotyping analysis and lineage division were performed by comparisons with the World Health Organization reference strains and reported lineage reference strains, respectively. Further phylogenetic analyses were performed to compare the genetic relationship. RESULTS: During 2010-2019, the domestic lineage 1E-L1 and multiple imported lineages of rubella viruses including 2B-L1, 1E-L2, and 2B-L2c were identified. Further analysis of the circulation trend of the different lineages indicated that 2 switches occurred among the lineages. The first shift was from lineage 1E-L1 to 2B-L1, which occurred around 2015-2016, followed by the lowest rubella incidence in 2017. The second shift was from lineage 2B-L1 to 1E-L2 and 2B-L2c, which occurred around 2018-2019, coinciding with rubella resurgence and the subsequent nationwide epidemic during 2018-2019. Insufficient genomic information worldwide made it impossible to trace the origin of the imported viruses. CONCLUSIONS: China was moving toward rubella elimination, as evidenced by the fact that previous endemic lineages were not detected. However, rubella reemerged in 2018 2019 due to the newly imported rubella viruses. Therefore, to realize the rubella elimination goal, joint efforts are required for all countries worldwide.

Exploitation of ammonia-inducible promoters for enzyme overexpression in Bacillus licheniformis.

Ammonium hydroxide is conventionally used as an alkaline reagent and cost-effective nitrogen source in enzyme manufacturing processes. However, few ammonia-inducible enzyme expression systems have been described thus far. In this study, genomic-wide transcriptional changes in Bacillus licheniformis CBBD302 cultivated in media supplemented with ammonia were analyzed, resulting in identification of 1443 differently expressed genes, of which 859 genes were upregulated and 584 downregulated. Subsequently, the nucleotide sequences of ammonia-inducible promoters were analyzed and their functionally-mediated expression of amyL, encoding an α-amylase, was shown. TRNA_RS39005 (copA), TRNA_RS41250 (sacA), TRNA_RS23130 (pdpX), TRNA_RS42535 (ald), TRNA_RS31535 (plp), and TRNA_RS23240 (dfp) were selected out of the 859 upregulated genes and each showed higher transcription levels (FPKM values) in the presence of ammonia and glucose than that of the control. The promoters, PcopA from copA, PsacA from sacA, PpdpX from pdpX, Pald from ald, and Pplp from plp, except Pdfp from dfp, were able to mediate amyL expression and were significantly induced by ammonia. The highest enzyme expression level was mediated by Pplp and represented 23% more α-amylase activity after induction by ammonia in a 5-L fermenter. In conclusion, B. licheniformis possesses glucose-independent ammonia-inducible promoters, which can be used to mediate enzyme expression and therefore enhance the enzyme yield in fermentations conventionally fed with ammonia for pH adjustment and nitrogen supply.

Limitation of thiamine pyrophosphate supply to growing Escherichia coli switches metabolism to efficient D-lactate formation.

Efficient production of D-lactate by engineered Escherichia coli entails balancing cell growth and product synthesis. To develop a metabolic switch to implement a desirable transition from cell growth to product fermentation, a thiamine auxotroph B0013-080A was constructed in a highly efficient D-lactate producer E. coli strain B0013-070. This was achieved by inactivation of thiE, a gene encoding a thiamine phosphate synthase for biosynthesis of thiamine monophosphate. The resultant mutant B0013-080A failed to grow on the medium in the absence of thiamine yet growth was restored when exogenous thiamine was provided. A linear relationship between cell mass formation and amount of thiamine supplemented was mathematically determined in a shake flask experiment and confirmed in a 7-L bioreactor system. This calculation revealed that ∼ 95-96 thiamine molecules per cell were required to satisfy cell growth. This relationship was employed to develop a novel fermentation process for D-lactate production by using thiamine as a limiting condition. A D-lactate productivity of 4.11 g · L(-1) · h(-1) from glycerol under microaerobic condition and 3.66 g · L(-1) · h(-1) from glucose under anaerobic condition was achieved which is 19.1% and 10.2% higher respectively than the parental strain. These results revealed a convenient and reliable method to control cell growth and improve D-lactate fermentation. This control strategy could be applied to other biotechnological processes that require optimal allocation of carbon between cell growth and product formation.

Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth.

UNLABELLED: L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD+-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the E. coli strain to produce L-lactate, three L-lactate dehydrogenase genes from different bacteria were cloned and expressed. The L-lactate producing strains, 090B1 (B0013-070, ΔldhA::diflldD::Pldh-ldhLca), 090B2 (B0013-070, ΔldhA::diflldD::Pldh-ldhStrb) and 090B3 (B0013-070, ΔldhA::diflldD::Pldh-ldhBcoa) were developed from a previously developed D-lactate over-producing strain, E. coli strain B0013-070 (ack-ptappspflBdldpoxBadhEfrdA) by: (1) deleting ldhA to block D-lactate formation, (2) deleting lldD to block the conversion of L-lactate to pyruvate, and (3) expressing an L-lactate dehydrogenase (L-LDH) to convert pyruvate to L-lactate under the control of the ldhA promoter. Fermentation tests were carried out in a shaking flask and in a 25-l bioreactor. Strains 090B1, 090B2 or 090B3 were shown to metabolize glucose to L-lactate instead of D-lactate. However, L-lactate yield and cell growth rates were significantly different among the metabolically engineered strains which can be attributed to a variation between temperature optimum for cell growth and temperature optimum for enzymatic activity of individual L-LDH. In a temperature-shifting fermentation process (cells grown at 37°C and L-lactate formed at 42°C), E. coli 090B3 was able to produce 142.2 g/l of L-lactate with no more than 1.2 g/l of by-products (mainly acetate, pyruvate and succinate) accumulated. In conclusion, the production of lactate by E. coli is limited by the competition relationship between cell growth and lactate synthesis. Enzymatic properties, especially the thermodynamics of an L-LDH can be effectively used as a factor to regulate a metabolic pathway and its metabolic flux for efficient L-lactate production. HIGHLIGHTS: The enzymatic thermodynamics was used as a tool for metabolic regulation. Minimizing the activity of L-lactate dehydrogenase in growth phase improved biomass accumulation. Maximizing the activity of L-lactate dehydrogenase improved lactate productivity in production phase.

Investigation into the impact of proton pump inhibitors on sertraline transport across the blood-brain barrier.

As a widely used antidepressant that works by inhibiting the reuptake of serotonin, sertraline exerts an antidepressant effect depending on its concentration in the brain, which might be limited by the blood-brain barrier (BBB). It is highly possible to combine proton pump inhibitors (PPIs) with sertraline in clinical trials. Nevertheless, the role played by PPIs in regulating the transport of sertraline across the BBB remains unclear. Here, the impact of PPIs on the distribution of sertraline in the brain and the mechanisms involved were investigated. A mouse brain distribution study showed that Omeprazole (OME), Pantoprazole (PAN), Ilaprazole (ILA), and Esomeprazole (ESO) increased the area under the brain concentration-time curves (AUC) for sertraline by 2.02-, 3.18-, 3.04-, and 4.21-fold, respectively, after the 14-day administration of PPIs. Besides, PPIs significantly increased the permeability of sertraline in brain perfusion experiments, with PAN having the highest rank order, followed by ILA, OME, and ESO. In the tail suspension test (TST), co-administration PPI groups showed significantly shorter immobility time than the control group. In vitro, four PPIs inhibited sertraline efflux in breast cancer resistance protein (BCRP)-overexpressing MDCKII cells, and showed a mixed inhibition type. In this study, PPIs were further found to inhibit the mRNA and protein expression of brain BCRP. To sum up, the findings of this study revealed that PPIs could enhance the brain distribution and antidepressant effect of sertraline, which may be attributed to the inhibition of BCRP expression at the BBB by PPIs.

DNA methylation-regulated miR-193a-3p dictates resistance of hepatocellular carcinoma to 5-fluorouracil via repression of SRSF2 expression.

Chemoresistance prevents effective cancer therapy and is rarely predictable prior to treatment, particularly for hepatocellular carcinoma (HCC). Following the chemoresistance profiling of eight HCC cell lines to each of nine chemotherapeutics, two cell lines (QGY-7703 as a sensitive and SMMC-7721 as a resistant cell line to 5-fluorouracil (5-FU) treatment) were systematically studied for mechanistic insights underpinning HCC 5-FU chemoresistance. Genomic profiling at both DNA methylation and microRNA (miR) levels and subsequent mechanistic studies illustrate a new mechanism for how DNA methylation-regulated miR-193a-3p dictates the 5-FU resistance of HCC cells via repression of serine/arginine-rich splicing factor 2 (SRSF2) expression. In turn, SRSF2 preferentially up-regulates the proapoptotic splicing form of caspase 2 (CASP2L) and sensitizes HCC cells to 5-FU. Forced changes of miR-193a-3p level reverse all of the phenotypic features examined, including cell proliferation, cell cycle progression, and 5-FU sensitivity, in cell culture and in nude mice. Importantly, the siRNA-mediated repression of SRSF2 phenocopies all of the miR-193a-3p mimic-triggered changes in QGY-7703. This newly identified miR-193a-3p-SRSF2 axis highlights a new set of companion diagnostics required for optimal 5-FU therapy of HCC, which involve assaying both the DNA methylation state of the miR-193a gene and the expression of miR-193a-3p and SRSF2 and the relative level of the proapoptotic versus antiapoptotic splicing forms of caspase 2 in clinical samples.

Purification, characterization and cloning of a thermotolerant isoamylase produced from Bacillus sp. CICIM 304.

A novel thermostable isoamylase, IAM, was purified to homogeneity from the newly isolated thermophilic bacterium Bacillus sp. CICIM 304. The purified monomeric protein with an estimated molecular mass of 100 kDa displayed its optimal temperature and pH at 70 °C and 6.0, respectively, with excellent thermostability between 30 and 70 °C and pH values from 5.5 to 9.0. Under the conditions of temperature 50 °C and pH 6.0, the K m and V max on glycogen were 0.403 ± 0.018 mg/mg and 0.018 ± 0.001 mg/(min mg), respectively. Gene encoding IAM, BsIam was identified from genomic DNA sequence with inverse PCRs. The open reading frame of the BsIam gene was 2,655 base pairs long and encoded a polypeptide of 885 amino acids with a calculated molecular mass of 101,155 Da. The deduced amino acid sequence of IAM shared less than 40 % homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. This enzyme showed its obvious superiority in the industrial starch conversion process.

Sexual Behavior and Perceived Loneliness in Elderly People Living with HIV in China during the COVID-19 Pandemic.

BACKGROUND: The proportion of elderly people living with HIV (PLHIV) is increasing in China. To advance targeted interventions and substantially improve their quality of life, we investigate indicators of loneliness and sexual behavior among elderly PLHIV in 10 districts/counties in China during the COVID-19 pandemic. METHODS: The demographic information and laboratory test results of the potential respondents were initially collected from the China Information System for Disease Control and Prevention. A two-stage stratified cluster sampling was used. The questionnaire survey was individually provided to all PLHIV aged +60. RESULTS: We recruited 1017 valid respondents with a median age of 66 years (interquartile range of 63-71), of which 776 (76.3%) were male. Overall, 341 respondents (33.5%) lived alone, and 304 (29.9%) felt lonely. A total of 726 respondents (71.4%) informed others of their HIV diagnosis. Among the 726 respondents, children were the most common group with whom the older people shared their HIV infection status, accounting for 82.9%. Approximately 20% of the older PLHIV engaged in sexual behavior in the last year, and 70% reported not using condoms. A significantly greater risk of loneliness was found among the females (AOR = 1.542, CI: 1.084, 2.193), those who suffered discrimination from informed people (AOR = 4.719, CI: 2.986, 7.459), were diagnosed <1 year prior (AOR = 2.061, CI: 1.345, 3.156), those living alone (AOR = 2.314, CI: 1.632, 3.280), those having no friends (AOR = 1.779, CI: 1.327, 2.386), and those who had a divorced or widowed marital status (AOR = 1.686, CI: 1.174, 2.421). CONCLUSIONS: Compared with non-lonely participants, the lonely participants were more likely to have a rural registered residence, a lower education level, no friends, be divorced or widowed, live alone, and lack knowledge of smartphones and reproductive health. The influence of COVID-19 had caused social activities to be more confined to the community, which impacts elderly HIV patients suffering from severe discrimination within families and communities.

Treatment failure and associated factors among people living with HIV on highly active antiretroviral therapy in mainland China: A systematic review and meta-analysis.

OBJECTIVE: Reducing the prevalence of treatment failure among people living with HIV (PLHIV) on highly active antiretroviral therapy (HAART) is crucial for improving individual health and reducing disease burden. This study aimed to assess existing evidence on treatment failure and its associated factors among PLHIV in mainland China. METHODS: We conducted a comprehensive search of PubMed, Web of Science, Cochrane Library, WanFang, China National Knowledge Infrastructure, and SinoMed databases. Relevant studies on treatment failure among PLHIV in mainland China until September 2022 were searched, including cross-sectional, case-control, and cohort studies. The primary outcome was treatment failure, and secondary outcomes were the potential influencing factors of treatment failure. We performed a meta-analysis to pool each outcome of interest, including meta-regression, subgroup, publication bias, and sensitivity analyses. RESULTS: A total of 81 studies were deemed eligible and included in the final meta-analysis. The pooled treatment failure prevalence among PLHIV in mainland China was 14.40% (95% confidence interval [CI]:12.30-16.63), of which the virological and immunological failure prevalence was 10.53% (95%CI:8.51-12.74) and 18.75% (95%CI:15.44-22.06), respectively. The treatment failure prevalence before and after 2016 was 18.96% (95%CI:13.84-24.67) and 13.19% (95%CI:10.91-15.64). Factors associated with treatment failure included good treatment adherence (odds ratio [OR] = 0.36, 95%CI:0.26-0.51), baseline CD4 counts>200 cells/µL (OR = 0.39, 95%CI:0.21-0.75), HAART regimens containing Tenofovir Disoproxil Fumarate (TDF) (OR = 0.70, 95%CI:0.54-0.92), WHO clinical stage III/IV (OR = 2.02, 95%CI:1.14-3.59) and age≥40 years (OR = 1.56, 95%CI:1.23-1.97). CONCLUSION: The prevalence of treatment failure among PLHIV receiving HAART in mainland China was low and tended to decline. Poor adherence, low baseline CD4 count, HAART regimens without TDF, advanced clinical stage, and old age were contributing factors for treatment failure. Relevant intervention programs are needed with increasing treatment adherence through behavioral intervention or precise intervention targeting older adults.

Etiological Study of Acute Conjunctivitis Caused by Human Adenovirus in Shanxi Province, China, between 2016 and 2019.

Human adenovirus (HAdV) is the primary cause of acute conjunctivitis. To improve our understanding of the etiology of adenoviral conjunctivitis, ocular samples were collected from 160 conjunctivitis cases in the Shanxi province of northern China between 2016 and 2019. Through preliminary identification, virus isolation, and type identification, a total of 63 HAdV isolates were obtained from the samples. Three species and seven types (HAdV-3, HAdV-4, HAdV-8, HAdV-37, HAdV-53, HAdV-64, and HAdV-85) were detected, with HAdV-64, HAdV-3, and HAdV-8 being the predominant types in 2016, 2018, and 2019, respectively. Further phylogenetic analysis indicated the relative genomic stability of seven HAdV-type strains, except for 4 HAdV-3 strains in 2018 with a novel amino acid insertion site (Pro) between P19 and S20 in the penton base gene. It is worth noting that the genomes of two Shanxi HAdV-85 strains from 2016 were almost identical to those of previously reported HAdV-85 strains that circulated in Japan in 2014 to 2018. China was the second country to sample and isolate HAdV-85, suggesting that HAdV-85 might be underreported as an ocular pathogen. Data obtained in this study provide valuable information on the prevalence of acute conjunctivitis caused by HAdV. IMPORTANCE HAdV types in cases of conjunctivitis in Shanxi province, China, in 2016 to 2019 showed evident diversity, with seven types (HAdV-3, HAdV-4, HAdV-8, HAdV-37, HAdV-53, HAdV-64, and HAdV-85) being identified, and relative genome stability of these viruses was observed. In addition, China was the second country to sample and isolate HAdV-85, which suggests that HAdV-85 might be underreported as an important pathogen associated with ocular infections. These results enhance the understanding of the etiology of adenoviral conjunctivitis and may aid in the development of prevention and control strategies for HAdV-related ocular infections in China.

Genetically switched D-lactate production in Escherichia coli.

During a fermentation process, the formation of the desired product during the cell growth phase competes with the biomass for substrates or inhibits cell growth directly, which results in a decrease in production efficiency. A genetic switch is required to precisely separate growth from production and to simplify the fermentation process. The ldhA promoter, which encodes the fermentative D-lactate dehydrogenase (LDH) in the lactate producer Escherichia coli CICIM B0013-070 (ack-pta pps pflB dld poxB adhE frdA), was replaced with the λ p(R) and p(L) promoters (as a genetic switch) using genomic recombination and the thermo-controllable strain B0013-070B (B0013-070, ldhAp::kan-cI(ts)857-p(R)-p(L)), which could produce two-fold higher LDH activity at 42°C than the B0013-070 strain, was created. When the genetic switch was turned off at 33°C, strain B0013-070B produced 10% more biomass aerobically than strain B0013-070 and produced only trace levels of lactate which could reduce the growth inhibition caused by oxygen insufficiency in large scale fermentation. However, 42°C is the most efficient temperature for switching on lactate production. The volumetric productivity of B0013-070B improved by 9% compared to that of strain B0013-070 when it was grown aerobically at 33°C with a short thermo-induction at 42°C and then switched to the production phase at 42°C. In a bioreactor experiment using scaled-up conditions that were optimized in a shake flask experiment, strain B0013-070B produced 122.8 g/l D-lactate with an increased oxygen-limited productivity of 0.89 g/g·h. The results revealed the effectiveness of using a genetic switch to regulate cell growth and the production of a metabolic compound.

Improvement of D-lactate productivity in recombinant Escherichia coli by coupling production with growth.

Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA pta pps pflB dld poxB adhE frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity of lactic acid compared to aerobic and O(2)-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O(2)-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results have revealed an approach for improving production of fermentative products in E. coli.

Fine tuning the transcription of ldhA for D-lactate production.

Fine tuning of the key enzymes to moderate rather than high expression levels could overproduce the desired metabolic products without inhibiting cell growth. The aims of this investigation were to regulate rates of lactate production and cell growth in recombinant Escherichia coli through promoter engineering and to evaluate the transcriptional function of the upstream region of ldhA (encoding fermentative lactate dehydrogenase in E. coli). Twelve ldhA genes with sequentially shortened chromosomal upstream regions were cloned in an ldhA deletion, E. coli CICIM B0013-080C (ack-pta pps pflB dld poxB adhE frdA ldhA). The varied ldhA upstream regions were further analyzed using program NNPP2.2 (Neural Network Promoter Prediction 2.2) to predict the possible promoter regions. Two-phase fermentations (aerobic growth and oxygen-limited production) of these strains showed that shortening the ldhA upstream sequence from 291 to 106 bp successively reduced aerobic lactate synthesis and the inhibition effect on cell growth during the first phase. Simultaneously, oxygen-limited lactate productivity was increased during the second phase. The putative promoter downstream of the -96 site of ldhA could function as a transcriptional promoter or regulator. B0013-080C/pTH-rrnB-ldhA8, with the 72-bp upstream segment of ldhA, could be grown at a high rate and achieve a high oxygen-limited lactate productivity of 1.09 g g(-1) h(-1). No transcriptional promoting region was apparent downstream of the -61 site of ldhA. We identified the latent transcription regions in the ldhA upstream sequence, which will help to understand regulation of ldhA expression.

Decoupling analysis of carbon emissions in China's power industry-based on ARDL model.

Electric power industry, as one of the main industries leading to the increase of China's carbon emissions, accounts for about 40% of the total carbon emissions. It is of great practical significance to study the influencing factors of carbon emission decoupling index in power industry and put forward relevant policy suggestions. Based on the decoupling index of China's electric power industry from 1995 to 2018, this paper explores the influence of each index on the decoupling index through the autoregressive- distributed lag model. It turns out that the policy will significantly change the rate of change of carbon emissions and the rate of economic growth, but the impact of the policy is extremely short-lived; power generation structure, environmental regulations, and total lighting value at night play a positive role in promoting the decoupling index, while thermal power fuel efficiency and power generation conversion ratio play a negative role in inhibiting the decoupling index. In addition, the influence of power generation structure, environmental regulations, and the total value of night light on decoupling index also has a lagging and cumulative effect. Therefore, we propose targeted policy recommendations for policy formulation, green development, and low carbon construction in China's power industry from different perspectives based on the findings of the study.

Pullulanase with high temperature and low pH optima improved starch saccharification efficiency.

Pullulanase, a starch debranching enzyme, is required for the preparation of high glucose/maltose syrup from starch. In order to expand its narrow reaction conditions and improve its application value, Bacillus naganoensis pullulanase (PulA) was mutated by site-directed mutagenesis and the biochemical characteristics of the mutants were studied. The mutant PulA-N3 with mutations at asparagine 467, 492 and 709 residues was obtained. It displayed the activity maximum at 60 °C and pH 4.5 and exceeded 90% activities between 45 and 60 °C and from pH 4.0 to pH 5.5, which was improved greatly compared with wild-type PulA. Its thermostability and acidic pH stability were also remarkably improved. Its catalytic rate (kcat/Vmax) was 2.76 times that of PulA. In the preparation of high glucose syrup, the DX (glucose content, %) values of glucose mediated by PulA-N3 and glucoamylase reached 96.08%, which were 0.82% higher than that of PulA. In conclusion, a new pullulanase mutant PulA-N3 was successfully developed, which has high debranching activity in a wide range of temperature and pH, thereby paving the way for highly efficient starch saccharification.