Production of polyhydroxyalkanoates by engineered Halomonas bluephagenesis using starch as a carbon source.

A novel α-amylase Amy03713 was screened and cloned from the starch utilization strain Vibrio alginolyticus LHF01. When heterologously expressed in Escherichia coli, Amy03713 exhibited the highest enzyme activity at 45 °C and pH 7, maintained >50 % of the enzyme activity in the range of 25-75 °C and pH 5-9, and sustained >80 % of the enzyme activity in 25 % (w/v) of NaCl solution, thus showing a wide range of adapted temperatures, pH, and salt concentrations. Halomonas bluephagenesis harboring amy03713 gene was able to directly utilize starch. With optimized amylase expression, H. bluephagenesis could produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). When cultured for PHB production, recombinant H. bluephagenesis was able to grow up to a cell dry weight of 11.26 g/L, achieving a PHB titer of 6.32 g/L, which is the highest titer that has been reported for PHB production from starch in shake flasks. This study suggests that Amy03713 is an ideal amylase for PHA production using starch as the carbon source in H. bluephagenesis.

Compound heterozygous mutations in the helicase RTEL1 causing Hoyeraal-Hreidarsson syndrome with Blake`s pouch cyst: a case report.

BACKGROUND: Telomeres inhibit DNA damage response at the ends of the chromosome to suppress cell cycle arrest as well as ensure genome stability. Dyskeratosis congenita (DC), a telomere-related disease, includes the classical triad involving oral leukoplakia, dysplastic nails, and lacy reticular pigment in the neck and/or upper chest. Hoyeraal-Hreidarrson syndrome (HHS), a severe manifestation of DC, frequently occurs during childhood, and patients with HHS often show short-term survival and thus do not exhibit all mucocutaneous manifestations or syndromic features. CASE: We report here a patient with HHS characterized by the proband`s clinical attributes, such as growth delay, bone marrow failure, microcephaly, defects in body development, and the absence of cerebellar hypoplasia combined with Blake`s pouch cyst. By using exome sequencing, novel compound heterozygous mutations (c.1451C > T and c.1266+3del78bp) were detected in the RTEL1 (regulator of telomere elongation helicase 1) gene. CONCLUSIONS: The DNA helicase RTEL1 plays a role in genome stability, DNA replication, telomere maintenance, and genome repair. Terminal restriction fragment length analysis revealed a significantly shorter telomere length of the proband. Our findings provided evidence that compound heterozygous RTEL1 mutations cause HHS.

Global Disease Burden and Attributable Risk Factor Analysis of Asthma in 204 Countries and Territories From 1990 to 2019.

PURPOSE: Asthma is a common chronic inflammatory respiratory tract disease with high morbidity and mortality. The global trends in asthma burden remain poorly understood, and asthma incidence has increased during the worldwide coronavirus disease 2019 (COVID-19) pandemic. This study aimed to provide a comprehensive view of the global distribution of asthma burden and its attributable risk factors from 1990 to 2019. METHODS: Based on the Global Burden of Disease Study 2019 Database, asthma incidence, deaths, disability-adjusted life years (DALYs), the corresponding age-standardized incidence rate (ASIR), age-standardized death rate (ASDR), age-standardized DALY rate, and estimated annual percentage change were analyzed according to age, sex, sociodemographic index (SDI) quintiles, and locations. Risk factors contributing to asthma deaths and DALYs were also investigated. RESULTS: Globally, the asthma incidence increased by 15%, but deaths and DALYs decreased. The corresponding ASIR, ASDR, and age-standardized DALY rate also decreased. The high SDI region had the highest ASIR, and the low SDI region had the highest ASDR. The ASDR and age-standardized DALY rate were negatively correlated with the SDI. The low-middle SDI region, particularly South Asia, showed the highest asthma-related deaths and DALYs. The incidence peak was under 9 years old, and more than 70% of all deaths occurred in the population over 60 years old. Smoking, occupational asthmagens, and a high body mass index were the main risk factors for asthma-related mortality and DALYs, and their distributions varied between sexes. CONCLUSIONS: Globally, the asthma incidence has increased since 1990. The greatest asthma burden is borne by the low-middle SDI region. The 2 groups that need special attention are those under 9 years old and those over 60 years old. Targeted strategies are needed to reduce the asthma burden based on geographic and sex-age characteristics. Our findings also provide a platform for further investigation into the asthma burden in the era of COVID-19.

Conversion of acetate and glyoxylate to fumarate by a cell-free synthetic enzymatic biosystem.

Fumarate is a value-added chemical that is widely used in food, medicine, material, and agriculture industries. With the rising attention to the demand for fumarate and sustainable development, many novel alternative ways that can replace the traditional petrochemical routes emerged. The in vitro cell-free multi-enzyme catalysis is an effective method to produce high value chemicals. In this study, a multi-enzyme catalytic pathway comprising three enzymes for fumarate production from low-cost substrates acetate and glyoxylate was designed. The acetyl-CoA synthase, malate synthase, and fumarase from Escherichia coli were selected and the coenzyme A achieved recyclable. The enzymatic properties and optimization of reaction system were investigated, reaching a fumarate yield of 0.34 mM with a conversion rate of 34% after 20 h of reaction. We proposed and realized the conversion of acetate and glyoxylate to fumarate in vitro using a cell-free multi-enzyme catalytic system, thus providing an alternative approach for the production of fumarate.

Engineering Vibrio alginolyticus as a novel chassis for PHB production from starch.

Vibrio alginolyticus LHF01 was engineered to efficiently produce poly-3-hydroxybutyrate (PHB) from starch in this study. Firstly, the ability of Vibrio alginolyticus LHF01 to directly accumulate PHB using soluble starch as the carbon source was explored, and the highest PHB titer of 2.06 g/L was obtained in 18 h shake flask cultivation. Then, with the analysis of genomic information of V. alginolyticus LHF01, the PHB synthesis operon and amylase genes were identified. Subsequently, the effects of overexpressing PHB synthesis operon and amylase on PHB production were studied. Especially, with the co-expression of PHB synthesis operon and amylase, the starch consumption rate was improved and the PHB titer was more than doubled. The addition of 20 g/L insoluble corn starch could be exhausted in 6-7 h cultivation, and the PHB titer was 4.32 g/L. To the best of our knowledge, V. alginolyticus was firstly engineered to produce PHB with the direct utilization of starch, and this stain can be considered as a novel host to produce PHB using starch as the raw material.

The novel HLA-A*02:1075 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-A*02:1075 differs from HLA-A*02:07:01:01 by one nucleotide in exon 5.

Factors Contributing to False-Negative Enzyme-Linked Immunospot Assay for Interferon-Gamma Results in Active Tuberculosis.

BACKGROUND: The enzyme-linked immunospot assay test (T-SPOT.TB) is effective for detecting TB, but there are still some persons with tuberculosis (TB) who have negative results. The aim of this study was to analyze the factors that contribute to false negative results in active TB patients. METHODS: We investigated the clinical trial and laboratory data of the active TB patients hospitalized in the First Affiliated Hospital of Xi'an Jiaotong University from May 2013 to November 2014. RESULTS: In 556 active TB patients, 198 infected sites were pulmonary, 358 infected sites were extra pulmonary, and 13.85% (77/556) had false negative T SPOT.TB results. The univariate and multivariate analysis showed that age and bone/joint TB were the factors contributing to the T-SPOT. TB false negative results and pleural TB were related to true positive results. CONCLUSIONS: The site of TB may significantly affect the sensitivity of T-SPOT.TB. False negative results of TSPOT.TB are associated with older age and bone/joint TB, so in older adult patients or those with bone/joint TB, the exclusion of TB should be regarded with caution when T-SPOT.TB results are negative.

[Expression of CD 86 on monocyte and B cell surface, the level of T(H)-derived cytokine and their correlation in children with asthma].

OBJECTIVE: To explore the role of T lymphocytes activation co-stimulation pathway and T lymphocyte subset activation in asthma pathogenesis. METHODS: The blood samples were taken from 35 asthma children (including 22 male and 13 female, age 11 months-9 years) and 31 normal children (including 19 male and 12 female, age 8 months-12 years). Direct immunofluorescence flow cytometry was used to detect the CD86 mean fluorescence intensity (MFI) on CD(14)(+) cell, CD(19)(+) cell percentage and CD19 and CD86 double positive cell percentage in PBMC activated by LPS. ELISA was used to detect the levels of IL-4, IL-13, IFN-gamma in culture supernatants of PBMC stimulated with PHA and plasma total IgE level. RESULTS: (1) The CD86 MFI on CD(14)(+) cell in asthma group was elevated significantly (31.8 +/- 9.2 vs 23.5 +/- 6.4, P < 0.01). (2) CD(19)(+) cell percentage and CD19 and CD86 double positive cell percentage in PBMC stimulated with LPS in asthma group were increased significantly (13.5 +/- 4.0 and 4.6 +/- 2.0 vs. 8.2 +/- 3.0 and 2.3 +/- 1.4, respectively, P < 0.01). The plasma total IgE level [186.6 (64.3 - 723.6)] was higher than that of control's [41.95 (0.9 - 115.2)]. (3) The levels of IL-4 and IL-13 in supernatants stimulated with PHA increased significantly [34.2 (2.8 - 70.0) and 1 239.0 (378.3 - 2 929.0) vs. 9.7 (2.0 - 46.2) and 683.2 (128.8 - 1 560.8) respectively, P < 0.01, P < 0.05]; and the level of IFN-gamma decreased significantly [12.16 (1.6 - 44.8) vs. 26.7 (2.1 - 99.5) P < 0.05]. (4) In asthma group there was a significantly positive correlation of CD86 MFI on CD(14)(+) cell with CD19 and CD86 double positive cell percentage (r = 0.644), there was a significantly positive correlation of CD(19)(+)CD(86)(+)cells percentage with plasma total IgE (r = 0.537); there was a significantly positive correlation of CD(19)(+)cells percentage with the levels of IL-4 and IL-13 (r = 0.607, 0.540 respectively); a significantly positive correlation of CD(19)(+)CD(86)(+) percentage cells with the levels of IL-4 and IL-13 was found (r = 0.617, 0.678, respectively). CONCLUSIONS: (1) The expression of CD86 on APC cells increased in children with acute asthma. (2) The response to mitogens of B lymphocyte and T(H2) but not T(H1) lymphocyte were increased significantly. The results suggest that CD86 and imbalance of T(H) subset may play an important role in asthma pathogenesis.