Enhancement of A82846B yield and proportion by overexpressing the halogenase gene in Amycolatopsis orientalis SIPI18099.

The glycopeptide antibiotic A82846B (chloroeremomycin) produced by Amycolatopsis orientalis is the precursor of the semi-synthetic antibiotic oritavancin. However, during the industrial production of A82846B, two major impurities, A82846A (63.6%) and A82846C (12%) which are structurally similar to A82846B (24.4%), are also produced. In this study, to improve the ratio of A82846B to A and C, the genes encoding halogenase in A82846B and vancomycin synthesis were integrated into A. orientalis SIPI18099 to test their halogenation ability, respectively. The results indicated that chal from the A82846B biosynthesis pathway was more efficient in reducing A and C factors. Moreover, by increasing the chal copy number, the proportion of A and C were gradually reduced while the titer and proportion of A82846B were improved. In a scaled-up industrial process, the proportion of A and C were decreased to 11.6% and 0.2% in the recombinant strain A.orientalis chal-3 with three gene copies of chal and the titers of A82846B (2.2 g/L) has increased by 2.8-folds compared to 780 mg/L produced by the parental strain, suggesting that the recombinant strain was suitable for the industrial production of A82846B with lower impurities.

[Extraction Method of Malaria Parasite DNA from Preserved Positive Blood Smears].

OBJECTIVE: To develop a method for DNA extraction from malaria parasites on preserved blood smears, to provide basis for research on malaria genetic traceability. METHODS: The improved DNA extraction kit (QIAamp DNA Mini Kit) was used to extract plasmodium DNA from 41 giemsa-stained blood smears, and the extraction was compared with that using the Chelex-100 and Na(2)HPO(4) methods. Nested PCR was used to amplify small subunit ribosomal RNA to identify Plasmodium parasite. The PCR products underwent sequencing and sequence alignment, to analyze the difference in PCR positive rates between blood smears prepared in the 1980s and in recent 10 years, between blood smears with and without deoil/decoloration, and between blood smears with different qualities. RESULTS: The total PCR positive rate for the improved kit method was 70.7% (29/41). The PCR positive rate for blood smears prepared in the 1980s and in recent 10 years was 78.6% (11/14) and 66.7% (18/27) respectively, with no significant difference (W=0.63, P>0.05). The PCR positive rate for blood smears with and with- out deoil/decoloration was 62.5% (15/24) and 82.4% (14/17) respectively, also with no significant difference (χ(2)= 1.89, P>0.05). However, the PCR positive rate was significantly higher in blood smears with high quality [93.3% (28/30)] than those with low quality [9.1%(1/1l)](=27.59, P<0.01). Sequence alignment showed that the PCR products were consistent with the target DNA fragments. However, DNA extracted using the Chelex-100 and Na(2)HPO(4) methods showed negative PCR results. CONCLUSIONS: DNA extracted from blood smears prepared in the 1980s using the improved Kit (QIAamp DNA Mini Kit) shows a high PCR positive rate. Besides, blood smear staining and use of oil for microscopic examination do not affect DNA extraction.

[Application of Nested PCR in the Diagnosis of Imported Plasmodium Ovale Infection].

OBJECTIVE: To identity Plasmodium ovale infection by 18S rRNA gene nested PCR. METHODS: Whole blood and filter paper blood samples of malaria patients in Shandong Province were collected during 2012-2013. The parasites were observed under a microscope with Giemsa staining. The genome DNA of blood samples were extracted as PCR templates. Genus- and species-specific primers were designed according to the Plasmodium 18S rRNA gene sequences. Plasmodium ovale-positive specimens were identified by nested PCR as well as verified by sequencing. RESULTS: There were 7 imported cases of P. ovale infection in the province during 2012-2013. Nested PCR results showed that the P. ovale specific band (800 bp) was amplified in all the 7 specimens. Blast results indicated that the PCR products were consistent with the Plasmodium ovale reference sequence in GenBank. CONCLUSION: Seven imported cases of ovale malaria in Shandong Province in 2012-2013 are confirmed by nested PCR.

Enterovirus infections are associated with white matter damage in neonates.

AIM: To explore the imaging findings of neonatal infants infected with enteroviruses. METHODS: A retrospective study was conducted on 12 patients who were diagnosed with encephalitis caused by enterovirus. Clinical presentation, cranial ultrasonography (cUS), magnetic resonance imaging (MRI) findings and neurodevelopment outcome of 12 cases were analysed. RESULTS: Twelve infants, with a gestational age of 35 to 39 weeks, presented at 36 to 41 weeks postmenstrual age with clinical symptoms of enterovirus infections. Ten of 12 neonatal infants had a fever and 4 of 12 presented with a sepsis-like illness. cUS in one preterm infant showed periventricular echogenicity. Neonatal MRI confirmed white matter changes in 12 infants. Follow-up of infants were 18 months. Outcome was variable with cerebral palsy in 2 infants and normal neurodevelopment outcome in 10 infants. CONCLUSIONS: Enterovirus may cause severe central nervous system infection in the neonatal period. The neuroimaging studies are informative and should be a part of care for infants with enteroviruses.

Virus load and virus shedding of SARS-CoV-2 and their impact on patient outcomes.

BACKGROUND: Understanding a virus shedding patterns in body fluids/secretions is important to determine the samples to be used for diagnosis and to formulate infection control measures. AIM: To investigate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shedding patterns and its risk factors. METHODS: All laboratory-confirmed coronavirus disease 2019 patients with complete medical records admitted to the Shenzhen Third People's Hospital from January 28, 2020 to March 8, 2020 were included. Among 145 patients (54.5% males; median age, 46.1 years), three (2.1%) died. The bronco-alveolar lavage fluid (BALF) had the highest virus load compared with the other samples. The viral load peaked at admission (3.3 × 108 copies) and sharply decreased 10 d after admission. RESULTS: The viral load was associated with prolonged intensive care unit (ICU) duration. Patients in the ICU had significantly longer shedding time compared to those in the wards (P < 0.0001). Age > 60 years [hazard ratio (HR) = 0.6; 95% confidence interval (CI): 0.4-0.9] was an independent risk factor for SARS-CoV-2 shedding, while chloroquine (HR = 22.8; 95%CI: 2.3-224.6) was a protective factor. CONCLUSION: BALF had the highest SARS-CoV-2 load. Elderly patients had higher virus loads, which was associated with a prolonged ICU stay. Chloroquine was associated with shorter shedding duration and increased the chance of viral negativity.

Congenital Hyperinsulinism in China: A Review of Chinese Literature Over the Past 15 Years.

OBJECTIVE: Congenital hyperinsulinism (CHI) is a rare but severe cause of hypoglycemia. The present study investigates the clinical presentation, therapeutic outcomes and genetic mutations of CHI in Chinese individuals over the past 15 years. METHODS: The authors retrospectively reviewed one case in their department and 206 cases reported from January 2002 to October 2016 in China. PubMed, Ovid Medline, Springer and Wanfang Database, CBMD database, and CKNI database were the sources used to collect the data. RESULTS: In total, 207 cases were recruited. Of these, the ages of 100 (48.3%) were within the 4th week after birth. Seventy-seven cases (37.2%) were born large for gestational age (LGA). Seizures occurred in 140 cases (67.6%). Among 140 cases (67.6%) who were administered diazoxide treatment, 90 (64.3%) were responsive. Seven cases (3.4%) received octreotide treatment and 19 cases (9.2%) underwent surgery. 63/129 cases (48.8%) were detected to have gene mutations, including ABCC8 (69.8%), KCNJ11 (12.7%), GLUD1, GCK, HADH, and HNF4A. Among the diazoxide-unresponsive cases, gene mutations were detected in 20/36 (55.6%) cases with ABCC8 and in 2 (5.6%) cases with KCNJ11. Among the diazoxide-responsive cases, gene mutations were detected in 8 patients with ABCC8, 4 with KCNJ11, 5 with GLUD1, and 1 with GCK. CONCLUSION: The present study indicates that most CHI cases occurred in neonates and that 1/3 of the cases were born LGA. ABCC8 and KCNJ11 are the most common gene mutations. More than half of the diazoxide-unresponsive CHI detected mutations are in ABCC8 and KCNJ11 genes. The GLUD1 gene mutations cause diazoxide-responsive CHI. Identifying the gene mutations can assist in the diagnosis and treatment of CHI.

SIRT3 inhibits cell proliferation in human gastric cancer through down-regulation of Notch-1.

SIRT3 is a member of the NAD+-dependent class III deacetylase sirtuin family and plays pivotal roles in regulating cellular functions. Accumulating evidence has recently demonstrated that SIRT3 may function as either oncogene or tumor suppressor in a panel of cancers. However, the biological function of SIRT3 in gastric cancer has been poorly characterized. The present study revealed that the mRNA and protein levels of SIRT3 were significantly reduced in human gastric cancer tissues and cell lines. In addition, overexpression of SIRT3 dramatically suppressed the proliferation ability and colony formation number of gastric cancer cells. By contrast, SIRT3 knockdown using small interfering RNA enhanced tumor cell growth and colony formation. On the molecular level, we found that SIRT3 inhibited the expression of Notch-1 both at the mRNA and protein levels in gastric cancer cells. Furthermore, Notch-1 overexpression diminished the inhibitory effects of SIRT3 on tumor cells proliferation. Taken together, these results demonstrated that SIRT3 suppressed the proliferation gastric cancer cells via down-regulation of Notch-1, which might provide novel therapeutic targets in the gastric cancer therapy.

[Cloning, expression, purification and identification of Toxoplasma gondii SAG2 gene in Escherichia coli].

OBJECTIVE: To construct a recombinant plasmid containing surface antigen 2(SAG2) gene of Toxoplasma gondii and express it in Escherichia coli. METHODS: The truncated SAG2 gene was amplified from the genomic DNA of T. gondii RH strain and cloned into plasmid pGEX-4T. Then the recombinant pGEX-4T-SAG2 was induced by IPTG and expressed in E. richia col BL21. The expressed proteins were analyzed by SDS-PAGE and purified, and the immunogenicity of the product was analyzed by Western blotting. RESULTS: The amplified SAG2 gene was about 561 bp, which was accorded to the expectation. The recombinant plasmid was constructed successfully by digested with double restriction enzyme and confirmed with DNA sequencing. SDS-PAGE and Western blotting showed the molecular weight of SAG2 fusion protein was about 47 ku, and the protein could be identified by GST-tag antibody. CONCLUSION: The truncated SAG2 gene of T. gondii has been successfully cloned and expressed in E. coli BL21 cells, and the recombinant protein has immunogenicity.

Pyrosequencing reveals fungal communities in the rhizosphere of Xinjiang Jujube.

Fungi are important soil components as both decomposers and plant symbionts and play a major role in ecological and biogeochemical processes. However, little is known about the richness and structure of fungal communities. DNA sequencing technologies allow for the direct estimation of microbial community diversity, avoiding culture-based biases. We therefore used 454 pyrosequencing to investigate the fungal communities in the rhizosphere of Xinjiang jujube. We obtained no less than 40,488 internal transcribed spacer (ITS) rDNA reads, the number of each sample was 6943, 6647, 6584, 6550, 6860, and 6904, and we used bioinformatics and multivariate statistics to analyze the results. The index of diversity showed greater richness in the rhizosphere fungal community of a 3-year-old jujube than in that of an 8-year-old jujube. Most operational taxonomic units belonged to Ascomycota, and taxonomic analyses identified Hypocreales as the dominant fungal order. Our results demonstrated that the fungal orders are present in different proportions in different sampling areas. Redundancy analysis (RDA) revealed a significant correlation between soil properties and the abundance of fungal phyla. Our results indicated lower fungal diversity in the rhizosphere of Xinjiang jujube than that reported in other studies, and we hope our findings provide a reference for future research.

Ni3S2/Ni-P bilayer coated on polyimide as a Pt- and TCO-free flexible counter electrode for dye-sensitized solar cells.

In this study, we reported an efficient, flexible, and low-cost (Pt-free and transparent conducting oxide (TCO)-free) counter electrode (CE) made of a polyimide (PI) substrate coated with a Ni3S2/Ni-P bilayer for dye-sensitized solar cells (DSCs). The bilayer Ni3S2/Ni-P hybrid film was deposited on a PI plastic substrate via a series of wet chemical/electrochemical processes. The bottom Ni-P layer was deposited on a PI to replace conventional TCO as a conductive layer, and the top Ni3S2 layer was employed as the electrocatalyst for I3(-) reduction. On the basis of the extensive electrochemical measurments, the as-prepared Ni3S2/Ni-P coated PI flexible CE demonstrated a Pt-like electrocatalytic for I3(-) reduction. As a result, the DSC assembled with the Ni3S2/Ni-P coated PI flexible CE exhibited an impressive photovoltaic conversion efficiency of 6.28% accompanied by a fill factor of 0.63 under 1 sun illumination (100 mW cm(-2), AM 1.5), which is comparative to that of the DSC based on the Pt coated indium tin oxide/polyethylene naphthalate (ITO/PEN) CE.

[Clinical observation on abdominal electroacupuncture for treatment of poststroke constipation].

OBJECTIVE: To compare therapeutic effects of abdominal electroacupuncture (EA) and western medicine on poststroke constipation. METHODS: Eighty cases were randomly divided into an EA group and a medication group, 40 cases in each group. The EA group were treated with EA at Daheng (SP 15), Fujie (SP 14), Tianshu (ST 25), Shuidao (ST 28), etc., once a day, 30 min each session, and the medication group with oral administration of 10 mg Cisapride, thrice each day. Seven days constituted one course. After 2 courses, clinical therapeutic effects were evaluated by cumulative scores of symptoms. RESULTS: The total effective rate of 92.5% in the EA group was significantly better than 72.5% in the medication group (P < 0.05). After treatment, the cumulative scores of clinical symptoms significantly decreased in the two groups (P < 0.05) and the improving degrees of symptoms in the EA group was significantly better than that in the medication group (P < 0.05). CONCLUSION: Abdominal electroacupuncture has a definite therapeutic effect on poststroke constipation, accelerating gastrointestinal movement.