Expression of flagellin FLjB derived from Salmonella enterica serovar typhimurium in Escherichia coli BL21

Flagellin FljB composes flagellar antigen (H:1,2) of S. Typhimurium. This kind of antigen increases immunogenicity of any conjugated antigen upon administration. Thus, it is supposed to have an enormous potentiality for vaccine development against bacterial infections and cancer diseases. fljB gene (1515 nucleotides) coding for mature FljB was amplified by PCR from genomic DNA of S. Typhimurium and inserted into pET32a(+) for expression in E. coli BL21. The protein FljB was well expressed under the fusion form with Trx, S-tag at N terminal and hexa-histidine at C terminal, thus the recombinant protein was abbreviated to TrxFljB. Study on the impact of temperature on the gene expression showed that TrxFljB was synthesized at lower level at 37oC comparing to the levels at 22oC and 25oC. 13% of the protein synthesized at 37oC was inclusion body. Lower temperatures used during induction phase increased the solubility of the recombinant protein. About 97% of TrxFljB synthesized at 25oC was soluble. IPTG concentration had a strong effect on the growth of freshly transformed cells but did not affect on the growth of stored and re-cultivated cells. The increase of IPTG concentration resulted in the decrease of the growth of freshly transformed cells and the TrxFljB productivity. However, 0.05 mM IPTG concentration was found to gain the full TrxFljB expression. TrxFljB productivity declined during storage of cells at 4oC and re-cultivation. At optimal condition, volumetric productivity of TrxFljB was about 300 mg/ l broth.

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus

<p>Abstract</p> <p>Background</p> <p><it>Salmonella enterica </it>serotype Enteritidis (SE) is considered to be one of the most potent pathogenic <it>Salmonella </it>serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in <it>Escherichia coli </it>by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in <it>Staphylococcus carnosus</it>, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes.</p> <p>Results</p> <p>Both SefA and H:gm were translocated to the outer membrane in <it>Escherichia coli</it>. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His₆) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from <it>Staphylococcus carnosus </it>suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis.</p> <p>Conclusion</p> <p>Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of <it>E. coli </it>for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in <it>Staphylococcus carnosus </it>shows that the distribution of the surface protein on each cell was comparatively very narrow in <it>E. coli</it>, the <it>E. coli </it>outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the <it>Escherichia coli </it>AIDA autotransporter efficiency.</p

In silico mining for alkaline enzymes from metagenomic dna data of gut microbes of the lower termite Coptotermes gestroi in Vietnam

The high alkaline proteases, lipases, cellulases and hemicellulases are important enzymes in research and industries. In this study, using the Alcapred software, the metagenomic DNA sequences of  the gut flora of Coptotermes gestroi were analyzed to identify the enzymes that were specifically adapted to alkaline condition. The results show that 737 of 943 ORFs (accounting for 72%) encoded proteases, 154 of 214 ORFs (holding 78%) encoded lipases and 338 of 575 ORFs (accounting for 59%) encoded cellulase and hemicelluase. All those enzymes were predicted to be alkaline enzymes. This study provide an overview picture of the alkaline enzyme groups of the gut flora of C. gestroi, and provide a good database for mining, isolation of the genes to produce recombinant enzymes

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

Expression of the recombinant single chain variable fragments recognizing blood antigen fused with thioredoxin in Escherichia coli

The technology of recombinant single chain variable fragments (scFvs) expression has been used in research, diagnosis and treatment of diseases. In the previous study, we studied the expression of a recombinant single chain variable fragment recognizing blood A antigen (antiA-scFv) in E. coli. However, the protein was insoluble form resulting in difficulty for purification, refolding and activity assesment. Here, we present the study on fused expression of the recombinant scFv -specific blood A antigen with thioredoxin (Trx) in the expression vector pET32a (+). The results showed that the Trx/antiA-scFv fusion protein was expressed with molecular weight of 49 kDa in a soluble form reaching 40% of the total recombinant protein. This result facilitates the optimal condition of soluble protein expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

A problem of self-isolation in Japan: The relationship between self-isolation and COVID-19 community case

Background: The Japanese government advised mild or asymptomatic　coronavirus disease-2019 (COVID-19) cases to self-isolate at home, while more severe individuals were treated at health posts. Poor compliance with self-isolation could be a potential reason for the new outbreak. Our study aimed to find out the correlation between the rising new cases of COVID-19 and home-based patients in Japan.Methods: A secondary data analysis study was conducted with the data from COVID-19-involved databases collected from Johns Hopkins University, Japanese Ministry of Health, Labour and Welfare, and Community Mobility Reports of Google. New community cases, stringency index, number of tests, and active cases were analyzed. Using a linear regression model, an independent variable was utilized for a given date to predict the future number of community cases.Results: Research results show that outpatient cases, the stringency, and Google Mobility Trend were all significantly associated with the number of COVID-19 community cases from the sixth day to the ninth day. The model predicting community cases on the eighth day (R2=0.8906) was the most appropriate showing outpatients, residential index, grocery and pharmacy index, retail and recreation index, and workplaces index were positively related (β1=24.2, 95% CI: 20.3–26.3, P<0.0001; β2=277.7, 95% CI: 171.8–408.2, P<0.0001; β3=112.4, 95% CI: 79.8–158.3, P<0.0001; β4=73.1, 95% CI: 53- 04.4, P<0.0001; β5=57.2, 95% CI: 25.2–96.8, P=0.001, respectively). In contrast, inpatients, park index, and adjusted stringency index were negatively related to the number of community cases (β6=-2.8, 95% CI: -3.9 – -1.6, P<0.0001; β7=-33, 95% CI: -43.6 – -27, P<0.0001; β8=-14.4, 95% CI: -20.1– -12, P<0.0001, respectively).Conclusion: Outpatient cases and indexes of Community Mobility Reports were associated with COVID-19 community cases

Results of acute cerebral infarction treatment with hyperbaric oxygen therapy, 2020–2022

Background: Cerebral stroke is the third leading cause of death after cardiovascular disease, cancerand the leading cause of disability for patients. Hyperbaric oxygen is a non-drug treatment that hasthe potential to improve brain function for patients with ischaemic stroke. The objective of this study wasto evaluate the results of treatment of acute cerebral infarction with hyperbaric oxygen therapy (HBOT).Materials and methods: This was a case-control study. One hundred ninety-five patients diagnosed withcerebral infarction, with signs of onset within 24 hours, were treated at the Centre for Underwater Medicineand Hyperbaric Oxygen of Vietnam National Institute of Maritime Medicine during the period from January2020 to December 2022. Study group included 100 patients with acute cerebral infarction treated witha combination of HBOT and medication and reference group included 95 patients treated by medicationonly (antiplatelets drugs, statins, control of associated risks factors)Results: After 7 days of treatment with hyperbaric oxygen (HBO), symptoms such as headache, dizziness,nausea, sensory disturbances, and Glasgow score of the study group improved better than that of the referencegroup (p &lt; 0.01). Movement recovery in the study group was better than the reference group:the percentage of patients with mild and moderate paralysis in the study group increased higher than thatof the reference group (86.0% and 68.4%), the degree of complete paralysis of the study group decreasedmore than that of the reference group (14.0% and 31.6%). The degree of independence in daily activitiesin the study group was better than the reference group. In the study group, the percentage of patients withcomplete independence in daily life increased from 27.0% to 84.0%. In the reference group, the rate of patientswho were independent in their daily activities increased from 37.9% to 51.6%. The average numberof treatment days of the study group was 10.32 ± 2.41 days and it the reference group 14.51 ± 3.24 days.Conclusions: Hyperbaric oxygen therapy is a non-drug treatment with many good effects in the treatmentof cerebral infarction, especially acute cerebral infarction. HBOT reduces and improves functional symptoms,improves mobility, and reduces treatment time for patients

NGHIÊN CỨU TỐI ƯU ĐIỀU KIỆN BIỂU HIỆN INTERLEUKIN-3 NGƯỜI DUNG HỢP VỚI PelB TRONG E. coli

Interleukin-3 (IL-3) is a multifunctional cytokine which modulates the proliferation, differentiation and maturation of various types of hematopoietic cells. Gene coding for IL-3 linked with pelB signal were incorporated into pET22b(+) for  expression of il3 gene in E. coli BL21; but IL-3 was synthesized at very low levels and still in fusion with PelB. To facilitate purification process, finding the appropriate fermentation conditions plays a key role in order to enhance gene il-3 expression and cleavage of PelB. In this study, we have optimized the conditions for the expression of IL-3, which included E. coli host strain JM109, LB cultivation medium, induction temperature was 25oC; induction with 0.05 mM IPTG at OD600 = 1. The cell biomass increases at about 2.3 times after optimization. IL-3 protein was expressed in the form of inclusion body and the PelB signal was cleaved. This result is conducive for purification of large amount of IL-3 to determine characteristics of protein. Interleukin-3 người (IL-3) là một cytokine đa chức năng tham gia vào các quá trình tự đổi mới, nhân lên, biệt hóa và trưởng thành của nhiều loại tế bào máu. Sau khi đưa gen il-3 gắn thêm tín hiệu tiết pelB vào vector pET22b(+) và tiến hành biểu hiện ở chủng E. coli BL21, chúng tôi nhận thấy IL-3 được tổng hợp ở mức rất thấp và còn gắn với PelB. Để thuận tiện cho khâu tinh sạch thì vấn đề then chốt là nghiên cứu tìm ra các điều kiện phù hợp làm tăng lượng IL-3 được tổng hợp, đồng thời cắt được PelB. Trong nghiên cứu này, chúng tôi so sánh khả năng sinh tổng hợp IL-3 của các chủng E. coli BL21, JM109, Soluble và Rossetta2; sau đó tối ưu hóa điều kiện biểu hiện gen il-3 về thành phần môi trường, nhiệt độ, nồng độ IPTG, thời điểm cảm ứng và kiểm tra trạng thái tồn tại của IL-3. Kết quả thu được cho thấy gen il-3 biểu hiện tốt và ổn định nhất ở chủng E. coli JM109. Dưới các điều kiện lên men thích hợp trong môi trường LB, ở 25oC, cảm ứng 0,05 mM IPTG tại OD600=1, IL-3 biểu hiện tốt, cắt khỏi PelB và tồn tại ở trạng thái không tan trong tế bào chất. Sinh khối tế bào tăng lên khoảng 2,3 lần sau khi tối ưu. Kết quả này là tiền đề cho bước tinh sạch lượng lớn IL-3 cho nghiên cứu tính chất của protein

How primary healthcare workers obtain information during consultations to aid safe prescribing in low-income and lower middle-income countries: a systematic review.

Background: We systematically reviewed the evidence on how primary healthcare workers obtain information during consultations to support decision-making for prescribing in low and lower middle-income countries. Methods: We searched electronic databases, consulted the Healthcare Information For All network, hand searched reference lists, ran citation searches of included studies and emailed authors of identified papers. Two reviewers extracted data and appraised quality with relevant tools. Results: Of 60 497 records found, 23 studies met our inclusion criteria. Fourteen studies were observational and nine were interventional. Frequently mentioned sources of information were books, leaflets, guidelines, aids and the internet. These sources were sometimes out of date and health workers reported being confused which to use. Internet access varied and even when it was available, use was limited by technical issues. Of the five electronic tools that were assessed, four had positive outcomes. Tools assisted prescribers with medicine selection and dosage calculations, which increased prescribing accuracy. The quality of reporting varied but was overall low. Discussion: Studies indicated a lack of up-to-date and relevant medicine information in low and lower middle-income settings. Internet-based sources appeared to be useful when it is possible to download content for offline use and to update when there is internet access. Electronic tools showed promise, but their accuracy needs to be validated and they should focus on giving actionable advice to guide prescribers. PROSPERO registration number: CRD42018091088

Prevalence of arterial hypertension in Vietnamese seafarers aboard merchant vessels: a cross-sectional study

Background: Hypertension is one of the leading causes of morbidity and mortality globally. It is a major riskfactor for major cardiovascular events such as stroke, myocardial infarction, heart failure, kidney failure,and blindness. The aim of this research is to assess the prevalence and some factors related to arterialhypertension on Vietnamese seafarers aboard merchant vessels.Materials and methods: Seven hundred eight Vietnamese seafarers working aboard merchant ships wereexamined at the Institute of Marine Medicine before going to sea during the period from January 2022to December 2022. It was a cross-sectional descriptive epidemiological study. The following parameterswere measured: blood pressure, height, weight, waist circumference, buttock circumference to assess theprevalence of hypertension, overweight, and obesity. Seafarers we directly interviewed about workplaceon ships and physical exercise, smoking tobacco, alcohol abuse, and anxiety symptoms to identify severalfactors associated with hypertension.Results: The prevalence of hypertension in seafarers was 32.9%, prehypertension 26.4%, overweight32.4%, obesity 13.3%, abdominal obesity 47.7%. Factors related to hypertension of seafarers included: jobduration at sea &gt; 10 years, odds ratio (OR) = 8.23 (95% confidence interval [CI] 4.34–17.27); non-officers,OR = 2.11 (95% CI 1.45–2.82); engine room crew, OR = 2.11 (95% CI 1.45–3.58); obesity, OR = 3.34(95% CI 2.15–5.63); abdominal obesity, OR = 9.12 (95% CI 4.23–18.45); current smoking, OR = 1.32(95% CI 1.02–1.99); irregular exercise, OR =1.43 (95% CI 1.03–2.18); anxiety symptoms, OR = 1.56 (95%CI 1.08–2.27).Conclusions: Hypertension is a health problem for Vietnamese seafarers. To minimise hypertension, seafarersneed to adjust their lifestyle, increase regular exercise and improve psychological issues on board