Khảo sát điều kiện trích ly và tinh sạch lectin từ đậu ma (Pueraria phaseoloides) bằng sắc ký ái lực

Nghiên cứu được thực hiện nhằm xác định điều kiện trích ly và tinh sạch lectin từ đậu ma Pueraria phaseoloides. Lectin đậu ma được trích ly cùng với dung dịch NaCl 0,9% ở các tỷ lệ (w/v), thời gian và nhiệt độ ủ khác nhau. Dịch chiết thô được tinh sạch bằng phương pháp tủa phân đoạn với muối ammonium sulfate, tiếp theo là sắc ký ái lực trên gel Sepharose D-galactose để cải thiện độ tinh sạch. Kết quả cho thấy lectin đậu ma đạt hiệu quả trích ly tối ưu với hoạt tính đặc hiệu đạt là 1.579 HAA (Hemagglutination assay)/mg ở tỉ lệ với dung môi trích ly là 1:4 (w/v), tại 50oC, trong 10 phút. Dịch trích lectin đậu ma sau khi tủa phân đoạn ở nồng độ muối 40% - 50% cho hiệu suất thu hồi 35,4% với độ tinh sạch tăng 6,38 lần so với dịch trích thô; trong khi phân đoạn F1 từ sắc ký ái lực cho hiệu suất thu hồi 9,85% với độ tinh sạch tăng 16,2 lần. Kết quả điện di SDS-PAGE xuất hiện hai băng protein có khối lượng phân tử 66,0 kDa và 56,0 kDa.

Lectin Activity in Commonly Consumed Plant-Based Foods: Calling for Method Harmonization and Risk Assessment

Lectins are ubiquitous proteins characterized through their ability to bind different types of carbohydrates. It is well known that active lectins from insufficiently prepared legumes can cause adverse human health effects. The objective of this study was to determine the activity of lectins in samples across plant families representing commercially available edible plants, and the feasibility of inactivating lectins through soaking and boiling. Lectins were extracted from the plant families Adoxaceae, Amaranthaceae, Cannabaceae, Fabaceae, Gramineae, Lamiaceae, Linaceae, Pedaliaceae, and Solanaceae. A hemagglutination assay based on non-treated or trypsin treated rabbit erythrocytes was used to measure the lectin activity. The results showed the highest lectin activity in species from the Fabaceae family and demonstrated that soaking and boiling have an effect on the levels of active lectins. This is the first large study that combines lectin activity obtained from two different assays with raw and processed edible plants. In addition, we examined the current risk assessment, and regulations necessary for an adequate official reporting of results. We encourage the scientific community to further explore this field and agree on harmonized methods for analysis and interpretation, and hope that our methodology can initiate this development.

Melioidosis: Laboratory Investigations and Association with Patient Outcomes

Melioidosis is an infection caused by the bacterium Burkholderia pseudomallei. The most common presentation is bacteremia occurring in 38–73% of all patients, and the mortality rate ranges from 9% to 42%. Although there is abundant data representing risk factors for infection and patient outcomes, there is limited information regarding laboratory investigations associated with bacteremia and mortality. We assessed a range of baseline and diagnostic investigations and their association with patient outcomes in a retrospective cohort study in Townsville, Australia. About 124 patients’ medical and laboratory records were reviewed between January 1, 1997 and December 31, 2020. Twenty-seven patients died and 87 patients were bacteremic. The presence of lymphopenia (< 1.5 × 109 cells/L) was the highest risk for bacteremia (relative risk [RR] 2.2; 95% CI: 1.3–3.7, P < 0.001). Factors associated with mortality included lymphopenia, (RR: 1.4; 95% CI: 1.2–1.6, P = 0.004); uremia (RR: 1.7; 95% CI: 1.1–2.5, P = 0.03); and an elevated international normalized ratio (RR: 1.5; 95% CI: 1.2–2.0, P = 0.006). Median incubation to positive blood culture result was 28 hours with 15/82 (18%) positive in ≤ 24 hours. For serological testing during admission only 53/121 (44%) were indirect hemagglutination assay positive, 67/120 (56%) enzyme immunoassay IgG positive, and 23/89 (26%) IgM positive. Simple baseline investigations at time of presentation may be used to stratify patients at high risk for both bacteremia and mortality. This information can be used as a decision aid for early intensive management.

Evaluation of In Ovo Antiviral Activities of Medicinal Flowers against Newcastle Disease Virus and Avian Influenza Virus

In Pakistan, the poultry industry is one of the rapidly growing industries. Due to lack of biosecurity measures, this is affected by some important infectious agents such as Avian Influenza virus (H9N2) and Newcastle disease virus (NDV) results in a huge economic loss. So, to control these losses discovery of new anti-viral drugs required to bring into line to fight against these infections. It is a general perception that the active components of medicinal plants have effective results against various infections like the influenza virus. The current therapeutic facilities need to be improved by investigating new antiviral drugs from natural resources to fight against viral infections. The present study was conducted on ethanolic extracts of seven different flowers to examine their antiviral activity against NDV and H9N2 in ovo using chicken embryonated egg inoculation. The spot agglutination and hemagglutination tests showed inhibitory effects of Rosa damascena Miller, Achillea millefolium, Woodfordia fruticosa Kurtz and Bombax ceiba L. against NDV as no agglutination observed. While the extracts of Taxacum officianale Weber, Hyssopus officianalis L. and Chrysanthemum cinerafolium (Trevis.) Vis. showed positive results for both spot agglutination and hemagglutination assay against NDV. However, both spot agglutination and hemagglutination assay showed inhibitory effect of all the flowers extracts against H9N2. The bioactive components such as alkaloids, ethers, terpenoids, etc. of each flower were analyzed through Gas chromatography mass spectrometry (GC-MS). The current results revealed that ethanolic extracts of these flowers possess strong antiviral activity because of their active ingredients. These ingredients should be isolated, commercialized and used for therapeutic purpose. © 2021 Friends Science Publishers

Comparison of Dot ELISA Using GroEL Recombinant Protein as an Antigen and an Indirect Hemagglutination Assay for Serodiagnosis of Melioidosis

Background: Melioidosis is a disease caused by the Burkholderia pseudomallei bacterium. The mortality rate of infected patients is quite high because the symptoms are similar to those of various diseases, making it difficult to diagnose clinically and preventing the immediate treatment with effective antibiotics that is required for the management of acute infections. To provide appropriate treatment, accurate and rapid diagnosis is required. Objective: The aims of this study were to develop Dot ELISA using purified GroEL B. pseudomallei recombinant protein as an antigen and to compare the newly developed assay with an indirect hemagglutination assay (IHA) for the diagnosis of melioidosis. Methods: The GroEL recombinant protein was purified by immobilized metal affinity chromatography before being used as an antigen. The optimal conditions of the Dot ELISA were determined and used for subsequent experiments. A total of 291 serum samples were evaluated by the established Dot ELISA and IHA, using the bacterial culture method as the gold standard of melioidosis diagnosis. Results: The results from Dot ELISA and IHA revealed sensitivity, specificity, and accuracy of 85.7% (Dot ELISA)/64.3% (IHA), 94.4%/85.5%, and 93.1%/82.5%, respectively. Conclusion: These results indicate that the Dot ELISA developed is an efficient, simple, rapid and cost-effective technique for the early diagnosis of melioidosis and can be used in a local laboratory without specialized equipment.

Zingerone improves the immune responses in an animal model of breast cancer

Objectives The potent anti-tumorigenic effects were attributed to ginger and there are some reports regarding the anti-cancer and immunomodulatory properties ginger-derived components. This study aimed to investigate the effects of zingerone on some immune-related parameters in an animal model of breast cancer. Methods The breast cancer was established in female BALB/c mice using a carcinogenic 4T1 cell line. At day 10 after cancer induction, tumor-bearing mice were divided into five groups and treated intraperitoneal (daily from days 11–30) with saline or zingerone (at doses 10, 20, 50 and 100 mg/kg/day). The mice were sacrificed on day 31 and the number of splenic Th1- and Treg cells, the expression of IFN-γ and TGF-β in the blood mononuclear cells, the antibody production against sheep red blood cell (SRBC) were determined using flow cytometry, real time-PCR and a standard hemagglutination assay, respectively. Results Zingerone at doses 50 and 100 mg/kg enhanced the number of splenic Th1 cells (p<0.03 and 0.007, respectively); at doses 10, 20, 50 and 100 mg/kg reduced the number of splenic Treg cells (p<0.02, 0.01, and 0.01, respectively), at doses 50 and 100 mg/kg enhanced the expression of IFN-γ (p<0.03), at doses 50 and 100 mg/kg reduced the expression of TGF-β, at doses 50 mg/kg reduced the titer of anti-SRBC antibody (p<0.05). Conclusions Zingerone improve the T cell-mediated and antibody responses in a mouse model of breast cancer. The immunotherapeutic potentials of zingerone in cancers need more considerations.

Investigation of the rapid immunochromatographic test performance in the diagnosis of syphilis; comparison of four serological methods

ObjectivesTo test the performance of the newly available rapid test for syphilis, we compared it with Treponema pallidum hemagglutination assay (TPHA). Additionally, we investigated the performance of rapid plasma reagin (RPR) and chemiluminescence microparticle immunoassays (CMIA) at our laboratory using TPHA as a gold standard.MethodsThe serum samples of 595 patients with the pre-diagnosis of syphilis were studied by four serological methods. The sensitivity, specificity, and predictive values of RPR, CMIA, and syphilis rapid test were assessed by utilizing TPHA as a gold standard for the diagnosis of syphilis.ResultsOf the patients, 6.2% (37/595) had positive RPR, 5.5% (33/595) had positive CMIA, 5.5% (33/595) had a positive rapid immunochromatographic method and 5% (30/595) had positive TPHA. When TPHA results were taken as the reference, the sensitivity of the rapid test for syphilis was 100%, the specificity was 99.5%, PPV was 90.9%, and NPV was 100.0%.ConclusionsIt was observed that the rapid test for syphilis used in the study was quite successful, its cost was appropriate, and the test was very fast and easy to apply. At the same time, the agreement between syphilis rapid test and TPHA was found to be excellent.

Sialylation and fucosylation changes of cytidine monophosphate-Nacetylneuraminic acid hydroxylase (CMAH) and glycoprotein, alpha1,3-galactosyltransferase(GGTA1) knockout pig erythrocyte membranes

The recent GGTA1 and CMAH DKO pigs have made it possible to resolve the immune barriers which are duo to xenoantigens on RBC such as αGal and Neu5Gc. Nevertheless, it still requires the detection of glycosylation alternation on the pig RBCs because even the minor changes would be unexpected xenoantigens.DKO RBC immune reactivity with human serum was assessed by hemagglutination assay. Glycosylation alteration of RBC membranes was characterized by NanoLC-Q-TOF-MS system and lectin blotting assay.Twelve GGTA1/CMAH DKO piglets were successfully produced. The immunoreactivity with human serum was remarkably reduced in DKO (less than 1:2 dilution), whereas wild type(WT) pigs showed agglutination (the least 1:256 dilution). The MS results showed that DKO increased neutral N-glycans as well as decreased total sialylated N-glycans, especially suggesting significant decrease of di-sialylated N-glycans (P < 0.05). Moreover, lectin blotting assay revealed that DKO pigs reduced the binding signals with AAL, AOL, LCA and SNA and increased the binding signal with MAL.DKO pigs decreased the expression of total fucosylation and sialylated N-glycans on the erythrocyte membrane. Our findings will support further investigation into DKO pig RBC glycosylation and contribute to uncover the roles of glycan changes for xenotransfusion.Summary statementTo detect glycosylation changes in red blood cells(RBC) of GGTA1/CMAH double knockout(DKO) pigs, comparative analysis of the glycan profiling was done.

Method for evaluating the neuraminidase activity of receptor-destroying enzyme (RDE) compounds using the influenza virus

Introduction. The classic hemagglutination inhibition reaction (RTGA) is used to determine the level of antiviral antibodies in human and animal serum specimens. During the performance of RTGA the tested sera must be treated with a receptor-destroying enzyme (RDE) to remove serum glycans that degrade the accuracy of the RTGA results. To optimize the amounts of RDE compounds used, it is necessary to know their real neuraminidase activity. This article describes a simple and economical method for testing the neuraminidase activity of receptor-destroying compounds using standard reagents and laboratory equipment.Aims of investigation. Design of an improved simple and convenient method for evaluating the neuraminidase activity using the flu virus.Material and methods. Here, we propose a convenient method for evaluating the activity of neuraminidase by double-fold dilution procedure with human or animal erythrocytes followed by hemagglutination assay with influenza A virus.Results and discussion. The method is based on the ability of neuraminidase to hydrolyze sialic acid residues on the cell surface of erythrocytes, that deprives red blood cells to be agglutinated with the flu virus, since these sialic glycans provide virus attachment and hemagglutination.Conclusion. The designed method allows the accurate measurement of the receptor-destroying (neuraminidase) activity of RDE compounds and the comparison of the compounds with each other. This test is necessary to optimize the RTGA protocol when monitoring blood sera of animals and humans after influenza infection and/or Acute Respiratory diseases (ARD). The designed method can be included in the guidelines of regulations for the RTGA protocol, which is used in different laboratories to monitor the epidemic process of influenza and ARD infections.