Venom-gland transcriptomics of the Malayan pit viper (Calloselasma rhodostoma) for identification, classification, and characterization of venom proteins

The Malayan pit viper (Calloselasma rhodostoma) is a hemotoxic snake widely found in Southeast Asia and is responsible for the majority of poisoning cases in this region, including Thailand. However, a comprehensive knowledge of the venom protein profile and classification, as well as novel venom proteins, of this viper is still limited. Recently, the detailed composition of several snake venoms has been discovered through the use of transcriptome analysis. Therefore, the aim of this study was to employ a next-generation sequencing platform and bioinformatics analysis to undertake venom-gland de novo transcriptomics of Malayan pit vipers. Furthermore, 21,272 functional coding genes were identified from 36,577 transcripts, of which 314 transcripts were identified as toxin proteins, accounting for 61.41% of total FPKM, which can be categorized into 22 toxin gene families. The most abundant are snake venom metalloproteinase kistomin (P0CB14) and zinc metalloproteinase/disintegrin (P30403), which account for 60.47% of total toxin FPKM and belong to the SVMP toxin family, followed by snake venom serine protease 1 (O13059) and Snaclec rhodocetin subunit beta (P81398), which account for 6.84% and 5.50% of total toxin FPKM and belong to the snake venom serine protease (SVSP) and Snaclec toxin family, respectively. Amino acid sequences of the aforementioned toxins were compared with those identified in other important medical hemotoxic snakes from Southeast Asia, including the Siamese Russell's viper (Daboia siamensis) and green pit viper (Trimeresurus albolabris), in order to analyze their protein homology. The results demonstrated that ranges of 58%–62%, 31%–60%, and 48%–59% identity was observed among the SVMP, Snaclec, and SVSP toxin families, respectively. Understanding the venom protein profile and classification is essential in interpreting clinical symptoms during human envenomation and developing potential therapeutic applications. Moreover, the variability of toxin families and amino acid sequences among related hemotoxic snakes found in this study suggests the use and development of universal antivenom for the treatment of envenomating patients is still challenging

Diagnostic Performance of Dengue NS1 and Antibodies by Serum Concentration Technique

Dengue infection has been a public health problem worldwide, especially in tropical areas. A lack of sensitive diagnostic methods in the early phase of the illness is one of the challenging problems in clinical practices. We, herein, analyzed 86 sera of acute febrile patients, from both dengue and non-dengue febrile illness, to study the diagnostic performance of dengue diagnostics. When compared with detection by Polymerase Chain Reaction (PCR), dengue NS1 detection by enzyme-linked immunosorbent assay (ELISA) had the highest sensitivity of 82.4% (with 94.3% specificity), while NS1 by rapid diagnostic test (RDT) had 76.5% sensitivity. IgM detection by ELISA and RDT showed only 27.5% and 17.9% sensitivity, respectively. The combination of NS1 and IgM in RDT yielded a sensitivity of 78.4%, with 97.1% specificity. One of the essential steps in making a diagnosis from patient samples is the preparation process. At present, a variety of techniques have been used to increase the number of analytes in clinical samples. In this study, we focused on the sample concentration method. The sera were concentrated three times with the ultrafiltration method using a 10 kDa molecular weight cut-off membrane. The results showed an increase in the sensitivity of RDT-NS1 detection at 80.4%, with 100% specificity. When combining NS1 and IgM detection, the concentration method granted RDT an 82.4% sensitivity, with 100% specificity. In conclusion, serum concentration by the ultrafiltration method is a simple and applicable technique. It could increase the diagnostic performance of point-of-care dengue diagnostics

A novel mechanism for the promotion of quercetin glycoside absorption by megalo α-1,6-glucosaccharide in the rat small intestine

The presence of α-1,6-glucosaccharide enhances absorption of water-soluble quercetin glycosides, a mixture of quercetin-3-O-β-D-glucoside (Q3G, 31.8%), mono (23.3%), di (20.3%) and more D-glucose adducts with α-1,4-linkage to D-glucose moiety of Q3G, in a ligated small intestinal loop of anesthetized rats. We enzymatically prepared α-1,6-glucosaccharides with different degrees of polymerization (DP) and separated them into a megaloisomaltosaccharide-containing fraction (M-IM, average DP = 11.0) and an oligoisomaltosaccharide-containing fraction (O-IM, average DP = 3.6). Luminal injection of either saccharide fractions promoted the absorption of total quercetin-derivatives from the small intestinal segment and this effect was greater for M-IM than O-IM addition. M-IM also increased Q3G, but not the quercetin aglycone, concentration in the water-phase of the luminal contents more strongly than O-IM. The enhancement of Q3G solubilization in the luminal contents may be responsible for the increases in the quercetin glucoside absorption promoted by α-1,6-glucosaccharides, especially M-IM. These results suggest that the ingestion of α-1,6-glucosaccharides promotes Q3G bioavailability

Ov-RPA–CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces

Abstract Background Opisthorchis viverrini infection is traditionally diagnosed using the Kato–Katz method and formalin ethyl–acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O. viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)–clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA–CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA–CRISPR/Cas assay to detect O. viverrini infection in field-collected human feces. Methods To detect O. viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA–CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA–CRISPR/Cas12a assay for detecting O. viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA–CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. Results The Ov-RPA–CRISPR/Cas12a assay exhibited high specificity for detecting O. viverrini DNA. On the basis of the detection limit, the assay could detect O. viverrini DNA at concentrations as low as 10−1 ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1 ng. To validate the Ov-RPA–CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O. viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA–CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. Conclusions The Ov-RPA–CRISPR/Cas12a assay developed in this study can successfully detect O. viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O. viverrini infection, marking an initial step in the development of point-of-care diagnosis. Graphical abstrac

False Positivity of Anti-SARS-CoV-2 Antibodies in Patients with Acute Tropical Diseases in Thailand

Serology remains a useful indirect method of diagnosing tropical diseases, especially in dengue infection. However, the current literature regarding cross-reactivity between SARS-CoV-2 and dengue serology is limited and revealed conflicting results. As a means to uncover relevant serological insight involving antibody classes against SARS-CoV-2 and cross-reactivity, anti-SARS-CoV-2 IgA, IgM, and IgG ELISA, based on spike and nucleocapsid proteins, were selected for a fever-presenting tropical disease patient investigation. The study was conducted at the Faculty of Tropical Medicine during March to December 2021. The study data source comprised (i) 170 non-COVID-19 sera from 140 adults and children presenting with acute undifferentiated febrile illness and 30 healthy volunteers, and (ii) 31 COVID-19 sera from 17 RT-PCR-confirmed COVID-19 patients. Among 170 non-COVID-19 samples, 27 were false positives (15.9%), of which IgA, IgM, and IgG cross-reactive antibody classes were detected in 18 (10.6%), 9 (5.3%), and 3 (1.8%) cases, respectively. Interestingly, one case exhibited both IgA and IgM false positivity, while two cases exhibited both IgA and IgG false positivity. The false positivity rate in anti-SARS-CoV-2 IgA and IgM was reported in adults with dengue infection (11.3% and 5%) and adults with other tropical diseases (16.7% and 13.3%). The urea dissociation method applied to mitigate false positivity resulted in significantly decreased ELISA-based false and true positives. In conclusion, the analysis of antibody against SARS-CoV-2 in sera of patients with different tropical diseases showed that high IgA and IgM false positivity thus potentially limits serological assay utility in fever-presenting patients in tropical areas

Combustion modelling for two-stroke cycle engines

SIGLEAvailable from British Library Document Supply Centre- DSC:DXN1331 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Cecal content weight, pH of cecal contents, and total SCFAs in cecal content of rats fed the control, S-IMO, L-IMO, or Dex diet.

<p>Values are expressed as means ± SEM (n = 6–7). Significant differences of the values the rats with or without 2% DSS treatment in each diet group (<i>P</i><0.05) were determined by an unpaired two-tailed Student’s <i>t</i>-test (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). Significant differences of the values against control group in each treatment (<i>P</i><0.05) were determined by Dunnett’s multiple comparison test (^†<i>P</i><0.05, ^††<i>P</i><0.01, ^†††<i>P</i><0.01).</p

Changes in DAI score and colon weight per length.

<p>(A) DAI was calculated by the sum of three clinical scores (stool consistency, rectal bleeding, and weight loss) during the DSS treatment period. Significant differences comparing scores to values on day 0 (before DSS treatment) were determined by Dunnett’s multiple comparison test (**<i>P</i><0.01, ***<i>P</i><0.001). (B) Colon weight per length with or without DSS treatment. Two-way ANOVA <i>P</i> values for the colon weight per length (diet and treatment) were 0.0047 for diet, <0.0001 for treatment, and 0.0053 for the interaction between diet and treatment. No significant difference was observed for strain. Significant differences between the untreated group and the 2% DSS-treated group were determined by an unpaired two-tailed Student’s <i>t</i>-test (**<i>P</i><0.01, ***<i>P</i><0.001). Values are expressed as means ± SEM (n = 5–7).</p