Biochemical and biological characterization of the venoms of Naja kaouthia and Naja mandalayensis from Myanmar and neutralization effects of BPI cobra antivenom.

Snakebite is a neglected public health issue, with many scientific and medical issues to be solved. Cobras are among the most common venomous snakes in Myanmar and are responsible for a considerable number of severe snakebite envenoming. There are three species of cobra (Naja kaouthia, Naja mandalayensis and Ophiophagus hannah) in Myanmar. The study aims to characterize the N. kaouthia and N. mandalayensis venoms and to investigate the efficacy of anti-cobra antivenom (BPI) against the two venoms. Protein components and fibrinogenolytic activity were determined by SDS-PAGE. Enzymatic activities for PLA2, protease and acetylcholinesterase were determined by spectrophotometric method. Anticoagulant activity was determined by recalcification time of citrated human plasma. Myotoxicity, necrotizing activity, median lethal dose (LD50) and median effective dose (ED50) were determined by WHO recommended methods. The SDS-PAGE displayed the proteins and enzymes containing in two venoms were different. N. kaouthia venom exhibited more in PLA2, acetylcholinesterase, anticoagulant, fibrinogenolytic and necrotizing activities than N. mandalayensis venom. N. mandalayensis venom had more protease activity and myotoxicity than N. kaouthia venom. The median lethal dose (LD50) of N. kaouthia and N. mandalayensis venom was 4.33 µg/mouse and 5.04 µg/mouse respectively. Both venoms induced fibrinogen Aα chain degradation in 30 min (N. kaouthia) and in 6 h (N. mandalayensis). The same median effective dose (ED50) (19.56 µg/mouse) showed that anti-NK antivenom can neutralize against lethal effect of N. mandalayensis venom. It can also neutralize the protease activity, anticoagulant activity and fibrinogenolytic activity of both venoms. Immunodiffusion and immunoblotting studies showed that the antivenom recognized its homologous venom (N. kaouthia) and cross-reacted against the heterologous venom (N. mandalayensis). The anti-NK antivenom is suitable to use for N. mandalayensis bite if monospecific antivenom is not available.

Evolutionary Dynamics of Whole-Genome Influenza A/H3N2 Viruses Isolated in Myanmar from 2015 to 2019.

This study aimed to analyze the genetic and evolutionary characteristics of the influenza A/H3N2 viruses circulating in Myanmar from 2015 to 2019. Whole genomes from 79 virus isolates were amplified using real-time polymerase chain reaction and successfully sequenced using the Illumina iSeq100 platforms. Eight individual phylogenetic trees were retrieved for each segment along with those of the World Health Organization (WHO)-recommended Southern Hemisphere vaccine strains for the respective years. Based on the WHO clades classification, the A/H3N2 strains in Myanmar from 2015 to 2019 collectively belonged to clade 3c.2. These strains were further defined based on hemagglutinin substitutions as follows: clade 3C.2a (n = 39), 3C.2a1 (n = 2), and 3C.2a1b (n = 38). Genetic analysis revealed that the Myanmar strains differed from the Southern Hemisphere vaccine strains each year, indicating that the vaccine strains did not match the circulating strains. The highest rates of nucleotide substitution were estimated for hemagglutinin (3.37 × 10-3 substitutions/site/year) and neuraminidase (2.89 × 10-3 substitutions/site/year). The lowest rate was for non-structural protein segments (4.19 × 10-5 substitutions/site/year). The substantial genetic diversity that was revealed improved phylogenetic classification. This information will be particularly relevant for improving vaccine strain selection.

Evolutionary analysis of human respiratory syncytial virus collected in Myanmar between 2015 and 2018.

We studied genetic variation in the second hypervariable region (HVR) of the G gene of human respiratory syncytial virus (HRSV) from 1701 nasal swab samples collected from outpatients with acute respiratory infections at two general hospitals in the cities Yangon and Pyinmana in Myanmar from 2015 to 2018. HRSV genotypes were characterized using phylogenetic trees constructed using the maximum likelihood method. Time-scale phylogenetic tree analyses were performed using the Bayesian Markov chain Monte Carlo method. In total, 244 (14.3%) samples were HRSV-positive and were classified as HRSV-A (n = 84, 34.4%), HRSV-B (n = 158, 64.8%), and co-detection of HRSV-A/HRSV-B (n = 2, 0.8%). HRSV epidemics occurred seasonally between July (1.9%, 15/785) and August (10.5%, 108/1028), with peak infections in September (35.8%, 149/416) and October (58.2%, 89/153). HRSV infection rate was higher in children ≥1 year of age than in those <1 year of age (70.5% vs. 29.5%). The most common HRSV symptoms in children were cough (80%-90%) and rhinorrhea (70%-100%). The predominant genotypes were ON1for HRSV-A (78%) and BA9 for HRSV-B (64%). Time to the most recent common ancestor was 2014 (95% highest posterior density [HPD], 2012-2015) for HRSV-A ON1 and 2009 (95% HPD, 2004-2012) for HRSV-B BA9. The mean evolutionary rate (substitutions/site/year) for HRSV-B (2.12 × 10-2, 95% HPD, 8.53 × 10-3-3.63 × 10-2) was slightly higher than that for HRSV-A (1.39 × 10-2, 95% HPD, 6.03 × 10-3-2.12 × 10-2). The estimated effective population size (diversity) for HRSV-A increased from 2015 to 2016 and declined in mid-2018, whereas HRSV-B diversity was constant in 2015 and 2016 and increased in mid-2017. In conclusion, the dominant HRSV-A and HRSV-B genotypes in Myanmar were ON1 and BA9, respectively, between 2015 and 2018. HRSV-B evolved slightly faster than HRSV-A and exhibited unique phylogenetic characteristics.

Dipterocarpus tuberculatus Roxb. Ethanol Extract Has Anti-Inflammatory and Hepatoprotective Effects In Vitro and In Vivo by Targeting the IRAK1/AP-1 Pathway.

Dipterocarpus tuberculatus Roxb. has been used traditionally as a remedy for many diseases, especially inflammation. Therefore, we analyzed and explored the mechanism of the anti-inflammatory effect of a Dipterocarpus tuberculatus Roxb. ethanol extract (Dt-EE). Dt-EE clearly and dose-dependently inhibited the expression of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1ß in lipopolysaccharide (LPS)-treated RAW264.7 cells. Also, Dt-EE suppressed the activation of the MyD88/TRIF-mediated AP-1 pathway and the AP-1 pathway related proteins JNK2, MKK4/7, and TAK1, which occurred as a result of inhibiting the kinase activity of IRAK1 and IRAK4, the most upstream factors of the AP-1 pathway. Finally, Dt-EE displayed hepatoprotective activity in a mouse model of hepatitis induced with LPS/D-galactosamine (D-GalN) through decreasing the serum levels of alanine aminotransferase and suppressing the activation of JNK and IRAK1. Therefore, our results strongly suggest that Dt-EE could be a candidate anti-inflammatory herbal medicine with IRAK1/AP-1 inhibitory and hepatoprotective properties.

Trends in and Barriers to Enrollment of Underrepresented Minority Students in a Pharmacy School.

Objective. To compare the mean national enrollment rates of underrepresented minority (URM) students in a pharmacy school with mean rates in California pharmacy schools, and identify barriers faced by URM students during the application process. Methods. The American Association of Colleges of Pharmacy (AACP) enrollment data from 2005 to 2014 were used to compare the demographics of California pharmacy schools with the average enrollment of URM students in pharmacy schools nationally. A survey was administered to students in the 2017 and 2018 classes at Touro University California College of Pharmacy to identify common barriers that students faced in pursuing pharmacy education. Results. The average enrollment of URM in pharmacy programs nationally was 12.3% in 2005, compared to 12.4% in 2014. The average enrollment of URM in California pharmacy schools was 9.4% in 2005 compared to 8.5% in 2014. The top barriers to pursuing pharmacy education that students reported included the cost of tuition (43.4%), prerequisite requirements (36.9%), and obtaining letters of recommendation (32.3%). Conclusion. The average enrollment of URM students in pharmacy schools nationally has remained higher than that in California pharmacy schools across the years studied. California pharmacy programs should develop strategies to alleviate the barriers identified and further diversify pharmacy education.

Comparison of Count Normalization Methods for Statistical Parametric Mapping Analysis Using a Digital Brain Phantom Obtained from Fluorodeoxyglucose-positron Emission Tomography.

OBJECTIVES: Alternative normalization methods were proposed to solve the biased information of SPM in the study of neurodegenerative disease. The objective of this study was to determine the most suitable count normalization method for SPM analysis of a neurodegenerative disease based on the results of different count normalization methods applied on a prepared digital phantom similar to one obtained using fluorodeoxyglucose-positron emission tomography (FDG-PET) data of a brain with a known neurodegenerative condition. METHODS: Digital brain phantoms, mimicking mild and intermediate neurodegenerative disease conditions, were prepared from the FDG-PET data of 11 healthy subjects. SPM analysis was performed on these simulations using different count normalization methods. RESULTS: In the slight-decrease phantom simulation, the Yakushev method correctly visualized wider areas of slightly decreased metabolism with the smallest artifacts of increased metabolism. Other count normalization methods were unable to identify this slightly decreases and produced more artifacts. The intermediate-decreased areas were well visualized by all methods. The areas surrounding the grey matter with the slight decreases were not visualized with the GM and VOI count normalization methods but with the Andersson. The Yakushev method well visualized these areas. Artifacts were present in all methods. When the number of reference area extraction was increased, the Andersson method better-captured the areas with decreased metabolism and reduced the artifacts of increased metabolism. In the Yakushev method, increasing the threshold for the reference area extraction reduced such artifacts. CONCLUSION: The Yakushev method is the most suitable count normalization method for the SPM analysis of neurodegenerative disease.

Phylogeographic analysis of human influenza A and B viruses in Myanmar, 2010-2015.

We investigated the circulation patterns of human influenza A and B viruses in Myanmar between 2010 and 2015 by analyzing full HA genes. Upper respiratory tract specimens were collected from patients with symptoms of influenza-like illness. A total of 2,860 respiratory samples were screened by influenza rapid diagnostic test, of which 1,577 (55.1%) and 810 (28.3%) were positive for influenza A and B, respectively. Of the 1,010 specimens that were positive for virus isolation, 370 (36.6%) were A(H1N1)pdm09, 327 (32.4%) were A(H3N2), 130 (12.9%) B(Victoria), and 183 (18.1%) were B(Yamagata) viruses. Our data showed that influenza epidemics mainly occurred during the rainy season in Myanmar. Our three study sites, Yangon, Pyinmana, and Pyin Oo Lwin had similar seasonality and circulating type and subtype of influenza in a given year. Moreover, viruses circulating in Myanmar during the study period were closely related genetically to those detected in Thailand, India, and China. Phylogeographic analysis showed that A(H1N1)pdm09 viruses in Myanmar originated from Europe and migrated to other countries via Japan. Similarly, A(H3N2) viruses in Myanmar originated from Europe, and disseminated to the various countries via Australia. In addition, Myanmar plays a key role in reseeding of influenza B viruses to Southeast Asia and East Asia as well as Europe and Africa. Thus, we concluded that influenza virus in Myanmar has a strong link to neighboring Asian countries, Europe and Oceania.

Begonia myanmarica (Begoniaceae), a new species from Myanmar, and molecular phylogenetics of Begonia sect. Monopteron.

BACKGROUND: A new species, Begonia myanmarica, was discovered from Myanmar and herein documented. Characterized by a single developed wing in the ovary/fruit, this species would be assigned to sect. Monopteron (sensu Doorenbos et al. in The sections of Begonia including descriptions, keys and species lists: studies in Begoniaceae VI. Wageningen Agricultural University, Wageningen, 1998) that is known by B. griffithiana and B. nepalensis from the Himalaya. To confirm its sectional assignment, we conducted morphological, phylogenetic and cytological studies. RESULTS: Morphological observations indicated that B. myanmarica was distinguishable from the two known species of sect. Monopteron by the leaf shape and size, 1-locular ovary, parietal placentation and chromosome number. Molecular phylogenetic analysis using nrITS sequences showed that B. myanmarica was not allied with the clade of sect. Monopteron, though both were nested within sect. Platycentrum-sect. Sphenanthera clade. CONCLUSIONS: Studies of morphology, molecular phylogenetics and cytology support the recognition of the new species, Begonia myanmarica, which is fully described and illustrated. Our results also indicate that B. myanmarica is not closely related to species previously assigned to sect. Monopteron, suggesting that the fruit morphology of a single developed wing in the ovary/fruit characterizing sect. Monopteron is homoplasious.

Flavonoid glycosides from the aerial parts of Acacia pennata in Myanmar.

Phytochemical investigations of the aerial parts of Acacia pennata (Mimosaceae) from Myanmar led to the isolation of five flavonoid glycosides and six known compounds. The new compounds were identified as (2R,3S)-3,5,7-trihdyroxyflavan-3-O-α-L-rhamnopyranoside, (2S)-5,7-dihydroxyflavan-7-O-ß-D-glucopyranoside-(4α → 8)-epiafzelechin-3-O-gallate, (2R)-4',7-dihydroxyflavan-(4α → 8)-(2R,3S)-3,5,7-trihdyroxyflavan-3″-O-α-L-rhamnopyranoside, 5,7-dihydroxyflavone 6-C-ß-boivinopyranosyl-7-O-ß-D-glucopyranoside, and 5,7-dihydroxyflavone 7-O-ß-D-glucopyranosyl-8-C-ß-boivinopyranoside based on interpretation of spectroscopic data.

Molecular Epidemiology of Dengue Viruses Co-circulating in Upper Myanmar in 2006.

To understand the molecular epidemiology of circulating dengue viruses (DENV) in Upper Myanmar, DENV isolation was attempted by inoculating the sera of a panel of 110 serum samples onto a C6/36 mosquito cell line. The samples were collected from dengue (DEN) patients admitted at Mandalay Children's Hospital in 2006. Infected culture fluids were subjected to a RT-PCR to detect the DENV genome. Three DENV strains were isolated. This was the first DENV isolation performed either in Mandalay or in Upper Myanmar. One strain belonged to DENV serotype-3 (DENV-3), and two other strains belonged to DENV serotype-4 (DEN-4). The sequence data for the envelope gene of these strains were used in a phylogenetic comparison of DENV-3 and DENV-4 from various countries. Phylogenetic analyses revealed that this DENV-3 strain was clustered within genotype II, and the two DENV-4 strains were clustered within genotype I in each serotype. The Myanmar strains were closely related to strains from the neighboring countries of Thailand and Bangladesh. These results are important for elucidating the trends of recent and future DEN outbreaks in Myanmar.

Two new phenolic glucosides from Lagerstroemia speciosa.

Two new phenolic glucosides, 1-O-benzyl-6-O-E-caffeoyl-ß-d-glucopyranoside and 1-O-(7S,8R)-guaiacylglycerol-(6-O-E-caffeoyl)-ß-d-glucopyranoside, were isolated from the aerial parts of Lagerstroemia speciosa, along with ten known compounds. The structures of the isolated compounds were determined based on 1D- and 2D-NMR, Q-TOF MS and optical rotation spectroscopic data. All of the compounds showed moderate inhibitory activities against nitric oxide production in lipopolysaccharide-treated RAW264.7 cells, with IC50 values of 69.5-83.3 µM.

Flavonoids from Symplocos racemosa.

A novel isoflavone glycoside, peseudobatigenin 7-O-[ß-d-apiofuranosyl-(1''''→5''')-O-ß-d-apiofuranosyl-(1'''→6'')]-ß-d-glucopyranoside, namely sympracemoside (1), was isolated from the aerial parts of Symplocos racemosa along with 15 known flavonoids (2-16). Their structures were characterized by Q-TOF mass, optical rotation, UV, 1D and 2D-NMR spectroscopic data. Compounds 3, 9, 16 showed moderate inhibitory activities against NO production with IC50 value of 88.2, 42.1 and 74.3 µM, respectively.

Syk/Src-targeted anti-inflammatory activity of Codariocalyx motorius ethanolic extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Codariocalyx motorius (Houtt.) H. Ohashi (Fabaceae) is one of several ethnopharmacologically valuable South Asian species prescribed as an herbal medicine for various inflammatory diseases. Due to the lack of systematic studies on this plant, we aimed to explore the inhibitory activity of Codariocalyx motorius toward inflammatory responses using its ethanolic extract (Cm-EE). MATERIALS AND METHODS: Lipopolysaccharide (LPS)-treated macrophages and a HCl/EtOH-induced gastritis model were used for evaluation of the anti-inflammatory activity of Cm-EE. HPLC and spectroscopic analysis were employed to identify potential active components. Mechanistic approaches to determine target enzymes included kinase assays, reporter gene assays, and overexpression of target enzymes. RESULTS: Cm-EE strongly suppressed nitric oxide (NO) and prostaglandin E2 (PGE2) release. Cm-EE-mediated inhibition was observed at the transcriptional level in the form of suppression of NF-κB (p65) translocation and activation. This extract also lowered the levels of phosphorylation of Src and Syk, their kinase activity, and their formation of signalling complexes by binding to the downstream enzyme p85/PI3K. In accord with these findings, the phosphorylation of p85 induced by overexpression of Src or Syk was also diminished by Cm-EE. Orally administered Cm-EE clearly inhibited gastritic ulcer formation and the phosphorylation of IκBα and Src in HCl/EtOH-treated stomachs of mice. By phytochemical analysis, luteolin and its glycoside, apigenin-7-O-glucuronide, and scutellarein-6-O-glucuronide were identified as major components of Cm-EE. Among these, it was found that luteolin was able to strongly suppress NO and PGE2 production under the same conditions. CONCLUSION: Syk/Src-targeted inhibition of NF-κB by Cm-EE could be a major anti-inflammatory mechanism contributing to its ethno pharmacological role as an anti-inflammatory herbal medicine.

Myrsine seguinii ethanolic extract and its active component quercetin inhibit macrophage activation and peritonitis induced by LPS by targeting to Syk/Src/IRAK-1.

ETHNOPHARMACOLOGICAL RELEVANCE: Myrsine seguinii H. LÉVEILLÉ (syn. Rapanea neriifolia) (Myrsinaceae) is a medicinal plants traditionally used in Myanmar to treat infectious and inflammatory diseases. Since none of reports have systematically demonstrated the anti-inflammatory activity of this plant, we aimed to mechanistically understand the regulatory roles of the plant in inflammatory responses using the ethanolic extract of Myrsine seguinii (Ms-EE). MATERIALS AND METHODS: Activated macrophages and peritonitis symptoms induced by lipopolysaccharide (LPS) were employed. HPLC analysis was used to identify active components. To characterize direct target enzymes, kinase assay was established. RESULTS: Ms-EE inhibited the production of nitric oxide (NO) and prostaglandin (PG)E2 in RAW264.7 cells and peritoneal macrophages stimulated by LPS. This extract suppressed the mRNA expression of the inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 genes by down-regulating the activation of nuclear factor (NF)-κB and activator protein (AP-1). Interestingly, it was found that Ms-EE can directly suppress the enzyme activities of Syk, Src, and interleukin-1 receptor-associated kinase-1 (IRAK-1). Similarly, orally administered Ms-EE inhibited the phosphorylation of Src and Syk in peritoneal exudate-derived cells prepared from peritonitis. Finally, HPLC analysis clearly demonstrated that quercetin is a major active component with suppressing activity on the release of inflammatory mediators (NO and PGE2), and the enzyme activities of Src, Syk, and IRAK-1. CONCLUSION: Ms-EE containing quercetin negatively modulates macrophage-mediated in vitro inflammatory responses and LPS-induced peritonitis by blocking the Src/Syk/NF-κB and IRAK-1/AP-1 pathways, which contributes to its major ethnopharmacological use as an anti-inflammatory herbal medicine.

Serological characterization of dengue virus infections observed among dengue hemorrhagic fever/dengue shock syndrome cases in upper Myanmar.

In Myanmar, dengue fever (DF)/dengue hemorrhagic fever (DHF) is one of the leading causes of morbidity and mortality among children. From Pyinmana Hospital in 2004 and Mandalay Children Hospital in 2006, 160 patients diagnosed clinically to have DHF/dengue shock syndrome (DSS) were examined for immunoglobulin M (IgM) and IgG levels. A focus reduction neutralization test was also used to determine primary or secondary dengue virus (DENV) infection. By using IgM-capture ELISA, 139 cases were confirmed as DENV infections. Of these IgM-positives, 94 samples were collected 7-24 days from the onset of illness, to which 13 (14%) and 81 (86%) were determined to be primary and secondary DENV infections, respectively. The 13 primary DENV infection cases were spread among the various severity groups (DHF grade I-IV and DSS) and represented age groups ranging from <1 year of age to 9 years of age. The patients in these primary infection cases showed a remarkably high IgM with a low IgG titer response compared with the secondary infection cases. No significant differences were observed in IgG titers with clinical severity. The data obtained in this study suggest that primary DENV infection cases exist certainly among DHF/DSS cases in Myanmar, and that additional mechanism(s) aside from the antibody-dependent enhancement mechanism could have influenced the clinical severity in DHF/DSS cases.

Dipterocarpus tuberculatus ethanol extract strongly suppresses in vitro macrophage-mediated inflammatory responses and in vivo acute gastritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Dipterocarpus tuberculatus Roxb. (Dipterocarpaceae) has been traditionally used to treat various inflammatory symptoms. However, no mechanistic studies on the anti-inflammatory actions of D. tuberculatus have been reported. This study is therefore aimed at exploring the anti-inflammatory effects of 95% ethanol extracts (Dt-EE) of this plant. MATERIALS AND METHODS: The regulatory activity of Dt-EE and its molecular mechanism on the release of nitric oxide (NO) and prostaglandin (PG)E2 in lipopolysaccharide (LPS)-treated macrophage-like RAW264.7 cells were elucidated by evaluating the activation of transcription factors and their upstream signals and by analyzing the kinase activities of target enzymes. Furthermore, to confirm its availability for oral use, an EtOH/HCl-induced acute gastritis model was tested with this extract. RESULTS: Dt-EE effectively suppressed LPS-mediated inflammatory responses such as the production of NO and PGE2 from macrophages in a dose-dependent manner. In particular, Dt-EE clearly blocked the activation of NF-κB by blocking the phosphorylation of its upstream enzymes IKK and Akt. Using a direct enzyme assay, Dt-EE was shown to block the enzyme activity of PDK1. Finally, this extract also remarkably ameliorated inflammatory lesions in the stomach induced by EtOH/HCl. CONCLUSION: Our data strongly suggest that Dt-EE can be considered as a novel anti-inflammatory remedy with PDK1/NF-κB inhibitory properties and can also be used to treat gastritis symptoms. In addition, our findings can serve as a basis for further phytochemical and pharmacological studies in the future.

Effect of Saccharomyces boulardii in the treatment of acute watery diarrhea in Myanmar children: a randomized controlled study.

This study was conducted to evaluate the efficacy of Saccharomyces boulardii in acute diarrhea. One hundred hospitalized children in Myanmar (age range = 3 months to 10 years) were included. Fifty were treated with S. boulardii for five days in addition to oral rehydration solution (ORS) and 50 were given ORS alone (control group) in an alternating order. The mean duration of diarrhea was 3.08 days in the S. boulardii group and 4.68 days (P < 0.05) in the control group. Stools had a normal consistency on day 3 in 38 (76%) of 50 patients in the S. boulardii group compared with only 12 (24%) of 50 in the control group (P = 0.019). On day 2, 27 (54%) of 50 had less than three stools per day in the S. boulardii group compared with only 15 (30%) of 50 in the control group (P = 0.019). Saccharomyces boulardii shortens the duration of diarrhea and normalizes stool consistency and frequency. The shortening of the duration of diarrhea results in a social and economic benefits.