Plasmodium falciparum populations from northeastern Myanmar display high levels of genetic diversity at multiple antigenic loci

Levels of genetic diversity of the malaria parasites and multiclonal infections are correlated with transmission intensity. In order to monitor the effect of strengthened malaria control efforts in recent years at the China-Myanmar border area, we followed the temporal dynamics of genetic diversity of three polymorphic antigenic markers msp1, msp2, and glurp in the Plasmodium falciparum populations. Despite reduced malaria prevalence in the region, parasite populations exhibited high levels of genetic diversity. Genotyping 258 clinical samples collected in four years detected a total of 22 PCR size alleles. Multiclonal infections were detected in 45.7% of the patient samples, giving a minimum multiplicity of infection of 1.41. The majority of alleles experienced significant temporal fluctuations through the years. Haplotype diversity based on the three-locus genotypes ranged from the lowest in 2009 at 0.33 to the highest in 2010 at 0.80. Sequencing of msp1 fragments from 36 random samples of five allele size groups detected 13 different sequences, revealing an additional layer of genetic complexity. This study suggests that despite reduced prevalence of malaria infections in this region, the parasite population size and transmission intensity remained high enough to allow effective genetic recombination of the parasites and continued maintenance of genetic diversity

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Quantitative detection of organic mercury in whole blood using derivatization and gas chromatography-negative chemical ionization-mass spectrometry

The Minamata disease, first identified in Japan in the 1950s, is caused by severe methylmercury (MeHg) poisoning. To prevent the development of this disease, routine evaluation of MeHg levels in blood samples is crucial. The purpose of this research was to explore the use of derivatization and capillary gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) for the quantitative detection of both organic and inorganic mercury in blood samples. Alkyl mercury in standard solutions was extracted as halide salts in toluene with hydrohalic acid. Fat contents in whole blood samples were removed by methyl isobutyl ketone and hexane using a cysteine/alkaline solution and then organic mercury was extracted as a bromide complex using toluene and cupper chloride solution. The linearity of the response ratio vs. concentration curves (R2) was 0.987 for methylmercury bromide and 0.990 for ethylmercury (EtHg) bromide, over the calibration range of 0.02 ng/mL to 20 ng/mL. The recovery of MeHg and EtHg was 67.1% and 49.3%, respectively. The concentrations of MeHg in whole blood samples determined using GC with an electron capture detector agreed with those determined using GC-NCI-MS, with a correlation coefficient of 0.923. The mean concentration of MeHg in a certified reference material (NMIJ CRM 7402-a) determined using GC-NCI-MS was 0.64 μg/g, comparable with the certified value of 0.58 μg/g. Our study demonstrates a simple and low-cost approach for analyzing mercury in biological samples, although further optimization is required given the relatively low recovery and the concern about the toxicity of methyl isobutyl ketone

Leonardite-derived humic substances are great adsorbents for cadmium

Adsorption is an important mechanism to immobilize cadmium (Cd) in soil, for which humic substances have a potential. However, commercial humic substances are either very acidic (pH = 2) or alkaline/Na+-enriched, making them less suitable for use in acid and saline soils. Here, we used leonardite to produce humic adsorbents HA (pH = 4.02), Ca-HA (pH = 10.9), and Ca-CPAM-HA (pH = 9.62) by using HCl, CaCl2, or CaCl2-polyacrylamide as a flocculant. Their elemental compositions, acidity, and spectroscopic properties were determined, and their Cd adsorption characteristics were assessed by batch kinetic and thermodynamic experiments at environmentally relevant concentrations. Further, HA was mixed with Cd-contaminated soils and incubated for a month to assess its effect on Cd immobilization. Good fitting of kinetic adsorption data into pseudo-second-order model, together with FTIR spectroscopic data, suggested the chemisorption mechanism by forming Cd(II)-carboxyl complexes. The maximum adsorption capacity derived from the Langmuir equation was 129, 114, and 110 mg Cd(II)/g for HA, Ca-HA, and Ca-CPAM-HA, respectively. These values are almost the same on carbon-normalized basis. HA reduced acetic acid extractable Cd by 31% or more. Besides their high propensity for Cd adsorption, humic adsorbents are inexpensive, safe, and beneficial to soil quality

Multidrug-Resistant Genotypes of Plasmodium falciparum, Myanmar

We performed a molecular epidemiologic survey of mutations associated with drug-resistance genes in Plasmodium falciparum in northeastern Myanmar. In this region, 3 highly mutated drug-resistance haplotypes and 1 associated with decreased quinine susceptibility were prevalent, which suggests that parasites may be resistant to multiple commonly used antimalarial drugs

In Vitro Sensitivity of Plasmodium falciparum Clinical Isolates from the China-Myanmar Border Area to Quinine and Association with Polymorphism in the Na+/H+ Exchanger ▿

Quinine resistance (QNR) in Plasmodium falciparum has been detected in many regions of the world where malaria is endemic. Genetic polymorphisms in at least four genes are implicated in QN susceptibility, and their significance often depends on the genetic background of the parasites. In this study, we have culture-adapted 60 P. falciparum clinical isolates from the China-Myanmar border and assessed their in vitro responses to QN. Our results showed that >50% of the parasite isolates displayed reduced sensitivity to QN, with a half-maximal inhibitory concentration (IC50) above 500 nM. Genotyping of pfcrt found that an overwhelming proportion of the parasite population had the chloroquine-resistant genotype, whereas pfmdr1 mutation genotypes and gene amplification were rare. Genotyping of the P. falciparum Na+/H+ exchanger gene (pfnhe1) at the minisatellite ms4760 locus identified 10 haplotypes. Haplotype 7, which harbors three copies of the DNNND repeat, was the most predominant, accounting for nearly half of the parasite isolates. Correlation studies did not reveal significant associations of the polymorphisms in pfcrt and pfmdr1 genes with QN response. However, the ms4760 haplotypes were highly associated with in vitro QN responses. In particular, parasite isolates with an increased DNNND copy number tended to have significantly reduced QN susceptibility, whereas parasite isolates with a higher NHNDNHNNDDD copy number had increased QN susceptibility. This study provided further support for the importance of pfnhe1 polymorphisms in influencing QNR in P. falciparum

Inhibition of sortase A by chalcone prevents Listeria monocytogenes infection

The critical role of sortase A in gram-positive bacterial pathogenicity makes this protein a good potential target for antimicrobial therapy. In this study, we report for the first time the crystal structure of Listeria monocytogenes sortase A and identify the active sites that mediate its transpeptidase activity. We also used a sortase A (SrtA) enzyme activity inhibition assay, simulation, and isothermal titration calorimetry analysis to discover that chalcone, an agent with little anti-L. monocytogenes activity, could significantly inhibit sortase A activity with an IC50 of 28.41 +/- 5.34 mu M by occupying the active site of SrtA. The addition of chalcone to a co-culture of L. monocytogenes and Caco-2 cells significantly inhibited bacterial entry into the cells and L. monocytogenes-mediated cytotoxicity. Additionally, chalcone treatment decreased the mortality of infected mice, the bacterial burden in target organs, and the pathological damage to L. monocytogenes-infected mice. In conclusion, these findings suggest that chalcone is a promising candidate for the development of treatment against L. monocytogenes infection. (C) 2016 Elsevier Inc. All rights reserved

An efficient high-speed counter-current chromatography method for the preparative separation of potential antioxidants from Paeonia lactiflora Pall. combination of in vitro evaluation and molecular docking

Paeonia lactiflora Pall., one of the most famous classical herbal medicine, has been used to treat diseases for over 1200 years. In this research, the functional ingredients were purified by online-switch two-dimensional high-speed counter-current chromatography combined with inner-recycling and continuous injection mode. The antioxidant activity was evaluated by investigating the 2,2 '-azobis (2-amidinopropane) dihydrochloride-induced oxidant damage in vitro and confirmed through molecular docking. n-Butanol/ethyl acetate/water (2:3:5, v/v) solvent system was used for the first-dimensional separation and optimized the sample loading. Two pure compounds and a polyphenol-enriched fraction were separated. The polyphenol-enriched fraction was separated with a solvent system n-hexane/ethyl acetate/methanol/water (2:8:4:6, v/v) with continuous injection mode. Five compounds were successfully separated, including gallic acid (1), methyl gallate (2), albiflorin (3), paeoniflorin (4), and ethyl gallate (5). Their structures were identified by mass spectrometry and NMR spectroscopy. The results from the antioxidant effect showed that albiflorin had stronger antioxidant activity. Molecular docking results indicated that the affinity energy of the identified compounds ranged from -3.79 to -8.22 kcal/mol and albiflorin showed the lowest affinity energy. Overall, all those findings suggested that the strong antioxidant capacity of albiflorin can be potentially used for the treatment of diseases caused by oxidation.N