Establishment of rat brain endothelial cells susceptible to rat cytomegalovirus ALL-03 infection

Endothelial cells have been implicated as key cells in promoting the pathogenesis and spread of cytomegalovirus (CMV) infection. This study describes the isolation and culture of rat brain endothelial cells (RBEC) and further evaluates the infectious potential of a Malaysian rat CMV (RCMV ALL-03) in these cultured cells. Brain tissues were mechanically fragmented, exposed to enzymatic digestion, purified by gradient density centrifugation, and cultured in vitro. Morphological characteristics and expression of von Willebrand factor (factor VIII-related antigen) verified the cells were of endothelial origin. RBEC were found to be permissive to the virus by cytopathic effects with detectable plaques formed within 7 d of infection. This was confirmed by electron microscopy examination which proved the existence of the viral particles in the infected cells. The susceptibility of the virus to these target cells under the experimental conditions described in this report provides a platform for developing a cell-culture-based experimental model for studies of RCMV pathogenesis and allows stimulation of further studies on host cell responses imposed by congenital viral infections.     

Synthesis,Structural and Antioxidant Studies of Some Novel N-Ethyl Phthalimide Esters

A series of N-ethyl phthalimide esters 4(a-n) were synthesized and characterized by spectroscopic studies. Further, the molecular structure of majority of compounds were analysed by single crystal X-ray diffraction studies. The X-ray analysis revealed the importance of substituents on the crystal stability and molecular packing. All the synthesized compounds were tested for in vitro antioxidant activity by DPPH radical scavenging, FRAP and CUPRAC methods. Few of them have shown good antioxidant activity.     

Prevalence and Patterns of Tobacco Use in Bangladesh from 2009 to 2012: Evidence from International Tobacco Control (ITC) Study

Background

Smoking and passive smoking are collectively the biggest preventable cause of death in Bangladesh, with major public health burden of morbidity, disability, mortality and community costs. The available studies of tobacco use in Bangladesh, however, do not necessarily employ nationally representative samples needed to monitor the problem at a national scale. This paper examines the prevalence and patterns of tobacco use among adults in Bangladesh and the changes over time using large nationally representative comparable surveys.

Methods

Using data from two enumerations of the International Tobacco Control (ITC) Bangladesh Project conducted in 2009 and 2012, prevalence estimates are obtained for all tobacco products by socio-economic determinants and sample types of over 90,000 individuals drawn from over 30,000 households. Household level sample weights are used to obtain nationally representative prevalence estimates and standard errors. Statistical tests of difference in the estimates between two time periods are based on a logistic regression model that accounts for the complex sampling design. Using a multinomial logit model, the time trend in tobacco use status is identified to capture the effects of macro level determinants including changes in tobacco control policies.

Results

Between 2009 and 2012, overall tobacco use went down from 42.4% to 36.3%. The decline is more pronounced with respect to smokeless tobacco use than smoking. The prevalence of exclusive cigarette smoking went up from 7.2% to 10.6%; exclusive bidi smoking remained stable at around 2%; while smoking both cigarette and bidi went down from 4.6% to 1.8%; exclusive smokeless tobacco use went down from 20.2% to 16.9%; and both smokeless tobacco use and smoking went down from 8.4% to 5.1%. In general, the prevalence of tobacco use is higher among men, increases from younger to older age groups, and is higher among poorer people. Smoking prevalence is the highest among the slum population, followed by the tribal population, the national population and the border area population, suggesting greater burden of tobacco use among the disadvantaged groups.

Conclusions

The overall decline in tobacco use can be viewed as a structural shift in the tobacco market in Bangladesh from low value products such as bidi and smokeless tobacco to high value cigarettes, which is expected with the growth in income and purchasing power of the general population. Despite the reduction in overall tobacco use, the male smoking prevalence in Bangladesh is still high at 37%. The world average of daily smoking among men is 31.1%. The Tobacco Control Act 2005 and the Amendment have yet to make a significant impact in curbing tobacco usage in Bangladesh. The findings in this paper further suggest that the tobacco control policies in Bangladesh need to include targeted interventions to restrain the use of particular types of tobacco products among specific demographic and socio-economic groups of the population, such as smoked tobacco among men, smokeless tobacco among women, and both smoked and smokeless tobacco among those living in rural areas, those in low socio-economic status and those belonging to the tribal and the slum population.     

C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms

The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD) and an equivalent phage optimized peptide (12/1) were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.     

Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester

This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.     

Evolution of habitat preference in 243 species of Bent‐toed geckos (Genus Cyrtodactylus Gray, 1827) with a discussion of karst habitat conservation

Understanding the processes that underpin adaptive evolutionary shifts within major taxonomic groups has long been a research directive among many evolutionary biologists. Such phenomena are best studied in large monophyletic groups that occupy a broad range of habitats where repeated exposure to novel ecological opportunities has happened independently over time in different lineages. The gekkonid genus Cyrtodactylus is just such a lineage with approximately 300 species that range from South Asia to Melanesia and occupy a vast array of habitats. Ancestral state reconstructions using a stochastic character mapping analysis of nine different habitat preferences were employed across a phylogeny composed of 76% of the known species of Cyrtodactylus. This was done in order to ascertain which habitat preference is the ancestral condition and from that condition, the transition frequency to more derived habitat preferences. The results indicate that a general habitat preference is the ancestral condition for Cyrtodactylus and the frequency of transitioning from a general habitat preference to anything more specialized occurs approximately four times more often than the reverse. Species showing extreme morphological and/or ecological specializations generally do not give rise to species bearing other habitat preferences. The evolution of different habitat preferences is generally restricted to clades that tend to occur in specific geographic regions. The largest radiations in the genus occur in rocky habitats (granite and karst), indicating that the transition from a general habitat preference to a granite or karst‐dwelling life style may be ecologically uncomplicated. Two large, unrelated clades of karst‐associated species are centered in northern Indochina and the largest clade of granite‐associated species occurs on the Thai‐Malay Peninsula. Smaller, independent radiations of clades bearing other habitat preferences occur throughout the tree and across the broad distribution of the genus. With the exception of a general habitat preference, the data show that karst‐associated species far out‐number all others (29.6% vs. 0.4%–10.2%, respectively) and the common reference to karstic regions as “imperiled arcs of biodiversity” is not only misleading but potentially dangerous. Karstic regions are not simply refugia harboring the remnants of local biodiversity but are foci of speciation that continue to generate the most speciose, independent, radiations across the genus. Unfortunately, karstic landscapes are some of the most imperiled and least protected habitats on the planet and these data continue to underscore the urgent need for their conservation.     

Does structural change in the zooplankton community affect larval fish feeding in anthropogenically disturbed tropical waters?

Environmental Biology of Fishes - Anthropogenic perturbations and climate change have altered the zooplankton community structure in the Klang Strait during the past 30&nbsp;years, in that the...     

First Record of Batrachochytrium dendrobatidis Infecting Four Frog Families from Peninsular Malaysia

The fungal pathogen Batrachochytrium dendrobatidis (Bd) infects amphibians on every continent where they occur and is linked to the decline of over 200 amphibian species worldwide. At present, only three published Bd surveys exist for mainland Asia, and Bd has been detected in South Korea alone. In this article, we report the first survey for Bd in Peninsular Malaysia. We swabbed 127 individuals from the six amphibian families that occur on Peninsular Malaysia, including two orders, 27 genera, and 47 species. We detected Bd on 10 out of 127 individuals from four of five states and five of 11 localities, placing the 95% confidence interval for overall prevalence at 4–14%. We detected no variation in Bd prevalence among regions, elevations, or taxonomic groups. The infection intensity ranged from 1 to 157,000 genome equivalents. The presence of Bd infections in native species without clinical signs of disease suggests that Bd may be endemic to the region. Alternately, Bd may have been introduced from non-native amphibians because of the substantial amphibian food trade in Peninsular Malaysia. Under both scenarios, management efforts should be implemented to limit the spread of non-native Bd and protect the tremendous amphibian diversity in Peninsular Malaysia.     

Molecular identification of Cryptosporidium parvum from avian hosts

Cryptosporidium species are protozoan parasites that infect humans and a wide variety of animals. This study was aimed at identifying Cryptosporidium species and genotypes isolated from avian hosts. A total of 90 samples from 37 different species of birds were collected throughout a 3-month period from April 2008 to June 2008 in the National Zoo of Kuala Lumpur, Malaysia. Prior to molecular characterization, all samples were screened for Cryptosporidium using a modified Ziehl-Neelsen staining technique. Subsequently samples were analysed with nested-PCR targeting the partial SSU rRNA gene. Amplicons were sequenced in both directions and used for phylogenetic analysis using Neighbour-Joining and Maximum Parsimony methods. Although 9 (10%) samples were positive for Cryptosporidium via microscopy, 8 (8.9%) produced amplicons using nested PCR. Phylogenetic trees identified all the isolates as Cryptosporidium parvum. Although C. parvum has not been reported to cause infection in birds, and the role of birds in this study was postulated mainly as mechanical transporters, these present findings highlight the significant public health risk posed by birds that harbour the zoonotic species of Cryptosporidium.     

A novel mechanism for complement activation at the surface of B cells following antigen binding

Coligation of CD21 with BCR on the surface of B cells provides a costimulatory signal essential for efficient Ab responses to T-dependent Ags. To achieve this, Ag must be directly linked to C3 fragments, but how this occurs in vivo is not fully understood. Using BCR transgenic mice, we demonstrated that C3 was deposited on the surface of B cells following both high- and moderate-affinity Ag binding. This was dependent on the specific binding of IgM to the BCR-bound Ag and can occur independently of soluble immune complex formation. Based on these data, we propose a novel model in which immune complexes can form directly on the surface of the B cell following Ag binding. This model has implications for our understanding of B lymphocyte activation.