Technical note: comparison of the Maresh reference data with the who international standard for normal growth in healthy children

The Maresh reference data on stature and long bone lengths in a sample of healthy middle-class children from Denver, Colorado [Maresh: Am J Dis Child 66 (1943) 227-257; Maresh: Am J Dis Child 89 (1955) 725-742; Maresh: Human growth and development (1970) p 155-200], have been used extensively by biological anthropologists to estimate juvenile age and body size using skeletal elements and to assess growth in skeletal series from different ethnic populations or archaeological cultural groups. How well these data reflect the potentially diverse growth patterns of healthy human populations from different geographic areas is unknown. Similarly, the efficacy of using the Maresh reference data to estimate stunting prevalence in prehistoric populations is unknown. This report presents the results from a comparison of the Maresh data on supine length and standing height to the World Health Organization (WHO) international child growth standard. The WHO growth standard is meant to depict typical human growth under optimal conditions and can be used to assess children worldwide, regardless of ethnicity and socioeconomic status. The results from this comparison indicate that although the Maresh reference data generally conform to the WHO standard, reflecting a normal human growth pattern, and therefore serve as a suitable reference for comparative studies of growth patterns, these reference data are not suitable for estimating stunting prevalence.     

Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract

Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR·RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.     

Tuberculosis incidence rates during 8 years of follow-up of an antiretroviral treatment cohort in South Africa: comparison with rates in the community

Background

Although antiretroviral therapy (ART) is known to be associated with time-dependent reductions in tuberculosis (TB) incidence, the long-term impact of ART on incidence remains imprecisely defined due to limited duration of follow-up and incomplete CD4 cell count recovery in existing studies. We determined TB incidence in a South African ART cohort with up to 8 years of follow-up and stratified rates according to CD4 cell count recovery. We compared these rates with those of HIV-uninfected individuals living in the same community.

Methodology/Principal Findings

Prospectively collected clinical data on patients receiving ART in a community-based cohort in Cape Town were analysed. 1544 patients with a median follow-up of 5.0 years (IQR 2.4–5.8) were included in the analysis. 484 episodes of incident TB (73.6% culture-confirmed) were diagnosed in 424 patients during 6506 person-years (PYs) of follow-up. The TB incidence rate during the first year of ART was 12.4 (95% CI 10.8–14.4) cases/100PYs and decreased to 4.92 (95% CI 3.64–8.62) cases/100PYs between 5 and 8 years of ART. During person-time accrued within CD4 cell strata 0–100, 101–200, 201–300, 301–400, 401–500, 501–700 and ≥700 cells/µL, TB incidence rates (95% CI) were 25.5 (21.6–30.3), 11.2 (9.4–13.5), 7.9 (6.4–9.7), 5.0 (3.9–6.6), 5.1 (3.8–6.8), 4.1 (3.1–5.4) and 2.7 (1.7–4.5) cases/100PYs, respectively. Overall, 75% (95% CI 70.9–78.8) of TB episodes were recurrent cases. Updated CD4 cell count and viral load measurements were independently associated with long-term TB risk. TB rates during person-time accrued in the highest CD4 cell count stratum (>700 cells/µL) were 4.4-fold higher that the rate in HIV uninfected individuals living in the same community (2.7 versus 0.62 cases/100PYs; 95%CI 0.58–0.65).

Conclusions/Significance

TB rates during long-term ART remained substantially greater than rates in the local HIV uninfected populations regardless of duration of ART or attainment of CD4 cell counts exceeding 700 cells/µL.     

Effects of biotic disturbances on forest carbon cycling in the United States and Canada

Forest insects and pathogens are major disturbance agents that have affected millions of hectares in North America in recent decades, implying significant impacts to the carbon (C) cycle. Here, we review and synthesize published studies of the effects of biotic disturbances on forest C cycling in the United States and Canada. Primary productivity in stands was reduced, sometimes considerably, immediately following insect or pathogen attack. After repeated growth reductions caused by some insects or pathogens or a single infestation by some bark beetle species, tree mortality occurred, altering productivity and decomposition. In the years following disturbance, primary productivity in some cases increased rapidly as a result of enhanced growth by surviving vegetation, and in other cases increased slowly because of lower forest regrowth. In the decades following tree mortality, decomposition increased as a result of the large amount of dead organic matter. Net ecosystem productivity decreased immediately following attack, with some studies reporting a switch to a C source to the atmosphere, and increased afterward as the forest regrew and dead organic matter decomposed. Large variability in C cycle responses arose from several factors, including type of insect or pathogen, time since disturbance, number of trees affected, and capacity of remaining vegetation to increase growth rates following outbreak. We identified significant knowledge gaps, including limited understanding of carbon cycle impacts among different biotic disturbance types (particularly pathogens), their impacts at landscape and regional scales, and limited capacity to predict disturbance events and their consequences for carbon cycling. We conclude that biotic disturbances can have major impacts on forest C stocks and fluxes and can be large enough to affect regional C cycling. However, additional research is needed to reduce the uncertainties associated with quantifying biotic disturbance effects on the North American C budget.     

Designing allosteric modulators for active conformational state of m-glutamate G-protein coupled receptors

G-protein coupled receptors (GPCRs) are found to be attractive drug targets for the treatment of various neuronal diseases. Allosteric modulators have their role in enhancing or suppressing the effect of glutamate on mGluRs. Structure of mGluR1 was generated with the help of Modeller software by considering human B2-adrenergic GPCR protein as template. Structure of various already known drug molecules were used for similarity search in the ZINC database and a large number of similar molecules were obtained, than filtering of these molecules were done by applying drug features. Molecules were screened by Molegro Virtual Docking program and numbers of novel molecules were generated by using LigBuilder software. Finally 16 novel drug candidates were selected, which were showing better results than the seed molecule and previously known modulators. These results will help in designing and synthesis of better drugs against diseases like Epilepsy and Parkinson's.     

Metabolic pathway analysis and molecular docking analysis for identification of putative drug targets in Toxoplasma gondii: novel approach

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that can infect a wide range of warm-blooded animals including humans. In humans and other intermediate hosts, toxoplasma develops into chronic infection that cannot be eliminated by host's immune response or by currently used drugs. In most cases, chronic infections are largely asymptomatic unless the host becomes immune compromised. Thus, toxoplasma is a global health problem and the situation has become more precarious due to the advent of HIV infections and poor toleration of drugs used to treat toxoplasma infection, having severe side effects and also resistance have been developed to the current generation of drugs. The emergence of these drug resistant varieties of T. gondii has led to a search for novel drug targets. We have performed a comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen T. gondii. The enzymes in the unique pathways of T. gondii, which do not show similarity to any protein from the host, represent attractive potential drug targets. We have listed out 11 such potential drug targets which are playing some important work in more than one pathway. Out of these, one important target is Glutamate dehydrogenase enzyme; it plays crucial part in oxidation reduction, metabolic process and amino acid metabolic process. As this is also present in the targets of tropical diseases of TDR (Tropical disease related Drug) target database and no PDB and MODBASE 3D structural model is available, homology models for Glutamate dehydrogenase enzyme were generated using MODELLER9v6. The model was further explored for the molecular dynamics simulation study with GROMACS, virtual screening and docking studies with suitable inhibitors against the NCI diversity subset molecules from ZINC database, by using AutoDock-Vina. The best ten docking solutions were selected (ZINC01690699, ZINC17465979, ZINC17465983, ZINC18141294_03, ZINC05462670, ZINC01572309, ZINC18055497_01, ZINC18141294, ZINC05462674 and ZINC13152284_01). Further the Complexes were analyzed through LIGPLOT. On the basis of Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds, specifically ZINC01690699 (as it has minimum energy score and one of the highest number of interactions with the active site residue), could be promising inhibitors for T. gondii using Glutamate dehydrogenase as Drug target.     

Single-molecule pull-down for studying protein interactions

This protocol describes a single-molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy and allows the probing of single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single-molecule fluorescence microscopy is used to probe the pulled-down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. Compared with western blot analysis, this ultrasensitive assay requires considerably less time and reagents and provides quantitative data. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5-2.5 h for data acquisition and analysis.