Polyphyly of Asian Tree Toads,Genus Pedostibes Günther, 1876 (Anura: Bufonidae), and the Description of a New Genus from Southeast Asia

The Asian Tree Toad genus Pedostibes, as currently understood, exhibits a conspicuously disjunct distribution, posing several immediate questions relating to the biogeography and taxonomy of this poorly known group. The type species, P. tuberculosus and P. kempi, are known only from India, whereas P. hosii, P. rugosus, and P. everetti are restricted to Southeast Asia. Several studies have shown that these allopatric groups are polyphyletic, with the Indian Pedostibes embedded within a primarily South Asian clade of toads, containing the genera Adenomus, Xanthophryne, and Duttaphrynus. Southeast Asian Pedostibes on the other hand, are nested within a Southeast Asian clade, which is the sister lineage to the Southeast Asian river toad genus Phrynoidis. We demonstrate that Indian and Southeast Asian Pedostibes are not only allopatric and polyphyletic, but also exhibit significant differences in morphology and reproductive mode, indicating that the Southeast Asian species’ are not congeneric with the true Pedostibes of India. As a taxonomic solution, we describe a new genus, Rentapia gen. nov. to accommodate the Southeast Asian species.     

Unmasking Heavily O-Glycosylated Serum Proteins Using Perchloric Acid: Identification of Serum Proteoglycan 4 and Protease C1 Inhibitor as Molecular Indicators for Screening of Breast Cancer

Heavily glycosylated mucin glycopeptides such as CA 27.29 and CA 15–3 are currently being used as biomarkers for detection and monitoring of breast cancer. However, they are not well detected at the early stages of the cancer. In the present study, perchloric acid (PCA) was used to enhance detection of mucin-type O-glycosylated proteins in the serum in an attempt to identify new biomarkers for early stage breast cancer. Sensitivity and specificity of an earlier developed sandwich enzyme-linked lectin assay were significantly improved with the use of serum PCA isolates. When a pilot case-control study was performed using the serum PCA isolates of normal participants (n = 105) and patients with stage 0 (n = 31) and stage I (n = 48) breast cancer, higher levels of total O-glycosylated proteins in sera of both groups of early stage breast cancer patients compared to the normal control women were demonstrated. Further analysis by gel-based proteomics detected significant inverse altered abundance of proteoglycan 4 and plasma protease C1 inhibitor in both the early stages of breast cancer patients compared to the controls. Our data suggests that the ratio of serum proteoglycan 4 to protease C1 inhibitor may be used for screening of early breast cancer although this requires further validation in clinically representative populations.     

Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness

Background

Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF).

Methods

We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge.

Results

Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS).

Conclusions

Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa.

Key Messages

Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS.

Trial Registration

Clinical Trials.gov NCT02284074     

Fasting Plasma Insulin at 5 Years of Age Predicted Subsequent Weight Increase in Early Childhood over a 5-Year Period—The Da Qing Children Cohort Study

Background

The association between hyperinsulinemia and obesity is well known. However, it is uncertain especially in childhood obesity, if initial fasting hyperinsulinemia predicts obesity, or obesity leads to hyperinsulinemia through insulin resistance.

Objective

To investigate the predictive effect of fasting plasma insulin on subsequent weight change after a 5-year interval in childhood.

Methods

424 Children from Da Qing city, China, were recruited at 5 years of age and followed up for 5 years. Blood pressure, anthropometric measurements, fasting plasma insulin, glucose and triglycerides were measured at baseline and 5 years later.

Results

Fasting plasma insulin at 5 years of age was significantly correlated with change of weight from 5 to 10 years (ΔWeight). Children in the lowest insulin quartile had ΔWeight of 13.08±0.73 kg compare to 18.39±0.86 in the highest insulin quartile (P<0.0001) in boys, and similarly 12.03±0.71 vs 15.80±0.60 kg (P<0.0001) in girls. Multivariate analysis showed that the predictive effect of insulin at 5 years of age on subsequent weight gain over 5 years remained statistically significant even after the adjustment for age, sex, birth weight, TV-viewing time and weight (or body mass index) at baseline. By contrast, the initial weight at 5 years of age did not predict subsequent changes in insulin level 5 years later. Children who had both higher fasting insulin and weight at 5 years of age showed much higher levels of systolic blood pressures, fasting plasma glucose, the homeostasis model assessment for insulin resistance (HOMA-IR) and triglycerides at 10 years of age.

Conclusions

Fasting plasma insulin at 5 years of age predicts weight gain and cardiovascular risk factors 5 year later in Chinese children of early childhood, but the absolute weight at 5 years of age did not predict subsequent change in fasting insulin.     

The formulation of life cycle impact assessment framework for Malaysia using Eco-indicator

Background, aim, and scope  

Life Cycle Assessment (LCA) is an emerging supporting tool designed to help practitioner in systematically assessing the environmental performance of selected product’s life cycle. A product’s life cycle includes the extraction of raw materials, production, and usage, and ends with waste treatment or disposal. Life cycle impact assessment (LCIA) as a part of LCA is a method used to derive the environmental burdens from selected product’s stages. LCIA is structured in classification, characterization, normalization and weighting. Presently most of the LCIA practices use European database to establish the characterization, normalization and weighting value. However, using these values for local LCA practice might not be able to reflect the actual Malaysian’s environmental scenario. The aim of this study is to create a Malaysian version of normalization and weighting value using the pollution database within Malaysia.     

Co-ordinated role of TLR3, RIG-I and MDA5 in the innate response to rhinovirus in bronchial epithelium

The relative roles of the endosomal TLR3/7/8 versus the intracellular RNA helicases RIG-I and MDA5 in viral infection is much debated. We investigated the roles of each pattern recognition receptor in rhinovirus infection using primary bronchial epithelial cells. TLR3 was constitutively expressed; however, RIG-I and MDA5 were inducible by 8-12 h following rhinovirus infection. Bronchial epithelial tissue from normal volunteers challenged with rhinovirus in vivo exhibited low levels of RIG-I and MDA5 that were increased at day 4 post infection. Inhibition of TLR3, RIG-I and MDA5 by siRNA reduced innate cytokine mRNA, and increased rhinovirus replication. Inhibition of TLR3 and TRIF using siRNA reduced rhinovirus induced RNA helicases. Furthermore, IFNAR1 deficient mice exhibited RIG-I and MDA5 induction early during RV1B infection in an interferon independent manner. Hence anti-viral defense within bronchial epithelium requires co-ordinated recognition of rhinovirus infection, initially via TLR3/TRIF and later via inducible RNA helicases.     

Perturbed heme binding is responsible for the blistering phenotype associated with mutations in the Caenorhabditis elegans dual oxidase 1 (DUOX1) peroxidase domain

Dual oxidase (DUOX) enzymes support a wide variety of essential reactions, from cellular signaling to thyroid hormone biosynthesis. In Caenorhabditis elegans, the DUOX system (CeDUOX1/2) plays a crucial role in innate immunity and in stabilizing the cuticle by forming tyrosine cross-links. The current model suggests that superoxide generated by CeDUOX1 at the C-terminal NADPH oxidase domain is rapidly converted to H(2)O(2). The H(2)O(2) is then utilized by the N-terminal peroxidase-like domain to cross-link tyrosines. We have now created a series of mutations in the isolated peroxidase domain, CeDUOX1(1-589). One set of mutations investigate the roles of a putative distal tyrosine (Tyr(105)) and Glu(238), a proposed covalent heme-binding residue. The results confirm that Glu(238) covalently binds to the heme group. A second set of mutations (G246D and D392N) responsible for a C. elegans blistering cuticle phenotype was also investigated. Surprisingly, although not among the catalytic residues, both mutations affected heme co-factor binding. The G246D mutant bound less total heme than the wild type, but a higher fraction of it was covalently bound. In contrast, the D392N mutant appears to fold normally but does not bind heme. Molecular dynamics simulations of a CeDUOX1(1-589) homology model implicate displacements of the proximal histidine residue as the likely cause. Both enzymes are structurally stable and through altered heme interactions exhibit partial or complete loss of tyrosine cross-linking activity, explaining the blistering phenotype. This result argues that the CeDUOX peroxidase domain is primarily responsible for tyrosine cross-linking.     

A-site residues move independently from P-site residues in all-atom molecular dynamics simulations of the 70S bacterial ribosome

The ribosome is a large macromolecular machine, and correlated motion between residues is necessary for coordinating function across multiple protein and RNA chains. We ran two all-atom, explicit solvent molecular dynamics simulations of the bacterial ribosome and calculated correlated motion between residue pairs by using mutual information. Because of the short timescales of our simulation (ns), we expect that dynamics are largely local fluctuations around the crystal structure. We hypothesize that residues that show coupled dynamics are functionally related, even on longer timescales. We validate our model by showing that crystallographic B-factors correlate well with the entropy calculated as part of our mutual information calculations. We reveal that A-site residues move relatively independently from P-site residues, effectively insulating A-site functions from P-site functions during translation.     

Sequence coevolution between RNA and protein characterized by mutual information between residue triplets

Coevolving residues in a multiple sequence alignment provide evolutionary clues of biophysical interactions in 3D structure. Despite a rich literature describing amino acid coevolution within or between proteins and nucleic acid coevolution within RNA, to date there has been no direct evidence of coevolution between protein and RNA. The ribosome, a structurally conserved macromolecular machine composed of over 50 interacting protein and RNA chains, provides a natural example of RNA/protein interactions that likely coevolved. We provide the first direct evidence of RNA/protein coevolution by characterizing the mutual information in residue triplets from a multiple sequence alignment of ribosomal protein L22 and neighboring 23S RNA. We define residue triplets as three positions in the multiple sequence alignment, where one position is from the 23S RNA and two positions are from the L22 protein. We show that residue triplets with high mutual information are more likely than residue doublets to be proximal in 3D space. Some high mutual information residue triplets cluster in a connected series across the L22 protein structure, similar to patterns seen in protein coevolution. We also describe RNA nucleotides for which switching from one nucleotide to another (or between purines and pyrimidines) results in a change in amino acid distribution for proximal amino acid positions. Multiple crystal structures for evolutionarily distinct ribosome species can provide structural evidence for these differences. For one residue triplet, a pyrimidine in one species is a purine in another, and RNA/protein hydrogen bonds are present in one species but not the other. The results provide the first direct evidence of RNA/protein coevolution by using higher order mutual information, suggesting that biophysical constraints on interacting RNA and protein chains are indeed a driving force in their evolution.     

Single Cell Analysis of Yeast Replicative Aging Using a New Generation of Microfluidic Device

A major limitation to yeast aging study has been the inability to track mother cells and observe molecular markers during the aging process. The traditional lifespan assay relies on manual micro-manipulation to remove daughter cells from the mother, which is laborious, time consuming, and does not allow long term tracking with high resolution microscopy. Recently, we have developed a microfluidic system capable of retaining mother cells in the microfluidic chambers while removing daughter cells automatically, making it possible to observe fluorescent reporters in single cells throughout their lifespan. Here we report the development of a new generation of microfluidic device that overcomes several limitations of the previous system, making it easier to fabricate and operate, and allowing functions not possible with the previous design. The basic unit of the device consists of microfluidic channels with pensile columns that can physically trap the mother cells while allowing the removal of daughter cells automatically by the flow of the fresh media. The whole microfluidic device contains multiple independent units operating in parallel, allowing simultaneous analysis of multiple strains. Using this system, we have reproduced the lifespan curves for the known long and short-lived mutants, demonstrating the power of the device for automated lifespan measurement. Following fluorescent reporters in single mother cells throughout their lifespan, we discovered a surprising change of expression of the translation elongation factor TEF2 during aging, suggesting altered translational control in aged mother cells. Utilizing the capability of the new device to trap mother-daughter pairs, we analyzed mother-daughter inheritance and found age dependent asymmetric partitioning of a general stress response reporter between mother and daughter cells.