A semiparametric modeling framework for potential biomarker discovery and the development of metabonomic profiles

Background  

The discovery of biomarkers is an important step towards the development of criteria for early diagnosis of disease status. Recently electrospray ionization (ESI) and matrix assisted laser desorption (MALDI) time-of-flight (TOF) mass spectrometry have been used to identify biomarkers both in proteomics and metabonomics studies. Data sets generated from such studies are generally very large in size and thus require the use of sophisticated statistical techniques to glean useful information. Most recent attempts to process these types of data model each compound's intensity either discretely by positional (mass to charge ratio) clustering or through each compounds' own intensity distribution. Traditionally data processing steps such as noise removal, background elimination and m/z alignment, are generally carried out separately resulting in unsatisfactory propagation of signals in the final model.     

An efficient field screening procedure for identifying transposants for constructing an Ac/Ds-based insertional-mutant library of rice.

An efficient system was developed, and several variables tested, for generating a large-scale insertional-mutagenesis population of rice. The most important feature in this improved Ac/Ds tagging system is that one can conveniently carry out large-scale screening in the field and select transposants at the seedling stage. Rice was transformed with a plasmid that includes a Basta-resistance gene (bar). After the Ds element is excised during transposition, bar becomes adjacent to the ubiquitin promoter, and the rice plant becomes resistant to the herbicide Basta. In principle, one can plant up to one million plants in the field and select those plants that survive after spraying with Basta. To test the utility of this system, 4 Ds starter lines were crossed with 14 different Ac plants, and many transposants were successfully identified after planting 134,285 F2 plants in the field. Over 2,800 of these transposants were randomly chosen for PCR analysis, and the results fully confirmed the reliability of the field screening procedure.     

Genetic loss of Faah compromises male fertility in mice

Marijuana is the most commonly used illicit drug. Although there is some indication that reproductive functions in males are impaired in chronic marijuana users, the genetic evidence and underlying causes remain largely unknown. Herein we show that genetic loss of Faah, which encodes fatty acid amide hydrolase (FAAH), results in elevated levels of anandamide, an endocannabinoid, in the male reproductive system, leading to compromised fertilizing capacity of sperm. This defect is rescued by superimposing deletion of cannabinoid receptor 1 (Cnr1). Retention of Faah(-/-) sperm on the egg zona pellucida provides evidence that the capacity of sperm to penetrate the zona barrier is hampered by elevated anandamide levels. Collectively, the results show that aberrant endocannabinoid signaling via CNR1 impairs normal sperm function. Besides unveiling a new regulatory mechanism of sperm function, this study has clinical significance in male fertility.     

Malarial infection develops mitochondrial pathology and mitochondrial oxidative stress to promote hepatocyte apoptosis

Activation of the mitochondrial apoptosis pathway by oxidative stress has been implicated in hepatocyte apoptosis during malaria. Because mitochondria are the source and target of reactive oxygen species (ROS), we have investigated whether hepatocyte apoptosis is linked to mitochondrial pathology and mitochondrial ROS generation during malaria. Malarial infection induces mitochondrial pathology by inhibiting mitochondrial respiration, dehydrogenases, and transmembrane potential and damaging the ultrastructure as evident from transmission electron microscopic studies. Mitochondrial GSH depletion and formation of protein carbonyl indicate that mitochondrial pathology is associated with mitochondrial oxidative stress. Fluorescence imaging of hepatocytes documents intramitochondrial superoxide anion (O₂^?) generation during malaria. O₂^? inactivates mitochondrial aconitase to release iron from iron–sulfur clusters, which forms the hydroxyl radical (OH) interacting with H₂O₂ produced concurrently. Malarial infection inactivates mitochondrial aconitase, and carbonylation of aconitase is evident from Western immunoblotting. The release of iron has been documented by fluorescence imaging of hepatocytes using Phen Green SK, and mitochondrial OH generation has been confirmed. During malaria, the depletion of cardiolipin and formation of the mitochondrial permeability transition pore favor cytochrome c release to activate caspase-9. Interestingly, mitochondrial OH generation correlates with the activation of both caspase-9 and caspase-3 with the progress of malarial infection, indicating the critical role of OH.     

Enhanced production and partial characterization of an extracellular polysaccharide from newly isolated Azotobacter sp. SSB81

A strain was selected by its highest extracellular polysaccharide (EPS) production ability compare to other isolates from the same rhizospheric soil. The selected strain was identified by 16S rDNA sequencing and designated as SSB81. Phylogenetic analysis of the gene sequence showed its close relatedness with Azotobacter vinelandii and Azotobacter salinestris. Maximum EPS (2.52 g l⁻¹) was recovered when the basal medium was supplemented with glucose (2.0%), riboflavin (1 mg l⁻¹) and casamino acid (0.2%). The EPS showed a stable viscosity level at acidic pH (3.0–6.5) and the pyrolysis temperature was found to be at 116.73 °C with an enthalpy (ΔH) of 1330.72 Jg⁻¹. MALDI TOF mass spectrometric result suggests that polymer contained Hex₅Pent₃ as oligomeric building subunit. SEM studies revealed that the polymer had a porous structure with small pore size distribution indicating the compactness of the polymer. This novel EPS may find possible application as a polymer for environmental bioremediation and biotechnological processes.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.     

Analysis of Endocytic Pathways in Drosophila Cells Reveals a Conserved Role for GBF1 in Internalization via GEECs

In mammalian cells, endocytosis of the fluid phase and glycosylphosphatidylinositol-anchored proteins (GPI-APs) forms GEECs (GPI-AP enriched early endosomal compartments) via an Arf1- and Cdc42-mediated, dynamin independent mechanism. Here we use four different fluorescently labeled probes and several markers in combination with quantitative kinetic assays, RNA interference and high resolution imaging to delineate major endocytic routes in Drosophila cultured cells. We find that the hallmarks of the pinocytic GEEC pathway are conserved in Drosophila and identify garz, the fly ortholog of the GTP exchange factor GBF1, as a novel component of this pathway. Live confocal and TIRF imaging reveals that a fraction of GBF1 GFP dynamically associates with ABD RFP (a sensor for activated Arf1 present on nascent pinosomes). Correspondingly, a GTP exchange mutant of GBF1 has altered ABD RFP localization in the evanescent field and is impaired in fluid phase uptake. Furthermore, GBF1 activation is required for the GEEC pathway even in the presence of Brefeldin A, implying that, like Arf1, it has a role in endocytosis that is separable from its role in secretion.     

Allosteric modulation bypasses the requirement for ATP hydrolysis in regenerating low affinity transition state conformation of human P-glycoprotein

ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653-11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP.Vi or 8-azido-ADP.Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP), resulting in dissociation of the latter. Regeneration of the [125I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [125I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [125I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (approximately 60 kDa) recognizable by a polyclonal antiserum specific for the NH2-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [125I]IAAP binding without any further nucleotide hydrolysis.     

Biofilm and metallo beta-lactamase production among the strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Acinetobacter</Emphasis> spp. at a Tertiary Care Hospital in Kathmandu,Nepal

Introduction

Pseudomonas aeruginosa and Acinetobacter spp. are found to be associated with biofilm and metallo-β-lactamase production and are the common causes of serious infections mainly in hospitalized patients. So, the main aims of this study were to determine the rates of biofilm production and metallo beta-lactamase production (MBL) among the strains of Pseudomonas aeruginosa and Acinetobacter spp. isolated from hospitalized patients.

Methods

A total of 85 P. aeruginosa isolates and 50 Acinetobacter spp. isolates isolated from different clinical specimens from patients admitted to Shree Birendra Hospital, Kathmandu, Nepal from July 2013 to May 2014 were included in this study. The bacterial isolates were identified with the help of biochemical tests. Modified Kirby-Bauer disc diffusion technique was used for antimicrobial susceptibility testing. Combined disc diffusion technique was used for the detection of MBL production, while Congo red agar method and tube adherence method were used for detection of biofilm production.

Results

Around 16.4% of P. aeruginosa isolates and 22% of the strains of Acinetobacter spp. were metallo β-lactamase producers. Out of 85 P. aeruginosa isolates, 23 (27.05%) were biofilm producers according to tube adherence test while, only 13 (15.29%) were biofilm producers as per Congo red agar method. Similarly, out of 50 Acinetobacter spp. 7 (14%) isolates were biofilm producers on the basis of tube adherence test, while only 5 (10%) were positive for biofilm production by Congo red agar method. Highest rates of susceptibility of P. aeruginosa as well as Acinetobacter spp. were seen toward colistin.

Conclusion

In our study, biofilm production and metallo beta-lactamase production were observed among Pseudomonas aeruginosa and Acinetobacter spp. However, no statistically significant association could be established between biofilm production and metallo beta-lactamase production.