Proteomic Profiles in Acute Respiratory Distress Syndrome Differentiates Survivors from Non-Survivors

Acute Respiratory Distress Syndrome (ARDS) continues to have a high mortality. Currently, there are no biomarkers that provide reliable prognostic information to guide clinical management or stratify risk among clinical trial participants. The objective of this study was to probe the bronchoalveolar lavage fluid (BALF) proteome to identify proteins that differentiate survivors from non-survivors of ARDS. Patients were divided into early-phase (1 to 7 days) and late-phase (8 to 35 days) groups based on time after initiation of mechanical ventilation for ARDS (Day 1). Isobaric tags for absolute and relative quantitation (iTRAQ) with LC MS/MS was performed on pooled BALF enriched for medium and low abundance proteins from early-phase survivors (n = 7), early-phase non-survivors (n = 8), and late-phase survivors (n = 7). Of the 724 proteins identified at a global false discovery rate of 1%, quantitative information was available for 499. In early-phase ARDS, proteins more abundant in survivors mapped to ontologies indicating a coordinated compensatory response to injury and stress. These included coagulation and fibrinolysis; immune system activation; and cation and iron homeostasis. Proteins more abundant in early-phase non-survivors participate in carbohydrate catabolism and collagen synthesis, with no activation of compensatory responses. The compensatory immune activation and ion homeostatic response seen in early-phase survivors transitioned to cell migration and actin filament based processes in late-phase survivors, revealing dynamic changes in the BALF proteome as the lung heals. Early phase proteins differentiating survivors from non-survivors are candidate biomarkers for predicting survival in ARDS.     

Hidden reservoirs of pathogens in dental settings

Nosocomial infections are a major concern to both clinicians and health care seekers. Investigations have suggested that laptops & mobile phones may contribute to cross-contamination and can serve as vehicles for infection transmission. Therefore, it is of interest to document the data on hidden reservoirs such as mobile phones and laptops of pathogens in dental settings at the Hazaribag College of dental sciences and Hospital, Jharkhand. The samples were collected from 25 laptops and 25 mobile phones from dentists working in a dental college in Hazaribag city. The samples were collected aseptically using sterile cotton swabs dipped in sterile saline by rotating the swabs on the keyboard surfaces of laptops and mobile phones, inoculated into Brain Heart Infusion broth, vortexed for 1 minute in Fischer Vortex Genie 2 on highest setting & streaked immediately on 5% sheep blood agar plates and were incubated at 37°C for 24 hours aerobically. The isolates were identified based on the colony morphology, colony characteristics and biochemical reactions. The bacterial species isolated were Staphylococcus aureus, Coagulase negative Staphylococcus, Bacillus species, Enterococci, Micrococci, and Pseudomonas etc. Predominant species isolated was Staphylococcus aureus and least was Micrococci. Higher percentage of organisms was found at the Department of Periodontics, Endodontics and least was found in Department of Public Health Dentistry. The percentage and type of organism isolated from keyboards of laptops and mobile phones were similar. Thus, laptops and mobile phones act as vehicles for transfer of potential pathogens associated with dental hospitals. Disinfecting the hands prior to examination of patients and disinfection of laptops and mobiles with alcohol wipes should be done to prevent nosocomial infections.     

Synthesis and pharmacological characterization of a potent,orally active p38 kinase inhibitor

Inhibitors of the MAP kinase p38 provide a novel approach for the treatment of osteoporosis, inflammatory disorders, and cancer. We have identified N-(3-tert-butyl-1-methyl-5-pyrazolyl)-N'-(4-(4-pyridinylmethyl)phenyl)urea as a potent and selective p38 kinase inhibitor in biochemical and cellular assays. This compound is orally active in two acute models of cytokine release (TNF-induced IL-6 and LPS-induced TNF) and a chronic model of arthritis (20-day murine collagen-induced arthritis).     

p38 kinase inhibitors for the treatment of arthritis and osteoporosis: thienyl, furyl, and pyrrolyl ureas

Inhibitors of the MAP kinase p38 are potentially useful for the treatment for osteoporosis, arthritis, and other inflammatory diseases. A series of thienyl, furyl, and pyrrolyl ureas has been identified as potent p38 inhibitors, displaying in vitro activity in the nanomolar range.     

Yttrium-90 delivered via a centering catheter and afterloader, given both before and after stent implantation, inhibits neointima formation in porcine coronary arteries

Background: Intracoronary radiation (IR) studies have shown reduction of neointima formation (NF). Extrapolation of animal studies with beta-radiation to clinical trials have shown variable results, which may be related to dosimetry, centering issues, and/or shielding of beta-rays by the stent metal. We examined the effect of yttrium-90 (90Y), a pure beta-emitter delivered via an automatic afterloader to a centering catheter, on the inhibition of NF in balloon-injured (BI) porcine coronary arteries as well as in arteries receiving 90Y either prior to or following stent implantation (SI).Methods: Twenty-three swine (44 coronary arteries) were studied. In the first study, IR (18 Gy at 1.2 mm from the balloon surface) was administered in 17 arteries following BI, while eight control arteries were subjected to BI only. In the second study, 10 swine (19 coronary arteries) underwent SI. IR (18 Gy) was administered in six arteries before and in eight arteries after SI, while five control arteries received SI only. The animals were sacrificed 2 weeks after BI and 4 weeks after SI. Their coronaries were perfusion fixed and stained, and vessel parameters (intimal area [IA] and medial fracture length [FL]) were analyzed by computer-aided histomorphometry.Results: Arteries subjected to IR following BI had less NF compared to controls (IA/FL=0.14+/-0.2 mm vs. 0.49+/-0.2 mm; P=0.003). IA was reduced significantly in the arteries receiving radiation before and after SI compared to controls (0.92+/-0.98 and 0.00+/-0/00 vs. 2.72+/-1.2 mm(2); P=0.014), despite similar SI in all groups.Conclusions: IR with 90Y delivered via a centering catheter is safe and effective with complete and homogenous inhibition of NF in the context of BI or SI in the porcine coronary model.     

Randomized controlled experiments in health and social sciences: Some conceptual issues

This brief article outlines some difficulties as well as benefits in conducting randomized controlled trials in social science settings especially in developing countries. Some of the historical developments are summarized and certain applications in health sciences are discussed from methodological and policy standpoints.     

Comprehensive analysis of protein-protein interactions between Arabidopsis MAPKs and MAPK kinases helps define potential MAPK signalling modules

The Arabidopsis genome encodes a 20-member gene family of mitogen-activated protein kinases (MPKs) but biological roles have only been identified for a small subset of these crucial signalling components. In particular, it is unclear how the MPKs may be organized into functional modules within the cell. To gain insight into their potential relationships, we used the yeast two-hybrid system to conduct a directed protein-protein interaction screen between all the Arabidopsis MPKs and their upstream activators (MAPK kinases; MKK). Novel interactions were also tested in vitro for enzyme-substrate functionality, using recombinant proteins. The resulting data confirm a number of earlier reported MKK-MPK relationships, but also reveal a more extensive pattern of interactions that should help to guide future analyses of MAPK signalling in plants.Key words: mitogen-activated protein kinase, mitogen-activated protein kinase kinase, yeast two-hybrid, phosphorylation, protein-protein interaction, ArabidopsisPlant genomes are notably rich in the number of protein kinase signalling components they encode^1–3 and it can therefore be anticipated that the associated signal transduction networks will be highly specialized and complex. Within the plant protein kinase super-family, the highly conserved Arabidopsis mitogen-activated protein kinases (MAPKs; MPKs) are represented by a 20-member family that is most closely related to the ERK class of metazoan MAPKs.⁴ This family includes three sub-families of MPKs whose activation domain carries a -TEY- motif, as well as a fourth, evolutionarily distinct -TDY- sub-family.^4,5 Dual-specificity MAPK kinases (MKK) serve as the canonical activators of MPKs through phosphorylation of both the threonine and tyrosine residues within the MPK activation loop -TXY- motif. The Arabidopsis genome encodes ten members of the MKK gene family, among which one (MKK10) lacks the fully conserved -S/T-X_3-5-S/T- motif that typifies eukaryotic MAPK kinases.⁵Numerous reports have provided evidence for the involvement of plant MPKs in a wide range of biotic and abiotic stress responses, as well as phytohormone signalling and developmental patterning, as recently reviewed in.⁶ However, defining functional MKK-MPK module combinations by connecting a particular activated MPK to a specific upstream MKK remains a challenge. Since there are precedents for activation of multiple MPKs by one MKK, as well as evidence for more than one MKK having the capability of activating a given MPK, there are many possible ways in which MKK-MPK signalling modules might potentially be configured. Phenotype-based forward genetic screens in Arabidopsis have provided relatively little insight into these relationships, with only one MPK (MPK4) being recovered as a loss-of-function mutant.⁷ The failure to recover mutations in the other MPK loci, or in any of the MKKs, in such screens could indicate that there is considerable functional redundancy within the MAPK signalling network, that the phenotypic consequences of a loss-of-function mutation are subtle or conditional, or that loss-of-function genotypes are non-viable.Since the nature of protein kinase/phosphatase activities depends on direct physical encounters between the enzyme and its target protein, we hypothesized that the ability of particular proteins to interact effectively with each other would define one level of specificity within the global Arabidopsis MKK/MPK network. To test this idea, we conducted a comprehensive directed yeast two-hybrid screen using the ten Arabidopsis MKKs as individual bait proteins, and each of the twenty MPKs as prey proteins. Several of the protein-protein interactions detected in this Y2H screen were also tested in direct phosphorylation assays in vitro, using recombinant proteins.Nine of the ten Arabidopsis MKK proteins were found to interact with at least one MPK protein in our Y2H assays (https://www.genevestigator.ethz.ch/gv/index.jsp). Its putative orthologue in Populus trichocarpa is similarly silent,^5,8 consistent with a gene that may be losing its biological functionality. Most other MKKs were found to interact with two or more MPK targets, and in several cases these results confirmed earlier reports of MKK-MPK interactions. For example, we found that MKK1 and MKK2, two closely related MAPKKs, both interacted with MPK 4 and with MPK11, a pair of paralogous MPKs. Interaction between MKK1 (MEK1) and MPK4 had already been observed in one of the first studies of plant MKK-MPK relationships,⁹ while a later study also found that MKK2 could interact with MPK4, among twelve MPKs surveyed.¹⁰ However, neither of these reports had examined MPK11. We could confirm by in vitro phosphorylation assays using “constitutively active” (CA) forms of recombinant MKK1 and MKK2 that both of these MKKs can phosphorylate recombinant MPK4, but, in contrast to the Y2H interaction pattern, both CAMKKs showed only very weak activity with MPK11 as a substrate (Fig. 2A and B).Open in a separate windowFigure 2Effects of incubation with recombinant GST-CAMKK proteins on protein phosphorylation of recombinant GST-MPKs. The constitutively active mutant forms of the MKKs were generated by QuickChange site-directed mutagenesis (Stratagene) and confirmed by sequencing. The conserved Ser or Thr residues in the activation loop in MKKs were replaced with acidic residues to create a “constitutively active” kinase (T218E and S224D for CAMKK1, T220D and T226E for CAMKK2, S221D and T227E for CAMKK6, S193E and S199D for CAMKK7, and S195E and S201E for CAMKK9). PCR amplicons of the full-length cDNAs corresponding to MPK2 (At1g59580), MPK4 (At4g01370), MPK6 (At2g43790), MPK10 (At3g59790), MPK11 (At1g01560), MPK13 (At1g07880), MPK17 (At2g01450), MPK20 (At2g42880), MKK1 (At4g26070), MKK2 (At4g29810), MKK6 (At5g56580), MKK7 (At1g18350) and MKK9 (At1g73500) were purified, digested with the appropriate restriction enzymes and subcloned in either the pGEX 4T-2 or pDEST15 vector, which expresses the recombinant protein with a N-terminal GST tag. Each of wild-type MAPK and mutant recombinant CAMKK1, CAMKK2, CAMKK6, CAMKK7 and CAMKK9 were expressed as glutathione S-transferase (GST) fusion proteins as previously described.¹⁹ For the in vitro phosphorylation assays, each GST-MPK (1 µg) was incubated in 25 µL of kinase reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM β-glycerolphosphate, 2 mM DTT, 10 mM MgCl₂, 0.1 mM Na₃VO₄, 0.1 mM ATP and 3 µCi of [γ-³²P] ATP) either with or without constitutively active GST-MKKs (0.3 µg) at 30°C for 30 min. The reaction was terminated by addition of concentrated SDS-PAGE sample buffer followed by boiling for 5 min. Reaction products were analyzed using SDS-PAGE, autoradiography, and CBB staining. (A) Phosphorylation of MPKs by incubation with CAMKK1. (B) Phosphorylation of MPKs by incubation with CAMKK2. (C) Phosphorylation of MPKs by incubation with CAMKK6. (D) Phosphorylation of MPK2 by incubation with CAMKK7. (E) Phosphorylation of MPKs by incubation with CAMKK9.

Table 1

Full-length cDNA clones corresponding to the open reading frame of each of the ten Arabidopsis MKK and twenty distinct MAPKs were isolated from Arabidopsis cDNA and cloned into a Gateway™ entry vector, either pENTR (Invitrogen) or pCR8 (Invitrogen)

Salinity-induced physiological and proteomic changes in Anabaena doliolum

This study is the first to offer information on salinity-induced inhibition of physiological variables, changes in proteome, and induction of glycolate metabolism in Anabaena doliolum. A significant reduction in O₂-evolution, carbon fixation, chlorophyll and NADPH/NADH level and increase in intracellular Na⁺ and respiration were observed following 150 mM NaCl treatment for 1 and 24 h. Interestingly, ATP content registered significant decrease after 1 h and recovery after 24 h treatment of 150 mM NaCl. Two-dimensional gel electrophoresis and MALDI-TOF MS detected a set of six proteins showing significant reproducible alterations, and homology with iron superoxide dismutase, superoxide dismutase (imported), phycocyanin alpha chain, elongation factor-Tu (EF-Tu), ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase of Nostoc PCC7120. Increased RuBisCO and decreased carbon fixation suggested operation of glycolate metabolism. This was confirmed by accumulation of free and phospho-glyceric acid, increase in glycolate oxidase activity, glycine, serine and ammonium contents. Since peroxide generated in this pathway cannot be scavenged due to sensitivity of catalase to NaCl the organism fails to acclimatize under salt stress.