In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-³H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-³H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.     

Mitochondrial dysfunction and genetic heterogeneity in chronic periodontitis

We performed an extensive study on mitochondrial dysfunction in chronic periodontitis (CP). Electron microscopic analysis of gingival cells revealed abnormal mitochondria in 60% of the patients. Mitochondrial membrane potential and oxygen consumption of gingival cells were reduced by 4 fold and 5.8 fold, respectively; whereas ROS production was increased by 18%. The genetic analysis by complete mitochondrial DNA sequencing revealed the identification of 14 novel mutations only in periodontal tissues but not in the blood, suggesting a role of oxidative stress on periodontal tissues. Thus, our functional and genetic analysis provided an evidence for the mitochondrial dysfunction in CP.     

Defective pro-IL-1β responses in macrophages from aged mice

Background

Cytokines regulated by the inflammasome pathway have been extensively implicated in various age-related immune pathologies. We set out to elucidate the contribution of the nod-like receptor protein 3 (NLRP3) inflammasome pathway to the previously described deficiencies in IL-1β production by macrophages from aged mice. We examined the production of pro-IL-1β and its conversion into IL-1β as two separate steps and compared these cytokine responses in bone marrow derived macrophages from young (6–8 weeks) and aged (18–24 months) C57BL/6 mice.

Findings

Relative to macrophages from young mice, macrophages from aged mice produced less pro-IL-1β after TLR4 stimulation with LPS. However upon activation of the NLRP3 inflammasome with ATP, macrophages from young and aged mice were able to efficiently convert and secrete intracellular pro-cytokines as functional cytokines.

Conclusions

Lower levels of IL-1β production are a result of slower and lower overall production of pro-IL-1β in macrophages from aged mice.

     

Synthesis and antibacterial activity of some novel imidazole-based dicationic quinolinophanes

Synthesis of novel quinoline based dicationic benzimidazolophanes and imidazolophanes incorporating various spacer units is described. Some of the quinolinophanes 1b, 3a, 3b and 4a exhibit good antibacterial activity against most of the human pathogenic bacteria in the tested concentrations as compared to the other cyclophanes as well as the test control, streptomycin.     

Biological removal of carcinogenic chromium(VI) using mixed Pseudomonas strains

The contamination of soil and wastewaters with Cr(VI) is a major problem. It has been suggested that microbial methods for Cr(VI) reduction are better than chemical methods, as they do not add other ions or toxic chemicals to the environment. In this study an aerobic reduction of Cr(VI) to Cr(III) by employing mixed Pseudomonas cultures isolated from a marshy land has been reported. The role of chromium concentration, temperature, pH and additives on the microbial reduction of Cr(VI) has been investigated. NADH was found to enhance the rate of reduction of Cr(VI). Complete reduction of chromium(VI) has been possible even at chromium(VI) concentrations of 300 ppm. Ions like SO(4)(2-) and poly-phenols inhibited the metabolic activity relating to Cr(VI) reduction. Under optimal conditions 100 mg/L of Cr(VI) was completely reduced within 180 min.     

Case-Mix,Care Processes,and Outcomes in Medically-Ill Patients Receiving Mechanical Ventilation in a Low-Resource Setting from Southern India: A Prospective Clinical Case Series

Background

Mechanical ventilation is a resource intensive organ support treatment, and historical studies from low-resource settings had reported a high mortality. We aimed to study the outcomes in patients receiving mechanical ventilation in a contemporary low-resource setting.

Methods

We prospectively studied the characteristics and outcomes (disease-related, mechanical ventilation-related, and process of care-related) in 237 adults mechanically ventilated for a medical illness at a teaching hospital in southern India during February 2011 to August 2012. Vital status of patients discharged from hospital was ascertained on Day 90 or later.

Results

Mean age of the patients was 40 ± 17 years; 140 (51%) were men. Poisoning and envenomation accounted for 98 (41%) of 237 admissions. In total, 87 (37%) patients died in-hospital; 16 (7%) died after discharge; 115 (49%) were alive at 90-day assessment; and 19 (8%) were lost to follow-up. Weaning was attempted in 171 (72%) patients; most patients (78 of 99 [79%]) failing the first attempt could be weaned off. Prolonged mechanical ventilation was required in 20 (8%) patients. Adherence to head-end elevation and deep vein thrombosis prophylaxis were 164 (69%) and 147 (62%) respectively. Risk of nosocomial infections particularly ventilator-associated pneumonia was high (57.2 per 1,000 ventilator-days). Higher APACHE II score quartiles (adjusted HR [95% CI] quartile 2, 2.65 [1.19–5.89]; quartile 3, 2.98 [1.24–7.15]; quartile 4, 5.78 [2.45–13.60]), and new-onset organ failure (2.98 [1.94–4.56]) were independently associated with the risk of death. Patients with poisoning had higher risk of reintubation (43% vs. 20%; P = 0.001) and ventilator-associated pneumonia (75% vs. 53%; P = 0.001). But, their mortality was significantly lower compared to the rest (24% vs. 44%; P = 0.002).

Conclusions

The case-mix considerably differs from other settings. Mortality in this low-resource setting is similar to high-resource settings. But, further improvements in care processes and prevention of nosocomial infections are required.     

Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment

Cell-intrinsic checkpoints limit the proliferative capacity of primary cells in response to telomere dysfunction. It is not known, however, whether telomere dysfunction contributes to cell-extrinsic alterations that impair stem cell function and organ homeostasis. Here we show that telomere dysfunction provokes defects of the hematopoietic environment that impair B lymphopoiesis but increase myeloid proliferation in aging telomerase knockout (Terc(-/-)) mice. Moreover, the dysfunctional environment limited the engraftment of transplanted wild-type hematopoietic stem cells (HSCs). Dysfunction of the hematopoietic environment was age dependent and correlated with progressive telomere shortening in bone marrow stromal cells. Telomere dysfunction impaired mesenchymal progenitor cell function, reduced the capacity of bone marrow stromal cells to maintain functional HSCs, and increased the expression of various cytokines, including granulocyte colony-stimulating factor (G-CSF), in the plasma of aging mice. Administration of G-CSF to wild-type mice mimicked some of the defects seen in aging Terc(-/-) mice, including impairment of B lymphopoiesis and HSC engraftment. Conversely, inhibition of G-CSF improved HSC engraftment in aged Terc(-/-) mice. Taken together, these results show that telomere dysfunction induces alterations of the environment that can have implications for organismal aging and cell transplantation therapies.     

Feather ornaments are dynamic traits in the Great Tit Parus major

In behavioural studies it has been common to quantify plumage colours or ornaments over a range of dates and link them to fitness characteristics without accounting for seasonal changes in these traits. Such changes are likely to be widespread among birds, yet we lack assessments of this variability within individuals. We studied both within‐ and between‐individual temporal changes in Great Tit Parus major ornaments, specifically the melanin‐based black breast stripe and the pigment‐free white cheek patch. During the non‐breeding season both ornaments varied. In juveniles and adult females, the area of the breast stripe first rose and then, from near the end of December, decreased. In adult males there was a linear decrease. In the cheek patch, the irregularity of the cheek borders showed either a linear (adults) or a non‐linear (juveniles) increase as the season progressed. In individuals repeatedly sampled during the same winter, the decrease in the size of the breast stripe was larger for males than females and there was an overall decrease in the regularity of the cheek borders. There was no relationship between the size of the breast stripe and the white cheek patch irregularities or the cheek patch area. These results imply that more attention should be paid to quantification, within individuals, of the components of expression of phenotypic traits. In addition, we suggest that further research should focus on explaining the causes and functions of ornament change.     

Aim2 deficiency in mice suppresses the expression of the inhibitory Fcgamma receptor (FcgammaRIIB) through the induction of the IFN-inducible p202, a lupus susceptibility protein

Murine Aim2 and Ifi202 genes (encoding for the Aim2 and p202 proteins) are members of the IFN-inducible Ifi200 gene family. The Aim2 deficiency in mice activates IFN signaling and stimulates the expression of the lupus susceptibility gene, the Ifi202, located within the NZB autoimmunity 2 (Nba2) interval. Given that the deficiency in the expression of the Fcgr2b gene (encoding for the inhibitory FcγRIIB receptor) is associated with increased lupus susceptibility in mice, we investigated whether the Aim2 protein could regulate the expression of Fcgr2b gene. In this article, we report that Aim2 deficiency in mice suppresses the expression of the FcγRIIB receptor. Interestingly, the Fcgr2b-deficient cells expressed increased levels of the IFN-β, activated IFN signaling, and expressed reduced levels of the Aim2 protein. Treatment of splenic cells with IFN-α or -γ reduced levels of the FcγRIIB mRNA and protein and also decreased the activity of the FcγRIIB p(-729/+585) Luc reporter. Moreover, levels of the FcγRIIB receptor were significantly higher in the Stat1-deficient splenic cells than in the wild-type cells. Accordingly, increased expression of IFN-β in lupus-prone B6.Nba2-ABC mice, as compared with non-lupus-prone C57BL/6 (B6) or B6.Nba2-C mice, was associated with reduced expression of the FcγRIIB receptor. Notably, overexpression of the p202 protein in cells decreased the expression of the Aim2 gene, activated the IFN response, and suppressed the expression of the Fcgr2b gene. These observations demonstrate that the expression of Aim2 protein is required to maintain the expression of the Fcgr2b gene and also predict epistatic interactions between the Ifi200 genes and the Fcgr2b gene within the Nba2 interval.