Calmodulin-Mediated Signal Transduction Pathways in Arabidopsis Are Fine-Tuned by Methylation

Calmodulin N-methyltransferase (CaM KMT) is an evolutionarily conserved enzyme in eukaryotes that transfers three methyl groups to a highly conserved lysyl residue at position 115 in calmodulin (CaM). We sought to elucidate whether the methylation status of CaM plays a role in CaM-mediated signaling pathways by gene expression analyses of CaM KMT and phenotypic characterization of Arabidopsis thaliana lines wherein CaM KMT was overexpressed (OX), partially silenced, or knocked out. CaM KMT was expressed in discreet spatial and tissue-specific patterns, most notably in root tips, floral buds, stamens, apical meristems, and germinating seeds. Analysis of transgenic plants with genetic dysfunction in CaM KMT revealed a link between the methylation status of CaM and root length. Plants with suppressed CaM methylation had longer roots and CaM KMT OX lines had shorter roots than wild type (Columbia-0). CaM KMT was also found to influence the root radial developmental program. Protein microarray analyses revealed a number of proteins with specificity for methylated forms of CaM, providing candidate functional intermediates between the observed phenotypes and the target pathways. This work demonstrates that the functionality of the large CaM family in plants is fine-tuned by an overarching methylation mechanism.     

Differential response of BDCA-1+ and BDCA-3+ myeloid dendritic cells to respiratory syncytial virus infection

Background

Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children, elderly, and immunocompromised individuals. Severe infection is associated with short- and long-term morbidity including pneumonia, recurrent wheezing, and abnormal pulmonary function, and several lines of evidence indicate that impaired adaptive immune responses during infection are critical in the pathophysiology of RSV-mediated disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract; however, few studies have examined the interactions between RSV and individual mDC subsets. In this study, we examined the effect of RSV on the functional response of primary mDC subsets (BDCA-1⁺ and BDCA-3⁺) isolated from peripheral blood.

Methods

BDCA-1⁺ and BDCA-3⁺ mDCs were isolated from the peripheral blood of healthy adults using FACS sorting. Donor-matched BDCA-1⁺ and BDCA-3⁺ mDCs were infected with RSV at a multiplicity of infection (MOI) of 5 for 40 hours. After infection, cells were analyzed for the expression of costimulatory molecules (CD86, CD80, and PD-L1), cytokine production, and the ability to stimulate allogenic CD4⁺ T cell proliferation.

Results

Both BDCA-1⁺ and BDCA-3⁺ mDCs were susceptible to infection with RSV and demonstrated enhanced expression of CD86, and the inhibitory costimulatory molecules CD80 and PD-L1. Compared to BDCA-3⁺ mDCs, RSV-infected BDCA-1⁺ mDC produced a profile of cytokines and chemokines predominantly associated with pro-inflammatory responses (IL-1β, IL-6, IL-12, MIP-1α, and TNF-α), and both BDCA-1⁺ and BDCA-3⁺ mDCs were found to produce IL-10. Compared to uninfected mDCs, RSV-infected BDCA-1⁺ and BDCA-3⁺ mDCs demonstrated a reduced capacity to stimulate T cell proliferation.

Conclusions

RSV infection induces a distinct pattern of costimulatory molecule expression and cytokine production by BDCA-1⁺ and BDCA-3⁺ mDCs, and impairs their ability to stimulate T cell proliferation.The differential expression of CD86 and pro-inflammatory cytokines by highly purified mDC subsets in response to RSV provides further evidence that BDCA-1⁺ and BDCA-3⁺ mDCs have distinct roles in coordinating the host immune response during RSV infection. Findings of differential expression of PD-L1 and IL-10 by infected mDCs, suggests possible mechanisms by which RSV is able to impair adaptive immune responses.     

Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial

We analyzed HIV-1 genome sequences from 68 newly infected volunteers in the STEP HIV-1 vaccine trial. To determine whether the vaccine exerted selective T cell pressure on breakthrough viruses, we identified potential T cell epitopes in the founder sequences and compared them to epitopes in the vaccine. We found greater distances to the vaccine sequence for sequences from vaccine recipients than from placebo recipients. The most significant signature site distinguishing vaccine from placebo recipients was Gag amino acid 84, a site encompassed by several epitopes contained in the vaccine and restricted by human leukocyte antigen (HLA) alleles common in the study cohort. Moreover, the extended divergence was confined to the vaccine components of the virus (HIV-1 Gag, Pol and Nef) and not found in other HIV-1 proteins. These results represent what is to our knowledge the first evidence of selective pressure from vaccine-induced T cell responses on HIV-1 infection in humans.     

Genetic and structural characterization of PvSERA4: potential implication as therapeutic target for Plasmodium vivax malaria

Plasmodium vivax malaria is geographically the most widely distributed and prevalent form of human malaria. The development of drug resistance by the parasite to existing drugs necessitates higher focus to explore and identify new drug targets. Plasmodial proteases have key roles in parasite biology and are involved in nutritional uptake, egress from infected reticulocytes, and invasion of the new target erythrocytes. Serine repeat antigens (SERA) of Plasmodium are parasite proteases that remain attractive drug targets and are important vaccine candidates due to their high expression profiles in the blood stages. SERA proteins have a unique putative papain-like cysteine protease motif that has either serine or cysteine in its active site. In P. vivax, PvSERA4 is the highest transcribed member of this multigene family. In this study, we have investigated the genetic polymorphism of PvSERA4 central protease domain and deduced its 3D model by homology modeling and also performed MD simulations to acquire refined protein structure. Sequence analysis of protease domain of PvSERA4 from Indian field isolates reveals that the central domain is highly conserved. The high sequence conservation of the PvSERA4 enzyme domain coupled with its high expression raises the possibility of it having a critical role in parasite biology and hence, being a reliable target for new selective inhibitor-based antimalarial chemotherapeutics. The 3D model showed the presence of an unusual antiparallel Beta hairpin motif between catalytic residues similar to hemoglobin binding motif of Plasmodial hemoglobinases. Our PvSERA4 model will aid in designing structure-based inhibitors against this enzyme.     

Non-Alcoholic Fatty Liver Disease Is Closely Associated with Sub-Clinical Inflammation: A Case-Control Study on Asian Indians in North India

Objectives

Association between sub-clinical inflammation and non-alcoholic fatty liver disease (NAFLD) has not been studied in Asian Indians. In this case-control study, we aimed to analyse association of NAFLD with the sub-clinical inflammation and metabolic profile in Asian Indians in north India.

Methods

Ultrasound diagnosed 120 cases of NAFLD were compared to 152 healthy controls without NAFLD. Anthropometric profile [body mass index (BMI), waist circumference (WC), hip circumference (HC)], high-sensitivity C-reactive protein (hs-CRP), metabolic profile [fasting blood glucose (FBG), lipid profile] and hepatic function tests [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] were recorded.

Results

Metabolic parameters [FBG, total cholesterol (TC), serum triglycerides (TG),low-density lipoprotein (LDL-c)], hs-CRP and prevalence of the metabolic syndrome were higher in cases as compared to controls (p-value<0.05 for all). The median (range) of hs-CRP (mg/L) for cases [2.6(0.2–13.4)] were significantly higher than in controls [1.4(0.03–11.4), p = 0.01]. Similarly, higher values of hs-CRP were obtained when subgroups of cases with obesity, abdominal obesity and the metabolic syndrome were compared to controls [2.75 (0.03–14.3) vs. 1.52 (0.04–14.3), p = 0.0010; 2.8 (0.03–14.3) vs. 1.5 (0.06–14.3), p = 0.0014 and 2.7 (0.5–14.3) vs. 1.6 (0.06–8.5), p = 0.0013, respectively. On multivariate logistic regression analysis BMI (p = 0.001), WC (p = 0.001), FBG (p = 0.002), TC (p = 0.008), TG (p = 0.002), blood pressure (p = 0.005), metabolic syndrome (p = 0.001) and hs-CRP (p = 0.003) were significantly and independently associated with NAFLD. After adjusting for significant variables, the association between high hs-CRP and NAFLD remained large and statistically significant [adjusted OR = 1.17, 95% confidence interval (CI) = 1.05–1.29]. An increase in 1 mg/dl of hs-CRP level calculated to increase the risk of developing NAFLD by 1.7 times as compared to controls after adjusting for significant variables associated with NAFLD.

Conclusions

In this cohort of Asian Indians in North India, presence of NAFLD showed independent relationships with sub-clinical inflammation.     

Surveillance of Travellers: An Additional Tool for Tracking Antimalarial Drug Resistance in Endemic Countries

Introduction

There are growing concerns about the emergence of resistance to artemisinin-based combination therapies (ACTs). Since the widespread adoption of ACTs, there has been a decrease in the systematic surveillance of antimalarial drug resistance in many malaria-endemic countries. The aim of this work was to test whether data on travellers returning from Africa with malaria could serve as an additional surveillance system of local information sources for the emergence of drug resistance in endemic-countries.

Methodology

Data were collected from travellers with symptomatic Plasmodium falciparum malaria returning from Senegal (n = 1,993), Mali (n = 2,372), Cote d’Ivoire (n = 4,778) or Cameroon (n = 3,272) and recorded in the French Malaria Reference Centre during the period 1996–2011. Temporal trends of the proportion of parasite isolates that carried the mutant genotype, pfcrt 76T, a marker of resistance to chloroquine (CQ) and pfdhfr 108N, a marker of resistance to pyrimethamine, were compared for travellers and within-country surveys that were identified through a literature review in PubMed. The in vitro response to CQ was also compared between these two groups for parasites from Senegal.

Results

The trends in the proportion of parasites that carried pfcrt 76T, and pfdhfr 108N, were compared for parasites from travellers and patients within-country using the slopes of the curves over time; no significant differences in the trends were found for any of the 4 countries. These results were supported by in vitro analysis of parasites from the field in Senegal and travellers returning to France, where the trends were also not significantly different.

Conclusion

The results have not shown different trends in resistance between parasites derived from travellers or from parasites within-country. This work highlights the value of an international database of drug responses in travellers as an additional tool to assess the emergence of drug resistance in endemic areas where information is limited.     

Protective effects of dietary EPA and DHA on ischemia–reperfusion-induced intestinal stress

The immunoregulatory effects of dietary omega-3 fatty acids are still not fully characterized. The aim of this study was to determine whether dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake limits intestinal ischemia–reperfusion (IR) injury. To test this, rats were fed either control or EPA/DHA supplemented diet for 3 weeks following which they underwent either a sham or an IR surgical protocol. A significant reduction in mucosal damage was observed after EPA/DHA supplemented diet as reflected by maintenance of total protein content. To address the underlying mechanisms of protection, we measured parameters of oxidative stress, intestinal and serological cytokines and intestinal eicosanoids. Interestingly, EPA/DHA fed animals displayed a higher activity of oxidative stress enzyme machinery, i.e., superoxide dismutase and catalase in addition to a reduction in total nitrate/nitrite content. While no changes in cytokines were observed, eicosanoid analyses of intestinal tissue revealed an increase in metabolites of the 12-lipoxygenase pathway following IR. Further, IR in EPA/DHA fed animals was accompanied by a significant increase of 17,18-epoxyeicosatetraenoic acid, 8-Iso prostaglandin F_3α and thromboxane B₃, by more than 12-, 6-, 3-fold, respectively. Thus, the data indicate that EPA/DHA supplementation may be able to reduce early intestinal IR injury by anti-oxidative and anti-inflammatory mechanisms.