Curative effect of 18β-glycyrrhetinic acid in experimental visceral leishmaniasis depends on phosphatase-dependent modulation of cellular MAP kinases

We earlier showed that 18β-glycyrrhetinic acid (GRA), a pentacyclic triterpenoid from licorice root, could completely cure visceral leishmaniasis in BALB/c mouse model. This was associated with induction of nitric oxide and proinflammatory cytokine production through the up regulation of NF-κB. In the present study we tried to decipher the underlying cellular mechanisms of the curative effect of GRA. Analysis of MAP kinase pathways revealed that GRA caused strong activation of p38 and to a lesser extent, ERK in bone marrow-derived macrophages (BMDM). Almost complete abrogation of GRA-induced cytokine production in presence of specific inhibitors of p38 and ERK1/2 confirmed the involvement of these MAP kinases in GRA-mediated responses. GRA induced mitogen- and stress-activated protein kinase (MSK1) activity in a time-dependent manner suggested that GRA-mediated NF-κB transactivation is mediated by p38, ERK and MSK1 pathway. As kinase/phosphatase balance plays an important role in modulating infection, the effect of GRA on MAPK directed phosphatases (MKP) was studied. GRA markedly reduced the expression and activities of three phosphatases, MKP1, MKP3 and protein phosphatase 2A (PP2A) along with a substantial reduction of p38 and ERK dephosphorylation in infected BMDM. Similarly in the in vivo situation, GRA treatment of L. donovani-infected BALB/c mice caused marked reduction of spleen parasite burden associated with concomitant decrease of individual phosphatase levels. However, activation of kinases also played an important role as the protective effect of GRA was significantly abrogated by pharmacological inhibition of p38 and ERK pathway. Curative effect of GRA may, therefore, be associated with restoration of proper cellular kinase/phosphatase balance, rather than modulation of either kinases or phosphatases.     

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination

Background

During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection.

Methods and Findings

To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject.

Conclusion

The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.     

Downregulation of yidC in Escherichia coli by Antisense RNA Expression Results in Sensitization to Antibacterial Essential Oils Eugenol and Carvacrol

Background

The rising drug resistance in pathogenic bacteria and inefficiency of current antibiotics to meet clinical requirements has augmented the need to establish new and innovative approaches for antibacterial drug discovery involving identification of novel antibacterial targets and inhibitors. Being obligatory for bacterial growth, essential gene products are considered vital as drug targets. The bacterial protein YidC is highly conserved among pathogens and is essential for membrane protein insertion due to which it holds immense potential as a promising target for antibacterial therapy.

Methods/Principal Findings

The aim of this study was to explore the feasibility and efficacy of expressed antisense-mediated gene silencing for specific downregulation of yidC in Escherichia coli. We induced RNA silencing of yidC which resulted in impaired growth of the host cells. This was followed by a search for antibacterial compounds sensitizing the YidC depleted cells as they may act as inhibitors of the essential protein or its products. The present findings affirm that reduction of YidC synthesis results in bacterial growth retardation, which warrants the use of this enzyme as a viable target in search of novel antibacterial agents. Moreover, yidC antisense expression in E. coli resulted in sensitization to antibacterial essential oils eugenol and carvacrol. Fractional Inhibitory Concentration Indices (FICIs) point towards high level of synergy between yidC silencing and eugenol/carvacrol treatment. Finally, as there are no known YidC inhibitors, the RNA silencing approach applied in this study put forward rapid means to screen novel potential YidC inhibitors.

Conclusions/Significance

The present results suggest that YidC is a promising candidate target for screening antibacterial agents. High level of synergy reported here between yidC silencing and eugenol/carvacrol treatment is indicative of a potential antibacterial therapy. This is the first report indicating that the essential gene yidC is a therapeutic target of the antibacterial essential oils eugenol and carvacrol in E. coli.     

Comparative structural modeling and docking studies of oxalate oxidase: Possible implication in enzyme supplementation therapy for urolithiasis

In humans oxalate is end product of protein metabolism, with no enzyme present to act on it. In conditions of its enhanced endogenous synthesis or increased absorption from the diet, oxalate accumulation leads to hyperoxaluria which can further lead to a number of pathological conditions including urolithiasis. Urolithiasis has been a perplexing problem due to its high incidence and rate of recurrence after treatment like Extracorporeal-shock wave lithotripsy (ESWL). Hence other prophylactic treatment becomes necessary. One of the newer approaches of curing such metabolic disorders is the enzyme supplementation therapy. Oxalate oxidase (OxOx) is a commonly occurring enzyme in plants, bacteria and fungi that catalyses oxidative cleavage of oxalate to CO(2) with reduction of dioxygen to H(2)O(2). Present study, used Hordeum vulgare OxOx crystal structure (PDB ID 2ET1A) as a template for constructing 3D models of OxOx from Triticum aestivum, Arabidopsis thaliana, Sclerotiana sclerotiarum. Similarly Homology models for isoforms Ceriporiopsis subvermispora 336, C. subvermispora 422 were constructed by using template Bacillus subtilis oxalate decarboxylase (Oxdc) (PDB ID 2UY8A) by comparative modeling approach in SWISS MODEL, MODELLER, 3D JIGSAW and GENO 3D program server. Based on overall stereochemical quality (PROCHECK, PROSA, VARIFY 3D), best models were selected, energy minimized, refined and characterized for active site in BioMed CaChe V 6.1 workspace. Selected models were further studied for structure function relationship with substrate (oxalate) and its analogue (glycolate) by using docking approach. Calculated interaction energy between the oxalate and constructed enzyme indicated that homology models for OxOx of T. aestivum, A. thaliana and S. sclerotiarum, can account for better regio-specificity of this enzyme towards oxalate. That supports the interested metabolism and thus may further implement in enzyme supplementation therapy for urolithiasis.     

Synthesis and biological evaluation of substituted 4-arylthiazol-2-amino derivatives as potent growth inhibitors of replicating Mycobacterium tuberculosis H₃₇Rv

In search of potential therapeutics for tuberculosis, we describe herein synthesis and biological evaluation of some substituted 4-arylthiazol-2-amino derivatives as modified analogues of the antiprotozoal drug Nitazoxanide (NTZ), which has recently been reported as potent inhibitor of Mtb H(37)Rv (Mtb MIC=52.12 μM) with an excellent ability to evade resistance. Among the synthesized derivatives, the two compounds 7a (MIC=15.28 μM) and 7c (MIC=17.03 μM) have exhibited about three times better Mtb growth inhibitory activity over NTZ and are free from any cytotoxicity (Vero CC(50) of 244 and 300 μM respectively). These two compounds represent promising leads for further optimization.     

A study to evaluate the effect of nootropic drug-piracetam on DNA damage in leukocytes and macrophages

Piracetam is a nootropic drug that protects neurons in neuropathological and age-related diseases and the activation and modulation of peripheral blood cells in patients with neuropathological conditions is well known. Therefore, in the present study, in vivo, ex vivo, and in vitro tests were conducted to investigate the effect of piracetam on leukocytes and macrophages. Lipopolysaccharide (LPS) causes oxidative DNA damage; thus, in the present study, LPS was used as a tool to induce DNA damage. In vivo experiments were conducted on Sprague Dawley rats, and piracetam (600mg/kg, oral) was provided for five consecutive days. On the fifth day, a single injection of LPS (10mg/kg, i.p.) was administered. Three hours after LPS injection, blood leukocytes and peritoneal macrophages were collected and processed, and a variety of different assays were conducted. Ex vivo treatments were performed on isolated rat blood leukocytes, and in vitro experiments were conducted on rat macrophage cell line J774A.1. Cell viability and the level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage were estimated in untreated (control) and piracetam-, LPS- and LPS+piracetam-treated leukocytes and macrophages. In vivo experiments revealed that rats pretreated with piracetam were significantly protected against LPS-induced increases in ROS levels and DNA damage. Ex vivo isolated leukocytes and J774A.1 cells treated with LPS exhibited augmented ROS levels and DNA damage, which were attenuated with piracetam treatment. Thus, the present study revealed the salutary effect of piracetam against LPS-induced oxidative stress and DNA damage in leukocytes and macrophages.     

Development of a molecular linkage map of pearl millet integrating DArT and SSR markers

Pearl millet is an important component of food security in the semi-arid tropics and is assuming greater importance in the context of changing climate and increasing demand for highly nutritious food and feed. Molecular tools have been developed and applied for pearl millet on a limited scale. However, the existing tool kit needs to be strengthened further for its routine use in applied breeding programs. Here, we report enrichment of the pearl millet molecular linkage map by exploiting low-cost and high-throughput Diversity Arrays Technology (DArT) markers. Genomic representation from 95 diverse genotypes was used to develop a DArT array with circa 7,000 clones following PstI/BanII complexity reduction. This array was used to genotype a set of 24 diverse pearl millet inbreds and 574 polymorphic DArT markers were identified. The genetic relationships among the inbred lines as revealed by DArT genotyping were in complete agreement with the available pedigree data. Further, a mapping population of 140 F₇ Recombinant Inbred Lines (RILs) from cross H 77/833-2 × PRLT 2/89-33 was genotyped and an improved linkage map was constructed by integrating DArT and SSR marker data. This map contains 321 loci (258 DArTs and 63 SSRs) and spans 1148 cM with an average adjacent-marker interval length of 3.7 cM. The length of individual linkage groups (LGs) ranged from 78 cM (LG 3) to 370 cM (LG 2). This better-saturated map provides improved genome coverage and will be useful for genetic analyses of important quantitative traits. This DArT platform will also permit cost-effective background selection in marker-assisted backcrossing programs as well as facilitate comparative genomics and genome organization studies once DNA sequences of polymorphic DArT clones are available.     

Nicotiana benthamiana phosphatidylinositol 4-kinase type II regulates chilli leaf curl virus pathogenesis

Geminiviruses are single-stranded DNA viruses that can cause significant losses in economically important crops. In recent years, the role of different kinases in geminivirus pathogenesis has been emphasized. Although geminiviruses use several host kinases, the role of phosphatidylinositol 4-kinase (PI4K) remains obscure. We isolated and characterized phosphatidylinositol 4-kinase type II from Nicotiana benthamiana (NbPI4KII) which interacts with the replication initiator protein (Rep) of a geminivirus, chilli leaf curl virus (ChiLCV). NbPI4KII-mGFP was localized into cytoplasm, nucleus or both. NbPI4KII-mGFP was also found to be associated with the cytoplasmic endomembrane systems in the presence of ChiLCV. Furthermore, we demonstrated that Rep protein directly interacts with NbPI4KII protein and influenced nuclear occurrence of NbPI4KII. The results obtained in the present study revealed that NbPI4KII is a functional protein kinase lacking lipid kinase activity. Downregulation of NbPI4KII expression negatively affects ChiLCV pathogenesis in N. benthamiana. In summary, NbPI4KII is a susceptible factor, which is required by ChiLCV for pathogenesis.