NDK/NME proteins: a host-pathogen interface perspective towards therapeutics.

No effective vaccine is available for any parasitic disease. The treatment to those is solely dependent on chemotherapy, which is always threatened due to development of drug resistance in bugs. This warrants identification of new drug targets. Here, we discuss Nucleoside diphosphate kinases (NDKs) of pathogens that alter host's intra and extracellular environment, as novel drug targets to simultaneously tackle multiple pathogens. NDKs having diverse functions, are highly conserved among prokaryotes and eukaryotes (the mammal NDKs are called NMEs [non-metastatic enzymes]). However, NDKs and NMEs have been separately analysed in the past for their structure and functions. The role of NDKs of pathogen in modulation of inflammation, phagocytosis, apoptosis, and ROS generation in host is known. Conversely, its combined contribution in host-pathogen interaction has not been studied yet. Through the sequence and domain analysis, we found that NDKs can be classified in two groups. One group comprised NMEs 1-4 and few NDKs of select essential protozoan parasites and the bacterium Mycobacterium tuberculosis. The other group included NME7 and the other NDKs of those parasites, posing challenges in the development of drugs specifically targeting pathogen NDKs, without affecting NME7. However, common drugs targeting group 2 NDKs of pathogens can be designed, as NME7 of group 2 is expressed only in ciliated host cells. This review thus analyses comparatively for the first time the structures and functions of human NMEs and pathogen NDKs and predicts the possibilities of NDKs as drug targets. In addition, pathogen NDKs have been now provided a nomenclature in alignment with the NMEs of humans.

Chemical Profiling of Chlorophytum comosum (Thunb.) Jaques by GC-MS/LC-ESIMS and its Antiproliferative Effects on Human Carcinoma Cell Lines.

BACKGROUND: Chlorophytum comosum, popularly known as Spider Ivy, is used as a medicinal plant in traditional Chinese medicine, however, its detailed chemical composition and biological activity are yet unexplored. OBJECTIVE: To carry out the phytochemical investigation on different parts of Chlorophytum comosum using GCMS/ LC-ESI-MS and evaluation of its antioxidant, hemolytic and antiproliferative potential on breast cancer (MCF-7), lung cancer (A549, H1299) and normal lung (L-132) cell lines. METHODS: Chemical constituents from aqueous roots and leaves extracts were identified using LC-ESI-MS/GCMS. The identified compounds were annotated based on the match of mass spectra with the literature using NIST 14 and METLIN databases. Antioxidant activity was studied using DPPH, FRAP and TPC assays. The antiproliferative effects of ethanolic roots and leaves extracts of Chlorophytum comosum were measured by MTT assay on breast cancer (MCF-7), lung cancer (A549 & H1299) and normal lung (L-132) cell lines. The toxicity studies of the extracts were carried out using Hemolysis assay. RESULTS: GC-MS analysis identified 34 metabolites in roots and 17 from leaves, while 17 compounds from roots and 7 from leaves were detected by LC-ESI-MS. Significant antiproliferative effects were observed on the A549 and MCF-7 cancer cell lines with IC50 values ranging from 56.86 µg/ml to 68.68 µg/ml while no marked response was observed against normal cell line L-132. CONCLUSION: Our study represents the first report on the detailed chemical composition and antiproliferative potential of Chlorophytum comosum against lung and breast cancer cell lines.

Interaction of CD80 with Neph1: a potential mechanism of podocyte injury.

BACKGROUND: The induction of CD80 on podocytes has been shown in animal models of podocyte injury and in certain cases of nephrotic syndrome. In a lipopolysaccharide (LPS)-induced mouse model of albuminuria, we have recently shown a signalling axis of LPS-myeloid cell activation-TNFα production-podocyte CD80 induction-albuminuria. Therefore, in this report, we investigated the cellular and molecular consequences of TNFα addition and CD80 expression on cultured podocytes. METHODS: A murine podocyte cell line was used for TNFα treatment and for over-expressing CD80. Expression and localization of various podocyte proteins was analysed by reverse transcriptase-polymerase chain reaction, western blotting and immunofluorescence. HEK293 cells were used to biochemically characterize interactions. RESULTS: Podocytes treated with LPS in vitro did not cause CD80 upregulation but TNFα treatment was associated with an increase in CD80 levels, actin derangement and poor wound healing. Podocytes stably expressing CD80 showed actin derangement and co-localization with Neph1. CD80 and Neph1 interaction was confirmed by pull down assays of CD80 and Neph1 transfected in HEK293 cells. CONCLUSION: Addition of TNFα to podocytes causes CD80 upregulation, actin reorganization and podocyte injury. Overexpressed CD80 and Neph1 interact via their extracellular domain. This interaction implies a mechanism of slit diaphragm disruption and possible use of small molecules that disrupt CD80-Neph1 interaction as a potential for treatment of nephrotic syndrome associated with CD80 upregulation.

Structural and Biochemical Characterization of Apicomplexan Inorganic Pyrophosphatases.

Inorganic pyrophosphatases (PPase) participate in energy cycling and they are essential for growth and survival of organisms. Here we report extensive structural and functional characterization of soluble PPases from the human parasites Plasmodium falciparum (PfPPase) and Toxoplasma gondii (TgPPase). Our results show that PfPPase is a cytosolic enzyme whose gene expression is upregulated during parasite asexual stages. Cambialistic PfPPase actively hydrolyzes linear short chain polyphosphates like PPi, polyP3 and ATP in the presence of Zn2+. A remarkable new feature of PfPPase is the low complexity asparagine-rich N-terminal region that mediates its dimerization. Deletion of N-region has an unexpected and substantial effect on the stability of PfPPase domain, resulting in aggregation and significant loss of enzyme activity. Significantly, the crystal structures of PfPPase and TgPPase reveal unusual and unprecedented dimeric organizations and provide new fundamental insights into the variety of oligomeric assemblies possible in eukaryotic inorganic PPases.

Japanese encephalitis virus invasion of cell: allies and alleys.

The mosquito-borne flavivirus, Japanese encephalitis virus (JEV), is the leading cause of virus-induced encephalitis globally and a major public health concern of several countries in Southeast Asia, with the potential to become a global pathogen. The virus is neurotropic, and the disease ranges from mild fever to severe hemorrhagic and encephalitic manifestations and death. The early steps of the virus life cycle, binding, and entry into the cell are crucial determinants of infection and are potential targets for the development of antiviral therapies. JEV can infect multiple cell types; however, the key receptor molecule(s) still remains elusive. JEV also has the capacity to utilize multiple endocytic pathways for entry into cells of different lineages. This review not only gives a comprehensive update on what is known about the virus attachment and receptor system (allies) and the endocytic pathways (alleys) exploited by the virus to gain entry into the cell and establish infection but also discusses crucial unresolved issues. We also highlight common themes and key differences between JEV and other flaviviruses in these contexts.

Corrigendum: Leishmanicidal activities of Artemisia annua leaf essential oil against Visceral Leishmaniasis.

[This corrects the article on p. 626 in vol. 5, PMID: 25505453.].

Apoptosis mediated leishmanicidal activity of Azadirachta indica bioactive fractions is accompanied by Th1 immunostimulatory potential and therapeutic cure in vivo.

BACKGROUND: Exploration of immunomodulatory antileishmanials of plant origin is now being strongly recommended to overcome the immune suppression evident during visceral leishmaniasis (VL) and high cost and toxicity associated with conventional chemotherapeutics. In accordance, we assessed the in vitro and in vivo antileishmanial and immunomodulatory potential of ethanolic fractions of Azadirachta indica leaves (ALE) and seeds (ASE). METHODS: A. indica fractions were prepared by sequential extraction of the powdered plant parts in hexane, ethanol and water. Erythrosin B staining was employed to appraise the anti-promastigote potential of ALE and ASE. Cytostatic or cytocidal mode of action was ascertained and alterations in parasite morphology were depicted under oil immersion light microscopy. Study of apoptotic correlates was performed to deduce the mechanism of induced cell death and anti-amastigote potential was assessed in Leishmania parasitized RAW 264.7 macrophages. In vivo antileishmanial effectiveness was evaluated in L. donovani infected BALB/c mice, accompanied by investigation of immunomodulatory potential of ALE and ASE. Adverse toxicity of the bioactive fractions against RAW macrophages was studied by MTT assay. In vivo side effects on the liver and kidney functions were also determined. Plant secondary metabolites present in ALE and ASE were analysed by Gas chromatography-mass spectrometry (GC-MS). RESULTS: ALE and ASE (500 µg ml(-1)) exhibited leishmanicidal activity in a time- and dose-dependent manner (IC50 34 and 77.66 µg ml(-1), respectively) with alterations in promastigote morphology and induction of apoptosis. ALE and ASE exerted appreciable anti-amastigote potency (IC50 17.66 and 24.66 µg ml(-1), respectively) that was coupled with profound in vivo therapeutic efficacy (87.76% and 85.54% protection in liver and 85.55% and 83.62% in spleen, respectively). ALE exhibited minimal toxicity with selectivity index of 26.10 whereas ASE was observed to be non-toxic. The bioactive fractions revealed no hepato- and nephro-toxicity. ALE and ASE potentiated Th1-biased cell-mediated immunity along with upregulation of INF-γ, TNF-α and IL-2 and decline in IL-4 and IL-10 levels. GC-MS analysis revealed several compounds that may have contributed to the observed antileishmanial effect. CONCLUSION: Dual antileishmanial and immunostimulatory efficacy exhibited by the bioactive fractions merits their use alone or as adjunct therapy for VL.

Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with ß-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

Leishmanicidal activities of Artemisia annua leaf essential oil against Visceral Leishmaniasis.

Visceral leishmaniasis (VL), the second-most dreaded parasitic disease after malaria, is currently endemic in 88 countries. Dramatic increases in the rates of infection, drug resistance, and non-availability of safe vaccines have highlighted the need for identification of novel and inexpensive anti-leishmanial agents from natural sources. In this study, we showed the leishmanicidal effect of essential oil from Artemisia annua leaves (AALEO) against Leishmania donovani in vitro and in vivo. AALEO was extracted by hydrodistillation and characterized by GC-MS, the most abundant compounds were found to be camphor (52.06 %) followed by ß-caryophyllene (10.95 %). AALEO exhibited significant leishmanicidal activity against L. donovani, with 50 % inhibitory concentration of 14.63 ± 1.49 µg ml(-1) and 7.3 ± 1.85 µg ml(-1), respectively, against the promastigotes and intracellular amastigotes. The effect was mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA nicking by TdT-mediated dUTP nick-end labeling assay, dyskinetoplastidy, cell cycle arrest at sub-G0-G1 phase, loss of mitochondrial membrane potential and reactive oxygen species generation in promastigotes and nitric oxide generation in ex vivo model. AALEO presented no cytotoxic effects against mammalian macrophages even at 200 µg ml(-1). Intra-peritoneal administration of AALEO (200 mg/ kg.b.w.) to infected BALB/c mice reduced the parasite burden by almost 90% in the liver and spleen with significant reduction in weight. There was no hepato- or nephro-toxicity as demonstrated by normal levels of serum enzymes. The promising antileishmanial activity shown by camphor-rich AALEO may provide a new lead in the treatment of VL.

Combining ability analysis for grain filling duration and yield traits in spring wheat (Triticum aestivum L. em. Thell.)

A diallel analysis of wheat (Triticum aestivum L. em. Thell) parents (n = 11) and their F1 (n = 55) and F2 (n = 55) offspring was carried out for the following four traits: grain filling duration (GFD), GFD for growing degree days (GDD), 1000 seed weight and seed yield per plant. Analysis of variance for general combining ability (GCA) and specific combining ability (SCA) displayed significant F1 and F2 general and specific combining ability effects for the four traits studied. For all the traits the GCA effects were relatively more important than the SCA effects, indicating that additive genetic effects were predominant. Crosses displaying high SCA effects for grain filling duration, seed weight and yield were observed to be derived from parents having various types of GCA effects (high x high, high x low, low x low and medium x low). The single seed descent method can be applied to exploit additive gene effects whereas dominance gene effects could be valuable in hybrid wheat breeding programs. Among the parents, genotypes from the International Maize and Wheat Improvement Center (Centro Internacional de Mejoramiento de Maíz y Trigo, CIMMYT) as well as South Asia were found to be superior general combiners for grain filling duration. Likewise, crosses involving diverse parents from CIMMYT and South Asia showed significant SCA effects for grain filling duration and other traits.