False-positive detection of Group B Streptococcus (GBS) in chromogenic media (Strep B Carrot Broth) due to presence of Enterococcus faecalis in High Vaginal swabs.

Introduction. Vaginal colonization of Group B Streptococcus (GBS) is associated with preterm births and neonatal sepsis. Thus routine screening of GBS in prenatal care is recommended.Hypothesis. Chromogenic media (carrot broth) aids in specific and rapid detection of GBS.Aim. To investigate the efficiency of Strep B Carrot Broth for detection of GBS in high vaginal swabs from pregnant women.Methods. In this study 201 vaginal swab samples were collected from pregnant women. Swabs were inoculated in chromogenic media (Strep B Carrot Broth). The positive and negative cultures were inoculated on blood agar and crome agar plates. The colonies were subjected to 16S rRNA sequencing and gene-specific PCR for confirmation. The Christie Atkins Munch Peterson (CAMP) and bile esculin agar (BEA) tests were used for biochemical confirmation. PCR was performed on genomic DNA isolated from uncultured vaginal swabs.Results. It was found that 20/201 (9.9 %) vaginal swab samples were positive in the Strep B Carrot Broth and 17/20 (85 %) and 19/20 (95 %) of these samples yielded colonies on blood agar and crome agar, respectively. Of the 181 carrot broth-negative samples, 1 (0.5 %) and 38 (20.9 %) yielded colonies on blood agar and crome agar plates, respectively. However, 16 s rRNA sequencing revealed that none of the 20 carrot broth-positive cultures were GBS and had sequence similarities to Enterococcus faecalis. This was also confirmed by using gene-specific PCR and BEA positivity. Furthermore, E. faecalis was detected by PCR in DNA isolated from 57 uncultured vaginal swabs samples, GBS could only be detected by PCR in four samples.Conclusion. Carrot broth-based culture can lead to false-positive detection due to the presence of E. faecalis. Thus GBS-positive results in carrot broth must be confirmed by the other molecular and biochemical tests before making a final diagnosis.

Sesamol Induces Apoptosis-Like Cell Death in Leishmania donovani.

Background: Visceral leishmaniasis (VL), caused by the protozoan parasite Leishmania donovani (L. donovani), is the most severe form of leishmaniasis. It is largely responsible for significant morbidity and mortality in tropical and subtropical countries. Currently, available therapeutics have lots of limitations including high-cost, adverse side-effects, painful route of administration, less efficacy, and resistance. Therefore, it is time to search for cheap and effective antileishmanial agents. In the present work, we evaluated the antileishmanial potential of sesamol against promastigotes as well as intracellular amastigotes. Further, we tried to work out its mechanism of antileishmanial action on parasites through different assays. Methodology: In vitro and ex vivo antileishmanial assays were performed to evaluate the antileishmanial potential of sesamol on L. donovani. Cytotoxicity was determined by MTT assay on human THP-1-derived macrophages. Sesamol-induced morphological and ultrastructural changes were determined by electron microscopy. H2DCFDA staining, JC-1dye staining, and MitoSOX red staining were performed for reactive oxygen assay (ROS), mitochondrial membrane potential, and mitochondrial superoxide, respectively. Annexin V/PI staining for apoptosis, TUNEL assay, and DNA laddering for studying sesamol-induced DNA fragmentation were performed. Conclusions: Sesamol inhibited the growth and proliferation of L. donovani promastigotes in a dose-dependent manner. It also reduced the intracellular parasite load without causing significant toxicity on host-macrophages. Overall, it showed antileishmanial effects through induction of ROS, mitochondrial dysfunction, DNA fragmentation, cell cycle arrest, and apoptosis-like cell death to parasites. Our results suggested the possible use of sesamol for the treatment of leishmaniasis after further in vivo validations.

Ubiquitin ligase TRUSS augments the expression of interleukin-10 via proteasomal processing of NF-κB1/p105 to NF-κB/p50.

The NF-κB/Rel family of transcription factors that play critical roles in a variety of cellular processes. Their production in the cell and physiological activation are tightly regulated. The proteasomal processing of inactive NF-κB1/p105 to active p50, with an anti-inflammatory role, is not well characterized. Here we show that ubiquitin ligase TRUSS is a mediator of transcriptional activation of anti-inflammatory cytokine IL-10 gene. Enforced expression of TRUSS led to enhanced IL-10 expression that could be inhibited in the presence of chemical inhibitors of NF-κB [BAY11-7082] and PI3K/Akt [LY249002] or after p65 overexpression. p50 was actively recruited on IL10 promoter in the presence of TRUSS but competed by p65 for binding. TRUSS facilitated the ubiquitination of NF-κB1/p105 and promoted its proteolytic processing to generate excess of p50. Our immune-histochemical studies confirmed enhanced expression of p105/p50 in the human HCC tumors. Further, the hepatic tumors of HCC patient as well as transgenic mice showed decreased levels of p50 as well as TRUSS and accumulation of p105. Thus, enhanced expression of IL-10 gene in the presence of TRUSS and regulation of NF-κB1/p105 processing could be an important regulatory mechanism for inflammatory response and tumorgenic transformation.

Sphingosine-1-phosphate signaling in Leishmania donovani infection in macrophages.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a crucial regulator of a wide array of cellular processes, such as apoptosis, cell proliferation, migration, and differentiation, but its role in Leishmania donovani infection is unknown. METHODOLOGY/ PRINCIPAL FINDINGS: In the present study, we observed that L. donovani infection in THP-1 derived macrophages (TDM) leads to decrease in the expression of S1pr2 and S1pr3 at mRNA level. We further observed that Leishmania infection inhibits the phosphorylation of sphingosine kinase 1 (sphK1) in a time-dependent manner. Exogenous S1P supplementation decreases L. donovani induced ERK1/2 phosphorylation and increases p38 phosphorylation in TDM, resulting in a decrease in the intracellular parasite burden in a dose-dependent manner. On the other hand, sphK inhibition by DMS increases ERK1/2 phosphorylation leading to increased IL-10 and parasite load. To gain further insight, cytokines expression were checked in S1P supplemented TDM and we observed increase in IL-12, while decrease IL-10 expression at mRNA and protein levels. In addition, treatment of antagonist of S1PR2 and S1PR3 such as JTE-013 and CAY10444 respectively enhanced Leishmania-induced ERK1/2 phosphorylation and parasite load. CONCLUSIONS: Our overall study not only reports the significant role of S1P signaling during L. donovani infection but also provides a novel platform for the development of new drugs against Leishmaniasis.

Leishmania donovani infection differentially regulates small G-proteins.

Leishmania is a protozoan parasite that resides and replicates in macrophages and causes leishmaniasis. The parasite alters the signaling cascade in host macrophages and evades the host machinery. Small G-proteins are GTPases, grouped in 5 different families that play a crucial role in the regulation of cell proliferation, cell survival, apoptosis, intracellular trafficking, and transport. In particular, the Ras family of small G-proteins has been identified to play a significant role in the cellular functions mentioned before. Here, we studied the differential expression of the most important small G-proteins during Leishmania infection. We found major changes in the expression of different isoforms of Ras, mainly in N-Ras. We observed that Leishmania donovani infection led to enhanced N-Ras expression, whereas it inhibited K-Ras and H-Ras expression. Furthermore, an active N-Ras pull-down assay showed enhanced N-Ras activity. L donovani infection also increased extracellular signal-regulated kinase 1/2 phosphorylation and simultaneously decreased p38 phosphorylation. In contrast, pharmacological inhibition of Ras led to reduction in the phosphorylation of extracellular signal-regulated kinase 1/2 and enhanced the phosphorylation of p38 in Leishmania-infected cells, which could lead to increased interleukin-12 expression and decreased interleukin-10 expression. Indeed, farnesylthiosalicyclic acid (a Ras inhibitor), when used at the effective level in L donovani-infected macrophages, reduced amastigotes in the host macrophages. Thus, upregulated N-Ras expression during L donovani infection could be a novel immune evasion strategy of Leishmania and would be a potential target for antileishmanial immunotherapy.

Identification of novel inhibitors against UDP-galactopyranose mutase to combat leishmaniasis.

Leishmania, a protozoan parasite that causes leishmaniasis, affects 1-2 million people every year worldwide. Leishmaniasis is a vector born disease and characterized by a diverse group of clinical syndromes. Current treatment is limited because of drug resistance, high cost, poor safety, and low efficacy. The urgent need for potent agents against Leishmania has led to significant advances in the development of novel antileishmanial drugs. ß-galactofuranose (ß-Galf) is an important component of Leishmanial cell surface matrix and plays a critical role in the pathogenesis of parasite. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf) which acts as the precursor for ß-Galf synthesis. Due to its absence in human, this enzyme is selected as the potential target in search of new antileishmanial drugs. Three dimensional protein structure model of Leishmania major UGM (LmUGM) has been homology modeled using Trypanosoma cruzi UGM (TcUGM) as a template. The stereochemistry was validated further. We selected already reported active compounds from PubChem database to target the LmUGM. Three compounds (6064500, 44570814, and 6158954) among the top hit occupied the UDP binding site of UGM suggested to work as a possible inhibitor for it. In vitro antileishmanial activity assay was performed with the top ranked inhibitor, 6064500. The 6064500 molecule has inhibited the growth of Leishmania donovani promastigotes significantly. Further, at similar concentrations it has exhibited significantly lesser toxicity than standard drug miltefosine hydrate in mammalian cells.

Sphingosine-1-phosphate signaling: unraveling its role as a drug target against infectious diseases.

Sphingosine-1-phosphate (S1P) signaling is reported in variety of cell types, including immune, endothelial and cancerous cells. It is emerging as a crucial regulator of cellular processes, such as apoptosis, cell proliferation, migration, differentiation and so on. This signaling pathway is initiated by the intracellular production and secretion of S1P through a cascade of enzymatic reactions. Binding of S1P to different S1P receptors (S1PRs) activates different downstream signaling pathways that regulate the cellular functions differentially depending upon the cell type. An accumulating body of evidence suggests that S1P metabolism and signaling is often impaired during infectious diseases; thus, its manipulation might be helpful in the treatment of such diseases. In this review, we summarize recent advances in our understanding of the S1P signaling pathway and its candidature as a novel drug target against infectious diseases.

Orchestration of membrane receptor signaling by membrane lipids.

Receptors on cell membrane bind to their respective ligands and transduce intracellular signals resulting in variety of effector functions. Membrane lipid composition determines the receptor signaling behavior, as the receptors assume different conformation to suit the biochemical milieu in its immediate vicinity in the membrane. Accordingly, these accommodate different signaling intermediates that dictate the course of intracellular signaling and the resulting effectors functions. In this review we provide an overview of how membrane lipids modulate membrane-properties, membrane-receptor functions and their significance in the host-pathogen interaction.