A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID).

The protein translocase of the mitochondrial inner membrane in Trypanosoma brucei, TbTIM17, forms a modular complex in association with several other trypanosome-specific proteins. To identify transiently interacting proximal partner(s) of TbTim17, we used Biotinylation Identification (BioID) by expressing a modified biotin ligase-TbTim17 (BirA∗-TbTim17) fusion protein in T. brucei. BirA∗-TbTim17 was targeted to mitochondria and assembled in the TbTIM complex. In the presence of biotin, BirA∗-TbTim17 biotinylated several mitochondrial proteins. Interestingly, TbHsp84/TbTRAP1, a mitochondrial Hsp90 homolog, was identified as the highest enriched biotinylated proteins. We validated that interaction and colocalization of TbTim17 and TbHsp84 in T. brucei mitochondria by coimmunoprecipitation analysis and confocal microscopy, respectively. TbTim17 association with TbTRAP1 increased several folds during denaturation/renaturation of mitochondrial proteins in vitro, suggesting TbTRAP1 acts as a chaperone for TbTim17 refolding. We demonstrated that knockdown of TbTRAP1 reduced cell growth and decreased the levels of the TbTIM17, TbTim62, and mitochondrial (m)Hsp70 complexes. However, ATPase, VDAC, and Atom69 complexes were minimally affected. Additionally, the steady state levels of TbTim17, TbTim62, and mHsp70 were reduced significantly, but Atom69, ATPase ß, and RBP16 were mostly unaltered due to TbTRAP1 knockdown. Quantitative proteomics analysis also showed significant reduction of TbTim62 along with a few other mitochondrial proteins due to TbTRAP1 knockdown. Finally, TbTRAP1 depletion did not hamper the import of the ectopically expressed TbTim17-2xMyc into mitochondria but reduced its assembly into the TbTIM17 complex, indicating TbTRAP1 is critical for assembly of TbTim17. This is the first report showing the role of TRAP1 in the TIM complex assembly in eukaryotes.

Quinolizidines as Novel SARS-CoV-2 Entry Inhibitors.

COVID-19, caused by the highly transmissible severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has rapidly spread and become a pandemic since its outbreak in 2019. We have previously discovered that aloperine is a new privileged scaffold that can be modified to become a specific antiviral compound with markedly improved potency against different viruses, such as the influenza virus. In this study, we have identified a collection of aloperine derivatives that can inhibit the entry of SARS-CoV-2 into host cells. Compound 5 is the most potent tested aloperine derivative that inhibited the entry of SARS-CoV-2 (D614G variant) spike protein-pseudotyped virus with an IC50 of 0.5 µM. The compound was also active against several other SARS-CoV-2 variants including Delta and Omicron. Results of a confocal microscopy study suggest that compound 5 inhibited the viral entry before fusion to the cell or endosomal membrane. The results are consistent with the notion that aloperine is a privileged scaffold that can be used to develop potent anti-SARS-CoV-2 entry inhibitors.

Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells.

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/ß-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/ß-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/ß-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3ß at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3ß was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of ß-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of ß-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating ß-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a ß-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the ß-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.

Optogenetically-induced multimerization of the dopamine transporter increases uptake and trafficking to the plasma membrane.

The dopamine transporter (DAT) is essential for the reuptake of the released neurotransmitter dopamine (DA) in the brain. Psychostimulants, methamphetamine and cocaine, have been reported to induce the formation of DAT multimeric complexes in vivo and in vitro. The interpretation of DAT multimer function has been primarily in the context of compounds that induce structural and functional modifications of the DAT, complicating the understanding of the significance of DAT multimers. To examine multimerization in the absence of DAT ligands as well as in their presence, we developed a novel, optogenetic fusion chimera of cryptochrome 2 and DAT with an mCherry fluorescent reporter (Cry2-DAT). Using blue light to induce Cry2-DAT multimeric protein complex formation, we were able to simultaneously test the functional contributions of DAT multimerization in the absence or presence of substrates or inhibitors with high spatiotemporal precision. We found that blue light-stimulated Cry2-DAT multimers significantly increased IDT307 uptake and MFZ 9-18 binding in the absence of ligands as well as after methamphetamine and nomifensine treatment. Blue light-induced Cry2-DAT multimerization increased colocalization with recycling endosomal marker Rab11 and had decreased presence in Rab5-positive early endosomes and Rab7-positive late endosomes. Our data suggest that the increased uptake and binding results from induced and rapid trafficking of DAT multimers to the plasma membrane. Our data suggest that DAT multimers may function to help maintain DA homeostasis.

Linking bacterial enterotoxins and alpha defensin 5 expansion in the Crohn's colitis: A new insight into the etiopathogenetic and differentiation triggers driving colonic inflammatory bowel disease.

Evidence link bacterial enterotoxins to apparent crypt-cell like cells (CCLCs), and Alpha Defensin 5 (DEFA5) expansion in the colonic mucosa of Crohn's colitis disease (CC) patients. These areas of ectopic ileal metaplasia, positive for Paneth cell (PC) markers are consistent with diagnosis of CC. Retrospectively, we: 1. Identified 21 patients with indeterminate colitis (IC) between 2000-2007 and were reevaluation their final clinical diagnosis in 2014 after a followed-up for mean 8.7±3.7 (range, 4-14) years. Their initial biopsies were analyzed by DEFA5 bioassay. 2. Differentiated ulcer-associated cell lineage (UACL) analysis by immunohistochemistry (IHC) of the CC patients, stained for Mucin 6 (MUC6) and DEFA5. 3. Treated human immortalized colonic epithelial cells (NCM460) and colonoids with pure DEFA5 on the secretion of signatures after 24hr. The control colonoids were not treated. 4. Treated colonoids with/without enterotoxins for 14 days and the spent medium were collected and determined by quantitative expression of DEFA5, CCLCs and other biologic signatures. The experiments were repeated twice. Three statistical methods were used: (i) Univariate analysis; (ii) LASSO; and (iii) Elastic net. DEFA5 bioassay discriminated CC and ulcerative colitis (UC) in a cohort of IC patients with accuracy. A fit logistic model with group CC and UC as the outcome and the DEFA5 as independent variable differentiator with a positive predictive value of 96 percent. IHC staining of CC for MUC6 and DEFA5 stained in different locations indicating that DEFA5 is not co-expressed in UACL and is therefore NOT the genesis of CC, rather a secretagogue for specific signature(s) that underlie the distinct crypt pathobiology of CC. Notably, we observed expansion of signatures after DEFA5 treatment on NCM460 and colonoids cells expressed at different times, intervals, and intensity. These factors are key stem cell niche regulators leading to DEFA5 secreting CCLCs differentiation 'the colonic ectopy ileal metaplasia formation' conspicuously of pathogenic importance in CC.

The rapid endocytic uptake of fetuin-A by adherent tumor cells is mediated by Toll-like receptor 4 (TLR4).

Fetuin-A is a serum glycoprotein synthesized and secreted into blood by the liver and whose main physiological function is the inhibition of ectopic calcification. However, a number of studies have demonstrated that it is a multifunctional protein. For example, endocytic uptake of fetuin-A by tumor cells resulting in rapid cellular adhesion and spreading has been reported. The precise uptake mechanism, however, has been elusive. The present studies were done to determine whether Toll-like receptor-4 (TLR4), which has been previously shown to be a receptor for fetuin-A and is commonly expressed in immune cells, could take part in the rapid uptake (< 3 min) of fetuin-A by tumor cells. Rapid uptake of fetuin-A was inhibited by the specific TLR4 inhibitor CLI-095 and also attenuated in TLR4 knockdown prostate tumor cells. Inhibition of TLR4 by CLI-095 also attenuated the rapid adhesion of tumor cells as well as invasion through a bed of Matrigel. The data suggest mechanisms by which TLR4 modulates the adhesion and growth of tumor cells.

Do Progestin-Only Contraceptives Contribute to the Risk of Developing Depression as Implied by Beta-Arrestin 1 Levels in Leukocytes? A Pilot Study.

We reported previously that reduction in beta-arrestin 1 (ß-AR 1) protein levels in peripheral blood mononuclear leukocytes (PBMC) significantly correlated with the severity of depressive symptoms in reproductive women. In this pilot study, we used ß-AR 1 protein levels in PBMC as a marker for developing depressive symptoms and the Hamilton Depression Rating Scale (HAM-D) scores to assess potential mood-related side effects of oral contraceptive use for routine birth control among women. We evaluated 29 women in this study. We enrolled the participants in three groups: Estrogen-progestin combination-oral contraceptives (COC, n = 10), progestin-only contraceptives (POC, n = 12), and non-hormonal or no contraceptives (NC, n = 7). We determined the ß-AR 1 protein levels in PBMCs by enzyme-linked immunosorbent assay (ELISA). We found that women in the POC group had significantly higher HAM-D scores compared to those in the COC (p < 0.0004) and NC (p < 0.004). The levels of ß-AR 1 protein were significantly attenuated in women in the POC group compared to women in the NC group (p = 0.03). Our findings suggest that the use of POC is a potential risk factor for developing depressive symptoms.

Extracellular histones are the ligands for the uptake of exosomes and hydroxyapatite-nanoparticles by tumor cells via syndecan-4.

The mechanisms by which exosomes (nano-vesicular messengers of cells) are taken up by recipient cells are poorly understood. We hypothesized that histones associated with these nanoparticles are the ligands which facilitate their interaction with cell surface syndecan-4 (SDC4) to mediate their uptake. We show that the incubation with fetuin-A (exosome-associated proteins) and histones mediates the uptake of exosomes that are normally not endocytosed. Similarly, hydroxyapatite-nanoparticles incubated with fetuin-A and histones (FNH) are internalized by tumor cells, while nanoparticles incubated with fetuin-A alone (FN) are not. The uptake of exosomes and FNH, both of which move to the perinuclear region of the cell, is attenuated in SDC4-knockdown cells. Data show that FNH can compete with exosomes for uptake and that both use SDC4 as uptake receptors.

Poly (ADP-Ribose) Polymerase-1 (PARP-1) Induction by Cocaine Is Post-Transcriptionally Regulated by miR-125b.

Cocaine exposure alters gene expression in the brain via methylation and acetylation of histones along with methylation of DNA. Recently, poly (ADP-ribose) polymerase-1 (PARP-1) catalyzed PARylation has been reported as an important regulator of cocaine-mediated gene expression. In this study, we report that the cellular microRNA "miR-125b" plays a key role for cocaine-induced PARP-1 expression. Acute and chronic cocaine exposure resulted in the downregulation of miR-125b concurrent with upregulation of PARP-1 in dopaminergic neuronal cells and nucleus accumbens (NAc) of mice but not in the medial prefrontal cortex (PFC) or ventral tegmental area (VTA). In silico analysis predicted a binding site of miR-125b in a conserved 3'-untranslated region (3'UTR) of the PARP-1 mRNA. Knockdown and overexpression studies showed that miR-125b levels negatively correlate with PARP-1 protein expression. Luciferase reporter assay using a vector containing the 3'UTR of PARP-1 mRNA confirmed regulation of PARP-1 by miR-125b. Specific nucleotide mutations within the binding site abrogated miR-125b's regulatory effect on PARP-1 3'UTR. Finally, we established that downregulation of miR-125b and concurrent upregulation of PARP-1 is dependent on binding of cocaine to the dopamine transporter (DAT). Collectively, these results identify miR-125b as a post-transcriptional regulator of PARP-1 expression and establish a novel mechanism underlying the molecular effects of cocaine action.

Mitochondrial protein Fus1/Tusc2 in premature aging and age-related pathologies: critical roles of calcium and energy homeostasis.

Decreased energy production and increased oxidative stress are considered to be major contributors to aging and aging-associated pathologies. The role of mitochondrial calcium homeostasis has also been highlighted as an important factor affecting different pathological conditions. Here, we present evidence that loss of a small mitochondrial protein Fus1 that maintains mitochondrial homeostasis results in premature aging, aging-associated pathologies, and decreased survival. We showed that Fus1KO mice develop multiple early aging signs including lordokyphosis, lack of vigor, inability to accumulate fat, reduced ability to tolerate stress, and premature death. Other prominent pathological changes included low sperm counts, compromised ability of adult stem cells to repopulate tissues, and chronic inflammation. At the molecular level, we demonstrated that mitochondria of Fus1 KO cells have low reserve respiratory capacity (the ability to produce extra energy during sudden energy demanding situations), and show significantly altered dynamics of cellular calcium response.Our recent studies on early hearing and memory loss in Fus1 KO mice combined with the new data presented here suggest that calcium and energy homeostasis controlled by Fus1 may be at the core of its aging-regulating activities. Thus, Fus1 protein and Fus1-dependent pathways and processes may represent new tools and targets for anti-aging strategies.

Fetuin-A (alpha 2HS glycoprotein) modulates growth, motility, invasion, and senescence in high-grade astrocytomas.

Glioblastomas (high-grade astrocytomas) are highly aggressive brain tumors with poor prognosis and limited treatment options. In the present studies, we have defined the role of fetuin-A, a liver-derived multifunctional serum protein, in the growth of an established glioblastoma cell line, LN229. We hereby demonstrate that these cells synthesize ectopic fetuin-A which supports their growth in culture in the absence of serum. We have demonstrated that a panel of tissue microarray (TMA) of glioblastomas also express ectopic fetuin-A. Knocking down fetuin-A using shRNA approach in LN229, significantly reduced their in vitro growth as well as growth and invasion in vivo. The fetuin-A knockdown subclones of LN229 (A and D) also had reduced motility and invasive capacity. Treatment of LN229 cells with asialofetuin (ASF), attenuated their uptake of labeled fetuin-A, and induced senescence in them. Interestingly, the D subclone that had ~90% reduction in ectopic fetuin-A, underwent senescence in serum-free medium which was blunted in the presence of purified fetuin-A. Uptake of labeled exosomes was attenuated in fetuin-A knockdown subclones A and D. Taken together, the studies demonstrate the impact of fetuin-A as significant node of growth, motility, and invasion signaling in glioblastomas that can be targeted for therapy.

Dopamine Transporter Activity Is Modulated by α-Synuclein.

The duration and strength of the dopaminergic signal are regulated by the dopamine transporter (DAT). Drug addiction and neurodegenerative and neuropsychiatric diseases have all been associated with altered DAT activity. The membrane localization and the activity of DAT are regulated by a number of intracellular proteins. α-Synuclein, a protein partner of DAT, is implicated in neurodegenerative disease and drug addiction. Little is known about the regulatory mechanisms of the interaction between DAT and α-synuclein, the cellular location of this interaction, and the functional consequences of this interaction on the basal, amphetamine-induced DAT-mediated dopamine efflux, and membrane microdomain distribution of the transporter. Here, we found that the majority of DAT·α-synuclein protein complexes are found at the plasma membrane of dopaminergic neurons or mammalian cells and that the amphetamine-mediated increase in DAT activity enhances the association of these proteins at the plasma membrane. Further examination of the interaction of DAT and α-synuclein revealed a transient interaction between these two proteins at the plasma membrane. Additionally, we found DAT-induced membrane depolarization enhances plasma membrane localization of α-synuclein, which in turn increases dopamine efflux and enhances DAT localization in cholesterol-rich membrane microdomains.

Complement protective epitopes and CD55-microtubule complexes facilitate the invasion and intracellular persistence of uropathogenic Escherichia coli.

BACKGROUND: Escherichia coli-bearing Dr-adhesins (Dr+ E. coli) cause chronic pyelonephritis in pregnant women and animal models. This chronic renal infection correlates with the capacity of bacteria to invade epithelial cells expressing CD55. The mechanism of infection remains unknown. METHODS: CD55 amino acids in the vicinity of binding pocket-Ser155 for Dr-adhesin were mutated to alanine and subjected to temporal gentamicin-invasion/gentamicin-survival assay in Chinese hamster ovary cells. CD55/microtubule (MT) responses were studied using confocal/electron microscopy, and 3-dimensional structure analysis. RESULTS: Mutant analysis revealed that complement-protective CD55-Ser165 and CD55-Phe154 epitopes control E. coli invasion by coregulating CD55-MT complex expression. Single-point CD55 mutations changed E. coli to either a minimally invasive (Ser165Ala) or a hypervirulent pathogen (Phe154Ala). Thus, single amino acid modifications with no impact on CD55 structure and bacterial attachment can have a profound impact on E. coli virulence. While CD55-Ser165Ala decreased E. coli invasion and led to dormant intracellular persistence, intracellular E. coli in CD55-Phe154Ala developed elongated forms (multiplying within vacuoles), upregulated CD55-MT complexes, acquired CD55 coat, and escaped phagolysosomal fusion. CONCLUSIONS: E. coli target complement-protective CD55 epitopes for invasion and exploit CD55-MT complexes to escape phagolysosomal fusion, leading to a nondestructive parasitism that allows bacteria to persist intracellularly.

mRNA and DNA PCR tests in cutaneous tuberculosis.

BACKGROUND: The microbiologic diagnosis of cutaneous tuberculosis is difficult because most lesions harbor only a small number of mycobacteria that cannot usually be detected by staining for the organism or by culture. Nucleic acid amplification tests based on the polymerase chain reaction (PCR) are potentially useful in this situation. AIMS: To evaluate the utility of mRNA PCR and DNA PCR in the diagnosis of cutaneous tuberculosis. METHODS: Biopsies from 28 cases of cutaneous tuberculosis and 19 controls with other diseases were subjected to microbiologic tests including direct smears for mycobacteria, culture and both mRNA PCR and DNA PCR. The laboratory was blinded to the clinical diagnosis. RESULTS: None of the patients or controls showed a positive reaction on mRNA PCR test. Seven of 28 cases and 5 out of 19 controls showed a positive result on DNA PCR test yielding a sensitivity of 25% and a specificity of 73.7%. CONCLUSION: The results of PCR tests in cutaneous tuberculosis should be interpreted in the light of clinical and histopathological findings.

Genital tuberculosis among infertile women and fertility outcome after antitubercular therapy.

OBJECTIVE: To compare modalities for diagnosing genital tuberculosis (GTB) and to assess fertility outcome after antitubercular therapy (ATT). METHODS: Infertile women underwent endometrial aspiration (EA) and peritoneal washing (PW) for histopathologic examination, PCR, and acid-fast bacilli (AFB) smear and culture of Mycobacterium tuberculosis; laparoscopy and hysteroscopy were also performed. Women with a positive laboratory test and/or laparoscopic finding classified as definitive/probable received ATT for 6 months. RESULTS: Of 196 women recruited, 187 underwent laparoscopy. Genital tuberculosis was diagnosed in 118 (60.2%). In 41.3%, EA PCR was positive; PW PCR was positive in 7.6%. The remaining laboratory tests were positive in a small number. Laparoscopy indicated definitive GTB in 9.1% and probable GTB in 37.4%. Among the 118 women treated for GTB, 22.9% conceived without in vitro fertilization; of these women, 74.1% had a positive EA PCR and 59.3% had a positive laparoscopy finding. A quarter of the women received ATT solely on the basis of the PCR result and 31.0% of these women conceived. CONCLUSION: No single test can detect all instances of GTB. A combination of tests is needed to increase the detection rate. Treatment given solely on the basis of a positive PCR result can result in conception.

Utility of reverse transcriptase PCR and DNA-PCR in the diagnosis of female genital tuberculosis.

This study was designed to test the utility of mRNA-based RT-PCR to detect viable bacilli, indicating active tubercular involvement, and DNA-PCR to detect present or past infection in the diagnosis of active female genital tuberculosis (TB) infection. A total of 200 subjects with complaints of infertility were enrolled in the study. Multiple sampling was done. One hundred and forty-three endometrial aspirate (EA), 94 peritoneal fluid/peritoneal washing (PF/PW) and six cornual biopsy (CB) specimens were collected for diagnosis using microscopy, culture, RT-PCR and DNA-PCR and results were compared with laparoscopic findings. RT-PCR and culture were concordant [positive in four (2.8%) EA specimens] signalling sampling from the site of active infection. Smear microscopy showed a poor detection rate while DNA-PCR showed high positivity. Sixty-one (44.85%) EA specimens, nine (9.57%) PF/PW specimens and two (33.33%) CB specimens were positive by DNA-PCR. Genital TB causing infertility (localized or secondary to TB elsewhere) can be picked up early by DNA-PCR, when it can be completely cured prior to the appearance of florid disease.

Leishmania-induced repression of selected non-coding RNA genes containing B-box element at their promoters in alternatively polarized M2 macrophages.

Leishmania is a group of parasitic protozoa that infect blood and tissue phagocytes including macrophages. We hypothesize that Leishmania is capable of establishing infection inside the macrophages because (a) they infect a subpopulation of macrophages; and (b) they "renovate" the macrophages before the establishment of infection. We found that only alternatively activated polarized M2 macrophages support Leishmania growth. Exposure of M2 macrophages to Leishmania promastigotes represses several selected RNA polymerase III (PolIII)-transcribed non-coding RNA (ncRNA) genes including those of 7SL RNA, vault RNA, and B2 RNA which have B-box element at their promoters. The B-box-binding transcription factor TFIIIC110 is down-regulated in Leishmania-exposed macrophages. Both the surface protease gp63 and the surface glycolipid LPG are required for the down-regulation of the ncRNAs in the M2 macrophages. We conclude that Leishmania surface gp63 collaborates with LPG to down-regulate TFIIIC110 in M2 macrophages to repress B-box containing ncRNA gene promoters.

Mechanism of down-regulation of RNA polymerase III-transcribed non-coding RNA genes in macrophages by Leishmania.

The parasitic protozoan Leishmania invades mammalian macrophages to establish infection. We reported previously that Leishmania manipulates the expression of several non-coding RNA genes (e.g. Alu RNA, B1 RNA, and signal recognition particle RNA) in macrophages to favor the establishment of their infection in the phagolysosomes of these cells (Ueda, Y., and Chaudhuri, G. (2000) J. Biol. Chem. 275, 19428-19432; Misra, S., Tripathi, M. K., and Chaudhuri, G. (2005) J. Biol. Chem. 280, 29364-29373). We report here the mechanism of this down-regulation. We found that the non-coding RNA (ncRNA) genes that are repressed by Leishmania infection in macrophages contain a "B-box" in their promoters and thus require the polymerase III transcription factor TFIIIC for their expression. We also found that Leishmania promastigotes through their surface protease (leishmanolysin or gp63) activate the thrombin receptor PAR1 in the macrophages. This activation of PAR1 raised the cytosolic concentration of Ca(2+) into the micromolar range, thereby activating the Ca(2+)-dependent protease µ-calpain. µ-Calpain then degraded TFIIIC110 to inhibit the expression of the selected ncRNA genes. Avirulent stocks of Leishmania not expressing surface gp63 failed to down-regulate ncRNAs in the exposed macrophages. Inhibition of PAR1 or calpain 1 in macrophages made them resistant to Leishmania infection. These data suggest that macrophage PAR1 and calpain 1 are potential drug targets against leishmaniasis.

Characterization of predominant Mycobacterium tuberculosis strains from different subpopulations of India.

The predominant strains from India belong to Central-Asian (CAS) and the East-African-Indian (EAI) clade of Mycobacterium tuberculosis. The two clades have also been shown to be geographically partitioned. The study of such strains may help to understand the characteristics that make M. tuberculosis an effective pathogen and its overrepresentation in certain populations. M. tuberculosis isolates characterized by spoligotyping under a population based tuberculosis study covering different regions from the North and South India were further analyzed by restriction fragment length polymorphism (RFLP) and by deletion analysis of M. tuberculosis specific deletion region 1 (TbD1). The genetic relationship of the two clades inferred using different genetic markers showed good correlation. In the North where the CAS clade predominates the isolates are characterized by presence of high IS6110 copy number and absence of TbD1 region whereas in the South where the EAI clade predominates the isolates are characterized by low copy number of IS6110 and presence of TbD1 region. The ancestral EAI strains were found to be less often associated with drug resistance or young age as compared to the CAS clade. The study highlights that the EAI lineage is well established in India and that CAS may be emerging or more recently introduced to India. The results depict a distinction in the lineage of strains from the North versus South India indicating a need to study if the pathogen has adapted to specific human populations.

Rapid detection of rifampin-resistant Mycobacterium tuberculosis directly from stained sputum smears using single-tube nested polymerase chain reaction deoxyribonucleic acid sequencing.

Microscopy is the mainstay of laboratory diagnosis of tuberculosis especially in resource poor countries. The World Health Organization has also recommended microscopy as the mainstay of diagnosis for directly observed treatment, short course. Using DNA extracts from Ziehl-Neelsen (ZN)-stained sputum smears, a single-tube nested polymerase chain reaction was optimized to confirm Mycobacterium tuberculosis complex and detect rifampin (RIF) resistance by sequencing, using a combination of novel (rpoB47 and rpoB158) and previously described (rpoB105 and rpoB293) primers. Carryover DNA was strictly monitored using several negative controls, and inhibition was ruled out by spiked controls. No such target was detected from negative controls and purified genomic DNA from other nontubercular mycobacteria. Resistance could be detected in 91.1% (51/56) slides. The results obtained were concordant with the 1% proportion method and DNA sequencing performed on culture isolates. Our results demonstrate that the method is suitable for rapid detection of susceptibility to RIF in acid-fast bacillus-positive ZN-stained slides obtained from patients suspected of harboring drug-resistant M. tuberculosis.