Repurposing approved protein kinase inhibitors as potent anti-leishmanials targeting Leishmania MAP kinases.

AIMS: Leishmaniasis, caused by the protozoan parasite poses a significant health burden globally. With a very few specific drugs, increased drug resistance it is important to look for drug repurposing along with the identification of pre-clinical candidates against visceral leishmaniasis. This study aims to identify potential drug candidates against visceral leishmaniasis by targeting leishmanial MAP kinases and screening FDA approved protein kinase inhibitors. MATERIALS AND METHODS: MAP kinases were identified from the Leishmania genome. 12 FDA approved protein kinase inhibitors were screened against Leishmania MAP kinases. Binding affinity, ADME and toxicity of identified drug candidates were profiled. The anti-proliferative effects and mechanism of action were assessed in Leishmania, including changes in cell morphology, flagellar length, cell cycle progression, reactive oxygen species (ROS) generation, and intra-macrophage parasitic burden. KEY FINDINGS: 23 MAP kinases were identified from the Leishmania genome. Sorafenib and imatinib emerged as repurposable drug candidates and demonstrated excellent anti-proliferative effects in Leishmania. Treatment with these inhibitors resulted in significant changes in cell morphology, flagellar length, and cell cycle arrest. Furthermore, sorafenib and imatinib promoted ROS generation and reduced intra-macrophage parasitic burden, and elicited anti-leishmanial activity in in vivo experimental VL models. SIGNIFICANCE: Collectively, these results imply involvement of MAP kinases in infectivity and survival of the parasite and can pave the avenue for repurposing sorafenib and imatinib as anti-leishmanial agents. These findings contribute to the exploration of new treatment options for visceral leishmaniasis, particularly in the context of emerging drug resistance.

Anticancer, antimicrobial and photocatalytic activities of a new pyrazole containing thiosemicarbazone ligand and its Co(III) and Ni(II) complexes: Synthesis, spectroscopic characterization and X-ray crystallography.

A new pyrazole based thiosemicarbazone ligand, 5-methyl-3-formylpyrazole-N(4)-isopropylthiosemicarbazone, (HMPzNHPri) (compound I), and its cobalt(III) and nickel(II) complexes, [Co(MPzNHPri)2]Cl (compound II) and [Ni(HMPzNHPri)2]Br2 (compound III), respectively, have been synthesized and characterized through various physico-chemical and spectroscopic studies. Both the reported Co(III) and Ni(II) complexes are cationic in nature and behave as 1:1 and 1:2 electrolytes in MeOH, respectively. Electronic spectral features of the complexes have classified them as distorted octahedral ones. IR spectral data (4000-450 cm-1) have suggested a monoprotic tridentate (NNS) function of compound I coordinating to the Co(III) ion via the pyrazolyl (tertiary) ring nitrogen, azomethine nitrogen and thiolato sulphur atom; while for compound III, compound I has been found to act as neutral NNS tridentate one, coordinating to Ni(II) via the pyrazolyl iminic nitrogen, azomethine nitrogen and thioketo sulphur. Structural features of all the compounds are confirmed by the single crystal X-ray data. All the compounds reported here have been found to exhibit significant photocatalytic activity towards degradation of Methylene Blue (MB) under UV radiation. Anticancer activity of all the three compounds against cancer cell lines (HeLa and A549) and a normal cell line (HEK293) have been investigated. Compound II has been found to be more efficient against the human cervical cancer cell (HeLa) and the lung cancer cell (A549) than compounds I and III. The ligand and both the complexes display potential activities against both gram-positive (Bacillus subtilis MTCC 7193) and gram-negative bacteria (E. coli MTCC 1610).

Modulation and Determination of the Status of Inflammasomes in Leishmania-Infected Macrophages.

Leishmania, an intra-macrophage kinetoplastid parasite, modulates a vast array of defensive mechanisms of the host macrophages to create a comfortable environment for their survival. When the host encounters intracellular pathogens, a multimeric protein complex called NLRP3 inflammasome gets turned on, leading to caspase-1 activation-mediated maturation of IL-1ß from its pro-form. However, Leishmania often manages to neutralize inflammasome activation by manipulating negative regulatory molecules of the host itself. Exhaustion of NLRP3 and pro-IL-1ß result from decreased NF-κB activity in infection, which was attributed to increased expression of A20, a negative regulator of NF-κB signalling. Moreover, reactive oxygen species, another key requirement for inflammasome activation, are inhibited by mitochondrial uncoupling protein 2 (UCP2) which is upregulated by Leishmania. Inflammasome activation is a complex event and procedures involved in monitoring inflammasome activation need to be accurate and error-free. In this chapter, we summarize the protocol that includes various experimental procedures required for the determination of the status of inflammasomes in Leishmania-infected macrophages.

Video parameters for action observation training in stroke rehabilitation: a scoping review.

PURPOSE: Action observation training (AOT) is a therapeutic approach used in stroke rehabilitation. Videos form the core of AOT, and knowledge of constituent parameters is essential to make the intervention robust and generalizable. Currently, there is a dearth of available information on video parameters to be used for AOT. Our purpose was to identify and describe the parameters that constitute AOT videos for stroke rehabilitation. METHOD: Electronic databases like PubMed, CINAHL, Scopus, Web of Science, ProQuest, and Ovid SP from inception to date according to PRISMA-ScR guidelines. Title, abstract, and full-text screening were done independently by two authors, with a third author for conflict resolution. Data on video parameters like length, quality, perspective, speed, screen size and distance, sound, and control videos were extracted. RESULTS: Seventy studies were included in this review. The most-reported parameters were video length (85.71%) and perspective of view (62.85%). Movement speed (7.14%) and sound (8.57%) were the least reported. Static landscapes or geometrical patterns were found suitable as control videos. CONCLUSION: Most video parameters except for length and perspective of view remain underreported in AOT protocols. Future studies with better descriptions of video parameters are required for comprehensive AOT interventions and result generalisation.

Videos shorter than 5 min may be preferred during action observation training (AOT) intervention in post-stroke.Egocentric view may be better for upper limb dexterity function and allocentric view for gross actions like walking.Choice of video disseminating device depends on its dimension as well as observer distance.Movement speed, video sound, and quality must be considered to obtain more comprehensive AOT videos.

Vesicle-Encapsulated Rolipram (PDE4 Inhibitor) and Its Anticancer Activity.

Vesicular carriers of drugs are popular for specific targeting and delivery. The most popular vesicles among these are liposomes. However, they suffer from some inherent limitations. In this work, alternative vesicles with enhanced stability, i.e., niosomes and bilosomes have been prepared, characterized, and their delivery efficiency studied. Bilosomes have the additional advantage of being able to withstand the harsh environment of the gastrointestinal tract (GIT). The taurine-derived bile salt (NaTC) was incorporated into the bilosome bilayer. The inspiration behind NaTC insertion is the recent reports on antiaging action and immune function of taurine. Fluorescence probing was used to study the vesicle environment. The entrapment and subsequent release of the important cAMP-specific PDE4 inhibitor/drug Rolipram, which has antibreast cancer properties, was assessed on the breast cancer cell line MCF-7. Rolipram has important therapeutic applications, one of the most significant in recent times being the treatment of Covid-19-triggered pneumonia and cytokine storms. As for cancer chemotherapy, the localization of drug, targeted delivery, and sustained release are extremely important issues, and it seemed worthwhile to explore the potential of the bilosomes and niosomes to entrap and release Rolipram. The important finding is that niosomes perform much better than bilosomes in the hormone-responsive breast cancer mileau MCF-7. Moreover, there was a 4-fold decrease in the IC50 of Rolipram encapsulated in niosomes compared to Rolipram alone. On the other hand, bilosome-encapsulated Rolipram shows higher IC50 value. The results can be further understood by molecular docking studies.

PDE4 inhibitor eliminates breast cancer stem cells via noncanonical activation of mTOR.

Ineffective cancer treatment is implicated in metastasis, recurrence, resistance to chemotherapy and radiotherapy, and evasion of immune surveillance. All these failures occur due to the persistence of cancer stem cells (CSCs) even after rigorous therapy, thereby rendering them as essential targets for cancer management. Contrary to the quiescent nature of CSCs, a gene profiler array disclosed that phosphatidylinositol-3-kinase (PI3K), which is known to be crucial for cell proliferation, differentiation, and survival, was significantly upregulated in CSCs. Since PI3K is modulated by cyclic adenosine 3',5' monophosphate (cAMP), analyses of cAMP regulation revealed that breast CSCs expressed increased levels of phosphodiesterase 4 (PDE4) in contrast to normal stem cells. In accordance, the effects of rolipram, a PDE4 inhibitor, were evaluated on PI3K regulators and signaling. The efficacy of rolipram was compared with paclitaxel, an anticancer drug that is ineffective in obliterating breast CSCs. Analyses of downstream signaling components revealed a switch between cell survival and death, in response to rolipram, specifically of the CSCs. Rolipram-mediated downregulation of PDE4A levels in breast CSCs led to an increase in cAMP levels and protein kinase A (PKA) expression. Subsequently, PKA-mediated upregulation of phosphatase and tensin homolog antagonized the PI3K/AKT/mTOR pathway and led to cell cycle arrest. Interestingly, direct yet noncanonical activation of mTOR by PKA, circumventing the influence of PI3K and AKT, temporally shifted the fate of CSCs toward apoptosis. Rolipram in combination with paclitaxel indicated synergistic consequences, which effectively obliterated CSCs within a tumor, thereby suggesting combinatorial therapy as a sustainable and effective strategy to abrogate breast CSCs for better patient prognosis.

Synthesis, bio-physical and anti-leishmanial studies of some novel indolo[3,2-a]phenanthridine derivatives.

Eight indolo[3,2-a]phenanthridine derivatives have been synthesized in a regioselective manner involving intramolecular Heck-type arylation as a key step. The compounds display interesting photophysical proprties and hence evaluated for their ability to interact with ct-DNA. Preliminary biophysical studies via UV and Fluorescence spectrophotometric titration with ct-DNA, and dye displacement studies with well known intercalator ethidium bromide and the groove binder Hoechst 33,258 reveal that the binding mode is probably minor groove binding. The prepared indolophenanthridine derivatives have also been evaluated as anti-leishmanial agents for the first time. MTT-assays for cell cytotoxicity against Leishmania promastigotes and Leishmania amastigotes were studied with the compounds 10b-f, 12-14 for the determination of their IC50 values. Cytotoxicity was determined using a murine RAW 264.7 cell line and human embryonic kidney cell line HEK 293. In L. donovani amastigote assay, compounds 10e, 10f and 12 showed good activity with relatively low cytotoxicity against RAW 264.7, resulting in acceptable selectivity indices. Selectivity index determination indicated compounds to be potent anti-leishmanial agents while 10b, 10c and 14 showed moderate selectivity index. Moreover, cell-cycle analysis of four different compounds 10b, 12, 13 and 14, representative of each group, was performed by FACS as an attempt to understand the mechanism of actions of these different sub-classes of the compounds on Leishmania.

Barriers leading to increased disability in neurologically challenged populations during COVID-19 pandemic: a scoping review.

PURPOSE: The objective of this scoping review was to get an overview of barriers emerging across the globe from the pandemic that are likely to increase the level of pre-existing disability status of neurologically challenged populations. METHODS: Database searches (PubMed/MEDLINE, CINAHL, ProQuest, Ovid, Scopus, and Web of Science) updated to December 2020 were conducted. Articles that identified challenges or barriers to neuro-rehabilitation, impact on disability status and health care services were included. Full-text articles limited to the English language with no restrictions on study design were included. Data was synthesized based on recurrent themes that were identified. RESULTS: Thirty-seven studies were included in this review. Neurological populations considered: stroke, multiple sclerosis, amyotrophic lateral sclerosis, parkinson's disease, autism, developmental disabilities, and those who required neurosurgical care. Barriers were grouped into categories as increased disease risk and complications, delayed or restricted access to neuro-rehabilitation, limited hospital access, telerehabilitation limitations, and shutdown of special centers of aid. CONCLUSIONS: COVID-19 pandemic has given rise to barriers that affect almost every aspect of healthcare and rehabilitation in neurologically challenged populations prompting an increase in their disability level. This can assist policymakers in designing mitigation strategies to minimize the detrimental effects on this vulnerable population.Implications for rehabilitationPandemic has led to the worsening of existing motor and non-motor symptoms, which need to be monitored, assessed and managed medically, and through rehabilitation in neurologically challenged populations.Notable decline of cognition and physical activity in neurologically challenged populations needs to be assessed and efforts to reverse these outcomes should be attempted.Rehabilitation services, hospital care and centers of aid need to be made more accessible for neurologically challenged populations with COVID-19 precautionary measures.Telemedicine and telerehabilitation need to be upgraded to enhance further face to face like interactions and for tracking of progressive disease.

Development and feasibility testing of action observation training videos in acute stroke survivors: Preliminary findings.

Background: Action observation training (AOT) is used for lower limb (LL) stroke rehabilitation in subacute and chronic stages, but concise information regarding the types of activities to be used and the feasibility of administration in the acute stroke population is unknown. The aim of this study was to develop and validate videos of appropriate activities for LL AOT and test administrative feasibility in acute stroke.   Method: A video inventory of LL activities was created after a literature survey and expert scrutiny. Five stroke rehabilitation experts validated the videos per domains of relevance, comprehension, clarity, camera position and brightness. LL AOT was then tested on ten individuals with acute stroke for uncovering barriers for clinical use in a feasibility study. Participants watched the activities and attempted imitation of the same. Determination of administrative feasibility was undertaken via participant interviews.   Results: Suitable LL activities for stroke rehabilitation were identified. Content validation of videos led to improvements in selected activities and video quality. Expert scrutiny led to further video processing to include different perspectives of view and speeds of projected movements. Barriers identified included inability to imitate actions shown in videos and increased distractibility for some participants.    Conclusion: A video catalogue of LL activities was developed and validated. AOT was deemed safe and feasible for acute stroke rehabilitation and may be used in future research and clinical practice.

Iron(III) Complex-Functionalized Gold Nanocomposite as a Strategic Tool for Targeted Photochemotherapy in Red Light.

Iron(III)-phenolate/carboxylate complexes exhibiting photoredox chemistry and photoactivated reactive oxygen species (ROS) generation at their ligand-to-metal charge-transfer (LMCT) bands have emerged as potential strategic tools for photoactivated chemotherapy. Herein, the synthesis, in-depth characterization, photochemical assays, and remarkable red light-induced photocytotoxicities in adenocarcinomic human immortalized human keratinocytes (HaCaT) and alveolar basal epithelial (A549) cells of iron(III)-phenolate/carboxylate complex of molecular formula, [Fe(L1)(L2)] (1), where L1 is bis(3,5 di-tert-butyl-2-hydroxybenzyl)glycine and L2 is 5-(1,2-dithiolan-3-yl)-N-(1,10-phenanthroline-5-yl)pentanamide, and the gold nanocomposite functionalized with complex 1 (1-AuNPs) are reported. There was a significant red shift in the UV-visible absorption band on functionalization of complex 1 to the gold nanoparticles (λmax: 573 nm, 1; λmax: 660 nm, 1-AuNPs), rendering the nanocomposite an ideal candidate for photochemotherapeutic applications. The notable findings in our present studies are (i) the remarkable cytotoxicity of the nanocomposite (1-AuNPs) to A549 (IC50: 0.006 µM) and HaCaT (IC50: 0.0075 µM) cells in red light (600-720 nm, 30 J/cm2) while almost nontoxic (IC50 > 500 µg/mL, 0.053 µM) in the dark, (ii) the nontoxicity of 1-AuNPs to normal human diploid fibroblasts (WI-38) or human peripheral lung epithelial (HPL1D) cells (IC50 > 500 µg/mL, 0.053 µM) both in the dark and red light signifying the target-specific anticancer activity of the nanocomposite, (iii) localization of 1-AuNPs in mitochondria and partly nucleus, (iv) remarkable red light-induced generation of reactive oxygen species (ROS: 1O2, •OH) in vitro, (v) disruption of the mitochondrial membrane due to enhanced oxidative stress, and (vi) caspase 3/7-dependent apoptosis. A similar cytotoxic profile of complex 1 was another key finding of our studies. Overall, our current investigations show a new red light-absorbing iron(III)-phenolate/carboxylate complex-functionalized gold nanocomposite (1-AuNPs) as the emerging next-generation iron-based photochemotherapeutic agent for targeted cancer treatment modality.

Evaluation of Modulators of cAMP-Response in Terms of Their Impact on Cell Cycle and Mitochondrial Activity of Leishmania donovani.

With the identification of novel cAMP binding effector molecules in Trypanosoma, the role of cAMP in kinetoplastida parasites gained an intriguing breakthrough. Despite earlier demonstrations of the role of cAMP in the survival of Leishmania during macrophage infection, there is essential need to specifically clarify the involvement of cAMP in various cellular processes in the parasite. In this context, we sought to gain a comprehensive understanding of the effect of cAMP analogs and cAMP-cyclic nucleotide phosphodiesterase (PDE) inhibitors on proliferation of log phase parasites. Administration of both hydrolyzable (8-pCPT-cAMP) and nonhydrolyzable analogs (Sp-8-pCPT-cAMPS) of cAMP resulted in a significant decrease of Leishmania proliferation. Among the various PDE inhibitors, etazolate was found to be potently antiproliferative. BrdU cell proliferation and K/N/F-enumeration microscopic study revealed that both cAMP analogs and selective PDE inhibitors resulted in significant cell cycle arrest at G1 phase with reduced S-phase population. Furthermore, careful examination of the flagellar motility patterns revealed significantly reduced coordinated forward flagellar movement of the promastigotes with a concomitant decrease in cellular ATP levels. Alongside, 8-pCPT-cAMP and PDE inhibitors etazolate and trequinsin showed marked reduction in mitochondrial membrane potential. Treatment of etazolate at subcytotoxic concentration to infected macrophages significantly reduced parasite burden, and administration of etazolate to Leishmania-infected BALB/c mice showed reduced liver and spleen parasite burden. Collectively, these results imply involvement of cAMP in various crucial processes paving the avenue for developing potent antileishmanial agent.

Role of leishmanial acidocalcisomal pyrophosphatase in the cAMP homeostasis in phagolysosome conditions required for intra-macrophage survival.

Exposure of Leishmania donovani to macrophage phagolysosome conditions (PC) (37°C and pH 5.5) led to increased intracellular cAMP and cAMP-mediated responses, which help in intra-macrophage survival pre-requisite for infectivity. In the absence of typical orthologs for G-proteins and G-protein coupled receptors, we sought to study the precise mechanisms for positive modulation of cAMP production during exposure to PC. Amongst two promastigote-stage specific membrane bound receptor adenylate cyclases (LdRAC-A and LdRAC-B), LdRAC-A appeared to function as a major cAMP generator following PC exposure. Pyrophosphate (PPi), an energy storage compound as well as a by-product of cAMP biosynthesis by adenylate cyclise, was found to be decreased following PC exposure. This may be due to microtubule and microfilament-driven translocation of acidocalcisomes near plasma membrane vicinity with concomitant increase of acidocalcisome membrane pyrophosphatase (LdV-H+PPase) and acidocalcisomal soluble pyrophosphatase (LdVSP1). Episomal over-expression and conditional silencing demonstrated regulatory role of V-H+PPase on cAMP trigger and consequent induction of resistance to macrophage-derived pro-oxidants and parasite killing. Furthermore, immunofluorescence analysis revealed possible co-localization of LdV-H+PPase and LdRAC-A during PC exposure. Collectively, these results suggest that translocation of acidocalcisome in membrane vicinity functions as a trigger for LdRAC-A-driven cAMP generation through depletion of PPi pool by LdV-H+PPase.

Acetylation of lysine 109 modulates pregnane X receptor DNA binding and transcriptional activity.

Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug-drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time. More recent mechanistic studies have shown that PXR is modulated by multiple PTMs. Herein we provide the first investigation of the role of acetylation in modulating PXR activity. Through LC-MS/MS analysis, we identified lysine 109 (K109) in the hinge as PXR's major acetylation site. Using various biochemical and cell-based assays, we show that PXR's acetylation status and transcriptional activity are modulated by E1A binding protein (p300) and sirtuin 1 (SIRT1). Based on analysis of acetylation site mutants, we found that acetylation at K109 represses PXR transcriptional activity. The mechanism involves loss of RXRα dimerization and reduced binding to cognate DNA response elements. This mechanism may represent a promising therapeutic target using modulators of PXR acetylation levels. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

A soluble phosphodiesterase in Leishmania donovani negatively regulates cAMP signaling by inhibiting protein kinase A through a two way process involving catalytic as well as non-catalytic sites.

Intracellular cAMP level and cAMP mediated responses are elevated when Leishmania are exposed to macrophage phagolysosome conditions (37 °C and pH 5.5). Phosphodiesterases play major role in cAMP regulation and in the present study we have cloned and characterized a 2.1 kb cytosolic isoform of phosphodiesterase from Leishmania donovani (LdPDED) which plays important role in cAMP homeostasis when the promastigotes are exposed to macrophage phagolysome conditions for converting to axenic amastigotes. Domain characterization suggested the presence of two pseudo-substrate sites similar to the ones present in the regulatory subunit of cAMP-dependent protein kinase A (PKA) and a putative PKA phosphorylation site at T(708) of C-terminus of LdPDED. Deletion constructs and site directed mutagenesis revealed the ability of LdPDED to interact with L. donovani PKA catalytic subunits (LdPKAC1 and LdPKAC2) resulting in inhibition of kinase activity in one hand and increase of phosphodiesterase activity through PKA mediated phosphorylation at putative phosphorylation site on the other hand. This study therefore identifies a unique phosphodiesterase in L. donovani which appears to regulate cAMP-dependent PKA signaling through a two way process.

Prostaglandin E2 negatively regulates the production of inflammatory cytokines/chemokines and IL-17 in visceral leishmaniasis.

Persistence of intracellular infection depends on the exploitation of factors that negatively regulate the host immune response. In this study, we elucidated the role of macrophage PGE2, an immunoregulatory lipid, in successful survival of Leishmania donovani, causative agent of the fatal visceral leishmaniasis. PGE2 production was induced during infection and resulted in increased cAMP level in peritoneal macrophages through G protein-coupled E-series prostanoid (EP) receptors. Among four different EPs (EP1-4), infection upregulated the expression of only EP2, and individual administration of either EP2-specific agonist, butaprost, or 8-Br-cAMP, a cell-permeable cAMP analog, promoted parasite survival. Inhibition of cAMP also induced generation of reactive oxygen species, an antileishmanial effector molecule. Negative modulation of PGE2 signaling reduced infection-induced anti-inflammatory cytokine polarization and enhanced inflammatory chemokines, CCL3 and CCL5. Effect of PGE2 on cytokine and chemokine production was found to be differentially modulated by cAMP-dependent protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC). PGE2-induced decreases in TNF-α and CCL5 were mediated specifically by PKA, whereas administration of brefeldin A, an EPAC inhibitor, could reverse decreased production of CCL3. Apart from modulating inflammatory/anti-inflammatory balance, PGE2 inhibited antileishmanial IL-17 cytokine production in splenocyte culture. Augmented PGE2 production was also found in splenocytes of infected mice, and administration of EP2 antagonist in mice resulted in reduced liver and spleen parasite burden along with host-favorable T cell response. These results suggest that Leishmania facilitates an immunosuppressive environment in macrophages by PGE2-driven, EP2-mediated cAMP signaling that is differentially regulated by PKA and EPAC.

The effect of including the C2 insert of nonmuscle myosin II-C on neuritogenesis.

The functional role of the C2 insert of nonmuscle myosin II-C (NM II-C) is poorly understood. Here, we report for the first time that the expression of the C2 insert-containing isoform, NM II-C1C2, is inducible in Neuro-2a cells during differentiation both at mRNA and protein levels. Immunoblot and RT-PCR analysis reveal that expression of NM II-C1C2 peaks between days 3 and 6 of differentiation. Localization of NM II-C1C2 in Neuro-2a cells suggests that the C2 insert-containing isoform is localized in the cytosol and along the neurites, specifically at the adherence point to substratum. Inhibition of endogenous NM II-C1C2 using siRNA decreases the neurite length by 43% compared with control cells treated with nonspecific siRNA. Time lapse image analysis reveals that neurites of C2-siRNA-treated cells have a net negative change in neurite length per minute, leading to a reduction of overall neurite length. During neuritogenesis, NM II-C1C2 can interact and colocalize with ß1-integrin in neurites. Altogether, these studies indicate that NM II-C1C2 may be involved in stabilizing neurites by maintaining their structure at adhesion sites.

Identification of a protein kinase A regulatory subunit from Leishmania having importance in metacyclogenesis through induction of autophagy.

cAMP-mediated responses act as modulators of environmental sensing and cellular differentiation of many kinetoplastidae parasites including Leishmania. Although cAMP synthesizing (adenylate cyclase) and degrading (phosphodiesterase) enzymes have been cloned and characterized from Leishmania, no cAMP-binding effector molecule has yet been identified from this parasite. In this study, a regulatory subunit of cAMP-dependent protein kinase (Ldpkar1), homologous to mammalian class I cAMP-dependent protein kinase regulatory subunit, has been identified from L. donovani. Further characterization suggested possible interaction of LdPKAR1 with PKA catalytic subunits and inhibition of PKA activity. This PKA regulatory subunit is expressed in all life cycle stages and its expression attained maximum level in stationary phase promastigotes, which are biochemically similar to the infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates LdPKAR1 expression and under starvation condition promastigotes overexpressing Ldpkar1 attained metacyclic features earlier than normal cells. Furthermore, Ldpkar1 overexpression accelerates autophagy, a starvation-induced cytological event necessary for metacyclogenesis and amastigote formation. Conditional silencing of Ldpkar1 delays the induction of autophagy in the parasite. The study, for the first time, reports the identification of a functional cAMP-binding effector molecule from Leishmania that may modulate important cytological events affecting metacyclogenesis.

Role of cAMP Signaling in the Survival and Infectivity of the Protozoan Parasite, Leishmania donovani.

Leishmania donovani, while invading macrophages, encounters striking shift in temperature and pH (from 22°C and pH 7.2 to 37°C and pH 5.5), which act as the key environmental trigger for differentiation, and increases cAMP level and cAMP-mediated responses. For comprehensive understanding of cAMP signaling, we studied the enzymes related to cAMP metabolism. A stage-specific and developmentally regulated isoform of receptor adenylate cyclase (LdRACA) showed to regulate differentiation-coupled induction of cAMP. The soluble acidocalcisomal pyrophosphatase, Ldvsp1, was the major isoform regulating cAMP level in association with LdRACA. A differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) might be related to infection establishment by shifting trypanothione pool utilization bias toward antioxidant defense. We identified and cloned a functional cAMP-binding effector molecule from L. donovani (a regulatory subunit of cAMP-dependent protein kinase, LdPKAR) that may modulate metacyclogenesis through induction of autophagy. This study reveals the significance of cAMP signaling in parasite survival and infectivity.

Pregnane X receptor activation induces FGF19-dependent tumor aggressiveness in humans and mice.

The nuclear receptor pregnane X receptor (PXR) is activated by a range of xenochemicals, including chemotherapeutic drugs, and has been suggested to play a role in the development of tumor cell resistance to anticancer drugs. PXR also has been implicated as a regulator of the growth and apoptosis of colon tumors. Here, we have used a xenograft model of colon cancer to define a molecular mechanism that might underlie PXR-driven colon tumor growth and malignancy. Activation of PXR was found to be sufficient to enhance the neoplastic characteristics, including cell growth, invasion, and metastasis, of both human colon tumor cell lines and primary human colon cancer tissue xenografted into immunodeficient mice. Furthermore, we were able to show that this PXR-mediated phenotype required FGF19 signaling. PXR bound to the FGF19 promoter in both human colon tumor cells and "normal" intestinal crypt cells. However, while both cell types proliferated in response to PXR ligands, the FGF19 promoter was activated by PXR only in cancer cells. Taken together, these data indicate that colon cancer growth in the presence of a specific PXR ligand results from tumor-specific induction of FGF19. These observations may lead to improved therapeutic regimens for colon carcinomas.