Allosteric Antagonist Modulation of TRPV2 by Piperlongumine Impairs Glioblastoma Progression.

The use of computational tools to identify biological targets of natural products with anticancer properties and unknown modes of action is gaining momentum. We employed self-organizing maps to deconvolute the phenotypic effects of piperlongumine (PL) and establish a link to modulation of the human transient receptor potential vanilloid 2 (hTRPV2) channel. The structure of the PL-bound full-length rat TRPV2 channel was determined by cryo-EM. PL binds to a transient allosteric pocket responsible for a new mode of anticancer activity against glioblastoma (GBM) in which hTRPV2 is overexpressed. Calcium imaging experiments revealed the importance of Arg539 and Thr522 residues on the antagonistic effect of PL and calcium influx modulation of the TRPV2 channel. Downregulation of hTRPV2 reduces sensitivity to PL and decreases ROS production. Analysis of GBM patient samples associates hTRPV2 overexpression with tumor grade, disease progression, and poor prognosis. Extensive tumor abrogation and long term survival was achieved in two murine models of orthotopic GBM by formulating PL in an implantable scaffold/hydrogel for sustained local therapy. Furthermore, in primary tumor samples derived from GBM patients, we observed a selective reduction of malignant cells in response to PL ex vivo. Our results establish a broadly applicable strategy, leveraging data-motivated research hypotheses for the discovery of novel means tackling cancer.

Molecular mechanism of TRPV2 channel modulation by cannabidiol.

Transient receptor potential vanilloid 2 (TRPV2) plays a critical role in neuronal development, cardiac function, immunity, and cancer. Cannabidiol (CBD), the non-psychotropic therapeutically active ingredient of Cannabis sativa, is an activator of TRPV2 and also modulates other transient receptor potential (TRP) channels. Here, we determined structures of the full-length rat TRPV2 channel in apo and CBD-bound states in nanodiscs by cryo-electron microscopy. We show that CBD interacts with TRPV2 through a hydrophobic pocket located between S5 and S6 helices of adjacent subunits, which differs from known ligand and lipid binding sites in other TRP channels. CBD-bound TRPV2 structures revealed that the S4-S5 linker plays a critical role in channel gating upon CBD binding. Additionally, nanodiscs permitted us to visualize two distinct TRPV2 apo states in a lipid environment. Together these results provide a foundation to further understand TRPV channel gating, their divergent physiological functions, and to accelerate structure-based drug design.

Evaluation of isosorbide-5-mononitrate as a cervical ripening agent prior to induced abortion in contrast to misoprostol- a randomized controlled trial.

OBJECTIVE: To determine whether vaginal application of 40 mg isosorbide-5-mononitrate (ISMN) has a comparable cervical ripening efficacy to and lesser side effects than 400 µg misoprostol in women scheduled for the first trimester induced abortion using a manual vacuum aspirator (MVA). METHODS: We conducted a prospective randomized open-label study in 70 women at 6-12 weeks of pregnancy at the R G Kar Medical College and Hospital, Kolkata, India, over a period of two years from 2015 to 2017. Forty milligrams of ISMN and 400 µg misoprostol were vaginally applied for cervical priming. The primary outcome measure was the cervical response assessed by the passage of the appropriate and largest sized MVA cannula through the internal os without resistance, at the beginning of the procedure. RESULTS: The base line cervical dilatation was found to be significantly higher in the misoprostol group than in the ISMN group (7.65±1.38 vs. 6.9±1.26 mm; P=0.025, 95% confidence interval, -1.4046 to -0.953). However, when the women were sub-analyzed based on parity, there was no statistically significant difference in the same parameters among the multigravid women. The need for further cervical dilatation was significantly higher in the ISMN group when the primigravid women were compared, although the multigravid women responded favorably to ISMN. CONCLUSION: In the primigravid women, misoprostol appears to exert a higher efficacy as a cervical ripening agent in contrast to ISMN. However, ISMN can be used in multigravid women for the same purpose as in this group, misoprostol did not show any significant improvement in efficacy over ISMN.

Cryo Electron Microscopy of TRP Channels.

Cryo electron microscopy (cryo-EM) is a powerful technique that can be used to elucidate the structural architecture of a protein molecule in a physiologically relevant environment. In this method, purified protein is frozen in its aqueous buffer in a thin layer of vitreous ice in which the biological macromolecules are embedded in various orientations. Images of this frozen sample are collected with an electron microscope, and the data is processed using different software algorithms resulting in high-resolution structures of the protein. Proteins in the presence of various ligands or other macromolecular complexes can also be studied by this method. Here, we present a protocol for the purification and vitrification of TRP channels for single particle cryo-EM.

Structural insights on TRPV5 gating by endogenous modulators.

TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery.

Structural insights into the molecular mechanism of mouse TRPA1 activation and inhibition.

Pain, though serving the beneficial function of provoking a response to dangerous situations, is an unpleasant sensory and emotional experience. Transient receptor potential ankyrin 1 (TRPA1) is a member of the transient receptor potential (TRP) cation channel family and is localized in "nociceptors," where it plays a key role in the transduction of chemical, inflammatory, and neuropathic pain. TRPA1 is a Ca2+-permeable, nonselective cation channel that is activated by a large variety of structurally unrelated electrophilic and nonelectrophilic chemical compounds. Electrophilic ligands are able to activate TRPA1 channels by interacting with critical cysteine residues on the N terminus of the channels via covalent modification and/or disulfide bonds. Activation by electrophilic compounds is dependent on their thiol-reactive moieties, accounting for the structural diversity of the group. On the other hand, nonelectrophilic ligands do not interact with critical cysteines on the channel, so the structural diversity of this group is unexplained. Although near-atomic-resolution structures of TRPA1 were resolved recently by cryo-electron microscopy, in the presence of both agonists and antagonists, detailed mechanisms of channel activation and inhibition by these modulators could not be determined. Here, we investigate the effect of both electrophilic and nonelectrophilic ligands on TRPA1 channel conformational rearrangements with limited proteolysis and mass spectrometry. Collectively, our results reveal that channel modulation results in conformational rearrangements in the N-terminal ankyrin repeats, the pre-S1 helix, the TRP-like domain, and the linker regions of the channel.

Transient Receptor Potential (TRP) Channels.

Transient Receptor Potential (TRP) channels are evolutionarily conserved integral membrane proteins. The mammalian TRP superfamily of ion channels consists of 28 cation permeable channels that are grouped into six subfamilies based on sequence homology (Fig. 6.1). The canonical TRP (TRPC) subfamily is known for containing the founding member of mammalian TRP channels. The vanilloid TRP (TRPV) subfamily has been extensively studied due to the heat sensitivity of its founding member. The melastatin-related TRP (TRPM) subfamily includes some of the few known bi-functional ion channels, which contain functional enzymatic domains. The ankyrin TRP (TRPA) subfamily consists of a single chemo-nociceptor that has been proposed to be a target for analgesics. The mucolipin TRP (TRPML) subfamily channels are found primarily in intracellular compartments and were discovered based on their critical role in type IV mucolipidosis (ML-IV). The polycystic TRP (TRPP) subfamily is a diverse group of proteins implicated in autosomal dominant polycystic kidney disease (ADPKD). Overall, this superfamily of channels is involved in a vast array of physiological and pathophysiological processes making the study of these channels imperative to our understanding of subcellular biochemistry.

Cryo-EM structure of 5-HT3A receptor in its resting conformation.

Serotonin receptors (5-HT3AR) directly regulate gut movement, and drugs that inhibit 5-HT3AR function are used to control emetic reflexes associated with gastrointestinal pathologies and cancer therapies. The 5-HT3AR function involves a finely tuned orchestration of three domain movements that include the ligand-binding domain, the pore domain, and the intracellular domain. Here, we present the structure from the full-length 5-HT3AR channel in the apo-state determined by single-particle cryo-electron microscopy at a nominal resolution of 4.3 Å. In this conformation, the ligand-binding domain adopts a conformation reminiscent of the unliganded state with the pore domain captured in a closed conformation. In comparison to the 5-HT3AR crystal structure, the full-length channel in the apo-conformation adopts a more expanded conformation of all the three domains with a characteristic twist that is implicated in gating.

Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM.

The transient receptor potential vanilloid 5 (TRPV5) channel is a member of the transient receptor potential (TRP) channel family, which is highly selective for Ca2+, that is present primarily at the apical membrane of distal tubule epithelial cells in the kidney and plays a key role in Ca2+ reabsorption. Here we present the structure of the full-length rabbit TRPV5 channel as determined using cryo-EM in complex with its inhibitor econazole. This structure reveals that econazole resides in a hydrophobic pocket analogous to that occupied by phosphatidylinositides and vanilloids in TRPV1, thus suggesting conserved mechanisms for ligand recognition and lipid binding among TRPV channels. The econazole-bound TRPV5 structure adopts a closed conformation with a distinct lower gate that occludes Ca2+ permeation through the channel. Structural comparisons between TRPV5 and other TRPV channels, complemented with molecular dynamics (MD) simulations of the econazole-bound TRPV5 structure, allowed us to gain mechanistic insight into TRPV5 channel inhibition by small molecules.

Structure of the full-length TRPV2 channel by cryo-EM.

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Šresolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels.

Mass drug administration for elimination of lymphatic filariasis: Recent experiences from a district of West Bengal, India.

BACKGROUND: Annual mass drug administration (MDA) with diethyl carbamazine (DEC) and Albendazole is the most cost-effective strategy to control lymphatic filariasis (LF). MATERIALS AND METHODS: The aim of the present study was to assess the coverage and the compliance of MDA, to elicit factors that influenced compliance, to document side-effects reported and to assess the awareness of the community regarding the disease and MDA program in Bankura district, West Bengal after 2012 round of MDA. Multistage cluster sampling method was adopted. Total four clusters; three villages and one urban municipality ward were selected. In each cluster, minimum 30 families were randomly selected and the head or any responsible family member was interviewed using a pre-designed, pre-tested schedule after taking written informed consent. Data were compiled and analyzed using SPSS 19.0. RESULTS: Total eligible population was 683 among which 98.8% received both the drugs. About 5% of the recipients took none of the drugs. More than two-thirds of the families took unsupervised dose. Drug compliance rate was significantly lower in urban (90.7%) than in the rural clusters (95.7%) (z = 2.46, P < 0.05). Effective coverage rate was significantly lower in urban than in the rural clusters (87.4% vs. 95.3%; z = 3.57, P < 0.01). Coverage compliance gap was higher in urban (5.7%) than in rural cluster (3.9%). Fear of side-effects was the main reason for non-compliance. Reported side-effects were few, mild, and transient. Around 60% of the surveyed families were aware about the MDA program whereas, 67% of them heard about LF. Only 41% families were provided information, education, and communication in last 15 days before MDA. Major sources of information for the surveyed families were leaflets (20.3%) and poster (9.8%). CONCLUSIONS: Widespread rural urban variation in performance status, poor social mobilization activities, lack of supervised dosing, and lack of knowledge of the community about the disease and the program are the major areas of concern.

Knowledge and Practice of Standard Precautions and Awareness Regarding Post-Exposure Prophylaxis for HIV among Interns of a Medical College in West Bengal, India.

OBJECTIVES: To assess the knowledge of interns on standard precautions and post-exposure prophylaxis for HIV, and identify the gap between knowledge and practice relating to standard precautions, as well as determining the perceived barriers against adherence to standard precautions. METHODS: The study was conducted on 130 interns of 2010-11 batch from a government-run medical college in Kolkata, India. All participants completed a self-administered questionnaire with items relating to basic components of standard precautions and post-exposure prophylaxis for HIV. The questionnaire also included open ended questions relating to reasons for non-adherence to the practice of standard precautions along with additional space for specific comments, if any. RESULTS: Poor adherence in the use of personal protective equipment, hand washing, safe handling and disposal of needles and sharp objects were found to be among the practices for which the interns expressed correct knowledge. While the main reasons for non-adherence were found to be clumsiness in handling needles, wearing gloves, feeling uncomfortable when wearing aprons, impracticality of regular hand-washing and non-availability of equipment. Although the majority of the respondents (84.6%) expressed awareness of washing sites of injured with soap and water, approximately 32.3% did not know that antiseptics could cause more damage. Also, only 63.8% expressed awareness of reporting any incidence of occupational exposure, while knowledge on post-exposure prophylaxis regimens was generally found to be poor. CONCLUSION: The considerable gap between knowledge and practice of standard precautions and inadequate knowledge of post-exposure prophylaxis emphasizes the need for continuous onsite training of interns with supportive supervision and monitoring of their activities.

Mental health, protective factors and violence among male adolescents: a comparison between urban and rural school students in West Bengal.

A cross-sectional study was conducted among 199 (104 urban, 95 rural) male students of classes VIII and IX, of two schools, in urban and rural areas of West Bengal to compare the prevalence of protective factors, mental health issues, and violence among the urban and rural adolescents using a self-administered questionnaire. Higher proportion of urban students than rural (67.3% vs. 62.5%) reported that their guardians understood their problems. Mental health issues like loneliness (17.3% vs. 9.8%), worry (17.3% vs. 10.7%), and suicidal thoughts (19.2% vs. 14.1%) were higher among urban students. Physical fight (53.8% vs. 11.6%), bullying (46.4% vs. 17%), physical attack by family members (46% vs. 17%), and by teachers (53% vs. 10.7%) were all more in urban adolescents. Mental health- and violence-related issues were prevalent more among urban students than those among rural students in spite of having more protective factors suggesting the need of frequent supervision, monitoring, and support of adolescents.

Patterns of alcohol consumption among male adults at a slum in Kolkata, India.

Globally, alcohol-abuse is a major cause of mortality and morbidity. Consumption of alcohol has increased in India in the recent decades. It is imperative to know the patterns of alcohol consumption among different types of consumers to launch a well-planned nationwide programme for the prevention and control of this devastating social pathology. This community-based, cross-sectional study was undertaken to identify the patterns of alcohol intake among different types of alcohol consumers and to assess the clinical signs of chronic harmful alcohol-use. A predesigned, pretested, semi-structured alcohol-use disorders identification test (AUDIT) questionnaire was used for interviewing males, aged >18 years, selected by random sampling from an updated household list of a randomly-selected sector of the service area of the Urban Health Centre in Chetla, Kolkata, West Bengal, India. Written informed consents were obtained from all the respondents. Relevant clinical examination for chronic harmful alcohol-use was done according to the AUDIT clinical screening procedures. The results revealed that 65.8% (150/228) were current consumers of alcohol; 14% were alcohol-dependents; 8% were hazardous or harmful consumers, and 78% were non-hazardous non-harmful consumers. The mean age of the respondents at the initiation of drinking alcohol was 20.8+5.9 years. Eighty-six percent of dependents (n=21) took both Indian-made foreign liquor and locally-made alcoholic beverages. The proportions of alcohol consumers who drank alone among alcohol-dependents, hazardous or harmful consumers, and non-hazardous non-harmful consumers were 71.4%, 50%, and 7.7% respectively, and the difference was significant (p<0.01). Forty-one percent of the consumers drank at public places and workplaces, which may be socially harmful. About 38% of the dependents purchased alcohol from unlicensed liquor shops. Only 16% expressed concerns for their drinking habit mainly to the past illness. The proportion of the concerned respondents was higher in the hazardous and harmful drinking patterns than in the non-hazardous non-harmful drinking pattern, and the difference was significant (p<0.05). About 62% of the dependents had clinical signs of chronic alcohol consumption. The presence of a considerable proportion of alcohol-dependents, the low mean age at initiation of drinking alcohol, and the habit of drinking in public places and workplaces are the main areas that need special emphasis by intervention programmes.

Performance evaluation of PPTCT (Prevention of parent to child transmission of HIV) programme: an experience from West Bengal.

BACKGROUND & OBJECTIVES: Prevention of parent-to-child transmission (PPTCT) services are an integral part of National AIDS Control Programme and their critical appraisal is necessary for improving quality care. The present study was conducted to evaluate the performance of PPTCT services in West Bengal during April, 2008 - March 2009 and April 2009 - March 2010 and identify gaps in service delivery for making suitable recommendations. METHODS: Data were collected from the Computerized Management Information System and validated by cross-checking records at each district. Focus group discussions (FGDs) were conducted among programme managers, counsellors and antenatal women attending the Integrated Counselling and Testing Centres. Performance indicators and outcomes of FGDs were analyzed. RESULTS: The proportion of antenatal women tested declined in 2009-2010 from 2008-2009 (64.3 to 63.8%). Proportions of counseled cases also declined (72.5 vs. 68.4%). HIV positivity rates among those tested were 0.13 and 0.14 per cent, respectively in two years. Proportion of mother-baby pairs receiving nevirapine prophylaxis was increased by 5 per cent. Medical colleges, and category A districts having high HIV prevalence provided better services. Follow up services of HIV-exposed birth cohorts were grossly unsatisfactory. INTERPRETATION & CONCLUSIONS: Gaps were identified at each step of service delivery for which capacity building, improvement of infrastructure including laboratory services and ensuring emergency labour room testing up to the sub-district level were imperative. Outsourcing follow up services to other community based organizations may also be considered.

Prevalence and health-seeking behavior of reproductive tract infection/sexually transmitted infections symptomatics: a cross-sectional study of a rural community in the Hooghly district of West Bengal.

A community-based, descriptive, cross-sectional study was conducted in Purushottampur village of the Singur block, Hooghly, using a pre-tested, semi-structured questionnaire, to find out the prevalence of RTI / STI symptomatics among the general population aged 15 to 49 years, and to assess their profiles and health-seeking behaviors. Prevalence of (Reproductive tract infection) RTI / STI (Sexually transmitted infections) symptoms in the last 12 months was found to be 13.9 and 13.6% among males and females, respectively. The most common symptom was dysuria and vaginal discharge among males and females, respectively. Almost half of the STI symptomatics (52% male, 50% female) did nothing for their symptoms. Better health-seeking behavior was observed among females. About 9.4% of the males and 47% of the females sought advice from the clinic / hospital / health workers. Strengthening of activities of the existing national program among the general population is needed to build a positive health-seeking behavior that will ensure success of the syndromic management of RTI / STI.