Structure-Activity Relationship Study of the High-Affinity Neuropeptide Y4 Receptor Positive Allosteric Modulator VU0506013.

Positive allosteric modulators targeting the Y4 receptor (Y4R), a G protein-coupled receptor (GPCR) involved in the regulation of satiety, offer great potential in anti-obesity research. In this study, we selected 603 compounds by using quantitative structure-activity relationship (QSAR) models and tested them in high-throughput screening (HTS). Here, the novel positive allosteric modulator (PAM) VU0506013 was identified, which exhibits nanomolar affinity and pronounced selectivity toward the Y4R in engineered cell lines and mouse descending colon mucosa natively expressing the Y4R. Based on this lead structure, we conducted a systematic SAR study in two regions of the scaffold and presented a series of 27 analogues with modifications in the N- and C-terminal heterocycles of the molecule to obtain insight into functionally relevant positions. By mutagenesis and computational docking, we present a potential binding mode of VU0506013 in the transmembrane core of the Y4R. VU0506013 presents a promising scaffold for developing in vivo tools to move toward anti-obesity drug research focused on the Y4R.

Knowledge, Attitude and Practice of Techniques of Face-mask Usage and Hand Hygiene among COVID-19 Positive Individuals at a COVID Care Centre in Central India: A Cross-sectional Comparative Study.

Most of the published reports of Knowledge, Attitude and Practice (KAP) surveys with regard to the COVID-19 pandemic are on healthy population or selective groups. We hypothesised that knowledge gap regarding COVID-appropriate behaviour (face-mask use technique and hand hygiene) was responsible for the spread of COVID-19 infection. The participants of our study were unique in the sense that they were already afflicted with COVID-19 infection before getting enrolled in the study. We conducted an online questionnaire-based survey among the COVID-19 positive patients admitted at the district COVID Care Centre at Vidisha, Madhya Pradesh, India to study the KAP of COVID-appropriate behaviour of individuals already afflicted with COVID-19. Two-hundred COVID-19 positive patients were approached, out of which 175 consented and participated in the survey, a response rate of >85.0%. The average knowledge score was 3.21±1.85 (out of 5). The average attitude score was 9.51±4.94 (out of 35). The average practice score was 12.4 (out of 72). Knowledge, attitude as well as practice scores were higher for the participants who were young (18 to 37 years of age), had higher education (university) and those with higher monthly income (>?10,000 per month). No significant difference was noted in these scores based on gender, and on the place of residence (rural vs. urban). Positive correlation was noted using Spearman's rank correlation coefficient for the practice of COVID-appropriate behaviour with higher knowledge and attitude scores. Overall, the KAP scores of our study participants were poor. Low knowledge scores were associated with still lower attitude scores for COVID-appropriate behaviour. The strong positive correlation was noted between knowledge, attitude and practice. The results of this KAP survey suggest the need to improve dissemination of knowledge and suitable modification of messaging strategies to improve attitude as well as practice of COVID-appropriate behaviour among the population.

Structure-activity relationship studies in a new series of 2-amino-N-phenylacetamide inhibitors of Slack potassium channels.

In this Letter we describe structure-activity relationship (SAR) studies conducted in five distinct regions of a new 2-amino-N-phenylacetamides series of Slack potassium channel inhibitors exemplified by recently disclosed high-throughput screening (HTS) hit VU0606170 (4). New analogs were screened in a thallium (Tl+) flux assay in HEK-293 cells stably expressing wild-type human (WT) Slack. Selected analogs were screened in Tl+ flux versus A934T Slack and other Slo family members Slick and Maxi-K and evaluated in whole-cell electrophysiology (EP) assays using an automated patch clamp system. Results revealed the series to have flat SAR with significant structural modifications resulting in a loss of Slack activity. More minor changes led to compounds with Slack activity and Slo family selectivity similar to the HTS hit.

Highly Selective Y4 Receptor Antagonist Binds in an Allosteric Binding Pocket.

Human neuropeptide Y receptors (Y1R, Y2R, Y4R, and Y5R) belong to the superfamily of G protein-coupled receptors and play an important role in the regulation of food intake and energy metabolism. We identified and characterized the first selective Y4R allosteric antagonist (S)-VU0637120, an important step toward validating Y receptors as therapeutic targets for metabolic diseases. To obtain insight into the antagonistic mechanism of (S)-VU0637120, we conducted a variety of in vitro, ex vivo, and in silico studies. These studies revealed that (S)-VU0637120 selectively inhibits native Y4R function and binds in an allosteric site located below the binding pocket of the endogenous ligand pancreatic polypeptide in the core of the Y4R transmembrane domains. Taken together, our studies provide a first-of-its-kind tool for probing Y4R function and improve the general understanding of allosteric modulation, ultimately contributing to the rational development of allosteric modulators for peptide-activated G protein-coupled receptors (GPCRs).

VU0606170, a Selective Slack Channels Inhibitor, Decreases Calcium Oscillations in Cultured Cortical Neurons.

Malignant migrating partial seizures of infancy is a rare, devastating form of epilepsy most commonly associated with gain-of-function mutations in the potassium channel, Slack. Not only is this condition almost completely pharmacoresistant, there are not even selective drug-like tools available to evaluate whether inhibition of these overactivated, mutant Slack channels may represent a viable path forward toward new antiepileptic therapies. Therefore, we used a high-throughput thallium flux assay to screen a drug-like, 100 000-compound library in search of inhibitors of both wild-type and a disease-associated mutant Slack channel. Using this approach, we discovered VU0606170, a selective Slack channel inhibitor with low micromolar potency. Critically, VU0606170 also proved effective at significantly decreasing the firing rate in overexcited, spontaneously firing cortical neuron cultures. Taken together, our data provide compelling evidence that selective inhibition of Slack channel activity can be achieved with small molecules and that inhibition of Slack channel activity in neurons produces efficacy consistent with an antiepileptic effect. Thus, the identification of VU0606170 provides a much-needed tool for advancing our understanding of the role of the Slack channel in normal physiology and disease as well as its potential as a target for therapeutic intervention.

Stress and interferon signalling-mediated apoptosis contributes to pleiotropic anticancer responses induced by targeting NGLY1.

BACKGROUND: Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. METHODS: We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. RESULTS: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. CONCLUSION: Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy.

Iterative Chemical Engineering of Vancomycin Leads to Novel Vancomycin Analogs With a High in Vitro Therapeutic Index.

Vancomycin is a glycopeptide antibiotic that inhibits transpeptidation during cell wall synthesis by binding to the D-Ala-D-Ala termini of lipid II. For long, it has been used as a last resort antibiotic. However, since the emergence of the first vancomycin-resistant enterococci in 1987, vancomycin resistance has become widespread, especially in hospitals. We have synthesized and evaluated 110 vancomycin analogs modified at the C-terminal carboxyl group of the heptapeptide moiety with R2NHR1NH2 substituents. Through iterative optimizations of the substituents, we identified vancomycin analogs that fully restore (or even exceed) the original inhibitory activity against vancomycin-resistant enterococci (VRE), vancomycin-intermediate (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) strains. The best analogs have improved growth inhibitory activity and in vitro therapeutic indices against a broad set of VRE and methicillin-resistant S. aureus (MRSA) isolates. They also exceed the activity of vancomycin against Clostridium difficile ribotypes. Vanc-39 and Vanc-42 have a low probability to provoke antibiotic resistance, and overcome different vancomycin resistance mechanisms (VanA, VanB, and VanC1).

Helicobacter pylori antigenic Lpp20 is a structural homologue of Tipα and promotes epithelial-mesenchymal transition.

BACKGROUND: Helicobacter pylori is a bacterium that affects about 50% of the world population and, despite being often asymptomatic, it is responsible of several gastric diseases, from gastritis to gastric cancer. The protein Lpp20 (HP1456) plays an important role in bacterium survival and host colonization, but the possibility that it might be involved in the etiology of H. pylori-related disorders is an unexplored issue. Lpp20 is a lipoprotein bound to the external membrane of the bacterium, but it is also secreted inside vesicles along with other two proteins of the same operon, i.e. HP1454 and HP1457. RESULTS: In this study we determined the crystal structure of Lpp20 and we found that it has a fold similar to a carcinogenic factor released by H. pylori, namely Tipα. We demonstrate that Lpp20 promotes cell migration and E-cadherin down-regulation in gastric cancer cells, two events recalling the epithelial-mesenchymal transition (EMT) process. Differently from Tipα, Lpp20 also stimulates cell proliferation. CONCLUSIONS: This identifies Lpp20 as a new pathogenic factor produced by H. pylori that promotes EMT and thereby the progression of cancer to the metastatic state.

Evaluation of the antibacterial and antibiofilm activities of novel CRAMP-vancomycin conjugates with diverse linkers.

We report the design, synthesis and antibacterial activity analysis of conjugates of vancomycin and cathelicidin-related antimicrobial peptides (CRAMP). Vancomycin inhibits the nascent peptidoglycan synthesis and is highly active against Gram-positive bacteria, whereas Gram-negative bacteria are generally insensitive due to a protective outer membrane. CRAMP is known to translocate across the Gram-negative outer membrane by a self-promoted uptake mechanism. Vancomycin-CRAMP conjugates were synthesized using click chemistry with diverse hydrophilic and hydrophobic linkers, with CRAMP functioning as a carrier peptide for the transfer of vancomycin through the outer membrane. Small hydrophobic linkers with an aromatic group result in the most active conjugates against planktonic Gram-negative bacteria, while maintaining the high activity of vancomycin against Gram-positive bacteria. These conjugates thus show a broad-spectrum activity, which is absent in CRAMP or vancomycin alone, and which is strongly improved compared to an equimolar mixture of CRAMP and vancomycin. In addition, these conjugates also show a strong inhibitory activity against S. Typhimurium biofilm formation.

New hybrid adaptive neuro-fuzzy algorithms for manipulator control with uncertainties- comparative study.

Control of an industrial robot includes nonlinearities, uncertainties and external perturbations that should be considered in the design of control laws. In this paper, some new hybrid adaptive neuro-fuzzy control algorithms (ANFIS) have been proposed for manipulator control with uncertainties. These hybrid controllers consist of adaptive neuro-fuzzy controllers and conventional controllers. The outputs of these controllers are applied to produce the final actuation signal based on current position and velocity errors. Numerical simulation using the dynamic model of six DOF puma robot arm with uncertainties shows the effectiveness of the approach in trajectory tracking problems. Performance indices of RMS error, maximum error are used for comparison. It is observed that the hybrid adaptive neuro-fuzzy controllers perform better than only conventional/adaptive controllers and in particular hybrid controller structure consisting of adaptive neuro-fuzzy controller and critically damped inverse dynamics controller.

Transcatheter closure of congenital muscular ventricular septal defect.

The success with occlusion devices for the closure of atrial septal defects and patent ductus arteriosus prompted the transcatheter closure of single and multiple muscular ventricular septal defects (VSD). The procedure for VSD was first attempted by Lock et al. in 1988 and devices originally designed for the closure of other intracardiac defects (Rashkind umbrella device, Lock clamshell, Cardioseal, coils, Sideris buttoned device etc.) were used with a variable success rate and a residual shunt. Recently, specially designed Amplatzer muscular VSD occluder and Sideris device are in use. The Amplatzer muscular VSD occluder has been undergoing clinical trial since 1998 after the animal experiments had shown 100% occlusion and complete endothelization at 3 months. The procedure was first attempted in August 1995 using the Rashkind umbrella device and since April 1998 only the Amplatzer muscular VSD occluder has been used. Of the149 patients who underwent transcatheter closure of VSD, 50 had muscular trabecular defects in various locations: mid-muscular, anterior, posterior, or apical. All cases were selected by detailed transthoracic and/or transesophageal echocardiography (TTE) and aneurysm of the muscular septum was observed in three of them. The age range was 3-28 years and the diameter of VSD was 4-11 mm. In all but one patient, the device was deployed from the venous side. Simultaneous TTE was done for proper positioning of the device and continuous electrocardiographic monitoring was also done for any arrhythmia/conduction defects. All patients were followed up every 3 months and received 3-5 mg/kg aspirin for 6 months. The procedure was successful in all patients. The Rashkind umbrella device (17 mm) was used in two and Amplatzer muscular VSD occluder (6-14 mm) in 48 patients. Forty-four devices were delivered by antegrade transvenous approach and six by the transjugular route. None had residual shunt, new aortic regurgitation, or tricuspid regurgitation. Transient complete heart block after 24 hours was noticed in one patient. On a follow-up of 2-90 months, the device was in position in all patients. There was no embolization of the device, and no late-conduction defects, infective endocarditis, or hemolysis. Transcatheter closure of muscular VSD is safe and efficacious, and should be considered as a procedure of choice as an alternative to surgery that avoids cardiopulmonary bypass.

Comparison of the effects of a cell saver and low-dose aprotinin on blood loss and homologous blood usage in patients undergoing valve surgery.

OBJECTIVE: To compare 2 important techniques of blood conservation, use of a cell saver and low-dose aprotinin, in terms of blood loss and homologous blood usage in patients undergoing cardiac valve surgery. DESIGN: Prospective, randomized. SETTING: Tertiary care hospital. PARTICIPANTS: Sixty adult patients undergoing elective valve surgery. INTERVENTIONS: The patients were divided into 3 groups of 20 each. In group 1, aprotinin in the dose of 30,000 KIU/kg was added to the pump prime, with a further dose of 15,000 KIU/kg added at the end of each hour of cardiopulmonary bypass. In group 2, a cell-saver system was used to collect all blood at the operation site for processing in preparation for subsequent reinfusion. Group 3 patients acted as a control group and underwent routine management, which included collection of autologous blood during the pre-cardiopulmonary bypass period. A hemoglobin of <8 g/dL was considered as an indication for bank blood transfusion in the postoperative period. MEASUREMENTS AND MAIN RESULTS: The chest tube drainage was significantly less in group 1 compared with groups 2 and 3, with total drainage (median [interquartile range]) amounting to 250 mL [105 to 325 mL] vs. 700 mL [525 to 910 mL] in group 2 and 800 mL [650 to 880 mL] in group 3 (p < 0.001). The patients in groups 1 and 2 required significantly less bank blood (median [interquartile range]) as compared with group 3 (350 mL [0 to 525 mL], 350 mL [0 to 350 mL], and 1050 mL [875 to 1050 mL]; p < 0.001), respectively. Cell saver provided 410 +/- 130 mL of hemoconcentrated blood in group 2. The average preoperative hemoglobin concentration was 11.3 g/dL, and it was around 9 g/dL on the 7th postoperative day. The hemoglobin concentration at various stages during hospitalization in all 3 groups was similar. CONCLUSIONS: Low-dose aprotinin and a cell saver are effective and comparable methods of blood conservation. Aprotinin helps by decreasing the postoperative drainage, and a cell saver helps by making the patient's own blood available for transfusion.

Mitral regurgitation following percutaneous transvenous mitral commissurotomy: a single-center experience.

BACKGROUND AND AIM OF THE STUDY: Percutaneous transvenous mitral commissurotomy (PTMC) has revolutionized the treatment of patients with symptomatic mitral stenosis and is now established as the procedure of choice. Despite high technical expertise in PTMC using the Inoue balloon, mitral regurgitation (MR) remains a major procedure-related complication. We retrospectively analyzed our data of PTMC using the Inoue balloon with regard to the incidence of MR, its likely causative mechanism, and follow up of these patients. METHODS: During the past ten years, PTMC was performed in 3,650 patients (median age 26 years; range: 8-76 years), of whom 910 (24.9%) were juveniles. Preprocedure mitral valve area (MVA) was 0.9 +/- 0.4 cm2 (range: 0.3-1.3 cm2); MR was mild in 1,396 cases (38.2%), moderate in 394 (10.8%) and severe in 22 (0.6%). None of the patients was rejected on the basis of echocardiographic score. RESULTS: The procedure was successful in 3,276 (89.8%), with post-procedure MVA of 1.7 +/- 0.6 cm2 (range: 1.4-2.6 cm2), and without development of any major complication. Severe MR was seen in 120 patients (3.3%), of whom 66 (1.8%) required urgent mitral valve replacement (MVR). Echocardiography in these latter patients showed leaflet rupture in 48 (72.7%), chordal rupture in 12 (18.2%) and excessive commissural tear in six (9.1%). Fifty-four patients (1.5%) with severe MR post PTMC were followed with medical treatment; echocardiography in these patients revealed chordal rupture in 40 (74.1%) and excessive commissural tear in 14 (25.9%). Follow up data were available in 49 patients (1.3%); 30 (0.8%) required MVR and 19 (0.5%) were in NYHA class II at a median follow up of 24 months. Moderate MR was seen in 188 cases (5.1%), with predominant causative mechanisms of excessive commissural tear in 120 (63.8%) and chordal rupture in 68 (36.2%). Severity of MR worsened in 30 cases (0.8%), of which 20 (0.6%) required elective MVR on follow up. MR decreased in 58 patients (1.6%), in whom excessive commissural tear was the causative mechanism. CONCLUSION: Significant MR (moderate or severe) after PTMC was seen in 308 patients (8.4%), of whom 116 (3.2%) required MVR urgently or on follow up. All patients with leaflet rupture during PTMC developed severe MR and required urgent MVR. There was a tendency for the severity of MR to decrease with time in cases where excessive commissural tear was the causative mechanism.

Transcatheter closure of atrial septal defect using self-expandable septal occluder.

Transcatheter closure of atrial septal defect is an accepted alternative to surgical closure. It was attempted in 63 patients (age range 1.5-55 years) using self-expandable Amplatzer septal occluder (AGA Med. Co., USA). The atrial septal anatomy was evaluated by transthoracic and multiplane transoesophageal echocardiography with special reference to septal margins and adjacent structures. The size of atrial septal defect on echocardiographic evaluation varied from 9-28 (17.5 +/- 4.7) mm. Fifty (79.4%) patients had adequate septal margins of 5 mm or larger, while remaining 13 (20.6%) had insufficient anterosuperior margin. Cardiac catheterisation revealed Qp/Qs ranging from 1.5 to 5.3 and balloon-stretched atrial septal defect diameter of 10-32 (20.3 +/- 5.3) mm. The procedure was overall successful in 62 (98.4%) patients and in all patients with insufficient anterosuperior margin. Embolisation of the device occurred in one (1.6%) patient within five minutes of the device release, which could not be retrieved non-surgically. Size of the device used was either same or preferably 1-3 mm more than the balloon-stretched atrial septal defect diameter. Total procedure time was 40-90 (59 +/- 12.4) minutes and the fluoroscopy time was 12-30 (17.3 +/- 4.2) minutes. Immediate post-procedure and pre-discharge echocardiography in patients with successful deployment of the device revealed complete abolition of shunt in 61 (98.4%) and trivial residual shunt in one (1.6%) patient. No patient developed atrioventricular valve regurgitation or cardiac arrhythmias. Thus, atrial septal defect closure using self-expandable septal occluder is a safe and efficacious procedure requiring a short procedural time. There is full control in the system for proper positioning or repositioning of the device with excellent technical success rate even in cases with insufficient anterosuperior septal margin.

Calcified subaortic left ventricular aneurysm causing right pulmonary artery obstruction in a child.

A 14-year old asymptomatic boy was diagnosed as having a large calcified subaortic left ventricular aneurysm that was found to obstruct flow across the right pulmonary artery during systole. Surgical patch closure of the neck of the aneurysm resulted in relief of obstruction. The case is reported for its rarity, massive calcification, absence of aortic regurgitation and dynamic compression of the right pulmonary artery.