LRRK2 dynamics analysis identifies allosteric control of the crosstalk between its catalytic domains.

The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

Role of the leucine-rich repeat protein kinase 2 C-terminal tail in domain cross-talk.

Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Preclinical evaluation of ADVM-062, a novel intravitreal gene therapy vector for the treatment of blue cone monochromacy.

Blue cone monochromacy (BCM) is a rare X-linked retinal disease characterized by the absence of L- and M-opsin in cone photoreceptors, considered a potential gene therapy candidate. However, most experimental ocular gene therapies utilize subretinal vector injection which would pose a risk to the fragile central retinal structure of BCM patients. Here we describe the use of ADVM-062, a vector optimized for cone-specific expression of human L-opsin and administered using a single intravitreal (IVT) injection. Pharmacological activity of ADVM-062 was established in gerbils, whose cone-rich retina naturally lacks L-opsin. A single IVT administration dose of ADVM-062 effectively transduced gerbil cone photoreceptors and produced a de novo response to long-wavelength stimuli. To identify potential first-in-human doses we evaluated ADVM-062 in non-human primates. Cone-specific expression of ADVM-062 in primates was confirmed using ADVM-062.myc, a vector engineered with the same regulatory elements as ADVM-062. Enumeration of human OPN1LW.myc-positive cones demonstrated that doses ≥3 × 1010 vg/eye resulted in transduction of 18%-85% of foveal cones. A Good Laboratory Practice (GLP) toxicology study established that IVT administration of ADVM-062 was well tolerated at doses that could potentially achieve clinically meaningful effect, thus supporting the potential of ADVM-062 as a one-time IVT gene therapy for BCM.

Capturing the domain crosstalk in full length LRRK2 and LRRK2RCKW.

LRRK2 is a multi-domain protein with three catalytically inert N-terminal domains (NtDs) and four C-terminal domains, including a kinase and a GTPase domain. LRRK2 mutations are linked to Parkinson's Disease. Recent structures of LRRK2RCKW and a full-length inactive LRRK2 (fl-LRRK2INACT) monomer revealed that the kinase domain drives LRRK2 activation. The LRR domain and also an ordered LRR- COR linker, wrap around the C-lobe of the kinase domain and sterically block the substrate binding surface in fl-LRRK2INACT. Here we focus on the crosstalk between domains. Our biochemical studies of GTPase and kinase activities of fl-LRRK2 and LRRK2RCKW reveal how mutations influence this crosstalk differently depending on the domain borders investigated. Furthermore, we demonstrate that removing the NtDs leads to altered intramolecular regulation. To further investigate the crosstalk, we used Hydrogen-Deuterium exchange Mass Spectrometry (HDX-MS) to characterize the conformation of LRRK2RCKW   and Gaussian Accelerated Molecular Dynamics (GaMD) to create dynamic portraits of fl-LRRK2 and LRRK2RCKW. These models allowed us to investigate the dynamic changes in wild type and mutant LRRK2s. Our data show that the a3ROC helix, the Switch II motif in the ROC domain, and the LRR-ROC linker play crucial roles in mediating local and global conformational changes. We demonstrate how these regions are affected by other domains in fl-LRRK2 and LRRK2RCKW and show how unleashing of the NtDs as well as PD mutations lead to changes in conformation and dynamics of the ROC and kinase domains which ultimately impact kinase and GTPase activities. These allosteric sites are potential therapeutic targets.

Dietary flavonoids-rich Citrus reticulata peel extract interacts with CREB signaling to suppress seizures and linked neurobehavioral impairments in a kindling mouse model.

Objectives: The citrus fruits peel contains a variety of bioactive metabolites that have shown multiple therapeutic effects. However, despite having substantial ethnomedicinal value, citrus peels remained underexplored and regarded as bio-waste. This present study was planned to investigate the effect of a characterized peel extract of Citrus reticulata c.v. (CRE) in pentylenetetrazole (PTZ)-induced kindling and associated cognitive and behavioral impairments in a mouse model.Methods: The kindled animals were treated daily with CRE (100 and 200 mg/kg) and challenged with a sub-effective dose of PTZ every 5th day to record the severity of seizures. In the end, different tests were performed to record behavioral and cognitive performance.Results: CRE-treated kindled animals showed a significant suppression in seizure severity following 20 days of the treatment. In the T-maze test, the extract treatment resulted in a marked increase in the spontaneous alternations, whereas it showed no change in anxiety behavior of kindled animals in the elevated plus-maze test. In both forced swim and tail suspension tests, CRE treatment demonstrated a considerable reduction in immobility time. However, no change in overall locomotion was observed in the open field test among all the groups. An increase in the hippocampal Creb and Bdnf expression and decreased glutamate-to-GABA ratio were observed in the CRE-treated kindled animals.Discussion: The results showed that CRE treatment suppresses epileptic seizures and associated cognitive deficits and depression-like behavior in kindled mice. The gene expression findings supported that the observed protective effects of CRE be due to its interaction with CREB signaling.

Do Interviews Really Matter in Generating Programs and Applicants' Rank Lists for the Match?

OBJECTIVE: A paucity of data exists on the role of the interview day in programs and applicants' final rank list. The objective of our study was to investigate the impact interview day has on our programs and our interviewees' final rank list. METHODS: For the 2020 appointment year, our program used an Electronic Residency Application System Application Scoring Tool and Interview Scoring Tool to generate the preliminary rank list for our pulmonary and critical care fellowship applicants. The final rank list was decided after interviewers' discussion during the program's rank list meeting. We aimed to correlate the preliminary and final lists. We also surveyed applicants on the importance of interview day in generating their rank list. RESULTS: The final and the preliminary rank lists were strongly correlated (rs(47) = 0.87, P < 0.001). There was a stronger correlation between the final rank and the rank based on the application score (rs(47) = 0.84, P < 0.001) than the rank based on the interview score (rs(47) = 0.64, P < 0.001). For the postinterview survey, 48 applicants were surveyed-20 replied with a response rate of 42% and 18 respondents (90%) rated the interview experience as important or very important in their rank list decisions. CONCLUSIONS: The programs rank list correlated more with the candidates' written application than their interview day performance; however, interview experience greatly influenced the applicants' rank lists. In the coronavirus disease 2019 pandemic, in which all interviews are virtual, programs should make diligent efforts to construct virtual interview days, given their importance to applicants in generating their final rank list for the match.

Combatting intracellular pathogens using bacteriophage delivery.

Intracellular pathogens reside in specialised compartments within the host cells restricting the access of antibiotics. Insufficient intracellular delivery of antibiotics along with several other resistance mechanisms weaken the efficacy of current therapies. An alternative to antibiotic therapy could be bacteriophage (phage) therapy. Although phage therapy has been in practice for a century against various bacterial infections, the efficacy of phages against intracellular bacteria is still being explored. In this review, we will discuss the advancement and challenges in phage therapy, particularly against intracellular bacterial pathogens. Finally, we will highlight the uptake mechanisms and approaches to overcome the challenges to phage therapy against intracellular bacteria.

Synthesis of Biologically Active Heterospirocycles through Iterative 1,3-Dipolar Cycloaddition Pathways.

We demonstrate the novel spiroannulation of exo-imines with 1,3-dipoles, for the first time, leading to 3D spirocycles with a secondary amine (NH) in the spiro-ring. The synthetic method described herein allows access to these previously unexplored heterospirocyclic cores that have application in the discovery of functional molecules for medicinal and materials science. This was demonstrated by discovering an unprecedented class of heterospirocycles with antimalarial activity against the human protozoan P. falciparum.

Apigenin reverses behavioural impairments and cognitive decline in kindled mice via CREB-BDNF upregulation in the hippocampus.

Objectives: Apigenin is the most common bioflavonoid known to be biologically active after systemic administration and show multiple pharmacological effects. The present study was designed to explore the role of apigenin in pentylenetetrazole (PTZ) kindling associated cognitive and behavioural impairments in the mouse model. Methods: The animals were kindled by injecting a sub-convulsive dose of PTZ (35 mg/kg) on every alternate day, followed by 20 days treatment with apigenin at two different doses (10 and 20 mg/kg). Seizure severity was assessed on every 5th, 10th, 15th, and 20th day during apigenin treatment after a PTZ injection, followed by analysis of cognitive and behavioural functions. Results: Apigenin treatment displayed insignificant effect on seizure severity in kindled mice at both the tested doses, in comparison to control. However, the treatment showed marked increase in per cent spontaneous alterations and decline in the anxiety index in T-maze and elevated plus maze tests, respectively. Apigenin-treated groups showed significant decrease in immobility period in both forced swim and tail suspension tests, without any change in the total locomotor activity in the open field test. Furthermore, increase in the hippocampus protein expression of brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), and phosphorylated CREB, with increased serotonin level were also observed in the treated animals. Discussion: The results of the present study showed that apigenin treatment prevents cognitive deficit and reverses behaviour impairments, without altering seizures severity in kindled mice. The observed effects can be attributed to CREB-BDNF upregulation in the hippocampus.

Preclinical Evaluation of ADVM-022, a Novel Gene Therapy Approach to Treating Wet Age-Related Macular Degeneration.

Inhibition of vascular endothelial growth factor, a key contributor to the choroidal neovascularization associated with wet age-related macular degeneration, is the mode of action of several approved therapies, including aflibercept, which requires frequent intravitreal injections to provide clinical benefit. Lack of compliance with the dosing schedule may result in recurrence of active wet macular degeneration, leading to irreversible vision impairment. Gene therapy providing sustained anti-vascular endothelial growth factor levels in the retina following a single injection could drastically reduce the treatment burden and improve visual outcomes. ADVM-022, an adeno-associated virus vector encoding aflibercept, is optimized for intravitreal delivery and strong protein expression. Here, we report the long-term expression and efficacy of ADVM-022-derived aflibercept in a laser-induced choroidal neovascularization model in non-human primates. Intravitreal administration of ADVM-022 was well tolerated and resulted in sustained aflibercept levels. In addition, ADVM-022 administration 13 months before lasering prevented the occurrence of clinically relevant choroidal neovascularization lesions, similar to animals that received a bolus of intravitreal aflibercept (standard of care) at the time of lesioning. These results demonstrate that a single intravitreal administration of ADVM-022 may provide a safe and effective long-term treatment option for wet macular degeneration and may ultimately improve patients' visual outcomes.

Renal Hemangioblastoma with Mixed Mullerian tumour of endometrium: A tale of two rare primary tumours.

INTRODUCTION: Renal hemangioblastoma (RH) is a very rare benign tumour. Hemangioblastoma most commonly occurs in the central nervous system (CNS), and only few cases of RH have been reported as they occur most commonly as asymptomatic masses found incidentally. Mixed Mullerian tumour (MMT) of the uterus is a rarer and aggressive form of uterine malignancy. The detection of two primary rare tumours incidentally is a rare entity. CASE PRESENTATION: A 50-year-old female presented with abnormal uterine bleeding which on endometrial sampling was diagnosed as a rare variety of endometrial cancer, i.e. MMT or uterine carcinosarcoma. On preoperative imaging, a renal mass was also detected which was highly vascular and was mimicking renal cell carcinoma (RCC). Fine needle aspiration cytology (FNAC) was done from the renal mass to differentiate between RCC and metastasis, but it showed only blood cells. Patient underwent staging laparotomy for endometrial cancer and frozen section examination of the renal mass which was inconclusive with few atypical cells, and thus, patient underwent radical nephrectomy too. Histopathological examination revealed it to be a RH which is a very rare benign tumour. DISCUSSION: RH is a rare benign tumour which does not require any treatment in majority of the patients. Only 26 cases of RH outside the CNS have been reported till date. MMT is a rare aggressive uterine tumour with an incidence of 1-2 % of all uterine neoplasms, which metastasizes early, and thus, early identification and treatment is the key. RH needs to be differentiated from RCC to avoid over treatment. Morphological findings are similar in RCC and RH; preoperative FNAC, PET scan, and intraoperative frozen section can be utilized to differentiate the two, in well-circumcised tumours and high index of suspicion. Occurrence of renal mass as an incidental finding in the preoperative work up of uterine malignancy directed us to the differentials of metastasis or another histologically distinct primary tumour. The presence of two rare primary tumours, i.e. RH and MMT in the same patient which are unrelated, is a rare entity.

Effect of γ-radiation on post-harvest storage life and quality of onion bulb under ambient condition.

Different doses of gamma Irradiation (0 Gy, 10 Gy, 60 Gy, 100 Gy and 200 Gy) were evaluated as potential treatment to extend the storage period and maintain the quality attributes of onion bulbs (cv. Punjab Noraya) at ambient temperature for a period of 144 days. It was observed that storage parameters such as physiological loss in weight, sprouting percentage, rotting percentage and quality traits like total soluble solids, firmness, color parameters, ascorbic acid and pyruvic acid varied significantly (p < 0.05) during storage period with respect to doses of irradiation. No rotting and sprouting were observed upto 24 days and 84 days, respectively in both gamma irradiated and un-irradiated bulbs. At 5 months of storage, the physiological loss in weight varied from 28.5 to 63.6% in all treatments. Physiological weight loss and rotting percentage were higher in the untreated (control) as well as bulbs radiated @ 10 Gy and 200 Gy. Firmness was better retained in the bulbs irradiated with gamma rays @ 120 Gy bulbs upto 84 days of storage. However, no clear-cut pattern for colour changes (L, a, b values) was observed with respect to the irradiation doses. TSS in bulbs decreased upto 36 days of storage and thereafter increased upto 48th day of storage irrespective of the dose of gamma irradiation. Ascorbic acid content of bulbs decreases significantly in all the irradiation treatments during storage but pyruvic acid initially increased, then decreased and again increased at the end of the storage period in un-irradiated and irradiated treatments. It is concluded that onion bulbs irradiated with gamma rays @ 120 Gy resulted in minimum loss in weight, rotting and sprouting while maintained best quality for 3 months of storage at ambient storage conditions.

The Claudin-like Protein HPO-30 Is Required to Maintain LAChRs at the C. elegans Neuromuscular Junction.

Communications across chemical synapses are primarily mediated by neurotransmitters and their postsynaptic receptors. There are diverse molecular systems to localize and regulate the receptors at the synapse. Here, we identify HPO-30, a member of the claudin superfamily of membrane proteins, as a positive regulator for synaptic localization of levamisole-dependent AChRs (LAChRs) at the Caenorhabditis elegans neuromuscular junction (NMJ). The HPO-30 protein localizes at the NMJ and shows genetic and physical association with the LAChR subunits LEV-8, UNC-29, and UNC-38. Using genetic and electrophysiological assays in the hermaphrodite C. elegans, we demonstrate that HPO-30 functions through Neuroligin at the NMJ to maintain postsynaptic LAChR levels at the synapse. Together, this work suggests a novel function for a tight junction protein in maintaining normal receptor levels at the NMJ.SIGNIFICANCE STATEMENT Claudins are a large superfamily of membrane proteins. Their role in maintaining the functional integrity of tight junctions has been widely explored. Our experiments suggest a critical role for the claudin-like protein, HPO-30, in maintaining synaptic levamisole-dependent AChR (LAChR) levels. LAChRs contribute to <20% of the acetylcholine-mediated conductance in adult Caenorhabditis elegans; however, they play a significant functional role in worm locomotion. This study provides a new perspective in the study of LAChR physiology.

Characterizing binding sites of heat responsive microRNAs and their expression pattern in heat stressed PBMCs of native cattle, exotic cattle and riverine buffaloes.

It is generally believed that due to evolutionary differences and adaptation to tropical conditions, Indian native cattle has superior heat tolerant ability than Bos taurus cattle. In the present study, 3'-UTR of two most important heat responsive genes i.e., heat shock protein 70.1 (HSP70.1) and heat shock factor- 1 (HSF-1) were sequence characterized in different breeds of Indian native cattle to identify the variations and miRNA binding sites. In addition, the impact of heat stress was assessed in a total of 57 PBMCs samples of native Sahiwal cows (Bos indicus), exotic Holstein cows (Bos taurus) and Murrah buffaloes (Bubalus bubalis) using various cellular parameters like cell viability, cytotoxicity and apoptosis. Further, expression profile of 12 heat responsive miRNAs were also evaluated in unstressed and stressed PBMCs to understand post transcriptional changes in native cows, exotic cows and Murrah buffaloes. The sequence data showed 3'-UTR of HSP70.1 gene of Indian cattle to be exactly similar to Bos taurus with no miRNA binding site. Whereas, sequencing of 3'-UTR of HSF-1 gene revealed 3 SNPs at positions G1762T; C1811T and C1983T with 7 well conserved miRNA binding sites. The impact of heat stress on various cellular parameters in terms of cell viability, cytotoxicity and apoptosis was highest in PBMCs of Holstein cows followed by Murrah buffaloes and Sahiwal cows. Further, in contrast to Holstein Frisian cows and Murrah buffaloes, the expression pattern of 12 heat responsive miRNAs, in heat stressed PBMCs of Sahiwal cows were quite distinct. There was a significant (p < 0.05) induction in expression of most of the miRNAs after heat stress in PBMCs of Sahiwal cows followed by a rapid decline. The distinct cellular response and pattern of miRNA expression across cattle types and buffaloes might be influencing their PBMCs tolerance level to heat stress.

Single Nucleotide Polymorphisms in B-Genome Specific UDP-Glucosyl Transferases Associated with Fusarium Head Blight Resistance and Reduced Deoxynivalenol Accumulation in Wheat Grain.

An in vitro spike culture method was optimized to evaluate Fusarium head blight (FHB) resistance in wheat (Triticum aestivum) and used to screen a population of ethyl methane sulfonate treated spike culture-derived variants (SCDV). Of the 134 SCDV evaluated, the disease severity score of 47 of the variants was ≤30%. Single nucleotide polymorphisms (SNP) in the UDP-glucosyltransferase (UGT) genes, TaUGT-2B, TaUGT-3B, and TaUGT-EST, differed between AC Nanda (an FHB-susceptible wheat variety) and Sumai-3 (an FHB-resistant wheat cultivar). SNP at 450 and 1,558 bp from the translation initiation site in TaUGT-2B and TaUGT-3B, respectively were negatively correlated with FHB severity in the SCDV population, whereas the SNP in TaUGT-EST was not associated with FHB severity. Fusarium graminearum strain M7-07-1 induced early expression of TaUGT-2B and TaUGT-3B in FHB-resistant SCDV lines, which were associated with deoxynivalenol accumulation and reduced FHB disease progression. At 8 days after inoculation, deoxynivalenol concentration varied from 767 ppm in FHB-resistant variants to 2,576 ppm in FHB-susceptible variants. The FHB-resistant SCDV identified can be used as new sources of FHB resistance in wheat improvement programs.

In vitro Evaluation and Comparison of Microleakage of Two Restorative Composite Resins in Class II Situations using Confocal Laser Scanning Microscopy.

AIM: The placement of composite in teeth is not an easy task and it poses many challenges. Microleakage is one of the factors that affects the success of any composite restoration. It influences the longevity of dental restorations. The present study was aimed to evaluate and compare microleakage of two restorative composites resins in class II cavities using confocal laser scanning microscopy (CLSM). MATERIALS AND METHODS: This was an in vitro study, which included 20 permanent mandibular first molar. On each tooth, 40 class II cavities were prepared with a carbide bur. The teeth were then randomly divided into two groups of 10 each. Group I included teeth in which SonicFill Bulk Fill composite was used. Group II included teeth in which Tetric EvoCeram Bulk Fill composite was used. The microleakage was measured using confocal microscopy at 10* magnification in the fluorescent mode by a scoring system. RESULTS: Estimation was done at cervical and occlusal levels, wherein group I included 10 teeth which were restored with SonicFill Bulk Fill composite and group II included 10 teeth which were restored with Tetric EvoCeram Bulk Fill composite. Microleakage was quantified on scoring basis, which was consecutively based on the dye penetration at different levels. Data thus obtained statistically revealed that microleakage was comparatively more in group II as compared with group I. In addition, it was somewhat similar on both cervical as well as occlusal regions. The difference was significant (p = 0.05). Comparison was also done at cervical and occlusal levels using Wilcoxon signed-rank test, which showed significant levels of differences (p > 0.05). CONCLUSION: There is more microleakage in Tetric EvoCeram Bulk Fill composite as compared with SonicFill Bulk Fill composite. CLINICAL SIGNIFICANCE: No material seems to totally eliminate microleakage in class II situations with gingival margin ended in dentine. However, CLSM is a useful tool in microleakage evaluation that could be used successfully to estimate the severity of microbial penetrations and material of choice as well.

Sulfur-Fluoride Exchange (SuFEx)-Mediated Synthesis of Sterically Hindered and Electron-Deficient Secondary and Tertiary Amides via Acyl Fluoride Intermediates.

Amide bond formation is one of the most executed reactions in chemistry and biology. This is largely due to the ubiquity of the amide functional group in biological molecules, natural products and pharmaceutically important drugs. We report here the development of "SuFExAmide": a new sulfur-fluoride exchange (SuFEx) click chemistry based protocol for the efficient amidation of carboxylic acids via acyl fluoride intermediates. We have developed benzene-1,3-disulfonyl fluoride as a cost effective, powerful and versatile coupling agent, which delivers challenging secondary and tertiary amides in excellent yields from sterically hindered and electron-deficient amines. The straightforward method offers significant benefits over existing protocols in terms of substrate scope, efficiency and ease of operation and is demonstrated by the synthesis of 44 amides, including GNF6702, an antiprotozoal drug candidate. In the majority of cases, the amide products are obtained in high yield without the need for excess reagents or chromatographic purification.

Recent Advances in Cyanamide Chemistry: Synthesis and Applications.

The application of alkyl and aryl substituted cyanamides in synthetic chemistry has diversified multi-fold in recent years. In this review, we discuss recent advances (since 2012) in the chemistry of cyanamides and detail their application in cycloaddition chemistry, aminocyanation reactions, as well as electrophilic cyanide-transfer agents and their unique radical and coordination chemistry.

Horner-Wadsworth-Emmons approach to piperlongumine analogues with potent anti-cancer activity.

Natural products with anti-cancer activity play a vital role in lead and target discovery. We report here the synthesis and biological evaluation of the plant-derived alkaloid, piperlongumine and analogues. Using a Horner-Wadsworth-Emmons coupling approach, a selection of piperlongumine-like compounds were prepared in good overall yield from a novel phosphonoacetamide reagent. A number of the compounds displayed potent anti-cancer activity against colorectal (HCT 116) and ovarian (IGROV-1) carcinoma cell lines, via a mechanism of action which may involve ROS generation. Contrary to previous reports, no selective action in cancer cell (MRC-5) was observed for piperlongumine analogues.

Dwindling of cardio damaging effect of isoproterenol by Punica granatum L. peel extract involve activation of nitric oxide-mediated Nrf2/ARE signaling pathway and apoptosis inhibition.

Punica granatum L. (Punicaceae) peel is often considered as a food waste in-spite of its high bioactive metabolite composition. Primarily it is rich in therapeutically active phenolics that act on multiple cellular sites, through diverse mechanisms. Hence, the present study was envisaged to investigate the effect of standardised peel extract of P. granatum against isoproterenol (ISO)-induced myocardial infarction (MI). ISO administration at a dose of 150 mg/kg; s.c., twice at 24 h interval resulted in electrocardiographic abnormalities with increased heart weight and myocardial tissue damage signifying MI. Pretreatment with the extract at 50, 100 and 200 mg/kg; p.o., for 21 days prior to ISO intoxication (30 min prior to intoxication on day 22 and 23) attenuated the observed changes, along with increased myocardial tissue superoxide dismutase activity, reduced glutathione and nitrite levels, and decreased lipid peroxidation. The extract treated groups also showed reduced serum marker enzymes of MI, showing maximum effect at highest tested dose. Immunohistochemical studies revealed increased myocardial expression of nuclear factor erythroid 2-related factor 2 (Nrf2), endothelial nitric oxide synthase (eNOS) and Bcl-2 proteins in the extract treated groups with decreased Bax expression. From the results it can be concluded that the extract pretreatment prevents ISO-induced MI through increased myocardial expression of eNOS, leading to nitric oxide-mediated Nrf2 activation, thus upregulating antioxidant mechanisms, along with inhibition of apoptosis.