Discovery of Anti-NRLP3 Inflammasome, Immunomodulatory Phytochemicals from the Extract of Habenaria intermediaD. Don: An Unexplored Plant Species.

The present study describes the isolation, identification, and quantification of biomarker compounds in plant extracts of Habenaria intermedia D. Don (Orchidaceae). The isolation of the compounds was carried out from H. intermedia D. Don by repeated column chromatography of petroleum ether and ethanol fractions of extract of tubers. These compounds were characterized by 1H and 13C NMR and mass spectral data. A new quantitative method was established by using high-performance liquid chromatography (HPLC)-PDA. As a result, seven compounds were isolated and characterized. This is the first report of isolation of these compounds from this plant species H. intermedia D.Don. Out of seven isolated compounds, five were used for the quantitative study. A reliable and suitable HPLC method was developed for the well-resolved chromatogram of compounds. The proposed method was applied successfully to the detection and quantification of compounds. This study also represents the immunomodulatory and anti-inflammasome biological studies of isolated natural products. Loroglossol (HBR-4) has been reported to possess immunomodulatory activity. The immunostimulating assay indicated that HBR-4 could significantly promote the cell proliferation, especially via IL-2, TNF-α, and IFN-γ secretion from spleen cells. These results suggested the potential utilization of HBR-4 as an attractive functional health supplement candidate for hypoimmunity population. Additionally, cyclophosphamide-induced immunosuppression was counteracted by treatment with HBR-4, revealing significant increase in hemagglutinating antibody responses and hemolytic antibody responses. The current work revealed the potential anti-inflammasome and immunomodulatory activities of H. intermedia D. Don compounds and validates the usage of this prominent Rasayna plant.

Flurbiprofen inhibits heme induced NLRP3 inflammasome in Berkeley sickle cell disease mice.

Sickle cell disease (SCD) is accompanied by several complications, which emanate from the sickling of erythrocytes due to a point mutation in the ß-globin chain of hemoglobin. Sickled erythrocytes are unable to move smoothly through small blood capillaries and therefore, cause vaso occlusion and severe pain. Apart from pain, continuous lysis of fragile sickled erythrocytes leads to the release of heme, which is a strong activator of the NLRP3 inflammasome, thus producing chronic inflammation in sickle cell disease. In this study, we identified flurbiprofen among other COX-2 inhibitors to be a potent inhibitor of heme-induced NLRP3 inflammasome. We found that apart from being a nociceptive agent, flurbiprofen exerts a strong anti-inflammatory effect by suppressing NF-κB signaling, which was evidenced by reduced levels of TNF-α and IL-6 in wild-type and sickle cell disease Berkeley mice models. Our data further demonstrated the protective effect of flurbiprofen on liver, lungs, and spleen in Berkeley mice. The current sickle cell disease pain management regime relies mainly on opiate drugs, which is accompanied by several side effects without modifying the sickle cell disease-related pathology. Considering the potent role of flurbiprofen in inhibiting NLRP3 inflammasome and other inflammatory cytokines in sickle cell disease, our data suggests that it can be explored further for better sickle cell disease pain management along with the possibility of disease modification.

Methoxy-naphthyl-Linked N-Benzyl Pyridinium Styryls as Dual Cholinesterase Inhibitors: Design, Synthesis, Biological Evaluation, and Structure-Activity Relationship.

The multifaceted nature of Alzheimer's disease (AD) indicates the need for multitargeted agents as potential therapeutics. Both cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), play a vital role in disease progression. Thus, inhibiting both ChEs is more beneficial than only one for effectively managing AD. The present study provides a detailed lead optimization of the e-pharmacophore-generated pyridinium styryl scaffold to discover a dual ChE inhibitor. A structure-activity relationship analysis indicated the importance of three structural fragments, methoxy-naphthyl, vinyl-pyridinium, and substituted-benzyl, in a dual ChE inhibitor pharmacophore. The optimized 6-methoxy-naphthyl derivative, 7av (SB-1436), inhibits EeAChE and eqBChE with IC50 values of 176 and 370 nM, respectively. The kinetic study has shown that 7av inhibits AChE and BChE in a non-competitive manner with ki values of 46 and 115 nM, respectively. The docking and molecular dynamics simulation demonstrated that 7av binds with the catalytic and peripheral anionic sites of AChE and BChE. Compound 7av also significantly stops the self-aggregation of Aß. The data presented herein indicate the potential of 7av for further investigation in preclinical models of AD.

Piperine analog PGP-41 treatment overcomes paclitaxel resistance in NCI/ADR-RES ovarian cells by inhibition of MDR1.

Chemoresistance is one of the leading causes of the failure of chemotherapy. Overexpression of P-glycoprotein (P-gp) in cancer cells is one of the most important contributing factors toward the development of chemoresistance. This study was designed to synthesize the derivatives of dihydronaphthyl and to evaluate the P-gp inhibition activity of these compounds. Among all the compounds, PGP-41 showed the most potent P-gp inhibition activity in colorectal adenocarcinoma LS-180 cells. This compound showed potent P-gp inhibition activity in chemoresistant ovarian cell line NCI/ADR-RES. Paclitaxel is one of the first lines of drugs for treating ovarian cancer and is a substrate of P-gp; therefore, NCI/ADR-RES cells are highly resistant to treatment with paclitaxel. Based on this information, we evaluated PGP-41 to overcome the paclitaxel resistance of NCI/ADR-RES cells. PGP-41 was able to sensitize the NCI/ADR-RES cells to the treatment of paclitaxel, which was evident by the reduced IC50 value of paclitaxel from 6.64 µM to 0.12 µM. The sensitization of NCI/ADR-RES cells by PGP-41 was comparable to that of elacridar and Zosuquidar. Further studies revealed that the PGP-41 exerts its effect by downregulating the expression of P-gp. Reduction of P-gp activity leads to the accumulation of higher intracellular concentration of paclitaxel, and thus allowing it to interact with its targets, which further helps in its increased efficacy. Paclitaxel was able to arrest the sensitized NCI/ADR-RES cells into G2M phase, which ultimately led to the induction of apoptotic proteins and the death of cancer cells. Being a different scaffold from zosuquidar and elacridar, further studies are required to develop PGP-41 into a potential drug to overcome chemoresistance in cancer cells.

Delayed KCNQ1/KCNE1 assembly on the cell surface helps IKs fulfil its function as a repolarization reserve in the heart.

KEY POINTS: In adult ventricular myocytes, the slow delayed rectifier (IKs ) channels are distributed on the surface sarcolemma, not t-tubules. In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools. KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into IKs channels on the cell surface. Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface. An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer. ABSTRACT: Slow delayed rectifier (IKs ) channels consist of KCNQ1 and KCNE1. IKs functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during ß-adrenergic stimulation. There has been much debate about how KCNQ1 and KCNE1 traffic in cells, where they associate to form IKs channels, and the distribution pattern of IKs channels relative to ß-adrenergic signalling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or IKs , to provide new insights into these issues. 'Retention-using-selected-hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulphide-bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. An in situ proximity ligation assay detected IKs channels in surface sarcolemma but not t-tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein-tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that, in cardiomyocytes, KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into IKs channels. This mode of delayed channel assembly helps IKs fulfil its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus-end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer.

Gender determination by morphometric analysis of mandibular ramus in sriganganagar population: A digital panoramic study.

BACKGROUND: Mandible is a dimorphic, dense compact bone that makes it very durable and well preserved in mass disasters for personnel identification. Mandibular ramus morphometric measurements can be used for gender determination using orthopantomogram (OPG) or on dry mandibles. AIM: To determine gender from morphometric analysis of mandibular ramus of 200 digital OPG of patients from Sriganganagar population. MATERIALS AND METHODS: The study was conducted on randomly selected digital OPG of 200 patients of both genders between the ages of 21 and 70 years taken using CS8000C machine from daily OPD. Morphometric analysis of mandibular ramus (maximum ramus breadth, minimum ramus breadth, condylar height, projective height of ramus, and coronoid height) was done twice by single maxillofacial radiologist independently at an interval of 1 day and mean of both the values were considered. The collected data was tabulated and analyzed using SPSS Software version 20 using independent t-test and discriminant function analysis. RESULTS: Out of total 200 subjects, 37% were male and 63% were female. Mean of minimum ramus breadth, maximum ramus height, and projected ramus height was noted significantly more among males while maximum ramus breadth was noted slightly higher in females. The overall accuracy for determining sex from mandibular ramus was found to be 77.6%, whereas for determining male and female, the accuracy was 78.4% and 76.8%, respectively. CONCLUSION: Mandibular ramus can be used for sexual dimorphism by morphometric analysis done on OPG among Sriganganagar population.

Evaluation of mandibular anterior nutrient canals in hypertensive and diabetes mellitus patients: A comparative radiographic study.

OBJECTIVE: Radiographic evaluation of nutrient canals (NCs) in the mandibular anterior region using intraoral periapical radiographs (IOPARs) and to determine whether they can be used as a potential marker for hypertension (HT) and diabetes mellitus (DM). MATERIALS AND METHODS: Randomly selected 600 patients of 21-60 years age group (Group I: 200 HT; Group II: 200 DM; Group III: Healthy subjects) were considered. The case history was recorded including details for DM and HT (duration, type, and medication). Blood pressure was measured, followed by blood examination for blood sugar levels. Selected patients were subjected to IOPARs using CS-2100C machine by the paralleling technique. Selected radiographs were evaluated by two observers independently for the presence/absence, number, and location of NCs between #33 and #43. Data obtained were tabulated and subjected to statistical analysis using SPSS 20.0 statistical software and intergroup reliability was checked using Cohen's kappa test. RESULTS: Evaluation of various parameters of NCs showed an insignificant interobserver bias. The incidence of NCs presence was noted maximum in Group II (93.5%), followed by Group I (88.5%) and III (44.5%). Of total 888 NCs found, maximum were found in Group I, followed by II and III. On comparing the incidence of NCs present among both genders and location in study groups, no statistical correlation was found. CONCLUSION: Statistically significant increase in the incidence and number of NCs in Group I and II compared to controls can act as an adjunct diagnostic marker for the detection of DM and HT; although, no significant correlation was obtained between gender and location of NCs in different study groups. Furthermore, there was no significant correlation was found between the severity of disease and incidence of the presence of NCs.

Evaluation of sexual dimorphism using permanent maxillary first molar in Sri Ganganagar population.

AIM OF STUDY: The aim is to evaluate existence of sexual dimorphism by variation in right and left permanent maxillary molars using buccolingual width (BLW) and mesio-distal width (MDW) measured intraorally and on study casts among Sri Ganganagar population. MATERIALS AND METHODS: Fifty patients (25 males and 25 females) with 17-25 years of age were selected. Impressions of maxillary arch were taken and the BLW and MDW were measured using digital Vernier calipers on study casts and intraorally. RESULTS: Highly significant correlation was found between MDW and BLW of both the maxillary permanent first molars for both genders (P < 0.05) intraorally. The MDW and BLW on study cast of both sides in both gender were more on left side in males while on right side in females. CONCLUSION: Left maxillary permanent first molar showed minimum mean difference of measurements on study cast and introrally than right, thus better predictor for gender dimorphism in forensics.