pH-Dependent Structure and Dynamics of the Catalytic Domains of Human Carbonic Anhydrase II and IX.

Extensive computer simulation studies have been carried out to probe the pH-dependent structure and dynamics of the two most efficient isoenzymes II and IX of human carbonic anhydrase (HCA) that control the pH in the human body. The equilibrium structure and hydration of their catalytic domains are found to be largely unaffected by the variation of pH in the range studied, in close agreement with the known experimental results. In contrast, a significant effect of the change in pH is observed for the first time on the local electrostatic potential of the active site walls and the dynamics of active site water molecules. We also report for the first time the free energy and kinetics of coupled fluctuations of orientation and protonation states of the well-known His-mediated proton shuttle (His-64) in both isozymes at pH 7 and 8. The transitions between different tautomers of in or out conformations of His-64 side chain range between 109 and 106 s-1 depending on pH. Possible implications of these results on conformation-dependent pKa of His-64 side chain and its role in driving the catalysis toward hydration of CO2 or dehydration of HCO3- with varying pH are discussed.

A glucuronated flavone TMMG spatially targets chondrocytes to alleviate cartilage degeneration through negative regulation of IL-1ß.

Chondrocytes are the only resident cell types that form the extracellular matrix of cartilage. Inflammation alters the anabolic and catabolic regulation of chondrocytes, resulting in the progression of osteoarthritis (OA). The potential of TMMG, a glucuronated flavone, was explored against the pathophysiology of OA in both in vitro and in vivo models. The effects of TMMG were evaluated on chondrocytes and the ATDC5 cell line treated with IL-1ß in an established in vitro inflammatory OA model. An anterior cruciate ligament transection (ACLT) model was used to simulate post-traumatic injury in vivo. Micro-CT and histological examination were employed to examine the micro-architectural status and cartilage alteration. Further, serum biomarkers were measured using ELISA to assess OA progression. In-vitro, TMMG reduced excessive ROS generation and inhibited pro-inflammatory IL-1ß secretion by mouse chondrocytes and macrophages, which contributes to OA progression. This expression pattern closely mirrored osteoclastogenesis prevention. In-vivo results show that TMMG prevented chondrocyte apoptosis and degradation of articular cartilage thickness, subchondral parameters, and elevated serum COMP, CTX-II, and IL-1ß which were significantly restored in 5 and 10 mg.kg-1day-1 treated animals and comparable to the positive control Indomethacin. In addition, TMMG also improved cartilage integrity and decreased the OARSI score by maintaining chondrocyte numbers and delaying ECM degradation. These findings suggest that TMMG may be a prospective disease-modifying agent that can mitigate OA progression.

Dendrimer-Mediated Delivery of Anticancer Drugs for Colon Cancer Treatment.

The third most common cancer worldwide is colon cancer (CC). Every year, there more cases are reported, yet there are not enough effective treatments. This emphasizes the need for new drug delivery strategies to increase the success rate and reduce side effects. Recently, a lot of trials have been done for developing natural and synthetic medicines for CC, among which the nanoparticle-based approach is the most trending. Dendrimers are one of the most utilized nanomaterials that are accessible and offer several benefits in the chemotherapy-based treatment of CC by improving the stability, solubility, and bioavailability of drugs. They are highly branched polymers, making it simple to conjugate and encapsulate medicines. Dendrimers have nanoscale features that enable the differentiation of inherent metabolic disparities between cancer cells and healthy cells, enabling the passive targeting of CC. Moreover, dendrimer surfaces can be easily functionalized to improve the specificity and enable active targeting of colon cancer. Therefore, dendrimers can be explored as smart nanocarriers for CC chemotherapy.

Signatures of high altitude adaptation in Tibeto-Burman tribes of the Darjeeling Hill Region.

OBJECTIVES: The long-term isolation, endogamy practices, and environmental adaptations have shaped the enormous human diversity in India. The genetic and morphological variations in mainland Indians are well studied. However, the data on the Indian Himalayan populations are scattered. Thus, the present study attempts to understand variations in the selected parameter among four Tibeto-Burman speaking ethnic tribal populations from the Darjeeling Hill Region (DHR) in the Eastern Himalaya Biodiversity Hotspot region of India. METHODS: A total of 178 healthy male individuals (Lepcha 98, Sherpa 31, Bhutia 27, and Tibetans 22) living at an altitudinal range of 1467-2258 m above the sea level were studied for the 10 parameters namely, weight (kg), height (cm), body mass index (BMI) (kg/m2 ) systolic and diastolic pressure (mm of Hg), pulse rate (per minute), saturation of peripheral oxygen (SPO2 ) (%), hemoglobin (g/dl), hematocrit (HCT) (%), and blood glucose (mg/dl). The data was statistically analyzed using analysis of variance and multiple linear regression methods. RESULTS: Our analysis revealed comparatively lower hemoglobin and HCT levels, and higher systolic and diastolic blood pressure in the Sherpas followed by the Tibetans. This may be reflecting the persistence of high-altitude adaptation signatures even in lowlands. Interestingly, the Tibetans differed significantly from other populations in terms of their higher body weight, height, and BMI. CONCLUSION: Thus, our study showed the persistence of high altitude signatures in Tibetans and Sherpa inhabited the DHR. Additionally, we also observed significant differences in the anthropometric and physiological parameters among the Tibeto-Burman populations of the DHR.

Design, synthesis and biological evaluation of new quinazolinone-benzopyran-indole hybrid compounds promoting osteogenesis through BMP2 upregulation.

In search of novel osteogenic entities, a series of twenty-seven quinazolinone-benzopyran-indole hybrids were designed and synthesized using molecular hybridization approach. All the compounds were scrutinized for their osteogenic potential, primarily based on alkaline phosphatase assay as one of the major anabolic markers. From the primary screening, four osteogenic compounds were sorted from the series and were found nontoxic to the osteoblasts. Further, increased osteoblast differentiation and osteogenic mRNA upregulations suggest compound 47 as the most potent osteoanabolic agent. Immunoblot and ELISA analysis demonstrated that compound 47 promotes osteogenesis via RUNX2 and BMP2 mediated non-canonical p38 pathway. In vivo studies in BALB/c mice inferred that compound 47 stimulates bone anabolism as evident from histological and gene expression studies at 5 mg. kg-1. day-1 dose. Furthermore, structural activity relationship (SAR) and pharmacokinetic studies suggest compound 47 as a BMP2 upregulator and a potential bone anabolic lead for combating future bone metabolic disorders.

Nanoparticles of naturally occurring PPAR-γ inhibitor betulinic acid ameliorates bone marrow adiposity and pathological bone loss in ovariectomized rats via Wnt/ß-catenin pathway.

AIMS: Postmenopausal osteoporosis is one of the world's biggest yet unnoticed health issues. After ovariectomy, declined estrogen level significantly contributes to the elevation of bone marrow adiposity and bone loss leading to osteoporosis. Therapeutics to prevent osteoporosis addressing various aspects are now in short supply. In this study we made an approach to synthesize nanoparticles of naturally occurring PPAR-γ inhibitor, betulinic acid (BA/NPs) and tested the same in altered bone metabolisms developed after ovariectomy. MAIN METHODS: The osteogenic efficacy of BA/NPs was established in human and rat primary osteoblast cells using qRT-PCR and immunoblot analysis. Furthermore, lineage allocations of multipotent bone marrow stromal cells were evaluated. Various aspects of altered bone metabolism after ovariectomy such as bone marrow adiposity and pathological bone loss were evaluated using µCT and histological assessments. KEY FINDINGS: BA/NPs exert potential osteogenic efficacy by modulating key osteogenic markers such as RUNX2 and BMP2. Mechanistically BA/NPs regulate osteoblastogenesis through Wnt/ß-catenin signaling. Further, BA/NPs showed the potential to inhibit the differentiation of multipotent BMSCs towards adipogenesis while favouring the osteogenic lineage via Wnt/ß-catenin pathway. In the in vivo study, increased bone marrow adiposity was reduced in ovariectomized rats after BA/NPs treatment as assessed by histology and µCT analysis. Loss of bone mineral density as a hallmark of pathological bone loss was also abrogated by BA/NPs. Furthermore, increased obesity after OVX was also prevented in BA/NPs treated animals. SIGNIFICANCE: Our findings imply that BA/NPs could be used further as a viable drug lead to counteract various pathophysiological challenges after menopause.

Structure and Dynamics of the Isozymes II and IX of Human Carbonic Anhydrase.

Human carbonic anhydrases (HCAs) are responsible for the pH control and sensing in our body and constitute key components in the central pH paradigm connected to cancer therapeutics. However, little or no molecular level studies are available on the pH-dependent stability and functional dynamics of the known isozymes of HCA. The main objective of this Article is to report the first bench-marking study on the structure and dynamics of the two most efficient isozymes, HCA II and IX, at neutral pH using classical molecular dynamics (MD) and constant pH MD (CpHMD) simulations combined with umbrella sampling, transition path sampling, and Markov state models. Starting from the known crystal structures of HCA II and the monomeric catalytic domain of HCA IX (labeled as HCA IX-c), we have generated classical MD and CpHMD trajectories (of length 1 µs each). In all cases, the overall stability, RMSD, and secondary structure segments of the two isozymes are found to be quite similar. Functionally important dynamics of these two enzymes have been probed in terms of active site hydration, coordination of the Zn(II) ion to a transient excess water, and the formation of putative proton transfer paths. The most important difference between the two isozymes is observed for the side-chain fluctuations of His-64 that is expected to shuttle an excess proton out of the active site as a part of the rate-determining intramolecular proton transfer reaction. The relative stability of the stable inward and outward conformations of the His-64 side-chain and the underlying free energy surfaces are found to depend strongly on the isozyme. In each case, a lower free energy barrier is detected between predominantly inward conformations from predominantly outward ones when simulated under constant pH conditions. The kinetic rate constants of interconversion between different free energy basins are found to span 107-108 s-1 with faster conformational transitions predicted at constant pH condition. The estimated rate constants and free energies are expected to validate if the fluctuation of the His-64 side-chain in HCA IX may have a significance similar to that known in the multistep catalytic cycle of HCA II.

A novel BMP2 secretagogue ameliorates glucocorticoid induced oxidative stress in osteoblasts by activating NRF2 dependent survival while promoting Wnt/ß-catenin mediated osteogenesis.

In our previous study, a novel BMP2 secretagogue was synthesized belonging to a class of galloyl conjugates of flavanones, with remarkable osteogenic potential that promoted bone regeneration. We aimed to establish the protective effect of our compound against bone loss that co-exists with excess Glucocorticoid (GC) therapy. GC therapy induces osteoblast damage leading to apoptosis by increasing reactive oxygen species (ROS). Our results delineate that compound 5e (a BMP2 secretagogue) activates NRF2 signalling to counter the disturbed cellular redox homeostasis and escalate osteoblast survival as assessed by Western blot and immunocytochemistry. Depletion of NRF2 by siRNA blocked activation of the NRF2/HO-1 pathway, magnified oxidative stress, increased apoptosis and abrogated the protective effects of compound 5e. 5e, on the other hand, increased ALP, mineralization activity, and promoted osteoblast differentiation by activating WNT/ß-catenin signalling in BMP2 dependent manner, validated by Western blot of WNT3A, SOST, GSK3-ß and ß-catenin nuclear translocation. Treatment of 5e in presence of BMP inhibitor noggin attenuated the osteogenic efficacy and minimized Wnt//ß-catenin signalling in presence of dexamethasone. Our compound prevents GC challenged trabecular and cortical bone loss assessed by micro-CT and promotes bone formation and osteocyte survival determined by calcein labelling and TUNEL assay in GC treated animals. The osteogenic potential of the compound was authenticated by bone turnover markers. On a concluding note, compounds with BMP upregulation can be potential therapeutics for the prevention and treatment of glucocorticoid-induced osteoporosis.

Benzofuran pyran hybrid prevents glucocorticoid induced osteoporosis in mice via modulation of canonical Wnt/ß-catenin signaling.

Glucocorticoid induced osteoporosis (GIOP) is the second most leading cause of osteoporosis. We have identified a compound, a benzofuran pyran hybrid compound 4e that has osteogenic potential and we wanted to assess its efficacy in GIOP in male mice. We assessed the effect of dexamethasone and compound 4e on primary osteoblasts using various cell based and immunofluorescence assays. For in vivo studies we administered methylprednisolone and compound 4e as a prophylactic measure in male Balb/c mice for 28 days and then evaluated the effect on bone microarchitecture by microCT, bone formation by histology along with clinically relevant bone markers. Compound 4e preserved osteoblast differentiation as evident by higher ALP positive cells and mineralization in compound treated groups. Compound 4e also increased the expression of osteogenic genes. This compound guarded ß-catenin expression both in vitro and in vivo as confirmed by western blot and immunofluorescence assays. This led to the preservation of bone microarchitecture and cortical thickness at 2.5 mg kg-1 and 5 mg kg-1 doses. Further compound 4e enhanced bone formation rate and regulated osteocyte death. The osteogenic potential of compound 4e was reflected by an increased level of serum marker osteocalcin and decreased levels of SOST and CTX-I. Overall, Compound 4e is able to overcome the catabolic effect of dexamethasone on bone by targeting the canonical WNT/ß-catenin signaling as evidenced by both in vitro and in vivo studies.

Caviunin glycoside (CAFG) from Dalbergia sissoo attenuates osteoarthritis by modulating chondrogenic and matrix regulating proteins.

ETHNOPHARMACOLOGICAL RELEVANCE: Dalbergia sissoo DC. (Indian rosewood or Sheesham) is a traditional medicinal plant, reported since time immemorial for its analgesic, anti-nociceptive, anti-inflammatory, and immuno-modulatory properties. D. sissoo DC (DS). is being used traditionally to cure joint inflammation and joint pain. AIM: To study the potential of DS leaves and its derived novel compound CAFG to treat the clinical symptoms of osteoarthritis (OA) and its underlying mechanism. METHODS: The chemical profile of DS extract (DSE) with isoflavonoids and isoflvaonoid glycosides from the DS was established by UHPLC-PDA and UHPLC-MS/MS. Monosodium iodoacetate (MIA) was injected into the knee joint to develop the OA model in rats. DSE was given orally for 28 days daily at 250 and 500 mg.kg-1day-1. For in-vitro experiments, chondrocytes isolated from joint articular cartilage were negatively induced with interleukin-1ß (IL-1ß) and CAFG was given to the cells as a co-treatment. RESULTS: Chondrocytes undergo apoptosis following inflammation and proteoglycan synthesis affected in MIA injected knees. DSE administration prevented these effects as assessed by H&E and Toluidine blue staining. Micro-CT analysis showed that subchondral bone loss was restored. DSE decreased elevated serum levels of cartilage-bone degradation (CTX-I, CTX-II, and COMP), inflammation markers IL-1ß, and matrix-degrading MMP-3 and 13. The effects of IL-1ß on gene expression of chondrocytes were reversed by CAFG treatment at 1 µM. CONCLUSION: Data showed that DSE protected joint cartilage and deterioration in subchondral bone in vivo while in in-vitro, its active ingredient CAFG prevented interleukin-1ß induced effects and inhibited OA. This finding suggest that DSE and CAFG could be used as a possible therapeutic to treat osteoarthritis.

Psychological, social and economic impact of COVID 19 on the working population of India: Exploratory factor analysis approach.

The purpose of this study is to unravel the effects of COVID-19 on the psychological, social, and economic well-being of the working population of India. To achieve the objectives of the study, an online survey was conducted, focusing on aspects like psycho-social well-being, safety, financial stability, and work from home implications. We have used exploratory factor analysis (EFA), t-test, and analysis of variance technique to find the underlying factors. The findings suggest that the female population of the society is more vulnerable to social-psychological and organizational stress. In terms of financial stability, private employees are more unstable as compared to government employees. Based on the standard of living, people of type 1 cities are more affected by the COVID-19 outbreak compared to the people of type 2 and type 3 cities. Hence, by and large, female employees, employees working in the private sector, and employees residing in type 1 cities are more likely to have the behavioral manifestation of negative psychological states caused by this pandemic. The findings will assist policymakers in understanding and devising appropriate policies considering the psycho-social and work-related economic issues faced by the working population of India during the COVID- 19 pandemic.

Synthesis and Evaluation of Galloyl Conjugates of Flavanones as BMP-2 Upregulators with Promising Bone Anabolic and Fracture Healing Properties.

The molecular hybridization concept led us to design a series of galloyl conjugates of flavanones that have potent osteoblast differentiation ability in vitro and promote bone formation in vivo. An array of in vitro studies, especially gene expression of osteogenic markers, evinced compound 5e as the most potent bone anabolic agent, found to be active at 1 pM, which was then further assessed for its osteogenic potential in vivo. From in vivo studies on rat calvaria and a fracture defect model, we inferred that compound 5e, at an oral dose of 5 mg/(kg day), increased the expression of osteogenic genes (RUNX2, BMP-2, Col1, and OCN) and the bone formation rate and significantly promoted bone regeneration at the fracture site, as evidenced by the increased bone volume/tissue fraction compared with vehicle-treated rats. Furthermore, structure-activity relationship studies and pharmacokinetic studies suggest 5e as a potential bone anabolic lead for future osteoporosis drug development.

Baicalin encapsulating lipid-surfactant conjugate based nanomicelles: Preparation, characterization and anticancer activity.

Lung cancer is one of the most common malignant tumors and emerged as one of the leading causes of cancer-related death worldwide. Surgical resection can be a curative treatment for early stage but the most of lung cancer patients are diagnosed at an advanced stage when the pulmonary tumor has been invaded beyond the respiratory system. Therefore, chemotherapy is suitable for curing metastasized tumor. Baicalin (BL) is a flavonoid which has been studied in the treatment of several types of cancer including lung cancer. However, its low solubility in water and non-specificity impede its practical utilization. Hence, we have reported a stearic acid and pluronic F68 conjugated nanomicelles (PF68-SA) system to improve therapeutic efficacy of BL. Solvent evaporation method was used to prepare the BL-loaded PF68-SA nanomicelles (BLNM). The designed BLNM were characterized for the particle size, surface charge, critical micelle concentration, colloidal stability, morphology, and total drug content. BLNM formulation showed improved toxicity of BL against A549 human lung cancer cells in cytotoxicity assay. Further, apoptosis study also depicted BLNM-induced cell death in A549 cells. Therefore, the synthesized fatty acid-modified polymeric nanomicellar system could be useful in overcoming the stability and low therapeutic efficacy issues of hydrophobic anticancer drugs like BL and delivering them to the cancer cells.

Benzofuran pyran compound rescues rat and human osteoblast from lipotoxic effect of palmitate by inhibiting lipid biosynthesis and promoting stabilization of RUNX2.

Obesity and ageing increases bone marrow fat which in turn is associated with lower bone mass. Marrow adipocytes by secreting cytokines, adipokines and free fatty acids change the bone marrow milieu and thus the number of osteoblasts. Palmitate is the common saturated fatty acid, an unavoidable ingredient we consume with food, which kindles cell apoptosis. Compound 4e is osteogenic in nature. We examine the effect of compound 4e in palmitate induced lipotoxicity in rat osteoblasts. Design of benzofuran Pyran hybrid compound (4e) was found to be effective in inhibiting palmitate induced cell apoptosis. In this study an in vitro model of palmitate was contrived. Anti-apoptotic effect of compound 4e was assessed by Annexin/PI and LDH (Lactate dehydrogenase) assay. Compound 4e also increased osteoblast differentiation and mineralization. It also increased expression of osteogenic markers (RUNX2 and BMP2), assessed by Real time PCR and immunofluorescence, which was impeded by palmitate. Acetyl Co-Carboxylase (ACC) and Fatty acid synthase (FAS), two prominent mediators of lipid biosynthesis were increased by palmitate exposure. Compound 4e modulated lipid biosynthesis by inhibiting ACC and FAS as reflected visually and after quantification of less lipid droplet formation suggesting that 4e is osteogenic and simultaneously anti-lipotoxic.

Effect of Obstructive Sleep Apnea on Condylar Malformation, Vertebral Column, and Head Posture: A Cephalometric Evaluation.

AIMS AND OBJECTIVES: Obstructive sleep apnea (OSA) is by far the most common sleep-related breathing disorder, affecting 2-4% of the adult population. The present study aims to compare the descriptive morphology of the cervical column in subjects with normal craniofacial morphology with those having condylar hypoplasia with OSA and to evaluate a positive correlation between the cervical columns, the cranial base angle, and the posture of the head and neck in subjects of condylar hypoplasia. MATERIALS AND METHODS: The present study comprised of lateral cephalogram of 40 subjects divided into two equal groups-control groups (n = 20) and OSA with condylar hypoplasia (n = 20). RESULTS AND OBSERVATION: The condylar hypoplasia group has fusion anomalies of 65% and 35% has a posterior arch deficiency. The cervical lordosis, inclination of the cervical column is found to have a positive statistically significant correlation in condylar hypoplasia subjects. CONCLUSION: Morphological deviations and deviation pattern of the cervical column occurred significantly more often in subjects with condylar hypoplasia as compared with normal craniofacial morphology which can be verified by the increased cranial base angle, cervical lordosis, the inclination of the upper cervical spine, and cranial base angle were positively correlated with a fusion of cervical column. CLINICAL SIGNIFICANCE: Specific types of craniofacial morphology and head postures such as a reduced posterior airway space, an abnormally long soft palate, a low position of the hyoid bone, and an extended head posture are considered predisposing factors of OSA. As posture of the head and neck is considered to be associated with OSA, OSA may be associated with fusion of the cervical column. Hence, to know the result of malformation in the cervical column prove to be important with regard to phenotypical subdivision, diagnosis, and treatment of OSA. HOW TO CITE THIS ARTICLE: Divya, Navit P, Singh R, et al. Effect of Obstructive Sleep Apnea on Condylar Malformation, Vertebral Column, and Head Posture: A Cephalometric Evaluation. Int J Clin Pediatr Dent 2020;13(S-1):S64-S68.

Adding further evidence for clinically significant anti-Leb antibody in a voluntary blood donor.

Herein, we report a case of naturally occurring anti-Leb alloantibody identified in the plasma of a first time voluntary blood donor. The immunohematology workup was done on the pilot sample tubes collected during blood donation by the conventional tube technique and using ID-Micro Column System Glass Beads card (anti-IgG, C3d; Ortho-Clinical Diagnostics, Raritan, New Jersey, USA). Blood group of the donor was confirmed to be B RhD positive, and the alloantibody in his plasma was identified as anti-Leb, having clinically significant characteristics. Since in this particular case, anti-Leb was IgM and IgG in nature, it was clinically significant and can lead to hemolytic transfusion reaction, especially if such fresh frozen plasma unit is transfused to Leb negative patients.

A Review of Theranostics Applications and Toxicities of Carbon Nanomaterials.

BACKGROUND: In the last few years, the use of modified Carbon Nanomaterials (CNMs) for theranostics (therapeutic and diagnosis) applications is a new and rapidly growing area in pharmacy and medical fields. Owing to this, their specific physicochemical behaviors like high stability, drug loading, surface area to volume ratio, with low toxicity and immunogenicity are mainly responsible to be considered those as smart nanomaterials. OBJECTIVES: This review describes the different dimensions of carbon-based nanocarriers including 0-D fullerene, 1-D Carbon Nanotubes (CNTs), and 2-D graphene and Graphene Oxide (GO) and their surface modification with different biocompatible and biodegradable molecules via covalent or non-covalent functionalization. The major focus of this article is on the different theranostics applications of CNMs like targeted drugs and genes delivery, photodynamic therapy, photothermal therapy, bioimaging, and biosensing. The therapeutic efficacy of drugs could be enhanced by delivering them directly on a specific site using different targeted ligands such as vitamins, peptide, carbohydrates, proteins, etc. A section of the article also discusses the toxicity of the CNMs to the living systems. CONCLUSIONS: In brief, this review article discusses the numerous theranostics applications and toxicities of CNMs.