Long term culturing of CHO cells: phenotypic drift and quality attributes of the expressed monoclonal antibody.

OBJECTIVE: Establishing cell lines with enhanced protein production requires a deep understanding of the cellular dynamics and cell line stability. The aim of the study is to investigate the impact of long term culturing (LTC) on cell morphology and altered cellular functions possibly leading to phenotypic drift, impacting product yield and quality. Study highlights the orthogonal cellular and analytical assay toolbox to define cell line stability for optimal culture performance and product quality. METHODS: We investigated recombinant monoclonal antibody (mAb) expressing CHO cells for 60 passages or 180 generations and assessed the cell growth characteristics and morphology by confocal and scanning electron microscopy. Quality attributes of expressed mAb is accessed by performing charge variants, glycan, and host cell protein analysis. RESULTS: We observed a 1.65-fold increase in viable cell population and 1.3-fold increase in cell specific growth rate. A 2.5-fold decrease in antibody titer and abatement of actin filament indicate cellular phenotypic drift. Mitochondrial membrane potential (∆ΨM) signified cell health and metabolic activity during LTC. Host cell protein production is reduced by 1.8-fold. Charge heterogeneity was perturbed with 12.5% and 43% reduction in abundance of acidic and basic charge variants respectively. Glycan profile indicated a decline in fucosylation with 17% increase in galactosylated species as compared with early passaged cells. CONCLUSION: LTC impinges on cellular phenotype as well as the quality of the expressed antibody, suggesting a defined subculturing limit to retain stable protein expression and cell morphology to achieve consistent product quality. Study signifies the changes in cellular and metabolic markers, suggesting cellular and analytical toolbox which could play a significant role in defining cell characteristics and ensured product quality.

Pilot study of population-based newborn screening for spinal muscular atrophy in New York state.

PurposeTo determine feasibility and utility of newborn screening for spinal muscular atrophy (SMA) in New York State.MethodsWe validated a multiplex TaqMan real-time quantitative polymerase chain reaction assay using dried blood spots for SMA. From January 2016 to January 2017, we offered, consented, and screened 3,826 newborns at three hospitals in New York City and tested newborns for the deletion in exon 7 of SMN1.ResultsNinety-three percent of parents opted in for SMA screening. Overall the SMA carrier frequency was 1.5%. We identified one newborn with a homozygous SMN1 deletion and two copies of SMN2, which strongly suggests the severe type 1 SMA phenotype. The infant was enrolled in the NURTURE clinical trial and was first treated with Spinraza at age 15 days. She is now age 12 months, meeting all developmental milestones, and free of any respiratory issues.ConclusionOur pilot study demonstrates the feasibility of population-based screening, the acceptance by families, and the benefit of newborn screening for SMA. We suggest that SMA be considered for addition to the national recommended uniform screening panel.

Structural studies on molecular mechanisms of Nelfinavir resistance caused by non-active site mutation V77I in HIV-1 protease.

BACKGROUND: The human immunodeficiency virus (HIV-1) is a retrovirus causing acquired immunodeficiency syndrome (AIDS), which has become a serious problem across the world and has no cure reported to date. Human immunodeficiency virus (HIV-1) protease is an attractive target for antiviral treatment and a number of therapeutically useful inhibitors have been designed against it. The emergence of drug resistant mutants of HIV-1 poses a serious problem for conventional therapies that have been used so far. Until now, thirteen protease inhibitors (PIs), major mutation sites and many secondary mutations have been listed in the HIV Drug Resistance Database. In this study, we have studied the effect of the V77I mutation in HIV-PR along with the co-occurring mutations L33F and K20T through multi-nanosecond molecular dynamics simulations. V77I is known to cause Nelfinavir (NFV) resistance in the subtype B population of HIV-1 protease. We have for the first time reported the effect of this clinically relevant mutation on the binding of Nelfinavir and the conformational flexibility of the protease. RESULTS: Two HIV-PR mutants have been considered in this study - the Double Mutant Protease (DBM) V77I-L33F and Triple Mutant Protease (TPM) V77I-K20T-L33F. The molecular dynamics simulation studies were carried out and the RMSD trajectories of the unliganded wild type and mutated protease were found to be stable. The binding affinity of NFV with wild type HIV-PR was very high with a Glide XP docking score of -9.3 Kcal/mol. NFV showed decreased affinity towards DBM with a docking score of -8.0 Kcal/mol, whereas its affinity increased towards TPM (Glide XP score: -10.3). Prime/MM-GBSA binding free energy of the wild type, DBM and TPM HIV-PR docked structures were calculated as -38.9, -11.1 and -42.6 Kcal/mol respectively. The binding site cavity volumes of wild type, DBM and TPM protease were 1186.1, 1375.5 and 1042.5 Å3 respectively. CONCLUSION: In this study, we have studied the structural roles of the two HIV-PR mutations by conducting molecular dynamics simulation studies of the wild type and mutant HIV-1 PRs. The present study proposes that DBM protease showed greater flexibility and the flap separation was greater with respect to the wild type protease. The cavity size of the MD-stabilized DBM was also found to be increased, which may be responsible for the decreased interaction of Nelfinavir with the cavity residues, thus explaining the decreased binding affinity. On the other hand, the binding affinity of NFV for TPM was found to be enhanced, accounted for by the decrease in cavity size of the mutant which facilitated strong interactions with the flap residues. The flap separation of TPM was less than the wild type protease and the decreased cavity size may be responsible for its lower resistance, and hence, may be the reason for its lower clinical relevance.

Abrogation of AuroraA-TPX2 by novel natural inhibitors: molecular dynamics-based mechanistic analysis.

INTRODUCTION: Cancer is characterized by uncontrolled cell growth and genetic instabilities. The human Aurora-A kinase protein plays a crucial role in spindle assembly during mitosis and is activated by another candidate oncogene, targeting protein for Xklp2 (TPX2). It has been proposed that dissociation of Aurora A-TPX2 complex leads to disruption of mitotic spindle apparatus, thereby preventing cell division and further tumor growth. MATERIALS AND METHODS: A large natural compound library was docked against the active site of Aurora A-TPX2 complex. The protein-ligand complexes were subjected to molecular dynamics simulation to ascertain their binding stability. The drug properties of the compounds were analyzed to observe their drug-like properties. RESULTS: The virtual screening of natural compound library yielded two high scoring compounds, the first compound CTOM [ZINC ID: 38143674] (Glide score: -9.49) was stable for 17 ns while the second TTOM (Glide score: -9.07) was stable for 15 ns. While CTOM interacted with His280, Thr288 of Aurora A and Tyr34, Lys38 of TPX2, TTOM interacted with Arg285 and Arg286 in addition to the residues involved with CTOM. CONCLUSIONS: We report two natural compounds as potential drugs leads for the disruption of this complex. These ligands show a preferable docking score and have many drugs like properties within in the range of 95% of known drugs. The study provides evidence that CTOM and TTOM can efficiently inhibit the TPX2-mediated activation of Aurora A. Thus, it paves way for an elaborate investigation and establishes the importance of computational approaches as time- and cost-effective techniques.

A novel isoform of the B cell tyrosine kinase BTK protects breast cancer cells from apoptosis.

Tyrosine kinases orchestrate key cellular signaling pathways and their dysregulation is often associated with cellular transformation. Several recent cases in which inhibitors of tyrosine kinases have been successfully used as anticancer agents have underscored the importance of this class of proteins in the development of targeted cancer therapies. We have carried out a large-scale loss-of-function analysis of the human tyrosine kinases using RNA interference to identify novel survival factors for breast cancer cells. In addition to kinases with known roles in breast and other cancers, we identified several kinases that were previously unknown to be required for breast cancer cell survival. The most surprising of these was the cytosolic, nonreceptor tyrosine kinase, Bruton's tyrosine kinase (BTK), which has been extensively studied in B cell development. Down regulation of this protein with RNAi or inhibition with pharmacological inhibitors causes apoptosis; overexpression inhibits apoptosis induced by Doxorubicin in breast cancer cells. Our results surprisingly show that BTK is expressed in several breast cancer cell lines and tumors. The predominant form of BTK found in tumor cells is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. This alternate form of BTK is expressed at significantly higher levels in tumorigenic breast cells than in normal breast cells. Since this protein is a survival factor for these cells, it represents both a potential marker and novel therapeutic target for breast cancer.

Stigma associated with genetic testing for rare diseases-causes and recommendations.

Rare disease (RD) is a term used to describe numerous, heterogeneous diseases that are geographically disparate. Approximately 400 million people worldwide live with an RD equating to roughly 1 in 10 people, with 71.9% of RDs having a genetic origin. RDs present a distinctive set of challenges to people living with rare diseases (PLWRDs), their families, healthcare professionals (HCPs), healthcare system, and societies at large. The possibility of inheriting a genetic disease has a substantial social and psychological impact on affected families. In addition to other concerns, PLWRDs and their families may feel stigmatized, experience guilt, feel blamed, and stress about passing the disease to future generations. Stigma can affect all stages of the journey of PLWRDs and their families, from pre-diagnosis to treatment access, care and support, and compliance. It adversely impacts the quality of life of RD patients. To better explore the impact of stigma associated with genetic testing for RDs, we conducted a literature search on PubMed and Embase databases to identify articles published on stigma and RDs from January 2013 to February 2023. There is a dearth of literature investigating the dynamics of stigma and RD genetic testing. The authors observed that the research into the implications of stigma for patient outcomes in low- and middle-income countries (LMICs) and potential interventions is limited. Herein, the authors present a review of published literature on stigma with a focus on RD genetic testing, the associated challenges, and possible ways to address these.

Patterns and Outcomes in Pediatric Abdominal Tuberculosis: A Single Centre Cohort Study.

INTRODUCTION: Abdominal tuberculosis presents in a variety of ways. Different testing modalities must be applied in addition to having a high clinical suspicion to diagnose and initiate therapy. Medications have a good response; however, morbidity has been seen following surgical management of complicated presentations like intestinal obstruction and perforation. There is a paucity of studies in the pediatric age group which evaluate response to the different treatment regimen and identify factors associated with poorer outcomes in children with abdominal tuberculosis. METHODS: Patient records of 75 children with abdominal tuberculosis at a single center were evaluated using a questionnaire, covering a 14-year period from 2007 to 2021. Demographic features, presenting signs and symptoms, investigations and treatment details were studied. In- person or telephonic follow-up was conducted to identify treatment outcomes. RESULTS: Incidence of abdominal TB was 7%, of all TB children with a mean age of 10.1 years. Mesenteric lymph nodes were involved in 67% and small intestine in 33% cases. Surgery was required in 22 children. 85% children completed treatment. Small intestine involvement had higher probability of undergoing surgery. Of the 70 children with complete follow up, 64 were well and 6 children succumbed to the disease. Older age, small intestine involvement and surgery were independently associated with higher mortality. CONCLUSION: Intestinal involvement is associated with greater need for surgical intervention and greater mortality. Adolescents have poorer outcomes. Further studies are required focusing on these individual subgroups to understand the patterns of presentation, causes for mortality and prevention. LEVEL OF EVIDENCE: Level 5.

Genodermatoses Picture: Difficulties Faced And Way Forward in Nepal.

Genodermatoses are group of genetic disorders that present with cutaneous manifestations. The exact prevalence on many of these conditions are unknown due to its rarity, need of specialized tests for diagnosis and lack of proper reporting system. Most of the patients are faced with life-long disability and associated stigma. There is a need for specialized centers for proper diagnosis of these conditions and a very elaborated yet simple reporting system in Nepal. These rare conditions should be kept in priority by the government in align with the sustainable development goals to ensure healthy-lives and promote well-being for all. A wider engagement of patient-led support groups might be useful in providing necessary information on the disease to the general population and alleviate the stigma associated with these diseases. Keywords: Epidermolysis bullosa; genodermatoses; rare diseases; Nepal.

A review of therapeutic failures in late-stage clinical trials.

INTRODUCTION: The process of drug approval involves extensive and expensive preclinical and clinical examination. Most drugs entering late-stage clinical trials get terminated for a variety of reasons including inability to achieve the primary endpoints or intolerable adverse effects. Only one-tenth of the drugs that enter clinical trials progress to Food and Drug Administration (FDA) regulatory submission. AREAS COVERED: This review offers insight into some of the attributes that may be responsible for a drug's failure in late-stage trials. Information from multiple open sources including PubMed articles published between 1989 and 2019, recent articles from authentic websites like www.ClinicalTrials.gov, www.fda.gov, and pharmaceutical news articles for the years between 2017 and 2021 were accumulated and summarized. Further, a few drug candidates that reached the phase III clinical trials but were discontinued at later stages have been presented as case studies. EXPERT OPINION: Ineluctable failures were observed due to insufficient knowledge about the mechanism of action where the disease progression stages are unclear. Other reasons were choice of patient population, late-stage treatment, and dosage. Adhering to the guidelines and recommendations provided by the regulatory authorities and learning from past failures, considerably reduce failure rates.

Incidence, Risk Factors, and Outcomes of Acute Kidney Injury in Preterm Neonates Hospitalized in the Neonatology Unit, North India: A Single-center Experience.

Acute kidney injury (AKI) is common in premature newborns and is associated with high mortality. It is unclear which risk factors lead to AKI in these neonates. We aimed to determine the incidence, risk factors, and outcomes of AKI in preterm neonates in the neonatal intensive care unit (NICU). They were screened and staged for AKI as per the amended neonatal criteria of Kidney Disease Improving Global Outcomes and followed up until discharge or death. Serum creatinine levels and urine output were measured. The incidence of AKI was 18.5% (37/200 neonates). The majority developed non-oliguric AKI. The risk factors significantly associated with AKI in neonates were the presence of sepsis, birth asphyxia, shock, respiratory distress syndrome, and hypothermia. The majority of neonates with AKI had a birthweight <1500 g and a gestational age of <32 weeks and had a higher risk of mortality, in contrast to than those without AKI. Mortality and NICU stay were significantly higher among those with Stage 3 AKI compared with Stage 2 and Stage 1 AKI. To prevent AKI and reduce the burden of high mortality in premature neonates, it is essential to prevent sepsis, birth asphyxia, and respiratory distress syndrome, as well as to detect shock and patent ductus arteriosus as early as possible. There is a need for good antenatal care to reduce the burden of prematurity.

Enhancing Equitable Access to Rare Disease Diagnosis and Treatment around the World: A Review of Evidence, Policies, and Challenges.

This document provides a comprehensive summary of evidence on the current situation of rare diseases (RDs) globally and regionally, including conditions, practices, policies, and regulations, as well as the challenges and barriers faced by RD patients, their families, and caregivers. The document builds on a review of academic literature and policies and a process of validation and feedback by a group of seven experts from across the globe. Panelists were selected based on their academic merit, expertise, and knowledge regarding the RD environment. The document is divided into five main sections: (1) methodology and objective; (2) background and context; (3) overview of the current situation and key challenges related to RDs covering six dimensions: burden of disease, patient journey, social impact, disease management, RD-related policies, and research and development; (4) recommendations; and (5) conclusions. The recommendations are derived from the discussion undertaken by the experts on the findings of this review and provide a set of actionable solutions to the challenges and barriers to improving access to RD diagnosis and treatment around the world. The recommendations can support critical decision-making, guiding efforts by a broad range of RDs stakeholders, including governments, international organizations, manufacturers, researchers, and patient advocacy groups.

A thematic study: impact of COVID-19 pandemic on rare disease organisations and patients across ten jurisdictions in the Asia Pacific region.

BACKGROUND: This study assesses the areas and extent of impact of the Coronavirus Disease of 2019 (COVID-19) pandemic on rare disease (RD) organisations in the Asia Pacific region. There is no existing literature that focuses on such impact on RD organisations in any jurisdictions, nor RD populations across multiple jurisdictions in the Asia Pacific region. A cross-sectional survey was distributed to RD organisations between April and May 2020. Quantitative and qualitative data on the impact of COVID-19 on RD organisations and patients were collected from the organisation representative's perspective. Qualitative data was analysed using thematic analysis. A follow-up focus group meeting was conducted in August 2020 to validate the survey findings and to discuss specific needs, support and recommendations for sustainable healthcare systems during the pandemic. RESULTS: A total of 80 RD organisations from Australia, Hong Kong Special Administrative Region of China, India, Japan, mainland China, Malaysia, New Zealand, the Philippines, Singapore and Taiwan participated in the study. Of all, 89% were concerned about the impact of pandemic on their organisations. Results indicate that 63% of the organisations functioned at a reduced capacity and 42% stated a decrease in funding as their biggest challenge. Overall, 95% believed their patients were impacted, particularly in healthcare access, social lives, physical health, psychological health and financial impact. Specifically, 43% identified the reduced healthcare access as their top impact, followed by 26% about the impact on daily living and social life. Focus group meeting discussed differential impact across jurisdictions and point towards telemedicine and digitalisation as potential solutions. CONCLUSIONS: This serves as the first study to assess the impact of COVID-19 on RD patients and organisations across multiple jurisdictions in the Asia Pacific region, identifying major themes on the impact on both RD patients and organisations. By including 80 organisations from ten jurisdictions, our study presents the most comprehensive assessment of the pandemic's impact to date. It highlights the need for mental health support and sheds light on moving towards telemedicine and digitalisation of organisation operation, which constitutes a sustainable model in times of pandemics and beyond.

Dielectric studies of wheat in powder form at microwave frequencies.

Dielectric constant and loss factor of Raj-4120 variety of Indian wheat were determined in powder form (grain size 125 to 150 micron) at room temperature. Microwaves at three different frequencies were employed in C-band, X-band and Ku-band respectively for investigating frequency dependence of dielectric parameters of the sample. Bulk dielectric values of the sample were determined by employing the dielectric mixture relations, such as, half power mixture equation, Landau and Lifshitz, Looyenga equation etc.

Recent developments and clinical applications of surgical glues: An overview.

Novel developments in surgical glues for sealing wounds, surgical incision and tissue healing was inspired by limitations related to other surgical techniques. Sealants or bioadhesives are considered as a promising candidate over sutures, staples for sealing of air and gas leakages with minimal scarring in different surgeries. Their physical, biological properties and adhesive strength have encouraged many surgeons for employing them in clinical practice. Commonly, various natural biopolymers such as fibrin, collagen, gelatin, albumin, chitosan are used for the preparation of surgical glues. These biopolymers mimic the mechanism of physiological interaction such as blood clotting process. Besides natural polymers, synthetic polymers are also incorporated in surgical glues. Several sealants or adhesives are available commercially and have been used for neurosurgery, orthopaedic, periodontal, ophthalmic, cardiovascular, pneumothoracic, gastrointestinal, plastic and reconstructive surgery. This article focuses on providing a comprehensive review on the development of surgical glues, their unique features and application in clinical practice. In addition to the advantages of these sealants, the insights on their limitations, especially biocompatibility and tissue elasticity have been covered. Although the use of surgical glues is increasing gradually, the next generation of products should be marketed after extensive clinical studies and adequate safety and efficacy data.

Structure, DNA/proteins binding, docking and cytotoxicity studies of copper(II) complexes with the first quinolone drug nalidixic acid and 2,2'­dipyridylamine.

This work presents the synthesis, structural characterization and biological affinity of the newly synthesized copper(II) complexes with the first antibacterial quinolone drug nalidixic acid (nal) or N-donor ligand 2,2'­dipyridylamine (bipyam). [Cu(II)(nal)(bipyam)Cl], (2) reveals a distorted square pyramidal based geometry in Cu(II) atom confirmed by X-ray crystallography technique. The theoretical stabilities and optimized structures of the complex were obtained from DFT calculations. The ability of the complexes to bind with calf thymus DNA (CT DNA) were investigated by electronic absorption, fluorescence, circular dichroism, and viscosity measurements techniques. The experimental results reveal that the complexes strongly interact with CT DNA via intercalative mode but complex 2 exhibits the highest affinity giving Kb=3.91±0.13×106, M-1. The fluorescence spectroscopy measurements show that both complexes have the superior ability to the replacement of EtBr from DNA-bound EtBr solution and bind to DNA through intercalative mode. Both complex also shows the superior affinity towards proteins with comparatively high binding constant values which have been further revealed by fluorescence spectroscopy measurements. Molecular docking analysis indicates that the interaction of the complexes and proteins are stabilized by hydrogen bonding and hydrophobic interaction. Furthermore, the results of in vitro cytotoxicity reveal that the complex 2 has excellent cytotoxicity than 1 against human breast cancer cell lines (MCF-7).

Weekly iron and folic acid supplementation with counseling reduces anemia in adolescent girls: a large-scale effectiveness study in Uttar Pradesh, India.

BACKGROUND: Weekly iron-folic acid supplementation in small-scale research trials and as administered in institutions has been demonstrated to be effective in reducing anemia in adolescent girls. OBJECTIVE: To assess the effectiveness of weekly iron-folic acid supplementation in a large-scale project in reducing the prevalence of anemia in adolescent girls. METHODS: The project provided weekly iron-folic acid tablets, family life education, and deworming tablets every 6 months to 150,700 adolescent school girls and non-schoolgirls of a total district population of 3,647,834. Consumption of the iron-folic acid tablets was supervised for schoolgirls but not for non-schoolgirls. Hemoglobin levels were assessed in a random sample of non-schoolgirls at 6 and 12 months and schoolgirls at 6 months. The effect of supplementation on the prevalence of anemia and the compliance rate were assessed over a 4-year period. RESULTS: In 4 years, the overall prevalence of anemia was reduced from 73.3% to 25.4%. Hemoglobin levels and anemia prevalence were influenced significantly at 6 months. No difference in the impact on hemoglobin or anemia prevalence was observed between supervised and unsupervised girls. Counseling on the positive effects of regular weekly iron-folic acid intake contributed to a high compliance rate of over 85%. The cost of implementation was US$0.36 per beneficiary per year. CONCLUSIONS: Weekly iron-folic acid supplementation combined with monthly education sessions and deworming every 6 months is cost-effective in reducing the prevalence of anemia in adolescent girls. Appropriate counseling, irrespective of supervision, is critical for achieving positive outcomes.

Dietary profile of sportswomen participating in team games at state/national level.

A cross sectional study was conducted toassess dietary profile of 100 Delhi based national / state level sportswomen, aged 18-25 years, participating in team games--volleyball, hockey, football and kabaddi. Mean energy intake was found to be 1471 +/- 479 Kcal. Only 24 percent of the sports women met the recommendations of 60-65 energy percent from carbohydrates and 87 percent were consuming more than 25 energy percent from fat. The mean macronutrients and micronutrient intakes of all the subjects were much lower than the recommendations. Improper food choices were also observed in majority. It becomes necessary to generate awareness among sports personnel regarding proper nutrition practices.

Pain scores in the management of postoperative pain in shoulder surgery.

PURPOSE: Recent public opinion has focused on the lay perception that postoperative pain control is inadequate. The Joint Commission on Accreditation of Healthcare Organizations has attempted to address this issue by mandating patient-reported numeric pain scores as part of postoperative pain assessment. No studies exist to validate these scores in orthopaedics. The purpose of this study was 3-fold: (1) to determine which preoperative variables were associated with elevated postoperative pain scores, (2) to identify whether pain scores correlated with more traditional signs and symptoms of pain, and (3) to identify whether patients with a high pain score (HPS) affect perioperative and postoperative complication rates. METHODS: We retrospectively evaluated 105 patients who underwent routine arthroscopic rotator cuff repair over a 12-month period. Correlations and linear regression analysis were used to assess factors associated with postoperative pain scores. Logistic regression analysis was used to construct a model of preoperative, demographic, surgical, and postoperative variables associated with HPSs. RESULTS: Wide variability existed in pain scores despite the similarity of the surgical stimulus. What subtle differences that were present in the surgical procedures had no bearing on pain scores. No correlation existed between pain scores and any physiologic correlate of pain. Pain scores were correlated with Workers' Compensation insurance, preoperative antianxiety medications, antidepressant medications, preoperative narcotics, and alcohol use. HPS patients had a longer recovery room stay (P < .05) and a higher incidence of postoperative nausea than those with a normal pain tolerance. HPS patients also had a significantly delayed return to work, as well as significantly lower Simple Shoulder Test scores at 6 weeks, although the final shoulder scores were similar to those in patients with a normal pain tolerance. CONCLUSIONS: Patient-reported pain scores have been mandated, but they vary widely among patients undergoing virtually the same procedure and correlate poorly with physiologic manifestations of pain. Pain scores tend to correlate with other types of variables, most of which would be expected by the experienced surgeon. The exclusive use of pain scores in postoperative pain management should be limited to prevent complications. LEVEL OF EVIDENCE: Level IV, prognostic case series.

GQSAR modeling and combinatorial library generation of 4-phenylquinazoline-2-carboxamide derivatives as antiproliferative agents in human Glioblastoma tumors.

BACKGROUND: TSPO translocator protein, encoded in humans by the Tspo gene plays a crucial role in mitochondria mediated apoptosis and necrotic cell death through its association with Mitochondrial Permeability Transition pore (MPTP). It has been shown that this function can be exploited as a potential treatment for human Glioblastoma Multiforme. In this study, a novel robust fragment based QSAR model has been developed for a series of 4-phenylquinazoline-2-carboxamides experimentally known to be ligands for TSPO, thus triggering apoptotic mechanism cascade. RESULTS: Model developed showed satisfactory statistical parameters for the experimentally reported dataset (r2=0.8259, q2=0.6788, pred_r2=0.8237 and F-test=37.9). Low standard error values (r2_se=0.253, q2_se=0.34, pred_r2_se=0.14) confirmed the accuracy of the generated model. The model obtained had 4 descriptors, namely, R1-Volume, R2-SsCH3E-index, R3-SsCH3count and R5-EpsilonR. Two of them had positive contribution while the other two had negative correlation. CONCLUSION: The high binding affinity and the presence of essential structural features in these compounds make them an ideal choice for the consideration as potent anti-GBM drugs. Activity predicted by GQSAR model reinforces their potential as worthy candidates for drugs against GBM. The detailed analysis carried out in this study provides a substantial basis for the prospective design and development of novel 4-phenylquinazoline-2-carboxamide compounds as TSPO ligands capable of inducing apoptosis in cancer cells.

Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells.

Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures. We determined specific mRNA levels in exfoliated buccal cells collected by cytologic brush, using a recently developed RNA-specific real-time quantitative reverse transcription-PCR strategy. In a pilot study, metabolic activity of exfoliated buccal cells was verified by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium assay in vitro. Transcriptional activity was observed, after timed in vivo exposure to mainstream tobacco smoke resulted in induction of CYP1B1 in serially collected buccal samples from the one subject examined. For a set of 11 subjects, mRNA expression of nine genes encoding carcinogen- and oxidant-metabolizing enzymes qualitatively detected in buccal cells was then shown to correlate with that in laser-microdissected lung from the same individuals (Chi2 = 52.91, P < 0.001). Finally, quantitative real-time reverse transcription-PCR assays for seven target gene (AhR, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, and GSTT1) and three reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, and 36B4] transcripts were performed on buccal specimens from 42 subjects. In multivariate analyses, gender, tobacco smoke exposure, and other factors were associated with the level of expression of CYP1B1, GSTP1, and other transcripts on a gene-specific basis, but substantial interindividual variability in mRNA expression remained unexplained. Within the power limits of this pilot study, gene expression signature was not clearly predictive of lung cancer case or control status. This noninvasive and quantitative method may be incorporated into high-throughput human applications for probing gene-environment interactions associated with cancer.