Therapeutic implication of MicroRNA-320a antagonist in attenuating blood clots formed during venous thrombosis.

Venous thrombosis (VT) is a complex multi-factorial disease and a major health concern worldwide. Its clinical implications include deep vein thrombosis (DVT) and pulmonary embolism (PE). VT pathogenesis involves intricate interplay of various coagulants and anti-coagulants. Growing evidences from epidemiological studies have shown that many non-coding microRNAs play significant regulatory role in VT pathogenesis by modulating expressions of large number of gene involved in blood coagulation. Present study aimed to investigate the effect of human micro RNA (hsa-miR)-320a antagonist on thrombus formation in VT. Surgery was performed on Sprague-Dawley (SD) rats, wherein the inferior vena cava (IVC) was ligated to introduce DVT. Animals were divided into four groups (n = 5 in each group); Sham controls (Sham), IVC ligated-DVT (DVT), IVC ligated-DVT + transfection reagent (DVT-NC) and IVC ligated-DVT + miR320a antagonist (DVT-miR-320a antagonist). IVC was dissected after 6 h and 24 h of surgery to estimate thrombus weight and coagulatory parameters such as levels of D-dimer, clotting time and bleeding time. Also, ELISA based biochemical assays were formed to assess toxicity of miRNA antagonist in animals. Our experimental analysis demonstrated that there was a marked reduction in size of thrombus in hsa-miR-320a antagonist treated animals, both at 6 h and 24 h. There was a marked reduction in D-dimer levels in hsa-miR-320a antagonist treated animals. Also, blood clotting time was delayed and bleeding time was increased significantly in hsa-miR-320a antagonist treated rats compared to the non-treated and Sham rats. There was no sign of toxicity in treated group compared to control animals. Hsa-miR-320a antagonist could be promising therapeutic target for management of VT.

RING finger E3 ligase, RNF138 inhibits osteoblast differentiation by negatively regulating Runx2 protein turnover.

A few ubiquitin ligases have been shown to target Runx2, the key osteogenic transcription factor and thereby regulate bone formation. The regulation of Runx2 expression and function are controlled both at the transcriptional and posttranslational levels. Really interesting new gene (RING) finger ubiquitin ligases of which RNF138 is a member are important players in the ubiquitin-proteasome system, contributing to the regulation of protein turnover and cellular processes. Here, we demonstrated that RNF138 negatively correlated with Runx2 protein levels in osteopenic ovariectomized rats which implied its role in bone loss. Accordingly, RNF138 overexpression potently inhibited osteoblast differentiation of mesenchyme-like C3H10T1/2 as well primary rat calvarial osteoblast (RCO) cells in vitro, whereas overexpression of catalytically inactive mutant RNF138Δ18-58 (lacks RING finger domain) had mild to no effect. Contrarily, RNF138 depletion copiously enhanced endogenous Runx2 levels and augmented osteogenic differentiation of C3H10T1/2 as well as RCOs. Mechanistically, RNF138 physically associates within multiple regions of Runx2 and ubiquitinates it leading to its reduced protein stability in a proteasome-dependent manner. Moreover, catalytically active RNF138 destabilized Runx2 which resulted in inhibition of its transactivation potential and physiological function of promoting osteoblast differentiation leading to bone loss. These findings underscore the functional involvement of RNF138 in bone formation which is primarily achieved through its modulation of Runx2 by stimulating ubiquitin-mediated proteasomal degradation. Thus, our findings indicate that RNF138 could be a promising novel target for therapeutic intervention in postmenopausal osteoporosis.

Influence of Root Canal Treatment on Oral-Health-Related Quality of Life (OHRQoL) in Saudi Patients: A Cross-Sectional Study.

Objective This cross-sectional study aimed to assess the influence of root canal treatment on the oral-health-related quality of life (OHRQoL) of patients in the Kingdom of Saudi Arabia (KSA). The data on KSA's population are significantly limited, highlighting the significance of additional research to be carried out in this particular field. Methods The study was conducted at the dental clinics of the College of Dentistry, Qassim University, from January to June 2022. A total of 112 patients who had undergone endodontic therapy (ET) for teeth with irreversible pulpitis were included. Demographic data and treatment procedures were recorded. The patients' responses to the OHIP-14 questionnaire were analyzed to assess their OHRQoL. The scores were converted into qualitative categories (good, moderate, poor) for classification. Results The majority of patients (59.8%) reported a good OHRQoL after ET. Physical pain was the only variable where more than 50% of patients reported experiencing pain. Older age and smoking status were significantly associated with a poorer OHRQoL. However, no significant gender differences in OHRQoL were observed. Conclusion ET had a positive impact on the OHRQoL of patients in Saudi Arabia. The study highlights the importance of considering patient-centered outcomes, such as OHRQoL, in assessing the success of root canal treatment. Further research with longitudinal designs and randomized controlled trials is needed to better understand the long-term effects of root canal treatment on patients' OHRQoL and to compare them with other dental treatments.

Effects of the Indian National Health Insurance Scheme (PM-JAY) on Hospitalizations, Out-of-pocket Expenditures and Catastrophic Expenditures.

India launched one of the world's largest health insurance programs, the Pradhan Mantri Jan Arogya Yojana (PM-JAY), targeting more than 500 million economically and socially disadvantaged Indians. PM-JAY is publicly funded and covers hospitalization costs in public and private facilities. We examine how PM-JAY has affected hospitalizations and out-of-pocket expenditures (OOPE), and given the high use of private health care in India, we compare these outcomes across public and private facilities. We conducted a household survey to collect data on socioeconomic and demographic information, health status and hospitalizations for more than 57,000 PM-JAY eligible individuals in six Indian states. Using multivariate regression models, we estimated whether PM-JAY was associated with any changes in hospitalizations, OOPE and catastrophic health expenditures (CHE) and whether these differed across public and private facilities. We found that PM-JAY was not associated with an increase in hospitalizations, but it increased the probability of visiting a private facility by 4.6% points (p < .05). PM-JAY was associated with a relative reduction of 13% in OOPE (p < .1) and 21% in CHE (p < .01). This was entirely driven by private facilities, where relative OOPE was reduced by 17% (p < .01) and CHE by 19% (p < .01). This implied that PM-JAY has shifted use from public to private hospitalizations. Given the complex healthcare system with the presence of parallel public and private systems in India, our study concludes that for economically and socially disadvantaged groups, PM-JAY contributes to improved access to secondary and tertiary care services from private providers.

Dexamethasone induces cancer mitigation and irreversible senescence in lung cancer cells via damaging cortical actin and sustained hyperphosphorylation of pRb.

Activation of the glucocorticoid receptors by its cognate ligand, dexamethasone (DEX) is commonly used as an adjuvant treatment in solid tumors. However, its direct effect on cancerous phenotype is not fully understood. We explored the effect and molecular mechanisms of DEX action in lung cancer. In in vitro experiments, DEX treatment causes decrease in migration, invasion and colony formation ability of A549 cells even at lower doses. DEX also decreased adhesion of A549 cells by reducing the formation of cortical actin. Treatment with RU486, a GR antagonist, indicated that these effects are partially mediated through GR. Further; DEX induces G0/G1 arrest of A549 cells. Mechanistically, DEX induces expression of both CDK inhibitors (p21Cip1, p27Kip1) and cyclin-dependent kinases (CDK4, CDK6). Due to this compensatory activation of CDKs and CDKIs, DEX induces the hyper phosphorylation state of Rb protein (pRb) leading to irreversible senescence as confirmed by ß-gal staining. Next, in clinical dataset of NSCLC (Non-small cell lung cancer), GR was lowly expressed in cancer patients as compared to the normal group, where higher expression of GR led to higher overall survival of NSCLC indicating for a protective role of GR. Interestingly, when combined with chemotherapeutic agents, DEX can modulate the drug-sensitivity of cells. Taken together, these data indicate that DEX through GR activation may suppress tumor growth by decreasing proliferation and inducing irreversible senescence and combination of standard chemotherapy and DEX can be a potential treatment for NSCLC.

Lysine Acetyltransferases (KATs) in Disguise: Diseases Implications.

Acetylation is one of the key post-translational protein modifications catalysed by the protein lysine acetyltransferases (KATs). KATs catalyse the transfer of acetyl groups to the epsilon-amino groups of lysine residues in histones and non-histone proteins. Because of its wide range of target proteins, KATs regulate many biological processes, and their aberrant activities may underlie several human diseases, including cancer, asthma, Chronic Obstructive Pulmonary Disease (COPD), and neurological disorders. Unlike most of the histone modifying enzymes, such as lysine methyltransferases, KATs do not possess any conserved domain like SET domain of lysine methyltransferases. However, almost all the major families of KATs are found to be transcriptional coactivators or adaptor proteins, with defined catalytic domains, called canonical KATs. Over the past two decades, a few proteins have been discovered to possess intrinsic KAT activity but are not classical coactivators. We would like to categorize them as non-canonical KATs (NC-KATs). These NC-KATs include general transcription factors TAFII250, mammalian TFIIIC complex, and mitochondrial protein GCN5L1, etc. This review focuses on our understanding, as well as controversies regarding non-canonical KATs, where we compare the structural and functional similarities and dissimilarities of non-canonical KATs with the canonical KATs. This review also highlights the potential role of NC-KATs in health and diseases.

Dexamethasone activates c-Jun NH2-terminal kinase (JNK) which interacts with GR and protects it from ubiquitin-mediated degradation in NSCLC cells.

Dexamethasone-mediated pharmacological activation of the glucocorticoid receptor (GR) is widely used in the treatment regimen of hematological malignancies and solid cancers. However, DEX sensitivity towards patients primarily depends on the endogenous protein levels of GR. We observed that DEX treatment leads to an increase in GR protein levels despite inhibition of neo-protein synthesis in non-small cell lung cancer (NSCLC) cells. Mechanistically, DEX-stimulation concomitantly increased the JNK phosphorylation and GR protein levels, however the JNK stimulation preceds GR upregulation. Moreover, we also observed that DEX-mediated phosphorylation is partially mediated by upregulation in MEKK1 phosphorylation. Further, GR protein levels were significantly decreased in JNK inhibitor (JNKi, SP600125) treated cells whereas MG132 treatment restored GR levels indicating that DEX induced JNK activity regulated the GR protein levels through proteasomal-degradation pathway. Next, we showed that DEX led to JNK activation which physically interacts with GR and protects it from ubiquitination-mediated degradation. Furthermore, at basal level GR interacts with JNK in cytoplasm whereas upon DEX stimulation GR and pJNK both localized to nucleus and interact with each other. Next, we show that JNK-mediated GR stabilization affects its nuclear transcriptional functional activity in NSCLC cells. In line with these in vitro data, patient dataset analysis also shows that increased levels of both JNK and GR contributes towards better prognosis of NSCLC patients. Taken together, our data shows that DEX treatment may lead to positive feedback regulation of GR by activating JNK and thus highlights importance of GR-JNK crosstalk in NSCLC.

Awareness of India's national health insurance scheme (PM-JAY): a cross-sectional study across six states.

The literature suggests that a first barrier towards accessing benefits of health insurance in low- and middle-income countries is lack of awareness of one's benefits. Yet, across settings and emerging schemes, limited scientific evidence is available on levels of awareness and their determinants. To fill this gap, we assessed socio-demographic and economic determinants of beneficiaries' awareness of the Pradhan Mantri Jan Arogya Yojana (PM-JAY), the national health insurance scheme launched in India in 2018, and their awareness of own eligibility. We relied on cross-sectional household (HH) survey data collected in six Indian states between 2019 and 2020. Representative data of HHs eligible for PM-JAY from 11 618 respondents (an adult representative from each surveyed HH) were used. We used descriptive statistics and multivariable logistic regression models to explore the association between awareness of PM-JAY and of one's own eligibility and socio-economic and demographic characteristics. About 62% of respondents were aware of PM-JAY, and among the aware, 78% knew that they were eligible for the scheme. Regression analysis confirmed that older respondents with a higher educational level and salaried jobs were more likely to know about PM-JAY. Awareness was lower among respondents from Meghalaya and Tamil Nadu. Respondents from Other Backward Classes, of wealthier socio-economic status or from Meghalaya or Gujarat were more likely to be aware of their eligibility status. Respondents from Chhattisgarh were less likely to know about their eligibility. Our study confirms that while more than half of the eligible population was aware of PM-JAY, considerable efforts are needed to achieve universal awareness. Socio-economic gradients confirm that the more marginalized are still less aware. We recommend implementing tailored, state-specific information dissemination approaches focusing on knowledge of specific scheme features to empower beneficiaries to demand their entitled services.

Out-of-Pocket expenditure and patient experience of care under-Indonesia's national health insurance: A cross-sectional facility-based study in six provinces.

BACKGROUND: Low- and middle-income countries worldwide are striving to achieve universal health coverage (UHC), frequently through expansion of statutory health insurance schemes. However, oftentimes evidence is lacking on progress towards quality patient-centred care and out-of-pocket expenditure (OOPE), particularly for poor population groups. We contribute patient-centred evidence examining patient experience and OOPE under JKN, the Indonesian social health insurance. METHODS: Using data from 2526 patient exit interviews conducted among JKN beneficiaries in 2015, we computed a summative patient experience measure from 14 experience items. We used descriptive statistics to assess patient experience and the probability, amount and components of OOPE. We applied a two-part model to examine the relationships between socio-demographics, facility types, and OOPE and an OLS regression on patient experience determinants. RESULTS: The mean patient experience measure was 11.7 out of 14 maximal points. Differences were observed between single items, with highest ratings on ease of understanding providers' language (97%) and lowest on waiting time (54%). OOPE were reported by 20% of patients with a mean equivalent to US$40, the most prevalent reason being medicines (61% of all OOPE). Considerable OOPE heterogeneity occurred by province and facility type. We found differentials in OOPE by gender (females paying more likely, but less) and subsidised JKN membership (same likelihood as non-subsidised, but paying less). CONCLUSION: Our findings suggest that during its early implementation, patients under JKN reported mostly positive patient experience yet a fifth incurred OOPE, mostly on medicines. Further patient-centred research is needed to ensure JKN's progress towards UHC.

A Study for Evaluation of Altered Mental Status Patients in Medicine Department.

Altered mental status (AMS) comprises a group of clinical symptoms rather than a specific diagnosis, and includes cognitive disorders, attention disorders, arousal disorders, and decreased level of consciousness. Patients often manifest vague symptoms, thus, AMS diagnosis and treatment are highly challenging for general medicine physicians. MATERIAL: This was an observational cross-sectional analytical study. This study will be conducted in Department of General Medicine, SMS Medical College and attached group of hospitals, Jaipur, Rajasthan, India. Duration of this study was 1 year. We analysed 150 cases in this study. OBSERVATION: We found that mean age for this study was 55.1 years. Major co-morbidity was Hypertension, ALD/CLD/PHTN, T2DM and carcinoma lung with 46.6%, 25.3%, 22.6% and 18% respectively. We found that 81.3% patients are non-alcoholic followed by 50% non-smoker. In our study 52.6% patients had structural Neurological aetiology followed by 17.3% of CO2 Narcosis, 10% patients had hepatic encephalopathy. 5.3% patients were having hypoglycemia and 4.6% were having hyponatremia. In brain MRI we found that 22.6% patients are of CVA ICH, 16.6% patients with CVA Infarct and 3.3% patients of hypoxic ischemic brain injury. CONCLUSION: In our study, we found that neurological causes for altered sensorium were more common than primary non-neurological diseases. Though neuroimaging was helpful in large number of patients, good history, thorough physical examination & laboratory reports also were vital in establishing diagnosis.

Nucleoporin-93 reveals a common feature of aggressive breast cancers: robust nucleocytoplasmic transport of transcription factors.

By establishing multi-omics pipelines, we uncover overexpression and gene copy-number alterations of nucleoporin-93 (NUP93), a nuclear pore component, in aggressive human mammary tumors. NUP93 overexpression enhances transendothelial migration and matrix invasion in vitro, along with tumor growth and metastasis in animal models. These findings are supported by analyses of two sets of naturally occurring mutations: rare oncogenic mutations and inactivating familial nephrotic syndrome mutations. Mechanistically, NUP93 binds with importins, boosts nuclear transport of importins' cargoes, such as ß-catenin, and activates MYC. Likewise, NUP93 overexpression enhances the ultimate nuclear transport step shared by additional signaling pathways, including TGF-ß/SMAD and EGF/ERK. The emerging addiction to nuclear transport exposes vulnerabilities of NUP93-overexpressing tumors. Congruently, myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK can inhibit tumor growth and metastasis. Our study sheds light on an emerging hallmark of advanced tumors, which derive benefit from robust nucleocytoplasmic transport.

Postoperative Analgesic Effect of Bupivacaine alone and with Dexmedetomidine in Wound Instillation for Lumbar Laminectomy: A Randomized Control Trial.

BACKGROUND: Dexmedetomidine has been used as an effective adjuvant to local anesthetics in peripheral nerve blocks and at the incision site. AIMS: We compared the postoperative analgesic effect of bupivacaine alone and in addition of dexmedetomidine to bupivacaine in wound instillation during lumbar laminectomy. SETTING AND DESIGN: This was a prospective, double-blind, randomized control trial. SUBJECTS AND METHODS: Sixty adults of the American Society of Anesthesiologists Grade I-II scheduled for elective lumbar laminectomy under general anesthesia were randomly allocated into two groups. Group B (control group) patients received wound instillation with 20 mL of 0.25% bupivacaine at the end of surgery and Group D patients received 2 µg.kg-1 dexmedetomidine diluted in 20 mL 0.25% bupivacaine as instillation over the incision site. If the NRS exceeded "4" at any point of time, rescue analgesia with injection diclofenac 75 mg deep intramuscular was administered. Postoperative pain score, duration of analgesia, total rescue analgesic required in the first 24 h, and side effects were compared between the groups. RESULTS: Demographic data were comparable in both the groups. Duration of analgesia (19.93 ± 3.2 in Group D vs. 12.13 ± 1.8 in Group B) was significantly more in Group D, number of analgesic demands were less in group D as compared to Group B, and total rescue analgesic required (62.51 ± 39.13 vs. 95.68 ± 33.5) was significantly less in Group D as compared to Group B. CONCLUSIONS: We conclude that dexmedetomidine 2 µg.kg-1 is an effective adjuvant to bupivacaine for wound instillation in terms of quality and duration of postoperative analgesia following lumbar laminectomy.

MicroRNAs in venous thrombo-embolism.

BACKGROUND: Venous Thrombo-embolism (VTE) is the major preventable cause of death and disability worldwide. It has the third highest incidence rate of hospital death after coronary artery disease (CAD) and stroke. With the establishment of Virchow's triad stating the major factors responsible for VTE including stasis, hypercoagulability and endothelial dysfunction, the last decade reported number of studies regarding its diagnosis and prophylaxis. Till date the most commonly used clinical marker for its diagnosis is the D-dimer test, detecting endogenous fibrinolysis. This test often gives false positive results and has low specificity. Other markers of coagulation are being used in combination with D-dimer; however, a reliable and sensitive biomarker is still needed for early and accurate diagnosis of VTE. Non-coding regulatory RNAs such as MicroRNAs (miRNAs) are small molecules that play a significant role in RNA silencing and post-transcriptional gene expression regulation. They can specifically bind to their target genes forming silencing complex, thereby inducing degradation and altered gene expression. A wide range of miRNAs have extensively been studied in a variety of cardiovascular diseases such as CAD, stroke, myocardial infarction (MI), atherosclerosis, obstructive sleep apnoea (OSA) and other complex diseases such as cancer. It has been demonstrated that circulating miRNAs have enormous potential to function as clinical diagnostic biomarkers for many diseases. This review comprehends recent studies establishing the inevitable role of miRNAs in pathogenesis of complex diseases with special emphasis on venous thrombosis. The differential expression pattern of these miRNAs shows a strong positive correlation with the manifestation of the pathological symptoms of diseases. Systematic consolidation of different miRNAs linked to VTE in various studies could be helpful in finding accurate diagnostic markers for thrombosis and may also find its place in VTE therapeutics. However, more extensive research and confirmatory experiments are needed to establish the role of these miRNAs as biomarkers.

Impact of India's National Tobacco Control Programme on bidi and cigarette consumption: a difference-in-differences analysis.

BACKGROUND: Despite the importance of decreasing tobacco use to achieve mortality reduction targets of the Sustainable Development Goals in low-income and middle-income countries (LMICs), evaluations of tobacco control programmes in these settings are scarce. We assessed the impacts of India's National Tobacco Control Programme (NTCP), as implemented in 42 districts during 2007-2009, on household-reported consumption of bidis and cigarettes. METHODS: Secondary analysis of cross-sectional data from nationally representative Household Consumer Expenditure Surveys (1999-2000; 2004-2005 and 2011-2012). Outcomes were: any bidi/cigarette consumption in the household and monthly consumption of bidi/cigarette sticks per person. A difference-in-differences two-part model was used to compare changes in bidi/cigarette consumption between NTCP intervention and control districts, adjusting for sociodemographic characteristics and time-based heterogeneity. FINDINGS: There was an overall decline in household-reported bidi and cigarette consumption between 1999-2000 and 2011-2012. However, compared with control districts, NTCP districts had no significantly different reductions in the proportions of households reporting bidi (adjusted OR (AOR): 1.03, 95% CI: 0.84 to 1.28) or cigarette (AOR: 1.01 to 95% CI: 0.82 to 1.26) consumption, or for the monthly per person consumption of bidi (adjusted coefficient: 0.07, 95% CI: -0.13 to 0.28) or cigarette (adjusted coefficient: -0.002, 95% CI: -0.26 to 0.26) sticks among bidi/cigarette consuming households. INTERPRETATION: Our findings indicate that early implementation of the NTCP may not have produced reductions in tobacco use reflecting generally poor performance against the Framework Convention for Tobacco Control objectives in India. This study highlights the importance of strengthening the implementation and enforcement of tobacco control policies in LMICs to achieve national and international child health and premature NCD mortality reduction targets.

Comparative evaluation of the effect of various irrigants and dry canal on electronic apex locators in locating simulated root perforations: an in vitro study / Avaliação comparativa do efeito de vários irrigantes e canal seco em localizadores apicais eletrônicos na localização de perfurações radiculares simuladas: um estudo in vitro

Objective: this study aimed to evaluate the effects of irrigants and dry canal on the accuracy of electronic apex locator (EAL) in locating simulated root perforations. Material and methods: twenty singlerooted, mandibular premolars were decoronated at CEJ, and the contents were removed with a barbed broach. The canals were instrumented up to a size of 15 K-file. The roots were artificially perforated at 4 mm from the anatomic apex. The actual length (AL) up to the perforation site was determined. The electronic length (EL) of perforations was obtained by Root ZX mini and iRoot in the dry canal and in the presence of 5.2% NaOCl, SmearOff, and 0.9% sodium chloride using a size 20 K-file. The differences between the EL and AL of the perforations were calculated. Statistical analyses using Friedman and Wilcoxon signed-rank tests were used to analyse the data with the level of significance set at p <0.05. Results: there were significant differences in different canal conditions with both Root ZX mini and i Root. Measurements in dry canals were significantly longer for both apex locators (p <0.05). Measurements with NaOCl were significantly shorter for both apex locators (p < 0.05). Both apex locators produced significantly accurate values for Saline and Smear OFF (p < 0.05). Conclusions: in this study, both Root ZX mini and i Root were affected by different canal conditions. The most accurate measurements were seen in the presence of saline and SmearOFF. (AU)

Objetivo: este estudo teve como objetivo avaliar os efeitos de irrigantes e canal seco na precisão do localizador apical eletrônico (EAL) em localizar perfurações radiculares simuladas. Material e métodos: vinte pré-molares inferiores unirradiculares tiveram suas coroas removidas na altura da JEC e o tecido pulpar removido com um extirpa nervos. Os canais foram instrumentados até a largura de uma lima k 15. As raízes foram perfuradas artificialmente a 4 mm do ápice anatômico. O comprimento real (AL) até o local da perfuração foi determinado. O comprimento eletrônico (EL) das perfurações foi obtido pelo Root ZX mini e iRoot no canal seco e na presença de 5,2% de NaOCl, SmearOff e 0,9% de cloreto de sódio usando uma lima K tamanho 20. As diferenças entre o EL e o AL das perfurações foram calculadas. Análises estatísticas, utilizando os testes de sinais por postos de Friedman e Wilcoxon, foram realizadas para analisar os dados com o nível de significância estabelecido em p < 0,05. Resultados: houve diferenças significativas nas diferentes condições do canal, tanto no Root ZX mini quanto no i Root. As medidas em canais secos foram significativamente maiores nos dois localizadores apicais (p < 0,05). As medidas com NaOCl foram significativamente mais curtas para os dois localizadores apicais (p<0,05). Ambos os localizadores apicais produziram valores significativamente precisos para Saline e Smear OFF (p < 0,05). Conclusões: neste estudo, tanto o Root ZX mini quanto o i Root foram afetados por diferentes condições do canal. As medidas mais precisa foram observadas na presença de soro fisiológico e SmearOFF. (AU)

ETS Proteins Bind with Glucocorticoid Receptors: Relevance for Treatment of Ewing Sarcoma.

The glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR's transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES.

The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

A Combination of Approved Antibodies Overcomes Resistance of Lung Cancer to Osimertinib by Blocking Bypass Pathways.

Purpose: Because of emergence of resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), no targeted treatments are available for patients with lung cancer who lose sensitivity due to new mutations or bypass mechanisms. We examined in animals and in vitro an alternative therapeutic approach making use of antibodies.Experimental Design: An osimertinib-sensitive animal model of lung cancer, which rapidly develops drug resistance, has been employed. To overcome compensatory hyperactivation of ERK, which we previously reported, an anti-EGFR antibody (cetuximab) was combined with other antibodies, as well as with a subtherapeutic dose of osimertinib, and cancer cell apoptosis was assayed.Results: Our animal studies identified a combination of three clinically approved drugs, cetuximab, trastuzumab (an anti-HER2 mAb), and osimertinib (low dose), as an effective and long-lasting treatment that is able to prevent onset of resistance to osimertinib. A continuous schedule of concurrent treatment was sufficient for effective tumor inhibition and for prevention of relapses. Studies employing cultured cells and analyses of tumor extracts indicated that the combination of two mAbs and a subtherapeutic TKI dose sorted EGFR and HER2 for degradation; cooperatively enhanced apoptosis; inhibited activation of ERK; and reduced abundance of several bypass proteins, namely MET, AXL, and HER3.Conclusions: Our in vitro assays and animal studies identified an effective combination of clinically approved drugs that might overcome resistance to irreversible TKIs in clinical settings. The results we present attribute the long-lasting effect of the drug combination to simultaneous blockade of several well-characterized mechanisms of drug resistance. Clin Cancer Res; 24(22); 5610-21. ©2018 AACR See related commentary by Fan and Yu, p. 5499.

SILAC identifies LAD1 as a filamin-binding regulator of actin dynamics in response to EGF and a marker of aggressive breast tumors.

Mutations mimicking growth factor-induced proliferation and motility characterize aggressive subtypes of mammary tumors. To unravel currently unknown players in these processes, we performed phosphoproteomic analysis on untransformed mammary epithelial cells (MCF10A) that were stimulated in culture with epidermal growth factor (EGF). We identified ladinin-1 (LAD1), a largely uncharacterized protein to date, as a phosphorylation-regulated mediator of the EGF-to-ERK pathway. Further experiments revealed that LAD1 mediated the proliferation and migration of mammary cells. LAD1 was transcriptionally induced, phosphorylated, and partly colocalized with actin stress fibers in response to EGF. Yeast two-hybrid, proximity ligation, and coimmunoprecipitation assays revealed that LAD1 bound to actin-cross-linking proteins called filamins. Cosedimentation analyses indicated that LAD1 played a role in actin dynamics, probably in collaboration with the scaffold protein 14-3-3σ (also called SFN). Depletion of LAD1 decreased the expression of transcripts associated with cell survival and inhibited the growth of mammary xenografts in an animal model. Furthermore, LAD1 predicts poor patient prognosis and is highly expressed in aggressive subtypes of breast cancer characterized as integrative clusters 5 and 10, which partly correspond to triple-negative and HER2-positive tumors. Thus, these findings reveal a cytoskeletal component that is critically involved in cell migration and the acquisition of oncogenic attributes in human mammary tumors.