Synthesis of N2-trans-isosafrole-dG-adduct Bearing DNAs and the Bypass Studies with Human TLS Polymerases κ and η.

Safrole is a natural product present in many plants and plant products, including spices and essential oils. During cellular metabolism, it converts to a highly reactive trans-isosafrole (SF) intermediate that reacts with genomic DNA and forms N2-SF-dG and N6-SF-dA DNA adducts, which are detected in the oral tissue of cancer patients with betel quid chewing history. To study the SF-induced carcinogenesis and to probe the role of low fidelity translesion synthesis (TLS) polymerases in bypassing SF adducts, herein, we report the synthesis of N2-SF-dG modified DNAs using phosphoramidite chemistry. The N2-SF-dG modification in the duplex DNA does not affect the thermal stability and retains the B-form of helical conformation, indicating that this adduct may escape the radar of common DNA repair mechanisms. Primer extension studies showed that the N2-SF-dG adduct is bypassed by human TLS polymerases hpolκ and hpolη, which perform error-free replication across this adduct. Furthermore, molecular modeling and dynamics studies revealed that the adduct reorients to pair with the incoming nucleotide, thus allowing the effective bypass. Overall, the results indicate that hpolκ and hpolη do not distinguish the N2-SF-dG adduct, suggesting that they may not be involved in the safrole-induced carcinogenicity.

Human Translesion Synthesis Polymerases polκ and polη Perform Error-Free Replication across N2-dG Methyleugenol and Estragole DNA Adducts.

The secondary metabolites of polypropanoids, methyleugenol (MEG), and estragole (EG), found in many herbs and spices, are commonly used as food flavoring agents and as ingredients in cosmetics. MEG and EG have been reported to cause hepatocarcinogenicity in rodents, human livers, and lung cells. The formation of N2-dG and N6-dA DNA adducts is primarily attributed to the carcinogenicity of these compounds. Therefore, these compounds have been classified as "possible human carcinogens" by the International Agency for Research on Cancer and "reasonably anticipated to be a human carcinogen" by the National Toxicology Program. Herein, we report the synthesis of the N2-MEG-dG and N2-EG-dG modified oligonucleotides to study the mutagenicity of these DNA adducts. Our studies show that N2-MEG-dG and N2-EG-dG could be bypassed by human translesion synthesis (TLS) polymerases hpolκ and hpolη in an error-free manner. The steady-state kinetics of dCTP incorporation by hpolκ across N2-MEG-dG and N2-EG-dG adducts show that the catalytic efficiencies (kcat/Km) were ∼2.5- and ∼4.4-fold higher, respectively, compared to the unmodified dG template. A full-length primer extension assay demonstrates that hpolκ exhibits better catalytic efficiency than hpolη. Molecular modeling and dynamics studies capturing pre-insertion, insertion, and post-insertion steps reveal the structural features associated with the efficient bypass of the N2-MEG-dG adduct by hpolκ and indicate the reorientation of the adduct in the active site allowing the successful insertion of the incoming nucleotide. Together, these results suggest that though hpolκ and hpolη perform error-free TLS across MEG and EG during DNA replication, the observed carcinogenicity of these adducts could be attributed to the involvement of other low fidelity polymerases.

Rare case of high-grade endometrial stromal sarcoma metastasising to inferior vena cava and right atrium.

Endometrial stromal sarcomas (ESSs) are a rare form of uterine malignancy representing <10% of all uterine sarcomas and <1% of all primary malignant tumours of the uterus. Invasion of the vascular system by low-grade ESS has been reported in the literature. Here we report the first case of a high-grade ESS invading the pelvic and gonadal vein and extending through the inferior vena cava to the right atrium, the diagnostic challenges and multidisciplinary management of the case.

Calcifying epithelial odontogenic tumour of the maxilla - A rare case report.

A calcifying epithelial odontogenic tumour (CEOT) is a rare benign odontogenic tumour, which was first described by J. J. Pindborg in 1955, hence it is also called the Pindborg tumour. It constitutes less than 1% of the entire odontogenic tumours and 0.4-3.0% of all intraosseous tumours. The origin of CEOT is still unclear. It may be derived from the oral epithelium, reduced enamel epithelium, stratum intermedium or remnants of the primitive dental lamina. Surgical management is usually enucleation and the recurrence rate is 15%. In this case report, we have described a case of CEOT that occurred in the left posterior region of the maxilla in a young adult male which clinically appeared as a peripheral lesion but radiological findings showed a central variant.

Knowledge, Attitudes, and Perceived Barriers of Healthcare Providers toward Early Mobilization of Adult Critically Ill Patients in Intensive Care Unit.

BACKGROUND: Early mobilization (EM) of critically ill adult patients in intensive care units (ICUs) is a newer concept. It is known to improve overall outcomes, but little is known regarding the attitude and knowledge of healthcare providers (HCPs) and multidisciplinary barriers to its use in the Indian scenario. AIMS AND OBJECTIVES: To study the knowledge and attitude of HCPs in ICU about the EM of adult patients who are critically ill and identify perceived barriers to the application of EM. MATERIALS AND METHODS: In a cross-sectional survey conducted in a tertiary care academic institute, the HCPs, namely, physicians, anesthetists, surgeons, nursing staff, and physiotherapists working in ICU were interviewed using a self-structured questionnaire. The data were presented as descriptive statistics. RESULTS: There was 80% response rate. The benefits of EM as shortened length of mechanical ventilation (MV) were acknowledged by 78% respondents and 54% believed that it maintains muscle strength. It was considered crucial by 44% respondents, who opined that it should be started as the patient's cardiorespiratory status stabilizes. The favorable attitudes observed were recognition of benefits for patients under MV exceeded the risks and readiness by physicians to reduce sedation levels and change the parameters of MV. The main barriers identified were the absence of written guidelines or protocols for EM, limited staff to mobilize patients, inadequate training of HCP to facilitate EM, excessive sedation, and medical instability. CONCLUSION: There exists an awareness of the benefits of EM and favorable attitudes to its application. However, the actual performance of EM was perceived as a challenge due to barriers identified in the study. HOW TO CITE THIS ARTICLE: Akhtar PM, Deshmukh PK. Knowledge, Attitudes, and Perceived Barriers of Healthcare Providers toward Early Mobilization of Adult Critically Ill Patients in Intensive Care Unit. Indian J Crit Care Med 2021;25(5):512-518.

Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells.

Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer (CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1 cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is epigenetically regulated by AR.

Insulin signaling pathway protects neuronal cell lines by Sirt3 mediated IRS2 activation.

Cellular stress like ER and oxidative stress are the principle causative agents of various proteinopathies. Multifunctional protein PARK7/DJ-1 provides protection against cellular stress. Recently, insulin/IGF also has emerged as a neuro-protective molecule. However, it is not known whether DJ-1 and insulin/IGF complement each other for cellular protection in response to stress. In this study, we show for the first time, that in human and mouse neuronal cell lines, down regulation of DJ-1 for 48 h leads to compensatory upregulation of insulin/IGF signaling (IIS) pathway genes, namely, insulin receptor, insulin receptor substrate, and Akt under normal physiological conditions as well as in cellular stress conditions. Moreover, upon exogenous supply of insulin there is a marked increase in the IIS components both at gene and protein levels leading to down regulation and inactivation of GSK3ß. By immunoprecipitation, it was observed that Sirt3 mediated deacetylation and activation of FoxO3a could not occur under DJ-1 downregulation. Transient DJ-1 downregulation also led to Akt mediated increased phosphorylation and nuclear exclusion of FoxO3a. When DJ-1 was downregulated increased interaction of Sirt3 with IRS2 was observed leading to its activation resulting in IIS upregulation. Thus, transient downregulation of DJ-1 leads to stimulation of IIS pathway by Sirt3 mediated IRS2 activation. Consequently, antiapoptotic program is triggered in neuronal cells via Akt-GSK3ß-FoxO3a axis. © 2018 BioFactors, 44(3):224-236, 2018.

Enhancing clinical measures of postural stability with wearable sensors.

About 30% of individuals over the age of 65, and 50% over age 80, fall at least once per year. Fall-related injuries cost the Canadian health care system $2.8 billion annually. Risk for falls in older adults is commonly assessed in the clinical environment using tools such as the Short Physical Performance Battery (SPPB), which include subjective assessments of postural sway while standing under various sensory conditions. This study uses wearable accelerometers and a force plate to quantify measures of postural stability during these tasks. Four participants were asked to maintain quiet stance in six different conditions, while their center of pressure (COP) and accelerations from six accelerometers were recorded. Standard deviations in signals were used as measures of postural sway. The sway observed in all sensors increased with the difficulty of the stance condition. Manipulation of vision and surface stiffness caused greater changes in sway in the AP than ML direction, while changes in stance configuration were more evident in the ML direction. Furthermore, the ankle sensor was the most sensitive in registering changes in sway when manipulating vision and surface stiffness (showing an increase of 236% over baseline values in AP sway with eyes closed and standing on foam), while the thigh was most sensitive to changes in stance width (showing an increase of 336% over baseline values in ML sway in the tandem stance condition). This study contributes in establishing the utility of wearable sensors for quantifying postural stability under various stance configurations in future studies with high-risk older adults.