Paraffin-embedded tissue as a source of RNA for gene expression analysis in oral malignancy

OBJECTIVE: To assess the feasibility of using archival oral mucosal tissue to examine gene expression at the ribonucleic acid (RNA) level.
MATERIALS AND METHODS: We describe the isolation of RNA from 8 nm sections of formalin-fixed paraffin-embedded oral mucosal tissue. RNA was reverse transcribed and three candidate genes amplified by polymerase chain reaction (PCR). The ribosomal protein S14 gene is a housekeeping gene which has been used as an internal standard in several quantitative PCR protocols. The thymidine kinase (TK) gene is expressed at low levels in most tissues and, with a well-documented genomic organisation, is a useful tool for discrimination between genomic DNA and cDNA. The RIa gene is reported to be overexpressed in many cancer cell lines, in malignant tissue and in vitro transformed cellS. RESULTS: The S14 gene, the TK gene and the RIα gene of the cAMP-dependent protein kinase (PKA) were amplified successfully from formalin-fixed paraffin-embedded tissue sections. The TK primer pair is a useful additional tool in the unambiguous identification of RNA-derived species.
CONCLUSION: RNA suitable for reverse transcribed (RT)-PCR was extracted from archival oral mucosal tissue. This should permit rapid sequence analysis of transcribed tumor suppressor genes and oncogenes in this material. Furthermore, the RT-PCR approach described may allow quantification of gene expression in oral mucosal archival material processed in a standard fashion.     

Thoracoscopic Dorsal Sympathectomy for Upper Limb Buerger's Disease

Background and Objectives:

Thromboangiitis obliterans is a common peripheral vascular disease in India. This study was conducted to assess the efficacy of thoracoscopic dorsal sympathectomy as a treatment for Buerger disease of the upper extremities.

Methods:

Thirty thoracoscopic dorsal sympathectomies (17 left- and 13 right-sided) were performed in a tertiary medical center in 5 women and 20 men (mean age, 41 years) between July 2010 and February 2013.

Results:

The mean operative time was 30 minutes, and the mean hospital stay was 52 hours. There were no complications. All patients had improvement in pain and were relapse-free after a mean follow-up period of 11.63 months.

Discussion:

Thoracoscopic dorsal sympathectomy reduces pain significantly by reducing peripheral resistance and promoting collateral development. The increased magnification of the thoracoscopic approach permits better visualization, ensuring complete excision and therefore good results. Thoracoscopic dorsal sympathectomy for Buerger disease of the upper limb is a safe and effective treatment.     

Stimulated tyrosine phosphorylation of phosphatidylinositol 3-kinase causes acidic pH-induced contraction in spontaneously hypertensive rat aorta

Acidic pH induced a contraction (APIC) in isolated aortas from spontaneously hypertensive (SHR) and Wistar Kyoto rats, but failed to produce any response in age-matched Wistar rat aorta. This study was conducted to test the hypothesis that tyrosine phosphorylation of proteins is a molecular mechanism underlying the APIC. Tyrosine kinase inhibitors, genistein and tyrphostin 23 inhibited the APIC in a concentration-dependent manner. APIC was inhibited by phosphatidylinositol 3-kinase (PI3-kinase) inhibitors, LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride] and wortmannin. Consistent with the results from tension measurement experiments, Western blot analysis showed that acidic pH induced an appreciable increment of tyrosine phosphorylation of 85-kDa protein (p85) in SHR aorta, which was completely inhibited by tyrphostin 23, whereas in Wistar rat aorta, the protein tyrosine phosphorylation was not observed. Further investigations using immunoprecipitation followed by Western blotting confirmed an increase in the tyrosine phosphorylation of p85. Analysis by SDS-polyacrylamide gel electrophoresis followed by silver staining of the gel revealed that amounts of multiple proteins with molecular sizes of 120, 130, 210, and 225 kDa were increased at acidic pH, which were immunoprecipitated with anti-phosphotyrosine antibody. Western blotting using a specific anti-PI3-kinase antibody identified the p85 as the regulatory subunit of PI3-kinase, whereas 120-, 130-, and 225-kDa proteins were identified by mass spectrometry as pro-alpha2 (I) collagen, collagen alpha1 (I) chain, and fibernectin I, respectively. As assayed by Western blotting using anti-myosin light chain (MLC) antibody, acidic pH induced a stimulation of MLC phosphorylation, and the stimulated MLC phosphorylation was abolished by tyrphostin 23 and LY-294002. These results suggest that acidic pH induces an increase in tyrosine phosphorylation of PI3-kinase, resulting in the MLC phosphorylation-dependent contraction of SHR aorta.     

Derivatives Form Better Lipoxygenase Inhibitors than Piperine: In Vitro and In Silico Study

Piperine is a secondary metabolite of black pepper. Its uses in medicine were already studied. However, its derivatives have not gained considerable attention. In the presented study, the Lipoxygenase (LOX) inhibitory activity of piperine and its derivatives, piperonylic acid, piperic acid, and piperonal have been assessed and compared by enzyme kinetics, ITC and molecular modeling experiments. The presented investigations expressed that all the studied compounds inhibited LOX by binding at its active site. The IC₅₀ values of these compounds were deduced from the kinetics data and found to be 85.79, 43.065, 45.17, and 50.78 μm for piperine, piperonylic acid, piperic acid, and piperonal, respectively. The binding free energies obtained from ITC experiments were −7.47, −8.33, −8.09, and −7.86 kcal/mol for piperine, piperonylic acid, piperic acid, and piperonal, respectively. Similarly, the glide scores obtained for piperine, piperonylic acid, piperic acid, and piperonal were −7.28, −10.32, −10.72, and −9.57 kcal/mol, respectively. The results of ITC and molecular modeling experiments suggested that piperonylic acid and piperonal exhibit stronger binding at the active site than piperine does. From the presented studies, it could be concluded that derivatives of piperine may be of higher significance than piperine for certain medicinal applications, implicating (Ayurvedic) fermented herbal drugs with piperine in them.     

An evaluation of two large scale demand side financing programs for maternal health in India: the MATIND study protocol

ABSTRACT: BACKGROUND: High maternal mortality in India is a serious public health challenge. Demand side financing interventions have emerged as a strategy to promote access to emergency obstetric care. Two such state run programs, Janani Suraksha Yojana (JSY)and Chiranjeevi Yojana (CY), were designed and implemented to reduce financial access barriers that preclude women from obtaining emergency obstetric care. JSY, a conditional cash transfer, awards money directly to a woman who delivers in a public health facility. This will be studied in Madhya Pradesh province. CY, a voucher based program, empanels private obstetricians in Gujarat province, who are reimbursed by the government to perform deliveries of socioeconomically disadvantaged women. The programs have been in operation for the last seven years.Methods/designsThe study outlined in this protocol will assess and compare the influence of the two programs on various aspects of maternal health care including trends in program uptake, institutional delivery rates, maternal and neonatal outcomes, quality of care, experiences of service providers and users, and cost effectiveness. The study will collect primary data using a combination of qualitative and quantitative methods, including facility level questionnaires, observations, a population based survey, in-depth interviews, and focus group discussions. Primary data will be collected in three districts of each province. The research will take place at three levels: the state health departments, obstetric facilities in the districts and among recently delivered mothers in the community. DISCUSSION: The protocol is a comprehensive assessment of the performance and impact of the programs and an economic analysis. It will fill existing evidence gaps in the scientific literature including access and quality to services, utilization, coverage and impact. The implementation of the protocol will also generate evidence to facilitate decision making among policy makers and program managers who currently work with or are planning similar programs in different contexts.     

Midwifery scope of practice among staff nurses: A grounded theory study in Gujarat,India

Backgroundmidwifery is a part of the nursing profession in India. This current study explores and describes the midwifery scope of practice among staff nurses.Methodsa grounded theory approach was used to develop a model. Twenty-eight service providers from the maternity sections of public health facilities, selected through purposive and theoretical sampling were interviewed in-depth. Unstructured observations in the labour wards were also used for developing the model.Findingsthe midwifery practice of staff nurses was limited in scope compared to international standards of midwifery. Their practice was circumstance driven, ranging from extended to marginal depending on the context. Their right to practice was not legally defined, but they were not specifically prohibited from practice. As a consequence, the staff nurses faced loss of skills, and deskilling when their practice was restricted. Their practice was perceived as risky, when the scope of practice was extended because it was not rightfully endorsed, the nurses having no officially recognized right to practice midwifery at that level. The clinical midwifery education of nursing and midwifery students was marginalized because the education of medical students was given priority, and the students only got exposed to the restricted practice of staff nurses.Conclusionsunclear definitions of the right to practice and the scope of practice have led to the un-utilized potential of staff nurses practising midwifery. This is detrimental because India faces an acute shortage of qualified personnel to meet the need in providing human resources for maternal health.