Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections

The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:

Understanding barriers to sexual communication

There is consistent empirical evidence to suggest that more open and positive sexual communication is a vital component of sexual relationships, but romantic partners tend to avoid sexual communication. Although clinical and theoretical writing has identified barriers to sexual communication, there is a relative paucity of empirical literature investigating specific barriers to sexual communication and whether these barriers are unique to sexual communication. We developed the Barriers to Communication Questionnaire, a measure that investigates the types of threat that are activated during couples’ sexual and nonsexual communication (Study 1) and the degree to which these threats are differentially activated across the two discussions (Study 2). In Study 1, we found that the same categories of threats were activated for both sexual and nonsexual conflict communication: threat to self, threat to partner, and threat to relationship. Study 2 revealed that threat to self is activated to a greater degree during sexual conflict communication compared to nonsexual conflict communication. The differential degree to which threat to self is activated during sexual and nonsexual communication provides a plausible explanation for why romantic partners tend to avoid sexual communication more so than nonsexual communication. The measure developed in the current study could be used clinically to identify the specific threats that are preventing an individual from communicating with the partner about needs and desires. In the research domain, the measure can be used to further investigate the causal association between emotional barriers and sexual communication.

Abstract 427: Micrornas Have Differential Degree of Expression in Pathological Cardiac Hypertrophy Compared to Physiological Cardiac Hypertrophy

Physiological and pathological left ventricular hypertrophies (LVH) are distinct processes that have differential pattern of gene expression. Based on initial stimuli, miRs expression levels can fluctuate and then cause a variance on their targets culminating in diverse cellular pathway activation. AIM: Here we compared miRs expression between pathological cardiac hypertrophy induced by transverse aortic constriction (TAC) and physiological cardiac hypertrophy induced by voluntary exercise in running wheels (EXE). METHODS: Adult male Balb/c mice (12-14 weeks old) mice were subjected to TAC or EXE protocol and data were evaluated at 7 (TAC-7D; EXE-7D) and 35 (TAC-35D; EXE-35D) days. Hypertrophy was measured by normalizing left ventricular weight to body weight (LVW/BW). We evaluated left ventricular expression levels of miRs: -26b, 27a, -143, -150, -195 and -499 by qRT-PCR in TAC and EXE groups. Comparisons between groups were performed by ANOVA with Bonferroni correction. Results are shown as mean±SEM. Results: Sedentary and Sham groups were similar among all variables tested. Animals subjected to TAC surgery demonstrated a greater hypertrophy than EXE animals at both time points (7D: 16% vs. 7%; 35D 26% vs 12%, p<0.05 for both). MiR-26b had increased levels in TAC group at both time points (7D: 1.14±0.1 vs 0.6±0.01; 35D: 4.8±1.4 vs 1.17±0.12; p<0.01 for both). We only detected an increase in miR-27a levels in TAC-7D compared to EXE-7D (2.7±1.0 vs 0.78±0.1, p <0.05). We identified an augmentation in miR-143 levels in TAC group at both time points (7D: 1.1±0.1 vs 0.75±0.1; 35D: 1.42±0.2 vs 0.9±0.1; p<0.05 for both). We detected an increase in miR-499 levels at both time points in TAC group (7D: 4.1±0.5 vs 0.67±0.2, p<0.001; 35D: 2.2±0.4 vs 0.9±0.2, p<0.01). We found an increase in miR-195 levels only in TAC-35D group compared to EXE-35D (2.6±0.3 vs 0.9±0.1, p<0.05). We did not notice any change in miR-150 levels neither at 7 days nor at 35 days. Conclusions: These preliminary data demonstrate a differential degree of miR expression between physiological and pathological hypertrophy. Further studies comparing physiological and pathological cardiac hypertrophy are necessary to find out the turning point that deviates heart from adaptive to maladaptive growth.

Comparative Study on Workplace Collaboration across the Leading Global Organizations in IT Sector

The significant role of work place collaboration has been widely felt across the globe due to diversity and highly changing organizational dynamics. Six components emerged from the relevant management and organizational behavior regarding how managers built unity through collaboration and create a conducive environment for work and productivity. They are known as –teaming, motivating subordinate, career counseling, communicating with subordinate, professional relationships, issues related to morale and quality of work life. The present piece of work is a study of work place collaboration on 60 employees belonging from 6 different organizations. . Self-assessment inventory on work place collaboration have been used to assess those six factors. The results revealed that there is a significant gap between “what is” and “what should be” across all the six components of work place collaboration. Correlation values also suggest the differential degree of correlation between the six factors for each organization. The findings have been discussed in terms of the organizational & IT Sector's perspectives.

Diagnostic Practice in a Psychiatric Unit

This study takes the view that although psychiatric diagnosis has frequently been criticized as being deficient in terms of both reliability and validity, as long as it remains in use as a classification scheme its limitation should continue to be investigated. In line with this approach the present investigation examined three questions: What are the frequencies of major diagnostic groupings on admission to hospital? Do these frequencies change by the time discharge arrives? Are there differences in the stability of initial diagnosis for the various diagnostic sub-groupings? It was found that the most frequently used admitting diagnoses in order were: affective psychoses, neuroses and schizophrenia. Both personality disorders and other psychoses were relatively rarely used. In general the same ranking also held on discharge. However some diagnoses applied on admission appeared much more stable than others. For example 80 percent of those patients initially diagnosed as schizophrenic remained so classified on discharge. In contrast the equivalent figure for diagnoses listed under ‘other conditions’ was 46 percent. Although practitioners often talk as though diagnosis is a meaningless ritual, in fact the present study suggests a differential degree to selection and consideration in their application of psychiatric categories.