Biplex quantitative PCR to detect transcriptionally active human papillomavirus 16 from patient saliva.

Head and neck cancers, particularly oropharyngeal cancers (OPC), have been increasingly associated with human papillomavirus (HPV) infections, specifically HPV16. The current methods for HPV16 detection primarily rely on p16 staining or PCR techniques. However, it is important to note the limitations of conventional PCR, as the presence of viral DNA does not always indicate an ongoing viral infection. Moreover, these tests heavily rely on the availability of tissue samples, which can present challenges in certain situations. In this study, we developed a RT-qPCR biplex approach to detect HPV16 oncogenes E6 and E7 RNA in saliva samples from OPC patients. Salivary supernatant was used as the liquid biopsy source. We successfully obtained RNA from salivary supernatant, preserving its integrity as indicated by the detection of several housekeeping genes. Our biplex approach accurately detected E6 and E7 RNA in HPV16-positive cell lines, tissues, and finally in OPC salivary samples. Importantly, the assay specifically targeted HPV16 and not HPV18. This biplexing technique allowed for reduced sample input without compromising specificity. In summary, our approach demonstrates the potential to detect viable HPV16 in saliva from OPC patients. Since the assay measures HPV16 RNA, it provides insights into the transcriptional activity of the virus. This could guide clinical decision-making and treatment planning for individuals with HPV-related OPC.

miRNA:miRNA Interactions: A Novel Mode of miRNA Regulation and Its Effect On Disease.

MicroRNAs (miRNAs) are known for their role in the post-transcriptional regulation of messenger RNA (mRNA). However, recent evidence has shown that miRNAs are capable of regulating non-coding RNAs, including miRNAs, in what is known as miRNA:miRNA interactions. There are three main models for the interplay between miRNAs. These involve direct interaction between two miRNAs, either in their mature or primary form, the subsequent changes in miRNA expression due to miRNA-directed transcriptional changes, and the cell-wide impact on miRNA and mRNA levels as a result of miRNA manipulation. Networks of mRNA and miRNA regulatory connections are invaluable to the discovery of miRNA:miRNA pathways, but this cannot be applied without consideration of the specific cell type or condition.In this chapter, we discuss what is understood about miRNA:miRNA interactions, their mechanisms and consequences in disease biology, and suggest further avenues of investigation based on current gaps in the literature and in our understanding of miRNA biology. We also address the pitfalls in contemporary methods relating to the identification of miRNA:miRNA interactions. Future work in this area may ultimately change the definitional role of miRNAs, and have far-reaching impacts on therapeutic and diagnostic developments.

Factors in the clinical learning environment that influence caring behaviors of undergraduate nursing students: An integrative review.

AIM: The aim is to explore literature on the influence of the clinical learning environment on caring behaviors of undergraduate nursing students. BACKGROUND: Caring is a fundamental aspect of nursing practice. However, factors of and in clinical learning environment that help shape the caring behaviors of nursing students as part of their education journey remain understudied. DESIGN: A modified version of Cooper's five-stage integrative review method was used. METHODS: Four databases (Cumulative Index of Nursing and Allied Health, PubMed, Scopus and Embase) were searched for research studies published from 2011 to 2021 in peer reviewed journals, written in English and addressing caring behaviors among nursing students in the clinical learning environment. A combination of keywords with Boolean operators was used including: "nursing students OR nursing undergraduates OR student nurses" and "clinical learning environment" AND "caring behaviors". Reporting followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement guidelines. RESULTS: Empirical evidence was drawn from 11 studies including seven qualitative designs, three quantitative designs and one mixed method design. The results of this review suggest that factors in the clinical learning environment influence nursing students' ability to develop caring behaviors. Specifically, the five themes of: [1] role modeling of clinical faculty and professional nurses, [2] creating a conducive clinical learning environment, [3] effective communication skills, [4] positive effect of simulation and [5] alternative clinical placements may facilitate the development of caring behaviors among nursing students. CONCLUSION: The findings highlight the factors in the clinical learning environment that influence nursing students' caring behaviors. Improving students' clinical learning experiences and implementing more effective role modeling and teaching strategies may advance their caring abilities. The information generated from this review provides evidence on how to enhance the clinical learning environment to develop students' caring behaviors, subsequently leading to more optimal patient outcomes.

miRNA interplay: mechanisms and consequences in cancer.

Canonically, microRNAs (miRNAs) control mRNA expression. However, studies have shown that miRNAs are also capable of targeting non-coding RNAs, including long non-coding RNAs and miRNAs. The latter, termed a miRNA:miRNA interaction, is a form of self-regulation. In this Review, we discuss the three main modes of miRNA:miRNA regulation: direct, indirect and global interactions, and their implications in cancer biology. We also discuss the cell-type-specific nature of miRNA:miRNA interactions, current experimental approaches and bioinformatic techniques, and how these strategies are not sufficient for the identification of novel miRNA:miRNA interactions. The self-regulation of miRNAs and their impact on gene regulation has yet to be fully understood. Investigating this hidden world of miRNA self-regulation will assist in discovering novel regulatory mechanisms associated with disease pathways.

Global miRNA to miRNA Interactions: Impacts for miR-21.

miRNAs inherently alter the cellular environment by regulating target genes. miRNAs may also regulate other miRNAs, with far-reaching influence on miRNA and mRNA expression. We explore this realm of small RNA regulation with a focus on the role of the oncogenic miR-21 and its impact on other miRNA species.

Developing a virus-microRNA interactome using cytoscape.

It is currently difficult to determine the effect of oncogenic viruses on the global function and regulation of pathways within mammalian cells. A thorough understanding of the molecular pathways and individual genes altered by oncogenic viruses is needed for the identification of targets that can be utilised for early diagnosis, prevention, and treatment methods. We detail a logical step-by-step guide to uncover viral-protein-miRNA interactions using publically available datasets and the network building program, Cytoscape. This method may be applied to identify specific pathways that are altered in viral infection, and contribute to the oncogenic transformation of cells. To demonstrate this, we constructed a gene regulatory interactome encompassing Human Papillomavirus Type 16 (HPV16) and its control of specific miRNAs. This approach can be broadly applied to understand and map the regulatory functions of other oncogenic viruses, and determine their role in altering the cellular environment in cancer. Availability and Implementation Cytoscape (Shannon et al. (2003), Smoot et al. (2010)) is freely available at https://cytoscape.org/. •This method allows for the analysis and visualization of large datasets to generate an interactome that integrates key players of molecular biology•This approach may be applied to any oncogenic virus to map its regulatory functions, and its secondary impact on gene regulation via microRNAs.

MicroRNA (miRNA)-to-miRNA Regulation of Programmed Cell Death 4 (PDCD4).

The regulation of tumor suppressor genes by microRNAs (miRNAs) is often demonstrated as a one-miRNA-to-one-target relationship. However, given the large number of miRNA sites within a 3' untranslated region (UTR), most targets likely undergo miRNA cooperation or combinatorial action. Programmed cell death 4 (PDCD4), an important tumor suppressor, prevents neoplastic events and is commonly downregulated in cancer. This study investigates the relationship between miRNA 21 (miR-21) and miR-499 in regulating PDCD4. This was explored using miRNA overexpression, mutational analysis of the PDCD4 3' UTR to assess regulation at each miRNA site, and 50% inhibitory concentration (IC50) calculations for combinatorial behavior. We demonstrate that the first miR-499 binding site within PDCD4 is inactive, but the two remaining sites are both required for PDCD4 suppression. Additionally, the binding of miR-21 to PDCD4 influenced miR-499 activity through an increase in its silencing potency and stabilization of its mature form. Furthermore, adjoining miRNA sites more than 35 nucleotides (nt) apart could potentially regulate thousands of 3' UTRs, similar to that observed between miR-21 and miR-499. The regulation of PDCD4 serves as a unique example of regulatory action by multiple miRNAs. This relationship was predicted to occur on thousands of targets and may represent a wider mode of miRNA regulation.

Human papillomavirus 16 E6 modulates the expression of miR-496 in oropharyngeal cancer.

Human papillomavirus (HPV), notably type 16, is a risk factor for up to 75% of oropharyngeal squamous cell carcinomas (SCC). It has been demonstrated that small non-coding RNAs known as microRNAs play a vital role in the cellular transformation process. In this study, we used an LNA array to further investigate the impact of HPV16 on the expression of microRNAs in oropharyngeal (tonsillar) cancer. A number of miRNAs were found to be deregulated, with miR-496 showing a four-fold decrease. Over-expression of the high risk E6 oncoprotein down-regulated miR-496, impacting upon the post-transcriptional control of the transcription factor E2F2. These HPV specific miRNAs were integrated with the HPV16 interactome to identify possible mechanistic pathways. These analyses provide insights into novel molecular interactions between HPV16 and miRNAs in oropharyngeal cancers.

MicroRNAs Regulating MicroRNAs in Cancer.

MicroRNAs (miRNA) are capable of self-regulation, termed miRNA to miRNA interaction. Very little is known about these interactions and their impact on the cellular milieu. We discuss known miRNA to miRNA interactions, potential mechanisms, and their role in cancer.

A 9-state analysis of designer stimulant, "bath salt," hospital visits reported to poison control centers.

STUDY OBJECTIVE: A new generation of designer stimulants marketed as "bath salts" emerged in late 2010. The goal is to describe the epidemiologic emergence of designer stimulants in 9 states in the Midwest. METHODS: A retrospective review of the National Poison Data System was performed between November 1, 2010, and November 30, 2011. Inclusion criteria were health care-evaluated bath salts or other synthetic stimulants exposures. Cases were excluded if the exposure was unrelated to a designer stimulant. Demographic and clinical characteristics of cases were calculated and differences in outcome and exposure by generation were examined. RESULTS: One thousand six hundred thirty-three patients met the inclusion criteria. Age ranged from 1 day to 61 years (mean=29.2 years), with 67.9% male patients. The most common clinical features were agitation (62.2%), tachycardia (55.2%), and hallucinations (32.7%). In addition to 15.5% of patients having a major medical effect, 0.6% died. Reason for use was primarily intentional abuse (88.5%). However, 0.7% of patients reported withdrawal. Treatment involved primarily benzodiazepines (58.5%), with 8.7% of patients being intubated. Baby Boomers were more likely to have a major medical outcome (24.2%) and to report injection as the method of administration (8.6%-12.9%). CONCLUSION: Synthetic stimulants rapidly swept across the Midwest, resulting in more than 1,600 patients seeking medical care. Serious medical effects or death was observed in 16.1% of cases. Older generations were more likely to inject and to have a major medical outcome.

Recruitment of transcription complexes to the beta-globin gene locus in vivo and in vitro.

Erythroid-specific, high level expression of the beta-globin genes is regulated by the locus control region (LCR), composed of multiple DNase I-hypersensitive sites and located far upstream of the genes. Recent studies have shown that LCR core elements recruit RNA polymerase II (pol II). In the present study we demonstrate the following: 1) pol II and other basal transcription factors are recruited to LCR core hypersensitive elements; 2) pol II dissociates from and re-associates with the globin gene locus during replication; 3) pol II interacts with the LCR but not with the beta-globin gene prior to erythroid differentiation in embryonic stem cells; and 4) the erythroid transcription factor NF-E2 facilitates the transfer of pol II from immobilized LCR constructs to a beta-globin gene in vitro. The data are consistent with the hypothesis that the LCR serves as the primary attachment site for the recruitment of macromolecular complexes involved in chromatin structure alterations and transcription of the globin genes.