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A hybridization assay for the detection of microRNA, miR21 in cancer cells using the bioluminescent enzyme Renilla luciferase (Rluc) as a label, has been developed. MicroRNAs are small RNAs found in plants, animals, and humans that perform key functions in gene silencing and affect early-stage cell development, cell differentiation, and cell death. miRNAs are considered useful early diagnostic and prognostic markers of cancer, candidates for therapeutic intervention, and targets for basic biomedical research. However, methods for highly sensitive and rapid detection of miRNA directly from samples such as cells that can serve as a suitable diagnostics platform are lacking. In that regard, the utilization of the bioluminescent label, Rluc, that offers the advantage of high signal-to-noise ratio, allows for the development of highly sensitive assays for the determination of miRNA in a variety of matrixes. In this paper, we have described the development of a competitive oligonucleotide hybridization assay for the detection of miR21 using the free miR21 and Rluc-labeled miR21 that competes to bind to an immobilized miR21 complementary probe. The miR21 microRNA chosen for this study is of biomedical significance because its levels are elevated in a variety of cancers. Using the optimized assay, a detection limit of 1 fmol was obtained. The assay was employed for the detection of miR21 in human breast adenocarcinoma MCF-7 cells and nontumorigenic epithelial MCF-10A cells. The comparison of miR21 expression level in two cell lines demonstrated higher expression of miR21 in breast cancer cell line MCF-7 compared to the nontumorigenic MCF-10A cells. Further, using the assay developed, the miR21 quantification could be performed directly in cell extracts. The hybridization assay was developed in a microplate format with a total assay time of 1.5 h and without the need for sample PCR amplification. The need for early molecular markers and their detection methods in cancer diagnosis is tremendous. The characteristics of the assay developed in this work show its suitability for early cancer diagnosis based on miRNA as a biomarker.

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IntroductionComparative data on the burden of atopic dermatitis (AD) in adults relative to the general population are limited. We performed a large-scale evaluation of the burden of disease among US adults with AD relative to matched non-AD controls, encompassing comorbidities, healthcare resource utilization (HCRU), and costs, using healthcare claims data. The impact of AD disease severity on these outcomes was also evaluated.MethodsAdult AD patients in the Commercial (n = 83,106), Medicare (n = 31,060), and Medi-Cal (n = 5550) databases were matched (1:1) to non-AD controls by demographic characteristics. AD patients were stratified by disease severity (higher, lower) using treatment as a surrogate measure of severity. The comorbidity burden, HCRU, and costs were evaluated during a 12-month follow-up period.ResultsIn the Commercial, Medicare, and Medi-Cal populations, patients with AD had a significantly higher overall comorbidity burden (P < 0.0001), an increased risk of asthma and allergic rhinitis (both P < 0.0001), higher HCRU (P < 0.05), and higher mean total per patient costs (Commercial: US$10,461 versus US$7187; Medicare: US$16,914 versus US$13,714; Medi-Cal; US$19,462 versus US$10,408; all P < 0.0001), compared with matched non-AD controls. Higher disease severity was associated with an increased comorbidity burden (P < 0.0001), HCRU (P < 0.05), and total costs (Commercial: US$14,580 versus US$7192; Medicare: US$21,779 versus US$12,490; Medi-Cal; US$22,123 versus US$16,639; all P < 0.0001) relative to lower severity disease.ConclusionIn this large-scale, healthcare claims database analysis, AD patients had a significantly higher comorbidity burden, HCRU, and costs compared with matched non-AD controls. Higher disease severity was associated with an even greater comorbidity and economic burden.FundingSanofi and Regeneron Pharmaceuticals, Inc.Electronic supplementary materialThe online version of this article (doi:10.1007/s12325-017-0582-z) contains supplementary material, which is available to authorized users.

The red fluorescent protein, DsRed, and a few of its mutants have been shown to bind copper ions resulting in quenching of its fluorescence. The response to Cu 2+ is rapid, selective, and reversible upon addition of a copper chelator. DsRed has been employed as an in vitro probe for Cu 2+ determination by us and other groups. It is also envisioned that DsRed can serve as an intracellular genetically encoded indicator of Cu 2+ concentration, and can be targeted to desired subcellular locations for Cu 2+ determination. However, no information has been reported yet regarding the mechanism of the fluorescence quenching of DsRed in the presence of Cu 2+ . In this work, we have performed spectroscopic investigations to determine the mechanism of quenching of DsRed fluorescence in the presence of Cu 2+ . We have studied the effect of Cu 2+ addition on two representative mutants of DsRed, specifically, DsRed-Monomer and DsRed-Express. Both proteins bind Cu 2+ with micromolar affinities. Stern-Volmer plots generated at different temperatures indicate a static quenching process in the case of both proteins in the presence of Cu 2+ . This mechanism was further studied using absorption spectroscopy. Stern-Volmer constants and quenching rate constants support the observation of static quenching in DsRed in the presence of Cu 2+ . Circular dichroism (CD)-spectroscopic studies revealed no effect of Cu 2+ -binding on the secondary structure or conformation of the protein. The effect of pH changes on the quenching of DsRed fluorescence in the presence of copper resulted in pKa values indicative of histidine and cysteine residue involvement in Cu 2+ binding.

Introduction: Typhoid fever is one of the most common public health problems in Nepal. It occurs in all parts of the world where water supplies and sanitation are sub-standard. In Dhulikhel hospital, this is one of the top acute febrile illnesses in inpatient department. The objectives of this study were to evaluate the clinical and laboratory parameters including culture and sensitivity, the response to therapy, and complications of enteric fever among child cases at Dhulikhel Hospital. . Statistical analysis was done with SPSS. Results: There were total of 138 cases of enteric fever admitted. There were 73 (53%) male and 65 (47%) female. Eighty-one percent were above five years of age. The most common clinical presentation was fever (100%) followed by headache and G I symptoms. Hepatomegaly was the most common sign seen among the cases and was seen in 110cases (79.71%). Most of the patients had normal WBC count 100 (72.46%) Widal test was positive in 70 (50.72%) cases and blood culture was positive in 52(37.68%) cases. Nalidixic acid was found to be resistant in 26 (50%) cases. Complications were seen in only 7 (5%) enteric fever cases. Conclusion: Typhoid fever is predominant in school going children in Nepal with slight male predominance. Fever lasting over 3 days followed by headache and GI symptoms are the major presenting symptoms. In making the diagnosis, the isolation of bacteria from blood is the "gold standard". Nalidixic acid resistant Salmonella typhi is on the increasing trend. Pneumonia was found to be the most common complication among all other complications seen in enteric cases. In Dhulikhel Hospital this is one of the top acute febrile illnesses in inpatient department.

Inappropriate dosing occurred among patients with normal and insufficient renal function. The consideration of clinical factors beyond renal function is necessary to reduce bleeding risk associated with DOAC therapy.

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