Single-Pair Fluorescence Resonance Energy Transfer Analysis of mRNA Transcripts for Highly Sensitive Gene Expression Profiling in Near Real Time

Expression analysis of mRNAs transcribed from certain genes can be used as important sources of biomarkers for <i>in vitro</i> diagnostics. While the use of reverse transcription quantitative PCR (RT-qPCR) can provide excellent analytical sensitivity for monitoring transcript numbers, more sensitive approaches for expression analysis that can report results in near real-time are needed for many critical applications. We report a novel assay that can provide exquisite limits-of-quantitation and consists of reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) to provide digital readout through molecular counting. For this assay, no PCR was employed, which enabled short assay turnaround times. To facilitate implementation of the assay, a cyclic olefin copolymer (COC) microchip, which was fabricated using hot embossing, was employed to carry out the LDR in a continuous flow format with online single-molecule detection following the LDR. As demonstrators of the assay’s utility, MMP-7 mRNA was expression profiled from several colorectal cancer cell lines. It was found that the RT-LDR/spFRET assay produced highly linear calibration plots even in the low copy number regime. Comparison to RT-qPCR indicated a better linearity over the low copy number range investigated (10–10 000 copies) with an <i>R</i>² = 0.9995 for RT-LDR/spFRET and <i>R</i>² = 0.98 for RT-qPCR. In addition, differentiating between copy numbers of 10 and 50 could be performed with higher confidence using RT-LDR/spFRET. To demonstrate the short assay turnaround times obtainable using the RT-LDR/spFRET assay, a two thermal cycle LDR was carried out on amphiphysin gene transcripts that can serve as important diagnostic markers for ischemic stroke. The ability to supply diagnostic information on possible stroke events in short turnaround times using RT-LDR/spFRET will enable clinicians to treat patients effectively with appropriate time-sensitive therapeutics

DataSheet_1_Kidney function and cardiovascular diseases: a large-scale observational and Mendelian randomization study.docx

BackgroundPrior observational studies have found an association between kidney function and cardiovascular diseases (CVDs). However, these studies did not investigate causality. Therefore, the aim of this study is to examine the causal relationship between kidney function and CVDs.MethodsWe utilized data from the eICU Collaborative Research Database (eICU-CRD) from the years 2014-2015 to evaluate the observational association between renal failure (RF) and CVDs. To investigate the causal effects of kidney function (estimated glomerular filtration rate [eGFR] and chronic kidney disease [CKD]) and CVDs (including atrial fibrillation [AF], coronary artery disease [CAD], heart failure [HF], any stroke [AS], and any ischemic stroke [AIS]), we conducted a two-sample bidirectional Mendelian randomization (MR) analysis.ResultsIn the observational analysis, a total of 157,883 patients were included. After adjusting for potential confounding factors, there was no significant association between baseline RF and an increased risk of developing CVDs during hospitalization [adjusted odds ratio (OR): 1.056, 95% confidence interval (CI): 0.993 to 1.123, P = 0.083]. Conversely, baseline CVDs was significantly associated with an increased risk of developing RF during hospitalization (adjusted OR: 1.189, 95% CI: 1.139 to 1.240, P ConclusionOur study provides evidence for causal effects of CVDs on kidney function. However, the evidence to support the causal effects of kidney function on CVDs is currently insufficient. Further mechanistic studies are required to determine the causality.</p

Palladium-Catalyzed Amidation of Aryl Halides Using 2‑Dialkylphosphino-2′-alkoxyl-1,1′-binaphthyl as Ligands

Palladium-catalyzed intermolecular C–N bond-forming reactions between aryl halides and amides are described using 2-dialkylphosphino-2′-alkoxyl-1,1′-binaphthyl, which is both bulky and electron-rich, as the ligand. A variety of amides, including aliphatic and aromatic primary amides, lactams, and carbamates, were viable substrates for the amidation, which exhibited good functional group compatibility. By tuning the substituents at the 2,2′-position of 1,1′-binaphthyl of the ligand, the palladium-catalyzed amidation of bulky aryl halides was realized and this coupling reaction was used to synthesize 2-amino-2′-methoxy-1,1′-binaphthyl in high yield

Cyclo[6]aramide-Tropylium Charge Transfer Complex as a Colorimetric Chemosensor for Differentiation of Intimate and Loose Ion Pairs

Shape-persistent iso-C₁₆-cyclo­[6]­aramide (<b>1</b>) was found to form a charge-transfer (CT) complex with aromatic carbonium tropylium (<b>Tr</b>⁺). The resulting CT complex was evidenced by both experimental results and theoretical calculations. Particularly, dibutylammonium salt with PF₆^– as the counterion can extrude <b>Tr</b>⁺ from the CT complex, but it cannot do so with Cl^–, thereby offering a visual approach to identify organic intimate ion pairs and loose ion pairs

DataSheet_1_Transcriptome analysis reveals effects of leukemogenic SHP2 mutations in biosynthesis of amino acids signaling.docx

Gain-of-function mutations of SHP2, especially D61Y and E76K, lead to the development of neoplasms in hematopoietic cells. Previously, we found that SHP2-D61Y and -E76K confer HCD-57 cells cytokine-independent survival and proliferation via activation of MAPK pathway. Metabolic reprogramming is likely to be involved in leukemogenesis led by mutant SHP2. However, detailed pathways or key genes of altered metabolisms are unknown in leukemia cells expressing mutant SHP2. In this study, we performed transcriptome analysis to identify dysregulated metabolic pathways and key genes using HCD-57 transformed by mutant SHP2. A total of 2443 and 2273 significant differentially expressed genes (DEGs) were identified in HCD-57 expressing SHP2-D61Y and -E76K compared with parental cells as the control, respectively. Gene ontology (GO) and Reactome enrichment analysis showed that a large proportion of DEGs were involved in the metabolism process. Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis showed that DEGs were the mostly enriched in glutathione metabolism and biosynthesis of amino acids in metabolic pathways. Gene Set Enrichment Analysis (GSEA) revealed that the expression of mutant SHP2 led to a significant activation of biosynthesis of amino acids pathway in HCD-57 expressing mutant SHP2 compared with the control. Particularly, we found that ASNS, PHGDH, PSAT1, and SHMT2 involved in the biosynthesis of asparagine, serine, and glycine were remarkably up-regulated. Together, these transcriptome profiling data provided new insights into the metabolic mechanisms underlying mutant SHP2-driven leukemogenesis.</p