Interleukin-6 autocrine signaling mediates melatonin MT(1/2) receptor-induced STAT3 Tyr(705) phosphorylation.

Melatonin receptors have previously been shown to elicit cellular signaling through the hematopoietic-specific G protein, G(16) . In the present study, we show that this functional coupling elicited biphasic stimulatory phosphorylation on STAT3 in recombinant MT(1) /Gα(16) cells and native Jurkat T cells (endogenously expressing MT(1) and Gα(16) ), with maximal Ser(727) phosphorylation occurring at 15min, while marked Tyr(705) phosphorylation became detectable only upon agonist treatment for 4 hr or more. By employing signal transducer and activator of transcription 3 (STAT3) phosphorylation-resistant mutants (STAT3-Y705F and STAT3-S727A), we further showed that the receptor-mediated STAT3 phosphorylations at Ser(727) and Tyr(705) were independent of each other. Results obtained from fractionation of 2-IMT-induced cells revealed that the Ser(727) and Tyr(705) phosphorylations were spatially distinct, with the former mainly situated in mitochondria and cytosol, while the latter was predominantly located in the nucleus. Further experiments revealed that the agonist-induced STAT3 phosphorylation at Tyr(705) was significantly suppressed by pretreatment with cycloheximide (a ribosome inhibitor), suggesting that de novo protein synthesis might play a critical role for this response. Using conditioned media obtained from 2-IMT-treated MT(1) /Gα(16) cells, multiplex immunoassays revealed that prolonged agonist treatment led to elevated productions of IL-6, GM-CSF and CXCL-8. Antibody against IL-6, but not those for GM-CSF and CXCL-8, effectively abolished the agonist-induced STAT3 Tyr(705) phosphorylation, suggesting the involvement of IL-6 in melatonin receptor-mediated STAT3 activation. Our results demonstrate that melatonin receptor/Gα(16) coupling is capable of triggering the production of cytokines including IL-6, and this autocrine loop may account for the subsequent STAT3 phosphorylation at Tyr(705) .

Longitudinal Competence Programs for Basic Point-of-Care Ultrasound in Critical Care: A Systematic Review.

BACKGROUND: Competence in point-of-care ultrasound (PoCUS) is widely recommended by several critical care societies. Despite numerous introductory short courses, very few doctors attain PoCUS competence because of the challenges in establishing longitudinal competence programs. RESEARCH QUESTION: To evaluate the methodologic quality of the literature on basic PoCUS competence processes in critical care. STUDY DESIGN AND METHODS: A systematic review to identify manuscripts meeting predefined inclusion criteria was performed using three medical databases (PubMed, OVID Embase, and Web of Science); using extra references from original articles, review articles, and expert panel guidelines; and by directly contacting authors for further information if required. The objectives, domains, and inclusion and exclusion criteria of the review were determined during discussions between experienced PoCUS educators. Data extraction and analyses were performed independently by three reviewers. RESULTS: Of the 5,408 abstracts extracted, 42 met the inclusion criteria for longitudinal PoCUS competence. Each study was described along four broad categories: general information, study design, and trainee characteristics; description of introductory course; description of longitudinal competence program; and grading of overall methodologic quality on a 4-point Likert scale. Thirty-nine studies (92.9%) were from a single center. Most studies lacked important details on study methodology such as prior ultrasound experience, pre- and postcourse tests, models for hands-on sessions, ratio of instructors to trainees, competence assessment criteria, number of scans performed by individual trainees, and formative and summative assessments. The studies were rated as follows: poor = 19 (45.2%), average = 15 (35.7%), good = 4 (9.5%), and excellent = 4 (9.5%). INTERPRETATION: Ther is very little high-quality evidence on PoCUS competence. To help frame policy guidelines to improve PoCUS education, there is a need for well-designed longitudinal studies on PoCUS competence. TRIAL REGISTRY: PROSPERO database; No.: CRD42018094033; URL: https://www.crd.york.ac.uk/PROSPERO/.

Basic echocardiography competence program in intensive care units: A multinational survey of intensive care units accredited by the College of Intensive Care Medicine.

In 2014, basic critical care echocardiography (BCCE) competence became a mandatory requirement for trainees registered with the College of Intensive Care Medicine (CICM). To determine the proportion of CICM intensive care units (ICUs) that conduct a BCCE competence program and to learn about the barriers/challenges and successful strategies, we conducted a survey of intensivists working in ICUs accredited by CICM for basic/advanced training in Australia, New Zealand, Hong Kong, Singapore, Ireland and India. Following consultations with content experts and a trial phase to improve clarity and minimise ambiguity, an 11-point questionnaire survey was sent to one intensivist from every CICM-accredited ICU by several methods. Participation was voluntary. Consent was implied. No incentives were offered. Results are reported as numbers and percentages. Of the 104 ICUs surveyed, 99 (95.1%) responded, with 75 (75.8%) having no BCCE teaching whatsoever. In the remaining 24 (24.2%) ICUs, the teaching process was widely variable. Only 5/99 (5.1%) ICUs provided a structured BCCE competence program through which trainees performed and archived BCCE scans, maintained a logbook and underwent formative and summative assessments for credentialling. Six more ICUs provided formative assessment but relied on external bodies for competence assessment. Overall, 20/99 (20.2%) ICUs allowed trainees to perform unsupervised scans for clinical management, even if they were not BCCE competent. Nineteen intensivists perceived management errors due to misinterpretation of echocardiographic findings. Very few CICM-accredited ICUs offer a structured BCCE competence program. To fulfil the objective of universal BCCE competence, potential solutions are presented.

A spatially addressable bead-based biosensor for simple and rapid DNA detection.

A simple glass-polymer-based biosensor that allows arrays of beads to be immobilized, separated and identified without any prior encoding is developed. To do so, distinct bead types that are conjugated with different oligonucleotide probes are sequentially spotted onto a polymeric matrix (or gel pad) on the surface of the device. The spotted beads are firmly immobilized to the gel pad, acquiring spatial codes that allow them to be identified. Throughput is enhanced by spotting different bead types onto hundreds of different gel pads. The bead-based biosensor was applied for the DNA-based detection of 10 model bacterial species and two single-nucleotide polymorphisms, and based on passive hybridization the final signals are obtained with single-mismatch specificity within 10 min.

Cancer-Associated Fibroblasts Regulate Tumor-Initiating Cell Plasticity in Hepatocellular Carcinoma through c-Met/FRA1/HEY1 Signaling.

Like normal stem cells, tumor-initiating cells (T-ICs) are regulated extrinsically within the tumor microenvironment. Because HCC develops primarily in the context of cirrhosis, in which there is an enrichment of activated fibroblasts, we hypothesized that cancer-associated fibroblasts (CAFs) would regulate liver T-ICs. We found that the presence of α-SMA(+) CAFs correlates with poor clinical outcome. CAF-derived HGF regulates liver T-ICs via activation of FRA1 in an Erk1,2-dependent manner. Further functional analysis identifies HEY1 as a direct downstream effector of FRA1. Using the STAM NASH-HCC mouse model, we find that HGF-induced FRA1 activation is associated with the fibrosis-dependent development of HCC. Thus, targeting the CAF-derived, HGF-mediated c-Met/FRA1/HEY1 cascade may be a therapeutic strategy for the treatment of HCC.

Comparison of Circulating Tumour Cells and Circulating Cell-Free Epstein-Barr Virus DNA in Patients with Nasopharyngeal Carcinoma Undergoing Radiotherapy.

Quantification of Epstein-Barr virus (EBV) cell-free DNA (cfDNA) is commonly used in clinical settings as a circulating biomarker in nasopharyngeal carcinoma (NPC), but there has been no comparison with circulating tumour cells (CTCs). Our study aims to compare the performance of CTC enumeration against EBV cfDNA quantitation through digital PCR (dPCR) and quantitative PCR. 74 plasma samples from 46 NPC patients at baseline and one month after radiotherapy with or without concurrent chemotherapy were analysed. CTCs were captured by microsieve technology and enumerated, while three different methods of EBV cfDNA quantification were applied, including an in-house qPCR assay for BamHI-W fragment, a CE-IVD qPCR assay (Sentosa ®) and a dPCR (Clarity™) assay for Epstein-Barr nuclear antigen 1 (EBNA1). EBV cfDNA quantitation by all workflows showed stronger correlation with clinical stage, radiological response and overall survival in comparison with CTC enumeration. The highest detection rate of EBV cfDNA in pre-treatment samples was seen with the BamHI-W qPCR assay (89%), followed by EBNA1-dPCR (85%) and EBNA1-qPCR (67%) assays. Overall, we show that EBV cfDNA outperforms CTC enumeration in correlation with clinical outcomes of NPC patients undergoing treatment. Techniques such as dPCR and target selection of BamHI-W may improve sensitivity for EBV cfDNA detection.

Evidence for glutamate as a neuroglial transmitter within sensory ganglia.

This study examines key elements of glutamatergic transmission within sensory ganglia of the rat. We show that the soma of primary sensory neurons release glutamate when depolarized. Using acute dissociated mixed neuronal/glia cultures of dorsal root ganglia (DRG) or trigeminal ganglia and a colorimetric assay, we show that when glutamate uptake by satellite glial cells (SGCs) is inhibited, KCl stimulation leads to simultaneous increase of glutamate in the culture medium. With calcium imaging we see that the soma of primary sensory neurons and SGCs respond to AMPA, NMDA, kainate and mGluR agonists, and selective antagonists block this response. Using whole cell patch-clamp technique, inward currents were recorded from small diameter (<30 µm) DRG neurons from intact DRGs (ex-vivo whole ganglion preparation) in response to local application of the above glutamate receptor agonists. Following a chronic constriction injury (CCI) of either the inferior orbital nerve or the sciatic nerve, glutamate expression increases in the trigeminal ganglia and DRG respectively. This increase occurs in neurons of all diameters and is present in the somata of neurons with injured axons as well as in somata of neighboring uninjured neurons. These data provides additional evidence that glutamate can be released within the sensory ganglion, and that the somata of primary sensory neurons as well as SGCs express functional glutamate receptors at their surface. These findings, together with our previous gene knockdown data, suggest that glutamatergic transmission within the ganglion could impact nociceptive threshold.

Spatially addressable bead-based biosensor for rapid detection of beta-thalassemia mutations.

A simple glass-polymer bead-based biosensor was validated for the detection of beta-thalassemia mutations. Different bead types, each carrying allele-specific probes targeting a particular mutation on the beta-globin gene, were immobilized and distinguished on the chip based on their spatial addresses. Genomic DNA samples carrying various single nucleotide transitions and transversions in the beta-globin gene were subjected to polymerase chain reaction and asymmetric amplification in the presence of Cy3-labeled primers, followed by hybridization onto the chip and detection under an epifluorescent microscope. Mutations that were heterozygous or homozygous were easily detected on the device based on the signal intensity difference (or similarity) between the wildtype and mutant probes. This device successfully detected all six common beta-globin gene mutations within 30 min. The number of targeted mutations on this chip can be easily expandable through the introduction of additional probe sets.

Rapid discrimination of single-nucleotide mismatches using a microfluidic device with monolayered beads.

A microfluidic device incorporating monolayered beads is developed for the discrimination of single-nucleotide mismatches, based on the differential dissociation kinetics between perfect match (PM) and mismatched (MM) duplexes. The monolayered beads are used as solid support for the immobilization of oligonucleotide probes containing a single-base variation. Target oligonucleotides hybridize to the probes, forming either PM duplexes or MM duplexes containing a single mismatch. Optimization studies show that PM and MM duplexes are easily discriminated based on their dissociation but not hybridization kinetics under an optimized buffer composition of 100mM NaCl and 50% formamide. Detection of single-nucleotide polymorphism (SNP) using the device is demonstrated within 8 min using four probes containing all the possible single-base variants. The device can easily be modified to integrate multiplexed detection, making high-throughput SNP detection possible.

Miniaturized platforms for the detection of single-nucleotide polymorphisms.

Conventional methods for detecting single-nucleotide polymorphisms (SNPs), the most common form of genetic variation in human beings, are mostly limited by their analysis time and throughputs. In contrast, advances in microfabrication technology have led to the development of miniaturized platforms that can potentially provide rapid high-throughput analysis at small sample volumes. This review highlights some of the recent developments in the miniaturization of SNP detection platforms, including microarray-based, bead-based microfluidic and microelectrophoresis-based platforms. Particular attention is paid to their ease of fabrication, analysis time, and level of throughput.

LabArray: real-time imaging and analytical tool for microarrays.

UNLABELLED: Microarrays have been used to perform high-throughput genetic analyses such as single-nucleotide polymorphisms detection and microbial genome analysis. Some of these analyses require real-time monitoring of the hybridization signals with respect to a varying experimental condition, such as temperature. However, current microarray imaging and analysis packages typically do not possess such real-time capabilities. Therefore, microarray image analyses are often time-consuming and labour-intensive. LabArray was developed to expedite such processes by enabling real-time monitoring of microarray signals. AVAILABILITY: LabArray is available at http://www.eng.nus.edu.sg/civil/Labarray/labarray.htm CONTACT: cveliuwt@nus.edu.sg SUPPLEMENTARY INFORMATION: Screenshots and instructions for use are available at the above website.

Evaluating single-base-pair discriminating capability of planar oligonucleotide microchips using a non-equilibrium dissociation approach.

The capability of planar rRNA-based oligonucleotide microarrays for single-base-pair discrimination was evaluated using an approach that compares the non-equilibrium dissociation profiles and dissociation temperatures (Tds) of all probe-target duplexes simultaneously. Three sets of 16S rRNA gene specific probes at different levels of specificity were used along with their counter probes for individual sets having either one or two mismatches (MM) to their targets at specific external (next to terminus) and various internal positions. Criteria based on the Td approach and a discrimination index (DI) were proven to be competent in discriminating PM from internal MM duplexes, but not always for external MM duplexes. Maximal DI for separating PM duplexes from ones with two and one internal MM usually occurred at temperatures approximately 5-10 degrees C and 10-15 degrees C, respectively, higher than the Tds of the PM duplexes. Washing buffer type and salt concentration, and MM number and position were shown statistically to affect dissociation profiles, Td, and single-base-pair discriminating capability. The reusability potential of the planar microchip was further demonstrated.