Short amplicon reverse transcription-polymerase chain reaction detects aberrant splicing in genes with low expression in blood missed by ribonucleic acid sequencing analysis for clinical diagnosis.

Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.

Quantification of the demands of cricket bowling and the relationship to injury risk: a systematic review.

BACKGROUND: Bowling in cricket is a complex sporting movement which, despite being well characterised, still produces a significant number of injuries each year. Fast bowlers are more likely to be injured than any other playing role. Frequency, duration, intensity and volume of bowling, which have been generalised as measurements of workload, are thought to be risk factors for injuries. Injury rates of fast bowlers have not reduced in recent years despite the implementation of various workload monitoring practices. OBJECTIVE: To identify the variables used to quantify frequency, intensity, time and volume of bowling; and evaluate relationships between these variables and injury risk. METHODS: Six online databases were systematically searched for studies on fast bowling that included terms related to workload. Population characteristics, variables relating to demand and their relationship to standardised definitions of physical activity were extracted from all included studies. RESULTS: Bowling workload is typically quantified through measures of frequency, duration, or indirect intensity, with few studies reporting on bowling volume. CONCLUSIONS: When reported on, volume was often described using imprecise or insufficient measures of intensity. There is a need to develop more appropriate measures of intensity during bowling and improve the quality of evidence to inform on bowling programme management practices.

Mechanical testing of glutaraldehyde cross-linked mitral valves. Part two: Elastic and viscoelastic properties of chordae tendineae.

The aim of this study was to assess whether the mechanical properties of mitral valve chordae tendineae are sensitive to being cross-linked under load. A total 64 chordae were extracted from eight porcine hearts. Two chordae (posterior basal) from each heart were subjected to uniaxial ramp testing and six chordae (two strut, two anterior basal and two posterior basal) were subjected to dynamic mechanical analysis over frequencies between 0.5 and 10 Hz. Chordae were either cross-linked in tension or cross-linked in the absence of loading. Chordae cross-linked under load transitioned from high to low extension at a lower strain than cross-linked unloaded chordae (0.07 cf. 0.22), with greater pre-transitional (30.8 MPa cf. 5.78 MPa) and post-transitional (139 MPa cf. 74.1 MPa) moduli. The mean storage modulus of anterior strut chordae ranged from 48 to 54 MPa for cross-linked unloaded chordae, as compared to 53-61 MPa cross-linked loaded chordae. The mean loss modulus of anterior strut chordae ranged from 2.3 to 2.9 MPa for cross-linked unloaded chordae, as compared to 3.8-4.8 MPa cross-linked loaded chordae. The elastic and viscoelastic properties of chordae following glutaraldehyde cross-linking are dependent on the inclusion/exclusion of loading during the cross-linking process; with loading increasing the magnitude of the material properties measured.

Mechanical testing of glutaraldehyde cross-linked mitral valves. Part one: In vitro mechanical behaviour.

The aim of this study was to perform an initial assessment, in vitro, of the feasibility of using a glutaraldehyde cross-linked porcine mitral valve to retain acute functionality, focusing on assessing mitral regurgitation. Six porcine hearts were tested using an in vitro simulator. Testing was repeated following cross-linking of mitral valves; where cross-linking was achieved by placing them in a glutaraldehyde solution. The simulator enabled systolic pressure on the ventricular side of the valve to be mimicked. Following testing, mitral valve leaflets underwent Scanning Electron Microscopy of the ventricular surface of both the anterior and posterior leaflets (1 cm2 samples). The peak pressure withstood by cross-linked valves was significantly lower than for untreated valves (108 mmHg cf. 128 mmHg for untreated valves; p < 0.05). The peak pressure was typically reached 0.5 s later than for the untreated valve. While both cross-linked and untreated valves exhibited endothelium denudation, the unfixed valve had less endothelial loss. Glutaraldehyde cross-linking of porcine mitral valves may be of potential value in assessing improved bioprosthetic mitral valve replacements. However, a more immobile valve exhibiting endothelial denudation (i.e. sclerosis) was a possible concerns identified following in vitro acute assessment.

Selective serotonin reuptake inhibitors inhibit human osteoclast and osteoblast formation and function.

BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants and one of the most commonly used medications. There is growing concern that SSRIs, which sequester in bone marrow at higher concentrations than brain or blood, increase bone fragility and fracture risk. However, their mechanism of action on human osteoclasts (OC) and osteoblasts (OB) differentiation remains unclear. METHODS: Expression of serotonin receptors (5-HTR), transporter (5-HTT), and tryptophan hydroxylase 1 (TPH1) was assessed in human OC (precursors and mature) and OB (nonmineralizing and mineralizing) by polymerase chain reaction. OC formation and resorption was measured in the presence of 5 SSRIs. OBs cultured with SSRIs for 28 days were assessed for alkaline phosphatase (ALP) and bone mineralization. Cell viability and apoptosis were determined by annexin V flow cytometry. RESULTS: OCs and OB expressed TPH1, 5-HTT, and 5-HTR1B. The 5-HTR2A was expressed only in OB, whereas 5-HTR2B expression increased from precursor to mature OC. All SSRIs (except citalopram) dose-dependently inhibited OC formation and resorption between 1 µmol/L and 10 µmol/L; order of potency: sertraline > fluoxetine > paroxetine > fluvoxamine > citalopram. Similarly, SSRIs (except citalopram) inhibited ALP and bone mineralization by OB but only at 30 µmol/L. Apoptosis was induced by SSRIs in OC and OB in an identical pattern to inhibitory effects. Serotonin treatment had no effect on either OC or OB parameters. CONCLUSIONS: These data demonstrate that SSRIs differentially inhibit bone cell function via apoptosis. This may explain the mechanisms of bone loss with chronic use and aid clinical choices.

Oncostatin M promotes bone formation independently of resorption when signaling through leukemia inhibitory factor receptor in mice.

Effective osteoporosis therapy requires agents that increase the amount and/or quality of bone. Any modification of osteoclast-mediated bone resorption by disease or drug treatment, however, elicits a parallel change in osteoblast-mediated bone formation because the processes are tightly coupled. Anabolic approaches now focus on uncoupling osteoblast action from osteoclast formation, for example, by inhibiting sclerostin, an inhibitor of bone formation that does not influence osteoclast differentiation. Here, we report that oncostatin M (OSM) is produced by osteoblasts and osteocytes in mouse bone and that it has distinct effects when acting through 2 different receptors, OSM receptor (OSMR) and leukemia inhibitory factor receptor (LIFR). Specifically, mouse OSM (mOSM) inhibited sclerostin production in a stromal cell line and in primary murine osteoblast cultures by acting through LIFR. In contrast, when acting through OSMR, mOSM stimulated RANKL production and osteoclast formation. A key role for OSMR in bone turnover was confirmed by the osteopetrotic phenotype of mice lacking OSMR. Furthermore, in contrast to the accepted model, in which mOSM acts only through OSMR, mOSM inhibited sclerostin expression in Osmr-/- osteoblasts and enhanced bone formation in vivo. These data reveal what we believe to be a novel pathway by which bone formation can be stimulated independently of bone resorption and provide new insights into OSMR and LIFR signaling that are relevant to other medical conditions, including cardiovascular and neurodegenerative diseases and cancer.