Early occupational intervention for people with low back pain in physically demanding jobs: A randomized clinical trial.

BACKGROUND: Occupational medicine seeks to reduce sick leave; however, evidence for an add-on effect to usual care is sparse. The objective of the GOBACK trial was to test whether people with low back pain (LBP) in physically demanding jobs and at risk of sick leave gain additional benefit from a 3-month complex intervention that involves occupational medicine consultations, a work-related evaluation and workplace intervention plan, an optional workplace visit, and a physical activity program, over a single hospital consultation and an MRI. METHODS AND FINDINGS: We enrolled people from the capital region of Denmark to an open-label, parallel-group randomized controlled trial with a superiority design from March 2014 through December 2015. In a hospital setting 305 participants (99 women) with LBP and in physically demanding jobs were randomized to occupational intervention (n = 153) or no additional intervention (control group; n = 152) added to a single hospital consultation giving a thorough explanation of the pain (i.e., clinical examination and MRI) and instructions to stay active and continue working. Primary outcome was accumulated sick leave days due to LBP during 6 months. Secondary outcomes were changes in neuropathic pain (painDETECT questionnaire [PDQ]), pain 0-10 numerical rating scale (NRS), Fear-Avoidance Beliefs Questionnaire (FABQ), Roland-Morris Disability Questionnaire (RMDQ), Short Form Health Survey (SF-36) for physical and mental health-related quality of life (HRQoL), and self-assessed ability to continue working (range 0-10). An intention-to-treat analysis of sick leave at 6 months showed no significant difference between groups (mean difference in days suggestively in favor of no additional intervention: 3.50 [95% CI -5.08 to 12.07], P = 0.42). Both groups showed significant improvements in average pain score (NRS), disability (RMDQ), fear-avoidance beliefs about physical activities and work (FABQ), and physical HRQoL (SF-36 physical component summary); there were no significant differences between the groups in any secondary outcome. There was no statistically significant improvement in neuropathic pain (PDQ score), mental HRQoL (SF-36 mental component summary), and self-assessed ability to stay in job. Four participants could not complete the MRI or the intervention due to a claustrophobic attack or accentuated back pain. Workplace visits may be an important element in the occupational intervention, although not always needed. A per-protocol analysis that included the 40 participants in the intervention arm who received a workplace visit as part of the additional occupational intervention did not show an add-on benefit in terms of sick leave (available cases after 6 months, mean difference: -0.43 days [95% CI -12.8 to 11.94], P = 0.945). The main limitations were the small number of sick leave days taken and that the comprehensive use of MRI may limit generalization of the findings to other settings, for example, general practice. CONCLUSIONS: When given a single hospital consultation and MRI, people in physically demanding jobs at risk of sick leave due to LBP did not benefit from a complex additional occupational intervention. Occupational interventions aimed at limiting biopsychological obstacles (e.g., fear-avoidance beliefs and behaviors), barriers in the workplace, and system barriers seem essential to reduce sick leave in patients with LBP. This study indicates that these obstacles and barriers may be addressed by thorough usual care. TRIAL REGISTRATION: Clinical Trials.gov: NCT02015572.

Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

Assessing sample and miRNA profile quality in serum and plasma or other biofluids.

MicroRNAs (miRNAs) constitute a class of small cellular RNAs (typically 21-23nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, although they are relatively few in number (less than 2000 human miRNAs). The high relative stability of miRNA in common clinical tissues and biofluids (e.g. plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. Furthermore miRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology. To facilitate discovery and clinical development of miRNA-based biomarkers, we developed a genome-wide Locked Nucleic Acid (LNA™)-based miRNA qPCR platform with unparalleled sensitivity and robustness. The platform allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Using this system, we have profiled thousands of biofluid samples including blood derived plasma and serum. An extensive quality control (QC) system has been implemented in order to secure technical excellence and reveal any unwanted bias coming from pre-analytical or analytical variables. We present our approaches to sample and RNA QC as well as data QC and normalization. Specifically we have developed normal reference ranges for circulating miRNAs in serum and plasma as well as a hemolysis indicator based on microRNA expression.

Test-retest reliability and agreement of lower-extremity kinematics captured in squatting and jumping preschool children using markerless motion capture technology.

The clinimetric properties of new technology should be evaluated in relevant populations before its implementation in research or clinical practice. Markerless motion capture is a new digital technology that allows for data collection in young children without some drawbacks commonly encountered with traditional systems. However, important properties, such as test-retest reliability, of this new technology have so far not been investigated. We recorded 63 preschool children using markerless motion capture (The Captury GmbH, Saarbrüken, Germany) while they performed squats and standing broad jumps. A retest session was conducted after 1 week. Recordings from the test session were processed twice to estimate the software-driven instrumental variability. Recordings from the first and second test sessions were compared to evaluate the week-to-week test-retest reliability. Statistical tests included 95% limits of agreement and intraclass correlations of absolute agreement (ICC). Jump length performance and four kinematic variables demonstrated acceptable instrumental variability (ICC > 0.76). The week-to-week reliability was excellent for jump length performance (ICC = 0.90) but poor to moderate (ICC < 0.55) for the kinematic variables. Our results indicate that preschool children exhibit considerable intra-individual kinematic variation from week-to-week during jump landings and squats. Consequently, we suggest that future work should explore individuals with persistent extreme kinematics over multiple test-sessions.

Improved microRNA quantification in total RNA from clinical samples.

microRNAs are small regulatory RNAs that are currently emerging as new biomarkers for cancer and other diseases. In order for biomarkers to be useful in clinical settings, they should be accurately and reliably detected in clinical samples such as formalin fixed paraffin embedded (FFPE) sections and blood serum or plasma. These types of samples represent a challenge in terms of microRNA quantification. A newly developed method for microRNA qPCR using Locked Nucleic Acid (LNA)-enhanced primers enables accurate and reproducible quantification of microRNAs in scarce clinical samples. Here we show that LNA-based microRNA qPCR enables biomarker screening using very low amounts of total RNA from FFPE samples and the results are compared to microarray analysis data. We also present evidence that the addition of a small carrier RNA prior to total RNA extraction, improves microRNA quantification in blood plasma and laser capture microdissected (LCM) sections of FFPE samples.

Optimising Repeated Exposure: Determining Optimal Stimulus Shape for Introducing a Novel Vegetable among Children.

Although it is well evident that a healthy diet rich in fruit and vegetables could prevent a number of major chronic diseases, national and international guidelines concerning their intake are not being reached by a large percentage of the population, including children. Thus, it is of interest to investigate how the consumption of this food group by children could be increased. The aim of this study was to examine the impact of serving style on the consumption of a raw snack vegetable (daikon) and the influence of its exposure on liking and intake of the vegetable. A group of 185 children 3-5 years old participated in the study. Two kindergartens served as intervention groups, while the third was assigned to be the control group of the study (n = 50). The intervention groups were repeatedly exposed to one of three different serving styles of daikon: sticks (n = 42), triangles (n = 46) or grated (n = 47), and they were all visited 7 times during the exposure period, on the same frequency (twice per week). Familiarity and liking of the target vegetable, daikon, and six other vegetables (cucumber, celery, celeriac, broccoli, cauliflower and beetroot) were measured at baseline, post-intervention and two follow up sessions (3- and 6-month) to investigate the likelihood of generalisation effects. Intake of daikon was measured at all control sessions and exposures. Moreover, children were asked to rank their favourite serving style of daikon and beetroot, among triangle, stick and grated, towards understanding the influence of shape on the efficacy of the exposure. The results revealed significant changes between liking and intake of daikon for the groups of triangles and sticks and the control group (p < 0.05). The group that received grated daikon did not show significant differences in liking and at intake levels during the exposures but performed well in the long-term. Throughout the exposure period, intake levels followed an overall increasing pattern, with all the groups to demonstrate a decrease of their intake at the last session, which was not found significant for the triangle group. Mere exposure was efficient towards increasing liking and intake of the novel vegetable with all the shapes to deliver positive results, but based on this study no particular serving style can be recommended.

Clinical Implications of Monitoring Circulating Tumor DNA in Patients with Colorectal Cancer.

Purpose: We investigated whether detection of ctDNA after resection of colorectal cancer identifies the patients with the highest risk of relapse and, furthermore, whether longitudinal ctDNA analysis allows early detection of relapse and informs about response to intervention.Experimental Design: In this longitudinal cohort study, we used massively parallel sequencing to identify somatic mutations and used these as ctDNA markers to detect minimal residual disease and to monitor changes in tumor burden during a 3-year follow-up period.Results: A total of 45 patients and 371 plasma samples were included. Longitudinal samples from 27 patients revealed ctDNA postoperatively in all relapsing patients (n = 14), but not in any of the nonrelapsing patients. ctDNA detected relapse with an average lead time of 9.4 months compared with CT imaging. Of 21 patients treated for localized disease, six had ctDNA detected within 3 months after surgery. All six later relapsed compared with four of the remaining patients [HR, 37.7; 95% confidence interval (CI), 4.2-335.5; P < 0.001]. The ability of a 3-month ctDNA analysis to predict relapse was confirmed in 23 liver metastasis patients (HR 4.9; 95% CI, 1.5-15.7; P = 0.007). Changes in ctDNA levels induced by relapse intervention (n = 19) showed good agreement with changes in tumor volume (κ = 0.41; Spearman ρ = 0.4).Conclusions: Postoperative ctDNA detection provides evidence of residual disease and identifies patients at very high risk of relapse. Longitudinal surveillance enables early detection of relapse and informs about response to intervention. These observations have implications for the postoperative management of colorectal cancer patients. Clin Cancer Res; 23(18); 5437-45. ©2017 AACR.

Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells.

This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca(v)) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between -60 and -10 mV. Blockade of K+ channels with TEA inhibited 83% of the current at +105 mV. Inhibition of KV channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K+ channels (BKCa) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was -32 mV and activation of BKCa with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca(v) blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca(v) 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K+-blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels.

Control of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases.

We tested the hypothesis that cGMP stimulates renin release through inhibition of the cAMP-specific phosphodiesterase 3 (PDE3) in isolated rat juxtaglomerular (JG) cells. In addition, we assessed the involvement of PDE4 in JG-cell function. JG cells expressed PDE3A and PDE3B, and the PDE3 inhibitor trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed, and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (C(m)) with a marked increase in C(m) at 1 micromol/L, no net change at 10 micromol/L, and a decrease at 100 micromol/L cAMP. cGMP also had a dual effect on C(m) at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on C(m). Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects of cAMP, cGMP, and trequinsin on C(m) and cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation of renin release by cGMP involves inhibition of PDE3 resulting in enhanced cAMP formation and activation of the cAMP sensitive protein kinase.

Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

Profiling microRNAs by real-time PCR.

A variety of physiological processes are associated with changes in microRNA (miRNA) expression. Analysis of miRNA has been applied to study normal physiology as well as diseased states including cancer. One major challenge in miRNA research is to accurately and practically determine the expression level of miRNAs in various experimental systems. Many genome-wide miRNA expression profiling studies have relied on microarrays technology, and frequently differentially expressed miRNAs have subsequently been confirmed with real-time quantitative PCR studies. Here, we describe how different primer strategies for first-strand cDNA synthesis and PCR amplification can affect measurements of miRNA expression levels. Overcoming the small nature of miRNAs is a difficult task as the short sequence available does not allow for designing primers using standard PCR primer design guidelines. Finally, we demonstrate how to determine differentially expressed miRNAs using a locked nucleic acid-based real-time PCR approach.

Coexpression of voltage-dependent calcium channels Cav1.2, 2.1a, and 2.1b in vascular myocytes.

Voltage-dependent Ca2+ channels Cav1.2 (L type) and Cav2.1 (P/Q type) are expressed in vascular smooth muscle cells (VSMCs) and are important for the contraction of renal resistance vessels. In the present study we examined whether native renal VSMCs coexpress L-, P-, and Q-type Ca2+ currents. The expression of both Cav2.1a (P-type) and Cav2.1b (Q-type) mRNA was demonstrated by RT-PCR in renal preglomerular vessels from rats and mice. Immunolabeling was performed on A7r5 cells, renal cryosections, and freshly isolated renal VSMCs with anti-Cav1.2 and anti-Cav2.1 antibodies. Conventional and confocal microscopy revealed expression of both channels in all of the smooth muscle cells. Whole-cell patch clamp on single preglomerular VSMCs from mice showed L-, P-, and Q-type currents. Blockade of the L-type currents by calciseptine (20 nmol/L) inhibited 35.6+/-3.9% of the voltage-dependent Ca2+ current, and blocking P-type currents (omega-agatoxin IVA 10 nmol/L) led to 20.2+/-3.0% inhibition, whereas 300 nmol/L of omega agatoxin IVA (blocking P/Q-type) inhibited 45.0+/-7.3%. In rat aortic smooth muscle cells (A7r5), blockade of L-type channels resulted in 28.5+/-6.1% inhibition, simultaneous blockade of L-type and P-type channels inhibited 58.0+/-11.8%, and simultaneous inhibition of L-, P-, and Q-type channels led to blockade (88.7+/-5.6%) of the Ca2+ current. We conclude that aortic and renal preglomerular smooth muscle cells express L-, P-, and Q-type voltage-dependent Ca2+ channels in the rat and mouse.

Activation of epithelial sodium channels by mouse channel activating proteases (mCAP) expressed in Xenopus oocytes requires catalytic activity of mCAP3 and mCAP2 but not mCAP1.

Mouse channel activating proteases 1, 2, and 3 (mCAP1, mCAP2, and mCAP3) were described recently as regulators of the epithelial sodium channel (ENaC). The mCAP are membrane-bound serine proteases that are synthesized as inactive proenzymes. To mature into active proteases, they undergo intramolecular cleavage by auto- and/or heterocatalytic processing. Specific antibodies against each mCAP were developed to distinguish between proenzyme and active protease by Western blot analysis. Various point mutations were introduced in the catalytic or protein-protein interacting domains of mCAP and wild-type and mutant enzymes were expressed in the Xenopus oocyte expression system to test for ability to activate ENaC. In mCAP3, an intact catalytic triad was necessary for activation of ENaC but not for intramolecular cleavage of the protease. This suggests a heterocatalytic mechanism. Mutating the catalytic triad of mCAP2 not only abolished ENaC activation completely but also impeded cleavage of the protease. Processing of mCAP2 therefore seems to be autocatalytic. Furthermore, mutations in conserved residues of mCAP2 located in two protein-protein interacting domains significantly modulated ENaC activation. Surprisingly, mCAP1 catalytically inactive mutants were still able to fully activate ENaC, and no evidence of mCAP1 intramolecular cleavage was seen. The presence of an intact glycosylphosphatidylinositol anchor, however, was required. It is concluded that auto- and heterocatalytic requirements are specific for each CAP and that endogenous partners are a necessity for activation of ENaC by mCAP in the Xenopus oocyte expression system.

A novel TMPRSS3 missense mutation in a DFNB8/10 family prevents proteolytic activation of the protein.

Pathogenic mutations in TMPRSS3, which encodes a transmembrane serine protease, cause non-syndromic deafness DFNB8/10. Missense mutations map in the low density-lipoprotein receptor A (LDLRA), scavenger-receptor cysteine-rich (SRCR), and protease domains of the protein, indicating that all domains are important for its function. TMPRSS3 undergoes proteolytic cleavage and activates the ENaC sodium channel in a Xenopus oocyte model system. To assess the importance of this gene in non-syndromic childhood or congenital deafness in Turkey, we screened for mutations affected members of 25 unrelated Turkish families. The three families with the highest LOD score for linkage to chromosome 21q22.3 were shown to harbor P404L, R216L, or Q398X mutations, suggesting that mutations in TMPRSS3 are a considerable contributor to non-syndromic deafness in the Turkish population. The mutant TMPRSS3 harboring the novel R216L missense mutation within the predicted cleavage site of the protein fails to undergo proteolytic cleavage and is unable to activate ENaC, thus providing evidence that pre-cleavage of TMPRSS3 is mandatory for normal function.

Role of T-type calcium channels in myogenic tone of skeletal muscle resistance arteries.

T-type calcium channels may be involved in the maintenance of myogenic tone. We tested their role in isolated rat cremaster arterioles obtained after CO(2) anesthesia and decapitation. Total RNA was analyzed by RT-PCR and Southern blotting for calcium channel expression. We observed expression of voltage-operated calcium (Ca(V)) channels Ca(V)3.1 (T-type), Ca(V)3.2 (T-type), and Ca(V)1.2 (L-type) in cremaster arterioles (n = 3 rats). Amplification products were observed only in the presence of reverse transcriptase and cDNA. Concentration-response curves of the relatively specific L-type blocker verapamil and the relatively specific T-type blockers mibefradil and nickel were made on cannulated vessels with either myogenic tone (75 mmHg) or a similar level of constriction induced by 30 mM K(+) at 35 mmHg. Mibefradil and nickel were, respectively, 162-fold and 300-fold more potent in inhibiting myogenic tone compared with K(+)-induced constriction [log(IC(50), M): mibefradil, basal -7.3 +/- 0.2 (n = 9) and K(+) -5.1 +/- 0.1 (n = 5); nickel, basal -4.1 +/- 0.2 (n = 5) and K(+) -1.6 +/- 0.5 (n = 5); means +/- SE]. Verapamil had a 17-fold more potent effect [log(IC(50), M): basal -6.6 +/- 0.1 (n = 5); K(+) -5.4 +/- 0.3 (n = 4); all log(IC(50)) P < 0.05, basal vs. K(+)]. These data suggest that T-type calcium channels are expressed and involved in maintenance of myogenic tone in rat cremaster muscle arterioles.

Local electric stimulation causes conducted calcium response in rat interlobular arteries.

The purpose of the present study was to investigate the conducted Ca(2+) response to local electrical stimulation in isolated rat interlobular arteries. Interlobular arteries were isolated from young Sprague-Dawley rats, loaded with fura 2, and attached to pipettes in a chamber on an inverted microscope. Local electrical pulse stimulation (200 ms, 100 V) was administered by means of an NaCl-filled microelectrode (0.7-1 M(Omega)) juxtaposed to one end of the vessel. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured with an image system at a site approximately 500 microm from the location of the electrode. The expression of mRNA for pore-forming units Ca(V)3.1 and Ca(V)3.2 of voltage-sensitive T-type channels was investigated by using RT-PCR. Current stimulation elicited a conducted [Ca(2+)](i) response. A positive electrode (relative to ground) increased [Ca(2+)](i) to 145 +/- 7% of baseline, whereas the response was absent when the electrode was negative. This response was not dependent on perivascular nerves, because the conducted response was unaffected by TTX (1 microM). The conducted [Ca(2+)](i) response was abolished by an ambient Ca(2+) free solution and blunted by nifedipine (1 microM). Rat interlobular arteries exhibited conducted [Ca(2+)](i) response to current stimulation. This response was dependent on Ca(2+) entry. L-type Ca(2+) channels may play a role in this process.