Statistical Considerations for Analyzing Ecological Momentary Assessment Data.

PURPOSE: The analysis of Ecological Momentary Assessment (EMA) data can be difficult to conceptualize due to the complexity of how the data are collected. The goal of this tutorial is to provide an overview of statistical considerations for analyzing observational data arising from EMA studies. METHOD: EMA data are collected in a variety of ways, complicating the statistical analysis. We focus on fundamental statistical characteristics of the data and general purpose statistical approaches to analyzing EMA data. We implement those statistical approaches using a recent study involving EMA. RESULTS: The linear or generalized linear mixed-model statistical approach can adequately capture the challenges resulting from EMA collected data if properly set up. Additionally, while sample size depends on both the number of participants and the number of survey responses per participant, having more participants is more important than the number of responses per participant. CONCLUSION: Using modern statistical methods when analyzing EMA data and adequately considering all of the statistical assumptions being used can lead to interesting and important findings when using EMA. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.17155961.

Medication state at the time of the offense: Medication noncompliance, insight and criminal responsibility.

This study used a mixed quantitative-qualitative methodology to examine whether mock jurors considered a defendant's meta-responsibility - specifically, the defendant's medication noncompliance and degree of insight into his/her schizophrenia - when determining the person's criminal responsibility. The degree of expert witness explanation regarding these factors was also varied. Participants (n = 173) were grouped into 30 juries, randomized across five conditions, and shown mock testimony and attorney arguments based on a real not guilty by reason of insanity court case. Linear mixed-modeling analysis showed that manipulations of medication compliance, insight, and expert testimony elaboration did not predict differential verdict and meta-responsibility outcomes. Nevertheless, qualitative exploration of focus groups from five juries (n = 29) indicated that participants across groups strongly considered meta-responsibility, but did so in a way that, along with a host of other considerations, suggested mock jurors were unable and/or unwilling to follow their duties as the triers of fact. Implications for legal participants, expert witnesses, and researchers are discussed.

Effect of Sex and Body Mass Index on Children's Physical Activity Intensity during Free Play at an Indoor Soft Play Center: An Exploratory Study.

Background: Indoor soft play can provide a safe but exciting physical activity opportunity regardless of environmental conditions. Relatively little is known about the quality or quantity of physical activity engaged in by children during indoor free soft play. The aim of this study was to evaluate the contribution indoor free soft play can make in enabling children to meet physical activity guidelines and to evaluate the effects of sex and body mass index category. Methods: Seventy-two boys and girls aged five to 10 years engaged in un-controlled indoor free soft play with a mean duration of 120.7 (27.1) min, during which physical activity was monitored using Actigraph accelerometers. Results: Children spent an average of 61.7 (24.2) min engaging in moderate to vigorous physical activity (MVPA) and 51.4% (n = 37) achieved the recommended 60 min of MVPA through the single visit to the indoor soft play center. Boys (68.3 (25.7) min) engaged in significantly (p < 0.05) more MVPA than girls (55.8 (21.4) min). Normal weight (65.7 (23.3) min) children engaged in significantly more MVPA than overweight children (48.0 (18.9) min). Conclusions: Attendance at a soft play indoor center has the potential to support children to engage in sufficient MVPA and overcome environmental factors that can restrict physical activity opportunities.

Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours.

Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy.

Gene expression signature of normal cell-of-origin predicts ovarian tumor outcomes.

The potential role of the cell-of-origin in determining the tumor phenotype has been raised, but not adequately examined. We hypothesized that distinct cells-of-origin may play a role in determining ovarian tumor phenotype and outcome. Here we describe a new cell culture medium for in vitro culture of paired normal human ovarian (OV) and fallopian tube (FT) epithelial cells from donors without cancer. While these cells have been cultured individually for short periods of time, to our knowledge this is the first long-term culture of both cell types from the same donors. Through analysis of the gene expression profiles of the cultured OV/FT cells we identified a normal cell-of-origin gene signature that classified primary ovarian cancers into OV-like and FT-like subgroups; this classification correlated with significant differences in clinical outcomes. The identification of a prognostically significant gene expression signature derived solely from normal untransformed cells is consistent with the hypothesis that the normal cell-of-origin may be a source of ovarian tumor heterogeneity and the associated differences in tumor outcome.

The functional properties of human slow skeletal troponin T isoforms in cardiac muscle regulation.

Human slow skeletal troponin T (HSSTnT) shares a high degree of homology with cardiac TnT (CTnT). Although the presence of HSSTnT has not been confirmed in the heart at the protein level, detectable levels of HSSTnT mRNA have been found. Whether HSSTnT isoforms are expressed transiently remains unknown. Because transient re-expression of HSSTnT may be a potential mechanism of regulating function, we explored the effect of HSSTnT on the regulation of cardiac muscle. At least three HSSTnT isoforms have been found to exist in slow skeletal muscle: HSSTnT1 (+exons 5 and 12), HSSTnT2 (+exon 5, -exon 12), and HSSTnT3 (-exons 5 and 12). Another isoform, HSSTnT hypothetical (Hyp) (-exon 5, +exon 12), has only been found at the mRNA level. Compared with HCTnT3 (adult isoform), Tn complexes containing HSSTnT1, -2, and -3 did not alter the actomyosin ATPase activation and inhibition in the presence and absence of Ca(2+), respectively. HSSTnTHyp was not evaluated as it did not form a Tn complex under a variety of conditions. Porcine papillary skinned fibers displaced with HSSTnT1, -2, or -3 and reconstituted with human cardiac troponin I and troponin C (HCTnI·TnC) complex showed a decrease in the Ca(2+) sensitivity of force development and an increase in maximal recovered force (HSSTnT1 and -3) compared with HCTnT3. In contrast, HSSTnTHyp showed an increase in the Ca(2+) sensitivity of force development. This suggests that re- or overexpression of specific SSTnT isoforms might have therapeutic potential in the failing heart because they increase the maximal force of contraction. In addition, circular dichroism and proteolytic digestion experiments revealed structural differences between HSSTnT isoforms and HCTnT3 and that HSSTnT1 is more susceptible to calpain and trypsin proteolysis than the other HSSTnTs. Overall, HSSTnT isoforms despite being homologues of CTnT may display distinct functional properties in muscle regulation.

The activity profile in international Taekwondo competition is modulated by weight category.

PURPOSE: To examine the activity profiles of elite male competitors during international Taekwondo competition in relation to fin, feather, and heavy weight categories. METHODS: Twelve male Taekwondo competitors equally representing fin, feather, and heavy weight divisions were studied during the 2005 World Taekwondo Championships using a time-motion system developed to analyze the activities and activity phases. The frequency and duration of activities were recorded and assimilated into four independent activity phases: fighting activity, preparatory activity, nonpreparatory activity and stoppage activity. The total number of exchanges and kicks were also calculated for each combat. RESULTS: For all weight groupings the mean ± SD fighting time was 1.7 ± 0.3 s, preparatory time 6.4 ± 2.1 s, nonpreparatory time 3.0 ± 0.6 s, referee stoppage time 2.8 ± 0.9 s and 28 ± 6 exchanges and 31 ± 7 kicks were performed. Differences in the mean fighting time (fin: 1.4 ± 0.2 s vs heavy: 1.8 ± 0.3 s; P = .03; effect size [ES] = 1.57), preparatory time (fin: 5.3 ± 1.0 s vs feather: 8.2 ± 2.6 s; P = .03; ES = 1.47) and the total number of exchanges (feather: 24 ± 6 vs heavy: 32 ± 5; P = .03; ES = 1.44) were identified between the weight categories. CONCLUSIONS: The activity profile in international Taekwondo competition was modulated by competitors' weight category. These findings suggest that conditioning sessions may need to be specialized to the requirements of specific weight categories.

Functional characterization of TNNC1 rare variants identified in dilated cardiomyopathy.

TNNC1, which encodes cardiac troponin C (cTnC), remains elusive as a dilated cardiomyopathy (DCM) gene. Here, we report the clinical, genetic, and functional characterization of four TNNC1 rare variants (Y5H, M103I, D145E, and I148V), all previously reported by us in association with DCM (Hershberger, R. E., Norton, N., Morales, A., Li, D., Siegfried, J. D., and Gonzalez-Quintana, J. (2010) Circ. Cardiovasc. Genet. 3, 155-161); in the previous study, two variants (Y5H and D145E) were identified in subjects who also carried MYH7 and MYBPC3 rare variants, respectively. Functional studies using the recombinant human mutant cTnC proteins reconstituted into porcine papillary skinned fibers showed decreased Ca(2+) sensitivity of force development (Y5H and M103I). Furthermore, the cTnC mutants diminished (Y5H and I148V) or abolished (M103I) the effects of PKA phosphorylation on Ca(2+) sensitivity. Only M103I decreased the troponin activation properties of the actomyosin ATPase when Ca(2+) was present. CD spectroscopic studies of apo (absence of divalent cations)-, Mg(2+)-, and Ca(2+)/Mg(2+)-bound states indicated that all of the cTnC mutants (except I148V in the Ca(2+)/Mg(2+) condition) decreased the α-helical content. These results suggest that each mutation alters the function/ability of the myofilament to bind Ca(2+) as a result of modifications in cTnC structure. One variant (D145E) that was previously reported in association with hypertrophic cardiomyopathy and that produced results in vivo in this study consistent with prior hypertrophic cardiomyopathy functional studies was found associated with the MYBPC3 P910T rare variant, likely contributing to the observed DCM phenotype. We conclude that these rare variants alter the regulation of contraction in some way, and the combined clinical, molecular, genetic, and functional data reinforce the importance of TNNC1 rare variants in the pathogenesis of DCM.

Fetal cardiac troponin isoforms rescue the increased Ca2+ sensitivity produced by a novel double deletion in cardiac troponin T linked to restrictive cardiomyopathy: a clinical, genetic, and functional approach.

A novel double deletion in cardiac troponin T (cTnT) of two highly conserved amino acids (Asn-100 and Glu-101) was found in a restrictive cardiomyopathic (RCM) pediatric patient. Clinical evaluation revealed the presence of left atrial enlargement and marked left ventricle diastolic dysfunction. The explanted heart examined by electron microscopy revealed myofibrillar disarray and mild fibrosis. Pedigree analysis established that this mutation arose de novo. The patient tested negative for six other sarcomeric genes. The single and double recombinant cTnT mutants were generated, and their functional consequences were analyzed in porcine skinned cardiac muscle. In the adult Tn environment (cTnT3 + cardiac troponin I), the single cTnT3-ΔN100 and cTnT3-ΔE101 mutations had opposing effects on the Ca(2+) sensitivity of force development compared with WT, whereas the double deletion cTnT3-ΔN100/ΔE101 increased the Ca(2+) sensitivity + 0.19 pCa units. In addition, cTnT3-ΔN100/ΔE101 decreased the cooperativity of force development, suggesting alterations in intrafilament protein-protein interactions. In the fetal Tn environment, (cTnT1 + slow skeletal troponin I), the single (cTnT1-ΔN110) and double (cTnT1-ΔN110/ΔE111) deletions did not change the Ca(2+) sensitivity compared with control. To recreate the patient's heterozygous genotype, we performed a reconstituted ATPase activity assay. Thin filaments containing 50:50 cTnT3-ΔN100/ΔE101:cTnT3-WT also increased the myofilament Ca(2+) sensitivity compared with WT. Co-sedimentation of thin filament proteins indicated that no significant changes occurred in the binding of Tn containing the RCM cTnT mutation to actin-Tm. This report reveals the protective role of Tn fetal isoforms as they rescue the increased Ca(2+) sensitivity produced by a cTnT-RCM mutation and may account for the lack of lethality during gestation.

Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach.

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.

Physiological responses and perceived exertion during international Taekwondo competition.

PURPOSE: To investigate the physiological responses and perceived exertion during international Taekwondo competition. METHODS: Eight male Taekwondo black belts (mean +/- SD, age 22 +/- 4 y, body mass 69.4 +/- 13.4 kg, height 1.82 +/- 0.10 m, competition experience 9 +/- 5 y) took part in an international-level Taekwondo competition. Each combat included three 2-min rounds with 30 s of recovery between each round. Heart rate (HR) was recorded at 5-s intervals during each combat. Capillary blood lactate samples were taken from the fingertip 1 min before competition, directly after each round and 1 min after competition. Competitors' rating of perceived exertion (RPE) was recorded for each round using Borg's 6-to-20 scale. RESULTS: HR (round 1: 175 +/- 15 to round 3: 187 +/- 8 beats x min(-1); P < .05), percentage of HR maximum (round 1: 89 +/- 8 to round 3: 96 +/- 5% HRmax; P < .05), blood lactate (round 1: 7.5 +/- 1.6 to round 3: 11.9 +/- 2.1 mmol x L(-1); P < .05) and RPE (round 1: 11 +/- 2 to round 3: 14 +/- 2; P < .05; mean +/- SD) increased significantly across rounds. CONCLUSIONS: International-level Taekwondo competition elicited near-maximal cardiovascular responses, high blood lactate concentrations, and increases in competitors' RPE across combat. Training should therefore include exercise bouts that sufficiently stimulate both aerobic and anaerobic metabolism.

Synthesis and evaluation of 2,5-dihydrochorismate analogues as inhibitors of the chorismate-utilising enzymes.

A library of 2,5-dihydrochorismate analogues were designed as inhibitors of the chorismate-utilising enzymes including anthranilate synthase, isochorismate synthase, salicylate synthase and 4-amino-4-deoxychorismate synthase. The inhibitors were synthesised in seven or eight steps from shikimic acid, sourced from star anise. The compounds exhibited moderate but differential inhibition against the four chorismate-utilising enzymes.

A functional and structural study of troponin C mutations related to hypertrophic cardiomyopathy.

Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca(2+) sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281-288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca(2+)] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca(2+) sensitivity with only A8V demonstrating lowered Ca(2+) sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca(2+). None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca(2+) affinity of site II measured by 2-(4'-(2''-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca(2+). Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca(2+) sensitivity of the myofilament; 2) the effects of the mutations on the Ca(2+) affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca(2+) sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca(2+) binding to cTnC.

A troponin T mutation that causes infantile restrictive cardiomyopathy increases Ca2+ sensitivity of force development and impairs the inhibitory properties of troponin.

Restrictive cardiomyopathy (RCM) is a rare disorder characterized by impaired ventricular filling with decreased diastolic volume. We are reporting the functional effects of the first cardiac troponin T (CTnT) mutation linked to infantile RCM resulting from a de novo deletion mutation of glutamic acid 96. The mutation was introduced into adult and fetal isoforms of human cardiac TnT (HCTnT3-DeltaE96 and HCTnT1-DeltaE106, respectively) and studied with either cardiac troponin I (CTnI) or slow skeletal troponin I (SSTnI). Skinned cardiac fiber measurements showed a large leftward shift in the Ca(2+) sensitivity of force development with no differences in the maximal force. HCTnT1-DeltaE106 showed a significant increase in the activation of actomyosin ATPase with either CTnI or SSTnI, whereas HCTnT3-DeltaE96 was only able to increase the ATPase activity with CTnI. Both mutants showed an impaired ability to inhibit the ATPase activity. The capacity of the CTnI.CTnC and SSTnI.CTnC complexes to fully relax the fibers after TnT displacement was also compromised. Experiments performed using fetal troponin isoforms showed a less severe impact compared with the adult isoforms, which is consistent with the cardioprotective role of SSTnI and the rapid onset of RCM after birth following the isoform switch. These data indicate that troponin mutations related to RCM may have specific functional phenotypes, including large leftward shifts in the Ca(2+) sensitivity and impaired abilities to inhibit ATPase and to relax skinned fibers. All of this would account for and contribute to the severe diastolic dysfunction seen in RCM.

Heart rate responses to Taekwondo training in experienced practitioners.

The purpose of this study was to evaluate the heart rate (HR) responses of specific Taekwondo training activities, practiced by experienced practitioners in a natural training environment. Eight male experienced Taekwondo practitioners, with 3- 13 years (5.4 +/- 3.2 years) experience took part in a 5-day Taekwondo training camp. Continuous HR measures were recorded at 5-second intervals during 6 training sessions; each session was observed and notated, and a diary of training activities was recorded. The HR responses were assimilated into 8 fundamental training activities for analysis: elastics, technical combinations, step sparring, pad work, forms, basic techniques and forms, sparring drills, and free sparring. Taekwondo training elicited HR into 64.7-81.4% of HR maximum (%HRmax). Moderate relative exercise intensities (64.7-69.4%HRmax) were elicited by elastics, technical combinations, and step sparring. The remaining 5 training activities elicited hard relative exercise intensities (74.7-81.4%HRmax). One-way repeated-measures analysis of variance with post hoc analysis revealed that elastics, technical combinations, and step sparring elicited significantly lower relative intensities than the remaining training activities (p < 0.05). Furthermore, forms, basic techniques and forms, sparring drills, and free sparring elicited significantly higher relative intensities than the remaining training activities (p < 0.05). In conclusion, all Taekwondo training activities in this study seemed suitable for cardiovascular conditioning, although different training activities stressed the cardiovascular system to different degrees. Practically, this suggests coaches need to structure Taekwondo training sessions based not only on the technical and tactical needs of practitioners but also in a manner that enables sufficient cardiovascular conditioning for competition.

Evidence that a novel thioesterase is responsible for polyketide chain release during biosynthesis of the polyether ionophore monensin.

Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the alpha/beta-hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N-acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.

Identification of 4-amino-4-deoxychorismate synthase as the molecular target for the antimicrobial action of (6s)-6-fluoroshikimate.

(6S)-6-Fluoroshikimate has antimicrobial activity. The molecular basis of this effect had not been identified, but there was speculation that (6S)-6-fluoroshikimate is first converted in vivo into 2-fluorochorismate, which then could inhibit 4-amino-4-deoxychorismate synthase (ADCS). 2-Fluorochorismate was prepared from E-fluorophosphoenolpyruvate and erythose-4-phosphate by the sequential reactions of DAHP synthase, dehydroquinate synthase, dehydroquinase, shikimate dehydrogenase, EPSP synthase, and chorismate synthase. Inhibition studies on ADCS showed that it was inhibited rapidly and irreversibly by 2-fluorochorismate. Electrospray mass spectrometry of the inactivated enzyme showed an additional mass of 198 +/- 10 Da. A novel peptide of 1087.6 Da was identified in the HPLC trace for the tryptic digest of 2-fluorochorismate-inactivated ADCS. Sequencing of this peptide by MS/MS showed that the peptide corresponded to residues 272-279 with a modification of 206.1 Da on Lys-274. This observation is particularly exciting in the context of a recent proposal for the catalytic mechanism of ADCS.

Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization.

The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway-specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently-proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin-linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.