Microstructural and Microvascular Phenotype of Sarcomere Mutation Carriers and Overt Hypertrophic Cardiomyopathy.

BACKGROUND: In hypertrophic cardiomyopathy (HCM), myocyte disarray and microvascular disease (MVD) have been implicated in adverse events, and recent evidence suggests that these may occur early. As novel therapy provides promise for disease modification, detection of phenotype development is an emerging priority. To evaluate their utility as early and disease-specific biomarkers, we measured myocardial microstructure and MVD in 3 HCM groups-overt, either genotype-positive (G+LVH+) or genotype-negative (G-LVH+), and subclinical (G+LVH-) HCM-exploring relationships with electrical changes and genetic substrate. METHODS: This was a multicenter collaboration to study 206 subjects: 101 patients with overt HCM (51 G+LVH+ and 50 G-LVH+), 77 patients with G+LVH-, and 28 matched healthy volunteers. All underwent 12-lead ECG, quantitative perfusion cardiac magnetic resonance imaging (measuring myocardial blood flow, myocardial perfusion reserve, and perfusion defects), and cardiac diffusion tensor imaging measuring fractional anisotropy (lower values expected with more disarray), mean diffusivity (reflecting myocyte packing/interstitial expansion), and second eigenvector angle (measuring sheetlet orientation). RESULTS: Compared with healthy volunteers, patients with overt HCM had evidence of altered microstructure (lower fractional anisotropy, higher mean diffusivity, and higher second eigenvector angle; all P<0.001) and MVD (lower stress myocardial blood flow and myocardial perfusion reserve; both P<0.001). Patients with G-LVH+ were similar to those with G+LVH+ but had elevated second eigenvector angle (P<0.001 after adjustment for left ventricular hypertrophy and fibrosis). In overt disease, perfusion defects were found in all G+ but not all G- patients (100% [51/51] versus 82% [41/50]; P=0.001). Patients with G+LVH- compared with healthy volunteers similarly had altered microstructure, although to a lesser extent (all diffusion tensor imaging parameters; P<0.001), and MVD (reduced stress myocardial blood flow [P=0.015] with perfusion defects in 28% versus 0 healthy volunteers [P=0.002]). Disarray and MVD were independently associated with pathological electrocardiographic abnormalities in both overt and subclinical disease after adjustment for fibrosis and left ventricular hypertrophy (overt: fractional anisotropy: odds ratio for an abnormal ECG, 3.3, P=0.01; stress myocardial blood flow: odds ratio, 2.8, P=0.015; subclinical: fractional anisotropy odds ratio, 4.0, P=0.001; myocardial perfusion reserve odds ratio, 2.2, P=0.049). CONCLUSIONS: Microstructural alteration and MVD occur in overt HCM and are different in G+ and G- patients. Both also occur in the absence of hypertrophy in sarcomeric mutation carriers, in whom changes are associated with electrocardiographic abnormalities. Measurable changes in myocardial microstructure and microvascular function are early-phenotype biomarkers in the emerging era of disease-modifying therapy.

Accurate diagnosis of apical hypertrophic cardiomyopathy using explainable advanced electrocardiogram analysis.

AIMS: Typical electrocardiogram (ECG) features of apical hypertrophic cardiomyopathy (ApHCM) include tall R waves and deep or giant T-wave inversion in the precordial leads, but these features are not always present. The ECG is used as the gatekeeper to cardiac imaging for diagnosis. We tested whether explainable advanced ECG (A-ECG) could accurately diagnose ApHCM. METHODS AND RESULTS: Advanced ECG analysis was performed on standard resting 12-lead ECGs in patients with ApHCM [n = 75 overt, n = 32 relative (<15 mm hypertrophy); a subgroup of which underwent cardiovascular magnetic resonance (n = 92)], and comparator subjects (n = 2449), including healthy volunteers (n = 1672), patients with coronary artery disease (n = 372), left ventricular electrical remodelling (n = 108), ischaemic (n = 114) or non-ischaemic cardiomyopathy (n = 57), and asymmetrical septal hypertrophy HCM (n = 126). Multivariable logistic regression identified four A-ECG measures that together discriminated ApHCM from other diseases with high accuracy [area under the receiver operating characteristic (AUC) curve (bootstrapped 95% confidence interval) 0.982 (0.965-0.993)]. Linear discriminant analysis also diagnosed ApHCM with high accuracy [AUC 0.989 (0.986-0.991)]. CONCLUSION: Explainable A-ECG has excellent diagnostic accuracy for ApHCM, even when the hypertrophy is relative, with A-ECG analysis providing incremental diagnostic value over imaging alone. The electrical (ECG) and anatomical (wall thickness) disease features do not completely align, suggesting that future diagnostic and management strategies may incorporate both features.

Modulating signalling lifetime to optimise a prototypical animal opsin for optogenetic applications.

Animal opsins are light activated G-protein-coupled receptors, capable of optogenetic control of G-protein signalling for research or therapeutic applications. Animal opsins offer excellent photosensitivity, but their temporal resolution can be limited by long photoresponse duration when expressed outside their native cellular environment. Here, we explore methods for addressing this limitation for a prototypical animal opsin (human rod opsin) in HEK293T cells. We find that the application of the canonical rhodopsin kinase (GRK1)/visual arrestin signal termination mechanism to this problem is complicated by a generalised suppressive effect of GRK1 expression. This attenuation can be overcome using phosphorylation-independent mutants of arrestin, especially when these are tethered to the opsin protein. We further show that point mutations targeting the Schiff base stability of the opsin can also reduce signalling lifetime. Finally, we apply one such mutation (E122Q) to improve the temporal fidelity of restored visual responses following ectopic opsin expression in the inner retina of a mouse model of retinal degeneration (rd1). Our results reveal that these two strategies (targeting either arrestin binding or Schiff-base hydrolysis) can produce more time-delimited opsin signalling under heterologous expression and establish the potential of this approach to improve optogenetic performance.

Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons.

There is no consensus on the best inhibitory optogenetic tool. Since Gi/o signalling is a native mechanism of neuronal inhibition, we asked whether Lamprey Parapinopsin ("Lamplight"), a Gi/o-coupled bistable animal opsin, could be used for optogenetic silencing. We show that short (405 nm) and long (525 nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, respectively, and that combining these wavelengths can be used to achieve intermediate levels of activity. These properties can be applied to produce switchable neuronal hyperpolarisation and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. Expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, with 405 and 525 nm stimuli producing responses of opposite sign in the output neurons of the retina. We conclude that bistable animal opsins can co-opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and reversible.

Precision measurement of cardiac structure and function in cardiovascular magnetic resonance using machine learning.

BACKGROUND: Measurement of cardiac structure and function from images (e.g. volumes, mass and derived parameters such as left ventricular (LV) ejection fraction [LVEF]) guides care for millions. This is best assessed using cardiovascular magnetic resonance (CMR), but image analysis is currently performed by individual clinicians, which introduces error. We sought to develop a machine learning algorithm for volumetric analysis of CMR images with demonstrably better precision than human analysis. METHODS: A fully automated machine learning algorithm was trained on 1923 scans (10 scanner models, 13 institutions, 9 clinical conditions, 60,000 contours) and used to segment the LV blood volume and myocardium. Performance was quantified by measuring precision on an independent multi-site validation dataset with multiple pathologies with n = 109 patients, scanned twice. This dataset was augmented with a further 1277 patients scanned as part of routine clinical care to allow qualitative assessment of generalization ability by identifying mis-segmentations. Machine learning algorithm ('machine') performance was compared to three clinicians ('human') and a commercial tool (cvi42, Circle Cardiovascular Imaging). FINDINGS: Machine analysis was quicker (20 s per patient) than human (13 min). Overall machine mis-segmentation rate was 1 in 479 images for the combined dataset, occurring mostly in rare pathologies not encountered in training. Without correcting these mis-segmentations, machine analysis had superior precision to three clinicians (e.g. scan-rescan coefficients of variation of human vs machine: LVEF 6.0% vs 4.2%, LV mass 4.8% vs. 3.6%; both P < 0.05), translating to a 46% reduction in required trial sample size using an LVEF endpoint. CONCLUSION: We present a fully automated algorithm for measuring LV structure and global systolic function that betters human performance for speed and precision.

Characteristics and outcomes of SARS-CoV-2 infection in Victorian children at a tertiary paediatric hospital.

AIM: Victoria experienced two 'waves' of COVID-19 between March and September 2020 and more cases than any other jurisdiction in Australia. Although world-wide reports of COVID-19 reflect that children are less likely to experience severe disease compared with adults, hospitalisations and deaths have been reported. We report testing and outcomes of children with SARS-CoV-2 infection presenting to a tertiary paediatric hospital in Melbourne. METHODS: We conducted a prospective cohort study at The Royal Children's Hospital (RCH), including all children and adolescents (aged 0-18 years) who presented and were tested for SARS-CoV-2 over a 6-month period, between 21 March 2020, up to the 21 September 2020. Detailed epidemiological and clinical data were recorded. RESULTS: A total of 19 708 tests for SARS-CoV-2 were performed in 14 419 patients. One hundred and eighty patients tested positive for SARS-CoV-2 (1.2%). 110 (61%) were symptomatic, 60 (33%) were asymptomatic and 10 (6%) were pre-symptomatic. Close contacts of a positive case were associated with a higher risk of a testing positive for SARS-CoV-2 (120/2027 (6%) vs. 60/14589 (0.4%), RD 5.5 (95% CI 4.5 to 6.5), P < 0.001). Eighteen (10%) SARS-CoV-2-positive patients were admitted to hospital with one patient requiring intensive care. All patients recovered fully with no deaths. CONCLUSION: In Victorian children presenting to a tertiary hospital, SARS-CoV-2 infection caused predominantly mild or asymptomatic infection, with most children not requiring hospitalisation.

Use of quantitative cardiovascular magnetic resonance myocardial perfusion mapping for characterization of ischemia in patients with left internal mammary coronary artery bypass grafts.

BACKGROUND: Quantitative myocardial perfusion mapping using cardiovascular magnetic resonance (CMR) is validated for myocardial blood flow (MBF) estimation in native vessel coronary artery disease (CAD). Following coronary artery bypass graft (CABG) surgery, perfusion defects are often detected in territories supplied by the left internal mammary artery (LIMA) graft, but their interpretation and subsequent clinical management is variable. METHODS: We assessed myocardial perfusion using quantitative CMR perfusion mapping in 38 patients with prior CABG surgery, all with angiographically-proven patent LIMA grafts to the left anterior descending coronary artery (LAD) and no prior infarction in the LAD territory. Factors potentially determining MBF in the LIMA-LAD myocardial territory, including the impact of delayed contrast arrival through the LIMA graft were evaluated. RESULTS: Perfusion defects were reported on blinded visual analysis in the LIMA-LAD territory in 27 (71%) cases, despite LIMA graft patency and no LAD infarction. Native LAD chronic total occlusion (CTO) was a strong independent predictor of stress MBF (B = - 0.41, p = 0.014) and myocardial perfusion reserve (MPR) (B = - 0.56, p = 0.005), and was associated with reduced stress MBF in the basal (1.47 vs 2.07 ml/g/min; p = 0.002) but not the apical myocardial segments (1.52 vs 1.87 ml/g/min; p = 0.057). Extending the maximum arterial time delay incorporated in the quantitative perfusion algorithm, resulted only in a small increase (3.4%) of estimated stress MBF. CONCLUSIONS: Perfusion defects are frequently detected in LIMA-LAD subtended territories post CABG despite LIMA patency. Although delayed contrast arrival through LIMA grafts causes a small underestimation of MBF, perfusion defects are likely to reflect true reductions in myocardial blood flow, largely due to proximal native LAD disease.

Tacrine - Benzothiazoles: Novel class of potential multitarget anti-Alzheimers drugs dealing with cholinergic, amyloid and mitochondrial systems.

A series of tacrine - benzothiazole hybrids incorporate inhibitors of acetylcholinesterase (AChE), amyloid ß (Aß) aggregation and mitochondrial enzyme ABAD, whose interaction with Aß leads to mitochondrial dysfunction, into a single molecule. In vitro, several of 25 final compounds exerted excellent anti-AChE properties and interesting capabilities to block Aß aggregation. The best derivative of the series could be considered 10w that was found to be highly potent and selective towards AChE with the IC50 value in nanomolar range. Moreover, the same drug candidate exerted absolutely the best results of the series against ABAD, decreasing its activity by 23% at 100 µM concentration. Regarding the cytotoxicity profile of highlighted compound, it roughly matched that of its parent compound - 6-chlorotacrine. Finally, 10w was forwarded for in vivo scopolamine-induced amnesia experiment consisting of Morris Water Maze test, where it demonstrated mild procognitive effect. Taking into account all in vitro and in vivo data, highlighted derivative 10w could be considered as the lead structure worthy of further investigation.

Ketamine Restores Thalamic-Prefrontal Cortex Functional Connectivity in a Mouse Model of Neurodevelopmental Disorder-Associated 2p16.3 Deletion.

2p16.3 deletions, involving heterozygous NEUREXIN1 (NRXN1) deletion, dramatically increase the risk of developing neurodevelopmental disorders, including autism and schizophrenia. We have little understanding of how NRXN1 heterozygosity increases the risk of developing these disorders, particularly in terms of the impact on brain and neurotransmitter system function and brain network connectivity. Thus, here we characterize cerebral metabolism and functional brain network connectivity in Nrxn1α heterozygous mice (Nrxn1α+/- mice), and assess the impact of ketamine and dextro-amphetamine on cerebral metabolism in these animals. We show that heterozygous Nrxn1α deletion alters cerebral metabolism in neural systems implicated in autism and schizophrenia including the thalamus, mesolimbic system, and select cortical regions. Nrxn1α heterozygosity also reduces the efficiency of functional brain networks, through lost thalamic "rich club" and prefrontal cortex (PFC) hub connectivity and through reduced thalamic-PFC and thalamic "rich club" regional interconnectivity. Subanesthetic ketamine administration normalizes the thalamic hypermetabolism and partially normalizes thalamic disconnectivity present in Nrxn1α+/- mice, while cerebral metabolic responses to dextro-amphetamine are unaltered. The data provide new insight into the systems-level impact of heterozygous Nrxn1α deletion and how this increases the risk of developing neurodevelopmental disorders. The data also suggest that the thalamic dysfunction induced by heterozygous Nrxn1α deletion may be NMDA receptor-dependent.

Prevalence and outcome of dual aortic stenosis and cardiac amyloid pathology in patients referred for transcatheter aortic valve implantation.

AIMS: Cardiac amyloidosis is common in elderly patients with aortic stenosis (AS) referred for transcatheter aortic valve implantation (TAVI). We hypothesized that patients with dual aortic stenosis and cardiac amyloid pathology (AS-amyloid) would have different baseline characteristics, periprocedural and mortality outcomes. METHODS AND RESULTS: Patients aged ≥75 with severe AS referred for TAVI at two sites underwent blinded bone scintigraphy prior to intervention (Perugini Grade 0 negative, 1-3 increasingly positive). Baseline assessment included echocardiography, electrocardiogram (ECG), blood tests, 6-min walk test, and health questionnaire, with periprocedural complications and mortality follow-up. Two hundred patients were recruited (aged 85 ± 5 years, 50% male). AS-amyloid was found in 26 (13%): 8 Grade 1, 18 Grade 2. AS-amyloid patients were older (88 ± 5 vs. 85 ± 5 years, P = 0.001), with reduced quality of life (EQ-5D-5L 50 vs. 65, P = 0.04). Left ventricular wall thickness was higher (14 mm vs. 13 mm, P = 0.02), ECG voltages lower (Sokolow-Lyon 1.9 ± 0.7 vs. 2.5 ± 0.9 mV, P = 0.03) with lower voltage/mass ratio (0.017 vs. 0.025 mV/g/m2, P = 0.03). High-sensitivity troponin T and N-terminal pro-brain natriuretic peptide were higher (41 vs. 21 ng/L, P < 0.001; 3702 vs. 1254 ng/L, P = 0.001). Gender, comorbidities, 6-min walk distance, AS severity, prevalence of disproportionate hypertrophy, and post-TAVI complication rates (38% vs. 35%, P = 0.82) were the same. At a median follow-up of 19 (10-27) months, there was no mortality difference (P = 0.71). Transcatheter aortic valve implantation significantly improved outcome in the overall population (P < 0.001) and in those with AS-amyloid (P = 0.03). CONCLUSIONS: AS-amyloid is common and differs from lone AS. Transcatheter aortic valve implantation significantly improved outcome in AS-amyloid, while periprocedural complications and mortality were similar to lone AS, suggesting that TAVI should not be denied to patients with AS-amyloid.

High content phenotypic screening identifies serotonin receptor modulators with selective activity upon breast cancer cell cycle and cytokine signaling pathways.

Heterogeneity in disease mechanisms between genetically distinct patients contributes to high attrition rates in late stage clinical drug development. New personalized medicine strategies aim to identify predictive biomarkers which stratify patients most likely to respond to a particular therapy. However, for complex multifactorial diseases not characterized by a single genetic driver, empirical approaches to identifying predictive biomarkers and the most promising therapies for personalized medicine are required. In vitro pharmacogenomics seeks to correlate in vitro drug sensitivity testing across panels of genetically distinct cell models with genomic, gene expression or proteomic data to identify predictive biomarkers of drug response. However, the vast majority of in vitro pharmacogenomic studies performed to date are limited to dose-response screening upon a single viability assay endpoint. In this article we describe the application of multiparametric high content phenotypic screening and the theta comparative cell scoring method to quantify and rank compound hits, screened at a single concentration, which induce a broad variety of divergent phenotypic responses between distinct breast cancer cell lines. High content screening followed by transcriptomic pathway analysis identified serotonin receptor modulators which display selective activity upon breast cancer cell cycle and cytokine signaling pathways correlating with inhibition of cell growth and survival. These methods describe a new evidence-led approach to rapidly identify compounds which display distinct response between different cell types. The results presented also warrant further investigation of the selective activity of serotonin receptor modulators upon breast cancer cell growth and survival as a potential drug repurposing opportunity.

Quantitative myocardial perfusion in coronary artery disease: A perfusion mapping study.

BACKGROUND: Cardiac MR stress perfusion remains a qualitative technique in clinical practice due to technical and postprocessing challenges. However, automated inline perfusion mapping now permits myocardial blood flow (MBF, ml/g/min) quantification on-the-fly without user input. PURPOSE: To investigate the diagnostic performance of this novel technique in detecting occlusive coronary artery disease (CAD) in patients scheduled to undergo coronary angiography. STUDY TYPE: Prospective, observational. SUBJECTS: Fifty patients with suspected CAD and 24 healthy volunteers. FIELD STRENGTH: 1.5T. SEQUENCE: "Dual" sequence multislice 2D saturation recovery. ASSESSMENT: All patients underwent cardiac MR with perfusion mapping and invasive coronary angiography; the healthy volunteers had MR with perfusion mapping alone. STATISTICAL TESTS: Comparison between numerical variables was performed using an independent t-test. Receiver operator characteristic (ROC) curves were generated for transmyocardial, endocardial stress MBF, and myocardial perfusion reserve (MPR, the stress:rest MBF ratio) to diagnose severe (>70%) stenoses as measured by 3D quantitative coronary angiography (QCA). ROC curves were compared by the method of DeLong et al. RESULTS: Compared with volunteers, patients had lower stress MBF and MPR even in vessels with <50% stenosis (2.00 vs. 3.08 ml/g/min, respectively). As stenosis severity increased (<50%, 50-70%, >70%), MBF and MPR decreased. To diagnose occlusive (>70%) CAD, endocardial and transmyocardial stress MBF were superior to MPR (area under the curve 0.92 [95% CI 0.86-0.97] vs. 0.90 [95% CI 0.84-0.95] and 0.80 [95% CI 0.72-0.87], respectively). An endocardial threshold of 1.31 ml/g/min provided a per-coronary artery sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 90%, 82%, 50%, and 98%, with a per-patient diagnostic performance of 100%, 66%, 57%, and 100%, respectively. DATA CONCLUSION: Perfusion mapping can diagnose occlusive CAD with high accuracy and, in particular, high sensitivity and NPV make it a potential "rule-out" test. LEVEL OF EVIDENCE: 1 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2019;50:756-762.

Novel Benzothiazole-based Ureas as 17ß-HSD10 Inhibitors, A Potential Alzheimer's Disease Treatment.

: It has long been established that mitochondrial dysfunction in Alzheimer's disease (AD) patients can trigger pathological changes in cell metabolism by altering metabolic enzymes such as the mitochondrial 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10), also known as amyloid-binding alcohol dehydrogenase (ABAD). We and others have shown that frentizole and riluzole derivatives can inhibit 17ß-HSD10 and that this inhibition is beneficial and holds therapeutic merit for the treatment of AD. Here we evaluate several novel series based on benzothiazolylurea scaffold evaluating key structural and activity relationships required for the inhibition of 17ß-HSD10. Results show that the most promising of these compounds have markedly increased potency on our previously published inhibitors, with the most promising exhibiting advantageous features like low cytotoxicity and target engagement in living cells.

Building a Global, Pediatric Vascular Access Registry: A Scoping Review of Trial Outcomes and Quality Indicators to Inform Evidence-Based Practice.

BACKGROUND: Internationally, there is a lack of comparative vascular access (VA) data for pediatric clinicians and organizations to benchmark outcomes, evaluate quality initiatives, and improve practice. A VA registry is needed to address these knowledge and data capture gaps. OBJECTIVES: To determine the range and heterogeneity of VA outcome measures or quality indicators reported in randomized controlled trials (RCTs) and clinical registries, to inform development of a homogeneous, reliable, minimum dataset for a pediatric VA registry. METHODS: Scoping review framework. A systematic search for RCTs reporting VA outcomes in pediatrics and neonates was undertaken in the Cochrane library, EMBASE, CINAHL, PubMed, MEDLINE, and EBSCO using a medical subject headings and key words related to VA and pediatrics. We included RCTs of children (0-18 years) reporting any VA outcome. We identified clinical registries reporting VA data in children (0-18) through web-based searches using key words related to VA and clinical or quality registries. Additional registries were identified through peer consultation. The frequency and scope of outcome measures and quality indicators were extracted from trials and registries and evaluated. RESULTS: From 93 RCTs included, 214 different VA measures were reported, reflecting 14 outcome domains. The most commonly reported outcome domains were insertion (44 RCTs; 47%), noninfectious complications (33 RCTs; 35%), and infectious complications (30 RCTs; 32%). Of the 22 registries identified, VA-associated infection was the main quality indicator routinely collected (12 registries; 55%). Outcomes such as mechanical complications and patient-reported outcomes were infrequently collected. LINKING EVIDENCE TO ACTION: Vascular access outcomes reported in pediatric and neonatal RCTs are highly heterogeneous. Internationally, clinical registries currently collect minimal VA data with the exception of infection outcomes. A core dataset of reliable, relevant measures to children and clinicians for VA device quality is needed. This will enable a VA registry that facilitates inter-institutional and international benchmarking.

Genome sequence, metabolic properties and cyanobacterial attachment of Porphyrobacter sp. HT-58-2 isolated from a filamentous cyanobacterium-microbial consortium.

Tolyporphins are structurally diverse tetrapyrrole macrocycles produced by the cyanobacterial culture HT-58-2. Although tolyporphins were discovered over 25 years ago, little was known about the microbiology of the culture. The studies reported herein expand the description of the community of predominantly alphaproteobacteria associated with the filamentous HT-58-2 cyanobacterium and isolate a dominant bacterium, Porphyrobacter sp. HT-58-2, for which the complete genome is established and growth properties are examined. Fluorescence in situ hybridization (FISH) analysis of the cyanobacterium-microbial community with a probe targeting the 16S rRNA of Porphyrobacter sp. HT-58-2 showed fluorescence emanating from the cyanobacterial sheath. Although genes for the biosynthesis of bacteriochlorophyll a (BChl a) are present in the Porphyrobacter sp. HT-58-2 genome, the pigment was not detected under the conditions examined, implying the absence of phototrophic growth. Comparative analysis of four Porphyrobacter spp. genomes from worldwide collection sites showed significant collinear gene blocks, with two inversions and three deletion regions. Taken together, the results enrich our understanding of the HT-58-2 cyanobacterium-microbial culture.

Quantitation of Tolyporphins, Diverse Tetrapyrrole Secondary Metabolites with Chlorophyll-Like Absorption, from a Filamentous Cyanobacterium-Microbial Community.

INTRODUCTION: Tolyporphins are unusual tetrapyrrole macrocycles produced by a non-axenic filamentous cyanobacterium (HT-58-2). Tolyporphins A-J, L, and M share a common dioxobacteriochlorin core, differ in peripheral substituents, and exhibit absorption spectra that overlap that of the dominant cyanobacterial pigment, chlorophyll a. Identification and accurate quantitation of the various tolyporphins in these chlorophyll-rich samples presents challenges. OBJECTIVE: To develop methods for the quantitative determination of tolyporphins produced under various growth conditions relative to that of chlorophyll a. METHODOLOGY: Chromatographic fractionation of large-scale (440 L) cultures afforded isolated individual tolyporphins. Lipophilic extraction of small-scale (25 mL) cultures, HPLC separation with an internal standard, and absorption detection enabled quantitation of tolyporphin A and chlorophyll a, and by inference the amounts of tolyporphins A-M. Absorption spectroscopy with multicomponent analysis of lipophilic extracts (2 mL cultures) afforded the ratio of all tolyporphins to chlorophyll a. The reported absorption spectral data for the various tolyporphins required re-evaluation for quantitative purposes. RESULTS AND DISCUSSION: The amount of tolyporphin A after 50 days of illumination ranged from 0.13 nmol/mg dry cells (media containing nitrate) to 1.12 nmol/mg (without nitrate), with maximum 0.23 times that of chlorophyll a. Under soluble-nitrogen deprivation after 35-50 days, tolyporphin A represents 1/3-1/2 of the total tolyporphins, and the total amount of tolyporphins is up to 1.8-fold that of chlorophyll a. CONCLUSIONS: The quantitative methods developed herein should facilitate investigation of the biosynthesis of tolyporphins (and other tetrapyrroles) as well as examination of other strains for production of tolyporphins. Copyright © 2017 John Wiley & Sons, Ltd.

Reach Out Churches: A Community-Based Participatory Research Pilot Trial to Assess the Feasibility of a Mobile Health Technology Intervention to Reduce Blood Pressure Among African Americans.

Innovative strategies are needed to reduce the hypertension epidemic among African Americans. Reach Out was a faith-collaborative, mobile health, randomized, pilot intervention trial of four mobile health components to reduce high blood pressure (BP) compared to usual care. It was designed and tested within a community-based participatory research framework among African Americans recruited and randomized from churches in Flint, Michigan. The purpose of this pilot study was to assess the feasibility of the Reach Out processes. Feasibility was assessed by willingness to consent (acceptance of randomization), proportion of weeks participants texted their BP readings (intervention use), number lost to follow-up (retention), and responses to postintervention surveys and focus groups (acceptance of intervention). Of the 425 church members who underwent BP screening, 94 enrolled in the study and 73 (78%) completed the 6-month outcome assessment. Median age was 58 years, and 79% were women. Participants responded with their BPs on an average of 13.7 (SD = 10.7) weeks out of 26 weeks that the BP prompts were sent. All participants reported satisfaction with the intervention. Reach Out, a faith-collaborative, mobile health intervention was feasible. Further study of the efficacy of the intervention and additional mobile health strategies should be considered.

Genome Sequence and Composition of a Tolyporphin-Producing Cyanobacterium-Microbial Community.

The cyanobacterial culture HT-58-2 was originally described as a strain of Tolypothrix nodosa with the ability to produce tolyporphins, which comprise a family of distinct tetrapyrrole macrocycles with reported efflux pump inhibition properties. Upon reviving the culture from what was thought to be a nonextant collection, studies of culture conditions, strain characterization, phylogeny, and genomics have been undertaken. Here, HT-58-2 was shown by 16S rRNA analysis to closely align with Brasilonema strains and not with Tolypothrix isolates. Light, fluorescence, and scanning electron microscopy revealed cyanobacterium filaments that are decorated with attached bacteria and associated with free bacteria. Metagenomic surveys of HT-58-2 cultures revealed a diversity of bacteria dominated by Erythrobacteraceae, 97% of which are Porphyrobacter species. A dimethyl sulfoxide washing procedure was found to yield enriched cyanobacterial DNA (presumably by removing community bacteria) and sequence data sufficient for genome assembly. The finished, closed HT-58-2Cyano genome consists of 7.85 Mbp (42.6% G+C) and contains 6,581 genes. All genes for biosynthesis of tetrapyrroles (e.g., heme, chlorophyll a, and phycocyanobilin) and almost all for cobalamin were identified dispersed throughout the chromosome. Among the 6,177 protein-encoding genes, coding sequences (CDSs) for all but two of the eight enzymes for conversion of glutamic acid to protoporphyrinogen IX also were found within one major gene cluster. The cluster also includes 10 putative genes (and one hypothetical gene) encoding proteins with domains for a glycosyltransferase, two cytochrome P450 enzymes, and a flavin adenine dinucleotide (FAD)-binding protein. The composition of the gene cluster suggests a possible role in tolyporphin biosynthesis.IMPORTANCE A worldwide search more than 25 years ago for cyanobacterial natural products with anticancer activity identified a culture (HT-58-2) from Micronesia that produces tolyporphins. Tolyporphins are tetrapyrroles, like chlorophylls, but have several profound structural differences that reside outside the bounds of known biosynthetic pathways. To begin probing the biosynthetic origin and biological function of tolyporphins, our research has focused on studying the cyanobacterial strain, about which almost nothing has been previously reported. We find that the HT-58-2 culture is composed of the cyanobacterium and a community of associated bacteria, complicating the question of which organisms make tolyporphins. Elucidation of the cyanobacterial genome revealed an intriguing gene cluster that contains tetrapyrrole biosynthesis genes and a collection of unknown genes, suggesting that the cluster may be responsible for tolyporphin production. Knowledge of the genome and the gene cluster sharply focuses research to identify related cyanobacterial producers of tolyporphins and delineate the tolyporphin biosynthetic pathway.

Conditions that promote primary human skeletal myoblast culture and muscle differentiation in vitro.

Conditions under which skeletal myoblasts are cultured in vitro are critical to growth and differentiation of these cells into mature skeletal myofibers. We examined several culture conditions that promoted human skeletal myoblast (HSkM) culture and examined the effect of microRNAs and mechanical stimulation on differentiation. Culture conditions for HSkM are different from those that enable rapid C2C12 myoblast differentiation. Culture on a growth factor-reduced Matrigel (GFR-MG)-coated surface in 2% equine serum-supplemented differentiation medium to promote HSkM differentiation under static conditions was compared with culture conditions used for C2C12 cell differentiation. Such conditions led to a >20-fold increase in myogenic miR-1, miR-133a, and miR-206 expression, a >2-fold increase in myogenic transcription factor Mef-2C expression, and an increase in sarcomeric α-actinin protein. Imposing ±10% cyclic stretch at 0.5 Hz for 1 h followed by 5 h of rest over 2 wk produced a >20% increase in miR-1, miR-133a, and miR-206 expression in 8% equine serum and a >35% decrease in 2% equine serum relative to static conditions. HSkM differentiation was accelerated in vitro by inhibition of proliferation-promoting miR-133a: immunofluorescence for sarcomeric α-actinin exhibited accelerated development of striations compared with the corresponding negative control, and Western blotting showed 30% more α-actinin at day 6 postdifferentiation. This study showed that 100 µg/ml GFR-MG coating and 2% equine serum-supplemented differentiation medium enhanced HSkM differentiation and myogenic miR expression and that addition of antisense miR-133a alone can accelerate primary human skeletal muscle differentiation in vitro.