Single-cell transcriptomics and data analyses for prokaryotes-Past, present and future concepts.

Transcriptomics, or more specifically mRNA sequencing, is a powerful tool to study gene expression at the single-cell level (scRNA-seq) which enables new insights into a plethora of biological processes. While methods for single-cell RNA-seq in eukaryotes are well established, application to prokaryotes is still challenging. Reasons for that are rigid and diverse cell wall structures hampering lysis, the lack of polyadenylated transcripts impeding mRNA enrichment, and minute amounts of RNA requiring amplification steps before sequencing. Despite those obstacles, several promising scRNA-seq approaches for bacteria have been published recently, albeit difficulties in the experimental workflow and data processing and analysis remain. In particular, bias is often introduced by amplification which makes it difficult to distinguish between technical noise and biological variation. Future optimization of experimental procedures and data analysis algorithms are needed for the improvement of scRNA-seq but also to aid in the emergence of prokaryotic single-cell multi-omics. to help address 21st century challenges in the biotechnology and health sector.

Multi-organ assessment in mainly non-hospitalized individuals after SARS-CoV-2 infection: The Hamburg City Health Study COVID programme.

AIMS: Long-term sequelae may occur after SARS-CoV-2 infection. We comprehensively assessed organ-specific functions in individuals after mild to moderate SARS-CoV-2 infection compared with controls from the general population. METHODS AND RESULTS: Four hundred and forty-three mainly non-hospitalized individuals were examined in median 9.6 months after the first positive SARS-CoV-2 test and matched for age, sex, and education with 1328 controls from a population-based German cohort. We assessed pulmonary, cardiac, vascular, renal, and neurological status, as well as patient-related outcomes. Bodyplethysmography documented mildly lower total lung volume (regression coefficient -3.24, adjusted P = 0.014) and higher specific airway resistance (regression coefficient 8.11, adjusted P = 0.001) after SARS-CoV-2 infection. Cardiac assessment revealed slightly lower measures of left (regression coefficient for left ventricular ejection fraction on transthoracic echocardiography -0.93, adjusted P = 0.015) and right ventricular function and higher concentrations of cardiac biomarkers (factor 1.14 for high-sensitivity troponin, 1.41 for N-terminal pro-B-type natriuretic peptide, adjusted P ≤ 0.01) in post-SARS-CoV-2 patients compared with matched controls, but no significant differences in cardiac magnetic resonance imaging findings. Sonographically non-compressible femoral veins, suggesting deep vein thrombosis, were substantially more frequent after SARS-CoV-2 infection (odds ratio 2.68, adjusted P < 0.001). Glomerular filtration rate (regression coefficient -2.35, adjusted P = 0.019) was lower in post-SARS-CoV-2 cases. Relative brain volume, prevalence of cerebral microbleeds, and infarct residuals were similar, while the mean cortical thickness was higher in post-SARS-CoV-2 cases. Cognitive function was not impaired. Similarly, patient-related outcomes did not differ. CONCLUSION: Subjects who apparently recovered from mild to moderate SARS-CoV-2 infection show signs of subclinical multi-organ affection related to pulmonary, cardiac, thrombotic, and renal function without signs of structural brain damage, neurocognitive, or quality-of-life impairment. Respective screening may guide further patient management.

PI3K/AKT signaling allows for MAPK/ERK pathway independency mediating dedifferentiation-driven treatment resistance in melanoma.

BACKGROUND: Current therapeutic management of advanced melanoma patients largely depends on their BRAF mutation status. However, the vast heterogeneity of the tumors hampers the success of therapies targeting the MAPK/ERK pathway alone. Dissecting this heterogeneity will contribute to identifying key players in the oncogenic progression to tailor more effective therapies. METHODS: We performed a comprehensive molecular and phenotypic characterization of a panel of patient-derived BRAFV600E-positive melanoma cell lines. Transcriptional profiling was used to identify groups of coregulated genes whose expression relates to an increased migratory potential and a higher resistance. RESULTS: A decrease in sensitivity to MAPK/ERK pathway inhibition with vemurafenib or trametinib corresponded with an increasing quiescence and migratory properties of the cells. This was accompanied by the loss of transcriptional signatures of melanocytic differentiation, and the gain of stem cell features that conferred highly-resistant/mesenchymal-like cells with increased xenobiotic efflux capacity. Nevertheless, targeting of the implicated ABC transporters did not improve the response to vemurafenib, indicating that incomplete BRAF inhibition due to reduced drug uptake is not a main driver of resistance. Rather, indifference to MAPK/ERK pathway inhibition arose from the activation of compensatory signaling cascades. The PI3K/AKT pathway in particular showed a higher activity in mesenchymal-like cells, conferring a lower dependency on MAPK/ERK signaling and supporting stem-like properties that could be reverted by dual PI3K/mTOR inhibition with dactolisib. CONCLUSIONS: In case of MAPK/ERK independency, therapeutic focus may be shifted to the PI3K/AKT pathway to overcome late-stage resistance in melanoma tumors that have acquired a mesenchymal phenotype. Video Abstract.

A novel yeast hybrid modeling framework integrating Boolean and enzyme-constrained networks enables exploration of the interplay between signaling and metabolism.

The interplay between nutrient-induced signaling and metabolism plays an important role in maintaining homeostasis and its malfunction has been implicated in many different human diseases such as obesity, type 2 diabetes, cancer, and neurological disorders. Therefore, unraveling the role of nutrients as signaling molecules and metabolites together with their interconnectivity may provide a deeper understanding of how these conditions occur. Both signaling and metabolism have been extensively studied using various systems biology approaches. However, they are mainly studied individually and in addition, current models lack both the complexity of the dynamics and the effects of the crosstalk in the signaling system. To gain a better understanding of the interconnectivity between nutrient signaling and metabolism in yeast cells, we developed a hybrid model, combining a Boolean module, describing the main pathways of glucose and nitrogen signaling, and an enzyme-constrained model accounting for the central carbon metabolism of Saccharomyces cerevisiae, using a regulatory network as a link. The resulting hybrid model was able to capture a diverse utalization of isoenzymes and to our knowledge outperforms constraint-based models in the prediction of individual enzymes for both respiratory and mixed metabolism. The model showed that during fermentation, enzyme utilization has a major contribution in governing protein allocation, while in low glucose conditions robustness and control are prioritized. In addition, the model was capable of reproducing the regulatory effects that are associated with the Crabtree effect and glucose repression, as well as regulatory effects associated with lifespan increase during caloric restriction. Overall, we show that our hybrid model provides a comprehensive framework for the study of the non-trivial effects of the interplay between signaling and metabolism, suggesting connections between the Snf1 signaling pathways and processes that have been related to chronological lifespan of yeast cells.

Impact of Myocardial Fibrosis on Left Ventricular Function Evaluated by Feature-Tracking Myocardial Strain Cardiac Magnetic Resonance in Competitive Male Triathletes With Normal Ejection Fraction.

BACKGROUND: To analyze the effect of myocardial fibrosis on left ventricular (LV) function evaluated by feature-tracking strain analysis by cine cardiac magnetic resonance (CMR) in competitive male triathletes with normal ejection fraction (EF).Methods and Results:78 asymptomatic male triathletes with >10 weekly training hours (43±11 years) and 28 male age-matched controls were studied by late gadolinium enhancement (LGE) and cine CMR. Global and segmental radial, longitudinal and circumferential strains were analyzed using feature-tracking cine CMR. Focal non-ischemic LGE was observed in 15 of 78 triathletes (19%, LGE+) with predominance in the basal inferolateral segments. LVEF was normal in LGE+ (62±6%) and LGE- triathletes (62±5%, P=0.958). In contrast, global radial strain was lower in LGE+ triathletes at 40±7% compared with LGE- triathletes (45±7%, P<0.05). Reduced segmental radial strain occurred either in LGE+ segments or in directly adjacent segments. Strain analysis revealed regional differences in controls, with the highest radial and longitudinal strain in the inferolateral segments, which were typically affected by fibrosis in LGE+ triathletes. CONCLUSIONS: Reduced global and regional radial strain suggests a negative effect of myocardial fibrosis on LV function in LGE+ triathletes with normal EF. The observed regional differences in controls with the highest radial and longitudinal strain in the inferolateral segments may explain the typical occurrence of fibrosis in this myocardial region in triathletes.

Prediction of the estimated 5-year risk of sudden cardiac death and syncope or non-sustained ventricular tachycardia in patients with hypertrophic cardiomyopathy using late gadolinium enhancement and extracellular volume CMR.

OBJECTIVES: To evaluate the ability of late gadolinium enhancement (LGE) and mapping cardiac magnetic resonance (CMR) including native T1 and global extracellular volume (ECV) to identify hypertrophic cardiomyopathy (HCM) patients at risk for sudden cardiac death (SCD) and to predict syncope or non-sustained ventricular tachycardia (VT). METHODS: A 1.5-T CMR was performed in 73 HCM patients and 16 controls. LGE size was quantified using the 3SD, 5SD and full width at half maximum (FWHM) method. T1 and ECV maps were generated by a 3(3)5 modified Look-Locker inversion recovery sequence. Receiver-operating curve analysis evaluated the best parameter to identify patients with increased SCD risk ≥4% and patients with syncope or non-sustained VT. RESULTS: Global ECV was the best predictor of SCD risk with an area under the curve (AUC) of 0.83. LGE size was significantly inferior to global ECV with an AUC of 0.68, 0.70 and 0.70 (all P < 0.05) for 3SD-, 5SD- and FWHM-LGE, respectively. Combined use of the SCD risk score and global ECV significantly improved the diagnostic accuracy to identify HCM patients with syncope or non-sustained VT. CONCLUSIONS: Combined use of the SCD risk score and global ECV has the potential to improve HCM patient selection, benefiting most implantable cardioverter defibrillators. KEY POINTS: • Global ECV identified the best HCM patients with increased SCD risk. • Global ECV performed equally well compared to a SCD risk score. • Combined use of the SCD risk score and global ECV improved test accuracy. • Combined use potentially improves selection of HCM patients for ICD implantation.

High Incidence of De Novo and Subclinical Atrial Fibrillation in Patients With Hypertrophic Cardiomyopathy and Cardiac Rhythm Management Device.

INTRODUCTION: Atrial fibrillation (AF) is an important prognostic parameter in patients with hypertrophic cardiomyopathy (HCM). Though cardiac rhythm management (CRM) devices (e.g., ICD, pacemaker or implantable loop recorder) can detect subclinical AF, data describing the incidence of AF are rare. We therefore investigated the incidence and clinical impact of de novo and subclinical AF detected by CRM devices in patients with HCM. METHODS AND RESULTS: In our retrospective single-center study, we included patients with HCM and need for CRM devices. The primary endpoint of the study was the incidence of clinical and subclinical de novo AF. During follow-up, patients were screened for adverse events like stroke, ventricular arrhythmia, heart failure, or death. From 192 HCM patients, 44 patients received a CRM device (38 ICDs, 5 pacemakers, 1 implantable loop recorder). In 14 of these patients (32%), AF had been documented before device implantation. Thirty (68%) patients were free from AF at the time of implantation. During a median follow-up of 595 days (interquartile range, 367-890 days), de novo AF was recorded in 16 of these 30 patients (53%). Fourteen (88%) of the 16 patients with de novo AF were free from any clinical symptoms, so these patients were classified to have subclinical AF. In logistic regression analysis, age was the only significant predictor for an increased risk of AF. CONCLUSION: AF is common in patients with HCM who need a CRM device. More than 50% of these patients develop de novo AF that was predominantly subclinical in our cohort.

Serum Matrix Metalloproteinases as Quantitative Biomarkers for Myocardial Fibrosis and Sudden Cardiac Death Risk Stratification in Patients With Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is associated with an increased risk of sudden cardiac death due to ventricular tachycardia (VT), and myocardial fibrosis reflects an important risk factor. Several matrix metalloproteinases (MMPs) and procollagen N-terminal propeptides (PNPs) are involved in collagen turnover and discussed as fibrosis biomarkers. We aimed to identify biomarkers that correlate with myocardial fibrosis in late-gadolinium-enhancement cardiac magnetic resonance imaging (LGE-CMR) and/or cardiac events (syncope, VT) in HCM patients. METHODS AND RESULTS: In 54 HCM patients (age 55.9 ± 14.3 y, 50% female) fibrosis was quantified by LGE-CMR. Serum concentrations of MMP-1, -2, -3, -9, and tissue inhibitor of MMP (TIMP) 1 were analyzed by means of enzyme-linked immunosorbent assay and PINP, PIIINP, and type I collagen C-terminal telopeptide (ICTP) concentrations by radioimmunoassay. MMP-9 was associated with fibrosis in LGE-CMR (mean increase 0.66 g/unit MMP9 [95% confidence interval [CI] 0.50-0.82]; P < .001) and with cardiac events in women (odds ratio [OR] 1.07 [1.01-1.12], P = .01) but not in men. Increased MMP-2 levels in women were associated with lower fibrosis (0.05 [-0.09 to -0.01]; P = .015). MMP-3 levels were positively associated with cardiac events (OR 1.13 [1.05-1.22]; P = .001) independently from fibrosis and sex. No association was detected for MMP-1, TIMP-1, PNPs, and ICTP. CONCLUSIONS: These data suggest that MMP-9 is a useful biomarker for fibrosis and cardiac events in female HCM patients, whereas MMP-3 is associated with a higher event rate independent from fibrosis and sex.

The E3 ubiquitin ligase Asb2ß is downregulated in a mouse model of hypertrophic cardiomyopathy and targets desmin for proteasomal degradation.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease with mutations in genes encoding sarcomeric proteins. Previous findings suggest deregulation of the ubiquitin proteasome system (UPS) in HCM in humans and in a mouse model of HCM (Mybpc3-targeted knock-in (KI) mice). In this study we investigated transcript levels of several muscle-specific E3 ubiquitin ligases in KI mice and aimed at identifying novel protein targets. METHODS AND RESULTS: Out of 9 muscle-specific E3 ligases, Asb2ß was found with the lowest mRNA level in KI compared to wild-type (WT) mice. After adenoviral-mediated Asb2ß transduction of WT neonatal mouse cardiomyocytes with either a WT or inactive Asb2ß mutant, desmin was identified as a new target of Asb2ß by mass spectrometry, co-immunoprecipitation and immunoblotting. Immunofluorescence analysis revealed a co-localization of desmin with Asb2ß at the Z-disk of the sarcomere. Knock-down of Asb2ß in cardiomyocytes resulted in higher desmin protein levels. Furthermore, desmin levels were higher in ventricular samples of HCM mice and patients than controls. CONCLUSIONS: This study identifies desmin as a new Asb2ß target for proteasomal degradation in cardiomyocytes and suggests that accumulation of desmin could contribute to UPS impairment in HCM mice and patients.

FHL2 expression and variants in hypertrophic cardiomyopathy.

Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.

Generation of bi-allelic MYBPC3 truncating mutant and isogenic control from an iPSC line of a patient with hypertrophic cardiomyopathy.

MYBPC3 is the most frequently affected gene in hypertrophic cardiomyopathy (HCM), which is an autosomal-dominant cardiac disease caused by mutations in sarcomeric proteins. Bi-allelic truncating MYBPC3 mutations are associated with severe forms of neonatal cardiomyopathy. We reprogrammed skin fibroblasts from a HCM patient carrying a heterozygous MYBPC3 truncating mutation into human induced pluripotent stem cells (iPSC) and used CRISPR/Cas9 to generate bi-allelic MYBPC3 truncating mutation and isogenic control hiPSC lines. All lines expressed pluripotency markers, had normal karyotype and differentiated into endoderm, ectoderm and cardiomyocytes in vitro. This set of three lines provides a useful tool to study HCM pathomechanisms.

Disease modeling of a mutation in α-actinin 2 guides clinical therapy in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2, encoding α-actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient-derived human-induced pluripotent stem cells (hiPSCs) and show that hiPSC-derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca2+ sensitivity, and also prolonged action potential duration and enhanced L-type Ca2+ current. The L-type Ca2+ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM-affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease-causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof-of-principle for the use of hiPSC for personalized treatment of cardiomyopathies.

The T-peak-to-T-end interval: a novel ECG marker for ventricular arrhythmia and appropriate ICD therapy in patients with hypertrophic cardiomyopathy.

INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is associated with an increased risk of sudden cardiac death (SCD) primarily due to ventricular arrhythmia (VA). In patients (pts.) with a high risk of SCD, the implantation of an intracardiac cardioverter defibrillator (ICD) is thus indicated. Previous studies suggest that a prolonged interval between the peak and the end of the T wave, T-peak to T-end (TpTe), is associated with an elevated risk of VA and SCD in various clinical settings. The aim of our study was to evaluate the association between TpTe and VA in HCM pts. with a previously implanted ICD. METHODS: In 40 HCM pts. (51.4 ± 16.4 years; 62.5% men), TpTe was measured using the baseline digital standard resting 12-lead ECG during sinus rhythm. VA was assessed by device follow-up. RESULTS: Within 41.8 ± 35.1 months, 7 (17.5%) pts. had VA leading to appropriate therapy (AT), 7 pts. (17.5%) had non-sustained VA, and 26 pts. (65.0%) had no VA. The maximum TpTe was significantly prolonged in pts. with VA leading to AT compared to pts. without VA (101.3 ± 19.6 vs. 79.9 ± 15.3 ms; p = 0.004). Maximum TpTe was associated with an elevated risk of VA leading to AT (hazard ratio per 10 ms increase 1.63; 95% CI 1.04-2.54; p = 0.031) and pts. with a maximum TpTe ≤ 78 ms were without any VA leading to AT during follow-up. There was no correlation of maximum TpTe to other clinical parameters in our patient cohort. CONCLUSION: A prolonged TpTe is associated with VA and AT in HCM. Our findings suggest that TpTe can possibly serve as a marker for ventricular arrhythmogenesis in pts. with HCM and assessment of TpTe might, therefore, optimize SCD risk stratification.

Myocardial Fibrosis in Competitive Triathletes Detected by Contrast-Enhanced CMR Correlates With Exercise-Induced Hypertension and Competition History.

OBJECTIVES: This study analyzed the presence of myocardial fibrosis detected by late gadolinium-enhancement (LGE) cardiac magnetic resonance (CMR) in correlation with the performance of competitive triathletes objectified by an exercise test and individual competition history. BACKGROUND: Myocardial fibrosis detected by LGE CMR has been reported to occur in 0% to 50% of asymptomatic athletes. However, the cause and mechanisms of myocardial fibrosis are unclear. METHODS: Eighty-three asymptomatic triathletes undergoing >10 training h per week (43 ± 10 years of age; 65% male) and 36 sedentary controls were studied by using LGE and extracellular volume (ECV) CMR. Parameters of physical fitness were measured by spiroergometry. Triathletes reported their lifetime competition results. RESULTS: LGE CMR revealed focal nonischemic myocardial fibrosis in 9 of 54 (17%) male triathletes (LGE+) but in none of the female triathletes (p < 0.05). LGE+ triathletes had higher peak exercise systolic blood pressure (213 ± 24 mm Hg) than LGE- triathletes (194 ± 26 mm Hg; p < 0.05). Furthermore, left ventricular mass index was higher in LGE+ triathletes (93 ± 7 g/m2) than in LGE- triathletes (84 ± 11 g/m2; p < 0.05). ECV in LGE- myocardium was higher in LGE+ triathletes (26.3 ± 1.8%) than in LGE- triathletes (24.4 ± 2.2%; p < 0.05). LGE+ triathletes completed longer cumulative distances in swimming and cycling races and participated more often in middle and Iron Man distances than LGE- triathletes. A cycling race distance of >1,880 km completed during competition had the highest accuracy to predict LGE, with an area under the curve value of 0.876 (p < 0.0001), resulting in high sensitivity (89%) and specificity (79%). Multivariate analysis identified peak exercise systolic blood pressure (p < 0.05) and the swimming race distance (p < 0.01) as independent predictors of LGE presence. CONCLUSIONS: Myocardial fibrosis in asymptomatic triathletes seems to be associated with exercise-induced hypertension and the race distances. There appears to be a safe upper limit, beyond which exercise may result in myocardial fibrosis.

Early segmental relaxation abnormalities in hypertrophic cardiomyopathy for differential diagnostic of patients with left ventricular hypertrophy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy (LVH). However, clinical signs can be subtle and differentiation from other causes of LVH is challenging. HYPOTHESIS: As diastolic dysfunction (DD) is an early sign in HCM, we aimed to find regional changes in relaxation pattern for differentiation from other entities of LVH. METHODS: In 148 patients (81 HCM, 55 arterial hypertension (AHT), 12 Fabry disease) and 63 healthy controls, relaxation patterns were assessed using regional tissue Doppler imaging. In 42 HCM patients, myocardial mass and fibrosis were quantified by cardiac magnetic resonance imaging and correlated with relaxation parameters. RESULTS: In HCM the septal to lateral isovolumic relaxation time (s/l IVRT) ratio was higher (1.5 ± 0.4) compared with AHT (1.1 ± 0.2), Fabry disease (1.0 ± 0.1), and controls (1.1 ± 0.2; P < 0.001), showing 77% sensitivity and 79% specificity to discriminate HCM-related LVH from other entities. The s/l IVRT ratio was independent of global DD in HCM (HCM with DD: 1.5 ± 0.5, n = 52; HCM without DD: 1.5 ± 0.3, n = 29) and remained significantly different from other entities in a subgroup of HCM patients with maximum wall thickness < 20 mm (s/l ratio: 1.5 ± 0.5, n = 28). Regional IVRT did not correlate with the corresponding segmental myocardial mass or amount of fibrosis in cardiac magnetic resonance imaging. CONCLUSIONS: HCM patients show a prolonged septal IVRT irrespective of the extent of LVH and even before developing global DD. The s/l IVRT ratio is significantly higher in HCM compared with AHT or Fabry disease, thus establishing segmental IVRT analysis as a potential parameter for differential diagnosis in LVH.

Colonization with Multidrug-Resistant Bacteria - On the Efficiency of Local Decolonization Procedures.

The effectiveness of a disinfectant-based decolonization strategy for multidrug-resistant bacteria like extended spectrum ß-lactamase (ESBL)-positive Gram-negative bacteria with or without additional fluoroquinolon and carbapenem resistance as well as vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus was assessed. Between 2011 and 2015, 25 patients from Libya, Syria, and the Ukraine with war traumata were treated at the Bundeswehr hospital Hamburg. The patients were heavily colonized and infected with multidrug-resistant bacteria, altogether comprising 371 distinct combinations of pathogens and isolation sites. Local disinfection was assessed for effectiveness regarding successful decolonization of multidrug-resistant bacteria. Altogether, 170 cases of successful decolonization were observed, comprising 95 (55.8%) such events at sampling sites that were accessible to disinfecting procedures. The remaining 75 (44.2%) decolonization events had to be considered as spontaneous. In contrast, 95 out of 172 (55.2%) colonized isolation sites that were accessible to disinfection procedures were successfully decolonized. Patient compliance with the enforced hygiene procedures was associated with decolonization success. Systemic antibiotic therapy did not relevantly affect isolation time. Disinfecting washing moderately supports local decolonization of multidrug-resistant pathogens in comparison with spontaneous decolonization rates if the patients' compliance with the applied hygiene procedures is ensured.

Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes.

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5' or 3' pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.

Orthostatic blood pressure test for risk stratification in patients with hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death (SCD) in young adults, mainly ascribed to ventricular tachycardia (VT). Assuming that VT is the major cause of (pre-) syncope in HCM patients, its occurrence is essential for SCD risk stratification and primarily preventive ICD-implantation. However, evidence of VT during syncope is often missing. As the differentiation of potential lethal causes for syncope such as VT from more harmless reasons is crucial, HCM patients were screened for orthostatic dysregulation by using a simple orthostatic blood pressure test. METHODS: Over 15 months (IQR [9;20]) 100 HCM patients (55.8±16.2 yrs, 61% male) were evaluated for (pre-)syncope and VT (24h-ECGs, device-memories) within the last five years. Eighty patients underwent an orthostatic blood pressure test. Logistic regression models were used for statistical analysis. RESULTS: In older patients (>40 yrs) a positive orthostatic test result increased the chance of (pre-) syncope by a factor of 63 (95%-CI [8.8; 447.9], p<0.001; 93% sensitivity, 95%-CI [76; 99]; 74% specificity, 95%-CI [58; 86]). No correlation with VT was shown. A prolonged QTc interval also increased the chance of (pre-) syncope by a factor of 6.6 (95%-CI [2.0; 21.7]; p=0.002). CONCLUSIONS: The orthostatic blood pressure test is highly valuable for evaluation of syncope and presyncope especially in older HCM patients, suggesting that orthostatic syncope might be more relevant than previously assumed. Considering the high complication rates due to ICD therapies, this test may provide useful information for the evaluation of syncope in individual risk stratification and may help to prevent unnecessary device implantations, especially in older HCM patients.