Lower affective empathy in oral contraceptive users: a cross-sectional fMRI study.

Evidence accumulates that oral contraceptive (OC) use modulates various socio-affective behaviors, including empathic abilities. Endogenous and synthetic sex hormones, such as estrogens and progestogens, bind to receptor sites in brain regions (i.e. frontal, limbic, and cerebellar) involved in socio-affective processing. Therefore, the aim of this study was to investigate the role of OC use in empathy. In a cross-sectional functional magnetic resonance imaging study, women in different hormonal states, including OC use (n = 46) or being naturally cycling in the early follicular (fNC: n = 37) or peri-ovulatory phase (oNC: n = 28), performed a visual, sentence-based empathy task. Behaviorally, OC users had lower empathy ratings than oNC women. Congruently, whole-brain analysis revealed significantly larger task-related activation of several brain regions, including the left dorsomedial prefrontal gyrus (dmPFG), left precentral gyrus, and left temporoparietal junction in oNC compared to OC women. In OC users, the activity of the left dmPFG and precentral gyrus was negatively associated with behavioral and self-reported affective empathy. Furthermore, empathy-related region-of-interest analysis indicated negative associations of brain activation with synthetic hormone levels in OC women. Overall, this multimodal, cross-sectional investigation of empathy suggests a role of OC intake in especially affective empathy and highlights the importance of including synthetic hormone levels in OC-related analyses.

Blood taken immediately after fatal resuscitation attempts yields higher quality DNA for genetic studies as compared to autopsy samples.

BACKGROUND: The out-of-hospital cardiac arrest (OHCA) in the young may be associated with a genetic predisposition which is relevant even for genetic counseling of relatives. The identification of genetic variants depends on the availability of intact genomic DNA. DNA from autopsy may be not available due to low autopsy frequencies or not suitable for high-throughput DNA sequencing (NGS). The emergency medical service (EMS) plays an important role to save biomaterial for subsequent molecular autopsy. It is not known whether the DNA integrity of samples collected by the EMS is better suited for NGS than autopsy specimens. MATERIAL AND METHODS: DNA integrity was analyzed by standardized protocols. Fourteen blood samples collected by the EMS and biomaterials from autopsy were compared. We collected 172 autopsy samples from different tissues and blood with postmortem intervals of 14-168 h. For comparison, DNA integrity derived from blood stored under experimental conditions was checked against autopsy blood after different time intervals. RESULTS: DNA integrity and extraction yield were higher in EMS blood compared to any autopsy tissue. DNA stability in autopsy specimens was highly variable and had unpredictable quality. In contrast, collecting blood samples by the EMS is feasible and delivered comparably the highest DNA integrity. CONCLUSIONS: Isolation yield and DNA integrity from blood samples collected by the EMS is superior in comparison to autopsy specimens. DNA from blood samples collected by the EMS on scene is stable at room temperature or even for days at 4 °C. We conclude that the EMS personnel should always save a blood sample of young fatal OHCA cases died on scene to enable subsequent genetic analysis.

Age-dependent development and clinical characteristics of an experimental parrot bornavirus-4 (PaBV-4) infection in cockatiels (Nymphicus hollandicus).

Parrot bornavirus (PaBV) is a pathogen often found in psittacine populations. Infected, clinically healthy carrier birds are of major importance for epidemiology, but the underlying pathomechanism of this carrier status is poorly understood. The age, implying the maturation status of the immune system, at the time of infection might be significant for the clinical outcome. Therefore, two groups of 11 cockatiels of different ages (adult and newly hatched) were inoculated with a PaBV-4 isolate intravenously. The trial lasted for 233 days and all birds were observed for clinical signs, PaBV-RNA shedding and anti-PaBV antibody production. At the end of the trial, histopathology, immunohistochemistry, PCR and virus re-isolation were performed. All 22 birds seroconverted and shed PaBV-RNA during the investigation period; the juvenile group earlier and more homogeneously. Nine of 11 birds of the adult group developed clinical signs; five birds died or had to be euthanized before the end of the study. In the juvenile group none of the birds developed clinical signs and only one bird died due to bacterial septicaemia. Eight birds of the adult group, but none of the juvenile group, showed a dilatation of the proventriculus. PaBV-RNA detection and virus re-isolation were successful in all birds. Immunohistochemically, PaBV antigen was found in all birds. Histopathology revealed mononuclear infiltrations in organs in birds of both groups, but the juveniles were less severely affected in the brain.Thus, PaBV infection at an age with a more naïve immune system makes the production of carrier birds more likely.RESEARCH HIGHLIGHTS PaBV infection at a young age might favour the development of carrier birds.Cockatiels infected at a very young age showed inflammation but no clinical signs.The juvenile group started seroconversion and PaBV-RNA shedding earlier.Seroconversion and PaBV-RNA shedding occurred more homogeneously in the juveniles.

Hemi- and Homozygous Loss-of-Function Mutations in DSG2 (Desmoglein-2) Cause Recessive Arrhythmogenic Cardiomyopathy with an Early Onset.

About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown and a genetic contribution might be underestimated. Here, we used a next-generation sequencing (NGS) approach and in addition single nucleotide polymor-phism (SNP) arrays for the genetic analysis of two independent index patients without familial medical history. Of note, this genetic strategy revealed a homozygous splice site mutation (DSG2-c.378+1G>T) in the first patient and a nonsense mutation (DSG2-p.L772X) in combination with a large deletion in DSG2 in the second one. In conclusion, a recessive inheritance pattern is likely for both cases, which might contribute to the hidden medical history in both families. This is the first report about these novel loss-of-function mutations in DSG2 that have not been previously identi-fied. Therefore, we suggest performing deep genetic analyses using NGS in combination with SNP arrays also for ACM index patients without obvious familial medical history. In the future, this finding might has relevance for the genetic counseling of similar cases.

De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents.

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient's myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

A homozygous DSC2 deletion associated with arrhythmogenic cardiomyopathy is caused by uniparental isodisomy.

AIMS: We aimed to unravel the genetic, molecular and cellular pathomechanisms of DSC2 truncation variants leading to arrhythmogenic cardiomyopathy (ACM). METHODS AND RESULTS: We report a homozygous 4-bp DSC2 deletion variant c.1913_1916delAGAA, p.Q638LfsX647hom causing a frameshift carried by an ACM patient. Whole exome sequencing and comparative genomic hybridization analysis support a loss of heterozygosity in a large segment of chromosome 18 indicating segmental interstitial uniparental isodisomy (UPD). Ultrastructural analysis of the explanted myocardium from a mutation carrier using transmission electron microscopy revealed a partially widening of the intercalated disc. Using qRT-PCR we demonstrated that DSC2 mRNA expression was substantially decreased in the explanted myocardial tissue of the homozygous carrier compared to controls. Western blot analysis revealed absence of both full-length desmocollin-2 isoforms. Only a weak expression of the truncated form of desmocollin-2 was detectable. Immunohistochemistry showed that the truncated form of desmocollin-2 did not localize at the intercalated discs. In vitro, transfection experiments using induced pluripotent stem cell derived cardiomyocytes and HT-1080 cells demonstrated an obvious absence of the mutant truncated desmocollin-2 at the plasma membrane. Immunoprecipitation in combination with fluorescence measurements and Western blot analyses revealed an abnormal secretion of the truncated desmocollin-2. CONCLUSION: In summary, we unraveled segmental UPD as the likely genetic reason for a small homozygous DSC2 deletion. We conclude that a combination of nonsense mediated mRNA decay and extracellular secretion is involved in DSC2 related ACM.

Reversal of Regioselectivity in Zinc-Dependent Medium-Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives.

The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4-phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4-phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

Human Induced Pluripotent Stem-Cell-Derived Cardiomyocytes as Models for Genetic Cardiomyopathies.

In the last few decades, many pathogenic or likely pathogenic genetic mutations in over hundred different genes have been described for non-ischemic, genetic cardiomyopathies. However, the functional knowledge about most of these mutations is still limited because the generation of adequate animal models is time-consuming and challenging. Therefore, human induced pluripotent stem cells (iPSCs) carrying specific cardiomyopathy-associated mutations are a promising alternative. Since the original discovery that pluripotency can be artificially induced by the expression of different transcription factors, various patient-specific-induced pluripotent stem cell lines have been generated to model non-ischemic, genetic cardiomyopathies in vitro. In this review, we describe the genetic landscape of non-ischemic, genetic cardiomyopathies and give an overview about different human iPSC lines, which have been developed for the disease modeling of inherited cardiomyopathies. We summarize different methods and protocols for the general differentiation of human iPSCs into cardiomyocytes. In addition, we describe methods and technologies to investigate functionally human iPSC-derived cardiomyocytes. Furthermore, we summarize novel genome editing approaches for the genetic manipulation of human iPSCs. This review provides an overview about the genetic landscape of inherited cardiomyopathies with a focus on iPSC technology, which might be of interest for clinicians and basic scientists interested in genetic cardiomyopathies.

A Screening Method for P450 BM3 Mutant Libraries Using Multiplexed Capillary Electrophoresis for Detection of Enzymatically Converted Compounds.

Capillary electrophoresis (CE) is an analytical method in which charged species are separated by attraction or repulsion performed in submillimeter diameter capillaries or micro- and nanofluidic channels through the application of a high voltage electric field. When capillary electrophoresis is assembled in a multicapillary instrument such as 96-well format (multiplexed), it becomes a powerful high-throughput system with the ability to simultaneously screen several types of samples like genetic mutations, metabolomes, kinase inhibitors, or enzymatic activities to name a few. The usage of a 96-multiplexed capillary electrophoresis system (96-MP-CE) represents a new platform for product-specific high-throughput screening of enzyme mutant libraries from directed evolution campaigns providing a comprehensive view on enzyme activity through the detection of all products formed. We describe the application of 96-MP-CE to screen mutant libraries of P450 BM3. MP-CE was used in directed evolution campaigns toward benzo-1,4-dioxane and α-isophorone.

Factors Influencing Vertical Transmission of Psittacine Bornavirus in Cockatiels (Nymphicus hollandicus).

The transmission of parrot bornavirus is still not fully understood. Although horizontal transmission through wounds can be one route, vertical transmission is still discussed. PaBV RNA and PaBV antigen were detected in psittacine embryos, but isolation of the virus failed, raising doubts about this route. In this study, cockatiels were infected either as adults (adult group) or during the first 6 days after hatching (juvenile group) and raised until sexual maturity to breed and lay eggs. A total of 92 eggs (adult group: 49, juvenile group: 43) were laid and incubated until day 17. The embryos and yolk samples were examined by RT-PCR for PaBV RNA and by infectivity assay for infectious virus. In the adult group, 14/31 embryos (45.2%) and 20/39 (51%) of the yolk samples demonstrated PaBV RNA in the PCR. Isolation of PaBV was not possible in any embryo of this group, but it was achieved in six yolk samples from one female. Anti-PaBV antibodies were detected in the yolk samples after seroconversion of all female parents. In the juvenile group, 22/29 embryos (74.9%) were positive for PaBV RNA. In 9/21 embryos (42.9%), PaBV isolation was possible. PaBV RNA was detected in 100% and infectious virus in 41% of the yolk samples. Anti-PaBV antibodies were detected in all yolk samples. For the first time, successful vertical transmission of PaBV was proven, but it seems to depend on the age when the parent birds are infected. Therefore, the age of the bird at time of infection may be an important factor in the occurrence of vertical transmission.

Tissue Distribution of Parrot Bornavirus 4 (PaBV-4) in Experimentally Infected Young and Adult Cockatiels (Nymphicus hollandicus).

Proventricular dilatation disease (PDD) caused by parrot bornavirus (PaBV) infection is an often-fatal disease known to infect Psittaciformes. The impact of age at the time of PaBV infection on organ lesions and tissue distribution of virus antigen and RNA remains largely unclear. For this purpose, tissue sections of 11 cockatiels intravenously infected with PaBV-4 as adults or juveniles, respectively, were examined via histology, immunohistochemistry applying a phosphoprotein (P) antibody directed against the bornaviral phosphoprotein and in situ hybridisation to detect viral RNA in tissues. In both groups of adult- and juvenile-infected cockatiels, widespread tissue distribution of bornaviral antigen and RNA as well as histologic inflammatory lesions were demonstrated. The latter appeared more severe in the central nervous system in adults and in the proventriculus of juveniles, respectively. During the study, central nervous symptoms and signs of gastrointestinal affection were only demonstrated in adult birds. Our findings indicate a great role of the age at the time of infection in the development of histopathological lesions and clinical signs, and thus provide a better understanding of the pathogenesis, possible virus transmission routes, and the development of carrier birds posing a risk to psittacine collections.

Pathogenic Variants in Cardiomyopathy Disorder Genes Underlie Pediatric Myocarditis-Further Impact of Heterozygous Immune Disorder Gene Variants?

Myocarditis is an inflammatory disease of the heart. Pediatric myocarditis with the dilated cardiomyopathy (DCM) phenotype may be caused by likely pathogenic or pathogenic genetic variants [(L)P] in cardiomyopathy (CMP) genes. Systematic analysis of immune disorder gene defects has not been performed so far. We analyzed 12 patients with biopsy-proven myocarditis and the DCM phenotype together with their parents using whole-exome sequencing (WES). The WES data were filtered for rare pathogenic variants in CMP (n = 89) and immune disorder genes (n = 631). Twelve children with a median age of 2.9 (1.0-6.8) years had a mean left ventricular ejection fraction of 28% (22-32%) and myocarditis was confirmed by endomyocardial biopsy. Patients with primary immunodeficiency were excluded from the study. Four patients underwent implantation of a ventricular assist device and subsequent heart transplantation. Genetic analysis of the 12 families revealed an (L)P variant in the CMP gene in 8/12 index patients explaining DCM. Screening of recessive immune disorder genes identified a heterozygous (L)P variant in 3/12 index patients. This study supports the genetic impact of CMP genes for pediatric myocarditis with the DCM phenotype. Piloting the idea that additional immune-related genetic defects promote myocarditis suggests that the presence of heterozygous variants in these genes needs further investigation. Altered cilium function might play an additional role in inducing inflammation in the context of CMP.

Pathogenic variants damage cell composition and single cell transcription in cardiomyopathies.

Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.

Mechanical circulatory support does not reduce advanced myocardial fibrosis in patients with end-stage heart failure.

AIMS: Mechanical unloading by ventricular assist devices (VADs) has become increasingly important for the therapy of end-stage heart failure during the last decade. However, VAD support was claimed to be associated with partial reverse remodelling. Unfortunately, the literature describes the contradictory effects of VAD systems on cardiac fibrosis, a hallmark of cardiac remodelling. To clarify these inconsistent results, the effects on cardiac fibrosis before and after mechanical unloading in 125 patients were examined. METHODS AND RESULTS: Left ventricular myocardial tissue from ischaemic or non-ischaemic cardiomyopathy patients undergoing VAD implantation and subsequent cardiac transplantation and non-failing hearts of the control group were analysed for 4-hydroxyproline (4OH-P) content as a marker for collagen protein. In addition, collagen cross-linking and mRNAs of collagens I and III and transforming growth factor beta-1 were measured. 4OH-P content was significantly increased in failing hearts compared with the control group and increased (P < 0.05) after mechanical unloading (nmol/mg tissue, mean ± standard deviation: 16.74 ± 9.68 vs. 7.75 ± 2.39 and 18.57 ± 9.19). However, plotting of the 4OH-P ratios (post/pre-VAD) against the collagen content pre-VAD could be fitted by non-linear regression. Collagen cross-linking correlated strongly with the total collagen content in pre- and post-VAD myocardium (r2  = 0.73 and 0.71, respectively). In contrast to the total collagen content, all three mRNAs of fibrotic genes were significantly down-regulated during VAD support when compared to pre-VAD. CONCLUSIONS: This investigation of a comparably large patient cohort revealed that cardiac fibrosis was strongly increased in heart failure and increased even after mechanical unloading. The mRNAs of collagens I and III are independently regulated from the collagen protein.

The Desmin (DES) Mutation p.A337P Is Associated with Left-Ventricular Non-Compaction Cardiomyopathy.

Here, we present a small Russian family, where the index patient received a diagnosis of left-ventricular non-compaction cardiomyopathy (LVNC) in combination with a skeletal myopathy. Clinical follow-up analysis revealed a LVNC phenotype also in her son. Therefore, we applied a broad next-generation sequencing gene panel approach for the identification of the underlying mutation. Interestingly, DES-p.A337P was identified in the genomes of both patients, whereas only the index patient carried DSP-p.L1348X. DES encodes the muscle-specific intermediate filament protein desmin and DSP encodes desmoplakin, which is a cytolinker protein connecting desmosomes with the intermediate filaments. Because the majority of DES mutations cause severe filament assembly defects and because this mutation was found in both affected patients, we analyzed this DES mutation in vitro by cell transfection experiments in combination with confocal microscopy. Of note, desmin-p.A337P forms cytoplasmic aggregates in transfected SW-13 cells and in cardiomyocytes derived from induced pluripotent stem cells underlining its pathogenicity. In conclusion, we suggest including the DES gene in the genetic analysis for LVNC patients in the future, especially if clinical involvement of the skeletal muscle is present.

Truncated titin proteins and titin haploinsufficiency are targets for functional recovery in human cardiomyopathy due to TTN mutations.

Heterozygous truncating variants in TTN (TTNtv), the gene coding for titin, cause dilated cardiomyopathy (DCM), but the underlying pathomechanisms are unclear and disease management remains uncertain. Truncated titin proteins have not yet been considered as a contributor to disease development. Here, we studied myocardial tissues from nonfailing donor hearts and 113 patients with end-stage DCM for titin expression and identified a TTNtv in 22 patients with DCM (19.5%). We directly demonstrate titin haploinsufficiency in TTNtv-DCM hearts and the absence of compensatory changes in the alternative titin isoform Cronos. Twenty-one TTNtv-DCM hearts in our cohort showed stable expression of truncated titin proteins. Expression was variable, up to half of the total titin protein pool, and negatively correlated with patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates, with deregulated ubiquitin-dependent protein quality control. We produced human induced pluripotent stem cell­derived cardiomyocytes (hiPSC-CMs), comparing wild-type controls to cells with a patient-derived, prototypical A-band-TTNtv or a CRISPR-Cas9­generated M-band-TTNtv. TTNtv-hiPSC-CMs showed reduced wild-type titin expression and contained truncated titin proteins whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR-Cas9, eliminating truncated titin proteins and raising wild-type titin content. Functional improvement also occurred when wild-type titin protein content was increased by proteasome inhibition. Our findings reveal the major pathomechanisms of TTNtv-DCM and can be exploited for new therapies to treat TTNtv-related cardiomyopathies.

Wounds as the Portal of Entrance for Parrot Bornavirus 4 (PaBV-4) and Retrograde Axonal Transport in Experimentally Infected Cockatiels (Nymphicus hollandicus).

In this study, we investigated the natural route of infection of psittacine bornavirus (PaBV), which is the causative agent of proventricular dilatation disease (PDD) in psittacines. We inoculated two infection groups through wounds with a PaBV-4 isolate. In nine cockatiels (Nymphicus hollandicus) we applied a virus suspension with a titer of 103 50% tissue culture infection dose (TCID50) via palatal lesions (Group P, P1-9). In a second group of three cockatiels, we applied a virus suspension with a titer of 104 TCID50 to footpad lesions (Group F, F1-3). In two cockatiels, the control (or "mock") group, we applied a virus-free cell suspension (Group M, M1-2) via palatal lesions. The observation period was 6 mo (Groups P and M) or 7 mo (Group F). We monitored PaBV-4 RNA shedding and seroconversion. At the end of the study, we examined the birds for the presence of inflammatory lesions, PaBV-4 RNA, and antigen in tissues, as well as virus reisolation of brain and crop material. We did not observe any clinical signs typical of PDD during this study. We also did not see seroconversion or PaBV RNA shedding in any bird during the entire investigation period, and virus reisolation was not successful. We only found PaBV-4 RNA in sciatic nerves, footpad tissue, skin, and in one sample from the intestine of Group F. In this group, the histopathology revealed mononuclear infiltrations mainly in skin and footpad tissue; immunohistochemistry showed positive reactions in spinal ganglia and in the spinal cord, and slightly in skin, footpad tissues, and sciatic nerves. In Groups P and M we found no viral antigen or specific inflammations. In summary, only the virus application on the footpad lesion led to detectable PaBV RNA, mononuclear infiltrations, and positive immunohistochemical reactions in tissues of the experimental birds. This could suggest that PaBV spreads via nervous tissue, with skin wounds as the primary entry route.

Distinct Myocardial Transcriptomic Profiles of Cardiomyopathies Stratified by the Mutant Genes.

Cardiovascular diseases are the number one cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well understood. Cardiomyopathies are primary diseases of the heart muscle and contribute to high rates of heart failure and sudden cardiac deaths. Here, we distinguished four different genetic cardiomyopathies based on gene expression signatures. In this study, RNA-Sequencing was used to identify gene expression signatures in myocardial tissue of cardiomyopathy patients in comparison to non-failing human hearts. Therefore, expression differences between patients with specific affected genes, namely LMNA (lamin A/C), RBM20 (RNA binding motif protein 20), TTN (titin) and PKP2 (plakophilin 2) were investigated. We identified genotype-specific differences in regulated pathways, Gene Ontology (GO) terms as well as gene groups like secreted or regulatory proteins and potential candidate drug targets revealing specific molecular pathomechanisms for the four subtypes of genetic cardiomyopathies. Some regulated pathways are common between patients with mutations in RBM20 and TTN as the splice factor RBM20 targets amongst other genes TTN, leading to a similar response on pathway level, even though many differentially expressed genes (DEGs) still differ between both sample types. The myocardium of patients with mutations in LMNA is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in PKP2 lead to a downregulation of genes of the extracellular matrix. Our results contribute to further understanding of the underlying molecular pathomechanisms aiming for novel and better treatment of genetic cardiomyopathies.

A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3.

The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450-1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.