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The environmental prevalence of antimicrobial resistance (AMR) has come into focus under the One Health concept. Wastewater treatment systems are among the significant sources of AMR in the environment. In such systems, it is uncertain to which extent antimicrobials present at sub-inhibitory concentrations constitute a selective pressure for bacterial maintenance and acquisition of antibiotic resistance (AR) genes. Here, we mapped AMR to inhibitors of folate biosynthesis in an aerated and a non-aerated horizontal subsurface flow treatment wetland receiving the same pre-treated municipal wastewater. General water characteristics and the concentrations of folate inhibitors were determined to define the ambient conditions over the longitudinal axis of the two treatment wetlands. Profiling of AMR as well as class 1 integrons, a carrier of AR genes against folate inhibitors and other antimicrobials, was conducted by cultivation-dependent and -independent methods. The wetlands achieved mean reductions of AR gene copy numbers in the effluents of at least 2 log, with the aerated system performing better. The folate inhibitors had no noticeable effect on the prevalence of respective AR genes. However, there was a transient increase of AR gene copy numbers and AR gene cassette composition in class 1 integrons in the aerated wetland. The comparison of all data from both wetlands suggests that higher levels of cellular stress in the aerated system promoted the mobility of AR genes via enhancing the activity of the DNA recombinase of the class 1 integron. The findings highlight that environmental conditions that modulate the activity of this genetic element can be more important for the fate of associated AR genes in treatment wetlands than the ambient concentration of the respective antimicrobial agents. By extrapolation, the results suggest that cellular stress also contributes to the mobility of AR gene in other wastewater treatment systems.

Integrity of mitochondrial DNA (mtDNA), encoding several subunits of the respiratory chain, is essential to maintain mitochondrial fitness. Mitochondria, as a central hub for metabolism, are affected in a wide variety of human diseases but also during normal ageing, where mtDNA integrity is compromised. Mitochondrial quality control mechanisms work at different levels, and mitophagy and its variants are critical to remove dysfunctional mitochondria together with mtDNA to maintain cellular homeostasis. Understanding the mechanisms governing a selective turnover of mutation-bearing mtDNA without affecting the entire mitochondrial pool is fundamental to design therapeutic strategies against mtDNA diseases and ageing. Here we show that mtDNA depletion after expressing a dominant negative version of the mitochondrial helicase Twinkle, or by chemical means, is due to an exacerbated mtDNA turnover. Targeting of nucleoids is controlled by Twinkle which, together with the mitochondrial transmembrane proteins ATAD3 and SAMM50, orchestrate mitochondrial membrane remodeling to form extrusions. mtDNA removal depends on autophagy and requires the vesicular trafficking protein VPS35 which binds to Twinkle-enriched mitochondrial subcompartments upon mtDNA damage. Stimulation of autophagy by rapamycin selectively removes mtDNA deletions which accumulated during muscle regenerationin vivo, but without affecting mtDNA copy number. With these results we unveil a new complex mechanism specifically targeting and removing mutant mtDNA which occurs outside the mitochondrial network. We reveal the molecular targets involved in a process with multiple potential benefits against human mtDNA related diseases, either inherited, acquired or due to normal ageing.

Rationale: In chronic heart failure, increased adrenergic activation contributes to structural remodeling and altered gene expression. Although adrenergic signaling alters histone modifications, it is unknown, whether it also affects other epigenetic processes, including DNA methylation and its recognition. Objective: The aim of this study was to identify the mechanism of regulation of the methyl-CpG–binding protein 2 (MeCP2) and its functional significance during cardiac pressure overload and unloading. Methods and Results: MeCP2 was identified as a reversibly repressed gene in mouse hearts after transverse aortic constriction and was normalized after removal of the constriction. Similarly, MeCP2 repression in human failing hearts resolved after unloading by a left ventricular assist device. The cluster miR-212/132 was upregulated after transverse aortic constriction or on activation of α 1 - and β 1 -adrenoceptors and miR-212/132 led to repression of MeCP2. Prevention of MeCP2 repression by a cardiomyocyte-specific, doxycycline-regulatable transgenic mouse model aggravated cardiac hypertrophy, fibrosis, and contractile dysfunction after transverse aortic constriction. Ablation of MeCP2 in cardiomyocytes facilitated recovery of failing hearts after reversible transverse aortic constriction. Genome-wide expression analysis, chromatin immunoprecipitation experiments, and DNA methylation analysis identified mitochondrial genes and their transcriptional regulators as MeCP2 target genes. Coincident with its repression, MeCP2 was removed from its target genes, whereas DNA methylation of MeCP2 target genes remained stable during pressure overload. Conclusions: These data connect adrenergic activation with a microRNA—MeCP2 epigenetic pathway that is important for cardiac adaptation during the development and recovery from heart failure.

Summary Ectopic lipid deposition and altered mitochondrial dynamics contribute to the development of obesity and insulin resistance. However, the mechanistic link between these processes remained unclear. Here we demonstrate that the C16:0 sphingolipid synthesizing ceramide synthases, CerS5 and CerS6, affect distinct sphingolipid pools and that abrogation of CerS6 but not of CerS5 protects from obesity and insulin resistance. We identify proteins that specifically interact with C16:0 sphingolipids derived from CerS5 or CerS6. Here, only CerS6-derived C16:0 sphingolipids bind the mitochondrial fission factor (Mff). CerS6 and Mff deficiency protect from fatty acid-induced mitochondrial fragmentation in vitro, and the two proteins genetically interact in vivo in obesity-induced mitochondrial fragmentation and development of insulin resistance. Our experiments reveal an unprecedented specificity of sphingolipid signaling depending on specific synthesizing enzymes, provide a mechanistic link between hepatic lipid deposition and mitochondrial fragmentation in obesity, and define the CerS6-derived sphingolipid/Mff interaction as a therapeutic target for metabolic diseases.

Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (F passive ) depend in part on titin isoform composition and phosphorylation. Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca 2+ -handling proteins, and chromatin-modifying enzymes in the heart, but has not been known to target titin. Objective: To elucidate whether CaMKII phosphorylates titin and modulates F passive in normal and failing myocardium. Methods and Results: Titin phosphorylation was assessed in CaMKIIδ/γ double-knockout (DKO) mouse, transgenic CaMKIIδC-overexpressing mouse, and human hearts, by Pro-Q-Diamond/Sypro-Ruby staining, autoradiography, and immunoblotting using phosphoserine-specific titin-antibodies. CaMKII-dependent site-specific titin phosphorylation was quantified in vivo by mass spectrometry using s table isotope labeling by amino acids in cell culture mouse heart mixed with wild-type (WT) or DKO heart. F passive of single permeabilized cardiomyocytes was recorded before and after CaMKII-administration. All-titin phosphorylation was reduced by >50% in DKO but increased by up to ≈100% in transgenic versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within the PEVK-domain of titin by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence. Phosphorylation at specific PEVK/titin N2B-unique sequence sites was decreased in DKO and amplified in transgenic versus WT hearts. F passive was elevated in DKO and reduced in transgenic compared with WT cardiomyocytes. CaMKII-administration lowered F passive of WT and DKO cardiomyocytes, an effect blunted by titin antibody pretreatment. Human end-stage failing hearts revealed higher CaMKII expression/activity and phosphorylation at PEVK/titin N2B-unique sequence sites than nonfailing donor hearts. Conclusions: CaMKII phosphorylates the titin springs at conserved serines/threonines, thereby lowering F passive . Deranged CaMKII-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.

The specification of distinct classes of neurones in the neural tube is governed by extracellular signals and transcriptional mediators including neurogenic bHLH- and homeobox-containing genes. Despite recent insights in the transcriptional code that controls generation of neuronal subtypes in the dorsal neural tube, the mechanisms that underlie regional identity in dorsal and lateral regions of the mammalian neural tube are not well understood. The homeobox gene Lbx1 is expressed in a subset of interneurons in the early neural tube and later in dorsal parts of the spinal cord. Here we show that Lbx1 is part of the machinery that controls formation of neurones in the dorsal horn. Targeted inactivation of Lbx1 leads to a severe reduction of neurones in the dorsal horn and to down-regulation of dorsal markers such as Lmx1a from E11.5 onwards. In contrast, Pax3, another dorsally expressed homeobox gene, was up-regulated in Lbx1 mutant mice at E18.5, suggesting the existence of a negative regulatory feedback loop. Lbx1/Pax3 double-mutant mice showed an enhanced Pax3 phenotype characterized by a completely open neural tube. We suggest that Lbx1 plays a critical role in the specification of dorsal interneurons and that Lbx1 and Pax3 might act together synergistically to promote neural tube closure.

The neuronal stem cell leukemia (NSCL) basic helix-loop-helix factors are neural cell-specific transcription factors. We have disrupted the NSCL-1 gene by homologous recombination and replaced the coding region with a beta-galactosidase reporter cassette to study the role of NSCL-1 in neuronal development and to follow the fate of NSCL-1 mutant cells. NSCL-1 mutant mice are viable and fertile on various genetic backgrounds and do not show any obvious signs of neurological malfunction. No differences in the distribution of NSCL-1 mutant or heterozygous neuronal cells were observed in the diencephalon, hippocampus, neocortex, and cerebellum at different stages of development. Likewise, no defects were found in the laminar organization of the cortex, and the distinct neuronal subpopulation appeared normal during development of the neocortex. Analysis of sensory neurons which strongly express NSCL-1 revealed that the spatiotemporal expression of neuronal differentiation factors, such as NeuroD and SCG-10, was not altered in developing distal and proximal cranial ganglia of mutant mice. In the cerebellum expression of NSCL-1 was confined to the proliferative and premigratory zone of the external granular layer and the internal granular layer. Interestingly, unlike cerebella of Math1(-/-) or NeuroD2(-/-) mice, NSCL-1-deficient mice have no obvious developmental defect, and neurons of the cerebellum appeared fully differentiated. Despite similar expression patterns of NSCL-1 and NSCL-2 in various areas of the diencephalon, including the arcuate nucleus and paraventricular nucleus, NSCL-1(-/-) mice are fertile and show no adult onset of obesity like NSCL-2 mutant mice. Double-mutant NSCL-1(-/-)-NSCL-2(-/-) mice do not show any additional obvious malformations of the central nervous system, although both genes are expressed in a largely overlapping pattern. Our results argue against a simple functional redundancy within the NSCL gene family.

Group D streptococci have rarely been associated with neonatal infections. We report a case of fulminant respiratory distress syndrome (RDS) caused by Streptococcus alactolyticus in a term neonate. Gram staining revealed gram-positive cocci and culture grew group D streptococci in samples taken from trachea, ear, and nasopharynx. Streptococcus alactolyticus was identified using automated microbial identification system (Vitek 2). Histopathology showed massive pulmonary inflammation with intra-alveolar granulocytosis and secondary pulmonary bleeding as etiology of fatal outcome. To our knowledge, this is first case presenting neonatal infection caused by Streptococcus alactolyticus.

Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (Fpassive) depend in part on titin isoform composition and phosphorylation. Ca2+/calmodulin-dependent protein kinase-IIδ (CaMKIIδ) phosphorylates ion channels, Ca2+-handling and myofilament proteins in the heart, but has not been known to target titin.Objective: To elucidate whether CaMKIIδ phosphorylates titin and regulates Fpassive in normal and failing myocardium.Methods and Results: Titin phosphorylation was assessed in CaMKIIδ/γ double-knockout (DKO) and transgenic CaMKIIδC-overexpressing (TG) mouse hearts, as well as human hearts, by Pro-Q-Diamond/Sypro-Ruby staining, autoradiography, and immunoblotting using phosphospecific titin antibodies. CaMKII-dependent site-specific titin phosphorylation was quantified in vivo by mass spectrometry using SILAC mouse heart tissue mixed with wildtype (WT) or DKO heart. Fpassive of single permeabilized cardiomyocytes was recorded before and after CaMKII administration. We detected hypophosphorylation of all-titin in DKO and hyperphosphorylation in TG compared to WT hearts. Conserved CaMKII-dependent phosphosites were identified within titin's PEVK-domain by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence (N2Bus). Phosphorylation at specific PEVK/N2Bus sites was decreased in DKO and amplified in TG versus WT hearts. Fpassive was elevated in DKO and reduced in TG compared to WT cardiomyocytes. CaMKII-administration lowered Fpassive of WT and DKO cardiomyocytes, an effect blunted by titin-phosphoantibody pretreatment. Human failing hypertrophic hearts revealed higher CaMKII expression/activity and phosphorylation at PEVK/N2Bus sites than nonfailing donor hearts.Conclusions: CaMKIIδ phosphorylates the titin springs at conserved serines/threonines, thereby lowering Fpassive. Deranged CaMKIIδ-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.