A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle.

BACKGROUND: Human iPSC-derived 3D-tissue-engineered-skeletal muscles (3D-TESMs) offer advanced technology for disease modelling. However, due to the inherent genetic heterogeneity among human individuals, it is often difficult to distinguish disease-related readouts from random variability. The generation of genetically matched isogenic controls using gene editing can reduce variability, but the generation of isogenic hiPSC-derived 3D-TESMs can take up to 6 months, thereby reducing throughput. METHODS: Here, by combining 3D-TESM and shRNA technologies, we developed a disease modelling strategy to induce distinct genetic deficiencies in a single hiPSC-derived myogenic progenitor cell line within 1 week. RESULTS: As proof of principle, we recapitulated disease-associated pathology of Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knock down of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes. Pathology in DMD-deficient 3D-TESMs was partially rescued by a candidate gene therapy treatment using micro-dystrophin, with similar efficacy compared to animal models. CONCLUSIONS: These results show that isogenic shRNA-based humanized 3D-TESM models provide a fast, cheap, and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.

Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes.

Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.

Ars2-containing bispecific, Fab- and IgG1-format BAR-bodies to target DLBCL cells.

Despite recent advances in the therapy of diffuse large B-cell lymphoma, not otherwise specified (DLBCL), around 30% of patients develop refractory disease or relapse after first-line treatment. Recently, Ars2 was reported as the auto-antigenic target of the B-cell receptor (BCR) in approximately 25% of activated B-cell DLBCL cases. Ars2 could be used to specifically target B cells expressing Ars2-reactive BCRs. However, the optimal therapeutic format to integrate Ars2 into has yet to be determined. To mimic therapeutic antibody formats, Ars2-containing bispecific and IgG1-like constructs (BCR antigens for reverse [BAR]-bodies) were developed. Two bispecific BAR-bodies connecting single-chain antibodies against CD16 or CD3 to the BCR-binding epitope of Ars2 were constructed. Both constructs showed strong binding to U2932 cells and induced effector cell-dependent and selective cytotoxicity against U2932 cells of up to 44% at concentrations of 20 µg/ml. Additionally, IgG1-format Ars2 BAR-bodies were constructed by replacing the variable heavy- and light-chain regions of a full-length antibody with the Ars2 epitope. IgG1-format Ars2 BAR-bodies also bound selectively to U2932 and OCI-Ly3 cells and induced selective cytotoxicity of up to 60% at 10 µg/ml. In conclusion, Ars2-containing bispecific and IgG1-format BAR-bodies both are new therapeutic formats to target DLBCL cells.

Organ Protection by Caloric Restriction Depends on Activation of the De Novo NAD+ Synthesis Pathway.

SIGNIFICANCE STATEMENT: AKI is a major clinical complication leading to high mortality, but intensive research over the past decades has not led to targeted preventive or therapeutic measures. In rodent models, caloric restriction (CR) and transient hypoxia significantly prevent AKI and a recent comparative transcriptome analysis of murine kidneys identified kynureninase (KYNU) as a shared downstream target. The present work shows that KYNU strongly contributes to CR-mediated protection as a key player in the de novo nicotinamide adenine dinucleotide biosynthesis pathway. Importantly, the link between CR and NAD+ biosynthesis could be recapitulated in a human cohort. BACKGROUND: Clinical practice lacks strategies to treat AKI. Interestingly, preconditioning by hypoxia and caloric restriction (CR) is highly protective in rodent AKI models. However, the underlying molecular mechanisms of this process are unknown. METHODS: Kynureninase (KYNU) knockout mice were generated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and comparative transcriptome, proteome and metabolite analyses of murine kidneys pre- and post-ischemia-reperfusion injury in the context of CR or ad libitum diet were performed. In addition, acetyl-lysin enrichment and mass spectrometry were used to assess protein acetylation. RESULTS: We identified KYNU as a downstream target of CR and show that KYNU strongly contributes to the protective effect of CR. The KYNU-dependent de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway is necessary for CR-associated maintenance of NAD+ levels. This finding is associated with reduced protein acetylation in CR-treated animals, specifically affecting enzymes in energy metabolism. Importantly, the effect of CR on de novo NAD+ biosynthesis pathway metabolites can be recapitulated in humans. CONCLUSIONS: CR induces the de novo NAD+ synthesis pathway in the context of IRI and is essential for its full nephroprotective potential. Differential protein acetylation may be the molecular mechanism underlying the relationship of NAD+, CR, and nephroprotection.

B-cell receptors of EBV-negative Burkitt lymphoma bind modified isoforms of autoantigens.

Burkitt lymphoma (BL) represents the most aggressive B-cell-lymphoma. Beside the hallmark of IG-MYC-translocation, surface B-cell receptor (BCR) is expressed, and mutations in the BCR pathway are frequent. Coincidental infections in endemic BL, and specific extra-nodal sites suggest antigenic triggers. To explore this hypothesis, BCRs of BL cell lines and cases were screened for reactivities against a panel of bacterial lysates, lysates of Plasmodium falciparum, a custom-made virome array and against self-antigens, including post-translationally modified antigens. An atypically modified, SUMOylated isoform of Bystin, that is, SUMO1-BYSL was identified as the antigen of the BCR of cell line CA46. SUMO1-BYSL was exclusively expressed in CA46 cells with K139 as site of the SUMOylation. Secondly, an atypically acetylated isoform of HSP40 was identified as the antigen of the BCR of cell line BL41. K104 and K179 were the sites of immunogenic acetylation, and the acetylated HSP40 isoform was solely present in BL41 cells. Functionally, addition of SUMO1-BYSL and acetylated HSP40 induced BCR pathway activation in CA46 and BL41 cells, respectively. Accordingly, SUMO1-BYSL-ETA' immunotoxin, produced by a two-step intein-based conjugation, led to the specific killing of CA46 cells. Autoantibodies directed against SUMO1-BYSL were found in 3 of 14 (21.4%), and autoantibodies against acetylated HSP40 in 1/14(7.1%) patients with sporadic Burkitt-lymphoma. No reactivities against antigens of the infectious agent spectrum could be observed. These results indicate a pathogenic role of autoreactivity evoked by immunogenic post-translational modifications in a subgroup of sporadic BL including two EBV-negative BL cell lines.

FGF21 modulates mitochondrial stress response in cardiomyocytes only under mild mitochondrial dysfunction.

The mitochondrial integrated stress response (mitoISR) has emerged as a major adaptive pathway to respiratory chain deficiency, but both the tissue specificity of its regulation, and how mitoISR adapts to different levels of mitochondrial dysfunction are largely unknown. Here, we report that diverse levels of mitochondrial cardiomyopathy activate mitoISR, including high production of FGF21, a cytokine with both paracrine and endocrine function, shown to be induced by respiratory chain dysfunction. Although being fully dispensable for the cell-autonomous and systemic responses to severe mitochondrial cardiomyopathy, in the conditions of mild-to-moderate cardiac OXPHOS dysfunction, FGF21 regulates a portion of mitoISR. In the absence of FGF21, a large part of the metabolic adaptation to mitochondrial dysfunction (one-carbon metabolism, transsulfuration, and serine and proline biosynthesis) is strongly blunted, independent of the primary mitoISR activator ATF4. Collectively, our work highlights the complexity of mitochondrial stress responses by revealing the importance of the tissue specificity and dose dependency of mitoISR.

Low kindlin-3 levels in osteoclasts of kindlin-3 hypomorphic mice result in osteopetrosis due to leaky sealing zones.

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist of densely packed podosomes tightly interconnected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3 (also known as Fermt3). In this study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of the kindlin-3 level of wild-type mice. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro-generated kindlin-3-hypomorphic osteoclast-like cells arrange their podosomes into adhesion patches and belts, but their podosome and actin organization is abnormal. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays, immunohistochemical staining and electron micrographs of bone sections showed that they fail to seal the resorption lacunae properly, which is required for secreted proteinases to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrin-mediated adhesion structures, such as sealing zones.

PERM1 interacts with the MICOS-MIB complex to connect the mitochondria and sarcolemma via ankyrin B.

Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB.

Operational nurse managers' perceptions on the competence of community service nurses in public settings in the Western Cape.

BACKGROUND: Community service nurses placed in the Western Cape Government public health facilities render essential healthcare to underserved populations. Anecdotal evidence from operational nurse managers indicated concerns that community service nurses may lack competence in basic required nursing competencies. OBJECTIVES: To investigate operational nurse managers' perceptions of the competence of community service nurses in public health facilities in the Western Cape. METHOD: A quantitative survey was conducted with an all-inclusive sample of 297 operational nurse managers in the Western Cape. A self-administered questionnaire with 65 questions with a 4-point rating scale was used to rate perceived competence of community service nurses across the South African Nursing Council (SANC) competencies. Descriptive and inferential statistics were calculated per competency domain. RESULTS: The survey (response rate: 59%) showed that the operational nurse managers perceived the community service nurses to be competent in the clinical patient care domain and mostly either developing proficiency or proficient in the SANC competencies of legal framework and ethical practice, interprofessional relationships, leadership, quality management and management competency domains. CONCLUSION: Community service nurses were found to be competent in the clinical patient care, possibly because of the integration of theory and practice focus of work-integrated learning in the programme. Education and practice supportive strategies for community service nurses should be developed to support the successful transition from students to community service nurses, especially around the development of research and critical thinking skills.

Phosphoproteomics of the developing heart identifies PERM1 - An outer mitochondrial membrane protein.

Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1- and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1-/- mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.

Exercise-dependent increases in protein synthesis are accompanied by chromatin modifications and increased MRTF-SRF signalling.

AIM: Resistance exercise increases muscle mass over time. However, the early signalling events leading to muscle growth are not yet well-defined. Here, we aim to identify new signalling pathways important for muscle remodelling after exercise. METHODS: We performed a phosphoproteomics screen after a single bout of exercise in mice. As an exercise model we used unilateral electrical stimulation in vivo and treadmill running. We analysed muscle biopsies from human subjects to verify if our findings in murine muscle also translate to exercise in humans. RESULTS: We identified a new phosphorylation site on Myocardin-Related Transcription Factor B (MRTF-B), a co-activator of serum response factor (SRF). Phosphorylation of MRTF-B is required for its nuclear translocation after exercise and is accompanied by the transcription of the SRF target gene Fos. In addition, high-intensity exercise also remodels chromatin at specific SRF target gene loci through the phosphorylation of histone 3 on serine 10 in myonuclei of both mice and humans. Ablation of the MAP kinase member MSK1/2 is sufficient to prevent this histone phosphorylation, reduce induction of SRF-target genes, and prevent increases in protein synthesis after exercise. CONCLUSION: Our results identify a new exercise signalling fingerprint in vivo, instrumental for exercise-induced protein synthesis and potentially muscle growth.

High-throughput proteomics fiber typing (ProFiT) for comprehensive characterization of single skeletal muscle fibers.

BACKGROUND: Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. Each fiber has a specific molecular expression of myosin heavy chain molecules (MyHC). So far, MyHCs are currently the best marker proteins for characterization of individual fiber types, and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers. METHODS: Herein, we describe a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited quantities of protein in single fibers, we developed a Proteomics high-throughput fiber typing (ProFiT) approach enabling profiling of MyHC in single fibers. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC, and phosphoproteomic analysis. RESULTS: Our fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of the Lys-6 incorporation rates in muscle fibers showed an increased turnover of slow fibers compared to fast fibers. In addition, labeling of mitochondrial respiratory chain complexes revealed a broad range of Lys-6 incorporation rates, depending on the localization of the subunits within distinct complexes. CONCLUSION: Overall, the ProFiT approach provides a versatile tool to rapidly characterize muscle fibers and obtain fiber-specific proteomes for different muscle groups.

LRPAP1 is a frequent proliferation-inducing antigen of BCRs of mantle cell lymphomas and can be used for specific therapeutic targeting.

The predominant usage of VH4-34 and V3-21 and reports of stereotyped CDR3s suggest a shared antigenic target of B-cell receptors (BCR) from mantle cell lymphomas (MCL). To identify the target antigens of MCL-BCRs, BCRs from 21 patients and seven MCL cell lines were recombinantly expressed and used for antigen screening. The BCRs from 8/21 patients and 2/7 MCL cell lines reacted specifically with the autoantigen low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1). High-titered and light chain-restricted anti-LRPAP1 serum antibodies were found in MCL patients, but not in controls. LRPAP1 induced proliferation by BCR pathway activation, while an LRPAP1-ETA' toxin-conjugate specifically killed MCL cells with LRPAP1-specific BCRs. Our results suggest a role of LRPAP1 in lymphomagenesis and maintenance of a considerable proportion of MCL cases by chronic autoantigenic stimulation, likely evolving from a chronic autoreactive B-cell response. Importantly, LRPAP1 can be used for a novel therapeutic approach that targets MCL with LRPAP1-reactive BCRs with high specificity.