The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

Bi-allelic GOT2 Mutations Cause a Treatable Malate-Aspartate Shuttle-Related Encephalopathy.

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

Zebrafish hhatla is involved in cardiac hypertrophy.

Cardiac hypertrophy is a compensatory response to pathological stimuli, ultimately progresses to cardiomyopathy, heart failure, or sudden death. Although many signaling pathways have been reported to be involved in the hypertrophic process, it is still not fully clear about the underlying molecular mechanisms for cardiac hypertrophy. Hedgehog acyltransferase-like (Hhatl), a sarcoplasmic reticulum-resident protein, exhibits high expression in the heart and muscle. However, the biological role of Hhatl in the heart remains unknown. In this study, we first found that the expression level of Hhatl is markedly decreased in cardiac hypertrophy. We further studied the role of hhatla, homolog of Hhatl with the zebrafish model. The depletion of hhatla in zebrafish leads to cardiac defects, as well as an enhanced level of hypertrophic markers. Besides, we found that calcineurin signaling participates in hhatla depletion-induced cardiac hypertrophy. Together, these results demonstrate a critical role for hhatla in cardiac hypertrophy.

ndufa7 plays a critical role in cardiac hypertrophy.

Cardiac hypertrophy is a common pathological change in patients with progressive cardiac function failure, which can be caused by hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) or arterial hypertension. Despite years of study, there is still limited knowledge about the underlying molecular mechanisms for cardiac hypertrophy. NDUFA7, a subunit of NADH:ubiquinone oxidoreductase (complex I), has been reported to be a novel HCM associated gene. However, the biological role of NDUFA7 in heart remains unknown. In this study, we found that NDUFA7 exhibited high expression in the heart, and its level was significantly decreased in mice model of cardiac hypertrophy. Moreover, we demonstrated that ndufa7 knockdown in developing zebrafish embryos resulted in cardiac development and functional defects, associated with increased expression of pathological hypertrophy biomarkers nppa (ANP) and nppb (BNP). Mechanistic study demonstrated that ndufa7 depletion promoted ROS production and calcineurin signalling activation. Moreover, NDUFA7 depletion contributed to cardiac cell hypertrophy. Together, these results report for the first time that ndufa7 is implicated in pathological cardiac hypertrophy.

Integrating glycomics and genomics uncovers SLC10A7 as essential factor for bone mineralization by regulating post-Golgi protein transport and glycosylation.

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.

Histone acetyltransferase 7 (KAT7)-dependent intragenic histone acetylation regulates endothelial cell gene regulation.

Although the functional role of chromatin marks at promoters in mediating cell-restricted gene expression has been well characterized, the role of intragenic chromatin marks is not well understood, especially in endothelial cell (EC) gene expression. Here, we characterized the histone H3 and H4 acetylation profiles of 19 genes with EC-enriched expression via locus-wide chromatin immunoprecipitation followed by ultra-high-resolution (5 bp) tiling array analysis in ECs versus non-ECs throughout their genomic loci. Importantly, these genes exhibit differential EC enrichment of H3 and H4 acetylation in their promoter in ECs versus non-ECs. Interestingly, VEGFR-2 and VEGFR-1 show EC-enriched acetylation across broad intragenic regions and are up-regulated in non-ECs by histone deacetylase inhibition. It is unclear which histone acetyltransferases (KATs) are key to EC physiology. Depletion of KAT7 reduced VEGFR-2 expression and disrupted angiogenic potential. Microarray analysis of KAT7-depleted ECs identified 263 differentially regulated genes, many of which are key for growth and angiogenic potential. KAT7 inhibition in zebrafish embryos disrupted vessel formation and caused loss of circulatory integrity, especially hemorrhage, all of which were rescued with human KAT7. Notably, perturbed EC-enriched gene expression, especially the VEGFR-2 homologs, contributed to these vascular defects. Mechanistically, KAT7 participates in VEGFR-2 transcription by mediating RNA polymerase II binding, H3 lysine 14, and H4 acetylation in its intragenic region. Collectively, our findings support the importance of differential histone acetylation at both promoter and intragenic regions of EC genes and reveal a previously underappreciated role of KAT7 and intragenic histone acetylation in regulating VEGFR-2 and endothelial function.

Dual effects of human neutrophil peptides in a mouse model of pneumonia and ventilator-induced lung injury.

BACKGROUND: Pneumonia is a major cause of high morbidity and mortality in critically illness, and frequently requires support with mechanical ventilation. The latter can lead to ventilator-induced lung injury characterized by neutrophil infiltration. The cationic human neutrophil peptides (HNP) stored in neutrophils can kill microorganisms, but excessive amount of HNP released during phagocytosis may contribute to inflammatory responses and worsen lung injury. Based on our previous work, we hypothesized that blocking the cell surface purinergic receptor P2Y6 will attenuate the HNP-induced inflammatory responses while maintaining their antimicrobial activity in pneumonia followed by mechanical ventilation. METHODS: Plasma HNP levels were measured in patients with pneumonia who received mechanical ventilation and in healthy volunteers. FVB littermate control and HNP transgenic (HNP+) mice were randomized to receive P. aeruginosa intranasally. The P2Y6 antagonist (MRS2578) or vehicle control was given after P. aeruginosa instillation. Additional mice underwent mechanical ventilation at either low pressure (LP) or high pressure (HP) ventilation 48 h after pneumonia, and were observed for 24 h. RESULTS: Plasma HNP concentration increased in patients with pneumonia as compared to healthy subjects. The bacterial counts in the bronchoalveolar lavage fluid (BALF) were lower in HNP+ mice than in FVB mice 72 h after P. aeruginosa instillation. However, upon receiving HP ventilation, HNP+ mice had higher levels of cytokines and chemokines in BALF than FVB mice. These inflammatory responses were attenuated by the treatment with MRS2578 that did not affect the microbial effects of HNP. CONCLUSIONS: HNP exerted dual effects by exhibiting antimicrobial activity in pneumonia alone condition while enhancing inflammatory responses in pneumonia followed by HP mechanical ventilation. Blocking P2Y6 can attenuate the inflammation without affecting the antibacterial property of HNP. The P2Y6 receptor may be a novel therapeutic target in attenuation of the leukocyte-mediated excessive host responses in inflammatory lung diseases.

Zebrafish heart failure models: opportunities and challenges.

Heart failure is a complex pathophysiological syndrome of pumping functional failure that results from injury, infection or toxin-induced damage on the myocardium, as well as genetic influence. Gene mutations associated with cardiomyopathies can lead to various pathologies of heart failure. In recent years, zebrafish, Danio rerio, has emerged as an excellent model to study human cardiovascular diseases such as congenital heart defects, cardiomyopathy, and preclinical development of drugs targeting these diseases. In this review, we will first summarize zebrafish genetic models of heart failure arose from cardiomyopathy, which is caused by mutations in sarcomere, calcium or mitochondrial-associated genes. Moreover, we outline zebrafish heart failure models triggered by chemical compounds. Elucidation of these models will improve the understanding of the mechanism of pathogenesis and provide potential targets for novel therapies.

Development of a zebrafish sepsis model for high-throughput drug discovery.

Sepsis is a leading cause of death worldwide. Current treatment modalities remain largely supportive. Intervention strategies focused on inhibiting specific mediators of the inflammatory host response have been largely unsuccessful, a consequence of an inadequate understanding of the complexity and heterogeneity of the innate immune response. Moreover, the conventional drug development pipeline is time consuming and expensive and the low success rates associated with cell-based screens underline the need for whole organism screening strategies, especially for complex pathological processes. Here, we established an LPS-induced zebrafish endotoxemia model, which exhibits the major hallmarks of human sepsis including, edema and tissue/organ damage, increased vascular permeability and vascular leakage accompanied by an altered expression of cellular junction proteins, increased cytokine expression, immune cell activation and ROS production, reduced circulation and increased platelet aggregation. We tested the suitability of the model for phenotype-based drug screening using three primary readouts: mortality, vascular leakage, and ROS production. Preliminary screening identified fasudil, a drug known to protect against vascular leakage in murine models, as a lead hit thereby validating the utility of our model for sepsis drug screens. This zebrafish sepsis model has the potential to rapidly analyze sepsis associated pathologies and cellular processes in the whole organism, as well as to screen and validate large numbers of compounds that can modify sepsis pathology in vivo.

Effect of empagliflozin on cardiac biomarkers in a zebrafish model of heart failure: clues to the EMPA-REG OUTCOME trial?

The sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin was recently reported to reduce heart failure-associated hospitalizations and cardiovascular mortality amongst individuals with type 2 diabetes at high cardiovascular risk. We sought to elucidate the underlying mechanism(s) for these protective effects using a validated zebrafish heart failure model to evaluate the impact of empagliflozin on the expression of biomarkers of heart failure and mortality. We used aristolochic acid (AA) to induce heart failure in developing cmlc2::GFP transgenic zebrafish embryos and monitored BNP signaling in nppb::Luc transgenic zebrafish with a luciferase reporter assay. Empagliflozin markedly reduced the morphological and functional cardiac changes induced by AA; dampened AA-enhanced expression of brain natriuretic peptide and atrial natriuretic peptide; and reduced embryonic mortality. Furthermore, morpholino-mediated knockdown of the slc5A2 gene mimicked the changes evoked by empagliflozin in developing zebrafish embryos previously exposed to AA. We report herein the first mechanistic data demonstrating a salutary benefit of SGLT2 inhibition on critical pathways of heart failure signaling. These findings provide important translational clues to the cardiovascular benefits documented in the EMPA-REG OUTCOME study.

Zebrafish models of major depressive disorders.

The zebrafish (Danio rerio) has emerged as a model species for translational research in various neuroscience areas, including depressive disorders. Because of their physiological (neuroanatomical, neuroendocrine, neurochemical) and genetic homology to mammals, robust phenotypes, and value in high-throughput genetic and chemical genetic screens, zebrafish are ideal for developing valid experimental models of major depression and discovering novel therapeutics. Behavioral testing approaches, such as approach-avoidance, cognitive, and social paradigms, are available in zebrafish and have utility in identifying depression-like indices in zebrafish in response to physiological, genetic, environmental, and/or psychopharmacological alterations. In addition, the high sensitivity of zebrafish to commonly prescribed psychotropic drugs supports the use of this model as an invaluable tool for pharmacological research and drug screening. This Review outlines the benefits of using the zebrafish model for depression studies and summarizes the current research in this field.

Fetal and infant exposure to the Chinese famine increases the risk of fatty liver disease in Chongqing, China.

BACKGROUND AND AIM: Early life exposure to the famine may affect the susceptibility to the metabolic disorders. The objective of this study was to examine the associations of early life exposure to the Chinese Famine (1959-1961) with the risk of fatty liver disease, obesity, hypertension, and hyperglycemia in adulthood. METHODS: We used the data of 10,935 adults born between 1956 and 1965 in Chongqing from check-up center at the 1st Affiliated Hospital of Chongqing Medical University in 2011. Logistic regression models were used to explore the effects of famine on the risk of fatty liver disease, obesity, hypertension, and hyperglycemia. RESULTS: In Chongqing, prevalences of fatty liver disease among adults in non-exposed (1956-1958 plus 1963-1965) group and born in 1959, 1960, 1961, and 1962 were 34.01%, 36.42%, 34.63%, 40.27%, and 36.09%, respectively. Prevalences of fatty liver disease, obesity, hypertension, and hyperglycemia were significantly higher in males versus females. The risk of fatty liver disease was about 1.375-fold (1.303 (95% confidence interval, 1.066-1.594) for male, 1.547(95% confidence interval, 1.195-2.002) for female) higher in subjects born in 1961, and 1.142-fold (1.117 (95% confidence interval, 0.957-1.305) for male, 1.242 (95% confidence interval, 1.003-1.537) for female) higher in subjects born in 1962 after adjustment of age and sex, compared with non-exposed subjects. Early life exposure to the Chinese famine was also associated with the increased risk of obesity and hyperglycemia. CONCLUSIONS: Exposure to the Chinese famine during fetal life and infancy was associated with an increased risk of fatty liver disease in adulthood.

Remote Ischemic Conditioning Prevents Lung and Liver Injury After Hemorrhagic Shock/Resuscitation: Potential Role of a Humoral Plasma Factor.

OBJECTIVE: To evaluate the efficacy of remote ischemic conditioning (RIC) on organ protection after hemorrhagic shock/resuscitation (S/R) in a murine model. BACKGROUND: Ischemia/reperfusion resulting from S/R contributes to multiple organ dysfunction in trauma patients. We hypothesized that RIC before shock (remote ischemic preconditioning), during shock (remote ischemic "PER"conditioning), or during resuscitation (remote ischemic "POST"conditioning) could confer organ protection. We also tested the effect of ischemic conditioned plasma on neutrophil migration in vivo using transgenic zebrafish models. METHODS: C57Bl/6 mice were subjected to S/R with or without hindlimb RIC. Serum levels of alanine aminotransferase and tumor necrosis factor-alpha, and liver tumor necrosis factor-alpha and interleukin 1ß mRNA were evaluated. In some experiments, lung protein leakage, cytokines, and myeloperoxidase activity were investigated. Plasma from mice subjected to RIC was microinjected into zebrafish, and neutrophil migration was assessed after tailfin transection or copper sulfate treatment. RESULTS: In mice subjected to S/R, remote ischemic preconditioning, remote ischemic "PER"conditioning, and remote ischemic "POST"conditioning each significantly reduced serum alanine aminotransferase and liver mRNA expression of tumor necrosis factor-alpha and interleukin 1ß and improved liver histology compared with control S/R mice. Lung injury and inflammation were also significantly reduced in mice treated with remote ischemic preconditioning. Zebrafish injected with plasma or dialyzed plasma (fraction >14 kDa) from ischemic conditioned mice had reduced neutrophil migration toward sites of injury compared with zebrafish injected with control plasma. CONCLUSIONS: RIC protects against S/R-induced organ injury, in part, through a humoral factor(s), which alters neutrophil function. The beneficial effects of RIC, performed during the S/R phase of care, suggest a role for its application early in the posttrauma period.

Modulation of chemokine gradients by apheresis redirects leukocyte trafficking to different compartments during sepsis, studies in a rat model.

INTRODUCTION: Prior work suggests that leukocyte trafficking is determined by local chemokine gradients between the nidus of infection and the plasma. We recently demonstrated that therapeutic apheresis can alter immune mediator concentrations in the plasma, protect against organ injury, and improve survival. Here we aimed to determine whether the removal of chemokines from the plasma by apheresis in experimental peritonitis changes chemokine gradients and subsequently enhances leukocyte localization into the infected compartment, and away from healthy tissues. METHODS: In total, 76 male adult Sprague-Dawley rats weighing 400 g to 600 g were included in this study. Eighteen hours after inducing sepsis by cecal ligation and puncture, we randomized these rats to apheresis or sham treatment for 4 hours. Cytokines, chemokines, and leukocyte counts from blood, peritoneal cavity, and lung were measured. In a separate experiment, we labeled neutrophils from septic donor animals and injected them into either apheresis or sham-treated animals. All numeric data with normal distributions were compared with one-way analysis of variance, and numeric data not normally distributed were compared with the Mann-Whitney U test. RESULTS: Apheresis significantly removed plasma cytokines and chemokines, increased peritoneal fluid-to-blood chemokine (C-X-C motif ligand 1, ligand 2, and C-C motif ligand 2) ratios, and decreased bronchoalveolar lavage fluid-to-blood chemokine ratios, resulting in enhanced leukocyte recruitment into the peritoneal cavity and improved bacterial clearance, but decreased recruitment into the lung. Apheresis also reduced myeloperoxidase activity and histologic injury in the lung, liver, and kidney. These Labeled donor neutrophils exhibited decreased localization in the lung when infused into apheresis-treated animals. CONCLUSIONS: Our results support the concept of chemokine gradient control of leukocyte trafficking and demonstrate the efficacy of apheresis to target this mechanism and reduce leukocyte infiltration into the lung.

rbm47, a novel RNA binding protein, regulates zebrafish head development.

BACKGROUND: Vertebrate trunk induction requires inhibition of bone morphogenetic protein (BMP) signaling, whereas vertebrate head induction requires concerted inhibition of both Wnt and BMP signaling. RNA binding proteins play diverse roles in embryonic development and their roles in vertebrate head development remain to be elucidated. RESULTS: We first characterized the human RBM47 as an RNA binding protein that specifically binds RNA but not single-stranded DNA. Next, we knocked down rbm47 gene function in zebrafish using morpholinos targeting the start codon and exon-1/intron-1 splice junction. Down-regulation of rbm47 resulted in headless and small head phenotypes, which can be rescued by a wnt8a blocking morpholino. To further reveal the mechanism of rbm47's role in head development, microarrays were performed to screen genes differentially expressed in normal and knockdown embryos. epcam and a2ml were identified as the most significantly up- and down-regulated genes, respectively. The microarrays also confirmed up-regulation of several genes involved in head development, including gsk3a, otx2, and chordin, which are important regulators of Wnt signaling. CONCLUSIONS: Altogether, our findings reveal that Rbm47 is a novel RNA-binding protein critical for head formation and embryonic patterning during zebrafish embryogenesis which may act through a Wnt8a signaling pathway.

Zebrafish cancer and metastasis models for in vivo drug discovery.

There is a great need for more efficient methods to discover new cancer therapeutics, as traditional drug development processes are slow and expensive. The use of zebrafish as a whole-organism screen is a time and cost-effective means of improving the efficiency and efficacy of drug development. This review features zebrafish genetic and cell transplantation models of cancer and metastasis, and current imaging and automation technologies that, together, will significantly advance the field of anti-cancer drug discovery.

Integrating single-cell and bulk RNA sequencing to predict prognosis and immunotherapy response in prostate cancer.

Prostate cancer (PCa) stands as a prominent contributor to morbidity and mortality among males on a global scale. Cancer-associated fibroblasts (CAFs) are considered to be closely connected to tumour growth, invasion, and metastasis. We explored the role and characteristics of CAFs in PCa through bioinformatics analysis and built a CAFs-based risk model to predict prognostic treatment and treatment response in PCa patients. First, we downloaded the scRNA-seq data for PCa from the GEO. We extracted bulk RNA-seq data for PCa from the TCGA and GEO and adopted "ComBat" to remove batch effects. Then, we created a Seurat object for the scRNA-seq data using the package "Seurat" in R and identified CAF clusters based on the CAF-related genes (CAFRGs). Based on CAFRGs, a prognostic model was constructed by univariate Cox, LASSO, and multivariate Cox analyses. And the model was validated internally and externally by Kaplan-Meier analysis, respectively. We further performed GO and KEGG analyses of DEGs between risk groups. Besides, we investigated differences in somatic mutations between different risk groups. We explored differences in the immune microenvironment landscape and ICG expression levels in the different groups. Finally, we predicted the response to immunotherapy and the sensitivity of antitumour drugs between the different groups. We screened 4 CAF clusters and identified 463 CAFRGs in PCa scRNA-seq. We constructed a model containing 10 prognostic CAFRGs by univariate Cox, LASSO, and multivariate Cox analysis. Somatic mutation analysis revealed that TTN and TP53 were significantly more mutated in the high-risk group. Finally, we screened 31 chemotherapeutic drugs and targeted therapeutic drugs for PCa. In conclusion, we identified four clusters based on CAFs and constructed a new CAFs-based prognostic signature that could predict PCa patient prognosis and response to immunotherapy and might suggest meaningful clinical options for the treatment of PCa.

N-acetylneuraminate pyruvate lyase controls sialylation of muscle glycoproteins essential for muscle regeneration and function.

Deleterious variants in N-acetylneuraminate pyruvate lyase (NPL) cause skeletal myopathy and cardiac edema in humans and zebrafish, but its physiological role remains unknown. We report generation of mouse models of the disease: NplR63C, carrying the human p.Arg63Cys variant, and Npldel116 with a 116-bp exonic deletion. In both strains, NPL deficiency causes drastic increase in free sialic acid levels, reduction of skeletal muscle force and endurance, slower healing and smaller size of newly formed myofibers after cardiotoxin-induced muscle injury, increased glycolysis, partially impaired mitochondrial function, and aberrant sialylation of dystroglycan and mitochondrial LRP130 protein. NPL-catalyzed degradation of sialic acid in the muscle increases after fasting and injury and in human patient and mouse models with genetic muscle dystrophy, demonstrating that NPL is essential for muscle function and regeneration and serves as a general marker of muscle damage. Oral administration of N-acetylmannosamine rescues skeletal myopathy, as well as mitochondrial and structural abnormalities in NplR63C mice, suggesting a potential treatment for human patients.

Acute removal of common sepsis mediators does not explain the effects of extracorporeal blood purification in experimental sepsis.

The effect of extracorporeal blood purification on clinical outcomes in sepsis is assumed to be related to modulation of plasma cytokine concentrations. To test this hypothesis directly, we treated rats that had a cecal ligation followed by puncture (a standard model of sepsis) with a modest dose of extracorporeal blood purification that did not result in acute changes in a panel of common cytokines associated with inflammation (TNF-α, IL-1ß, IL-6, and IL-10). Pre- and immediate post-treatment levels of these cytokines were unchanged compared to the sham therapy of extracorporeal circulation without blood purifying sorbent. The overall survival to 7 days, however, was significantly better in animals that received extracorporeal blood purification compared to those with a sham procedure. This panel of common plasma cytokines along with alanine aminotransferase and creatinine was significantly lower 72 h following extracorporeal blood purification compared to sham-treated rats. Thus, the effects of this procedure on organ function and survival do not appear to be due solely to immediate changes in the usual measured circulating cytokines. These results may have important implications for the design and conduct of future trials in sepsis including defining alternative targets for extracorporeal blood purification and other therapies.