MYCBPAP is a central apparatus protein required for centrosome-nuclear envelope docking and sperm tail biogenesis in mice.

The structure of the sperm flagellar axoneme is highly conserved across species and serves the essential function of generating motility to facilitate the meeting of spermatozoa with the egg. During spermiogenesis, the axoneme elongates from the centrosome, and subsequently the centrosome docks onto the nuclear envelope to continue tail biogenesis. Mycbpap is expressed predominantly in mouse and human testes and conserved in Chlamydomonas as FAP147. A previous cryo-electron microscopy analysis has revealed the localization of FAP147 to the central apparatus of the axoneme. Here, we generated Mycbpap knockout mice and demonstrated the essential role of Mycbpap in male fertility. Deletion of Mycbpap led to disrupted centrosome-nuclear envelope docking and abnormal flagellar biogenesis. Furthermore, we generated transgenic mice with tagged MYCBPAP, which restored the fertility of Mycbpap knockout males. Interactome analyses of MYCBPAP using Mycbpap transgenic mice unveiled binding partners of MYCBPAP including central apparatus proteins such as CFAP65 and CFAP70 that constitute the C2a projection and centrosome-associated proteins such as CCP110. These findings provide insights into a MYCBPAP-dependent regulation of the centrosome-nuclear envelope docking and sperm tail biogenesis.

Enhancing immunity against Candida albicans infections through TIGIT knockout.

T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is an inhibitory receptor expressed by T and natural killer cells. Here, we used TIGIT knockout (KO) mice to demonstrate that mouse TIGIT directly interacts with Candida albicans. Reduced fungal growth and colonization were observed when TIGIT-KO splenocytes were co-cultured with C. albicans compared to the wild type (WT). In a systemic candidiasis model, TIGIT-KO mice exhibited improved survival and reduced body weight loss compared to WT mice. Organ-specific fungal burden assessment revealed significantly lower fungal loads in the kidneys, spleen, and lungs of TIGIT-KO mice. Finally, we show that the agglutinin-like sequence proteins ALS6, ALS7, and ALS9 of C. albicans are ligands for TIGIT and that the absence of these proteins abolishes the TIGIT effect in vivo. Our results identify the significance of TIGIT in modulating host defense against C. albicans and highlight the potential therapeutic implications for C. albicans infections. IMPORTANCE: Our results identify the significance of T cell immunoreceptor with immunoglobulin and ITIM domain in modulating host defense against Candida albicans and highlight the potential therapeutic implications for C. albicans infections.

RAB37-mediated autophagy guards ovarian homeostasis and function.

Loss of ovarian homeostasis is associated with ovary dysfunction and female diseases; however, the underlying mechanisms responsible for the establishment of homeostasis and its function in the ovary have not been fully elucidated. Here, we showed that conditional knockout of Rab37 in oocytes impaired macroautophagy/autophagy proficiency in the ovary and interfered with follicular homeostasis and ovary development in mice. Flunarizine treatment upregulated autophagy, thus rescuing the impairment of follicular homeostasis and ovarian dysfunction in rab37 knockout mice by reprogramming of homeostasis. Notably, both the E2F1 and EGR2 transcription factors synergistically activated Rab37 transcription and promoted autophagy. Thus, RAB37-mediated autophagy ensures ovary function by maintaining ovarian homeostasis.

Podocyte Cell-Specific Npr1 is Required for Blood Pressure and Renal Homeostasis in Male and Female Mice: Role of Sex-Specific Differences.

Atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA), stimulating natriuresis and diuresis and reducing blood pressure (BP), but the role of ANP/NPRA signaling in podocytes (highly specialized epithelial cells covering the outer surfaces of renal glomerular capillaries) remains unclear. This study aimed to determine the effect of conditional deletion of podocyte (PD)-specific Npr1 (encoding NPRA) gene knockout (KO) in male and female mice. Tamoxifen-treated wild-type control (PD Npr1 f/f; WT), heterozygous (PD-Cre-Npr1 f/+; HT), and knockout (PD-Cre-Npr1 f/-; KO) mice were fed a normal-, low-, or high-salt diet for 4 weeks. Podocytes isolated from HT and KO male and female mice showed complete absence of Npr1 mRNA and NPRA protein compared to WT mice. BP, plasma creatinine, plasma sodium, urinary protein, and albumin/creatinine ratio were significantly increased, while plasma total protein, albumin, creatinine clearance, and urinary sodium levels were significantly reduced in the HT and KO male and female mice compared to WT mice. These changes were significantly greater in males than females. On a normal-salt diet, glomerular filtration rate (GFR) was significantly decreased in PD Npr1 HT and KO male and female mice compared with WT mice. Immunofluorescence of podocin and synaptopodin were also significantly reduced in HT and KO mice compared to WT mice. These observations suggest that in podocytes, ANP/NPRA signaling may be crucial in the maintenance and regulation of glomerular filtration and BP and serve as a biomarker of renal function in a sex-dependent manner.

Eighteen genes primarily expressed in the testis are not required for male fertility in mice.

There are approximately 20,000 protein-coding genes in humans and mice. More than 1000 of these genes are predominantly expressed in the testis or are testis-specific and thought to play an important role in male reproduction. Through the production of gene knockout mouse models and phenotypic evaluations, many genes essential for spermatogenesis, sperm maturation, and fertilization have been discovered, greatly contributing to the elucidation of their molecular mechanisms. On the other hand, there are many cases in which single-gene knockout models do not affect fertility, indicating that tissue-specific genes are not always critical. Here, we selected 18 genes whose mRNA expression is restricted to the testis or higher than in other tissues, but whose function in male reproduction is unknown. We then created single-gene KO mouse models using the CRISPR/Cas9 system. The established KO males were subjected to mating tests and screened for effects on fecundity, revealing that these genes were not essential for spermatogenesis and male fertility. This knowledge will contribute to understanding the functions of genes characteristic of the testis and identify the cause of male infertility.

Joint GWAS and WGCNA Identify Genes Regulating the Isoflavone Content in Soybean Seeds.

Isoflavone is a secondary metabolite of the soybean phenylpropyl biosynthesis pathway with physiological activity and is beneficial to human health. In this study, the isoflavone content of 205 soybean germplasm resources from 3 locations in 2020 showed wide phenotypic variation. A joint genome-wide association study (GWAS) and weighted gene coexpression network analysis (WGCNA) identified 33 single-nucleotide polymorphisms and 11 key genes associated with soybean isoflavone content. Gene ontology enrichment analysis, gene coexpression, and haplotype analysis revealed natural variations in the Glyma.12G109800 (GmOMT7) gene and promoter region, with Hap1 being the elite haplotype. Transient overexpression and knockout of GmOMT7 increased and decreased the isoflavone content, respectively, in hairy roots. The combination of GWAS and WGCNA effectively revealed the genetic basis of soybean isoflavone and identified potential genes affecting isoflavone synthesis and accumulation in soybean, providing a valuable basis for the functional study of soybean isoflavone.

HepG2 PMM2-CDG knockout model: A versatile platform for variant and therapeutic evaluation.

Phosphomannomutase 2 deficiency (PMM2-CDG), the most frequent congenital disorder of glycosylation, is an autosomal recessive disease caused by biallelic pathogenic variants in the PMM2 gene. There is no cure for this multisystemic syndrome. Some of the therapeutic approaches that are currently in development include mannose-1-phosphate replacement therapy, drug repurposing, and the use of small chemical molecules to correct folding defects. Preclinical models are needed to evaluate the efficacy of treatments to overcome the high lethality of the available animal model. In addition, the number of variants with unknown significance is increasing in clinical settings. This study presents the generation of a cellular disease model by knocking out the PMM2 gene in the hepatoma HepG2 cell line using CRISPR-Cas9 gene editing. The HepG2 knockout model accurately replicates the PMM2-CDG phenotype, exhibiting a complete absence of PMM2 protein and mRNA, a 90% decrease in PMM enzymatic activity, and altered ICAM-1, LAMP1 and A1AT glycoprotein patterns. The evaluation of PMM2 disease-causing variants validates the model's utility for studying new PMM2 clinical variants, providing insights for diagnosis and potentially for evaluating therapies. A CRISPR-Cas9-generated HepG2 knockout model accurately recapitulates the PMM2-CDG phenotype, providing a valuable tool for assessing disease-causing variants and advancing therapeutic strategies.

Proteomic Analysis of Rap1A GTPase Signaling-Deficient C57BL/6 Mouse Pancreas and Functional Studies Identify an Essential Role of Rap1A in Pancreas Physiology.

Ras-related Rap1A GTPase is implicated in pancreas ß-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein ß (Ero1lß), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lß and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15-30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function.

The Role of the MiR-181 Family in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fourth-leading cause of cancer-related death worldwide. Due to the high mortality rate in HCC patients, discovering and developing novel systemic treatment options for HCC is a vital unmet medical need. Among the numerous molecular alterations in HCCs, microRNAs (miRNAs) have been increasingly recognised to play critical roles in hepatocarcinogenesis. We and others have recently revealed that members of the microRNA-181 (miR-181) family were up-regulated in some, though not all, human cirrhotic and HCC tissues-this up-regulation induced epithelial-mesenchymal transition (EMT) in hepatocytes and tumour cells, promoting HCC progression. MiR-181s play crucial roles in governing the fate and function of various cells, such as endothelial cells, immune cells, and tumour cells. Previous reviews have extensively covered these aspects in detail. This review aims to give some insights into miR-181s, their targets and roles in modulating signal transduction pathways, factors regulating miR-181 expression and function, and their roles in HCC.

Endocannabinoids and their receptors modulate endometriosis pathogenesis and immune response.

Endometriosis (EM), characterized by the presence of endometrial-like tissue outside the uterus, is the leading cause of chronic pelvic pain and infertility in females of reproductive age. Despite its high prevalence, the molecular mechanisms underlying EM pathogenesis remain poorly understood. The endocannabinoid system (ECS) is known to influence several cardinal features of this complex disease including pain, vascularization, and overall lesion survival, but the exact mechanisms are not known. Utilizing CNR1 knockout (k/o), CNR2 k/o, and wild-type (WT) mouse models of EM, we reveal contributions of ECS and these receptors in disease initiation, progression, and immune modulation. Particularly, we identified EM-specific T cell dysfunction in the CNR2 k/o mouse model of EM. We also demonstrate the impact of decidualization-induced changes on ECS components, and the unique disease-associated transcriptional landscape of ECS components in EM. Imaging mass cytometry (IMC) analysis revealed distinct features of the microenvironment between CNR1, CNR2, and WT genotypes in the presence or absence of decidualization. This study, for the first time, provides an in-depth analysis of the involvement of the ECS in EM pathogenesis and lays the foundation for the development of novel therapeutic interventions to alleviate the burden of this debilitating condition.

Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes.

The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.

Generation of bone-specific lysyl hydroxylase 2 knockout mice and their phenotypes.

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of type I collagen. This modification is critical for the formation of stable hydroxylysine-aldehyde derived collagen cross-links, thus, for the stability of collagen fibrils. Though dysfunction of LH2 causes Bruck syndrome, recessive osteogenesis imperfecta with joint contracture, the molecular mechanisms by which LH2 affects bone formation are still not well understood. Since the Plod2 knockout mice are embryonically lethal, we generated bone-specific LH2 conditional knockout mice (bsLH2-cKO) using the osteocalcin-Cre/loxP system, and evaluated phenotypes of femurs. LH2 mRNA and protein levels assessed by qPCR, immunohistochemistry and Data Independent Acquisition proteomics were all markedly low in bsLH2-cKO femurs when compared to controls. Lysine hydroxylation of both carboxy- and amino-terminal telopeptides of an α1(I) chain were significantly diminished resulting in reduction of the hydroxylysine-aldehyde derived cross-links. The collagen fibrils in bsLH2-cKO appeared to be thicker, often fused and irregular when compared to controls. In addition, bone mineral density and mechanical properties of bsLH2-cKO femurs were significantly impaired. Taken together, these data demonstrate that LH2-catalyzed modification and consequent cross-linking of collagen are critical for proper bone formation and mechanical strength.

The effects of age and dysfunction on meibomian gland population dynamics.

PURPOSE: While meibomian gland dysfunction (MGD) is widely recognized as a major cause of evaporative dry eye disease, little is known about normal gland differentiation and lipid synthesis or the mechanism underlying gland atrophy and abnormal lipid secretion. The purpose of this study was to use single-cell and spatial transcriptomics to probe changes in cell composition, differentiation, and gene expression associated with two murine models of MGD: age-related gland atrophy in wild-type mice and altered meibum quality in acyl-CoA wax alcohol acyltransferase 2 (Awat2) knockout (KO) mice. METHODS: Young (6 month) and old (22 month) wild type, C57Bl/6 mice and young (3 month) and old (13 month) Awat2 KO mice were used in these studies. For single-cell analysis, the tarsal plate was dissected from the upper and lower eyelids, and single cells isolated and submitted to the UCI Genomic Core, while for the spatial analysis frozen tissue sections were shipped to Resolve Biosciences on dry ice and sections probed in duplicate using a meibomian gland specific, 100 gene Molecular Chartography panel. RESULTS: Analysis of gene expression patterns identified the stratified expression of lipogenic genes during meibocyte differentiation, which may control the progressive synthesis of meibum lipids; an age-related decrease in meibocytes; and increased immune cell infiltration. Additionally, we detected unique immune cell populations in the Awat2 KO mouse suggesting activation of psoriasis-like, inflammatory pathways perhaps caused by ductal dilation and hyperplasia. CONCLUSION: Together these findings support novel mechanism controlling gland function and dysfunction.

Serotonin transporter knockout in rats reduces beta- and gamma-band functional connectivity between the orbitofrontal cortex and amygdala during auditory discrimination.

The orbitofrontal cortex and amygdala collaborate in outcome-guided decision-making through reciprocal projections. While serotonin transporter knockout (SERT-/-) rodents show changes in outcome-guided decision-making, and in orbitofrontal cortex and amygdala neuronal activity, it remains unclear whether SERT genotype modulates orbitofrontal cortex-amygdala synchronization. We trained SERT-/- and SERT+/+ male rats to execute a task requiring to discriminate between two auditory stimuli, one predictive of a reward (CS+) and the other not (CS-), by responding through nose pokes in opposite-side ports. Overall, task acquisition was not influenced by genotype. Next, we simultaneously recorded local field potentials in the orbitofrontal cortex and amygdala of both hemispheres while the rats performed the task. Behaviorally, SERT-/- rats showed a nonsignificant trend for more accurate responses to the CS-. Electrophysiologically, orbitofrontal cortex-amygdala synchronization in the beta and gamma frequency bands during response selection was significantly reduced and associated with decreased hubness and clustering coefficient in both regions in SERT-/- rats compared to SERT+/+ rats. Conversely, theta synchronization at the time of behavioral response in the port associated with reward was similar in both genotypes. Together, our findings reveal the modulation by SERT genotype of the orbitofrontal cortex-amygdala functional connectivity during an auditory discrimination task.

Efficient and rapid one-step method to generate gene deletions in Streptococcus pyogenes.

Streptococcus pyogenes or Group A Streptococcus (GAS) remains a significant infectious problem around the world, particularly in low- and middle-income settings. Moreover, a recent invasive GAS infection (iGAS) upsurge has been observed in high-income settings. However, to date, no vaccine is available. Finding a good vaccine antigen and understanding the role of virulence factors in GAS infections have been hampered, in part, by technical difficulties to transform the many different strains and generate knockout mutants. Using colE1-type plasmid as a suicide vector, we have set up a method allowing the generation of non-polar mutants of GAS in 3 days. IMPORTANCE: Group A Streptococcus (GAS) is a major human pathogen, causing diseases ranging from mild and superficial infections of the skin and pharyngeal epithelium to severe systemic and invasive diseases. Since June 2022, several European countries, the US, and Australia are facing an upsurge of invasive life-threatening GAS infections. Finding a good vaccine antigen and understanding the role of virulence factors in GAS infections have been hampered, in part, by technical difficulties to transform the many different GAS strains and generate knockout mutants. Moreover, these tools must be adapted to a large range of different strains, since GAS are divided into more than 260 emm-types (M-type). We have set up a method allowing the generation of non-polar mutants of GAS in 3 days and in diverse backgrounds, which contrasts with previously published protocols.

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

FDX2, an iron-sulfur cluster assembly factor, is essential to prevent cellular senescence, apoptosis or ferroptosis of ovarian cancer cells.

Recent studies reveal that biosynthesis of iron-sulfur clusters (Fe-Ss) is essential for cell proliferation, including that of cancer cells. Nonetheless, it remains unclear how Fe-S biosynthesis functions in cell proliferation/survival. Here, we report that proper Fe-S biosynthesis is essential to prevent cellular senescence, apoptosis or ferroptosis, depending on cell context. To assess these outcomes in cancer, we developed an ovarian cancer line with conditional KO of FDX2, a component of the core Fe-S assembly complex. FDX2 loss induced global down-regulation of Fe-S-containing proteins and Fe2+ overload, resulting in DNA damage and p53 pathway activation, and driving the senescence program. p53-deficiency augmented DNA damage responses upon FDX2 loss, resulting in apoptosis rather than senescence. FDX2 loss also sensitized cells to ferroptosis, as evidenced by compromised redox homeostasis of membrane phospholipids (PLs). Our results suggest that p53 status and PL homeostatic activity are critical determinants of diverse biological outcomes of Fe-S deficiency in cancer cells.

Generation of an Armcx1 Conditional Knockout Mouse.

Armadillo repeat-containing X-linked protein-1 (Armcx1) is a poorly characterized transmembrane protein that regulates mitochondrial transport in neurons. Its overexpression has been shown to induce neurite outgrowth in embryonic neurons and to promote retinal ganglion cell (RGC) survival and axonal regrowth in a mouse optic nerve crush model. In order to evaluate the functions of endogenous Armcx1 in vivo, we have created a conditional Armcx1 knockout mouse line in which the entire coding region of the Armcx1 gene is flanked by loxP sites. This Armcx1fl line was crossed with mouse strains in which Cre recombinase expression is driven by the promoters for ß-actin and Six3, in order to achieve deletion of Armcx1 globally and in retinal neurons, respectively. Having confirmed deletion of the gene, we proceeded to characterize the abundance and morphology of RGCs in Armcx1 knockout mice aged to 15 months. Under normal physiological conditions, no evidence of aberrant retinal or optic nerve development or RGC degeneration was observed in these mice. The Armcx1fl mouse should be valuable for future studies investigating mitochondrial morphology and transport in the absence of Armcx1 and in determining the susceptibility of Armcx1-deficient neurons to degeneration in the setting of additional heritable or environmental stressors.

Lowering an ER stress-regulated long noncoding RNA protects mice from diabetes and isolated pancreatic islets from cell death.

We investigated the role of the endoplasmic reticulum (ER) stress-regulated long noncoding RNA (lncRNA) lncMGC in pancreatic islets and the pathology of type 1 diabetes (T1D), as well as the potential of lncMGC-based therapeutics. In vivo, blood glucose levels (BGLs) and HbA1c were significantly lower in lncMGC-knockout (KO)-streptozotocin (STZ)-treated diabetic mice compared to wild-type STZ. Antisense oligonucleotides (GapmeR) targeting lncMGC significantly attenuated insulitis and BGLs in T1D NOD mice compared to GapmeR-negative control (NC). GapmeR-injected T1D Akita mice showed significantly lower BGLs compared to Akita-NC mice. hlncMGC-GapmeR lowered BGLs in partially humanized lncMGC (hlncMGC)-STZ mice compared to NC-injected mice. CHOP (ER stress regulating transcription factor) and lncMGC were upregulated in islets from diabetic mice but not in lncMGC-KO and GapmeR-injected diabetic mice, suggesting ER stress involvement. In vitro, hlncMGC-GapmeR increased the viability of isolated islets from human donors and hlncMGC mice and protected them from cytokine-induced apoptosis. Anti-ER stress and anti-apoptotic genes were upregulated, but pro-apoptotic genes were down-regulated in lncMGC KO mice islets and GapmeR-treated human islets. Taken together, these results show that a GapmeR-targeting lncMGC is effective in ameliorating diabetes in mice and also preserves human and mouse islet viability, implicating clinical translation potential.

Carnitine O-octanoyltransferase (CROT) deficiency in mice leads to an increase of omega-3 fatty acids.

Introduction: Carnitine O-octanoyltransferase (CROT) is a well-established peroxisomal enzyme involved in liver fatty acid oxidation, but less is known about its recently discovered role in promoting vascular calcification, and whether CROT-dependent liver metabolism contributes to the latter. To date, CROT function in the context of calcification potential has been conducted in the dyslipidemic low-density lipoprotein receptor-deficient (Ldlr-/-) mice. Objectives: To differentiate peroxisome and CROT-dependent lipid biology from that of lipoprotein-mediated lipid biology, we therefore conducted a metabolomic analysis of the liver and plasma of normolipidemic CROT-deficient (Crot-/-) mice. Methods: We performed LC-MS-based metabolomics on liver and plasma derived from Crot-/- and Crot +/- mice and sibling Crot+/+ mice, using a dual-phase metabolite extraction protocol, and multiple LC-MS acquisition strategies. Results: We identified between 79 to 453 annotated metabolites from annotated metabolites from liver samples, and 117 to 424 annotated metabolites from plasma samples. Through differential abundance analysis, we determined that omega-3 fatty acids such as EPA, DPA, and DHA were higher in the liver of Crot-/- and Crot +/- mice than Crot+/+ mice. EPA were higher in plasma of Crot-/- mice than Crot+/+ mice. We also determined that the anti-inflammatory dicarboxylic acids, tetradecanedioic acid and azelaic acid, were higher in the plasma of CROT-deficient mice. Conclusion: Our study associated genetic CROT deletion with increased levels of anti-inflammatory molecules in mouse liver and plasma. These results suggest a potential mechanism for anti-calcification effects of CROT suppression and the potential use of omega-3 fatty acids as biomarkers for future CROT inhibition therapies.