14-3-3 binding motif phosphorylation disrupts Hdac4-organized condensates to stimulate cardiac reprogramming.

Cell fate conversion is associated with extensive post-translational modifications (PTMs) and architectural changes of sub-organelles, yet how these events are interconnected remains unknown. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 is identified in pivotal functional proteins for iCM reprogramming, including transcription factors and chromatin modifiers. Akt1 kinase and protein phosphatase 2A are the key writer and key eraser of the PC14-3-3 code, respectively. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolishes reprogramming. We discover that key PC14-3-3-embedded factors, such as histone deacetylase 4 (Hdac4), Mef2c, and Foxo1, form Hdac4-organized inhibitory nuclear condensates. PC14-3-3 activation disrupts Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a PTM code could be a general mechanism for stimulating cell reprogramming.

Hepatic danger signaling triggers TREM2+ macrophage induction and drives steatohepatitis via MS4A7-dependent inflammasome activation.

Metabolic dysfunction-associated steatohepatitis (MASH), formerly known as nonalcoholic steatohepatitis (NASH), is an advanced stage of metabolic fatty liver disease. The pathogenic mechanisms of MASH center on hepatocyte injury and the ensuing immune response within the liver microenvironment. Recent work has implicated TREM2+ macrophages in various disease conditions, and substantial induction of TREM2+ NASH-associated macrophages (NAMs) serves as a hallmark of metabolic liver disease. Despite this, the mechanisms through which NAMs contribute to MASH pathogenesis remain poorly understood. Here, we identify membrane-spanning 4-domains a7 (MS4A7) as a NAM-specific pathogenic factor that exacerbates MASH progression in mice. Hepatic MS4A7 expression was strongly induced in mouse and human MASH and associated with the severity of liver injury. Whole-body and myeloid-specific ablation of Ms4a7 alleviated diet-induced MASH pathologies in male mice. We demonstrate that exposure to lipid droplets (LDs), released upon injury of steatotic hepatocytes, triggered NAM induction and exacerbated MASH-associated liver injury in an MS4A7-dependent manner. Mechanistically, MS4A7 drove NLRP3 inflammasome activation via direct physical interaction and shaped disease-associated cell states within the liver microenvironment. This work reveals the LD-MS4A7-NLRP3 inflammasome axis as a pathogenic driver of MASH progression and provides insights into the role of TREM2+ macrophages in disease pathogenesis.

Sotagliflozin attenuates liver-associated disorders in cystic fibrosis rabbits.

Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene lead to CF, a life-threating autosomal recessive genetic disease. While recently approved Trikafta dramatically ameliorates CF lung diseases, there is still a lack of effective medicine to treat CF-associated liver disease (CFLD). To address this medical need, we used a recently established CF rabbit model to test whether sotagliflozin, a sodium-glucose cotransporter 1 and 2 (SGLT1/2) inhibitor drug that is approved to treat diabetes, can be repurposed to treat CFLD. Sotagliflozin treatment led to systemic benefits to CF rabbits, evidenced by increased appetite and weight gain as well as prolonged lifespan. For CF liver-related phenotypes, the animals benefited from normalized blood chemistry and bile acid parameters. Furthermore, sotagliflozin alleviated nonalcoholic steatohepatitis-like phenotypes, including liver fibrosis. Intriguingly, sotagliflozin treatment markedly reduced the otherwise elevated endoplasmic reticulum stress responses in the liver and other affected organs of CF rabbits. In summary, our work demonstrates that sotagliflozin attenuates liver disorders in CF rabbits and suggests sotagliflozin as a potential drug to treat CFLD.

XBP1-mediated transcriptional regulation of SLC5A1 in human epithelial cells in disease conditions.

BACKGROUND: Sodium-Glucose cotransporter 1 and 2 (SGLT1/2) belong to the family of glucose transporters, encoded by SLC5A1 and SLC5A2, respectively. SGLT2 is almost exclusively expressed in the renal proximal convoluted tubule cells. SGLT1 is expressed in the kidneys but also in other organs throughout the body. Many SGLT inhibitor drugs have been developed based on the mechanism of blocking glucose (re)absorption mediated by SGLT1/2, and several have gained major regulatory agencies' approval for treating diabetes. Intriguingly these drugs are also effective in treating diseases beyond diabetes, for example heart failure and chronic kidney disease. We recently discovered that SGLT1 is upregulated in the airway epithelial cells derived from patients of cystic fibrosis (CF), a devastating genetic disease affecting greater than 70,000 worldwide. RESULTS: In the present work, we show that the SGLT1 upregulation is coupled with elevated endoplasmic reticulum (ER) stress response, indicated by activation of the primary ER stress senor inositol-requiring protein 1α (IRE1α) and the ER stress-induced transcription factor X-box binding protein 1 (XBP1), in CF epithelial cells, and in epithelial cells of other stress conditions. Through biochemistry experiments, we demonstrated that the spliced form of XBP1 (XBP1s) acts as a transcription factor for SLC5A1 by directly binding to its promoter region. Targeting this ER stress → SLC5A1 axis by either the ER stress inhibitor Rapamycin or the SGLT1 inhibitor Sotagliflozin was effective in attenuating the ER stress response and reducing the SGLT1 level in these cellular model systems. CONCLUSIONS: The present work establishes a causal relationship between ER stress and SGLT1 upregulation and provides a mechanistic explanation why SGLT inhibitor drugs benefit diseases beyond diabetes.

Serine synthesis via reversed SHMT2 activity drives glycine depletion and acetaminophen hepatotoxicity in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects one-third of the global population. Understanding the metabolic pathways involved can provide insights into disease progression and treatment. Untargeted metabolomics of livers from mice with early-stage steatosis uncovered decreased methylated metabolites, suggesting altered one-carbon metabolism. The levels of glycine, a central component of one-carbon metabolism, were lower in mice with hepatic steatosis, consistent with clinical evidence. Stable-isotope tracing demonstrated that increased serine synthesis from glycine via reverse serine hydroxymethyltransferase (SHMT) is the underlying cause for decreased glycine in steatotic livers. Consequently, limited glycine availability in steatotic livers impaired glutathione synthesis under acetaminophen-induced oxidative stress, enhancing acute hepatotoxicity. Glycine supplementation or hepatocyte-specific ablation of the mitochondrial SHMT2 isoform in mice with hepatic steatosis mitigated acetaminophen-induced hepatotoxicity by supporting de novo glutathione synthesis. Thus, early metabolic changes in MASLD that limit glycine availability sensitize mice to xenobiotics even at the reversible stage of this disease.

SWI/SNF Complex in Vascular Smooth Muscle Cells and Its Implications in Cardiovascular Pathologies.

Mature vascular smooth muscle cells (VSMC) exhibit a remarkable degree of plasticity, a characteristic that has intrigued cardiovascular researchers for decades. Recently, it has become increasingly evident that the chromatin remodeler SWItch/Sucrose Non-Fermentable (SWI/SNF) complex plays a pivotal role in orchestrating chromatin conformation, which is critical for gene regulation. In this review, we provide a summary of research related to the involvement of the SWI/SNF complexes in VSMC and cardiovascular diseases (CVD), integrating these discoveries into the current landscape of epigenetic and transcriptional regulation in VSMC. These novel discoveries shed light on our understanding of VSMC biology and pave the way for developing innovative therapeutic strategies in CVD treatment.

14-3-3 binding motif phosphorylation disrupts Hdac4 organized condensates to stimulate cardiac reprogramming.

Cell fate conversion is associated with extensive epigenetic and post translational modifications (PTMs) and architectural changes of sub-organelles and organelles, yet how these events are interconnected remains unknown. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 was identified in pivotal functional proteins for iCM reprogramming, including transcription factors and epigenetic factors. Akt1 kinase and PP2A phosphatase were a key writer and eraser of the PC14-3-3 code, respectively. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolished reprogramming. We discovered that key PC14-3-3 embedded factors, such as Hdac4, Mef2c, Nrip1, and Foxo1, formed Hdac4 organized inhibitory nuclear condensates. Notably, PC14-3-3 activation disrupted Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a post-translational modification code could be a general mechanism for stimulating cell reprogramming and organ regeneration. Highlights: A PC14-3-3 (phosphorylation code in 14-3-3 binding motifs) is identified in pivotal functional proteins, such as HDAC4 and Mef2c, that stimulates iCM formation.Akt1 kinase and PP2A phosphatase are a key writer and a key eraser of the PC14-3-3 code, respectively, and PC14-3-3 code activation can replace Mef2c and Gata4 in cardiac reprogramming.PC14-3-3 activation disrupts Hdac4 organized condensates which results in releasing multiple 14-3-3 motif embedded proteins from the condensates to stimulate cardiac reprogramming.Sub-organelle dynamics and function regulated by a post-translational modification code could be a general mechanism in stimulating cell reprogramming and organ regeneration.

ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells.

BACKGROUND: In eukaryotic cells, DNA double strand breaks (DSB) are primarily repaired by canonical non-homologous end joining (c-NHEJ), homologous recombination (HR) and alternative NHEJ (alt-NHEJ). Zinc finger and SCAN domain containing 4 (ZSCAN4), sporadically expressed in 1-5% mouse embryonic stem cells (mESCs), is known to regulate genome stability by promoting HR. RESULTS: Here we show that ZSCAN4 promotes DNA repair by acting with Poly (ADP-ribose) polymerase 1 (PARP1), which is a key member of the alt-NHEJ pathway. In the presence of PARP1, ZSCAN4-expressing mESCs are associated with lower extent of endogenous or chemical induced DSB comparing to ZSCAN4-negative ones. Reduced DSBs associated with ZSCAN4 are abolished by PARP1 inhibition, achieved either through small molecule inhibitor or gene knockout in mESCs. Furthermore, PARP1 binds directly to ZSCAN4, and the second ⍺-helix and the fourth zinc finger motif of ZSCAN4 are critical for this binding. CONCLUSIONS: These data reveal that PARP1 and ZSCAN4 have a protein-protein interaction, and shed light on the molecular mechanisms by which ZSCAN4 reduces DSB in mESCs.

Single-Molecule Spatial Transcriptomics of Human Thoracic Aortic Aneurysms Uncovers Calcification-Related CARTPT-Expressing Smooth Muscle Cells.

BACKGROUND: Although single-cell RNA-sequencing is commonly applied to dissect the heterogeneity in human tissues, it involves the preparation of single-cell suspensions via cell dissociation, causing loss of spatial information. In this study, we employed high-resolution single-cell transcriptome imaging to reveal rare smooth muscle cell (SMC) types in human thoracic aortic aneurysm (TAA) tissue samples. METHODS: Single-molecule spatial distribution of transcripts from 140 genes was analyzed in fresh-frozen human TAA samples with region and sex-matched controls. In vitro studies and tissue staining were performed to examine human CART prepropeptide (CARTPT) regulation and function. RESULTS: We captured thousands of cells per sample including a spatially distinct CARTPT-expressing SMC subtype enriched in male TAA samples. Immunoassays confirmed human CART (cocaine- and amphetamine-regulated transcript) protein enrichment in male TAA tissue and truncated CARTPT secretion into cell culture medium. Oxidized low-density lipoprotein, a cardiovascular risk factor, induced CARTPT expression, whereas CARTPT overexpression in human aortic SMCs increased the expression of key osteochondrogenic transcription factors and reduced contractile gene expression. Recombinant human CART treatment of human SMCs further confirmed this phenotype. Alizarin red staining revealed calcium deposition in male TAA samples showing similar localization with human CART staining. CONCLUSIONS: Here, we demonstrate the feasibility of single-molecule imaging in uncovering rare SMC subtypes in the diseased human aorta, a difficult tissue to dissociate. We identified a spatially distinct CARTPT-expressing SMC subtype enriched in male human TAA samples. Our functional studies suggest that human CART promotes osteochondrogenic switch of aortic SMCs, potentially leading to medial calcification of the thoracic aorta.

Myeloid BAF60a deficiency alters metabolic homeostasis and exacerbates atherosclerosis.

Atherosclerosis, a leading health concern, stems from the dynamic involvement of immune cells in vascular plaques. Despite its significance, the interplay between chromatin remodeling and transcriptional regulation in plaque macrophages is understudied. We discovered the reduced expression of Baf60a, a component of the switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex, in macrophages from advanced plaques. Myeloid-specific Baf60a deletion compromised mitochondrial integrity and heightened adhesion, apoptosis, and plaque development. BAF60a preserves mitochondrial energy homeostasis under pro-atherogenic stimuli by retaining nuclear respiratory factor 1 (NRF1) accessibility at critical genes. Overexpression of BAF60a rescued mitochondrial dysfunction in an NRF1-dependent manner. This study illuminates the BAF60a-NRF1 axis as a mitochondrial function modulator in atherosclerosis, proposing the rejuvenation of perturbed chromatin remodeling machinery as a potential therapeutic target.

NAD+ rescues aging-induced blood-brain barrier damage via the CX43-PARP1 axis.

Blood-brain barrier (BBB) function deteriorates during aging, contributing to cognitive impairment and neurodegeneration. It is unclear what drives BBB leakage in aging and how it can be prevented. Using single-nucleus transcriptomics, we identified decreased connexin 43 (CX43) expression in cadherin-5+ (Cdh5+) cerebral vascular cells in naturally aging mice and confirmed it in human brain samples. Global or Cdh5+ cell-specific CX43 deletion in mice exacerbated BBB dysfunction during aging. The CX43-dependent effect was not due to its canonical gap junction function but was associated with reduced NAD+ levels and mitochondrial dysfunction through NAD+-dependent sirtuin 3 (SIRT3). CX43 interacts with and negatively regulates poly(ADP-ribose) polymerase 1 (PARP1). Pharmacologic inhibition of PARP1 by olaparib or nicotinamide mononucleotide (NMN) supplementation rescued NAD+ levels and alleviated aging-associated BBB leakage. These findings establish the endothelial CX43-PARP1-NAD+ pathway's role in vascular aging and identify a potential therapeutic strategy to combat aging-associated BBB leakage with neuroprotective implications.

XBP1-mediated transcriptional regulation of SLC5A1 in human epithelial cells in disease conditions.

Background: sodium-dependent glucose cotransporter 1 and 2 (SGLT1/2) belong to the family of glucose transporters, encoded by SLC5A1 and SLC5A2, respectively. SGLT-2 is almost exclusively expressed in the renal proximal convoluted tubule cells. SGLT-1 is expressed in the kidneys but also in other organs throughout the body. Many SGLT inhibitor drugs have been developed based on the mechanism of blocking glucose (re)absorption mediated by SGLT1/2, and several have gained major regulatory agencies' approval for treating diabetes. Intriguingly these drugs are also effective in treating diseases beyond diabetes, for example heart failure and chronic kidney disease. We recently discovered that SGLT-1 is upregulated in the airway epithelial cells derived from patients of cystic fibrosis (CF), a devastating genetic disease affecting greater than 70,000 worldwide. Results: in the present work, we show that the SGLT-1 upregulation is coupled with elevated endoplasmic reticulum (ER) stress response, indicated by activation of the primary ER stress senor inositol-requiring protein 1a (IRE1a) and the ER stress-induced transcription factor X-box binding protein 1 (XBP1), in CF epithelial cells, and in epithelial cells of other stress conditions. Through biochemistry experiments, we demonstrated that XBP1 acts as a transcription factor for SLC5A1 by directly binding to its promoter region. Targeting this ER stress → SLC5A1 axis by either the ER stress inhibitor Rapamycin or the SGLT-1 inhibitor Sotagliflozin was effective in attenuating the ER stress response and reducing the SGLT-1 levels in these cellular model systems. Conclusions: the present work establishes a causal relationship between ER stress and SGLT-1 upregulation and provides a mechanistic explanation why SGLT inhibitor drugs benefit diseases beyond diabetes.

Computational redesign of cytochrome P450 CYP102A1 for highly stereoselective omeprazole hydroxylation by UniDesign.

Cytochrome P450 CYP102A1 is a prototypic biocatalyst that has great potential in chemical synthesis, drug discovery, and biotechnology. CYP102A1 variants engineered by directed evolution and/or rational design are capable of catalyzing the oxidation of a wide range of organic compounds. However, it is difficult to foresee the outcome of engineering CYP102A1 for a compound of interest. Here, we introduce UniDesign as a computational framework for enzyme design and engineering. We tested UniDesign by redesigning CYP102A1 for stereoselective metabolism of omeprazole (OMP), a proton pump inhibitor, starting from an active but nonstereoselective triple mutant (TM: A82F/F87V/L188Q). To shift stereoselectivity toward (R)-OMP, we computationally scanned three active site positions (75, 264, and 328) for mutations that would stabilize the binding of the transition state of (R)-OMP while destabilizing that of (S)-OMP and picked three variants, namely UD1 (TM/L75I), UD2 (TM/A264G), and UD3 (TM/A328V), for experimentation, based on computed energy scores and models. UD1, UD2, and UD3 exhibit high turnover rates of 55 ± 4.7, 84 ± 4.8, and 79 ± 5.7 min-1, respectively, for (R)-OMP hydroxylation, whereas the corresponding rates for (S)-OMP are only 2.2 ± 0.19, 6.0 ± 0.68, and 14 ± 2.8 min-1, yielding an enantiomeric excess value of 92, 87, and 70%, respectively. These results suggest the critical roles of L75I, A264G, and A328V in steering OMP in the optimal orientation for stereoselective oxidation and demonstrate the utility of UniDesign for engineering CYP102A1 to produce drug metabolites of interest. The results are discussed in the context of protein structures.

Generating endogenous Myh11-driven Cre mice for sex-independent gene deletion in smooth muscle cells.

Specific and efficient smooth muscle cell-targeted (SMC-targeted) gene deletion is typically achieved by pairing SMMHC-CreERT2-Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreERT2, is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreERT2 exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing bacterial artificial chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreERT2-Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11-driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the CreNLSP2A or CreERT2-P2A sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre-knockin mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive (Myh11-CreNLSP2A) and inducible (Myh11-CreERT2-P2A) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11-CreERT2-RAD mice and the Itga8-CreERT2 mouse models, our models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.

Liver-specific in vivo base editing of Angptl3 via AAV delivery efficiently lowers blood lipid levels in mice.

BACKGROUND: Gene editing has emerged as an exciting therapeutic development platform for numerous genetic and nongenetic diseases. Targeting lipid-modulating genes such as angiopoietin-related protein 3 (ANGPTL3) with gene editing offers hope for a permanent solution to lower cardiovascular disease risks associated with hypercholesterolemia. RESULTS: In this study, we developed a hepatocyte-specific base editing therapeutic approach delivered by dual adeno-associated virus (AAV) to enable hepatocyte-specific targeting of Angptl3 to lower blood lipid levels. Systemic AAV9-mediated delivery of AncBE4max, a cytosine base editor (CBE), targeting mouse Angptl3 resulted in the installation of a premature stop codon in Angptl3 with an average efficiency of 63.3 ± 2.3% in the bulk liver tissue. A near-complete knockout of the ANGPTL3 protein in the circulation were observed within 2-4 weeks following AAV administration. Furthermore, the serum levels of triglyceride (TG) and total cholesterol (TC) were decreased by approximately 58% and 61%, respectively, at 4 weeks after treatment. CONCLUSIONS: These results highlight the promise of liver-targeted Angptl3 base editing for blood lipid control.

A ubiquitination-mediated degradation system to target 14-3-3-binding phosphoproteins.

The phosphorylation of 14-3-3 binding motif is involved in many cellular processes. A strategy that enables targeted degradation of 14-3-3-binding phosphoproteins (14-3-3-BPPs) for studying their functions is highly desirable for basic research. Here, we report a phosphorylation-induced, ubiquitin-proteasome-system-mediated targeted protein degradation (TPD) strategy that allows specific degradation of 14-3-3-BPPs. Specifically, by ligating a modified von Hippel-Lindau E3-ligase with an engineered 14-3-3 bait, we generated a protein chimera referred to as Targeted Degradation of 14-3-3-binding PhosphoProtein (TDPP). TDPP can serve as a universal degrader for 14-3-3-BPPs based on the specific recognition of the phosphorylation in 14-3-3 binding motifs. TDPP shows high efficiency and specificity to a difopein-EGFP reporter, general and specific 14-3-3-BPPs. TDPP can also be applied for the validation of 14-3-3-BPPs. These results strongly support TDPP as a powerful tool for 14-3-3 related research.

Recent progress in polymeric gene vectors: Delivery mechanisms, molecular designs, and applications.

Gene therapy and gene delivery have drawn extensive attention in recent years especially when the COVID-19 mRNA vaccines were developed to prevent severe symptoms caused by the corona virus. Delivering genes, such as DNA and RNA into cells, is the crucial step for successful gene therapy and remains a bottleneck. To address this issue, vehicles (vectors) that can load and deliver genes into cells are developed, including viral and non-viral vectors. Although viral gene vectors have considerable transfection efficiency and lipid-based gene vectors become popular since the application of COVID-19 vaccines, their potential issues including immunologic and biological safety concerns limited their applications. Alternatively, polymeric gene vectors are safer, cheaper, and more versatile compared to viral and lipid-based vectors. In recent years, various polymeric gene vectors with well-designed molecules were developed, achieving either high transfection efficiency or showing advantages in certain applications. In this review, we summarize the recent progress in polymeric gene vectors including the transfection mechanisms, molecular designs, and biomedical applications. Commercially available polymeric gene vectors/reagents are also introduced. Researchers in this field have never stopped seeking safe and efficient polymeric gene vectors via rational molecular designs and biomedical evaluations. The achievements in recent years have significantly accelerated the progress of polymeric gene vectors toward clinical applications.

PRDM16 deficiency in vascular smooth muscle cells aggravates abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is usually asymptomatic until life-threatening complications occur, predominantly involving aortic rupture. Currently, no drug-based treatments are available, primarily due to limited understanding of AAA pathogenesis. The transcriptional regulator PR domain-containing protein 16 (PRDM16) is highly expressed in the aorta, but its functions in the aorta are largely unknown. By RNA-seq analysis, we found that vascular smooth muscle cell-specific (VSMC-specific) Prdm16-knockout (Prdm16SMKO) mice already showed extensive changes in the expression of genes associated with extracellular matrix (ECM) remodeling and inflammation in the abdominal aorta under normal housing conditions without any pathological stimuli. Human AAA lesions displayed lower PRDM16 expression. Periadventitial elastase application to the suprarenal region of the abdominal aorta aggravated AAA formation in Prdm16SMKO mice. During AAA development, VSMCs undergo apoptosis because of both intrinsic and environmental changes, including inflammation and ECM remodeling. Prdm16 deficiency promoted inflammation and apoptosis in VSMCs. A disintegrin and metalloproteinase 12 (ADAM12) is a gelatinase that can degrade various ECMs. We found that ADAM12 is a target of transcriptional repression by PRDM16. Adam12 knockdown reversed VSMC apoptosis induced by Prdm16 deficiency. Our study demonstrated that PRDM16 deficiency in VSMCs promoted ADAM12 expression and aggravates AAA formation, which may provide potential targets for AAA treatment.

DT-109 ameliorates nonalcoholic steatohepatitis in nonhuman primates.

Nonalcoholic steatohepatitis (NASH) prevalence is rising with no pharmacotherapy approved. A major hurdle in NASH drug development is the poor translatability of preclinical studies to safe/effective clinical outcomes, and recent failures highlight a need to identify new targetable pathways. Dysregulated glycine metabolism has emerged as a causative factor and therapeutic target in NASH. Here, we report that the tripeptide DT-109 (Gly-Gly-Leu) dose-dependently attenuates steatohepatitis and fibrosis in mice. To enhance the probability of successful translation, we developed a nonhuman primate model that histologically and transcriptionally mimics human NASH. Applying a multiomics approach combining transcriptomics, proteomics, metabolomics, and metagenomics, we found that DT-109 reverses hepatic steatosis and prevents fibrosis progression in nonhuman primates, not only by stimulating fatty acid degradation and glutathione formation, as found in mice, but also by modulating microbial bile acid metabolism. Our studies describe a highly translatable NASH model and highlight the need for clinical evaluation of DT-109.

Generation of Rabbit Chimeras by Eight-Cell Stage Embryo Injection.

Pluripotent stem cell (PSC) injection to the blastocyst stage embryos is a widely used method to evaluate the pluripotency through chimeric contribution. It is routinely used to produce transgenic mice. However, PSC injection to the blastocyst stage embryos in rabbits is challenging. At this stage, the in vivo developed rabbit blastocysts possess a thick mucin layer that is inhibitory for microinjection, whereas in vitro developed rabbit blastocysts that lack such mucin layer often fail to implant after embryo transfer. In this chapter, we describe a detailed protocol of rabbit chimera production through mucin-free eight-cell stage embryo injection procedure.