Mechanistic insights into DNA binding and cleavage by a compact type I-F CRISPR-Cas system in bacteriophage.

CRISPR-Cas systems are widespread adaptive antiviral systems used in prokaryotes. Some phages, in turn, although have small genomes can economize the use of genetic space to encode compact or incomplete CRISPR-Cas systems to inhibit the host and establish infection. Phage ICP1, infecting Vibrio cholerae, encodes a compact type I-F CRISPR-Cas system to suppress the antiphage mobile genetic element in the host genome. However, the mechanism by which this compact system recognizes the target DNA and executes interference remains elusive. Here, we present the electron cryo-microscopy (cryo-EM) structures of both apo- and DNA-bound ICP1 surveillance complexes (Aka Csy complex). Unlike most other type I surveillance complexes, the ICP1 Csy complex lacks the Cas11 subunit or a structurally homologous domain, which is crucial for dsDNA binding and Cas3 activation in other type I CRISPR-Cas systems. Structural and functional analyses revealed that the compact ICP1 Csy complex alone is inefficient in binding to dsDNA targets, presumably stalled at a partial R-loop conformation. The presence of Cas2/3 facilitates dsDNA binding and allows effective dsDNA target cleavage. Additionally, we found that Pseudomonas aeruginosa Cas2/3 efficiently cleaved the dsDNA target presented by the ICP1 Csy complex, but not vice versa. These findings suggest a unique mechanism for target dsDNA binding and cleavage by the compact phage-derived CRISPR-Cas system.

Nuclear GAPDH in cortical microglia mediates cellular stress-induced cognitive inflexibility.

We report a mechanism that underlies stress-induced cognitive inflexibility at the molecular level. In a mouse model under subacute cellular stress in which deficits in rule shifting tasks were elicited, the nuclear glyceraldehyde dehydrogenase (N-GAPDH) cascade was activated specifically in microglia in the prelimbic cortex. The cognitive deficits were normalized with a pharmacological intervention with a compound (the RR compound) that selectively blocked the initiation of N-GAPDH cascade without affecting glycolytic activity. The normalization was also observed with a microglia-specific genetic intervention targeting the N-GAPDH cascade. At the mechanistic levels, the microglial secretion of High-Mobility Group Box (HMGB), which is known to bind with and regulate the NMDA-type glutamate receptors, was elevated. Consequently, the hyperactivation of the prelimbic layer 5 excitatory neurons, a neural substrate for cognitive inflexibility, was also observed. The upregulation of the microglial HMGB signaling and neuronal hyperactivation were normalized by the pharmacological and microglia-specific genetic interventions. Taken together, we show a pivotal role of cortical microglia and microglia-neuron interaction in stress-induced cognitive inflexibility. We underscore the N-GAPDH cascade in microglia, which causally mediates stress-induced cognitive alteration.

Porcine Kidney Organoids Derived from Naïve-like Embryonic Stem Cells.

The scarcity of donor kidneys greatly impacts the survival of patients with end-stage renal failure. Pigs are increasingly becoming potential organ donors but are limited by immunological rejection. Based on the human kidney organoid already established with the CHIR99021 and FGF9 induction strategy, we generated porcine kidney organoids from porcine naïve-like ESCs (nESCs). The derived porcine organoids had a tubule-like constructure and matrix components. The porcine organoids expressed renal markers including AQP1 (proximal tubule), WT1 and PODO (podocyte), and CD31 (vascular endothelial cells). These results imply that the organoids had developed the majority of the renal cell types and structures, including glomeruli and proximal tubules. The porcine organoids were also identified to have a dextran absorptive function. Importantly, porcine organoids have a certain abundance of vascular endothelial cells, which are the basis for investigating immune rejection. The derived porcine organoids might serve as materials for immunosuppressor screening for xenotransplantation.

GPER-dependent estrogen signaling increases cardiac GCN5L1 expression.

Reversible lysine acetylation regulates the activity of cardiac metabolic enzymes, including those controlling fuel substrate metabolism. Mitochondrial-targeted GCN5L1 and SIRT3 have been shown to regulate the acetylation status of mitochondrial enzymes, but the role that lysine acetylation plays in driving metabolic differences between male and female hearts is not currently known. In this study, we describe a significant difference in GCN5L1 levels between male and female mouse hearts, and in the hearts of women between post- and premenopausal age. We further find that estrogen drives GCN5L1 expression in a cardiac cell line and uses pharmacological approaches to determine the mechanism to be G protein-coupled estrogen receptor (GPER) activation, via translational regulation.NEW & NOTEWORTHY We demonstrate here for the first time that mitochondrial protein acetylation is increased in female hearts, associated with an increase in GCN5L1 levels through a GPER-dependent mechanism. These findings reveal a new potential mediator of divergent cardiac mitochondrial function between men and women.

USF3 modulates osteoporosis risk by targeting WNT16, RANKL, RUNX2, and two GWAS lead SNPs rs2908007 and rs4531631.

Osteoporotic fractures cause major morbidity and mortality in the aging population. Genome-wide association studies (GWAS) have identified USF3 as the novel susceptibility gene of osteoporosis. However, the functional role in bone metabolism and the target gene of the basic helix-loop-helix transcription factor USF3 are unclear. Here, we show that USF3 enhances osteoblast differentiation and suppresses osteoclastogenesis in cultured human osteoblast-like U-2OS cells. Mechanistic studies revealed that transcription factor USF3 antagonistically interacts with anti-osteogenic TWIST1/TCF12 heterodimer in the WNT16 and RUNX2 promoter, and counteracts CREB1 and JUN/FOS in the RANKL promoter. Importantly, the osteoporosis GWAS variant g.1744A>G (rs2908007A>G) located in the WNT16 promoter confers G-allele-specific transcriptional modulation by USF3, TWIST1/TCF12 and TBX5/TBX15, and USF3 transactivates the osteoclastogenesis suppressor WNT16 promoter activity and antagonizes the repression of WNT16 by TWIST1 and TCF12. The risk G allele of osteoporosis GWAS variant g.3260A>G (rs4531631A>G) in the RANKL promoter facilitates the binding of CREB1 and JUN/FOS and enhances transactivation of the osteoclastogenesis contributor RANKL that is inhibited by USF3. Our findings uncovered the functional mechanisms of osteoporosis novel GWAS-associated gene USF3 and lead single nucleotide polymorphisms rs2908007 and rs4531631 in the regulation of bone formation and resorption.

Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease.

Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States.

The inner cell mass of the pre-implantation blastocyst consists of the epiblast and hypoblast from which embryonic stem cells (ESCs) and extra-embryonic endoderm (XEN) stem cells, respectively, can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of its in vivo tissue origin. We have developed a novel approach for deriving porcine XEN (pXEN) cells via culturing the blastocysts with a chemical cocktail culture system. The pXEN cells were positive for XEN markers, including Gata4, Gata6, Sox17, and Sall4, but not for pluripotent markers Oct4, Sox2, and Nanog. The pXEN cells also retained the ability to undergo visceral endoderm (VE) and parietal endoderm (PE) differentiation in vitro. The maintenance of pXEN required FGF/MEK+TGFß signaling pathways. The pXEN cells showed a stable phenotype through more than 50 passages in culture and could be established repeatedly from blastocysts or converted from the naïve-like ESCs established in our lab. These cells provide a new tool for exploring the pathways of porcine embryo development and differentiation and providing further reference to the establishment of porcine ESCs with potency of germline chimerism and gamete development.

CaMKII exacerbates heart failure progression by activating class I HDACs.

BACKGROUND: Persistent cardiac Ca2+/calmodulin dependent Kinase II (CaMKII) activation plays an essential role in heart failure development. However, the molecular mechanisms underlying CaMKII induced heart failure progression remains incompletely understood. Histone deacetylases (HDACs) are critical for transcriptional responses to stress, and contribute to expression of pathological genes causing adverse ventricular remodeling. Class I HDACs, including HDAC1, HDAC2 and HDAC3, promote pathological cardiac hypertrophy, whereas class IIa HDACs suppress cardiac hypertrophy. While it is known that CaMKII deactivates class IIa HDACs to enhance cardiac hypertrophy, the role of CaMKII in regulating class I HDACs during heart failure progression is unclear. METHODS AND RESULTS: CaMKII increases the deacetylase activity of recombinant HDAC1, HDAC2 and HDAC3 via in vitro phosphorylation assays. Phosphorylation sites on HDAC1 and HDAC3 are identified with mass spectrometry. HDAC1 activity is also increased in cardiac-specific CaMKIIδC transgenic mice (CaMKIIδC-tg). Beyond post-translational modifications, CaMKII induces HDAC1 and HDAC3 expression. HDAC1 and HDAC3 expression are significantly increased in CaMKIIδC-tg mice. Inhibition of CaMKII by overexpression of the inhibitory peptide AC3-I in the heart attenuates the upregulation of HDAC1 after myocardial infarction surgery. Importantly, a potent HDAC1 inhibitor Quisinostat improves downregulated autophagy genes and cardiac dysfunction in CaMKIIδC-tg mice. In addition to Quisinostat, selective class I HDACs inhibitors, Apicidin and Entinostat, HDAC3 specific inhibitor RGFP966, as well as HDAC1 and HDAC3 siRNA prevent CaMKII overexpression induced cardiac myocyte hypertrophy. CONCLUSION: CaMKII activates class I HDACs in heart failure, which may be a central mechanism for heart failure progression. Selective class I HDACs inhibition may be a novel therapeutic avenue to alleviate CaMKII hyperactivity induced cardiac dysfunction.

BOLA (BolA Family Member 3) Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension.

BACKGROUND: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. METHODS: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. RESULTS: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. CONCLUSIONS: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development.

Computational and functional analyses of T2D GWAS SNPs for transcription factor binding.

Genome-wide association studies (GWASs) have successfully identified numerous non-coding genetic variants for type 2 diabetes (T2D), but the functional roles underlying these non-coding variants remain largely unknown. The effects of T2D GWAS lead SNPs on transcriptional factors binding motifs were firstly analyzed via JASPAR, followed by functional validations including dual-luciferase reporter assays, biotin-based DNA pull-down assays, real-time quantitative PCR, and western blotting. The results showed that GWAS SNP rs4430796 conferred T allele specific transcriptional enhancer activity via a PAX6 binding element, and upregulated the expression of HNF1B. GWAS SNP rs4607103 showed a bidirectional modulation of ADAMTS9-AS2 and ADAMTS9 by TCF7L2 in a T allele-specific manner. GWAS SNP rs849135 conferred C allele-specific bidirectional transcriptional enhancer activity via a CREB1 binding element. Our findings have uncovered the functional mechanisms of three T2D GWAS SNPs via affecting the binding of transcription factors, providing new insights into the genetics and molecular pathogenesis of T2D.

Derivation of novel naive-like porcine embryonic stem cells by a reprogramming factor-assisted strategy.

The establishment of ungulate embryonic stem cells (ESCs) has been notoriously difficult via a conventional approach. We combined a traditional ESC culture method with reprogramming factors to assist the establishment of porcine naive-like ESCs (nESCs). Pig embryonic fibroblasts were transfected with a tetracycline-inducible vector carrying 4 classic mouse reprogramming factors, followed by somatic cell nuclear transfer and culturing to the blastocyst stage. Then, the inner cell mass was isolated and seeded in culture medium. The naive-like ESCs had characteristic verys similar to those of mouse ESCs and showed no signs of altered morphology or differentiation, even after 130 passages. They depended on leukemia inhibitory factor signals for maintenance of pluripotency, and the female cell lines had low expression of the X-inactive specific transcript gene and no histone H3 lysine 27 trimethylation spot. Notably, the ESCs differentiated into 3 germ layers in vitro and could be induced to undergo directional neural and kidney precursor differentiation under defined conditions, and the ESCs could keep proliferating after doxycycline was removed. nESCs can be established, and the well-characterized ESC lines will be useful for the research of transgenic pig models for human disease.-Zhang, M., Wang, C., Jiang, H., Liu, M., Yang, N., Zhao, L., Hou, D., Jin, Y., Chen, Q., Chen, Y., Wang, J., Dai, Y., Li, R. Derivation of novel naive-like porcine embryonic stem cells by a reprogramming factor-assisted strategy.

Loss of GCN5L1 in cardiac cells disrupts glucose metabolism and promotes cell death via reduced Akt/mTORC2 signaling.

GCN5L1 regulates protein acetylation and mitochondrial energy metabolism in diverse cell types. In the heart, loss of GCN5L1 sensitizes the myocardium to injury from exposure to nutritional excess and ischemia/reperfusion injury. This phenotype is associated with the reduced acetylation of metabolic enzymes and elevated mitochondrial reactive oxygen species (ROS) generation, although the direct molecular targets of GCN5L1 remain largely unknown. In this study, we sought to determine the mechanism by which GCN5L1 impacts energy substrate utilization and mitochondrial health. We find that hypoxia and reoxygenation (H/R) leads to a reduction in cell viability and Akt phosphorylation in GCN5L1 knockdown AC16 cardiomyocytes, in parallel with elevated glucose utilization and impaired fatty acid use. We demonstrate that glycolysis is uncoupled from glucose oxidation under normoxic conditions in GCN5L1-depleted cells. We show that GCN5L1 directly binds to the Akt-activating mTORC2 component Rictor, and that loss of Rictor acetylation is evident in GCN5L1 knockdown cells. Finally, we show that restoring Rictor acetylation in GCN5L1-depleted cells reduces mitochondrial ROS generation and increases cell survival in response to H/R. These studies suggest that GCN5L1 may play a central role in energy substrate metabolism and cell survival via the regulation of Akt/mTORC2 signaling.

The Performance of Gene Expression Signature-Guided Drug-Disease Association in Different Categories of Drugs and Diseases.

A gene expression signature (GES) is a group of genes that shows a unique expression profile as a result of perturbations by drugs, genetic modification or diseases on the transcriptional machinery. The comparisons between GES profiles have been used to investigate the relationships between drugs, their targets and diseases with quite a few successful cases reported. Especially in the study of GES-guided drugs-disease associations, researchers believe that if a GES induced by a drug is opposite to a GES induced by a disease, the drug may have potential as a treatment of that disease. In this study, we data-mined the crowd extracted expression of differential signatures (CREEDS) database to evaluate the similarity between GES profiles from drugs and their indicated diseases. Our study aims to explore the application domains of GES-guided drug-disease associations through the analysis of the similarity of GES profiles on known pairs of drug-disease associations, thereby identifying subgroups of drugs/diseases that are suitable for GES-guided drug repositioning approaches. Our results supported our hypothesis that the GES-guided drug-disease association method is better suited for some subgroups or pathways such as drugs and diseases associated with the immune system, diseases of the nervous system, non-chemotherapy drugs or the mTOR signaling pathway.

Cardiac-specific deletion of GCN5L1 restricts recovery from ischemia-reperfusion injury.

GCN5L1 regulates mitochondrial protein acetylation, cellular bioenergetics, reactive oxygen species (ROS) generation, and organelle positioning in a number of diverse cell types. However, the functional role of GCN5L1 in the heart is currently unknown. As many of the factors regulated by GCN5L1 play a major role in ischemia-reperfusion (I/R) injury, we sought to determine if GCN5L1 is an important nexus in the response to cardiac ischemic stress. Deletion of GCN5L1 in cardiomyocytes resulted in impaired myocardial post-ischemic function and increased infarct development in isolated work-performing hearts. GCN5L1 knockout hearts displayed hallmarks of ROS damage, and scavenging of ROS restored cardiac function and reduced infarct volume in vivo. GCN5L1 knockdown in cardiac-derived AC16 cells was associated with reduced activation of the pro-survival MAP kinase ERK1/2, which was also reversed by ROS scavenging, leading to restored cell viability. We therefore conclude that GCN5L1 activity provides an important protection against I/R induced, ROS-mediated damage in the ischemic heart.

Adropin treatment restores cardiac glucose oxidation in pre-diabetic obese mice.

Exposure to a high fat (HF) diet promotes increased fatty acid uptake, fatty acid oxidation and lipid accumulation in the heart. These maladaptive changes impact cellular energy metabolism and may promote the development of cardiac dysfunction. Attempts to increase cardiac glucose utilization have been proposed as a way to reverse cardiomyopathy in obese and diabetic individuals. Adropin is a nutrient-regulated metabolic hormone shown to promote glucose oxidation over fatty acid oxidation in skeletal muscle homogenates in vitro. The focus of the current study was to investigate whether adropin can regulate substrate metabolism in the heart following prolonged exposure to a HF diet in vivo. Mice on a long-term HF diet received serial intraperitoneal injections of vehicle or adropin over three days. Cardiac glucose oxidation was significantly reduced in HF animals, which was rescued by acute adropin treatment. Significant decreases in cardiac pyruvate dehydrogenase activity were observed in HF animals, which were also reversed by adropin treatment. In contrast to previous studies, this change was unrelated to Pdk4 expression, which remained elevated in both vehicle- and adropin-treated HF mice. Instead, we show that adropin modulated the expression of the mitochondrial acetyltransferase enzyme GCN5L1, which altered the acetylation status and activity of fuel metabolism enzymes to favor glucose utilization. Our findings indicate that adropin exposure leads to increased cardiac glucose oxidation under HF conditions, and may provide a future therapeutic avenue in the treatment of diabetic cardiomyopathy.

The protein acetylase GCN5L1 modulates hepatic fatty acid oxidation activity via acetylation of the mitochondrial ß-oxidation enzyme HADHA.

Sirtuin 3 (SIRT3) deacetylates and activates several mitochondrial fatty acid oxidation enzymes in the liver. Here, we investigated whether the protein acetylase GCN5 general control of amino acid synthesis 5-like 1 (GCN5L1), previously shown to oppose SIRT3 activity, is involved in the regulation of hepatic fatty acid oxidation. We show that GCN5L1 abundance is significantly up-regulated in response to an acute high-fat diet (HFD). Transgenic GCN5L1 overexpression in the mouse liver increased protein acetylation levels, and proteomic detection of specific lysine residues identified numerous sites that are co-regulated by GCN5L1 and SIRT3. We analyzed several fatty acid oxidation proteins identified by the proteomic screen and found that hyperacetylation of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α (HADHA) correlates with increased GCN5L1 levels. Stable GCN5L1 knockdown in HepG2 cells reduced HADHA acetylation and increased activities of fatty acid oxidation enzymes. Mice with a liver-specific deletion of GCN5L1 were protected from hepatic lipid accumulation following a chronic HFD and did not exhibit hyperacetylation of HADHA compared with WT controls. Finally, we found that GCN5L1-knockout mice lack HADHA that is hyperacetylated at three specific lysine residues (Lys-350, Lys-383, and Lys-406) and that acetylation at these sites is significantly associated with increased HADHA activity. We conclude that GCN5L1-mediated regulation of mitochondrial protein acetylation plays a role in hepatic metabolic homeostasis.

LncRNA ZBTB40-IT1 modulated by osteoporosis GWAS risk SNPs suppresses osteogenesis.

Previous genome-wide linkage and association studies have identified an osteoporosis-associated locus at 1p36 that harbors SNPs rs34920465 and rs6426749. The 1p36 locus also comprises the WNT4 gene with known role in bone metabolism and functionally unknown ZBTB40/lncRNA ZBTB40-IT1 genes. How these might interact to contribute to osteoporosis susceptibility is not known. In this study, we show that lncRNA ZBTB40-IT1 is able to suppress osteogenesis and promote osteoclastogenesis by regulating the expression of WNT4, RUNX2, OSX, ALP, COL1A1, OPG and RANKL in U-2OS and hFOB1.19 cell lines, whereas ZBTB40 plays an opposite role in bone metabolism. Treatment with parathyroid hormone significantly downregulates the expression of ZBTB40-IT1 in U-2OS cell lines. ZBTB40 can suppress ZBTB40-IT1 expression but has no response to parathyroid hormone treatment. Dual-luciferase assay and biotin pull-down assay demonstrate that osteoporosis GWAS lead SNPs rs34920465-G and rs6426749-C alleles can respectively bind transcription factors JUN::FOS and CREB1, and upregulate ZBTB40 and ZBTB40-IT1 expression. Our study discovers the critical role of ZBTB40 and lncRNA ZBTB40-IT1 in bone metabolism, and provides a mechanistic basis for osteoporosis GWAS lead SNPs rs34920465 and rs6426749.

Disabling of nephrogenesis in porcine embryos via CRISPR/Cas9-mediated SIX1 and SIX4 gene targeting.

SIX1 and SIX4 genes play critical roles in kidney development. We evaluated the effect of these genes on pig kidney development by generating SIX1-/- and SIX1-/- /SIX4-/- pig foetuses using CRISPR/Cas9 and somatic cell nuclear transfer. We obtained 3 SIX1-/- foetuses and 16 SIX1-/- /SIX4-/- foetuses at different developmental stages. The SIX1-/- foetuses showed a migration block of the left kidney and a smaller size for both kidneys. The ureteric bud failed to form the normal branching and collecting system. Abnormal expressions of kidney development-related genes (downregulation of PAX2, PAX8, and BMP4 and upregulation of EYA1 and SALL1) were also observed in SIX1-/- foetal kidneys and confirmed in vitro in porcine kidney epithelial cells (PK15) following SIX1 gene deletion. The SIX1-/- /SIX4-/- foetuses exhibited more severe phenotypes, with most foetuses showing retarded development at early stages of gestation. The kidney developed only to the initial stage of metanephros formation. These results demonstrated that SIX1 and SIX4 are key genes for porcine metanephros development. The creation of kidney-deficient porcine foetuses provides a platform for generating human kidneys inside pigs using blastocyst complementation.

Validation of goose liver fat measurement by QCT and CSE-MRI with biochemical extraction and pathology as reference.

OBJECTIVES: This study aimed to validate the accuracy and reliability of quantitative computed tomography (QCT) and chemical shift encoded magnetic resonance imaging (CSE-MRI) to assess hepatic steatosis. METHODS: Twenty-two geese with a wide range of hepatic steatosis were collected. After QCT and CSE-MRI examinations, the liver of each goose was removed and samples were taken from the left lobe, upper and lower half of the right lobe for biochemical measurement and histology. Fat percentages by QCT and proton density fat fraction by MRI (MRI-PDFF) were measured within the sample regions of biochemical measurement and histology. The accuracy of QCT and MR measurements were assessed through Spearman correlation coefficients (r) and Passing and Bablok regression equations using biochemical measurement as the "gold standard". RESULTS: Both QCT and MRI correlated highly with chemical extraction [r = 0.922 (p < 0.001) and r = 0.949 (p < 0.001) respectively]. Chemically extracted triglyceride was accurately predicted by both QCT liver fat percentages (Y = 0.6 + 0.866 × X) and by MRI-PDFF (Y = -1.8 + 0.773 × X). CONCLUSIONS: QCT and CSE-MRI measurements of goose liver fat were accurate and reliable compared with biochemical measurement. KEY POINTS: • QCT and CSE-MRI can measure liver fat content accurately and reliably • Histological grading of hepatic steatosis has larger sampling variability • QCT and CSE-MRI have potential in the clinical setting.