A multiomics approach reveals evidence for phenylbutyrate as a potential treatment for combined D,L-2- hydroxyglutaric aciduria.

PURPOSE: To identify therapies for combined D, L-2-hydroxyglutaric aciduria (C-2HGA), a rare genetic disorder caused by recessive variants in the SLC25A1 gene. METHODS: Patients C-2HGA were identified and diagnosed by whole exome sequencing and biochemical genetic testing. Patient derived fibroblasts were then treated with phenylbutyrate and the functional effects assessed by metabolomics and RNA-sequencing. RESULTS: In this study, we demonstrated that C-2HGA patient derived fibroblasts exhibited impaired cellular bioenergetics. Moreover, Fibroblasts form one patient exhibited worsened cellular bioenergetics when supplemented with citrate. We hypothesized that treating patient cells with phenylbutyrate (PB), an FDA approved pharmaceutical drug that conjugates glutamine for renal excretion, would reduce mitochondrial 2-ketoglutarate, thereby leading to improved cellular bioenergetics. Metabolomic and RNA-seq analyses of PB-treated fibroblasts demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of PB, an increased level of phenylacetylglutamine in patient cells was consistent with the drug acting as 2-ketoglutarate sink. CONCLUSION: Our pre-clinical studies suggest that citrate supplementation has the possibility exacerbating energy metabolism in this condition. However, improvement in cellular bioenergetics suggests phenylbutyrate might have interventional utility for this rare disease.

Loss of the mitochondrial citrate carrier, Slc25a1/CIC disrupts embryogenesis via 2-Hydroxyglutarate.

Biallelic germline mutations in the SLC25A1 gene lead to combined D/L-2-hydroxyglutaric aciduria (D/L-2HGA), a fatal systemic disease uniquely characterized by the accumulation of both enantiomers of 2-hydroxyglutaric acid (2HG). How SLC25A1 deficiency contributes to D/L-2HGA and the role played by 2HG is unclear and no therapy exists. Both enantiomers act as oncometabolites, but their activities in normal tissues remain understudied. Here we show that mice lacking both SLC25A1 alleles exhibit developmental abnormalities that mirror human D/L-2HGA. SLC25A1 deficient cells undergo premature senescence, suggesting that loss of proliferative capacity underlies the pathogenesis of D/L-2HGA. Remarkably, D- and L-2HG directly induce senescence and treatment of zebrafish embryos with the combination of D- and L-2HG phenocopies SLC25A1 loss, leading to developmental abnormalities in an additive fashion relative to either enantiomer alone. Metabolic analyses further demonstrate that cells with dysfunctional SLC25A1 undergo mitochondrial respiratory deficit and remodeling of the metabolism and we propose several strategies to correct these defects. These results reveal for the first time pathogenic and growth suppressive activities of 2HG in the context of SLC25A1 deficiency and suggest that targeting the 2HG pathway may be beneficial for the treatment of D/L-2HGA.

Mitochondrial ROS Triggers KIN Pathogenesis in FAN1-Deficient Kidneys.

Karyomegalic interstitial nephritis (KIN) is a genetic adult-onset chronic kidney disease (CKD) characterized by genomic instability and mitotic abnormalities in the tubular epithelial cells. KIN is caused by recessive mutations in the FAN1 DNA repair enzyme. However, the endogenous source of DNA damage in FAN1/KIN kidneys has not been identified. Here we show, using FAN1-deficient human renal tubular epithelial cells (hRTECs) and FAN1-null mice as a model of KIN, that FAN1 kidney pathophysiology is triggered by hypersensitivity to endogenous reactive oxygen species (ROS), which cause chronic oxidative and double-strand DNA damage in the kidney tubular epithelial cells, accompanied by an intrinsic failure to repair DNA damage. Furthermore, persistent oxidative stress in FAN1-deficient RTECs and FAN1 kidneys caused mitochondrial deficiencies in oxidative phosphorylation and fatty acid oxidation. The administration of subclinical, low-dose cisplatin increased oxidative stress and aggravated mitochondrial dysfunction in FAN1-deficient kidneys, thereby exacerbating KIN pathophysiology. In contrast, treatment of FAN1 mice with a mitochondria-targeted ROS scavenger, JP4-039, attenuated oxidative stress and accumulation of DNA damage, mitigated tubular injury, and preserved kidney function in cisplatin-treated FAN1-null mice, demonstrating that endogenous oxygen stress is an important source of DNA damage in FAN1-deficient kidneys and a driver of KIN pathogenesis. Our findings indicate that therapeutic modulation of kidney oxidative stress may be a promising avenue to mitigate FAN1/KIN kidney pathophysiology and disease progression in patients.

Are Human Learners Capable of Learning Arbitrary Language Structures.

The artificial grammar learning paradigm is a classic method of investigating the influence of universal constraints on shaping learning biases on language acquisition. While this method has been used extensively by linguists to test theoretical claims in generative grammar, one of the most prevalent frameworks in language acquisition, several studies have questioned whether artificial grammar learning reflects language acquisition enough to allow us to use it to draw inferences about the validity of universal constraints, particularly those arising from phonetic naturalness. The current study tests whether artificial grammar learning shows the effect of one robust phonetic naturalness constraint: the restriction on nasal harmony patterns arising from the sonority hierarchy. Nasal harmony is of particular interest because it is one of the few types of harmony that occurs between consonants and vowels, which is an under-researched topic. The results, contrary to the skeptical concerns, showed that participants (n = 120) could learn an artificial grammar involving a natural pattern, but could not learn one corresponding to an arbitrary/phonetically unmotivated pattern in the same way or to the same degree. This study contributes epistemic support to the large body of work using artificial grammar experiments to test phonological operations.

A multiomics approach to understanding pathology of Combined D,L-2- Hydroxyglutaric Aciduria and phenylbutyrate as potential treatment.

Combined D, L-2-Hydroxyglutaric Aciduria (D,L-2HGA) is a rare genetic disorder caused by recessive mutations in the SLC25A1 gene that encodes the mitochondrial citrate carrier protein (CIC). SLC25A1 deficiency leads to a secondary increase in mitochondrial 2-ketoglutarate that, in turn, is reduced to neurotoxic 2-hydroxyglutarate. Clinical symptoms of Combined D,L-2HGA include neonatal encephalopathy, respiratory insufficiency and often with death in infancy. No current therapies exist, although replenishing cytosolic stores by citrate supplementation to replenish cytosolic stores has been proposed. In this study, we demonstrated that patient derived fibroblasts exhibited impaired cellular bioenergetics that were worsened with citrate supplementation. We hypothesized treating patient cells with phenylbutyrate, an FDA approved pharmaceutical drug, would reduce mitochondrial 2-ketoglutarate, leading to improved cellular bioenergetics including oxygen consumption and fatty acid oxidation. Metabolomic and RNA-seq analyses demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of phenylbutyrate, detected levels of phenylacetylglutamine was consistent with the drug acting as 2-ketoglutarate sink in patient cells. Our pre-clinical studies suggest citrate supplementation is unlikely to be an effective treatment of the disorder. However, cellular bioenergetics suggests phenylbutyrate may have interventional utility for this rare disease.

Glutamine energy substrate anaplerosis increases bone density in the Pahenu2 classical PKU mouse in the absence of phenylalanine restriction.

Osteopenia is an under-investigated clinical presentation of phenylalanine hydroxylase (PAH)-deficient phenylketonuria (PKU). While osteopenia is not fully penetrant in human PKU, the Pahenu2 mouse is universally osteopenic and ideal to study the phenotype. We determined Pahenu2 mesenchymal stem cells (MSCs) are developmentally impaired in the osteoblast lineage. Moreover, we determined energy dysregulation and oxidative stress contribute to the osteoblast developmental deficit. The MSC preferred substrate glutamine (Gln) was applied to enhance energy homeostasis. In vitro Pahenu2 MSCs, in the context of 1200 µM Phe, respond to Gln with increased in situ alkaline phosphatase activity indicating augmented osteoblast differentiation. Oximetry applied to Pahenu2 MSCs in osteoblast differentiation show Gln energy substrate increases oxygen consumption, specifically maximum respiration and respiratory reserve. For 60 days post-weaning, Pahenu2 animals received either no intervention (standard lab chow), amino acid defined chow maintaining plasma Phe at ~200 µM, or standard lab chow where ad libitum water was a 2% Gln solution. Bone density was assessed by microcomputed tomography and bone growth assessed by dye labeling. Bone density and dye labeling in Phe-restricted Pahenu2 was indistinguishable from untreated Pahenu2. Gln energy substrate provided to Pahenu2, in the context of uncontrolled hyperphenylalaninemia, present increased bone density and dye labeling. These data provide further evidence that Pahenu2 MSCs experience a secondary energy deficit that is responsive both in vitro and in vivo to Gln energy substrate and independent of hyperphenylalaninemia. Energy support may have effect to treat human PKU osteopenia and elements of PKU neurologic disease resistant to standard of care systemic Phe reduction. Glutamine energy substrate anaplerosis increased Pahenu2 bone density and improved in vitro MSC function in the context of hyperphenylalaninemia in the classical PKU range.

Treatment of VLCAD-Deficient Patient Fibroblasts with Peroxisome Proliferator-Activated Receptor δ Agonist Improves Cellular Bioenergetics.

Background: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive disease that prevents the body from utilizing long-chain fatty acids for energy, most needed during stress and fasting. Symptoms can appear from infancy through childhood and adolescence or early adulthood, and include hypoglycemia, recurrent rhabdomyolysis, myopathy, hepatopathy, and cardiomyopathy. REN001 is a peroxisome-proliferator-activated receptor delta (PPARδ) agonist that modulates the expression of the genes coding for fatty acid ß-oxidation enzymes and proteins involved in oxidative phosphorylation. Here, we assessed the effect of REN001 on VLCAD-deficient patient fibroblasts. Methods: VLCAD-deficient patient and control fibroblasts were treated with REN001. Cells were harvested for gene expression analysis, protein content, VLCAD enzyme activity, cellular bioenergetics, and ATP production. Results: VLCAD-deficient cell lines responded differently to REN001 based on genotype. All cells had statistically significant increases in ACADVL gene expression. Small increases in VLCAD protein and enzyme activity were observed and were cell-line- and dose-dependent. Even with these small increases, cellular bioenergetics improved in all cell lines in the presence of REN001, as demonstrated by the oxygen consumption rate and ATP production. VLCAD-deficient cell lines containing missense mutations responded better to REN001 treatment than one containing a duplication mutation in ACADVL. Discussion: Treating VLCAD-deficient fibroblasts with the REN001 PPARδ agonist results in an increase in VLCAD protein and enzyme activity, and a decrease in cellular stress. These results establish REN001 as a potential therapy for VLCADD as enhanced expression may provide a therapeutic increase in total VLCAD activity, but suggest the need for mutation-specific treatment augmented by other treatment measures.

Persistent DNA damage underlies tubular cell polyploidization and progression to chronic kidney disease in kidneys deficient in the DNA repair protein FAN1.

Defective DNA repair pathways contribute to the development of chronic kidney disease (CKD) in humans. However, the molecular mechanisms underlying DNA damage-induced CKD pathogenesis are not well understood. Here, we investigated the role of tubular cell DNA damage in the pathogenesis of CKD using mice in which the DNA repair protein Fan1 was knocked out. The phenotype of these mice is orthologous to the human DNA damage syndrome, karyomegalic interstitial nephritis (KIN). Inactivation of Fan1 in kidney proximal tubule cells sensitized the kidneys to genotoxic and obstructive injury characterized by replication stress and persistent DNA damage response activity. Accumulation of DNA damage in Fan1 tubular cells induced epithelial dedifferentiation and tubular injury. Characteristic to KIN, cells with chronic DNA damage failed to complete mitosis and underwent polyploidization. In vitro and in vivo studies showed that polyploidization was caused by the overexpression of DNA replication factors CDT1 and CDC6 in FAN1 deficient cells. Mechanistically, inhibiting DNA replication with Roscovitine reduced tubular injury, blocked the development of KIN and mitigated kidney function in these Fan1 knockout mice. Thus, our data delineate a mechanistic pathway by which persistent DNA damage in the kidney tubular cells leads to kidney injury and development of CKD. Furthermore, therapeutic modulation of cell cycle activity may provide an opportunity to mitigate the DNA damage response induced CKD progression.

Comparative metabolomics in the Pahenu2 classical PKU mouse identifies cerebral energy pathway disruption and oxidative stress.

Classical phenylketonuria (PKU, OMIM 261600) owes to hepatic deficiency of phenylalanine hydroxylase (PAH) that enzymatically converts phenylalanine (Phe) to tyrosine (Tyr). PKU neurologic phenotypes include impaired brain development, decreased myelination, early onset mental retardation, seizures, and late-onset features (neuropsychiatric, Parkinsonism). Phe over-representation is systemic; however, tissue response to hyperphenylalaninemia is not consistent. To characterize hyperphenylalaninemia tissue response, metabolomics was applied to Pahenu2 classical PKU mouse blood, liver, and brain. In blood and liver over-represented analytes were principally Phe, Phe catabolites, and Phe-related analytes (Phe-conjugates, Phe-containing dipeptides). In addition to Phe and Phe-related analytes, the metabolomic profile of Pahenu2 brain tissue evidenced oxidative stress responses and energy dysregulation. Glutathione and homocarnosine anti-oxidative responses are apparent Pahenu2 brain. Oxidative stress in Pahenu2 brain was further evidenced by increased reactive oxygen species. Pahenu2 brain presents an increased NADH/NAD ratio suggesting respiratory chain complex 1 dysfunction. Respirometry in Pahenu2 brain mitochondria functionally confirmed reduced respiratory chain activity with an attenuated response to pyruvate substrate. Glycolysis pathway analytes are over-represented in Pahenu2 brain tissue. PKU pathologies owe to liver metabolic deficiency; yet, Pahenu2 liver tissue shows neither energy disruption nor anti-oxidative response. Unique aspects of metabolomic homeostasis in PKU brain tissue along with increased reactive oxygen species and respiratory chain deficit provide insight to neurologic disease mechanisms. While some elements of assumed, long standing PKU neuropathology are enforced by metabolomic data (e.g. reduced tryptophan and serotonin representation), energy dysregulation and tissue oxidative stress expand mechanisms underlying neuropathology.

Phenylketonuria oxidative stress and energy dysregulation: Emerging pathophysiological elements provide interventional opportunity.

Phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is rightfully considered the paradigm treatable metabolic disease. Dietary substrate restriction (i.e. phenylalanine (Phe) restriction) was applied >60 years ago and remains the primary PKU management means. The traditional model of PKU neuropathophysiology dictates blood Phe over-representation directs asymmetric blood:brain barrier amino acid transport through the LAT1 transporter with subsequent increased cerebral Phe concentration and low concentrations of tyrosine (Tyr), tryptophan (Trp), leucine (Leu), valine (Val), and isoleucine (Ile). Low Tyr and Trp concentrations generate secondary serotonergic and dopaminergic neurotransmitter paucities, widely attributed as drivers of PKU neurologic phenotypes. White matter disease, a central PKU characteristic, is ascribed to Phe-mediated tissue toxicity. Impaired cerebral protein synthesis, by reduced concentrations of non-Phe large neutral amino acids, is another cited pathological mechanism. The PKU amino acid transport model suggests Phe management should be more efficacious than is realized, as even early identified, continuously treated patients that retain therapy compliance into adulthood, demonstrate neurologic disease elements. Reduced cerebral metabolism was an early-recognized element of PKU pathology. Legacy data (late 1960's to mid-1970's) determined the Phe catabolite phenylpyruvate inhibits mitochondrial pyruvate transport. Respirometry of Pahenu2 cerebral mitochondria have attenuated respiratory chain complex 1 induction in response to pyruvate substrate, indicating reduced energy metabolism. Oxidative stress is intrinsic to PKU and Pahenu2 brain tissue presents increased reactive oxygen species. Phenylpyruvate inhibits glucose-6-phosphate dehydrogenase that generates reduced niacinamide adenine dinucleotide phosphate the obligatory cofactor of glutathione reductase. Pahenu2 brain tissue metabolomics identified increased oxidized glutathione and glutathione disulfide. Over-represented glutathione disulfide argues for reduced glutathione reductase activity secondary to reduced NADPH. Herein, we review evidence of energy and oxidative stress involvement in PKU pathology. Data suggests energy deficit and oxidative stress are features of PKU pathophysiology, providing intervention-amenable therapeutic targets to ameliorate disease elements refractory to standard of care.

Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development.

Mammalian nephrons originate from a population of nephron progenitor cells, and changes in these cells' transcriptomes contribute to the cessation of nephrogenesis, an important determinant of nephron number. To characterize microRNA (miRNA) expression and identify putative cis-regulatory regions, we collected nephron progenitor cells from mouse kidneys at embryonic day 14.5 and postnatal day zero and assayed small RNA expression and transposase-accessible chromatin. We detect expression of 1104 miRNA (114 with expression changes), and 46,374 chromatin accessible regions (2103 with changes in accessibility). Genome-wide, our data highlight processes like cellular differentiation, cell migration, extracellular matrix interactions, and developmental signaling pathways. Furthermore, they identify new candidate cis-regulatory elements for Eya1 and Pax8, both genes with a role in nephron progenitor cell differentiation. Finally, we associate expression-changing miRNAs, including let-7-5p, miR-125b-5p, miR-181a-2-3p, and miR-9-3p, with candidate cis-regulatory elements and target genes. These analyses highlight new putative cis-regulatory loci for miRNA in nephron progenitors.

Phenylalanine hydroxylase deficient phenylketonuria comparative metabolomics identifies energy pathway disruption and oxidative stress.

Classical phenylketonuria (PKU, OMIM 261600) owes to hepatic deficiency of phenylalanine hydroxylase (PAH) that enzymatically converts phenylalanine (Phe) to tyrosine (Tyr). PKU neurologic phenotypes include impaired brain development, decreased myelination, early onset mental retardation, seizures, and late-onset features (neuropsychiatric, Parkinsonism). PAH deficiency leads to systemic hyperphenylalaninemia; however, the impact of Phe varies between tissues. To characterize tissue response to hyperphenylalaninemia, metabolomics was applied to tissue from therapy noncompliant classical PKU patients (blood, liver), the Pahenu2 classical PKU mouse (blood, liver, brain) and the PAH deficient pig (blood, liver, brain, cerebrospinal fluid). In blood, liver, and CSF from both patients and animal models over-represented analytes were principally Phe, Phe catabolites, and Phe-related analytes (conjugates, Phe-containing dipeptides). In addition to Phe and Phe-related analytes, the metabolomic profile of PKU brain tissue (mouse, pig) evidenced oxidative stress responses and energy dysregulation. In Pahenu2 and PKU pig brain tissues, anti-oxidative response by glutathione and homocarnosine is apparent. Oxidative stress in Pahenu2 brain was further demonstrated by increased reactive oxygen species. In Pahenu2 and PKU pig brain, an increased NADH/NAD ratio suggests a respiratory chain dysfunction. Respirometry in PKU brain mitochondria (mouse, pig) functionally confirmed reduced respiratory chain activity. Glycolysis pathway analytes are over-represented in PKU brain tissue (mouse, pig). PKU pathologies owe to liver metabolic deficiency; yet, PKU liver tissue (mouse, pig, human) shows neither energy disruption nor anti-oxidative response. Unique aspects of metabolomic homeostasis in PKU brain tissue along with increased reactive oxygen species and respiratory chain deficit provide insight to neurologic disease mechanisms. While some elements of assumed, long standing PKU neuropathology are enforced by metabolomic data (e.g. reduced tryptophan and serotonin representation), energy dysregulation and tissue oxidative stress expand mechanisms underlying neuropathology.

Mesenchymal stem cell energy deficit and oxidative stress contribute to osteopenia in the Pahenu2 classical PKU mouse.

Osteopenia occurs in a subset of phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) patients. While osteopenia is not fully penetrant in patients, the Pahenu2 classical PKU mouse is universally osteopenic, making it an ideal model of the phenotype. Pahenu2 Phe management, with a Phe-fee amino acid defined diet, does not improve bone density as histomorphometry metrics remain indistinguishable from untreated animals. Previously, we demonstrated Pahenu2 mesenchymal stem cells (MSCs) display impaired osteoblast differentiation. Oxidative stress is recognized in PKU patients and PKU animal models. Pahenu2 MSCs experience oxidative stress determined by intracellular superoxide over-representation. The deleterious impact of oxidative stress on mitochondria is recognized. Oximetry applied to Pahenu2 MSCs identified mitochondrial stress by increased basal respiration with concurrently reduced maximal respiration and respiratory reserve. Proton leak secondary to mitochondrial complex 1 dysfunction is a recognized superoxide source. Respirometry applied to Pahenu2 MSCs, in the course of osteoblast differentiation, identified a partial complex 1 deficit. Pahenu2 MSCs treated with the antioxidant resveratrol demonstrated increased mitochondrial mass by MitoTracker green labeling. In hyperphenylalaninemic conditions, resveratrol increased in situ alkaline phosphatase activity suggesting partial recovery of Pahenu2 MSCs osteoblast differentiation. Up-regulation of oxidative energy production is required for osteoblasts differentiation. Our data suggests impaired Pahenu2 MSC developmental competence involves an energy deficit. We posit energy support and oxidative stress reduction will enable Pahenu2 MSC differentiation in the osteoblast lineage to subsequently increase bone density.

Endothelial-Derived miR-17∼92 Promotes Angiogenesis to Protect against Renal Ischemia-Reperfusion Injury.

BACKGROUND: Damage to the renal microvasculature is a hallmark of renal ischemia-reperfusion injury (IRI)-mediated AKI. The miR-17∼92 miRNA cluster (encoding miR-17, -18a, -19a, -20a, -19b-1, and -92a-1) regulates angiogenesis in multiple settings, but no definitive role in renal endothelium during AKI pathogenesis has been established. METHODS: Antibodies bound to magnetic beads were utilized to selectively enrich for renal endothelial cells from mice. Endothelial-specific miR-17∼92 knockout (miR-17∼92endo-/- ) mice were generated and given renal IRI. Mice were monitored for the development of AKI using serum chemistries and histology and for renal blood flow using magnetic resonance imaging (MRI) and laser Doppler imaging. Mice were treated with miRNA mimics during renal IRI, and therapeutic efficacies were evaluated. RESULTS: miR-17, -18a, -20a, -19b, and pri-miR-17∼92 are dynamically regulated in renal endothelial cells after renal IRI. miR-17∼92endo-/- exacerbates renal IRI in male and female mice. Specifically, miR-17∼92endo-/- promotes renal tubular injury, reduces renal blood flow, promotes microvascular rarefaction, increases renal oxidative stress, and promotes macrophage infiltration to injured kidneys. The potent antiangiogenic factor thrombospondin 1 (TSP1) is highly expressed in renal endothelium in miR-17∼92endo-/- after renal IRI and is a target of miR-18a and miR-19a/b. miR-17∼92 is critical in the angiogenic response after renal IRI, which treatment with miR-18a and miR-19b mimics can mitigate. CONCLUSIONS: These data suggest that endothelial-derived miR-17∼92 stimulates a reparative response in damaged renal vasculature during renal IRI by regulating angiogenic pathways.

Neighborhood-Level Public Facilities and COVID-19 Transmission: A Nationwide Geospatial Study In China

Individual-level studies on the coronavirus disease 2019 (COVID-19) have proliferated; however, research on neighborhood-level factors associated with COVID-19 is limited. We gathered the geographic data of all publically released COVID-19 cases in China and used a case-control (1:4 ratio) design to investigate the association between having COVID-19 cases in a neighborhood and number and types of public facilities nearby. Having more restaurants, shopping centers, hotels, living facilities, recreational facilities, public transits, educational institutions, and health service facilities was associated with significantly higher odds of having COVID-19 cases in a neighborhood. The associations for restaurants, hotels, reactional and education facilities were more pronounced in cities with fewer than six million people than those in larger cities. Our results have implications for designing targeted prevention strategies at the neighborhood level to reduce the burden of COVID-19.

Loss of miR-17~92 results in dysregulation of Cftr in nephron progenitors.

We have previously demonstrated that loss of miR-17~92 in nephron progenitors in a mouse model results in renal hypodysplasia and chronic kidney disease. Clinically, decreased congenital nephron endowment because of renal hypodysplasia is associated with an increased risk of hypertension and chronic kidney disease, and this is at least partly dependent on the self-renewal of nephron progenitors. Here, we present evidence for a novel molecular mechanism regulating the self-renewal of nephron progenitors and congenital nephron endowment by the highly conserved miR-17~92 cluster. Whole transcriptome sequencing revealed that nephron progenitors lacking this cluster demonstrated increased Cftr expression. We showed that one member of the cluster, miR-19b, is sufficient to repress Cftr expression in vitro and that perturbation of Cftr activity in nephron progenitors results in impaired proliferation. Together, these data suggest that miR-19b regulates Cftr expression in nephron progenitors, with this interaction playing a role in appropriate nephron progenitor self-renewal during kidney development to generate normal nephron endowment.

Facile fabrication of superporous and biocompatible hydrogel scaffolds for artificial corneal periphery.

Biocompatible and highly porous network hydrogel scaffolds were fabricated for the development of artificial cornea (AC) periphery/skirt that could be used to enhance the long-term retention of the implants. In this study, a series of hydrogel scaffolds for this application was fabricated from the photo-polymerization of a mixture of poly(ethylene glycol) (PEG)- and poloxamer (P407)-based macromer solutions in dichloromethane in which solvent-induced phase separation (SIPS) arose to form scaffolds with macroporous structure and high water content. The overall porosity ranging from 20% to 75% and open/closed pore structure of the hydrogel scaffolds could be finely tuned by varying the ratio of P407/PEG in the macromer solution and solvent type. The total porosity and open-cell structure of the macropores in the synthesized hydrogel scaffolds affected the swelling behavior, dynamic properties such as the storage moduli of the hydrogels as well as their degradation rates. Based on the subcutaneous implantation in rats, superporous hydrogel scaffolds induced the formation of thinner fibrous capsules around the implants and showed less inflammatory reaction, suggesting that the hydrogel scaffolds made from SIPS exhibited good cytocompatibility. The combined results of swelling ratio, porosity, physical strength and subcutaneous implant tests indicated that the superporous hydrogels with porosity >50% showed potentials to be used for cornea periphery application.

[Effects of dimethylamine tetracycline combined with tea polyphenols on the levels of IL-1ß and IL-17F in patients with early peri-implant soft tissue inflammation].

PURPOSE: Using impact test to study energy absorption ability of mouthguard splints of different thickness and materials. METHODS: In this experiment, group 1 was BIOPLAST 5 mm splint, group 2 was BIOPLAST 4 mm splint, group 3 was BIOPLAST 3 mm splint，group 4 was BIOPLAST 2 mm splint and group 7 was BIOPLAST 1 mm splint. Group 5 was ERKOLOC-PRO 2 mm splint, group 6 was DURAN 2 mm splint. Mouthguard splints were clashed by impact head from different heights. Digital Image Correlation System was used to record the process of the test. Images were transferred to data processing software, to analyze incident speed, incident depth and rebound height of the impact head. Absorbed energy and energy absorption ratio were calculated to analyze energy absorption ability of mouthguard splints of different thickness and materials. The data were analyzed using SPSS 22.0 software package for one-way analysis of variance and LSD. RESULTS: The higher impact head fell, the larger energy absorption ratio was. There was significant difference among group 1, 2, 3, 4, 7 in average energy absorption ratio（P<0.05）, and group 7 was the largest. There was significant difference among group 4, 5, 6 in average energy absorption ratio（P<0.05）, among which group 4 and group 5 were larger. CONCLUSIONS: 3 mm splint is good enough to be used to make mouthguard, which is also thinner and more comfortable. Splint of soft material is more suitable for mouthguard than splint of hard material.

Small non-coding RNA expression in mouse nephrogenic mesenchymal progenitors.

MicroRNAs (miRNAs) are small non-coding RNAs that are essential for the regulation of gene expression and play critical roles in human health and disease. Here we present comprehensive miRNA profiling data for mouse nephrogenic mesenchymal progenitors, a population of cells enriched for nephron progenitors that give rise to most cell-types of the nephron, the functional unit of the kidney. We describe a miRNA expression in nephrogenic mesenchymal progenitors, with 162 miRNAs differentially expressed in progenitors when compared to whole kidney. We also annotated 49 novel miRNAs in the developing kidney and experimentally validated 4 of them. Our data are available as a public resource, so that it can be integrated into future studies and analyzed in the context of other functional and epigenomic data in kidney development. Specifically, it will be useful in the effort to shed light on molecular mechanisms underlying processes essential for normal kidney development, like nephron progenitor specification, self-renewal and differentiation.