Apparent Phenotype Research Articles

In mouse kidney endogenous transmembrane serine protease 2 (TMPRSS2) contributes to proteolytic processing and activation of the epithelial sodium channel (ENaC)

Regulation of ENaC in the distal nephron is essential for sodium homeostasis and blood pressure control. A unique feature of ENaC is its complex proteolytic processing leading to channel activation due to release of inhibitory tracts from its α- and γ-subunits. In particular, fully cleaved γ-ENaC is critical for high channel activity. However, the physiologically relevant proteases involved remain elusive. Using Xenopus laevis oocytes and H441 airway epithelial cells, we recently demonstrated that TMPRSS2 proteolytically activates ENaC by cleaving the channel’s γ-subunit ( J Biol Chem 2022 Jun;298(6):102004). Here we investigate whether TMPRSS2 also contributes to proteolytic ENaC regulation in the kidney. Using mRNA sequencing, we demonstrated that mouse cortical collecting duct (mCCD cl1 ) cells express TMPRSS2 at a high level unaffected by aldosterone, an important ENaC stimulating hormone. TMPRSS2 knockdown by CRISPR/Cas9 technology significantly reduced fully cleaved γ-ENaC in membrane-enriched fractions of mCCD cl1 cell lysates (n=5). Apical application of chymotrypsin significantly stimulated ENaC-mediated short circuit current ( I SC ) in TMPRSS2-knockdown cells, but not in control cells (2.0 ± 1.1 μA/cm² vs. 0.2 ± 0.6 μA/cm²; n=13-14; p<0.001; mean ± SD). Moreover, the stimulatory effect of aldosterone on I SC was impaired in TMPRSS2-knockdown cells compared to control cells (9.1 ± 4.0 μA/cm² vs. 18.3 ± 4.0 μA/cm²; n=7-8; p<0.01). After aldosterone treatment I SC stimulation by chymotrypsin was enhanced in TMPRSS2-knockdown cells (5.3 ± 1.9 μA/cm²; n=7) but undetectable in control cells (−0.8 ± 1.6 μA/cm²; n=8). These findings indicate that TMPRSS2 deficiency impairs proteolytic ENaC activation in mCCD cl1 cells. To confirm this in an in vivo model, we used constitutive TMPRSS2 knockout mice ( Tmprss2 -/- ). Using a combination of RNAscope technology and immunofluorescence, we demonstrated high expression of Tmprss2 mRNA in renal tubular cells, including cells with β-ENaC protein staining. Importantly, the ratio of fully to partially cleaved γ-ENaC was significantly reduced in membrane-enriched fractions of whole-kidney lysates from Tmprss2 -/- mice (n=10). Maintained on a standard diet, Tmprss2 -/- mice had no apparent renal phenotype with plasma aldosterone levels similar to those of Tmprss2 +/+ mice (120 ± 43 pg/ml in Tmprss2 -/- vs. 92 ± 17 pg/mL in Tmprss2 +/+ ; n=7-10). Moreover, the ability of Tmprss2 -/- mice to reduce urinary sodium excretion in response to four days of low sodium diet was fully preserved. However, under low sodium diet plasma aldosterone increased to significantly higher levels in Tmprss2 -/- mice compared to controls (752 ± 146 pg/ml in Tmprss2 -/- vs. 268 ± 55 pg/ml in Tmprss2 +/+ ; n=7-11; p<0.01). This is most likely a compensatory response to compromised proteolytic ENaC activation due to TMPRSS2 deficiency. We conclude that TMPRSS2 plays a physiological role in proteolytic ENaC processing and activation in the kidney. This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 509149993, TRR 374 (subproject A4 to A.I. and C.K.) and the Bayerisches Staatsministerium für Wissenschaft und Kunst (Bavarian Ministry of Art and Science), project VI-Corona-Forschung (subproject 07 to M.B. and T.G). A.K. was supported by the Interdisziplinäres Zentrum für klinische Forschung (IZKF) Erlangen. S.W. was supported by the Bundesministerium für Bildung und Forschung (BMBF, Federal Ministry of Education and Research, Germany), project SENSE-CoV2, 01KI20172A. P.S. and F.A. were funded by the IZKF Tübingen. The authors declare no conflicts of interest. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

3' Untranslated Region Structural Elements in CYP24A1 Are Associated With Infantile Hypercalcemia Type 1.

Loss-of-function mutations in the CYP24A1 protein-coding region causing reduced 25 hydroxyvitamin D (25OHD) and 1,25 dihydroxyvitamin D (1,25(OH)2 D) catabolism have been observed in some cases of infantile hypercalcemia type 1 (HCINF1), which can manifest as nephrocalcinosis, hypercalcemia and adult-onset hypercalciuria, and renal stone formation. Some cases present with apparent CYP24A1 phenotypes but do not exhibit pathogenic mutations. Here, we assessed the molecular mechanisms driving apparent HCINF1 where there was a lack of CYP24A1 mutation. We obtained blood samples from 47 patients with either a single abnormality of no obvious cause or a combination of hypercalcemia, hypercalciuria, and nephrolithiasis as part of our metabolic and stone clinics. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine serum vitamin D metabolites and direct sequencing to confirm CYP24A1 genotype. Six patients presented with profiles characteristic of altered CYP24A1 function but lacked protein-coding mutations in CYP24A1. Analysis upstream and downstream of the coding sequence showed single nucleotide variants (SNVs) in the CYP24A1 3' untranslated region (UTR). Bioinformatics approaches revealed that these 3' UTR abnormalities did not result in microRNA silencing but altered the CYP24A1 messenger RNA (mRNA) secondary structure, which negatively impacted translation. Our experiments showed that mRNA misfolding driven by these 3' UTR sequence-dependent structural elements was associated with normal 25OHD but abnormal 1,25(OH)2 D catabolism. Using CRISPR-Cas9 gene editing, we developed an in vitro mutant model for future CYP24A1 studies. Our results form a basis for future studies investigating structure-function relationships and novel CYP24A1 mutations producing a semifunctional protein. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Downregulation of CDC25C in NPCs Disturbed Cortical Neurogenesis.

Cell division regulators play a vital role in neural progenitor cell (NPC) proliferation and differentiation. Cell division cycle 25C (CDC25C) is a member of the CDC25 family of phosphatases which positively regulate cell division by activating cyclin-dependent protein kinases (CDKs). However, mice with the Cdc25c gene knocked out were shown to be viable and lacked the apparent phenotype due to genetic compensation by Cdc25a and/or Cdc25b. Here, we investigate the function of Cdc25c in developing rat brains by knocking down Cdc25c in NPCs using in utero electroporation. Our results indicate that Cdc25c plays an essential role in maintaining the proliferative state of NPCs during cortical development. The knockdown of Cdc25c causes early cell cycle exit and the premature differentiation of NPCs. Our study uncovers a novel role of CDC25C in NPC division and cell fate determination. In addition, our study presents a functional approach to studying the role of genes, which elicit genetic compensation with knockout, in cortical neurogenesis by knocking down in vivo.

Mutations in the NXF-1:NXT-1 mRNA export complex affect gene-expression driven by the hsp-16.41 promoter.

The NXF-1 : NXT-1 heterodimer is essential for the nuclear export of mRNA. Here we describe three new alleles of nxf-1 and one allele of nxt-1 isolated from a forward genetic screen. These mutations cause no apparent phenotype under normal growth conditions, but partially suppress the lethality caused by heat-shock induced expression of the PEEL-1 toxin from P hsp-16.41 :: peel-1 . There is also decreased expression of P hsp-16.41 ::eGFP in an nxf-1 mutant. We propose that NXF-1 : NXT-1 influences the expression of heat-shock activated genes due to a role in the recruitment of the hsp-16.41 promoter to the nuclear pore complex during heat-shock.

Difluoromethyl-1,3,4-oxadiazoles are slow-binding substrate analog inhibitors of histone deacetylase 6 with unprecedented isotype selectivity

Histone deacetylase 6 (HDAC6) is an attractive drug development target because of its role in the immune response, neuropathy, and cancer. Knockout mice develop normally and have no apparent phenotype, suggesting that selective inhibitors should have an excellent therapeutic window. Unfortunately, current HDAC6 inhibitors have only moderate selectivity and may inhibit other HDAC subtypes at high concentrations, potentially leading to side effects. Recently, substituted oxadiazoles have attracted attention as a promising novel HDAC inhibitor chemotype, but their mechanism of action is unknown. Here, we show that compounds containing a difluoromethyl-1,3,4-oxadiazole (DFMO) moiety are potent and single-digit nanomolar inhibitors with an unprecedented greater than 104-fold selectivity for HDAC6 over all other HDAC subtypes. By combining kinetics, X-ray crystallography, and mass spectrometry, we found that DFMO derivatives are slow-binding substrate analogs of HDAC6 that undergo an enzyme-catalyzed ring opening reaction, forming a tight and long-lived enzyme-inhibitor complex. The elucidation of the mechanism of action of DFMO derivatives paves the way for the rational design of highly selective inhibitors of HDAC6 and possibly of other HDAC subtypes as well with potentially important therapeutic implications.

Aggregation‐prone human tau accelerates lifespan and motility decline in a Caenorhabditis elegans model of tau polymerization

AbstractBackgroundTau aggregates is a key molecular hallmark of Alzheimer’s disease and other Alzheimer’s Disease‐Related Disorders. In these neurodegenerative disorders tau is aggregated in areas of the brain that exhibit high levels of synaptic and neuronal degeneration. Most animal models focus on neuronal death as is an apparent phenotype; however, we lack an in vivo model to observe the events that occur early in pathology compared to the endpoint (neuronal death). Thus, we want to build a model using C. elegans to understand the factors that initiate tau polymerization and toxicity.MethodTo this end, we have generated a transgenic C. elegans model expressing different variants of the longest human tau isoform (hTau40) tagged to GFP. We used a pan‐neuronal promoter to express wild‐type hTau40 and several aggregation‐prone hTau40 variants. We also used mutated alleles to test the conditions under which wild‐type tau is induced to aggregate or can exacerbate the phenotype observed under the aggregation‐prone tau variants. We used confocal microscopy to assess the accumulation of tau‐GFP within neurons in an age‐dependent manner. In addition, we used a combination of lifespan, behavior and biochemical assays to evaluate the role of tau‐GFP intensity/location with the overall healthspan of the nematode.ResultWe observed differences in the accumulation and presentation of tau variants can be measured in vivo using GFP. Wild‐type tau‐GFP appears smooth and filamentous and localized to axons and cell bodies. On the contrary, the aggregation‐prone 3PO‐tau is punctate in axons with extensive inclusions of GFP in cell bodies which increases in an age‐dependent manner. The expression of aggregation‐prone tau variants appears to correlate with the decreased lifespan and decreased motility, which is an indication that tau aggregation within the cell bodies is toxic.ConclusionThese results suggests that we can use our model to identify factors that can regulate tau aggregation in vivo. Additionally, the further development of this C. elegans model can be use to question early physiological changes that occur early vs. late in the disease while identifying or validating possible risks factors.

Quantitative Proteomics Analysis Reveals That Cyclooxygenase-2 Modulates Mitochondrial Respiratory Chain Complex IV in Cardiomyocytes.

The biochemical mechanisms of cell injury and myocardial cell death after myocardial infarction remain unresolved. Cyclooxygenase 2 (COX-2), a key enzyme in prostanoid synthesis, is expressed in human ischemic myocardium and dilated cardiomyopathy, but it is absent in healthy hearts. To assess the role of COX-2 in cardiovascular physiopathology, we developed transgenic mice that constitutively express functional human COX-2 in cardiomyocytes under the control of the α-myosin heavy chain promoter. These animals had no apparent phenotype but were protected against ischemia-reperfusion injury in isolated hearts, with enhanced functional recovery and diminished cellular necrosis. To further explore the phenotype of this animal model, we carried out a differential proteome analysis of wild-type vs. transgenic cardiomyocytes. The results revealed a tissue-specific proteomic profile dominated by mitochondrial proteins. In particular, an increased expression of respiratory chain complex IV proteins was observed. This correlated with increased catalytic activity, enhanced respiratory capacity, and increased ATP levels in the heart of COX-2 transgenic mice. These data suggest a new link between COX-2 and mitochondria, which might contribute to the protective cardiac effects of COX-2 against ischemia-reperfusion injury.

Generation and Characterization of a Novel Angelman Syndrome Mouse Model with a Full Deletion of the Ube3a Gene.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by deficits in maternally inherited UBE3A. The disease is characterized by intellectual disability, impaired motor skills, and behavioral deficits, including increased anxiety and autism spectrum disorder features. The mouse models used so far in AS research recapitulate most of the cardinal AS characteristics. However, they do not mimic the situation found in the majority of AS patients who have a large deletion spanning 4–6 Mb. There is also a large variability in phenotypes reported in the available models, which altogether limits development of therapeutics. Therefore, we have generated a mouse model in which the Ube3a gene is deleted entirely from the 5′ UTR to the 3′ UTR of mouse Ube3a isoform 2, resulting in a deletion of 76 kb. To investigate its phenotypic suitability as a model for AS, we employed a battery of behavioral tests directed to reveal AS pathology and to find out whether this model better mirrors AS development compared to other available models. We found that the maternally inherited Ube3a-deficient line exhibits robust motor dysfunction, as seen in the rotarod and DigiGait tests, and displays abnormalities in additional behavioral paradigms, including reduced nest building and hypoactivity, although no apparent cognitive phenotype was observed in the Barnes maze and novel object recognition tests. The AS mice did, however, underperform in more complex cognition tasks, such as place reversal in the IntelliCage system, and exhibited a different circadian rhythm activity pattern. We show that the novel UBE3A-deficient model, based on a whole-gene deletion, is suitable for AS research, as it recapitulates important phenotypes characteristic of AS. This new mouse model provides complementary possibilities to study the Ube3a gene and its function in health and disease as well as possible therapeutic interventions to restore function.

Low bone mass and impaired fracture healing in mouse models of Trisomy21 (Down syndrome).

Individuals with Down syndrome (DS), the result of trisomy of human chromosome Hsa21 (Ts21), present with an array of skeletal abnormalities typified by altered craniofacial features, short stature and low bone mineral density (BMD). While bone deficits progress with age in both sexes, low bone mass is more pronounced in DS men than women and osteopenia appears earlier. In the current study, the reproductive hormone status (FSH, LH, testosterone) of 17 DS patients (males, ages range 19-52years) was measured. Although testosterone was consistently low, the hypothalamic-pituitary-gonadal axis was intact with corresponding rises in FSH and LH. To provide further insight into the heterogeneity of the bone mass in DS, the skeletal phenotypes of three of the most used murine DS models, Ts65Dn (Ts65), TC1, and Dp16(Yey1) (Dp16) were characterized and contrasted. Evaluation of the bone phenotype of both male and female 3-month-old Dp16 mice demonstrated sexual dimorphism, with low bone mass apparent in males, as it is in Ts65, but not in female Dp16. In contrast, male TC1 mice had no apparent bone phenotype. To determine whether low bone mass in DS impacted fracture healing, fractures of the middle phalanx (P2) digits were generated in both male and female Dp16 mice at 15weeks of age, an age where the sexually dimorphic low BMD persisted. Fracture healing was assessed via in vivo microCT over (13weeks) 93days post fracture (DPF). At 93 DPF, 0% of DS male (n=12) or female (n=8) fractures healed, compared to 50% of the male (n=28) or female (n=8) WT littermate fractures. MicroCT revealed periosteal unbridged mineralized callus formation across the fracture gap in Dp16 mice, which was confirmed by subsequent histology. These studies provide the first direct evidence of significantly impaired fracture healing in the setting of DS.

PSMC1 variant causes a novel neurological syndrome.

Proteasome 26S, the eukaryotic proteasome, serves as the machinery for cellular protein degradation. It is composed of the 20S core particle and one or two 19S regulatory particles, composed of a base and a lid. To date, several human diseases have been associated with mutations within the 26S proteasome subunits; only one of them affects a base subunit. We now delineate an autosomal recessive syndrome of failure to thrive, severe developmental delay and intellectual disability, spastic tetraplegia with central hypotonia, chorea, hearing loss, micropenis and undescended testes, as well as mild elevation of liver enzymes. None of the affected individuals achieved verbal communication or ambulation. Ventriculomegaly was evident on MRI. Homozygosity mapping combined with exome sequencing revealed a disease‐associated p.I328T PSMC1 variant. Protein modeling demonstrated that the PSMC1 variant is located at the highly conserved putative ATP binding and hydrolysis domain, and is suggested to interrupt a hydrophobic core within the protein. Fruit flies in which we silenced the Drosophila ortholog Rpt2 specifically in the eye exhibited an apparent phenotype that was highly rescued by the human wild‐type PSMC1, yet only partly by the mutant PSMC1, proving the functional effect of the p.I328T disease‐causing variant.

Cystic fibrosis transmembrane regulator correction attenuates heart failure-induced lung inflammation.

Heart failure (HF) affects 64 million people worldwide. Despite advancements in prevention and therapy, quality of life remains poor for many HF patients due to associated target organ damage. Pulmonary manifestations of HF are well-established. However, difficulties in the treatment of HF patients with chronic lung phenotypes remain as the underlying patho-mechanistic links are still incompletely understood. Here, we aim to investigate the cystic fibrosis transmembrane regulator (CFTR) involvement in lung inflammation during HF, a concept that may provide new mechanism-based therapies for HF patients with pulmonary complications. In a mouse model of HF, pharmacological CFTR corrector therapy (Lumacaftor (Lum)) was applied systemically or lung-specifically for 2 weeks, and the lungs were analyzed using histology, flow cytometry, western blotting, and qPCR. Experimental HF associated with an apparent lung phenotype characterized by vascular inflammation and remodeling, pronounced tissue inflammation as evidenced by infiltration of pro-inflammatory monocytes, and a reduction of pulmonary CFTR+ cells. Moreover, the elevation of a classically-activated phenotype of non-alveolar macrophages coincided with a cell-specific reduction of CFTR expression. Pharmacological correction of CFTR with Lum mitigated the HF-induced downregulation of pulmonary CFTR expression and increased the proportion of CFTR+ cells in the lung. Lum treatment diminished the HF-associated elevation of classically-activated non-alveolar macrophages, while promoting an alternatively-activated macrophage phenotype within the lungs. Collectively, our data suggest that downregulation of CFTR in the HF lung extends to non-alveolar macrophages with consequences for tissue inflammation and vascular structure. Pharmacological CFTR correction possesses the capacity to alleviate HF-associated lung inflammation.

AAV-mediated delivery of osteoblast/osteoclast-regulating miRNAs for osteoporosis therapy

Osteoporosis occurs due to a dysregulation in bone remodeling, a process requiring both bone-forming osteoblasts and bone-resorbing osteoclasts. Current leading osteoporosis therapies suppress osteoclast-mediated bone resorption but show limited therapeutic effects because osteoblast-mediated bone formation decreases concurrently. We developed a gene therapy strategy for osteoporosis that simultaneously promotes bone formation and suppresses bone resorption by targeting two microRNAs (miRNAs)—miR-214-3p and miR-34a-5p. We modulated the expression of these miRNAs using systemically delivered recombinant adeno-associated viral (rAAV) vectors targeting the bone. rAAV-mediated overexpression of miR-214-3p or inhibition of miR-34a-5p in the skeleton resulted in bone loss in adult mice, resembling osteoporotic bones. Conversely, rAAV-mediated inhibition of miR-214-3p or overexpression of miR-34a-5p reversed bone loss in mouse models for postmenopausal and senile osteoporosis by increasing osteoblast-mediated bone formation and decreasing osteoclast-mediated bone resorption. Notably, these mice did not show any apparent pathological phenotypes in non-skeletal tissues. Mechanistically, inhibiting miR-214-3p upregulated activating transcription factor 4 in osteoblasts and phatase and tensin homolog in osteoclasts, while overexpressing miR-34a-5p downregulated Notch1 in osteoblasts and TGF-β-induced factor homeobox 2 in osteoclasts. In summary, bone-targeting rAAV-mediated regulation of miR-214-3p or miR-34a-5p is a promising new approach to treat osteoporosis, while limiting adverse effects in non-skeletal tissues.

Remission of obesity and insulin resistance is not sufficient to restore mitochondrial homeostasis in visceral adipose tissue

Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, proteomic, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most obesity and overnutrition-related pathological features were successfully reverted, we observed a high degree of metabolic dysfunction in visceral white adipose tissue, characterized by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and an apparent global healthy phenotype, obesity triggered a cascade of events in visceral adipose tissue progressing from mitochondrial metabolic and proteostatic alterations to widespread cellular stress, which compromises its biosynthetic and recycling capacity. In humans, weight loss after bariatric surgery showed a transcriptional signature in visceral adipose tissue similar to our mouse model of obesity reversion. Overall, our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in visceral adipose tissue.

PB2295: EVANS SYNDROME AND PHENOTYPIC HETEROGENEITY OF SASH3 GERMLINE LOSS-OF-FUNCTION MUTATIONS BEYOND X-LINKED IMMUNODEFICIENCY

Background: “Evans syndrome (ES)” denotes a heterogeneous disease entity, defined by two or more autoimmune cytopenias, due to mostly unknown pathogenic mechanisms. Further, ES is increasingly recognized in pediatric patients with defined inborn errors of immunity (IEIs) including pathogenic variants in CTLA4, STAT3, TNFRSF6, PIK3CD, and KRAS. Loss-of-function mutations in Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3) have recently been reported as a novel IEI entity in four patients with X-linked combined immunodeficiency with immune dysregulation including immune cytopenias. SASH3 is an X-chromosomal encoded adaptor protein that is exclusively expressed in lymphocytes. Although knowledge of the exact molecular function of this protein is scarce, humans and mice lacking expression of SASH3 show defects in proximal and distal T-cell receptor signalling. Aims: We aimed to dissect aberrant immune cell functionality in SASH3-deficient patients to further enhance our understanding of pathogenic mechanisms in patients with ES. Methods: We performed whole exome sequencing, deep clinical phenotyping and immunophenotyping in a patient with ES and his clinically asymptomatic brother. Results: Our index patient presented with severe ES and recurrent infections at the age of 8 years. Immune phenotyping of his peripheral mononuclear cells (PBMCs) showed transient CD4+ T- and B-cell lymphopenia and reduced T-cell proliferation upon PHA and SEB stimulation. Furthermore, the patient had hypogammaglobulinemia, and the ‘transitory CD21 low B-cells’ were significantly increased, reflecting impaired lymphocyte maturation possibly causing the autoreactive phenotype of the disease. Interestingly, the patient developed large hemorrhagic splenic cysts during ITP associated bleeding diathesis, and thus underwent splenectomy, which led to a complete remission of ES. Using whole-exome sequencing, we discovered a germline nonsense mutation in SASH3 (c.862C>T;p.Arg288Ter) in a now 16 years-old patient with ES. Surprisingly, this maternally inherited mutation was also identified in the hitherto clinically inconspicuous younger brother. The identified mutation is identical to the one reported in two males in the discovery of SASH3 deficiency. Divergently, the now 8-year-old brother who is also carrying the same variant has no clinically apparent phenotype until now. Notably, disease onset occurred earlier (i.e., between age 2 and 5 years) in the previously reported patients compared to our patients. Summary/Conclusion: We present the 5th and 6th patient with germline hemizygous deleterious variants in SASH3. Thus, this study expands the clinical phenotype and underlines a marked phenotypic heterogeneity as illustrated in siblings bearing the same mutation. Future studies will need to address comprehensively the phenotypic spectrum of the disease and genetic and non-genetic modifiers of clinical phenotypes and severity.

Hypoxia-inducible factor expression is related to apoptosis and cartilage degradation in temporomandibular joint osteoarthritis

BackgroundIt remains unclear etiology of cartilaginous tissues in osteoarthritis (OA) lesions. In this study, we hypothesized the accumulation of hypoxia-inducible factor (HIF) and activated apoptosis relate to condylar cartilage degeneration in vivo.MethodsMalocclusion stress was applied for 2 weeks, 4 weeks and 8 weeks to induce an OA-like lesion animal model in rats. Histological analysis was performed by H&E staining and Safranin O/fast green staining. The expression levels of protein in condylar cartilage were examined by immunostaining to evaluate cartilage degeneration.ResultsWe found apparent histological phenotypes associated with degeneration in the occlusion disorder (OD) stress group. The OD group at 4 weeks and 8 weeks had obviously reduced expression of Aggrecan (Acan) and type II collagen (Col II) in cartilage. In contrast, the OD groups had higher levels of ADAM metallopeptidase with thrombospondin type 5 (ADAMTS5) and matrix metallopeptidase 13 (MMP13) in the condylar cartilage than the control group. Moreover, the OD group cartilage had prominent degenerative changes with reduced levels of hypoxia inducible factor 1 alpha (HIF1α) and increased levels of hypoxia inducible factor 2 alpha (HIF2α) and the apoptosis factor Caspase3 in condylar cartilage at 8 weeks.ConclusionThus, abnormal hypoxic conditions inducing Occlusion disorder stress results in cartilage degeneration. opposite expression patterns of HIF1α and HIF2α could be involved in the pathogenesis of condylar cartilage degeneration and chondrocyte apoptosis. HIF2α may provide a potential negative feedback mechanism for HIF1α during cartilage damage.

A Cross-Tissue Investigation of Molecular Targets and Physiological Functions of Nsun6 Using Knockout Mice.

The 5-methylcytosine (m5C) modification on an mRNA molecule is deposited by Nsun2 and its paralog Nsun6. While the physiological functions of Nsun2 have been carefully studied using gene knockout (KO) mice, the physiological functions of Nsun6 remain elusive. In this study, we generated an Nsun6-KO mouse strain, which exhibited no apparent phenotype in both the development and adult stages as compared to wild-type mice. Taking advantage of this mouse strain, we identified 80 high-confident Nsun6-dependent m5C sites by mRNA bisulfite sequencing in five different tissues and systematically analyzed the transcriptomic phenotypes of Nsun6-KO tissues by mRNA sequencing. Our data indicated that Nsun6 is not required for the homeostasis of these organs under laboratory housing conditions, but its loss may affect immune response in the spleen and oxidoreductive reaction in the liver under certain conditions. Additionally, we further investigated T-cell-dependent B cell activation in KO mice and found that Nsun6 is not essential for the germinal center B cell formation but is associated with the formation of antibody-secreting plasma cells. Finally, we found that Nsun6-mediated m5C modification does not have any evident influence on the stability of Nsun6 target mRNAs, suggesting that Nsun6-KO-induced phenotypes may be associated with other functions of the m5C modification or Nsun6 protein.

Differential Etv2 threshold requirement for endothelial and erythropoietic development.

SUMMARYEndothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.

Comparative Analysis of L-Fucose Utilization and Its Impact on Growth and Survival of Campylobacter Isolates

Campylobacter jejuni and Campylobacter coli were previously considered asaccharolytic, but are now known to possess specific saccharide metabolization pathways, including L-fucose. To investigate the influence of the L-fucose utilization cluster on Campylobacter growth, survival and metabolism, we performed comparative genotyping and phenotyping of the C. jejuni reference isolate NCTC11168 (human isolate), C. jejuni Ca1352 (chicken meat isolate), C. jejuni Ca2426 (sheep manure isolate), and C. coli Ca0121 (pig manure isolate), that all possess the L-fucose utilization cluster. All isolates showed enhanced survival and prolonged spiral cell morphology in aging cultures up to day seven in L-fucose-enriched MEMα medium (MEMαF) compared to MEMα. HPLC analysis indicated L-fucose utilization linked to acetate, lactate, pyruvate and succinate production, confirming the activation of the L-fucose pathway in these isolates and its impact on general metabolism. Highest consumption of L-fucose by C. coli Ca0121 is conceivably linked to its enhanced growth performance up to day 7, reaching 9.3 log CFU/ml compared to approximately 8.3 log CFU/ml for the C. jejuni isolates. Genetic analysis of the respective L-fucose clusters revealed several differences, including a 1 bp deletion in the Cj0489 gene of C. jejuni NCTC11168, causing a frameshift in this isolate resulting in two separate genes, Cj0489 and Cj0490, while no apparent phenotype could be linked to the presumed frameshift in this isolate. Additionally, we found that the L-fucose cluster of C. coli Ca0121 was most distant from C. jejuni NCTC11168, but confirmation of links to L-fucose metabolism associated phenotypic traits in C. coli versus C. jejuni isolates requires further studies.

Characterization of eosinophilic esophagitis variants by clinical, histological, and molecular analyses: A cross-sectional multi-center study.

ObjectivePhysicians are increasingly confronted with patients presenting with symptoms of esophageal dysfunction resembling eosinophilic esophagitis (EoE), but absence of significant esophageal eosinophilia. The purpose of this study was to characterize and classify this group of EoE variants.DesignPatients from six EoE‐centers with symptoms of esophageal dysfunction, but peak eosinophil counts of <60/mm2 (<15/hpf) in esophageal biopsies and absence of gastro‐esophageal reflux disease (GERD) were included. Clinical, endoscopic, (immuno)‐histological, and molecular features were determined and compared with EoE, GERD, and healthy controls.ResultsWe included 69 patients with EoE variants. Endoscopic abnormalities were found in 53.6%. We identified three histological subtypes: EoE‐like esophagitis (36/69, 52.2%), lymphocytic esophagitis (14/69, 20.3%), and non‐specific esophagitis (19/69, 27.5%). Immunohistochemistry revealed—in contrast to EoE—no significant increase in inflammatory cell infiltrates compared with GERD and healthy controls, except for lymphocytes in lymphocytic esophagitis. EoE‐typical Th2‐response was absent in all EoE variants. However, considerable structural changes were detected based on histology and protein expression. Using next generation mRNA sequencing, we found the three EoE variants to have distinct molecular fingerprints partially sharing pronounced traits of EoE. Hierarchical sample clustering of RNA sequencing data confirmed the presence of an EoE‐like (characterized by eotaxin‐3 expression), non‐specific, and lymphocytic variant cluster (characterized by CD3 cells and TSLP expression).ConclusionAll EoE variants are clinically and histologically active conditions despite the absence of esophageal eosinophilia. EoE variants appear to be part of a disease spectrum, where classical EoE represents the most common and apparent phenotype.

Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates

Bioactive oxylipins play multiple roles during inflammation and in the immune response, with termination of their actions partly dependent on the activity of yet-to-be characterized dehydrogenases. Here, we report that human microsomal dehydrogenase reductase 9 (DHRS9, also known as SDR9C4 of the short-chain dehydrogenase/reductase (SDR) superfamily) exhibits a robust oxidative activity toward oxylipins with hydroxyl groups located at carbons C9 and C13 of octadecanoids, C12 and C15 carbons of eicosanoids, and C14 carbon of docosanoids. DHRS9/SDR9C4 is also active toward lipid inflammatory mediator dihydroxylated Leukotriene B4 and proresolving mediators such as tri-hydroxylated Resolvin D1 and Lipoxin A4, although notably, with lack of activity on the 15-hydroxyl of prostaglandins. We also found that the SDR enzymes phylogenetically related to DHRS9, i.e., human SDR9C8 (or retinol dehydrogenase 16), the rat SDR9C family member known as retinol dehydrogenase 7, and the mouse ortholog of human DHRS9 display similar activity toward oxylipin substrates. Mice deficient in DHRS9 protein are viable, fertile, and display no apparent phenotype under normal conditions. However, the oxidative activity of microsomal membranes from the skin, lung, and trachea of Dhrs9−/− mice toward 1 μM Leukotriene B4 is 1.7- to 6-fold lower than that of microsomes from wild-type littermates. In addition, the oxidative activity toward 1 μM Resolvin D1 is reduced by about 2.5-fold with DHRS9-null microsomes from the skin and trachea. These results strongly suggest that DHRS9 might play an important role in the metabolism of a wide range of bioactive oxylipins in vivo.