Macro-based collagen quantification and segmentation in picrosirius red-stained heart sections using light microscopy.

Picrosirius red staining constitutes an important and broadly used tool to visualize collagen and fibrosis in various tissues. Although multiple qualitative and quantitative analysis methods to evaluate fibrosis are available, many require specialized devices and software or lack objectivity and scalability. Here, we aimed to develop a versatile and powerful "QuantSeg" macro in the FIJI image processing software capable of automated, robust, and quick collagen quantification in cardiac tissue from light micrographs. To examine different patterns of fibrosis, an optional segmentation algorithm was implemented. To ensure the method's validity, we quantified the collagen content in a set of wild-type versus plakoglobin-knockout murine hearts exhibiting extensive fibrosis using both the macro and an established, fluorescence microscopy-based method, and compared results. To demonstrate the capabilities of the segmentation feature, rat hearts were examined post-myocardial infarction. We found the QuantSeg macro to robustly detect the differences in fibrosis between knockout and control hearts. In sections with low collagen content, the macro yielded more consistent results than using the fluorescence microscopy-based technique. With its wide range of output parameters, ease of use, cost effectiveness, and objectivity, the QuantSeg macro has the potential to become an established method for analysis of PSR-stained tissue. The novel segmentation feature allows for automated evaluation of different patterns of cardiac fibrosis for the first time.

Novel Ca-Chelating Peptides from Protein Hydrolysate of Antarctic Krill (Euphausia superba): Preparation, Characterization, and Calcium Absorption Efficiency in Caco-2 Cell Monolayer Model.

Antarctic krill (Euphausia superba) is the world's largest resource of animal proteins and is thought to be a high-quality resource for future marine healthy foods and functional products. Therefore, Antarctic krill was degreased and separately hydrolyzed using flavourzyme, pepsin, papain, and alcalase. Protein hydrolysate (AKH) of Antarctic krill prepared by trypsin showed the highest Ca-chelating rate under the optimized chelating conditions: a pH of 8.0, reaction time of 50 min, temperature of 50 °C, and material/calcium ratio of 1:15. Subsequently, fourteen Ca-chelating peptides were isolated from APK by ultrafiltration and a series of chromatographic methods and identified as AK, EAR, AEA, VERG, VAS, GPK, SP, GPKG, APRGH, GVPG, LEPGP, LEKGA, FPPGR, and GEPG with molecular weights of 217.27, 374.40, 289.29, 459.50, 275.30, 300.36, 202.21, 357.41, 536.59, 328.37, 511.58, 516.60, 572.66, and 358.35 Da, respectively. Among fourteen Ca-chelating peptides, VERG presented the highest Ca-chelating ability. Ultraviolet spectrum (UV), Fourier Transform Infrared (FTIR), and scanning electron microscope (SEM) analysis indicated that the VERG-Ca chelate had a dense granular structure because the N-H, C=O and -COOH groups of VERG combined with Ca2+. Moreover, the VERG-Ca chelate is stable in gastrointestinal digestion and can significantly improve Ca transport in Caco-2 cell monolayer experiments, but phytate could significantly reduce the absorption of Ca derived from the VERG-Ca chelate. Therefore, Ca-chelating peptides from protein hydrolysate of Antarctic krill possess the potential to serve as a Ca supplement in developing healthy foods.

Mechanistic Insights of the LEMD2 p.L13R Mutation and Its Role in Cardiomyopathy.

BACKGROUND: Nuclear envelope proteins play an important role in the pathogenesis of hereditary cardiomyopathies. Recently, a new form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane protein LEMD2 was discovered. The aim was to unravel the molecular mechanisms of mutant LEMD2 in the pathogenesis of cardiomyopathy. METHODS: We generated a Lemd2 p.L13R knock-in mouse model and a corresponding cell model via CRISPR/Cas9 technology and investigated the cardiac phenotype as well as cellular and subcellular mechanisms of nuclear membrane rupture and repair. RESULTS: Knock-in mice developed a cardiomyopathy with predominantly endocardial fibrosis, left ventricular dilatation, and systolic dysfunction. Electrocardiograms displayed pronounced ventricular arrhythmias and conduction disease. A key finding of knock-in cardiomyocytes on ultrastructural level was a significant increase in nuclear membrane invaginations and decreased nuclear circularity. Furthermore, increased DNA damage and premature senescence were detected as the underlying cause of fibrotic and inflammatory remodeling. As the p.L13R mutation is located in the Lap2/Emerin/Man1 (LEM)-domain, we observed a disrupted interaction between mutant LEMD2 and BAF (barrier-to-autointegration factor), which is required to initiate the nuclear envelope rupture repair process. To mimic increased mechanical stress with subsequent nuclear envelope ruptures, we investigated mutant HeLa-cells upon electrical stimulation and increased stiffness. Here, we demonstrated impaired nuclear envelope rupture repair capacity, subsequent cytoplasmic leakage of the DNA repair factor KU80 along with increased DNA damage, and recruitment of the cGAS (cyclic GMP-AMP synthase) to the nuclear membrane and micronuclei. CONCLUSIONS: We show for the first time that the Lemd2 p.L13R mutation in mice recapitulates human dilated cardiomyopathy with fibrosis and severe ventricular arrhythmias. Impaired nuclear envelope rupture repair capacity resulted in increased DNA damage and activation of the cGAS/STING/IFN pathway, promoting premature senescence. Hence, LEMD2 is a new player inthe disease group of laminopathies.

Hypertriglyceridemic waist phenotype and risk of chronic kidney disease in community-dwelling adults aged 60 years and older in Tianjin, China: a 7-year cohort study.

BACKGROUND: The hypertriglyceridemic waist (HTGW) phenotype has been proposed to be related to the occurrence and progression of chronic kidney disease (CKD). The ageing trend of the Chinese population continues to intensify, and elderly individuals are at high risk of CKD. The purpose of this study was to investigate the cross-sectional and longitudinal associations between the HTGW phenotype and the risk of CKD by following community-dwelling adults aged 60 years and older in Tianjin, China, for 7 years. METHODS: This study was an observational cohort study conducted between 2013 and 2019. Of 2050 participants aged 60 years and older who underwent an annual health examination in 2013, 1605 individuals with complete data were enrolled in the cross-sectional analysis. Among them, 1271 individuals were observed until 2019. Detailed follow-up records were available for 816 participants, of whom 600 participants without CKD at baseline were eligible for inclusion in the retrospective analysis. The HTGW phenotype was defined as a waist circumference of 90 cm or more and triglyceride concentrations of 2.0 mmol/L or more in males or a waist circumference of 85 cm or more and triglyceride concentrations of 1.5 mmol/L or more in females. CKD was defined as an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73m2 and/or proteinuria (urinary albumin-to-creatinine ratio (ACR) ≥ 30 mg/g). Multivariable logistic regression analyses were performed to evaluate the relationship between the HTGW phenotype and CKD. RESULTS: In 2013, the prevalence of CKD among older adults was 31.03%, and the prevalence of CKD in the HTGW phenotype group was 37.81%. Over a 7-year observation period, 195 individuals developed CKD, with an incidence rate of 32.50%. Statistically significant associations were observed between the HTGW phenotype and CKD in older adults in both cross-sectional surveys and retrospective analyses, with odds ratios and 95% confidence intervals of 1.38 (95% CI: 1.03-1.86, P = 0.033) and 2.27 (95% CI: 1.30-3.97, P = 0.004), respectively, after adjustment for confounders. CONCLUSIONS: In this community-based cohort study, the HTGW phenotype was confirmed to be independently associated with an increased risk of prevalent and incident CKD in older adults aged 60 years and above in Tianjin, China.

Sestrin1 exerts a cytoprotective role against oxygen-glucose deprivation/reoxygenation-induced neuronal injury by potentiating Nrf2 activation via the modulation of Keap1.

Sestrin1 (Sesn1) acts as a stress-inducible protein that performs a remarkable cytoprotective function upon diverse cellular stresses. However, whether Sesn1 exerts a cytoprotective role in neurons following cerebral ischemia/reperfusion injury is unknown. The goal of this work was to evaluate the role of Sesn1 in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury in vitro. The induction of Sesn1 was found in neurons exposed to OGD/R treatment. The silencing of Sesn1 rendered neurons more vulnerable to OGD/R injury, while the up-regulation of Sesn1 ameliorated OGD/R-induced neuronal injury by reducing apoptosis and the generation of reactive oxygen species (ROS). Furthermore, the up-regulation of Sesn1 promoted the activity of the nuclear factor-erythroid 2-related factor 2 (Nrf2) by down-regulating the expression of the Kelchlike ECH-associated protein 1 (Keap1). The restoration of Keap1 or the suppression of Nrf2 remarkably abolished the Sesn1-induced neuroprotection effects in OGD/R-exposed neurons. In summary, our work indicates that Sesn1 is a remarkable neuroprotective protein that potentiates Nrf2 activation via Keap1 to ameliorate OGD/R-induced injury.

FBW7 Mediates Senescence and Pulmonary Fibrosis through Telomere Uncapping.

Tissue stem cells undergo premature senescence under stress, promoting age-related diseases; however, the associated mechanisms remain unclear. Here, we report that in response to radiation, oxidative stress, or bleomycin, the E3 ubiquitin ligase FBW7 mediates cell senescence and tissue fibrosis through telomere uncapping. FBW7 binding to telomere protection protein 1 (TPP1) facilitates TPP1 multisite polyubiquitination and accelerates degradation, triggering telomere uncapping and DNA damage response. Overexpressing TPP1 or inhibiting FBW7 by genetic ablation, epigenetic interference, or peptidomimetic telomere dysfunction inhibitor (TELODIN) reduces telomere uncapping and shortening, expanding the pulmonary alveolar AEC2 stem cell population in mice. TELODIN, synthesized from the seventh ß strand blade of FBW7 WD40 propeller domain, increases TPP1 stability, lung respiratory function, and resistance to senescence and fibrosis in animals chronically exposed to environmental stress. Our findings elucidate a pivotal mechanism underlying stress-induced pulmonary epithelial stem cell senescence and fibrosis, providing a framework for aging-related disorder interventions.

Generation of two patient-derived iPSC lines from siblings (LIBUCi001-A and LIBUCi002-A) and a genetically modified iPSC line (JMUi001-A-1) to mimic dilated cardiomyopathy with ataxia (DCMA) caused by a homozygous DNAJC19 mutation.

Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from mutations in DNAJC19. Two patient-derived dermal fibroblast cell lines of siblings with the same homozygous splice acceptor site mutation in DNAJC19 (NM_145261.4):c.130-1G>C were reprogrammed into induced pluripotent stem cell (iPSC) lines (LIBUCi001-A and LIBUCi002-A) using non-integrative Sendai virus. Additionally, a third DNAJC19tv (truncation variant) iPSC line (JMUi001-A-1) was generated by CRISPR/Cas9 in healthy control iPSCs (JMUi001-A). All three DCMA iPSC lines present normal karyotypes, high expression of pluripotency markers and the capacity to differentiate into cells of all three germ layers.

Plasma triglyceride levels and central obesity predict the development of kidney injury in Chinese community older adults.

Objective: Despite the achievement of blood glucose, blood pressure targets, the risk for kidney injury remains high among older adults. This observational retrospective study investigated whether high TG or high WC contribute to this high residual risk for kidney injury. Methods: A total of 843 elderly from Dongli Community, Tianjin, China, we selected 666 individuals with a baseline estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m2 and negative microalbuminuria completing a 3-year follow-up. At baseline, subjects were grouped according to the levels of TG and WC. The primary outcome was the incidence of kidney injury, defined as low eGFR (eGFR <60 mL/min/1.73 m2) or reduced eGFR (eGFR reduced >25%) or UACR ≥30 mg/g. Results: Overall, 6.01% developed low eGFR, 11.11% reduced eGFR, 25.98% UACR ≥30 mg/g, and 3.45% low eGFR and UACR ≥30mg/g after 3-year follow-up. TG ≥1.7 mmol/L increased the risk of eGFR <60 mL/min/1.73 m2 by 1.44-fold, of UACR ≥30 mg/g by 32%, and of developing both abnormality by 1.41-fold in model 1; further adjustment for potential confounders factors, the association is slightly weakened in model 2 and 3; WC (≥90 cm in men and ≥85 cm in women) were associated with a 1.68-fold higher risk of eGFR <60 mL/min/1.73 m2 and a 1.43-fold risk of UACR ≥30 mg/g and a 1.89-fold risk of developing both abnormality in model 1. Further adjustment for potential confounders factors, the association is slightly weakened in model 2 and 3. Conclusions: In a population of Chinese community-dwelling older adults, high TG and central obesity were risk factors for the development of kidney injury over 3 years.

Characterization of a Unique Form of Arrhythmic Cardiomyopathy Caused by Recessive Mutation in LEMD2.

Nuclear envelope proteins have been shown to play an important role in the pathogenesis of inherited dilated cardiomyopathy. Here, we present a remarkable cardiac phenotype caused by a homozygous LEMD2 mutation in patients of the Hutterite population with juvenile cataract. Mutation carriers develop arrhythmic cardiomyopathy with mild impairment of left ventricular systolic function but severe ventricular arrhythmias leading to sudden cardiac death. Affected cardiac tissue from a deceased patient and fibroblasts exhibit elongated nuclei with abnormal condensed heterochromatin at the periphery. The patient fibroblasts demonstrate cellular senescence and reduced proliferation capacity, which may suggest an involvement of LEM domain containing protein 2 in chromatin remodeling processes and premature aging.

TGF-beta receptor mediated telomerase inhibition, telomere shortening and breast cancer cell senescence.

Human telomerase reverse transcriptase (hTERT) plays a central role in telomere lengthening for continuous cell proliferation, but it remains unclear how extracellular cues regulate telomerase lengthening of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces the hTERT gene repression in a BMPRII receptor- and Smad3-dependent manner in human breast cancer cells. Chonic exposure of human breast cancer cells to BMP7 results in short telomeres, cell senescence and apoptosis. Mutation of the BMPRII receptor, but not TGFbRII, ACTRIIA or ACTRIIB receptor, inhibits BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres and continued cell proliferation. Expression of hTERT prevents BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging by a mechanism involving BMPRII receptor- and Smad3-mediated repression of the hTERT gene.