Cell Function Research Articles

Golgi-restored vesicular replenishment retards bone aging and empowers aging bone regeneration

Healthy aging is a common goal for humanity and society, and one key to achieving it is the rejuvenation of senescent resident stem cells and empowerment of aging organ regeneration. However, the mechanistic understandings of stem cell senescence and the potential strategies to counteract it remain elusive. Here, we reveal that the aging bone microenvironment impairs the Golgi apparatus thus diminishing mesenchymal stem cell (MSC) function and regeneration. Interestingly, replenishment of cell aggregates-derived extracellular vesicles (CA-EVs) rescues Golgi dysfunction and empowers senescent MSCs through the Golgi regulatory protein Syntaxin 5. Importantly, in vivo administration of CA-EVs significantly enhanced the bone defect repair rate and improved bone mass in aging mice, suggesting their therapeutic value for treating age-related osteoporosis and promoting bone regeneration. Collectively, our findings provide insights into Golgi regulation in stem cell senescence and bone aging, which further highlight CA-EVs as a potential rejuvenative approach for aging bone regeneration.

Bioelectric stimulation outperforms brain derived neurotrophic factor in promoting neuronal maturation

Neuronal differentiation and maturation are crucial for developing research models and therapeutic applications. Brain-derived neurotrophic factor (BDNF) is a widely used biochemical stimulus for promoting neuronal maturation. However, the broad effects of biochemical stimuli on multiple cellular functions limit their applicability in both in vitro models and clinical settings. Electrical stimulation (ES) offers a promising physical method to control cell fate and function, but it is hampered by lack of standard and optimised protocols. In this study, we demonstrate that ES outperforms BDNF in promoting neuronal maturation in human neuroblastoma SH-SY5Y. Additionally, we address the question regarding which ES parameters regulate biological responses. The neuronal differentiation and maturation of SH-SY5Y cells were tested under several pulsed ES regimes. We identified accumulated charge and effective electric field time as novel criteria for determining optimal ES regimes. ES parameters were obtained using electrochemical characterisation and equivalent circuit modelling. Our findings show that neuronal maturation in SH-SY5Y cells correlates with the amount of accumulated charge during ES. Higher charge accumulation (~ 50 mC/h) significantly promotes extensive neurite outgrowth and ramification, and enhances the expression of synaptophysin, yielding effects exceeding those of BDNF. In contrast, fewer charge injection to the culture (~ 0.1 mC/h) minimally induces maturation but significantly increases cell proliferation. Moreover, ES altered the concentration and protein cargo of secreted extracellular vesicles (EV). ES with large enough accumulated charge significantly enriched EV proteome associated with neural development and function. These results demonstrate that each ES regime induces distinct cellular responses. Increased accumulated charge facilitates the development of complex neuronal morphologies and axonal ramification, outperforming exogenous neurotrophic factors. Controlled ES methods are immediately applicable in creating mature neuronal cultures in vitro with minimal chemical intervention.

Mesenchymal stem cells from perinatal tissues promote diabetic wound healing via PI3K/AKT activation

BackgroundDiabetic foot ulcers (DFUs) represent a major complication of diabetes, often leading to poor healing outcomes with conventional treatments. Mesenchymal stem cell (MSC) therapies have emerged as a promising alternative, given their potential to modulate various pathways involved in wound healing. This study evaluates and compares the therapeutic potential of MSCs derived from perinatal tissues—human umbilical cord MSCs (hUCMSCs), human chorionic villi MSCs (hCVMSCs), and human decidua basalis MSCs (hDCMSCs)—in a diabetic wound healing model.MethodsWe performed in vitro and in vivo studies to compare the efficacy of hUCMSCs, hCVMSCs, and hDCMSCs. Mass spectrometry was used to analyze the secreted proteins of the MSCs. We incorporated the MSCs into a polyethylene glycol diacrylate (PEGDA) and sodium alginate (SA) hydrogel matrix with collagen I (Col-I) to evaluate their effects on wound healing.ResultsAll three types of MSCs promoted wound healing, with hUCMSCs and hCVMSCs showing stronger effects compared to hDCMSCs. Both hUCMSCs and hCVMSCs demonstrated robust wound healing kinetics, with enhanced keratinocyte proliferation (KRT14+/Ki67+ cells), maturation (KRT10/KRT14 ratio), and angiogenesis. In vitro studies demonstrated that the MSC-derived secretome enhanced keratinocyte proliferation and migration, endothelial cell function and stem cell recruitment, indicating robust paracrine effects. Mass spectrometry revealed a conserved set of proteins including THBS1 (thrombospondin 1), SERPINE1 (serpin family E member 1), ANXA1 (annexin A1), LOX (lysyl oxidase), and ITGB1 (integrin beta-1) which are involved in extracellular matrix (ECM) organization and wound healing, with the PI3K/AKT signaling pathway playing a central role. The PEGDA/SA/Col-I hydrogel demonstrated a unique balance of mechanical and biological properties and an optimal environment for MSC viability and function. Application of either hUCMSC- or hCVMSC-laden hydrogels resulted in accelerated wound closure, improved re-epithelialization, increased collagen deposition, and enhanced vascularization in vivo. ConclusionsMSCs From perinatal tissues particularly hUCMSCs and hCVMSCs significantly enhance diabetic wound healing through PI3K/AKT pathway activation while hDCMSCs exhibited weaker efficacy. The PEGDA/SA/Col-I hydrogel supports MSC viability and function offering a promising scaffold for DFU treatment. These findings underscore the potential of specific perinatal MSCs and optimized hydrogel formulations in advancing diabetic wound care.

Type 1 Diabetes Genetic Risk Scores: History, Application and Future Directions.

To review the genetics of type 1 diabetes (T1D) and T1D genetic risk scores, focusing on their development, research and clinical applications, and future directions. More than 90 genetic loci have been linked to T1D risk, with approximately half of the genetic risk attributable to the human leukocyte antigen (HLA) locus, along with non-HLA loci that have smaller effects to disease risk. The practical use of T1D genetic risk scores simplifies the complex genetic information, within the HLA and non-HLA regions, by combining the additive effect and interactions of single nucleotide polymorphisms (SNPs) associated with risk. Genetic risk scores have proven to be useful in various aspects, including classifying diabetes (e.g., distinguishing between T1D vs. neonatal, type 2 or other diabetes types), predicting the risk of developing T1D, assessing the prognosis of the clinical course (e.g., determining the risk of developing insulin dependence and glycemic control), and research into the heterogeneity of diabetes (e.g., atypical diabetes). However, there are gaps in our current knowledge including the specific sets of genes that regulate transition between preclinical stages of T1D, response to disease modifying therapies, and other outcomes of interest such as persistence of beta cell function. Several T1D genetic risk scores have been developed and shown to be valuable in various contexts, from classification of diabetes to providing insights into its etiology and predicting T1D risk across different stages of T1D. Further research is needed to develop and validate T1D genetic risk scores that are effective across all populations and ancestries. Finally, barriers such as cost, and training of medical professionals have to be addressed before the use of genetic risk scores can be incorporated into routine clinical practice.

Waist-to-hip ratio better reflect beta-cell function and predicts diabetes risk in adult with overweight or obesity.

Controversy remains as to which obesity measures better predict type 2 diabetes (T2D) risk in overweight or obese individuals. The objective of this study is to determine which commonly used obesity measures better reflect beta cell function and predict T2D risk in participants with overweight or obesity and to validate the findings using prospective cohort data. Cross-sectional data from the Obesity Clinic of the Xiangya Hospital of the Central South University and prospective cohort from UK Biobank. BMI, waist circumference (WC), waist-to-hip ratio (WHpR), and waist-to-height ratio (WHtR) were measured. Primary outcomes included beta cell function indices in the cross-sectional study and the occurrence of diabetes obtained from UK Biobank data. One thousand four hundred and ninety-seven participants with overweight or obesity (median age 29 years, 41% males) and 322,023 UK Biobank participants without diabetes at baseline (mean age 56.83 years, 50.4% males) were studied. WHpR had a stronger association with beta cell function and central body fat distribution than the other three obesity measures irrespective of glucometabolic states. WHpR associated positively with diabetes risk in participants using the hazard ratio scale (HR per SD increase of WHpR, 2.311, 95% CI 2.250-2.374). WHpR is a superior index in reflecting central body obesity, estimating beta cell function, and predicting T2D risk in people with overweight or obesity compared to BMI, WC, and WHtR.

Single cell transcriptome profiling reveals pathogenesis of bullous pemphigoid

Bullous pemphigoid (BP) triggers profound functional changes in both immune and non-immune cells in the skin and circulation, though the underlying mechanisms remain unclear. In this study, we conduct single-cell transcriptome analysis of lesional and non-lesional skin, as well as blood samples from BP patients. In lesional skin, non-immune cells upregulate pathways related to metabolism, wound healing, immune activation, and cell migration. LAMP3+DCs from cDC2 show stronger pro-inflammatory signatures than those from cDC1, and VEGFA+ mast cells, crucial for BP progression, are predominantly in lesional skin. As BP patients transition from active to remission stages, blood B cell function shifts from differentiation and memory formation to increased type 1 interferon signaling and reduced IL-4 response. Blood CX3CR1+ ZNF683+ and LAG3+ exhausted T cells exhibit the highest TCR expansion among clones shared with skin CD8+T cells, suggesting their role in fueling skin CD8+T cell clonal expansion. Clinical BP severity correlates positively with blood NK cell IFN-γ production and negatively with amphiregulin (AREG) production. NK cell-derived AREG mitigates IFN-γ-induced keratinocyte apoptosis, suggesting a crucial balance between AREG and IFN-γ in BP progression. These findings highlight functional shifts in BP pathology and suggest potential therapeutic targets.

Regulation of NK cell development, maturation, and antitumor responses by the nuclear receptor NR2F6

Natural killer (NK) cell development and functionality rely on precise regulation by specific transcription factors (TFs). Our study demonstrates that the nuclear orphan receptor NR2F6 represses the expression of the activating receptor NKp46, an established key player in NK cell-mediated cytotoxicity during infection and tumor rejection. Despite normal NK cell development in the bone marrow, germline Nr2f6-deficient mice exhibit impaired terminal maturation of NK cells in the periphery. Short-term NK cell responses to lipopolysaccharide (LPS) activation, independent of NKp46, are subsequently reduced in Nr2f6-deficient mice. Conventional type 1 dendritic cells (cDC1) and macrophage populations are decreased in spleens of Nr2f6-deficient mice, subsequently, IL-15-dependent NK cell priming is limited. Administration of exogenous IL-15 in vitro and as IL-15 complex in vivo can compensate for these deficits, promoting terminal maturation of NK cells in Nr2f6-deficient mice. Subsequent transcriptome analysis reveals significant changes in gene expression profiles of NK cells from IL-15 complex treated Nr2f6-deficient mice, with notable alterations in essential NK genes such as Klrg1, Prdm1, Stat5a, Zeb2, and Prf1. Consequently, Nr2f6-deficient IL-15 complex-treated NK cells raise enhanced effector responses of IFNγ, Perforin, and Granzyme B upon ex vivo activation. Of importance, Nr2f6-deficient mice are protected against MHC-I negative B16-F10 melanoma lung metastasis formation, especially with IL-15 complex treatment, indicating the potential of NR2F6 to affect NKp46-dependent NK cell-mediated tumor surveillance. The therapeutic targeting of NR2F6 may be a promising strategy for boosting NKp46-dependent NK-cell-mediated tumor surveillance and metastasis.

Transcriptomic profiles of single-cell autophagy-related genes (ATGs) in lung diseases

Autophagy related genes (ATGs) play essential roles in maintaining cellular functions, although biological and pathological alterations of ATG phenotypes remain poorly understood. To address this knowledge gap, we utilized the single-cell sequencing technology to elucidate the transcriptomic atlas of ATGs in lung diseases, with a focus on lung epithelium and lymphocytes. This study conducted a comprehensive investigation into RNA profiles of ATGs in the lung tissues obtained from healthy subjects and patients with different lung diseases through single-cell RNA sequencing (scRNA-seq), including COVID-19 related acute lung damage, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), systemic sclerosis (SSC), and lung adenocarcinoma (LUAD). Our findings revealed significant variations of ATGs expression across lung epithelial cell subsets, e.g., over-expression of MAPK8 in basal cells, ATG10 in club cells, and BCL2 in a goblet cell subset. The changes of autophagy-related pathways varied between lung epithelial and lymphocyte subsets. We identified the disease-associated changes in ATG expression, including significant alterations in BCL2, BCL2L1, PRKCD, and PRKCQ in inflammatory lung diseases (COPD and IPF), and MAP2K7, MAPK3, and RHEB in lung cancer (LUAD), as compared to normal lung tissues. Key ligand-receptor pairs (e.g., CD6-ALCAM, CD99-CD99) and signaling pathways (e.g., APP, CD74) might serve as biomarkers for lung diseases. To evaluate ATGs responses to external challenges, we examined ATGs expression in different epithelial cell lines exposed to cigarette smoking extract (CSE), lysophosphatidylcholine (lysoPC), lipopolysaccharide (LPS), and cholesterol at various doses and durations. Notable changes were observed in CFLAR, EIF2S1, PPP2CA, and PPP2CB in A549 and H1299 against CSE and LPS. The heterogeneity of ATGs expression was dependent on cell subsets, pathologic conditions, and challenges, as well as varied among cellular phenotypes, functions, and behaviors, and the severity of lung diseases. In conclusion, our data might provide new insights into the roles of ATGs in epithelial biology and pulmonary disease pathogenesis, with implications for disease progression and prognosis.Graphical 1. The transcriptomic profiles of autophagy-related genes (ATGs) varied among pulmonary epithelial and lymphocyte subsets in lung diseases.2. ATGs play essential roles in the maintenance of pulmonary cell functions and intercellular interactions between lung epithelia and lymphocytes by reprogramming the metabolisms and the energy balance.3. ATGs can be the candidates of biology-specific biomarkers and therapeutic targets for pulmonary diseases.

Caffeic acid stimulates migration and invasion of human trophoblast HTR-8/SVneo cells.

The placenta is a transient organ essential for development of the fetus. Adequate invasion of trophoblast cells, specialized cells of the placenta, is of utmost importance for the establishment and maintenance of healthy pregnancy. Caffeic acid (CA), one of the most abundantly present hydroxycynamic acids in everyday human diet, exhibits various physiological effects such as antioxidant, anti-inflammatory and anticancer activities including an inhibitory effect on migration and invasion of different cancer cell types. There are not many studies on CA safety in human pregnancy. Therefore, the aim of this research was to investigate the potential of CA to affect trophoblast cell function. We evaluated adhesion, migration and invasion of human trophoblast HTR-8/SVneo cells following CA treatment by functional assays. Furthermore, expression of molecular mediators of these processes such as integrin α1, α5 and β1 subunits and matrix metalloproteinase (MMP)-2 and MMP-9 was evaluated at the mRNA level by qPCR and the protein level by cell-based ELISA assay or zymography. Our results showed that 24 h treatment with 10 μM CA stimulated migration and invasion of HTR-8/SVneo cells as well as expression of the integrin α1 subunit. Furthermore, treatment with 100 μM CA stimulated expression of MMP2 and MMP9 mRNA in the treated HTR-8/SVneo cells as well as secretion of MMP-9. According to obtained results, we can conclude that CA could have the potential to affect processes important for placentation. However, further research is needed to elucidate all aspects of potential CA effects on placental function and pregnancy as a whole.

Endoplasmic reticulum stress-related CLIP4 plays a procarcinogenic role in hepatocellular carcinoma: an integrated analysis

ObjectiveTo explore the potential of endoplasmic reticulum stress (ERS)-associated protein CLIP4 as a biomarker for hepatocellular carcinoma (HCC) and the underlying mechanism.MethodsTCGA public database and a tissue microarray were used to investigate the molecular characteristics of CLIP4 and its association with disease. TCGA-LIHC dataset was used for single-gene differential expression analysis, single-gene correlation analysis, functional enrichment analysis, immune infiltration analysis, and DNA methylation analysis. RNA-seq, immunohistochemistry, western blotting, and RT-qPCR were used to verify the effect of ERS on CLIP4 expression. Public databases and miRNA-seq data were used to explore the TF-miRNA-CLIP4 regulatory network. CCK-8, colony formation, EdU staining, wound-healing, Transwell, western blotting and RT-qPCR were used to detect the effects of CLIP4 on the proliferation, migration and epithelial-mesenchymal transition (EMT) of HCC cells.ResultsAnalysis of TCGA datasets and tissue microarrays demonstrated that elevated CLIP4 expression was associated with poor prognosis in HCC. Enrichment analysis revealed that CLIP4 is involved in the immune response, cell adhesion, and EMT. There was a positive correlation between CLIP4 expression and the infiltration of the majority of immune cells, immunomodulators, and chemokines. Furthermore, the DNA methylation pattern of CLIP4 was found to have significant prognostic value. ERS was found to significantly upregulate CLIP4 expression. In addition, the ERS-RELA-miR-222-5p-CLIP4 transcriptional network was constructed to clarify the role of CLIP4. Cell function experiments confirmed that it promotes the proliferation, migration, and EMT of HCC cells.ConclusionsCLIP4 is a potential immune-related oncogenic molecule in HCC. ERS regulates the expression of CLIP4, and CLIP4 promotes the proliferation, migration, and EMT of HCC cells.

Targeting MDM2 affects spastin protein levels and functions: implications for HSP treatment

Spastin is a microtubule (MT) severing enzyme that regulates several cell functions associated with MT dynamics. A reduction in spastin protein levels is responsible for approximately 40% of cases of Hereditary Spastic Paraplegia (HSP), a neurodegenerative disease. Currently, there is no cure for HSP but strategies to induce a recovery of spastin levels are emerging as potential therapeutic approaches. Here, we show that MDM2 interacts with spastin MT-interacting and trafficking (MIT) domain. By biochemical and functional experiments, we demonstrate that MDM2 binds spastin and regulates its levels in a post-transcriptional manner independently of the E3 ubiquitin ligase activity. Of relevance, treatment of spastin-deficient cells with the MDM2 inhibitor Nutlin-3a can restore spastin levels and functions, such as cytokinetic abscission and sorting of transferrin receptor. These findings identify MDM2 as a novel interactor of spastin and a potential druggable regulator of its protein levels.

Investigation of the role and mechanism of dapagliflozin in mitigating renal injury in rats afflicted with diabetic kidney disease.

The etiology of diabetic kidney disease (DKD) is multifaceted, with hyperglycemia, inflammation, oxidative stress, and fibrosis recognized as key contributors to renal damage in individuals with DKD. Clinical evidence suggests that dapagliflozin not only reduces blood glucose levels but also demonstrates superior efficacy in ameliorating pancreatic islet cell injury while preserving cardiac and renal function. However, the precise underlying mechanism has been poorly elucidated in the current literature. In this study, a DKD rat model was established by administering a single intraperitoneal injection of streptozotocin (STZ) to investigate the renoprotective properties of dapagliflozin and its underlying mechanisms. The findings of this study indicate that dapagliflozin enhanced pancreatic islet cell function, lowered blood glucose levels, and significantly reduced biochemical markers and renal pathological damage in DKD rats. Dapagliflozin also exerted anti-inflammatory, antioxidant, and antifibrotic effects by inhibiting the activation of the p38 MAPK/NF-κB pathway, enhancing the activity of the SIRT1/Akt/GSK-3β/Nrf2/HO-1 signaling pathway, and inhibiting the over-activation of the TGF-β1/Smad2/3 signaling pathway. These effects led to a reduction in renal injury and improved renal function in DKD rats.

Pigment epithelium-derived factor (PEDF) Promotes Survival and Contraction of Myoepithelial Cells in Lacrimal Gland.

PEDF is critical for general health of the ocular surface. In order to study mechanisms of PEDF's action in lacrimal gland (LG) secretion, these visual structures were studied in a PEDF deficient (Pedf-/-) mouse model using biochemical, histochemical, and morphometric analyses. In Pedf-/- animals there were several ocular surface and LG disturbances not seen in controls. Notably, changes in body and LG weight, corneal sensitivity, tear film, ocular surface damage, and size of acini comprising the LG were evident. Survival of myoepithelial cells (MECs) surrounding the acini showed a PEDF survival dependence as there were significant reduction in MEC-specific P63 cells and Bcl2 expression levels, and increased TUNEL positive cells in PEDF deficient mice. Expression levels of the major contractile MEC proteins, α-SMA, calponin, and keratin 14, were also reduced with PEDF gene deletion and MECs contraction apparatus impaired, since oxytocin significantly reduced acinar area in controls but had no effect in PEDF deficient LGs, although the oxytocin receptor (OXTR) was expressed in both PEDF genotypes. These findings suggest that PEDF is essential to MECs survival and contractile function, and tear homeostasis on the ocular surface. Treatment with PEDF is likely to alleviate ocular-related conditions in diseases associated with dry eye as well as promote healthy MEC cell function in other secretory glands of the body.

Modulation of the thiol redox proteome by sugarcane ash-derived silica nanoparticles: insights into chronic kidney disease of unknown etiology

IntroductionChronic kidney disease of unknown etiology (CKDu) is an epidemic which is increasingly prevalent among agricultural workers and nearby communities, particularly those involved in the harvest of sugarcane. While CKDu is likely multifactorial, occupational exposure to silica nanoparticles (SiNPs), a major constituent within sugarcane ash, has gained increased attention as a potential contributor. SiNPs have high potential for generation of reactive oxygen species (ROS), and their accumulation in kidney could result in oxidative stress induced kidney damage consistent with CKDu pathology.MethodsIn order to characterize the impact of sugarcane ash derived (SAD) SiNPs on human kidney proximal convoluted tubule (PCT) cells and identify potential mechanisms of toxicity, HK-2 cells were exposed to treatments of either pristine, manufactured, 200 nm SiNPs or SAD SiNPs and changes to cellular energy metabolism and redox state were determined. To determine how the cellular redox environment may influence PCT cell function and toxicity, the redox proteome was examined using cysteine-targeted click chemistry proteomics.ResultsPristine, 200 nm SiNPs induced minimal changes to energy metabolism and proteomic profiles in vitro while treatment with SAD SiNPs resulted in mitochondrial membrane hyperpolarization, inhibited mitochondrial respiration, increased reactive oxygen species generation, and redox proteomic trends suggesting activation of aryl hydrocarbon receptor (AHR) and other signaling pathways with known roles in mitochondrial inhibition and CKD progression.ConclusionResults suggest that PCT cell exposure to SAD SiNPs could promote glycolytic and fibrotic shifts consistent with CKDu pathology via oxidative stress-mediated disruption of redox signaling pathways.

Non-lethal sonodynamic therapy mitigates hypertensive renal fibrosis through the PI3K/AKT/mTORC1-autophagy pathway

Hypertension constitutes a significant public health concern, characterized by a high incidence and mortality rate. Hypertensive kidney disease is a prevalent complication associated with hypertension and is the second leading cause of end-stage renal disease (ESRD). Renal fibrosis linked to hypertension has emerged as the third leading cause of disease in dialysis patients. Autophagy activity is crucial for maintaining homeostasis, vitality, and physiological function of kidney cells, while also protecting the kidneys from fibrosis. The deficiency of autophagy will increase the sensitivity of the kidney to the damage, leading to impaired renal function, accumulation of damaged mitochondria and more severe of renal fibrosis. However, enhancing autophagy by activating the PI3K/AKT, AMPK, and mTOR pathways, improves podocyte injury and renal pathological changes, and ameliorates renal function. Current clinical interventions aimed at halting or reversing renal fibrosis in hypertensive patients are notably limited in their efficacy. Here, we present Non-lethal Sonodynamic Therapy (NL-SDT), in which ultrasound is used to activate locally sonosensitizers, thereby stimulating the production of reactive oxygen species for the purpose of modulating cell function or fate, as a novel methodology to inhibit progression of hypertensive renal fibrosis.To confirm whether NL-SDT can reduce hypertensive renal fibrosis and its mechanism. The mice model of hypertensive renal fibrosis was established by using osmotic minipumps (Alzet model 2004, Cupertino, CA) equipped with angiotensin-II (Ang II). The pumps were implanted in mice, ensuring constant infusion of Ang II at a dose of 1.0 µg/kg per minute for 4 weeks. The mice were exposed to 0.4 W/cm2 intensity ultrasonic radiation for 15 min at 4 h post injection of sinoporphyrin sodium (DVDMS) (4 mg/kg) into the caudal vein was repeated weekly for 4 treatments. The kidney from mice was stained with masson’s trichrome staining for collagen fiber expression, while alpha-smooth muscle actin (α-SMA) expression was determined via immunohistochemical staining. The protein levels of fibrosis parameters (α-SMA, collagen I, vimentin), pathway-related proteins (PI3K, AKT, mTORC1) and autophagy-related protein LC3B were determined using western blotting. Intracellular reactive oxygen species (ROS) levels were detected using DCFH-DA probe. Immunofluorescence was also used to observe the expression of α-SMA and E-cadherin in cells. Pathway-related protein inhibitors (the autophagy-related inhibitor 3-methyladenine (3-MA), chloroquine (CQ), ROS inhibitor N-acetyl-L-cysteine (NAC) were applied, and autophagosome changes were observed under transmission electron microscopy. Immunofluorescence was used to observe LC3 spot formation within cells.We obtained the following results via animal and cellular research. In vivo, (1) The collagen area of renal tissue was increased significantly in Ang II group (50.6%). The positive expression of α-SMA was increased significantly (37.8%). (2) The collagen area decreased after NL-SDT treatment (34.8%). The expression of α-SMA was decreased too (48.9%). The expression of LC3B increased in NL-SDT group. (3) The effect of NL-SDT on reducing renal fibrosis can be changed by rapamycin and CQ. In vitro. (1) The expression of α-SMA, collagen I and vimentin were increased significantly in TGF-β1-induced NRK-52E cells. (2) The increase of autophagosomes was observed in TGF-β1-induced NRK-52E cells after NL-SDT. The levels of ROS were increased after NL-SDT (24.8%). The effect of NL-SDT on autophagy was reversed after administration of NAC. The expression of PI3K, P-AKT and P-mTORC1 was decreased in TGF-β1-induced NRK-52E cells after NL-SDT. NL-SDT inhibited the transition of epithelial cells into myofibroblasts by activating PI3K-AKT-mTORC1-autophagy pathway in TGF-β1-induced NRK-52E cells. (3) The administration of the pathway inhibitors showed a reciprocal effect on NL-SDT-inhibited epithelial-mesenchymal transition (EMT).(1) NL-SDT reduced blood pressure temporarily in mice model of hypertensive renal fibrosis induced by Ang II. (2) NL-SDT alleviated renal fibrosis in mice model of hypertensive renal fibrosis induced by Ang II. (3) NL-SDT promoted autophagy by inhibiting PI3K-AKT-mTORC1 signaling pathway and alleviated renal fibrosis in mice model of hypertensive renal fibrosis induced by Ang II. NL-SDT is a non-invasive and efficacious regimen to inhibit renal fibrosis. It may be a new approach for clinical treatment of renal fibrosis, delaying or reducing the occurrence of ESRD.

Anti-inflammatory treatment using alpha melanocyte stimulating hormone (α-MSH) does not alter osteoblasts differentiation and fracture healing

BackgroundAlpha-melanocyte-stimulating-hormone (α-MSH) has been identified as a new anti-inflammatory treatment compound in rheumatoid arthritis (RA) and other inflammatory diseases. However, its direct effect on bone cell differentiation or on bone regeneration, which is an inflammatory process, too, has not been investigated, yet. Bone tissue is significantly affected in inflammatory joint diseases. Additionally, inflammatory signaling is essential -in bone regeneration during fracture healing. Therefore, we evaluated the impact of α-MSH-treatment on bone forming cells in an inflammatory setting in vitro and as a treatment approach in a murine fracture healing model in vivo.MethodsThe influence of α-MSH treatment and melanocortin-receptor expression patterns was investigated in vitro in the presence of either IL-1β or/and TNF-α as an inflammatory stimulus. Osteoblast cell function was evaluated by analyzing proliferation and mineralisation capacities. Using quantitative real time PCR, we analyzed mRNA expression of receptors. To explore the impact of α-MSH on bone regeneration in vivo, treatment with α-MSH or NaCl (control) was performed in a murine fracture-healing model using a closed femur fracture stabilized with an intramedullary implant (female, n = 6–8 mice per group).Resultsα-MSH-treatment did not impair either proliferation nor mineralisation of osteoblastic cells under native or inflammatory conditions (no significant differences found). All four melanocortin receptor-molecules were expressed in murine osteoblastic cells but in very limited amounts and this did not change upon treatment with inflammatory cytokines or α-MSH or both at the same time. Callus formation in fractured femurs of α-MSH-treated mice was slightly delayed at day 14 post fracture with regard to less cartilage formation (NaCl: 19.9%; α-MSH: 13.5%) and soft tissue remodeling (NaCl: 15.2%; α-MSH: 19.5%) but these results were not significantly different and fracture healing overall occurred in a regular way.Conclusionα-MSH has no negative impact on bone or bone-forming cells in native, inflammatory, or regenerative contexts. We can conclude from our results, that treatment of inflammatory diseases using α-MSH does not interfere significantly with bone regeneration in a murine fracture model and therefore treatment with α-MSH could be continued without negative effects on bone formation and bone regeneration in patients.Clinical trial numberNot applicable.

The transmembrane protein Syndecan is required for stem cell survival and maintenance of their nuclear properties.

Tissue maintenance is underpinned by resident stem cells whose activity is modulated by microenvironmental cues. Using Drosophila as a simple model to identify regulators of stem cell behaviour and survival in vivo, we have identified novel connections between the conserved transmembrane proteoglycan Syndecan, nuclear properties and stem cell function. In the Drosophila midgut, Syndecan depletion in intestinal stem cells results in their loss from the tissue, impairing tissue renewal. At the cellular level, Syndecan depletion alters cell and nuclear shape, and causes nuclear lamina invaginations and DNA damage. In a second tissue, the developing Drosophila brain, live imaging revealed that Syndecan depletion in neural stem cells results in nuclear envelope remodelling defects which arise upon cell division. Our findings reveal a new role for Syndecan in the maintenance of nuclear properties in diverse stem cell types.

Serum uric acid to creatinine ratio as a predictor of insulin resistance, β cell function, and metabolic syndrome in normal Korean adults: a cross-sectional study

BackgroundThis study aimed to determine the relations between serum uric acid to creatinine ratio(SUA/Cr) and insulin resistance, pancreatic β cell function, and outbreak of metabolic syndrome (MetS) in normal Korean participants.Materials and methodsThis study included 14,984 participants without diabetes mellitus or gout who participated in the 2019–2021 Korea National Health and Nutrition Examination Survey. To evaluate insulin resistance and β cell function, the homeostasis model assessment (HOMA) was used. Insulin resistance was suggested by HOMA-IR, and β cell function was presented as HOMA-β. Multivariate logistic linear regression analysis was used to identify the factors affecting HOMA-IR, HOMA-β, and MetS. Cut-off values of SUA/Cr to predict insulin resistance, β cell dysfunction, and MetS risk were also been suggested.ResultsConsequent to dividing SUA/Cr into tertiles, the higher the SUA/Cr, the higher the HOMA-IR and dysfunction of β cell, and the rate of MetS increased (p < 0.05). SUA/Cr was associated with insulin resistance, β cell function, and existence of MetS (adjusted odds ratio [OR]; 1.231 [95% confidence interval [CI]; 1.204–1.259], 1.033 [1.011–1.057], and 1.065 [1.026–1.106], respectively). In addition, the group with the clinical significance was the 3rd tertile. In this group, insulin resistance, β cell dysfunction, and MetS risk could be predicted when SUA/Cr value was 8.2716, 8.8710, and 7.9762, respectively. Based on the total number of people, meaningful SUA/Cr values were 7.0175, 6.7925, and 6.9369.ConclusionsThe SUA/Cr may be a useful marker for predicting the insulin resistance, β cell function and incidence of MetS in normal Korean participants.

Functional Characterization of Parallel Fiber-Purkinje Cell Synapses in Two Friedreich’s Ataxia Mouse Models

Friedreich ataxia (FRDA) is an autosomal recessive disorder caused by GAA expansions in the FXN gene, which codes for the protein frataxin (FXN). These mutations reduce FXN expression, leading to mitochondrial dysfunction and multisystemic disease. Accumulating evidence suggests that neuronal dysfunction, rather than neuronal death, may drive the neurological phenotypes of FRDA, but the mechanisms underlying such neurological phenotypes remain unclear. To investigate the neural circuit basis of this dysfunction, we employed field recordings to measure Purkinje cell (PC) function and synaptic properties along with western blotting and immunohistochemistry to determine their density and structure in two established FRDA mouse models, the shRNA-frataxin (FRDAkd) and the frataxin knock in-knockout (KIKO) mice. Western blotting demonstrated subtle changes in mitochondrial proteins and only a modest reduction in the density of calbindin positive cells PCs in the cerebellar cortex of the FRDAkd mice, with no change in the density of PCs in the KIKO mice. Though PC density differed slightly in the two models, field recordings of parallel fiber-PC synapses in the molecular layer demonstrated concordant hypo-excitability of basal synaptic transmission and impairments of long-term plasticity using induction protocols associated with both potentiation and depression of synaptic strength. These results indicate that synaptic instability might be a common feature in FRDA mouse models.Graphical

Full-length mRNA sequencing resolves novel variation in 5' UTR length for genes expressed during human CD4 T-cell activation.

Isoform sequencing (Iso-Seq) uses long-read technology to produce highly accurate full-length reads of mRNA transcripts. Visualization of individual mRNA molecules can reveal new details of transcript variation within understudied portions of mRNA, such as the 5' untranslated region (UTR). Differential 5' UTRs may contain motifs, upstream open reading frames (uORFs), and secondary structures that can serve to regulate translation or further indicate changes in promoter usage, where transcriptional control may impact protein expression levels. To begin to explore isoform variation during T-cell activation, we generated the first Iso-Seq reference transcriptome of activated human CD4 T cells. Within this dataset, we discovered many novel splice- and end-variant transcripts. Remarkably, one in every eight genes expressed in our dataset was found to have a notable proportion of transcripts with 5' UTR lengthened by over 100bp compared to the longest corresponding UTR within the Gencode dataset. Among these end-variant transcripts, two novel isoforms were identified for CXCR5, a chemokine receptor associated with T follicular helper cell (Tfh) function and differentiation. When investigated in a model cell system, these lengthened UTR conferred reduced transcript stability and, for one of these isoforms, short uORFs introduced by the added length altered protein expression kinetics. This study highlights instances in which current reference databases are incomplete relative to the information obtained by long-read sequencing of intact mRNA. Iso-Seq is thus a promising approach to better understanding the plasticity of promoter usage, alternative splicing, and UTR sequences that influence RNA stability and translation efficiency.