Heparin prevents caspase-11-dependent septic lethality independent of anticoagulant properties.

Heparin, a mammalian polysaccharide, is a widely used anticoagulant medicine to treat thrombotic disorders. It is also known to improve outcomes in sepsis, a leading cause of mortality resulted from infection-induced immune dysfunction. Whereas it is relatively clear how heparin exerts its anticoagulant effect, the immunomodulatory mechanisms enabled by heparin remain enigmatic. Here, we show that heparin prevented caspase-11-dependent immune responses and lethality in sepsis independent of its anticoagulant properties. Heparin or a chemically modified form of heparin without anticoagulant function inhibited the alarmin HMGB1-lipopolysaccharide (LPS) interaction and prevented the macrophage glycocalyx degradation by heparanase. These events blocked the cytosolic delivery of LPS in macrophages and the activation of caspase-11, a cytosolic LPS receptor that mediates lethality in sepsis. Survival was higher in septic patients treated with heparin than those without heparin treatment. The identification of this previously unrecognized heparin function establishes a link between innate immune responses and coagulation.

Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy.

Diabetic nephropathy (DN) is one of the most important comorbidities for diabetic patients, which is the main factor leading to end-stage renal disease. Heparin analogs can delay the progression of DN, but the mechanism is not fully understood. In this study, we found that low molecular weight heparin therapy significantly upregulated some downstream proteins of the peroxisome proliferator-activated receptor (PPAR) signaling pathway by label-free quantification of the mouse kidney proteome. Through cell model verification, low molecular weight heparin can protect the heparan sulfate of renal tubular epithelial cells from being degraded by heparanase that is highly expressed in a high-glucose environment, enhance the endocytic recruitment of fatty acid-binding protein 1, a coactivator of the PPAR pathway, and then regulate the activation level of intracellular PPAR. In addition, we have elucidated for the first time the molecular mechanism of heparan sulfate and fatty acid-binding protein 1 interaction. These findings provide new insights into understanding the role of heparin in the pathogenesis of DN and developing corresponding treatments.

Impact of heparanase-2 (Hpa2) on cancer and inflammation: Advances and paradigms.

HPSE2, the gene-encoding heparanase 2 (Hpa2), is mutated in urofacial syndrome (UFS), a rare autosomal recessive congenital disease attributed to peripheral neuropathy. Hpa2 lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase (Hpa1), yet it exhibits a high affinity toward HS, thereby inhibiting Hpa1 enzymatic activity. Hpa2 regulates selected genes that promote normal differentiation, tissue homeostasis, and endoplasmic reticulum (ER) stress, resulting in antitumor, antiangiogenic, and anti-inflammatory effects. Importantly, stress conditions induce the expression of Hpa2, thus establishing a feedback loop, where Hpa2 enhances ER stress which, in turn, induces Hpa2 expression. In most cases, cancer patients who retain high levels of Hpa2 survive longer than patients bearing Hpa2-low tumors. Experimentally, overexpression of Hpa2 attenuates the growth of tumor xenografts, whereas Hpa2 gene silencing results in aggressive tumors. Studies applying conditional Hpa2 knockout (cHpa2-KO) mice revealed an essential involvement of Hpa2 contributed by the host in protecting against cancer and inflammation. This was best reflected by the distorted morphology of the Hpa2-null pancreas, including massive infiltration of immune cells, acinar to adipocyte trans-differentiation, and acinar to ductal metaplasia. Moreover, orthotopic inoculation of pancreatic ductal adenocarcinoma (PDAC) cells into the pancreas of Hpa2-null vs. wild-type mice yielded tumors that were by far more aggressive. Likewise, intravenous inoculation of cancer cells into cHpa2-KO mice resulted in a dramatically increased lung colonization reflecting the involvement of Hpa2 in restricting the formation of a premetastatic niche. Elucidating Hpa2 structure-activity-relationships is expected to support the development of Hpa2-based therapies against cancer and inflammation.

Fibroblast growth factor 23-mediated regulation of osteoporosis: Assessed via Mendelian randomization and in vitro study.

Despite numerous investigations on the influence of fibroblast growth factor 23 (FGF23), α-Klotho and FGF receptor-1 (FGFR1) on osteoporosis (OP), there is no clear consensus. Mendelian randomization (MR) analysis was conducted on genome-wide association studies (GWASs)-based datasets to evaluate the causal relationship between FGF23, α-Klotho, FGFR1 and OP. The primary endpoint was the odds ratio (OR) of the inverse-variance weighted (IVW) approach. Furthermore, we stably transfected FGF23-mimic or siRNA-FGF23 into human bone marrow mesenchymal stem cells (hBMSCs) in culture and determined its cell proliferation and the effects on osteogenic differentiation. Using MR analysis, we demonstrated a strong correlation between serum FGF23 levels and Heel- and femoral neck-BMDs, with subsequent ORs of 0.919 (95% CI: 0.860-0.983, p = 0.014) and 0.751 (95% CI: 0.587-0.962; p = 0.023), respectively. The expression levels of FGF23 were significantly increased in femoral neck of patients with OP than in the control cohort (p < 0.0001). Based on our in vitro investigation, after overexpression of FGF23, compared to the control group, the BMSC's proliferation ability decreased, the expression level of key osteogenic differentiation genes (RUNX2, OCN and OSX) significantly reduced, mineralized nodules and ALP activity significantly decreased. After silencing FGF23, it showed a completely opposite trend. Augmented FGF23 levels are causally associated with increased risk of OP. Similarly, FGF23 overexpression strongly inhibits the osteogenic differentiation of hBMSCs, thereby potentially aggravating the pathological process of OP.

Vitamin D inhibits ferroptosis and mitigates the kidney injury of prediabetic mice by activating the Klotho/p53 signaling pathway.

Diabetic nephropathy (DN) is a serious public health problem worldwide, and ferroptosis is deeply involved in the pathogenesis of DN. Prediabetes is a critical period in the prevention and control of diabetes and its complications, in which kidney injury occurs. This study aimed to explore whether ferroptosis would induce kidney injury in prediabetic mice, and whether vitamin D (VD) supplementation is capable of preventing kidney injury by inhibiting ferroptosis, while discussing the potential mechanisms. High-fat diet (HFD) fed KKAy mice and high glucose (HG) treated HK-2 cells were used as experimental subjects in the current study. Our results revealed that serious injury and ferroptosis take place in the kidney tissue of prediabetic mice; furthermore, VD intervention significantly improved the kidney structure and function in prediabetic mice and inhibited ferroptosis, showing ameliorated iron deposition, enhanced antioxidant capability, reduced reactive oxygen species (ROS) and lipid peroxidation accumulation. Meanwhile, VD up-regulated Klotho, solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression, and down-regulated p53, transferrin receptor 1 (TFR1) and Acyl-Coenzyme A synthetase long-chain family member 4 (ACSL4) expression. Moreover, we demonstrated that HG-induced ferroptosis is antagonized by treatment of VD and knockdown of Klotho attenuates the protective effect of VD on ferroptosis in vitro. In conclusion, ferroptosis occurs in the kidney of prediabetic mice and VD owns a protective effect on prediabetic kidney injury, possibly by via the Klotho/p53 pathway, thus inhibiting hyperglycemia-induced ferroptosis.

The current and emerging Klotho-enhancement strategies.

Klotho is well known as a gene with antiaging properties. It has membrane and soluble forms, providing a unique system that controls various metabolic processes essential to health and disease. Klotho deficiency has been revealed to be associated with various aging-related disorders. Based on its various known and unknown protective properties, upregulating the Klotho gene may be a possible therapeutic and/or preventive approach in aging-related complications. Some agents, such as hormonal compounds, renin-angiotensin system inhibitors, antioxidants, peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, statins, vitamin D receptor agonists, antioxidants, anti-inflammatory agents, mammalian target of rapamycin (mTOR) signaling inhibitors, and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitors, can possibly lead to the upregulation and elevation of Klotho levels. Demethylation and deacetylation of the Klotho gene can also be considered other possible Klotho-enhancement methods. Some emerging techniques, such as RNA modifications, gene therapy, gene editing, and exosome therapy, probably have the potential to be applied for increasing Klotho. In the present study, these current and emerging Klotho-enhancement strategies and their underlying mechanisms were comprehensively reviewed, which could highlight some potential avenues for future research.

Klotho accelerates the progression of polycystic ovary syndrome through promoting granulosa cell apoptosis and inflammation†.

The morbidity of polycystic ovary syndrome (PCOS) is in highly increasing rate nowadays. PCOS not only affects the fertility in women, but also threatens the health of whole life. Hence, to find the prognostic risk factors is of great value. However, the effective predictors in clinical practice of PCOS are still in blackness. In this study, we found Klotho (KL) was increased in follicular fluid (FF) and primary luteinized granulosa cells (GCs) from PCOS patients with hyperandrogenism. Furthermore, we found follicular KL was negatively correlated with numbers of mature oocytes, and positively correlated with serum testosterone, LH, and LH/FSH levels menstrual cycle and number of total antral follicles in PCOS patients. In primary luteinized GCs, the increased KL was accompanied with upregulation of cell apoptosis and inflammation-related genes. In ovaries of PCOS mice and cultured human KGN cell line, KL was up-regulated and accompanied by apoptosis, inflammation, and mitochondrial dysfunction. Therefore, our findings suggest new mechanisms for granulosa cell injury and revealed to target inhibit KL maybe a new therapeutic strategy for treatment of PCOS.

Pentoxifylline ameliorates subclinical atherosclerosis progression in patients with type 2 diabetes and chronic kidney disease: a randomized pilot trial.

BACKGROUND: Diabetic kidney disease (DKD) is associated with a higher risk of cardiovascular disease (CVD). Pentoxifylline (PTF), a nonselective phosphodiesterase inhibitor with anti-inflammatory, antiproliferative, and antifibrotic actions, has demonstrated renal benefits in both clinical trials and meta-analyses. The present work aimed to study the effects of PTF on the progression of subclinical atherosclerosis (SA) in a population of patients with diabetes and moderate to severe chronic kidney disease (CKD). METHODS: In this open-label, randomized controlled, prospective single-center pilot study the evolution of carotid intima-media thickness (CIMT) and ankle-brachial index (ABI) were determined in 102 patients with type 2 diabetes mellitus and CKD assigned to PTF, aspirin or control groups during 18 months. We also determined the variations in the levels of inflammatory markers and Klotho (KL), a protein involved in maintaining cardiovascular health, and their relationship with the progression of SA. RESULTS: Patients treated with PTF presented a better evolution of CIMT, increased KL mRNA levels in peripheral blood cells (PBCs) and reduced the inflammatory state. The progression of CIMT values was inversely related to variations in KL both in serum and mRNA expression levels in PBCs. Multiple regression analysis demonstrated that PTF treatment and variations in mRNA KL expression in PBCs, together with changes in HDL, were significant determinants for the progression of CIMT (adjusted R2 = 0.24, P < 0.001) independently of traditional risk factors. Moreover, both variables constituted protective factors against a worst progression of CIMT [OR: 0.103 (P = 0.001) and 0.001 (P = 0.005), respectively]. CONCLUSIONS: PTF reduced SA progression assessed by CIMT variation, a beneficial effect related to KL gene expression in PBCs. TRIAL REGISTRATION: The study protocol code is PTF-AA-TR-2009 and the trial was registered on the European Union Drug Regulating Authorities Clinical Trials (EudraCT #2009-016595-77). The validation date was 2010-03-09.

Genomic editing of intronic enhancers unveils their role in fine-tuning tissue-specific gene expression in Arabidopsis thaliana.

Enhancers located in introns are abundant and play a major role in the regulation of gene expression in mammalian species. By contrast, the functions of intronic enhancers in plants have largely been unexplored and only a handful of plant intronic enhancers have been reported. We performed a genome-wide prediction of intronic enhancers in Arabidopsis thaliana using open chromatin signatures based on DNase I sequencing. We identified 941 candidate intronic enhancers associated with 806 genes in seedling tissue and 1,271 intronic enhancers associated with 1,069 genes in floral tissue. We validated the function of 15 of 21 (71%) of the predicted intronic enhancers in transgenic assays using a reporter gene. We also created deletion lines of three intronic enhancers associated with two different genes using CRISPR/Cas. Deletion of these enhancers, which span key transcription factor binding sites, did not abolish gene expression but caused varying levels of transcriptional repression of their cognate genes. Remarkably, the transcriptional repression of the deletion lines occurred at specific developmental stages and resulted in distinct phenotypic effects on plant morphology and development. Clearly, these three intronic enhancers are important in fine-tuning tissue- and development-specific expression of their cognate genes.

Functional metagenomic screening identifies an unexpected ß-glucuronidase.

The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for ß-glucuronidase activity. We identified SN243, a genuine ß-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase 3 family member. This glycoside hydrolase family contains only one recently added ß-glucuronidase, showing that a functional metagenomic approach can shed light on assignments that are currently 'unpredictable' by bioinformatics. Kinetic analyses of SN243 characterized it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous glycoside hydrolase 3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre-volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.

Efficiency of the alpha-hairpinin SmAMP-X gene promoter from Stellaria media plant depends on selection of transgenic approach.

The antimicrobial activity of the alpha-HAIRPININ ANTIMICROBIAL PEPTIDE X (SmAMP-X gene, GenBank acc. No. HG423454.1) from Stellaria media plant has been shown in vitro. Here, we isolated the SmAMP-X gene promoter and found two genomic sequences for the promoter (designated pro-SmAMP-X and pro-SmAMP-X-Ψ2) with 83% identity in their core and proximal regions. We found that the abilities of these promoters to express the uidA reporter and the nptII selectable marker differ according to the structural organization of T-DNA in the binary vector used for plant transformation. Analysis of Agrobacterium-infiltrated Nicotiana benthamiana leaves, transgenic Arabidopsis thaliana lines, and transgenic Solanum tuberosum plants revealed that both promoters in the pCambia1381Z and pCambia2301 binary vectors generate 42-100% of the ß-glucuronidase (GUS) activity generated by the CaMV35S promoter. According to 5'-RACE (rapid amplification of cDNA ends) analysis, both plant promoters are influenced by the CaMV35S enhancer used to express selectable markers in the T-DNA region of pCambia1381Z and pCambia2301. The exclusion of CaMV35S enhancer from the T-DNA region significantly reduces the efficiency of pro-SmAMP-X-Ψ2 promoter for GUS production. Both promoters in the pCambia2300 vector without CaMV35S enhancer in the T-DNA region weakly express the nptII selectable marker in different tissues of transgenic N. tabacum plants and enable selection of transgenic cells in media with a high concentration of kanamycin. Overall, promoter sequences must be functionally validated in binary vectors lacking CaMV35S enhancer.

α-Klotho prevents diabetic retinopathy by reversing the senescence of macrophages.

BACKGROUND: Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus (DM) and a significant cause of acquired blindness in the working-age population worldwide. Aging is considered as an important risk factor for DR development. Macrophages in aged mice bear typical M2 marker proteins but simultaneously express a pro-inflammatory factor profile. This may explain why the level of intraocular inflammation does not decrease during proliferative diabetic retinopathy (PDR) despite the occurrence of neovascularization and fibrosis (M2 activation). α-Klotho (KL) was originally discovered as a soluble anti-aging factor, which is mainly expressed in kidney tubular epithelium, choroid plexus in the brain and secreted in the blood. However, the role of KL in DR pathophysiology has not been previously reported. METHODS: Type 1 (streptozotocin [STZ]-induced) and type 2 (a high-fat diet along with a low dose of STZ) diabetic mouse models were established and injected with or without KL adenovirus via the tail vein for 12 weeks. Vldlr-/- mice were injected intravitreally with or without soluble KL protein from P8 to P15. The retinal structure and function were analyzed by electroretinogram and optical coherence tomography. The neovascular lesions were analyzed by retinal flat mount and RPE flat mount. The senescence markers, macrophage morphology, and KL expression levels were detected by immunofluorescence staining. A cell model was constructed using RAW264.7 cells stimulated by 4-hydroxynonenal (4HNE) and transfected with or without KL adenovirus. The senescence-associated secretory phenotypes were detected by qRT-PCR. Senescence was detected by SA-ß-Gal staining. Serum, aqueous humor, and vitreous humor KL levels of proliferative diabetic retinopathy (PDR) patients were measured by enzyme-linked immunosorbent assay. Quantitative proteomics and bioinformatics were applied to predict the change of proteins and biological function after overexpression of KL in macrophages. The effects of KL on the HECTD1 binding to IRS1 were analyzed by bioinformatics, molecular docking, and Western Blot. RESULTS: Serum, aqueous humor, and vitreous humor KL levels were lower in patients with PDR than in those with cataracts. KL relieved the retinal structure damage, improved retina function, and inhibited retinal senescence in diabetic mice. KL administration attenuated the neovascular lesions in VLDLR-/- mice by decreasing the secretion of VEGFA and FGF2 from macrophages. KL also protected RAW264.7 cells from 4HNE-induced senescence. Additionally, it inhibited E3 ubiquitin ligase HECTD1 expression in both diabetic mouse peripheral blood mononuclear cells and 4HNE-treated RAW264.7 cells. KL inhibited HECTD1 binding to IRS1 and reduced the ubiquitination of IRS1. CONCLUSIONS: Macrophage aging is involved in DM-induced retinopathy. KL alleviates DM-induced retinal macrophage senescence by downregulating HECTD1 and decreasing IRS1 ubiquitination and degradation. Meanwhile, KL administration attenuated the neovascular lesions by altering the activation state of macrophages and decreasing the expression of VEGFA and FGF2.

Association of elevated cerebrospinal fluid levels of the longevity protein α-Klotho with a delayed onset of cognitive impairment in Parkinson's disease patients.

BACKGROUND AND PURPOSE: Parkinson's disease (PD) is an age-related condition characterized by substantial phenotypic variability. Consequently, pathways and proteins involved in biological aging, such as the central aging pathway comprising insulin-like growth factor 1-α-Klotho-sirtuin 1-forkhead box O3-peroxisome proliferator-activated receptor γ, may potentially influence disease progression. METHODS: Cerebrospinal fluid (CSF) levels of α-Klotho in 471 PD patients were examined. Of the 471 patients, 96 carried a GBA1 variant (PD GBA1), whilst the 375 non-carriers were classified as PD wild-type (PD WT). Each patient was stratified into a CSF α-Klotho tertile group based on the individual level. Kaplan-Meier survival curves and Cox regression analysis stratified by tertile groups were conducted. These longitudinal data were available for 255 patients. Follow-up times reached from 8.4 to 12.4 years. The stratification into PD WT and PD GBA1 was undertaken to evaluate potential continuum patterns, particularly in relation to CSF levels. RESULTS: Higher CSF levels of α-Klotho were associated with a significant later onset of cognitive impairment. Elevated levels of α-Klotho in CSF were linked to higher Montreal Cognitive Assessment scores in male PD patients with GBA1 mutations. CONCLUSIONS: Our results indicate that higher CSF levels of α-Klotho are associated with a delayed cognitive decline in PD. Notably, this correlation is more prominently observed in PD patients with GBA1 mutations, potentially reflecting the accelerated biological aging profile characteristic of individuals harboring GBA1 variants.

Klotho: molecular mechanisms and emerging therapeutics in central nervous system diseases.

Klotho is recognized as an aging-suppressor protein that is implicated in a variety of processes and signaling pathways. The anti-inflammatory, anti-apoptotic, anti-oxidant, and anti-tumor bioactivities of klotho have extended its application in neurosciences and made the protein popular for its lifespan-extending capacity. Furthermore, it has been demonstrated that klotho levels would reduce with aging and numerous pathologies, particularly those related to the central nervous system (CNS). Evidence supports the idea that klotho can be a key therapeutic target in CNS diseases such as amyotrophic lateral sclerosis, Parkinson's disease, stroke, and Alzheimer's disease. Reviewing the literature suggests that the upregulation of klotho expression regulates various signaling pathways related to autophagy, oxidative stress, inflammation, cognition, and ferroptosis in neurological disorders. Therefore, it has been of great interest to develop drugs or agents that boost or restore klotho levels. In this regard, the present review was designed and aimed to gather the delegated documents regarding the therapeutic potential of Klotho in CNS diseases focusing on the molecular and cellular mechanisms.

Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.

α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling.

The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23-FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling.

Expression analysis of genes encoding extracellular leucine-rich repeat proteins in Arabidopsis thaliana.

Leucine-rich repeat (LRR)-containing proteins have been identified in diverse species, including plants. The diverse intracellular and extracellular LRR variants are responsible for numerous biological processes. We analyzed the expression patterns of Arabidopsis thaliana extracellular LRR (AtExLRR) genes, 10 receptor-like proteins, and 4 additional genes expressing the LRR-containing protein by a promoter: ß-glucuronidase (GUS) study. According to in silico expression studies, several AtExLRR genes were expressed in a tissue- or stage-specific and abiotic/hormone stress-responsive manner, indicating their potential participation in specific biological processes. Based on the promoter: GUS assay, AtExLRRs were expressed in different cells and organs. A quantitative real-time PCR investigation revealed that the expressions of AtExLRR3 and AtExLRR9 were distinct under various abiotic stress conditions. This study investigated the potential roles of extracellular LRR proteins in plant growth, development, and response to various abiotic stresses.

Production of bilirubin via whole-cell transformation utilizing recombinant Corynebacterium glutamicum expressing a ß-glucuronidase from Staphylococcus sp. RLH1.

Bilirubin, a key active ingredient of bezoars with extensive clinical applications in China, is produced through a chemical process. However, this method suffers from inefficiency and adverse environmental impacts. To address this challenge, we present a novel and efficient approach for bilirubin production via whole-cell transformation. In this study, we employed Corynebacterium glutamicum ATCC13032 to express a ß-glucuronidase (StGUS), an enzyme from Staphylococcus sp. RLH1 that effectively hydrolyzes conjugated bilirubin to bilirubin. Following the optimization of the biotransformation conditions, a remarkable conversion rate of 79.7% in the generation of bilirubin was obtained at temperate 40 °C, pH 7.0, 1 mM Mg2+ and 6 mM antioxidant NaHSO3 after 12 h. These findings hold significant potential for establishing an industrially viable platform for large-scale bilirubin production.

The Analysis of Serum Klotho Protein Level and Related Gene Polymorphism in Osteoporotic Fracture of Elderly Patients with Osteoporosis.

Objective: Klotho protein level are reported to play important roles in the osteoporosis. To investigate the correlation between serum Klotho protein level and related gene (Klotho G395-A gene) polymorphism and osteoporotic fracture in elderly patients with osteoporosis. Methods: A total of 62 elderly patients with osteoporosis admitted to the Department of Orthopedics of our hospital from January 2021 to June 2022 were included in the study group. Another 62 elderly patients without osteoporosis who underwent a physical examination at the same time were selected as the control group. Patients in the study group were divided into group A (n = 23, osteoporotic fracture) and group B (n = 39, osteoporotic fracture) according to the occurrence of osteoporotic fracture. Serum Klotho protein level was detected in all patients, and its related gene (Klotho G395-A gene) polymorphism was analyzed. After fasting in the morning (fasting for more than 8 hours), 3-5 ml venous blood was collected and immediately placed in a centrifuge tube. Serum was separated and serum Klotho protein level was measured by enzyme-linked immunosorbent assay kit. Polymorphism typing was performed by Taqman allele-specific hybridization analysis. At the same time, general information (gender, age, body mass index, systolic blood pressure, diastolic blood pressure, glycated glucose protein, low-density lipoprotein cholesterol, bone mineral density) was collected. The differences in general data, serum Klotho protein level and Klotho G395-A gene polymorphism between the study group and the control group were analyzed. Spearman analysis was used to analyze the correlation between general data, serum Klotho protein level and Klotho G395-A gene and osteoporotic fracture. Logistic analysis was used to analyze the independent risk factors of osteoporotic fracture. Results: There was no significant difference of the sex, systolic blood pressure (SBP), diastolic blood pressure (DBP), Klotho G395-A genotype GG and alleles A and G between the study group and the control group. There was significant difference of body mass index (BMI), glycated glucose protein, low-density lipoprotein cholesterol (LDL-C), bone mineral density, serum Klotho protein level and Klotho G395-A genotype AA and AG were between the study group and the control group. Gender, age, glycated glucose protein and Klotho G395-A genotype AA were positively correlated with osteoporotic fracture (P < .05), while bone mineral density was negatively correlated with osteoporotic fracture (P < .05). There was no correlationship between the serum Klotho protein level and the incidence of osteoporotic fracture (P > .05). Logistic analysis showed that age, bone mineral density and Klotho G395-A genotype AA were independent risk factors for osteoporotic fracture. Conclusion: The level of serum Klotho protein and related gene polymorphisms are both related to osteoporotic fracture in elderly patients with osteoporosis. It is significant to reduce the incidence of osteoporotic fractures. In future, more experiments are needed to explore the underlying mechanism.

Metformin enhances the survival ability of ovarian granulosa cells in polycystic ovary syndrome by promoting LINC00548 to suppress androgen receptor/klotho pathway.

BACKGROUND: Metformin (Met) has been reported to play the key role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the precise mechanisms underlying the actions of Met in PCOS remain incompletely understood. This study aimed to confirm the mechanism of Met interacting with a long non-coding RNA LINC00548 in PCOS. METHODS: Ovarian granulosa cells (OGCs) were incubated 500 nM dihydrotestosterone (DHT) to construct PCOS in vitro model and then were treated 20 µM Met. A series of cell experiments including Cell Counting Kit-8, Terminal uridine nucleotide end labeling, and flow cytometry were performed to confirm the changes of OGC survival ability. Quantitative real-time polymerase chain reaction was conducted to determine the levels of LINC00548, whereas Western blotting was applied to determine the levels of androgen receptor (AR) and klotho. RESULTS: Met improved the cell viability and suppressed cell apoptosis in DHT-treated OGCs. LINC00548 downregulated in DHT-treated OGCs was upregulated by Met, and its overexpression further enhanced the positive effects of Met on the survival ability of DHT-treated OGCs. In addition, Met could induce the upregulation of LINC00548 to suppress the activation of AR/klotho pathway in DHT-treated OGCs. CONCLUSION: Overall, this study discovers that Met enhances the survival ability of OGCs in PCOS by elevating LINC00548 expression to suppress AR/klotho pathway.