Expression Of Catabolic Genes Research Articles

The MKK3 MAPK cascade integrates temperature and after-ripening signals to modulate seed germination

The timing of seed germination is controlled by the combination of internal dormancy and external factors. Temperature is a major environmental factor for seed germination. The permissive temperature range for germination is narrow in dormant seeds and expands during after-ripening (AR) (dormancy release). Quantitative trait loci analyses of preharvest sprouting in cereals have revealed that MKK3, a mitogen-activated protein kinase (MAPK) cascade protein, is a negative regulator of grain dormancy. Here, we show that the MAPKKK19/20-MKK3-MPK1/2/7/14 cascade modulates the germination temperature range in Arabidopsis seeds by elevating the germinability of the seeds at sub- and supraoptimal temperatures. The expression of MAPKKK19 and MAPKKK20 is induced around optimal temperature for germination in after-ripened seeds but repressed in dormant seeds. MPK7 activation depends on the expression levels of MAPKKK19/20, with expression occurring under conditions permissive for germination. Abscisic acid (ABA) and gibberellin (GA) are two major phytohormones which are involved in germination control. Activation of the MKK3 cascade represses ABA biosynthesis enzyme gene expression and induces expression of ABA catabolic enzyme and GA biosynthesis enzyme genes, resulting in expansion of the germinable temperature range. Our data demonstrate that the MKK3 cascade integrates temperature and AR signals to phytohormone metabolism and seed germination.

Nuclear receptor signaling via NHR-49/MDT-15 regulates stress resilience and proteostasis in response to reproductive and metabolic cues.

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in Caenorhabditis elegans We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, an ortholog of mammalian peroxisome proliferator-activated receptor α (PPARα), regulates stress resilience and proteostasis downstream from embryo integrity and other pathways that influence lipid homeostasis and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing intertissue pathway in somatic cells, triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49, together with its coactivator, MDT-15, contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer. Our findings indicate that NHR-49 also contributes to stress resilience in other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting, and that increased NHR-49 activity is sufficient to improve proteostasis and stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.

Multi-omic analyses reveal key sectors of jasmonate-mediated defense responses in rice.

The phytohormone jasmonate (JA) plays a central role in plant defenses against biotic stressors. However, our knowledge of the JA signaling pathway in rice (Oryza sativa) remains incomplete. Here, we integrated multi-omic data from three tissues to characterize the functional modules involved in organizing JA-responsive genes. In the core regulatory sector, MYC2 transcription factor transcriptional cascades are conserved. in different species but with distinct regulators (e.g. bHLH6 in rice)., in which genes are early expressed across all tissues. In the feedback sector, MYC2 also regulates the expression of JA repressor and catabolic genes, providing negative feedback that truncates the duration of JA responses. For example, the MYC2-regulated NAC (NAM, ATAF1/2 and CUC2) transcription factor genes NAC1, NAC3, and NAC4 encode proteins that repress JA signaling and herbivore resistance. In the tissue-specific sector, many late-expressed genes are associated with the biosynthesis of specialized metabolites that mediate particular defensive functions. For example, the terpene synthase gene TPS35 is specifically induced in the leaf sheath and TPS35 functions in defense against oviposition by brown planthoppers and the attraction of this herbivore's natural enemies. Thus, by characterizing core, tissue-specific, and feedback sectors of JA-elicited defense responses, this work provides a valuable resource for future discoveries of key JA components in this important crop.

17β-estradiol activates SOX6 to balance the anabolism and catabolism via estrogen receptor 2 in chondrocyte.

We investigated the influence of 17β-estradiol (17β-E2) on cartilage extracellular matrix (ECM) homeostasis in postmenopausal women. We focused on the roles of estrogen receptors (ESR) and SOX6 in 17β-E2-mediated stimulation of ECM metabolism during chondrocyte (CH) degeneration. We compared the expression of anabolic genes (collagen II and aggrecan) and catabolic genes (MMPs and TIMPs) in IL-1β-induced CH degeneration in vitro, with and without 17β-E2 supplementation. We separately silenced the SOX6, ESR1, and ESR2 genes in CHs to determine their impact on 17β-E2 treatment. Additionally, we used Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and luciferase assays to investigate protein-DNA interactions within ESR2 and SOX6-promoter complexes. After three days of IL-1β treatment, ESR1/2, SOX6, collagen II, aggrecan, and TIMP1/3 were decreased, while MMP3/9/13 were increased. The addition of 17β-E2 partially reversed these effects, but silencing SOX6, ESR1, or ESR2 weakened the protective effects of 17β-E2. Silencing ESR2, but not ESR1, abolished the upregulation of SOX6 induced by 17β-E2. ESR2 was found to bind the SOX6 promoter and regulate SOX6 expression. 17β-E2 upregulates SOX6 through ESR2 mediation, and the synergistic effect of 17β-E2 and ESR2 on SOX6 balances ECM metabolism in CHs.

Nutrients, surfactants, and aeration in constructed wetlands affect bacterial persistence and metabolic activity during the remediation of crude oil-contaminated water

The use of constructed wetlands (CWs) is one of the best options to treat wastewater. In CWs, microorganisms play a major role in the degradation of organic pollutants but the concentration of nutrients, surfactant, and aeration (NSA) in oil-contaminated water is one of the factors that affect the persistence and metabolic functioning of hydrocarbon-degrading microorganisms. In the present investigation, the influence of the addition of NSA on the persistence of the augmented bacteria, copy of (alkane hydroxylase gene) alkB gene, and its expression level in the water, soil, and plants of CWs were evaluated. The CWs mesocosms were developed by the vegetation of Typha latifolia and Cyperus laevigatus and inoculated with the bacterial consortium (Pseudomonas putida TYRI39, Acinetobacter junii TYRH47, Acinetobacter sp. CYRH17, Pseudomonas sp. CYSI27, and Pseudomonas sp. TYRH42). The mesocosms were provided with nutrients (20 mg l− 1 N, 2.6 mg l− 1 P, and 16.4 mg l− 1 K) in liquid form, surfactant Tween-20 (0.2%, v/v) in liquid form, and aeration (≥ 7.0 ± 1 mg l− 1 DO) using aeration pump. The addition of NSA in CWs enhanced the persistence and metabolic functioning of the inoculated bacteria in the water, rhizospheric soil, and plants. The maximum hydrocarbon removal (97%) was observed in the water treated by CWs having C. laevigatus, bacteria, and NSA, and it is correlated with the copy numbers of alkB and its expression level. The application of NSA in CWs not only improved bacterial persistence and catabolic gene expression but also increased plant development and hydrocarbon removal.

Investigation of the morphological, physiological, biochemical, and catabolic characteristics and gene expression under drought stress in tolerant and sensitive genotypes of wild barley [Hordeum vulgare subsp. spontaneum (K. Koch) Asch. & Graebn.

BackgroundBarley (H. vulgare L.) is an important cereal crop cultivated across various climates globally. Barley and its ancestor (H. vulgare subsp. spontaneum) are an economically valuable model for genetic research and improvement. Drought, among various abiotic stresses, is a substantial threat to agriculture due to its unpredictable nature and significant impact on crop yield.ResultsThis study was conducted in both greenhouse and laboratory settings. Prior to the study, wild barley accessions were pre-selected based on their sensitivity or tolerance to drought as determined from fieldwork in the 2020–2021 and 2021–2022 cropping seasons. The effects of three levels of drought stress were evaluated (control, 90–95% field capacity [FC]; mild stress, 50–55% FC; and severe stress, 25–30% FC). Several parameters were assessed, including seedling and root growth, enzymatic activity (CAT, SOD, POD), soluble protein levels, chlorophyll content, carotenoids, abaxial and adaxial stomatal density and dimensions, and relative gene expression of Dhn1, SOD, POD, and CAT. Drought stress significantly increased enzyme activities, especially at 25–30% FC, and more in the tolerant genotype. On the other hand, sensitive genotypes showed a notable increase in stomatal density. Under drought stress, there was a general decline in seedling and root growth, protein content, chlorophyll and carotenoids, and stomatal dimensions. Importantly, gene expression analysis revealed that Dhn1, SOD, POD, and CAT were upregulated under drought, with the highest expression levels observed in the drought-tolerant genotype under severe stress conditions (25–30% FC).ConclusionsOur investigation highlights the distinct morphological, physiological, biochemical, and gene-expression profiles of drought-resistant and drought-sensitive wild barley genotypes under varying degrees of drought.

Abstract B047: Inhibition of the polyamine pathway: a novel therapeutic approach to treat aggressive pediatric medulloblastoma

Abstract Medulloblastoma is the most common pediatric malignant brain tumor. Polyamines, critical intracellular polycations governing key cell processes such as DNA replication, translation, and cell proliferation, are frequently upregulated in cancer. We previously showed that the polyamine pathway is critical to the growth and survival of pediatric diffuse midline gliomas and MYC amplified neuroblastoma. Given the activity of polyamine inhibition in DMG, and given the critical role of MYC in medulloblastoma, we sought to investigate the potential of polyamine pathway inhibition as a therapeutic approach for medulloblastoma. We compared the expression of regulators of the polyamine pathway in a cohort of pediatric medulloblastoma tumors from patients enrolled in the Zero Childhood Cancer (ZERO) Personalized Medicine Program and found significant over-expression compared with normal fetal brain samples. Analysis of Cavalli dataset of 763 medulloblastoma samples, found that high expression of polyamine biosynthetic genes predicted poor overall survival, and low expression of catabolic flux genes significantly correlated with better outcomes. We next treated medulloblastoma cells treated with a polyamine synthesis inhibitor, DFMO, and found that led to a compensatory increase in polyamine transporter protein expression. Conversely, treatment with the polyamine transport inhibitor AMXT 1501 led to compensatory increase in ODC1, the key polyamine synthetic enzyme. Dual targeting of the polyamine pathway, (DFMO and AMXT 1501) showed a potent and synergistic inhibition of cell proliferation and reduced the clonogenic potential of medulloblastoma cells in soft agar clonogenic assays. Flow cytometric analysis of Annexin V-stained cells showed that treatment with DFMO and AMXT 1501 in combination led to induction of apoptosis. Furthermore, we found that dual polyamine inhibition potently sensitized medulloblastoma cells, particularly Group 3, to the chemotherapeutic agent SN-38, leading to complete eradication of medulloblastoma cells in both cytotoxicity assays and clonogenic assays at nanomolar chemotherapeutic concentrations. We are currently validating this treatment strategy in aggressive orthotopic medulloblastoma models. Our findings suggest that inhibiting both polyamine synthesis and uptake holds promise for sensitizing highly aggressive pediatric medulloblastoma tumors to chemotherapy. Currently, AMXT 1501 combined with DFMO is undergoing evaluation in a Phase I clinical trial for adults (NCT05500508). Building on these promising results, we are developing a Phase I/II clinical trial for children with aggressive brain tumors through the international CONNECT consortium. Citation Format: Aaminah Khan, Anjana Gopalakrishnan, Jie Liu, Mark R. Burns, Maria Tsoli, David S. Ziegler. Inhibition of the polyamine pathway: a novel therapeutic approach to treat aggressive pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B047.

Effects of Diclofenac Etalhyaluronate (SI-613/ONO-5704) on Cartilage Degeneration in Arthritic Rats and Inflammatory Cytokine-Stimulated Human Chondrocytes.

Cartilage degeneration is a key feature of osteoarthritis (OA) and rheumatoid arthritis and is thought to negatively impact patients' quality of life. Diclofenac etalhyaluronate (DEH, SI-613/ONO-5704) is a hyaluronic acid (HA) derivative chemically bound to diclofenac (DF) that has been reported to improve OA symptoms; however, its effect on cartilage degeneration remains unknown. In the present study, we investigated the chondroprotective effect of DEH in rats with collagen-induced arthritis and interleukin-1β-stimulated human chondrocytes. Rats with collagen-induced arthritis were administered DEH and HA intra-articularly, and DF orally. Knee joint swelling, histological scores of articular cartilage, and inflammatory (Il1b) and catabolic (Mmp3 and Mmp13) gene expression in the synovial tissue and cartilage were evaluated. In vitro direct effects of DEH on matrix metalloproteinase (MMP)-3 and MMP-13 expression were examined in interleukin-1β-stimulated human chondrocytes. In a rat model of collagen-induced arthritis, a single intra-articular dose of DEH inhibited knee joint inflammation and cartilage degeneration. Daily oral administration of DF had similar effects. Conversely, HA administered as a single intra-articular dose had no effect. Only DEH inhibited Mmp3 gene expression in the cartilage, whereas DEH and DF inhibited Mmp3 and Mmp13 mRNA expression in the synovial tissue. In interleukin-1β-stimulated human chondrocytes, DEH and HA inhibited MMP-3 and MMP-13 production, whereas DF had no effect. In this study, we demonstrated the chondroprotective effect of DEH in rats with collagen-induced arthritis and in interleukin-1β-stimulated human chondrocytes. Thus, DEH may suppress cartilage degeneration in patients with musculoskeletal diseases, such as OA.

Medium-Chain Fatty Acid Feeding Reduces Oxidation and Causes Panacinar Steatosis in Livers of Neonatal Pigs

BackgroundMedium-chain fatty acids (MCFAs) are commonly used to enhance the caloric content of infant formulas. We previously reported that pigs fed MCFA developed hepatic steatosis when compared to those fed isocaloric long-chain fatty acid (LCFA) rich formula. ObjectivesThe objectives of this study were to investigate: 1) whether MCFA and LCFA feeding affect hepatic fatty acid oxidation, and 2) how fat type alters the expression of hepatic fatty acid metabolic genes. MethodsTwenty-six, 7-d-old pigs were fed a low-energy control (CONT) formula, or 2 isocaloric high-energy formulas rich in LCFA or MCFA for 22 days. Livers were collected for examining ex vivo fatty acid oxidation, fatty acid content, and mRNA expression of fatty acid metabolic genes. ResultsLiver fat was 20% for pigs in the MCFA compared with 2.9% and 4.6% for those in the CONT and LCFA groups (P < 0.05). MCFA-fed pigs had greater amounts of hepatic laurate, myristate, palmitate, and palmitoleate (14, 34, 49, and 9.3 mg · g-1) than those fed LCFA and CONT (1.8, 1.9, 19, 1.5 mg · g-1) formulas (P ≤ 0.05). Hepatic laurate and palmitate oxidation was reduced for pigs fed MCFA (29 mmol · mg-1 · h-1) compared with those fed CONT (54 mmol · mg-1 · h-1) and LCFA (51 mmol · mg-1 · h-1) formulas (P < 0.05). Expression of fatty acid synthase 3 (FASN-3), fatty acid binding protein 1 (FABP-1), and acetyl-CoA carboxylase 1 (ACACA-1) were 8-, 6-, and 2-fold greater for pigs in the MCFA than those in the LCFA and CONT groups (P < 0.05). ConclusionsFeeding MCFA resulted in hepatic steatosis compared with an isocaloric formula rich in LCFA. Steatosis occurred concomitantly with reduced fatty acid oxidation but greater mRNA expression of fatty acid synthetic and catabolic genes.

Characterization of granulation in citrus “Harumi” fruit during postharvest storage

Granulation is a serious physiological disorder in citrus fruit, which impairs the fruit quality and causes economic losses to the citrus industry. ‘Harumi’ ((Citrus unshiu × C. sinensis) × C. reticulate), a mandarin-type variety, is prone to granulation, especially after long-term storage. In the present study, to elucidate the molecular mechanism of granulation, physiochemical changes were investigated in the juice sacs of ‘Harumi’ during the granulation process. The results showed that large size fruit (L fruit, transverse diameter of 8.5 - 10.0 cm) was prone to granulation, while small size fruit (S fruit, transverse diameter of 5.0 - 6.5 cm) was not. In L fruit, lignin content increased rapidly, which was accompanied by the reduction of the content of sugars (fructose and glucose) and organic acid (citric acid) during the granulation process. Moreover, the occurrence of granulation inhibited carotenoid biosynthesis in the ‘Harumi’ fruit. In L fruit, the content of the major carotenoids, β-cryptoxanthin, all-trans-violaxanthin, and 9-cis-violaxanthin, were decreased during the granulation process, and as a result, total carotenoid content in L fruit was 75.8% lower than that in S fruit in the fifth month after harvest. The real-time PCR results showed that the expression of carotenoid biosynthetic genes (CitPSY, CitPDS, CitZDS, CitLCYb2, and CitHYb) was down-regulated, while the expression of carotenoid catabolic genes (CitNCED2 and CitNCED3) was up-regulated in L fruit during the granulation process. This study is the first to investigate the carotenoid metabolism during the granulation process, which will provide new insights into exploring the causes of granulation in citrus fruit.

IMPACT OF MINCING TECHNIQUES ON CHONDROCYTE VIABILITY IN BOVINE ARTICULAR CARTILAGE: A COMPARATIVE STUDY BETWEEN COMMERCIALLY AVAILABLE SHAVERS AND SCALPEL MINCING

Mincing cartilage with commercially available shavers is increasingly used for treating focal cartilage defects. This study aimed to compare the impact of mincing bovine articular cartilage using different shaver blades on chondrocyte viability.Bovine articular cartilage was harvested using a scalpel or three different shaver blades (2.5 mm, 3.5 mm, or 4.2 mm) from a commercially available shaver. The cartilage obtained with a scalpel was minced into fragments smaller than 1 mm3. All four conditions were cultivated in a culture medium for seven days. After Day 1 and Day 7, metabolic activity, RNA isolation, and gene expression of anabolic (COL2A1, ACAN) and catabolic genes (MMP1, MMP13), Live/Dead staining and visualization using confocal microscopy, and flow cytometric characterization of minced cartilage chondrocytes were measured.The study found that mincing cartilage with shavers significantly reduced metabolic activity after one and seven days compared to scalpel mincing (p&lt;0.001). Gene expression of anabolic genes was reduced, while catabolic genes were increased after day 7 in all shaver conditions. The MMP13/COL2A1 ratio was also increased in all shaver conditions. Confocal microscopy revealed a thin line of dead cells at the lesion site with viable cells below for the scalpel mincing and a higher number of dead cells diffusely distributed in the shaver conditions. After seven days, there was a significant decrease in viable cells in the shaver conditions compared to scalpel mincing (p&lt;0.05). Flow cytometric characterization revealed fewer intact cells and proportionally more dead cells in all shaver conditions compared to the scalpel mincing.Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing. This indicates that mincing cartilage with a shaver should be considered a matrix rather than a cell therapy. Further experimental and clinical studies are required to standardize the mincing process with a shaver.Acknowledgements: This study received unrestricted funding from KARL STORZ SE &amp; Co. KG.

PH-sensing G protein-coupled orphan receptor GPR68 is expressed in human cartilage and correlates with degradation of extracellular matrix during OA progression

Background Osteoarthritis (OA) is a debilitating joints disease affecting millions of people worldwide. As OA progresses, chondrocytes experience heightened catabolic activity, often accompanied by alterations in the extracellular environment’s osmolarity and acidity. Nevertheless, the precise mechanism by which chondrocytes perceive and respond to acidic stress remains unknown. Recently, there has been growing interest in pH-sensing G protein-coupled receptors (GPCRs), such as GPR68, within musculoskeletal tissues. However, function of GPR68 in cartilage during OA progression remains unknown. This study aims to identify the role of GPR68 in regulation of catabolic gene expression utilizing an in vitro model that simulates catabolic processes in OA. Methods We examined the expression of GPCR by analyzing high throughput RNA-Seq data in human cartilage isolated from healthy donors and OA patients. De-identified and discarded OA cartilage was obtained from joint arthroplasty and chondrocytes were prepared by enzymatic digestion. Chondrocytes were treated with GPR68 agonist, Ogerin and then stimulated IL1β and RNA isolation was performed using Trizol method. Reverse transcription was done using the cDNA synthesis kit and the expression of GPR68 and OA related catabolic genes was quantified using SYBR® green assays. Results The transcriptome analysis revealed that pH sensing GPCR were expressed in human cartilage with a notable increase in the expression of GPR68 in OA cartilage which suggest a potential role for GPR68 in the pathogenesis of OA. Immunohistochemical (IHC) and qPCR analyses in human cartilage representing various stages of OA indicated a progressive increase in GPR68 expression in cartilage associated with higher OA grades, underscoring a correlation between GPR68 expression and the severity of OA. Furthermore, IHC analysis of Gpr68 in murine cartilage subjected to surgically induced OA demonstrated elevated levels of GPR68 in knee cartilage and meniscus. Using IL1β stimulated in vitro model of OA catabolism, our qPCR analysis unveiled a time-dependent increase in GPR68 expression in response to IL1β stimulation, which correlates with the expression of matrix degrading proteases suggesting the role of GPR68 in chondrocytes catabolism and matrix degeneration. Using pharmacological activator of GPR68, our results further showed that GPR68 activation repressed the expression of MMPs in human chondrocytes. Conclusions Our results demonstrated that GPR68 was robustly expressed in human cartilage and mice and its expression correlates with matrix degeneration and severity of OA progression in human and surgical model. GPR68 activation in human chondrocytes further repressed the expression of MMPs under OA pathological condition. These results identify GPR68 as a possible therapeutic target in the regulation of matrix degradation during OA.

Characterization of human placenta-derived exosome (pExo) as a potential osteoarthritis disease modifying therapeutic

ObjectiveHuman placenta-derived exosomes (pExo) were generated, characterized, and evaluated as a therapeutic candidate for the treatment of osteoarthritis (OA).MethodspExo was generated from full-term human placenta tissues by sequential centrifugation, purification, and sterile filtration. Upon analysis of particle size, cytokine composition, and exosome marker expression, pExo was further tested in cell-based assays to examine its effects on human chondrocytes. In vivo therapeutic efficacies were evaluated in a medial meniscal tear/medial collateral ligament tear (MCLT + MMT) rat model, in which animals received pExo injections intraarticularly and weight bearing tests during in-life stage while histopathology and immunohistochemistry were performed as terminal endpoints.ResultspExo displayed typical particle size, expressed maker proteins of exosome, and contained proteins with pro-proliferative, pro-anabolic, anti-catabolic, or anti-inflammatory activities. In vitro, pExo promoted chondrocyte migration and proliferation dose-dependently, which may involve its activation of cell growth-related signaling pathways. Expression of inflammatory and catabolic genes induced in a cellular OA model was significantly suppressed by pExo. In the rat OA model, pExo alleviated pain burden, restored cartilage degeneration, and downregulated expressions of pro-inflammatory, catabolic, or apoptotic proteins in a dose-dependent manner.ConclusionsOur study demonstrates that pExo has multiple potential therapeutic effects including symptom control and disease modifying characteristics. This may make it an attractive candidate for further development as an anti-OA therapeutic.

Proteomics analysis of C2C12 myotubes treated with atrophy inducing cancer cell-derived factors.

Cancer-associated cachexia is a wasting syndrome that results in dramatic loss of whole-body weight, predominantly due to loss of skeletal muscle mass. It has been established that cachexia inducing cancer cells secrete proteins and extracellular vesicles (EVs) that can induce muscle atrophy. Though several studies examined these cancer-cell derived factors, targeting some of these components have shown little or no clinical benefit. To develop new therapies, understanding of the dysregulated proteins and signaling pathways that regulate catabolic gene expression during muscle wasting is essential. Here, we sought to examine the effect of conditioned media (CM) that contain secreted factors and EVs from cachexia inducing C26 colon cancer cells on C2C12 myotubes using mass spectrometry-based label-free quantitative proteomics. We identified significant changes in the protein profile of C2C12 cells upon exposure to C26-derived CM. Functional enrichment analysis revealed enrichment of proteins associated with inflammation, mitochondrial dysfunction, muscle catabolism, ROS production, and ER stress in CM treated myotubes. Furthermore, strong downregulation in muscle structural integrity and development and/or regenerative pathways were observed. Together, these enriched proteins in atrophied muscle could be utilized as potential muscle wasting markers and the dysregulated biological processes could be employed for therapeutic benefit in cancer-induced muscle wasting.

CTRP13 ablation improves systemic glucose and lipid metabolism

ObjectiveTissue crosstalk mediated by secreted hormones underlies the integrative control of metabolism. We previously showed that CTRP13/C1QL3, a secreted protein of the C1q family, can improve glucose metabolism and insulin action in vitro and reduce food intake and body weight in mice when centrally delivered. A role for CTRP13 in regulating insulin secretion in isolated islets has also been demonstrated. It remains unclear, however, whether the effects of CTRP13 on cultured cells and in mice reflect the physiological function of the protein. Here, we use a loss-of-function mouse model to address whether CTRP13 is required for metabolic homeostasis. MethodsWT and Ctrp13 knockout (KO) mice fed a standard chow or a high-fat diet were subjected to comprehensive metabolic phenotyping. Transcriptomic analyses were carried out on visceral and subcutaneous fat, liver, and skeletal muscle to identify pathways altered by CTRP13 deficiency. RNA-seq data was further integrated with the Metabolic Syndrome in Man (METSIM) cohort data. Adjusted regression analysis was used to demonstrate that genetic variation of CTRP13 expression accounts for a significant proportion of variance between differentially expressed genes (DEGs) in adipose tissue and metabolic traits in humans. ResultsContrary to expectation, chow-fed Ctrp13-KO male mice had elevated physical activity, lower body weight, and improved lipid handling. On a high-fat diet (HFD), Ctrp13-KO mice of either sex were consistently more active and leaner. Loss of CTRP13 reduced hepatic glucose output and improved glucose tolerance, insulin sensitivity, and triglyceride clearance, though with notable sex differences. Consistent with the lean phenotype, transcriptomic analyses revealed a lower inflammatory profile in visceral fat and liver. Reduced hepatic steatosis was correlated with the suppression of lipid synthesis and enhanced lipid catabolism gene expression. Visceral fat had the largest number of DEGs and mediation analyses on the human orthologs of the DEGs suggested the potential causal contribution of CTRP13 to human metabolic syndrome. ConclusionsOur results suggest that CTRP13 is a negative metabolic regulator, and its deficiency improves systemic metabolic profiles. Our data also suggest the reduction in circulating human CTRP13 levels seen in obesity and diabetes may reflect a compensatory physiologic response to counteract insulin resistance.

Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate-bead culture model.

Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model. After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis. Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment. WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.

Cyclic loading induces anabolic gene expression in ACLs in a load-dependent and sex-specific manner.

Anterior cruciate ligament (ACL) injuries are historically thought to be a result of a single acute overload or traumatic event. However, recent studies suggest that ACL failure may be a consequence of fatigue damage. Additionally, the remodeling response of ACLs to fatigue loading is unknown. Therefore, the objective of this study was to investigate the remodeling response of ACLs to cyclic loading. Furthermore, given that women have an increased rate of ACL rupture, we investigated whether this remodeling response is sex specific. ACLs were harvested from male and female New Zealand white rabbits and cyclically loaded in a tensile bioreactor mimicking the full range of physiological loading (2, 4, and 8 MPa). Expression of markers for anabolic and catabolic tissue remodeling, as well as inflammatory cytokines, was quantified using quantitative reverse transcriptionpolymerase chain reaction. We found that the expression of markers for tissue remodeling of the ACL is dependent on the magnitude of loading and is sex specific. Male ACLs activated an anabolic response to cyclic loading at 4 MPa but turned off remodeling at 8 MPa. These data support the hypothesis that noncontact ACL injury may be a consequence of failed tissue remodeling and inadequate repair of microtrauma resulting from elevated loading. Compared to males, female ACLs failed to increase anabolic gene expression with loading and exhibited higher expression of catabolic genes at all loading levels, which may explain the increased rate of ACL tears in women. Together, these data provide insight into load-induced ACL remodeling and potential causes of tissue rupture.

Glycerol 3-phosphate phosphatase/PGPH-2 counters metabolic stress and promotes healthy aging via a glycogen sensing-AMPK-HLH-30-autophagy axis in C. elegans

Metabolic stress caused by excess nutrients accelerates aging. We recently demonstrated that the newly discovered enzyme glycerol-3-phosphate phosphatase (G3PP; gene Pgp), which operates an evolutionarily conserved glycerol shunt that hydrolyzes glucose-derived glycerol-3-phosphate to glycerol, counters metabolic stress and promotes healthy aging in C. elegans. However, the mechanism whereby G3PP activation extends healthspan and lifespan, particularly under glucotoxicity, remained unknown. Here, we show that the overexpression of the C. elegans G3PP homolog, PGPH-2, decreases fat levels and mimics, in part, the beneficial effects of calorie restriction, particularly in glucotoxicity conditions, without reducing food intake. PGPH-2 overexpression depletes glycogen stores activating AMP-activate protein kinase, which leads to the HLH-30 nuclear translocation and activation of autophagy, promoting healthy aging. Transcriptomics reveal an HLH-30-dependent longevity and catabolic gene expression signature with PGPH-2 overexpression. Thus, G3PP overexpression activates three key longevity factors, AMPK, the TFEB homolog HLH-30, and autophagy, and may be an attractive target for age-related metabolic disorders linked to excess nutrients.

Immunomodulatory potential of mesenchymal stromal cell-derived extracellular vesicles in chondrocyte inflammation.

Osteoarthritis (OA) affects a large percentage of the population worldwide. Current surgical and nonsurgical concepts for treating OA only result in symptom-modifying effects. However, there is no disease-modifying therapy available. Extracellular vesicles released by mesenchymal stem/stromal cells (MSC-EV) are promising agents to positively influence joint homeostasis in the osteoarthritic surroundings. This pilot study aimed to investigate the effect of characterized MSC-EVs on chondrogenesis in a 3D chondrocyte inflammation model with the pro-inflammatory cytokine TNFα. Bovine articular chondrocytes were expanded and transferred into pellet culture at passage 3. TNFα, human MSC-EV preparations (MSC-EV batches 41.5-EVi1 and 84-EVi), EVs from human platelet lysate (hPL4-EV), or the combination of TNFα and EVs were supplemented. To assess the effect of MSC-EVs in the chondrocyte inflammation model after 14 days, DNA, glycosaminoglycan (GAG), total collagen, IL-6, and NO release were quantified, and gene expression of anabolic (COL-II, aggrecan, COMP, and PRG-4), catabolic (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5), dedifferentiation (COL-I), hypertrophy (COL-X, VEGF), and inflammatory (IL-8) markers were analyzed; histological evaluation was performed using safranin O/Fast Green staining and immunohistochemistry of COL I and II. For statistical evaluation, nonparametric tests were chosen with a significance level of p < 0.05. TNFα supplementation resulted in catabolic stimulation with increased levels of NO and IL-6, upregulation of catabolic gene expression, and downregulation of anabolic markers. These findings were supported by a decrease in matrix differentiation (COL-II). Supplementation of EVs resulted in an upregulation of the chondrogenic marker PRG-4. All MSC-EV preparations significantly increased GAG retention per pellet. In contrast, catabolic markers and IL-8 expression were upregulated by 41.5-EVi1. Regarding protein levels, IL-6 and NO release were increased by 41.5-EVi1. Histologic and immunohistochemical evaluations indicated a higher differentiation potential of chondrocytes treated with 84-EVi. MSC-EVs can positively influence chondrocyte matrix production in pro-inflammatory surroundings, but can also stimulate inflammation. In this study MSC-EV 41.5-EVi1 supplementation increased chondrocyte inflammation, whereas MSC-84-EVi supplementation resulted a higher chondrogenic potential of chondrocytes in 3D pellet culture. In summary, the selected MSC-EVs exhibited promising chondrogenic effects indicating their significant potential for the treatment of OA; however, the functional heterogeneity in MSC-EV preparations has to be solved.

GPRC5B protects osteoarthritis by regulation of autophagy signaling.

Osteoarthritis (OA) is one of the most common chronic diseases in the world. However, current treatment modalities mainly relieve pain and inhibit cartilage degradation, but do not promote cartilage regeneration. In this study, we show that G protein-coupled receptor class C group 5 member B (GPRC5B), an orphan G-protein-couple receptor, not only inhibits cartilage degradation, but also increases cartilage regeneration and thereby is protective against OA. We observed that Gprc5b deficient chondrocytes had an upregulation of cartilage catabolic gene expression, along with downregulation of anabolic genes invitro. Furthermore, mice deficient in Gprc5b displayed a more severe OA phenotype in the destabilization of the medial meniscus (DMM) induced OA mouse model, with upregulation of cartilage catabolic factors and downregulation of anabolic factors, consistent with our invitro findings. Overexpression of Gprc5b by lentiviral vectors alleviated the cartilage degeneration in DMM-induced OA mouse model by inhibiting cartilage degradation and promoting regeneration. We also assessed the molecular mechanisms downstream of Gprc5b that may mediate these observed effects and identify the role of protein kinase B (AKT)-mammalian target of rapamycin (mTOR)-autophagy signaling pathway. Thus, we demonstrate an integral role of GPRC5B in OA pathogenesis, and activation of GPRC5B has the potential in preventing the progression of OA.