Expression Of Catabolic Genes Research Articles

Effects of iron overload in human joint tissue explant cultures and animal models.

Osteoarthritis (OA) is a prevalent degenerative joint disease affecting over 530 million individuals worldwide. Recent studies suggest a potential link between iron overload, a condition characterised by the excessive accumulation of iron in the body, and the onset of OA. Iron is essential for various biological processes, and any disruption in its homeostasis can trigger significant health effects, including OA. This study aimed to elucidate the effects of excess iron on joint tissue and the underlying mechanisms associated with excess iron and OA development. Human articular cartilage (n = 6) and synovium (n = 4) were collected from patients who underwent total knee arthroplasty. Cartilage and synovium explants were incubated with a gradually increasing concentration of ferric ammonium citrate for 3days respectively. The effects of iron homeostasis in tissue explants were analysed using a Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). To further study the effects of iron excess on OA initiation and development, male 3-week-old Hfe-/- and 5-week-old Tfr2-/- mice, animal models of hereditary haemochromatosis were established. Littermate wild-type mice were fed a high-iron diet to induce dietary overload. All animals were sacrificed at 8weeks of age, and knee joints were harvested for histological analysis. The LA-ICP-MS analysis unveiled changes in the elemental composition related to iron metabolism, which included alterations in FTH1, FPN1, and HAMP within iron(III)-treated cartilage explants. While chondrocyte viability remained stable under different iron concentrations, ex vivo treatment with a high concentration of Fe3+ increased the catabolic gene expression of MMP13. Similar alterations were observed in the synovium, with added increases in GAG content and inflammation markers. In vivo studies further supported the role of iron overload in OA development as evidenced by spontaneous OA symptoms, proteoglycan loss, increased Mankin scores, synovial thickening, and enhanced immunohistochemical expression of MMP13, ADAMTS5, and P21 in Hfe-/-, Tfr2-/-, and diet-induced iron overload mouse models. Our findings elucidate the specific pathways through which excess iron accelerates OA progression and highlights potential targets for therapeutic intervention aimed at modulating iron levels to mitigate OA symptoms. KEY MESSAGES: Iron overload alters joint iron metabolism, increasing OA markers in cartilage and synovium. High iron levels in mice accelerate OA, highlighting genetic and dietary impacts. Excess iron prompts chondrocyte iron storage response, signalling potential OA pathways. Iron dysregulation linked to increased cartilage degradation and synovial inflammation. Our findings support targeted therapies for OA based on iron modulation strategies.

Development of a novel rabbit model for femoroacetabular impingement through surgically induced acetabular overcoverage.

There is a lack of validated small animal models for femoroacetabular impingement (FAI) that induce intra-articular lesions and cause osteoarthritis (OA) progression. The gene expression profile of articular cartilage in patients with FAI has not been characterized in animal studies. The purpose of this study is to describe a novel rabbit model for FAI with validated induction of intra-articular lesions and OA progression and to characterize the gene expression pattern in impinged cartilage using this model. Thirty 6-month-old New Zealand White rabbits underwent unilateral endobutton implant placement at the acetabular rim to surgically create overcoverage. Radiological assessment confirmed secure placement of endobutton at the acetabular rim for all operated hips with a mean alteration in lateral center-edge angle (ΔLCEA) of 16.2 ± 6.6°. Gross inspection revealed secondary cartilage injuries in the anterosuperior region of the femoral head for the operated hips. Cartilage injuries were shown to exacerbate with increased impingement duration, as demonstrated by the modified Outerbridge scores and Mankin scores. Immunostaining and quantitative real-time polymerase chain reaction revealed elevated expression of inflammatory, anabolic and catabolic genes in impinged cartilage. RNA sequencing analysis of cartilage tissue revealed a distinct transcriptome profile and identified C-KIT, CD86, and CD68 as central markers. Our study confirmed that the novel rabbit FAI model created acetabular overcoverage and produced articular cartilage injury at the impingement zone. Cartilage from the impingement zone demonstrated a heightened metabolic state, corroborating with the gene expression pattern observed in patients with FAI.

Changes in Skeletal Muscle Atrophy over Time in a Rat Model of Adenine-Induced Chronic Kidney Disease

This study evaluated changes over time in skeletal muscle atrophy, expressions of skeletal muscle anabolic and catabolic genes, and mitochondrial activity by skeletal muscle type in an adenine-induced chronic kidney disease (CKD) model. A CKD model was successfully established by feeding male Wistar rats a 0.75% adenine diet for 4 weeks starting at 8 weeks of age. Control and CKD groups were sacrificed at 12 and 20 weeks of age. The back muscles were analyzed histologically, and succinate dehydrogenase (SDH) staining was performed to evaluate mitochondrial activity. Gene expressions of myogenic determination gene number 1 and myogenin as indicators of muscle anabolism, atrogin-1 and muscle RING-finger protein-1 (MuRF1) as indicators of muscle catabolism, and peroxisome proliferator-activated receptor-γ coactivator-1-α as a marker of mitochondrial biogenesis were assessed. Type I and type II muscle cross-sectional areas (CSAs) were decreased at 12 weeks, but type I muscle CSA was recovered at 20 weeks. SDH staining was lower in CKD than in control rats at 12 weeks, but no significant difference was observed at 20 weeks. Increased expressions of myogenin, atrogin-1, and MuRF-1 were observed only at 12 weeks, but no differences were observed at 20 weeks. The adenine-induced CKD rat model appears to show changes in muscle atrophy over time.

Efficacy and safety of SKCPT in patients with knee osteoarthritis: A multicenter, randomized, double-blinded, active-controlled phase III clinical trial

Ethnopharmacological relevanceOsteoarthritis (OA) is the most prevalent type of arthritis worldwide and a leading cause of years lost to pain and disability. Among the current pharmacological treatments for OA, symptomatic slow-acting drugs for OA (SYSADOA) induce pain relief and aim to improve joint function by relieving inflammation while causing fewer gastrointestinal and cardiovascular adverse events than non-steroidal anti-inflammatory drugs (NSAIDs). SKCPT is a herbal SYSADOA formulated from Clematis mandshurica, Trichosanthes kirilowii, and Prunella vulgaris powdered extracts. This preparation has been shown to induce cartilage protection and anti-inflammatory effects in preclinical studies and inhibit glycosaminoglycan degradation and catabolic gene expression in human OA chondrocytes and cartilage. Aim of the studyWe aimed to evaluate the non-inferiority of SKCPT to celecoxib and safety for treating knee OA. Materials and methodsThis multicenter, randomized, double-blind, phase III clinical trial enrolled adults with primary knee OA who were randomized (1:1) to SKCPT 300 mg twice daily or celecoxib 200 mg once daily for 12 weeks. ResultsIn total, 278 patients were assigned to treatment (SKCPT, 136; celecoxib, 142) for approximately 12 weeks. The primary endpoint was the mean change of Korean Western Ontario and McMaster Universities Osteoarthritis Index (K-WOMAC) pain subscale scores from baseline to Day 84. The mean change (least squares [LS] mean ± standard error) from baseline to Day 84 was −23.74 ± 1.48 for SKCPT and −25.88 ± 1.44 for celecoxib. The two-sided 95% confidence interval of the difference (LS mean) between groups was [−1.94, 6.20], confirming that the upper limit was less than the non-inferiority margin of 10. Additionally, there were no significant differences in the secondary endpoints (mean changes of K-WOMAC pain, physical, stiffness subscale, and total score, and the frequency and number of doses of rescue medications) between groups at all time points. Differences between groups in adverse events and adverse drug reactions were not significant, and no serious adverse events occurred. ConclusionsSKCPT efficacy was non-inferior, and its safety profile was similar, to celecoxib. Building on previous results showing that SYSADOA reduce NSAID intake, the present results suggest that the SYSADOA SKCPT could effectively replace NSAIDs in knee OA treatment while avoiding long-term side effects.

Disruption of branched-chain amino acid homeostasis promotes the progression of DKD via enhancing inflammation and fibrosis-associated epithelial-mesenchymal transition

Background and aimsThe disrupted homeostasis of branched-chain amino acids (BCAAs, including leucine, isoleucine, and valine) has been strongly correlated with diabetes with a potential causal role. However, the relationship between BCAAs and diabetic kidney disease (DKD) remains to be established. Here, we show that the elevated BCAAs from BCAAs homeostatic disruption promote DKD progression unexpectedly as an independent risk factor. Methods and resultsSimilar to other tissues, the suppressed BCAAs catabolic gene expression and elevated BCAAs abundance were detected in the kidneys of type 2 diabetic mice and individuals with DKD. Genetic and nutritional studies demonstrated that the elevated BCAAs from systemic disruption of BCAAs homeostasis promoted the progression of DKD. Of note, the elevated BCAAs promoted DKD progression without exacerbating diabetes in the animal models of type 2 DKD. Mechanistic studies demonstrated that the elevated BCAAs promoted fibrosis-associated epithelial-mesenchymal transition (EMT) by enhancing the activation of proinflammatory macrophages through mTOR signaling. Furthermore, pharmacological enhancement of systemic BCAAs catabolism using small molecule inhibitor attenuated type 2 DKD. Finally, the elevated BCAAs also promoted DKD progression in type 1 diabetic mice without exacerbating diabetes. ConclusionBCAA homeostatic disruption serves as an independent risk factor for DKD and restoring BCAA homeostasis pharmacologically or dietarily represents a promising therapeutic strategy to ameliorate the progression of DKD.

The Expression of Toll-like Receptors in Cartilage Endplate Cells: A Role of Toll-like Receptor 2 in Pro-Inflammatory and Pro-Catabolic Gene Expression.

The vertebral cartilage endplate (CEP), crucial for intervertebral disc health, is prone to degeneration linked to chronic low back pain, disc degeneration, and Modic changes (MC). While it is known that disc cells express toll-like receptors (TLRs) that recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), it is unclear if CEP cells (CEPCs) share this trait. The CEP has a higher cell density than the disc, making CEPCs an important contributor. This study aimed to identify TLRs on CEPCs and their role in pro-inflammatory and catabolic gene expression. Gene expression of TLR1-10 was measured in human CEPs and expanded CEPCs using quantitative polymerase chain reaction. Additionally, surface TLR expression was measured in CEPs grouped into non-MC and MC. CEPCs were stimulated with tumor necrosis factor alpha, interleukin 1 beta, small-molecule TLR agonists, or the 30 kDa N-terminal fibronectin fragment. TLR2 signaling was inhibited with TL2-C29, and TLR2 protein expression was measured with flow cytometry. Ex vivo analysis found all 10 TLRs expressed, while cultured CEPCs lost TLR8 and TLR9 expression. TLR2 expression was significantly increased in MC1 CEPCs, and its expression increased significantly after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer upregulated TLR2 protein expression. The TLR2/1 and TLR2/6 ligands upregulated pro-inflammatory genes and matrix metalloproteases (MMP1, MMP3, and MMP13), and TLR2 inhibition inhibited their upregulation. Endplate resorptive capacity of TLR2 activation was confirmed in a CEP explant model. The expression of TLR1-10 in CEPCs suggests that the CEP is susceptible to PAMP and DAMP stimulation. Enhanced TLR2 expression in MC1, and generally in CEPCs under inflammatory conditions, has pro-inflammatory and pro-catabolic effects, suggesting a potential role in disc degeneration and MC.

A repeated strike loading organ culture model for studying compression-associated chronic disc degeneration.

Mechanical stress has been viewed as one of the key risk factors in accelerating the intervertebral disc degeneration process. The goal of the present study was to employ a repeated strike loading bovine caudal disc system to elucidate the pathophysiological impacts of cumulative mechanical stress on the disc. The discs in the model groups were subjected to two different mechanical stresses: one strike loading or repeated strike loading. The following indices were analyzed: histological morphology, glycosaminoglycan release, disc height, cell viability, apoptosis-related protein expression, and catabolism-related gene expression. Both mechanical stress modes induced degenerative changes in the discs by day 11, such as clefts and delamination of the annulus fibrosus; they increased glycosaminoglycan release. Cell viability was significantly decreased and catabolic gene expression was significantly up-regulated in the degenerative loading group and repeated strike loading group by day 9. These alterations remained evident in the annulus fibrosus tissue of the repeated strike loading group on day 11.Our data suggests that the repeated strike loading model adopted in this study could lead to degenerative changes in the disc organ model. Annulus fibrosus cells displayed a more noticeable response to mechanical stress damage and a slower recovery process, suggesting that the annulus fibrosus serves as a pivotal factor in disc degeneration due to mechanical stress injuries. The study also indicates that due to the gradual self-repair of intervertebral disc cells after injury, it is necessary to apply repeated strike loading on the disc at specific intervals when researching the repair of chronic disc injuries.

Altered extracellular matrix and mechanotransduction gene expression in rat bone tissue following long-term estrogen deficiency.

Osteoporosis is primarily associated with bone loss, but changes in bone tissue matrix composition and osteocyte mechanotransduction have also been identified. However, the molecular mechanisms underlying these changes and their relation to bone loss are not fully understood. The objectives of this study were to (1) conduct comprehensive temporal gene expression analyses on cortical bone tissue from ovariectomized rats, with a specific focus on genes known to govern matrix degradation, matrix production, and mechanotransduction, and (2) correlate these findings with bone mass, trabecular and cortical microarchitecture, and mineral and matrix composition. Microarray data revealed 35 differentially expressed genes in the cortical bone tissue of the ovariectomized cohort. We report that catabolic gene expression abates after the initial accelerated bone loss period, which occurs within the first 4wk of estrogen deficiency. However, in long-term estrogen deficiency, we report increased expression of genes associated with extracellular matrix deposition (Spp1, COL1A1, COL1A2, OCN) and mechanotransduction (Cx43) compared with age-matched controls and short-term estrogen deficiency. These changes coincided with increased heterogeneity of mineral-to-matrix ratio and collagen maturity, to which extracellular matrix markers COL1A1 and COL1A2 were positively correlated. Interestingly, mineral heterogeneity and collagen maturity, exhibited a negative correlation with PHEX and IFT88, associated with mechanosensory cilia formation and Hedgehog (Hh) signaling. This study provides the first insight into the underlying mechanisms governing secondary mineralization and heterogeneity of matrix composition of bone tissue in long-term estrogen deficiency. We propose that altered mechanobiological responses in long-term estrogen deficiency may play a role in these changes.

Catabolic gene indices of hydrocarbon diminution in Ultisol treated with cropped Bacillus altitudinis-amendments

This study evaluated catabolic gene expression as an index of hydrocarbon breakdown in ultisol treated with Bacillus altitudinis-Cropped Biofertilizers derived from Biosolid and Brewer Spent Grain (BSG) using phenotypic and molecular methods. We observed significant reductions in Total Petroleum Hydrocarbons (TPH) concentrations with both amendments, albeit the biosolid based amendment was markedly more effective. Concurrently, there was a substantial increase in hydrocarbon-degrading bacteria. Notably, the bulk of Operational Taxonomic Units (OTUs) in biosolid (95.77 %) and BSG (93.00 %) amended Ultisol were Proteobacteria, Acidobacteria, Actinobacteria, and Planctomycetes, which are key players in hydrocarbon bioremediation. We observed the presence of eleven hydrocarbon-degrading genes through M5nr analysis. These genes encompass essential catalysts for aliphatic hydrocarbon degradation and hydrocarbon desulfurization. Notably, these enzymes/genes include Bacterial Flavin-bound Monooxygenase (AlmA), Alkane Monooxygenase (AlkM), Alpha Ketoglutarate Dependent Dioxygenase (alkB), Propane Monooxygenase (PrM), Cytochrome P450 (cP450), Methane Monooxygenase/Ammonia Monooxygenase (MMO/AMO) subunits A, B, and C, Alkane Sulfonate Monooxygenase (ssuD), Alkane Sulfonates Transport System Permease Protein (ssuC), Dibenzothiophene Monooxygenase (dszC), Dibenzothiophene-Sulfone Monooxygenase (dszA), and Dibenzothiophene-5,5-Dioxide Monooxygenase (dszB). These genes play pivotal roles in the degradation of aliphatic hydrocarbons and hydrocarbon desulfurization. Interestingly, unique gene expression patterns were observed for each amendment, with Actinomycetales and Bulkhoderiales orders expressing the majority of identified genes. These findings have revealed the amendment-specific microbial and genetic alterations and, the diversity and potential of the annotated genes induced by the Bacillus altitudinis-Cropped Biofertilizers (biosolid and BSG amendments) for effective remediation of hydrocarbon-contaminated soils.

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.

Multiomic 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 multiomic 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.