RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Bioelectric medicine: unveiling the therapeutic potential of micro-current stimulation.

Bioelectric medicine (BEM) refers to the use of electrical signals to modulate the electrical activity of cells and tissues in the body for therapeutic purposes. In this review, we particularly focused on the microcurrent stimulation (MCS), because, this can take place at the cellular level with sub-sensory application unlike other stimuli. These extremely low-level currents mimic the body's natural electrical activity and are believed to promote various physiological processes. To date, MCS has limited use in the field of BEM with applications in several therapeutic purposes. However, recent studies provide hopeful signs that MCS is more scalable and widely applicable than what has been used so far. Therefore, this review delves into the landscape of MCS, shedding light on the multifaceted applications and untapped potential of MCS in the realm of healthcare. Particularly, we summarized the hierarchical mediation from cell to whole body responses by MCS including its physiological applications. Our final objective of this review is to contribute to the growing body of literature that unveils the captivating potential of BEM, with MCS poised at the intersection of technological innovation and the intricacies of the human body.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

Background: It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Methods: Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. Results: E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. Conclusion: E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

Protective Effects of Cereal Grain Extracts on Alcohol-Induced Hepatocyte Damage.

This study investigated the protective effects of cereal grains on alcohol-induced hepatocyte damage. Cereal grains were extracted with methanol, and their radical scavenging properties and total phenolic contents were examined. Black rice extract exhibited the highest total polyphenol content and radical scavenging capacity. Treatment with sorghum extract increased the viability of cells exposed to alcohol by up to 81.6%. All cereal grain extracts decreased reactive oxygen species and malondialdehyde production and glutathione depletion in HepG2 cells exposed to ethanol. In particular, black rice and sorghum extracts exhibited greater antioxidant effects than other cereal grains. Treatment with black rice extract increased the levels of alanine aminotransferase and aspartate aminotransferase of alcohol-exposed cells to control levels. Overall, black rice extract showed a greater protective effect compared with other cereal grains against alcohol exposure in HepG2 cells and could improve alcohol-induced liver problems.

Comparison of the Antioxidant Potency of Four Triterpenes of Centella asiatica against Oxidative Stress.

We comparatively evaluated the antioxidant properties of key triterpenes from Centella asiatica, including asiatic acid (AA), asiaticoside, madecassic acid, and madecassoside, in several cell types, including skin fibroblasts, macrophages, hepatocytes, and endothelial cells, under conditions promoting oxidative stress. AA conferred the highest viability on Hs68 cells exposed to ultraviolet B (UVB) irradiation. Triterpene pretreatment attenuated the UVB-induced generation of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as the UVB-induced depletion of glutathione (GSH) in skin fibroblasts. AA most potently inhibited UVB-induced MMP generation, resulting in increased intracellular collagen levels. Pretreatment with triterpenes, particularly AA, significantly improved cell viability and attenuated TBHP-induced levels of ROS, alanine aminotransferase, and aspartate aminotransferase in HepG2 cells. Triterpenes attenuated ROS levels and reduced MDA and GSH expression in EA.hy926 cells. In RAW264.7 macrophages, production of nitric oxide, tumor necrosis factor-α, and interleukin-6 (indicators of LPS-induced oxidative damage) was significantly reduced by treatment with any of the triterpenes. Statistical analyses of triterpene biological activities using principal component analysis and hierarchical clustering revealed that AA exerted the greatest overall influence and showed remarkable activity in Hs68 and HepG2 cells.

Discovery of a Novel Bioactive Compound in Orange Peel Polar Fraction on the Inhibition of Trimethylamine and Trimethylamine N-Oxide through Metabolomics Approaches and In Vitro and In Vivo Assays: Feruloylputrescine Inhibits Trimethylamine via Suppressing cntA/B Enzyme.

This study compares the inhibitory effects of orange peel polar fraction (OPP) and orange peel nonpolar fraction (OPNP) on trimethylamine (TMA) and trimethylamine N-oxide (TMAO) production in response to l-carnitine treatment in vivo and in vitro. Metabolomics is used to identify bioactive compounds. The research demonstrates that the OPP effectively regulates atherosclerosis-related markers, TMA and TMAO in plasma and urine, compared to the OPNP. Our investigation reveals that these inhibitory effects are independent of changes in gut microbiota composition. The effects are attributed to the modulation of cntA/B enzyme activity and FMO3 mRNA expression in vitro. Moreover, OPP exhibits stronger inhibitory effects on TMA production than OPNP, potentially due to its higher content of feruloylputrescine, which displays the highest inhibitory activity on the cntA/B enzyme and TMA production. These findings suggest that the OPP containing feruloylputrescine has the potential to alleviate cardiovascular diseases by modulating cntA/B and FMO3 enzymes without directly influencing gut microbiota composition.

The secreted protein Amuc_1409 from Akkermansia muciniphila improves gut health through intestinal stem cell regulation.

Akkermansia muciniphila has received great attention because of its beneficial roles in gut health by regulating gut immunity, promoting intestinal epithelial development, and improving barrier integrity. However, A. muciniphila-derived functional molecules regulating gut health are not well understood. Microbiome-secreted proteins act as key arbitrators of host-microbiome crosstalk through interactions with host cells in the gut and are important for understanding host-microbiome relationships. Herein, we report the biological function of Amuc_1409, a previously uncharacterised A. muciniphila-secreted protein. Amuc_1409 increased intestinal stem cell (ISC) proliferation and regeneration in ex vivo intestinal organoids and in vivo models of radiation- or chemotherapeutic drug-induced intestinal injury and natural aging with male mice. Mechanistically, Amuc_1409 promoted E-cadherin/ß-catenin complex dissociation via interaction with E-cadherin, resulting in the activation of Wnt/ß-catenin signaling. Our results demonstrate that Amuc_1409 plays a crucial role in intestinal homeostasis by regulating ISC activity in an E-cadherin-dependent manner and is a promising biomolecule for improving and maintaining gut health.

Targeted Learning with an Undersmoothed Lasso Propensity Score Model for Large-Scale Covariate Adjustment in Healthcare Database Studies.

Lasso regression is widely used for large-scale propensity score (PS) estimation in healthcare database studies. In these settings, previous work has shown that undersmoothing (overfitting) Lasso PS models can improve confounding control, but it can also cause problems of non-overlap in covariate distributions. It remains unclear how to select the degree of undersmoothing when fitting large-scale Lasso PS models to improve confounding control while avoiding issues that can result from reduced covariate overlap. Here, we used simulations to evaluate the performance of using collaborative-controlled targeted learning to data-adaptively select the degree of undersmoothing when fitting large-scale PS models within both singly and doubly robust frameworks to reduce bias in causal estimators. Simulations showed that collaborative learning can data-adaptively select the degree of undersmoothing to reduce bias in estimated treatment effects. Results further showed that when fitting undersmoothed Lasso PS-models, the use of cross-fitting was important for avoiding non-overlap in covariate distributions and reducing bias in causal estimates.

Assessment of Six Blackberry Cultivars Using a Combination of Metabolomics, Biological Activity, and Network Pharmacology Approaches.

Blackberries have gained considerable attention due to their high antioxidant content and potential health benefits. This study compared the metabolite profiles of six blackberry cultivars and investigated their biological activities. The metabolites extracted from blackberries were analyzed using metabolomics, and their biological activities and mechanisms were confirmed using in vitro models and network pharmacology. Among the cultivars examined, "Kiowa" ripe berries exhibited the highest antioxidant and anti-inflammatory activities. These effects were primarily attributed to the accumulation of flavonoids (quercitrin and luteolin) and anthocyanin (cyanidin 3-O-glucoside) in the phenylpropanoid pathway. Furthermore, our research identified 13 blackberry metabolites that interacted with 31 genes, including AKT1, CASP3, JUN, MAPK8, NOS3, NQO1, and HMOX1 which play roles in reducing oxidative stress, protecting cells from damage, and suppressing inflammation. These findings suggest that blackberry metabolites, such as quercitrin, luteolin, and cyanidin 3-O-glucoside, may exert therapeutic effects by modulating specific genes and pathways associated with antioxidant and anti-inflammatory responses. This research is promising not only for plant breeders but also for those interested in harnessing the health-promoting properties of blackberries.

Comparative analysis of LC-MS/MS and real-time PCR assays for efficient detection of potential allergenic silkworm.

The silkworm (Bombyx mori) has long been valued food and feed in East Asia for its abundant nutritional and medicinal attributes, conversely, it can elicit allergic responses in susceptible individuals. Therefore, the development of silkworm detection method is required to avert allergenic incidents. In this study, two methodologies, tandem mass spectrometry (LC-MS/MS) and real-time PCR, were developed to achieve effective silkworm detection. These methods exhibited exceptional sensitivity in identifying silkworm presence in processed foods. Furthermore, model cookies spiked with silkworm were used to validate the sensitivities of LC-MS/MS (0.0005%) and real-time PCR (0.001%). Overall, these techniques were useful for trace silkworm detection in food products; therefore, they may help prevent allergic reactions. To the best of our knowledge, this study represents the first comparison of LC-MS/MS and real-time PCR methods for silkworm detection, marking an important contribution to the field. Data are available from ProteomeXchange under identifier PXD042494.

Strawberry soluble solids QTL with inverse effects on yield.

Sugars are the main drivers of strawberry sweetness, and understanding their genetic control is of critical importance for breeding. Large-scale genome-wide association studies were performed in two populations totaling 3399 individuals evaluated for soluble solids content (SSC) and fruit yield. Two stable quantitative trait loci (QTL) on chromosome 3B and 6A for SSC were identified. Favorable haplotypes at both QTL for SSC decreased yield, though optimal allelic combinations were identified with reduced impacts on yield. Metabolites in the starch and sucrose metabolism pathway were characterized and quantified for 23 contrasting genotypes in leaves, white fruit, and red fruit. Variations in sucrose concentrations/efflux indicated genetic variation underlying sucrose accumulation and transportation during fruit ripening. Integration of genome-wide association studies and expression quantitative locus mapping identified starch synthase 4 (FxaC_10g00830) and sugar transporter 2-like candidate genes (FxaC_21g51570) within the respective QTL intervals. These results will enable immediate applications in genomics-assisted breeding for flavor and further study of candidate genes underlying genetic variation of sugar accumulation in strawberry fruit.

5-aminosalicylic acid suppresses osteoarthritis through the OSCAR-PPARγ axis.

Osteoarthritis (OA) is a progressive and irreversible degenerative joint disease that is characterized by cartilage destruction, osteophyte formation, subchondral bone remodeling, and synovitis. Despite affecting millions of patients, effective and safe disease-modifying osteoarthritis drugs are lacking. Here we reveal an unexpected role for the small molecule 5-aminosalicylic acid (5-ASA), which is used as an anti-inflammatory drug in ulcerative colitis. We show that 5-ASA competes with extracellular-matrix collagen-II to bind to osteoclast-associated receptor (OSCAR) on chondrocytes. Intra-articular 5-ASA injections ameliorate OA generated by surgery-induced medial-meniscus destabilization in male mice. Significantly, this effect is also observed when 5-ASA was administered well after OA onset. Moreover, mice with DMM-induced OA that are treated with 5-ASA at weeks 8-11 and sacrificed at week 12 have thicker cartilage than untreated mice that were sacrificed at week 8. Mechanistically, 5-ASA reverses OSCAR-mediated transcriptional repression of PPARγ in articular chondrocytes, thereby suppressing COX-2-related inflammation. It also improves chondrogenesis, strongly downregulates ECM catabolism, and promotes ECM anabolism. Our results suggest that 5-ASA could serve as a DMOAD.

smdi: an R package to perform structural missing data investigations on partially observed confounders in real-world evidence studies.

Objectives: Partially observed confounder data pose a major challenge in statistical analyses aimed to inform causal inference using electronic health records (EHRs). While analytic approaches such as imputation are available, assumptions on underlying missingness patterns and mechanisms must be verified. We aimed to develop a toolkit to streamline missing data diagnostics to guide choice of analytic approaches based on meeting necessary assumptions. Materials and methods: We developed the smdi (structural missing data investigations) R package based on results of a previous simulation study which considered structural assumptions of common missing data mechanisms in EHR. Results: smdi enables users to run principled missing data investigations on partially observed confounders and implement functions to visualize, describe, and infer potential missingness patterns and mechanisms based on observed data. Conclusions: The smdi R package is freely available on CRAN and can provide valuable insights into underlying missingness patterns and mechanisms and thereby help improve the robustness of real-world evidence studies.

Protective Effect of Whole Wheat on Muscle Atrophy in C2C12 Cells via Akt/FoxO1 Signaling Pathways.

Skeletal muscles are important for body movement, postural maintenance, and energy metabolism. Muscle atrophy is caused by various factors, including lack of exercise, age, genetics, and malnutrition, leading to the loss of muscle mass. The Akt/FoxO signaling pathway plays a key role in the regulation of muscle protein synthesis and degradation. Whole wheat contains functional ingredients that may indirectly contribute to muscle health and function and can help prevent or slow the progression of muscle atrophy. In this study, the protective effects of three wheat cultivars (Seodun, Ol, and Shinmichal 1) against hydrogen peroxide-induced muscle atrophy in C2C12 cells were investigated. We found that whole-wheat treatment reduced reactive oxygen species production, prevented glutathione depletion, and increased myotube diameter, thereby reducing muscle atrophy by activating myoblast differentiation. Generally, "Shinmichal 1" exhibited the highest activation of the Akt/FoxO signaling pathway. In contrast, "Seodun" showed similar or slightly higher activities than those of the H2O2-treated only group. In conclusion, whole wheat exerts a protective effect against muscle atrophy by activating the Akt/FoxO signaling pathway. This study indicates that whole wheat may help prevent muscle atrophy.

Chemically-defined and scalable culture system for intestinal stem cells derived from human intestinal organoids.

Three-dimensional human intestinal organoids (hIO) are widely used as a platform for biological and biomedical research. However, reproducibility and challenges for large-scale expansion limit their applicability. Here, we establish a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC3D-hIO) within hIO derived from human pluripotent stem cells. The intrinsic self-organisation property of ISC3D-hIO, combined with air-liquid interface culture in a minimally defined medium, forces ISC3D-hIO to differentiate into the intestinal epithelium with cellular diversity, villus-like structure, and barrier integrity. Notably, ISC3D-hIO is an ideal cell source for gene editing to study ISC biology and transplantation for intestinal diseases. We demonstrate the intestinal epithelium differentiated from ISC3D-hIO as a model system to study severe acute respiratory syndrome coronavirus 2 viral infection. ISC3D-hIO culture technology provides a biological tool for use in regenerative medicine and disease modelling.

Trends in new and persistent opioid use in older adults with and without cancer.

BACKGROUND: The impact of ongoing efforts to decrease opioid use on patients with cancer remains undefined. Our objective was to determine trends in new and additional opioid use in patients with and without cancer. METHODS: This retrospective cohort study used data from Surveillance, Epidemiology, and End Results program-Medicare for opioid-naive patients with solid tumor malignancies diagnosed from 2012 through 2017 and a random sample of patients without cancer. We identified 238 470 eligible patients with cancer and further focused on 4 clinical strata: patients without cancer, patients with metastatic cancer, patients with nonmetastatic cancer treated with surgery alone ("surgery alone"), and patients with nonmetastatic cancer treated with surgery plus chemotherapy or radiation therapy ("surgery+"). We identified new, early additional, and long-term additional opioid use and calculated the change in predicted probability of these outcomes from 2012 to 2017. RESULTS: New opioid use was higher in patients with cancer (46.4%) than in those without (6.9%) (P < .001). From 2012 to 2017, the predicted probability of new opioid use was more stable in the cancer strata (relative declines: 0.1% surgery alone; 2.4% surgery+; 8.8% metastatic cancer), than in the noncancer stratum (20.0%) (P < .001 for each cancer to noncancer comparison). Early additional use declined among surgery patients (‒14.9% and ‒17.5% for surgery alone and surgery+, respectively) but was stable among patients with metastatic disease (‒2.8%, P = .50). CONCLUSIONS: Opioid prescribing declined over time at a slower rate in patients with cancer than in patients without cancer. Our study suggests important but tempered effects of the changing opioid climate on patients with cancer.

Preclinical Study of Dual-Wavelength Light-Emitting Diode Therapy in an Osteoarthritis Rat Model.

OBJECTIVE: To evaluate the efficacy of light-emitting diode (LED) and their dual-wavelengths as a treatment strategy for osteoarthritis. METHODS: We induced osteoarthritis in male Sprague-Dawley rats by intra-articular injection of sodium iodoacetate into the right rear knee joint. The animals with lesions were divided into an untreated group and an LED-treated group (n=7 each). In the LED-treated group, the lesioned knee was irradiated with lasers (850 and 940 nm) and dose (3.15 J/cm2) for 20 minutes per session, twice a week for 4 weeks. Knee joint tissues were stained and scanned using an in vivo micro-computed tomography (CT) scanner. Serum interleukin (IL)-6 and IL-18 levels were determined using enzyme-linked immuno-sorbent assay. Several functional tests (lines crossed, rotational movement, rearing, and latency to remain rotating rod) were performed 24 hours before LED treatment and at 7, 14, 21, and 28 days after treatment. RESULTS: LED-treated rats showed improved locomotor function and suppressed matrix-degrading cytokines. Micro-CT images indicated that LED therapy had a preserving effect on cartilage and cortical bone. CONCLUSION: LED treatment using wavelengths of 850 and 940 nm resulted in significant functional, anatomical, and histologic improvements without adverse events in a rat model. Further research is required to determine the optimal wavelength, duration, and combination method, which will maximize treatment effectiveness.

Synergistic Effect of Madecassoside and Rosmarinic Acid Against Ultraviolet B-Induced Photoaging in Human Skin Fibroblasts.

Madecassoside (MD) and rosmarinic acid (RA) are well-known compounds with wound healing and antiaging effects. We demonstrated the synergistic protective activity of the MD-RA combination in Hs68 cells against ultraviolet B (UVB)-induced photoaging. The cell viabilities of MD, RA, and MD-RA combinations at various ratios (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, v/v) were measured to compare their protective effects against UVB radiation. The synergistic interaction between MD and RA was confirmed using a combination index. The strongest effect of the MD-RA combination was observed at a ratio of 3:7. The combination of MD-RA 3:7 exerted a synergistic effect against UVB-induced changes in cell viability, as well as superoxide dismutase activity, reactive oxygen species, glutathione, catalase activity, and malondialdehyde levels. Moreover, the inhibitory effect of the MD-RA combination (3:7) on matrix metalloproteinases and total collagen production was higher than that of MD or RA alone. These results demonstrated that the MD-RA combination (3:7) generated a strong synergistic effect against UVB-induced photoaging in Hs68 cells. Overall, our results provide scientific evidence to support the development of a new combination therapy for skin protection against UVB-induced photoaging through the synergistic interaction between MD and RA. These natural compounds are promising options for antiaging and skin protection in the cosmetic and pharmaceutical industries.

Identification of Polymethoxyflavones (PMFs) from Orange Peel and Their Inhibitory Effects on the Formation of Trimethylamine (TMA) and Trimethylamine-N-oxide (TMAO) Using cntA/B and cutC/D Enzymes and Molecular Docking.

This study investigates the inhibitory effects of polymethoxyflavones (PMFs) on enzymes involved in the production of trimethylamine (TMA) and trimethylamine-N-oxide (TMAO). PMFs were isolated from Valencia orange peel and identified using column separation and NMR techniques. The findings reveal that nobiletin and 3,6,7,8,2',5'-hexamethoxyflavone significantly suppress cntA/B and cutC/D, respectively. Furthermore, 3,6,7,8,2',5'-hexamethoxyflavone decreases the level of TMAO formation by suppressing the FMO3 mRNA level. This study elucidates that specific structural features of PMFs can contribute to their interactions with enzymes. Our study represents the first demonstration of the ability of PMFs to mitigate the risk of cardiovascular disease (CVD) by inhibiting enzymes responsible for TMA production, which are generated by gut microbiomes. Furthermore, we introduce a novel model system utilizing TMA-induced HepG2 cells to assess and compare the inhibitory effects of PMFs on TMAO production. These findings could pave the way for the development of novel therapeutic approaches to manage CVD.