Loss of PBX1 function in Leydig cells causes testicular dysgenesis and male sterility.

Leydig cells are essential components of testicular interstitial tissue and serve as a primary source of androgen in males. A functional deficiency in Leydig cells often causes severe reproductive disorders; however, the transcriptional programs underlying the fate decisions and steroidogenesis of these cells have not been fully defined. In this study, we report that the homeodomain transcription factor PBX1 is a master regulator of Leydig cell differentiation and testosterone production in mice. PBX1 was highly expressed in Leydig cells and peritubular myoid cells in the adult testis. Conditional deletion of Pbx1 in Leydig cells caused spermatogenic defects and complete sterility. Histological examinations revealed that Pbx1 deletion impaired testicular structure and led to disorganization of the seminiferous tubules. Single-cell RNA-seq analysis revealed that loss of Pbx1 function affected the fate decisions of progenitor Leydig cells and altered the transcription of genes associated with testosterone synthesis in the adult testis. Pbx1 directly regulates the transcription of genes that play important roles in steroidogenesis (Prlr, Nr2f2 and Nedd4). Further analysis demonstrated that deletion of Pbx1 leads to a significant decrease in testosterone levels, accompanied by increases in pregnenolone, androstenedione and luteinizing hormone. Collectively, our data revealed that PBX1 is indispensable for maintaining Leydig cell function. These findings provide insights into testicular dysgenesis and the regulation of hormone secretion in Leydig cells.

Genomics-based identification of a cold adapted clade in Deinococcus.

BACKGROUND: Microbes in the cold polar and alpine environments play a critical role in feedbacks that amplify the effects of climate change. Defining the cold adapted ecotype is one of the prerequisites for understanding the response of polar and alpine microbes to climate change. RESULTS: Here, we analysed 85 high-quality, de-duplicated genomes of Deinococcus, which can survive in a variety of harsh environments. By leveraging genomic and phenotypic traits with reverse ecology, we defined a cold adapted clade from eight Deinococcus strains isolated from Arctic, Antarctic and high alpine environments. Genome-wide optimization in amino acid composition and regulation and signalling enable the cold adapted clade to produce CO2 from organic matter and boost the bioavailability of mineral nitrogen. CONCLUSIONS: Based primarily on in silico genomic analysis, we defined a potential cold adapted clade in Deinococcus and provided an updated view of the genomic traits and metabolic potential of Deinococcus. Our study would facilitate the understanding of microbial processes in the cold polar and alpine environments.

In Vivo Exposure Pathways of Ambient Magnetite Nanoparticles Revealed by Machine Learning-Aided Single-Particle Mass Spectrometry.

Nanosized ultrafine particles (UFPs) from natural and anthropogenic sources are widespread and pose serious health risks when inhaled by humans. However, tracing the inhaled UFPs in vivo is extremely difficult, and the distribution, translocation, and metabolism of UFPs remain unclear. Here, we report a label-free, machine learning-aided single-particle inductively coupled plasma mass spectrometry (spICP-MS) approach for tracing the exposure pathways of airborne magnetite nanoparticles (MNPs), including external emission sources, and distribution and translocation in vivo using a mouse model. Our results provide quantitative analysis of different metabolic pathways in mice exposed to MNPs, revealing that the spleen serves as the primary site for MNP metabolism (84.4%), followed by the liver (11.4%). The translocation of inhaled UFPs across different organs alters their particle size. This work provides novel insights into the in vivo fate of UFPs as well as a versatile and powerful platform for nanotoxicology and risk assessment.

The Proteomic Landscape of Monocytes in Response to Colorectal Cancer Cells.

Colorectal cancer (CRC) involves a complex interaction between tumor cells and immune cells, notably monocytes, leading to immunosuppression. This study explored these interactions using in vitro coculture systems of THP-1 cells and CRC cell lines, employing quantitative proteomics to analyze protein changes in monocytes. Multiple analytical methods were utilized to delineate the altered proteomic landscape, identify key proteins, and their associated functional pathways for comprehensive data analysis. Differentially expressed proteins (DEPs) were selected and validated by cross-referencing them with publicly available TCGA and GEO data sets to explore their potential clinical significance. Our analysis identified 161 up-regulated and 130 down-regulated DEPs. The enrichment results revealed impairments in adhesion and innate immune functions in monocytes, potentially facilitating cancer progression. The down-regulation of FN1, THSB1, and JUN may contribute to these impairments. Furthermore, the overexpression of ADAMTSL4, PRAM1, GPNMB, and NPC2 on monocytes was associated with unfavorable prognostic outcomes in CRC patients, suggesting potential biomarkers or therapeutic targets. This study illustrated the proteomic landscape of monocytes in response to CRC cells, providing clues for future investigations of the crosstalk between cancer cells and monocytes within the tumor microenvironment.

Unveiling Atomic-Scaled Local Chemical Order of High-Entropy Intermetallic Catalyst for Alkyl-Substitution-Dependent Alkyne Semihydrogenation.

High-entropy intermetallic (HEI) nanocrystals, composed of multiple elements with an ordered structure, are of immense interest in heterogeneous catalysis due to their unique geometric and electronic structures and the cocktail effect. Despite tremendous efforts dedicated to regulating the metal composition and structures with advanced synthetic methodologies to improve the performance, the surface structure, and local chemical order of HEI and their correlation with activity at the atomic level remain obscure yet challenging. Herein, by determining the three-dimensional (3D) atomic structure of quinary PdFeCoNiCu (PdM) HEI using atomic-resolution electron tomography, we reveal that the local chemical order of HEI regulates the surface electronic structures, which further mediates the alkyl-substitution-dependent alkyne semihydrogenation. The 3D structures of HEI PdM nanocrystals feature an ordered (intermetallic) core enclosed by a disordered (solid-solution) shell rather than an ordered surface. The lattice mismatch between the core and shell results in apparent near-surface distortion. The chemical order of the intermetallic core increases with annealing temperature, driving the electron redistribution between Pd and M at the surface, but the surface geometrical (chemically disordered) configurations and compositions are essentially unchanged. We investigate the catalytic performance of HEI PdM with different local chemical orders toward semihydrogenation across a broad range of alkynes, finding that the electron density of surface Pd and the hindrance effect of alkyl substitutions on alkynes are two key factors regulating selective semihydrogenation. We anticipate that these findings on surface atomic structure will clarify the controversy regarding the geometric and/or electronic effects of HEI catalysts and inspire future studies on tuning local chemical order and surface engineering toward enhanced catalysts.

SRSF3 Knockdown Inhibits Lipopolysaccharide-Induced Inflammatory Response in Macrophages.

Serine/arginine-rich splicing factor 3 (SRSF3), the smallest member of the SR protein family, serves multiple roles in RNA processing, including splicing, translation, and stability. Recent studies have shown that SRSF3 is implicated in several inflammatory diseases. However, its impact on macrophage inflammation remains unclear. Herein, we determined the expression of SRSF3 in inflammatory macrophages and found that the level of SRSF3 was increased in macrophages within atherosclerotic plaques, as well as in RAW-264.7 macrophages stimulated by lipopolysaccharides. Moreover, the downregulation of SRSF3 suppressed the levels of inflammatory cytokines by deactivating the nuclear factor κB (NFκB) pathway. Furthermore, the alternative splicing of myeloid differentiation protein 2 (MD2), a co-receptor of toll-like receptor 4 (TLR4), is regulated by SRSF3. The depletion of SRSF3 increased the level of the shorter MD2B splicing variants, which contributed to inflammatory inhibition in macrophages. In conclusion, our findings imply that SRSF3 regulates lipopolysaccharide-stimulated inflammation, in part by controlling the alternative splicing of MD2 mRNA in macrophages.

Histone modification in psoriasis: Molecular mechanisms and potential therapeutic targets.

Psoriasis is an immune-mediated, inflammatory disease. Genetic and environmental elements are involved in the nosogenesis of this illness. Epigenetic inheritance serves as the connection between genetic and environmental factors. Histone modification, an epigenetic regulatory mechanism, is implicated in the development of numerous diseases. The basic function of histone modification is to regulate cellular functions by modifying gene expression. Modulation of histone modification, such as regulation of enzymes pertinent to histone modification, can be an alternative approach for treating some diseases, including psoriasis. Herein, we reviewed the regulatory mechanisms and biological effects of histone modifications and their roles in the pathogenesis of psoriasis.

Pulmonary interstitial lesions in pemphigus mouse model: Verifying pemphigus may not be only limited to skin and mucosa.

Interstitial lung disease (ILD) has been identified as a prevalent complication and significant contributor to mortality in individuals with pemphigus. In this study, a murine model of pemphigus was developed through the subcutaneous administration of serum IgG obtained from pemphigus patients, allowing for an investigation into the association between pemphigus and ILD. Pulmonary interstitial lesions were identified in the lungs of a pemphigus mouse model through histopathology, RT-qPCR and Sircol assay analyses. The severity of these lesions was found to be positively associated with the concentration of IgG in the injected serum. Additionally, DIF staining revealed the deposition of serum IgG in the lung tissue of pemphigus mice, indicating that the subcutaneous administration of human IgG directly impacted the lung tissue of the mice, resulting in damage. This study confirms the presence of pulmonary interstitial lesions in the pemphigus mouse model and establishes a link between pemphigus and ILD.

Pectin: Health-promoting properties as a natural galectin-3 inhibitor.

Galectin-3 has a variety of important pathophysiological significance in the human body. Much evidence shows that the abnormal expression of galectin-3 is related to the formation and development of many diseases. Pectin is mostly obtained from processed citrus fruits and apples and is a known natural inhibitor of galactin-3. A large number of peels produced each year are discarded, and it is necessary to recycle some of the economically valuable active compounds in these by-products to reduce resource waste and environmental pollution. By binding with galectin-3, pectin can directly reduce the expression level of galectin-3 on the one hand, and regulate the expression level of cytokines by regulating certain signaling pathways on the other hand, to achieve the effect of treating diseases. This paper begins by presenting an overview of the basic structure of pectin, subsequently followed by a description of the structure of galectin-3 and its detrimental impact on human health when expressed abnormally. The health effects of pectin as a galectin-3 inhibitor were then summarized from the perspectives of anticancer, anti-inflammatory, ameliorating fibrotic diseases, and anti-diabetes. Finally, the challenges and prospects of future research on pectin are presented, which provide important references for expanding the application of pectin in the pharmaceutical industry or developing functional dietary supplements.

Preparation and characterization of a novel humanized collagen III with repeated fragments of Gly300-Asp329.

Recombinant human collagens have attracted intensive interest in the past two decades, demonstrating considerable potential in medicine, tissue engineering, and cosmetics. Several humanized recombinant collagens have been produced, exhibiting similar characteristics as the native species. To get insight into the structural and bioactive properties of different parts of collagen, in this study, the segment of Gly300-Asp329 of type III collagen was first adopted and repeated 18 times to prepare a novel recombinant collagen (named rhCLA). RhCLA was successfully expressed in E. coli, and a convenient separation procedure was established through reasonably combining alkaline precipitation and acid precipitation, yielding crude rhCLA with a purity exceeding 90%. Additionally, a polishing purification step utilizing cation exchange chromatography was developed, achieving rhCLA purity surpassing 98% and an overall recovery of approximately 120 mg/L culture. Simultaneously, the contents of endotoxin, nucleic acids, and host proteins were reduced to extremely low levels. This fragmented type III collagen displayed a triple-helical structure and gel-forming capability at low temperatures. Distinct fibrous morphology was also observed through TEM analysis. In cell experiments, rhCLA exhibited excellent biocompatibility and cell adhesion properties. These results provide valuable insights for functional studies of type III collagen and a reference approach for the large-scale production of recombinant collagens.

High-intensity focused ultrasound ablation in the treatment of fumarate hydratase-deficient uterine leiomyoma.

OBJECTIVE: This study aimed to explore the efficacy and safety of high-intensity focused ultrasound (HIFU) ablation for treating fumarate hydratase (FH)-deficient uterine leiomyomas. METHOD: Ten patients with FH-deficient uterine leiomyomas treated with HIFU ablation at the Third Xiangya Hospital from July 2017 to January 2023 were enrolled in this study. The effectiveness and adverse effects of HIFU were analyzed. RESULTS: The median age of the patients who received HIFU was 32.0 years (range: 28-41 years). Only 2 patients had solitary uterine leiomyomas, whereas the remaining 8 patients had multiple uterine leiomyomas. The median diameter of the largest myoma was 56 mm (range: 41-99 mm). Magnetic resonance imaging showed that the FH-deficient uterine leiomyomas of 8 patients presented as mixed intensity on T2WI, that of one patient was hypointense, and that of another patient was hyperintense on T2WI. All patients successfully underwent HIFU ablation in one session without severe adverse effects. The median nonperfusion volume ratio (NPVR) was 40% (30.0%-78.0%) after HIFU treatment. Four patients had NPVR ≥70%. At 3-month follow-up after HIFU ablation, the clinical symptoms of 5 of the 8 patients with symptoms before treatment were relieved. Six months after treatment, 4 of the 8 patients with symptoms were still in remission. All patients received reintervention by March 2024. The reintervention rates were 20%, 70%, and 90% at 12, 24, and 36 months, respectively, after HIFU ablation. CONCLUSION: HIFU is a safe and feasible treatment for FH-deficient uterine leiomyomas, and most patients show effective results in the short term after treatment. However, the reintervention rates are high, and the long-term effects are limited.

Autophagy Regulates Chromatin Ubiquitination in DNA Damage Response through Elimination of SQSTM1/p62.

Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, and loss of autophagy has been linked to increased genome instability. Here, we report that loss of autophagy is coupled to reduced histone H2A ubiquitination after DNA damage. p62/SQSTM1, which accumulates in autophagy-defective cells, directly binds to and inhibits nuclear RNF168, an E3 ligase essential for histone H2A ubiquitination and DNA damage responses. As a result, DNA repair proteins such as BRCA1, RAP80, and Rad51 cannot be recruited to the sites of DNA double-strand breaks (DSBs), which impairs DSB repair. Moreover, nuclear-localized p62 increased the sensitivity of tumor cells to radiation both in vitro and in vivo, and this required its interaction with RNF168. Our findings indicate that autophagy-deficiency-induced p62 accumulation results in inhibition of histone ubiquitination and highlight the complex relationship between autophagy and the DNA damage response.

Changes of microbial communities and metabolites in the fermentation of persimmon vinegar by bioaugmentation fermentation.

To evaluate the effects of bioaugmentation fermentation inoculated with one ester-producing strain (Wickerhamomyces anomalus ZX-1) and two strains of lactic acid bacteria (Lactobacillus plantarum CGMCC 24035 and Lactobacillus acidophilus R2) for improving the flavor of persimmon vinegar, microbial community, flavor compounds and metabolites were analyzed. The results of microbial diversity analysis showed that bioaugmentation fermentation significantly increased the abundance of Lactobacillus, Saccharomyces, Pichia and Wickerhamomyces, while the abundance of Acetobacter, Apiotrichum, Delftia, Komagataeibacter, Kregervanrija and Aspergillus significantly decreased. After bioaugmentation fermentation, the taste was softer, and the sensory irritancy of acetic acid was significantly reduced. The analysis of HS-SPME-GC-MS and untargeted metabolomics based on LC-MS/MS showed that the contents of citric acid, lactic acid, malic acid, ethyl lactate, methyl acetate, isocitrate, acetoin and 2,3-butanediol were significantly increased. By multivariate analysis, 33 differential metabolites were screened out to construct the correlation between the differential metabolites and microorganisms. Pearson correlation analysis showed that methyl acetate, ethyl lactate, betaine, aconitic acid, acetoin, 2,3-butanediol and isocitrate positively associated with Wickerhamomyces and Lactobacillus. The results confirmed that the quality of persimmon vinegar was improved by bioaugmentation fermentation.

PANI/GO and Sm co-modified Ti/PbO2 dimensionally stable anode for highly efficient amoxicillin degradation: Performance assessment, impact parameters and degradation mechanism.

The abuse and uncontrolled discharge of antibiotics present a severe threat to environment and human health, necessitating the development of efficient and sustainable treatment technology. In this work, we employ a facile one-step electrodeposition method to prepare polyaniline/graphite oxide (PANI/GO) and samarium (Sm) co-modified Ti/PbO2 (Ti/PbO2-PANI/GO-Sm) electrode for the degradation of amoxicillin (AMX). Compared with traditional Ti/PbO2 electrode, Ti/PbO2-PANI/GO-Sm electrode exhibits more excellent oxygen evolution potential (2.63 V) and longer service life (56 h). In degradation experiment, under optimized conditions (50 mg L-1 AMX, 20 mA cm-2, pH 3, 0.050 M Na2SO4, 25 °C), Ti/PbO2-PANI/GO-Sm electrode achieves remarkable removal efficiencies of 88.76% for AMX and 79.92% for chemical oxygen demand at 90 min. In addition, trapping experiment confirms that ·OH plays a major role in the degradation process. Based on theoretical calculation and liquid chromatography-mass spectrometer results, the heterocyclic portion of AMX molecule is more susceptible to ·OH attacks. Thus, this novel electrode offers a sustainable and efficient solution to address environmental challenges posed by antibiotic-contaminated wastewater.

The effect of deviations in sintering temperature on the translucency and color of multi-layered zirconia.

OBJECT: This study aimed to investigate the changes in the translucency and color of four different multi-layered zirconia materials when the sintering temperature were inaccurate. MATERIALS AND METHODS: Two hundred zirconia samples (11 × 11 × 1.0 mm) of four multi-layered zirconia, Upcera TT-GT (UG), Upcera TT-ML (UM), Cercon xt ML (CX), and Lava Esthetic (LE), were divided into five subgroups according to the sintering temperature: L1 (5% lower temperature), L2 (2.5% lower temperature), R (recommended sintering temperature), H2 (2.5% higher temperature), H1 (5% higher temperature). After sintering, color coordinates were measured. Then the translucency parameter (TP) values, and the color differences (between the inaccurate sintering temperature and the recommended temperature) of each zirconia specimen were calculated. Statistical analysis was performed by using three-way ANOVA tests, the one-way ANOVA, and Tukey's post hoc test. RESULTS: Three-way ANOVA results showed that material type, sintering temperature, specimen section, and their interactions significantly influenced the TP values (except for the interactions of specimen section and sintering temperature) (P < .05). TP values of zirconia specimens were significantly different in the inaccurate sintering temperatures (P < .05), except for the cervical and body sections of UG group (P > .05). Compared with recommended sintering temperature, higher sintering temperature caused higher TP values for CX, but lower for LE. Three-way ANOVA results showed that material type, sintering temperature, and their interactions significantly influenced the ∆E00 values (P < .05). There were no significant differences in ∆E00 values of UM and CX groups at different inaccurate sintering temperatures, and were clinical imperception (except for UM-L1) (∆E00 < 1.25). ∆E00 values of all zirconia specimens showed clinically acceptable (∆E00 < 2.23). CONCLUSION: The deviations in sintering temperature significantly influenced the translucency and color of tested multi-layered zirconia. The trends of translucency in the multi-layered zirconia depended on material type and the color changes of all zirconia materials were clinically acceptable at inaccurate sintering temperatures.

Special issue "The advance of solid tumor research in China": Participants with a family history of cancer have a higher participation rate in low-dose computed tomography for lung cancer screening.

We aimed to determine participation in low-dose computed tomography (LDCT) of individuals with a family history of common cancers in a population-based screening program to provide timely evidence in high-risk populations in China. The analysis was conducted using data from the Cancer Screening Program in Urban China (CanSPUC), which recruited 282 377 participants aged 40 to 74 years from eight cities in the Henan province. Using the CanSPUC risk score system, 55 428 participants were evaluated to have high risk for lung cancer and were recommended for LDCT. We calculated the overall and group-specific participation rates using family history of common cancers and compared differences in participation rates between different groups. Odds ratios (ORs) and 95% confidence intervals were derived by multivariable logistic regression. Of the 55 428 participants, 22 260 underwent LDCT (participation rate, 40.16%). Family history of lung, esophageal, stomach, liver and colorectal cancer was associated with increased participation in LDCT screening. The odds of participants with a family history of one, two, three and four or more cancer cases undergoing LDCT screening were 1.9, 2.7, 2.8 and 3.5 times, respectively, than those without a family history of cancer. Compared to those without a history of cancer, participation in LDCT gradually increased as the number of cancer cases in the family increased (P < .001). Our findings suggest that there is room for improvement in lung cancer screening given the relatively low participation rate. Lung cancer screening in populations with a family history of cancer may improve efficiency and cost-effectiveness; however, this requires further verification.

Changes in sex hormones and their interactions are related to pain perception between different menstrual subphases.

Whether sex hormones are related to pain perception across the menstrual cycle is unclear. We examined changes in experimental pain perception in healthy young females between the early to midfollicular subphase (emF) and the midluteal subphase (mL) and explored the role of sex hormones. Sixty-six participants were involved in the study. We tested pressure pain, cold pain, ischemic pain, and needle pain, while at the same time we measured sex hormones levels in two menstrual subphases. Only the right ulna pressure test showed a significant reduction in pain threshold (PPTh3) during the mL. The absolute change of PPTh3 (PPTh3mL - PPTh3emF) was related to the absolute change of prolactin. The relative change of the range of pain tolerance for pressure pain of the right ulna (RPT3rc) was related to the relative change of progesterone (Prc) and estradiol (E2rc) levels, and the interaction effects showed that at Prc ≤ 30, E2rc was positively correlated with RPT3rc. The same, the relative change of pressure pain tolerance of the pulp of the middle finger on the right hand (PPTo4rc) was related to E2rc and Prc, and the results of the interaction between E2rc and Prc suggest that when E2rc is ≤0.8, Prc is positively correlated with PPTo4rc. Two different formulas were applied in this study and showed inconsistent results. Most pain tests showed no difference between the two subphases of the menstrual cycle. Only the relative changes of the PPTo4 and RPT3 are related to the E2rc and Prc, respectively, between menstrual subphases in an interactive way in healthy young women.

Advances in Development of mRNA-Based Therapeutics.

Recently, mRNA-based therapeutics have been greatly boosted since the development of novel technologies of both mRNA synthesis and delivery system. Promising results were showed in both preclinical and clinical studies in the field of cancer vaccine, tumor immunotherapy, infectious disease prevention and protein replacement therapy. Recent advancements in clinical trials also encouraged scientists to attempt new applications of mRNA therapy such as gene editing and cell programming. These studies bring mRNA therapeutics closer to real-world application. Herein, we provide an overview of recent advances in mRNA-based therapeutics.

Synergetic interaction of capsid proteins for virus-like particles assembly of foot-and-mouth disease virus (serotype O) from the inclusion bodies.

Recombinant virus-like particles (VLP) with single capsid protein have been successfully produced through prokaryotic system, but for those with multiple capsid proteins such as the foot-and-mouth disease virus (FMDV), this approach is more challenging. In this study, in vitro assembly of FMDV VLP was investigated with its capsids VP1, VP2 and VP3 separately expressed as inclusion bodies. After extraction and solubilization, three capsids were purified in denatured state through a flow-through model, increasing its purity to 90%. VLP assembly for FMDV was observed after diluting the mixture of denatured capsids in the ration of 1: 1: 1, while no VLP appeared if the separately diluted and refolded capsids were co-incubated. This result suggests certain synergetic interactions exist among the three capsids, which are crucial for FMDV VLP assembly. Sodium chloride and capsid protein concentration both greatly affect the assembling efficiency. After purification through size exclusion chromatography, VLP with similar diameter and morphology as inactivated FMDV were obtained, which elicited high IgG titers and B cell activation when vaccinated in mouse. It could also induce specific humoral and cellular immune responses in splenocytes proliferative experiments. Our study demonstrated the feasibility of in vitro assembling FMDV VLP from inclusion bodies of VP1, VP2 and VP3 for the first time.

Recent progress in plant-derived polysaccharides with prebiotic potential for intestinal health by targeting gut microbiota: a review.

Natural products of plant origin are of high interest and widely used, especially in the food industry, due to their low toxicity and wide range of bioactive properties. Compared to other plant components, the safety of polysaccharides has been generally recognized. As dietary fibers, plant-derived polysaccharides are mostly degraded in the intestine by polysaccharide-degrading enzymes secreted by gut microbiota, and have potential prebiotic activity in both non-disease and disease states, which should not be overlooked, especially in terms of their involvement in the treatment of intestinal diseases and the promotion of intestinal health. This review elucidates the regulatory effects of plant-derived polysaccharides on gut microbiota and summarizes the mechanisms involved in targeting gut microbiota for the treatment of intestinal diseases. Further, the structure-activity relationships between different structural types of plant-derived polysaccharides and the occurrence of their prebiotic activity are further explored. Finally, the practical applications of plant-derived polysaccharides in food production and food packaging are summarized and discussed, providing important references for expanding the application of plant-derived polysaccharides in the food industry or developing functional dietary supplements.