Association between plant-based dietary index and gallstone disease: A cross sectional study from NHANES.

BACKGROUND: The relationship between plant-based diets and gallstone disease has been debated. This study aimed to shed light on the association between plant-based dietary index and the risk of developing gallstone disease. METHODS: Eligible participants were selected from National Health and Nutrition Examination Survey (NHANES) 2017-2020. Three plant-based diet indexes (PDI, healthy PDI, unhealthy PDI) were calculated using data from two NHANES 24-h dietary recall interviews. Restricted Cubic Spline and multivariate logistic regression were used to analyze the associations. Subgroup analysis was adopted to make the results more robust. RESULTS: A total of 5673 eligible participants were analyzed. After adjusting for various confounding variables, uPDI was positively associated with gallstone disease (OR = 1.53, 95%CI: 1.02-2.29). No association was found between PDI/hPDI and gallstone disease (p > 0.05). The results of subgroup analysis did not show any positive association between uPDI and gallstones in specific groups. CONCLUSION: Our study shows that the elevated uPDI are linked to a higher risk of gallstone disease.

Catalyst: Fast and flexible modeling of reaction networks.

We introduce Catalyst.jl, a flexible and feature-filled Julia library for modeling and high-performance simulation of chemical reaction networks (CRNs). Catalyst supports simulating stochastic chemical kinetics (jump process), chemical Langevin equation (stochastic differential equation), and reaction rate equation (ordinary differential equation) representations for CRNs. Through comprehensive benchmarks, we demonstrate that Catalyst simulation runtimes are often one to two orders of magnitude faster than other popular tools. More broadly, Catalyst acts as both a domain-specific language and an intermediate representation for symbolically encoding CRN models as Julia-native objects. This enables a pipeline of symbolically specifying, analyzing, and modifying CRNs; converting Catalyst models to symbolic representations of concrete mathematical models; and generating compiled code for numerical solvers. Leveraging ModelingToolkit.jl and Symbolics.jl, Catalyst models can be analyzed, simplified, and compiled into optimized representations for use in numerical solvers. Finally, we demonstrate Catalyst's broad extensibility and composability by highlighting how it can compose with a variety of Julia libraries, and how existing open-source biological modeling projects have extended its intermediate representation.

Retraction Notice to: YAP1 Inhibition in HUVECs Is Associated with Released Exosomes and Increased Hepatocarcinoma Invasion and Metastasis.

[This retracts the article DOI: 10.1016/j.omtn.2020.05.021.].

Intra-tumor heterogeneity and prognostic risk signature for hepatocellular carcinoma based on single-cell analysis.

Intra-tumor heterogeneity poses a serious challenge in the treatment of cancer, including hepatocellular carcinoma (HCC). Recent developments in single-cell RNA sequencing (scRNA-seq) make it possible to examine the heterogeneity of tumor cells. The Gene Expression Omnibus (GEO) database was retrieved to obtain scRNA-seq data of 13 HCC and 8 para cancer samples, and the cells were clustered using FindNeighbors and FindClusters functions. Cell subsets were defined using the "Enricher" function of the clusterProfiler package. Monocle was used to predict cell developmental trajectory. The LIMMA package included in the R program was utilized to detect differentially expressed genes (DEGs) between HCC and paracancerous tissues. Univariate Cox analysis and Least Absolute and Selection Operator (Lasso) Cox regression analysis were conducted to establish a risk assessment model. Thirteen cell subpopulations were identified from the sequencing data of 64,634 single cells. Four cell subgroups (dendritic cells, hepatocytes, liver bud hepatic cells, and liver progenitor cells) in tumor tissues were highly enriched. Between HCC and para cancer tissues, 3024 DEGs were identified, and 641 were specific markers of four cell subgroups. To develop a prognostic risk model, 9 genes among the 641 genes were selected. In the training and validation sets, the model demonstrated a higher 5-year AUC and independently served as a prognostic marker as confirmed by multivariate and univariate Cox analyses. This study revealed the characteristics of different cell subpopulations of immune cells and tumor cells from the HCC microenvironment. We established a novel nine-gene prognostic model to determine the death risk of HCC patients. The discoveries in this research improved the current knowledge of HCC heterogeneity and may inspire future research.

Brain fingerprints along the language hierarchy.

Recent studies have shown that the brain functional connectome constitutes a unique fingerprint that allows the identification of individuals from a group. However, what information encoded in the brain that makes us unique remains elusive. Here, we addressed this issue by examining how individual identifiability changed along the language hierarchy. Subjects underwent fMRI scanning during rest and when listening to short stories played backward, scrambled at the sentence level, and played forward. Identification for individuals was performed between two scan sessions for each task as well as between the rest and task sessions. We found that individual identifiability tends to increase along the language hierarchy: the more complex the task is, the better subjects can be distinguished from each other based on their whole-brain functional connectivity profiles. A similar principle is found at the functional network level: compared to the low-order network (the auditory network), the high-order network is more individualized (the frontoparietal network). Moreover, in both cases, the increase in individual identifiability is accompanied by the increase in inter-subject variability of functional connectivities. These findings advance the understanding of the source of brain individualization and have potential implications for developing robust connectivity-based biomarkers.

Associations between HIV testing and multilevel stigmas among gay men and other men who have sex with men in nine urban centers across the United States.

BACKGROUND: Complex manifestation of stigma across personal, community, and structural levels and their effect on HIV outcomes are less understood than effects in isolation. Yet, multilevel approaches that jointly assesses HIV criminalization and personal sexual behavior stigma in relation to HIV testing have not been widely employed or have only focused on specific subpopulations. The current study assesses the association of three types of MSM-related sexual behavior-related stigma (family, healthcare, general social stigma) measured at both individual and site levels and the presence/absence of laws criminalizing HIV transmission with HIV testing behaviors to inform HIV surveillance and prevention efforts among HIV-negative MSM in a holistic and integrated way. METHODS: We included nine National HIV Behavioral Surveillance (NHBS) 2017 sites: Baltimore, MD; Denver, CO; Detroit, MI; Houston, TX; Long Island/Nassau-Suffolk, NY; Los Angeles, CA; Portland, OR; San Diego, CA; and Virginia Beach and Norfolk, VA. Multivariable generalized hierarchical linear modeling was used to examine how sexual behavior stigmas (stigma from family, anticipated healthcare stigma, general social stigma) measured at the individual and site levels and state HIV criminalization legislation (no, HIV-specific, or sentence-enhancement laws) were associated with past-year HIV testing behaviors across sites (n = 3,278). RESULTS: The majority of MSM across sites were tested for HIV in the past two years (n = 2,909, 95.4%) with the average number of times tested ranging from 1.79 (SD = 3.11) in Portland, OR to 4.95 (SD = 4.35) in Los Angeles, CA. In unadjusted models, there was a significant positive relationship between stigma from family and being tested for HIV in the past two years. Site-level HIV-specific criminalization laws were associated with an approximate 5% reduction in the prevalence of receiving any HIV test in the past two years after individual level stigma and sociodemographic covariate adjustments (PR = 0.94, 95% CI, 0.90-0.99). CONCLUSIONS: Structural barriers faced by MSM persist and ending the HIV epidemic in the US requires a supportive legal environment to ensure effective engagement in HIV services among MSM. Home-based solutions, such as self-testing, used to deliver HIV testing may be particularly important in punitive settings while legal change is advocated for on the community and state levels.

Association of Serum Complement C1q and C3 Level with Age-Related Macular Degeneration in Women.

PURPOSE: To investigate the association between serum complement components and age-related macular degeneration (AMD). PATIENTS AND METHODS: A total of 118 AMD patients and age- and sex-matched 106 control subjects were included. Demographic data and the level of serum complement component (C)1q, C3 and C4 were evaluated. Based on sex, the subjects were stratified into male and female subgroups. RESULTS: The level of C1q (226.31±45.33mg/dL) was significantly higher and C3 (121.14±15.76mg/dL) was significantly lower than that in control group (200.03±38.54mg/dL) (128.42±19.81mg/dL) in the female AMD patients (p = 0.005, p = 0.045). Logistic regression showed that increased C1q (OR = 1.132, p = 0.016) and decreased C3 (OR = 0.960, p = 0.048) were independent risk factors for female AMD patients. No statistical significance was observed in the male. CONCLUSION: Increased C1q and decreased C3 were associated with increased risk of AMD, suggesting that the complement classical pathway probably be involved in AMD, especially in female.

Metrics of Sexual Behavior Stigma Among Cisgender Men Who Have Sex With Men in 9 Cities Across the United States.

Men who have sex with men (MSM) in the United States are stigmatized for their same-sex practices, which can lead to risky sexual behavior, potentiating risk for human immunodeficiency virus (HIV) infection. Improved measurement is necessary for accurately reporting and mitigating sexual behavior stigma. We added 13 sexual behavior stigma items to local surveys administered in 2017 at 9 sites in the Centers for Disease Control and Prevention's National HIV Behavioral Surveillance system, which uses venue-based, time-sampling procedures to survey cisgender MSM in US Census Metropolitan Statistical Areas. We performed exploratory factor analytical procedures on site-specific (Baltimore, Maryland; Denver, Colorado; Detroit, Michigan; Houston, Texas; Nassau-Suffolk, New York; Portland, Oregon; Los Angeles, California; San Diego, California; and Virginia Beach-Norfolk, Virginia) and pooled responses to the survey items. A 3-factor solution-"stigma from family" (α = 0.70), "anticipated health-care stigma" (α = 0.75), and "general social stigma" (α = 0.66)-best fitted the pooled data and was the best-fitting solution across sites. Findings demonstrate that MSM across the United States experience sexual behavior stigma similarly. The results reflect the programmatic utility of enhanced stigma measurement, including tracking trends in stigma over time, making regional comparisons of stigma burden, and supporting evaluation of stigma-mitigation interventions among MSM across the United States.

Stiff neural ordinary differential equations.

Neural Ordinary Differential Equations (ODEs) are a promising approach to learn dynamical models from time-series data in science and engineering applications. This work aims at learning neural ODEs for stiff systems, which are usually raised from chemical kinetic modeling in chemical and biological systems. We first show the challenges of learning neural ODEs in the classical stiff ODE systems of Robertson's problem and propose techniques to mitigate the challenges associated with scale separations in stiff systems. We then present successful demonstrations in stiff systems of Robertson's problem and an air pollution problem. The demonstrations show that the usage of deep networks with rectified activations, proper scaling of the network outputs as well as loss functions, and stabilized gradient calculations are the key techniques enabling the learning of stiff neural ODEs. The success of learning stiff neural ODEs opens up possibilities of using neural ODEs in applications with widely varying time-scales, such as chemical dynamics in energy conversion, environmental engineering, and life sciences.

CISD2 Promotes Resistance to Sorafenib-Induced Ferroptosis by Regulating Autophagy in Hepatocellular Carcinoma.

PURPOSE: Sorafenib is a multi-kinase inhibitor that is used as a standard treatment for advanced hepatocellular carcinoma (HCC). However, the mechanism of sorafenib resistance in HCC is still unclear. It has been shown that CISD2 expression is related to the progression and poor prognosis of HCC. Here, we show a new role for CISD2 in sorafenib resistance in HCC. METHODS: Bioinformatic analysis was used to detect the expression of negative regulatory genes of ferroptosis in sorafenib-resistant samples. The concentration gradient method was used to establish sorafenib-resistant HCC cells. Western blot was used to detect the protein expression of CISD2, LC3, ERK, PI3K, AKT, mTOR, and Beclin1 in HCC samples. Quantitative real-time PCR (qPCR) was used to detect gene expression. CISD2 shRNA and Beclin1 shRNA were transfected to knock down the expression of the corresponding genes. Cell viability was detected by a CCK-8 assay. ROS were detected by DCFH-DA staining, and MDA and GSH were detected with a Lipid Peroxidation MDA Assay Kit and Micro Reduced Glutathione (GSH) Assay Kit, respectively. Flow cytometry was used to detect apoptosis and the levels of ROS and iron ions. RESULTS: CISD2 was highly expressed in HCC cells compared with normal cells and was associated with poor prognosis in patients. Knockdown of CISD2 promoted a decrease in the viability of drug-resistant HCC cells. CISD2 knockdown promoted sorafenib-induced ferroptosis in resistant HCC cells. The levels of ROS, MDA, and iron ions increased, but the change in GSH was not obvious. Knockdown of CISD2 promoted uncontrolled autophagy in resistant HCC cells. Inhibition of autophagy attenuated CISD2 knockdown-induced ferroptosis. The autophagy promoted by CISD2 knockdown was related to Beclin1. When CISD2 and Beclin1 were inhibited, the effect on ferroptosis was correspondingly weakened. CONCLUSION: Inhibition of CISD2 promoted sorafenib-induced ferroptosis in resistant cells, and this process promoted excessive iron ion accumulation through autophagy, leading to ferroptosis. The combination of CISD2 inhibition and sorafenib treatment is an effective therapeutic strategy for resistant HCC.

Association between Red Blood Cell Distribution Width and Diabetic Retinopathy: A 5-Year Retrospective Case-Control Study.

PURPOSE: Red blood cell distribution width (RDW) has been regarded as an emerging biomarker of the general population and cardiovascular disease. In this study, we aimed to evaluate the association between RDW and diabetic retinopathy (DR). METHODS: This case-control study included 167 patients with DR, 131 patients with diabetes mellitus (DM), and 170 age- and sex-matched healthy controls from April 2014 to May 2019. Demographic data, laboratory parameters, and ocular examinations were collected. RESULTS: RDW values of the DR group were significantly higher than those of the healthy control (p < 0.001) and DM group (p=0.002). A similar trend was observed when RDW was compared among the 3 groups with respect to age and gender. Logistic regression analysis has shown the OR of RDW was 3.791 (2.33-6.168; p < 0.001) against the control group and was 1.348 (0.997-1.823; p=0.047) against the DM group. CONCLUSION: RDW values were significantly elevated in DR patients, and an elevated RDW was associated with an increased incidence of DR in patients with DM.

LINC00511 drives invasive behavior in hepatocellular carcinoma by regulating exosome secretion and invadopodia formation.

BACKGROUND: Tumor cells are known to release large numbers of exosomes containing active substances that participate in cancer progression. Abnormally expressed long noncoding RNAs (lncRNAs) have been confirmed to regulate multiple processes associated with tumor progression. However, the mechanism by which lncRNAs affect exosome secretion remains unclear. METHODS: The underlying mechanisms of long noncoding RNA LINC00511 (LINC00511) regulation of multivesicular body (MVB) trafficking, exosome secretion, invadopodia formation, and tumor invasion were determined through gene set enrichment analysis (GSEA), immunoblotting, nanoparticle tracking analysis, confocal colocalization analysis, electron microscopy, and invasion experiments. RESULTS: We revealed that the tumorigenesis process is associated with a significant increase in vesicle secretion in hepatocellular carcinoma (HCC). Additionally, LINC00511 was significantly more highly expressed in HCC tissues and is related to vesicle trafficking and MVB distribution. We also found that in addition to the formation of invadopodia in HCC progression, abnormal LINC00511 induces invadopodia formation in HCC cells by regulating the colocalization of vesicle associated membrane protein 7 (VAMP7) and synaptosome associated protein 23 (SNAP23) to induce the invadopodia formation, which are key secretion sites for MVBs and control exosome secretion. Finally, we revealed that LINC0051-induced invadopodia and exosome secretion were involved in tumor progression. CONCLUSIONS: Our experiments revealed novel findings on the relationship between LINC00511 dysregulation in HCC and invadopodia production and exosome secretion. This is a novel mechanism by which LINC00511 regulates invadopodia biogenesis and exosome secretion to further promote cancer progression.

Radiometric Performance Evaluation of FY-4A/AGRI Based on Aqua/MODIS.

Fengyun-4A (FY-4A) is the first satellite of the Chinese second-generation geostationary orbit meteorological satellites (FY-4). The Advanced Geostationary Radiation Imager (AGRI), onboard FY-4A does not load with high-precision calibration facility in visible and near infrared (VNIR) channel. As a consequence, it is necessary to comprehensively evaluate its radiometric performance and quantitatively describe the attenuation while using its VNIR data. In this paper, the radiometric performance at VNIR channels of FY-4A/AGRI is evaluated based on Aqua/MODIS data using the deep convective cloud (DCC) target. In order to reduce the influence of view angle and spectral response difference, the bi-directional reflectance distribution function (BRDF) correction and spectral matching have been performed. The evaluation result shows the radiometric performance of FY-4A/AGRI: (1) is less stable and with obvious fluctuations; (2) has a lower radiation level because of 24.99% lower compared with Aqua/MODIS; 3) has a high attenuation with 9.11% total attenuation over 2 years and 4.0% average annual attenuation rate. After the evaluation, relative radiometric normalization between AGRI and MODIS in VNIR channel is performed and the procedure is proved effective. This paper proposed a more reliable reference for the quantitative applications of FY-4A data.

Neutrophil-To-Lymphocyte Ratio as a Potential Biomarker of Neovascular Glaucoma.

Purpose: To evaluate blood count-derived inflammation indexes as a detection or predictive marker for neovascular glaucoma (NVG) secondary to retinal vein occlusion (RVO) and diabetic retinopathy (DR).Methods: This was a retrospective, case-control study design. The level of white blood cell (WBC), neutrophil (N), neutrophil/lymphocyte ratio (NLR), and lymphocyte/monocyte ratio (LMR) were evaluated in NVG patients secondary to RVO or DR.Results: A significant difference was found in those biomarkers between control group and NVG secondary to RVO or DR. Logistic regression analysis revealed these indexes were associated with the risk of NVG in DR and RVO patients. Multiple linear regression analysis showed a significant correlation between NLR and visual fields mean deviation in the NVG-RVO group.Conclusions: This study indicated that WBC, N, NLR, and LMR were related to NVG, and NLR may be useful as an potential inflammation biomarker indicating the risk and severity for NVG secondary to RVO.

IKKß activation promotes amphisome formation and extracellular vesicle secretion in tumor cells.

Intracellular organelle cross-talk is a new and important research area. Under stress conditions, the coordinated action of the autophagy and endosomal systems in tumor cells is essential for maintaining cellular homeostasis and survival. The activation of the IκB kinase (IKK) complex is also involved in the regulation of stress and homeostasis in tumor cells. Here, we try to explore the effects of constitutively active IKKß subunits (CA-IKKß) on autophagy and endosomal system interactions. We confirm that CA-IKKß induces accumulation of autophagosomes and their fusion with MVBs to form amphisomes in cancer cells, and also drives the release of EVs containing autophagy components through an amphisome-dependent mechanism. We further demonstrate that CA-IKKß inhibits the expression of RAB7, thereby weakening the lysosomal-dependent degradation pathway. CA-IKKß also induces phosphorylation of SNAP23 at Ser95 instead of Ser110, which further promotes amphisome-plasma membrane fusion and sEV secretion. These results indicate that CA-IKKß drives the formation and transport of amphisomes, thereby regulating tumor cell homeostasis, which may illuminate a special survival mechanism in tumor cells under stress.

Focus on the morphogenesis, fate and the role in tumor progression of multivesicular bodies.

Multivesicular bodies (MVBs) are endosome organelles that are gradually attracting research attention. Initially, MVBs were considered as important components of the endosomal-lysosomal degradation pathway. In recent years, with an increase in extracellular vesicle (EV) research, the biogenesis, fate, and pathological effects of MVBs have been increasingly studied. However, the mechanisms by which MVBs are sorted to the lysosome and plasma membrane remain unclear. In addition, whether the trafficking of MVBs can determine whether exosomes are released from cells, the factors are involved in cargo loading and regulating the fate of MVBs, and the roles that MVBs play in the development of disease are unknown. Consequently, this review focuses on the mechanism of MVB biogenesis, intraluminal vesicle formation, sorting of different cargoes, and regulation of their fate. We also discuss the mechanisms of emerging amphisome-dependent secretion and degradation. In addition, we highlight the contributions of MVBs to the heterogeneity of EVs, and their important roles in cancer. Thus, we attempt to unravel the various functions of MVBs in the cell and their multiple roles in tumor progression. Video Abstract.

YAP1 Inhibition in HUVECs Is Associated with Released Exosomes and Increased Hepatocarcinoma Invasion and Metastasis.

Hepatocellular carcinoma is one of the most common gastrointestinal malignancies. Anti-angiogenesis therapies have recently demonstrated promise in the treatment of malignancies, although early treatment benefits may be accompanied by metastasis over time. Additional and more effective anti-angiogenic treatment modalities are therefore needed. We previously found that Yes-associated protein 1 (YAP1) expression is increased in hepatocellular carcinoma (HCC), particularly around tumor-associated blood vessels, suggesting a role in angiogenesis. The YAP1 inhibitor verteporfin is presently in anti-angiogenic clinical trials for the treatment of various cancers. Depleted YAP1 from vascular endothelial cells effectively reduced proliferation and tube formation, validating its utility as an anti-angiogenesis target. We also showed that YAP1 depletion or inhibition in vascular endothelial cells leads to increased release of exosomes containing the long non-coding RNA (lncRNA) MALAT1 into the tumor microenvironment. Direct exosomal transfer of MALAT1 to hepatic cells leads to increased hepatic cell invasion and migration via activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. These observations may explain the occurrence of distant tumor metastasis with YAP1-associated anti-angiogenic therapy over time. It provides insight into new pathways and treatment paradigms that may be targeted to increase the long-term success of anti-angiogenic therapies.

MicroRNA-375 Targets ATG14 to Inhibit Autophagy and Sensitize Hepatocellular Carcinoma Cells to Sorafenib.

PURPOSE: Sorafenib has revolutionized treatment of hepatocellular carcinoma (HCC), but its efficacy is limited by drug resistance. Autophagy is the process by which cellular components are transported to lysosomes for degradation, which promotes energy production and production of macromolecular precursors. Studies have suggested that the cytoprotective function of autophagy may contribute to chemoresistance or targeted drug resistance in cancer cells. We investigated the effects of miR-375 and autophagy-related protein 14, and their interrelationships, on sorafenib efficacy. METHODS: Cell viability was measured using the MTT assay, and apoptosis was evaluated using flow cytometry. Colony formation assay was performed to determine changes in cell number. Real-time PCR and Western blotting were performed to quantify the expression of key genes and proteins. Immunofluorescence and transmission electron microscopy were used to detect autophagy. Dual-luciferase reporter assays were used to verify a direct target. RESULTS: We characterized the relationship between sorafenib and autophagy. We showed that inhibition of autophagy enhanced sensitivity of HCC to sorafenib and showed that miR-375 was important in this process. Finally, we showed that miR-375 affected sensitivity of HCC cells to sorafenib through regulation of ATG14. CONCLUSION: We showed that miR-375 sensitized HCC cells to sorafenib by blocking sorafenib-induced autophagy. We also showed that ATG14 was a direct autophagy-related target of miR-375. These findings indicated that miR-375-ATG14 was important in the development of sorafenib resistance in HCC.

LncRNA NEAT1 promotes autophagy via regulating miR-204/ATG3 and enhanced cell resistance to sorafenib in hepatocellular carcinoma.

Long noncoding RNAs (lncRNAs) has been acknowledged in tumorigenesis gradually because of the great importance in different cancers. LncRNA nuclear enriched abundant transcript 1 (NEAT1) is a novel lncRNA and has been reported to promote multiple cancer progression. However, the biological roles of NEAT1 in hepatocellular carcinoma (HCC) is not cleared nowadays. In the present research, the level of NEAT1 was found to be upregulated in HCC by The Cancer Genome Atlas. In addition, NEAT1 expression is negatively correlated with the survival rate in HCC. Further investigation revealed that NEAT1 upregulation inhibited sorafenib efficacy and promoted autophagy. We found that NEAT1 could be a sponge for microRNA-204 (miR-204) and inhibits its level to upregulate ATG3 expression. In addition to the above, we demonstrated that miR-204 mimics also attenuated tumor autophagy. And rescue assays demonstrated that NEAT1 promotes HCC autophagy through modulating miR-204/ATG3 pathway. Collectively, this study first demonstrated that a novel NEAT1/miR-204/ATG3 signaling regulates HCC progression.

Autophagy: A novel mechanism of chemoresistance in cancers.

The success of targeted drug therapy for cancer patients has attracted extensive attention from academia and society. However, the rapid development of acquired drug resistance is becoming a major challenge. Autophagy, as an essential homeostatic and catabolic process, is crucial for the degradation or recycling of proteins and cellular components. Autophagy has a crucial role in several cellular functions and its dysregulation is associated with tumorigenesis, tumor-stroma interactions, and resistance to cancer therapy. A growing body of evidence shows that in multiple types of cancer, autophagy is also a key regulator in the tumor microenvironment and the cellular drug response. However, our understanding of the process of autophagy remains incompletely. In this review, we identify the role of autophagy and describe recent advances in the identification of the mechanism by which autophagy is implicated in drug resistance, with a focus on the mode of action, and validation as potential therapeutics.