Identification of inositol monophosphatase as a broad-spectrum antiviral target of ivermectin.

Ivermectin has broad-spectrum antiviral activities. Despite the failure in clinical application of COVID-19, it can serve as a lead compound for the development of more effective broad-spectrum antivirals, for which a better understanding of its antiviral mechanisms is essential. We thus searched for potential novel targets of ivermectin in host cells by label-free thermal proteomic profiling using Huh-7 cells. Inositol monophosphatase (IMPase) was found among the proteins with shifted thermal stability by ivermectin. Ivermectin could inhibit IMPase activity and reduce cellular myo-inositol and phosphatidylinositol-4-phosphate levels. On the other hand, inositol could impair the antiviral activity of ivermectin and lithium, an IMPase inhibitor with known antiviral activity. As phosphatidylinositol phosphate is crucial for the replication of many RNA viruses, inhibition of cellular myo-inositol biosynthesis may be an important antiviral mechanism of ivermectin. Hence, inhibition of IMPase could serve as a potential target for broad-spectrum antiviral development.

Identification and characterization of ARID1A-interacting proteins in renal tubular cells and their molecular regulation of angiogenesis.

BACKGROUND: Defects and deficiency of AT-rich interactive domain-containing protein 1A (ARID1A) encoded by a tumor suppressor gene ARID1A have recently been suggested to get involved in angiogenesis, a crucial process in carcinogenesis. However, molecular mechanisms of ARID1A deficiency to induce angiogenesis in kidney cancer remain underinvestigated. METHODS: We performed large-scale identification of ARID1A protein interactors in renal tubular epithelial cells (RTECs) using immunoprecipitation (IP) followed by nanoLC-ESI-LTQ-Orbitrap tandem mass spectrometry (MS/MS). Their roles in angiogenesis were investigated using various assays. RESULTS: A total of 74 ARID1A-interacting proteins were identified. Protein-protein interactions analysis revealed that these identified proteins interacted directly or indirectly with ARID1A. Among them, the direct interaction between ARID1A and ß-actin was validated by IP and reciprocal IP followed by Western blotting. Small interfering RNA (siRNA) was used for single and double knockdowns of ARID1A and ACTB. Semi-quantitative RT-PCR demonstrated that deficiency of ARID1A, but not ACTB, significantly affected expression of angiogenesis-related genes in RTECs (VEGF and FGF2 were increased, whereas PDGF and EGF were decreased). However, the knockdowns did not affect TGFB1 and FGF1 levels. The quantitative mRNA expression data of VEGF and TGFB1 were consistent with the secreted levels of their protein products as measured by ELISA. Only secreted products derived from ARID1A-deficient RTECs significantly increased endothelial cells (ECs) migration and tube formation. Some of the other carcinogenic features could also be confirmed in the ARID1A-deficient RTECs, including increased cell migration and chemoresistance. Double knockdowns of both ARID1A and ACTB did not enhance the effects of single ARID1A knockdown in all assays. CONCLUSIONS: We report herein a large dataset of the ARID1A-interacting proteins in RTECs using an IP-MS/MS approach and confirm the direct interaction between ARID1A and ß-actin. However, the role of ARID1A deficiency in angiogenesis is independent of ß-actin.

Roles of heat-shock protein 90 and its four domains (N, LR, M and C) in calcium oxalate stone-forming processes.

Human heat-shock protein 90 (HSP90) has four functional domains, including NH2-terminal (N), charged linker region (LR), middle (M) and COOH-terminal (C) domains. In kidney stone disease (or nephrolithiasis/urolithiasis), HSP90 serves as a receptor for calcium oxalate monohydrate (COM), which is the most common crystal to form kidney stones. Nevertheless, roles of HSP90 and its four domains in kidney stone formation remained unclear and under-investigated. We thus examined and compared their effects on COM crystals during physical (crystallization, growth and aggregation) and biological (crystal-cell adhesion and crystal invasion through extracellular matrix (ECM)) pathogenic processes of kidney stone formation. The analyses revealed that full-length (FL) HSP90 obviously increased COM crystal size and abundance during crystallization and markedly promoted crystal growth, aggregation, adhesion onto renal cells and ECM invasion. Comparing among four individual domains, N and C domains exhibited the strongest promoting effects, whereas LR domain had the weakest promoting effects on COM crystals. In summary, our findings indicate that FL-HSP90 and its four domains (N, LR, M and C) promote COM crystallization, crystal growth, aggregation, adhesion onto renal cells and invasion through the ECM, all of which are the important physical and biological pathogenic processes of kidney stone formation.

Systematic comparisons of various markers for mast cell activation in RBL-2H3 cells.

Mast cell activation plays a key role in various allergic diseases and anaphylaxis. Several methods/techniques can be used for detection of mast cell activation. However, there was no previous systematic evaluation to compare the efficacy of each method/technique. The present study thus systematically compared various markers for mast cell activation induced by IgE cross-linking. The widely used RBL-2H3 mast cells were sensitized with anti-DNP (dinitrophenyl) IgE overnight and activated with DNP-BSA (bovine serum albumin) for up to 4 h. The untreated cells and those with anti-DNP IgE sensitization but without DNP-BSA activation served as the controls. Intracellular calcium level gradually increased to ~2-fold at 1 h, reached its peak (~5-fold) at 2 h, and returned to the basal level at 3-h post-activation. The increases in cellular tryptase level (by Western blotting) (~0.3- to 0.4-fold) and average cell size (~2.5-fold) and decrease of nucleus/cytoplasm ratio (~0.4- to 0.5-fold) were marginal at all time-points. By contrast, ß-hexosaminidase release and CD63 expression (by both flow cytometry and immunofluorescence detection/localization), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence detection/localization) stably and obviously increased (~10-fold as compared with the untreated control and sensitized-only cells or detectable only after activation). Based on these data, the stably obvious increases (by ≥ 10-fold) in ß-hexosaminidase release, CD63 expression (by both flow cytometry and immunofluorescence staining), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence staining) are recommended as the markers of choice for the in vitro study of mast cell activation using RBL-2H3 cells.

Kidney stone proteomics: an update and perspectives.

INTRODUCTION: Main problems of kidney stone disease are its increasing prevalence and high recurrence rate after calculi removal in almost all areas around the globe. Despite enormous efforts in the past, its pathogenic mechanisms remain unclear and need further elucidations. Proteomics has thus become an essential tool to unravel such sophisticated disease mechanisms at cellular, subcellular, molecular, tissue, and whole organism levels. AREAS COVERED: This review provides abrief overview of kidney stone disease followed by updates on proteomics for investigating urinary stone modulators, matrix proteins, cellular responses to different types/doses of calcium oxalate (CaOx) crystals, sex hormones and other stimuli, crystal-cell interactions, crystal receptors, secretome, and extracellular vesicles (EVs), all of which lead to better understanding of the disease mechanisms. Finally, the future challenges and translation of these obtained data to the clinic are discussed. EXPERT OPINION: Knowledge from urinary proteomics for exploring the important stone modulators (either inhibitors or promoters) will be helpful for early detection of asymptomatic cases for prompt prevention of symptoms, complications, and new stone formation. Moreover, these modulators may serve as the new therapeutic targets in the future for successful treatment and prevention of kidney stone disease by medications or other means of intervention.

Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N-LR-M-C) domains.

Heat shock protein 90 (HSP90) plays essential roles in the normal physiology and comprises four distinct domains, including NH2-terminal (N), charged linker region (LR), middle (M), and COOH-terminal (C) domains, all of which regulate HSP90 biological functions. We reported herein detailed protocols to produce recombinant full-length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET-32b(+) expression vector. All HSP90 constructs were expressed as soluble Trx-His-S tagged proteins after induction with 0.25 mM isopropyl-ß-d-thiogalactopyranoside (IPTG) at 18 °C overnight and further purified by affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) resin. The enterokinase (EK) digestion was optimized for efficient cleavage of the Trx-His-S tag from each HSP90 construct by varying concentrations of EK (0.5-1 U) and urea (0-3 M). Each HSP90 construct was highly purified and approximately 0.1-1 mg proteins were obtained from 100 ml of bacterial culture. All the purified HSP90 constructs were successfully confirmed by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and their secondary structure was quantified using attenuated total reflection - Fourier-transform infrared (ATR-FTIR) spectroscopy. Our expression and purification protocols would facilitate further structural and functional studies of human HSP90.

ARID1A knockdown triggers epithelial-mesenchymal transition and carcinogenesis features of renal cells: role in renal cell carcinoma.

Down-regulation/mutation of AT-rich interactive domain 1A (ARID1A), a novel tumor suppressor gene, has been reported in various cancers. Nevertheless, its role in renal cell carcinoma (RCC) remained unclear and underinvestigated. We thus evaluated carcinogenesis effects of ARID1A knockdown in nonmalignant Madin-Darby canine kidney (MDCK) renal cells using small interfering RNA (siRNA) against ARID1A (siARID1A). The siARID1A-transfected cells had decreased cell death, increased cell proliferation, and cell cycle shift (from G0/G1 to G2/M) compared with those transfected with controlled siRNA (siControl). Additionally, the siARID1A-transfected cells exhibited epithelial-mesenchymal transition (EMT) shown by greater spindle index, increased mesenchymal markers (fibronectin/vimentin), and decreased epithelial markers (E-cadherin/zonula occludens-1). Moreover, the siARID1A-transfected cells had increases in migratory activity, nuclear size, self-aggregated multicellular spheroid size, invasion capability, chemoresistance (to docetaxel), Snail family transcriptional repressor 1 expression, and TGF-ß1 secretion. All of these siARID1A-knockdown effects on the carcinogenic features were reproducible in malignant RCC (786-O) cells, which exhibited a higher degree of carcinogenic phenotypes compared with the nonmalignant MDCK cells. Finally, immunohistochemistry showed obvious decrease in ARID1A protein expression in human RCC tissues (n = 23) compared with adjacent normal renal tissues (n = 23). These data indicate that ARID1A down-regulation triggers EMT and carcinogenesis features of renal cells in vitro, and its role in RCC could be proven in human tissues.-Somsuan, K., Peerapen, P., Boonmark, W., Plumworasawat, S., Samol, R., Sakulsak, N., Thongboonkerd, V. ARID1A knockdown triggers epithelial-mesenchymal transition and carcinogenesis features of renal cells: role in renal cell carcinoma.

Protective Cellular Mechanism of Estrogen Against Kidney Stone Formation: A Proteomics Approach and Functional Validation.

Females have less incidence/prevalence of kidney stone disease than males. Estrogen thus may serve as the protective factor but with unclear mechanism. This study explores cellular mechanism underlying such stone preventive mechanism of estrogen. Madin darby canine kidney (MDCK) renal tubular cells are incubated with or without 20 nm 17ß-estradiol for 7 days. Comparative proteomics reveals 58 differentially expressed proteins in estrogen-treated versus control cells that are successfully identified by nanoLC-ESI-Q-TOF-MS/MS. Interestingly, these altered proteins are involved mainly in "binding and receptor," "metabolic process," and "migration and healing" networks. Functional investigations demonstrate reduction of calcium oxalate (CaOx) crystal-binding capability of the estrogen-treated cells consistent with the decreased levels of annexin A1 and α-enolase (the known CaOx crystal-binding receptors) on the cell surface. High-calcium and high-oxalate challenge initially enhances surface expression of annexin A1 and α-enolase, respectively, both of which return to their basal levels by estrogen. Additionally, estrogen reduces intracellular ATP level and promotes cell migration and tissue healing. Taken together, estrogen causes changes in cellular proteome of renal tubular cells that lead to decreased surface expression of CaOx crystal receptors, reduced intracellular metabolism, and enhanced cell proliferation and tissue healing, all of which may contribute, at least in part, to stone prevention.

Protein Network Analysis and Functional Studies of Calcium Oxalate Crystal-Induced Cytotoxicity in Renal Tubular Epithelial Cells.

Our previous expression study has reported a set of proteins with altered levels in renal tubular cells after exposure to calcium oxalate monohydrate (COM) crystals, which are the main composition of kidney stones. However, their functional significance remained largely unknown. In this study, protein network analysis revealed that the significantly altered proteins induced by COM crystals were involved mainly in three main functional networks, including i) cell proliferation and wound healing; ii) oxidative stress and mitochondrial function; and iii) cellular junction complex and integrity. Cell proliferation and wound healing assays showed that the COM-treated cells had defective proliferation and tissue healing capability, respectively. Oxyblot analysis demonstrated accumulation of the oxidized proteins, whereas intracellular ATP level was significantly increased in the COM-treated cells. Additionally, level of zonula occludens-1 (ZO-1), a tight junction protein, was significantly decreased, consistent with the significant declines in transepithelial resistance (TER) and level of RhoA signaling molecule in the COM-treated cells. These findings indicate significant perturbations in mitochondrial and oxidative stress axis that cause defective cell proliferation, tissue healing capability, junctional protein complex, and cellular integrity of renal tubular epithelial cells exposed to COM crystals that may play important roles in kidney stone pathogenesis.

Alterations of proteins in MDCK cells during acute potassium deficiency.

Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury.

Physiologic changes of urinary proteome by caffeine and excessive water intake.

BACKGROUND: Diurnal variations and physiologic changes of urinary proteome have been suggested in the urinary proteomics field. However, no clear evidence has been demonstrated. The present study thus aimed to define changes in urinary proteome by physiological stimuli, i.e. caffeine intake and excessive water drinking, both of which cause physiologic diuresis. METHODS: Urine samples were collected from 30 healthy individuals under three different conditions: (i) morning void as the control; (ii) after drinking a cup of coffee; and (iii) after drinking 1 L of water within 20 min. Thereafter, differentially excreted proteins were analyzed by 2-DE proteomics approach and validated by Western blotting and ELISA. RESULTS: Spot matching, quantitative intensity analysis, and ANOVA followed by Tukey's post-hoc multiple comparisons and the Bonferroni correction revealed significant differences in levels of five protein spots among three different conditions. These proteins were identified by quadrupole time-of-flight mass spectrometry (Q-TOF MS) and/or MS/MS analyses as kininogen 1 isoform 3, ß-actin, prostaglandin D synthase (PGDS), fibrinogen α-chain and immunoglobulin light chain. Among these, the decreased level of immunoglobulin was successfully validated by Western blotting and ELISA. CONCLUSIONS: These data indicated that caffeine intake and excessive water drinking could affect urinary excretion of some proteins and may affect urinary proteome analysis.

Chromosome-centric Human Proteome Project (C-HPP): Chromosome 12.

Following an official announcement of the Chromosome-centric Human Proteome Project (C-HPP), the Chromosome 12 (Ch12) Consortium has been established by five representative teams from five Asian countries including Thailand (Siriraj Hospital, Mahidol University), Singapore (National University of Singapore), Taiwan (Academia Sinica), Hong Kong (The Chinese University of Hong Kong), and India (Institute of Bioinformatics). We have worked closely together to extensively and systematically analyze all missing and known proteins encoded by Ch12 for their tissue/cellular/subcellular localizations. The target organs/tissues/cells include kidney, brain, gastrointestinal tissues, blood/immune cells, and stem cells. In the later phase, post-translational modifications and functional significance of Ch12-encoded proteins as well as their associations with human diseases (i.e., immune diseases, metabolic disorders, and cancers) will be defined. We have collaborated with other chromosome teams, Human Kidney and Urine Proteome Project (HKUPP), AOHUPO Membrane Proteomics Initiative, and other existing HUPO initiatives in the Biology/Disease-Based Human Proteome Project (B/D-HPP) to delineate functional roles and medical implications of Ch12-encoded proteins. The data set to be obtained from this multicountry consortium will be an important piece of the jigsaw puzzle to fulfill the missions and goals of the C-HPP and the global Human Proteome Project (HPP).

The upregulation of lamin A/C as a compensatory mechanism during tight junction disruption in renal tubular cells mediated by calcium oxalate crystals.

Calcium oxalate monohydrate (COM), the most important crystal causing kidney stone disease, upregulates lamin A/C but downregulates zonula occludens-1 (ZO-1) in renal tubular cells. While roles for F-actin and α-tubulin and their association with ZO-1 are known to regulate COM-mediated tight junction (TJ) disruption, roles of lamin A/C and its interplay with ZO-1 in COM kidney stone model remain unclear and are thus the objectives of this study. Lamin A/C was knocked down in MDCK cells by silencing RNA specific for LMNA (siLMNA). Both wild-type (WT) and siLMNA cells were treated with COM for 48-h compared with the untreated (control) cells. Western blotting and immunofluorescence staining revealed upregulated lamin A/C and downregulated ZO-1 in the COM-treated WT cells. siLMNA successfully reduced lamin A/C expression in both control and COM-treated cells. Nonetheless, siLMNA did not reverse the effect of COM on the decreases in ZO-1 and transepithelial resistance, but further reduced their levels in both control and COM-treated cells. Protein-protein interaction analysis demonstrated that two cytoskeletal proteins (actin and tubulin) served as the linkers to connect lamin A/C with ZO-1 and occludin (both of which are the TJ proteins). Altogether, these data implicate that lamin A/C and ZO-1 are indirectly associated to control TJ function, and ZO-1 expression is regulated by lamin A/C. Moreover, COM-induced upregulation of lamin A/C most likely serves as a compensatory mechanism to cope with the downregulation of ZO-1 during COM-mediated TJ disruption.

StoneMod 2.0: Database and prediction of kidney stone modulatory proteins.

Stone modulators are various kinds of molecules that play crucial roles in promoting/inhibiting kidney stone formation. Several recent studies have extensively characterized the stone modulatory proteins with the ultimate goal of preventing kidney stone formation. Herein, we introduce the StoneMod 2.0 database (https://www.stonemod.org), which has been dramatically improved from the previous version by expanding the number of the modulatory proteins in the list (from 32 in the initial version to 17,130 in this updated version). The stone modulatory proteins were recruited from solid experimental evidence (via PubMed) and/or predicted evidence (via UniProtKB, QuickGO, ProRule, STITCH and OxaBIND to retrieve calcium-binding and oxalate-binding proteins). Additionally, StoneMod 2.0 has implemented a scoring system that can be used to determine the likelihood and to classify the potential stone modulatory proteins as either "solid" (modulator score ≥ 50) or "weak" (modulator score < 50) modulators. Furthermore, the updated version has been designed with more user-friendly interfaces and advanced visualization tools. In addition to the monthly scheduled update, the users can directly submit their experimental evidence online anytime. Therefore, StoneMod 2.0 is a powerful database with prediction scores that will be very useful for many future studies on the stone modulatory proteins.

The inhibitory effects of epigallocatechin-3-gallate on calcium oxalate monohydrate crystal growth, aggregation and crystal-cell adhesion.

Epigallocatechin-3-gallate (EGCG), a predominant phytochemical in tea plant, has been reported to prevent kidney stone formation but with vague mechanism. We investigated modulatory effects of EGCG (at 0.1-100 µM) on calcium oxalate monohydrate (COM) crystals at various stages of kidney stone development. EGCG significantly increased crystal size (at 1-100 µM), but decreased crystal number (at 10-100 µM), resulting in unchanged crystal mass and volume. Interestingly, EGCG at 10-100 µM caused morphological change of the crystals from typical monoclinic prismatic to coffee-bean-like shape, which represented atypical/aberrant form of COM as confirmed by attenuated total reflection - Fourier transform infrared (ATR-FTIR) spectroscopy. EGCG at all concentrations significantly inhibited crystal growth in a concentration-dependent manner. However, only 100 µM and 10-100 µM of EGCG significantly inhibited crystal aggregation and crystal-cell adhesion, respectively. Immunofluorescence staining (without permeabilization) revealed that surface expression of heat shock protein 90 (HSP90) (a COM crystal receptor) on MDCK renal cells was significantly decreased by 10 µM EGCG, whereas other surface COM receptors (annexin A1, annexin A2, enolase 1 and ezrin) remained unchanged. Immunoblotting showed that 10 µM EGCG did not alter total level of HSP90 in MDCK cells, implicating that its decreased surface expression was due to translocation. Our data provide a piece of evidence explaining mechanism underlying the anti-lithiatic property of EGCG by inhibition of COM crystal growth, aggregation and crystal-cell adhesion via reduced surface expression of HSP90, which is an important COM crystal receptor.

Resveratrol inhibits calcium oxalate crystal growth, reduces adhesion to renal cells and induces crystal internalization into the cells, but promotes crystal aggregation.

Resveratrol is a natural phenolic compound that belongs to stilbenoid group found in diverse plants. Health benefits and therapeutic potentials of resveratrol have been widely recognized in various diseases. In kidney stone disease, it can alleviate oxalate-induced hyperproduction of free radicals in renal epithelial cells. Nevertheless, its direct effects on calcium oxalate (CaOx) crystal, which is the major stone component, remained unclear. This study therefore addressed the direct effects of resveratrol (at 1, 10 or 100 µM) on each step of CaOx kidney stone formation. The results revealed that resveratrol had no significant effects on CaOx crystallization. However, resveratrol significantly decreased CaOx crystal growth and adhesion to renal epithelial cells at all concentrations, and induced crystal internalization into the cells (a process related to crystal degradation by endolysosomes) in a concentration-dependent manner. On the other hand, resveratrol promoted crystal aggregation. These data indicate that resveratrol serves as a dual modulator on CaOx stone formation. While it inhibits CaOx stone development by reducing crystal growth and adhesion to renal cells and by inducing crystal internalization into the cells, resveratrol promotes crystal aggregation, which is one of the mechanisms leading to kidney stone formation.

Large-scale identification of calcium oxalate stone inhibitory proteins in normal human urine.

Recent evidence has shown that proteins in normal human urine can inhibit calcium oxalate (CaOx) kidney stone formation. Herein, we performed fast protein liquid chromatography (FPLC) to fractionate normal human urinary proteins using anion-exchange (DEAE) and size-exclusion (Superdex 200) materials. FPLC fractions (F1-F15) were examined by CaOx crystallization, growth, aggregation and crystal-cell adhesion assays. The fractions with potent inhibitory activities against CaOx crystals were then subjected to mass spectrometric protein identification. The data revealed that 13 of 15 fractions showed inhibitory activities in at least one crystal assay. Integrating CaOx inhibitory scores demonstrated that F6, F7 and F8 had the most potent inhibitory activities. NanoLC-ESI-Qq-TOF MS/MS identified 105, 93 and 53 proteins in F6, F7 and F8, respectively. Among them, 60 were found in at least two fractions and/or listed among known inhibitors with solid experimental evidence in the StoneMod database (https://www.stonemod.org). Interestingly, 10 of these 60 potential inhibitors have been reported with lower urinary levels in CaOx stone formers compared with healthy (non-stone) individuals, strengthening their roles as potent CaOx stone inhibitors. Our study provides the largest dataset of potential CaOx stone inhibitory proteins that will be useful for further elucidations of stone-forming mechanisms and ultimately for therapeutic/preventive applications.

Proteomic insight towards key modulating proteins regulated by the aryl hydrocarbon receptor involved in ovarian carcinogenesis and chemoresistance.

Gynecological malignancies pose a severe threat to female lives. Ovarian cancer (OC), the most lethal gynecological malignancy, is clinically presented with chemoresistance and a higher relapse rate. Several studies have highly correlated the incidence of OC to exposure to environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a process mainly mediated through activating the aryl hydrocarbon receptor (AhR). We have previously reported that exposure of OC cells to TCDD, an AhR activator, significantly modulated the expression of several genes that play roles in stemness and chemoresistance. However, the effect of AhR activation on the whole OC cell proteome aiming at identifying novel druggable targets for both prevention and treatment intervention purposes remains unrevealed. For this purpose, we conducted a comparative proteomic analysis of OC cells A2780 untreated/treated with TCDD for 24 h using a mass spectrometry-based label-free shotgun proteomics approach. The most significantly dysregulated proteins were validated by Western blot analysis. Our results showed that upon AhR activation by TCDD, out of 2598 proteins identified, 795 proteins were upregulated, and 611 were downregulated. STRING interaction analysis and KEGG-Reactome pathway analysis approaches identified several significantly dysregulated proteins that were categorized to be involved in chemoresistance, cancer progression, invasion and metastasis, apoptosis, survival, and prognosis in OC. Importantly, selected dysregulated genes identified by the proteomic study were validated at the protein expression levels by Western blot analysis. In conclusion, this study provides a better understanding of the the cross-talk between AhR and several other molecular signaling pathways and the role and involvement of AhR in ovarian carcinogenesis and chemoresistance. Moreover, the study suggests that AhR is a potential therapeutic target for OC prevention and maintenance. SIGNIFICANCE: To our knowledge, this is the first study that investigates the role and involvement of AhR and its regulated genes in OC by performing a comparative proteomic analysis to identify the critical proteins with a modulated expression upon AhR activation. We found AhR activation to play a tumor-promoting and chemoresistance-inducing role in the pathogenesis of OC. The results of our study help to devise novel therapeutics for better management and prevention and open the doors to finding novel biomarkers for the early detection and prognosis of OC.

Protein network analysis and functional enrichment via computational biotechnology unravel molecular and pathogenic mechanisms of kidney stone disease.

Mass spectrometry-based proteomics has been extensively applied to current biomedical research. From such large-scale identification of proteins, several computational tools have been developed for determining protein-protein interactions (PPI) network and functional significance of the identified proteins and their complex. Analyses of PPI network and functional enrichment have been widely applied to various fields of biomedical research. Herein, we summarize commonly used tools for PPI network analysis and functional enrichment in kidney stone research and discuss their applications to kidney stone disease (KSD). Such computational approach has been used mainly to investigate PPI networks and functional significance of the proteins derived from urine of patients with kidney stone (stone formers), stone matrix, Randall's plaque, renal papilla, renal tubular cells, mitochondria and immune cells. The data obtained from computational biotechnology leads to experimental validation and investigations that offer new knowledge on kidney stone formation processes. Moreover, the computational approach may also lead to defining new therapeutic targets and preventive strategies for better outcome in KSD management.

Kidney Stone Prevention.

Kidney stone disease (KSD) (alternatively nephrolithiasis or urolithiasis) is a global health care problem that affects people in almost all of developed and developing countries. Its prevalence has been continuously increasing with a high recurrence rate after stone removal. Although effective therapeutic modalities are available, preventive strategies for both new and recurrent stones are required to reduce physical and financial burdens of KSD. To prevent kidney stone formation, its etiology and risk factors should be first considered. Low urine output and dehydration are the common risks of all stone types, whereas hypercalciuria, hyperoxaluria, and hypocitraturia are the major risks of calcium stones. In this article, up-to-date knowledge on strategies (nutrition-based mainly) to prevent KSD is provided. Important roles of fluid intake (2.5-3.0 L/d), diuresis (>2.0-2.5 L/d), lifestyle and habit modifications (for example, maintain normal body mass index, fluid compensation for working in high-temperature environment, and avoid cigarette smoking), and dietary management [for example, sufficient calcium at 1000-1200 mg/d, limit sodium at 2 or 3-5 g/d of sodium chloride (NaCl), limit oxalate-rich foods, avoid vitamin C and vitamin D supplements, limit animal proteins to 0.8-1.0 g/kg body weight/d but increase plant proteins in patients with calcium and uric acid stone and those with hyperuricosuria, increase proportion of citrus fruits, and consider lime powder supplementation] are summarized. Moreover, uses of natural bioactive products (for example, caffeine, epigallocatechin gallate, and diosmin), medications (for example, thiazides, alkaline citrate, other alkalinizing agents, and allopurinol), bacterial eradication, and probiotics are also discussed.