Garcinia dulcis Extract Alleviates Inflammation in Kidney and Liver of the 2-Kidney-1-Clip Hypertensive rat.

Garcinia dulcis (GD) extract possesses anti-hypertensive property that are poorly characterized. This study aimed to investigate an anti-inflammatory effect of GD flower extract in the 2-kidney-1-clip (2K1C) hypertensive compared to sham operative (SO) rat. Male Wistar rats were divided into 2 groups; the 2K1C group in which a silver clip was placed around renal artery to induce hypertension, and the SO normotensive group. Four weeks later, each group of rats were further divided into 2 subgroups, each subgroup was orally gavaged of either corn oil (vehicle) or 50 mg/kg BW GD extract daily for 4 weeks. The malondialdehyde (MDA) levels in serum, liver, and kidney were determined. Hematoxylin and eosin staining was carried out for histological examination, Periodic acid - Schiff staining for glomerular injury, Masson's trichrome staining for renal fibrosis, and immunohistochemistry for either tumor necrosis factor alpha (TNF-α) or endothelial nitric oxide synthase (eNOS) investigation. Taken together, our results demonstrated that GD flower extract decreased the MDA level in both serum and liver and kidney tissue and suppressed the expression of TNF-α in both liver and kidney of 2K1C hypertensive rats. Mesangial cell proliferation, expansion of mesangial matrix, widening Bowman's capsule space, congestion of glomerular capillary and vessel, cloudy swelling of renal tubular epithelial cell, and renal fibrosis were observed in the kidneys of 2K1C rats. Therefore, we concluded that GD flower extract can alleviate liver and kidney inflammation in which partially attenuates the glomerular injury in the 2K1C rat.

Whole Genome Sequencing Reveals Antimicrobial Resistance and Virulence Genes of Both Pathogenic and Non-Pathogenic B. cereus Group Isolates from Foodstuffs in Thailand.

Members of the Bacillus cereus group are spore-forming Gram-positive bacilli that are commonly associated with diarrheal or emetic food poisoning. They are widespread in nature and frequently present in both raw and processed food products. Here, we genetically characterized 24 B. cereus group isolates from foodstuffs. Whole-genome sequencing (WGS) revealed that most of the isolates were closely related to B. cereus sensu stricto (12 isolates), followed by B. pacificus (5 isolates), B. paranthracis (5 isolates), B. tropicus (1 isolate), and "B. bingmayongensis" (1 isolate). The most detected virulence genes were BAS_RS06430, followed by bacillibactin biosynthesis genes (dhbA, dhbB, dhbC, dhbE, and dhbF), genes encoding the three-component non-hemolytic enterotoxin (nheA, nheB, and nheC), a gene encoding an iron-regulated leucine-rich surface protein (ilsA), and a gene encoding a metalloprotease (inhA). Various biofilm-associated genes were found, with high prevalences of tasA and sipW genes (matrix protein-encoding genes); purA, purC, and purL genes (eDNA synthesis genes); lytR and ugd genes (matrix polysaccharide synthesis genes); and abrB, codY, nprR, plcR, sinR, and spo0A genes (biofilm transcription regulator genes). Genes related to fosfomycin and beta-lactam resistance were identified in most of the isolates. We therefore demonstrated that WGS analysis represents a useful tool for rapidly identifying and characterizing B. cereus group strains. Determining the genetic epidemiology, the presence of virulence and antimicrobial resistance genes, and the pathogenic potential of each strain is crucial for improving the risk assessment of foodborne B. cereus group strains.

Production and immunological characterization of the novel single-chain variable fragment (scFv) antibodies against the epitopes on Opisthorchis viverrini cathepsin F (OvCatF).

BACKGROUND: Opisthorchis viverrini infection is a significant health problem in several countries, especially Southeast Asia. The infection causes acute gastro-hepatic symptoms and also long-term infection leading to carcinogenesis of an aggressive bile duct cancer (cholangiocarcinoma; CCA). Hence, the early diagnosis of O. viverrini infection could be the way out of this situation. Still, stool examination by microscopic-based methods, the current diagnostic procedure is restricted by low parasite egg numbers in the specimen and unprofessional laboratorians. The immunological procedure provides a better chance for diagnosis of the infection. Hence, this study aims to produce single-chain variable fragment (scFv) antibodies for use as a diagnostic tool for O. viverrini infection. METHODS: This study uses phage display technologies to develop the scFv antibodies against O. viverrini cathepsin F (OvCatF). The OvCatF-deduced amino acid sequence was analyzed and predicted for B-cell epitopes used for short peptide synthesis. The synthetic peptides were used to screen the phage library simultaneously with OvCatF recombinant protein (rOvCatF). The potentiated phages were collected, rescued, and reassembled in XL1-blue Escherichia coli (E. coli) as a propagative host. The positive clones of phagemids were isolated, and the single-chain variable (scFv) fragments were sequenced, computationally predicted, and molecular docked. The complete scFv fragments were digested from the phagemid, subcloned into the pOPE101 expression vector, and expressed in XL1-blue E. coli. Indirect ELISA and Western analysis were used to verify the detection efficiency. RESULTS: The scFv phages specific to OvCatF were successfully isolated, subcloned, and produced as a recombinant protein. The recombinant scFv antibodies were purified and refolded to make functional scFv. The evaluation of specific recognition of the particular epitopes and detection limit results by both computational and laboratory performances demonstrated that all three recombinant scFv antibodies against OvCatF could bind specifically to rOvCatF, and the lowest detection concentration in this study was only one hundred nanograms. CONCLUSION: Our produced scFv antibodies will be the potential candidates for developing a practical diagnostic procedure for O. viverrini infection in humans in the future.

Cardiovascular Protective Effect of Garcinia dulcis Flower Acetone Extract in 2-Kidney-1-Clip Hypertensive Rats.

Morelloflavone and camboginol are bioactive compounds purified from Garcinia dulcis (GD), which has anti-inflammatory and antihypertensive properties. The objective of this study was to examine the cardiovascular protective effect of GD flower acetone extract in 2-kidney-1-clip (2K1C) hypertensive rats. Male Wistar rats underwent 2K1C or sham operation (SO) and were housed for 4 weeks. Each group of rats, then, was further divided into 2 subgroups receiving oral administration of either 50 mg/kg BW GD extract or corn oil (vehicle) daily for 4 weeks. Noninvasive blood pressure (BP) and body weight were measured weekly throughout the study. Subsequently, the invasive measurement of arterial BP and the heart rate were determined in all anesthetized rats. The baroreceptor reflex sensitivity (BRS) was investigated by injection of either phenylephrine or sodium nitroprusside for bradycardia or tachycardia response, respectively. Histological examination of the heart and thoracic aorta was also performed in order to investigate the general morphology and the tumor necrosis factor alpha (TNF-α) expression. We found that the GD flower extract significantly diminished the BP and restored the impaired BRS. Moreover, it also decreased the TNF-α expression in the cardiac muscle and thoracic aorta of 2K1C when compared to the SO group. Taken together, our data showed that GD flower extract exhibits the cardiovascular protective effect in the 2K1C hypertensive rats.

Coproprevalence, seroprevalence, and geographic distribution of Fasciola spp. infection in beef and dairy cattle in Pak Chong highland, Nakhon-Ratchasima Province, Northeast Thailand.

Fasciola spp. is a major livestock parasite, especially in cattle, water buffalo, sheep, and goats. Infection reduces animal productivity, e.g., meat, dairy, wool and might cause death. In Thailand, reports of Fasciola spp. infection in livestock, especially dairy and beef cattle, are sparse. Pakchong district in Nakhon Ratchasima province is one of Thailand's largest farming areas for dairy and beef cattle, but the prevalence of Fasciola spp. infection has never been reported in this district. The landscape of this area is mainly a hilltop plateau with many water sources suitable for the development of lymnaeid snails, the intermediate host of Fasciola spp., which are essential for the parasite life cycle. This study surveyed the copro- and seroprevalence of Fasciola spp. infection in dairy and beef cattle farmed in Pakchong district by microscope-based examination, PCR, and indirect ELISA. Associated risk factors and geographic information data were collected and analyzed. Paired stool and serum samples were collected from 102 dairy cattle and 99 beef cattle from April to November 2021. Sample analyses demonstrated a high prevalence of Fasciola spp. infection, especially in beef cattle. The overall copro-prevalence was 5.97%, with 0.99% in dairy cattle and 11.11% in beef cattle. The overall seroprevalence was 23.88%, with 2.94% in dairy cattle and 45.45% in beef cattle. Moreover, the data indicated that infection status was not correlated with animal sex and age whereas consumption of natural grasses, water resources, housing floor, and farming system were significant risk factors. Data analysis by a geographic information system (GIS) demonstrated that an associated risk could be farmed in lowering areas, especially in Chan Thuck, Nong Sa Rai, and Khlong Muang subdistricts. In conclusion, this study reports the prevalence of Fasciola spp. infection in cattle in a major farming area of Thailand which could be beneficial for designing parasite control policies in this region as well as adapting this knowledge to other Fasciola spp. endemic areas.

Cystatins from the Human Liver Fluke Opisthorchis viverrini: Molecular Characterization and Functional Analysis.

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1-6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3-6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3-9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated.

Ethyl Acetate Extract of Halymenia durvillei Induced Apoptosis, Autophagy, and Cell Cycle Arrest in Colorectal Cancer Cells.

Colorectal cancer is one of the most death-dealing cancers. However, conventional cancer treatments still have side effects. Therefore, novel chemotherapeutic agents with less side effects are still in search. A marine red seaweed, Halymenia durvillei, is recently interested in its anticancer effects. This study investigated the anticancer effect of ethyl acetate extract of H. durvillei (HDEA) on HT-29 colorectal cancer cells in association with the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. HDEA-treated HT-29 and OUMS-36 cells were used for cell viability tests by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay. The effects of HDEA on apoptosis and cell cycle were evaluated. The nuclear morphology and mitochondrial membrane potential (ΔΨm) were observed by Hoechst 33342 and JC-1 staining, respectively. The gene expression of PI3K, AKT, and mTOR genes was evaluated using a real-time semiquantitative reverse transcription-polymerase chain reaction. The corresponding protein expressions were assessed by western blot analysis. The result revealed that the cell viability of treated HT-29 cells diminished while that of OUMS-36 cells was non-significant. By the down-regulation of cyclin-dependent ki-nase 4 and cyclin D1, HDEA-treated HT-29 cells were arrested in the G0/G1 phase. By the up-regulation of cleaved poly(adenosine diphosphate-ribose) polymerase, caspase-9, caspase-8, caspase-3, and Bax, HDEA-treated HT-29 cells underwent apoptosis, but suppressed Bcl-2, disrupted nuclear morphology and ΔΨm. Furthermore, treated HT-29 cells underwent autophagy by up-regulation of light chain 3-II and beclin-1. Lastly, HDEA suppressed the expression of PI3K, AKT, and mTOR. Therefore, HDEA exerts anticancer effects against HT-29 cells, confirmed by apoptosis, autophagy, and cell cycle arrest induction via regulation of the PI3K/AKT/mTOR signaling pathway.

Current prevalence and geographic distribution of helminth infections in the parasitic endemic areas of rural Northeastern Thailand.

BACKGROUND: Helminth infection is a global health issue that not only causes acute helminthiasis but long-term infection may lead to complicated symptoms as well as severe complications. The World Health Organization cooperated with the Ministry of Public Health in many countries, particularly where high prevalence, spending a lot of resources for limiting the infection. In Thailand, the incidence of parasitic helminth infections was continuously declined in the last few decades according to several campaigns for parasitic elimination. However, the rural community in the northeast of Thailand where the highest prevalence of the country still needs to be monitored. This present study aims to report the current prevalence of parasitic helminth infections in Nakhon Ratchasima and Chaiyaphum provinces where sharing a huge area of the northeastern region of Thailand but only a few studies have been published. METHODS: The stool specimens were collected from 11,196 volunteers and processed by modified Kato-Katz thick smear, PBS-ethyl acetate concentration techniques, and PCR. The epidemiological data were collected, analyzed, and used for generating of parasitic hotspots. RESULTS: The results indicated that O. viverrini remains the major parasite in this area with a total prevalence of 5.05% followed by Taenia spp., Hookworms, T. trichiura, and Echinostoma spp., respectively. Mueang district of Chaiyaphum province has the highest prevalence especially O. viverrini with a prevalence of 7.15% that higher than the latest national surveillance. Interestingly, the prevalence of O. viverrini was hugely reported (more than 10%) in five subdistricts. The geographic localization of O. viverrini infections revealed that a lot of water reservoirs such as the lakes or branches of the river in the two-most prevalent subdistricts. Our finding indicated that gender and age were insignificantly different. CONCLUSION: This finding suggested that the parasitic helminth infection in the rural areas of northeast of Thailand remains high and the housing location is a major contributing factor for the parasitic infection.

Type I Cystatin Derived from Fasciola gigantica Suppresses Macrophage-Mediated Inflammatory Responses.

There is an inverse relationship between the high incidence of helminth infection and the low incidence of inflammatory disease. Hence, it may be that helminth molecules have anti-inflammatory effects. Helminth cystatins are being extensively studied for anti-inflammatory potential. Therefore, in this study, the recombinant type I cystatin (stefin-1) of Fasciola gigantica (rFgCyst) was verified to have LPS-activated anti-inflammatory potential, including in human THP-1-derived macrophages and RAW 264.7 murine macrophages. The results from the MTT assay suggest that rFgCyst did not alter cell viability; moreover, it exerted anti-inflammatory activity by decreasing the production of proinflammatory cytokines and mediators, including IL-1ß, IL-6, IL-8, TNF-α, iNOS, and COX-2 at the gene transcription and protein expression levels, as determined by qRT-PCR and Western blot analysis, respectively. Further, the secretion levels of IL-1ß, IL-6, and TNF-α determined by ELISA and the NO production level determined by the Griess test were decreased. Furthermore, in Western blot analysis, the anti-inflammatory effects involved the downregulation of pIKKα/ß, pIκBα, and pNF-κB in the NF-κB signaling pathway, hence reducing the translocation from the cytosol into the nucleus of pNF-κB, which subsequently turned on the gene of proinflammatory molecules. Therefore, cystatin type 1 of F. gigantica is a potential candidate for inflammatory disease treatment.

Production and Immunological Characterization of scFv Specific to Epitope of Opisthorchis viverrini Rhophilin-Associated Tail Protein 1-like (OvROPN1L).

(1) Background: Opisthorchis viverrini is a significant health problem in the Mekong subregion of Southeast Asia, causing aggressive cholangiocarcinoma. Current diagnostic procedures do not cover early diagnosis and low infection. Hence, an effective diagnostic tool is still required. Immunodiagnosis seems promising, but attempts to generate monoclonal antibodies have not yet been successful. This study aims to develop a single-chain variable antibody fragment (scFv) against Rhophilin-associated tail protein 1-like (ROPN1L), the sperm-specific antigen of adult O. viverrini, which has not been reported elsewhere. (2) Methods: The target epitope for phage screening was L3-Q13 of OvROPN1L, which showed the highest antigenicity to human opisthorchiasis analyzed in a previous study. This peptide was commercially synthesized and used for phage library screening. The isolated phage was produced in a bacterial expression system and tested for specificity in vitro and in silico. (3) Results: One of fourteen phages, named scFv anti-OvROPN1L-CL19, significantly bound to rOvROPN1L compared with non-infected hamster fecal extracts. This phage clone was successfully produced and purified using Ni-NTA chromatography. Indirect ELISA demonstrated that scFv anti-OvROPN1L-CL19 has a high reactivity with O. viverrini-infected hamster fecal extracts (12 wpi, n = 6) in comparison with non-infected hamster fecal extracts (0 wpi, n = 6), while the polyclonal rOvROPN1L antibodies did not show such a difference. Molecular modeling and docking confirmed our in vitro findings. (4) Conclusion: scFv anti-OvROPN1L-CL19 could be used as an effective material for developing O. viverrini-immunodiagnostic procedures in the future.

Anti-Inflammatory Effect of Garcinol Extracted from Garcinia dulcis via Modulating NF-κB Signaling Pathway.

Garcinia is a significant medicinal plant with many beneficial phytoconstituents, including garcinol. This study investigated the anti-inflammatory effect of garcinol isolated from Garcinia dulcis fruit in LPS-activated THP-1 and Raw 264.7 macrophages. The results demonstrated that the low concentration of garcinol did not alter cell viability. Furthermore, co-incubation of garcinol with LPS inhibited the production of pro-inflammatory cytokines, including TNF-α, IL-8, IL-6, IL-1ß, and pro-inflammatory mediators, including iNOS and COX-2 at the mRNA and protein expression levels. Garcinol also decreased the secretion of TNF-α, IL-6, IL-1ß, PGE2, and NO. Moreover, the anti-inflammatory effects involved an alteration in the NF-κB signaling pathway. Downregulation of pIKKα/ß, pIκBα, and pNF-κB was observed, hence reducing the translocation of pNF-κB from the cytosol into the nucleus, which subsequently decreased the production of pro-inflammatory molecules. Therefore, garcinol isolated from Garcinia dulcis is a potential candidate as an anti-inflammatory agent for inflammation-related disease treatment.

Garcinia dulcis Flower Extract Alters Gut Microbiota and Fecal Metabolomic Profiles of 2K1C Hypertensive Rats.

Garcinia dulcis (GD) extract has been found to have anti-hypertensive properties in animal studies. GD can also alter the colonic microbiota of rats. However, the effects of GD on changes in the gut microbiota and metabolomic profiles of normotensive and hypertensive rats are currently unknown. The purpose of this study was to evaluate changes in the gut microbiota and metabolomic profiles of 2-kidneys-1 clip (2K1C) hypertensive rats after feeding with GD flower extract. Rats were randomly divided into the following 4 groups: sham operation (SO) receiving corn oil (CO) (SO + CO), SO receiving GD (SO + GD), 2K1C receiving corn oil (2K1C + CO) and 2K1C receiving GD (2K1C + GD). Body weight (BW) and systolic blood pressure (SBP) were measured weekly throughout the study. Gut microbiota and fecal metabolites were measured from fresh fecal contents. Alpha diversity results demonstrated a similar microbial richness and diversity between groups. Linear discriminant analysis (LDA) effect size (LEfSe) suggested that GD treatment affected gut microbial community structure in both hypertensive and normotensive rats. Feeding rats with GD caused metabolic alterations that rendered 2K1C + GD rats similar to SO + CO and SO + GD rats. Findings suggest that the impact of GD on gut microbiota and metabolite profiles may be related to its anti-hypertensive properties.

Molecular Cloning and Characterization of a Fasciola gigantica Nuclear Receptor Subfamily 1 (FgNR1).

Fasciola gigantica, a giant liver fluke, causes tremendous loss to the livestock economy in several regions throughout the world. The situation of drug resistance has been emerging increasingly; therefore, novel drugs and drug targets need to be discovered. The adult F. gigantica inhabits the major bile ducts where bile salts accumulate­these are steroid-like molecules that mediate several physiological processes in organisms through interacting with their specific nuclear receptors. However, the molecular mechanism of the interaction in the parasitic organisms have not been clearly understood. In this study, putative nuclear receptor subfamily 1 of F. gigantica (FgNR1) was identified. Nucleotide and amino acid sequences of the FgNR1 homolog were obtained from the transcriptome of F. gigantica and predicted for properties and functions using bioinformatics. The full-length cDNA was cloned and expressed in the bacterial expression system and then used for immunization. Western analysis and immunolocalization suggested that FgNR1 could be detected in the crude worm antigens and was highly expressed in the caeca and testes of the adult parasite. Moreover, the bile could significantly activate the expression of FgNR1 in cultured parasites. Our results indicated that FgNR1 has high potential for the development of a novel anthelminthic drug in the future.

Daily consumption of monosodium glutamate pronounced hypertension and altered renal excretory function in normotensive and hypertensive rats.

This study aimed to investigate the effects of monosodium glutamate (MSG) on the levels of arterial blood pressure (ABP) and renal excretory function. Male Wistar rats were divided into 2 groups (n = 24 each) namely sham operation (SO) and 2-kidneys-1-clip (2K1C) to develop the normotensive and hypertensive model, respectively. Four weeks after the operation, each group of rats were further divided into 4 subgroups (n = 6 each) which were orally administered of either distilled water or MSG at the doses of 80, 160, or 320 mg/kg BW/day once a day for 8 weeks. The body weight, the 24-hour water intake, and the 24-hour urine output were recorded weekly. Then, each rat was anesthetized and the ABP was measured via carotid artery. The renal excretory function was examined by using the clearance technique to measure the levels of the glomerular filtration rate and the renal blood flow. The levels of serum malondialdehyde (MDA) as a marker of oxidative stress were analyzed. The expression of tumor necrosis factor alpha (TNF-α) in the kidneys was also investigated using immunohistochemistry. It was found that all doses of MSG significantly increased the ABP in both SO and 2K1C groups. All doses of MSG significantly increased the serum MDA levels and the expression of TNF-α in the kidneys of the SO groups. Long-term intake of 320 mg/kg BW MSG significantly decreased the renal excretion of salt and water in both SO and 2K1C groups. As a whole, this study demonstrated that MSG consumption contributed to an increase in oxidative stress which could lead to alterations in the cardiovascular and renal function.

Evaluation of indirect-ELISA using eluted antigens from Trichinella spiralis muscle larvae for diagnosis of swine trichinellosis.

Trichinellosis is caused by Trichinella spiralis muscle larvae (TsML), which is transmitted to human when they eat infected raw or undercooked meat. T. spiralis infection is detected by an enzyme-linked immunosorbent assay (ELISA) using excretory-secretory antigens (ESAg); however, the preparation of ESAg is challenging, and yields are low, which hampers screening efforts. In this study, crude somatic antigens (CSAg) of TsML with molecular weights (MWs) of 43, 79 and 101 kDa have been identified in swine trichinellosis sera with less cross-reaction with uninfected sera and other parasitic infected sera. After that, the CSAg at MWs of 43, 79 and 101 kDa (TsCSAg-43, TsCSAg-79, and TsCSAg-101, respectively) were isolated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The eluted antigens were analyzed by IgG-ELISA for sensitivity and specificity, and specific antigens from the three regions were identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The sensitivity of IgG-ELISA using the three eluted antigens was 100% with specificities of 97.77%, 95.54%, 90.63% and for TsCSAg-43, TsCSAg-79, and TsCSAg-101, respectively. The LC-MS-MS results of immunomics showed that 18/20 spots of the antigens with MWs of 43, 79, and 101 kDa represent 11 different proteins identified. TsCSAg-43 showed the highest specificity, indicating that the specific proteins identified, including 45 kDa antigen-trichina [fragment], DNA topoisomerase 2-alpha antigen targeted by protective antibodies, and a conserved hypothetical protein (gi339234223), should be developed and produced in large volumes for further immunodiagnostic studies.

Camboginol and Morelloflavone from Garcinia dulcis (Roxb.) Kurz Flower Extract Promote Autophagic Cell Death against Human Glioblastoma Cells through Endoplasmic Reticulum Stress.

Garcinia dulcis is a tropical plant native to Southeast Asia that is traditionally used as a folk remedy to cure several pathological symptoms. Camboginol and morelloflavone have been revealed by previous studies as the principal bioactive compounds from the flower extract of G. dulcis. The disease-preventing properties of camboginol or morelloflavone, including anti-cancer, from various parts of G. dulcis have been revealed by recent studies. Glioblastoma is the aggressive malignant stage of brain cancer and suffers from chemotherapeutic resistance. This study aimed to test the anti-cancer effect of G. dulcis flower extract against the proliferation of A172 human glioblastoma cells. The extract had cytotoxic activity and promoted cell cycle arrest at the S and G2/M phases. Autophagic cell death was promoted by cytotoxic concentrations of the extract, as observed by enhancing autophagic flux and the expression of autophagic markers. Autophagic cell death induced by the extract might be associated with endoplasmic reticulum (ER) stress. Conclusively, it was indicated by this study that the extract from the flower of G. dulcis had a protective effect against the proliferation of A172 human glioblastoma cells through the induction of ER stress-mediated cytotoxic autophagy.

Efficiency of the Stool-PCR Test Targeting NADH Dehydrogenase (Nad) Subunits for Detection of Opisthorchis viverrini Eggs.

Opisthorchis viverrini infection is the major parasitic infection problem in Southeast Asian countries, and long-term infection will lead to cholangiocarcinoma (CCA), the bile duct cancer. The early diagnosis of O. viverrini infection may interrupt the progression of the opisthorchiasis and other related illnesses, especially CCA. The current diagnostic procedure is stool examination by microscope-based methods such as direct smear and concentration techniques but it is limited by low parasite egg numbers. The molecular diagnosis prompts the chance to evaluate the light infection with low number of parasite eggs but is currently inconvenient for routine use due to special equipment requirement and unstable sensitivities. Our present study aims to establish the efficiency of OvNad subunits, the mitochondrial gene, for introducing as a potential diagnostic target by conventional PCR, the cheapest and easiest molecular procedure. A total of 166 stool samples were investigated microscopically by the PBS-ethyl acetate concentration technique (PECT); 75 samples were O. viverrini positive with 28 samples that were positive with single parasite (hookworm, A. lumbricoides, S. stercoralis, Taenia spp., and T. trichiura), 11 samples were with mixed infection, and 52 samples were without parasite detection. The detection limits of OvNad subunits were evaluated in artificially spiked samples containing 0, 1, 5, 10, 20, 50, and 100 Ov-eggs. The result suggested that the best detection efficacy was of OvNad5 that had exact detection limits at only 5 eggs. In the PCR amplification of OvNad subunits, there exist 100% specificities with varied sensitivities from 64%, 88%, 80%, and 100% of OvNad1, OvNad2, OvNad4, and OvNad5, respectively. OvNad subunits were amplified specifically without cross reactivity with the other collected parasites. Our study established that OvNad subunits, especially OvNad5, are the potent candidates for PCR amplification of stool containing Ov-eggs with high confidential sensitivity, specificity, PPV, and NPV even in the light infection that would be a benefit for developing as a routine diagnosis of O. viverrini infection.

Suppression of Cholangiocarcinoma Cell Growth and Proliferation by Atractylodes lancea (Thunb) DC. through ERK-Signaling Cascade.

OBJECTIVE: The study aimed to investigate the inhibitory effects of AL on the ERK signaling molecules (ERK, p-ERK, cyclin D, and eIF4B) and the growth and proliferation of CCA cells. MATERIALS AND METHODS: The viability of the three CCA cell lines CL-6, HuCCT1, and HuH28 was determined using MTT assay. The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was investigated. The protein extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed by using RT-PCR. RESULTS: The potency of cytotoxic activity of AL (by MTT assay) was about three times higher than the standard drug 5-fluorouracil. The IC50 (concentration that inhibits cell growth by 50%) of AL for the CL-6, HuCCT-1 and HuH28 cell lines were 29.77±6.64, 35.45±4.96, and 35.32±6.69 µg/mL (mean+SD), respectively. The cells were exposed to AL extract at the IC50 for 0, 12, 24, 48, and 72 hours in the absence and presence of Ras/ERK inhibitors (salirasib and XMD8-92). Protein expression was determined by Western blot analysis. The results suggested the lack of significant inhibitory effect of AL on ERK at 48 and 72 hours of exposure in all CCA cell types. On the other hand, a significant inhibitory effect was observed with p-ERK expression in all CCA cell types. Cyclin D was significantly down-regulated at 72 hours of exposure in all cell types with different potencies. The expression of eIF4B was markedly inhibited in HuCCT-1 but slightly inhibited in CL-6 and HuH28 cells. Real-time PCR analysis revealed significant down-regulation of ERK following 72 hours of AL exposure in the HuCCT1 and HuH28, but not CL-6 cell. CONCLUSION: The ERK signaling cascade and downstream molecules are potential targets of action of AL in CCA.

Improvement of a PCR-based method for the detection of Opisthorchis viverrini eggs in human stool samples by targeting internal transcribed spacer-2 (ITS-2), cytochrome oxidase subunit 1 (cox1), and cytochrome b (cyb).

Opisthorchis viverrini infection causes various complications in patients, ranging from asymptomatic to severe chronic disease including cholangiocarcinoma (CCA). O. viverrini is endemic in Southeast Asia and acting as a risk for CCA. Early diagnosis of O. viverrini infection can reduce the number of CCA cases. The routine diagnosis for opisthorchiasis is direct wet smear, sometimes coupled with concentration techniques, which has limitations when investigating light infection or if done by laboratorians with lack of experiences. PCR-based methods have been established for the detection of O. viverrini egg DNA from stool samples, but have never fully succeeded for light infections when compared to wet smear concentration techniques. This study aims to improve the PCR-based method for detection of O. viverrini eggs in stool samples by targeting the genes ITS-2, cox1, and cyb. The results reveal higher sensitivity than conventional concentration techniques, with all newly designed primers. ITS-2 has an overall sensitivity of 76.9% with 66.7% in the samples with < 50 EPG, while cox1 has shown 96.2% overall sensitivity and 94.1% in the same EPG intervals. Interestingly, the new pointing target, cyb, has shown 100% sensitivity in all egg intervals in this study, particularly for light infections (EPG less than 100). No cross-reactivity was found in Taenia spp., Trichuris trichiura, Ascaris lumbricoides, Capillaria philippinensis, and hookworm. The procedure is convenient, with shorter steps compared to previous reports, and it appears appropriate for use in the diagnosis of light infection with O. viverrini. These three genes are good candidates for use in PCR-based detection of the parasite eggs. Further testing with a larger cluster of samples is however necessary.

Anticancer Activity of Fucoidan via Apoptosis and Cell Cycle Arrest on Cholangiocarcinoma Cell.

OBJECTIVE: Many previous studies reported that fucoidan has antitumor activities. The objective of the present study was to determine the cytotoxic effects and related mechanisms of cell death induced by fucoidan extracted from Fucus vesiculosus on CL-6 cholangiocarcinoma cell. METHODS: CL-6 and OUMS cells were treated with 0, 100, 200, and 300 µg/mL of fucoidan. MTT assay was used to determine cytotoxicity. Flow cytometry-based assay was used to examine the distribution of apoptosis and cell cycle. The changes in nuclear morphology were determined using Hoechst 33,342 staining. Mitochondrial membrane potential (ΔΨm) was evaluated using the JC-1 kit. The apoptotic, anti-apoptotic, and cell cycle-related proteins study were examined by Western blot analysis. RESULTS: The relative viable cell number of treated CL-6 cells was decreased but no effect was observed in OUMS normal cells. Furthermore, treated cells were arrested in the G0/G1 phase with down-regulation of cyclin D1 and CDK4. Annexin V/PI staining with flow cytometry analysis suggested that fucoidan could induce apoptosis in CL-6 cells. Western blot study revealed the up-regulation of apoptotic markers including Bax, cleaved PARP, cleaved caspase-3, but down-regulation of anti-apoptotic markers,  cl-2. Moreover, fucoidan could induce nuclear fragmentation and chromatin condensation with alteration of ΔΨm.  Conclusion: Fucoidan exerts antitumor properties against CL-6 cholangiocarcinoma cells illustrated by the induction of apoptosis and cell cycle arrest.
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