Publications by authors named "Roytrakul Sittiruk"

228 Publications

Kallikrein-11, in Association with Coiled-Coil Domain Containing 25, as a Potential Prognostic Marker for Cholangiocarcinoma with Lymph Node Metastasis.

Molecules 2021 May 22;26(11). Epub 2021 May 22.

Centre of Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, KhonKaen University, Khon Kaen 40002, Thailand.

Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes. Currently, the treatment and prognosis for CCA are mostly poor. Recently, we have reported that coiled-coil domain containing 25 (CCDC25) protein level in the sera may be a diagnostic marker for CCA. Subsequently, we identified three binding proteins of CCDC25 and found that kallikrein-11 (KLK11) expression was highest among those binding proteins. In this study, we investigated CCDC25 and KLK11 expression in CCA and adjacent normal tissues ( = 18) using immunohistochemistry. The results demonstrated that the expressions of CCDC25 and KLK11 in CCA tissues were both significantly higher than the adjacent tissues ( < 0.001 and = 0.001, respectively). Then, using GEPIA bioinformatics analysis, KLK11 mRNA was significantly overexpressed in CCA tumor tissues compared with normal tissues ( < 0.05). Moreover, CCDC25 expression was positively correlated with KLK11 expression in CCA with lymph node metastasis ( = 0.028, r = 0.593). An analysis for the interaction of KLK11 with CCDC25 and other proteins, using STRING version 11.0, revealed that CCDC25 and KLK11 correlated with metastasis-related proteins. In addition, Kaplan-Meier survival curve analysis revealed that a high expression of KLK11 was associated with the poor prognosis of CCA. In conclusion, KLK11 is, as a binding protein for CCDC25, possibly involved in the metastatic process of CCA. KLK11 may be used as a prognostic marker for CCA.
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http://dx.doi.org/10.3390/molecules26113105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196963PMC
May 2021

Evaluation of osteochondral-like tissues using human freeze-dried cancellous bone and chondrocyte sheets to treat osteochondral defects in rabbits.

Biomater Sci 2021 May 21. Epub 2021 May 21.

Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand. and Department of Chemical Engineering, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.

Human freeze-dried cancellous bone combined with human chondrocyte sheets have recently been used to construct an osteochondral-like tissue, which resembled a cartilage layer on a subchondral bone layer. Nevertheless, the efficacy of these human tissues in a xenogeneic model has been rarely reported. Therefore, this study aimed to evaluate the potential of human freeze-dried cancellous bones combined with human chondrocyte sheets for the treatment of osteochondral defects in rabbits. The key roles of the extracellular matrix (ECM) and released cytokines in these tissues in osteochondral repair were also assessed. Triple-layered chondrocyte sheets were constructed using a temperature-responsive culture surface. Then, they were placed onto cancellous bone to form chondrocyte sheet-cancellous bone tissues. The immunostaining of collagen type II (COL2) and the proteomic analysis of the human tissues were carried out before the transplantation. In our in vitro study, the triple-layered chondrocyte sheets adhered well on the cancellous bone, and the COL2 expression was apparent throughout the tissue structures. From the proteomic analysis results, it was found that the major function of the secreted proteins found in these tissues was protein binding. The distinct pathways were focal adhesion and the ECM-receptor interaction pathways. Among the highly expressed proteins, laminin-alpha 5 (LAMA5) and fibronectin (FN) not only played roles in the protein binding and ECM-receptor interaction, but also were involved in the cytokine-mediated signaling pathway. At 12 weeks after xenogeneic transplantation, compared to the control group, the defects treated with the chondrocyte sheets showed more hyaline-like cartilage tissue, as indicated by the abundance of safranin-O and COL2 with a partial collagen type I (COL1) expression. At 4, 8, and 12 weeks, compared to the defects treated with the cancellous bone, the staining of safranin-O and COL2 was more apparent in the defects treated with the chondrocyte sheet-cancellous bone tissues. Therefore, the human chondrocyte sheets and chondrocyte sheet-cancellous bone tissues provide a potential treatment for rabbit femoral condyle defect. LAMA5 and FN found in these human xenografts and their culture media might play key roles in the ECM-receptor interaction and might be involved in the cytokine-mediated signaling pathway during tissue repair.
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http://dx.doi.org/10.1039/d1bm00239bDOI Listing
May 2021

Bioinformatic Prediction of Signaling Pathways for Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and Its Role in Cholangiocarcinoma Cells.

Molecules 2021 Apr 29;26(9). Epub 2021 Apr 29.

Centre of Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, KhonKaen University, KhonKaen 40002, Thailand.

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) is involved in the DNA damage repair pathways and associates with the metastasis of several human cancers. However, the signaling pathway of APEX1 in cholangiocarcinoma (CCA) has never been reported. In this study, to predict the signaling pathways of APEX1 and related proteins and their functions, the effects of APEX1 gene silencing on APEX1 and related protein expression in CCA cell lines were investigated using mass spectrometry and bioinformatics tools. Bioinformatic analyses predicted that APEX1 might interact with cell division cycle 42 (CDC42) and son of sevenless homolog 1 (SOS1), which are involved in tumor metastasis. RNA and protein expression levels of APEX1 and its related proteins, retrieved from the Gene Expression Profiling Interactive Analysis (GEPIA) and the Human Protein Atlas databases, revealed that their expressions were higher in CCA than in the normal group. Moreover, higher levels of APEX1 expression and its related proteins were correlated with shorter survival time. In conclusion, the signaling pathway of APEX1 in metastasis might be mediated via CDC42 and SOS1. Furthermore, expression of APEX1 and related proteins is able to predict poor survival of CCA patients.
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http://dx.doi.org/10.3390/molecules26092587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125001PMC
April 2021

Streptococcus suis serotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PLoS One 2021 4;16(5):e0249682. Epub 2021 May 4.

Department of Veterinary Public Health, Faculty of Veterinary Sciences, Chulalongkorn University, Bangkok, Thailand.

Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249682PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096114PMC
May 2021

Bioactive peptide isolated from sesame seeds inhibits cell proliferation and induces apoptosis and autophagy in leukemic cells.

EXCLI J 2021 23;20:709-721. Epub 2021 Mar 23.

Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Nakhon Pathom 73170, Thailand.

Leukemia is the most common type of hematological malignancies. Several natural products including bioactive peptides have been explored and studied for their anti-leukemic activities. In the present study, anti-leukemic peptide, IGTLILM (IM-7), was isolated and identified from the protein hydrolysate of sesame seeds by reverse phase-solid phase extraction, off-gel fractionation and nano LC-MS/MS. The cytotoxic effects of IM-7 were studied in MOLT-4 and NB4 acute leukemic cell lines using an MTT assay. The induction of apoptosis and autophagy was investigated by flow cytometry using Annexin V-FITC/PI staining and anti-LC3/FITC antibodies, respectively. The mRNA alterations of apoptotic and autophagic-related genes were determined by reverse transcription-quantitative PCR. The present study found that IM-7 inhibited the proliferation of MOLT-4 and NB4 cells in dose-dependent manner, but it showed a minimal effect on healthy mononuclear cells. IM-7 activated apoptosis and autophagy through the upregulation of CASP3, ULK1 and BECN1 and the downregulation of BCL2. In addition, IM-7 enhanced the cytotoxic effect of the anti-leukemic drug, daunorubicin. The findings suggested that IM-7 was potent to suppress the proliferation of MOLT-4 and NB4 leukemic cells and induce apoptosis and autophagy through the regulation of caspase 3-Bcl-2 and ULK1-Beclin1, respectively.
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http://dx.doi.org/10.17179/excli2021-3406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073838PMC
March 2021

Analysis of the Protein-Protein Interaction Network Identifying c-Met as a Target of Gigantol in the Suppression of Lung Cancer Metastasis.

Cancer Genomics Proteomics 2021 May-Jun;18(3):261-272

Cell-based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand;

Background/aim: c-Met (mesenchymal-epithelial transition factor) facilitates cancer progression and is recognized as a promising drug target. The molecular target of gigantol from Dendrobium draconis in suppressing cancer metastasis is largely unknown.

Materials And Methods: Proteins affected by gigantol treatment were subjected to proteomic and bioinformatic analysis. Protein-Protein interaction (PPI) networks were constructed by the Search Tool for the Retrieval of Interacting Genes (STRING). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database and hub gene were used to enrich the dominant pathways. Western blot analysis and immunofluorescence were used to validate the effect of gigantol on the target protein and signaling.

Results: Gigantol down-regulates 41 adhesion proteins and 39-migratory proteins, while it up-regulates 30 adhesion-related proteins and 22 proteins controlling cell migration. The key components of our constructed PPI network comprised 41 proteins of cell adhesion enriched in 40 nodes with 25 edges, 39 proteins of cell migration enriched in 39 nodes with 76 edges in down-regulated proteins, 30 proteins of cell adhesion enriched in 30 nodes with 21 edges, and 22 proteins of cell migration enriched in 22 nodes with 22 edges in up-regulated protein. c-Met was identified as a central protein of the PPI network in the largest degree. KEGG mapper further suggested that c-Met, PI3K, and AKT were the regulatory proteins affected by gigantol. To confirm, the effects of gigantol on c-Met, the p-PI3K, PI3K, p-AKT, and AKT proteins were investigated by western blotting and the results showed a consistent effect of gigantol in the suppression of the c-Met/PI3K/AKT signal. Next, immunofluorescence showed a dramatic decrease in c-Met, PI3K and AKT activation in response to gigantol.

Conclusion: c-Met is an important target of gigantol treatment in lung cancer cells. Gigantol suppresses metastasis-related cell motility through decreasing c-Met resulting in PI3K/AKT signaling disruption.
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http://dx.doi.org/10.21873/cgp.20257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126329PMC
February 2021

Caspase-3, a shrimp phosphorylated hemocytic protein is necessary to control YHV infection.

Fish Shellfish Immunol 2021 Jul 14;114:36-48. Epub 2021 Apr 14.

Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Phutthamonthon 4 Rd, Salaya, Nakhon Pathom, 73170, Thailand. Electronic address:

By using immunohistochemistry detection, yellow head virus (YHV) was found to replicate in granule-containing hemocytes including semi-granular hemocytes (SGC) and granular hemocytes (GC) during the early phase (24 h post injection) of YHV-infected shrimp. Higher signal of YHV infection was found in GC more than in SGC. Comparative phosphoproteomic profiles between YHV-infected and non-infected GC reveal a number of phosphoproteins with different expression levels. The phosphoprotein spot with later on identified as caspase-3 in YHV-infected GC is most interesting. Blocking caspase-3 function using a specific inhibitor (Ac-DEVD-CMK) demonstrated high replication of YHV and consequently, high shrimp mortality. The immunohistochemistry results confirmed the high viral load in shrimp that caspase-3 activity was blocked. Caspase-3 is regulated through a variety of posttranslational modifications, including phosphorylation. Analysis of phosphorylation sites of shrimp caspase-3 revealed phosphorylation sites at serine residue. Taken together, caspase-3 is a hemocytic protein isolated from shrimp granular hemocytes with a role in anti-YHV response and regulated through the phosphorylation process.
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http://dx.doi.org/10.1016/j.fsi.2021.04.007DOI Listing
July 2021

Targeting AKT/mTOR and Bcl-2 for Autophagic and Apoptosis Cell Death in Lung Cancer: Novel Activity of a Polyphenol Compound.

Antioxidants (Basel) 2021 Mar 29;10(4). Epub 2021 Mar 29.

Cell-Based Drug and Health Products Development Research Unit, Chulalongkorn University, Bangkok 10330, Thailand.

Autophagic cell death (ACD) is an alternative death mechanism in resistant malignant cancer cells. In this study, we demonstrated how polyphenol stilbene compound PE5 exhibits potent ACD-promoting activity in lung cancer cells that may offer an opportunity for novel cancer treatment. Cell death caused by PE5 was found to be concomitant with dramatic autophagy induction, as indicated by acidic vesicle staining, autophagosome, and the LC3 conversion. We further confirmed that the main death induction caused by PE5 was via ACD, since the co-treatment with an autophagy inhibitor could reverse PE5-mediated cell death. Furthermore, the defined mechanism of action and upstream regulatory signals were identified using proteomic analysis. Time-dependent proteomic analysis showed that PE5 affected 2142 and 1996 proteins after 12 and 24 h of treatment, respectively. The crosstalk network comprising 128 proteins that control apoptosis and 25 proteins involved in autophagy was identified. Protein-protein interaction analysis further indicated that the induction of ACD was via AKT/mTOR and Bcl-2 suppression. Western blot analysis confirmed that the active forms of AKT, mTOR, and Bcl-2 were decreased in PE5-treated cells. Taken together, we demonstrated the novel mechanism of PE5 in shifting autophagy toward cell death induction by targeting AKT/mTOR and Bcl-2 suppression.
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http://dx.doi.org/10.3390/antiox10040534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066183PMC
March 2021

The Proteomics and Metabolomics Analysis for Screening the Molecular Targets of Action of β-Eudesmol in Cholangiocarcinoma.

Asian Pac J Cancer Prev 2021 Mar 1;22(3):909-918. Epub 2021 Mar 1.

Graduate Program in Bioclinical Sciences, Chulabhorn International College of Medicine, Thammasat University, Paholyothin Road, Klonglung, Pathumthani Thailand.

Objective: β-eudesmol is the active compound isolated from Atractylodes lancea (Thunb) D.C. The actions of this compound against cholangiocarcinoma (CCA) cells include anti-angiogenesis and anti-cell proliferation and growth. For more understanding of the molecular targets of action of β-eudesmol, the CCA cells (CL-6) were exposed to β-eudesmol for 24 and 48 hours.

Methods: Proteins and metabolites from the intra- and extra-cellular components of the CL-6 cells were extracted and identified by LC-MS/MS.  Protein analysis was performed using the Venn diagram (protein grouping), PANTHER (gene ontology), and STITCH software (protein-protein interaction). Metabolite analysis including their interactions with proteins, was performed using MetaboAnalyst software.

Results: The analysis showed that the actions of β-eudesmol were associated with various biological processes particularly apoptosis and cell cycle. These included blood coagulation, wound healing, DNA repair, PI3K-Akt signaling pathway, immune system process, MAPK cascade, urea cycle, purine metabolism, ammonia recycling, and methionine metabolism.

Conclusion: Possible molecular targets of action of β-eudesmol against CL-6 for cell apoptosis induction were TNFRSf6, cytochrome C, BAX3, DHCR24, CD29, and ATP.  On the other hand, possible targets for cell cycle arrest induction were CDKN2B, MLF1, TFDP2, CDK11-p110, and nicotinamide.
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http://dx.doi.org/10.31557/APJCP.2021.22.3.909DOI Listing
March 2021

A Novel Peptide Derived from Ginger Induces Apoptosis through the Modulation of p53, BAX, and BCL2 Expression in Leukemic Cell Lines.

Planta Med 2021 Jun 23;87(7):560-569. Epub 2021 Mar 23.

Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Nakhon Pathom, Thailand.

Despite the efficacy of chemotherapy, the adverse effects of chemotherapeutic drugs are considered a limitation of leukemia treatment. Therefore, a chemotherapy drug with minimal side effects is currently needed. One interesting molecule for this purpose is a bioactive peptide isolated from plants since it has less toxicity to normal cells. In this study, we extracted protein from the rhizome and performed purification to acquire the peptide fraction with the highest cytotoxicity using ultrafiltration, reverse-phase chromatography, and off-gel fractionation to get the peptide fraction that contained the highest cytotoxicity. Finally, a novel antileukemic peptide, P2 (sequence: RALGWSCL), was identified from the highest cytotoxicity fraction. The P2 peptide reduced the cell viability of NB4, MOLT4, and Raji cell lines without an effect on the normal peripheral blood mononuclear cells. The combination of P2 and daunorubicin significantly decreased leukemic cell viability when compared to treatment with either P2 or daunorubicin alone. In addition, leukemic cells treated with P2 demonstrated increased apoptosis and upregulation of caspase 3, 8, and 9 gene expression. Moreover, we also examined the effects of P2 on p53, which is the key regulator of apoptosis. Our results showed that treatment of leukemic cells with P2 led to the upregulation of p53 and Bcl-2-associated X protein, and the downregulation of B-cell lymphoma 2, indicating that p53 is involved in apoptosis induction by P2. The results of this study are anticipated to be useful for the development of P2 as an alternative drug for the treatment of leukemia.
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http://dx.doi.org/10.1055/a-1408-5629DOI Listing
June 2021

Proteomic analysis of drug-susceptible and multidrug-resistant nonreplicating Beijing strains of cultured .

Biochem Biophys Rep 2021 Jul 9;26:100960. Epub 2021 Mar 9.

Department of Microbiology and Research and Diagnostic Center for Emerging Infectious Diseases (RCEID), Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.

The existence of latent tuberculosis infection (LTBI) is one of the main obstacles hindering eradication of tuberculosis (TB). To better understand molecular mechanisms and explore biomarkers for the pathogen during LTBI, we cultured strains of () under stress conditions, mimicking those in the host granuloma intracellular environment, to induce entry into the non-replicating persistence stage. The stresses included hypoxia, low pH (5.0), iron deprivation (100 μM of 2, 2'-dipyridyl) and nutrient starvation (10% M7H9 medium). Three Mtb strains were studied: two clinical isolates (drug-susceptible Beijing (BJ) and multidrug-resistant Beijing (MDR-BJ) strains) and the reference laboratory strain, H37Rv. We investigated the proteomics profiles of these strains cultured in stressful conditions and then validated the findings by transcriptional analysis. NarJ (respiratory nitrate reductase delta chain) was significantly up-regulated at the protein level and the mRNA level in all three Mtb strains. The narJ gene is a member of the narGHJI operon encoding all nitrate reductase subunits, which play a role in nitrate metabolism during the adaptation of to stressful intracellular environments and the subsequent establishment of latent TB. The identification of up-regulated mRNAs and proteins of under stress conditions could assist development of biomarkers, drug targets and vaccine antigens.
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http://dx.doi.org/10.1016/j.bbrep.2021.100960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960788PMC
July 2021

Salivary Proteomic Analysis of Canine Oral Melanoma by MALDI-TOF Mass Spectrometry and LC-Mass Spectrometry/Mass Spectrometry.

Methods Mol Biol 2021 ;2265:429-445

Biochemistry Unit, Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.

Canine oral melanoma (COM) is a common oral cancer with aggressiveness and high metastasis. A tumor in a dog's mouth makes it difficult to be observed at the early-clinical stage. Salivary biomarkers may be useful for early detection, prognosis, and monitoring of therapies. In addition, salivary collection is a simple and non-invasive technique. The present study describes how to identify salivary biomarkers in COM using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) approaches. Western blot analysis has been used to confirm the protein expression. The sequence of expressed protein from western blot has been verified by LC-MS/MS.
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http://dx.doi.org/10.1007/978-1-0716-1205-7_31DOI Listing
April 2021

Black rice cultivar from Java Island of Indonesia revealed genomic, proteomic, and anthocyanin nutritional value.

Acta Biochim Pol 2021 Mar;68(1):55-63

1Research Center of Smart Molecule of Natural Genetics Resources, Brawijaya University, Indonesia; 2Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Indonesia.

Black rice is considered to be functional food containing anthocyanins as bioactive compounds. This study examined the genomic and proteomic patterns in local black rice from Java Island, Indonesia, with attention to the mechanism of anthocyanin synthesis. Three kinds of black rice from Java Island, including black rice from East Java (BREJ), black rice from Central Java (BRCJ), and black rice from West Java (BRWJ), were studied in comparison to white rice (WREJ) and red rice (RREJ). Genomic profiling was done by simple sequence repeat (SSR) analysis, and sequencing of red coleoptile (Rc) and glycosyltransferase (GT) genes, followed by in silico analysis. Total anthocyanin was investigated by ultra-high performance liquid chromatography- diode array detector (UHPLC-DAD). The proteomic profiles were determined by liquid-chromatography and mass spectrometry of tryptic peptides. The SSR profiles showed a specific band in each black rice variant. The Rc gene exon-2 sequences were similar in the three black rice cultivars. The GT gene sequence was identified as a new variant that correlates with the purple stem, leaf, bran, and whole grain morphology seen exclusively in the BRWJ cultivar. The anthocyanin composition in Java black rice is diverse. The highest cyanidin level was seen in BRWJ and the highest level of peonidin-3-O-glucoside in BREJ. Proteomic profiling of the black rice cultivars demonstrated that the expression of proteins that might be related to the levels of anthocyanin synthesis varied. These studies conclude that the genomic, proteomic and anthocyanins composition of Java black rice cultivars may be used the improvement of their functional nutrition values.
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http://dx.doi.org/10.18388/abp.2020_5386DOI Listing
March 2021

Enhanced contextual fear memory in peroxiredoxin 6 knockout mice is associated with hyperactivation of MAPK signaling pathway.

Mol Brain 2021 02 25;14(1):42. Epub 2021 Feb 25.

Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan.

Fear dysregulation is one of the symptoms found in post-traumatic stress disorder (PTSD) patients. The functional abnormality of the hippocampus is known to be implicated in the development of such pathology. Peroxiredoxin 6 (PRDX6) belongs to the peroxiredoxin family. This antioxidant enzyme is expressed throughout the brain, including the hippocampus. Recent evidence reveals that PRDX6 plays an important role in redox regulation and the modulation of several signaling molecules involved in fear regulation. Thus, we hypothesized that PRDX6 plays a role in the regulation of fear memory. We subjected a systemic Prdx6 knockout (Prdx6) mice to trace fear conditioning and observed enhanced fear response after training. Intraventricular injection of lentivirus-carried mouse Prdx6 into the 3rd ventricle reduced the enhanced fear response in these knockout mice. Proteomic analysis followed by validation of western blot analysis revealed that several proteins in the MAPK pathway, such as NTRK2, AKT, and phospho-ERK1/2, cPLA2 were significantly upregulated in the hippocampus of Prdx6 mice during the retrieval stage of contextual fear memory. The distribution of PRDX6 found in the astrocytes was also observed throughout the hippocampus. This study identifies PRDX6 as a participant in the regulation of fear response. It suggests that PRDX6 and related molecules may have important implications for understanding fear-dysregulation associated disorders like PTSD.
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http://dx.doi.org/10.1186/s13041-021-00754-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908735PMC
February 2021

Chemosensitizing activity of peptide from Lentinus squarrosulus (Mont.) on cisplatin-induced apoptosis in human lung cancer cells.

Sci Rep 2021 Feb 18;11(1):4060. Epub 2021 Feb 18.

Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand.

The limitations of cisplatin, a standard chemotherapy for lung cancer, have been documented with serious adverse effects and drug resistance. To address the need for novel therapy, this study firstly reveals the potential of peptide from Lentinus squarrosulus (Mont.) as a chemotherapeutic adjuvant for cisplatin treatment. The purified peptide from L. squarrosulus aqueous extracts was obtained after eluting with 0.4 M NaCl through FPLC equipped with anion exchange column. Preincubation for 24 h with 5 µg/mL of the peptide at prior to treatment with 5 µM cisplatin significantly diminished %cell viability in various human lung cancer cells but not in human dermal papilla and proximal renal cells. Flow cytometry indicated the augmentation of cisplatin-induced apoptosis in lung cancer cells pretreated with peptide from L. squarrosulus. Preculture with the peptide dramatically inhibited colony formation in lung cancer cells derived after cisplatin treatment. Strong suppression on integrin-mediated survival was evidenced with the diminution of integrins (β1, β3, β5, α5, αV) and down-stream signals (p-FAK/FAK, p-Src/Src, p-Akt/Akt) consequence with alteration of p53, Bax, Blc-2 and Mcl-1 in cisplatin-treated lung cancer cells preincubated with peptide from L. squarrosulus. These results support the development of L. squarrosulus peptide as a novel combined chemotherapy with cisplatin for lung cancer treatment.
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http://dx.doi.org/10.1038/s41598-021-83606-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892851PMC
February 2021

A novel peptide isolated from garlic shows anticancer effect against leukemic cell lines via interaction with Bcl-2 family proteins.

Chem Biol Drug Des 2021 May 1;97(5):1017-1028. Epub 2021 Mar 1.

Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Nakhon pathom, Thailand.

Leukemia is a group of cancer caused by the abnormal proliferation and differentiation of hematopoietic stem cells. Efforts geared toward effective therapeutic strategies with minimal side effects are underway. Peptides derived from natural resources have recently gained special attention as alternative chemotherapeutic agents due to their minimal adverse effects. In the present study, the aim was to isolate peptides from garlic (Allium sativum) and investigate their anticancer activity against leukemic cell lines. The protein extract of A. sativum was pepsin-digested to obtain protein hydrolysate followed by sequential purification methods. A novel anticancer peptide, VKLRSLLCS (VS-9), was identified and characterized by mass spectrometric analysis. The peptide was demonstrated to significantly inhibit the cell proliferation of MOLT-4 and K562 leukemic cell lines while exhibiting minimal inhibition against normal PBMC. Particularly, VS-9 could induce apoptosis and upregulate mRNA levels of caspase 3, caspase 8, caspase 9, and Bax while downregulating Bcl-2, Bcl-xL, and Bcl-w. Molecular docking of VS-9 with the anti-apoptotic Bcl-2 protein family suggested that VS-9 could bind the binding groove of the BH3 domain on target proteins. Protein-peptide interaction analysis by affinity chromatography and LC-MS/MS further showed that VS-9 could bind Bcl-2 proteins. Results suggest VS-9 as a potential garlic-derived novel anticancer peptide possessing apoptosis-inducing properties against leukemic cell lines via anti-apoptotic Bcl-2 protein family.
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http://dx.doi.org/10.1111/cbdd.13831DOI Listing
May 2021

Comparative secretome analysis between salinity-tolerant and control Chlamydomonas reinhardtii strains.

Planta 2021 Feb 16;253(3):68. Epub 2021 Feb 16.

Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Ngamwongwan Rd., Bangkok, 10900, Thailand.

Main Conclusion: Secretome analysis of a salt-tolerant and control Chlamydomonas reinhardtii revealed 514 differentially expressed proteins. Membrane transport and trafficking, signal transduction and channel proteins were up-regulated in the ST secretome. Salinity is a major abiotic stress that limits crop production worldwide. Multiple adverse effects have been reported in many living organisms exposed to high-saline concentrations. Chlamydomonas reinhardtii is known for secreting proteins in response to many environmental stresses. A salinity-tolerant (ST) strain of Chlamydomonas has been developed, whose cells were able to grow at 300 mM NaCl. The current study analyzed the secretomes of ST grown in TAP medium supplemented with 300 mM NaCl and the laboratory strain CC-503 grown in TAP medium without NaCl supplement. In total, 514 secreted proteins were identified of which 203 were up-regulated and 110 were down-regulated. Bioinformatic analysis predicted 168 proteins to be secreted or in the conventional secretory pathway. Out of these, 70 were up-regulated, while 51 proteins were down-regulated. Proteins involved in membrane transport and trafficking, signal transduction and channel proteins were altered in their expression in the ST secretome, suggesting the response of saline stress acts toward not only the intracellular pool of proteins but also the extracellular proteins. This also suggested that the secreted proteins might have roles in the extracellular space. Signal peptide (SP) prediction revealed that almost 40% of the predicted secreted proteins contained a signal peptide; however, a high proportion of proteins lacked an SP, suggesting that these proteins might be secreted through an unconventional protein secretion pathway.
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http://dx.doi.org/10.1007/s00425-021-03583-7DOI Listing
February 2021

Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells.

Biochem Biophys Rep 2021 Mar 12;25:100903. Epub 2021 Jan 12.

Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand.

Background: Previous studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown.

Methods: We generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics.

Results: PC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP.

Conclusions: Suppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells.

General Significance: Our results highlight the possibility of the use of PC as an anti-cancer drug target.
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http://dx.doi.org/10.1016/j.bbrep.2020.100903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806519PMC
March 2021

Identification of proteins responsive to heterologous protein production in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656.

Yeast 2021 May 14;38(5):316-325. Epub 2021 Jan 14.

Microbial Cell Factory Research Team, Biorefinery and Bioproduct Technology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand.

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label-free comparative proteomic approach. Four hundred sixty-four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%-20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%-25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.
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http://dx.doi.org/10.1002/yea.3548DOI Listing
May 2021

Antifungal Activity and Molecular Mechanisms of Partial Purified Antifungal Proteins from against .

J Fungi (Basel) 2020 Dec 3;6(4). Epub 2020 Dec 3.

Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.

Antifungal proteins (AFPs) are able to inhibit a wide spectrum of fungi without significant toxicity to the hosts. This study examined the antifungal activity of AFPs isolated from a Thai medicinal plant, against the human pathogenic fungus . This dimorphic fungus causes systemic infections in immunocompromised individuals and is endemic in Southeast Asian countries. The crude protein extract inhibited the growth of . The anti- activity was completely lost when treated with proteinase K and pepsin, indicating that the antifungal activity was dependent on a protein component. The total protein extract from was partially purified by size fractionation to ≤10, 10-30, and ≥30 kDa fractions and tested for the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). All fractions showed anti- activity with the MIC and MFC values of 32 to 128 μg/mL and >128 μg/mL, respectively. In order to determine the mechanism of inhibition, all fractions were tested with mutant strains affected in G-protein signaling and cell wall integrity pathways. The anti- activity of the 10-30 kDa fraction was abrogated by deletion of and , the genes encoding alpha subunits of heterotrimeric G-proteins, indicating that the inhibitory effect is related to intracellular signaling through G-proteins. The work demonstrates that antifungal proteins isolated from represent sources for novel drug development.
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http://dx.doi.org/10.3390/jof6040333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761713PMC
December 2020

Characterization of a novel yeast phase-specific antigen expressed during in vitro thermal phase transition of Talaromyces marneffei.

Sci Rep 2020 12 3;10(1):21169. Epub 2020 Dec 3.

Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand.

Talaromyces marneffei is a dimorphic fungus that has emerged as an opportunistic pathogen particularly in individuals with HIV/AIDS. Since its dimorphism has been associated with its virulence, the transition from mold to yeast-like cells might be important for fungal pathogenesis, including its survival inside of phagocytic host cells. We investigated the expression of yeast antigen of T. marneffei using a yeast-specific monoclonal antibody (MAb) 4D1 during phase transition. We found that MAb 4D1 recognizes and binds to antigenic epitopes on the surface of yeast cells. Antibody to antigenic determinant binding was associated with time of exposure, mold to yeast conversion, and mammalian temperature. We also demonstrated that MAb 4D1 binds to and recognizes conidia to yeast cells' transition inside of a human monocyte-like THP-1 cells line. Our studies are important because we demonstrated that MAb 4D1 can be used as a tool to study T. marneffei virulence, furthering the understanding of the therapeutic potential of passive immunity in this fungal pathogenesis.
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http://dx.doi.org/10.1038/s41598-020-78178-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713699PMC
December 2020

Proteomic Examination for Gluconeogenesis Pathway-Shift during Polyhydroxyalkanoate Formation in Grown on Glycerol.

Bioengineering (Basel) 2020 Dec 1;7(4). Epub 2020 Dec 1.

Proteomics Research Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Khlong Luang, Pathumthani 12120, Thailand.

Because of availability and inexpensive, glycerol can be considered as a suitable raw material for polyhydroxyalkanoate (PHA) production with bacterial fermentation. Nevertheless, compared to the production of glucose as a raw precursor, PHA produced from glycerol by was found to produce lower PHA with low bacterial growth rates. According to our study, was able to synthesize glucose-like intermediates from glycerol via gluconeogenesis. This resulted in a decrease of the cell dry weight and the yield of PHA polymers, especially in the active cell growth phase. It was indicated that glycerol used as a carbon source of the PHA synthesis pathway has glucogenesis-shift, which causes a decrease of the PHA content and productivity. In this research, we investigated the proteins that were closely expressed with the increase of the intracellular PHA and glucose content. For solving the above problem, the proteins inside the bacterial cells were analyzed and compared to the database proteins via mass spectrometry. The proteins were isolated by 1-D SDS-polyacrylamide gel electrophoresis (PAGE) technique and identified by the liquid chromatography mass spectrometry (LC-MS) technique. By using bioinformatics validation, a total number of 1361 proteins were examined and found in the culture bacterial cells. Selective protein expression was correlated with the amount of PHA at each cultivation time and generating glucose by studying the 1361 proteins was elucidated in proteomic information. The results of the cluster of proteins were found to contain 93 proteins using the multiple array viewer (MEV) program with the KMS data analysis model. Protein species with the same expression pattern for PHA and six proteins with similar expression patterns were found to be correlated with generating glucose content. The associations of the two protein groups were then determined through a Stitch program. The protein and chemical associations were analyzed both directly and indirectly through different databases. The proteins of interest were found with research data linked between glycerol and glucose. Five protein types are connecting to glucose and glycerol shift pathway, two of which are glycosyl hydrolase (H16_B1563) and short-chain dehydrogenase (H16_B0687), both of which are enzymes used to break the bonds of complex sugars, possibly related to the partial conversion of glycerol to glucose. The two proteins found in the strains used in the H16 experiment give rise to the break down the bonds of α,α-1,1-glucoside of malto-oligosyltrehalose and short-chain sugar molecules such as mannitol (CHO), respectively. In this research, finding the associated expression proteins which is involved in changing the pathway of gluconeogenesis shift to PHA synthesis will be useful information on genetically modifying microorganisms to produce PHA more efficiently, leading to reduction of the production costs.
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http://dx.doi.org/10.3390/bioengineering7040154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712004PMC
December 2020

Autophagy and apoptosis induction by sesamin in MOLT-4 and NB4 leukemia cells.

Oncol Lett 2021 Jan 12;21(1):32. Epub 2020 Nov 12.

Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Nakhon Pathom 73170, Thailand.

Sesamin, the major furofuran lignan found in the seeds of L., has been investigated for its various medicinal properties. In the present study, the anti-leukemic effects of sesamin and its underlying mechanisms were investigated in MOLT-4 and NB4 acute leukemic cells. Leukemic cells were treated with various concentrations of sesamin. Cell viability was determined using an MTT assay. Flow cytometry using Annexin V-FITC/PI staining and anti-LC3/FITC antibodies was applied to detect the level of apoptosis and autophagy, respectively. Reverse transcription-quantitative PCR was performed to examine the alterations in the mRNA expression of apoptotic and autophagic genes. In addition, bioinformatics tools were used to predict the possible interactions between sesamin and its targets. The results revealed that sesamin inhibited MOLT-4 and NB4 cell proliferation in a dose-dependent manner. In addition, sesamin induced both apoptosis and autophagy. In sesamin-treated cells, the gene expression levels of caspase 3 and unc-51 like autophagy activating kinase 1 (ULK1) were upregulated, while those of mTOR were downregulated compared with in the control. Notably, the protein-chemical interaction network indicated that caspase 3, mTOR and ULK1 were the essential factors involved in the effects of sesamin treatment, as with anticancer agents, such as rapamycin, AZD8055, Torin1 and 2. Overall, the findings of the present study suggested that sesamin inhibited MOLT-4 and NB4 cell proliferation, and induced apoptosis and autophagy through the regulation of caspase 3 and mTOR/ULK1 signaling, respectively.
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http://dx.doi.org/10.3892/ol.2020.12293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693381PMC
January 2021

A shotgun proteomic approach reveals novel potential salivary protein biomarkers for asthma.

J Asthma 2020 Dec 7:1-13. Epub 2020 Dec 7.

Department of Oral Biology, Faculty of Dentistry, Thammasat University (Rangsit Campus), Klong Luang, Prathumthani, Thailand.

Objective: The aim of this study was to determine if there is an association between the salivary protein profile and disease control in asthma.

Methods: Thirty asthmatic patients (17 adults and 13 children) participated in this study. Saliva samples were collected from healthy subjects, controlled and uncontrolled asthmatics. Individual samples from each group were combined to form a pooled sample, from which proteomic analysis was performed using gel-based quantitative proteomics.

Results: Fourteen out of thirty asthmatics were classified to be controlled asthma. Most of asthmatics received inhaled corticosteroids as the controller medications. SDS-PAGE showed predominant bands at high molecular weight in asthmatic saliva compared to that of the controls. Shotgun proteomic analyses indicated that 193 salivary proteins were expressed in both controlled and uncontrolled asthmatics. They were predicted to associate with proteins involved in pathogenesis of asthma including IL-5, IL-6, MCP-1, VEGF, and periostin and asthma medicines (Cromolyn, Nedocromil, and Theophylline). Nucleoside diphosphate kinase (NME1-NME2) only expressed in controlled asthmatics whereas polycystic kidney and hepatic disease 1 (PKHD1)/fibrocystin, zinc finger protein 263 (ZNF263), uncharacterized LOC101060047 (ENSG00000268865), desmoglein 2 (DSG2) and S100 calcium binding protein A2 (S100A2) were only found in uncontrolled asthma. Therefore, the six proteins were associated with disease control in children and adults with asthma.

Conclusion: Our findings suggest that NME1-NME2, PKHD1, ZNF 263, uncharacterized LOC101060047, DSG 2 and S100 A2 in saliva are associated with disease control in asthma.
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http://dx.doi.org/10.1080/02770903.2020.1850773DOI Listing
December 2020

Metaproteomic investigation of functional insight into special defined microbial starter on production of fermented rice with melanogenesis inhibition activity.

PLoS One 2020 4;15(11):e0241819. Epub 2020 Nov 4.

Department of Microbiology, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, Thailand.

Fermentation of rice grains requires diverse metabolic enzymes to be synchronously synthesized by the microbial community. Although many studies have used a metaproteomic approach to investigate the roles of microorganisms in improving the flavor of fermented foods, their roles in producing compounds with biological activity have not yet been reported. In a previous study the ferment obtained from unpolished black rice (UBR) fermented with a defined microbial starter (De-E11), comprised of Rhizopus oryzae, Saccharomycopsis fibuligera, Saccharomyces cerevisiae, and Pediococcus pentosaceus, (fermented UBR; FUBR) showed a strong melanogenesis inhibition activity in B16F10 melanoma cells. Hence, in this study, the roles of these microorganisms in producing the melanogenesis inhibitor(s) in FUBR was investigated using a metaproteomic approach. The melanogenesis inhibition activity of the FUBR liquid (FR-Liq) was found to increase with longer fermentation times. R. oryzae and S. cerevisiae were the major hosts of proteins related to the biosynthesis of melanogenesis inhibitor(s) in the FUBR. During fermentation, the enzymes involved in the degradation of UBR and in the carbohydrate metabolic process were identified. These enzymes were associated with the process of releasing of bioactive compound(s) from UBR and the synthesis of organic acids from the microorganisms, respectively. In addition, enzymes involved in the synthesis of some known melanogenesis inhibitor(s) and in the degradation of the melanogenesis stimulator (arsenate) were detected. Varying the combination of microorganisms in the De-E11 starter to produce the FR-Liq revealed that all four microorganisms were required to produce the most potent melanogenesis inhibition activity. Taken together with the metaproteomics results, this suggested that the microorganisms in De-E11 synchronously synthesize the FR-Liq with melanogenesis inhibition activity. In conclusion, this information on the metaproteome in FUBR will increase our understanding of the microbial metabolic modes and could lead to knowledge-based improvements in the fermented rice process to produce melanogenesis inhibitor(s).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241819PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641363PMC
December 2020

Andrographolide and Its 14-Aryloxy Analogues Inhibit Zika and Dengue Virus Infection.

Molecules 2020 Oct 30;25(21). Epub 2020 Oct 30.

School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 211816, China.

Andrographolide is a labdene diterpenoid with potential applications against a number of viruses, including the mosquito-transmitted dengue virus (DENV). In this study, we evaluated the anti-viral activity of three 14-aryloxy analogues (ZAD-1 to ZAD-3) of andrographolide against Zika virus (ZIKV) and DENV. Interestingly, one analogue, ZAD-1, showed better activity against both ZIKV and DENV than the parental andrographolide. A two-dimension (2D) proteomic analysis of human A549 cells treated with ZAD-1 compared to cells treated with andrographolide identified four differentially expressed proteins (heat shock 70 kDa protein 1 (HSPA1A), phosphoglycerate kinase 1 (PGK1), transketolase (TKT) and GTP-binding nuclear protein Ran (Ran)). Western blot analysis confirmed that ZAD-1 treatment downregulated expression of HSPA1A and upregulated expression of PGK1 as compared to andrographolide treatment. These results suggest that 14-aryloxy analogues of andrographolide have the potential for further development as anti-DENV and anti-ZIKV agents.
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http://dx.doi.org/10.3390/molecules25215037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662321PMC
October 2020

Characterization of a Novel Peptide from Pathogenic and Its Cytotoxic Effect.

Pathogens 2020 Oct 30;9(11). Epub 2020 Oct 30.

Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.

Leptospirosis is a zoonotic infectious disease caused by pathogenic species. Virulence proteins have been shown to be key determinants of the pathogenesis of pathogenic . A specific peptide at a mass-to-charge ratio of 7000 Da was identified in whole cells using matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. This peptide was specifically present in pathogenic and in clinical isolates. We report here the characterization of this specific peptide using a proteomics approach. This peptide was significantly matched to a hypothetical conserved protein (LA2458) with a calculated molecular weight of 7140.136 Da containing a tellurite-resistance domain at its C terminus (TerB-C). The amino acid sequences revealed the presence of hydrophobic transmembrane portions and two linear B-cell epitopes. Despite its low abundance, this synthetic peptide demonstrated dose-dependent cytotoxicity toward African green monkey kidney (Vero) cells via the apoptosis pathway. The concentration of the peptide 100 µM induced about 50% of cell death after a 24 h exposure. This peptide could be useful for the diagnosis of leptospirosis and the study of pathogenesis.
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http://dx.doi.org/10.3390/pathogens9110906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692646PMC
October 2020

High glucose-ROS conditions enhance the progression in cholangiocarcinoma via upregulation of MAN2A2 and CHD8.

Cancer Sci 2021 Jan 29;112(1):254-264. Epub 2020 Nov 29.

Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand.

Diabetes is a major risk factor in the development and progression of several cancers including cholangiocarcinoma (CCA). However, the molecular mechanism by which hyperglycemia potentiates progression of CCA is not clearly understood. Here, we showed that a high glucose condition significantly increased reactive oxygen species (ROS) production and promoted aggressive phenotypes of CCA cells, including proliferation and migration activities. Mannosidase alpha class 2a member 2 (MAN2A2), was upregulated at both mRNA and protein levels in a high glucose- and ROS-dependent manner. In addition, cell proliferation and migration were significantly reduced by MAN2A2 knockdown. Based on our proteome and in silico analyses, we further found that chromodomain helicase DNA-binding protein 8 (CHD8) was induced by ROS signaling and regulated MAN2A2 expression. Overexpression of CHD8 increased MAN2A2 expression, while CHD8 knockdown dramatically reduced proliferation and migration as well as MAN2A2 expression in CCA cells. Moreover, both MAN2A2 and CHD8 were highly expressed with positive correlation in CCA tumor tissues. Collectively, these data suggested that high glucose conditions promote CCA progression through ROS-mediated upregulation of MAN2A2 and CHD8. Thus, glucose metabolism is a promising therapeutic target to control tumor progression in patients with CCA and diabetes.
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http://dx.doi.org/10.1111/cas.14719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780024PMC
January 2021

Gigantol Targets MYC for Ubiquitin-proteasomal Degradation and Suppresses Lung Cancer Cell Growth.

Cancer Genomics Proteomics 2020 Nov-Dec;17(6):781-793

Cell-Based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand

Background: Gigantol is a pharmacologically active bibenzyl compound exerting potential anticancer activities. At non-toxic concentrations, it reduces cancer stem cell properties and tumorigenicity. The mechanisms of the effects of gigantol on cancer cell growth are largely unknown. This study aimed to unravel the molecular profile and identify the prominent molecular mechanism of the effects of gigantol in controlling lung cancer cell proliferation.

Materials And Methods: Proteomics and bioinformatics analysis were used accompanied by experimental molecular pharmacology approaches.

Results: Gigantol exhibited antiproliferative effects on human lung cancer cells confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide proliferation assay and colony growth assay. The protein profile in response to gigantol treatment associated with regulation of cell proliferation was analyzed to determine the prominent protein targets. Among the significant hub proteins, MYC, an important proto-oncogene and proliferation-promoting transcription factor, was down-regulated with the highest number of protein-protein interactions. MYC down-regulation was confirmed by western blot analysis. The up-stream regulator of MYC, Glycogen synthase kinase 3 beta (GSK3β) was found to be responsible for MYC destabilization mediated by gigantol. Gigantol facilitated GSK3β function and resulted in the increase of MYC-ubiquitin complex as evaluated by immunoprecipitation.

Conclusion: Gigantol was found to inhibit lung cancer proliferation through induction of GSK3β-mediated MYC ubiquitin-proteasome degradation. These data suggest gigantol to be a promising candidate for novel strategy in inhibition of lung cancer.
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http://dx.doi.org/10.21873/cgp.20232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675646PMC
August 2020