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<p>ELISAs were evaluated for IgM and IgG antibodies using sera from melioidosis patients (N... more <p>ELISAs were evaluated for IgM and IgG antibodies using sera from melioidosis patients (N = 45) and healthy donors (N = 45) on the pre-coated plate with 15 μg/ml of rFliC. Box plots show OD at 450 nm of rFliC-specific IgG (A) and IgM (B) in different groups of subjects. All data in box plots are presented as 25<sup>th</sup> and 75<sup>th</sup> percentile boundaries in the box with the median line within the box; the whiskers indicate the 10<sup>th</sup> and 90<sup>th</sup> percentiles. (C) Receiver Operating Characteristics (ROC) plots.</p
BioMed research international, 2018
Increased expression levels of both mitochondrial citrate transporter (CTP) and plasma membrane c... more Increased expression levels of both mitochondrial citrate transporter (CTP) and plasma membrane citrate transporter (PMCT) proteins have been found in various cancers. The transported citrates by these two transporter proteins provide acetyl-CoA precursors for the de novo lipogenesis (DNL) pathway to support a high rate of cancer cell viability and development. Inhibition of the DNL pathway promotes cancer cell apoptosis without apparent cytotoxic to normal cells, leading to the representation of selective and powerful targets for cancer therapy. The present study demonstrates that treatments with CTP inhibitor (CTPi), PMCT inhibitor (PMCTi), and the combination of CTPi and PMCTi resulted in decreased cell viability in two hepatocellular carcinoma cell lines (HepG2 and HuH-7). Treatment with citrate transporter inhibitors caused a greater cytotoxic effect in HepG2 cells than in HuH-7 cells. A lower concentration of combined CTPi and PMCTi promotes cytotoxic effect compared with eith...
Journal of clinical microbiology, Jan 30, 2018
Melioidosis is a fatal infectious disease caused by the environmental bacterium It is highly ende... more Melioidosis is a fatal infectious disease caused by the environmental bacterium It is highly endemic in Asia and northern Australia but neglected in many other tropical countries. Melioidosis patients have a wide range of clinical manifestations and definitive diagnosis requires bacterial culture which can be time-consuming. A reliable rapid serological tool is greatly needed for disease surveillance and diagnosis. We previously demonstrated by ELISA that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we have developed a rapid immunochromatography test (ICT) using Hcp1 as the target antigen (Hcp1-ICT). We evaluated this test for specific antibody detection using serum samples obtained from 4 groups of human subjects includingi 487 culture-confirmed melioidosis patients from four hospitals in northeast Thailand; ii 202 healthy donors from northeast Thailand; iii 90 U.S. healthy donors and iv 207 patients infected with oth...
PloS one, 2018
Burkholderia pseudomallei is a flagellated Gram-negative bacterium which is the causative agent o... more Burkholderia pseudomallei is a flagellated Gram-negative bacterium which is the causative agent of melioidosis. The disease poses a major public health problem in tropical regions and diabetes is a major risk factor. The high mortality rate of melioidosis is associated with severe sepsis which involves the overwhelming production of pro-inflammatory cytokines. Bacterial flagellar protein (flagellin) activates Toll-like receptor 5 (TLR5)-mediated innate immune signaling pathways and induces adaptive immune response. However, previous studies of TLR5 signaling in melioidosis have been performed using recombinant flagellin from Salmonella Typhimurium instead of B. pseudomallei. This study aimed to investigate human innate immune response and antibody response against a recombinant B. pseudomallei flagellin (rFliC). We prepared B. pseudomallei rFliC and used it to stimulate HEK-BlueTM-hTLR5 and THP1-DualTM cells to assess TLR5 activation. Subsequently, whole blood stimulation assays wit...
The American journal of tropical medicine and hygiene, Jan 19, 2018
Burkholderia pseudomallei, the etiologic agent of melioidosis, is an important but under-recogniz... more Burkholderia pseudomallei, the etiologic agent of melioidosis, is an important but under-recognized cause of disease in the tropics. Although first described over a century ago as a septicemic illness associated with morphine addicts in Rangoon, Burma, there is little information regarding the incidence of melioidosis in present-day Myanmar. To address this issue, we used two recently developed and validated serological assays to detect B. pseudomallei-specific antibodies in 124 serum samples obtained from febrile patients in the delta region of Myanmar. Using cutoff values derived from culture-confirmed melioidosis cases in neighboring Thailand, 3.2% of the samples exhibited reactivity profiles consistent with active B. pseudomallei infections. Collectively, these findings indicate that melioidosis likely represents a significant cause of morbidity and mortality in Myanmar and support the need for further studies to assess the true burden of disease in this country.
PLOS Neglected Tropical Diseases, 2017
Background Melioidosis is a severe disease caused by Burkholderia pseudomallei. Clinical manifest... more Background Melioidosis is a severe disease caused by Burkholderia pseudomallei. Clinical manifestations are diverse and acute infections require immediate treatment with effective antibiotics. While culture is the current diagnostic standard, it is time-consuming and has low sensitivity. In endemic areas, inaccessibility to biosafety level 3 facilities and a lack of good serodiagnostic tools can impede diagnosis and disease surveillance. Recent studies have suggested that O-polysaccharide (OPS) and hemolysin co-regulated protein 1 (Hcp1) are promising target antigens for serodiagnosis of melioidosis. Methodology/Principle findings We evaluated rapid ELISAs using crude antigens, purified OPS and Hcp1 to measure antibody levels in three sets of sera: (i) 419 serum samples from melioidosis patients, Thai and U.S. healthy donors, (ii) 120 serum samples from patients with other bacterial infections, and (iii) 423 serum samples from 200 melioidosis patients obtained upon admission and at 12 and 52 weeks post-recovery. We observed significantly higher antibody levels using the crude antigen prepared from wild type B. pseudomallei K96243 compared to that of an OPS-mutant. The areas under receiver operator characteristics (AUROCCs) for diagnosis were compared for individual Hcp1-ELISA or OPS-ELISA or combined Hcp1/OPS-ELISA. For Thai donors, AUROCCs were highest and comparable between the Hcp1-ELISA and the combined Hcp1/OPS-ELISA (0.95 versus 0.94). For U.S. donors, the AUROCC was highest for the combined Hcp1/OPS-ELISA (0.96). Significantly higher seropositivity was observed in diabetic patients compared to those without diabetes for both the Hcp1-ELISA (87.3% versus 69.7%) and OPS-ELISA (88.1% versus 60.6%). Although antibody levels for Hcp1 were highest upon admission, the titers declined by week 52 post-recovery.
FEBS Open Bio, May 14, 2018
Suppression of the expression or activities of enzymes that are involved in the synthesis of de n... more Suppression of the expression or activities of enzymes that are involved in the synthesis of de novo lipogenesis (DNL) in cancer cells triggers cell death via apoptosis. The plasma membrane citrate transporter (PMCT) is the initial step that translocates citrate from blood circulation into the cytoplasm for de novo long‐chain fatty acids synthesis. This study investigated the antitumor effect of the PMCT inhibitor (PMCTi) in inducing apoptosis by inhibiting the DNL pathway in HepG2 cells. The present findings showed that PMCTi reduced cell viability and enhanced apoptosis through decreased intracellular citrate levels, which consequently caused inhibition of fatty acid and triacylglycerol productions. Thus, as a result of the reduction in fatty acid synthesis, the activity of carnitine palmitoyl transferase‐1 (CPT‐1) was suppressed. Decreased CPT‐1 activity also facilitated the disruption of mitochondrial membrane potential (ΔΨm) leading to stimulation of apoptosis. Surprisingly, primary human hepatocytes were not affected by PMCTi. Increased caspase‐8 activity as a consequence of reduction in fatty acid synthesis was also found to cause disruption of ΔΨm. In addition, apoptosis induction by PMCTi was associated with an enhanced reactive oxygen species generation. Taken together, we suggest that inhibition of the DNL pathway following reduction in citrate levels is an important regulator of apoptosis in HepG2 cells via suppression of CPT‐1 activity. Thus, targeting the DNL pathway mediating CPT‐1 activity by PMCTi may be a selective potential anticancer therapy.
<p>ELISAs were evaluated for IgM and IgG antibodies using sera from melioidosis patients (N... more <p>ELISAs were evaluated for IgM and IgG antibodies using sera from melioidosis patients (N = 45) and healthy donors (N = 45) on the pre-coated plate with 15 μg/ml of rFliC. Box plots show OD at 450 nm of rFliC-specific IgG (A) and IgM (B) in different groups of subjects. All data in box plots are presented as 25<sup>th</sup> and 75<sup>th</sup> percentile boundaries in the box with the median line within the box; the whiskers indicate the 10<sup>th</sup> and 90<sup>th</sup> percentiles. (C) Receiver Operating Characteristics (ROC) plots.</p
BioMed research international, 2018
Increased expression levels of both mitochondrial citrate transporter (CTP) and plasma membrane c... more Increased expression levels of both mitochondrial citrate transporter (CTP) and plasma membrane citrate transporter (PMCT) proteins have been found in various cancers. The transported citrates by these two transporter proteins provide acetyl-CoA precursors for the de novo lipogenesis (DNL) pathway to support a high rate of cancer cell viability and development. Inhibition of the DNL pathway promotes cancer cell apoptosis without apparent cytotoxic to normal cells, leading to the representation of selective and powerful targets for cancer therapy. The present study demonstrates that treatments with CTP inhibitor (CTPi), PMCT inhibitor (PMCTi), and the combination of CTPi and PMCTi resulted in decreased cell viability in two hepatocellular carcinoma cell lines (HepG2 and HuH-7). Treatment with citrate transporter inhibitors caused a greater cytotoxic effect in HepG2 cells than in HuH-7 cells. A lower concentration of combined CTPi and PMCTi promotes cytotoxic effect compared with eith...
Journal of clinical microbiology, Jan 30, 2018
Melioidosis is a fatal infectious disease caused by the environmental bacterium It is highly ende... more Melioidosis is a fatal infectious disease caused by the environmental bacterium It is highly endemic in Asia and northern Australia but neglected in many other tropical countries. Melioidosis patients have a wide range of clinical manifestations and definitive diagnosis requires bacterial culture which can be time-consuming. A reliable rapid serological tool is greatly needed for disease surveillance and diagnosis. We previously demonstrated by ELISA that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we have developed a rapid immunochromatography test (ICT) using Hcp1 as the target antigen (Hcp1-ICT). We evaluated this test for specific antibody detection using serum samples obtained from 4 groups of human subjects includingi 487 culture-confirmed melioidosis patients from four hospitals in northeast Thailand; ii 202 healthy donors from northeast Thailand; iii 90 U.S. healthy donors and iv 207 patients infected with oth...
PloS one, 2018
Burkholderia pseudomallei is a flagellated Gram-negative bacterium which is the causative agent o... more Burkholderia pseudomallei is a flagellated Gram-negative bacterium which is the causative agent of melioidosis. The disease poses a major public health problem in tropical regions and diabetes is a major risk factor. The high mortality rate of melioidosis is associated with severe sepsis which involves the overwhelming production of pro-inflammatory cytokines. Bacterial flagellar protein (flagellin) activates Toll-like receptor 5 (TLR5)-mediated innate immune signaling pathways and induces adaptive immune response. However, previous studies of TLR5 signaling in melioidosis have been performed using recombinant flagellin from Salmonella Typhimurium instead of B. pseudomallei. This study aimed to investigate human innate immune response and antibody response against a recombinant B. pseudomallei flagellin (rFliC). We prepared B. pseudomallei rFliC and used it to stimulate HEK-BlueTM-hTLR5 and THP1-DualTM cells to assess TLR5 activation. Subsequently, whole blood stimulation assays wit...
The American journal of tropical medicine and hygiene, Jan 19, 2018
Burkholderia pseudomallei, the etiologic agent of melioidosis, is an important but under-recogniz... more Burkholderia pseudomallei, the etiologic agent of melioidosis, is an important but under-recognized cause of disease in the tropics. Although first described over a century ago as a septicemic illness associated with morphine addicts in Rangoon, Burma, there is little information regarding the incidence of melioidosis in present-day Myanmar. To address this issue, we used two recently developed and validated serological assays to detect B. pseudomallei-specific antibodies in 124 serum samples obtained from febrile patients in the delta region of Myanmar. Using cutoff values derived from culture-confirmed melioidosis cases in neighboring Thailand, 3.2% of the samples exhibited reactivity profiles consistent with active B. pseudomallei infections. Collectively, these findings indicate that melioidosis likely represents a significant cause of morbidity and mortality in Myanmar and support the need for further studies to assess the true burden of disease in this country.
PLOS Neglected Tropical Diseases, 2017
Background Melioidosis is a severe disease caused by Burkholderia pseudomallei. Clinical manifest... more Background Melioidosis is a severe disease caused by Burkholderia pseudomallei. Clinical manifestations are diverse and acute infections require immediate treatment with effective antibiotics. While culture is the current diagnostic standard, it is time-consuming and has low sensitivity. In endemic areas, inaccessibility to biosafety level 3 facilities and a lack of good serodiagnostic tools can impede diagnosis and disease surveillance. Recent studies have suggested that O-polysaccharide (OPS) and hemolysin co-regulated protein 1 (Hcp1) are promising target antigens for serodiagnosis of melioidosis. Methodology/Principle findings We evaluated rapid ELISAs using crude antigens, purified OPS and Hcp1 to measure antibody levels in three sets of sera: (i) 419 serum samples from melioidosis patients, Thai and U.S. healthy donors, (ii) 120 serum samples from patients with other bacterial infections, and (iii) 423 serum samples from 200 melioidosis patients obtained upon admission and at 12 and 52 weeks post-recovery. We observed significantly higher antibody levels using the crude antigen prepared from wild type B. pseudomallei K96243 compared to that of an OPS-mutant. The areas under receiver operator characteristics (AUROCCs) for diagnosis were compared for individual Hcp1-ELISA or OPS-ELISA or combined Hcp1/OPS-ELISA. For Thai donors, AUROCCs were highest and comparable between the Hcp1-ELISA and the combined Hcp1/OPS-ELISA (0.95 versus 0.94). For U.S. donors, the AUROCC was highest for the combined Hcp1/OPS-ELISA (0.96). Significantly higher seropositivity was observed in diabetic patients compared to those without diabetes for both the Hcp1-ELISA (87.3% versus 69.7%) and OPS-ELISA (88.1% versus 60.6%). Although antibody levels for Hcp1 were highest upon admission, the titers declined by week 52 post-recovery.
FEBS Open Bio, May 14, 2018
Suppression of the expression or activities of enzymes that are involved in the synthesis of de n... more Suppression of the expression or activities of enzymes that are involved in the synthesis of de novo lipogenesis (DNL) in cancer cells triggers cell death via apoptosis. The plasma membrane citrate transporter (PMCT) is the initial step that translocates citrate from blood circulation into the cytoplasm for de novo long‐chain fatty acids synthesis. This study investigated the antitumor effect of the PMCT inhibitor (PMCTi) in inducing apoptosis by inhibiting the DNL pathway in HepG2 cells. The present findings showed that PMCTi reduced cell viability and enhanced apoptosis through decreased intracellular citrate levels, which consequently caused inhibition of fatty acid and triacylglycerol productions. Thus, as a result of the reduction in fatty acid synthesis, the activity of carnitine palmitoyl transferase‐1 (CPT‐1) was suppressed. Decreased CPT‐1 activity also facilitated the disruption of mitochondrial membrane potential (ΔΨm) leading to stimulation of apoptosis. Surprisingly, primary human hepatocytes were not affected by PMCTi. Increased caspase‐8 activity as a consequence of reduction in fatty acid synthesis was also found to cause disruption of ΔΨm. In addition, apoptosis induction by PMCTi was associated with an enhanced reactive oxygen species generation. Taken together, we suggest that inhibition of the DNL pathway following reduction in citrate levels is an important regulator of apoptosis in HepG2 cells via suppression of CPT‐1 activity. Thus, targeting the DNL pathway mediating CPT‐1 activity by PMCTi may be a selective potential anticancer therapy.