A novel use of a geometric morphometric technique to distinguish human parasite eggs of twelve different species.

Copro-microscopic diagnostic methods are the most common approach for screening patients with parasitic infections. However, expertise is required to identify helminthic eggs from fecal specimens. Consequently, new methods are required to support accurate species identification. Novel technologies have recently been developed for the classification of organisms, including geometric morphometric (GM) approaches. In this study, the outline-based GM approach was used to distinguish the eggs of 12 common human parasite species, including Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, hookworm, Capillaria philippinensis, Opisthorchis spp., Fasciola spp., Paragonimus spp., Schistosoma mekongi, Taenia spp., Hymenolepis diminuta and Hymenolepis nana. The GM analysis revealed that the size cannot be used as the main variable in the identification of parasite species at the egg stage, producing only 30.18% overall accuracy. However, comparisons of shape based on the Mahalanobis distances between pairs of parasite species showed significant differences in all pairs (p < 0.05). The shape analysis produced 84.29% overall accuracy. This is the first time that outline-based GM has been preliminarily confirmed as a valuable approach to support copro-microscopic analysis, in order to effectively screen helminth eggs. However, further studies with a larger set of helminth eggs and artefacts should be carried out to increase confidence in the identification of parasite species in the absence of local experts.

Intraspecific Genetic Variation of Anisakis typica in Indian Mackerel Caught from the Gulf of Thailand, Samut Songkhram Province.

Anisakis nematodes infecting Indian mackerel (Rastrelliger kanagurta) were initially discovered in Thailand in our preliminary investigation. Nevertheless, the species of Anisakis collected has not been determined nor has its genetic variation been researched. Thus, this study aimed to molecularly identify the species of Anisakis specimens using the internal transcribed spacer (ITS) region of ribosomal DNA sequences. In addition, the intraspecific genetic variation was also determined using mitochondrial cytochrome oxidase subunit II (COII) gene sequences. The phylogenetic relationships of the ITS region classified all samples into Anisakis typica; however, the genetic variation between them could not be distinguished. By contrast, the phylogenetic tree analysis of the COII region identified all samples as A. typica, with 17 different haplotypes by 66 polymorphic sites and five of the substitutions resulted in amino acid change. Additionally, the distribution pattern of the COII region can be separated into two groups between South America and Asian countries. All our haplotypes belong to Asian countries. Compared with the two genetic markers used in this investigation, COII appears to be a better candidate for studying genetic variation sensitive to environmental changes and intermediate or definitive host behavioral changes.

Phenotypic and genotypic characterization of Thai isolates of Plasmodium falciparum after an artemisinin resistance containment project.

BACKGROUND: In Thailand, artemisinin-based combination therapy (ACT) has been used to treat uncomplicated falciparum malaria since 1995. Unfortunately, artemisinin resistance has been reported from Thailand and other Southeast Asian countries since 2003. Malarone®, a combination of atovaquone-proguanil (ATQ-PG), has been used to cease artemisinin pressure in some areas along Thai-Cambodia border, as part of an artemisinin resistance containment project since 2009. This study aimed to determine genotypes and phenotypes of Plasmodium falciparum isolates collected from the Thai-Cambodia border after the artemisinin resistance containment project compared with those collected before. RESULTS: One hundred and nine of P. falciparum isolates collected from Thai-Cambodia border from Chanthaburi and Trat provinces during 1988-2016 were used in this study. Of these, 58 isolates were collected after the containment. These parasite isolates were characterized for in vitro antimalarial sensitivities including chloroquine (CQ), quinine (QN), mefloquine (MQ), piperaquine (PPQ), artesunate (AS), dihydroartemisinin (DHA), ATQ and PG and genetic markers for drug resistance including the Kelch13 (k13), Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1) and cytochrome b (cytb) genes. Mean CQ, QN, MQ, PPQ and AS IC50s of the parasite isolates collected from 2009 to 2016 exhibited significantly higher than those of parasites collected before 2009. Approximately 57% exhibited in vitro MQ resistance. Approximately 94% of the isolates collected from 2009 to 2016 contained the pfmdr1 184F allele. Mutations of the k13 gene were detected in approximately 90% of the parasites collected from 2009 to 2016 which were significantly higher than the parasite isolates collected before. No ATQ-resistant genotype and phenotype of P. falciparum were found among the isolates collected after the containment project. CONCLUSIONS: Although the containment project had been implemented in this area, the expansion of artemisinin-resistant parasites did not decline. In addition, reduced sensitivity of the partner drugs of ACT including MQ and PPQ were identified.

PFMDR1 POLYMORPHISMS INFLUENCE ON IN VITRO SENSITIVITY OF THAI PLASMODIUM FALCIPARUM ISOLATES TO PRIMAQUINE, SITAMAQUINE AND TAFENOQUINE.

Primaquine (PQ), an 8-aminoquinoline, is considered a tissue schizonticide drug for radical cure in vivax and ovale malaria, with minimal impact on asexual erythrocytic stages at therapeutic concentrations. Tafenoquine (TQ), a new 8-aminoquinoline analog of PQ, is active against both malaria parasite tissue and blood stages and is being promoted as a drug candidate for antimalarial chemotherapy and chemoprophylaxis and potential transmission blocking against Plasmodium vivax and P. falciparum. This study compared in vitro sensitivity of Thai P. falciparum isolates against three 8-aminoquinolines, PQ, TQ and sitamaquine (SQ), a related 8-aminoquinoline and assessed the importance of pfmdr1 polymorphism on the in vitro response. Seventy-eight laboratory adapted Thai P. falciparum isolates were evaluated for in vitro sensitivity to the three 8-aminoquinolines using a radioisotopic assay, and pfmdr1 polymorphisms were determined using PCR-based methods. All three drugs have weak antiplasmodial activity against asexual erythrocytic stage with SQ being the most potent by almost 10 folds. Cross susceptibility was observed in all three 8-aminoquinolines. Parasites containing pfmdr1 86Y, 184Y or 1034S allele exhibit significantly higher PQ IC50. TQ sensitivity was reduced in those parasites containing pfmdr1 86Y, 1034S or 1042N allele. However, there was no significant influence of pfmdr1 alleles on SQ sensitivity. The data highlight unique differences among three representative 8-aminoquinoline drugs that may be useful in understanding their potential utility in antimalarial development.

Distribution of pfmdr1 polymorphisms in Plasmodium falciparum isolated from Southern Thailand.

BACKGROUND: Drug resistance in Plasmodium falciparum is a major problem in malaria control especially along the Thai-Myanmar and Thai-Cambodia borders. To date, a few molecular markers have been identified for anti-malarial resistance in P. falciparum, including the P. falciparum chloroquine resistance transporter (pfcrt) and the P. falciparum multidrug resistance 1 (pfmdr1). However no information is available regarding the distribution pattern of these gene polymorphisms in the parasites from the Thai-Malaysia border. This study was conducted to compare the distribution pattern of the pfcrt and pfmdr1 polymorphisms in the parasites from the lower southern provinces, Thai-Malaysia border and the upper southern provinces, Thai-Myanmar border. In addition, in vitro sensitivities of anti-malarial drugs including chloroquine, mefloquine, quinine, and artesunate were determined. METHODS: In all, 492 P. falciparum-positive blood samples were collected from the lower southern provinces: Songkhla, Yala and Narathiwas. From the upper southern part of Thailand, Ranong and Chumphon, 66 samples were also collected. Polymorphisms of the pfcrt and the pfmdr1 gene were determined using PCR techniques. In vitro sensitivities of anti-malarial drugs were determined using radioisotopic method. RESULTS: All parasites from both areas contained the pfcrt 76 T allele. The pfmdr1 86Y allele was significantly more common in the parasites isolated from the lower southern areas. In contrast, the pfmdr1 184F allele was predominant among the parasites from the upper southern areas especially Ranong. In addition, the parasites from Ranong contained higher copy numbers than the parasites from other provinces. All adapted parasite isolates exhibited CQ-resistant phenotype. Neither QN nor MQ resistance was detected in these isolates. CONCLUSION: The parasites from Thai-Malaysia border exhibited different resistant patterns compared to other areas along the international border of Thailand. This information will be useful for anti-malarial drug policy in Thailand.

Investigating the impact of climate and seasonality on mosquito (Diptera: Culicidae) vector populations in the connecting areas of the Tenasserim range forests in Thailand.

Mosquito-borne diseases pose a significant public health challenge globally. Our study focused on the seasonal diversity of mosquito species in the connecting areas of the Tenasserim (also known as Tanaosri) range forests in Thailand. Additionally, we employed the geometric morphometric technique to assess variations in wing size and shape among five predominant mosquito species. Throughout the study period, we collected a total of 9,522 mosquitoes, encompassing 42 species across eight genera. In these connecting areas of forests, the Simpson index and Shannon species diversity index were recorded at 0.86 and 2.36, respectively, indicating a high level of mosquito diversity. Our analysis using the Analysis of Similarities (ANOSIM) test showed significant seasonal differences in mosquito communities, with an R-value of 0.30 (p < 0.05) in the lower connecting areas and 0.37 (p < 0.05) in the upper connecting areas. Additionally, canonical correspondence analyses showed that the abundance of each mosquito species is influenced by various climate factors. Phenotypic analyses of wing size and shape have deepened our understanding of local adaptation and the seasonal pressures impacting these vectors. Notably, most species exhibited larger wing sizes in the dry season compared to other seasons. Additionally, seasonal assessments of wing shape in five predominant mosquito species revealed significant differences across seasonal populations (p < 0.05). Ongoing monitoring of these populations is crucial to enhancing our understanding of the seasonal effects on mosquito abundance and physiological adaptations. These insights are essential for developing more effective strategies to manage mosquito-borne diseases.

Polymorphisms of the pfmdr1 but not the pfnhe-1 gene is associated with in vitro quinine sensitivity in Thai isolates of Plasmodium falciparum.

BACKGROUND: The emergence of Plasmodium falciparum resistance to most currently used anti-malarial drugs is a major problem in malaria control along the Thai-Myanmar and Thai-Cambodia borders. Quinine (QN) with tetracycline/doxycycline has been used as the second-line treatment for uncomplicated falciparum malaria. In addition, QN monotherapy has been the first-line treatment for falciparum malaria in pregnant women. However, reduced in vitro and in vivo responses to QN have been reported. To date, a few genetic markers for QN resistance have been proposed including Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum Na+/H+ exchanger (pfnhe-1). This study was to investigate the role of the pfmdr1 and pfnhe-1 gene on in vitro QN sensitivity in Thai isolates of P. falciparum. METHODS: Eighty-five Thai isolates of P. falciparum from the Thai-Myanmar and Thai-Cambodia borders from 2003-2008 were determined for in vitro QN sensitivity using radioisotopic assay. Polymorphisms of the pfmdr1 and pfnhe-1 gene were determined by PCR-RFLP and sequence analysis. Associations between the in vitro QN sensitivity and the polymorphisms of the pfmdr1 and pfnhe-1 gene were evaluated. RESULTS: The mean QN IC50 was 202.8 nM (range 25.7-654.4 nM). Only four isolates were QN resistant when the IC50 of >500 nM was used as the cut-off point. Significant associations were found between the pfmdr1 mutations at codons N86Y and N1042D and in vitro QN sensitivity. However, no associations with the number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the microsatellite ms4760 of the pfnhe-1 gene were identified. CONCLUSION: Data from the present study put doubt regarding the pfnhe-1 gene as to whether it could be used as the suitable marker for QN resistance in Thailand. In contrast, it confirms the influence of the pfmdr1 gene on in vitro QN sensitivity.

Genetic Diversity, Haplotype Relationships, and kdr Mutation of Malaria Anopheles Vectors in the Most Plasmodium knowlesi-Endemic Area of Thailand.

Plasmodium knowlesi, a malaria parasite that occurs naturally in long-tailed macaques, pig-tailed macaques, and banded leaf monkeys, is currently regarded as the fifth of the human malaria parasites. We aimed to investigate genetic diversity based on the cytochrome c oxidase subunit I (COI) gene, detect Plasmodium parasites, and screen for the voltage-gated sodium channel (VGSC)-mutation-mediated knockdown resistance (kdr) of Anopheles mosquitoes in Ranong province, which is the most P. knowlesi-endemic area in Thailand. One hundred and fourteen Anopheles females belonging to eight species, including An. baimaii (21.05%), An. minimus s.s. (20.17%), An. epiroticus (19.30%), An. jamesii (19.30%), An. maculatus s.s. (13.16%), An. barbirostris A3 (5.26%), An. sawadwongporni (0.88%), and An. aconitus (0.88%), were caught in three geographical regions of Ranong province. None of the Anopheles mosquitoes sampled in this study were infected with Plasmodium parasites. Based on the sequence analysis of COI sequences, An. epiroticus had the highest level of nucleotide diversity (0.012), followed by An. minimus (0.011). In contrast, An. maculatus (0.002) had the lowest level of nucleotide diversity. The Fu's Fs and Tajima's D values of the Anopheles species in Ranong were all negative, except the Tajima's D values of An. minimus (0.077). Screening of VGSC sequences showed no presence of the kdr mutation of Anopheles mosquitoes. Our results could be used to further select effective techniques for controlling Anopheles populations in Thailand's most P. knowlesi-endemic area.

Geometric morphometrics versus DNA barcoding for the identification of malaria vectors Anopheles dirus and An. baimaii in the Thai-Cambodia border.

Anopheles (Cellia) dirus Peyton & Harrison and Anopheles baimaii Sallum & Peyton are sibling species within the Dirus complex belonging to the Leucosphyrus group, and have been incriminated as primary vectors of malaria in Thailand. In the present study, DNA barcoding and geometric morphometrics were used to distinguish between An. dirus and An. baimaii in the international border areas, Trat Province, eastern Thailand. Our results revealed that DNA barcoding based on the cytochrome c oxidase subunit I gene could not be used to distinguish An. dirus from An. baimaii. The overlapping values between intra- and interspecific genetic divergence indicated no barcoding gap present for An. dirus and An. baimaii (ranging from 0 to 0.99%). However, the results of the geometric morphometric analysis based on the wing shape clearly distinguished An. dirus and An. baimaii, with 92.42% of specimens assigned to the correct species. We concluded that geometric morphometrics is an effective tool for the correct species identification of these two malaria vectors. Our findings could be used to make entomological surveillance information more accurate, leading to further effective mosquito control planning in Thailand and other countries in Southeast Asia.

Mitochondrial DNA barcoding of mosquito species (Diptera: Culicidae) in Thailand.

The correct identification of mosquito species is important for effective mosquito vector control. However, the standard morphological identification of mosquito species based on the available keys is not easy with specimens in the field due to missing or damaged morphological features during mosquito collections, often leading to the misidentification of morphologically indistinguishable. To resolve this problem, we collected mosquito species across Thailand to gather genetic information, and evaluated the DNA barcoding efficacy for mosquito species identification in Thailand. A total of 310 mosquito samples, representing 73 mosquito species, were amplified using mitochondrial cytochrome c oxidase subunit I (COI) primers. The average maximum intraspecific genetic variation of the 73 mosquito species was 1% ranged from 0-5.7%. While, average minimum interspecific genetic variation (the distance to the nearest neighbour) of the 73 mosquito species was 7% ranged from 0.3-12.9%. The identification of success rates based on the "Best Match," "Best Close Match," and "All Species Barcodes" methods were 97.7%, 91.6%, and 81%, respectively. Phylogenetic analyses of Anopheles COI sequences demonstrated a clear separation between almost all species (except for those between An. baimaii and An. dirus), with high bootstrap support values (97%-99%). Furthermore, phylogenetic analyses revealed potential sibling species of An. annularis, An. tessellatus, and An. subpictus in Thailand. Our results indicated that DNA barcoding is an effective molecular approach for the accurate identification of mosquitoes in Thailand.

Geometric morphometric and molecular techniques for discriminating among three cryptic species of the Anopheles barbirostris complex (diptera: culicidae) in Thailand.

Anopheles members of the Barbirostris complex are important vectors of malaria in Thailand. However, they are morphologically indistinguishable because they are closely related species. In this study, wing geometric morphometrics (GM) and DNA barcoding based on the cytochrome c oxidase subunit 1 (C O I) gene were applied to differentiate cryptic species of the Barbirostris complex in Thailand. Three cryptic species of the Barbirostris complex, Anopheles dissidens (19.44%), Anopheles saeungae (24.54%), and Anopheles wejchoochotei (56.02%) were initially identified using the multiplex polymerase chain reaction assay. DNA barcoding analyses showed low intraspecific distances (range, 0.27%-0.63%) and high interspecific distances (range, 1.92%-3.68%), consistent with the phylogenetic analyses that showed clear species groups. While wing size and shape analyses based on landmark-based GM indicated differences between three species (p < 0.05). The cross-validated reclassification revealed that the overall efficacy of wing size analysis for species identification of the Barbirostris complex was less than the wing shape analysis (56.43% vs. 74.29% total performance). Therefore, this study's results are guidelines for applying modern techniques to identify members within the Barbirostris complex, which are still difficult to distinguish by morphology-based identification and contribute to further appropriate malaria control.

The patterns of mutation and amplification of Plasmodium falciparum pfcrt and pfmdr1 genes in Thailand during the year 1988 to 2003.

The study investigated the patterns of pfmdr1 and pfcrt genetic polymorphisms in Plasmodium falciaprum isolates collected from Thailand during the periods 1988-1993 (35 isolates), and 2003 (21 isolates). Pfcrt polymorphisms were almost universal for the mutations at codons K76T, A220S, Q271E, N326S, and R371I. All parasites displayed the chloroquine (CQ)-resistant phenotypes. This data suggested that pfcrt gene was sufficient to CQ resistance but did not mediate level of resistance. The prevalence [number of isolates (%)] of pfmdr1 polymorphisms at codons N86Y, Y184F, S1034C, N1042D and D1246Y were five (9%), 48 (86%), ten (18%), and 15 (27%), respectively. All isolates carried the wild-type nucleotide at position 1246. Results support the role of pfmdr1 in modulating susceptibilities of the P. falciparum to CQ, QN, and MQ. The frequencies of the S1034C and N1042D pfmdr1 polymorphisms and number of gene copy were significantly different in isolates collected during the two periods, with a trend of increasing prevalence of wild-type genotypes and number of gene copy from 1988 to 2003. The prominent pattern of pfmdr1 at codons 86/184/1034/1042/1246 was NFSND, with prevalence increasing from 40% to 95% during the 10-year period.

Drug resistance and in vitro susceptibility of Plasmodium falciparum in Thailand during 1988-2003.

The aim of the present study was to investigate antimalarial drug pressure resulting from the clinical use of different antimalarials in Thailand. The phenotypic diversity of the susceptibility profiles of antimalarials, i.e., chloroquine (CQ), quinine (QN), mefloquine (MQ), and artesunate (ARS) in Plasmodium falciparum isolates collected during the period from 1988 to 2003 were studied. P. falciparum isolates from infected patients were collected from the Thai-Cambodian border area at different time periods (1988-1989, 1991-1992, and 2003), during which 3 different patterns of drug use had been implemented: MQ + sulphadoxine (S) + pyrimethamine (P), MQ alone and MQ + ARS, respectively. The in vitro drug susceptibilities were investigated using a method based on the incorporation of [(3)H] hypoxanthine. A total of 50 isolates were tested for susceptibilities to CQ, QN, MQ, and ARS. Of these isolates, 19, 16, and 15 were adapted during the periods 1988-1989, 1991-1993, and 2003, respectively. P. falciparum isolates collected during the 3 periods were resistant to CQ. Sensitivities to MQ declined from 1988 to 2003. In contrast, the parasite was sensitive to QN, and similar sensitivity profile patterns were observed during the 3 time periods. There was a significantly positive but weak correlation between the IC(50) values of CQ and QN, as well as between the IC(50) values of QN and MQ. Drug pressure has impact on sensitivity of P. falciparum to MQ. A combination therapy of MQ and ARS is being applied to reduce the parasite resistance, and also increasing the efficacy of the drug.

Characterization of Plasmodium knowlesi dihydrofolate reductase-thymidylate synthase and sensitivity to antifolates.

Malaria caused by an infection of Plasmodium knowlesi can result in high parasitemia and deaths. Therefore, effective and prompt treatment is necessary to reduce morbidity and mortality. The study aims to characterize P. knowlesi dihydrofolate reductase-thymidylate synthase enzyme (PkDHFR-TS) and its sensitivity to antifolates. The putative Pkdhfr gene was PCR amplified from field isolates collected from the Southern Thailand. Molecular analysis showed 11 polymorphisms in the dhfr domain of the bifunctional dhfr-ts gene. Of these, 1 polymorphism was a non-synonymous substitution (R34L) that had previously been reported but not associated with antifolate resistance. The recombinant PkDHFR-TS enzyme was found to be sensitive to standard antifolates-pyrimethamine and cycloguanil-as well as P218, a registered candidate drug currently first in human clinical trial. Results suggest that antifolates class of compounds should be effective against P. knowlesi infection.

In Vitro Sensitivity of Pyronaridine in Thai Isolates of Plasmodium falciparum.

Pyronaridine, a Mannich base antimalarial agent with a high activity against chloroquine-resistant Plasmodium falciparum, has been combined with artesunate as a new artemisinin based combination therapy (ACT). Pyronaridine-artesunate combination could be one of the choices for the treatment of uncomplicated falciparum malaria in multidrug-resistant areas including Thailand. The aim of this study was to determine in vitro sensitivity and cross-resistance pattern of pyronaridine in Thai isolates of P. falciparum. In addition, the influence of resistant genes concerning in vitro pyronaridine sensitivity was determined. The mean pyronaridine 50% inhibitory concentration (IC50) of 118 parasite isolates was 5.6 ± 3.1 nM (range = 0.2-15.4 nM) with a significant positive correlation with artesunate IC50 (r = 0.246, P = 0.008) and amodiaquine IC50 (r = 0.220, P = 0.042) and a significant negative correlation with quinine IC50 (r = -0.185, P = 0.047). Parasites containing the pfmdr1 86Y allele exhibited significantly reduced pyronaridine sensitivity compared with those with the pfmdr1 N86 allele (7.6 ± 3.3 nM and 5.4 ± 3.0 nM, respectively, P = 0.032, independent t test); however, the difference may not be clinically relevant. Pyronaridine-artesunate could be the candidate ACT to treat multidrug-resistant falciparum malaria in Thailand with careful monitoring.

Influence of the pfmdr1 Gene on In Vitro Sensitivities of Piperaquine in Thai Isolates of Plasmodium falciparum.

Piperaquine combined with dihydroartemisinin is one of the artemisinin derivative combination therapies, which can replace artesunate-mefloquine in treating uncomplicated falciparum malaria in Thailand. The aim of this study was to determine the in vitro sensitivity of Thai Plasmodium falciparum isolates against piperaquine and the influence of the pfmdr1 gene on in vitro response. One hundred and thirty-seven standard laboratory and adapted Thai isolates of P. falciparum were assessed for in vitro piperaquine sensitivity. Polymorphisms of the pfmdr1 gene were determined by polymerase chain reaction methods. The mean and standard deviation of the piperaquine IC50 in Thai isolates of P. falciparum were 16.7 ± 6.3 nM. The parasites exhibiting chloroquine IC50 of ≥ 100 nM were significantly less sensitive to piperaquine compared with the parasite with chloroquine IC50 of < 100 nM. No significant association between the pfmdr1 copy number and piperaquine IC50 values was found. In contrast, the parasites containing the pfmdr1 86Y allele exhibited significantly reduced piperaquine sensitivity. Before nationwide implementation of dihydroartemisinin-piperaquine as the first-line treatment in Thailand, in vitro and in vivo evaluations of this combination should be performed especially in areas where parasites containing the pfmdr1 86Y allele are predominant such as the Thai-Malaysian border.

Disruption of endocytic trafficking protein Rab7 impairs invasiveness of cholangiocarcinoma cells.

BACKGROUND: Alterations and mutations of endo-lysosomal trafficking proteins have been associated with cancer progression. Identification and characterization of endo-lysosomal trafficking proteins in invasive cholangiocarcinoma (CCA) cells may benefit prognosis and drug design for CCA. OBJECTIVE: To identify and characterize endo-lysosomal trafficking proteins in invasive CCA. METHODS: A lysosomal-enriched fraction was isolated from a TNF-α induced invasive CCA cell line (KKU-100) and uninduced control cells and protein identification was performed with nano-LC MS/MS. Novel lysosomal proteins that were upregulated in invasive CCA cells were validated by real-time RT-PCR. We selected Rab7 for further studies of protein level using western blotting and subcellular localization using immunofluorescence. The role of Rab7 in CCA invasion was determined by siRNA gene knockdown and matrigel transwell assay. RESULTS: Rab7 mRNA and protein were upregulated in invasive CCA cells compared with non-treated controls. Immunofluorescence studies demonstrated that Rab7 was expressed predominantly in invasive CCA cells and was localized in the cytoplasm and lysosomes. Suppression of Rab7 translation significantly inhibited TNF-α-induced cell invasion compared to non-treated control (p= 0.044). CONCLUSIONS: Overexpression of Rab7 in CCA cells was associated with cell invasion, supporting Rab7 as a novel candidate for the development of diagnostic and therapeutic strategies for CCA.

Comparison of two in vitro sensitivity tests for Plasmodium falciparum.

The main purpose of the study was to compare the in vitro sensitivity results obtained from the two widely-used in vitro systems: (1) standard WHO micro-technique based on schizont maturation inhibition using fresh isolates (M-I), and (2) micro-technique based on incorporation of [3H]-hypoxanthine using culture-adapted isolates (M-II). The study was conducted during 1998 and 2002. A total of 473 Plasmodium falciparum isolates were collected from five highly malaria endemic areas of Thailand, ie, Mae Sot district, Tak (north-western), Kanchanaburi (western), Ranong (south-western), Ratchaburi (south-western) and Chantaburi (eastern) Provinces. The antimalarials tested were: mefloquine, quinine, chloroquine, artemisinin and dihydroartemisinin. The sensitivity results for mefloquine obtained from the two methods were significantly different from each other. The IC50 values for M-II was less than M-I. The median (95%C.I.) IC50 value for mefloquine using the M-II method was significantly lower [696.47 (393.11-1,233.2) nM] than for M-I [3,955.4 (1,035.61-5,108.9) nM]. The in vitro sensitivity results for quinine were significantly different from each other. The median (95% C.I.) IC50 value for M-II [161 (42-351) nM] was 2.5-fold that of M-I [66 (24-450) nM].

ALCAM is a Novel Cytoplasmic Membrane Protein in TNF-α Stimulated Invasive Cholangiocarcinoma Cells.

BACKGROUND: Cholangiocarcinoma (CCA), or bile duct cancer, is incurable with a high mortality rate due to a lack of effective early diagnosis and treatment. Identifying cytoplasmic membrane proteins of invasive CCA that facilitate cancer progression would contribute toward the development of novel tumor markers and effective chemotherapy. MATERIALS AND METHODS: An invasive CCA cell line (KKU-100) was stimulated using TNF-α and then biotinylated and purified for mass spectrometry analysis. Novel proteins expressed were selected and their mRNAs expression levels were determined by real-time RT-PCR. In addition, the expression of ALCAM was selected for further observation by Western blot analysis, immunofluorescent imaging, and antibody neutralization assay. RESULTS: After comparing the proteomics profile of TNF-α induced invasive with non-treated control cells, over-expression of seven novel proteins was observed in the cytoplasmic membrane of TNF-α stimulated CCA cells. Among these, ALCAM is a novel candidate which showed significant higher mRNA- and protein levels. Immunofluorescent assay also supported that ALCAM was expressed on the cell membrane of the cancer, with increasing intensity associated with TNF-α. CONCLUSIONS: This study indicated that ALCAM may be a novel protein candidate expressed on cytoplasmic membranes of invasive CCA cells that could be used as a biomarker for development of diagnosis, prognosis, and drug or antibody-based targeted therapies in the future.

Up-regulation of AKAP13 and MAGT1 on cytoplasmic membrane in progressive hepatocellular carcinoma: a novel target for prognosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers and is associated with high mortality worldwide. The current gold standards for HCC surveillance are detection of serum α-fetoprotein (AFP) and ultrasonography; however, non-specificity of AFP and ultrasonography has frequently been reported. Therefore, alternative tools, especially novel specific tumor markers, are required. In this study, cytoplasmic membrane proteins were isolated from phorbol 12-myristate 13-acetate (PMA)-induced invasive HepG2 cells and identified using nano-scale liquid chromatographic tandem mass spectrometry (NLC-MS/MS) with comparison to non-treated controls. The results showed that two proteins, magnesium transporter protein 1 (MAGT1) and A-kinase anchor protein 13 (AKAP13), were highly expressed in PMA-treated HepG2 cells. This up-regulation was confirmed by real-time RT-PCR, western blot analysis, and immunofluorescent staining studies. Furthermore, evaluation of MAGT1 and AKAP13 expression in clinical HCC tissues by immunohistochemistry suggested that both proteins were strongly expressed in tumor tissues with significantly higher average immunoreactive scores of Remmele and Stegner (IRS) than in non-tumor tissues (P ≤ 0.005). In conclusion, the expression levels of MAGT1 and AKAP13 in HCC may be potential biomarkers for the diagnosis and prognosis of this cancer.