Significant Divergence in Sensitivity to Antimalarial Drugs between Neighboring Plasmodium falciparum Populations along the Eastern Border of Myanmar.

Malaria parasites in different areas where malaria is endemic display different levels of resistance to antimalarial drugs as the result of varied drug use histories. To provide updated knowledge of drug sensitivities during the malaria elimination phase in Southeast Asia, an epicenter of multidrug resistance, we determined in vitro susceptibilities of culture-adapted Plasmodium falciparum isolates from two eastern border regions (Wa and Kachin) of Myanmar to 10 drugs. Despite their close proximity, the Kachin parasites displayed higher 50% inhibitory concentrations than the Wa parasites to chloroquine, piperaquine, naphthoquine, mefloquine, quinine, pyrimethamine, pyronaridine, lumefantrine, and dihydroartemisinin. Genotyping of genes associated with drug resistance also showed significant differences in the prevalence rates of mutant alleles between the two regions. Particularly, major pfdhfr mutations mediating pyrimethamine resistance and the pfdhps A437G mutation had significantly higher frequencies in the Kachin parasites (P < 0.005). Moreover, when pfdhfr and pfdhps were considered together, the wild-type allele was found only in the Wa samples (22.6%). In addition, the pfmdr1 Y184F mutation reached 38.7% in the Kachin parasites, compared to 9.7% in the Wa parasites, whereas N86Y was only detected in the Wa parasites, at 22.6%. Furthermore, the F446I mutation and all mutations in the propeller domain of the PfK13 gene were significantly more frequent in the Kachin parasites. Collectively, this work demonstrates that even in spatially closely separated regions, parasites can exhibit drastic differences in drug sensitivities and genetic makeups underlying drug resistance, which may reflect regionally different drug histories and genetic drift of these isolated parasite populations.

Microgeographic Heterogeneity of Border Malaria During Elimination Phase, Yunnan Province, China, 2011-2013.

To identify township-level high-risk foci of malaria transmission in Yunnan Province, China, along the international border, we retrospectively reviewed data collected in hospitals and clinics of 58 townships in 4 counties during 2011-2013. We analyzed spatiotemporal distribution, especially hot spots of confirmed malaria, using geographic information systems and Getis-Ord Gi*(d) cluster analysis. Malaria incidence, transmission seasonality, and Plasmodium vivax:P. falciparum ratio remained almost unchanged from 2011 to 2013, but heterogeneity in distribution increased. The number of townships with confirmed malaria decreased significantly during the 3 years; incidence became increasingly concentrated within a few townships. High-/low-incidence clusters of P. falciparum shifted in location and size every year, whereas the locations of high-incidence P. vivax townships remained unchanged. All high-incidence clusters were located along the China-Myanmar border. Because of increasing heterogeneity in malaria distribution, microgeographic analysis of malaria transmission hot spots provided useful information for designing targeted malaria intervention during the elimination phase.

Genetic diversity of the Pvk12 gene in Plasmodium vivax from the China-Myanmar border area.

BACKGROUND: Plasmodium falciparum resistance to artemisinin emerged in the Greater Mekong Sub-region has been associated with mutations in the propeller domain of the kelch gene Pfk13. METHODS: Here the polymorphisms in Pvk12 gene, the orthologue of Pfk13 in Plasmodium vivax, were determined by PCR and sequencing in 262 clinical isolates collected in recent years (2012-2015) from the China-Myanmar border area. RESULTS: Sequencing of full-length Pvk12 genes from these isolates identified three synonymous mutations (N172N, S360S, S697S) and one non-synonymous mutation M124I, all of which were at very low prevalence (2.0-3.1%). Moreover, these mutations were non-overlapping between the two study sites on both sides of the border. Molecular evolutionary analysis detected signature of purifying selection on Pvk12. CONCLUSIONS: There is no direct evidence that Pvk12 is involved in artemisinin resistance in P. vivax, but it remains a potential candidate requiring further investigation. Continuous monitoring of potential drug resistance in this parasite is needed in order to facilitate the regional malaria elimination campaign.

[Leptin-mediated ERK Signaling Pathway Promotes the Transformation 
of Rat Alveolar Type II Epithelial Cells Induced by Yunnan Tin Mine Dust].

BACKGROUND: Currently, a significant number of miners are involved in mining operations at the Gejiu tin mine in Yunnan. This occupational setting is associated with exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons, and radioactive radon, thereby significantly elevating the risk of lung cancer. This study aims to investigate the involvement of leptin-mediated extracellular regulated protein kinase (ERK) signaling pathway in the malignant transformation of rat alveolar type II epithelial cells induced by Yunnan tin mine dust. METHODS: Immortalized rat alveolar cells type II (RLE-6TN) cells were infected with Yunnan tin mine dust at a concentration of 200 µg/mL for nine consecutive generations to establish the infected cell model, which was named R200 cells. The cells were cultured normally, named as R cells. The expression of leptin receptor in both cell groups was detected using the Western blot method. The optimal concentration of leptin and mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) on R200 cells was determined using the MTT method. Starting from the 20th generation, the cells in the R group were co-cultured with leptin, while the cells in the R200 group were co-cultured with the MEK inhibitor U0126. The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining. Additionally, concanavalin A (ConA) was utilized to detect any morphological differences, and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells. The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method. RESULTS: Both the cells in the R group and R200 group express leptin receptor OB-R. Compared to the R200 group, the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect. The proliferation of R200 cells infected with the virus is inhibited by 30 µmol/L U0126, and a statistically significant divergence was seen when compared to the control group (P<0.05). Starting from the 25th generation, the cell morphology of the leptin-induced R200 group (R200L group) underwent changes, leading to malignant transformation observed at the 30th generation. The characteristics of malignant transformation became evident by the 40th generation in the R200L group. In contrast, the other groups showed agglutination of P40 cells, and the speed of cell aggregation increased with an increase in ConA concentration. Notably, the R200L group exhibited faster cell aggregation compared to the U0126-induced R200 (R200LU) group. Additionally, the cells in the R200L group were capable of forming clones starting from P30, with a colony formation rate of 2.25‰±0.5‰. However, no clonal colonies were observed in the R200LU group and R200 group. The expression of phosphorylated extracellular signal-regulated kinase (pERK) was enhanced in cells of the R200L group. However, when the cells in the R200L group were treated with U0126, a blocking agent, the phosphorylation level of pERK decreased. CONCLUSIONS: Leptin can promote the malignant transformation of lung epithelial cells infected by mine dust, and the ERK signaling pathway may be necessary for the transformation of alveolar type II epithelial cells induced by Yunnan tin mine dust.

Gastrodin ameliorates cognitive dysfunction in diabetes by inhibiting PAK2 phosphorylation.

Diabetes is associated with higher prevalence of cognitive dysfunction, while the underlying mechanism is still elusive. In this study, we aim to explore the potential mechanism of diabetes-induced cognitive dysfunction and assess the therapeutic effects of Gastrodin on cognitive dysfunction. Diabetes was induced by a single injection of streptozotocin. The Morris Water Maze Test was employed to assess the functions of spatial learning and memory. Transcriptome was used to identify the potential factors involved. Western blot and immunofluorescence were applied to detect the protein expression. Our results have shown that spatial learning was impaired in diabetic rats, coupled with damaged hippocampal pyramidal neurons. Gastrodin intervention ameliorated the spatial learning impairments and neuronal damages. Transcriptomics analysis identified differential expression genes critical for diabetes-induced hippocampal damage and Gastrodin treatment, which were further confirmed by qPCR and western blot. Moreover, p21 activated kinase 2 (PAK2) was found to be important for diabetes-induced hippocampal injury and its inhibitor could promote the survival of primary hippocampal neurons. It suggested that PAK2 pathway may be involved in cognitive dysfunction in diabetes and could be a therapeutic target for Gastrodin intervention.

Different In Vitro Drug Susceptibility Profile of Plasmodium falciparum Isolates from Two Adjacent Areas of Northeast Myanmar and Molecular Markers for Drug Resistance.

The Greater Mekong Subregion (GMS) is the epicenter of antimalarial drug resistance. We determined in vitro susceptibilities to 11 drugs of culture-adapted Plasmodium falciparum isolates from adjacent areas (Laiza and Muse) along the China−Myanmar border. Parasites from this region were highly resistant to chloroquine and pyrimethamine but relatively sensitive to other antimalarial drugs. Consistently, the Dd2-like pfcrt mutations were fixed or almost fixed in both parasite populations, and new mutations mediating piperaquine resistance were not identified. Similarly, several mutations related to pfdhfr and pfdhps were also highly prevalent. Despite their geographical proximity, malaria parasites from Laiza showed significantly higher in vitro resistance to artemisinin derivatives, naphthoquine, pyronaridine, lumefantrine, and pyrimethamine than parasites from Muse. Likewise, the pfdhfr N51I, pfdhps A581G, pfmrp1 H785N, and pfk13 F446I mutations were significantly more frequent in Laiza than in Muse (p < 0.05). For the pfmdr1 mutations, Y184F was found only in Laiza (70%), whereas F1226Y was identified only in Muse (31.8%). Parasite isolates from Laiza showed a median RSA value of 5.0%, significantly higher than the 2.4% in Muse. Altogether, P. falciparum parasite populations from neighboring regions in the GMS may diverge substantially in their resistance to several antimalarial drugs. This information about different parasite populations will guide antimalarial treatment policies to effectively manage drug resistance during malaria elimination.

Collagen Membrane for Guided Bone Regeneration in Dental and Orthopedic Applications.

Treatment of cortical bone defects is a clinical challenge. Guided bone regeneration (GBR), commonly used in oral and maxillofacial dental surgery, may show promise for orthopedic applications in repair of cortical bone defects. However, a limitation in the use of GBR for cortical bone defects is the lack of an ideal scaffold that provides sufficient mechanical support to bridge the cortical bone with minimal interference in the repair process. We have developed a new collagen membrane, CelGro™, for use in GBR. We report the material characterization of CelGro and evaluate the performance of CelGro in translational preclinical and clinical studies. The results show CelGro has a bilayer structure of different fiber alignment and is composed almost exclusively of type I collagen. CelGro was found to be completely acellular and free from xenoantigen, α-gal (galactose-alpha-1,3-galactose). In the preclinical study of a rabbit cortical bone defect model, CelGro demonstrated enhanced bone-remodeling activity and cortical bone healing. Microcomputed tomography evaluation showed early bony bridging over the defect area 30 days postoperatively, and nearly complete restoration of mature cortical bone at the bone defect site 60 days postoperatively. Histological analysis 60 days after surgery further confirmed that CelGro enables bridging of the cortical bone defect by induction of newly formed cortical bone. Compared to a commercially available collagen membrane, Bio-Gide®, CelGro showed much better cortical alignment and reduced porosity at the defect interface. As selection of orthopedic patients with cortical bone defects is complex, we conducted a clinical study evaluating the performance of CelGro in guided bone regeneration around dental implants. CelGro was used in GBR procedures in a total of 16 implants placed in 10 participants. Cone-beam computed tomography images show significantly increased bone formation both horizontally and vertically, which provides sufficient support to stabilize implants within 4 months. Together, the findings of our study demonstrate that CelGro is an ideal membrane for GBR not only in oral and maxillofacial reconstructive surgery but also in orthopedic applications (Clinical Trial ID ACTRN12615000027516).

Polymorphism of Antifolate Drug Resistance in Plasmodium vivax From Local Residents and Migrant Workers Returned From the China-Myanmar Border.

Drug-resistant Plasmodium vivax malaria impedes efforts to control, eliminate, and ultimately eradicate malaria in Southeast Asia. P. vivax resistance to antifolate drugs derives from point mutations in specific parasite genes, including the dihydropteroate synthase (pvdhps), dihydrofolate reductase (pvdhfr), and GTP cyclohydrolase I (pvgch1) genes. This study aims to investigate the prevalence and spread of drug resistance markers in P. vivax populating the China-Myanmar border. Blood samples were collected from symptomatic patients with acute P. vivax infection. Samples with single-clone P. vivax infections were sequenced for pvdhps and pvdhfr genes and genotyped for 6 flanking microsatellite markers. Copy number variation in the pvgch1 gene was also examined. Polymorphisms were observed in six different codons of the pvdhps gene (382, 383, 512, 549, 553, and 571) and six different codons of the pvdhfr gene (13, 57, 58, 61, 99, 117) in two study sites. The quadruple mutant haplotypes 57I/L/58R/61M/117T of pvdhfr gene were the most common (comprising 76% of cases in Myitsone and 43.7% of case in Laiza). The double mutant haplotype 383G/553G of pvdhps gene was also prevalent at each site (40.8% and 31%). Microsatellites flanking the pvdhfr gene differentiated clinical samples from wild type and quadruple mutant genotypes (FST= 0.259-0.3036), as would be expected for a locus undergoing positive selection. The lack of copy number variation of pvgch1 suggests that SP-resistant P. vivax may harbor alternative mechanisms to secure sufficient folate.

[Expression of fragile histidine triad (FHIT) protein and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust].

OBJECTIVE: To study the expression and significance of fragile histidine triad (FHIT) and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust. METHODS: Every second generation of immortalized human bronchial epithelial cells (BEAS-2B) and human embryo lung fibroblasts (WI-38) were exposed to 100 µg/ml Yunnan tin mine dust for 72 h, until the ninth generation. The cells were subsequently co-cultured from the 11th generation. Experimental setup: B group, B (W) group, B (W 100) group, B100 group, B100 (W) group, B100 (W100) group. The expressions of FHIT and Ki-67 in epithelial cells were determined by the method of immunocytochemistry at the 16th, 26th and 36th generation. The percentage of Ki-67 positive cells was calculated as proliferation index. RESULTS: The expression of FHIT was observed in BEAS-2B cells. The expression levels of FHIT among B group, B (W) group and B (W 100) group had not instinctive difference. At the 16th generation, the expression of FHIT in the B100 group was decreased compared with that in the B group and the expression of FHIT between B100 (W) group and B100 (W100) group was lower than that in the B100 group. At the 26th generation, the expression of FHIT was decreased compared with that at the 16th generation in the B100, B100 (W) and B100 (W100) groups. However, At the 36th generation, positive expression were observed again in the B100, B100 (W) and B100 (W100) groups and the expression levels were in incremental order. At the 16th, 26th and 36th generation, the proliferation indexes of B group, B (W) group and B (W 100) group were all < 3%. The proliferation indexes of B100, B100 (W) and B100 (W100) were increased step by step with the generation elongation. CONCLUSIONS: FHIT could be a target at which Yunnan tin mine dust induces transformation of BEAS-2B cells. The proliferation activation of BEAS-2B cells can be improved by Yunnan tin mine dust.

Periplaneta americana Extracts Accelerate Liver Regeneration via a Complex Network of Pathways.

Successful recovery from hepatectomy is partially contingent upon the rate of residual liver regeneration. The traditional Chinese medicines known as Periplaneta americana extracts (PAEs) positively influence wound healing by promoting tissue repair. However, the effect of PAEs on liver regeneration is unknown. We used a mouse liver regeneration model after 70% partial hepatectomy (PH) and a hepatocyte culture to determine whether PAEs can promote liver regeneration as effectively as skin regeneration and establish their modes of action. L02 cells were divided into serum-starved control (NC) and three PAEs (serum starvation + 0.1 mg/ml, 0.5 mg/ml, or 1 mg/ml PAEs) groups. L02 cell proliferation was assessed at 24 h, 48 h, and 72 h by CCK-8 assay. Forty male C57 mice were randomly divided into control (NC), normal saline (NS), PAEs400 (400 mg/kg/d), and PAEs800 (800 mg/kg/d) groups (n = 10 per group). The NS and both PAEs groups were administered normal saline and PAEs, respectively, by gavage for 10 days. Two hours after the tenth gavage, the NS and both PAEs groups were subjected to 70% PH and the residual liver was harvested after 48 h. The hepatic regeneration rate was evaluated and hepatocyte proliferation was estimated by immunohistochemical (IHC) staining for Ki-67. Twelve DEG libraries (three samples per group) were prepared and sequencing was performed in an Illumina HiSeq 2000 (Mus_musculus) at the Beijing Genomics Institute. The genes expressed in the liver tissues and their expression profiles were analyzed by bioinformatics. KEGG was used to annotate, enrich, and analyze the pathways. PAEs promoted hepatocyte proliferation in vitro and in vivo and accelerated mouse liver regeneration after 70% PH. The screening criteria were fold change (FC) ≥ 2 and q-value < 0.001. We identified 1,092 known DEGs in PAEs400 and PAEs800. Of these, 153 were categorized in cellular processes. The KEGG analysis revealed that the aforementioned DEGs participated in several signaling pathways closely associated with cell proliferation including PI3K-Akt, MAPK, Apelin, Wnt, FoxO, mTOR, Ras, VEGF, ErbB, Hippo, and AMPK. It was concluded that PAEs can effectively improve liver regeneration via the synergistic activation of different signaling pathways.

Ex vivo susceptibilities of Plasmodium vivax isolates from the China-Myanmar border to antimalarial drugs and association with polymorphisms in Pvmdr1 and Pvcrt-o genes.

BACKGROUND: Vivax malaria is an important public health problem in the Greater Mekong Subregion (GMS), including the China-Myanmar border. Previous studies have found that Plasmodium vivax has decreased sensitivity to antimalarial drugs in some areas of the GMS, but the sensitivity of P. vivax to antimalarial drugs is unclear in the China-Myanmar border. Here, we investigate the drug sensitivity profile and genetic variations for two drug resistance related genes in P. vivax isolates to provide baseline information for future drug studies in the China-Myanmar border. METHODOLOGY/PRINCIPAL FINDINGS: A total of 64 P. vivax clinical isolates collected from the China-Myanmar border area were assessed for ex vivo susceptibility to eight antimalarial drugs by the schizont maturation assay. The medians of IC50 (half-maximum inhibitory concentrations) for chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate, artemether, dihydroartemisinin were 84.2 nM, 34.9 nM, 4.0 nM, 22.3 nM, 41.4 nM, 2.8 nM, 2.1 nM and 2.0 nM, respectively. Twelve P. vivax clinical isolates were found over the cut-off IC50 value (220 nM) for chloroquine resistance. In addition, sequence polymorphisms in pvmdr1 (P. vivax multidrug resistance-1), pvcrt-o (P. vivax chloroquine resistance transporter-o), and difference in pvmdr1 copy number were studied. Sequencing of the pvmdr1 gene in 52 samples identified 12 amino acid substitutions, among which two (G698S and T958M) were fixed, M908L were present in 98.1% of the isolates, while Y976F and F1076L were present in 3.8% and 78.8% of the isolates, respectively. Amplification of the pvmdr1 gene was only detected in 4.8% of the samples. Sequencing of the pvcrt-o in 59 parasite isolates identified a single lysine insertion at position 10 in 32.2% of the isolates. The pvmdr1 M908L substitutions in pvmdr1 in our samples was associated with reduced sensitivity to chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate and dihydroartemisinin. CONCLUSIONS: Our findings depict a drug sensitivity profile and genetic variations of the P. vivax isolates from the China-Myanmar border area, and suggest possible emergence of chloroquine resistant P. vivax isolates in the region, which demands further efforts for resistance monitoring and mechanism studies.

[Expression of p14(ARF) and E2F-1 in lung cancer in Gejiu and Xuanwei regions of Yunnan Province].

OBJECTIVE: To investigate the correlations between p14(ARF) and E2F-1, and the role of their alterations in the tumorigenesis of the lung cancer in Gejiu and Xuanwei regions in Yunnan Province for providing the important experiment basis in revealing the molecular mechanism and looking for new markers for early diagnosis of lung cancer. METHODS: The expression of p14(ARF) and E2F-1 was detected at theirs protein level by Immunohistochemistry S-P method in 30 specimens of lung cancer of Gejiu tin miners, 30 specimens of lung cancer of Xuanwei peasants and 20 specimens of normal lung tissue. E2F-1 mRNA was detected by ISH in 25 specimens of lung cancer of Gejiu tin miners, 25 specimens of lung cancer of Xuanwei peasants and 10 specimens of normal lung tissue. The positive signals were quantitatively analysed by HPIAS-100. RESULTS: The positive unit (PU) of p14(ARF) and E2F-1 was 16.44 +/- 4.85 and 47.39 +/- 5.43 in Gejiu group, and 16.79 +/- 3.55 and 48.15 +/- 9.11 in Xuanwei group. Expression of p14(ARF) and E2F-1 protein in lung cancer of Gejiu and Xuanwei were statistically different compared with that in the normal lung (P < 0.01) respectively; The PU of E2F-1 mRNA was 48.58 +/- 7.75 in Gejiu group, and 49.41 +/- 8.53 in Xuanwei group, which was higher than that in normal tissue group. The differences were significant (P < 0.01). There was positive correlation between the expression of E2F-1 protein and E2F-1 mRNA in Gejiu group, Xuanwei group and normal group (P < 0.01, r = 0.833). The expression of p14(ARF) protein was significantly negatively correlated with the expression of E2F-1 protein (P < 0.01, r = -0.830). CONCLUSION: There is the over-expression of E2F-1 gene and the deletion of p14(ARF) gene in the tumorigenesis of the lung cancer in Gejiu and Xuanwei regions in Yunnan Province. Over-expression of E2F-1 protein in lung cancer may be caused by enhanced transcription.

[Expression of hTERT and Mad1 in lung cancer in Gejiu and Xuanwei of Yunnan Province].

OBJECTIVE: To study the expression of hTERT mRNA and Mad1 protein in lung cancer of Gejiu and Xuanwei and normal lung tissue and to investigate their correlations with lung cancer. METHODS: Mad1 protein was detected by immunohistochemistry S-P method, and hTERT message RNA (mRNA) was detected by in situ hybridization (ISH) in 40 specimens of lung cancer of Gejiu Tin miners and 20 specimens of lung cancer of Xuanwei peasants and 20 specimens of normal lung tissue. The positive signals were quantitatively analyzed by HPIAS-100. RESULTS: The positive unit (PU) of Mad1 protein was 16.77 +/- 6.01 in Gejiu Tin Miners lung cancer group, and 19.36 +/- 4.54 in Xuanwei peasant lung cancer group, compared with the normal lung tissue (46.05 +/- 7.26). The difference was highly significant (P < 0.01); The PU of hTERT mRNA was 72.10 +/- 13.07 in Gejiu Tin miners lung cancer group, and 74.20 +/- 15.17 in Xuanwei peasant lung cancer group, which was higher than that in normal tissue group (10.70 +/- 2.21). The difference was significant (P < 0.01). The expression of Mad1 protein was negatively correlated with the expression hTERT mRNA (P < 0.05, r = 0.9881, r = -0.999). CONCLUSION: Reduced hTERT mRNA expression may play an important role in the occurrence of lung cancer. The expression of hTERT mRNA and deletion of Mad1 protein are closely related to pathogenesis of lung cancer.

[Interaction between malignant transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust].

OBJECTIVE: To study the interaction between transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust. METHODS: (1) The immortalized human bronchial epithelial cell line BEAS-2B and human embryo lung fibroblast cell line WI-38 were grown in MEM medium containing 5% and 10% FBS, respectively, at 37 degrees C and 5% CO2 with saturated humidity. The cells were subcultured every 6 days. BEAS-2B cells and WI-38 cells were induced with Yunnan tin mine dust on every other generation at the concentration of 100 microg/ml. From the 11th generation, the cells were co-cultured. Epithelial cell transformation was tested using concanavalin A (ConA) agglutination and anchorage-independent growth assays. The cell cycles were analyzed through flow cytometry. The expressions of alpha-SMA in fibroblasts were determined with immunocytochemistry. RESULTS: (1) Cell morphology of mine dust-exposed epithelial cells began to transform at the 28th generation. Similar transformations were observed with mine dust-induced epithelial cells co-cultured with fibroblasts from the 20th generation and mine dust-induce epithelial cells co-cultured with mine dust-induced fibroblasts from the 16th generation. ConA agglutination assay and anchorage-independent growth assays were negative in normal BEAS-2B cells. At the 26 th generation, the agglutination test result of the mine dust-exposed epithelial cells was positive. Co-cultured with fibroblasts and mine dust-exposed fibroblasts, the agglutination time of the mine dust-exposed epithelial cells became short. Epithelial cell anchorage-independent growth assay was positive for mine dust-exposed epithelial cells co-cultured with fibroblasts at the 36th generations and for mine dust-exposed epithelial cells co-cultured with mine dust-exposed fibroblasts at the 26th generations. The clone formation rate of the 26th generation was 6.00 per thousand +/- 1.00 per thousand and 15.33 per thousand +/- 2.52 per thousand respectively, with the significant differences (P < 0.05). With generation adding, the portion of S phase increased for mine dust-exposed epithelial cells. (2) At the 26th generations, fibroblasts expressed alpha-SMA. Co-cultured with epithelial cell, the alpha-SMA expression of fibroblasts increased. Especially, positive cell numbers and intensity of staining dramatically increased with generation adding. CONCLUSIONS: (1) The tin mine dust can induce malignant transformation of human pulmonary epithelial cells BEAS-2B and activation of fibroblasts WI-38. (2) The epithelial cells are major target in carcinogenesis induced by Yunnan tin mine dust. (3) Transformation of epithelia and activation of fibroblasts co-evolve in the developing process of induced lung cancer by Yunnan tin mine dust.

Alterations of 63 hub genes during lingual carcinogenesis in C57BL/6J mice.

To identify potential biomarkers of lingual cancer, 75 female C57BL/6J mice were subjected to 16-week oral delivery of 4-nitroquinoline-1-oxide (4NQO; 50 mg/L), with 10 mice used as controls. Lingual mucosa samples representative of normal tissue (week 0) and early (week 12) and advanced (week 28) tumorigenesis were harvested for microarray and methylated DNA immunoprecipitation sequencing (MeDIP-Seq). Combined analysis with Short Time-series Expression Miner (STEM), the Cytoscape plugin cytoHubba, and screening of differentially expressed genes enabled identification of 63 hub genes predominantly altered in the early stage rather than the advanced stage. Validation of microarray results was carried out using qRT-PCR. Of 63 human orthologous genes, 35 correlated with human oral squamous cell carcinoma. KEGG analysis showed "pathways in cancer", involving 13 hub genes, as the leading KEGG term. Significant alterations in promoter methylation were confirmed at Tbp, Smad1, Smad4, Pdpk1, Camk2, Atxn3, and Cdh2. HDAC2, TBP, and EP300 scored ≥10 on Maximal Clique Centrality (MCC) in STEM profile 11 and were overexpressed in human tongue cancer samples. However, expression did not correlate with smoking status, tumor differentiation, or overall survival. These results highlight potentially useful candidate biomarkers for lingual cancer prevention, diagnosis, and treatment.

Longitudinal surveillance of drug resistance in Plasmodium falciparum isolates from the China-Myanmar border reveals persistent circulation of multidrug resistant parasites.

Multidrug-resistant Plasmodium falciparum in the Greater Mekong Subregion of Southeast Asia is a major threat to malaria elimination and requires close surveillance. In this study, we collected 107 longitudinal clinical samples of P. falciparum in 2007-2012 from the malaria hypoendemic region of the China-Myanmar border and measured their in vitro susceptibilities to 10 antimalarial drugs. Overall, parasites had significantly different IC50 values to all the drugs tested as compared to the reference 3D7 strain. Parasites were also genotyped in seven genes that were associated with drug resistance including pfcrt, pfmdr1, pfmrp1, pfdhfr, pfdhps, pfnhe1, and PfK13 genes. Despite withdrawal of chloroquine and antifolates from treating P. falciparum, parasites remained highly resistant to these drugs and mutations in pfcrt, pfdhfr, and pfdhps genes were highly prevalent and almost reached fixation in the study parasite population. Except for pyronaridine, quinine and lumefantrine, all other tested drugs exhibited significant temporal variations at least between some years, but only chloroquine and piperaquine had a clear temporal trend of continuous increase of IC50s. For the pfmrp1 gene, several mutations were associated with altered sensitivity to a number of drugs tested including chloroquine, piperaquine, lumefantrine and dihydroartemisinin. The association of PfK13 mutations with resistance to multiple drugs suggests potential evolution of PfK13 mutations amid multidrug resistance genetic background. Furthermore, network analysis of drug resistance genes indicated that certain haplotypes associated multidrug resistance persisted in these years, albeit there were year-to-year fluctuations of the predominant haplotypes.

[Expression of survivin and PCNA in lung cancer cells induced by Gejiu mineral powder].

BACKGROUND: Gejiu in Yunnan Province is a region where the incidence of lung cancer is high among the miners of tin mine. Our previous research team successfully induced the malignant transformation of immortalized human bronchial epithelial cells (BEAS-2B) by Gejiu mineral powder in vitro. The objective of this study is to explore the role of survivin and proliferating cell nuclear antigen (PCNA) in the pathway of the malignant transformation of BEAS-2B induced by Gejiu mineral powder. METHODS: Protein expression of survivn and PCNA in BEAS-2B cells and its malignant transformation cells was respectively evaluated by immunofluorescence cytochemistry staining technique and immunohistochemistry. RESULTS: (1)The expression of survivin protein was negative in BEAS-2B cells and was positive in its malignant transformation cells by immunofluorescence cytochemistry staining techniques. And the protein level of PCNA was low in BEAS-2B cells and high in its malignant transformation cells. (2)The positive expression of survivin protein in BEAS-2B cells (0/20) was significantly lower than that in its malignant transformation cells (85%, 17/20) by immunohistochemistry (P < 0.001). And the labelling index (LI) of PCNA in BEAS-2B cells was significantly lower than that in its malignant transformation cells (P < 0.001). LI of PCNA in the malignant transformation cells of BEAS-2B with positive expression of survivin was significantly higher than that with negative expression of survivin (P < 0.001). CONCLUSIONS: (1)The up-regulation expression of survivin in malignant transforma- tion cells of BEAS-2B suggests that survivin may play an important role in the pathway of malignant transformation in BEAS-2B cells induced by Gejiu mineral powder. It may give a proof for revealing the carcinogenesis of lung cancer in Gejiu miner. Survivin may be identified as a novel potential diagnostic and therapeutic target of lung cancer in Gejiu miner. (2)The up-regulation expression of PCNA in malignant transformation cells of BEAS-2B suggests that cell proliferation may play an important role in the pathway of malignant transformation. (3)Survivin may promote cell proliferation mediated by PCNA. Survivn and PCNA may play synergetic roles in the process of malignant transformation in BEAS-2B cells induced by Gejiu mineral powder.

[Pathological study of lung cancer induced by Yunnan tin mine dusts in F344 rats].

OBJECTIVE: To set up animal models of the lung cancer induced by Yunnan tin mineral dusts (no radon) in F344 rats and to explore the process of carcinogenesis and pathologic alterations in various stages of malignant transformation in the animal models. METHODS: One hundred and ninety F344 rats were randomly divided into Yunnan tin mineral dust group (100 rats), furfural physiological saline group (30 rats), physiological saline group (30 rats) and normal control group (30 rats). The intratracheal instillation with mass fraction of 6% suspension liquid mixture Yunnan tin mineral dusts, volume fraction of 2% furfural physiological saline and physiological saline 0.2 ml was performed in the rates once per week respectively except normal control group. Then the rats were sacrificed in batch periodically after one week. The last rat was exposed to the tin mine dusts for 100 weeks. The morphological process and tumor formation were dynamically observed under LM and TEM. Immunohistochemistry detection of cytokeratin of High MW and low MW was used for tumor classification. Pollak stein was used to evaluate the development of fibrosis of lung in the rats. RESULTS: Bronchoalveolar inflammation occurred in the early stage after the intratracheal instillation of Yunnan tin mineral dust was performed in F344 rates. Along with reduction of inflammation, collagen fibrils increased at alveolar interstices. Simple hyperplasia, papillary hyperplasia and metaplasia of the epithelial cells in alveolar and bronchi were observed, followed by atypical adenomatous hyperplasia and squamous dysplasia. Lung cancer was induced in the end. Among the 14 cases of lung cancer, 9 cases were adenocarcinoma, 2 squamous cell carcinoma and 3 mixed carcinoma. No lung cancer occurred in other three control groups. There was a significant difference in the malignancy rate between the experimental group and the three control groups (P < 0.01). The squamous metaplasia and squamous carcinoma were found in alveoli that expressed cytokeratin of High MW. Lung fibrosis was found in 31 cases of in the tin mineral dust group. The greater the mineral dust deposit was, the more serious the alveolar fibrosis was. CONCLUSION: Yunnan tin mineral dusts without radon induce lung cancer in rates. The adenocarcinoma and squamous carcinomas induced in F344 rat lung can occur in the alveoli. The further study on whether type II alveolar epithelial cells are the origin cells of adenocarcinoma and some peripheral squamous lung carcinomas is worthwhile.

Genetic diversity of Plasmodium falciparum populations in southeast and western Myanmar.

BACKGROUND: The genetic diversity of malaria parasites reflects the complexity and size of the parasite populations. This study was designed to explore the genetic diversity of Plasmodium falciparum populations collected from two southeastern areas (Shwekyin and Myawaddy bordering Thailand) and one western area (Kyauktaw bordering Bangladesh) of Myanmar. METHODS: A total of 267 blood samples collected from patients with acute P. falciparum infections during 2009 and 2010 were used for genotyping at the merozoite surface protein 1 (Msp1), Msp2 and glutamate-rich protein (Glurp) loci. RESULTS: One hundred and eighty four samples were successfully genotyped at three genes. The allelic distributions of the three genes were all significantly different among three areas. MAD20 and 3D7 were the most prevalent alleles in three areas for Msp1 and Msp2, respectively. The Glurp allele with a bin size of 700-750 bp was the most prevalent both in Shwekyin and Myawaddy, whereas two alleles with bin sizes of 800-850 bp and 900-1000 bp were the most prevalent in the western site Kyauktaw. Overall, 73.91% of samples contained multiclonal infections, resulting in a mean multiplicity of infection (MOI) of 1.94. Interestingly, the MOI level presented a rising trend with the order of Myawaddy, Kyauktaw and Shwekyin, which also paralleled with the increasing frequencies of Msp1 RO33 and Msp2 FC27 200-250 bp alleles. Msp1 and Msp2 genes displayed higher levels of diversity and higher MOI rates than Glurp. PCR revealed four samples (two from Shwekyin and two from Myawaddy) with mixed infections of P. falciparum and P. vivax. CONCLUSIONS: This study genotyped parasite clinical samples from two southeast regions and one western state of Myanmar at the Msp1, Msp2 and Glurp loci, which revealed high levels of genetic diversity and mixed-strain infections of P. falciparum populations at these sites. The results indicated that malaria transmission intensity in these regions remained high and more strengthened control efforts are needed. The genotypic data provided baseline information for monitoring the impacts of malaria elimination efforts in the region.

A more appropriate white blood cell count for estimating malaria parasite density in Plasmodium vivax patients in northeastern Myanmar.

The conventional method of estimating parasite densities employ an assumption of 8000 white blood cells (WBCs)/µl. However, due to leucopenia in malaria patients, this number appears to overestimate parasite densities. In this study, we assessed the accuracy of parasite density estimated using this assumed WBC count in eastern Myanmar, where Plasmodium vivax has become increasingly prevalent. From 256 patients with uncomplicated P. vivax malaria, we estimated parasite density and counted WBCs by using an automated blood cell counter. It was found that WBC counts were not significantly different between patients of different gender, axillary temperature, and body mass index levels, whereas they were significantly different between age groups of patients and the time points of measurement. The median parasite densities calculated with the actual WBC counts (1903/µl) and the assumed WBC count of 8000/µl (2570/µl) were significantly different. We demonstrated that using the assumed WBC count of 8000 cells/µl to estimate parasite densities of P. vivax malaria patients in this area would lead to an overestimation. For P. vivax patients aged five years and older, an assumed WBC count of 5500/µl best estimated parasite densities. This study provides more realistic assumed WBC counts for estimating parasite densities in P. vivax patients from low-endemicity areas of Southeast Asia.