Polymorphic microsatellite loci isolated from Cervus unicolor (Cervidae) show inbreeding in a domesticated population of Taiwan Sambar deer.

Primers for eight microsatellites were developed; they successfully amplified DNA from 20 domesticated Formosan Sambar deer (Cervus unicolor swinhoei). All loci were polymorphic, with 10-19 alleles per locus. The average observed heterozygosity across loci and samples was 0.310, ranging from 0 to 0.750 at each locus. All loci but one, CU18, deviated from Hardy-Weinberg equilibrium due to excessive homozygosity in these domesticated broodstocks, reflecting inbreeding. These microsatellite loci will be useful, not only for assessment of population structure and genetic variability, but also for conservation of wild deer populations in Taiwan.

Physicochemical properties of radiopaque dicalcium silicate cement as a root-end filling material in an acidic environment.

AIM: To investigate the effect of two solutions differing by pH (7.4 and 4.0) on the physicochemical properties of a radiopaque dicalcium silicate cement. METHODOLOGY: The cement was prepared by hand-mixing the dicalcium silicate powder with distilled water in a liquid-to-powder ratio of 0.4 mL g(-1) . A total of 253 cement specimens with dimension of 6 mm (diameter) × 3 mm (height) were used. The morphology, weight loss, porosity and diametral tensile strength of the cement were evaluated after soaking in a solution for different time intervals, in addition to pH changes in the cement-immersed solutions. RESULTS: After soaking in a pH 7.4 solution for 1 day, the particle size of precipitated apatite spherulites on the cement surfaces was greater than that obtained in a pH 4.0 solution. Solution pH did not result in a significant difference (P > 0.05) in diametral tensile strength of cement specimens at the same soaking time-point. On day 30, the sample was associated with a weight loss of 0.8% in a pH 4.0 solution, whereas in a pH 7.4 solution, a weight increase of 0.2% occurred. A greater porosity of the cement soaked in a pH 4.0 was found compared with that in the solution with pH 7.4. Soaking time affected significantly (P < 0.05) the porosity, weight change and strength of the cements in an acidic environment more than at pH 7.4. CONCLUSIONS: High apatite-forming activity and low degradation were the characteristics of a radiopaque dicalcium silicate cement.

Population structure and demographic history of Sicyopterus japonicus (Perciformes; Gobiidae) in Taiwan inferred from mitochondrial control region sequences.

The amphidromous goby Sicyopterus japonicus is distributed throughout southern Taiwan and Japan. Larvae of this freshwater fish go through a long marine stage. This migratory mode influences population genetic structure. We examined the genetic diversity, population differentiation, and demographic history of S. japonicus based on the mitochondrial DNA control region. We identified 102 haplotypes from 107 S. japonicus individuals from 22 populations collected from Taiwan and Islet Lanyu. High mean haplotype diversity (h = 0.999) versus low nucleotide diversity (θπ = 0.008) was detected across populations. There was low correspondence between clusters identified in the neighbor-joining tree and geographical region, as also indicated by AMOVA and pairwise F(ST) estimates. Both mismatch distribution analysis and Tajima's D test indicated that S. japonicus likely experienced a demographic expansion. Using a Bayesian skyline plot approach, we estimated the time of onset of the expansion of S. japonicus at 135 kyr (during the Pleistocene) and the time of stable effective population size at approximately 2.5 kyr (last glacial maximum). Based on these results, we suggest 1) a panmictic population at the oceanic planktonic larval stage, mediated by the Kuroshio current; 2) a long planktonic marine stage and long period of dispersal, which may have permitted efficient tracking of environmental shifts during the Pleistocene; and 3) a stable, constant population size ever since the last glacial maximum.

Endoscope-assisted transoral excision of a huge parapharyngeal pleomorphic adenoma.

The treatment of choice for a parapharyngeal pleomorphic adenoma is total surgical resection. We describe an endoscope-assisted transoral excision of a huge parapharyngeal pleomorphic adenoma, and discuss the benefits of this type of operation.

Multiple factors have shaped the phylogeography of Chinese spiny loach Cobitis sinensis in Taiwan as inferred from mitochondrial DNA variation.

Mitochondrial DNA cytochrome b sequences (1140 bp) in 61 specimens of Chinese spiny loach Cobitis sinensis from 12 drainages in Taiwan were identified as two major clades, exhibiting a southern and a northern distribution, north of TzengWen and south of TzengWen (including TzengWen), respectively. The divergence time between these two phylogroups was estimated at 7.34-9.06 million years before present (B.P.), but these two phylogroups were formed c. 3.41-4.23 and 2.22-2.75 M B.P., respectively. Moreover, geological events have been recalculated that Taiwan Island emerged above sea level at an estimate of c. 4-5 M B.P., and quickly became its present shape at c. 2 M B.P. through mountain building. These results suggest that these two major clades of C. sinensis in Taiwan might originate from two different continental populations, since the island's initial isolation in the Pliocene. Within southern Taiwan, the initial colonization was hypothesized to be in KaoPing River, followed by its northward dispersal. The high divergence between KaoPing and TzengWen was influenced by glaciations and landforms. Within north Taiwan, the colonization was from the Miaoli Plateau through western Taiwan to north-eastern and northern Taiwan. This dispersal pattern is concordant with the previously proposed hypothesis. Apparently, both geological and phylogeographic evidence suggested that river capture of the upper Takia River by the LanYang River promoted range expansion in freshwater fishes and also indicated that the Central Range within Taiwan did not act as a barrier to the dispersal of C. sinensis.

Evaluation of methanogenic treatment of TMAH (tetra-methyl ammonium hydroxide) in a full-scale TFT-LCD wastewater treatment process.

This study evaluated TMAH biodegradation under methanogenic conditions. Under methanogenic conditions, a sludge from a full-scale UASB treating TFT-LCD wastewater was able to degrade 2,000 mg/L of TMAH within 10 h and attained a specific degradation rate of 19.2 mgTMAH/gVSS-h. Furthermore, several chemicals including some surfactants, DMSO, and sulfate were examined for their potential inhibitory effects on TMAH biodegradation under methanogenic conditions. The results indicated that surfactant S1 (up to 2%) and DMSO (up to 1,000 mg/L) presented negligible inhibitory effects on TMAH degradation, while surfactant S2 (0.2-1%) might inhibit methanogenic reaction without any TMAH degradation for 3-5 h. At sulfate concentrations higher than 300 mg/L, a complete inhibition of methanogenic reaction and TMAH biodegradation was observed. Results from cloning and sequencing of archaeal 16S rRNA gene fragments showed that Methanosarcina barkeri and Methanosarcina mazei were the dominant methanogens in the UASB treating TMAH-containing TFT-LCD wastewater.

Low-energy helium-neon laser induces melanocyte proliferation via interaction with type IV collagen: visible light as a therapeutic option for vitiligo.

BACKGROUND: The treatment of vitiligo remains a challenge for clinical dermatologists. We have previously shown that the helium-neon laser (He-Ne laser, 632.8 nm) is a therapeutic option for treatment of this depigmentary disorder. OBJECTIVES: Addressing the intricate interactions between melanocytes, the most important cellular component in the repigmentation scheme of vitiligo, and their innate extracellular matrix collagen type IV, the current study aimed to elucidate the effects of the He-Ne laser on melanocytes. METHODS: Cultured melanocytes were irradiated with the He-Ne laser. Relevant biological parameters including cell attachment, locomotion and growth were evaluated. In addition, the potentially involved molecular pathways were also determined. RESULTS: Our results show that in addition to suppressing mobility but increasing attachment to type IV collagen, the He-Ne laser stimulates melanocyte proliferation through enhanced alpha2beta1 integrin expression. The expression of phosphorylated cyclic-AMP response element binding protein (CREB), an important regulator of melanocyte growth, was also upregulated by He-Ne laser treatment. Using a specific mitochondrial uncoupling agent [carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)], the proliferative effect of the He-Ne laser on melanocytes was abolished and suppression of melanocyte growth was noted. CONCLUSIONS: In summary, we have demonstrated that the He-Ne laser imparts a growth stimulatory effect on functional melanocytes via mitochondria-related pathways and proposed that other minor pathways including DNA damage may also be inflicted by laser treatment on irradiated cells. More importantly, we have completed the repigmentation scheme of vitiligo brought about by He-Ne laser light in vitro and provided a solid theoretical basis regarding how the He-Ne laser induces recovery of vitiligo in vivo.

Renal Function Changes Under Everolimus Plus Cyclosporine or Everolimus Plus Tacrolimus After Heart Transplantation.

Everolimus (EVR) can be used with calcineurin inhibitors to reduce the risk of renal dysfunction, with similar immunosuppressive effect. In this study, we compared renal function after heart transplantation (HT) under EVR with cyclosporine (CSA) or tacrolimus (TAC). Between 2004 and 2014, EVR with CSA or TAC was used in 117 HT at the National Taiwan University Hospital. After HT, all patients received corticosteroid, EVR (C0 target 3-8 ng/mL) and CSA (C0 blood level 100-200 ng/mL), or TAC (Co blood level 5-10 ng/mL). Renal function was evaluated before HT, every month after HT for up to 1 year, and then every 3 months for up to 2 years. Blood-drug levels of EVR, CSA, and TAC were also monitored simultaneously with renal function. The estimated mean glomerular filtration rate (eGFR) was 76.5 mL/min/1.73 m2 before HT. After HT, the eGFR was 64 mL/min/1.73 m2 at the third month, and 64 mL/min/1.73 m2 at the end of first year. The difference was significant between pre-HT and post-HT (P = .00) during the first year. No significant differences were noted between the CSA and TAC groups. Careful monitoring of blood-drug level and renal function is crucial after heart transplantation. It is concluded that under close monitoring blood-drug level and renal function, it is possible to reach acceptable postoperative renal function with no difference of renal function between EVR plus CSA and EVR plus TAC.

Outcomes of Heart Transplant Recipients With Preexisting Malignancies.

BACKGROUND: Ensuring careful selection of heart transplant recipients with pretransplant malignancies (PTM) has been suggested in several retrospective studies. However, cancer survival rates continue to increase and we still lack outcomes data on PTM patients who have undergone heart transplantation (HT) within the Asian region. Herein we report pretransplant characteristics and outcomes among PTM patients with HT. METHODS: A total of 354 patients underwent HT from January 2004 to January 2016. Eight of these patients had a history malignancy that was being treated before transplantation. Posttransplant outcomes and clinical characteristics were collected and possible prognostic factors analyzed. RESULTS: The median age of the patients with a preexisting malignancy was 60 years. The PTM group included 5 males and 3 females, with a median duration of follow-up of 43 months. In this group there were 2 patients with lymphoma after chemotherapy, 1 with colon cancer postoperatively, and 1 was on chemotherapy. In the other 4 patients, nasopharyngeal cancer, thyroid cancer, breast cancer, and endometrial cancer were identified, and each had undergone treatment. Only 1 premalignancy patient, with nasopharyngeal cancer, had disease recurrence. The 5-year overall survival of these patients was 50.0 ± 17.7%, but 5-year survival for those without PTM was 68.7 ± 2.0%. CONCLUSION: PTM was 2.3% in our cohort. PTM is associated with an increased risk of all-cause mortality. Thus, our findings suggest careful consideration when selecting PTM patients for HT.

Human T-lymphotropic Virus Type II Infection inVietnamese Thalassemic Patients.

Anti-human T-lymphotropic virus type I/II (HTLV-I/II) antibodies were screened by particle agglutination test in a total of 66 patients with thalassemia major who received multiple transfusion from paid donors at the Blood Transfusion Hematology Center of Ho Chi Minh City in South Vietnam. HTLV-II infection was confirmed in 6 patients (9.1%) by Western blot analysis and/or polymerase chain reaction. Phylogenetic analysis revealed that long terminal repeat sequences of HTLV-II proviruses from 5 thalassemic patients in Vietnam belonged to the same phylogenetic subgroup of HTLV-IIb as those from intravenous drug abusers in North America and Europe. These data shed light on the route of introducing HTLV-II into Vietnam.

Relapse of imported vivax malaria despite standard-dose primaquine therapy: an investigation with molecular genotyping analyses.

Taiwan CDC investigated four cases of recurrent imported vivax malaria during 2003-2010. Molecular genotyping results and the lack of inter-episodes travel history indicated that two of the patients, who acquired vivax malaria in Indonesia and the Solomon Islands, respectively, suffered relapses after an interval of 3-4 months, despite completing standard-dose primaquine therapy (30 mg/day for 14 days) for the first episode. Treatment with a higher dose of primaquine (60 mg/day for 14 days) prevented further relapse in both patients. This finding calls for further monitoring of the therapeutic efficacy of primaquine in treating Plasmodium vivax acquired in southeast Asia and Oceania.

Contrasting phylogeographical patterns between mainland and island taxa of the Pinus luchuensis complex.

Species whose geographical distribution encompasses both mainland and island populations provide an ideal system for examining isolation and genetic divergence. In this study, paternally transmitted chloroplast DNA (cpDNA) and maternally transmitted mitochondrial DNA (mtDNA) were used to estimate population structure and phylogeography of Pinus luchuensis, a species found in eastern China (ssp. hwangshanensis), Taiwan (ssp. taiwanensis), and the Ryukyu Archipelago (ssp. luchuensis). Gene genealogies of both mtDNA and cpDNA reveal two major lineages. Molecular dating indicates that these lineages diverged before the colonization of P. luchuensis subspecies in Taiwan and the Ryukyu Archipelago. Both mtDNA and cpDNA show a lack of correspondence between molecular phylogeny and subspecies designation. Phylogeographical analysis suggests that paraphyly of the subspecies is the result of recent divergence rather than secondary contacts. In spite of the short divergence history of P. luchuensis on islands, the island populations show the same degree of genetic divergence as mainland populations. Low levels of genetic diversity in the mainland ssp. hwangshanensis suggest demographic bottlenecks. In contrast, the high heterogeneity of genetic composition for island populations is likely to be associated with a history of multiple colonization from the mainland. The spatial apportionment of organelle DNA polymorphisms is consistent with a pattern of stepwise colonization on island populations.

Population structure of wild bananas, Musa balbisiana, in China determined by SSR fingerprinting and cpDNA PCR-RFLP.

Both demographic history and dispersal mechanisms influence the apportionment of genetic diversity among plant populations across geographical regions. In this study, phylogeography and population structure of wild banana, Musa balbisiana, one of the progenitors of cultivated bananas and plantains in China were investigated by an analysis of genetic diversity of simple sequence repeat (SSR) fingerprint markers and cpDNA PCR-RFLP. A chloroplast DNA (cpDNA) genealogy of 21 haplotypes identified two major clades, which correspond to two geographical regions separated by the Beijiang and Xijiang rivers, suggesting a history of vicariance. Significant genetic differentiation was detected among populations with cpDNA markers, a result consistent with limited seed dispersal in wild banana mediated by foraging of rodents. Nuclear SSR data also revealed significant geographical structuring in banana populations. In western China, however, there was no detected phylogeograpahical pattern, possibly due to frequent pollen flow via fruit bats. In contrast, populations east of the Beijiang River and the population of Hainan Island, where long-range soaring pollinators are absent, are genetically distinct. Colonization-extinction processes may have influenced the evolution of Musa populations, which have a metapopulation structure and are connected by migrating individuals. Effective gene flow via pollen, estimated from the nuclear SSR data, is 3.65 times greater than gene flow via seed, estimated from cpDNA data. Chloroplast and nuclear DNAs provide different insights into phylogeographical patterns of wild banana populations and, taken together, can inform conservation practices.

Phylogeography of the Qinghai-Tibetan Plateau endemic Juniperus przewalskii (Cupressaceae) inferred from chloroplast DNA sequence variation.

The vegetation of the northeast Qinghai-Tibetan Plateau is dominated by alpine meadow and desert-steppe with sparse forests scattered within it. To obtain a better understanding of the phylogeography of one constituent species of the forests in this region, we examined chloroplast trnT-trnF and trnS-trnG sequence variation within Juniperus przewalskii, a key endemic tree species. Sequence data were obtained from 392 trees in 20 populations covering the entire distribution range of the species. Six cpDNA haplotypes were identified. Significant population subdivision was detected (G(ST) = 0.772, N(ST) = 0.834), suggesting low levels of recurrent gene flow among populations and significant phylogeographic structure (N(ST) > G(ST), P < 0.05). Eight of the nine disjunct populations surveyed on the high-elevation northeast plateau were fixed for a single haplotype (A), while the remaining, more westerly population, contained the same haplotype at high frequency together with two low frequency haplotypes (C and F). In contrast, most populations that occurred at lower altitudes at the plateau edge were fixed or nearly fixed for one of two haplotypes, A or E. However, two plateau edge populations had haplotype compositions different from the rest. In one, four haplotypes (A, B, D and E) were present at approximately equivalent frequencies, which might reflect a larger refugium in the area of this population during the last glacial period. Phylogenetic analysis indicated that the most widely distributed haplotype A is not ancestral to other haplotypes. The contrasting phylogeographic structures of the haplotype-rich plateau edge area and the almost haplotype-uniform plateau platform region indicate that the plateau platform was recolonized by J. przewalskii during the most recent postglacial period. This is supported by the findings of a nested clade analysis, which inferred that postglacial range expansion from the plateau edge followed by recent fragmentation is largely responsible for the present-day spatial distribution of cpDNA haplotypes within the species.

Lineage sorting accounting for the disassociation between chloroplast and mitochondrial lineages in oaks of southern France.

Dumolin-Lapégue et al. (Mol. Biol. Evol. 15: 1321-1331. 1998) suggested that recurrent inversions of a 4-bp sequence of the mtDNA nad4-1/2 locus due to intramolecular recombination were responsible for the disassociation of chloroplast and mitochondrial genomes of French oaks. Based on their PCR-RFLP (PCR-restriction fragment length polymorphism) data obtained from three noncoding spacers, a minimum spanning network representing the phylogeny of the cpDNA was reconstructed. The mapping of alleles b and c of the mtDNA nad4-1/2 locus on the cpDNA network revealed a nonrandom distribution, which contradicted the expected patterns when repeated, and ongoing inversions had been occurring. The fact that polymorphisms (a mixed c + d type) were mostly restricted to the interior nodes of the network, which represented ancient haplotypes and geographically coincided with probable glacial refugia in southern Europe, agreed with a migrant-pool model. Evidence of a widespread pattern of polymorphism distribution indicated that mtDNA haplotypes were likely to be more ancient than the cpDNA haplotypes. Lineage sorting, due to relative age of cpDNA vs. mtDNA, plus the specific migratory mode, which recruited colonists from a random sample of resource populations during glacial expansion (thereby extending the lineage sorting period, LSP), may have resulted in the disassociation of chloroplast and mitochondrial genomes in oaks.

Removal of cadmium ions in wastewater through biosorption.

Cadmium ions in wastewater can be removed by biosorption on to Acinetobacter calca var. antratus, isolated from a wastewater sample from a copper refinery. There are two equillibrium states: the first, which is reached quickly, is probably direct adsorption, and the second may be due to the release of ion adsorption materials by lysed cells.

Binding affinity and functional significance of NIT2 and NIT4 binding sites in the promoter of the highly regulated nit-3 gene, which encodes nitrate reductase in Neurospora crassa.

In the filamentous fungus Neurospora crassa, both the global-acting regulatory protein NIT2 and the pathway-specific regulatory protein NIT4 are required to turn on the expression of the nit-3 gene, which encodes nitrate reductase, the first enzyme in the nitrate assimilatory pathway. Three NIT2 binding sites and two NIT4 binding sites have been identified in the 1.3-kb nit-3 promoter region via mobility shift and footprinting experiments with NIT2-beta-galactosidase and NIT4-beta-Gactosidase fusion proteins. Quantitative mobility shift assays were used to examine the affinity of individual NIT2 binding sites for the native NIT2 protein present in N. crassa nuclear extracts. In vivo analysis of nit-3 promoter 5' deletion constructs and individual NIT2 and NIT4 binding-site deletions or mutations revealed that all of the NIT2 and NIT4 binding sites are required for the full level of expression of the nit-3 gene. A cluster of two NIT2 and two NIT4 binding sites located more than 1 kb upstream of the translational start site is required for nit-3 expression, and one NIT2 binding site and one NIT4 site, which are immediately adjacent to each other, are of particular functional importance. A significant NIT2-NIT4 protein-protein interaction might occur upon their binding to nearby sites.

DNA recognition by the NIT2 nitrogen regulatory protein: importance of the number, spacing, and orientation of GATA core elements and their flanking sequences upon NIT2 binding.

NIT2, a global positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes in the nitrogen regulation circuit. NIT2 binding sites in the promoter region of the nit-3, alc, and lao genes are very different in sequence context except for the presence of at least two copies of a GATA core sequence. Changing a single nucleotide of only one of two closely spaced GATA core elements abolished NIT2 binding, demonstrating their importance for NIT2 binding. The effect of altering the number, orientation, or spacing of paired GATA elements and the importance of 5'- and 3'-flanking sequences upon NIT2 binding were examined. Strong binding sites for a NIT2-beta GAL fusion protein appear to contain at least two GATA elements, which can have varied spacing but must be within a certain effective distance, approximately 30 bp, of each other. Surprisingly, the orientation of GATA elements and their flanking sequences have only modest effects upon NIT2 binding.

Molecular evolution and phylogeny of the atpB-rbcL spacer of chloroplast DNA in the true mosses.

The nucleotide variation of a noncoding region between the atpB and rbcL genes of the chloroplast genome was used to estimate the phylogeny of 11 species of true mosses (subclass Bryidae). The A+T rich (82.6%) spacer sequence is conserved with 48% of bases showing no variation between the ingroup and outgroup. Rooted at liverworts, Marchantia and Bazzania, the monophyly of true mosses was supported cladistically and statistically. A nonparametric Wilcoxon Signed-Ranks test Ts statistic for testing the taxonomic congruence showed no significant differences between gene trees and organism trees as well as between parsimony trees and neighbor-joining trees. The reconstructed phylogeny based on the atpB-rbcL spacer sequences indicated the validity of the division of acrocarpous and pleurocarpous mosses. The size of the chloroplast spacer in mosses fits into an evolutionary trend of increasing spacer length from liverworts through ferns to seed plants. According to the relative rate tests, the hypothesis of a molecular clock was supported in all species except for Thuidium, which evolved relatively fast. The evolutionary rate of the chloroplast DNA spacer in mosses was estimated to be (1.12 +/- 0.019) x 10(-10) nucleotides per site per year, which is close to the nonsynonymous substitution rates of the rbcL gene in the vascular plants. The constrained molecular evolution (total nucleotide substitutions, K approximately 0.0248) of the chloroplast DNA spacer is consistent with the slow evolution in morphological traits of mosses. Based on the calibrated evolutionary rate, the time of the divergence of true mosses was estimated to have been as early as 220 million years ago.

Characterization and phylogenetic analysis of a novel hepatitis D virus strain discovered by restriction fragment length polymorphism analysis.

The hepatitis D virus (HDV) genotypes in 46 HDV-infected patients and 12 prostitutes were screened with Xhol restriction fragment length polymorphism (RFLP) analysis of reverse transcription PCR products of viral genomes and verified by phylogenetic analysis. The amplificates of three (6.5%) patients and two (17%) prostitutes showed a novel RFLP pattern different from those of the three known genotypes. Complete HDV genomic sequence identities between isolates with a novel RFLP and the HDV genotypes I, II and III were 72.3, 77.2 and 63.0%, respectively. Importantly, divergence was mostly seen in various regions related to replication or packaging. The novel isolates formed a monophyletic group (P < 0.05) and were most closely related to genotype II.