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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Chloroplast DNA phylogeography of Cunninghamia konishii (Cupressaceae), an endemic conifer of Taiwan.

In this study, we investigated the genetic structure and phylogeographic pattern of the genus Cunninghamia, a member of the Cupressaceae restricted to mainland China and Taiwan, based on sequences of the trnD-trnT noncoding spacer of the chloroplast DNA. Maternal inheritance of chloroplasts was determined experimentally. No paternal leakage was detected. Both parsimony and neighbor-joining analyses revealed the polyphyly of Cunninghamia konishii, populations of which were nested in clades of C. lanceolata from mainland China. The nucleotide diversity of chloroplast DNA sequences within C. konishii (0.0118) was higher than that between species (0.0104), which agrees with a previous allozyme investigation. Based on mutational differences between sequences, a minimum spanning network consisting of five clades was constructed. Significant genetic differentiation (phiST = 0.130, P < 0.001) was detected between the clades based on AMOVA analyses. We infer several possible refugia in the Yunnan, Zhejiang, and Guangdong provinces of south China, all located in the minimum network as interior nodes. We also infer possible migration routes of Cunninghamia populations. The phylogeographic pattern shown in the reconstructed network suggests that the present-day Cunninghamia populations in Taiwan were derived from six different sources in continental Asia via long-distance seed dispersal. A migrant-pool model explains the heterogeneous composition of the organelle DNA in Taiwan's populations and the low differentiation between populations of Taiwan and China (phiCT = 0.012, P = 0.454). In contrast with the genetic heterogeneity within geographic populations, many local populations have attained coalescence at the trnD-trnT alleles, which has led to significant differentiation at the population level.

Organelle DNA phylogeography of Cycas taitungensis, a relict species in Taiwan.

The phylogegraphic pattern of Cycas taitungensis, an endemic species with two remaining populations in Taiwan, was investigated based on genetic variability and phylogeny of the atpB-rbcL noncoding spacer of chloroplast DNA (cpDNA) and the ribosomal DNA (rDNA) internal transcribed spacer (ITS) of mitochondrial DNA (mtDNA). High levels of genetic variation at both organelle loci, due to frequent intramolecular recombination, and low levels of genetic differentiation were detected in the relict gymnosperm. The apportionment of genetic variation within and between populations agreed with a migrant-pool model, which describes a migratory pattern with colonists recruited from a random sample of earlier existing populations. Phylogenies obtained from cpDNA and mtDNA were discordant according to neighbour-joining analyses. In total four chlorotypes (clades I-IV) and five mitotypes (clades A-E) were identified based on minimum spanning networks of each locus. Significant linkage disequilibrium in mitotype-chlorotype associations excluded the possibility of the recurrent homoplasious mutations as the major force causing phylogenetic inconsistency. The most abundant chlorotype I was associated with all mitotypes and the most abundant mitotype C with all chlorotypes; no combinations of rare mitotypes with rare chlorotypes were found. According to nested clade analyses, such nonrandom associations may be ascribed to relative ages among alleles associated with the geological history through which cycads evolved. Nested in networks as interior nodes coupled with wide geographical distribution, the most dominant cytotypes of CI and EI may represent ancestral haplotypes of C. taitungensis with a possible long existence prior to the Pleistocene glacial maximum. In contrast, rare chlorotypes and mitotypes with restricted and patchy distribution may have relatively recent origins. Newly evolved genetic elements of mtDNA, with a low frequency, were likely to be associated with the dominant chlorotype, and vice versa, resulting in the nonrandom mitotype-chlorotype associations. Paraphyly of CI and EI cytotypes, leading to the low level of genetic differentiation between cycad populations, indicated a short period for isolation, which allowed low possibilities of the attainment of coalescence at polymorphic ancestral alleles.

Phylogeography of Kandelia candel in East Asiatic mangroves based on nucleotide variation of chloroplast and mitochondrial DNAs.

Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to long-range dispersal. To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia candel based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplast DNA (cpDNA) atpB-rbcL spacer, cpDNA trnL-trnF spacer, and mitochondrial DNA (mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals. Three data sets suggested consistent phylogenies recovering two differentiated lineages corresponding to geographical regions, i.e. northern South-China-Sea + East-China-Sea region and southern South-China-Sea region (Sarawak). Phylogenetically, the Sarawak population was closely related to the Ranong population of western Peninsula Malaysia instead of other South-China-Sea populations, indicating its possible origin from the Indian Ocean Rim. No geographical subdivision was detected within the northern geographical region. An analysis of molecular variance (AMOVA) revealed low levels of genetic differentiation between and within mainland and island populations (phiCT = 0.015, phiSC = 0.037), indicating conspicuous long-distance seedling dispersal across oceans. Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations as the major force causing phylogenetic discrepancy between mtDNA and the trnL-trnF spacer within the northern region. Instead, relative ages of alleles contributed to non-random chlorotype-mitotype associations and tree inconsistency. Widespread distribution and random associations (chi2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes indicated the panmixis of populations of the northern geographical region as a whole. In contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration.

Mitochondrial DNA phylogeography of Acrossocheilus paradoxus (Cyprinidae) in Taiwan.

Nucleotide sequences of 3' end of the cytochrome b gene, tRNA genes, D-loop control region, and the 5' end of the 12S rRNA of mitochondrial DNA (mtDNA) were used to assess the genetic and phylogeographic structure of Acrossocheilus paradoxus populations, a Cyprinidae fish of Taiwan. A hierarchical examination of populations in 12 major streams from three geographical regions using an analysis of molecular variance (AMOVA) indicates high genetic differentiation both among populations (PhiST = 0.511, P < 0.001) and among regions (PhiCT = 0.368, P < 0.001). Limited migration largely contributed to the genetic differentiation. High nucleotide diversity (1.13%) and haplotype diversity (0.80%) were detected among populations. The degree of genetic differentiation was correlated with geographical distance between populations, a result consistent with the one-dimensional stepping stone models. A neighbour-joining tree recovered by (DAMBE) supports the pattern of isolation by distance and reveals a closer relationship between populations of the central and southern regions. A minimum spanning network based on nucleotide substitutions reflected migration routes from populations of the central region to the northern and southern regions, respectively. Postglacial colonization and expansion can explain the phylogeographical pattern. Single and ancient migration events may have allowed the northern region to attain the monophyly of mtDNA alleles. In contrast, most populations within geographical regions are either paraphyletic or polyphyletic due to the relatively shorter time period for coalescence. Both low haplotype number and genetic variability suggest a bottleneck event in the Chingmei population of northern Taiwan. Based on coalescence theory, the monophyly of the Tungkang population of the southern region may be associated with a founder event.

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.

Detection and viral nucleotide sequence analysis of transfusion-transmitted virus infection in acute fulminant and non-fulminant hepatitis.

The impact of transfusion-transmitted virus (TTV) infection on acute fulminant and non-fulminant hepatitis is unclear. In this study, serum samples from 164 patients with acute hepatitis of various aetiologies, from 34 asymptomatic hepatitis B virus carriers and from 202 healthy adults were tested for TTV DNA by the semiconserved nested polymerase chain reaction. TTV viraemia was prevalent in patients with acute hepatitis C, in patients with acute hepatitis D virus superinfection and in patients with non-A-E hepatitis (27-30%) but the incidence was not significantly different from that of healthy controls (31 of 202, 15.3%). There were no significant differences in gender, age, presence of hepatitis G virus, the occurrence of fulminant hepatitis, or in serum albumin, bilirubin or alanine aminotransferase levels (9/30 vs 35/134) between patients with or without TTV viraemia. Seven of the nine TTV-positive patients with fulminant hepatitis were co-infected with hepatitis C, D or E. TTV clones from 18 subjects, with or without fulminant hepatitis, were sequenced and analysed phylogenetically. Eleven (61. 1%) belonged to TTV group 1, six (33.3%) to TTV group 2 and one to TTV group 3. No particular strain of TTV was associated with fulminant hepatitis. In summary, in Taiwan, TTV is prevalent in the general population as well as in patients with liver diseases. TTV plays an insignificant role in acute fulminant and non-fulminant hepatitis. Its influence on outcome requires a longitudinal study.

Purification, cloning and sequence analyses for pro-metalloprotease-disintegrin variants from Deinagkistrodon acutus venom and subclassification of the small venom metalloproteases.

Acidic and basic hemorrhagic metalloproteases were purified from the venom of Deinagkistrodon acutus (from Fujian Province, China) using gel filtration and anion exchange on FPLC and reversed-phase HPLC. Their hemorrhagic activities and N-terminal sequences were characterized. Extensive screening of the venom gland cDNA after PCR amplification resulted in the identification and sequencing of a total of seven cDNA clones encoding the multidomain precursors of six acidic and one alkaline low molecular mass metalloproteases. Two of the precursors contain a processable disintegrin domain. Disintegrins of 5 kDa were also purified from the venom. The partial amino-acid sequences and molecular masses determined by electrospray ionization mass spectrometry of the purified proteins specifically match those deduced from two of the cDNA sequences. Moreover, phylogenetic analyses based on 30 complete sequences of low molecular mass venom metalloproteases revealed that they may be classified into three functional subtypes: acidic hemorrhagins, basic and moderate hemorrhagins, and nonhemorrhagic enzymes. Subtype-specific amino-acid substitutions in the C-terminal regions of the enzymes were highlighted to explore the structure-activity relationships of the enzymes.