Seasonal hematology and plasma biochemistry reference range values of the yellow-marginated box turtle (Cuora flavomarginata).

The yellow-marginated box turtle (Cuora flavomarginata) is critically endangered and very few studies have been performed to aid the conservation effort. This study reports the first complete analysis of blood parameters for this species and will provide a reference for future conservation studies. Hematology and biochemistry reference range values were established from 52 (18 males and 35 females) clinically healthy yellow-marginated box turtles (C. flavomarginata). In order to investigate the gender and seasonal differences in these ranges, blood samples were collected from the jugular veins of the turtles in January, April, July, and October to represent the four seasons. To detect significant variation (P < 0.05), data were analyzed using repeated measurements of analysis of variance. Significant gender differences were observed in red blood cell mass parameters, absolute eosinophils, absolute monocytes, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, calcium, uric acid, cholesterol, creatine kinase, glucose, phosphorus (P), total protein, and triglycerides. Marked seasonal variations were noted in red blood cell mass parameters, absolute monocytes, albumin, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, uric acid, creatine kinase, creatinine, glucose, P, and triglycerides. Primary reasons for the differences between gender and season were associated with reproduction and temperature change. Cuora flavomarginata has lower alanine aminotransferase values compared with other chelonians.

Heterogeneous capture-recapture models with covariates: a partial likelihood approach for closed populations.

In practice, when analyzing data from a capture-recapture experiment it is tempting to apply modern advanced statistical methods to the observed capture histories. However, unless the analysis takes into account that the data have only been collected from individuals who have been captured at least once, the results may be biased. Without the development of new software packages, methods such as generalized additive models, generalized linear mixed models, and simulation-extrapolation cannot be readily implemented. In contrast, the partial likelihood approach allows the analysis of a capture-recapture experiment to be conducted using commonly available software. Here we examine the efficiency of this approach and apply it to several data sets.

Genome-Wide Microarray Analysis Suggests Transcriptomic Response May Not Play a Major Role in High- to Low-Altitude Acclimation in Harvest Mouse (Micromys minutus).

The harvest mouse (Micromys minutus) is a small rodent species with a wide range of vertical distribution in Taiwan, extending from the sea level to 3100 m altitude. This species has recently suffered from habitat loss in high-altitude areas due to orchard cultivation, which may have resulted in mouse migration from high to low altitude. To investigate whether there is any physiological mechanism involved in altitude acclimation, rat cDNA microarray was used to compare transcriptomic patterns of the skeletal muscle tissues taken from individuals native to the high-altitude environment and those transferred to the low-altitude captive site. Of the 23,188 genes being analyzed, 47 (33 up-regulated and 14 down-regulated) were found to have differential expression (fold change > 4 or < -4, ANOVA p < 0.05). However, after multiple testing correction with a false discovery rate (FDR), only the result for Tnfrsf12a was found to be statistically significant (fold change = 13, FDR p < 0.05). The result was confirmed by quantitative polymerase chain reaction (q-PCR). The expression of Tnfrsf12a possibly relates to the skeletal muscle biology and thus can be correlated with altitude acclimation. However, finding only one gene transcript with significant alteration suggests that transcriptomic response may not play a major role in high- to low-altitude acclimation in harvest mouse.

Draft Genome Sequence of Carbapenem-Resistant Klebsiella pneumoniae XPY20 Collected from a Bloodstream Infection Patient.

Bloodstream infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) strains have been a severe problem with high clinical costs and high mortality rates. The blaKPC-2-producing CRKP strain XPY20 was collected from the blood of a patient. The genome characteristics and antimicrobial resistance mechanisms were determined using next-generation sequencing.

Simultaneous emergence and rapid spread of three OXA-23 producing Acinetobacter baumannii ST208 strains in intensive care units confirmed by whole genome sequencing.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a common nosocomial bacterial pathogen with limited treatment options. CRAB outbreaks are disastrous for critically ill patients. This study investigated carbapenemase-produced A. baumannii outbreaks in a tertiary hospital. Although multiple outbreaks were suggested by pulse-field gel electrophoresis, the genetic lineages and evolution between these isolates were not clear. To investigate the genomic epidemiology of these outbreaks and to reveal possible transmission routes, whole genome sequences (WGS) were compared and analyzed. From the WGS data, thirty isolates had the same sequence type (ST208); acquired resistance genes and chromosome resistant genes were detected and were responsible for multidrug resistance. A phylogenetic tree of single-nucleotide polymorphisms (SNPs) compared to the earliest index isolate found that three outbreaks had emerged and disseminated simultaneously. Of these, <10 SNPs were detected within the cluster, whereas at least 600 SNPs were found between the clusters. The probable transmission routes of outbreaks were generated combined with the genetic distance of isolates and patient epidemiological data. In conclusion, WGS was a convenient and accurate monitoring method for genomic epidemiologic investigation of outbreaks, and the genomic surveillance of multidrug-resistant bacterial pathogens would be a powerful warning system for the surveillance and prevention of outbreaks.

Systematic revision of the Taiwanese genus Kurixalus members with a description of two new endemic species (Anura, Rhacophoridae).

Two new species of rhacophorid tree frog were identified in Taiwan. In both new taxa, derived reproductive characteristics of laying eggs in tree holes and oophagous tadpoles are shared with Kurixalus eiffingeri, but they are divergent from each other in molecular genetics, mating calls, and tadpole and adult morphology. The morphological characteristics and the molecular phylogenetic evidence support the hypothesis that the two new species, Kurixalus berylliniris sp. n. and Kurixalus wangi sp. n., are both monophyletic lineages.

Determination of theophylline concentration in serum by chemiluminescent immunoassay.

OBJECTIVE: This study aimed to establish chemiluminescent immunoassay (CLIA) for quantitative determination of theophylline levels in human serum. METHODS: To measure the concentration of theophylline (n=122) and evaluate the assay. RESULTS: The linear range of the CLIA method was 0.51-40 mg/L (Y=1.02X+0.44, r=0.995). The intra and inter CV (coefficient variance) of CLIA were 3.20% and 3.57%, respectively. The average recovery rate was 102.3%. This method was free from interference by brilirubin (<200 micromol/L), hemoglobin (<10 g/L), and triglycerides (<15 mmol/L). CONCLUSION: This method is simple, convenient and precise for clinical pharmacokinetics study of theophylline.

[The method of wavelength calibration in high resolution absorption spectroscopy].

A new method for the calibration of wavelength in a measurement of high resolution molecular absorption spectra by referring the standard spectrum of iodine molecule is presented in this paper. Segment offset is employed in the calibration of wavelength, while the data smoothing, baseline determining and peak fitting are applied to preprocess the spectral data. A visual software is provided for automatically calibrating wavelength with a friendly style of mutual dialogue between human and computer. The software has been used to calibrate the absorption spectra of N2+ molecular ion in the visible region 16,422-17,896 cm-1, the absolute accuracy is about 0.006 cm-1.

Validating a rapid, real-time, PCR-based direct mutation detection assay for preimplantation genetic diagnosis.

Although co-amplification of polymorphic microsatellite markers is the current gold standard for preimplantation genetic diagnosis (PGD) of single-gene disorders (SGD), this approach can be hampered by the lack of availability of informative markers. We recently (2011) devised a novel in-house assay for PGD of aromatic L-amino acid decarboxylase deficiency, based on an amplification refractory mutation system and quantitative PCR (ARMS-qPCR). The objective of the present study was to verify ARMS-qPCR in a cohort of 20 PGD cycles with a diverse group of SGDs (15 couples at risk for 10 SGDs). Day-3 cleavage-stage embryos were subjected to biopsy and genotyping, followed by fresh embryo transfer (FET). The diagnostic rate was 82.9%; unaffected live births were achieved in 9 of 20 FET cycles (45%), with only one false negative (among 54 transferred embryos). Overall, the ARMS-qPCR had frequent allele-dropout (ADO), rendering it inappropriate as the sole diagnostic method (despite a favorable live-birth rate). Regardless, it has the potential to complement the current gold-standard methodology, especially when trophectoderm biopsy becomes a preferred option and genotyping needs to be timely enough to enable FET.

Genomic analyses of the Formosan harvest mouse (Micromys minutus) and comparisons to the brown Norway rat (Rattus norvegicus) and the house mouse (Mus musculus).

The harvest mouse, Micromys minutus (MMIN), has a very wide range of distribution (from the British Isles across the Euroasian continent to Japan and Taiwan). We studied an isolated population of MMIN in Taiwan, which is at the southeastern margin of the species' geographic distribution, and compared its genetic complement with those of the same species previously reported from other geographic locations and with two model rodent species, the house mouse (Mus musculus) and the brown Norway rat (Rattus norvegicus). The diploid number (2N) of MMIN was 68, consistent with that reported for other populations. However, variations were noted in the fundamental number (FN) and the shape and banding patterns of the individual chromosomes among populations. The FN of MMIN was estimated to be 72, including 2 bi-armed autosomes, 31 one-armed autosomes, and one pair of one-armed sex chromosomes. Here, we propose the first ideogram for MMIN. C-banding, Ag-NOR, and the locations of 18S rRNA gene sequences (MMIN chromosomes no. 10, 14, 19, 29, 31, 33, and X) mapped by fluorescence in situ hybridization (FISH) are also reported. Additionally, we compared the 18S rDNA sequences and performed cross-species X chromosome painting (FISH) for M. minutus, M. musculus, and R. norvegicus. The results indicate that both genetic elements are rather conserved across species. Thus, implications for the phylogenetic position of Micromys were limited.

Identification of novel Bartonella spp. in bats and evidence of Asian gray shrew as a new potential reservoir of Bartonella.

Many studies indicated that small mammals are important reservoirs for Bartonella species. Using molecular methods, several studies have documented that bats could harbor Bartonella. This study was conducted to investigate the relationship of Bartonella spp. identified in bats and small mammals living in the same ecological environment. During May 2009 and March 2010, a total of 102 blood specimens were collected. By whole blood culture and molecular identification, a total of 6 bats, 1 rodent and 9 shrews were shown to be infected by Bartonella species. After sequencing and phylogenetic analyses of the sequences of gltA, ftsZ, rpoB and ribC genes, these specific isolates from bats were not similar to the known Bartonella species (the similarity values were less than 91.2%, 90.5%, 88.8%, and 82.2%, respectively); these isolates formed an independent clade away from other known Bartonella type strains. The Bartonella spp. isolated from small mammals, which were closely related to Bartonella tribocorum, Bartonella elizabethae, Bartonella grahamii, Bartonella rattimassiliensis and Bartonella queenslandensis, were similar to the findings in previous studies worldwide. Therefore, the results implied that the species of Bartonella strains isolated from small mammals were different from those identified in bats. Our results strongly suggested that the bat isolate could be a new Bartonella species. This study is also the first one to isolate Bartonella organisms from Asian gray shrews, Crocidura attenuata tanakae.

Use of a cytogenetic whole-genome comparison to resolve phylogenetic relationships among three species: implications for mammalian systematics and conservation biology.

The objective was to apply a novel modification of a genome-wide, comparative cytogenetic technique (comparative genomic hybridization, comparative genomic hybridization (CGH)), to study species belonging to the myrmecophagous (ant/termite eating) mammalian orders/superorders (Pholidota, Tubulidentata, Carnivora, and Xenarthra), as a model for other applications in mammalian systematics and conservation biology. In this study, CGH was applied to high-quality metaphase spreads of pangolin (Pholidota), using probes of sloth and canine (Xenarthra and Carnivora, respectively) genomic DNA labeled with different fluorophores, thereby facilitating analysis of the visible color spectrum on pangolin karyotypes. Our results posited that pholidotes are closer to carnivores than to xenarthrans, which confirmed the current consensus that myrmecophagy in these mammalian lineages was more likely because of homoplasy (convergent evolution) than being an ancestral character. Since the modified CGH technique used is genome-wide, has chromosome-level resolution, and does not need full genome sequencing, it has considerable potential in systematics and other fields.

Genome-wide gene expression analysis implicates the immune response and lymphangiogenesis in the pathogenesis of fetal chylothorax.

Fetal chylothorax (FC) is a rare condition characterized by lymphocyte-rich pleural effusion. Although its pathogenesis remains elusive, it may involve inflammation, since there are increased concentrations of proinflammatory mediators in pleural fluids. Only a few hereditary lymphedema-associated gene loci, e.g. VEGFR3, ITGA9 and PTPN11, were detected in human fetuses with this condition; these cases had a poorer prognosis, due to defective lymphangiogenesis. In the present study, genome-wide gene expression analysis was conducted, comparing pleural and ascitic fluids in three hydropic fetuses, one with and two without the ITGA9 mutation. One fetus (the index case), from a dizygotic pregnancy (the cotwin was unaffected), received antenatal OK-432 pleurodesis and survived beyond the neonatal stage, despite having the ITGA9 mutation. Genes and pathways involved in the immune response were universally up-regulated in fetal pleural fluids compared to those in ascitic fluids. Furthermore, genes involved in the lymphangiogenesis pathway were down-regulated in fetal pleural fluids (compared to ascitic fluid), but following OK-432 pleurodesis, they were up-regulated. Expression of ITGA9 was concordant with overall trends of lymphangiogenesis. In conclusion, we inferred that both the immune response and lymphangiogenesis were implicated in the pathogenesis of fetal chylothorax. Furthermore, genome-wide gene expression microarray analysis may facilitate personalized medicine by selecting the most appropriate treatment, according to the specific circumstances of the patient, for this rare, but heterogeneous disease.

Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals.

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.

Preimplantation and prenatal genetic diagnosis of aromatic L-amino acid decarboxylase deficiency with an amplification refractory mutation system-quantitative polymerase chain reaction.

OBJECTIVES: To develop a diagnostic platform for preimplantation genetic diagnosis (PGD) and prenatal genetic diagnosis (PND) to prevent births of aromatic L-amino acid decarboxylase deficiency (AADC) patients. MATERIALS AND METHODS: Five Taiwanese families carrying AADC were enrolled. A novel technique, amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR), was developed for both of PGD and PND. For PGD, blastomere biopsies of day-3 cleavage-stage embryos were subjected to ARMS-qPCR. Villi, cultured amniocytes, or both were used to confirm the PGD result; this approach could also be used as the sole method for PND after in vivo conception). RESULTS: Unaffected live births were achieved in four of the five families, except one with ongoing PGD. The ARMS-qPCR correctly classified blastomeres (from day-3 cleavage-stage embryos) as affected (homozygous mutant), carrier (heterozygous for mutant and wild-type alleles), or normal (homozygous wild-type) within 1 working day. CONCLUSIONS: To our knowledge, this is the first report of successful PGD of AADC. The molecular technique we devised (ARMS-qPCR) was applicable for PGD as well as PND of AADC. Furthermore, it has great potential for similar applications in other monogenic disorders.

Phylogenetic relationships in genus Niviventer (Rodentia: Muridae) in China inferred from complete mitochondrial cytochrome b gene.

Chinese species of the genus Niviventer, predominantly distributed in the southeastern Tibetan Plateau and in Taiwan, are a diverse group and have not yet received a thorough molecular phylogenetic analysis. Here, we reconstructed the phylogenetic relationships of 32 specimens representing nine Chinese species of Niviventer, based on sequences of the complete mitochondrial cytochrome b gene. Maximum parsimony, maximum likelihood and Bayesian analysis resulted in three consistent trees, each supported by high bootstrap values. The results showed that the Niviventer species included here are monophyletic. The nine species were classified into three distinct clades: clade A with Niviventer brahma, N. confucianus, N. coxingi, N. culturatus, N. eha and N. fulvescens; clade B with N. andersoni and N. excelsior; clade C with N. cremoriventer. Our results also suggested that N. culturatus should be a valid species rather than a subspecies of N. confucianus. Divergence times among species were calibrated according to the middle-late Pleistocene (1.2-0.13 Mya) fossil records of N. confucianus. The results demonstrated that the first radiation event of the genus Niviventer occurred in early Pleistocene (about 1.66 Mya), followed by the divergence of clades A and B at about 1.46 Mya. Most of the extant Niviventer species appeared during early to middle Pleistocene (about 1.29-0.67 Mya). These divergence times are coincidental with the last uplift events of the Tibetan Plateau, Kun-Huang movement, Pleistocene glaciations and the vicariant formation of Taiwan Strait. Consequently geographical events and Pleistocene glaciations have played a great role in the diversification of Niviventer.