Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

Comparison of Optical Coherence Tomography Measurement Reproducibility between Children and Adults

Purpose

To compare the reproducibility of SD-OCT (spectral-domain optical coherence tomography) measurements of RNFL (retinal nerve fiber layer) and macular thickness between children and adults.

Methods

Seventy-one eyes of 71 children and 71 eyes of 71 adults were prospectively enrolled. RNFL and macular thicknesses were measured by one operator, with a brief rest between measurements. The two measurements were obtained using the eye tracking and retest function of Spectralis SD-OCT. Reproducibility was evaluated with reference to COVs (coefficients of variation) and ICCs (intraclass correlation coefficients). The ICC values of the RNFL and macular thicknesses were compared, respectively between the two groups, by Fisher’s z-test.

Results

The RNFL and macular thicknesses did not differ between the two groups. The COVs of the RNFL measurements ranged from 0.945 to 4.531% in the children group and from 0.496 to 1.391% in the adults group. In most of the RNFL sectors, the ICCs of the children group (range: 0.731–0.987) were significantly lower than those of the adults group (range: 0.986–0.993). The COVs of the macular measurements ranged from 0.496 to 1.157% in the children group and from 0.275 to 0.656% in the adults group. The ICCs (range: 0.860–0.974) in the children group, significantly lower than for the adults (range: 0.989–0.995), in all of the macular sectors.

Conclusions

The reproducibility of SD-OCT RNFL and macular measurements for children was excellent, albeit statistically lower than that for adults.     

The Computerized Table Setting Test for Detecting Unilateral Neglect

Background

Patients with unilateral neglect fail to respond normally to stimuli on the left side. To facilitate the evaluation of unilateral spatial neglect, we developed a new application that runs on a tablet device and investigated its feasibility in stroke patients.

Methods

We made the computerized table setting test (CTST) to run on the tablet computer. Forty acute ischemic stroke patients (20 patients with right hemispheric infarction with neglect, 10 patients with right hemispheric infarction without neglect, and 10 patients with left hemispheric infarction) and 10 healthy controls were prospectively enrolled to validate the CTST. The test requires subjects to set a table by dragging 12 dishes located below the table on the tablet screen. The horizontal deviation of the 12 dishes from the midline of the table, the selection tendency measured by the sequence of the dish selection, and the elapsed time for table setting were calculated automatically.

Results

Parameters measured by the CTST were correlated with the results of conventional neglect tests. The horizontal deviation was significantly higher in patients with right hemispheric infarction with neglect compared with the other groups. The selection tendency and elapsed time also were significantly different in patients with right hemispheric infarction with neglect compared with the left hemispheric infarction and control groups, but were similar to those with right hemispheric infarction without neglect.

Conclusions

The CTST is feasible to administer and comparable with conventional neglect tests. This new application may be useful for the initial diagnosis and follow-up of neglect patients.     

Activation of brain steroidogenesis and neurogenesis during the gonadal differentiation in protandrous black porgy,Acanthopagrus schlegelii

The early brain development, at the time of gonadal differentiation was investigated using a protandrous teleost, black porgy. This natural model of monosex juvenile fish avoids the potential complexity of sexual dimorphism. Brain neurogenesis was evaluated by histological analyses of the diencephalon, at the time of testicular differentiation (in fish between 90 and 150 days after hatching). Increases in the number of both Nissl‐stained total brain cells, and Pcna‐immunostained proliferative brain cells were observed in specific area of the diencephalon, such as ventromedialis thalami and posterior preoptic area, revealing brain cell proliferation. qPCR analyses showed significantly higher expression of the radial glial cell marker blbp and neuron marker bdnf. Strong immunohistochemical staining of Blbp and extended cellular projections were observed. A peak expression of aromatase (cyp19a1b), as well as an increase in estradiol (E₂) content were also detected in the early brain. These data demonstrate that during gonadal differentiation, the early brain exhibits increased E₂ synthesis, cell proliferation, and neurogenesis. To investigate the role of E₂ in early brain, undifferentiated fish were treated with E₂ or aromatase inhibitor (AI). E₂ treatment upregulated brain cyp19a1b and blbp expression, and enhanced brain cell proliferation. Conversely, AI reduced brain cell proliferation. Castration experiment did not influence the brain gene expression patterns and the brain cell number. Our data clearly support E₂ biosynthesis in the early brain, and that brain E₂ induces neurogenesis. These peak activity patterns in the early brain occur at the time of gonad differentiation but are independent of the gonads. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 121–136, 2016     

Validation and Application of a Commercial Quantitative Real-Time Reverse Transcriptase-PCR Assay in Investigation of a Large Dengue Virus Outbreak in Southern Taiwan

BackgroundAccurate, rapid, and early diagnosis of dengue virus (DENV) infections is essential for optimal clinical care. Here, we evaluated the efficacy of the quantitative real-time PCR (qRT-PCR)-LightMix dengue virus EC kit for DENV detection using samples from a dengue outbreak in Taiwan in 2015.MethodsSera from patients with suspected DENV infection were analyzed and compared using the LightMix kit, a Dengue NS1 Ag + Ab Combo kit for detection of NS1 antigen and DENV-specific IgM and IgG antibodies, and an “in-house” qualitative DENV-specific RT-PCR assay.ResultsA total of 8,989, 8,954, and 1581 samples were subjected to NS1 antigen detection, IgM and IgG detection, and LightMix assays, respectively. The LightMix assay yielded a linear curve for viral loads (VL) between 10² and 10⁶ copies/reaction, and the minimum detection limits for DENV serotype 1 (DENV1) and DENV2, DENV3, and DENV4 were 1, 10, and 100 focus forming units (FFU)/mL, respectively. There was 88.9% concordance between the results obtained using the NS1 antigen combo kit and by LightMix analysis, and the diagnostic sensitivity and specificity of the two methods were 89.4 and 100%, and 84.7 and 100%, respectively. Notably, fatal cases were attributed to DENV2 infection, and 79.5% (27/34) of these cases occurred in patients ≥ 71 years of age. Among these older patients, 82.3% (14/17) were NS1/IgM/IgG (+/-/-), exhibiting VLs between 10⁶–10⁹ copies/mL, which was markedly higher than the rate observed in the other age groups.ConclusionsThe LightMix assay was effective for early diagnosis of DENV infection. Our data indicate that high VLs during primary infection in elderly patients may be a positive predictor for severe illness, and may contribute to high mortality rates.     

Understanding the Physicochemical Characteristics and the Improved Enzymatic Saccharification of Corn Stover Pretreated with Aqueous and Gaseous Ammonia

Physicochemical characteristics of corn stover pretreated by soaking in aqueous ammonia (SAA) and low-moisture anhydrous ammonia (LMAA) were compared and investigated. The glucan digestibility of the treated biomass reached 90 % (SAA) and 84 % (LMAA). The LMAA pretreatment enhanced the digestibility by cleaving cross-linkages between cell wall components, whereas the SAA pretreatment additionally improved the digestibility by efficiently removing a major portion of the lignin under mild reaction conditions without significant loss of carbohydrates. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and gel permeation chromatography (GPC) revealed the structural and chemical transformations of lignin during the pretreatments. Both pretreatments effectively cleaved ferulate cell wall cross-linking that is associated with the recalcitrance of grass lignocellulosics toward enzymatic saccharification. Extracted lignin from SAA pretreatment was extensively depolymerized but retained “native” character, as evidenced by the retention of β-ether linkages.     

L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice

New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR)-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (T_CM) were then genetically modified to express an anti-L1-CAM CAR (CE7R), which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R⁺ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p.) administration of CE7R⁺ T_CM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R⁺ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer.     

Skin Autofluorescence Is Associated with Endothelial Dysfunction in Uremic Subjects on Hemodialysis

Background

Elevated levels of advanced glycation end products (AGEs) within tissues may contribute to endothelial dysfunction, an early indicator of atherosclerosis. We aimed to investigate whether levels of skin AGEs could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.

Methods and Results

One hundred and nineteen uremic patients on hemodialysis and 57 control subjects with moderate-to-high cardiovascular risk factors and without chronic kidney disease (CKD) were enrolled. We used ultrasound to measure flow-mediated vasodilation (FMD). An AGE reader measured skin autoflurorescence (AF). We then compared differences in FMD and skin AF values between the two groups. The uremic subjects had significantly higher levels of skin AF (3.47±0.76 AU vs. 2.21±0.45 arbitrary units; P<0.01) and significantly lower levels of FMD (4.79%±1.88% vs. 7.19%±2.17%; P<0.01) than the non-CKD subjects. After adjusting for all potential covariates, we found that skin AF level independently predicted FMD in both the hemodialysis and the non-CKD groups. In the hemodialysis group, skin AF ≥ 3.05 arbitrary units predicted abnormal FMD at a sensitivity of 87.9% and a specificity of 78.6% (P<0.01).

Conclusions

Skin AF could be a useful marker to predict endothelial dysfunction in uremic subjects on hemodialysis.     

iMet-Q: A User-Friendly Tool for Label-Free Metabolomics Quantitation Using Dynamic Peak-Width Determination

Efficient and accurate quantitation of metabolites from LC-MS data has become an important topic. Here we present an automated tool, called iMet-Q (intelligent Metabolomic Quantitation), for label-free metabolomics quantitation from high-throughput MS1 data. By performing peak detection and peak alignment, iMet-Q provides a summary of quantitation results and reports ion abundance at both replicate level and sample level. Furthermore, it gives the charge states and isotope ratios of detected metabolite peaks to facilitate metabolite identification. An in-house standard mixture and a public Arabidopsis metabolome data set were analyzed by iMet-Q. Three public quantitation tools, including XCMS, MetAlign, and MZmine 2, were used for performance comparison. From the mixture data set, seven standard metabolites were detected by the four quantitation tools, for which iMet-Q had a smaller quantitation error of 12% in both profile and centroid data sets. Our tool also correctly determined the charge states of seven standard metabolites. By searching the mass values for those standard metabolites against Human Metabolome Database, we obtained a total of 183 metabolite candidates. With the isotope ratios calculated by iMet-Q, 49% (89 out of 183) metabolite candidates were filtered out. From the public Arabidopsis data set reported with two internal standards and 167 elucidated metabolites, iMet-Q detected all of the peaks corresponding to the internal standards and 167 metabolites. Meanwhile, our tool had small abundance variation (≤0.19) when quantifying the two internal standards and had higher abundance correlation (≥0.92) when quantifying the 167 metabolites. iMet-Q provides user-friendly interfaces and is publicly available for download at http://ms.iis.sinica.edu.tw/comics/Software_iMet-Q.html.     

Cross-Modal and Intra-Modal Characteristics of Visual Function and Speech Perception Performance in Postlingually Deafened,Cochlear Implant Users

Evidence of visual-auditory cross-modal plasticity in deaf individuals has been widely reported. Superior visual abilities of deaf individuals have been shown to result in enhanced reactivity to visual events and/or enhanced peripheral spatial attention. The goal of this study was to investigate the association between visual-auditory cross-modal plasticity and speech perception in post-lingually deafened, adult cochlear implant (CI) users. Post-lingually deafened adults with CIs (N = 14) and a group of normal hearing, adult controls (N = 12) participated in this study. The CI participants were divided into a good performer group (good CI, N = 7) and a poor performer group (poor CI, N = 7) based on word recognition scores. Visual evoked potentials (VEP) were recorded from the temporal and occipital cortex to assess reactivity. Visual field (VF) testing was used to assess spatial attention and Goldmann perimetry measures were analyzed to identify differences across groups in the VF. The association of the amplitude of the P1 VEP response over the right temporal or occipital cortex among three groups (control, good CI, poor CI) was analyzed. In addition, the association between VF by different stimuli and word perception score was evaluated. The P1 VEP amplitude recorded from the right temporal cortex was larger in the group of poorly performing CI users than the group of good performers. The P1 amplitude recorded from electrodes near the occipital cortex was smaller for the poor performing group. P1 VEP amplitude in right temporal lobe was negatively correlated with speech perception outcomes for the CI participants (r = -0.736, P = 0.003). However, P1 VEP amplitude measures recorded from near the occipital cortex had a positive correlation with speech perception outcome in the CI participants (r = 0.775, P = 0.001). In VF analysis, CI users showed narrowed central VF (VF to low intensity stimuli). However, their far peripheral VF (VF to high intensity stimuli) was not different from the controls. In addition, the extent of their central VF was positively correlated with speech perception outcome (r = 0.669, P = 0.009). Persistent visual activation in right temporal cortex even after CI causes negative effect on outcome in post-lingual deaf adults. We interpret these results to suggest that insufficient intra-modal (visual) compensation by the occipital cortex may cause negative effects on outcome. Based on our results, it appears that a narrowed central VF could help identify CI users with poor outcomes with their device.