Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10² CFU/ml in pure cultures and ≥10³ CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Antimicrobial Non-Susceptibility of Escherichia coli from Outpatients and Patients Visiting Emergency Rooms in Taiwan

Longitudinal nationwide surveillance data on antimicrobial non-susceptibility and prevalence of extended-spectrum β-lactamases (ESBLs) as well as AmpC β-lactamases producers among Escherichia coli from different sources in the community settings are limited. Such data may impact treatment practice. The present study investigated E. coli from outpatients and patients visiting emergency rooms collected by the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program. A total of 3481 E. coli isolates were studied, including 2153 (61.9%) from urine and 1125 (32.3%) from blood samples. These isolates were collected biennially between 2002 and 2012 from a total of 28 hospitals located in different geographic regions of Taiwan. Minimum inhibitory concentrations (MIC) were determined using methods recommended by the Clinical Laboratory Standards Institute (CLSI). The prevalence and factors associated with the presence of ESBL and AmpC β-lactamase-producers were determined. Significant increases in non-susceptibility to most β-lactams and ciprofloxacin occurred during the study period. By 2012, non-susceptibility to cefotaxime and ciprofloxacin reached 21.1% and 26.9%, respectively. The prevalence of ESBL- and AmpC- producers also increased from 4.0% and 5.3%, respectively, in 2002–2004, to 10.7% for both in 2010–2012 (P < 0.001). The predominant ESBL and AmpC β-lactamase genes were CTX-M and CMY-types, respectively. Non-susceptibility of urine isolates to nitrofurantoin remained at around 8% and to fosfomycin was low (0.7%) but to cefazolin (based on the 2014 CLSI urine criteria) increased from 11.5% in 2002–2004 to 23.9% in 2010–2012 (P <0.001). Non-susceptibility of isolates from different specimen types was generally similar, but isolates from elderly patients were significantly more resistant to most antimicrobial agents and associated with the presence of ESBL- and AmpC- β-lactamases. An additional concern is that decreased ciprofloxacin susceptibility (MIC 0.12–1 mg/L) was as high as 25% in isolates from all age groups, including those from pediatric patients. Our data indicated that there is a need to re-evaluate appropriate treatment selection for community-acquired infections in Taiwan. Identification of community reservoirs of multidrug-resistant E. coli is also warranted.     

Mean growth rate when rare is not a reliable metric for persistence of species

The coexistence of many species within ecological communities poses a long‐standing theoretical puzzle. Modern coexistence theory (MCT) and related techniques explore this phenomenon by examining the chance of a species population growing from rarity in the presence of all other species. The mean growth rate when rare, , is used in MCT as a metric that measures persistence properties (like invasibility or time to extinction) of a population. Here we critique this reliance on and show that it fails to capture the effect of temporal random abundance variations on persistence properties. The problem becomes particularly severe when an increase in the amplitude of stochastic temporal environmental variations leads to an increase in , since at the same time it enhances random abundance fluctuations and the two effects are inherently intertwined. In this case, the chance of invasion and the mean extinction time of a population may even go down as increases.     

The immune response to mouse hepatitis virus: expression of monocyte procoagulant activity and plasminogen activator during infection in vivo

After infection with 10(3) plaque-forming units of mouse hepatitis virus strain 3 (MHV-3) in vivo, peripheral blood mononuclear cells and splenic cells expressed procoagulant activity (PCA) in a pattern directly correlating with susceptibility to disease. Mononuclear cells from BALB/cJ mice, a strain which is fully susceptible to MHV-3, expressed a greater than 500-fold increase in PCA. PCA was first detected within 12 hr of infection; prior to histologic evidence of disease and viral replication, it reached maximal levels 48 hr post-infection (p.i.) and persisted until the death of the animals 5 to 7 days p.i. Mononuclear cells from C3HeB/FeJ mice expressed a significant but lesser titer of PCA, with elevated PCA persisting throughout the chronically infected state until death of the animals 4 to 6 mo p.i. Basal levels of PCA were detected in mononuclear cells from fully resistant A/J mice despite the presence of large amounts of virus in livers, spleens, and sera from these animals. When mononuclear cells expressing high PCA were subfractionated, monocytes were found to be the cellular source of greater than 96% of the PCA activity. Increased plasminogen activator activity was found in monocytes from resistant A/J mice at the time when PCA was markedly elevated in BALB/cJ and C3HeB/FeJ mice. This activity persisted for 5 to 7 days p.i., but was undetectable 10 days p.i. at a time when the mice had cleared the virus from their blood streams. These observations suggest that monocyte PCA may be important in the pathogenesis of MHV-3 disease, whereas the production of monocyte plasminogen activators may contribute to resistance of A/J mice to MHV-3-induced liver disease.     

Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis

Although Aurora B is important in cleavage furrow ingression and completion during cytokinesis, the mechanism by which kinase activity is targeted to the cleavage furrow and the molecule(s) responsible for this process have remained elusive. Here, we demonstrate that an essential mitotic kinesin MKlp2 requires myosin-II for its localization to the equatorial cortex, and this event is required to recruit Aurora B to the equatorial cortex in mammalian cells. This recruitment event is also required to promote the highly focused accumulation of active RhoA at the equatorial cortex and stable ingression of the cleavage furrow in bipolar cytokinesis. Specifically, in drug-induced monopolar cytokinesis, targeting Aurora B to the cell cortex by MKlp2 is essential for cell polarization and furrow formation. Once the furrow has formed, MKlp2 further recruits Aurora B to the growing furrow. This process together with continuous Aurora B kinase activity at the growing furrow is essential for stable furrow propagation and completion. In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis. This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis. Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.     

Role of HLA-DP Polymorphisms on Chronicity and Disease Activity of Hepatitis B Infection in Southern Chinese

Background and Aims

The association between HLA-DP single nucleotide polymorphisms (SNPs) and chronic hepatitis B virus (HBV) infection varies between different populations. We aimed to study the association between HLA-DP SNPs and HBV infection and disease activity in the Chinese population of Hong Kong.

Methods

We genotyped SNPs rs3077 (near HLA-DPA1) and rs9277378 and rs3128917 (both near HLA-DPB1) in 500 HBV carriers (hepatitis B surface antigen [HBsAg]-positive), 245 non-HBV infected controls (HBsAg- and antibody to hepatitis B core protein [anti-HBc]-negative), and 259 subjects with natural HBV clearance (HBsAg-negative, anti-HBc-positive). Inactive HBV carriers state was defined by HBV DNA levels <2,000 IU/ml and persistently normal alanine aminotransferase level for least 12 months.

Results

Compared to the non-HBV infected subjects, the HBV carriers had a significantly lower frequency of the rs3077 T allele (p = 0.0040), rs9277378 A allele (p = 0.0068) and a trend for lower frequency of rs3128917 T allele (p = 0.054). These alleles were associated with an increased chance of HBV clearance (rs3077: OR = 1.41, p = 0.0083; rs9277378: OR = 1.61, p = 0.00011; rs3128917: OR = 1.54, p = 0.00017). Significant associations between HLA-DP genotypes and HBV clearance were also found under different genetic models. Haplotype TAT was associated with an increased chance of HBV clearance (OR = 1.64, p = 0.0013). No association was found between these SNPs and HBV disease activity.

Conclusion

HLA-DP SNPs rs3077, rs9277378 and rs3128917 were associated with chronicity of HBV disease in the Chinese. Further studies are required to determine whether these SNPs influence the disease endemicity in different ethnic populations.     

Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypo-diploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by -D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.     

Nitrite inhalation in rats elevates tissue NOS III expression and alters tyrosine nitration and phosphorylation

Organic nitrites are nitric oxide (NO) donors that are used predominantly as inhalant drugs of abuse and have been shown to have immunomodulating effects. NO donors can modulate NOS activity and expression, thus altering the level of endogenous NO production. NO can react with superoxide (O(*)(2)(-)) to form peroxynitrite (ONOO(-)), which can nitrate tyrosine residues in proteins and alter tyrosine phosphorylation. We investigated the effects of inhaled isobutyl nitrite (ISBN) on NOS expression, tyrosine nitration, and tyrosine phosphorylation in selected organs of rats. Following exposures of 109 and 1517 ppm ISBN for 4 h, the lung, spleen, liver, and kidney were removed and assayed by SDS-PAGE for NOS III (eNOS), NOS II (iNOS), nitrotyrosine (NT)- and phosphotyrosine (PT)-immunoreactive proteins using specific antibodies. ISBN at 1517 ppm, but not 109 ppm, caused an increase in NOS III expression in the liver and kidney, but not in the lung and spleen. No apparent effect on NOS II expression was observed in these organs. The expressions of NT and PT protein bands (30-200 kDa) were increased in the liver and kidney, but not in the lung and spleen. This increase in NT persisted for 24 h post-exposure. Increased NOS III expression in the liver and kidney may promote peroxynitrite formation and contribute to the increase in NT and PT immunoreactivity. ISBN inhalation may thus cause changes in cellular signaling involving tyrosine phosphorylation. These findings may suggest a mechanistic basis for the apparent immunotoxicity associated with nitrite abuse.     

Forward chemical genetic screens in Arabidopsis identify genes that influence sensitivity to the phytotoxic compound sulfamethoxazole

The rise of high mountain chains is widely seen as one of the factors driving rapid diversification of land plants and the formation of biodiversity hotspots. Supporting evidence was reported for the impact of the rapid rise of the Andean mountains but this hypothesis has so far been less explored for the impact of the “roof of the world”. The formation of the Himalaya, and especially the rise of the Qinghai–Tibetan Plateau in the recent 20 million years, altered the monsoon regimes that dominate the current climates of South East Asia. Here, we infer the hypothesis that the rise of Himalaya had a strong impact on the plant diversity in the biodiversity hotspot of the Southwest Chinese Mountains. Our analyses of the diversification pattern of the derived fern genus Lepisorus recovered evidence for changes in plant diversity that correlated with the strengthening of South East Asian monsoon. Southwest China or Southwest China and Japan was recovered as the putative area of origin of Lepisorus and enhancing monsoon regime were found to shape the early diversification of the genus as well as subsequent radiations during the late Miocene and Pliocene. We report new evidence for a coincidence of plant diversification and changes of the climate caused by the uplift of the Himalaya. These results are discussed in the context of the impact of incomplete taxon sampling, uncertainty of divergence time estimates, and limitations of current methods used to assess diversification rates.     

MRI study of minor physical anomaly in childhood autism implicates aberrant neurodevelopment in infancy

Background

MPAs (minor physical anomalies) frequently occur in neurodevelopmental disorders because both face and brain are derived from neuroectoderm in the first trimester. Conventionally, MPAs are measured by evaluation of external appearance. Using MRI can help overcome inherent observer bias, facilitate multi-centre data acquisition, and explore how MPAs relate to brain dysmorphology in the same individual. Optical MPAs exhibit a tightly synchronized trajectory through fetal, postnatal and adult life. As head size enlarges with age, inter-orbital distance increases, and is mostly completed before age 3 years. We hypothesized that optical MPAs might afford a retrospective ‘window’ to early neurodevelopment; specifically, inter-orbital distance increase may represent a biomarker for early brain dysmaturation in autism.

Methods

We recruited 91 children aged 7–16; 36 with an autism spectrum disorder and 55 age- and gender-matched typically developing controls. All children had normal IQ. Inter-orbital distance was measured on T1-weighted MRI scans. This value was entered into a voxel-by-voxel linear regression analysis with grey matter segmented from a bimodal MRI data-set. Age and total brain tissue volume were entered as covariates.

Results

Intra-class coefficient for measurement of the inter-orbital distance was 0.95. Inter-orbital distance was significantly increased in the autism group (p = 0.03, 2-tailed). The autism group showed a significant relationship between inter-orbital distance grey matter volume of bilateral amygdalae extending to the unci and inferior temporal poles.

Conclusions

Greater inter-orbital distance in the autism group compared with healthy controls is consistent with infant head size expansion in autism. Inter-orbital distance positively correlated with volume of medial temporal lobe structures, suggesting a link to “social brain” dysmorphology in the autism group. We suggest these data support the role of optical MPAs as a “fossil record” of early aberrant neurodevelopment, and potential biomarker for brain dysmaturation in autism.