Requirement for Phe36 for DNA binding and mismatch repair by Escherichia coli MutS protein

The MutS family of DNA repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. Photocrosslinking and mutational studies previously identified a highly conserved Phe residue at the N-terminus of Thermus aquaticus MutS protein that is critical for mismatch recognition in vitro. Here, a mutant Escherichia coli MutS protein harboring a substitution of Ala for the corresponding Phe36 residue is assessed for proficiency in mismatch repair in vivo and DNA binding and ATP hydrolysis in vitro. The F36A protein is unable to restore mismatch repair proficiency to a mutS strain as judged by mutation to rifampicin or reversion of a specific point mutation in lacZ. The F36A protein is also severely deficient for binding to heteroduplexes containing an unpaired thymidine or a G:T mismatch although its intrinsic ATPase activity and subunit oligomerization are very similar to that of the wild-type MutS protein. Thus, the F36A mutation appears to confer a defect specific for recognition of insertion/deletion and base pair mismatches.     

Susceptibilities to Amphotericin B, Fluconazole and Voriconazole of Trichosporon Clinical Isolates

A total of 35 Trichosporon isolates were collected from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) project from 1999 to 2006, and their identifications as well as drug susceptibilities were determined. The most frequently isolated species was T. asahii (62.9%), and the most common clinical sample that yielded Trichosporon isolates was urine (37.1%). The etiology of all seven invasive trichosporonosis was T. asahii. For the 22 T. asahii isolates, the MIC(50) and MIC(90) for amphotericin B were 0.25 and 1 μg/mL, respectively. Those for fluconazole were 2 and 4 μg/mL, respectively, and for voriconazole 0.031 and 0.063 μg/mL, respectively. When the intraclass correlation coefficients (ICCs) and agreements were calculated, we found that the MICs of fluconazole obtained from different methods were similar and the inter-method discrepancies were low. Nevertheless, no unanimous MIC of amphotericin B and voriconazole was obtained among different methods.     

IL-31 Associated with Coronary Artery Lesion Formation in Kawasaki Disease

Background

Kawasaki disease (KD) is known to be associated with T help (Th) 2 reaction and subsequently allergic diseases. Interleukin-31 (IL-31) has also been reported to be involved in Th2 mediated diseases such as allergic diseases. However, the role of IL-31 in KD has not been previously reported. The aim of this study is to investigate whether IL-31 is associated with KD and its clinical outcome.

Material

A total of 78 KD patients who met the criteria of KD were enrolled in this study as well as 20 age-matched controls. Plasma samples were conducted to measure IL-31 before intravenous immunoglobulin (IVIG) treatment (KD1), within 3 days after IVIG treatment (KD2) and at least 3 weeks after IVIG treatment (KD3) by utilizing enzyme-linked immunosorbent assay (ELISA).

Result

Our findings showed that IL-31 expression was higher in KD patients after IVIG treatment significantly (KD2>KD1: 1265.0±199.3 vs. 840.2±152.5 pg/ml, p<0.0001). Further analysis revealed that IL-31 level was significantly higher in KD patients with coronary artery lesion (CAL) (656.6±139.5 vs. 1373.0±422.0 pg/ml, p = 0.04) before IVIG treatment (KD1). There were no significant differences between the IVIG resistance and IVIG responsiveness groups.

Conclusion

IL-31 was increased after IVIG treatment in patients with KD and was significantly associated with CAL formation. The results from this study may help to identify a novel risk factor for predicting KD and CAL formation.     

Quantification of Bird-to-Bird and Bird-to-Human Infections during 2013 Novel H7N9 Avian Influenza Outbreak in China

From February to May, 2013, 132 human avian influenza H7N9 cases were identified in China resulting in 37 deaths. We developed a novel, simple and effective compartmental modeling framework for transmissions among (wild and domestic) birds as well as from birds to human, to infer important epidemiological quantifiers, such as basic reproduction number for bird epidemic, bird-to-human infection rate and turning points of the epidemics, for the epidemic via human H7N9 case onset data and to acquire useful information regarding the bird-to-human transmission dynamics. Estimated basic reproduction number for infections among birds is 4.10 and the mean daily number of human infections per infected bird is 3.16*10⁻⁵ [3.08*10⁻⁵, 3.23*10⁻⁵]. The turning point of 2013 H7N9 epidemic is pinpointed at April 16 for bird infections and at April 9 for bird-to-human transmissions. Our result reveals very low level of bird-to-human infections, thus indicating minimal risk of widespread bird-to-human infections of H7N9 virus during the outbreak. Moreover, the turning point of the human epidemic, pinpointed at shortly after the implementation of full-scale control and intervention measures initiated in early April, further highlights the impact of timely actions on ending the outbreak. This is the first study where both the bird and human components of an avian influenza epidemic can be quantified using only the human case data.     

Determinism of bacterial metacommunity dynamics in the southern East China Sea varies depending on hydrography

Spatial variation of communities composition (metacommunities) results from multiple assembly mechanisms, including environmental filtering and dispersal; however, whether and why the relative importance of the assembly mechanisms in shaping bacterial metacommunity changes through time in marine pelagic systems remains poorly studied. Here, we applied the elements of metacommunity structure framework and the variation partitioning framework to examine whether temporal variation of hydrographic conditions influences bacterioplankton metacommunity dynamics in the southern East China Sea (ECS). The spatiotemporal variation of bacterial communities composition was revealed using 454 pyrosequencing of 16S rDNA. In addition to the whole bacterial community, we analyzed four dominant taxonomic groups (Cyanobacteria, Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria) separately. Our analyses indicate that, considering the whole community level, the determinism of metacommunity structure varied among seasons. When the degree of connectivity was low (December), the metacommunity exhibited random distribution and was explained mainly by the environmental component. However, Clementsian metacommunity was found at intermediate connectivity (May), during which the environmental and spatial predictors were both significant. When connectivity was high (August), a random distribution pattern was found and no significant effect of environmental filtering or dispersal limitation was detected. Nevertheless, when considering different taxonomic groups, the differences in metacommunity dynamics among groups were found. Our results suggest that the driving forces of metacommunity dynamics varied depending on hydrography, as the degrees of environmental heterogeneity and connectivity among habitat patches were determined by circulation pattern. Moreover, mechanisms varied among different taxonomic groups, suggesting that differential dispersal capacity among taxonomic groups should be integrated into community assembly studies.     

The Trypanosoma cruzi protease cruzain mediates immune evasion

Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min) and no increase in ～P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min) stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.     

Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression

Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm² in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (^·OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H₂O₂), ^·OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H₂O₂ was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow. J. Cell. Physiol. 175:156–162, 1998. © 1998 Wiley-Liss, Inc.     

A novel, topologically constrained DNA molecule containing a double Holliday junction: design, synthesis, and initial biochemical characterization

The double Holliday junction (dHJ) is a central intermediate to homologous recombination, but biochemical analysis of the metabolism of this structure has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a novel dHJ substrate that consists of two small, double stranded DNA circles conjoined by two Holliday junctions (HJs). Its biochemical synthesis is based on the production of two pairs of single stranded circles from phagemids, followed by their sequential annealing with reverse gyrase. The sequence between the two HJs is identical on both strands, allowing the HJs to migrate without the generation of unpaired regions of DNA, whereas the distance between the HJs is on the order of gene conversion tracts thus far measured in Drosophila and mouse model systems. The structure of this substrate also provides similar topological constraint as would occur in an endogenous dHJ. Digestion of the dHJ substrate by T7 endonuclease I resolves the substrate into crossover and non-crossover products, as predicted by the Szostak model of double strand break repair. This substrate will greatly facilitate the examination of the mechanism of resolution of double Holliday junctions.     

Mesd encodes an LRP5/6 chaperone essential for specification of mouse embryonic polarity

Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. Here, we demonstrate that mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3(-)(/)(-) embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling.