bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of cystic fibrosis transmembrane conductance regulator membrane trafficking: not just from the endoplasmic reticulum to the Golgi
Authors: Farinha Carlos M. , Matos Paulo , Amaral Margarida D.
Published: June 2013

Biogenesis of cystic fibrosis transmembrane conductance regulator (CFTR) starts with its cotranslational insertion into the membrane of the endoplasmic reticulum (ER) and core glycosylation. These initial events are followed by a complex succession of steps with the main goal of checking the overall quality of CFTR conformation in order to promote its exit from the ER through the secretory pathway. Failure to pass the various checkpoints of the ER quality control targets the most frequent disease‐causing mutant protein (F508del‐CFTR) for premature degradation. For wild‐type CFTR that exits the ER, trafficking through the Golgi is the major site for glycan processing, although nonconventional trafficking pathways have also been described for CFTR. Once CFTR is at the cell surface, its stability is also controlled by multiple protein interactors, including Rab proteins, Rho small GTPases, and PDZ proteins. These regulate not only anterograde trafficking to the cell surface, but also endocytosis and recycling, thus achieving fine and tight modulation of CFTR plasma membrane levels. Exciting recent data have related autophagy and epithelial differentiation to the regulation of CFTR trafficking. Herein, we review the various checkpoints of the complex quality control along the secretory trafficking pathway and the associated pathways that are starting to be explored for the benefit of cystic fibrosis patients.
Low‐temperature atmospheric pressure plasma enhanced tooth whitening: the next‐generation technology
Authors: Claiborne D , McCombs G , Lemaster M , Akman MA , Laroussi M
Published: June 2013

Objectives

To evaluate the safety and effectiveness of the plasma pencil (PP) device in conjunction with H2O2 gel. The purpose of this study was to determine whether LTAPP delivered using the PP would enhance the tooth‐whitening process while causing no thermal threat.

Methods

The study consisted of thirty extracted human teeth that were randomized into two groups: Group I received LTAPP plus 36% H2O2 gel at 10, 15 and 20 min and Group II received 36% H2O2 gel only at the same time intervals. Tooth surface temperature was measured periodically throughout the experiment using a non‐contact thermometer. Digital photographs were taken pre‐ and post‐treatment and transferred to Adobe Photoshop for comparison, using the CIELAB Color Value System. Only L* (lightness) values were evaluated in this study. Data were analysed using descriptive statistics and t‐test at the 0.05 level.

Results

The results revealed a statistically significant difference in mean CIE L* values after exposure to LTAPP plus 36% H2O2 gel, compared with 36% H2O2 only, in the 10‐ and 20‐min groups (P = 0.0003 and 0.0103, respectively). The temperature in both treatment groups remained under 80°F throughout the study, which is below the thermal threat for vital tooth bleaching.

Conclusion

Utilizing PP device in conjunction with 36% H2O2 safely accelerates and enhances the tooth‐whitening process.
NaOH treatment reduces capsaicin content and pungency of ‘Makedoniko’ pepper (Capsicum annuum L.)
Authors: Kosmidou Elisavet , Kefalas Panagiotis , Gerasopoulos Dimitrios
Published: June 2013

Pickled peppers are preferred by certain consumer groups at reduced pungency. To investigate the reduction in pepper pungency and capsaicin content, fresh ‘Makedoniko’ hot peppers were treated with 0, 0.5 or 1% NaOH before preserved in acid brine for 60 days. Pepper physicochemical traits were monitored following NaOH treatment and during preservation. Capsaicin content was measured in the beginning and at the end of preservation, and the peppers were also tested organoleptically. Preservation in acid brine induced the typical green–yellow product colour, increased firmness and decreased the dry mass in all treatments. NaOH‐treated pepper increased phenolics, chlorophyll and antioxidant content in dry mass during preservation faster than the control. Capsaicin content was reduced by 25 or 50% following 0.5 or 1% NaOH treatment, respectively. Pepper organoleptic pungency was also decreased following 1% NaOH treatment. NaOH reduces capsaicin content and pungency of ‘Makedoniko’ pepper, with minor effects on its processed product quality.
The Parkinson's disease‐associated gene PINK1 protects neurons from ischemic damage by decreasing mitochondrial translocation of the fission promoter Drp1
Authors: Zhao Yanxin , Chen Fangzhe , Chen Shufen , Liu Xueyuan , Cui Mei , Dong Qiang
Published: June 2013

Our previous study has shown that PTEN‐induced novel kinase 1 (PINK1) knocking down significantly induced mitochondrial fragmentation. Although PINK1 is proved to be associated with autosomal recessive parkinsonism and its function in this chronic pathological process is widely studied, its role in acute energy crisis such as ischemic stroke is poorly known. In this study by employing an oxygen–glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. Human PINK1, two PINK1 mutants W437X and K219M, or Pink1 shRNA were transduced before OGD using lentiviral delivery. Our results showed that over‐expression of wild‐type PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential. PINK1 over‐expression also reversed OGD increased mitochondrial fragmentation, and suppressed the translocation of the mitochondrial fission protein dynamin‐related protein 1 (Drp1) from the cytosol to the mitochondria. Transduction of the mutant PINK1 failed to provide any protective effect, while knockdown of Pink1 significantly increased the severity of OGD‐induced neuronal damage. Importantly, inhibition of Drp1 reversed the effects of knocking down Pink1 on neuronal death and ATP production in response to OGD. This study demonstrates that PINK1 prevents ischemic damage in neurons by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia‐induced mitochondrial fission. These novel findings implicate a pivotal role of PINK1 regulated mitochondrial dynamics in the pathology of ischemic stroke.

image

In this study by employing an oxygen–glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. We indicated that PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia‐induced mitochondrial fission.
E‐cadherin downregulation and Twist overexpression since early stages of oral carcinogenesis
Authors: de Freitas Silva Brunno Santos , Yamamoto‐Silva Fernanda Paula , Pontes Hélder Antônio Rebelo , Pinto Júnior Décio dos Santos
Published: June 2013

There is some evidence of Twist participation in oral carcinogenesis; however, little is known about its interaction with E‐cadherin in oral squamous cell carcinoma (OSCC) development. This experimental study included an immunohistochemical analysis of Twist and E‐cadherin proteins in paraffin‐embedded specimens of oral leukoplakia (OL), OSCC, and normal oral mucosa. In addition, it was also performed a Western blot and double‐immunofluorescence analysis of Twist and E‐cadherin expression in OSCC cell lines. Significant differences in Twist and E‐cadherin immunoexpression were observed between normal oral mucosa and OL, with an inverse relation since the earliest stages of oral dysplasia (r = −0,512; P < 0.001). Western blot and double‐immunofluorescence analysis showed differences in Twist and E‐cadherin expression among human oral keratinocytes and OSCC cell lines suggesting that downregulation of E‐cadherin occurs in a dependent manner of Twist in OSCC. Our results showed a possible value of Twist and E‐cadherin in the prediction of risk of oral epithelium malignant transformation.
Depending on the stage of hepatosteatosis, p53 causes apoptosis primarily through either DRAM‐induced autophagy or BAX
Authors: Liu Kai , Lou Jinli , Wen Tao , Yin Jiming , Xu Bin , Ding Wei , Wang Anna , Liu Daojie , Zhang Chao , Chen Dexi , Li Ning
Published: June 2013

Background & Aims

Apoptosis mediated by p53 plays a pathological role in the progression of hepatosteatosis. It is noteworthy that p53 can promote the expression of damage‐regulated autophagy modulator (DRAM), an inducer of autophagy‐mediated apoptosis. However, the relationship between p53‐mediated apoptosis and autophagy in hepatosteatosis remains elusive. This study aimed to examine how p53 orchestrates autophagy and apoptosis to affect hepatosteatosis.

Methods

HepG2 cells were treated with oleic acid (OA) for 24 h to induce hepatosteatosis. Mice were fed a high‐fat diet for 20 or 40 weeks to induce hepatosteatosis.

Results

OA induced a dose‐dependent increase in steatosis severity and apoptosis. OA also induced autophagy, which was a critical inducer of apoptosis in mild steatosis induced by 400 μM OA, but not in the more severe steatosis induced by 800 and 1200 μM OA. p53 inhibition by siRNA mostly blocked OA‐induced apoptosis and autophagy. Moreover, OA‐induced autophagy was DRAM‐dependent and primarily occurred in the mitochondria (mitophagy), where DRAM was localized. In severe steatosis induced by 1200 μM OA, apoptosis was mainly dependent on p53‐induced expression of BAX, which was also localized to the mitochondria. Our in vivo study showed that p53 expression increased in both mild and severe hepatosteatosis. Increased DRAM expression and autophagy were identified in mild hepatosteatosis, whereas greater BAX expression was observed in severe hepatosteatosis.

Conclusions

p53 may induce apoptosis via different mechanisms. DRAM‐mediated mitophagy is a primary apoptotic inducer in mild hepatosteatosis, whereas p53‐induced BAX expression mainly induces apoptosis in severe hepatosteatosis.
Determination of ethyl glucuronide in hair improves evaluation of long‐term alcohol abstention in liver transplant candidates
Authors: Sterneck Martina , Yegles Michel , Rothkirch von Gregor , Staufer Katharina , Vettorazzi Eik , Schulz Karl‐Heinz , Tobias Niels , Graeser Christian , Fischer Lutz , Nashan Bjoern , Andresen‐Streichert Hilke
Published: June 2013

Background & Aims

Prior to listing patients for Orthotopic liver transplantation (OLT) an abstention period of 6 months is required. Ethyl glucuronide in the hair is a new reliable marker for the assessment of alcohol consumption. Here, the diagnostic value of determining the ethyl glucuronide concentration in the hair of liver transplant candidates was evaluated.

Methods

In 63 transplant candidates with alcoholic liver cirrhosis and 25 control patients with cirrhosis of other aetiologies alcohol markers, i.e. hEtG, urine EtG, blood ethanol, methanol and carbohydrate deficient transferrin were determined in parallel to an interview with a psychologist.

Results

A total of 19 (30%) transplant candidates admitted alcohol consumption within the last 6 months, while 39/63 (62%) were positive for at least one alcohol marker. In 52% of the 44 candidates denying alcohol consumption, abstention was disproved by detecting at least one positive alcohol marker, in 83% of cases by a positive hEtG result. In the control patients stating abstention from alcohol all hEtG tests were negative. No impact of renal or liver function on hEtG results was detected. A specificity of 98% and a positive predictive value of 92% were calculated for testing hEtG in proximal hair segment and applying a cut‐off of 30 pg/mg.

Conclusions

In 52% of patients denying alcohol consumption within the last 6 months, alcohol abstention was disproved, in 83% of cases by hEtG testing. Therefore, hEtG is a promising new marker for the evaluation of long‐term alcohol abstention in liver transplant candidates.
miR146a impairs the IFN‐induced anti‐HBV immune response by downregulating STAT1 in hepatocytes
Authors: Hou Zhao H. , Han Qiu J. , Zhang Cai , Tian Zhi G. , Zhang Jian
Published: June 2013

Background & Aims

Previous studies have shown that hepatitis B virus (HBV) interferes with host antiviral immunity via multiple pathways. In clinical practice, interferon resistance is a serious issue for treatment of HBV infection. Now, miRNAs have been reported to be widely involved in antiviral immunity and have become a novel tool to study virus–host interaction. We question whether miRNAs play a role in HBV‐induced interferon resistance in hepatocytes.

Methods

MiRNAs levels in HepG2 and HepG2.2.15 cells were compared by qRT‐PCR. The effects of miR146a on HBV infection were characterized by interference miR146a level, followed by the quantification of HBV mRNA, DNA and antigens. We employed qRT‐PCR and western blot to study the effects of miR146a on the IFN‐α signalling pathway. The miR146a promoter activity was validated by a luciferase reporter assay.

Results

HBV infection impaired IFN‐α signalling pathway in hepatocytes. MiR146a was upregulated in HBV+ HepG2.2.15 cells, and the transcriptional activity of miR146a in HepG2.2.15 cells was increased compared with HepG2 cells. HBV infection, especially the introduction of HBx, induced miR146a expression in vitro. Moreover, miR146a attenuated the production of type I interferon‐induced antiviral factors. Low STAT1 levels were noticed in HBV+ HCC cells, and the luciferase reporter assay showed that STAT1 was post‐transcriptionally downregulated by miR146a. Furthermore, the silencing of miR146a by antisense inhibitors enhanced IFN‐α‐mediated anti‐HBV efficiency.

Conclusions

Our findings demonstrate that HBV infection promotes miR146a transcription, which represses STAT1 and results in interferon resistance. These observations reveal a novel role for miR146a in HBV immunopathogenesis, and provide a potential target for the therapeutic recovery of IFN‐α‐induced anti‐HBV effects.
Tfh and plasma cells are correlated with hypergammaglobulinaemia in patients with autoimmune hepatitis
Authors: Ma Liang , Qin Junjie , Ji Huifan , Zhao Pingwei , Jiang Yanfang
Published: June 2013

Background

This study aimed at examining the numbers of different subsets of circulating Tfh and B cells in patients with new onset autoimmune hepatitis (AIH).

Methods

A total of 17 patients with new onset AIH and 17 age‐/gender‐matched healthy controls (HC) were examined for the numbers of ICOS+, PD‐1+ and IL‐21+ Tfh cells, CD27+, CD38+, CD95+, CD86+ and IL‐10+ B cells were quantified by flow cytometry. The concentrations of serum IL‐21 and IL‐10 were examined.

Results

In comparison with that in the HC, significantly increased numbers of circulating CD38+, CD86+ or CD95+ B cells, ICOS+ and PD‐1+ Tfh cells and increased levels of serum IL‐21, but reduced numbers of CD27+, IL‐10+ B cells were detected in the patients. The concentrations of serum IL‐21 and IL‐10 were positively correlated with the numbers of CD4+CXCR5+ TFH and CD19+CD5+CD1d+ B cells respectively. The numbers of ICOS+ or PD‐1+ Tfh cells were correlated positively with CD86+ or CD95+ B cells in those patients respectively. The numbers of CD38+ B cells, ICOS+ or PD‐1+ Tfh cells were correlated positively with the concentrations of serum IgG or IgM in the patients respectively; the concentrations of serum IL‐21 were correlated positively with serum IgG, IgA and IgM and the concentrations of serum IL‐10 were correlated negatively with serum IgG and IgM in the patients.

Conclusion

Circulating activated Tfh and plasma cells may be associated with hypergammaglobulinaemia during the pathogenic process of AIH in humans.
Relaxin decreases the severity of established hepatic fibrosis in mice
Authors: Bennett Robert G. , Heimann Dean G. , Singh Sudhir , Simpson Ronda L. , Tuma Dean J.
Published: June 2013

Background & Aims

Hepatic fibrosis is characterized by excess collagen deposition, decreased extracellular matrix degradation and activation of the hepatic stellate cells. The hormone relaxin has shown promise in the treatment of fibrosis in a number of tissues, but the effect of relaxin on established hepatic fibrosis is unknown. The aim of this study was to determine the effect of relaxin on an in vivo model after establishing hepatic fibrosis

Methods

Male mice were made fibrotic by carbon tetrachloride treatment for 4 weeks, followed by treatment with two doses of relaxin (25 or 75 μg/kg/day) or vehicle for 4 weeks, with continued administration of carbon tetrachloride.

Results

Relaxin significantly decreased total hepatic collagen and smooth muscle actin content at both doses, and suppressed collagen I expression at the higher dose. Relaxin increased the expression of the matrix metalloproteinases MMP13 and MMP3, decreased the expression of MMP2 and tissue inhibitor of metalloproteinase 2 (TIMP2) and increased the overall level of collagen‐degrading activity. Relaxin decreased TGFβ‐induced Smad2 nuclear localization in mouse hepatic stellate cells.

Conclusions

The results suggest that relaxin reduced collagen deposition and HSC activation in established hepatic fibrosis despite the presence of continued hepatic insult. This reduced fibrosis was associated with increased expression of the fibrillar collagen‐degrading enzyme MMP13, decreased expression of TIMP2, and enhanced collagen‐degrading activity, and impaired TGFβ signalling, consistent with relaxin's effects on activated fibroblastic cells. The results suggest that relaxin may be an effective treatment for the treatment of established hepatic fibrosis.
Notch signaling mediates melanoma–endothelial cell communication and melanoma cell migration
Authors: Howard Jason D. , Moriarty Whei F. , Park JinSeok , Riedy Katherine , Panova Izabela P. , Chung Christine H. , Suh Kahp‐Yang , Levchenko Andre , Alani Rhoda M.
Published: June 2013

Stromal and cellular components within the tumor microenvironment significantly influence molecular signals mediating tumor growth and progression. We recently performed a screen to evaluate critical mediators of melanoma–endothelial communication and identified several molecular pathways associated with these cellular networks, including Notch3. Here, we evaluate the nature of melanoma–endothelial communication mediated by Notch3 and its functional significance. We find that Notch3 is specifically upregulated in melanoma–endothelial cell cocultures and is functionally associated with increased Notch signaling in melanoma cells. Furthermore, induced Notch3 signaling in melanoma cell lines leads to enhanced tumor cell migration without associated increases in tumor cell growth. Additionally, Notch3 expression is specifically associated with malignant patient samples and is not evident in benign nevi. We conclude that Notch3 mediates melanoma–endothelial cell communication and tumor cell migration and may serve as a meaningful therapeutic target for this aggressive malignancy.
Improving the Milk Supply Chain in Developing Countries: Analysis, Insights, and Recommendations
Authors: Mu Liying , Dawande Milind , Mookerjee Vijay
Published: June 2013

Quality issues in milk—arising primarily from deliberate adulteration by producers—have been reported in several developing countries. In the milk supply chain, a station buys raw milk from a number of producers, mixes the milk and sells it to a firm (that then sells the processed milk to end consumers). We study a non‐cooperative game between a station and a population of producers. Apart from penalties on proven low‐quality producers, two types of incentives are analyzed: confessor rewards for low‐quality producers who confess and quality rewards for producers of high‐quality milk. Contrary to our expectations, whereas (small) confessor rewards can help increase both the quality of milk and the station's profit, quality rewards can be detrimental. We examine two structures based on the ordering of individual and mixed testing of milk: pre‐mixed individual testing (first test a fraction of producers individually and then [possibly] perform a mixed test on the remaining producers) and post‐mixed individual testing (first test the mixed milk from all producers and then test a fraction of producers individually). Whereas pre‐mixed individual testing can be socially harmful, a combination of post‐mixed individual testing and other incentives achieves a desirable outcome: all producers supply high‐quality milk with only one mixed test and no further testing by the station.
Interruption and Forgetting in Knowledge‐Intensive Service Environments
Authors: Froehle Craig M. , White Denise L.
Published: June 2013

An increasing barrier to productivity in knowledge‐intensive work environments is interruptions. Interruptions stop the current job and can induce forgetting in the worker. The induced forgetting can cause re‐work; to complete the interrupted job, additional effort and time is required to return to the same level of job‐specific knowledge the worker had attained prior to the interruption. This research employs primary observational and process data gathered from a hospital radiology department as inputs into a discrete‐event simulation model to estimate the effect of interruptions, forgetting, and re‐work. To help mitigate the effects of interruption‐induced re‐work, we introduce and test the operational policy of sequestering, where some service resources are protected from interruptions. We find that sequestering can improve the overall productivity and cost performance of the system under certain circumstances. We conclude that research examining knowledge‐intensive operations should explicitly consider interruptions and the forgetting rate of the system's human workers or models will overestimate the system's productivity and underestimate its costs.
The Effect of Competition on the Efficient–Responsive Choice
Authors: Wang Tong , Thomas Douglas J. , Rudi Nils
Published: June 2013

In determining their operations strategy, a firm chooses whether to be responsive or efficient. For firms competing in a market with uncertain demand and varying intensity of substitutability for the competitor's product, we characterize the responsive or efficient choice in equilibrium. To focus first on the competitive implications, we study a model where a firm can choose to be responsive at no additional fixed or marginal cost. We find that competing firms will choose the same configuration (responsive or efficient), and responsiveness tends to be favorable when demand uncertainty is high or when product competition is not too strong. Intense competition can drive firms to choose to be efficient rather than responsive even when there is no additional cost of being responsive. In such a case, both firms would be better off by choosing to be responsive but cannot credibly commit. We extend the basic model to study the impact of endogenized production timing, multiple productions and product holdback (or, equivalently, postponed production). For all these settings, we find structurally similar results; firms choose the same configuration, and the firms may miss Pareto‐improvements. Furthermore, through extensions to the basic model, we find that greater operational flexibility can make responsiveness look less attractive in the presence of product competition. In contrast to our basic model and other extensions, we find it is possible for one firm to be responsive while the other is efficient when there is either a fixed cost or variable cost premium associated with responsive delivery.
Carlactone‐independent seedling morphogenesis in Arabidopsis
Authors: Scaffidi Adrian , Waters Mark T. , Ghisalberti Emilio L. , Dixon Kingsley W. , Flematti Gavin R. , Smith Steven M.
Published: June 2013

Strigolactone hormones are derived from carotenoids via carlactone, and act through the α/β–hydrolase D14 and the F–box protein D3/MAX2 to repress plant shoot branching. While MAX2 is also necessary for normal seedling development, D14 and the known strigolactone biosynthesis genes are not, raising the question of whether endogenous, canonical strigolactones derived from carlactone have a role in seedling morphogenesis. Here, we report the chemical synthesis of the strigolactone precursor carlactone, and show that it represses Arabidopsis shoot branching and influences leaf morphogenesis via a mechanism that is dependent on the cytochrome P450 MAX1. In contrast, both physiologically active Z–carlactone and the non‐physiological E isomer exhibit similar weak activity in seedlings, and predominantly signal through D14 rather than its paralogue KAI2, in a MAX2‐dependent but MAX1‐independent manner. KAI2 is essential for seedling morphogenesis, and hence this early‐stage development employs carlactone‐independent morphogens for which karrikins from wildfire smoke are specific surrogates. While the commonly employed synthetic strigolactone GR24 acts non‐specifically through both D14 and KAI2, carlactone is a specific effector of strigolactone signalling that acts through MAX1 and D14.
Adaptation to acidic soil is achieved by increased numbers of cis‐acting elements regulating ALMT1 expression in Holcus lanatus
Authors: Chen Zhi Chang , Yokosho Kengo , Kashino Miho , Zhao Fang‐Jie , Yamaji Naoki , Ma Jian Feng
Published: June 2013

Yorkshire fog (Holcus lanatus), which belongs to the Poaceae family and is a close relative of the agronomic crop oat (Avena sativa), is a widely adaptable grass species that is able to grow on highly acidic soils with high levels of Al, but the mechanism underlying the high Al tolerance is unknown. Here, we characterized two accessions of H. lanatus collected from an acid plot (soil pH 3.6, HL–A) and a neutral plot (pH 7.1, HL–N) in terms of Al tolerance, organic acid anion secretion and related gene expression. In response to Al (pH 4.5), the HL–A roots secreted approximately twice as much malate as the HL–N roots, but there was no difference in citrate secretion. Cloning of the gene HlALMT1 responsible for malate secretion showed that the encoded amino acid sequence did not differ between two accessions, but the expression level in the outer cell layers of the HL–A roots was twice as high as in the HL–N roots. This difference was not due to the genomic copy number, but was due to the number of cis‐acting elements for an Al‐responsive transcription factor (HlART1) in the promoter region of HlALMT1, as demonstrated by both a yeast one‐hybrid assay and a transient assay in tobacco protoplasts. Furthermore, introduction of HlALMT1 driven by the HL–A promoter into rice resulted in significantly more Al‐induced malate secretion than introduction of HlALMT1 driven by the HL–N promoter. These findings indicate that the adaptation of H. lanatus to acidic soils may be achieved by increasing number of cis‐acting elements for ART1 in the promoter region of the HlALMT1 gene, enhancing the expression of HlALMT1 and the secretion of malate.
Discovery of Plant Phenolic Compounds That Act as Type III Secretion System Inhibitors or Inducers of the Fire Blight Pathogen, Erwinia amylovora
Authors: Khokhani Devanshi , Zhang Chengfang , Li Yan , Wang Qi , Zeng Quan , Yamazaki Akihiro , Hutchins William , Zhou Shan-Shan , Chen Xin , Yang Ching-Hong
Published: June 2013

Erwinia amylovora
causes a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized by
E. amylovora
in order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer,
trans
-2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through the

rsmB
Ea

-RsmA
Ea
system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS of
E. amylovora
. In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens.
Dry/Wet Cycles Change the Activity and Population Dynamics of Methanotrophs in Rice Field Soil
Authors: Ma Ke , Conrad Ralf , Lu Yahai
Published: June 2013

The methanotrophs in rice field soil are crucial in regulating the emission of methane. Drainage substantially reduces methane emission from rice fields. However, it is poorly understood how drainage affects microbial methane oxidation. Therefore, we analyzed the dynamics of methane oxidation rates, composition (using terminal restriction fragment length polymorphism [T-RFLP]), and abundance (using quantitative PCR [qPCR]) of methanotrophpmoAgenes (encoding a subunit of particulate methane monooxygenase) and their transcripts over the season and in response to alternate dry/wet cycles in planted paddy field microcosms.In situmethane oxidation accounted for less than 15% of total methane production but was enhanced by intermittent drainage. The dry/wet alternations resulted in distinct effects on the methanotrophic communities in different soil compartments (bulk soil, rhizosphere soil, surface soil). The methanotrophic communities of the different soil compartments also showed distinct seasonal dynamics. In bulk soil, potential methanotrophic activity and transcription ofpmoAwere relatively low but were significantly stimulated by drainage. In contrast, however, in the rhizosphere and surface soils, potential methanotrophic activity andpmoAtranscription were relatively high but decreased after drainage events and resumed after reflooding. While type II methanotrophs dominated the communities in the bulk soil and rhizosphere soil compartments (and to a lesser extent also in the surface soil), it was thepmoAof type I methanotrophs that was mainly transcribed under flooded conditions. Drainage affected the composition of the methanotrophic community only minimally but strongly affected metabolically active methanotrophs. Our study revealed dramatic dynamics in the abundance, composition, and activity of the various type I and type II methanotrophs on both a seasonal and a spatial scale and showed strong effects of dry/wet alternation cycles, which enhanced the attenuation of methane flux into the atmosphere.
Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates
Authors: Sultana Reshma , McBain Andrew J. , O'Neill Catherine A.
Published: June 2013

In this study, we investigated whether probiotic lysates can modify the tight-junction function of human primary keratinocytes. The keratinocytes were grown on cell culture inserts and treated with lysates from
Bifidobacterium longum
,
Lactobacillus plantarum
,
Lactobacillus reuteri
,
Lactobacillus fermentum
, or
Lactobacillus rhamnosus
GG. With the exception of
L. fermentum
(which decreased cell viability), all strains markedly enhanced tight-junction barrier function within 24 h, as assessed by measurements of transepithelial electrical resistance (TEER). However,
B. longum
and
L. rhamnosus
GG were the most efficacious, producing dose-dependent increases in resistance that were maintained for 4 days. These increases in TEER correlated with elevated expression of tight-junction protein components. Neutralization of Toll-like receptor 2 abolished both the increase in TEER and expression of tight-junction proteins induced by
B. longum
, but not
L. rhamnosus
GG. These data suggest that some bacterial strains increase tight-junction function via modulation of protein components but the different pathways involved may vary depending on the bacterial strain.
Molecular Epidemiology of Newcastle Disease in Mexico and the Potential Spillover of Viruses from Poultry into Wild Bird Species
Authors: Cardenas Garcia Stivalis , Navarro Lopez Roberto , Morales Romeo , Olvera Miguel A. , Marquez Miguel A. , Merino Ruben , Miller Patti J. , Afonso Claudio L.
Published: June 2013

Newcastle disease, one of the most important health problems that affects the poultry industry around the world, is caused by virulent strains of Newcastle disease virus. Newcastle disease virus is considered to be endemic in several countries in the Americas, including Mexico. In order to control Newcastle disease outbreaks and spread, intensive vaccination programs, which include vaccines formulated with strains isolated at least 60 years ago, have been established. These vaccines are dissimilar in genotype to the virulent Newcastle disease viruses that had been circulating in Mexico until 2008. Here, 28 isolates obtained between 2008 and 2011 from different regions of Mexico from free-living wild birds, captive wild birds, and poultry were phylogenetically and biologically characterized in order to study the recent epidemiology of Newcastle disease viruses in Mexico. Here we demonstrate that, until recently, virulent viruses from genotype V continued to circulate and evolve in the country. All of the Newcastle disease viruses of low virulence, mostly isolated from nonvaccinated free-living wild birds and captive wild birds, were highly similar to LaSota (genotype II) and PHY-LMV42 (genotype I) vaccine strains. These findings, together with the discovery of two virulent viruses at the Mexican zoo, suggest that Newcastle disease viruses may be escaping from poultry into the environment.
Oral Administration of Live Exopolysaccharide-Producing Pediococcus parvulus, but Not Purified Exopolysaccharide, Suppressed Enterobacteriaceae without Affecting Bacterial Diversity in Ceca of Mice
Authors: Lindström Cecilia , Xu Jie , Öste Rickard , Holst Olle , Molin Göran
Published: June 2013

Growing evidence indicates that the gut microbiota could have an important role in the development of diet- and lifestyle-induced diseases. It has been shown that modulation of the gut microbiota by means of probiotics and prebiotics could improve host health. An oat-based product fermented by the exopolysaccharide (EPS)-producing organism
Pediococcus parvulus
2.6 has been reported to have a bifidogenic effect. To find out whether the effect could be attributed to the EPS or the bacterium, mice were fed a diet supplemented with 2% purified EPS or 10
8
CFU/g of live
P. parvulus
2.6 for 6 weeks. Both supplementations altered the gut microbiota composition but in different directions. Purified EPS not only significantly lowered the microbial diversity (
P
< 0.001) but decreased the bifidobacterial population (
P
= 0.01). In contrast, the live EPS-producing bacterium
P. parvulus
2.6 antagonized
Enterobacteriaceae
without disturbing the homeostasis of the cecal microbiota.
Diversity of Bacterial Endosymbionts Associated with Macrosteles Leafhoppers Vectoring Phytopathogenic Phytoplasmas
Authors: Ishii Yoshiko , Matsuura Yu , Kakizawa Shigeyuki , Nikoh Naruo , Fukatsu Takema
Published: June 2013

Here, we investigate the endosymbiotic microbiota of the
Macrosteles
leafhoppers
M. striifrons
and
M. sexnotatus
, known as vectors of phytopathogenic phytoplasmas. PCR, cloning, sequencing, and phylogenetic analyses of bacterial 16S rRNA genes identified two obligate endosymbionts, “
Candidatus
Sulcia muelleri” and “
Candidatus
Nasuia deltocephalinicola,” and five facultative endosymbionts,
Wolbachia
,
Rickettsia
,
Burkholderia
,
Diplorickettsia
, and a novel bacterium belonging to the
Rickettsiaceae
, from the leafhoppers. “
Ca
. Sulcia muelleri” and “
Ca
. Nasuia deltocephalinicola” exhibited 100% infection frequencies in the host species and populations and were separately harbored within different bacteriocytes that constituted a pair of coherent bacteriomes in the abdomen of the host insects, as in other deltocephaline leafhoppers.
Wolbachia
,
Rickettsia
,
Burkholderia
,
Diplorickettsia
, and the novel
Rickettsiaceae
bacterium exhibited infection frequencies at 7%, 31%, 12%, 0%, and 24% in
M. striifrons
and at 20%, 0%, 0%, 20%, and 0% in
M. sexnotatus
, respectively. Although undetected in the above analyses, phytoplasma infections were detected in 16% of
M. striifrons
and 60% of
M. sexnotatus
insects by nested PCR of 16S rRNA genes. Two genetically distinct phytoplasmas, namely, “
Candidatus
Phytoplasma asteris,” associated with aster yellows and related plant diseases, and “
Candidatus
Phytoplasma oryzae,” associated with rice yellow dwarf disease, were identified from the leafhoppers. These results highlight strikingly complex endosymbiotic microbiota of the
Macrosteles
leafhoppers and suggest ecological interactions between the obligate endosymbionts, the facultative endosymbionts, and the phytopathogenic phytoplasmas within the same host insects, which may affect vector competence of the leafhoppers.
Organization of Organelles within Hyphae of Ashbya gossypii Revealed by Electron Tomography
Authors: Gibeaux Romain , Hoepfner Dominic , Schlatter Ivan , Antony Claude , Philippsen Peter
Published: June 2013

Ashbya gossypii
grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in
A. gossypii
. Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (∼200, ∼150, and ∼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.
The Vibrio parahaemolyticus Small RNA RyhB Promotes Production of the Siderophore Vibrioferrin by Stabilizing the Polycistronic mRNA
Authors: Tanabe Tomotaka , Funahashi Tatsuya , Nakao Hiroshi , Maki Jun , Yamamoto Shigeo
Published: June 2013

High-affinity iron acquisition in
Vibrio parahaemolyticus
is mediated by the cognate siderophore vibrioferrin. We have previously reported that the vibrioferrin biosynthesis operon (
pvs
Op) is regulated at the transcriptional level by the iron-responsive repressor Fur (T. Tanabe, T. Funahashi, H. Nakao, S. Miyoshi, S. Shinoda, and S. Yamamoto, J. Bacteriol. 185:6938–6949, 2003). In this study, we identified the Fur-regulated small RNA RyhB and the RNA chaperone Hfq protein as additional regulatory proteins of vibrioferrin biosynthesis. We found that vibrioferrin production was greatly impaired in both the
ryhB
and
hfq
deletion mutants, and a TargetRNA search (
http://snowwhite.wellesley.edu/targetRNA/index2.html
) revealed that the 5′-untranslated region of
pvs
Op mRNA (
pvs
Op 5′-UTR) contains a potential base-pairing region required for the formation of the RyhB-
pvs
Op 5′-UTR duplex. An electrophoresis mobility shift assay indicated that RyhB can directly bind to the
pvs
Op 5′-UTR with the aid of Hfq. Rifampin chase experiments indicated that the half-life of
pvs
Op mRNA in the
ryhB
and
hfq
mutants was approximately 3-fold shorter than that in the parental strain, suggesting that both RyhB and Hfq are engaged in the stabilization of
pvs
Op mRNA. Chrome azurol S assays followed by electrophoresis mobility shift assays and rifampin chase experiments carried out for mutant strains indicated that base pairing between RyhB and the
pvs
Op 5′-UTR results in an increase in the stability of
pvs
Op mRNA, thereby leading to the promotion of vibrioferrin production. It is unprecedented that RyhB confers increased stability on a polycistronic mRNA involved in siderophore biosynthesis as a direct target.
Effect of an Oxygen-Tolerant Bifurcating Butyryl Coenzyme A Dehydrogenase/Electron-Transferring Flavoprotein Complex from Clostridium difficile on Butyrate Production in Escherichia coli
Authors: Aboulnaga El-Hussiny , Pinkenburg Olaf , Schiffels Johannes , El-Refai Ahmed , Buckel Wolfgang , Selmer Thorsten
Published: June 2013

The butyrogenic genes from
Clostridium difficile
DSM 1296
T
have been cloned and expressed in
Escherichia coli
. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in
E. coli
and kinetically characterized
in vitro
. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host.
In vitro
and
in vivo
studies demonstrated that
C. difficile
possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD
+
-oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from
C. difficile
is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (

R

hodobacter
n
itrogen
f
ixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family
Authors: Schürmann Marc , Hirsch Beatrice , Wübbeler Jan Hendrik , Stöveken Nadine , Steinbüchel Alexander
Published: June 2013

The
act
gene of
Variovorax paradoxus
TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act
TBEA6
(2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3′-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn
5
::
mob
-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn
5
::
mob
was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated Act
TBEA6
to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. Act
TBEA6
was heterologously expressed in
Escherichia coli
Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent
V
max
of 44.6 μmol min
−1
mg
−1
for succinyl-CoA, which corresponds to a turnover number of 36.0 s
−1
per subunit of Act
TBEA6
. For 3SP, the apparent
V
max
was determined as 46.8 μmol min
−1
mg
−1
, which corresponds to a turnover number of 37.7 s
−1
per subunit of Act
TBEA6
. The apparent

K
m

values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the
V. paradoxus
Δ
act
mutant did not reproduce the phenotype of the Tn
5
::
mob-
induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn
5
::
mob
-induced mutant with pBBR1MCS5::
acd
DPN7
partially restored growth on 3SP, which indicated a polar effect of the Tn
5
::
mob
transposon on
acd
TBEA6
, located downstream of
act
TBEA6
.
Exercise and the platelet activator calcium chloride both influence the growth factor content of platelet-rich plasma (PRP): overlooked biochemical factors that could influence PRP treatment
Authors: Hamilton Bruce , Tol Johannes L , Knez Wade , Chalabi Hakim
Published: June 2013

Background

There is strong evidence that exercise affects platelet haemostasis factors, but this potential effect on growth factor concentrations in platelet-rich plasma (PRP) has never been studied. In addition, there is a paucity of studies focusing on the effects of activating agents used in conjunction with PRP. The first aim of this study was to evaluate the effect of exercise on platelet and platelet-derived growth factors (PDGF)-AB, hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) concentrations in PRP. The second aim was to study the effect of the activating agent calcium chloride (CaCl2) on growth factor concentration in relation to different exercise states.

Methods

Controlled laboratory study. Ten healthy participants performed 1 h of submaximal exercise with blood being withdrawn immediately pre, post and 18 h following. PRP was prepared in each condition in both an activated CaCl2and non-activated form. Concentrations of PDGF-AB, HGF, IGF-1 and VEGF were evaluated using standard ELISA systems.

Results

Exercise had no significant effect on platelet concentration, but significantly suppressed both VEGF and PDGF-AB concentrations. Exercise state had no significant effect on IGF-1 or HGF concentration. Activation with CaCl2resulted in a significant increase in PDGF-AB and IGF-1 concentrations, unchanged VEGF and significantly reduced HGF concentrations.

Conclusions

Exercise significantly impacts on PDGFs in PRP with significantly reduced concentrations of VEGF and PDFG-AB. Furthermore, the activation of PRP with CaCl2results in a differentiated GF release from platelets. These relevant factors can potentially influence outcome in daily clinical practice and are recommended to be accounted for in future study design.