bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of microsatellite markers by transcriptome sequencing in two species of Amorphophallus (Araceae)
Authors: Zheng Xingfei , Pan Cheng , Diao Ying , You Yongning , Yang Chaozhu , Hu Zhongli
Published: July 2013

Background

Amorphophallus is a genus of perennial plants widely distributed in the tropics or subtropics of West Africa and South Asia. Its corms contain a high level of water-soluble glucomannan; therefore, it has long been used as a medicinal herb and food source. Genetic studies of Amorphophallus have been hindered by a lack of genetic markers. A large number of molecular markers are required for genetic diversity study and improving disease resistance in Amorphophallus. Here, we report large scale of transcriptome sequencing of two species: Amorphophallus konjac and Amorphophallus bulbifer using deep sequencing technology, and microsatellite (SSR) markers were identified based on these transcriptome sequences.

Results

cDNAs of A. konjac and A. bulbifer were sequenced using Illumina HiSeq™ 2000 sequencing technology. A total of 135,822 non-redundant unigenes were assembled from about 9.66 gigabases, and 19,596 SSRs were identified in 16,027 non-redundant unigenes. Di-nucleotide SSRs were the most abundant motif (61.6%), followed by tri- (30.3%), tetra- (5.6%), penta- (1.5%), and hexa-nucleotides (1%) repeats. The top di- and tri-nucleotide repeat motifs included AG/CT (45.2%) and AGG/CCT (7.1%), respectively. A total of 10,754 primer pairs were designed for marker development. Of these, 320 primers were synthesized and used for validation of amplification and assessment of polymorphisms in 25 individual plants. The total of 275 primer pairs yielded PCR amplification products, of which 205 were polymorphic. The number of alleles ranged from 2 to 14 and the polymorphism information content valued ranged from 0.10 to 0.90. Genetic diversity analysis was done using 177 highly polymorphic SSR markers. A phenogram based on Jaccard’s similarity coefficients was constructed, which showed a distinct cluster of 25 Amorphophallus individuals.

Conclusion

A total of 10,754 SSR markers have been identified in Amorphophallus using transcriptome sequencing. One hundred and seventy-seven polymorphic markers were successfully validated in 25 individuals. The large number of genetic markers developed in the present study should contribute greatly to research into genetic diversity and germplasm characterization in Amorphophallus.
Differential impacts of juvenile hormone, soldier head extract and alternate caste phenotypes on host and symbiont transcriptome composition in the gut of the termite Reticulitermes flavipes
Authors: Sen Ruchira , Raychoudhury Rhitoban , Cai Yunpeng , Sun Yijun , Lietze Verena-Ulrike , Boucias Drion G , Scharf Michael E
Published: July 2013

Background

Termites are highly eusocial insects and show a division of labor whereby morphologically distinct individuals specialize in distinct tasks. In the lower termite Reticulitermes flavipes (Rhinotermitidae), non-reproducing individuals form the worker and soldier castes, which specialize in helping (e.g., brood care, cleaning, foraging) and defense behaviors, respectively. Workers are totipotent juveniles that can either undergo status quo molts or develop into soldiers or neotenic reproductives. This caste differentiation can be regulated by juvenile hormone (JH) and primer pheromones contained in soldier head extracts (SHE). Here we offered worker termites a cellulose diet treated with JH or SHE for 24-hr, or held them with live soldiers (LS) or live neotenic reproductives (LR). We then determined gene expression profiles of the host termite gut and protozoan symbionts concurrently using custom cDNA oligo-microarrays containing 10,990 individual ESTs.

Results

JH was the most influential treatment (501 total ESTs affected), followed by LS (24 ESTs), LR (12 ESTs) and SHE treatments (6 ESTs). The majority of JH up- and downregulated ESTs were of host and symbiont origin, respectively; in contrast, SHE, LR and LS treatments had more uniform impacts on host and symbiont gene expression. Repeat “follow-up” bioassays investigating combined JH + SHE impacts in relation to individual JH and SHE treatments on a subset of array-positive genes revealed (i) JH and SHE treatments had opposite impacts on gene expression and (ii) JH + SHE impacts on gene expression were generally intermediate between JH and SHE.

Conclusions

Our results show that JH impacts hundreds of termite and symbiont genes within 24-hr, strongly suggesting a role for the termite gut in JH-dependent caste determination. Additionally, differential impacts of SHE and LS treatments were observed that are in strong agreement with previous studies that specifically investigated soldier caste regulation. However, it is likely that gene expression outside the gut may be of equal or greater importance than gut gene expression.
Characterising the aetiology of severe acute gastroenteritis among patients visiting a hospital in Qatar using real-time polymerase chain reaction
Authors: Al-Thani Asma , Baris Maide , Al-Lawati Najah , Al-Dhahry Said
Published: July 2013

Background

Acute gastroenteritis (AGE) remains a common cause of clinic visits and hospitalizations, though its aetiology has not been determined in Qatar.

Methods

We performed a prospective, emergency department–based study of 288 children and adults with AGE. Stool specimens were collected at presentation from June to November 2009. Faecal specimens were tested, using real-time PCR, for a panel of four viral (norovirus, adenovirus, astrovirus and rotavirus) and bacterial pathogens.

Results

Viral and bacterial pathogens were detected in 131 (45.5%) and 34 (12.2%) of the 288 patients recruited. The most commonly detected pathogens were norovirus (28.5%), rotavirus (10.4%), followed by adenovirus (6.25%) and astrovirus (0.30%). Norovirus was the most commonly detected viral pathogen amongst all the age groups with an almost even distribution in all age groups. Rotavirus and adenovirus were more common in children under 5 yr of age. Astrovirus was found in only one person.

Conclusions

Viruses, especially noroviruses, are associated with severe diarrhoea in children and adults in Qatar. Further studies to confirm the findings and to explore the causes of illness among patients from whom a pathogen cannot be determined are needed.
Overexpression of YAP 1 contributes to progressive features and poor prognosis of human urothelial carcinoma of the bladder
Authors: Liu Jian-Ye , Li Yong-Hong , Lin Huan-Xin , Liao Yi-Ji , Mai Shi-Juan , Liu Zhou-Wei , Zhang Zhi-Ling , Jiang Li-Juan , Zhang Jia-Xing , Kung Hsiang-Fu , Zeng Yi-Xin , Zhou Fang-Jian , Xie Dan
Published: July 2013

Background

Yes-associated protein 1 (YAP 1), the nuclear effector of the Hippo pathway, is a key regulator of organ size and a candidate human oncogene in multiple tumors. However, the expression dynamics of YAP 1 in urothelial carcinoma of the bladder (UCB) and its clinical/prognostic significance are unclear.

Methods

In this study, the methods of quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry (IHC) were utilized to investigate mRNA/ protein expression of YAP 1 in UCBs. Spearman’s rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data.

Results

Up-regulated expression of YAP 1 mRNA and protein was observed in the majority of UCBs by qRT-PCR and Western blotting, when compared with their paired normal bladder tissues. By IHC, positive expression of YAP 1 was examined in 113/213 (53.1%) of UCBs and in 6/86 (7.0%) of normal bladder specimens tissues. Positive expression of YAP 1 was correlated with poorer differentiation, higher T classification and higher N classification (P < 0.05). In univariate survival analysis, a significant association between positive expression of YAP 1 and shortened patients’ survival was found (P < 0.001). In different subsets of UCB patients, YAP 1 expression was also a prognostic indicator in patients with grade 2 (P = 0.005) or grade 3 (P = 0.046) UCB, and in patients in pT1 (P = 0.013), pT2-4 (P = 0.002), pN- (P < 0.001) or pT2-4/pN- (P = 0.004) stage. Importantly, YAP 1 expression (P = 0.003) together with pT and pN status (P< 0.05) provided significant independent prognostic parameters in multivariate analysis.

Conclusions

Our findings provide evidences that positive expression of YAP 1 in UCB may be important in the acquisition of an aggressive phenotype, and it is an independent biomarker for poor prognosis of patients with UCB.
Portuguese propolis disturbs glycolytic metabolism of human colorectal cancer in vitro
Authors: Valença Isabel , Morais-Santos Filipa , Miranda-Gonçalves Vera , Ferreira Ana Margarida , Almeida-Aguiar Cristina , Baltazar Fátima
Published: July 2013

Background

Propolis is a resin collected by bees from plant buds and exudates, which is further processed through the activity of bee enzymes. Propolis has been shown to possess many biological and pharmacological properties, such as antimicrobial, antioxidant, immunostimulant and antitumor activities. Due to this bioactivity profile, this resin can become an alternative, economic and safe source of natural bioactive compounds.

Antitumor action has been reported in vitro and in vivo for propolis extracts or its isolated compounds; however, Portuguese propolis has been little explored. The aim of this work was to evaluate the in vitro antitumor activity of Portuguese propolis on the human colon carcinoma cell line HCT-15, assessing the effect of different fractions (hexane, chloroform and ethanol residual) of a propolis ethanol extract on cell viability, proliferation, metabolism and death.

Methods

Propolis from Angra do Heroísmo (Azores) was extracted with ethanol and sequentially fractionated in solvents with increasing polarity, n-hexane and chloroform. To assess cell viability, cell proliferation and cell death, Sulforhodamine B, BrDU incorporation assay and Anexin V/Propidium iodide were used, respectively. Glycolytic metabolism was estimated using specific kits.

Results

All propolis samples exhibited a cytotoxic effect against tumor cells, in a dose- and time-dependent way. Chloroform fraction, the most enriched in phenolic compounds, appears to be the most active, both in terms of inhibition of viability and cell death. Data also show that this cytotoxicity involves disturbance in tumor cell glycolytic metabolism, seen by a decrease in glucose consumption and lactate production.

Conclusion

Our results show that Portuguese propolis from Angra do Heroísmo (Azores) can be a potential therapeutic agent against human colorectal cancer.
Modest additive effects of integrated vector control measures on malaria prevalence and transmission in western Kenya
Authors: Zhou Guofa , Afrane Yaw A , Dixit Amruta , Atieli Harrysone E , Lee Ming-Chieh , Wanjala Christine L , Beilhe Leila B , Githeko Andrew K , Yan Guiyun
Published: July 2013

Background

The effect of integrating vector larval intervention on malaria transmission is unknown when insecticide-treated bed-net (ITN) coverage is very high, and the optimal indicator for intervention evaluation needs to be determined when transmission is low.

Methods

A post hoc assignment of intervention-control cluster design was used to assess the added effect of both indoor residual spraying (IRS) and Bacillus-based larvicides (Bti) in addition to ITN in the western Kenyan highlands in 2010 and 2011. Cross-sectional, mass parasite screenings, adult vector populations, and cohort of active case surveillance (ACS) were conducted before and after the intervention in three study sites with two- to three-paired intervention-control clusters at each site each year. The effect of larviciding, IRS, ITNs and other determinants of malaria risk was assessed by means of mixed estimating methods.

Results

Average ITN coverage increased from 41% in 2010 to 92% in 2011 in the study sites. IRS intervention had significant added impact on reducing vector density in 2010 but the impact was modest in 2011. The effect of IRS on reducing parasite prevalence was significant in 2011 but was seasonal specific in 2010. ITN was significantly associated with parasite densities in 2010 but IRS application was significantly correlated with reduced gametocyte density in 2011. IRS application reduced about half of the clinical malaria cases in 2010 and about one-third in 2011 compare to non-intervention areas.

Conclusion

Compared with a similar study conducted in 2005, the efficacy of the current integrated vector control with ITN, IRS, and Bti reduced three- to five-fold despite high ITN coverage, reflecting a modest added impact on malaria transmission. Additional strategies need to be developed to further reduce malaria transmission.
A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients
Authors: Akada Junko , Kamei Shuichi , Ito Akane , Ito Moe , Kitagawa Takao , Furumoto Hiroko , Kato Yukari , Tamesa Michiko , Takashima Motonari , Shirai Mutsunori , Yamano Hirofumi , Oka Masaaki , Kuramitsu Yasuhiro , Nakamura Kazuyuki
Published: July 2013

Background

We report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals.

Results

Five cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera.

Conclusion

This protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.
Metallothionein 2A inhibits NF-κB pathway activation and predicts clinical outcome segregated with TNM stage in gastric cancer patients following radical resection
Authors: Pan Yuanming , Huang Jiaqiang , Xing Rui , Yin Xin , Cui Jiantao , Li Wenmei , Yu Jun , Lu Youyong
Published: July 2013

Background

Metallothionein 2A (MT2A) as a stress protein, plays a protective role in gastric mucosal barrier. Its role in the development of gastric cancer (GC) is unclear. The mechanism of MT2A will be investigated in gastric tumorigenesis.

Methods

MT2A expression was detected in 973 gastric specimens. The biological function was determined through ectopic expressing MT2Ain vitroandin vivo. The possible downstream effectors of MT2A were investigated in NF-κB signaling. The protein levels of MT2A, IκB-α and p-IκB-α (ser32/36) expression were analyzed in a subset of 258 patients by IHC staining. The prognostic effects of MT2A, status of IκB-α and TNM stage were evaluated using the Kaplan-Meier method and compared using the log-rank test.

Results

Decreased MT2A expression was detected in cell lines and primary tumors of GC. In clinical data, loss of MT2A (MT2A + in Normal (n =171, 76.0%); Intestinal metaplasia (n = 118, 50.8%); GC (n = 684. 22.4%,P < 0.001)) was associated with poor prognosis (P < 0.001), advanced TNM stage (P = 0.05), and down-regulation of IκB-α expression (P < 0.001). Furthermore, MT2A was the independent prognostic signature segregated from the status of IκB-α and pathological features. In addition, MT2A inhibited cell growth through apoptosis and G2/M arrest, which negatively regulated NF-κB pathway through up-regulation of IκB-α and down-regulation of p-IκB-α and cyclin D1 expression.

Conclusions

MT2A might play a tumor suppressive activity through inhibiting NF-κB signaling and may be a prognostic biomarker and potential target for individual therapy of GC patients.
On the hierarchical variational inclusion problems in Hilbert spaces
Authors: Chang Shih-sen , Kim Jong Kyu , Joseph Lee HW , Chun Chi Kin
Published: July 2013

The purpose of this paper is by using Maingé’s approach to study the existence and approximation problem of solutions for a class of hierarchical variational inclusion problems in the setting of Hilbert spaces. As applications, we solve the convex programming problems and quadratic minimization problems by using the main theorems. Our results extend and improve the corresponding recent results announced by many authors.

MSC:47J05, 47H09, 49J25.
A network-based approach for predicting key enzymes explaining metabolite abundance alterations in a disease phenotype
Authors: Pey Jon , Tobalina Luis , Prada J de Cisneros Joaquín , Planes Francisco J
Published: July 2013

Background

The study of metabolism has attracted much attention during the last years due to its relevance in various diseases. The advance in metabolomics platforms allows us to detect an increasing number of metabolites in abnormal high/low concentration in a disease phenotype. Finding a mechanistic interpretation for these alterations is important to understand pathophysiological processes, however it is not an easy task. The availability of genome scale metabolic networks and Systems Biology techniques open new avenues to address this question.

Results

In this article we present a novel mathematical framework to find enzymes whose malfunction explains the accumulation/depletion of a given metabolite in a disease phenotype. Our approach is based on a recently introduced pathway concept termed Carbon Flux Paths (CFPs), which extends classical topological definition by including network stoichiometry. Using CFPs, we determine the Connectivity Curve of an altered metabolite, which allows us to quantify changes in its pathway structure when a certain enzyme is removed. The influence of enzyme removal is then ranked and used to explain the accumulation/depletion of such metabolite. For illustration, we center our study in the accumulation of two metabolites (L-Cystine and Homocysteine) found in high concentration in the brain of patients with mental disorders. Our results were discussed based on literature and found a good agreement with previously reported mechanisms. In addition, we hypothesize a novel role of several enzymes for the accumulation of these metabolites, which opens new strategies to understand the metabolic processes underlying these diseases.

Conclusions

With personalized medicine on the horizon, metabolomic platforms are providing us with a vast amount of experimental data for a number of complex diseases. Our approach provides a novel apparatus to rationally investigate and understand metabolite alterations under disease phenotypes. This work contributes to the development of Systems Medicine, whose objective is to answer clinical questions based on theoretical methods and high-throughput “omics” data.
Genetic lineages of undifferentiated-type gastric carcinomas analysed by unsupervised clustering of genomic DNA microarray data
Authors: Sonoda Ayano , Mukaisho Ken-ichi , Nakayama Takahisa , Diem Vo Thi Ngoc , Hattori Takanori , Andoh Akira , Fujiyama Yoshihide , Sugihara Hiroyuki
Published: July 2013

Background

It is suspected that early gastric carcinoma (GC) is a dormant variant that rarely progresses to advanced GC. We demonstrated that the dormant and aggressive variants of tubular adenocarcinomas (TUBs) of the stomach are characterized by loss of MYC and gain of TP53 and gain of MYC and/or loss of TP53, respectively. The aim of this study is to determine whether this is also the case in undifferentiated-type GCs (UGCs) of different genetic lineages: one with a layered structure (LS+), derived from early signet ring cell carcinomas (SIGs), and the other, mostly poorly differentiated adenocarcinomas, without LS but with a minor tubular component (TC), dedifferentiated from TUBs (LS−/TC+).

Methods

Using 29 surgically resected stomachs with 9 intramucosal and 20 invasive UGCs (11 LS+ and 9 LS−/TC+), 63 genomic DNA samples of mucosal and invasive parts and corresponding reference DNAs were prepared from formalin-fixed, paraffin-embedded tissues with laser microdissection, and were subjected to array-based comparative genomic hybridization (aCGH), using 60K microarrays, and subsequent unsupervised, hierarchical clustering. Of 979 cancer-related genes assessed, we selected genes with mean copy numbers significantly different between the two major clusters.

Results

Based on similarity in genomic copy-number profile, the 63 samples were classified into two major clusters. Clusters A and B, which were rich in LS+ UGC and LS−/TC+ UGC, respectively, were discriminated on the basis of 40 genes. The aggressive pattern was more frequently detected in LS−/TC+ UGCs, (20/26; 77%), than in LS+UGCs (17/37; 46%; P = 0.0195), whereas no dormant pattern was detected in any of the UGC samples.

Conclusions

In contrast to TUBs, copy number alterations of MYC and TP53 exhibited an aggressive pattern in LS+ SIG at early and advanced stages, indicating that early LS+ UGCs inevitably progress to an advanced GC. Cluster B (enriched in LS−/TC+) exhibited more frequent gain of driver genes and a more frequent aggressive pattern than cluster A, suggesting potentially worse prognosis in UGCs of cluster B.
Early brain computed tomography findings are associated with outcome in patients treated with therapeutic hypothermia after out-of-hospital cardiac arrest
Authors: Kim Soo Hyun , Choi Seung Pill , Park Kyu Nam , Youn Chun Song , Oh Sang Hoon , Choi Se Min
Published: July 2013

Background

This study evaluated the association between the results of immediate brain computed tomography (CT) scans and outcome in patients who were treated with therapeutic hypothermia after cardiac arrest. The evaluation was based on the changes in the ratio of gray matter to white matter.

Methods

A total of 167 patients who were successfully resuscitated after cardiac arrest from March 2009 to December 2011 were included in this study. We selected 51 patients who received a brain CT scan within 1 hour after the return of spontaneous circulation (ROSC) and who had been treated with therapeutic hypothermia. Circular regions of measurement (10 mm2) were placed over regions of interest (ROIs), and the average attenuations in gray matter (GM) and white matter (WM) were recorded in the basal ganglia, at the level of the centrum semiovale and in the high convexity area. Three GM-to-WM ratios (GWRs) were calculated: one for the basal ganglia, one for the cerebrum and the average of the two. The neurological outcomes were assessed using the Cerebral Performance Category (CPC) scale at the time of hospital discharge, and a good neurological outcome was defined as a CPC score of 1 or 2.

Results

The average GWR was the strongest predictor of poor neurological outcome as determined using receiver operating characteristic curves (basal ganglia area under the curve (AUC) = 0.716; cerebrum AUC = 0.685; average AUC = 0.747). An average GWR < 1.14 predicted a poor neurological outcome with a sensitivity of 13.3% (95% confidence interval (CI) 3.8-30.7), a specificity of 100% (95% CI 83.9-100), a positive predictive value of 100% (95% CI 2.5-100), and a negative predictive value of 44.7% (CI 28.9-58.9).

Conclusions

Our study demonstrated that low GWRs in the immediate brain CT scans of patients treated with therapeutic hypothermia after ROSC were associated with poor neurological outcomes. Immediate brain CT scans could help predict outcome after cardiac arrest.
Transcription factor NFκB regulates the expression of the histone deacetylase SIRT1
Authors: Katto Judith , Engel Nicole , Abbas Wasim , Herbein Georges , Mahlknecht Ulrich
Published: July 2013

Background

The NAD-dependent protein deacetylase SIRT1 has a wide range of different targets, which may be regulated either directly through deacetylation and thus potentially altering their activity or localization or indirectly by deacetylation of histones, which in turn alters their transcription rate and availability. SIRT1 is therefore involved in the regulation of many different and fundamental cellular processes such as apoptosis, metabolism, differentiation and cell cycle arrest. It is also involved in the regulation of resistance of cells against oxidative stress and longevity under conditions of caloric restriction. Even though the targets and role of SIRT1 have been studied quite intensively, only little is known about the mechanisms affecting SIRT1 transcriptional regulation. The nuclear factor NFκB is a well-studied and widely known transcription factor, which is involved in the regulation of many important cellular activities. The regulation of NFκB by SIRT1 has been reported recently, but it is, however, still unknown whether a feedback mechanism affects the regulation of SIRT1 too, particularly in view of the fact that putative NFκB binding sites within the SIRT1 promoter suggest just that.

Results

In the study presented herein we show that there is activation of the SIRT1 promoter by overexpression of different NFκB subunits. Direct binding of NFκB to the SIRT1 promoter can be demonstrated by an electrophoretic mobility shift assay. Further investigations indicated enhanced expression of SIRT1 on the mRNA levels in cells overexpressing NFκB. A functional assay showed that acetylation of one of the main target proteins of SIRT1 is reduced in these cells.

Conclusions

These finding together indicate SIRT1 expression to be regulated in a positive feedback loop by NFκB. The putative binding sites for NFκB found within the SIRT1 promoter appears to be functional and several NFκB subunits are able to enhance the expression of SIRT1 if they are overexpressed.
Bone: The final frontier for Staphylococcus aureus penetration in chronic rhinosinusitis
Authors: Pezato Rogério , Bottura Luciano , de Paula Santos Rodrigo , Voegels Richard Louis , Bachi Andre Luis Lacerda , Gregório Luis Carlos
Published: July 2013

The superantigenic properties of Staphylococcus aureus have been implicated in increasing the inflammatory process in airway diseases. Local formation of IgE antibodies against staphylococcal enterotoxins by secondary lymphoid tissue in nasal polyps has been demonstrated. Staphylococcus aureus is known to colonize the nasal mucosa, and has been found invading the nasal submucosa and intracellularly.

Objective

To evaluate the limits of Staphylococcus aureus invasion in the upper airway.

Material and methods

Inferior turbinate samples from 3 patients without sinus disease, 6 ethmoid samples from patients with chronic rhinosinusitis with nasal polyposis, and 6 ethmoid samples from patients with chronic rhinosinusitis without nasal polyposis were studied. A fluorescein-labeled PNA probe against Staphylococcus aureus was used to test for the presence of the bacterium in bone (after decalcification) and mucosa.

Results

We found Staphylococcus aureus invading the nasal submucosa in patients with nasal polyposis, but no cases of Staphylococcus aureus positivity in bone. In conclusion, we cannot support the hypothesis of nasal bone as a reservoir for Staphylococcus aureus, releasing massive amounts of staphylococcal enterotoxins and eliciting an inflammatory reaction, as occurs with the nasal mucosa.
β-Keto esters from ketones and ethyl chloroformate: a rapid, general, efficient synthesis of pyrazolones and their antimicrobial, in silico and in vitro cytotoxicity studies
Authors: Ragavan Ramasamy Venkat , Kumar Kalavathi Murugan , Vijayakumar Vijayaparthasarathi , Sarveswari Sundaramoorthy , Ramaiah Sudha , Anbarasu Anand , Karthikeyan Sivashanmugam , Giridharan Periyasamy , Kumari Nalilu Suchetha
Published: July 2013

Background

Pyrazolones are traditionally synthesized by the reaction of β-keto esters with hydrazine and its derivatives. There are methods to synthesize β-keto esters from esters and aldehydes, but these methods have main limitation in varying the substituents. Often, there are a number of methods such as acylation of enolates in which a chelating effect has been employed to lock the enolate anion using lithium and magnesium salts; however, these methods suffer from inconsistent yields in the case of aliphatic acylation. There are methods to synthesize β-keto esters from ketones like caboxylation of ketone enolates using carbon dioxide and carbon monoxide sources in the presence of palladium or transition metal catalysts. Currently, the most general and simple method to synthesize β-keto ester is the reaction of dimethyl or ethyl carbonate with ketone in the presence of strong bases which also requires long reaction time, use of excessive amount of reagent and inconsistent yield. These factors lead us to develop a simple method to synthesize β-keto esters by changing the base and reagent.

Results

A series of β-keto esters were synthesized from ketones and ethyl chloroformate in the presence of base which in turn are converted to pyrazolones and then subjected to cytotoxicity studies towards various cancer cell lines and antimicrobial activity studies towards various bacterial and fungal strains.

Conclusion

The β-keto esters from ethyl chloroformate was successfully attempted, and the developed method is simple, fast and applicable to the ketones having the alkyl halogens, protecting groups like Boc and Cbz that were tolerated and proved to be useful in the synthesis of fused bicyclic and tricyclic pyrazolones efficiently using cyclic ketones. Since this method is successful for different ketones, it can be useful for the synthesis of pharmaceutically important pyrazolones also. The synthesized pyrazolones were subjected to antimicrobial, docking and cytotoxicity assay against ACHN (human renal cell carcinoma), Panc-1 (human pancreatic adenocarcinoma) and HCT-116 (human colon cancer) cell line, and lead molecules have been identified. Some of the compounds are found to have promising activity against different bacterial and fungal strains tested.
Insulin Hypersecretion in Islets From Diet-Induced Hyperinsulinemic Obese Female Mice Is Associated With Several Functional Adaptations in Individual β-Cells
Authors: Gonzalez Alejandro , Merino Beatriz , Marroquí Laura , Ñeco Patricia , Alonso-Magdalena Paloma , Caballero-Garrido Ernesto , Vieira Elaine , Soriano Sergi , Gomis Ramon , Nadal Angel , Quesada Ivan
Published: July 2013

Insulin resistance and hyperinsulinemia are generally associated with obesity. Obese nondiabetic individuals develop a compensatory β-cell response to adjust insulin levels to the increased demand, maintaining euglycemia. Although several studies indicate that this compensation relies on structural changes, the existence of β-cell functional adaptations is incompletely understood. Here, we fed female mice with a high-fat diet (HFD) for 12 weeks. These animals became obese, hyperinsulinemic, insulin-resistant, and mildly glucose-intolerant while fed, and fasting glycemia was comparable in HFD and control mice. Islets from HFD animals exhibited increased β-cell mass and hypertrophy. Additionally, they had enhanced insulin gene expression and content and augmented glucose-induced insulin secretion. Electrophysiological examination of β-cells from both groups showed no differences in KATP channel open probability and conductance. However, action potentials elicited by glucose had larger amplitude in obese mice. Glucose-induced Ca2+ signals in intact islets, in isolated β-cells, and individual β-cells within islets were also increased in HFD mice. Additionally, a higher proportion of glucose-responsive cells was present in obese mice. In contrast, whole-cell Ca2+ current densities were similar in both groups. Capacitance measurements showed that depolarization-evoked exocytosis was enhanced in HFD β-cells compared with controls. Although this augment was not significant when capacitance increases of the whole β-cell population were normalized to cell size, the exocytotic output varied significantly when β-cells were distributed by size ranges. All these findings indicate that β-cell functional adaptations are present in the islet compensatory response to obesity.
Regulation of EGFR trafficking and cell signaling by Sprouty2 and MIG6 in lung cancer cells
Authors: Walsh Alice M. , Lazzara Matthew J.
Published: July 2013

The duration and specificity of epidermal growth factor receptor (EGFR) activation and signaling are determinants of cellular decision processes and are tightly regulated by receptor dephosphorylation, internalization and degradation. In addition, regulatory proteins that are upregulated or activated post-transcriptionally upon receptor activation may initiate feedback loops that play crucial roles in spatiotemporal regulation of signaling. We examined the roles of Sprouty2 (SPRY2) and mitogen-inducible gene 6 (MIG6), two feedback regulators of EGFR trafficking and signaling, in lung cancer cells with or without EGFR-activating mutations. These mutations are of interest because they confer unusual cellular sensitivity to EGFR inhibition through a mechanism involving an impairment of EGFR endocytosis. We found that the endocytosis of wild-type and mutant EGFR was promoted by SPRY2 knockdown and antagonized by MIG6 knockdown. SPRY2 knockdown also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation, EGFR expression, and EGFR recycling. In a cell line expressing mutant EGFR, this effect on ERK led to a marked increase in cell death response to EGFR inhibition. The effects of SPRY2 knockdown on EGFR endocytosis and recycling were primarily the result of the concomitant change in EGFR expression, but this was not true for the observed changes in ERK phosphorylation. Thus, our study demonstrates that SPRY2 and MIG6 are important regulators of wild-type and mutant EGFR trafficking and points to an EGFR expression-independent function of SPRY2 in the regulation of ERK activity that may impact cellular sensitivity to EGFR inhibitors, especially in the context of EGFR mutation.
MiR-19b/20a/92a regulates the self-renewal and proliferation of gastric cancer stem cells
Authors: Wu Qiong , Yang Zhiping , Wang Fang , Hu Sijun , Yang Li , Shi Yongquan , Fan Daiming
Published: July 2013

Human gastric cancers contain a population of gastric cancer stem cells (GCSCs) that can undergo self-renewal and multipotent differentiation. GCSCs can be enriched by EpCAM+/CD44+ gastric cancer cells. However, the mechanisms underlying how GCSCs balance self-renewal and differentiation remain to be explored. Because miRNAs can regulate cancer cell fates, we compared tumorspheric cancer cells enriched for GCSCs with more differentiated cells in terms of miRNA expression. We found that the miR-17-92 cluster members miR-19b, miR-20a and miR-92a were gradually reduced during the differentiation of GCSCs. Herein, we speculated that miR-17-92 members might function as regulators to sustain the self-renewal ability of GCSCs. By down-regulating miR-19b, miR-20a and miR-92a in EpCAM+/CD44+ GCSCs, or over-expressing them in EpCAM-/CD44- non-GCSC populations, we found that miR-19b, miR-20a and miR-92a could sustain the self-renewal function of GCSCs. Furthermore, we found that miR-19b, miR-20a and miR-92a could also promote the proliferation of gastric cancer cells. Moreover, miR-17-92 targeted the E2F1 and HIPK1 proteins, which suppressed Wnt-β-catenin signaling. A real-time PCR analysis of miR-19b, miR-20a and miR-92a expression in 97 gastric cancer specimens suggested that miR-92a could be used as an independent prognostic factor in gastric cancer. This study indicated that several members of the miR-17-92 cluster,miR-19b, miR-20a and miR-92a, might play significant roles in the development of gastric cancer stem cells and that miR-92a has the potential to be used as a predictive prognostic marker in gastric cancer.
Damage response of XRCC1 at sites of DNA single strand breaks is regulated by phosphorylation and ubiquitylation after degradation of poly(ADP-ribose)
Authors: Wei Leizhen , Nakajima Satoshi , Hsieh Ching-Lung , Kanno Shinichiro , Masutani Mitsuko , Levine Arthur S. , Yasui Akira , Lan Li
Published: July 2013

Single-strand breaks (SSBs) are the most common type of oxidative DNA damage and they are related to aging and many genetic diseases. The scaffold protein for repair of SSBs, XRCC1, accumulates at sites of poly(ADP-ribose) (pAR) synthesized by PARP, but it is retained at sites of SSBs after pAR degradation. How XRCC1 responds to SSBs after pAR degradation and how this affects repair progression are not well understood. We found that XRCC1 dissociates from pAR and is translocated to sites of SSBs dependent on its BRCTII domain and the function of PARG. In addition, phosphorylation of XRCC1 is also required for the proper dissociation kinetics of XRCC1 because (1) phosphorylation sites mutated in XRCC1 (X1 pm) cause retention of XRCC1 at sites of SSB for a longer time compared to wild type XRCC1; and (2) phosphorylation of XRCC1 is required for efficient polyubiquitylation of XRCC1. Interestingly, a mutant of XRCC1, LL360/361DD, which abolishes pAR binding, shows significant upregulation of ubiquitylation, indicating that pARylation of XRCC1 prevents the poly-ubiquitylation. We also found that the dynamics of the repair proteins DNA polymerase beta, PNK, APTX, PCNA and ligase I are regulated by domains of XRCC1. In summary, the dynamic damage response of XRCC1 is regulated in a manner that depends on modifications of polyADP-ribosylation, phosphorylation and ubiquitylation in live cells.
Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain
Authors: Huang Xiaojun , Liu Ying , Wang Ruiwu , Zhong Xiaowei , Liu Yingjie , Koop Andrea , Chen S. R. Wayne , Wagenknecht Terence , Liu Zheng
Published: July 2013

Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595–CFP and K4269–YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating.
Mechanical tuning of the moth ear: distortion-product otoacoustic emissions and tympanal vibrations
Authors: Mora Emanuel C. , Cobo-Cuan Ariadna , Macías Frank , Pérez Martha , Nowotny Manuela , Kössl Manfred
Published: July 2013

The mechanical tuning of the ear in the moth E. pugione was investigated by distortion-products otoacoustic emissions (DPOAE) and laser Doppler vibrometry (LDV). DPOAE-audiograms were assessed using a novel protocol that may be advantageous for noninvasive auditory studies in insects. To evoke DPOAE, two-tone stimuli within frequency and level ranges that generated a large matrix of values (960 frequency-level combinations) were used to examine the acoustic space in which the moth tympanum shows its best mechanical and acoustical responses. The DPOAE-tuning curve derived from the response matrix resembles that obtained previously by electrophysiology, is V-shaped, tuned to frequencies between 25 and 45 kHz with low Q10dB values of 1.21 ± 0.26. In addition, while using a comparable stimulation regime, mechanical distortion in the displacement of the moth's tympanal membrane at the stigma was recorded with a laser Doppler vibrometer. The corresponding mechanical vibration audiograms were compared to DPOAE audiograms. Both types of audiograms have comparable shape but most of the mechanical response fields are shifted towards lower frequencies. We showed for the first time in moths that distortion-product otoacoustic emissions have a pronounced analogy in the vibration of the tympanic membrane where they may originate. Our work supports previous studies that point to the stigma (and the internally associated transduction machinery) as an important place of sound amplification in the moth ear, but also suggests a complex mechanical role for the rest of the transparent zone.
Sensitivity of ventilation and brain metabolism to ammonia exposure in rainbow trout,Oncorhynchus mykiss
Authors: Zhang Li , Nawata Michele , Wood Chris
Published: July 2013

Ammonia has been documented as a respiratory gas which stimulates ventilation, and is sensed by peripheral neuroepithelial cells (NECs) in the gills in ammoniotelic rainbow trout. However, the hyperventilatory response is abolished in trout chronically exposed (1+ months) to high environmental ammonia (HEA, 250 μmol l-1 (NH4)2SO4). This study investigates whether the brain is involved in the acute sensitivity of ventilation to ammonia, and whether changes in brain metabolism are related to the loss of hyperventilatory responses in trout chronically exposed to HEA (“HEA trout”). Hyperventilation (via increased ventilatory amplitude rather than rate), and increased total ammonia concentration ([TAmm]) in brain tissue were induced in parallel by acute HEA exposure in control trout in a concentration-series experiment (500, 750, 1000 μmol l-1 (NH4)2SO4), but these inductions were abolished in HEA trout. Ventilation was correlated more closely to [TAmm] in brain rather than to [TAmm] in plasma or cerebrospinal fluid (CSF). The close correlation of hyperventilation and increased brain [TAmm] also occurred in control trout acutely exposed to HEA in a time series analysis (500 μmol l-1 (NH4)2SO4 - 15, 30, 45, and 60 min), as well as in a methionine sulfoxamine (MSOX) pre-injection experiment (to inhibit glutamine synthetase, GSase). These correlations consistently suggest that brain [TAmm] is involved in the hyperventilatory responses to ammonia in trout. The MSOX treatments, together with measurements of GSase activity, TAmm, glutamine, and glutamate concentrations in brain tissue, were conducted in both the control and HEA trout. These experiments revealed that GSase plays an important role in transferring ammonia to glutamate to make glutamine in trout brain, thereby attenuating the elevation of brain [TAmm] following HEA exposure, and that glutamate concentration is reduced in HEA trout. The mRNAs for the ammonia channel proteins Rhbg, Rhcg1, and Rhcg2 were expressed in trout brain, and the expression of Rhbg and Rhcg2 increased in HEA trout, potentially as a mechanism to facilitate the efflux of ammonia. In summary, the brain appears to be involved in the sensitivity of ventilation to ammonia, and brain ammonia levels are regulated metabolically in trout.
A conserved fertility signal despite population variation in the cuticular chemical phenotype of the trap-jaw antOdontomachus brunneus
Authors: Smith Adrian A. , Millar Jocelyn G. , Hanks Lawrence M. , Suarez Andrew V.
Published: July 2013

Contact pheromones in the form of cuticular hydrocarbons are widespread among insects. Eusocial insects present a special challenge for understanding the evolution of the cuticular hydrocarbon profile because this blend is responsible for multiple distinct roles such as nestmate recognition and signalling fertility status. This study investigates these two signalling roles of the hydrocarbon profile in the trap-jaw ant Odontomachus brunneus Patton. We demonstrate that the cuticular hydrocarbon profile is highly variable across populations and provide evidence that these differences are used for nestmate discrimination. Through manipulative experiments we also show that (Z)-9-nonacosene (Z9:C29) is used as a fertility signal and its role is conserved across populations. Our data demonstrate that both fertility and nestmate signalling influence the cuticular hydrocarbon profile and specifically the relative abundance of Z9:C29 on the cuticle of O. brunneus. Our study suggests that natural selection works on the cuticular chemical profile through multiple regulatory pathways, diversifying nestmate signals while conserving fertility signals.
Seasonality influences cuticle melanization and immune defense in a cricket: support for a temperature-dependent immune investment hypothesis in insects
Authors: Fedorka Kenneth M. , Copeland Emily K. , Winterhalter Wade E.
Published: July 2013

To improve thermoregulation in colder environments, insects are expected to darken their cuticles with melanin via the phenoloxidase cascade; a phenomenon predicted by the thermal melanin hypothesis. However, the phenoloxidase cascade also plays a significant role in insect immunity, leading to the additional hypothesis that the thermal environment indirectly shapes immune function via direct selection on cuticle color. Support for the latter hypothesis comes from the cricket Allonemobius socius (Scudder), where cuticle darkness and immune-related phenoloxidase activity increase with latitude. However, thermal environments vary seasonally as well as geographically, suggesting that seasonal plasticity in immunity may also exist. Although seasonal fluctuations in vertebrate immune function are common (due to flux in breeding or resource abundance), seasonality in invertebrate immunity has not been widely explored. We addressed this possibility by rearing crickets in simulated summer and fall environments and assayed their cuticle color and immune function. Prior to estimating immunity, crickets were placed in a common environment to minimize metabolic rate differences. Fall-like individuals exhibited darker cuticles, a greater phenoloxidase activity and greater resistance to the bacteria Serratia marcescens. These data support the hypothesis that changes in the thermal environment modify cuticle color, which indirectly shapes immune investment through pleiotropy. This hypothesis may represent a widespread mechanism governing immunity in numerous systems, considering that most insects operate in seasonally and geographically variable thermal environments.