bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence-based case report: multiple thrombotic episodes associated with lenalidomide and dexamethasone therapy for multiple myeloma
Authors: Rushworth Gordon F. , Leslie Stephen J. , Forsyth Peter , Vincent Claire
Published: January 2012

Lenalidomide in combination with dexamethasone is a treatment for patients with relapsed or refractory myeloma. Although this combination demonstrates a high level of efficacy, it further exacerbates the hypercoaguable state that exists within myeloma. Thromboprophylactic regimen require careful selection and if warfarin is chosen, assiduous monitoring is required to ensure it will be clinically effective. We report the case of one patient who experienced multiple thrombotic events despite anticoagulant or antiplatelet thromboprophylaxis and review the contributing factors.
Delineation of antigen-specific and antigen-nonspecific CD8+ memory T-cell responses after cytokine-based cancer immunotherapy
Authors: Tietze Julia K. , Wilkins Danice E. C. , Sckisel Gail D. , Bouchlaka Myriam N. , Alderson Kory L. , Weiss Jonathan M. , Ames Erik , Bruhn Kevin W. , Craft Noah , Wiltrout Robert H. , Longo Dan L. , Lanier Lewis L. , Blazar Bruce R. , Redelman Doug , Murphy William J.
Published: January 2012

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8+ T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8+ T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8+ T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8+ CD25− cells within the tumor site. These findings demonstrate that memory CD8+ T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.
The endothelial protein C receptor (PROCR) Ser219Gly variant and risk of common thrombotic disorders: a HuGE review and meta-analysis of evidence from observational studies
Authors: Dennis Jessica , Johnson Candice Y. , Adediran Adeniyi Samuel , de Andrade Mariza , Heit John A. , Morange Pierre-Emmanuel , Trégouët David-Alexandre , Gagnon France
Published: January 2012

The endothelial protein C receptor (EPCR) limits thrombus formation by enhancing activation of the protein C anticoagulant pathway, and therefore may play a role in the etiology of thrombotic disorders. The rs867186 single-nucleotide polymorphism in the PROCR gene (g.6936A > G, c.4600A > G), resulting in a serine-to-glycine substitution at codon 219, has been associated with reduced activation of the protein C pathway, although its association with thrombosis risk remains unclear. The present study is a highly comprehensive systematic review and meta-analysis, including unpublished genome-wide association study results, conducted to evaluate the evidence for an association between rs867186 and 2 common thrombotic outcomes, venous thromboembolism (VTE) and myocardial infarction (MI), which are hypothesized to share some etiologic pathways. MEDLINE, EMBASE, and HuGE Navigator were searched through July 2011 to identify relevant epidemiologic studies, and data were summarized using random-effects meta-analysis. Twelve candidate genes and 13 genome-wide association studies were analyzed (11 VTE and 14 MI, including 37 415 cases and 84 406 noncases). Under the additive genetic model, the odds of VTE increased by a factor of 1.22 (95% confidence interval, 1.11-1.33, P < .001) for every additional copy of the G allele. No evidence for association with MI was observed.
Up-regulation of a HOXA-PBX3 homeobox-gene signature following down-regulation of miR-181 is associated with adverse prognosis in patients with cytogenetically abnormal AML
Authors: Li Zejuan , Huang Hao , Li Yuanyuan , Jiang Xi , Chen Ping , Arnovitz Stephen , Radmacher Michael D. , Maharry Kati , Elkahloun Abdel , Yang Xinan , He Chunjiang , He Miao , Zhang Zhiyu , Dohner Konstanze , Neilly Mary Beth , Price Colles , Lussier Yves A. , Zhang Yanming , Larson Richard A. , Le Beau Michelle M. , Caligiuri Michael A. , Bullinger Lars , Valk Peter J. M. , Delwel Ruud , Lowenberg Bob , Liu Paul P. , Marcucci Guido , Bloomfield Clara D. , Rowley Janet D. , Chen Jianjun
Published: January 2012

Increased expression levels of miR-181 family members have been shown to be associated with favorable outcome in patients with cytogenetically normal acute myeloid leukemia. Here we show that increased expression of miR-181a and miR-181b is also significantly (P < .05; Cox regression) associated with favorable overall survival in cytogenetically abnormal AML (CA-AML) patients. We further show that up-regulation of a gene signature composed of 4 potential miR-181 targets (including HOXA7, HOXA9, HOXA11, and PBX3), associated with down-regulation of miR-181 family members, is an independent predictor of adverse overall survival on multivariable testing in analysis of 183 CA-AML patients. The independent prognostic impact of this 4-homeobox-gene signature was confirmed in a validation set of 271 CA-AML patients. Furthermore, our in vitro and in vivo studies indicated that ectopic expression of miR-181b significantly promoted apoptosis and inhibited viability/proliferation of leukemic cells and delayed leukemogenesis; such effects could be reversed by forced expression of PBX3. Thus, the up-regulation of the 4 homeobox genes resulting from the down-regulation of miR-181 family members probably contribute to the poor prognosis of patients with nonfavorable CA-AML. Restoring expression of miR-181b and/or targeting the HOXA/PBX3 pathways may provide new strategies to improve survival substantially.
A neuronal death model: overexpression of neuronal intermediate filament protein peripherin in PC12 cells
Authors: Lee Wen-Ching , Chen Yun-Yu , Kan Daphne , Chien Chung-Liang
Published: January 2012

Background

Abnormal accumulation of neuronal intermediate filament (IF) is a pathological indicator of some neurodegenerative disorders. However, the underlying neuropathological mechanisms of neuronal IF accumulation remain unclear. A stable clone established from PC12 cells overexpressing a GFP-Peripherin fusion protein (pEGFP-Peripherin) was constructed for determining the pathway involved in neurodegeneration by biochemical, cell biology, and electronic microscopy approaches. In addition, pharmacological approaches to preventing neuronal death were also examined.

Results

Results of this study showed that TUNEL positive reaction could be detected in pEGFP-Peripherin cells. Swollen mitochondria and endoplasmic reticulum (ER) were seen by electron microscopy in pEGFP-Peripherin cells on day 8 of nerve growth factor (NGF) treatment. Peripherin overexpression not only led to the formation of neuronal IF aggregate but also causes aberrant neuronal IF phosphorylation and mislocation. Western blots showed that calpain, caspase-12, caspase-9, and caspase-3 activity was upregulated. Furthermore, treatment with calpain inhibitor significantly inhibited cell death.

Conclusions

These results suggested that the cytoplasmic neuronal IF aggregate caused by peripherin overexpression may induce aberrant neuronal IF phosphorylation and mislocation subsequently trapped and indirectly damaged mitochondria and ER. We suggested that the activation of calpain, caspase-12, caspase-9, and caspase-3 were correlated to the dysfunction of the ER and mitochondria in our pEGFP-Peripherin cell model. The present study suggested that pEGFP-Peripherin cell clones could be a neuronal death model for future studies in neuronal IFs aggregate associated neurodegeneration.
Core module biomarker identification with network exploration for breast cancer metastasis
Authors: Yang Ruoting , Daigle Bernie J , Petzold Linda R , Doyle Francis J
Published: January 2012

Background

In a complex disease, the expression of many genes can be significantly altered, leading to the appearance of a differentially expressed "disease module". Some of these genes directly correspond to the disease phenotype, (i.e. "driver" genes), while others represent closely-related first-degree neighbours in gene interaction space. The remaining genes consist of further removed "passenger" genes, which are often not directly related to the original cause of the disease. For prognostic and diagnostic purposes, it is crucial to be able to separate the group of "driver" genes and their first-degree neighbours, (i.e. "core module") from the general "disease module".

Results

We have developed COMBINER: COre Module Biomarker Identification with Network ExploRation. COMBINER is a novel pathway-based approach for selecting highly reproducible discriminative biomarkers. We applied COMBINER to three benchmark breast cancer datasets for identifying prognostic biomarkers. COMBINER-derived biomarkers exhibited 10-fold higher reproducibility than other methods, with up to 30-fold greater enrichment for known cancer-related genes, and 4-fold enrichment for known breast cancer susceptible genes. More than 50% and 40% of the resulting biomarkers were cancer and breast cancer specific, respectively. The identified modules were overlaid onto a map of intracellular pathways that comprehensively highlighted the hallmarks of cancer. Furthermore, we constructed a global regulatory network intertwining several functional clusters and uncovered 13 confident "driver" genes of breast cancer metastasis.

Conclusions

COMBINER can efficiently and robustly identify disease core module genes and construct their associated regulatory network. In the same way, it is potentially applicable in the characterization of any disease that can be probed with microarrays.
Fine scale analysis of gene expression in Drosophila melanogaster gonads reveals Programmed cell death 4 promotes the differentiation of female germline stem cells
Authors: Cash Amy C , Andrews Justen
Published: January 2012

Background

Germline stem cells (GSCs) are present in the gonads of Drosophila females and males, and their proper maintenance, as well as their correct differentiation, is essential for fertility and fecundity. The molecular characterization of factors involved in maintenance and differentiation is a major goal both in Drosophila and stem cell research. While genetic studies have identified many of these key factors, the use of genome-wide expression studies holds the potential to greatly increase our knowledge of these pathways.

Results

Here we report a genome-wide expression study that uses laser cutting microdissection to isolate germline stem cells, somatic niche cells, and early differentiating germ cells from female and male gonads. Analysis of this data, in association with two previously published genome-wide GSC data sets, revealed sets of candidate genes as putatively expressed in specific cell populations. Investigation of one of these genes, CG10990 the Drosophila ortholog of mammalian Programmed cell death 4 (Pdcd4), reveals expression in female and male germline stem cells and early differentiating daughter cells. Functional analysis demonstrates that while it is not essential for oogenesis or spermatogenesis, it does function to promote the differentiation of GSCs in females. Furthermore, in females, Pdcd4 genetically interacts with the key differentiation gene bag of marbles (bam) and the stem cell renewal factor eIF4A, suggesting a possible pathway for its function in differentiation.

Conclusions

We propose that Pdcd4 promotes the differentiation of GSC daughter cells by relieving the eIF4A-mediated inhibition of Bam.
Rates of evolution in stress-related genes are associated with habitat preference in two Cardamine lineages
Authors: Ometto Lino , Li Mingai , Bresadola Luisa , Varotto Claudio
Published: January 2012

Background

Elucidating the selective and neutral forces underlying molecular evolution is fundamental to understanding the genetic basis of adaptation. Plants have evolved a suite of adaptive responses to cope with variable environmental conditions, but relatively little is known about which genes are involved in such responses. Here we studied molecular evolution on a genome-wide scale in two species of Cardamine with distinct habitat preferences: C. resedifolia, found at high altitudes, and C. impatiens, found at low altitudes. Our analyses focussed on genes that are involved in stress responses to two factors that differentiate the high- and low-altitude habitats, namely temperature and irradiation.

Results

High-throughput sequencing was used to obtain gene sequences from C. resedifolia and C. impatiens. Using the available A. thaliana gene sequences and annotation, we identified nearly 3,000 triplets of putative orthologues, including genes involved in cold response, photosynthesis or in general stress responses. By comparing estimated rates of molecular substitution, codon usage, and gene expression in these species with those of Arabidopsis, we were able to evaluate the role of positive and relaxed selection in driving the evolution of Cardamine genes. Our analyses revealed a statistically significant higher rate of molecular substitution in C. resedifolia than in C. impatiens, compatible with more efficient positive selection in the former. Conversely, the genome-wide level of selective pressure is compatible with more relaxed selection in C. impatiens. Moreover, levels of selective pressure were heterogeneous between functional classes and between species, with cold responsive genes evolving particularly fast in C. resedifolia, but not in C. impatiens.

Conclusions

Overall, our comparative genomic analyses revealed that differences in effective population size might contribute to the differences in the rate of protein evolution and in the levels of selective pressure between the C. impatiens and C. resedifolia lineages. The within-species analyses also revealed evolutionary patterns associated with habitat preference of two Cardamine species. We conclude that the selective pressures associated with the habitats typical of C. resedifolia may have caused the rapid evolution of genes involved in cold response.
Analysis of genome-wide association study data using the protein knowledge base
Authors: Ballouz Sara , Liu Jason Y , Oti Martin , Gaeta Bruno , Fatkin Diane , Bahlo Melanie , Wouters Merridee A
Published: January 2012

Background

Genome-wide association studies (GWAS) aim to identify causal variants and genes for complex disease by independently testing a large number of SNP markers for disease association. Although genes have been implicated in these studies, few utilise the multiple-hit model of complex disease to identify causal candidates. A major benefit of multi-locus comparison is that it compensates for some shortcomings of current statistical analyses that test the frequency of each SNP in isolation for the phenotype population versus control.

Results

Here we developed and benchmarked several protocols for GWAS data analysis using different in-silico gene prediction and prioritisation methodologies. We adopted a high sensitivity approach to the data, using less conservative statistical SNP associations. Multiple gene search spaces, either of fixed-widths or proximity-based, were generated around each SNP marker. We used the candidate disease gene prediction system Gentrepid to identify candidates based on shared biomolecular pathways or domain-based protein homology. Predictions were made either with phenotype-specific known disease genes as input; or without a priori knowledge, by exhaustive comparison of genes in distinct loci. Because Gentrepid uses biomolecular data to find interactions and common features between genes in distinct loci of the search spaces, it takes advantage of the multi-locus aspect of the data.

Conclusions

Results suggest testing multiple SNP-to-gene search spaces compensates for differences in phenotypes, populations and SNP platforms. Surprisingly, domain-based homology information was more informative when benchmarked against gene candidates reported by GWA studies compared to previously determined disease genes, possibly suggesting a larger contribution of gene homologs to complex diseases than Mendelian diseases.
The architecture and ppGpp-dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression
Authors: Ramachandran Vinoy K , Shearer Neil , Jacob Jobin J , Sharma Cynthia M , Thompson Arthur
Published: January 2012

Background

Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (STEX), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wild-type S. Typhimurium and a ppGpp null strain under growth conditions which model STEX. In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium STEX primary transcriptome than previously recognised.

Results

Here we report the precise mapping of transcriptional start sites (TSSs) for 78% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34% and 20% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment.

Conclusions

The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research.
Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression
Authors: Vining Kelly J , Pomraning Kyle R , Wilhelm Larry J , Priest Henry D , Pellegrini Matteo , Mockler Todd C , Freitag Michael , Strauss Steven H
Published: January 2012

Background

DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood.

Results

We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation.

Conclusions

We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.
Centromere-associated repeat arrays on Trypanosoma brucei chromosomes are much more extensive than predicted
Authors: Echeverry Maria C , Bot Christopher , Obado Samson O , Taylor Martin C , Kelly John M
Published: January 2012

Background

African trypanosomes belong to a eukaryotic lineage which displays many unusual genetic features. The mechanisms of chromosome segregation in these diploid protozoan parasites are poorly understood. Centromeres in Trypanosoma brucei have been localised to chromosomal regions that contain an array of ~147 bp AT-rich tandem repeats. Initial estimates from the genome sequencing project suggested that these arrays ranged from 2 - 8 kb. In this paper, we show that the centromeric repeat regions are much more extensive.

Results

We used a long-range restriction endonuclease mapping approach to more accurately define the sizes of the centromeric repeat arrays on the 8 T. brucei chromosomes where unambiguous assembly data were available. The results indicate that the sizes of the arrays on different chromosomes vary from 20 to 120 kb. In addition, we found instances of length heterogeneity between chromosome homologues. For example, values of 20 and 65 kb were obtained for the arrays on chromosome 1, and 50 and 75 kb for chromosome 5.

Conclusions

Our results show that centromeric repeat arrays on T. brucei chromosomes are more similar in size to those of higher eukaryotes than previously suspected. This information provides a firmer framework for investigating aspects of chromosome segregation and will allow epigenetic features associated with the process to be more accurately mapped.
Learning generative models of molecular dynamics
Authors: Razavian Narges Sharif , Kamisetty Hetunandan , Langmead Christopher J
Published: January 2012

We introduce three algorithms for learning generative models of molecular structures from molecular dynamics simulations. The first algorithm learns a Bayesian-optimal undirected probabilistic model over user-specified covariates (e.g., fluctuations, distances, angles, etc). L
1 reg-ularization is used to ensure sparse models and thus reduce the risk of over-fitting the data. The topology of the resulting model reveals important couplings between different parts of the protein, thus aiding in the analysis of molecular motions. The generative nature of the model makes it well-suited to making predictions about the global effects of local structural changes (e.g., the binding of an allosteric regulator). Additionally, the model can be used to sample new conformations. The second algorithm learns a time-varying graphical model where the topology and parameters change smoothly along the trajectory, revealing the conformational sub-states. The last algorithm learns a Markov Chain over undirected graphical models which can be used to study and simulate kinetics. We demonstrate our algorithms on multiple molecular dynamics trajectories.
Delayed larval development in Anopheles mosquitoes deprived of Asaiabacterial symbionts
Authors: Chouaia Bessem , Rossi Paolo , Epis Sara , Mosca Michela , Ricci Irene , Damiani Claudia , Ulissi Ulisse , Crotti Elena , Daffonchio Daniele , Bandi Claudio , Favia Guido
Published: January 2012

Background

In recent years, acetic acid bacteria have been shown to be frequently associated with insects, but knowledge on their biological role in the arthropod host is limited. The discovery that acetic acid bacteria of the genus Asaia are a main component of the microbiota of Anopheles stephensi makes this mosquito a useful model for studies on this novel group of symbionts. Here we present experimental results that provide a first evidence for a beneficial role of Asaia in An. stephensi.

Results

Larvae of An. stephensi at different stages were treated with rifampicin, an antibiotic effective on wild-type Asaia spp., and the effects on the larval development were evaluated. Larvae treated with the antibiotic showed a delay in the development and an asynchrony in the appearance of later instars. In larvae treated with rifampicin, but supplemented with a rifampicin-resistant mutant strain of Asaia, larval development was comparable to that of control larvae not exposed to the antibiotic. Analysis of the bacterial diversity of the three mosquito populations confirmed that the level of Asaia was strongly decreased in the antibiotic-treated larvae, since the symbiont was not detectable by PCR-DGGE (denaturing gradient gel electrophoresis), while Asaia was consistently found in insects supplemented with rifampicin plus the antibiotic-resistant mutant in the diet, and in those not exposed to the antibiotic.

Conclusions

The results here reported indicate that Asaia symbionts play a beneficial role in the normal development of An. stephensi larvae.
Detection and characterization of Wolbachia infections in laboratory and natural populations of different species of tsetse flies (genus Glossina)
Authors: Doudoumis Vangelis , Tsiamis George , Wamwiri Florence , Brelsfoard Corey , Alam Uzma , Aksoy Emre , Dalaperas Stelios , Abd-Alla Adly , Ouma Johnson , Takac Peter , Aksoy Serap , Bourtzis Kostas
Published: January 2012

Background

Wolbachia is a genus of endosymbiotic α-Proteobacteria infecting a wide range of arthropods and filarial nematodes. Wolbachia is able to induce reproductive abnormalities such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization and male killing, thus affecting biology, ecology and evolution of its hosts. The bacterial group has prompted research regarding its potential for the control of agricultural and medical disease vectors, including Glossina spp., which transmits African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals.

Results

In the present study, we employed a Wolbachia specific 16S rRNA PCR assay to investigate the presence of Wolbachia in six different laboratory stocks as well as in natural populations of nine different Glossina species originating from 10 African countries. Wolbachia was prevalent in Glossina morsitans morsitans, G. morsitans centralis and G. austeni populations. It was also detected in G. brevipalpis, and, for the first time, in G. pallidipes and G. palpalis gambiensis. On the other hand, Wolbachia was not found in G. p. palpalis, G. fuscipes fuscipes and G. tachinoides. Wolbachia infections of different laboratory and natural populations of Glossina species were characterized using 16S rRNA, the wsp (Wolbachia Surface Protein) gene and MLST (Multi Locus Sequence Typing) gene markers. This analysis led to the detection of horizontal gene transfer events, in which Wobachia genes were inserted into the tsetse flies fly nuclear genome.

Conclusions

Wolbachia infections were detected in both laboratory and natural populations of several different Glossina species. The characterization of these Wolbachia strains promises to lead to a deeper insight in tsetse flies-Wolbachia interactions, which is essential for the development and use of Wolbachia-based biological control methods.
Horizontal transmission of the symbiotic bacterium Asaia sp. in the leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae)
Authors: Gonella Elena , Crotti Elena , Rizzi Aurora , Mandrioli Mauro , Favia Guido , Daffonchio Daniele , Alma Alberto
Published: January 2012

Background

Bacteria of the genus Asaia have been recently recognized as secondary symbionts of different sugar-feeding insects, including the leafhopper Scaphoideus titanus, vector of Flavescence dorée phytoplasmas. Asaia has been shown to be localized in S. titanus gut, salivary glands and gonoducts and to be maternally transmitted to the progeny by an egg smearing mechanism. It is currently not known whether Asaia in S. titanus is transmitted by additional routes. We performed a study to evaluate if Asaia infection is capable of horizontal transmission via co-feeding and venereal routes.

Results

A Gfp-tagged strain of Asaia was provided to S. titanus individuals to trace the transmission pathways of the symbiotic bacterium. Co-feeding trials showed a regular transfer of bacterial cells from donors to recipients, with a peak of frequency after 72 hours of exposure, and with concentrations of the administrated strain growing over time. Venereal transmission experiments were first carried out using infected males paired with uninfected females. In this case, female individuals acquired Gfp-labelled Asaia, with highest infection rates 72-96 hours after mating and with increasing abundance of the tagged symbiont over time. When crosses between infected females and uninfected males were conducted, the occurrence of “female to male” transmission was observed, even though the transfer occurred unevenly.

Conclusions

The data presented demonstrate that the acetic acid bacterial symbiont Asaia is horizontally transmitted among S. titanus individuals both by co-feeding and venereal transmission, providing one of the few direct demonstrations of such a symbiotic transfer in Hemiptera. This study contributes to the understanding of the bacterial ecology in the insect host, and indicates that Asaia evolved multiple pathways for the colonization of S. titanus body.
GDNF protects enteric glia from apoptosis: evidence for an autocrine loop
Authors: Steinkamp Martin , Gundel Heike , Schulte Nadine , Spaniol Ulrike , Pflueger Carolin , Zizer Eugen , von Boyen Georg BT
Published: January 2012

Background

Enteric glia cells (EGC) play an important role in the maintenance of intestinal mucosa integrity. During the course of acute Crohn's disease (CD), mucosal EGC progressively undergo apoptosis, though the mechanisms are largely unknown. We investigated the role of Glial-derived neurotrophic factor (GDNF) in the regulation of EGC apoptosis.

Methods

GDNF expression and EGC apoptosis were determined by immunofluorescence using specimen from CD patients. In primary rat EGC cultures, GDNF receptors were assessed by western blot and indirect immunofluorescence microscopy. Apoptosis in cultured EGC was induced by TNF-α and IFN-γ, and the influence of GDNF on apoptosis was measured upon addition of GDNF or neutralizing anti-GDNF antibody.

Results

Increased GDNF expression and Caspase 3/7 activities were detected in in specimen of CD patients but not in healthy controls. Moreover, inactivation of GDNF sensitized in EGC cell to IFN-γ/TNF-α induced apoptosis.

Conclusions

This study proposes the existence of an autocrine anti-apoptotic loop in EGC cells which is operative in Crohn's disease and dependent of GDNF. Alterations in this novel EGC self-protecting mechanism could lead to a higher susceptibility towards apoptosis and thus contribute to disruption of the mucosal integrity and severity of inflammation in CD.
NKG2D ligand tumor expression and association with clinical outcome in early breast cancer patients: an observational study
Authors: de Kruijf Esther M , Sajet Anita , van Nes Johanna GH , Putter Hein , Smit Vincent THBM , Eagle Robert A , Jafferji Insiya , Trowsdale John , Liefers Gerrit Jan , van de Velde Cornelis JH , Kuppen Peter JK
Published: January 2012

Background

Cell surface NKG2D ligands (NKG2DL) bind to the activating NKG2D receptor present on NK cells and subsets of T cells, thus playing a role in initiating an immune response. We examined tumor expression and prognostic effect of NKG2DL in breast cancer patients.

Methods

Our study population (n = 677) consisted of all breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed paraffin-embedded tumor tissue was immunohistochemically stained with antibodies directed against MIC-A/MIC-B (MIC-AB), ULBP-1, ULBP-2, ULBP-3, ULBP-4, and ULBP-5.

Results

NKG2DL were frequently expressed by tumors (MIC-AB, 50% of the cases; ULBP-1, 90%; ULBP-2, 99%; ULBP-3, 100%; ULBP-4, 26%; ULBP-5, 90%) and often showed co-expression: MIC-AB and ULBP-4 (p = 0.043), ULBP-1 and ULBP-5 (p = 0.006), ULBP-4 and ULBP-5 (p < 0.001). MIC-AB (p = 0.001) and ULBP-2 (p = 0.006) expression resulted in a statistically significant longer relapse free period (RFP). Combined expression of these ligands showed to be an independent prognostic parameter for RFP (p < 0.001, HR 0.41). Combined expression of all ligands showed no associations with clinical outcome.

Conclusions

We demonstrated for the first time that NKG2DL are frequently expressed and often co-expressed in breast cancer. Expression of MIC-AB and ULBP-2 resulted in a statistically significant beneficial outcome concerning RFP with high discriminative power. Combination of all NKG2DL showed no additive or interactive effect of ligands on each other, suggesting that similar and co-operative functioning of all NKG2DL can not be assumed. Our observations suggest that among driving forces in breast cancer outcome are immune activation on one site and tumor immune escape on the other site.
Increase of ATP-sensitive potassium (KATP) channels in the heart of type-1 diabetic rats
Authors: Chen Zhih-Cherng , Cheng Yung-Ze , Chen Li-Jen , Cheng Kai-Chun , Li Yin- Xiao , Cheng Juei- Tang
Published: January 2012

Background

An impairment of cardiovascular function in streptozotocin (STZ)-diabetic rats has been mentioned within 5 days-to-3 months of induction. ATP-sensitive potassium (KATP) channels are expressed on cardiac sarcolemmal membranes. It is highly responsive to metabolic fluctuations and can have effects on cardiac contractility. The present study attempted to clarify the changes of cardiac KATP channels in diabetic disorders.

Methods

Streptozotocin-induced diabetic rats and neonatal rat cardiomyocytes treated with a high concentration of glucose (a D-glucose concentration of 30 mM was used and cells were cultured for 24 hr) were used to examine the effect of hyperglycemia on cardiac function and the expression of KATP channels. KATP channels expression was found to be linked to cardiac tonic dysfunction, and we evaluated the expression levels of KATP channels by Western blot and Northern blot analysis.

Results

The result shows diazoxide produced a marked reduction of heart rate in control group. Furthermore, the methods of Northern blotting and Western blotting were employed to identify the gene expression of KATP channel. Two subunits of cardiac KATP channel (SUR2A and kir 6.2) were purchased as indicators and showed significantly decreased in both diabetic rats and high glucose treated rat cardiac myocytes. Correction of hyperglycemia by insulin or phlorizin restored the gene expression of cardiac KATP in these diabetic rats.

Conclusions

Both mRNA and protein expression of cardiac KATP channels are decreased in diabetic rats induced by STZ for 8 weeks. This phenomenon leads to result in desensitization of some KATP channel drugs.
Small surfactant-like peptides can drive soluble proteins into active aggregates
Authors: Zhou Bihong , Xing Lei , Wu Wei , Zhang Xian-En , Lin Zhanglin
Published: January 2012

Background

Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli) cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos) when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF) and a short beta structure peptide ELK16 (LELELKLKLELELKLK) have a similar property.

Results

In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6) were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used), Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same), Bacillus pumilus xylosidase (XynB), and green fluorescent protein (GFP), and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM) and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red) to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core.

Conclusions

This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in vivo, and can be explored for production of functional biopolymers with detergent or other interfacial activities.
Robust, small-scale cultivation platform for Streptomyces coelicolor
Authors: Sohoni Sujata Vijay , Bapat Prashant Madhusudan , Lantz Anna Eliasson
Published: January 2012

Background

For fermentation process and strain improvement, where one wants to screen a large number of conditions and strains, robust and scalable high-throughput cultivation systems are crucial. Often, the time lag between bench-scale cultivations to production largely depends on approximate estimation of scalable physiological traits. Microtiter plate (MTP) based screening platforms have lately become an attractive alternative to shake flasks mainly because of the ease of automation. However, there are very few reports on applications for filamentous organisms; as well as efforts towards systematic validation of physiological behavior compared to larger scale are sparse. Moreover, available small-scale screening approaches are typically constrained by evaluating only an end point snapshot of phenotypes.

Results

To address these issues, we devised a robust, small-scale cultivation platform in the form of MTPs (24-square deepwell) for the filamentous bacterium Streptomyces coelicolor and compared its performance to that of shake flasks and bench-scale reactors. We observed that re-designing of medium and inoculum preparation recipes resulted in improved reproducibility. Process turnaround time was significantly reduced due to the reduction in number of unit operations from inoculum to cultivation. The incorporation of glass beads (ø 3 mm) in MTPs not only improved the process performance in terms of improved oxygen transfer improving secondary metabolite production, but also helped to transform morphology from pellet to disperse, resulting in enhanced reproducibility. Addition of MOPS into the medium resulted in pH maintenance above 6.50, a crucial parameter towards reproducibility. Moreover, the entire trajectory of the process was analyzed for compatibility with bench-scale reactors. The MTP cultivations were found to behave similar to bench-scale in terms of growth rate, productivity and substrate uptake rate and so was the onset of antibiotic synthesis. Shake flask cultivations however, showed discrepancy with respect to morphology and had considerably reduced volumetric production rates of antibiotics.

Conclusion

We observed good agreement of the physiological data obtained in the developed MTP platform with bench-scale. Hence, the described MTP-based screening platform has a high potential for investigation of secondary metabolite biosynthesis in Streptomycetes and other filamentous bacteria and the use may significantly reduce the workload and costs.
Alpha-tocotrienol is the most abundant tocotrienol isomer circulated in plasma and lipoproteins after postprandial tocotrienol-rich vitamin E supplementation
Authors: Fairus Syed , Nor Rosnah M , Cheng Hwee M , Sundram Kalyana
Published: January 2012

Background

Tocotrienols (T3) and tocopherols (T), both members of the natural vitamin E family have unique biological functions in humans. T3 are detected in circulating human plasma and lipoproteins, although at concentrations significantly lower than α-tocopherol (α-T). T3, especially α-T3 is known to be neuropotective at nanomolar concentrations and this study evaluated the postprandial fate of T3 and α-T in plasma and lipoproteins.

Methods

Ten healthy volunteers (5 males and 5 females) were administered a single dose of vitamin E [526 mg palm tocotrienol-rich fraction (TRF) or 537 mg α-T] after 7-d pre-conditioning on a T3-free diet. Blood was sampled at baseline (fasted) and 2, 4, 5, 6, 8, and 24 h after supplementation. Concentrations of T and T3 isomers in plasma, triacylglycerol-rich particles (TRP), LDL, and HDL were measured at each postprandial interval.

Results

After TRF supplementation, plasma α-T3 and γ-T3 peaked at 5 h (α-T3: 4.74 ± 1.69 μM; γ-T3: 2.73 ± 1.27 μM). δ-T3 peaked earlier at 4 h (0.53 ± 0.25 μM). In contrast, α-T peaked at 6 h (30.13 ± 2.91 μM) and 8 h (37.80 ± 3.59 μM) following supplementation with TRF and α-T, respectively. α-T was the major vitamin E isomer detected in plasma, TRP, LDL, and HDL even after supplementation with TRF (composed of 70% T3). No T3 were detected during fasted states. T3 are detected postprandially only after TRF supplementation and concentrations were significantly lower than α-T.

Conclusions

Bio-discrimination between vitamin E isomers in humans reduces the rate of T3 absorption and affects their incorporation into lipoproteins. Although low absorption of T3 into circulation may impact some of their physiological functions in humans, T3 have biological functions well below concentration noted in this study.
Lack of EGFR mutations benefiting gefitinib treatment in adenocarcinoma of esophagogastric junction
Authors: Wang Wen-Ping , Wang Kang-Ning , Gao Qiang , Chen Long-Qi
Published: January 2012

Background

The epidermal growth factor receptor (EGFR) inhibitor, gefitinib, has been reported to successfully treat advanced non-small cell lung cancer patients with genetic mutations in EGFR. The aim of this study was to investigate the existence of EGFR mutations in carcinoma of esophagogastric junction, and also to explore the possibility of treating carcinoma of esophagogastric junction using gefitinib.

Methods

From Aug. 2009 to Jun. 2010, 65 patients with carcinoma of esophagogastric junction underwent surgical resection. The tumor tissue and corresponding blood specimens were collected from all cases. The DNA was extracted and PCR amplification was accomplished based on designed primers for exons 18, 19, 20, and 21. EGFR exons 18, 19, 20 and 21 of both cancer cell and white blood cell were finally successfully sequenced.

Results

In exon 20, a variant from CAG to CAA at codon 787 (2361G-> A) was identified in 19 patients, which was a genomic variation of EGFR since it was found in both cancer tissue and white blood cells. This EGFR alteration was a synonymous single nucleotide polymorphism (SNP) since CAA and CAG were encoding the same amino-acid of Glutamine (Q787Q, NCBI database 162093G > A, SNP ID: rs10251977). No genetic alteration was found in exons 18, 19 or 21.

Conclusions

Adenocarcinoma of esophagogastric junction rarely presents EGFR mutation, especially gefitinib-associated mutations such as L858R, or delE746-A750. This means that the gefitinib-based gene target therapy should not be recommended for treating carcinoma of esophagogastric junction.
Complications as indicators of quality assurance after 401 consecutive colorectal cancer resections: the importance of surgeon volume in developing colorectal cancer units in India
Authors: Shetty Guruprasad S , Bodhankar Yashodhan D , Ingle Sachin , Thakkar Rohan G , Goel Mahesh , Shukla Parul J , Shrikhande Shailesh V
Published: January 2012

Background

The low incidence of colorectal cancer in India, coupled with absence of specialized units, contribute to lack of relevant data arising from the subcontinent. We evaluated the data of the senior author to better define the requirements that would enable development of specialized units in a country where colorectal cancer burden is increasing.

Methods

We retrospectively analyzed data of 401 consecutive colorectal resections from a prospective database of the senior author. In addition to patient demographics and types of resections, perioperative data like intraoperative blood loss, duration of surgery, complications, re-operation rates and hospital stay were recorded and analyzed.

Results

The median age was 52 years (10-86 years). 279 were males and 122 were females. The average duration of surgery was 220.32 minutes (range 50 - 480 min). The overall complication rate was 12.2% (49/401) with a 1.2% (5/401) mortality rate. The patients having complications had an increase in their median hospital stay (from 10.5 days to 23.4 days) and the re-operation rate in them was 51%. The major complications were anastomotic leaks (2.5%) and stoma related complications (2.7%).

Conclusions

This largest ever series from India compares favorably with global standards. In a nation where colorectal cancer is on the rise, it is imperative that high volume centers develop specialized units to train future specialist colorectal surgeons. This would ensure improved quality assurance and delivery of health care even to outreach, low volume centers.
Gelatin-layered and multi-sized porous β-tricalcium phosphate for tissue engineering scaffold
Authors: Kim Sung-Min , Yi Soon-Aei , Choi Seong-Ho , Kim Kwang-Mahn , Lee Yong-Keun
Published: January 2012

The multi-sized porous β-tricalcium phosphate scaffolds were fabricated by freeze drying followed by slurry coating using a multi-sized porous sponge as a template. Then, gelatin was dip coated on the multi-sized porous β-tricalcium phosphate scaffolds under vacuum. The mechanical and biological properties of the fabricated scaffolds were evaluated and compared to the uniformly sized porous scaffolds and scaffolds that were not coated by gelatin. The compressive strength was tested by a universal testing machine, and the cell viability and differentiation behavior were measured using a cell counting kit and alkaline phosphatase activity using the MC3T3-E1 cells. In comparison, the gelatin-coated multi-sized porous β-tricalcium phosphate scaffold showed enhanced compressive strength. After 14 days, the multi-sized pores were shown to affect cell differentiation, and gelatin coatings were shown to affect the cell viability and differentiation. The results of this study demonstrated that the multi-sized porous β-tricalcium phosphate scaffold coated by gelatin enhanced the mechanical and biological strengths.