151 research outputs found
Targeted Next-Generation Sequencing Validates the Use of Diagnostic Biopsies as a Suitable Alternative to Resection Material for Mutation Screening in Colorectal Cancer
Background: Mutation testing in the context of neoadjuvant therapy must be performed on biopsy samples. Given the issue of tumour heterogeneity, this raises the question of whether the biopsies are representative of the whole tumour. Here we have compared the mutation profiles of colorectal biopsies with their matched resection specimens.
Methods: We performed next-generation sequencing (NGS) analysis on 25 paired formalin-fixed, paraffin-embedded colorectal cancer biopsy and primary resection samples. DNA was extracted and analysed using the TruSight tumour kit, allowing the interrogation of 26 cancer driver genes. Samples were run on an Illumina MiSeq. Mutations were validated using quick-multiplex-consensus (QMC)-polymerase chain reaction (PCR) in conjunction with high resolution melting (HRM). The paired biopsy and resection tumour samples were assessed for presence or absence of mutations, mutant allele frequency ratios, and allelic imbalance status.
Results: A total of 81 mutations were detected, in ten of the 26 genes in the TruSight kit. Two of the 25 paired cases were wild-type across all genes. The mutational profiles, allelic imbalance status, and mutant allele frequency ratios of the paired biopsy and resection samples were highly concordant (88.75–98.85%), with all but three (3.7%) of the mutations identified in the resection specimens also being present in the biopsy specimens. All 81 mutations were confirmed by QMC-PCR and HRM analysis, although four low-level mutations required a co-amplification at lower denaturation temperature (COLD)-PCR protocol to enrich for the mutant alleles.
Conclusions: Diagnostic biopsies are adequate and reliable materials for molecular testing by NGS. The use of biopsies for molecular screening will enhance targeted neoadjuvant therapy
Lobar and segmental liver atrophy associated with hilar cholangiocarcinoma and the impact of hilar biliary anatomical variants: a pictorial essay
The radiological features of lobar and segmental liver atrophy and compensatory hypertrophy associated with biliary obstruction are important to recognise for diagnostic and therapeutic reasons. Atrophied lobes/segments reduce in volume and usually contain crowded dilated bile ducts extending close to the liver surface. There is often a “step” in the liver contour between the atrophied and non-atrophied parts. Hypertrophied right lobe or segments enlarge and show a prominently convex or “bulbous” visceral surface. The atrophied liver parenchyma may show lower attenuation on pre-contrast computed tomography (CT) and CT intravenous cholangiography (CT-IVC) and lower signal intensity on T1-weighted magnetic resonance imaging (MRI). Hilar biliary anatomical variants can have an impact on the patterns of lobar/segmental atrophy, as the cause of obstruction (e.g. cholangiocarcinoma) often commences in one branch, leading to atrophy in that drainage region before progressing to complete biliary obstruction and jaundice. Such variants are common and can result in unusual but explainable patterns of atrophy and hypertrophy. Examples of changes seen with and without hilar variants are presented that illustrate the radiological features of atrophy/hypertrophy
Resource allocation within the National AIDS Control Program of Pakistan: a qualitative assessment of decision maker's opinions
BACKGROUND: Limited resources, whether public or private, demand prioritisation among competing needs to maximise productivity. With a substantial increase in the number of reported HIV cases, little work has been done to understand how resources have been distributed and what factors may have influenced allocation within the newly introduced Enhanced National AIDS Control Program of Pakistan. The objective of this study was to identify perceptions of decision makers about the process of resource allocation within Pakistan's Enhanced National AIDS Control Program. METHODS: A qualitative study was undertaken and in-depth interviews of decision makers at provincial and federal levels responsible to allocate resources within the program were conducted. RESULTS: HIV was not considered a priority issue by all study participants and external funding for the program was thought to have been accepted because of poor foreign currency reserves and donor agency influence rather than local need. Political influences from the federal government and donor agencies were thought to manipulate distribution of funds within the program. These influences were thought to occur despite the existence of a well-laid out procedure to determine allocation of public resources. Lack of collaboration among departments involved in decision making, a pervasive lack of technical expertise, paucity of information and an atmosphere of ad hoc decision making were thought to reduce resistance to external pressures. CONCLUSION: Development of a unified program vision through a consultative process and advocacy is necessary to understand goals to be achieved, to enhance program ownership and develop consensus about how money and effort should be directed. Enhancing public sector expertise in planning and budgeting is essential not just for the program, but also to reduce reliance on external agencies for technical support. Strengthening available databases for effective decision making is required to make financial allocations based on real, rather than perceived needs. With a large part of HIV program funding dedicated to public-private partnerships, it becomes imperative to develop public sector capacity to administer contracts, coordinate and monitor activities of the non-governmental sector
Dramatic Co-Activation of WWOX/WOX1 with CREB and NF-κB in Delayed Loss of Small Dorsal Root Ganglion Neurons upon Sciatic Nerve Transection in Rats
BACKGROUND:Tumor suppressor WOX1 (also named WWOX or FOR) is known to participate in neuronal apoptosis in vivo. Here, we investigated the functional role of WOX1 and transcription factors in the delayed loss of axotomized neurons in dorsal root ganglia (DRG) in rats. METHODOLOGY/PRINCIPAL FINDINGS:Sciatic nerve transection in rats rapidly induced JNK1 activation and upregulation of mRNA and protein expression of WOX1 in the injured DRG neurons in 30 min. Accumulation of p-WOX1, p-JNK1, p-CREB, p-c-Jun, NF-kappaB and ATF3 in the nuclei of injured neurons took place within hours or the first week of injury. At the second month, dramatic nuclear accumulation of WOX1 with CREB (>65% neurons) and NF-kappaB (40-65%) occurred essentially in small DRG neurons, followed by apoptosis at later months. WOX1 physically interacted with CREB most strongly in the nuclei as determined by FRET analysis. Immunoelectron microscopy revealed the complex formation of p-WOX1 with p-CREB and p-c-Jun in vivo. WOX1 blocked the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation in vitro. In contrast, WOX1 enhanced promoter activation governed by c-Jun, Elk-1 and NF-kappaB. WOX1 directly activated NF-kappaB-regulated promoter via its WW domains. Smad4 and p53 were not involved in the delayed loss of small DRG neurons. CONCLUSIONS/SIGNIFICANCE:Rapid activation of JNK1 and WOX1 during the acute phase of injury is critical in determining neuronal survival or death, as both proteins functionally antagonize. In the chronic phase, concurrent activation of WOX1, CREB, and NF-kappaB occurs in small neurons just prior to apoptosis. Likely in vivo interactions are: 1) WOX1 inhibits the neuroprotective CREB, which leads to eventual neuronal death, and 2) WOX1 enhances NF-kappaB promoter activation (which turns to be proapoptotic). Evidently, WOX1 is the potential target for drug intervention in mitigating symptoms associated with neuronal injury
Efficiency of Purine Utilization by Helicobacter pylori: Roles for Adenosine Deaminase and a NupC Homolog
The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase
Immunodetection of nmt55/p54(nrb) isoforms in human breast cancer
BACKGROUND: We previously identified and characterized a novel 55 kDa nuclear protein, termed nmt55/p54(nrb), whose expression was decreased in a subset of human breast tumors. The objective of this study was to determine if this reduced expression in human breast tumors was attributed to the regulation of mRNA transcription or the presence of altered forms of this protein. RESULTS: Northern blot analysis and ribonuclease protection assay indicated that nmt55/p54(nrb) mRNA is expressed at varying levels in estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast tumors suggesting that reduced expression of nmt55/p54(nrb) protein in ER- tumors was not due to transcriptional regulation. To determine if multiple protein isoforms are expressed in breast cancer, we utilized Western blot and immunohistochemical analyses, which revealed the expression of an nmt55/p54(nrb) protein isoform in a subset of ER+ tumors. This subset of ER+ human breast tumors expressed an altered form of nmt55/p54(nrb) that was undetectable with an amino-terminal specific antibody suggesting that this isoform contains alterations or modifications within the amino terminal domain. CONCLUSIONS: Our study indicates that nmt55/p54(nrb) protein is post-transcriptionally regulated in human breast tumors leading to reduced expression in ER- tumors and the expression of an amino terminal altered isoform in a subset of ER+ tumors. The potential involvement of nmt55/p54(nrb) in RNA binding and pre-mRNA splicing may be important for normal cell growth and function; thus, loss or alteration of protein structure may contribute to tumor growth and progression
Organization and molecular evolution of a disease-resistance gene cluster in coffee trees
<p>Abstract</p> <p>Background</p> <p>Most disease-resistance (R) genes in plants encode NBS-LRR proteins and belong to one of the largest and most variable gene families among plant genomes. However, the specific evolutionary routes of NBS-LRR encoding genes remain elusive. Recently in coffee tree (<it>Coffea arabica</it>), a region spanning the <it>S</it><sub><it>H</it></sub><it>3 </it>locus that confers resistance to coffee leaf rust, one of the most serious coffee diseases, was identified and characterized. Using comparative sequence analysis, the purpose of the present study was to gain insight into the genomic organization and evolution of the <it>S</it><sub><it>H</it></sub><it>3 </it>locus.</p> <p>Results</p> <p>Sequence analysis of the <it>S</it><sub><it>H</it></sub><it>3 </it>region in three coffee genomes, E<sup>a </sup>and C<sup>a </sup>subgenomes from the allotetraploid <it>C. arabica </it>and C<sup>c </sup>genome from the diploid <it>C. canephora</it>, revealed the presence of 5, 3 and 4 R genes in E<sup>a</sup>, C<sup>a</sup>, and C<sup>c </sup>genomes, respectively. All these R-gene sequences appeared to be members of a CC-NBS-LRR (CNL) gene family that was only found at the <it>S</it><sub><it>H</it></sub><it>3 </it>locus in <it>C. arabica</it>. Furthermore, while homologs were found in several dicot species, comparative genomic analysis failed to find any CNL R-gene in the orthologous regions of other eudicot species. The orthology relationship among the <it>S</it><sub><it>H</it></sub><it>3</it>-CNL copies in the three analyzed genomes was determined and the duplication/deletion events that shaped the <it>S</it><sub><it>H</it></sub><it>3 </it>locus were traced back. Gene conversion events were detected between paralogs in all three genomes and also between the two sub-genomes of <it>C. arabica</it>. Significant positive selection was detected in the solvent-exposed residues of the <it>S</it><sub><it>H</it></sub><it>3</it>-CNL copies.</p> <p>Conclusion</p> <p>The ancestral <it>S</it><sub><it>H</it></sub><it>3</it>-CNL copy was inserted in the <it>S</it><sub><it>H</it></sub><it>3 </it>locus after the divergence between Solanales and Rubiales lineages. Moreover, the origin of most of the <it>S</it><sub><it>H</it></sub><it>3</it>-CNL copies predates the divergence between <it>Coffea </it>species. The <it>S</it><sub><it>H</it></sub><it>3</it>-CNL family appeared to evolve following the birth-and-death model, since duplications and deletions were inferred in the evolution of the <it>S</it><sub><it>H</it></sub><it>3 </it>locus. Gene conversion between paralog members, inter-subgenome sequence exchanges and positive selection appear to be the major forces acting on the evolution of <it>S</it><sub><it>H</it></sub><it>3</it>-CNL in coffee trees.</p
Systematic review of the evidence relating FEV1 decline to giving up smoking
<p>Abstract</p> <p>Background</p> <p>The rate of forced expiratory volume in 1 second (FEV<sub>1</sub>) decline ("beta") is a marker of chronic obstructive pulmonary disease risk. The reduction in beta after quitting smoking is an upper limit for the reduction achievable from switching to novel nicotine delivery products. We review available evidence to estimate this reduction and quantify the relationship of smoking to beta.</p> <p>Methods</p> <p>Studies were identified, in healthy individuals or patients with respiratory disease, that provided data on beta over at least 2 years of follow-up, separately for those who gave up smoking and other smoking groups. Publications to June 2010 were considered. Independent beta estimates were derived for four main smoking groups: never smokers, ex-smokers (before baseline), quitters (during follow-up) and continuing smokers. Unweighted and inverse variance-weighted regression analyses compared betas in the smoking groups, and in continuing smokers by amount smoked, and estimated whether beta or beta differences between smoking groups varied by age, sex and other factors.</p> <p>Results</p> <p>Forty-seven studies had relevant data, 28 for both sexes and 19 for males. Sixteen studies started before 1970. Mean follow-up was 11 years. On the basis of weighted analysis of 303 betas for the four smoking groups, never smokers had a beta 10.8 mL/yr (95% confidence interval (CI), 8.9 to 12.8) less than continuing smokers. Betas for ex-smokers were 12.4 mL/yr (95% CI, 10.1 to 14.7) less than for continuing smokers, and for quitters, 8.5 mL/yr (95% CI, 5.6 to 11.4) less. These betas were similar to that for never smokers. In continuing smokers, beta increased 0.33 mL/yr per cigarette/day. Beta differences between continuing smokers and those who gave up were greater in patients with respiratory disease or with reduced baseline lung function, but were not clearly related to age or sex.</p> <p>Conclusion</p> <p>The available data have numerous limitations, but clearly show that continuing smokers have a beta that is dose-related and over 10 mL/yr greater than in never smokers, ex-smokers or quitters. The greater decline in those with respiratory disease or reduced lung function is consistent with some smokers having a more rapid rate of FEV<sub>1 </sub>decline. These results help in designing studies comparing continuing smokers of conventional cigarettes and switchers to novel products.</p
Combining Next-Generation Sequencing Strategies for Rapid Molecular Resource Development from an Invasive Aphid Species, Aphis glycines
Aphids are one of the most important insect taxa in terms of ecology, evolutionary biology, genetics and genomics, and interactions with endosymbionts. Additionally, many aphids are serious pest species of agricultural and horticultural plants. Recent genetic and genomic research has expanded molecular resources for many aphid species, including the whole genome sequencing of the pea aphid, Acrythosiphon pisum. However, the invasive soybean aphid, Aphis glycines, lacks in any significant molecular resources.Two next-generation sequencing technologies (Roche-454 and Illumina GA-II) were used in a combined approach to develop both transcriptomic and genomic resources, including expressed genes and molecular markers. Over 278 million bp were sequenced among the two methods, resulting in 19,293 transcripts and 56,688 genomic sequences. From this data set, 635 SNPs and 1,382 microsatellite markers were identified. For each sequencing method, different soybean aphid biotypes were used which revealed potential biotype specific markers. In addition, we uncovered 39,822 bp of sequence that were related to the obligatory endosymbiont, Buchnera aphidicola, as well as sequences that suggest the presence of Hamiltonella defensa, a facultative endosymbiont.Molecular resources for an invasive, non-model aphid species were generated. Additionally, the power of next-generation sequencing to uncover endosymbionts was demonstrated. The resources presented here will complement ongoing molecular studies within the Aphididae, including the pea aphid whole genome, lead to better understanding of aphid adaptation and evolution, and help provide novel targets for soybean aphid control
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