A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

BACKGROUND: Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results. Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC. METHODS: RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted. RESULTS: The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 10(8 )copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR. CONCLUSION: The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens

Mutations associated with progression in follicular lymphoma predict inferior outcomes at diagnosis: Alliance A151303

Follicular lymphoma (FL) is clinically heterogeneous, with select patients tolerating extended watch-and-wait, whereas others require prompt treatment, suffer progression of disease within 24 months of treatment (POD24), and/or experience aggressive histologic transformation (t-FL). Because our understanding of the relationship between genetic alterations in FL and patient outcomes remains limited, we conducted a clinicogenomic analysis of 370 patients with FL or t-FL (from Cancer and Leukemia Group B/Alliance trials 50402/50701/50803, or real-world cohorts from Washington University School of Medicine, Cleveland Clinic, or University of Miami). FL subsets by grade, stage, watch-and-wait, or POD24 status did not differ by mutation burden, whereas mutation burden was significantly higher in relapsed/refractory (rel/ref) FL and t-FL than in newly diagnosed (dx) FL. Nonetheless, mutation burden in dx FL was not associated with frontline progression-free survival (PFS). CREBBP was the only gene more commonly mutated in FL than in t-FL yet mutated CREBBP was associated with shorter frontline PFS in FL. Mutations in 20 genes were more common in rel/ref FL or t-FL than in dx FL, including 6 significantly mutated genes (SMGs): STAT6, TP53, IGLL5, B2M, SOCS1, and MYD88. We defined a mutations associated with progression (MAP) signature as ≥2 mutations in these 7 genes (6 rel/ref FL or t-FL SMGs plus CREBBP). Patients with dx FL possessing a MAP signature had shorter frontline PFS, revealing a 7-gene set offering insight into FL progression risk potentially more generalizable than the m7-Follicular Lymphoma International Prognostic Index (m7-FLIPI), which had modest prognostic value in our cohort. Future studies are warranted to validate the poor prognosis associated with a MAP signature in dx FL, potentially facilitating novel trials specifically in this high-risk subset of patients

Cross-talk between Hippo and Wnt signalling pathways in intestinal crypts : insights from an agent-based model

Intestinal crypts are responsible for the total cell renewal of the lining of the intestines; this turnover is governed by the interplay between signalling pathways and the cell cycle. The role of Wnt signalling in cell proliferation and differentiation in the intestinal crypt has been extensively studied, with increased signalling found towards the lower regions of the crypt. Recent studies have shown that the Wnt signalling gradient found within the crypt may arise as a result of division-based spreading from a Wnt ‘reservoir’ at the crypt base. The discovery of the Hippo pathway’s involvement in maintaining crypt homeostasis is more recent; a mechanistic understanding of Hippo pathway dynamics, and its possible cross-talk with the Wnt pathway, remains lacking. To explore how the interplay between these pathways may control crypt homeostasis, we extended an ordinary differential equation model of the Wnt signalling pathway to include a phenomenological description of Hippo signalling in single cells, and then coupled it to a cell-based description of cell movement, proliferation and contact inhibition in agent-based simulations. Furthermore, we compared an imposed Wnt gradient with a division-based Wnt gradient model. Our results suggest that Hippo signalling affects the Wnt pathway by reducing the presence of free cytoplasmic β-catenin, causing cell cycle arrest. We also show that a division-based spreading of Wnt can form a Wnt gradient, resulting in proliferative dynamics comparable to imposed-gradient models. Finally, a simulated APC double mutant, with misregulated Wnt and Hippo signalling activity, is predicted to cause monoclonal conversion of the crypt

Ultra-deep sequencing reveals the mutational landscape of classical Hodgkin lymphoma

UNLABELLED: The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to approximately 1,000× median depth of coverage. An orthogonal error-corrected sequencing approach verified \u3e95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7, novel stop gain mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. As a further application of our exome sequencing, we attempted to identify expressed somatic single-nucleotide variants (SNV) in single-nuclei RNA sequencing (snRNA-seq) data generated from a patient in our cohort. Our snRNA analysis identified a clear cluster of cells containing a somatic SNV identified in our deep exome data. This cluster has differentially expressed genes that are consistent with genes known to be dysregulated in HRS cells (e.g., PIM1 and PIM3). The cluster also contains cells with an expanded B-cell clonotype further supporting a malignant phenotype. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells and demonstrates the feasibility of snRNA-seq in the context of cHL. These studies provide the foundation for the further analysis of genomic variants in large cohorts of patients with cHL. SIGNIFICANCE: Our data demonstrate the utility of ultra-deep exome sequencing in uncovering somatic variants in Hodgkin lymphoma, creating new opportunities to define the genes that are recurrently mutated in this disease. We also show for the first time the successful application of snRNA-seq in Hodgkin lymphoma and describe the expression profile of a putative cluster of HRS cells in a single patient

Increased variability in ApcMin/+ intestinal tissue can be measured with microultrasound

Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic

Tpl2 is required for VEGF-A-stimulated signal transduction and endothelial cell function

New blood vessel sprouting (angiogenesis) and vascular physiology are fundamental features of metazoan species but we do not fully understand how signal transduction pathways regulate diverse vascular responses. The vascular endothelial growth factor (VEGF) family bind membrane-bound receptor tyrosine kinases (VEGFRs), which trigger multiple signal transduction pathways and diverse cellular responses. We evaluated whether the MAP3K family member and proto-oncoprotein Tpl2 (MAP3K8) regulates basal and VEGF-A-stimulated signal transduction in endothelial cells. Notably, stimulation with exogenous VEGF-A increased Tpl2 mRNA levels and consequently de novo protein synthesis. Depletion of Tpl2 levels reveals a role in both basal and VEGF-A-stimulated endothelial cell responses, including endothelial-leukocyte interactions, monolayer permeability and new blood vessel formation. Under basal conditions, Tpl2 modulates a signal transduction cascade resulting in phosphorylation of a nuclear transcription factor (ATF-2) and altered endothelial gene expression, a pathway previously identified as crucial in VEGF-dependent vascular responses. Loss of Tpl2 expression or activity impairs signal transduction through Akt, eNOS and ATF-2, broadly impacting on endothelial function. Our study now provides a mechanism for Tpl2 as a central component of signal transduction pathways in the endothelium

Emergence and Genetic Variation of Neuraminidase Stalk Deletions in Avian Influenza Viruses

When avian influenza viruses (AIVs) are transmitted from their reservoir hosts (wild waterfowl and shorebirds) to domestic bird species, they undergo genetic changes that have been linked to higher virulence and broader host range. Common genetic AIV modifications in viral proteins of poultry isolates are deletions in the stalk region of the neuraminidase (NA) and additions of glycosylation sites on the hemagglutinin (HA). Even though these NA deletion mutations occur in several AIV subtypes, they have not been analyzed comprehensively. In this study, 4,920 NA nucleotide sequences, 5,596 HA nucleotide and 4,702 HA amino acid sequences were analyzed to elucidate the widespread emergence of NA stalk deletions in gallinaceous hosts, the genetic polymorphism of the deletion patterns and association between the stalk deletions in NA and amino acid variants in HA. Forty-seven different NA stalk deletion patterns were identified in six NA subtypes, N1–N3 and N5–N7. An analysis that controlled for phylogenetic dependence due to shared ancestry showed that NA stalk deletions are statistically correlated with gallinaceous hosts and certain amino acid features on the HA protein. Those HA features included five glycosylation sites, one insertion and one deletion. The correlations between NA stalk deletions and HA features are HA-NA-subtype-specific. Our results demonstrate that stalk deletions in the NA proteins of AIV are relatively common. Understanding the NA stalk deletion and related HA features may be important for vaccine and drug development and could be useful in establishing effective early detection and warning systems for the poultry industry