Increased angiogenesis and blood vessel maturation in acellular collagen-heparin scaffolds containing both FGF2 and VEGF

An important issue in tissue engineering is the vascularisation of the implanted construct, which often takes several weeks. In vivo, the growth factors VEGF and FGF2 show a combined effect on both angiogenesis and maturation of blood vessels. Therefore, we hypothesise that the addition of these growth factors to an acellular construct increases blood vessel formation and maturation. To systematically evaluate the contribution of each scaffold component with respect to tissue response and in particular to blood vessel formation, five porous scaffolds were prepared and characterised, viz.: collagen, collagen with heparin, and collagen with heparin plus one or two growth factors (rrFGF2 and rrVEGF). Scaffolds were subcutaneously implanted in 3 months old Wistar rats. Of all scaffolds tested, the one with a combination of growth factors displayed the highest density of blood vessels (type IV collagen) and most mature blood vessels (smooth muscle actin). In addition, no hypoxic cells were found in this scaffold at day 7 and 21 (hypoxia inducible factor 1-alpha). These results indicate that the addition of both FGF2 and VEGF to an acellular construct enhances an early mature vasculature. This opens prospects for (acellular) tissue-engineered constructs in conditions as ischaemic heart disease or diabetic ulcers.     

Assemblathon 1: a competitive assessment of de novo short read assembly methods

Low-cost short read sequencing technology has revolutionized genomics, though it is only just becoming practical for the high-quality de novo assembly of a novel large genome. We describe the Assemblathon 1 competition, which aimed to comprehensively assess the state of the art in de novo assembly methods when applied to current sequencing technologies. In a collaborative effort, teams were asked to assemble a simulated Illumina HiSeq data set of an unknown, simulated diploid genome. A total of 41 assemblies from 17 different groups were received. Novel haplotype aware assessments of coverage, contiguity, structure, base calling, and copy number were made. We establish that within this benchmark: (1) It is possible to assemble the genome to a high level of coverage and accuracy, and that (2) large differences exist between the assemblies, suggesting room for further improvements in current methods. The simulated benchmark, including the correct answer, the assemblies, and the code that was used to evaluate the assemblies is now public and freely available from http://www.assemblathon.org/.     

Natural reinfection with respiratory syncytial virus does not boost virus-specific T-cell immunity

To determine the role of respiratory syncytial virus (RSV)-specific cell-mediated immunity during natural reinfection, we investigated whether RSV-specific T-cell responses protect against reinfection and, subsequently, whether reinfection boosts virus-specific memory. In a cohort of 55 infants who were hospitalized for RSV bronchiolitis, RSV-specific lymphoproliferative responses in the peripheral blood were measured at three time-points: on admission, 4 wk after admission, and 1 y later, after the second winter season. Memory was defined as a stimulation index (SI) >2. During the second winter season, nasal secretions were collected in every case of a runny nose. Reinfection was diagnosed if immunofluorescence or PCR was positive for RSV. Virus-specific memory was found in one child on admission for primary RSV infection, whereas 4 wk later 44 infants (80%) had memory. Reinfection with RSV was found in 23 infants (43%) during the second winter season. After the second season, memory was found in 20 infants (38%). No differences in SI after the second winter season were found between infants with and without reinfection (2.3 versus 2.1). However, a highly significant correlation was found between SI measured 4 wk after primary RSV infection and SI after the second winter season (r = 0.40, p = 0.001). In conclusion, RSV-specific T-cell responses did not provide protection against reinfection. Moreover, reinfection did not boost RSV-specific T-cell proliferation. To explain both findings, it is hypothesized that RSV-specific T cells fail to expand in vivo upon reinfection.     

An evaluation of the challenges to developing tumor BRCA1 and BRCA2 testing methodologies for clinical practice

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin‐fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter‐laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next‐generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.     

Exploring the potential association among sleep disturbances,cognitive impairments,and immune activation in 22q11.2 deletion syndrome

22q11.2 deletion syndrome (22q11.DS) is a neurogenetic disorder caused by a microdeletion in chromosome 22. Its phenotype includes high rates of psychiatric disorders, immune system abnormalities, and cognitive impairments. We assessed the quality of sleep in 22q11.2DS and its potential link to inflammatory markers and cognitive deficits. Thirty‐three 22q11.2DS individuals and 24 healthy controls were studied. Sleep parameters were assessed by the Pittsburgh sleep quality index (PSQI) questionnaire and correlated with serum cytokine levels and cognitive functioning, measured using the Penn computerized neurocognitive battery (CNB). The 22q11.2DS individuals had significantly worse sleep quality scores than the controls, unrelated to the psychiatric or physical comorbidities common to 22q11.2DS. Interleukin 6 levels were correlated with the overall score of the PSQI questionnaire for nonpsychotic 22q11.2DS participants only. Several domains of the CNB were associated with poorer sleep quality, suggesting that cognitive impairments in 22q11.2DS may be at least partially explained by poor sleep quality. Our findings confirm sleep impairments in individuals with 22q11.2DS, which might negatively affect their cognitive functioning, and corroborate a potential role of immunological pathways in the 22q11.2DS neuro‐phenotype.     

A model for predicting occult carotid artery stenosis: screening is justified in a selected population

OBJECTIVES: The diagnosis and treatment of carotid artery disease is an integral part of stroke prevention. However, a population of patients who would benefit from screening for carotid artery stenosis has not been well defined. As part of an institutional stroke-screening program, a modified, rapid duplex scan was developed to evaluate patients for occult carotid stenosis. The goal of this study was to evaluate risk factors predictive of carotid stenosis in a selected population, and to identify patients who would benefit from carotid screening. METHODS: Patients were eligible for the study if they were >60 years of age and had a history of hypertension, heart disease, current smoking, or family history of stroke. A modified carotid duplex scan that had been previously validated against formal duplex scanning was utilized; this involved visualization of the carotid bulb and proximal internal carotid artery where Doppler flow velocities were obtained and recorded. RESULTS: Screening was performed on 394 patients. Thirty-eight patients (9.6%) had either unilateral or bilateral carotid stenosis of > or =50%. Risk factors evaluated included smoking, hypertension, cardiac disease, or hypercholesterolemia. If none of these risk factors was present, the incidence of carotid stenosis was 1.8%. This increased to 5.8% with one risk factor, 13.5% with two risk factors, and 16.7% with three risk factors. Two of three patients with all four risk factors had carotid stenosis (66.7%). Logistic regression and prespecified contrast statements for multiple comparisons were used to assess the relationship between the presence of risk factors and occult carotid artery stenosis. The presence of any one of these risk factors was associated with a statistically significant increase in the presence of occult carotid stenosis (P <.01). This was also statistically significant for the presence of any two risk factors (P <.01) or three risk factors (P <.05). CONCLUSION: The prevalence of carotid stenosis significantly increases with the presence of one or more identifiable demographic risk factors in a selected population. Assuming the diagnosis and treatment of carotid stenosis are fundamental to stroke prevention, screening for carotid artery disease is justified in this group of patients.     

Genomic Profiling of Intrahepatic Cholangiocarcinoma: Refining Prognosis and Identifying Therapeutic Targets

Background

The molecular alterations that drive tumorigenesis in intrahepatic cholangiocarcinoma (ICC) remain poorly defined. We sought to determine the incidence and prognostic significance of mutations associated with ICC among patients undergoing surgical resection.

Methods

Multiplexed mutational profiling was performed using nucleic acids that were extracted from 200 resected ICC tumor specimens from 7 centers. The frequency of mutations was ascertained and the effect on outcome was determined.

Results

The majority of patients (61.5 %) had no genetic mutation identified. Among the 77 patients (38.5 %) with a genetic mutation, only a small number of gene mutations were identified with a frequency of >5 %: IDH1 (15.5 %) and KRAS (8.6 %). Other genetic mutations were identified in very low frequency: BRAF (4.9 %), IDH2 (4.5 %), PIK3CA (4.3 %), NRAS (3.1 %), TP53 (2.5 %), MAP2K1 (1.9 %), CTNNB1 (0.6 %), and PTEN (0.6 %). Among patients with an IDH1-mutant tumor, approximately 7 % were associated with a concurrent PIK3CA gene mutation or a mutation in MAP2K1 (4 %). No concurrent mutations in IDH1 and KRAS were noted. Compared with ICC tumors that had no identified mutation, IDH1-mutant tumors were more often bilateral (odds ratio 2.75), while KRAS-mutant tumors were more likely to be associated with R1 margin (odds ratio 6.51) (both P < 0.05). Although clinicopathological features such as tumor number and nodal status were associated with survival, no specific mutation was associated with prognosis.

Conclusions

Most somatic mutations in resected ICC tissue are found at low frequency, supporting a need for broad-based mutational profiling in these patients. IDH1 and KRAS were the most common mutations noted. Although certain mutations were associated with ICC clinicopathological features, mutational status did not seemingly affect long-term prognosis.