Macropinocytotic uptake and infection of human epithelial cells with species B2 adenovirus type 35

The human adenovirus serotype 35 (HAdV-35, short Ad35) causes kidney and urinary tract infections, and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here, we show that infectious entry of Ad35 into HeLa, human kidney HK-2 cells and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate, and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180 which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chemicals against the serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3 or the sodium-proton exchange inhibitor EIPA blocked the endocytic uptake of Ad35. Dominant-negative proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1 or the Pak1 effector C-terminal binding protein 1 (CtBP1) potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy and live cell imaging showed that Ad35 colocalized with fluid phase markers in large endocytic structures that were positive for CD46, alpha v integrins and also CtBP1. Our results extend earlier observations with HAdV-3 (Ad3), and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells

Dislocation of total hip replacement in patients with fractures of the femoral neck: A prospective cohort study of 713 consecutive hips

Background Total hip replacement is increasingly used in active, relatively healthy elderly patients with fractures of the femoral neck. Dislocation of the prosthesis is a severe complication, and there is still controversy regarding the optimal surgical approach and its influence on stability. We analyzed factors influencing the stability of the total hip replacement, paying special attention to the surgical approach

4th ESPT summer school: precision medicine and personalised health

In September 2018, the European Society of Pharmacogenomics and Personalised Therapy (ESPT), with the support of the Swiss Personalized Health Network (SPHN), organized its 4th biennial summer school, entitled 'Precision Medicine and Personalised Health' (Campus Biotech, Geneva, Switzerland; www.esptsummerschool.eu/ ). The school's comprehensive and innovative educational program aimed to address the fundamentals of pharmacogenomics, the latest knowledge on established and new concepts in the field of precision medicine, as well as its advanced clinical applications in personalized health. The school consisted of 31 lectures, eight interactive workshops, visits to genome center and poster presentations, involving 40 speakers from distinguished international faculties. The meeting was a resounding success by generating informal environments between more than 80 participants from 26 different countries

Promoter methylation and large intragenic rearrangements of DPYD are not implicated in severe toxicity to 5-fluorouracil-based chemotherapy in gastrointestinal cancer patients

<p>Abstract</p> <p>Background</p> <p>Severe toxicity to 5-fluorouracil (5-FU) based chemotherapy in gastrointestinal cancer has been associated with constitutional genetic alterations of the dihydropyrimidine dehydrogenase gene (<it>DPYD</it>).</p> <p>Methods</p> <p>In this study, we evaluated <it>DPYD </it>promoter methylation through quantitative methylation-specific PCR and screened <it>DPYD </it>for large intragenic rearrangements in peripheral blood from 45 patients with gastrointestinal cancers who developed severe 5-FU toxicity. <it>DPYD </it>promoter methylation was also assessed in tumor tissue from 29 patients</p> <p>Results</p> <p>Two cases with the IVS14+1G > A exon 14 skipping mutation (c.1905+1G > A), and one case carrying the 1845 G > T missense mutation (c.1845G > T) in the DPYD gene were identified. However, <it>DPYD </it>promoter methylation and large <it>DPYD </it>intragenic rearrangements were absent in all cases analyzed.</p> <p>Conclusions</p> <p>Our results indicate that <it>DPYD </it>promoter methylation and large intragenic rearrangements do not contribute significantly to the development of 5-FU severe toxicity in gastrointestinal cancer patients, supporting the need for additional studies on the mechanisms underlying genetic susceptibility to severe 5-FU toxicity.</p

Role of Cellular Heparan Sulfate Proteoglycans in Infection of Human Adenovirus Serotype 3 and 35

Species B human adenoviruses (Ads) are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs). We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection

The value of episodic, intensive blood glucose monitoring in non-insulin treated persons with type 2 diabetes: Design of the Structured Testing Program (STeP) Study, a cluster-randomised, clinical trial [NCT00674986]

<p>Abstract</p> <p>Background</p> <p>The value and utility of self-monitoring of blood glucose (SMBG) in non-insulin treated T2DM has yet to be clearly determined. Findings from studies in this population have been inconsistent, due mainly to design differences and limitations, including the prescribed frequency and timing of SMBG, role of the patient and physician in responding to SMBG results, inclusion criteria that may contribute to untoward floor effects, subject compliance, and cross-arm contamination. We have designed an SMBG intervention study that attempts to address these issues.</p> <p>Methods/design</p> <p>The Structured Testing Program (STeP) study is a 12-month, cluster-randomised, multi-centre clinical trial to evaluate whether poorly controlled (HbA1c ≥ 7.5%), non-insulin treated T2DM patients will benefit from a comprehensive, integrated physician/patient intervention using structured SMBG in US primary care practices. Thirty-four practices will be recruited and randomly assigned to an active control group (ACG) that receives enhanced usual care or to an enhanced usual care group plus structured SMBG (STG). A total of 504 patients will be enrolled; eligible patients at each site will be randomly selected using a defined protocol. Anticipated attrition of 20% will yield a sample size of at least 204 per arm, which will provide a 90% power to detect a difference of at least 0.5% in change from baseline in HbA1c values, assuming a common standard deviation of 1.5%. Differences in timing and degree of treatment intensification, cost effectiveness, and changes in patient self-management behaviours, mood, and quality of life (QOL) over time will also be assessed. Analysis of change in HbA1c and other dependent variables over time will be performed using both intent-to-treat and per protocol analyses. Trial results will be available in 2010.</p> <p>Discussion</p> <p>The intervention and trial design builds upon previous research by emphasizing appropriate and collaborative use of SMBG by both patients and physicians. Utilization of per protocol and intent-to-treat analyses facilitates a comprehensive assessment of the intervention. Use of practice site cluster-randomisation reduces the potential for intervention contamination, and inclusion criteria (HbA1c ≥ 7.5%) reduces the possibility of floor effects. Inclusion of multiple dependent variables allows us to assess the broader impact of the intervention, including changes in patient and physician attitudes and behaviours.</p> <p>Trial Registration</p> <p>Current Controlled Trials NCT00674986.</p

Effects of metal-on-metal wear on the host immune system and infection in hip arthroplasty

Methods We reviewed the available literature on the influence of degradation products of MOM bearings in total hip arthroplasties on infection risk. Results Wear products were found to influence the risk of infection by hampering the immune system, by inhibiting or accelerating bacterial growth, and by a possible antibiotic resistance and heavy metal co-selection mechanism. Interpretation Whether or not the combined effects of MOM wear products make MOM bearings less or more prone to infection requires investigation in the near future

Intragenic deletions and a deep intronic mutation affecting pre-mRNA splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5-fluorouracil toxicity

Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme acting in the catabolism of the widely used antineoplastic agent 5-fluorouracil (5FU). DPD deficiency is known to cause a potentially lethal toxicity following administration of 5FU. Here, we report novel genetic mechanisms underlying DPD deficiency in patients presenting with grade III/IV 5FU-associated toxicity. In one patient a genomic DPYD deletion of exons 21–23 was observed. In five patients a deep intronic mutation c.1129–5923C>G was identified creating a cryptic splice donor site. As a consequence, a 44 bp fragment corresponding to nucleotides c.1129–5967 to c.1129–5924 of intron 10 was inserted in the mature DPD mRNA. The deleterious c.1129–5923C>G mutation proved to be in cis with three intronic polymorphisms (c.483 + 18G>A, c.959–51T>G, c.680 + 139G>A) and the synonymous mutation c.1236G>A of a previously identified haplotype. Retrospective analysis of 203 cancer patients showed that the c.1129–5923C>G mutation was significantly enriched in patients with severe 5FU-associated toxicity (9.1%) compared to patients without toxicity (2.2%). In addition, a high prevalence was observed for the c.1129–5923C>G mutation in the normal Dutch (2.6%) and German (3.3%) population. Our study demonstrates that a genomic deletion affecting DPYD and a deep intronic mutation affecting pre-mRNA splicing can cause severe 5FU-associated toxicity. We conclude that screening for DPD deficiency should include a search for genomic rearrangements and aberrant splicing

Sequencing technologies and genome sequencing

The high-throughput - next generation sequencing (HT-NGS) technologies are currently the hottest topic in the field of human and animals genomics researches, which can produce over 100 times more data compared to the most sophisticated capillary sequencers based on the Sanger method. With the ongoing developments of high throughput sequencing machines and advancement of modern bioinformatics tools at unprecedented pace, the target goal of sequencing individual genomes of living organism at a cost of $1,000 each is seemed to be realistically feasible in the near future. In the relatively short time frame since 2005, the HT-NGS technologies are revolutionizing the human and animal genome researches by analysis of chromatin immunoprecipitation coupled to DNA microarray (ChIP-chip) or sequencing (ChIP-seq), RNA sequencing (RNA-seq), whole genome genotyping, genome wide structural variation, de novo assembling and re-assembling of genome, mutation detection and carrier screening, detection of inherited disorders and complex human diseases, DNA library preparation, paired ends and genomic captures, sequencing of mitochondrial genome and personal genomics. In this review, we addressed the important features of HT-NGS like, first generation DNA sequencers, birth of HT-NGS, second generation HT-NGS platforms, third generation HT-NGS platforms: including single molecule Heliscope™, SMRT™ and RNAP sequencers, Nanopore, Archon Genomics X PRIZE foundation, comparison of second and third HT-NGS platforms, applications, advances and future perspectives of sequencing technologies on human and animal genome research