Relationships between components of blood pressure and cardiovascular events in patients with stable coronary artery disease and hypertension

Observational studies have shown a J-shaped relationship between diastolic blood pressure (BP) and cardiovascular events in hypertensive patients with coronary artery disease. We investigated whether the increased risk associated with low diastolic BP reflects elevated pulse pressure (PP). In 22 672 hypertensive patients with coronary artery disease from the CLARIFY registry (Prospective Observational Longitudinal Registry of Patients With Stable Coronary Artery Disease), followed for a median of 5.0 years, BP was measured annually and averaged. The relationships between PP and diastolic BP, alone or combined, and the primary composite outcome (cardiovascular death or myocardial infarction) were analyzed using multivariable Cox proportional hazards models. Adjusted hazard ratios for the primary outcome were 1.62 (95% confidence interval [CI], 1.40–1.87), 1.00 (ref), 1.07 (95% CI, 0.94–1.21), 1.54 (95% CI, 1.32–1.79), and 2.34 (95% CI, 1.95–2.81) for PP<45, 45 to 54 (reference), 55 to 64, 65 to 74, and ≥75 mm Hg, respectively, and 1.50 (95% CI, 1.31–1.72), 1.00 (reference), and 1.58 (95% CI, 1.42–1.77) for diastolic BPs of <70, 70 to 79 (ref), and ≥80 mm Hg, respectively. In a cross-classification analysis between diastolic BP and PP, the relationship between diastolic BP and the primary outcome remained J-shaped when the analysis was restricted to patients with the lowest-risk PP (45–64 mm Hg), with adjusted hazard ratios of 1.53 (95% CI, 1.27–1.83), 1.00 (ref), and 1.54 (95% CI, 1.34–1.75) in the <70, 70 to 79 (reference), and ≥80 mm Hg subgroups, respectively. The J-shaped relationship between diastolic BP and cardiovascular events in hypertensive patients with coronary artery disease persists in patients within the lowest-risk PP range and is therefore unlikely to be solely the consequence of an increased PP reflecting advanced vascular disease

Your Proof Fails? Testing Helps to Find the Reason

Applying deductive verification to formally prove that a program respects its formal specification is a very complex and time-consuming task due in particular to the lack of feedback in case of proof failures. Along with a non-compliance between the code and its specification (due to an error in at least one of them), possible reasons of a proof failure include a missing or too weak specification for a called function or a loop, and lack of time or simply incapacity of the prover to finish a particular proof. This work proposes a new methodology where test generation helps to identify the reason of a proof failure and to exhibit a counter-example clearly illustrating the issue. We describe how to transform an annotated C program into C code suitable for testing and illustrate the benefits of the method on comprehensive examples. The method has been implemented in STADY, a plugin of the software analysis platform FRAMA-C. Initial experiments show that detecting non-compliances and contract weaknesses allows to precisely diagnose most proof failures.Comment: 11 pages, 10 figure

Pan-HA antibodies for influenza detection and quantification

The influenza virus imposes a heavy burden for society in terms of health and economy. Influenza is an elusive enveloped virus due to antigenic shift and drift of two surface proteins: neuraminidase (NA) and hemagglutinin (HA). As a result, new strains emerge every year which require seasonal vaccination for protection. Furthermore, large vaccine quantities are urgently needed in case of pandemics. Theoretically, vaccines against a new strain can be manufactured in as little as three weeks with certain platforms and technologies. However, vaccine quantification and release are still relying on the use of the Single Radial Immunodiffusion (SRID) assay using a strain-specific antibody to calculate HA concentration. This is a major limitation because it can take up to three months to generate the reagents necessary to run the SRID assay, including the strain-specific antibody. Hence, one of the major hurdles in the process of influenza vaccine production is the quantification of HA which is critical to establish proper dosing. To circumvent the need for strain-specific antibodies, we have produced two monoclonal antibodies (F211-11H12-3 and F211-10A9-2) against a highly conserved peptide sequence found within the HA molecule (1). Multiple strains belonging to 13 different influenza A subtypes, as well as 6 strains belonging to B lineages were detected by Western blot and dot blot. Overall, mAb F211-11H12-3 recognizes preferentially influenza A subtype 1, while the mAb F211-10A9-2 has a higher affinity for influenza A subtype 2. Therefore, all strains tested could be detected when both mAb are combined and used as a cocktail. Next, we performed quantitative dot blots by generating a standard curve ranging from 160ng/ml to 20µg/ml HA. This method is simple, easy to implement and highly reproducible. In-process samples as well as purified material can be quantified by dot blot after denaturation with urea. Even though the SRID is the only assay approved by regulatory agencies, quantitative dot blots can be used during manufacturing to optimize and monitor the production process. Finally, ELISA is widely used for quantification and preliminary data demonstrates that samples can be quantified with the pan-HA mAbs. In conclusion, a pan-HA antibody cocktail was generated against a highly conserved peptide sequence of influenza. Viruses produced in eggs and mammalian cells from 40 different strains were detected by Western blot. Reproducible quantification was achieved by dot blot using the two mAbs and an appropriate calibrating standard. The combination of pan-HA antibodies with an immunoassay such as the dot blot assay could accelerate process development and help establish new generation quantification methods for influenza. As the field is looking for flexible and versatile solutions to shift away from the SRID assay and strain-specific antibodies, the development of broad-spectrum antibodies offers a long-awaited alternative. 1) Chun et al, Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins, Vaccine (26), pp 6068-6076, 2008

Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Viral Vaccine (CVV) as reported by the 2015 WHO Informal Consultation report titled “Influenza Vaccine Response during the Start of a Pandemic”. In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza A/Puerto Rico/8/34 H1N1 strain, and advances in the large-scale transfection of suspension cultured HEK-293 cells. Transfection in shake flasks was performed using 1ug of total plasmid and 1x106 cells/mL. The supernatant was harvested after 48 hpt and used to infect a new shake flasks at 1x106 cells/mL for virus amplification. 3-L bioreactor was inoculated and transfected at 1x106 cells/mL with 1ug of total plasmid and harvested after 48hpt and the virus generated was amplified in shake flask. Quantification by TCID50, SRID, Dot-blot and TRPS were performed as well as characterization by TEM and HA and NA sequencing. Small-scale transfection in shake flasks generated 1.5x105 IVP/mL after 48 hpt and 1x107 IVP/mL after 96 hpi. For large-scale experiment a 3-L controlled stirred tank bioreactor resulted in supernatant (P0) virus titer of 5x104 IVP/mL and 2.8x107 IVP/mL after only one amplification (P1) in HEK-293 suspension cells. We demonstrate the efficent generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Thus, this innovative approach is better suited to rationally design and mass produce the CVV within timelines dictated by pandemic situations and produce effective responsiveness than previous methodolog

Charcot-Marie-Tooth disease type 2CC due to NEFH variants causes a progressive, non-length-dependent, motor-predominant phenotype

Objective: Neurofilaments are the major scaffolding proteins for the neuronal cytoskeleton, and variants in NEFH have recently been described to cause axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC). Methods: In this large observational study, we present phenotype–genotype correlations on 30 affected and 3 asymptomatic mutation carriers from eight families. Results: The majority of patients presented in adulthood with motor-predominant and lower limb-predominant symptoms and the average age of onset was 31.0±15.1 years. A prominent feature was the development of proximal weakness early in the course of the disease. The disease progressed rapidly, unlike other Charcot-Marie-Tooth disease (CMT) subtypes, and half of the patients (53%) needed to use a wheelchair on average 24.1 years after symptom onset. Furthermore, 40% of patients had evidence of early ankle plantarflexion weakness, a feature which is observed in only a handful of CMT subtypes. Neurophysiological studies and MRI of the lower limbs confirmed the presence of a non-length-dependent neuropathy in the majority of patients. All families harboured heterozygous frameshift variants in the last exon of NEFH, resulting in a reading frameshift to an alternate open reading frame and the translation of approximately 42 additional amino acids from the 3' untranslated region (3′-UTR). Conclusions: This phenotype–genotype study highlights the unusual phenotype of CMT2CC, which is more akin to spinal muscular atrophy rather than classic CMT. Furthermore, the study will enable more informative discussions on the natural history of the disease and will aid in NEFH variant interpretation in the context of the disease’s unique molecular genetics

The pattern and diagnostic criteria of sensory neuronopathy: a case–control study

Acquired sensory neuronopathies encompass a group of paraneoplastic, dysimmune, toxic or idiopathic disorders characterized by degeneration of peripheral sensory neurons in dorsal root ganglia. As dorsal root ganglia cannot easily be explored, the clinical diagnosis of these disorders may be difficult. The question as to whether there exists a common clinical pattern of sensory neuronopathies, allowing the establishment of validated and easy-to-use diagnostic criteria, has not yet been addressed. In this study, logistic regression was used to construct diagnostic criteria on a retrospective study population of 78 patients with sensory neuronopathies and 56 with other sensory neuropathies. For this, sensory neuronopathy was provisionally considered as unambiguous in 44 patients with paraneoplastic disorder or cisplatin treatment and likely in 34 with a dysimmune or idiopathic setting who may theoretically have another form of neuropathy. To test the homogeneity of the sensory neuronopathy population, likely candidates were compared with unambiguous cases and then the whole population was compared with the other sensory neuropathies population. Criteria accuracy was checked on 37 prospective patients referred for diagnosis of sensory neuropathy. In the study population, sensory neuronopathy showed a common clinical and electrophysiological pattern that was independent of the underlying cause, including unusual forms with only patchy sensory loss, mild electrical motor nerve abnormalities and predominant small fibre or isolated lower limb involvement. Logistic regression allowed the construction of a set of criteria that gave fair results with the following combination: ataxia in the lower or upper limbs + asymmetrical distribution + sensory loss not restricted to the lower limbs + at least one sensory action potential absent or three sensory action potentials <30% of the lower limit of normal in the upper limbs + less than two nerves with abnormal motor nerve conduction study in the lower limbs

Performance of the CMS Cathode Strip Chambers with Cosmic Rays

The Cathode Strip Chambers (CSCs) constitute the primary muon tracking device in the CMS endcaps. Their performance has been evaluated using data taken during a cosmic ray run in fall 2008. Measured noise levels are low, with the number of noisy channels well below 1%. Coordinate resolution was measured for all types of chambers, and fall in the range 47 microns to 243 microns. The efficiencies for local charged track triggers, for hit and for segments reconstruction were measured, and are above 99%. The timing resolution per layer is approximately 5 ns

Membrane Protein Location-Dependent Regulation by PI3K (III) and Rabenosyn-5 in Drosophila Wing Cells

The class III phosphatidylinositol-3 kinase (PI3K (III)) regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate (PI(3)P). While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein β-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of β-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes

Aristolochic Acid I Induced Autophagy Extenuates Cell Apoptosis via ERK 1/2 Pathway in Renal Tubular Epithelial Cells

Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. However, limited information is available about autophagy in aristolochic acid (AA) nephropathy. In this study, we investigated the role of autophagy and related signaling pathway during progression of AAI-induced injury to renal tubular epithelial cells (NRK52E cells). The results showed that autophagy in NRK52E cells was detected as early as 3–6 hrs after low dose of AAI (10 µM) exposure as indicated by an up-regulated expression of LC3-II and Beclin 1 proteins. The appearance of AAI-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in NRK52E cells provided further evidence for autophagy. However, cell apoptosis was not detected until 12 hrs after AAI treatment. Blockade of autophagy with Wortmannin or 3-Methyladenine (two inhibitors of phosphoinositede 3-kinases) or small-interfering RNA knockdown of Beclin 1 or Atg7 sensitized the tubular cells to apoptosis. Treatment of NRK52E cells with AAI caused a time-dependent increase in extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity, but not c-Jun N-terminal kinase (JNK) and p38. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 resulted in a decreased AAI-induced autophagy that was accompanied by an increased apoptosis. Taken together, our study demonstrated for the first time that autophagy occurred earlier than apoptosis during AAI-induced tubular epithelial cell injury. Autophagy induced by AAI via ERK1/2 pathway might attenuate apoptosis, which may provide a protective mechanism for cell survival under AAI-induced pathological condition