Cloning and genomic characterization of Felis domesticus papillomavirus type 1

A novel papillomavirus was cloned from hyperkeratotic cutaneous lesions of a Persian domestic cat. The Felis domesticus papillomavirus (FdPV-1) genome counts 8300 bp and has a typical genome structure with an early region (E1, E2, E4, E6, E7), a late region (L1, L2), and a noncoding upstream regulatory region (URR or NCR1) between the end of L1 and the beginning of E6. The FdPV-1 also shows an unusual second noncoding region (NCR2) of 1.3 kb, situated between the end of E2 and the beginning of L2. This NCR2 is uniquely related to a similar region in the canine oral papillomavirus (COPV). Phylogenetic analysis places FdPV-1 together with COPV, the cottontail rabbit papillomavirus, human papillomavirus type 1 (HPV-1), and HPV-63 in the group of the benign cutaneous papillomaviruses. The position of FdPV-1 in the phylogenetic tree allows us to hypothesize that already in an early phase of the papillomavirus molecular evolution, a split occurred into viruses with a dual tropism primarily for cutaneous epithelia but also secondarily for mucosal surfaces, and viruses with a specific monotropism for mucosal surfaces. The close relationship between FdPV-1 and COPV, and between their Canidae and Felidae hosts, supports the hypothesis that papillomaviruses have speciated and coevolved together with their hosts throughout vertebrate evolution. A papillomavirus mutation rate of 0.73 to 0.96 x 10(-8) nucleotide substitutions per base per year was calculated.     

Analysis of a complex analgesic formulation by high-performance liquid chromatography with column-switching

An isocratic method is described which allows the complete separation of acetylsalicylic acid, paracetamol, caffeine, carbromal, bromisoval and codeine as well as the potential impurities salicylic acid, diacetyl-p-aminophenol and acetylcodeine. A column-switching technique was developed employing two columns (10 cm and 25 cm) containing 7-mum Zorbax C(8) material. The separation is better and faster than that obtained with a gradient elution method.     

Plant cytokinin analogues with inhibitory activity on cyclin-dependent kinases exert their antiproliferative effect through induction of apoptosis initiated by the mitochondrial pathway: determination by a multiparametric flow cytometric analysis

OBJECTIVE: Regulation of the cell cycle by cyclin-dependent kinase (CDK) activity occurs at multiple levels and is often altered in human cancers. Therefore, CDK activity has been targeted for drug discovery, and a number of small molecules have now been identified as CDK inhibitors. Plant cytokinin analogues with CDK inhibitory activity and antiproliferative effects were studied to characterize the cellular basis of the cytotoxic effect. METHODS: The IC(50) value (concentration at which 50% of the cell proliferation is inhibited) and AC(50) value (concentration at which 50% of the cell population is apoptotic) were determined by flow cytometry and microscopy, respectively. A new multiparametric flow cytometric analysis was used to study the sequence of different apoptotic events. In this assay, analysis of phosphatidylserine exposure, mitochondrial membrane depolarization, activation of caspases and DNA condensation were combined. RESULTS: Treatment of Jurkat and KG1 cells with the CDK inhibitors results in a decrease of viable cells and a parallel increase in percentage of apoptotic cells. Apoptosis was accompanied by a rapid decrease of mitochondrial membrane potential, which precedes DNA condensation, exposure of phosphatidylserine and activation of caspases. CONCLUSIONS: The main cellular mechanism of the antiproliferative effect of plant cytokinin analogues with CDK inhibitory activity is the induction of apoptosis. The multiparametric flow cytometric technique allowed to follow the kinetics of various aspects of apoptotic cell changes and demonstrated that cytokinin analogue-induced apoptosis starts through the mitochondrial pathway. This technique could also become of value for the rapid screening of pro-apoptotic properties of chemotherapeutic compounds.     

Effects of Fecal Microbiota Transplantation on Composition in Mice with CKD

Background: Chronic kidney disease (CKD) is a renal disorder characterized by the accumulation of uremic toxins with limited strategies to reduce their concentrations. A large amount of data supports the pivotal role of intestinal microbiota in CKD complications and as a major source of uremic toxins production. Here, we explored whether fecal microbiota transplantation (FMT) could be attenuated in metabolic complication and uremic toxin accumulation in mice with CKD. Methods: Kidney failure was chemically induced by a diet containing 0.25% (w/w) of adenine for four weeks. Mice were randomized into three groups: control, CKD and CKD + FMT groups. After four weeks, CKD mice underwent fecal microbiota transplantation (FMT) from healthy mice or phosphate buffered saline as control. The gut microbiota structure, uremic toxins plasmatic concentrations, and metabolic profiles were explored three weeks after transplantation. Results: Associated with the increase of alpha diversity, we observed a noticeable improvement of gut microbiota disturbance, after FMT treatment. FMT further decreased p-cresyl sulfate accumulation and improved glucose tolerance. There was no change in kidney function. Conclusions: These data indicate that FMT limited the accumulation of uremic toxins issued from intestinal cresol pathway by a beneficial effect on gut microbiota diversity. Further studies are needed to investigate the FMT efficiency, the timing and feces amount for the transplantation before, to become a therapeutic option in CKD patients.     

Nusinersen treatment significantly improves hand grip strength,hand motor function and MRC sum scores in adult patients with spinal muscular atrophy types 3 and 4

Journal of Neurology - Nusinersen recently became available as the first treatment for Spinal Muscular Atrophy (SMA) and data on its effectiveness and safety in adult SMA patients are still scarce....     

Unbiased plasma proteomics for novel diagnostic biomarkers in cardiovascular disease: identification of quiescin Q6 as a candidate biomarker of acutely decompensated heart failure

Aims Biochemical marker testing has improved the evaluation and management of patients with cardiovascular diseases over the past decade. Natriuretic peptides (NPs), used in clinical practice to assess cardiac dysfunction, exhibit many limitations, however. We used an unbiased proteomics approach for the discovery of novel diagnostic plasma biomarkers of heart failure (HF). Methods and results A proteomics pipeline adapted for very low-abundant plasma proteins was applied to clinical samples from patients admitted with acute decompensated HF (ADHF). Quiescin Q6 (QSOX1), a protein involved in the formation of disulfide bridges, emerged as the best performing marker for ADHF (with an area under the receiver operator characteristic curve of 0.86, 95% confidence interval: 0.79-0.92), and novel isoforms of NPs were also identified. Diagnostic performance of QSOX1 for ADHF was confirmed in 267 prospectively collected subjects of whom 76 had ADHF. Combining QSOX1 to B-type NP (BNP) significantly improved diagnostic accuracy for ADHF by particularly improving specificity. Using thoracic aortic constriction in rats, QSOX1 was specifically induced within both left atria and ventricles at the time of HF onset. Conclusion The novel biomarker QSOX1 accurately identifies ADHF, particularly when combined with BNP. Through both clinical and experimental studies we provide lines of evidence for a link between ADHF and cardiovascular production of QSOX1.     

Protein-Bound Uremic Toxins Stimulate Crosstalk between Leukocytes and Vessel Wall

Leukocyte activation and endothelial damage both contribute to cardiovascular disease, a major cause of morbidity and mortality in CKD. Experimental in vitro data link several protein-bound uremic retention solutes to the modulation of inflammatory stimuli, including endothelium and leukocyte responses and cardiovascular damage, corroborating observational in vivo data. However, the impact of these uremic toxins on the crosstalk between endothelium and leukocytes has not been assessed. This study evaluated the effects of acute and continuous exposure to uremic levels of indoxylsulfate (IS), p-cresylsulfate (pCS), and p-cresylglucuronide (pCG) on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using intravital microscopy. Superfusion with IS induced strong leukocyte adhesion, enhanced extravasation, and interrupted blood flow, whereas pCS caused a rapid increase in leukocyte rolling. Superfusion with pCS and pCG combined caused impaired blood flow and vascular leakage but did not further enhance leukocyte rolling over pCS alone. Intravenous infusion with IS confirmed the superfusion results and caused shedding of heparan sulfate, pointing to disruption of the glycocalyx as the mechanism likely mediating IS-induced flow stagnation. These results provide the first clear in vivo evidence that IS, pCS, and pCG exert proinflammatory effects that contribute to vascular damage by stimulating crosstalk between leukocytes and vessels.Cardiovascular disease remains the most important cause of death among patients with CKD,¹ and it is associated with a baseline inflammatory status.^2,3 Atherosclerosis is highly prevalent and advances more rapidly in individuals with renal dysfunction compared with the general population.^4–6 A key role in the development of atherosclerosis is played by leukocyte–endothelial interactions.²CKD is characterized by the progressive retention of a host of solutes. A substantial number of these compounds is protein-bound.^7,8 The indole indoxylsulfate (IS) and the phenolic conjugates p-cresylsulfate (pCS) and p-cresylglucuronide (pCG) are prototype members of this group. Observational data associate these solutes with enhanced cardiovascular damage and progression of kidney failure, and in in vitro experiments, several underlying isolated molecular mechanisms support the link with these observational data.^8–12 Although for all three compounds, one of two key mechanisms of vascular damage (i.e., leukocyte activation or endothelial dysfunction) have been shown in separate in vitro models, they were, to the best of our knowledge, never directly assessed by evaluating the complicated crosstalk between endothelium and leukocytes in an in vivo situation. Intravital microscopy permits in vivo visualization of leukocyte recruitment in translucent tissues in real time. This technique was already applied successfully by our group to study harmful effects of peritoneal dialysis solutions on peritoneal membrane physiology.¹³The present study evaluates the effects of an acute peritoneal superfusion of the protein-bound uremic compounds IS, pCS, and pCG and also, continuous intravenous infusion of IS on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using this intravital microscopic method.