Nitisinone Arrests but Does Not Reverse Ochronosis in Alkaptonuric Mice.

Alkaptonuria (AKU) is an ultrarare autosomal recessive disorder resulting from a deficiency of homogentisate 1,2 dioxygenase (HGD), an enzyme involved in the catabolism of phenylalanine and tyrosine. Loss of HGD function prevents metabolism of homogentisic acid (HGA), leading to increased levels of plasma HGA and urinary excretion. Excess HGA becomes deposited in collagenous tissues and subsequently undergoes polymerisation, principally in the cartilages of loaded joints, in a process known as ochronosis. This results in an early-onset, devastating osteoarthropathy for which there is currently no effective treatment. We recently described the natural history of ochronosis in a murine model of AKU, demonstrating that deposition of ochronotic pigment begins very early in life and accumulates with age. Using this model, we were able to show that lifetime treatment with nitisinone, a potential therapy for AKU, was able to completely prevent deposition of ochronotic pigment. However, although nitisinone has been shown to inhibit ochronotic deposition, whether it can also facilitate removal of existing pigment has not yet been examined. We describe here that midlife administration of nitisinone to AKU mice arrests further deposition of ochronotic pigment in the tibiofemoral joint, but does not result in the clearance of existing pigment. We also demonstrate the dose-dependent response of plasma HGA to nitisinone, highlighting its efficacy for personalised medicine, where dosage can be tailored to the individual AKU patient

Biochemical and genetic analysis of butyrylcholinesterase (BChE) in a family, due to prolonged neuromuscular blockade after the use of succinylcholine

Butyrylcholinesterase (BChE) is a plasma enzyme that catalyzes the hydrolysis of choline esters, including the muscle-relaxant succinylcholine and mivacurium. Patients who present sustained neuromuscular blockade after using succinylcholine usually carry BChE variants with reduced enzyme activity or an acquired BChE deficiency. We report here the molecular basis of the BCHE gene underlying the slow catabolism of succinylcholine in a patient who underwent endoscopic nasal surgery. We measured the enzyme activity of BChE and extracted genomic DNA in order to study the promoter region and all exons of the BCHE gene of the patient, her parents and siblings. PCR products were sequenced and compared with reference sequences from GenBank. We detected that the patient and one of her brothers have two homozygous mutations: nt1615 GCA > ACA (Ala539Thr), responsible for the K variant, and nt209 GAT > GGT (Asp70Gly), which produces the atypical variant A. Her parents and two of her brothers were found to be heterozygous for the AK allele, and another brother is homozygous for the normal allele. Sequence analysis of exon 1 including 5′UTR showed that the proband and her brother are homozygous for –116GG. The AK/AK genotype is considered the most frequent in hereditary hypocholinesterasemia (44%). This work demonstrates the importance of defining the phenotype and genotype of the BCHE gene in patients who are subjected to neuromuscular block by succinylcholine, because of the risk of prolonged neuromuscular paralysis

The languages of peace during the French religious wars

The desirability of peace was a common topos in sixteenth-century political rhetoric, and the duty of the king to uphold the peace for the benefit of his subjects was also a long-established tradition. However, the peculiar circumstances of the French religious wars, and the preferred royal policy of pacification, galvanized impassioned debate among both those who supported and those who opposed confessional coexistence. This article looks at the diverse ways in which peace was viewed during the religious wars through an exploration of language and context. It draws not only on the pronouncements of the crown and its officials, and of poets and jurists, but also on those of local communities and confessional groups. Opinion was not just divided along religious lines; political imperatives, philosophical positions and local conditions all came into play in the arguments deployed. The variegated languages of peace provide a social and cultural dimension for the contested nature of sixteenth-century French politics. However, they could not restore harmony to a war-torn and divided kingdom

CNVassoc: Association analysis of CNV data using R

Background: Copy number variants (CNV) are a potentially important component of the genetic contribution to risk of common complex diseases. Analysis of the association between CNVs and disease requires that uncertainty in CNV copy-number calls, which can be substantial, be taken into account; failure to consider this uncertainty can lead to biased results. Therefore, there is a need to develop and use appropriate statistical tools. To address this issue, we have developed CNVassoc, an R package for carrying out association analysis of common copy number variants in population-based studies. This package includes functions for testing for association with different classes of response variables (e.g. class status, censored data, counts) under a series of study designs (case-control, cohort, etc) and inheritance models, adjusting for covariates. The package includes functions for inferring copy number (CNV genotype calling), but can also accept copy number data generated by other algorithms (e.g. CANARY, CGHcall, IMPUTE). Results: Here we present a new R package, CNVassoc, that can deal with different types of CNV arising from different platforms such as MLPA o aCGH. Through a real data example we illustrate that our method is able to incorporate uncertainty in the association process. We also show how our package can also be useful when analyzing imputed data when analyzing imputed SNPs. Through a simulation study we show that CNVassoc outperforms CNVtools in terms of computing time as well as in convergence failure rate. Conclusions: We provide a package that outperforms the existing ones in terms of modelling flexibility, power, convergence rate, ease of covariate adjustment, and requirements for sample size and signal quality. Therefore, we offer CNVassoc as a method for routine use in CNV association studiesThis work has been supported by the Spanish Ministry of Science and Innovation (MTM2008-02457 to JRG, BIO2009-12458 to RD-U and statistical genetics network MTM2010-09526-E (subprograma MTM) to JRG, IS, GL and RD-U). GL is supported by the Juan de la Cierva Program of the Spanish Ministry of Science and Innovation

Third generation cephalosporin use in a tertiary hospital in Port of Spain, Trinidad: need for an antibiotic policy

BACKGROUND: Tertiary care hospitals are a potential source for development and spread of bacterial resistance being in the loop to receive outpatients and referrals from community nursing homes and hospitals. The liberal use of third-generation cephalosporins (3GCs) in these hospitals has been associated with the emergence of extended-spectrum beta- lactamases (ESBLs) presenting concerns for bacterial resistance in therapeutics. We studied the 3GC utilization in a tertiary care teaching hospital, in warded patients (medical, surgical, gynaecology, orthopedic) prescribed these drugs. METHODS: Clinical data of patients (≥ 13 years) admitted to the General Hospital, Port of Spain (POSGH) from January to June 2000, and who had received 3GCs based on the Pharmacy records were studied. The Sanford Antibiotic Guide 2000, was used to determine appropriateness of therapy. The agency which procures drugs for the Ministry of Health supplied the cost of drugs. RESULTS: The prevalence rate of use of 3GCs was 9.5 per 1000 admissions and was higher in surgical and gynecological admissions (21/1000) compared with medical and orthopedic (8 /1000) services (p < 0.05). Ceftriaxone was the most frequently used 3GC. Sixty-nine (36%) patients without clinical evidence of infection received 3Gcs and prescribing was based on therapeutic recommendations in 4% of patients. At least 62% of all prescriptions were inappropriate with significant associations for patients from gynaecology (p < 0.003), empirical prescribing (p < 0.48), patients with undetermined infection sites (p < 0.007), and for single drug use compared with multiple antibiotics (p < 0.001). Treatment was twice as costly when prescribing was inappropriate CONCLUSIONS: There is extensive inappropriate 3GC utilization in tertiary care in Trinidad. We recommend hospital laboratories undertake continuous surveillance of antibiotic resistance patterns so that appropriate changes in prescribing guidelines can be developed and implemented. Though guidelines for rational antibiotic use were developed they have not been re-visited or encouraged, suggesting urgent antibiotic review of the hospital formulary and instituting an infection control team. Monitoring antibiotic use with microbiology laboratory support can promote rational drug utilization, cut costs, halt inappropriate 3GC prescribing, and delay the emergence of resistant organisms. An ongoing antibiotic peer audit is suggested

The Arabidopsis RNA Polymerase II Carboxyl Terminal Domain (CTD) Phosphatase-Like1 (CPL1) is a biotic stress susceptibility gene

© 2018, The Author(s). Crop breeding for improved disease resistance may be achieved through the manipulation of host susceptibility genes. Previously we identified multiple Arabidopsis mutants known as enhanced stress response1 (esr1) that have defects in a KH-domain RNA-binding protein and conferred increased resistance to the root fungal pathogen Fusarium oxysporum. Here, screening the same mutagenized population we discovered two further enhanced stress response mutants that also conferred enhanced resistance to F. oxysporum. These mutants also have enhanced resistance to a leaf fungal pathogen (Alternaria brassicicola) and an aphid pest (Myzus persicae), but not to the bacterial leaf pathogen Pseudomonas syringae. The causal alleles in these mutants were found to have defects in the ESR1 interacting protein partner RNA Polymerase II Carboxyl Terminal Domain (CTD) Phosphatase-Like1 (CPL1) and subsequently given the allele symbols cpl1-7 and cpl1-8. These results define a new role for CPL1 as a pathogen and pest susceptibility gene. Global transcriptome analysis and oxidative stress assays showed these cpl1 mutants have increased tolerance to oxidative stress. In particular, components of biotic stress responsive pathways were enriched in cpl1 over wild-type up-regulated gene expression datasets including genes related to defence, heat shock proteins and oxidative stress/redox state processes

Advanced Technologies for Oral Controlled Release: Cyclodextrins for oral controlled release

Cyclodextrins (CDs) are used in oral pharmaceutical formulations, by means of inclusion complexes formation, with the following advantages for the drugs: (1) solubility, dissolution rate, stability and bioavailability enhancement; (2) to modify the drug release site and/or time profile; and (3) to reduce or prevent gastrointestinal side effects and unpleasant smell or taste, to prevent drug-drug or drug-additive interactions, or even to convert oil and liquid drugs into microcrystalline or amorphous powders. A more recent trend focuses on the use of CDs as nanocarriers, a strategy that aims to design versatile delivery systems that can encapsulate drugs with better physicochemical properties for oral delivery. Thus, the aim of this work was to review the applications of the CDs and their hydrophilic derivatives on the solubility enhancement of poorly water soluble drugs in order to increase their dissolution rate and get immediate release, as well as their ability to control (to prolong or to delay) the release of drugs from solid dosage forms, either as complexes with the hydrophilic (e.g. as osmotic pumps) and/ or hydrophobic CDs. New controlled delivery systems based on nanotechonology carriers (nanoparticles and conjugates) have also been reviewed

Factors affecting the yield of microRNAs from laser microdissectates of formalin-fixed tissue sections

<p>Abstract</p> <p>Background</p> <p>Quantification of microRNAs in specific cell populations microdissected from tissues can be used to define their biological roles, and to develop and deploy biomarker assays. In this study, a number of variables were examined for their effect on the yield of microRNAs in samples obtained from formalin-fixed paraffin-embedded tissues by laser microdissection.</p> <p>Results</p> <p>MicroRNA yield was improved by using cresyl violet instead of hematoxylin-eosin to stain tissue sections in preparation for microdissection, silicon carbide instead of glass fiber as matrix in RNA-binding columns, and overnight digestion of dissected samples with proteinase K. Storage of slides carrying stained tissue sections at room temperature for up to a week before microdissection, and storage of the microdissectates at room temperature for up to a day before RNA extraction did not adversely affect microRNA yield.</p> <p>Conclusions</p> <p>These observations should be of value for the efficient isolation of microRNAs from microdissected formalin-fixed tissues with a flexible workflow.</p

Drosophila muscleblind Codes for Proteins with One and Two Tandem Zinc Finger Motifs

Muscleblind-like proteins, Muscleblind (Mbl) in Drosophila and MBNL1-3 in vertebrates, are regulators of alternative splicing. Human MBNL1 is a key factor in the etiology of myotonic dystrophy (DM), a muscle wasting disease caused by the occurrence of toxic RNA molecules containing CUG/CCUG repeats. MBNL1 binds to these RNAs and is sequestered in nuclear foci preventing it from exerting its normal function, which ultimately leads to mis-spliced mRNAs, a major cause of the disease. Muscleblind-proteins bind to RNAs via N-terminal zinc fingers of the Cys3-His type. These zinc fingers are arranged in one (invertebrates) or two (vertebrates) tandem zinc finger (TZF) motifs with both fingers targeting GC steps in the RNA molecule. Here I show that mbl genes in Drosophila and in other insects also encode proteins with two TZF motifs, highly similar to vertebrate MBNL proteins. In Drosophila the different protein isoforms have overlapping but possibly divergent functions in vivo, evident by their unequal capacities to rescue the splicing defects observed in mbl mutant embryos. In addition, using whole transcriptome analysis, I identified several new splicing targets for Mbl in Drosophila embryos. Two of these novel targets, kkv (krotzkopf-verkehrt, coding for Chitin Synthase 1) and cora (coracle, coding for the Drosophila homolog of Protein 4.1), are not muscle-specific but expressed mainly in epidermal cells, indicating a function for mbl not only in muscles and the nervous system