Principles for management of hip fracture for older adults taking direct oral anticoagulants: an international consensus statement.

Hip fracture is a common serious injury among older adults, yet the management of hip fractures for patients taking direct oral anticoagulants remains inconsistent worldwide. Drawing from a synthesis of available evidence and expert opinion, best practice approaches for managing patients with a hip fracture and who are taking direct oral anticoagulants pre-operatively were considered by a working group of the Fragility Fracture Network Hip Fracture Audit Special Interest Group. The literature and related clinical guidelines were reviewed and a two-round modified Delphi study was conducted with a panel of experts from 16 countries and involved seven clinical specialities. Four consensus statements were achieved: peripheral nerve blocks can reasonably be performed on presentation for patients with hip fracture who are receiving direct oral anticoagulants; hip fracture surgery can reasonably be performed for patients taking direct oral anticoagulants < 36 h from last dose; general anaesthesia could reasonably be administered for patients with hip fracture and who are taking direct oral anticoagulants < 36 h from last dose (assuming eGFR > 60 ml.min-1.1.73 m-2); and it is generally reasonable to consider recommencing direct oral anticoagulants (considering blood loss and haemoglobin) < 48 h after hip fracture surgery. No consensus was achieved regarding timing of spinal anaesthesia. The consensus statements were developed to aid clinicians in their decision-making and to reduce practice variations in the management of patients with hip fracture and who are taking direct oral anticoagulants. Each statement will need to be considered specific to each individual patient's treatment.

A novel Android app to evaluate and enhance auditory and tactile temporal thresholds.

With this work, we introduce a novel Android app designed to monitor and enhance auditory and tactile temporal sensitivity. To assess the app's reliability, we tested its technical performance evaluating stimuli production's accuracy (i.e., onset, offset, and duration of stimulation). To validate the app with participants we generated temporal intervals, using either sounds or vibratory stimuli, by implementing two versions of a Two-Alternative Forced-Choice (2AFC) task. Auditory and tactile temporal sensitivity of 12 participants was evaluated using this procedure. To investigate whether temporal abilities could be enhanced using the app, participants were then divided into two groups: one group was trained for four days on the auditory temporal task, while the other was trained for four days on the tactile temporal task. Results suggest that the app can i) effectively measure auditory and tactile temporal thresholds and ii) be used to enhance temporal abilities through perceptual learning. The accessibility of the experimental protocols, combined with our findings, fosters the app's involvement in rehabilitation programs, for example, with a specific focus on sensory disabilities that are associated with temporal deficits (e.g., deafness and Parkinson).Clinical Relevance- The current work introduces a novel app that can be used to monitor and improve temporal abilities, in both the auditory and the tactile modalities.

Autonomy and competence satisfaction as resources for facing chronic pain disability in adolescence: a self-determination perspective.

This study aimed to test empirically the developmental goal pursuit model of paediatric chronic pain, which draws upon Self-Determination Theory for understanding risks and resources for living with chronic pain. This study examined the relationship between basic psychological need satisfaction (i.e. the satisfaction of the needs for autonomy, relatedness and competence) and the fear-avoidance model of pain in adolescents suffering from chronic pain. Hundred and twenty adolescents (mean age = 14.52, 71.6% female), receiving treatment through paediatric pain centres for chronic pain, were enrolled. Adolescents completed measures of basic psychological need satisfaction, fear and avoidance of pain, and pain-related functional impairment. Path analyses model indicated that higher levels of autonomy and competence satisfaction were associated with lower levels of functional disability, through the mediation of fear and avoidance of pain. Relatedness satisfaction was not significatively related to fear of pain, avoidance, and functional disability. The integration of Self-Determination Theory in the paediatric pain literature may further our understanding of potential resources for decreasing functional disability in children living with chronic pain.

Distribution of Antibiotic Resistance Genes among the Phylogroups of Escherichia coli in Diarrheic Calves and Chickens Affected by Colibacillosis in Tehran, Iran.

Antibiotic resistance occurs in the endogenous flora of exposed population in addition to pathogenic bacteria. This study was conducted to evaluate the distribution of antibiotic resistance genes among 63 isolates of Escherichia coli of Escherichia coli (E. coli) in diarrheic calves and poultry. According to the results, B1 and B2 were the most prevalent phylogroups of E. coli in calves and poultry carcasses, respectively. Antimicrobial resistance was observed in 76% of the isolates, and 62% of the strains were multi-drug resistant. Antibiotic resistance in E. coli strains obtained from calves strains was significantly higher than those obtained from poultries. Additionally, the strains of B1 and D phylogroups had the highest and lowest antimicrobial resistance, respectively. At least one encoding gene for integrone was detected in 23 strains (36.5%) and Class I integron had the highest prevalence. Accordingly, this study gave baseline information on the magnitude of the resistance problem and its genetic background in E. coli from domesticated animals of the Tehran, Iran. Moreover, the power of oligonucleotide array technology in the discrimination of different genotypes during a short time was confirmed in this study.

Pulmonary hypertension is associated with higher mortality in cystic fibrosis patients awaiting lung transplantation.

AIM: Pulmonary hypertension (PH) is frequently found in patients with advanced parenchymal lung diseases. In advanced stages, cystic fibrosis (CF) patients can develop PH and eventually cor pulmonale. Little is known about the prevalence of PH in CF patients and its impact on outcome. METHODS: We retrospectively studied a large cohort of CF patients evaluated for lung transplantation between 1995 and 2010. All the patients underwent right heart catheterization as part of the evaluation. We included 179 unique consecutive adult CF patients. Age was 24±9 years and 45.8% were women. RESULTS: Eighty-seven patients were transplanted (48.6%) and 65 died (36.3%) while waiting for LT. By right heart catheterization, 38.5% of the patients had PH (mean ≥25 mm Hg). PaCO(2) (P=0.045) and forced vital capacity (P=0.023) were independent predictors of PH in CF patients. The median survival (free of lung transplantation) was 13.4 months. After adjusting for several covariates, the presence of PH significantly increased mortality (hazard ratio, HR) (P<0.001). Pulmonary vascular resistance was associated with mortality (P=0.03). When both PH and PVR were included in the model, only PH predicted mortality. CONCLUSION: Pulmonary hypertension of mild degree is frequently found in CF patients with advanced lung disease and its presence significantly worsens survival.

Pulmonary hypertension is associated with higher mortality in cystic fibrosis patients awaiting lung transplantation.

AIM:Pulmonary hypertension (PH) is frequently found in patients with advanced parenchymal lung diseases. In advanced stages, cystic fibrosis (CF) patients can develop PH and eventually cor pulmonale. Little is known about the prevalence of PH in CF patients and its impact on outcome. METHODS: We retrospectively studied a large cohort of CF patients evaluated for lung transplantation between 1995 and 2010. All the patients underwent right heart catheterization as part of the evaluation. We included 179 unique consecutive adult CF patients. Age was 24±9 years and 45.8% were women. RESULTS:Eighty-seven patients were transplanted (48.6%) and 65 died (36.3%) while waiting for LT. By right heart catheterization, 38.5% of the patients had PH (mean ≥25 mm Hg). PaCO2 (P=0.045) and forced vital capacity (P=0.023) were independent predictors of PH in CF patients. The median survival (free of lung transplantation) was 13.4 months. After adjusting for several covariates, the presence of PH significantly increased mortality (hazard ratio, HR) (P<0.001). Pulmonary vascular resistance was associated with mortality (P=0.03). When both PH and PVR were included in the model, only PH predicted mortality. CONCLUSION: Pulmonary hypertension of mild degree is frequently found in CF patients with advanced lung disease and its presence significantly worsens survival.

One test microbial diagnostic microarray for identification of Mycoplasma mycoides subsp. mycoides and other Mycoplasma species.

The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri strains. Specifically, we observed that some Italian Mycoplasma mycoides subsp. mycoides strains were positive for two out of the three Mycoplasma mycoides subsp. capri genes, differently from what has been observed for other European or African Mycoplasma mycoides subsp. mycoides strains. This study highlighted the use of microarray technology as a simple and effective method for a single-step identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. The opportunity to discriminate several mycoplasmas in a single analysis enhances diagnostic rapidity and may represent a useful tool to screen occasionally mycoplasmas affecting animal farming in territories where diagnostic laboratory support is limited. The heat-map of the hybridization results of the comparative genomic hybridizations DNA-designed chip clearly indicates that the microarray performs well for the identification of the tested Mycoplasma mycoides subsp. mycoides reference and field strains, discriminating them from other mycoplasmas.

Mutations in the motor and stalk domains of KIF5A in spastic paraplegia type 10 and in axonal Charcot-Marie-Tooth type 2.

Spastic paraplegia type 10 (SPG10) is an autosomal dominant form of hereditary spastic paraplegia (HSP) due to mutations in KIF5A, a gene encoding the neuronal kinesin heavy chain implicated in anterograde axonal transport. KIF5A mutations were found in both pure and complicated forms of the disease; a single KIF5A mutation was also detected in a CMT2 patient belonging to an SPG10 mutant family. To confirm the involvement of the KIF5A gene in both CMT2 and SPG10 phenotypes and to define the frequency of KIF5A mutations in an Italian HSP patient population, we performed a genetic screening of this gene in a series of 139 HSP and 36 CMT2 affected subjects. We identified five missense changes, four in five HSP patients and one in a CMT2 subject. All mutations, including the one segregating in the CMT2 patient, are localized in the kinesin motor domain except for one, falling within the stalk domain and predicted to generate protein structure destabilization. The results obtained indicate a KIF5A mutation frequency of 8.8% in the Italian HSP population and identify a region of the kinesin protein, the stalk domain, as a novel target for mutation. In addition, the mutation found in the CMT2 patient strengthens the hypothesis that CMT2 and SPG10 are the extreme phenotypes resulting from mutations in the same gene.

The genetic features of 24 patients affected by familial and sporadic hemiplegic migraine.

Familial hemiplegic migraine (FHM) is the only migraine subtype for which a monogenic mode of inheritance, autosomal dominant has been clearly established. It is genetically heterogeneous and at least three different genes exist (CACNA1A, ATP1A2, and SCN1A), the so-called FHM1, FHM2, and FHM3 genes, respectively. Sporadic hemiplegic migraine (SHM) is a disorder, in which some patients may have their pathophysiology identical to FHM, but others, possibly the majority, may have different pathophysiology, probably related to the mechanisms of typical migraine with aura. In our study, we have screened the DNA of 24 patients affected by FHM and SHM. Only in three patients, 2 sporadic and 1 familial cases, we have described genetic mutations, all of them in the ATP1A2 gene. In our opinion, these results demonstrate a more frequent involvement of the ATP1A2 gene not only in the sporadic form, but probably also in the Italian FHM patients without permanent cerebellar signs. Moreover, the absence of CACNA1A, ATP1A2 and SCN1A mutations in the other 12 familial cases suggests the involvement of still unknown genes.

Impact of gavage dosing procedure and gastric content on adverse respiratory effects and mortality in rat toxicity studies.

Unscheduled mortality preceded by adverse respiratory clinical signs in rats dosed by oral gavage may not only be caused by technical gavage error or systemic toxicity but may also be caused by gastro-esophageal reflux and subsequent aspiration of high concentrations of drug formulation. In a 3 week oral gavage rat toxicity study for an early drug development compound, preterminal deaths (approximately 20% of animals) at high doses (≥1000 mg kg(-1) ) and concentrations (≥60 mg ml(-1) ) were preceded by recurrent dyspnea, rales or excessive salivation, without evidence of accidental intrapulmonary gavage error. Histological evaluation revealed extensive necrosis and inflammatory changes in the upper respiratory tract, especially in the nasal turbinates and/or nasopharynx. The presence of food particles in inflammatory exudates suggested a retrograde aspiration of stomach content with test formulation via the nasopharyngeal duct into the posterior region of the nose. In contrast, no mortality or adverse respiratory effects were observed in rats following 2 week intravenous administration at comparable exposures or oral gavage administration at lower concentrations (≤20 mg ml(-1) ). In a pharmacology study, the compound caused a dose-dependent increase in gastric content (partly due to inhibition of gastric emptying), providing a pharmacological basis for the suspected gavage-mediated gastroesophageal reflux. Reducing the dose volume and dosing fasted animals substantially reduced or eliminated the respiratory effects and mortality at the high test article concentrations, demonstrating that the adverse effects are related to the gavage method.

The GST domain of GDAP1 is a frequent target of mutations in the dominant form of axonal Charcot Marie Tooth type 2K.

BACKGROUND: Mutations in GDAP1 associate with demyelinating (CMT4A) and axonal (CMT2K) forms of CMT. While CMT4A shows recessive inheritance, CMT2K can present with either recessive (AR-CMT2K) or dominant segregation pattern (AD-CMT2K), the latter being characterised by milder phenotypes and later onset. The majority of the GDAP1 mutations are associated with CMT4A and AR-CMT2K, with only four heterozygous mutations identified in AD-CMT2K. METHODS: We screened GDAP1 gene in a series of 43 index patients, 39 with CMT2 and 4 with intermediate CMT, with sporadic and familial occurrence of the disease. RESULTS: Three novel mutations were identified in three families with dominant segregation of the disease: two missense changes, p.Arg226Ser and p.Ser34Cys, affecting the GST domain of the GDAP1 protein and a novel deletion (c.23delAG) leading to early truncation of the protein upstream the GST domain. Wide variability in clinical presentation is shared by all three families mostly in terms of age at onset and disease severity. A rare variant p.Gly269Arg, located within the GST domain, apparently acts as phenotype modulator in the family carrying the deletion. CONCLUSION: The results obtained reveal a GDAP1 mutation frequency of 27% in the dominant families analysed, a figure still unreported for this gene, thus suggesting that GDAP1 involvement in dominant CMT2 might be higher than expected.

Novel splice-site mutations and a large intragenic deletion in PLA2G6 associated with a severe and rapidly progressive form of infantile neuroaxonal dystrophy.

Infantile neuroaxonal dystrophy, INAD, is a severe progressive psychomotor disorder with infantile onset and characterized by the presence of axonal spheroids throughout the central and peripheral nervous systems. A subset of INAD patients shows also brain iron accumulation which represents instead the distinctive feature of the idiopathic neurodegeneration with brain iron accumulation, NBIA. These diseases share the same causative gene, PLA2G6, encoding iPLA2-VIA, a calcium-independent phospholipase. Mutations that lead to a complete absence of protein are associated with a severe INAD profile, while compound heterozygous mutations with possibly a residual protein activity are instead associated with the less severe NBIA phenotype. Here we describe two INAD patients both with an unusually rapid disease progression and a peculiar neuroradiological presentation in one of them. Compound heterozygosity for a large intragenic deletion and a nonsense mutation was found in one of them while the other is carrying two novel splice-site mutations. Breakpoint-sequence analysis suggests a non-allelic-homologous-recombination (NAHR) event, probably underlying the rearrangement. These findings, while supporting the genotype-phenotype correlation already observed in INAD patients, provide the first sequence characterization of a genomic rearrangement in PLA2G6 gene, thus orienting the search for missing mutant alleles in PLA2G6 related diseases.

Assessment of the genotoxic and carcinogenic risks of p-nitrophenol when it is present as an impurity in a drug product.

According to the 2008 US FDA (draft) and 2006 EMEA guidance documents for genotoxic impurities, an impurity that is positive in an in vitro genotoxicity study, in the absence of in vivo genotoxicity or carcinogenicity data, should be treated as genotoxic and typically controlled to 1.5 microg/day for chronic use. For p-nitrophenol (PNP), existing study results (i.e., positive in vitro clastogenicity in mammalian cells, no information on its in vivo genotoxicity, and negative with respect to carcinogenicity in a dermal mouse study with no confirmation of systemic exposure) indicated that it should be considered genotoxic and exposure as a drug impurity limited. Therefore, to more completely characterize the genotoxic potential of PNP (consistent with the guidance documents), in vivo mouse micronucleus and dermal pharmacokinetic bridging studies were conducted. In the micronucleus study, PNP was negative, demonstrating that the reported in vitro clastogenicity is not present in vivo. In the pharmacokinetic study, PNP was well absorbed dermally, validating the negative dermal carcinogenicity assessment. These results indicate that PNP should be considered a non-genotoxic impurity and, as a drug impurity, a threshold limit of 4 mg/day would be set (per ICH Q3C). This threshold limit is higher than the EPA reference dose (listed in the 2006 Edition of the Drinking Water Standards and Health Advisories), so if present at such levels, the specification limits for PNP should be determined on a case-by-case basis, based on risk-benefit.

Amino acid changes in the amino terminus of the Na,K-adenosine triphosphatase alpha-2 subunit associated to familial and sporadic hemiplegic migraine.

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura inherited with an autosomal dominant pattern. Here, we report the genetic analysis of four families and one sporadic case with hemiplegic migraine (HM) in whom we searched for mutations in the three genes associated with the disease CACNA1A, ATP1A2 and SCN1A. Two novel amino acid changes p.Arg65Trp and p.Tyr9Asn, in the Na,K-adenosine triphosphatase (ATPase) alpha-2 subunit encoded by the ATP1A2 gene, were found in one FHM family and in the sporadic case, respectively. These mutations are peculiar for their location in the extreme N-terminus, an uncommon mutation target in this protein. Low frequency of migraine attacks in all our mutant patients with low complexity of the associated aura symptoms in the sporadic case is also observed. Besides the two novel mutations, the data here reported confirm the involvement of ATP1A2 gene in the sporadic form of HM, while the negative results on the other families tested for all genes known in HM strengthen the hypothesis of the existence of at least another locus involved in FHM.

Metabolism of a novel phosphodiesterase-IV inhibitor (V11294) by human hepatic cytochrome P450 forms.

1. The metabolism of a novel phosphodiesterase-IV inhibitor (V11294) was studied in human liver microsomal and cytosol preparations and in cDNA-expressed human hepatic CYP forms. 2. Human liver microsomes, but not cytosol, catalysed the NADPH-dependent metabolism of V11294 to V10331 (formed by hydroxylation of the cyclopentyl ring), V10332 (N-desethyl V11294) and V11689 (formed by hydroxylation of the isopropyl side chain). In addition, smaller amounts of a secondary metabolite V11690 (which can be formed from either V10332 or V11689) were also produced. 3. Kinetic analysis of V11294 metabolism to V10331, V10332 and V11689 in two preparations of pooled human liver microsomes revealed average K(m) = 2.5, 8.1 and 3.9 micro M, respectively. 4. The metabolism of V11294 was determined with a characterized bank of 16 individual human liver microsomal preparations employing a V11294 substrate concentration of 8 micro M (i.e. approximately the K(m) for V10332 formation and around twice the K(m) for V10331 and V11689 formation). Good correlations (r(2) = 0.570-0.903) were observed between V10331, V10332 and V11689 formation and markers of CYP3A forms. In contrast, poorer correlations (r(2) = 0.0002-0.428) were observed with markers of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 5. Using human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP forms, V11294 (8 micro M) was metabolized by cDNA-expressed CYP3A4 to V10331, V10332 and V11689, with lower amounts of V11690 also being formed. Lower rates of V11294 metabolism to some V11294 metabolites were also observed with cDNA-expressed CYP2C9, CYP2C19 and CYP2D6, whereas only very low or undetectable rates of V11294 metabolism were observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8 and CYP2E1. 6. The metabolism of V11294 (8 micro M) to V10331, V10332 and V11689 was markedly inhibited by the CYP3A mechanism-based inhibitor troleandomycin. In contrast, V11294 metabolism was not significantly affected by inhibitors of CYP1A2, CYP2C9, CYP2D6 and CYP2E1 or by the CYP2C19 substrate S-mephenytoin. 7. In summary, by correlation analysis, chemical inhibition studies and the use of cDNA-expressed CYPs, V11294 metabolism in human liver to V10331, V10332 and V11689 appears to be primarily catalysed by CYP3A forms.

Identification of cytochrome P-450 isoforms responsible for cis-tramadol metabolism in human liver microsomes.

The metabolism of cis-tramadol has been studied in human liver microsomes and in cDNA-expressed human cytochrome P-450 (CYP) isoforms. Human liver microsomes catalyzed the NADPH-dependent metabolism of tramadol to the two primary tramadol metabolites, namely, O-desmethyl-tramadol (metabolite M1) and N-desmethyl-tramadol (metabolite M2). In addition, tramadol was also metabolized to two minor secondary metabolites (each comprising < or =3.0% of total tramadol metabolism), namely, N,N-didesmethyl-tramadol (metabolite M3) and N,O-didesmethyl-tramadol (metabolite M5). Kinetic analysis revealed that multiple CYP enzymes were involved in the metabolism of tramadol to both M1 and M2. For the high-affinity enzymes involved in M1 and M2 formation, K(m) values were 116 and 1021 microM, respectively. Subsequent reaction phenotyping studies were performed with a tramadol substrate concentration of 250 microM. In studies with characterized human liver microsomal preparations, good correlations were observed between tramadol metabolism to M1 and M2 and enzymatic markers of CYP2D6 and CYP2B6, respectively. Tramadol was metabolized to M1 by cDNA-expressed CYP2D6 and to M2 by CYP2B6 and CYP3A4. Tramadol metabolism in human liver microsomes to M1 and M2 was markedly inhibited by the CYP2D6 inhibitor quinidine and the CYP3A4 inhibitor troleandomycin, respectively. In summary, this study demonstrates that cis-tramadol can be metabolized to tramadol metabolites M1, M2, M3, and M5 in human liver microsomal preparations. By kinetic analysis and the results of the reaction phenotyping studies, tramadol metabolism in human liver is catalyzed by multiple CYP isoforms. Hepatic CYP2D6 appears to be primarily responsible for M1 formation, whereas M2 formation is catalyzed by CYP2B6 and CYP3A4.