Identification of extracellular vesicles and characterization of miRNA expression profiles in human blastocoel fluid.

In this study, for the first time, we demonstrated the presence of microRNAs and extracellular vesicles in human blastocoel fluid. The bioinformatic and comparative analyses identified the biological function of blastocoel fluid microRNAs and suggested a potential role inside the human blastocyst. We found 89 microRNAs, expressed at different levels, able to regulate critical signaling pathways controlling embryo development, such as pluripotency, cell reprogramming, epigenetic modifications, intercellular communication, cell adhesion and cell fate. Blastocoel fluid microRNAs reflect the miRNome of embryonic cells and their presence, associated with the discovery of extracellular vesicles, inside blastocoel fluid, strongly suggests their important role in mediating cell communication among blastocyst cells. Their characterization is important to better understand the earliest stages of embryogenesis and the complex circuits regulating pluripotency. Moreover, blastocoel fluid microRNA profiles could be influenced by blastocyst quality, therefore, microRNAs might be used to assess embryo potential in IVF cycles.

Acute endovascular treatment (< 48 hours) of uncoilable ruptured aneurysms at non-branching sites using silk flow-diverting devices.

A blood blister-like (BBL) or dissecting aneurysm should be carefully considered if located at a non-branching site of the supra-clinoid internal carotid artery (ICA). Several surgical and endovascular treatment methods have been proposed but they all carry a relatively high risk of morbidity and mortality. This study evaluated the effectiveness of a novel Silk flow-diverting device (SFD) placed in the early acute stage. Three patients presenting with acute subarachnoid haemorrhage caused by small blister-like aneurysms of the carotid siphon were treated within 48 hours after admission by placement of SFDs. More than one device was placed to cover the lesion. None of the patients were premedicated and started anti-platelet therapy during the procedure. All aneurysms were successfully occluded. A good outcome was observed in two out of three treated patients. No thromboembolic or haemorrhagic event occurred during or after the procedures, or during follow-up (6-14 months). SFD prevented rebleeding and the use of these devices could be proposed as an option to treat fragile uncoilable BBL aneurysms, even in the early acute phase without anti-platelet premedication. Larger studies and long-terms results are necessary.

Short communication: Correlation of on-site inspection and laboratory milk testing results for Wisconsin grade A dairy farms in 2007 and 2008.

This study examined whether regulatory on-site dairy farm inspection results correlated with reported laboratory somatic cell count (SCC), standard plate count (SPC), and beta-lactam drug residue (DR) results for individual farms. Results were obtained for Wisconsin grade A dairy farms in 2007 and 2008 (>11,000 farms, >1.4 million data points). The proportion of farms failing an on-site inspection ranged from 12% for farms that had never failed an SCC test (>750,000 cells/mL), an SPC test (>100,000 cfu/mL), or a DR test (drug detected) to 55% for farms that had failed at least 1 of each type of test. Conditional probability analysis showed that the probability of a farm failing an on-site farm inspection was higher if the farm had failed a DR test and increased as the proportion of samples failing SCC or SPC or both increased. However, the statistical correlations were weak (R

Unilateral oculomotor nerve palsy: unusual sign of hydrocephalus.

We report an unusual case of hydrocephalus in which unilateral oculomotor nerve paralysis was the predominant sign. Misinterpretation of such an atypical clinical sign may lead to inappropriate therapy. We outline the role of intracranial pressure monitoring as an adjunctive diagnostic tool and we suggest a presumptive mechanism to explain the correlation between enlarged ventricles and 3(rd) nerve dysfunction. A 16-year-old boy presented with a complete left oculomotor nerve palsy associated with imaging findings of dilated ventricles and Dandy-Walker variant cystic malformation. Monitoring of intracranial pressure through a ventricular catheter was undertaken. In the first phase (no cerebrospinal fluid drainage [CSF] drainage) mean intracranial pressure (ICP) values were >0 mmHg. A second phase (with progressively longer CSF draining) further defined the diagnosis. A ventriculo-peritoneal shunt was then placed and the nerve function returned to normal within few days. Third cranial nerve dysfunction as a predominant sign of hydrocephalus is very rare and may raise doubts as to the real significance of the imaging findings of enlarged ventricles. In this ground, ICP monitoring is a safe and helpful diagnostic tool that can afford a more accurate evaluation and proper treatment. The supposed mechanism of 3(rd) nerve dysfunction was bending/stretching of the nerve.

A diagnostic flowchart, including TCD, Xe-CT and angiography, to improve the diagnosis of vasospasm critically affecting cerebral blood flow in patients with subarachnoid haemorrhage, sedated and ventilated.

The aim of this study was to prospectively evaluate a clinical protocol including transcranial doppler (TCD), Xenon-CT (Xe-CT) and angiography, for the detection of vasospasm leading to critical reductions of regional cerebral blood flow (rCBF) in both ventilated and sedated SAH patients, i.e. patients in whom clinical evaluation was not possible. Seventy-six patients were prospectively included in a surveillance protocol for daily TCD vasospasm monitoring. When TCD showed a V(mean) above 120 cm/sec in the middle cerebral artery (MCA), patients underwent Xe-CT study. If rCBF in the MCA was reduced to below 20 ml/100 g/min or if there was a reduction in the rCBF with significant asymmetry between the two MCAs, angiography was performed. Conversely, further Xe-CT and angiography were not obtained unless the TCD V(mean) values reached values above 160 cm/sec. In 35 patients, V(mean) attained values above 120 cm/sec, but only in five of them, rCBF was suggestive of vasospasm, and angiography confirmed the diagnosis in four. The protocol suggests that in sedated and ventilated patients, detection of a critical rCBF reduction due to vasospasm is possible to allow for more specific treatment and to reduce undue medical complications.

Regional cerebral blood flow levels as measured by xenon-CT in vascular territorial low-density areas after subarachnoid hemorrhage are not always ischemic.

INTRODUCTION: The aim of this study was to assess regional cerebral blood flow (rCBV) in areas of CT hypoattenuation appearing in the postoperative period in patients treated for aneurysmal subarachnoid hemorrhage (SAH) using xenon-enhanced CT scanning (Xe-CT). METHODS: We analyzed 15 patients (5 male and 10 female; mean age 49.7+/-12.1 years) with SAH on CT performed on admission to hospital and who showed a low-density area within a well-defined vascular territory on CT scans after clipping or coiling of a saccular aneurysm. All zones of hypoattenuation were larger than 1 cm(2) and showed signs of a mass effect suggesting a subacute phase of evolution. Two aneurysms were detected in two patients. Aneurysms were located in the middle cerebral artery (n=7), in the anterior communicating artery (n=6), in the internal carotid artery (n=3), and in the posterior communicating artery (n=1). Treatments were surgical (n=8), endovascular (n=2) or both (n=1). A total of 36 Xe-CT studies were performed and rCBF values were measured in two different regions of interest (ROI): the low-density area, and an area of normal-appearing brain tissue located symmetrically in the contralateral hemisphere. RESULTS: rCBF levels were significantly lower in the low-density area than in the contralateral normal-appearing area (P<0.01). In the low-density areas, irreversible ischemia (CBF <10 ml/100 g per minute) was present in 11/36 lesions (30.6%), ischemic penumbra (CBF 10-20 ml/100 g per minute) and oligemia (CBF 20-34 ml/100 g per minute) in 8/36 lesions (22.2%), relative hyperemia (CBF 34-55 ml/100 g per minute) in 7/36 lesions (19.4%), and absolute hyperemia (CBF >55 ml/100 g per minute) in 2/36 lesions (5.6%). CONCLUSION: Our study confirmed that rCBF is reduced in new low-density lesions related to specific vascular territories. However, only about one-third of the lesions showed rCBF levels consistent with irreversible ischemia and in a relatively high proportion of lesions, rCBF levels indicated penumbral, oligemic and hyperemic areas.

Residual feed intake of purebred Angus steers: effects on meat quality and palatability.

Relationships between residual feed intake (RFI) and other performance variables were determined using 54 purebred Angus steers. Individual feed intake and BW gain were recorded during a 70-d post-weaning period to calculate RFI. After the 70-d post-weaning test, steers were fed a finishing ration to a similar fat thickness (FT), transported to a commercial facility, and slaughtered. A subsample of carcasses (n = 32) was selected to examine the relationships among RFI, meat quality, and palatability. Steers were categorized into high (> 0.5 SD above the mean; n = 16), medium (mid; +/- 0.5 SD from the mean; n = 21), and low (< 0.5 SD below the mean; n = 17) RFI groups. No differences were detected in ADG, initial BW, and d 71 BW among the high, mid, and low RFI steers. Steers from the high RFI group had a greater DMI (P = 0.004) and feed conversion ratio (FCR; DMI:ADG; P = 0.002) compared with the low RFI steers. Residual feed intake was positively correlated with DMI (r = 0.54; P = 0.003) and FCR (r = 0.42; P = 0.002), but not with initial BW, d 71 BW, d 71 ultrasound FT, initial ultrasound LM area, d 71 ultrasound LM area, or ADG. The FCR was positively correlated with initial BW (r = 0.46; P = 0.0005), d 71 BW (r = 0.34; P = 0.01), and DMI (r = 0.40; P = 0.003) and was negatively correlated with ADG (r = -0.65; P = 0.001). There were no differences among RFI groups for HCW, LM area, FT, KPH, USDA yield grade, marbling score, or quality grade. Reflectance color b* scores of steaks from high RFI steers were greater (P = 0.02) than those from low RFI steers. There was no difference between high and low RFI groups for LM calpastatin activity. Warner-Bratzler shear force and sensory panel tenderness and flavor scores of steaks were similar across RFI groups. Steaks from high RFI steers had lower (P = 0.04) off-flavor scores than those from low RFI steers. Cook loss percentages were greater (P = 0.005) for steaks from low RFI steers than for those from mid RFI steers. These data support current views that RFI is independent of ADG, but is correlated with DMI and FCR. Importantly, the data also support the hypothesis that there is no relationship between RFI and beef quality in purebred Angus steers.

Cerebral blood flow in mean cerebral artery low density areas is not always ischemic in patients with aneurysmal subarachnoid hemorrhage--relationship with neurological outcome.

Aneurysmal subarachnoid hemorrhage (SAH) can be complicated by reduction of regional cerebral blood flow (rCBF) from large conductance vessels leading to focal edema appearing as an area of hypoattenuation on CT. In this study we included 29 patients with SAH due to aneurysmal rupture, having 36 CT low density areas within the middle cerebral artery territory in whom a total of 56 Xenon-CT (Xe-CT) studies were performed. Collectively, we evaluated 70 hypoattenuated areas. rCBF levels were measured in two different regions of interest drawn manually on the CT scan, one in the low density area and the other in a corresponding contralateral area of normal-appearing brain tissue. In the low density area (22.6 +/- 22.7 ml/100 gr/min) rCBF levels were significantly lower than in the contralateral area (32.8 +/- 17.1 7 ml/100 gr/min) (p = 0.0007). In the injured areas deep ischemia (CBF < 6 ml/ 100 g/min) was present in only 25.7% of Xe-CT studies, suggesting that hypodense areas are not always ischemic, whereas in 43.7% of the lesions/Xe-CT studies we found hyperemic values. Patients with a better outcome had hyperemic lesions, suggesting brain tissue recovery in injured areas.

Mutation analysis of two candidate genes for premature ovarian failure, DACH2 and POF1B.

BACKGROUND: Balanced X;autosome translocations interrupting the 'critical region' of the long arm of the human X chromosome are often associated with premature ovarian failure (POF). However, the mechanisms leading to X-linked ovarian dysfunction are largely unknown, as the majority of the X chromosome breakpoints have been mapped to gene-free genomic regions. A few genes have been found to be interrupted, but their role has never been clarified. METHODS AND RESULTS: By fine mapping of the X chromosome breakpoint of an X;autosome balanced translocation, we identified a new interrupted gene, POF1B. We performed a mutation analysis of POF1B and of another gene previously identified, DACH2, localized approximately 700 kb distal in Xq21, in a cohort of >200 Italian POF patients. Rare mutations were found in patients in both genes. CONCLUSIONS: Our findings could not demonstrate any involvement of POF1B, but suggest that rare mutations in the DACH2 gene may have a role in the POF phenotype.

Comparison of commercially available ELISA kits with human sera-based detection methods for peanut allergens in foods.

Undeclared peanut allergens as contaminants in foodstuffs represent a major health problem for sensitized persons. Various immunochemical techniques are employed to detect and quantify peanut allergens. There is an urgent need to compare and standardize those test systems to enable comparable allergen analyses of foodstuffs, comparable studies, and consequent and consistent measures against the presence of hidden peanut allergens. The present study compared commercially available peanut ELISA kits with human sera-based immunoassay techniques (dot blotting and Western blotting), enabling semiquantitative and quantitative detection, and identification of peanut contaminants in foodstuffs. Additionally, the effect of conventional roasting conditions on the detection and quantification of peanut with the selected methods was investigated.

Safety and immunogenicity of a recombinant hemagglutinin vaccine for H5 influenza in humans.

Recent outbreaks of avian influenza in humans have demonstrated the need for vaccines for influenza viruses with pandemic potential. Recombinant hemagglutinins are an attractive option for such vaccines because they do not require handling potentially highly pathogenic influenza viruses for vaccine production. In order to evaluate the immunogenicity, optimum dosing and timing of administration of a recombinant baculovirus-expressed H5 HA (rH5) in humans, 147 healthy adults were assigned randomly to receive intramuscular rH5 as two doses of 25, 45 or 90 microg each, one dose of 90 microg followed by a dose of 10 microg, or two doses of placebo, at intervals between doses of 21, 28 or 42 days. All doses of rH5 were well tolerated. The rH5 vaccine was modestly immunogenic at high dose. Neutralizing antibody responses to a titer of 1:80 or greater were seen in 23% (14/60) of individuals after a single dose of 90 microg, and in 52% (15/29) after two doses of 90 microg. Varying intervals between doses from 21 to 42 days had no significant effect on antibody responses to vaccination. These results suggest that baculovirus-expressed H5 HA can induce functional antibody in individuals who have not had prior exposure to H5 viruses, but that further studies to improve the immunogenicity of the vaccine are needed.

Species-differences in disposition and reductive metabolism of methoxymorpholinodoxorubicin (PNU 152243), a new potential anticancer agent.

Plasma pharmacokinetics, excretion balance and urinary metabolites of methoxymorpholino doxorubicin (MMDX) were investigated in male and female rats and in female dogs after i.v. administration of the(14)C-labelled drug. The mean total recovery of radioactivity in 96 h (urine plus faeces) was approximately 74 and 60% dose in male and female rats, respectively, while in female dogs approximately 72% dose was recovered in 336 h. Most of the radioactivity was present in faeces, with the urinary elimination accounting for only 3-4% dose in rats and dogs. These data suggest that biliary excretion is an important route of elimination of MMDX and/or its metabolites in both species. No differences were observed in the urinary metabolic profile of male and female rats. Two main peaks were present in radiochromatograms of urine from rats and dogs, i.e. MMDX and its 13-dihydro metabolite (MMDX-ol), accounting for approximately 25 and 20% of total radioactivity in 0-24-h urine in rats and 30 and 36% in dogs. The MMDX-ol/MMDX ratio in dog urine was higher than that observed in rat urine. No aglycones were detected in the urine samples from either species. In the rat, the plasma concentration-time profile suggested that the disposition of MMDX, MMDX-ol and total radioactivity is not sex-dependent. MMDX was the major species present in the systemic circulation; its AUC (0-96 h) accounted for 70% of total plasma radioactivity with the sum of AUC (MMDX) plus AUC (MMDX-ol) accounting for 77% of total radioactivity. In the dog, the sum of AUC (MMDX) plus AUC (MMDX-ol) amounted to 8% of radioactivity AUC(0-t(z) indicating that an important proportion of other(s) unknown metabolite(s) is present in dog plasma. Plasma levels of MMDX-ol in the rat were approximately 10-fold lower than those of the parent compound, whereas they were three times higher than those of MMDX in the dog. These data show that the reduction of the 13-keto group of MMDX is species-dependent, and occurs preferentially in the dog compared to the rat.

Evaluation of the genetic stability of the temperature-sensitive PB2 gene mutation of the influenza A/Ann Arbor/6/60 cold-adapted vaccine virus.

A single-gene reassortant bearing the PB2 gene of the A/Ann Arbor/6/60 cold-adapted virus in the background of the A/Korea/82 (H3N2) wild-type virus is a temperature-sensitive (ts) virus with an in vitro shutoff temperature of 38 degrees C. A single mutation at amino acid (aa) at 265 (Asp-Ser) of the PB2 protein is responsible for the ts phenotype. This ts single-gene PB2 reassortant virus was serially passaged at elevated temperatures in Madin-Darby canine kidney cells to generate ts+ phenotypic revertant viruses. Four ts+ phenotypically revertant viruses were derived independently, and each possessed a shutoff temperature for replication in vitro of > 40 degrees C. Each of the four phenotypically revertant viruses replicated efficiently in the upper and lower respiratory tracts of mice and hamsters, unlike the PB2 single-gene reassortant virus, confirming that the ts phenotype was responsible for the attenuation of this virus in rodents. Mating the ts+ revertants with wild-type virus yielded ts progeny in high frequency, indicating that the loss of ts phenotype was due to a suppressor mutation which was mapped to the PA gene in each of the four independently derived ts phenotypic revertants. Nucleotide sequence analysis confirmed the absence of new mutations on the PB2 gene and the presence of predicted amino acid changes in the PA proteins of the revertant viruses. These studies suggest that single amino acid changes at aa 245 (Glu-Lys) or 347 (Asp-Asn) of the PA protein can completely suppress the ts and attenuation phenotypes specified by the Asp-Ser mutation at aa 265 of the PB2 protein of the A/Ann Arbor/6/60 cold-adapted virus.

Disposition and urinary metabolic pattern of cabergoline, a potent dopaminergic agonist, in rat, monkey and man.

1. The disposition and urinary metabolic pattern of 14C-cabergoline was studied in rat, monkey and man after oral administration of the labelled drug. 2. In all species radioactivity was mainly excreted in faeces, with urinary excretion accounting for 11, 13 and 22% of the dose in rat, monkey and man, respectively. 3. After oral treatment, biliary excretion of radioactivity in rat accounted for 19% of the dose within 24 h. 4. Unchanged drug in 0-24-h urine samples of rat, monkey and man amounted to 20, 9 and 10% of urinary radioactivity, respectively. In the 24-72-h urine samples of all species the relative percentage of unchanged drug increased compared with that measured in the 0-24-h urine. 5. The main metabolite was the acid derivative (FCE 21589), which in 0-24-h urine samples of rat, monkey and man accounted for 30, 21 and 41% of urinary radioactivity, respectively. 6. Other metabolites identified in urine of all species resulted from hydrolysis of the urea moiety, the loss of the 3-dimethylaminopropyl group and the deallylation of the piperidine nitrogen.

In vivo glucuronidation in rat and humans of 5,6-dihydro-7-(1H-imidazol-1-yl)-naphthalene-2-carboxylic acid, a selective inhibitor of thromboxane synthase.

The metabolites of 5,6-dihydro-7-(1H-imidazol-1-yl)-naphthalene-2-carboxylic acid, FCE 22178, a new thromboxane synthase inhibitor, were investigated in urine of rats and healthy volunteers after a single oral dose of 10 mg/kg and 400 mg, respectively, of the tritium-labeled drug. Cumulative urinary excretion of radioactivity after 4 days amounted to 64.6% and 91.0% of the dose in rat and humans, respectively. Urinary fractions of 0-24 hr, accounting for 61.8% and 79.5% of the dose, were analyzed by radio-HPLC with direct injection. Following incubation with beta-glucuronidase both in the presence and absence of saccharo-1,4-beta-lactone, a specific inhibitor of this enzyme, a metabolite was identified as a glucuronoconjugate of FCE 22178. The recovery of the glucuronide in the rat and man amounted to approximately 30% and almost 100% of urinary radioactivity, respectively. Control incubations showed a complete deglucuronidation in the case of rat urine compared with less than 10% in human urine. Addition of saccharo-1,4-beta-lactone abolished this phenomenon, suggesting the presence of an endogenous beta-glucuronidase in rat urine. Further identification of the only metabolite present in human urine by tandem MS analysis confirmed the structure of the acyl glucuronide of FCE 22178.

Metabolic fate of FCE 22716, a new antihypertensive agent, and of its N-oxide (FCE 24220) in the rat.

[3H]-FCE 22716 and [3H]-FCE 24220 were given both orally and intravenously to the rat. Radioactivity was mainly eliminated by the faecal route after oral administration in both cases. After intravenous administration, renal excretion was twice the faecal one in the case of FCE 22716, whereas for FCE 24220 the two routes were equal. In urine FCE 22716 was eliminated almost completely unchanged after both oral and intravenous administration. FCE 24220 was extensively reduced to FCE 22716 after oral administration, whereas after intravenous treatment, this reduction, although important, was not complete.

Comparison of the disposition and of the metabolic pattern of Reboxetine, a new antidepressant, in the rat, dog, monkey and man.

The purpose of this study was to compare the disposition and the metabolic pattern of Reboxetine in several species, including man. [14C]-Reboxetine was given orally to the rat, the dog, the monkey (5 mg/kg) and man (2 and 4 mg/kg). Radioactivity was eliminated both by the renal and faecal route in the rat and the dog, mainly in urine in the monkey and man. Reboxetine was extensively metabolized. A number of urinary metabolites were quantified by radio-HPLC and tentatively identified by comparison with the retention times of reference compounds. Suggested routes of metabolic transformation are: 2-O-dealkylation; hydroxylation of the ethoxyphenoxy ring; oxidation of the morpholine ring; morpholine ring-opening; and combinations of these. Metabolites were partially or completely conjugated with glucuronic acid and/or sulphuric acid.

Absorption, disposition, and urinary metabolism of 14C-rifabutin in rats.

14C-rifabutin was given orally (25 mg/kg) and intravenously (i.v.) (10 mg/kg) to female Sprague-Dawley rats. Radioactivity was eliminated by both the renal and fecal routes, amounting to 44.49 and 43.39% of the dose, respectively, in urine and feces at 96 h after the oral dose and to 47.81 and 40.76% of the dose, respectively, in urine and feces after the i.v. dose. Differences between the two routes of administration were negligible. Tissue distribution of radioactivity after the oral dose was investigated by the combustion technique. At 2 h, the highest concentration of radioactivity was observed in the liver, followed by the lung, abdominal adipose tissue, and spleen, whereas at 72 h, the sequence was abdominal adipose tissue, liver, spleen, bone marrow, and lung. Brain levels of radioactivity were very low. The results of whole-body autoradiography after i.v. administration confirmed the above. Whole-body autoradiography of pregnant rats showed higher concentrations of radioactivity in the uterus than in the placenta and trace levels in the fetuses up to 8 h. Radioactivity was absent in the amniotic fluid. The urinary metabolism was studied by radio-high-pressure liquid chromatography. Rifabutin accounted for 7.4 and 7.2% of the dose in 0- to 48-h urine after oral and i.v. administration, respectively. Metabolites 31-OH rifabutin and 25-O-deacetyl rifabutin amounted to 4.3 and 1.6% of the dose, respectively, after oral administration and to 2.6 and 0.7% of the dose, respectively, after i.v. administration. The remaining urinary radioactivity was mainly due to polar compounds.

Animal pharmacokinetics and interspecies scaling of FCE 22101, a penem antibiotic.

The pharmacokinetics of FCE 22101 were studied in rats, rabbits, Cynomolgus monkeys and dogs after intravenous administration. Pertinent pharmacokinetic parameters were determined according to a two-compartment model and correlated among species as an exponential function of body weight, thereby allowing an estimation of pharmacokinetic parameters corresponding to a 70 kg man. Allometric equations, including data on humans reported in the literature, were also established and used to study similarities and differences in the disposition of FCE 22101 among species. The pharmacokinetic profile of FCE 22101 was consistent with the principles of allometry in all animal species studied (and in man) with the exception of the Cynomolgus monkey, in which clearance of FCE 22101 was slower than expected.