Identification and disruption of bacteria associated with sheep scab mites-novel means of control?

Psoroptes ovis mites, which cause psoroptic mange (sheep scab), were investigated to identify potential bacterial targets for endosymbiont control of sheep scab. In addition, transmission of bacteria to the sheep skin was investigated through the characterisation of bacteria present in P. ovis faecal trails and on the fleece environment by internal transcribed spacer (ITS) sequencing. A diverse range of bacteria was identified in addition to a potential endosymbiont candidate, Comamonas sp, which was detected in P. ovis by both ITS PCR and endosymbiont-specific PCR. Disruption of these bacteria within P. ovis, through the use of antibiotics, was explored; with significant reduction in mean mite survival when administered antibiotic diets compared with controls (LR4 = 23.12, P < 0.001). The antibiotic treatments also significantly affected the bacterial density (CFU/mite) within P. ovis, indicating that mite survival may be linked to the bacterial communities that they harbour. Although antibiotics are not suitable for practical application, these results suggest disrupting bacteria associated with P. ovis should be further investigated for novel control.

[Indications of intraoperative ultrasound in head and neck surgery]. / Indikationen für den intraoperativen Ultraschall bei Eingriffen im Kopf-Hals-Bereich.

BACKGROUND: A possible use of B-scan sonography arises from the difficulty in transferring information by means of imaging to the intraoperative situation, which is now possible with navigation systems in complicated surgical procedures in the field of otolaryngology. A solution to this problem offers the intraoperative use of ultrasonography for orientation in soft tissue surgery. PATIENTS AND METHOD: A prospective study involving 115 patients in total entailed scanning with a small part linear and fingertip probe with either 10 and 7.5 MHZ. An ultrasound endoscope featuring a 7.5 MHZ convex probe was used to image endolarygeal processes. RESULTS: Indications included panendoscopies, parotidectomies, submandibulectomies, lymph node exstirpations and abscess incisions. The colour doppler sonography was used in reconstructive surgery involving microvascular transplants. The display of soft tissue tumours provided information about tumour size as well as demarcation or infiltration of neighbouring structures. The fingertip probe and the ultrasound endoscopy served to evaluate areas that were morphologically difficult to access. After clamping the radial artery when harvesting the forearm flap, a sufficient perfusion of the thumb and later the sufficiency of the vascular anastomosis could be verified. CONCLUSION: The intraoperative use of sonography is an inexpensive non-invasive procedure that can be performed by the surgeon himself and allows quick and reliable orientation during difficult operations.

Whole genome association study identifies polymorphisms associated with QT prolongation during iloperidone treatment of schizophrenia.

Administration of certain drugs (for example, antiarrhythmics, antihistamines, antibiotics, antipsychotics) may occasionally affect myocardial repolarization and cause prolongation of the QT interval. We performed a whole genome association study of drug-induced QT prolongation after 14 days of treatment in a phase 3 clinical trial evaluating the efficacy, safety and tolerability of a novel atypical antipsychotic, iloperidone, in patients with schizophrenia. We identified DNA polymorphisms associated with QT prolongation in six loci, including the CERKL and SLCO3A1 genes. Each single nucleotide polymorphism (SNP) defined two genotype groups associated with a low mean QT change (ranging from -0.69 to 5.67 ms depending on the SNP) or a higher mean QT prolongation (ranging from 14.16 to 17.81 ms). The CERKL protein is thought to be part of the ceramide pathway, which regulates currents conducted by various potassium channels, including the hERG channel. It is well established that inhibition of the hERG channel can prolong the QT interval. SLCO3A1 is thought to play a role in the translocation of prostaglandins, which have known cardioprotective properties, including the prevention of torsades de pointes. Our findings also point to genes involved in myocardial infarction (PALLD), cardiac structure and function (BRUNOL4) and cardiac development (NRG3). Results of this pharmacogenomic study provide new insight into the clinical response to iloperidone, developed with the goal of directing therapy to those patients with the optimal benefit/risk ratio.

Association of the NPAS3 gene and five other loci with response to the antipsychotic iloperidone identified in a whole genome association study.

A whole genome association study was performed in a phase 3 clinical trial conducted to evaluate a novel antipsychotic, iloperidone, administered to treat patients with schizophrenia. Genotypes of 407 patients were analyzed for 334,563 single nucleotide polymorphisms (SNPs). SNPs associated with iloperidone efficacy were identified within the neuronal PAS domain protein 3 gene (NPAS3), close to a translocation breakpoint site previously observed in a family with schizophrenia. Five other loci were identified that include the XK, Kell blood group complex subunit-related family, member 4 gene (XKR4), the tenascin-R gene (TNR), the glutamate receptor, inotropic, AMPA 4 gene (GRIA4), the glial cell line-derived neurotrophic factor receptor-alpha2 gene (GFRA2), and the NUDT9P1 pseudogene located in the chromosomal region of the serotonin receptor 7 gene (HTR7). The study of these polymorphisms and genes may lead to a better understanding of the etiology of schizophrenia and of its treatment. These results provide new insight into response to iloperidone, developed with the ultimate goal of directing therapy to patients with the highest benefit-to-risk ratio.

Benchtop NMR for Online Reaction Monitoring of the Biocatalytic Synthesis of Aromatic Amino Alcohols.

Online analytics provides insights into the progress of an ongoing reaction without the need for extensive sampling and offline analysis. In this study, we investigated benchtop NMR as an online reaction monitoring tool for complex enzyme cascade reactions. Online NMR was used to monitor a two-step cascade beginning with an aromatic aldehyde and leading to an aromatic amino alcohol as the final product, applying two different enzymes and a variety of co-substrates and intermediates. Benchtop NMR enabled the concentration of the reaction components to be detected in buffered systems in the single-digit mM range without using deuterated solvent. The concentrations determined via NMR were correlated with offline samples analyzed via uHPLC and displayed a good correlation between the two methods. In summary, benchtop NMR proved to be a sensitive, selective and reliable method for online reaction monitoring in (multi-step) biosynthesis. In future, online analytic systems such as the benchtop NMR devices described might not only enable direct monitoring of the reaction, but may also form the basis for self-regulation in biocatalytic reactions.

Self-reported falls and fall-related injuries among persons aged>or=65 years--United States, 2006.

PROBLEM: In 2005, 15,802 persons aged>or=65 years died from fall injuries. How many older adults seek outpatient treatment for minor or moderate fall injuries is unknown. METHOD: To estimate the percentage of older adults who fell during the preceding three months, the Centers for Disease Control and Prevention (CDC) analyzed data from two questions about falls included in the 2006 Behavioral Risk Factor Surveillance System (BRFSS) survey. RESULTS: Approximately 5.8 million (15.9%) persons aged>or=65 years reported falling at least once during the preceding three months, and 1.8 million (31.3%) of those who fell sustained an injury that resulted in a doctor visit or restricted activity for at least one day. DISCUSSION: This report presents the first national estimates of the number and proportion of persons reporting fall-related injuries associated with either doctor visits or restricted activity. SUMMARY: The prevalence of falls reinforces the need for broader use of scientifically proven fall-prevention interventions. IMPACT ON INDUSTRY: Falls and fall-related injuries represent an enormous burden to individuals, society, and to our health care system. Because the U.S. population is aging, this problem will increase unless we take preventive action by broadly implementing evidence-based fall prevention programs. Such programs could appreciably decrease the incidence and health care costs of fall injuries, as well as greatly improve the quality of life for older adults.

Clinical feasibility test on a minimally invasive laser therapy system in microsurgery of nerves.

The clinical feasibility test described here evaluates the basis for a laser therapy system that enables tumour tissue to be separated from nerves in a minimally invasive manner. It was first investigated whether, using an Er:YAG laser, laser-induced nerve (specifically, facial nerve) responses in the rabbit in vivo can be reliably detected with the hitherto standard monitoring techniques. Peripherally recordable neuromuscular signals (i.e. compound action potentials, CAPs) were used to monitor nerve function and to establish a feedback loop. The first occurrence of laser-evoked CAPs was taken as the criterion for deciding when to switch off the laser. When drawing up criteria governing the control and termination of the laser application, the priority was the maintenance of nerve function. Five needle-electrode arrays specially developed for this purpose, each with a miniature preamplifier, were then placed into the facial musculature instead of single-needle electrodes. The system was tested in vivo under realistic surgical conditions (i.e. facial-nerve surgery in the rabbit). This modified multi-channel electromyography (EMG) system enabled laser-evoked CAPs to be detected that have amplitudes 10 times smaller than those picked up by commercially available systems. This optimization, and the connection of the neuromuscular unit with the Er:YAG laser via the electrode array to create a feedback loop, were designed to make it possible to maintain online control of the laser ablation process in the vicinity of neuronal tissue, thus ensuring that tissue excision is both reliable and does not affect function. Our results open up new possibilities in minimally invasive surgery near neural structures.

The effects of terbium on the accumulation of cisplatin in human ovarian cancer cells.

In this investigation, we report a relationship between the terbium (Tb3+) binding protein and the accumulation of cisplatin in human ovarian cancer cells. The number of Tb3+ binding sites in cisplatin-resistant C13+ cells is significantly greater by 79% than those in cisplatin-sensitive 2008 cells. Exposure to Tb3+ also increased the cellular accumulation of cisplatin. The accumulation of cisplatin as a function of the Tb3+ concentration in the C13+ cells (0.85%/microM Tb3+) was significantly greater than the accumulation of cisplatin in 2008 cells with respect to Tb3+ (0.46%/microM Tb3+). The number of Tb3+ binding sites in revertant RH4 cells was similar to that in 2008 cells. The RH4 cells were less sensitive to the stimulatory effects of Tb3+ than the C13+ cells. Our results show that the Tb3+ binding protein correlates with cisplatin resistance, and the receptor binding of Tb3+ increases the accumulation of cisplatin in cisplatin-resistant cells.

Atypical pulmonary and neurologic complications of amiodarone in the same patient. Report of a case and review of the literature.

Atypical manifestations of pulmonary toxicity and previously unreported autonomic nervous system dysfunction complicating amiodarone therapy were observed in a patient being treated for sustained ventricular tachycardia. Pulmonary and hepatic nodules on computed tomographic scan masquerading as metastatic carcinoma were initially noted. Focal infiltrates and a large left pleural effusion mimicking infection, malignant neoplasm, or collagen vascular disease became manifest at a later stage. Autonomic dysfunction presented as incapacitating orthostatic hypotension and persisted for six weeks after amiodarone withdrawal. The pleuropulmonary toxic effects were reversible on discontinuation of amiodarone therapy, and resolution was hastened by short-course steroid treatment.

Free thyroxine concentrations by equilibrium dialysis and chemiluminescent immunoassays in 13 hypothyroid dogs positive for thyroglobulin antibody.

OBJECTIVE: To determine if concentrations of free thyroxine (FT4) measured by semi-automated chemiluminescent immunoassay (CLIA) correspond to FT4 determined by equilibrium dialysis (ED) in hypothyroid dogs positive for thyroglobulin antibody (TGA). ANIMALS: Thirteen TGA-positive dogs classified as hypothyroid based on subnormal FT4 concentrations by ED. METHODS: Qualitative assessment of canine TGA was performed using an enzyme-linked immunosorbent assay. Serum total thyroxine and total triiodothyronine concentrations were measured by radioimmunoassay. Serum FT4 concentration was determined by ED, and also by semi-automated CLIA for human FT4 (FT4h) and veterinary FT4 (FT4v). Canine thyroid stimulating hormone concentration was measured by semi-automated CLIA. RESULTS: Each dog's comprehensive thyroid profile supported a diagnosis of hypothyroidism. For detection of hypothyroidism, sensitivities of CLIA for FT4h and FT4v were 62% (95% CI, 32-85%) and 75% (95% CI, 36-96%), respectively, compared to FT4 by ED. Five of 13 (38%) dogs had FT4h and 2 of 8 (25%) dogs had FT4v concentrations by CLIA that were increased or within the reference range. Percentage of false-negative test results for FT4 by CLIA compared to ED was significantly (P < .0001 for FT4h and P < .001for FT4v) higher than the hypothesized false-negative rate of 0%. CONCLUSIONS AND CLINICAL IMPORTANCE: Caution should be exercised in screening dogs for hypothyroidism using FT4 measured by CLIA alone. Some (25-38%) TGA-positive hypothyroid dogs had FT4 concentrations determined by CLIA that did not support a diagnosis of hypothyroidism.

Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice.

Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.

Osteoblastic response to the defective matrix in the osteogenesis imperfecta murine (oim) mouse.

This work examines the cellular pathophysiology associated with the weakened bone matrix found in a murine model of osteogenesis imperfecta murine (oim). Histomorphometric analysis of oim/oim bone showed significantly diminished bone mass, and the osteoblast and osteoclast histomorphometric parameters were increased in the oim/oim mice, compared with wild-type (+/+) mice. To assess osteoblast activity, a rat Col1a1 promoter linked to the chloramphenicol acetyltransferase reporter transgene was bred into the oim model. At 8 d and 1 month of age, no difference in transgene activity between oim and control mice was observed. However, at 3 months of age, chloramphenicol acetyl transferase activity was elevated in oim/oim;Tg/Tg, compared with +/+;Tg/Tg and oim/+;Tg/Tg. High levels of urinary pyridinoline crosslinks in the oim/oim;Tg/Tg mice were present at all ages, reflecting continuing high bone resorption. Our data portray a state of ineffective osteogenesis in which the mutant mouse never accumulates a normal quantity of bone matrix. However, it is only after the completion of the rapid growth phase that the high activity of the oim/oim osteoblast can compensate for the high rate of bone resorption. This relationship between bone formation and resorption may explain why the severity of osteogenesis imperfecta decreases after puberty is completed. The ability to quantify high bone turnover and advantages of using a transgene that reflects osteoblast lineage activity make this a useful model for studying interventions designed to improve the bone strength in osteogenesis imperfecta.

Differential regulation of COL2A1 expression in developing and mature chondrocytes.

To investigate the regulation of type II collagen gene expression in cells undergoing chondrogenic differentiation, we have employed a 5-kbp genomic fragment of the human type II collagen gene which contains 1.8kbp of upstream sequences, the transcription start site, the first exon and 3 kbp of intronic sequences, fused to either lac Z or chloramphenicol acetyl transferase-reporter gene. Transient expression studies revealed a parallel increase in transgene activity and endogenous type II collagen mRNA levels during the onset of the cartilage differentiation of limb mesenchymal cells in high-density micromass cultures. At later periods in culture, however, the transgene activity declines, although steady-state levels of type II collagen mRNA are reported to continue to increase (Kosher et al.: J. Cell. Biol. 102: 1151-1156, 1986; Kravis and Upholt. Dev. Biol. 108: 164-172, 1985). In addition, the activity of the transgene is seven-fold higher at the onset of chondrogenic differentiation in micromass cultures that in well differentiated sternal chondrocytes, although similar levels of type II collagen transcripts are found in these cells. Furthermore, deletions of intronic segments resulted in greater drop in activity of the constructs in differentiating chondrocytes in micromass cultures than in mature sternal chondrocytes. The expression of the construct in transgenic mice is higher at the onset of chondrogenic differentiation and in newly differentiated chondrocytes than in more mature differentiated chondrocytes. Based on these observations, it appears that the mechanisms involved in the regulation of the type II collagen gene at the onset of chondrocyte differentiation are different from those resulting in the maintenance of its expression in fully differentiated chondrocytes.

A novel method for DEAE-dextran mediated transfection of adherent primary cultured human macrophages.

Primary cultured human macrophages are difficult to transfect. We have developed a DEAE-dextran DNA transfection method that mediates the reproducible transfection of primary cultured adherent human macrophages. Three factors essential for successful transfection were identified: DEAE-dextran concentration, the quantity of DNA per transfection and the incubation time of the macrophages with the transfection medium. Maximum levels of luciferase expression were attained within 24 h and maintained for at least 56 h after transfection. While serum in the transfection medium attenuated transfection, the treatment of the macrophages with chloroquine, DMSO, or glycerol did not enhance transfection within this system. A CMV enhancer/promoter mediated substantially greater luciferase expression in the macrophages than either HIV or RSV LTRs. DEAE-dextran facilitated superior transfection compared to either cationic liposome and calcium phosphate methods, and was more practical compared to electroporation for multiple transfections. This transfection protocol provides a simple, inexpensive, reproducible method for the evaluation of gene expression in primary cultured adherent human macrophages.

Binding characteristics of kappa opioids in rat brain. A comparison of in vitro binding paradigms.

Putative kappa binding was investigated in homogenates of the brain of the rat using [3H]ethylketocylazocine and [3H]diprenorphine under conditions where mu and delta sites were blocked. Under blocked conditions, the binding of [3H]ethylketocyclazocine labelled a single site, as defined by kinetic and equilibrium analysis, which showed high affinity for dynorphin 1-17, U50,488H, and benzomorphan compounds. However, blocked binding of [3H]diprenorphine showed a biphasic dissociation curve, and did not show high affinity for the specific kappa agonists dynorphin 1-17 or U50,488H. It is proposed that blocked [3H]ethylketocyclazocine is the more appropriate paradigm to study putative kappa binding, while blocked [3H]diprenorphine may label additional non-mu/non-delta sites.

Electrocardiographic and electrophysiologic effects of pirmenol in ventricular tachycardia.

Twenty patients with ventricular tachycardia refractory to drug treatment underwent electrophysiologic study with pirmenol. Patients ranged in age from 39 to 84 years (mean 61); the presenting arrhythmia was sustained ventricular tachycardia in 15, nonsustained ventricular tachycardia in 3 and ventricular fibrillation in 2. After discontinuation of all antiarrhythmic drugs (for at least 5 half-lives) and assessment of electrocardiographic and electrophysiologic parameters in the drug-free state, patients underwent comprehensive intracardiac electrophysiologic evaluation with intravenous pirmenol (mean dose 195 +/- 46 mg). Programmed ventricular stimulation began at least 30 minutes after pirmenol infusion was started in each patient. There was significant shortening of sinus cycle length in all patients, from 746 +/- 155 to 683 +/- 107 ms (mean +/- standard deviation). In 7 patients in whom ventricular tachycardia could not be induced after intravenous pirmenol, an oral pirmenol regimen was begun. The dosage was 200 or 250 mg (both 3 times/day) in 2 and 5 patients, respectively. Seven hours after the third dose of oral drug was given, these patients underwent repeat electrophysiologic testing. Intravenous and oral pirmenol significantly prolonged the PR, QT, QTc and JT intervals compared with baseline. Intravenous pirmenol also significantly prolonged the QRS interval compared with baseline. Oral pirmenol significantly prolonged the sinus node recovery time compared with intravenous pirmenol. Intravenous pirmenol significantly increased the HV interval compared with control; oral pirmenol did not demonstrate a significant prolongation of the HV interval, but this is due to the smaller number of patients studied while taking oral drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensory stimulation increases cortical expression of the fragile X mental retardation protein in vivo.

Fragile X syndrome is a common cause of mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), an RNA binding protein whose function remains unclear. Recent in vitro work has demonstrated that the protein is translated near the synapse in an activity dependent manner [33]. We therefore asked whether expression of FMRP might be altered by neuronal activity in vivo. Using immunoblots of different sub-cellular fractions of the rat somatosensory cortex, we show that the levels of FMRP increase significantly following unilateral whisker stimulation, a model of experience dependent plasticity. This increase is greatest between 2 and 8 h after the stimulus and is seen in both a synaptosomal fraction as well as a sub-cellular fraction enriched for polyribosomal complexes. In contrast, detectable levels of FMRP within the somatosensory cortex show either a decrease or no change after a kainic acid induced seizure compared to water treated controls. Our findings demonstrate that FMRP expression levels are modulated in vivo in response to neuronal activity and suggest a role for FMRP in activity dependent plasticity.

Induction of transcription factors in somatosensory cortex after tactile stimulation.

Immediate early response genes have been shown to be inducible in the central nervous system after a variety of stimuli. Induction of these transcription factors in cerebral cortex by a physiological stimulus had not previously been demonstrated. In this study, tactile stimuli induced multiple transcription factors in the somatosensory cortex. Adult male rats were lightly anesthetized with urethane. Tactile stimuli was delivered by a paint brush gently stroking an animals whiskers on one side of its face for a 15 min period. Two h later, the animals were sacrificed. Cortex contralateral to the stimulation was compared with ipsilateral cortex using antibodies raised against immediate early response gene products NGFI-A, NGFI-B, and c-fos. The different transcription factors showed slightly different patterns of response to the tactile stimulus. However, the induction of immunohistochemical staining was most prominent in layer 4 with all antibodies under study. This increase in the number of cell bodies stained was less robust than that seen in the somatosensory cortex after a seizure, and showed more of a predominance in layer 4 cells. These data demonstrate that physiologic stimulation can induce immediate early response genes in cortical cells, and that multiple immediate early response genes react to a stimulus.

Seizures and sensory stimulation result in different patterns of brain derived neurotrophic factor protein expression in the barrel cortex and hippocampus.

Brain-derived neurotrophic factor (BDNF) is important for the development and trophic support of neurons, and may be involved in controlling axonal sprouting and synaptic plasticity. In order to investigate the activity-dependent regulation of the BDNF gene, BDNF expression was examined within the rat somatosensory cortex (SSC) and hippocampus following vibrissae stimulation, kainic acid induced seizure, and pentylenetetrazol (PTZ) induced seizure. The specific goals of this study were to determine the time course and magnitude of BDNF's activity-dependent expression, and to compare the expression patterns of three commonly used neuronal activation paradigms. Our results demonstrate three novel observations. First, the patterns of BDNF protein expression are dependent upon the neuronal stimulation model used. Both unilateral whisker stimulation (a model of experience dependent plasticity) and kainic acid induced seizure were able to increase the levels of BDNF protein within the SSC and hippocampus. In contrast, PTZ induced seizure did not increase BDNF protein levels in either tissue. Second, there is a dissociation between BDNF mRNA and protein levels following PTZ induced seizure. PTZ seizures resulted in strong increases of BDNF mRNA levels without corresponding increases of the protein. Finally, whisker stimulation resulted in an unexpected increase in BDNF mRNA and protein levels within the hippocampus. These results suggest specific types of neuronal activity can regulate gene expression differently. Furthermore, temporal and spatial differences between the expression of BDNF protein and mRNA levels suggest that the BDNF gene is regulated at the level of translation as well as transcription.