Two new mouse models of Gjb1-associated Charcot-Marie-Tooth disease type 1X.

BACKGROUND: Charcot-Marie-Tooth disease type 1X is caused by mutations in GJB1, which is the second most common gene associated with inherited peripheral neuropathy. The GJB1 gene encodes connexin 32 (CX32), a gap junction protein expressed in myelinating glial cells. The gene is X-linked, and the mutations cause a loss of function. AIMS: A large number of disease-associated variants have been identified, and many result in mistrafficking and mislocalization of the protein. An existing knockout mouse lacking Gjb1 expression provides a valid animal model of CMT1X, but the complete lack of protein may not fully recapitulate the disease mechanisms caused by aberrant CX32 proteins. To better represent the spectrum of human CMT1X-associated mutations, we have generated a new Gjb1 knockin mouse model. METHODS: CRISPR/Cas9 genome editing was used to produce mice carrying the R15Q mutation in Gjb1. In addition, we identified a second allele with an early frame shift mutation in codon 7 (del2). Mice were analyzed using clinically relevant molecular, histological, neurophysiological, and behavioral assays. RESULTS: Both alleles produce protein detectable by immunofluorescence in Schwann cells, with some protein properly localizing to nodes of Ranvier. However, both alleles also result in peripheral neuropathy with thinly myelinated and demyelinated axons, as well as degenerating and regenerating axons, predominantly in distal motor nerves. Nerve conduction velocities were only mildly reduced at later ages and compound muscle action potential amplitudes were not reduced. Levels of neurofilament light chain in plasma were elevated in both alleles. The del2 mice have an onset at ~3 months of age, whereas the R15Q mice had a later onset at 5-6 months of age, suggesting a milder loss of function. Both alleles performed comparably to wild type littermates in accelerating rotarod and grip strength tests of neuromuscular performance. INTERPRETATION: We have generated and characterized two new mouse models of CMT1X that will be useful for future mechanistic and preclinical studies.

Time-resolved relaxation and fragmentation of polycyclic aromatic hydrocarbons investigated in the ultrafast XUV-IR regime.

Polycyclic aromatic hydrocarbons (PAHs) play an important role in interstellar chemistry and are subject to high energy photons that can induce excitation, ionization, and fragmentation. Previous studies have demonstrated electronic relaxation of parent PAH monocations over 10-100 femtoseconds as a result of beyond-Born-Oppenheimer coupling between the electronic and nuclear dynamics. Here, we investigate three PAH molecules: fluorene, phenanthrene, and pyrene, using ultrafast XUV and IR laser pulses. Simultaneous measurements of the ion yields, ion momenta, and electron momenta as a function of laser pulse delay allow a detailed insight into the various molecular processes. We report relaxation times for the electronically excited PAH*, PAH+* and PAH2+* states, and show the time-dependent conversion between fragmentation pathways. Additionally, using recoil-frame covariance analysis between ion images, we demonstrate that the dissociation of the PAH2+ ions favors reaction pathways involving two-body breakup and/or loss of neutral fragments totaling an even number of carbon atoms.

Neural differences in the temporal cascade of reactive and proactive control for bilinguals and monolinguals.

This study explored differences in sustained top-down attentional control (i.e., proactive control) and spontaneous types of control (i.e., reactive control) in bilingual and monolingual speakers. We modified a Color-Word Stroop task to varying levels of conflict and included switching trials in addition to more "traditional" inhibition Stroop conditions. The task was administered during scalp electroencephalography (EEG) to evaluate the temporal course of cognitive control during trials. The behavioral Stroop effect was observed across the whole sample; however, there were no differences in accuracy or response time between the bilingual and monolingual groups. Event-related potentials (ERPs) were calculated for the N200, N450, and conflict Sustained Potential (SP). On the pure-blocked incongruent trials, the bilingual group displayed reduced signal during interference suppression (N450) and increased later signal, as indexed by the conflict SP. On the mixed-block incongruent trials, both the bilinguals and monolinguals displayed increased later signal at the conflict SP. This suggests that proactive control may be a default mode for bilinguals on tasks requiring inhibition. In the switching trials, that place high demands on the executive control component of shifting, the language groups did not differ. Overall, these results suggest processing differences between bilinguals and monolinguals extend beyond early response inhibition processes. Greater integration of proactive and reactive control may be needed to sort conflicting language environments for bilinguals, which may be transferring to domain-general mechanisms.

Towards area wide management of insect vectored viruses of tomatoes in the Bowen district.

The Bowen region of Northern Queensland is an important winter production area for tomatoes in Australia. There are three economically important viruses in the region that affect tomato, Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Potato leafroll virus (PLRV), which are vectored by whiteflies, thrips and aphids, respectively. An area wide management approach is required to lower the primary inoculum throughout the district. To this end, we undertook investigations into the virus incidence and alternative hosts for the virus and vectors in different cropping regions throughout the district, as well as local management options such as insecticide application and possible non-host cover crops for the wet-season break in production. The initial incidence of Potato leafroll virus was very high, most probably due to abnormal weather patterns for the district, and has ceased to be a problem. Tomato yellow leaf curl virus is a continual problem even at the beginning of the season, indicating large reservoir host(s) in the environment. Only four alternative hosts have been identified: Stachytarpheta jamaicensis (TSWV), Solanum americanum (PLRV and TYLCV) Trianthema portulacastrum (TYLCV), and Amaranthus viridis(TLYCV). Different insecticide and application options were trialled for protection against Tomato yellow leaf curl virus, with the best possible option yielding marketable fruit more than ninety percent of a resistant hybrid. A trial of yield vs time of infection of TYLCV found that whitefly exclusion for 6 weeks post-transplant yielded an average increase of nearly three kilograms of marketable fruit per plant. A number of pulse crops have been confirmed as non-hosts of tomato yellow leaf curl for use as cover crops in the wet-season break. Most of the production has moved to dual resistant TYLCV/TSWV hybrids, though an area wide management program still needs to be established to reduce the primary inoculum throughout the district, giving growers more varietal options, especially early in the season.

Incidence of sexually transmitted infections in HIV-infected and HIV-uninfected adolescents in the USA.

Little is known about the incidence of bacterial sexually transmitted infections (STIs) among HIV-infected versus HIV-uninfected adolescents. This secondary analysis of a national, multisite study included adolescents aged 12-18 years who were behaviourally HIV-infected (n = 346) or HIV-uninfected but at-risk (n = 182). Incidence rates of bacterial STIs (gonorrhoea, chlamydia [CT] and trichomonas [TV; women]) were calculated using Poisson modelling. Factors associated with incident STIs were explored using Cox proportional hazards modelling. HIV-infected versus HIV-uninfected women had higher TV incidence (1.3 versus 0.6/100 person-months; P = 0.002). HIV-uninfected versus HIV-infected women had higher CT incidence (1.6 versus 1.1/100 person-months; P = 0.04). Among women, demographic, behavioural and HIV-related factors were associated with incident STIs. Among men, there were no differences in incident STIs. In this first analysis comparing STI incidence between HIV-infected and HIV-uninfected adolescents, bacterial STI incidence among women significantly differed by HIV status, and factors associated with incident STIs varied by STI and HIV status.

Adolescents' agreement to test for HIV when different testing methods are offered.

Offering rapid HIV testing improves rates of testing in adults, but little is known about whether offering adolescents a choice of testing methods increases rates of testing. The aims of the study were to determine rates of HIV testing in adolescents when different testing methods were offered and explore factors associated with agreement to be tested for HIV. Participants (n= 200, sexually experienced 13-22 year olds) were recruited from an urban adolescent clinic, completed a 99-item theory-based survey and were offered their choice of venipuncture, rapid fingerstick or rapid oral fluid HIV testing. Approximately half (49.5%) agreed to HIV testing. Male gender, parental completion of high school, intention to test for HIV if offered by clinician and higher perceived likelihood of current HIV infection were independently associated with agreement to test. Combining new strategies, such as opt-out testing, with routine testing may be needed to improve rates of adolescent HIV testing.

Formation and relaxation of RbHe exciplexes on He nanodroplets studied by femtosecond pump and picosecond probe spectroscopy.

Vibrationally resolved photoionization spectra of RbHe exciplexes forming on He nanodroplets are recorded using femtosecond pump-probe spectroscopy with amplitude-shaped probe pulses. The time-evolution of the spectra reveals an exciplex formation time ~10 ps followed by vibrational relaxation extending up to ≳ 1 ns. This points to an indirect, time-delayed desorption process of RbHe off the He surface.

Multiplexed microsatellite recovery using massively parallel sequencing.

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356,958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5 M (USD).

Coherent transients in the femtosecond photoassociation of ultracold molecules.

We demonstrate the photoassociation of ultracold rubidium dimers using coherent femtosecond pulses. Starting from a cloud of ultracold rubidium atoms, electronically excited rubidium molecules are formed with shaped photoassociation pump pulses. The excited state molecules are projected with a time-delayed probe pulse onto molecular ion states which are detected in a mass spectrometer. Coherent transient oscillations of the excited state population are observed in the wings of the pump pulse, in agreement with the time-dependent solution of the Schrödinger equation of the excitation process.

Parentage and relatedness in polyandrous comb-crested jacanas using ISSRs.

In this article we present the first analysis of parentage and relatedness in a natural vertebrate population, using Intersimple Sequence Repeat (ISSR) markers. Thus, 28 ISSR markers were used in a study of a sex-role reversed, simultaneously polyandrous shorebird from northeastern Australia, the comb-crested jacana (Irediparra gallinacea). Assessment of parentage was based on comparison of field observations, novel bands, individual-specific bands found in 7/9 males and 4/6 females, and a 99% CI exclusion criteria. Integrating results from these approaches resulted in confirmation of paternity in all 36 chicks. In only one case (2.8% of chicks) was a co-mate assigned paternity. Thus, comb-crested jacanas appear to be genetically monogamous. These results showed resemblance to sequentially polyandrous birds but differed from the simultaneously polyandrous wattled jacana ( Jacana jacana; Emlen et al. 1998). A significant relationship between relatedness and ISSR similarity resulted in recognition that 14/15 adults sampled may be related to at least one other adult by 0.25 or more. Lack of dispersal may be explained by physical limitations and adequate regional habitat. ISSRs proved to be simple and helpful in resolving these issues.

Evaluation of suspected appendicitis in children using limited helical CT and colonic contrast material.

OBJECTIVE: Colonic contrast material evaluation of suspected appendicitis in pediatric patients is technically more challenging than in adults because less intraabdominal fat is present. To determine the accuracy and feasibility of focused CT for pediatric patients, we carried out this retrospective investigation. MATERIALS AND METHODS: Between November 1995 and July 1999, 199 pediatric patients (1-18 years old; mean age, 12 years) were examined with focused CT in the emergency division for suspected appendicitis. The findings on CT were compared with the findings at surgery, pathology, and clinical follow-up. RESULTS: There were 64 true-positive CT scans, two false-negative, 128 true-negative, one false-positive, and four indeterminate. Seventy-four patients underwent appendectomy, with a negative appendectomy rate of 9%. One hundred twenty-five patients without appendicitis were treated nonoperatively. The true-positive rate was 32%, true-negative rate was 64%, sensitivity was 97%, specificity was 99%, positive predictive value was 98%, negative predictive value was 98%, and overall accuracy was 96%. Pediatric patients tolerated the procedure well. Colonic contrast material saved time and provided improved identification of the cecum and appendix. In 62 patients without appendicitis, focused CT provided alternative diagnoses. CONCLUSION: Focused CT appears to be nearly as accurate in pediatric patients as in adults. Focused CT provided alternative diagnoses in 48% of the patients for whom CT findings were negative for appendicitis.

Somatic delivery of catecholamines in the striatum attenuate parkinsonian symptoms and widen the therapeutic window of oral sinemet in rats.

Guidelines for clinical transplantation studies for Parkinson's disease emphasize that transplants should be considered as an adjunct to systemic L-DOPA, yet few preclinical studies have specifically assessed the potential of transplants as an adjunct to the clinical gold standard treatment. The objectives of the present study were to determine if encapsulated PC12 cells implanted in rats with severe unilateral dopamine depletions: (i) have a direct therapeutic effect on measures of parkinsonian symptoms; and/or (ii) increase the therapeutic window of oral sinemet in this model. Rats with severe unilateral dopamine depletions received striatal implants of encapsulated PC12 cells producing dopamine and L-DOPA. These rats were tested on a battery of behavioral measures of parkinsonian symptoms, at a range of doses of oral sinemet (0, 12, 24, and 36 mg/kg). Stereotypies/dyskinesias were also quantified after high doses of oral sinemet (36 and 50 mg/kg). The results confirm that parkinsonian symptoms can be quantified in rats with severe dopamine depletions, and the validity and clinical relevance of these measures are supported by the fact that the clinical gold standard treatment, oral sinemet, attenuates these parkinsonian symptoms. Somatic delivery of dopamine and L-DOPA, directly to the dopamine-depleted striatum, also attenuates parkinsonian symptoms. In fact, the magnitude of the therapeutic effect produced by continuous, site-specific, somatic delivery of dopamine and L-DOPA was larger than the effect produced by acute, systemic, oral sinemet. The beneficial effects of oral sinemet and striatal implants of catecholamine-producing devices were additive, but there were no adverse effects related to striatal catecholamine-producing devices, and these devices did not increase the adverse effects related to oral sinemet. Therefore, striatal implants of catecholamine-producing devices have direct therapeutic effects which are fairly robust, and they widen the therapeutic window of oral sinemet.

Assessment of factors regulating serum growth hormone binding protein in pigs.

These studies were conducted to examine the influence of several variables on the growth hormone binding protein (GHBP) activity in serum of pigs. Continuous long-term porcine somatotropin (pST) injections (daily for 6 to 7 wk) increased GHBP activity (P less than .05). However, periodic short-term pST injections (daily, every 2nd d, or every 4th d for 2 wk) did not cause a significant change in GHBP levels (P greater than .40). Although fasting seems to reduce liver GH receptors, no difference was observed between fed animals and animals fasted for 5 d (P greater than .30). Between 0 and 6 mo of age, boar and gilt serum GHBP activity were not significantly different from each other but increased with age in both sexes (P less than .0001). There was no significant correlation between serum GHBP and BW at 6 mo of age in this study (P greater than .30). In pregnant sows, GHBP concentrations were highest at the beginning (d 72) of the third trimester (P less than .05). Growth hormone receptor activity reported by other researchers and GHBP activity in this study seem to vary similarly except during fasting, which may indicate alternate regulation of either the GHBP or the GH receptor.

Ultrastructural differentiation of sodium butyrate-treated human pancreatic adenocarcinoma cell lines.

The differentiation effects of sodium butyrate were examined in a series of human pancreatic adenocarcinoma cell lines: Panc-1, a poorly differentiated cell line; HPAF, a pleomorphic cell line isolated in this laboratory; and two clones of the parental HPAF cell line, well-differentiated CD11 and less-differentiated CD18. Incubation with 2 mM sodium butyrate induced a dramatic decrease in cell proliferation and saturation densities in culture and an increase in alkaline phosphatase activity. Of particular interest, incubation with sodium butyrate also caused a number of morphologic alterations in these cells, attributed to an induction of secretory differentiation. Following sodium butyrate treatment, CD18 cells were virtually indistinguishable from the more highly differentiated CD11 cells as evidenced by an increase in the number of profiles of rough endoplasmic reticulum and of Golgi. Intercellular and intracytoplasmic lumens, whose appearance is quite common in CD11 cells but nonexistent in untreated CD18 cells, appeared in these cells following only 5 days of sodium butyrate treatment. An increase in the cytoplasmic secretory elements was also observed in sodium butyrate-treated Panc-1 cells; however, lumen formation never occurred in these cells.

Carotid endarterectomy: a ten-year analysis of outcome and cost of treatment.

Between 1978 and 1988, 215 patients with an average age of 67 years, underwent 246 carotid endarterectomies. Two hundred ten (85.4%) patients were symptomatic, and 36 (14.6%) were asymptomatic. Six patients (2.4%) had a postoperative stroke, and all had immediate reoperation. One of these patients died (30 day mortality rate, 0.4% for the series), and two (0.8%) recovered completely, whereas three (1.2%) had a mild permanent neurologic deficit. Two patients (0.8%) had nonfatal myocardial infarction. Mean follow-up of 42.2 months (range, 1 to 126 months) was achieved. At 5 and 8 years actuarial survival rates of 82% and 66% and stroke-free survival rates of 67% and 37% were observed. Actuarial stroke free rates of 90% at 5 and 8 years were noted. By introducing and observing guidelines that required preoperative study of most clearly defined classes of patients before admission for surgical treatment, the average length of stay for carotid endarterectomy was lowered from 9.5 days in the first 5 years of the study to 5.8 days in the second 5 years (p = 0.001). Average hospital charges, expressed in constant dollars, decreased from $3113 in the first 5 years to $2620 in the second 5 years (p = 0.02) despite an 88% inflationary increase in medical consumer price index. This experience shows that the length of hospitalization of patients with carotid endarterectomy can be reduced and the cost of admission lowered without untoward effect on perioperative morbidity and mortality rates.

Infected urachal cysts.

Infected urachal cysts present with protean clinical manifestations. Diagnosis is frequently difficult in spite of the use of modern genitourinary radiologic imaging techniques. Patients with intra-abdominal or pelvic symptoms are frequently misdiagnosed. Surgical excision is the treatment of choice for infected urachal cysts.

The effect of parainfluenza 3 infection on guinea pig basophil and lung mast cell histamine release.

The guinea pig infected with parainfluenza 3 (P-3) has provided an animal model to study mechanisms of virus-induced asthma and airway hyperreactivity. Evidence to identify the mechanisms by which P-3 infection enhances airway hyperreactivity, however, has not been established. The present study evaluated the effect of P-3 infection on histamine release (HR) from isolated peripheral blood guinea pig basophils and lung mast cells. Basophil HR was determined in animals made basophilic with 13 daily intraperitoneal injections of sheep blood. On Day 10 of the sensitization, blood was obtained from all animals, and leukocyte HR was determined to sheep gammaglobulin (SG), which served as the secretagogue. After these baseline studies, the animals were separated into two groups; one was insufflated with P-3 virus and the other with growth medium that did not contain virus. Four days later, animals were killed, basophils and lung mast cells were isolated, HR was determined, and lung tissue was cultured for the presence of virus. In basophils isolated from animals proven to be infected with P-3 virus, we found significantly more histamine released in response to the lower doses of SG and a significant shift in the -log EC50 dose of this antigen causing HR in these cells. Experiments conducted with the calcium ionophore A23187 as the basophil secretagogue in the same experimental design did not demonstrate similar HR findings. Furthermore, a defect in calcium disposition did not account for the enhanced HR from basophils isolated from infected animals. HR from pulmonary mast cells was similar in cells isolated from P-3-infected and control insufflated animals.(ABSTRACT TRUNCATED AT 250 WORDS)