Imaging Dendritic Spines in Caenorhabditis elegans.

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Field Evaluations of Sulfuryl Fluoride Fumigation for Control of the Common Bed Bug (Hemiptera: Cimicidae), Using a 1.9× Dosage Factor in Motor Vehicles and Filled Cargo Trailers.

This study investigated the efficacy of using Vikane gas fumigant (sulfuryl fluoride) at the 1.9× dosage rate for eliminating bed bugs (Cimex lectularius L.) in two challenging infestation situations: personal vehicles, and confined spaces densely packed with personal belongings. The vehicles used in this study were large minivans with seating that folded into the floor. The confined spaces were cargo trailers filled to 85% capacity with books, furniture, and other household items. Each van and trailer was equipped with ~90 sentinel bed bugs consisting of three groups of 9-11 bed bug eggs, 10 nymphs, and 10 adults. The Vikane Fumiguide calculator was used to determine the target dosage (g-h/m3) to apply in each replicate (e.g., one van or trailer). Sulfuryl fluoride concentrations were measured throughout the fumigation process using a Spectros SF-ReportIR. Concentration readings were input into the Fumiguide to determine when the accumulated dosage (g-h/m3) was achieved, and when aeration should be initiated. After aeration was complete, the sentinel bed bugs were removed from the replicates and bed bug nymph and adult mortality was recorded. Bed bug eggs were monitored for 23 d to determine latent mortality. Fumigated bed bug mortality for each replication was 100% regardless of life stage. Latent mortality was observed in a single bed bug egg, but the first instar never fully eclosed. This study determined that fumigation with sulfuryl fluoride at the 1.9× dosage factor is an effective method for eliminating resistant bed bugs from vehicles and personal belongings in densely packed situations.

Cognitive processing speed in multiple sclerosis clinical practice: association with patient-reported outcomes, employment and magnetic resonance imaging metrics.

BACKGROUND AND PURPOSE: To analyze the relationship between cognitive processing speed, patient-reported outcome measures (PROMs), employment and magnetic resonance imaging (MRI) metrics in a large multiple sclerosis cohort. METHODS: Cross-sectional clinical data, PROMs, employment and MRI studies within 90 days of completion of the Processing Speed Test (PST), a technology-enabled adaptation of the Symbol Digit Modalities Test, were collected. MRI was analyzed using semi-automated methods. Correlations of PST score with PROMs and MRI metrics were examined using Spearman's rho. Wilcoxon rank sum testing compared MRI metrics across PST score quartiles and linear regression models identified predictors of PST performance. Effects of employment and depression were also investigated. RESULTS: In 721 patients (mean age 47.6 ± 11.4 years), PST scores were significantly correlated with all MRI metrics, including cord atrophy and deep gray matter volumes. Linear regression demonstrated self-reported physical disability, cognitive function, fatigue and social domains (adjusted R2  = 0.44, P < 0.001) as the strongest clinical predictors of PST score, whereas that of MRI variables included T2 lesion volume, whole-brain fraction and cord atrophy (adjusted R2  = 0.42, P < 0.001). An inclusive model identified T2 lesion volume, whole-brain fraction, self-reported upper extremity function, cognition and social participation as the strongest predictors of PST score (adjusted R2  = 0.51, P < 0.001). There was significant effect modification by depression on the relationship between self-reported cognition and PST performance. Employment status was associated with PST scores independent of age and physical disability. CONCLUSION: The PST score correlates with PROMs, MRI measures of focal and diffuse brain injury, and employment. The PST score is a feasible and meaningful measure for routine multiple sclerosis care.

Multiple Sclerosis Performance Test: validation of self-administered neuroperformance modules.

BACKGROUND AND PURPOSE: The purpose was to determine the test-retest reliability, practice effects, convergent validity and sensitivity to multiple sclerosis (MS) disability of neuroperformance subtests from the patient self-administered Multiple Sclerosis Performance Test (MSPT) designed to assess low contrast vision (Contrast Sensitivity Test, CST), upper extremity motor function (Manual Dexterity Test, MDT) and lower extremity motor function (Walking Speed Test, WST) and to introduce the concept of regression-based norms to aid clinical interpretation of performance scores using the MSPT cognition test (Processing Speed Test, PST) as an example. METHODS: Substudy 1 assessed test-retest reliability, practice effects and convergent validity of the CST, MDT and WST in 30 MS patients and 30 healthy controls. Substudy 2 examined sensitivity to MS disability in over 600 MS patients as part of their routine clinic assessment. Substudy 3 compared performance on the PST in research volunteers and clinical samples. RESULTS: The CST, MDT and WST were shown to be reliable, valid and sensitive to MS outcomes. Performance was comparable to technician-administered testing. PST performance was poorer in the clinical sample compared with the research volunteer sample. CONCLUSIONS: The self-administered MSPT neuroperformance modules produce reliable, objective metrics that can be used in clinical practice and support outcomes research. Published studies which require patient voluntary consent may underestimate the rate of cognitive dysfunction observed in a clinical setting.

Grazing Management and Buffer Strip Impact on Nitrogen Runoff from Pastures Fertilized with Poultry Litter.

Nitrogen runoff from pastures fertilized with animal manure, such as poultry litter, can result in accelerated eutrophication. The objective of this study was to evaluate the long-term effects of grazing management and buffer strips on N runoff from pastures fertilized with poultry litter. A 12-yr study was conducted on 15 small watersheds in Booneville, AR, using five management practices: continuous grazing, haying, rotational grazing, rotational grazing with an unfertilized buffer strip, and rotational grazing with a fenced unfertilized riparian buffer. Poultry litter was applied annually at a rate of 5.6 Mg ha. Concentrations and loads of total N, NO-N, NH-N, organic N, and total organic C in runoff varied intra- and interannually and coincided with precipitation trends. Overall, the greatest component of total N in runoff was organic N. Rotational grazing resulted in the highest concentrations and loads of all forms of N in runoff compared with other treatments, including the continuously grazed paddocks, which were grazed almost twice as much. Total organic C concentrations and loads in runoff were also higher from rotationally grazed watersheds than other treatments. Rotational grazing is considered a best management practice that typically reduces soil erosion; hence, the mechanism by which it caused higher N and C runoff is unclear. Nitrogen runoff losses from rotationally grazed pastures were reduced by 44% with unfertilized buffer strips, by 54% with fenced unfertilized riparian buffers, and by 52% by converting pastures to hayfields.

Genetic epidemiology of malignant hyperthermia in the UK.

BACKGROUND: Gaps in our understanding of genetic susceptibility to malignant hyperthermia (MH) limit the application and interpretation of genetic diagnosis of the condition. Our aim was to define the prevalence and role of variants in the three genes implicated in MH susceptibility in the largest comprehensively phenotyped MH cohort worldwide. METHODS: We initially included one individual from each positive family tested in the UK MH Unit since 1971 to detect variants in RYR1, CACNA1S, or STAC3. Screening for genetic variants has been ongoing since 1991 and has involved a range of techniques, most recently next generation sequencing. We assessed the pathogenicity of variants using standard guidelines, including family segregation studies. The prevalence of recurrent variants of unknown significance was compared with the prevalence reported in a large database of sequence variants in low-risk populations. RESULTS: We have confirmed MH susceptibility in 795 independent families, for 722 of which we have a DNA sample. Potentially pathogenic variants were found in 555 families, with 25 RYR1 and one CACNA1S variants previously unclassified recurrent variants significantly over-represented (P<1×10-7) in our cohort compared with the Exome Aggregation Consortium database. There was genotype-phenotype discordance in 86 of 328 families suitable for segregation analysis. We estimate non-RYR1/CACNA1S/STAC3 susceptibility occurs in 14-23% of MH families. CONCLUSIONS: Our data provide current estimates of the role of variants in RYR1, CACNA1S, and STAC3 in susceptibility to MH in a predominantly white European population.

Assessing the pathogenicity of RYR1 variants in malignant hyperthermia.

BACKGROUND: . Missense variants in the ryanodine receptor 1 gene ( RYR1 ) are associated with malignant hyperthermia but only a minority of these have met the criteria for use in predictive DNA diagnosis. We examined the utility of a simplified method of segregation analysis and a functional assay for determining the pathogenicity of recurrent RYR1 variants associated with malignant hyperthermia. METHODS: . We identified previously uncharacterised RYR1 variants found in four or more malignant hyperthermia families and conducted simplified segregation analyses. An efficient cloning and mutagenesis strategy was used to express ryanodine receptor protein containing one of six RYR1 variants in HEK293 cells. Caffeine-induced calcium release, measured using a fluorescent calcium indicator, was compared in cells expressing each variant to that in cells expressing wild type ryanodine receptor protein. RESULTS.: We identified 43 malignant hyperthermia families carrying one of the six RYR1 variants. There was segregation of genotype with the malignant hyperthermia susceptibility phenotype in families carrying the p.E3104K and p.D3986E variants, but the number of informative meioses limited the statistical significance of the associations. HEK293 functional assays demonstrated an increased sensitivity of RyR1 channels containing the p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I compared with wild type, but cells expressing p.D3986E had a similar caffeine sensitivity to cells expressing wild type RyR1. CONCLUSIONS: . Segregation analysis is of limited value in assessing pathogenicity of RYR1 variants in malignant hyperthermia. Functional analyses in HEK293 cells provided evidence to support the use of p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I for diagnostic purposes but not p.D3986E.

High-Precision Half-Life Measurements for the Superallowed ß

Precision measurements of superallowed Fermi ß-decay transitions, particularly for the lightest superallowed emitters ^{10}C and ^{14}O, set stringent limits on possible scalar current contributions to the weak interaction. In the present work, a discrepancy between recent measurements of the ^{10}C half-life is addressed through two high-precision half-life measurements, via γ-ray photopeak and ß counting, that yield consistent results for the ^{10}C half-life of T_{1/2}=19.2969±0.0074 s and T_{1/2}=19.3009±0.0017 s, respectively. The latter is the most precise superallowed ß-decay half-life measurement reported to date and the first to achieve a relative precision below 10^{-4}. A fit to the world superallowed ß-decay data including the ^{10}C half-life measurements reported here yields b_{F}=-0.0018±0.0021 (68% C.L.) for the Fierz interference term and C_{S}/C_{V}=+0.0009±0.0011 for the ratio of the weak scalar to vector couplings assuming left-handed neutrinos.

Attitudinal barriers to participation in oncology clinical trials: factor analysis and correlates of barriers.

Patient participation in cancer clinical trials is low. Little is known about attitudinal barriers to participation, particularly among patients who may be offered a trial during an imminent initial oncology consult. The aims of the present study were to confirm the presence of proposed subscales of a recently developed cancer clinical trial attitudinal barriers measure, describe the most common cancer clinical trials attitudinal barriers, and evaluate socio-demographic, medical and financial factors associated with attitudinal barriers. A total of 1256 patients completed a survey assessing demographic factors, perceived financial burden, prior trial participation and attitudinal barriers to clinical trials participation. Results of a factor analysis did not confirm the presence of the proposed four attitudinal barriers subscale/factors. Rather, a single factor represented the best fit to the data. The most highly-rated barriers were fear of side-effects, worry about health insurance and efficacy concerns. Results suggested that less educated patients, patients with non-metastatic disease, patients with no previous oncology clinical trial participation, and patients reporting greater perceived financial burden from cancer care were associated with higher barriers. These patients may need extra attention in terms of decisional support. Overall, patients with fewer personal resources (education, financial issues) report more attitudinal barriers and should be targeted for additional decisional support.

Analysis of long-term outcomes for combined pars plana vitrectomy (PPV) and glaucoma tube shunt surgery in eyes with advanced glaucoma.

PURPOSE: To analyze 12- and 24-month visual acuity, intraocular pressure, and complications associated with combined pars plana vitrectomy (PPV) and glaucoma tube shunt placement in eyes with glaucoma. PATIENTS AND METHODS: A retrospective chart review was performed of patients with advanced glaucoma who underwent combined PPV and tube shunt surgery from 2006 to 2010. A minimum of 12 months of follow-up was required for their inclusion in the study. Visual acuity, intraocular pressure, complications, and number of glaucoma medications at 1 and 2 years postoperatively were analyzed. RESULTS: Twenty-eight eyes met the inclusion and exclusion criteria. Baseline visual acuity was 20/200 or worse in 14/28 eyes (50.0%) and 20/40 or better in 2/28 eyes (7.1%). Visual acuity remained 20/200 or worse in 50.0% (P=0.921) and 44.4% (P=0.973) of eyes after 1 and 2 years postoperatively, respectively. At baseline, the mean intraocular pressure was 30.4 mm Hg. There was significant improvement in mean IOP at 1 year (14.7 mm Hg, P=0.001) and at 2 years (15.2 mm Hg, P=0.001) postoperatively. Baseline number of glaucoma medications averaged 3.0±1.09 (SD), and improved to 1.8±1.28 (SD) at 1 year (P=0.0002) and to 1.4±1.33 at 2 years (P<0.0001) postoperatively. CONCLUSION: In this retrospective interventional case series, surgical management of advanced glaucoma with a combination of PPV and glaucoma tube shunt resulted in significantly reduced IOP and glaucoma medications at 1 and 2 years postoperatively.

Looking at ocriplasmin as a new option in eye disease.

Vitreomacular traction (VMT) syndrome and macular hole (MH) are disease processes that are caused by anomalous separation of the vitreous from the macula. Until now, management of these conditions has involved vitreoretinal surgery. Clinical trials have shown that a single intravitreal microplasmin (ocriplasmin) injection can induce separation of the vitreous from the macular surface and thereby relieve the tractional changes that contribute to vision loss from VMT and MH. Herein, we summarize the results of these trials which demonstrate that a significant number of intravitreal ocriplasmin-treated patients have resolution of their vitreoretinal interface abnormalities without the need for vitreoretinal surgery.

Technical note: development of a quantitative PCR method for monitoring strain dynamics during yogurt manufacture.

Yogurt starter cultures may consist of multiple strains of Lactobacillus delbrueckii ssp. bulgaricus (LB) and Streptococcus thermophilus (ST). Conventional plating methods for monitoring LB and ST levels during yogurt manufacture do not allow for quantification of individual strains. The objective of the present work was to develop a quantitative PCR method for quantification of individual strains in a commercial yogurt starter culture. Strain-specific primers were designed for 2 ST strains (ST DGCC7796 and ST DGCC7710), 1 LB strain (DGCC4078), and 1 Lactobacillus delbrueckii ssp. lactis strain (LL; DGCC4550). Primers for the individual ST and LB strains were designed to target unique DNA sequences in clustered regularly interspersed short palindromic repeats. Primers for LL were designed to target a putative mannitol-specific IIbC component of the phosphotransferase system. Following evaluation of primer specificity, standard curves relating cell number to cycle threshold were prepared for each strain individually and in combination in yogurt mix, and no significant differences in the slopes were observed. Strain balance data was collected for yogurt prepared at 41 and 43°C to demonstrate the potential application of this method.

Retinal tears after posterior vitreous detachment and vitreous hemorrhage in patients on systemic anticoagulants.

UNLABELLED: AIMS OR PURPOSE: To determine the rate of retinal tears (RTs) after posterior vitreous detachment (PVD) and vitreous hemorrhage (VH) in patients on systemic anticoagulants. METHODS: In all, 260 eyes of 260 patients with an acute PVD and VH were followed for evidence of an RT or detachment. Patients were divided into those taking systemic anticoagulants and those not taking anticoagulants. RESULTS: A total of 137 patients (53%) were taking anticoagulants, 123 (47%) were not. Overall, 72% of patients not taking any anticoagulant had evidence of an RT, whereas 46% of patients taking an anticoagulant had an RT (P-value 0.0002). Also, 37% of patients not taking an anticoagulant had a retinal detachment (RD), whereas 23% of patients taking any anticoagulant had an RD (P-value 0.01). CONCLUSIONS: In patients with an acute PVD and VH using anticoagulants, RTs and RDs were common. Anticoagulation status may be an important contributing factor in predicting the incidence of an RT or detachment.

Change in quality of life in patients with relapsing-remitting multiple sclerosis over 2 years in relation to other clinical parameters: results from a trial of intramuscular interferon {beta}-1a.

BACKGROUND: A randomized, placebo-controlled, multicenter study of weekly intramuscular injections of interferon beta-1a (IFNß-1a) in relapsing-remitting multiple sclerosis included the Sickness Impact Profile (SIP), a validated measure of patient-reported quality of life (QoL). OBJECTIVE: To demonstrate the impact of moderate to severe SIP disability at baseline and change in QoL as measured by SIP over 2 years in relation to other study parameters. METHODS: In 158 patients, SIP scores were determined at baseline and 2 years. Scores were correlated with disease progression and treatment. RESULTS: Patients who experienced disability progression, as defined by Expanded Disability Status Scale (EDSS) and annualized relapse rate, during the study demonstrated significant worsening in Physical SIP scores compared with patients who did not progress (p=0.031). In patients with low SIP scores, indicating moderate or severe disability at baseline, treatment with IFNß-1a significantly improved Physical SIP subscores. CONCLUSIONS: Patients with disability progression defined using EDSS, the physician-derived primary outcome measure, had Physical SIP scores indicating worsening disability, validating the physician-derived primary outcome measure using patient self-report. Treatment with IFNß-1a had beneficial effects on QoL in patients with worse SIP scores at baseline.

Cloning of genes encoding colicin E2 in Lactococcus lactis subspecies lactis and evaluation of the colicin-producing transformants as inhibitors of Escherichia coli O157:H7 during milk fermentation.

Colicin E2 (ColE2) is a proteinaceous bacterial toxin produced by some strains of Escherichia coli and other members of the Enterobacteriaceae that exhibits inhibitory activity against some strains of E. coli O157:H7. A 2.0-kb DNA fragment, containing the ColE2 structural gene ceaB and immunity gene ceiB from E. coli NCTC 50133 (pColE2-P9), was cloned into the lactococcal plasmid vector pNZ2103. The lysis gene, celB, was not cloned. The plasmid, pLR-E2, encoding the cloned genes was transformed into E. coli DH5α and Lactococcus lactis ssp. lactis LM0230 and PN-1 using electroporation. The bacteriocin ColE2 was expressed in transformants of both E. coli and L. lactis ssp. lactis. Secretion of ColE2 into media was verified by spot-on-lawn assays and measurement of ColE2 activity in the growth medium of transformants. The level of ColE2 produced by transformants containing pLR-E2 was similar to that produced by the parental strain, E. coli NCTC 50133 (pColE2-P9). Evaluation of a ColE2-producing transformant of L. lactis ssp. lactis as a starter culture revealed that, although ColE2 was produced by transformants and could be detected in milk during fermentation, the inhibitory activity of ColE2 against E. coli O157:H7 was significantly decreased in milk compared with buffered growth medium.

Autofluorescence imaging can identify preinvasive or clinically occult lesions in fallopian tube epithelium: a promising step towards screening and early detection.

BACKGROUND: Optical imaging systems are robust, portable, relatively inexpensive, and have proven utility in detecting precancerous lesions in the lung, esophagus, colon, oral cavity and cervix. We describe the use of light-induced endogenous fluorescence (autofluorescence) in identifying preinvasive and occult carcinomas in ex vivo samples of human fallopian tube (FT) epithelium. METHODS: Women undergoing surgery for an i) ovarian mass, ii) a history suggestive of hereditary breast-ovarian cancer, or iii) known serous ovarian cancer following neoadjuvant chemotherapy (NAC) were approached for informed consent. Immediately following surgery, FT's were photographed in reflectance and fluorescence at high resolution. Images included: (1) white-light reflectance of luminal/epithelial surface; (2) narrow-band green reflectance (570 nm) (3) green autofluorescence (405/436 nm excitation); and (4) blue autofluorescence (405 nm excitation). Areas revealing a loss of natural tissue fluorescence or marked increase in tissue microvasculature were recorded and compared to final histopathologic diagnosis (SEE-FIM protocol). RESULTS: Fifty-six cases involving one or both fallopian tubes underwent reflectance and fluorescence visualization. Nine cases were excluded, either secondary to non-ovarian primary pathology (7) or excessive trauma (2) rendering tissue interpretation impossible. Of the 47 cases remaining, there were 11 high grade serous (HGS) and 9 non-serous ovarian carcinomas undergoing primary debulking surgery, 5 serous carcinomas having received NAC, 8 benign ovarian tumors, and 14 women undergoing risk-reducing bilateral salpingo-oophorectomy (RRBSO). Methodology was feasible, efficient, and reproducible. TIC or carcinoma was identified in 7/11 HGS, 3/5 NAC, and 1/14 RRBSO. Optical images were reviewed to determine test positive or negative based on standardized criteria. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for the entire cohort (73%; 83%; 57%; 91%) and in a subgroup that excluded non-serous histology (87.5%; 92%; 78%; 96%). CONCLUSIONS: Abnormal FT lesions can be identified using ex vivo optical imaging technologies. With this platform, we will move towards genomic interrogation of identified lesions, and developing in vivo screening modalities via falloposcopy.

Factors that influence the employment status of people with multiple sclerosis: a multi-national study.

Paid employment is valuable for society and for the individual. A diagnosis of a chronic illness such as multiple sclerosis (MS) can influence a person's employment status. Previous studies have reported that demographic and disease-related aspects can predict whether a person with MS will leave their job. The aim of the study was to assess the factors that people with MS believe to contribute to their employment status and to determine whether any of these differentiate people with MS who are employed from those who are not employed. A multinational questionnaire assessed aspects related to employment that facilitate or hinder job maintenance. Data was collected in 18 European countries. A total of 1,141 questionnaires were completed. Of those responding, 694 (61%) subjects were employed and 477 (39%) were unemployed. The items that significantly differentiated the groups were related to MS symptoms, workplace environment and financial considerations. While MS influences employment status for many people who face difficult symptoms, aspects like a flexible work schedule and financial security are important and perhaps key to promoting job maintenance among people with MS.