Optogenetic TDP-43 nucleation induces persistent insoluble species and progressive motor dysfunction in vivo.

TDP-43 is a predominantly nuclear DNA/RNA binding protein that is often mislocalized into insoluble cytoplasmic inclusions in post-mortem patient tissue in a variety of neurodegenerative disorders including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD). The underlying causes of TDP-43 proteinopathies remain unclear, but recent studies indicate the formation of these protein assemblies is driven by aberrant phase transitions of RNA deficient TDP-43. Technical limitations have prevented our ability to understand how TDP-43 proteinopathy relates to disease pathogenesis. Current animal models of TDP-43 proteinopathy often rely on overexpression of wild-type TDP-43 to non-physiological levels that may initiate neurotoxicity through nuclear gain of function mechanisms, or by the expression of disease-causing mutations found in only a fraction of ALS patients. New technologies allowing for light-responsive control of subcellular protein crowding provide a promising approach to drive intracellular protein aggregation, as we have previously demonstrated in vitro. Here we present a model for the optogenetic induction of TDP-43 proteinopathy in Drosophila that recapitulates key features of patient pathology, including detergent insoluble cytoplamsic inclusions and progressive motor dysfunction.

Stocking density, milking duration, and lying times of lactating cows on Canadian freestall dairy farms.

Lying time is an important measure of cow comfort, and the lying behavior of dairy cattle can now be recorded automatically with the use of accelerometers. To assess the effect that stall stocking density and the time that cows spend away from the home pen being milked has on the lying behavior of Holstein cattle, a total of 111 commercial freestall dairy farms were visited in Canada. Accelerometers were used to automatically record the lying behavior of 40 focal cows per farm. Total duration of lying, lying bout frequency, and the mean duration of lying bouts were calculated. Pen population was the total number of cows in the pen. To calculate stall stocking density (%) the number of cows in the pen and the number of useable stalls were counted and multiplied by 100, and the length × width of the pen was divided by the number of cows in the pen to calculate area/cow (m(2)). Time away from the pen per day was recorded from when the first cow in each pen was taken out of the home pen for milking until the last cow returned to the home pen after milking, and this time was multiplied by daily milking frequency. The median value for lying duration at the farm level was 10.6h/d, with 10.5 lying bouts/d, and a median lying bout duration of 1.2h. Stall stocking density ranged from 52.2 to 160.0%, with very few farms (7%) stocking at greater than 120%. Although stall stocking density was not significantly correlated with lying behavior, the results showed that no farm with stocking density greater that 100% achieved an average herd lying duration of 12h/d or higher, whereas 21.6% of farms with a stocking density of 100% or less did achieve the target lying time of ≥ 12 h/d, as recommended by the Canadian Code of Practice (χ(2)=4.86, degrees of freedom = 1). Area/cow (m(2)) was not correlated with any aspect of lying behavior, but regardless of space per cow, pen population was correlated with daily frequency and duration of lying bouts. As the number of cows in the pen increased, lying daily bout frequency increased (correlation coefficient = 0.24) and lying bout duration decreased (correlation coefficient = -0.30). Lying behavior was affected by the time the cows were away from the pen being milked. When cows were away from the pen for longer than 3.7h/d, no farm achieved the recommended herd median lying time of 12h/d or longer. These results suggest that providing 1 stall for each cow in the pen and minimizing time away from the pen are important factors if cattle are to achieve the recommended daily lying duration of 12h/d.

NUP62 localizes to ALS/FTLD pathological assemblies and contributes to TDP-43 insolubility.

A G4C2 hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of ALS and FTLD (C9-ALS/FTLD) with cytoplasmic TDP-43 inclusions observed in regions of neurodegeneration. The accumulation of repetitive RNAs and dipeptide repeat protein (DPR) are two proposed mechanisms of toxicity in C9-ALS/FTLD and linked to impaired nucleocytoplasmic transport. Nucleocytoplasmic transport is regulated by the phenylalanine-glycine nucleoporins (FG nups) that comprise the nuclear pore complex (NPC) permeability barrier. However, the relationship between FG nups and TDP-43 pathology remains elusive. Our studies show that nuclear depletion and cytoplasmic mislocalization of one FG nup, NUP62, is linked to TDP-43 mislocalization in C9-ALS/FTLD iPSC neurons. Poly-glycine arginine (GR) DPR accumulation initiates the formation of cytoplasmic RNA granules that recruit NUP62 and TDP-43. Cytoplasmic NUP62 and TDP-43 interactions promotes their insolubility and NUP62:TDP-43 inclusions are frequently found in C9orf72 ALS/FTLD as well as sporadic ALS/FTLD postmortem CNS tissue. Our findings indicate NUP62 cytoplasmic mislocalization contributes to TDP-43 proteinopathy in ALS/FTLD.

Effects of providing total mixed rations indoors and on pasture on the behavior of lactating dairy cattle and their preference to be indoors or on pasture.

Grazing is considered a normal behavior for dairy cattle, although they may not be able to meet their nutritional requirements from grazing alone, and so to sustain higher yields requires access to a total mixed ration (TMR). The study aim was to provide dairy cows with access to TMR indoors and on pasture to establish influence on behavior and preference for each location. The study took place from August to November, 2009, using 36 late-lactation Holstein-Friesian dairy cows. The cows were allocated to 1 of 3 26-d study periods (n=12×3). Within each period the cows were further divided into a control (n=6) or treatment (n=6) group using a crossover design, where the cows were changed between the control and treatment group after 13 d. Treatment cows had access to TMR indoors and on pasture, whereas control cows only had access to TMR indoors. Following a.m. and p.m. milkings the cows were taken to a point equidistant between indoors and pasture and given the choice of going to pasture (1.5 ha) or to a freestall barn. Between milkings the cows had free access between the locations. Initial choice was recorded and a video camera was used to record time spent in each location. Behavior observations were recorded to establish how the cows spent their time during the day. To determine what factors influenced preference, weather conditions, milk yield, body condition score, and lameness were recorded. Initially, the cows chose indoors following milking (96.4±0.80%). Overall, the cows expressed a partial preference for pasture (71.1±1.82%), which was different from 100, 50, and 0%. Study period influenced preference with cows spending less time on pasture as the season progressed (86.7 vs. 68.3 vs. 58.3% for study periods 1, 2, and 3, respectively). Providing the cows with TMR outdoors did not affect pasture use, but resulted in an increase in TMR intake of 2.2±0.41 kg of dry matter/d. The cows spent more time on pasture as the temperature-humidity index indoors (55.6±0.92) and outdoors (54.6±0.82) increased, but rainfall and milk yield did not influence preference. Cows with lameness score >1.5 spent more time indoors (35.4±4.52 vs. 25.2±2.64% for cows with >1.5 vs. ≤1.5 lameness score, respectively). In conclusion, the cows expressed a partial preference for pasture, which was not influenced by providing TMR on pasture.

Gastropod Urosalpinx: pH of Accessory Boring Organ while Boring.

Recent development of a microelectrode has enabled the first continuous recording of the pH of the secretion of the normally functioning accessory boring organ of the shell-boring predatory snail Urosalpinx. The recording was made in an incomplete borehole in a glass-shell model. The minimum pH recorded was 3.8; hitherto the secretion had been considered neutral.

Grass silage particle size when fed with or without maize silage alters performance, reticular pH and metabolism of Holstein-Friesian dairy cows.

The particle size of the forage has been proposed as a key factor to ensure a healthy rumen function and maintain dairy cow performance, but little work has been conducted on ryegrass silage (GS). To determine the effect of chop length of GS and GS:maize silage (MS) ratio on the performance, reticular pH, metabolism and eating behaviour of dairy cows, 16 multiparous Holstein-Friesian cows were used in a 4×4 Latin square design with four periods each of 28-days duration. Ryegrass was harvested and ensiled at two mean chop lengths (short and long) and included at two ratios of GS:MS (100:0 or 40:60 dry matter (DM) basis). The forages were fed in mixed rations to produce four isonitrogenous and isoenergetic diets: long chop GS, short chop GS, long chop GS and MS and short chop GS and MS. The DM intake (DMI) was 3.2 kg/day higher (P<0.001) when cows were fed the MS than the GS-based diets. The short chop length GS also resulted in a 0.9 kg/day DM higher (P<0.05) DMI compared with the long chop length. When fed the GS:MS-based diets, cows produced 2.4 kg/day more (P<0.001) milk than when fed diets containing GS only. There was an interaction (P<0.05) between chop length and forage ratio for milk yield, with a short chop length GS increasing yield in cows fed GS but not MS-based diets. An interaction for DM and organic matter digestibility was also observed (P<0.05), where a short chop length GS increased digestibility in cows when fed the GS-based diets but had little effect when fed the MS-based diet. When fed the MS-based diets, cows spent longer at reticular pH levels below pH 6.2 and pH 6.5 (P<0.01), but chop length had little effect. Cows when fed the MS-based diets had a higher (P<0.05) milk fat concentration of C18 : 2n-6 and total polyunsaturated fatty acids compared with when fed the GS only diets. In conclusion, GS chop length had little effect on reticular pH, but a longer chop length reduced DMI and milk yield but had little effect on milk fat yield. Including MS reduced reticular pH, but increased DMI and milk performance irrespective of the GS chop length.

Drosophila Ref1/ALYREF regulates transcription and toxicity associated with ALS/FTD disease etiologies.

RNA-binding proteins (RBPs) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the underlying disease mechanisms remain unclear. In an unbiased screen in Drosophila for RBPs that genetically interact with TDP-43, we found that downregulation of the mRNA export factor Ref1 (fly orthologue to human ALYREF) mitigated TDP-43 induced toxicity. Further, Ref1 depletion also reduced toxicity caused by expression of the C9orf72 GGGGCC repeat expansion. Ref1 knockdown lowered the mRNA levels for these related disease genes and reduced the encoded proteins with no effect on a wild-type Tau disease transgene or a control transgene. Interestingly, expression of TDP-43 or the GGGGCC repeat expansion increased endogenous Ref1 mRNA levels in the fly brain. Further, the human orthologue ALYREF was upregulated by immunohistochemistry in ALS motor neurons, with the strongest upregulation occurring in ALS cases harboring the GGGGCC expansion in C9orf72. These data support ALYREF as a contributor to ALS/FTD and highlight its downregulation as a potential therapeutic target that may affect co-existing disease etiologies.

Flow dependence of valve area in aortic stenosis: relation to valve morphology.

OBJECTIVES: We sought to develop an index of flow dependence of valve area in aortic valve (AoV) stenosis and to determine whether this index is related to structural characteristics of the diseased valve. BACKGROUND: Many studies of AoV stenosis using Gorlin or continuity equation methods have demonstrated flow dependence (an increase in valve area with increased flow). Variation in flow dependence between patients despite similar flow rates remains unexplained. METHODS: Dobutamine Doppler echocardiography was used to calculate flow rate and valve area by the continuity equation in 27 patients with aortic stenosis. For each patient the slope of the regression line of valve area to flow rate was determined (slope of flow dependence). Transesophageal echocardiography was used to evaluate features of valve morphology potentially related to the etiology of AoV stenosis and the mechanism of flow dependence. RESULTS: Mean slope of flow dependence was 0.28 cm2/100 ml per s (range -0.06 to 0.53); flow dependence was significantly >0 in 21 patients and was lower for bicuspid valves (slope 0.21 cm2/100 ml per s) than for tricuspid valves with <10% commissural fusion (slope 0.35, p < 0.01). Off-center/ovoid orifices demonstrated the least flow dependence (slope 0.19), whereas star-shaped orifices showed the most (slope 0.36, p < 0.01). Greater flow dependence was related to a lower percentage of commissural fusion (r = -0.46, p = 0.02) as well as diffuse sclerosis, primarily involving the cusp bodies, rather than localized sclerosis, with involvement of cusp margins. CONCLUSIONS: The slope of flow dependence of valve area in AoV stenosis differs markedly between patients. More flow dependence was associated with tricuspid valves and the morphologic features characteristic of calcific AoV stenosis, whereas less flow dependence was associated with bicuspid valves and the features of rheumatic disease.

Value Driven Outcomes (VDO): a pragmatic, modular, and extensible software framework for understanding and improving health care costs and outcomes.

OBJECTIVE: To develop expeditiously a pragmatic, modular, and extensible software framework for understanding and improving healthcare value (costs relative to outcomes). MATERIALS AND METHODS: In 2012, a multidisciplinary team was assembled by the leadership of the University of Utah Health Sciences Center and charged with rapidly developing a pragmatic and actionable analytics framework for understanding and enhancing healthcare value. Based on an analysis of relevant prior work, a value analytics framework known as Value Driven Outcomes (VDO) was developed using an agile methodology. Evaluation consisted of measurement against project objectives, including implementation timeliness, system performance, completeness, accuracy, extensibility, adoption, satisfaction, and the ability to support value improvement. RESULTS: A modular, extensible framework was developed to allocate clinical care costs to individual patient encounters. For example, labor costs in a hospital unit are allocated to patients based on the hours they spent in the unit; actual medication acquisition costs are allocated to patients based on utilization; and radiology costs are allocated based on the minutes required for study performance. Relevant process and outcome measures are also available. A visualization layer facilitates the identification of value improvement opportunities, such as high-volume, high-cost case types with high variability in costs across providers. Initial implementation was completed within 6 months, and all project objectives were fulfilled. The framework has been improved iteratively and is now a foundational tool for delivering high-value care. CONCLUSIONS: The framework described can be expeditiously implemented to provide a pragmatic, modular, and extensible approach to understanding and improving healthcare value.

Cerebral oxygenation measured by near-infrared spectroscopy: comparison with jugular bulb oximetry.

BACKGROUND: Near-infrared spectroscopy is a potential tool for measuring adequacy of cerebral oxygenation during cardiac operations. The cerebral microcirculation is predominantly venous (by volume) and therefore regional cerebral oxygenation measured by near-infrared spectroscopy should reflect jugular bulb venous saturations. METHODS: We compared simultaneous regional cerebral oxygenation and jugular bulb venous saturation measurements in 40 children (median age, 4.5 years; range 2 weeks to 14.5 years) in the cardiac catheter laboratory (n = 29) and during cardiac operations (n = 11). RESULTS: For all patients combined the correlation between regional cerebral oxygenation and jugular bulb venous saturation was 0.69 (p < 0.0001) and was similar for the two groups. For individual children undergoing cardiac operations excellent correlations were obtained (r = 0.78 to 0.96; median, 0.91). However, at low values of jugular bulb venous saturation, regional cerebral oxygenation tended to run high, whereas the converse was true for high values of jugular bulb venous saturation. CONCLUSIONS: These findings suggest that near-infrared spectroscopy may be a useful tool for assessing intravascular cerebral oxygenation during pediatric cardiac operations. Prospective studies of neurologic outcome will be required to establish the value of this technique for assessing the adequacy of cerebral protection.

Plaque minerals and caries experience: associations and interrelationships.

Inverse associations were demonstrated between DMFT and total calcium, phosphorus, magnesium, strontium, potassium, and fluoride concentrations in individual plaques. Lithium, strontium, fluoride, and zinc contents were inversely related to plaque mass. Consistent interdependence patterns between elements indicate the operation of a mineral level regulating mechanism, effective under diverse environmental conditions.

Physiologic consequences of training.

Systematic exercise training results in changes in skeletal muscle that increase oxidative capacity and vascular conductance, which lead to an increase in maximal A-VO2. Also, maximal cardiac output is increased, largely because of an increase in maximal stroke volume. Heart rate is decreased at rest and during submaximal exercise because of increased parasympathetic tone and the effect of increased stroke volume on reflex sympathetic tone. The increases in maximal A-VO2 and cardiac output result in increased maximal VO2, the hallmark of the dynamically trained individual. Predominant static exercise training results in fewer increases in maximal VO2 and more local muscle strength enhancements. The systematic application of cross-training can increase both cardiovascular and strength parameters, leading to enhanced athletic performance.

Audit of the effect of a nurse run asthma clinic on workload and patient morbidity in a general practice.

The aim of this study was to assess the effect of a nurse run asthma clinic on practice workload and the morbidity of patients in a four partner general practice. One hundred and fifteen asthmatic patients were studied and comparisons were made between the 12 months prior to the introduction of the clinic and the first 12 months after the clinic started. Morbidity was measured in terms of: the number of courses of oral steroids, the number of emergency salbutamol nebulizations, and the number of days lost from work or school. The number of consultations with the general practitioners were recorded. The repeat prescribing register was also monitored throughout the study to examine the effect of the clinic on prescribing in the practice. Consultations with general practitioners fell from a total of 818 to 414 during the study period (P less than 0.001). This was offset by 496 consultations with the nurse in the first 12 months of the clinic. As a result of attending the nurse clinic significant reductions (P less than 0.01) were found in the patients' requirements for courses of oral steroids, acute nebulizations and days lost from work or school. The results for the 46 children were similar to those for the 69 adults, confirming that the asthma clinic was effective for all ages. The clinic coincided with an increase in the number of patients receiving regular bronchodilator therapy and prophylactic medication. Eighty per cent of patients had their medication modified as a result of attending the clinic. The cost of prescribing remained remarkably stable.(ABSTRACT TRUNCATED AT 250 WORDS)

Betal chewing and caries experience in New Guinea.

The prevalence of dental caries and variables relating to the betel-chewing habit were examined in a sample of 301 rural New Guineans aged 12--24 years. Consistent inverse associations were demonstrated between caries experience and the intensity of betel chewing for individuals and aggregates of subjects. The results indicate that betel chewing confers a degree of protection against caries.

Sinusitis and the acute orbit in children.

Acute orbital infection is an uncommon condition which is often secondary to acute sinusitis. Although it can present in any age group it is most prevalent in children and may cause impaired vision, blindness, intracranial complications and death. This paper documents the experience at the Royal Liverpool Childrens Hospital, Alder Hey, from 1973 to 1989. Clinical details were recorded retrospectively from the hospital case notes. Sixty-eight children had orbital sepsis of whom 30 had associated acute sinusitis. Of these 30 children, orbital sepsis was always unilateral with a preference for the left side; ten had diplopia of whom four had a sub-periosteal abscess which was subsequently drained. There were no serious complications although two children had diplopia for two to three months.

A survey of patients seen in consultant clinics in conservative dentistry at Edinburgh Dental Hospital in 1990.

A baseline study of referrals by letter to a department of conservative dentistry staffed purely by honorary consultants was conducted for one year. Distinct differences were found between the majority of patients referred from general practice and those referred from other sources. Principal reasons for referral were problems of tooth replacement, appearance, endodontics and tooth wear. A predominance of female patients was seen in all groups except those complaining of tooth wear. 'Endodontic' patients featured the highest proportion of requests for hospital treatment. Complex restorative treatment was provided for the majority (70%) of referred patients, and about 10% received treatment from dental students. The rates of referral showed evidence of dependence on travelling distance to the referral centre but not on the availability of local GDPs. This emphasises the importance of maintaining existing specialist dental referral services in major population centres and the development of new services where appropriate.

Evaluation of peak flow and symptoms only self management plans for control of asthma in general practice.

OBJECTIVE: To compare a peak flow self management plan for asthma with a symptoms only plan. DESIGN: Randomisation to one of the self management plans and follow up for a year. SETTING: Four partner, rural training practice in Norfolk. SUBJECTS: 115 Patients (46 children and 69 adults) with asthma who were having prophylactic treatment for asthma and attending a nurse run asthma clinic. MAIN OUTCOME MEASURES: The number of doctor consultations, courses of oral steroids, and short term nebulised salbutamol treatments and the number of patients who required doctor consultations, courses of oral steroids, and short term nebulised salbutamol. RESULTS: Both self management plans produced significant reductions in the outcome measures but there were no significant differences in the degree of improvement between the groups. The results were similar for children and adults. The proportions of patients requiring a doctor consultation fell from 98% (50/51) to 66% (34/51) in the peak flow group and from 97% (62/64) to 53% (34/64) in the symptoms only group and the proportions requiring oral steroids from 73% (34/46) to 47% (21/46) and 52% (31/60) to 12% (7/60). The median number of doctor consultations was reduced from 8.0 to 2.0 in the peak flow group and from 4.5 to 1.0 in the symptoms only group. CONCLUSIONS: The peak flow meter was not the crucial ingredient in the improved illness of the two groups. Teaching patients the importance of their symptoms and the appropriate action to take when their asthma deteriorates is the key to effective management of asthma. Simply prescribing peak flow meters without a system of self management and regular review will be unlikely to improve patient care.

Metastatic carcinoma in the neck from an occult primary lesion.

Over an 11-year period, from January 1980 to December 1990, 23 patients with metastatic carcinoma of the neck were admitted to Addington Hospital with an unknown primary lesion. More than 50% of patients presented with disease in the N3 nodal stage. Sixteen patients had squamous cell carcinoma, 6 undifferentiated carcinoma and 1 adenocarcinoma. The diagnoses were made by means of fine-needle aspiration cytology and histological assessment of the neck nodes. Twenty-one patients were treated with radiotherapy and 2 with surgery. The primary tumour became apparent in only 4 (17%) patients, 7, 8, 9, and 12 months respectively after initial presentation. The sites were the base of tongue in 2, the pyriform fossa in 1 and the scalp in the remaining patient. The overall 3-year and 5-year survival rates were 26% and 17% respectively.

Preference and behavior of lactating dairy cows given free access to pasture at two herbage masses and two distances.

A number of factors influence dairy cow preference to be indoors or at pasture. The study reported here investigated whether herbage mass and distance affects preference and if continuously housed cows exhibited behavioral and production differences compared to cows that had free access to pasture. Dairy cows (n = 16) were offered a free choice of being in cubicle housing (1.5 cubicles/cow) or at pasture with a high (3,000 ± 200 kg DM/ha) vs. low (1,800 ± 200 kg DM/ha) herbage mass. A control group (n = 16) was confined to cubicle housing for the duration of the study. Each herbage mass was offered at either a near (38 m) or far (254 m) distance in a 2 × 2 factorial crossover design to determine motivation to access pasture. Overall, dairy cows expressed a partial preference to be at pasture, spending 68.7% of their time at pasture. This was not affected (P > 0.05) by herbage mass. Both grass intake (P = 0.001) and grazing time (P = 0.039) was greater when cows were offered the high herbage mass. Neither total mixed ration intake (P > 0.05) nor milk yield (P > 0.05) was affected by herbage mass or distance. Additionally, no interaction existed between herbage mass and distance (P > 0.05). Distance affected preference: overall time on pasture was greater at the near distance (P = 0.002); however, nighttime use was not affected by distance (P = 0.184). Housed cows produced less milk than free-choice cows and this was potentially due to a combination of decreased lying time in housed cows (P < 0.001) and grass intake (1.22 kg/d) in free-choice cows. This study shows that herbage mass is not a major factor driving dairy cow preference for pasture, but distance does affect preference for pasture during the day. Additionally, there are clear production and welfare benefits for providing cows with a choice to be at pasture or cubicle housing over being continuously housed. Further research is necessary to quantify the effect of lying time on milk yields.

Lyn kinase mediates cell motility and tumor growth in EGFRvIII-expressing head and neck cancer.

PURPOSE: EGF receptor variant III (EGFRvIII) has been detected in several cancers in which tumors expressing this truncated growth factor receptor show more aggressive behavior. The molecular mechanisms that contribute to EGFRvIII-mediated tumor progression that are amenable to targeted therapy are incompletely understood. The present study aimed to better define the role of Src family kinases (SFKs) in EGFRvIII-mediated cell motility and tumor growth of head and neck squamous cell carcinomas (HNSCC). EXPERIMENTAL DESIGN: HNSCC models expressing EGFRvIII were treated with dasatinib, a pharmacologic inhibitor of SFKs. RESULTS: SFK inhibition significantly decreased cell proliferation, migration, and invasion of EGFRvIII-expressing HNSCC cells. Administration of dasatinib to mice bearing EGFRvIII-expressing HNSCC xenografts resulted in a significant reduction of tumor volume compared with controls. Immunoprecipitation with anti-c-Src, Lyn, Fyn, and Yes antibodies followed by immunoblotting for phosphorylation of the SFK activation site (Y416) showed specific activation of Lyn kinase in EGFRvIII-expressing HNSCC cell lines and human HNSCC tumor specimens. Selective inhibition of Lyn using siRNA decreased cell migration and invasion of EGFRvIII-expressing HNSCCs compared with vector control cells. CONCLUSIONS: These findings show that Lyn mediates tumor progression of EGFRvIII-expressing HNSCCs in which strategies to inhibit SFK may represent an effective therapeutic strategy.