The Integration of Proteome-Wide PTM Data with Protein Structural and Sequence Features Identifies Phosphorylations that Mediate 14-3-3 Interactions.

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called "14-3-3 site-finder". We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.

Heterogeneous mass distribution of the rubble-pile asteroid (101955) Bennu.

The gravity field of a small body provides insight into its internal mass distribution. We used two approaches to measure the gravity field of the rubble-pile asteroid (101955) Bennu: (i) tracking and modeling the spacecraft in orbit about the asteroid and (ii) tracking and modeling pebble-sized particles naturally ejected from Bennu's surface into sustained orbits. These approaches yield statistically consistent results up to degree and order 3, with the particle-based field being statistically significant up to degree and order 9. Comparisons with a constant-density shape model show that Bennu has a heterogeneous mass distribution. These deviations can be modeled with lower densities at Bennu's equatorial bulge and center. The lower-density equator is consistent with recent migration and redistribution of material. The lower-density center is consistent with a past period of rapid rotation, either from a previous Yarkovsky-O'Keefe-Radzievskii-Paddack cycle or arising during Bennu's accretion following the disruption of its parent body.

R-spondin 2 mediates neutrophil egress into the alveolar space through increased lung permeability.

OBJECTIVE: R-spondin 2 (RSPO2) is required for lung morphogenesis, activates Wnt signaling, and is upregulated in idiopathic lung fibrosis. Our objective was to investigate whether RSPO2 is similarly important in homeostasis of the adult lung. While investigating the characteristics of bronchoalveolar lavage in RSPO2-deficient (RSPO2-/-) mice, we observed unexpected changes in neutrophil homeostasis and vascular permeability when compared to control (RSPO2+/+) mice at baseline. Here we quantify these observations to explore how tonic RSPO2 expression impacts lung homeostasis. RESULTS: Quantitative PCR (qPCR) analysis demonstrated significantly elevated myeloperoxidase (MPO) expression in bronchoalveolar lavage fluid (BALF) cells from RSPO2-/- mice. Likewise, immunocytochemical (ICC) analysis demonstrated significantly more MPO+ cells in BALF from RSPO2-/- mice compared to controls, confirming the increase of infiltrated neutrophils. We then assessed lung permeability/barrier disruption via Fluorescein Isothiocyanate (FITC)-dextran instillation and found a significantly higher dextran concentration in the plasma of RSPO2-/- mice compared to identically treated RSPO2+/+ mice. These data demonstrate that RSPO2 may be crucial for blood-gas barrier integrity and can limit neutrophil migration from circulation into alveolar spaces associated with increased lung permeability and/or barrier disruption. This study indicates that additional research is needed to evaluate RSPO2 in scenarios characterized by pulmonary edema or neutrophilia.

Assessment of Instrumental Activities of Daily Living in Older Adults with Subjective Cognitive Decline Using the Virtual Reality Functional Capacity Assessment Tool (VRFCAT).

BACKGROUND: Continuing advances in the understanding of Alzheimer's disease progression have inspired development of disease-modifying therapeutics intended for use in preclinical populations. However, identification of clinically meaningful cognitive and functional outcomes for individuals who are, by definition, asymptomatic remains a significant challenge. Clinical trials for prevention and early intervention require measures with increased sensitivity to subtle deficits in instrumental activities of daily living (IADL) that comprise the first functional declines in prodromal disease. Validation of potential endpoints is required to ensure measure sensitivity and reliability in the populations of interest. OBJECTIVES: The present research validates use of the Virtual Reality Functional Capacity Assessment Tool (VRFCAT) for performance-based assessment of IADL functioning in older adults (age 55+) with subjective cognitive decline. DESIGN: Cross-sectional validation study. SETTING: All participants were evaluated on-site at NeuroCog Trials, Durham, NC, USA. PARTICIPANTS: Participants included 245 healthy younger adults ages 20-54 (131 female), 247 healthy older adults ages 55-91 (151 female) and 61 older adults with subjective cognitive decline (SCD) ages 56-97 (45 female). MEASURES: Virtual Reality Functional Capacity Assessment Tool; Brief Assessment of Cognition App; Alzheimer's Disease Cooperative Study Prevention Instrument Project - Mail-In Cognitive Function Screening Instrument; Alzheimer's Disease Cooperative Study Instrumental Activities of Daily Living - Prevention Instrument, University of California, San Diego Performance-Based Skills Assessment - Validation of Intermediate Measures; Montreal Cognitive Assessment; Trail Making Test- Part B. RESULTS: Participants with SCD performed significantly worse than age-matched normative controls on all VRFCAT endpoints, including total completion time, errors and forced progressions (p≤0001 for all, after Bonferonni correction). Consistent with prior findings, both groups performed significantly worse than healthy younger adults (age 20-54). Participants with SCD also performed significantly worse than controls on objective cognitive measures. VRFCAT performance was strongly correlated with cognitive performance. In the SCD group, VRFCAT performance was strongly correlated with cognitive performance across nearly all tests with significant correlation coefficients ranging from 0.3 to 0.7; VRFCAT summary measures all had correlations greater than r=0.5 with MoCA performance and BAC App Verbal Memory (p<0.01 for all). CONCLUSIONS: Findings suggest the VRFCAT provides a sensitive tool for evaluation of IADL functioning in individuals with subjective cognitive decline. Strong correlations with cognition across groups suggest the VRFCAT may be uniquely suited for clinical trials in preclinical AD, as well as longitudinal investigations of the relationship between cognition and function.

Tuning the endothelial response: differential release of exocytic cargos from Weibel-Palade bodies.

Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation. The molecules that contribute to these processes are co-stored in secretory granules. How can the cells control release of granule content to allow differentiated responses? Selected agonists recruit an exocytosis-linked actin ring to boost release of a subset of cargo. SUMMARY: Background Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease.

A partially differentiated interior for (1) Ceres deduced from its gravity field and shape.

Remote observations of the asteroid (1) Ceres from ground- and space-based telescopes have provided its approximate density and shape, leading to a range of models for the interior of Ceres, from homogeneous to fully differentiated. A previously missing parameter that can place a strong constraint on the interior of Ceres is its moment of inertia, which requires the measurement of its gravitational variation together with either precession rate or a validated assumption of hydrostatic equilibrium. However, Earth-based remote observations cannot measure gravity variations and the magnitude of the precession rate is too small to be detected. Here we report gravity and shape measurements of Ceres obtained from the Dawn spacecraft, showing that it is in hydrostatic equilibrium with its inferred normalized mean moment of inertia of 0.37. These data show that Ceres is a partially differentiated body, with a rocky core overlaid by a volatile-rich shell, as predicted in some studies. Furthermore, we show that the gravity signal is strongly suppressed compared to that predicted by the topographic variation. This indicates that Ceres is isostatically compensated, such that topographic highs are supported by displacement of a denser interior. In contrast to the asteroid (4) Vesta, this strong compensation points to the presence of a lower-viscosity layer at depth, probably reflecting a thermal rather than compositional gradient. To further investigate the interior structure, we assume a two-layer model for the interior of Ceres with a core density of 2,460-2,900 kilograms per cubic metre (that is, composed of CI and CM chondrites), which yields an outer-shell thickness of 70-190 kilometres. The density of this outer shell is 1,680-1,950 kilograms per cubic metre, indicating a mixture of volatiles and denser materials such as silicates and salts. Although the gravity and shape data confirm that the interior of Ceres evolved thermally, its partially differentiated interior indicates an evolution more complex than has been envisioned for mid-sized (less than 1,000 kilometres across) ice-rich rocky bodies.

Cow- and farm-level risk factors for lameness on dairy farms with automated milking systems.

Lameness is a major concern to animal health and welfare within the dairy industry. Our objectives were to describe the prevalence of lameness in high-producing cows on farms with automated milking systems (AMS) and to identify the main risk factors for lameness at the animal and farm level. We visited 36 AMS farms across Canada and Michigan. Farm-level factors related to stall design, bedding use, flooring, and stocking rates were recorded by trained observers. Cows were scored for lameness, leg injuries, body condition (BCS), and body size (hip width and rump height; n=1,378; 25-40 cows/farm). Mean herd prevalence of clinical lameness was 15% (range=2.5-46%). Stall width relative to cow size and parity was found to be the most important factor associated with lameness. Not fitting the average stall width increased the odds of being lame 3.7 times in primiparous cows. A narrow feed alley [<430cm; odds ratio (OR)=1.9], obstructed lunge space (OR=1.7), a low BCS (OR=2.1 for BCS ≤2.25 compared with BCS 2.75-3.0), and presence of hock lesions (OR=1.6) were also identified as important risk factors for lameness. Only 1 of 36 farms had stalls of adequate width and length for the cows on their farm. For lameness prevention, it can be concluded that more emphasis needs be placed on either building stalls of appropriate width or selecting for smaller-framed cows that fit the existing stalls.

Lying times of lactating cows on dairy farms with automatic milking systems and the relation to lameness, leg lesions, and body condition score.

Lying down and resting are important for optimal cow health, welfare, and production. In comparison with free stall farms with a milking parlor, farms with automated milking systems (AMS) may place less constraint on how long cows can lie down. However, few studies report lying times on AMS farms. The aims of this study were to describe the variation in lying times of dairy cows in AMS farms and to understand how much of the variation in individual lying times is related to cow-level factors, including lameness, the presence of hock and knee lesions, and body condition score (BCS). We visited 36 farms in Canada (Quebec: n = 10; Ontario: n = 10; British Columbia: n = 4; and Alberta: n = 5), and the United States (Michigan: n = 7). Gait scores, presence of hock and knee lesions, and BCS were recorded for 40 Holstein cows from each herd. Parity and days in milk were retrieved from farm records. Lying time was recorded across 4d using accelerometers (n = 1,377). Multivariable analysis was performed. Of scored cows, 15.1% were lame (i.e., obviously limping; 203 of 1,348 cows). Knee lesions were found in 27.1% (340 of 1,256 cows) and hock lesions were found in 30.8% (421 of 1,366 cows) of the animals. Daily lying time varied among cows. Cows spent a median duration of 11.4 h/d lying down (25th-75th percentile = 9.7-12.9 h), with a lying bout frequency of 9.5 bouts/d (25th-75th percentile = 7.5-12 bouts/d) and a median bout duration of 71 min (25th-75th percentile = 58-87 min/bout). Lameness was associated with cows lying down for 0.6 h/d longer in fewer, longer bouts. Increased lying time was also associated with increased parity, later stage of lactation and higher BCS. Older cows (parity ≥ 3) spent about 0.5 h/d more lying down compared with parity 1 cows, and cows with BCS ≥ 3.5 lay down on average 1 h/d longer than cows with BCS ≤ 2.25. Hock lesions were associated with shorter lying times in univariable models, but no associations were found in the multivariable models. We concluded that only a small proportion of the variation between cows in lying time is explained by lameness, leg lesions, and BCS.

Testing Refrigeration Trucks for the Emergency Evacuation of Companion Animals.

The purpose of this study was to quantify the changes in oxygen (O2) and carbon dioxide (CO2) in sealed refrigerator trucks scheduled to be used for transporting companion animals (dogs and cats) during an emergency evacuation. A total of 122 nonhuman animals (total weight = 1,248 kg) housed in individual crates were loaded into a 16-m refrigeration truck. Once they were loaded, the doors were closed and the percentages of O2 and CO2 were measured every 5 min by O2 and CO2 analyzers, and they were used to quantify the changes in gas pressure in the sealed truck. CO2 had a much higher-than-predicted increase, and O2 had a higher-than-predicted decrease. These 2 pressures in combination with the functionality of the respiratory system will limit the animal's ability to load O2, and over time, they will initiate asphyxia or suffocation. Over time, the partial pressure of oxygen (PO2) in the sealed truck will decrease, causing hypoxia, and the partial pressure of carbon dioxide (PCO2) will increase, causing hypercapnia.

Seasonal acclimatization determined by non-invasive measurements of coat insulation.

Seasonal acclimatization in terrestrial mammals in the Northern Hemisphere involves changes in coat insulation. It is more economical to provide increased insulation than increased heat production for protection against the cold. This study was done to test a technique for the non-invasive measurement of mammal coat insulation and to measure coat insulation over several seasons on captive exotics. The working hypothesis was that species that have no coat or have a coat that does not change seasonally do not acclimatize seasonally. Three surface temperature readings were measured from the torso area. The insulation was calculated using measured metabolic rates and body temperature when possible. The African elephants, giraffe and okapi did not acclimatize with average maximum insulation values of 0.256°Cm(2) W(-1) . The Amur tigers and mountain goats acclimatized to seasonal ambient conditions by increasing the insulation values of the hair coats in the cold with an average maximum insulation values of 0.811°Cm(2) W(-1) . The cold adapted species are more than three times more insulated in the cold than the equatorial species. The husbandry implications of exotics that have no ability to acclimatize to Northern Hemisphere seasonal ambient changes are profound. Giraffe, African elephants, and okapi when exposed to cold conditions with ambient air temperatures below 21°C will use body energy reserves to maintain a heat balance and will require housing that provides ambient conditions of 21°C.

Unmeasured costs of haemophilia: the economic burden on families with children with haemophilia.

INTRODUCTION AND OBJECTIVES: Although economic evaluations of haemophilia-related care have highlighted both the health care payer and societal perspectives, the costs to families with children with haemophilia have not been examined. This study determined the costs incurred by families of children with haemophilia, attending a haemophilia treatment centre (HTC), servicing a large geographical area in Eastern Canada. METHODS: Families recorded all direct and indirect costs associated with haemophilia-related care for a year. Costs incurred to receive care at the HTC and local health care centres were compared. The relationship between distance to the HTC and costs was modelled using linear regression. RESULTS: Participants included 31/45 children (68%) from 27 families attending the HTC. Median age was 12 years (range: 0.5-17 years); 24/31 (77%) had severe haemophilia. The median distance to the HTC and local health care facility was 230 km (range: 7-600 km) and 33.5 km (range: 2-400 km) respectively. Due to this difference in distance, 23/31 (74%) children do not attend the HTC for management of acute haemorrhage. The median annual total cost per family to attend the HTC is $775.93 (range: $200.00-$5741.00). The total cost to attend the HTC increases by $2.16 (95% CI 1.24-3.9) per kilometer from the HTC. The median total annual cost of haemophilia-related care per family is $1222.50 (range: $396.00-$8037.00). CONCLUSION: Families incur high costs related to haemophilia care. The distance to the HTC is a barrier to care. Improving access to HTCs is paramount in improving haemophilia-related outcomes.

Relationship between locomotor play of dairy calves and their weight gains and energy intakes around weaning.

Play behaviour has been proposed as a measure of good welfare in growing animals and locomotor play by calves is often reduced after weaning off milk. Adjusting weaning age according to individual calves' abilities to eat solid feed maintains energy intake and weight gain during weaning. We investigated the effects of this method of weaning on locomotor play of calves and the relationship between locomotor play and energy intake and weight gains. We measured the running behaviour of 56 Holstein heifer calves before and after weaning. Calves were housed in groups of eight, fed milk, grain starter and hay from automated feeders. Weaning began when their voluntary intake of grain starter reached either 200 or 400 g/day, and weaning was completed when starter intake reached either 800 or 1600 g/day. Before weaning, older calves ran less than young ones; and the duration of running correlated with weight gains and digestible energy intake. Immediately after weaning, digestible energy intake and locomotor play decreased but no correlation was observed between these variables. One week after weaning, the duration of running was correlated with both energy intake and weight gain. Digestible energy intake increased but locomotor play continued to decrease. The amount of running a calf does after weaning partly reflects energy intake and weight gain, supporting suggestions that locomotor play is good indicator of welfare and fitness of growing animals. However, the decline in locomotor play following weaning is not solely due to decreased energy intake.

Swiss ball enhances lumbar multifidus activity in chronic low back pain.

OBJECTIVES: This study examined the effects of sitting surfaces on the cross-sectional area of lumbar multifidus (LM) in patients with Chronic Low Back Pain (CLBP) and healthy controls (HC). DESIGN: Cross-Sectional Case Controlled Study. SETTING: Isle of Man Institute of Sport. PARTICIPANTS: 40 age and sex matched, sporting participants aged 18-45 years, recruited from private physiotherapy practice patients (n = 20 CLBP, 16 male, 4 female, and n = 20 healthy controls, 16 males and 4 females). MAIN OUTCOME MEASURES: Cross-sectional area of LM was measured using rehabilitative ultrasound imaging. RESULTS: Swiss Ball (SB) was more effective at stimulating LM than a Stable Surface (SS) in both groups: CLBP:SB:12.3 (cm(2)) (SD:3.6), SS:10.15 (SD:2.6), p < 0.0001; HC:SB:12.5 (SD:2.7), SS:11.3 (SD:2.9), p < 0.0001). No significant differences between groups were noted. No differences between left and right side cross-sectional areas between or within groups were noted. CONCLUSION: Cross-sectional area of LM increased as the lability of the surface increased, demonstrating that SB was more effective at stimulating LM activity than a non-labile surface. This confirms current clinical practice and supports the use of a labile surface in spinal rehabilitation. The lack of LM asymmetry within and between groups is discussed.

The effect of pioglitazone and extended-release niacin on HDL-cholesterol in diabetes patients in a real-world setting.

OBJECTIVE: This retrospective study was designed to assess the effects of the combination of pioglitazone and extended-release niacin on the lipid panel, particularly HDL-cholesterol, when used in patients with type 2 diabetes in an endocrinology specialty practice. METHODS: The electronic medical records of 434 adult patients with type 2 diabetes receiving extended-release niacin and pioglitazone were screened for review. Patients with type 2 diabetes and hyperlipidemia were included for review if they received the combination of pioglitazone at doses ≥ 15 mg/day and extended-release niacin (Niaspan) at doses ≥ 1000 mg/day for ≥6 months. Statistical analysis used paired t-tests with p < 0.05 as statistically significant. Both ANOVA and the Tukey-Kramer test for multiple comparisons (α = 0.05) were also used. RESULTS: A total of 47 patients, 83% were men with average age of 58, met all eligibility criteria for the study. Compared with baseline, a statistically significant increase in HDL-C (+ 25.13%, p < 0.0001) was observed at the conclusion of combination therapy. The HDL-C levels progressively increased with duration of combination treatment, and were not correlated with concomitant statin use. Significant decreases in total cholesterol and triglycerides were detected, and HbA1c decreased 0.84% during combination therapy for all therapies combined. CONCLUSION: The combination of pioglitazone and extended-release niacin in patients with type 2 diabetes and hyperlipidemia, used in commonly prescribed doses for at least 6 months, resulted in statistically significant improvements in HDL-C, total cholesterol, and triglycerides, and did not result in deteriorations in glycemic control.

Tyrosine-derived polycarbonate-silica xerogel nanocomposites for controlled drug delivery.

Biodegradable polymer-ceramic composites offer significant potential advantages in biomedical applications where the properties of either polymers or ceramics alone are insufficient to meet performance requirements. Here we demonstrate the highly tunable mechanical and controlled drug delivery properties accessible with novel biodegradable nanocomposites prepared by non-covalent binding of silica xerogels and co-polymers of tyrosine-poly(ethylene glycol)-derived poly(ether carbonate). The Young's moduli of the nanocomposites exceed by factors of 5-20 times those of the co-polymers or of composites made with micron scale silica particles. Increasing the fraction of xerogel in the nanocomposites increases the glass transition temperature and the mechanical strength, but decreases the equilibrium water content, which are all indicative of strong non-covalent interfacial interactions between the co-polymers and the silica nanoparticles. Sustained, tunable controlled release of both hydrophilic and hydrophobic therapeutic agents from the nanocomposites is demonstrated with two clinically significant drugs, rifampicin and bupivacaine. Bupivacaine exhibits an initial small burst release followed by slow release over the 7 day test period. Rifampicin release fits the diffusion-controlled Higuchi model and the amount released exceeds the dosage required for treatment of clinically challenging infections. These nanocomposites are thus attractive biomaterials for applications such as wound dressings, tissue engineering substrates and stents.

Minimum cost of transport in Asian elephants: do we really need a bigger elephant?

Body mass is the primary determinant of an animal's energy requirements. At their optimum walking speed, large animals have lower mass-specific energy requirements for locomotion than small ones. In animals ranging in size from 0.8 g (roach) to 260 kg (zebu steer), the minimum cost of transport (COT(min)) decreases with increasing body size roughly as COT(min)∝body mass (M(b))(-0.316±0.023) (95% CI). Typically, the variation of COT(min) with body mass is weaker at the intraspecific level as a result of physiological and geometric similarity within closely related species. The interspecific relationship estimates that an adult elephant, with twice the body mass of a mid-sized elephant, should be able to move its body approximately 23% cheaper than the smaller elephant. We sought to determine whether adult Asian and sub-adult African elephants follow a single quasi-intraspecific relationship, and extend the interspecific relationship between COT(min) and body mass to 12-fold larger animals. Physiological and possibly geometric similarity between adult Asian elephants and sub-adult African elephants caused body mass to have a no effect on COT(min) (COT(min)∝M(b)(0.007±0.455)). The COT(min) in elephants occurred at walking speeds between 1.3 and ∼1.5 m s(-1), and at Froude numbers between 0.10 and 0.24. The addition of adult Asian elephants to the interspecific relationship resulted in COT(min)∝M (-0.277±0.046)(b). The quasi-intraspecific relationship between body mass and COT(min) among elephants caused the interspecific relationship to underestimate COT(min) in larger elephants.

Preliminary evidence for biologic activity of toceranib phosphate (Palladia(®)) in solid tumours.

The purpose of this study was to provide an initial assessment of the potential biologic activity of toceranib phosphate (Palladia®, Pfizer Animal Health, Madison, NJ, USA) in select solid tumours in dogs. Cases in which toceranib was used to treat dogs with apocrine gland anal sac adenocarcinoma (AGASACA), metastatic osteosarcoma (OSA), thyroid carcinoma, head and neck carcinoma and nasal carcinoma were included. Clinical benefit (CB) was observed in 63/85 (74%) dogs including 28/32 AGASACA [8 partial response (PR), 20 stable disease (SD)], 11/23 OSAs (1 PR and 10 SD), 12/15 thyroid carcinomas (4 PR and 8 SD), 7/8 head and neck carcinomas [1 complete response (CR), 5 PR and 1 SD] and 5/7 (1 CR and 4 SD) nasal carcinomas. For dogs experiencing CB, the median dose of toceranib was 2.8 mg kg(-1) , 36/63 (58.7%) were dosed on a Monday/Wednesday/Friday basis and 47/63 (74.6%) were treated 4 months or longer. Although these data provide preliminary evidence that toceranib exhibits CB in dogs with certain solid tumours, future prospective studies are necessary to define its true activity.

Posthumous diagnosis of long QT syndrome from neonatal screening cards.

BACKGROUND: Molecular autopsy in sudden unexplained death in the young (SUDY) victims cannot usually be performed if tissue suitable for DNA extraction is not retained at autopsy. OBJECTIVE: The purpose of this study was to assess the feasibility and clinical value of posthumous genetic testing for long QT syndrome (LQTS) using residual material from the neonatal screening (Guthrie) card in SUDY victims. METHODS: Twenty-one cases were investigated up to 13 years after death. Deaths occurred at <1 year in one, 1-18 years in 18, and 19-35 years in two patients. Guthrie cards were 3-39 years old. DNA was extracted, and amplicons corresponding to the coding regions of the LQTS genes 1, 2, 3, 5, and 6 underwent either denaturing high-performance liquid chromatography screening or direct DNA sequencing. RESULTS: Adequate DNA was extracted in every case, although repeated purification and amplification was often required. Rare variants were detected in six of 19 cases undergoing diagnostic screening. Four (21%) are considered to be pathological and have been used for family screening: R243C and H455Y in KCNQ1 in 12-year-old and 13-year-old boys, respectively, and Q81H and S621R in KCNH2 in 21-month and 28-year-old females, respectively. Variants of uncertain significance were R1047L in KCNH2 in a 2-year-old girl and S38G in KCNE1 in a 19-month-old boy. Point mutation tests for previously identified familial LQTS mutations revealed a positive result in both cases: E146K in KCNQ1 and exon 6-4del in KCNH2. CONCLUSION: Residual material from Guthrie cards collected for newborn metabolic screening can be used as a reliable source of DNA for the posthumous diagnosis of LQTS decades after SUDY, although purification and amplification of DNA is time intensive.

On optical depth profiling using confocal Raman spectroscopy.

Until 2006 the performance of confocal Raman spectroscopy depth profiling was typically described and modeled through the application of geometrical optics, including refraction at the surface, to explain the degree of resolution and the precise form of the depth profile obtained from transparent and semicrystalline materials. Consequently a range of techniques, physical and analytical, was suggested to avoid the errors thus encountered in order to improve the practice of Raman spectroscopy, if not the understanding of the underlying mechanisms. These approaches were completely unsuccessful in accounting for the precise form of the depth profile, the fact that spectra obtained from laminated samples always contain characteristic peaks from all materials present both well above and below the focal point and that spectra can be obtained when focused some 40 mum above the sample surface. This paper provides further evidence that the physical processes underlying Raman spectroscopy are better modeled and explained through the concept of an extended illuminated volume contributing to the final Raman spectrum and modeled through a photon scattering approach rather than a point focus ray optics approach. The power of this numerical model lies in its ability to incorporate, simultaneously, the effects of degree of refraction at the surface (whether using a dry or oil objective lens), the degree of attenuation due to scatter by the bulk of the material, the Raman scattering efficiency of the material, and surface roughness effects. Through this we are now able to explain why even removing surface aberration and refraction effects through the use of oil immersion objective lenses cannot reliably ensure that the material sampled is only that at or close to the point of focus of the laser. Furthermore we show that the precise form of the depth profile is affected by the degree of flatness of the surface of the sample. Perhaps surprisingly, we show that the degree of flatness of the material surface is, in fact, more important than obtaining a precise refractive index match between the immersion oil and the material when seeking a high-quality depth profile or Raman spectrum from within a transparent or semicrystalline material, contrary to accepted norms that samples for interrogation by Raman spectroscopy require little preparation.