Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.

Contingency theories of leadership: how might we use them in clinical practice?

This article explores how contingency theories of leadership (pragmatic theories that note 'no one size fits all') can be used by multidisciplinary health-care teams to improve communication and patient care.

The Cornell Net Carbohydrate and Protein System: Updates to the model and evaluation of version 6.5.

New laboratory and animal sampling methods and data have been generated over the last 10 yr that had the potential to improve the predictions for energy, protein, and AA supply and requirements in the Cornell Net Carbohydrate and Protein System (CNCPS). The objectives of this study were to describe updates to the CNCPS and evaluate model performance against both literature and on-farm data. The changes to the feed library were significant and are reported in a separate manuscript. Degradation rates of protein and carbohydrate fractions were adjusted according to new fractionation schemes, and corresponding changes to equations used to calculate rumen outflows and postrumen digestion were presented. In response to the feed-library changes and an increased supply of essential AA because of updated contents of AA, a combined efficiency of use was adopted in place of separate calculations for maintenance and lactation to better represent the biology of the cow. Four different data sets were developed to evaluate Lys and Met requirements, rumen N balance, and milk yield predictions. In total 99 peer-reviewed studies with 389 treatments and 15 regional farms with 50 different diets were included. The broken-line model with plateau was used to identify the concentration of Lys and Met that maximizes milk protein yield and content. Results suggested concentrations of 7.00 and 2.60% of metabolizable protein (MP) for Lys and Met, respectively, for maximal protein yield and 6.77 and 2.85% of MP for Lys and Met, respectively, for maximal protein content. Updated AA concentrations were numerically higher for Lys and 11 to 18% higher for Met compared with CNCPS v6.0, and this is attributed to the increased content of Met and Lys in feeds that were previously incorrectly analyzed and described. The prediction of postruminal flows of N and milk yield were evaluated using the correlation coefficient from the BLUP (R(2)BLUP) procedure or model predictions (R(2)MDP) and the concordance correlation coefficient. The accuracy and precision of rumen-degradable N and undegradable N and bacterial N flows were improved with reduced bias. The CNCPS v6.5 predicted accurate and precise milk yield according to the first-limiting nutrient (MP or metabolizable energy) with a R(2)BLUP=0.97, R(2)MDP=0.78, and concordance correlation coefficient=0.83. Furthermore, MP-allowable milk was predicted with greater precision than metabolizable energy-allowable milk (R(2)MDP=0.82 and 0.76, respectively, for MP and metabolizable energy). Results suggest a significant improvement of the model, especially under conditions of MP limitation.

Three-dimensional cell manipulation and patterning using dielectrophoresis via a multi-layer scaffold structure.

Cell manipulation is imperative to the areas of cellular biology and tissue engineering, providing them a useful tool for patterning cells into cellular patterns for different analyses and applications. This paper presents a novel approach to perform three-dimensional (3D) cell manipulation and patterning with a multi-layer engineered scaffold. This scaffold structure employed dielectrophoresis as the non-contact mechanism to manipulate cells in the 3D domain. Through establishing electric fields via this multi-layer structure, the cells in the medium became polarized and were attracted towards the interior part of the structure, forming 3D cellular patterns. Experiments were conducted to evaluate the manipulation and the patterning processes with the proposed structure. Results show that with the presence of a voltage input, this multi-layer structure was capable of manipulating different types of biological cells examined through dielectrophoresis, enabling automatic cell patterning in the time-scale of minutes. The effects of the voltage input on the resultant cellular pattern were examined and discussed. Viability test was performed after the patterning operation and the results confirmed that majority of the cells remained viable. After 7 days of culture, 3D cellular patterns were observed through SEM. The results suggest that this scaffold and its automated dielectrophoresis-based patterning mechanism can be used to construct artificial tissues for various tissue engineering applications.

Retention rate of Gabapentin in children with intractable epilepsies at 1 year.

Gabapentin (GAB) is a newer second-line antiepileptic drug (AED) used in children. This is a multi-centre retrospective observational study of the efficacy, tolerability and retention rate in 105 children, aged 0-17.5 years (mean 10.1) over a 14 year period. The median age of epilepsy onset was 2.5 years (range 0-14.6). 72% started GAB as at least the 3rd AED, with 43% having been withdrawn from at least 2 AEDs. 77% had focal and 52% symptomatic epilepsies. The maintenance doses for GAB ranged 6.0-87.3 mg/kg/day (mean 43.7). The study comprised 157 person-treatment years for GAB. GAB was well tolerated with 55% remaining on treatment beyond 1 year. No serious adverse events were reported whilst on GAB, but 39% reported possibly and probably related adverse events. Seizure improvement (<50% seizure frequency compared to baseline) at more than 12 months of treatment, was reported in 35% of patients starting GAB, including 6% who remained seizure free. The results demonstrated the efficacy and tolerability of GAB in children with difficult to treat epilepsies, and a good response to treatment beyond 12 months, in both focal and generalised epilepsies.

Retention rate of clobazam, topiramate and lamotrigine in children with intractable epilepsies at 1 year.

UNLABELLED: Clobazam (CLB), Topiramate (TOP) and Lamotrigine (LAM) are newer second-line antiepileptic drugs (AEDs) used in children. This is a single-centre retrospective observational study of the efficacy, tolerability and retention rate in 224 separate treatment episodes in 194 children, aged 0.1-16.7 years (median 9.4) over an 8 year period. The median age of epilepsy onset was 3.3 years (range 0-15.1). 79% started CLB, TOP or LAM as at least the 3rd AED, with 39% having been withdrawn from at least 2 AEDs. 53% had generalised and 37% idiopathic epilepsies. The maintenance doses for CLB ranged 0.12-3.50 mg/kg/day (mean 0.7); for TOP 0.45-32.0 mg/kg/day (mean 7.1) and for LAM 1.13-16.0 mg/kg/day (mean 5.6). The study comprised 75 person-treatment years for CLB, 56 for TOP, 124 for LAM. RESULTS: CLB, TOP and LAM were well tolerated with 51%, 37% and 69% remaining on treatment beyond 1 year respectfully. 1 serious adverse event for CLB (inducing seizures) and 2 for LAM (rashes) were reported, and 60%, 47% and 39% had possibly and probably related adverse events for CLB, TOP and LAM respectively. Beyond 12 months seizure improvement (< 50% seizure frequency compared to baseline) was reported in 43%, 35% and 44% on CLB, TOP and LAM, including 5% and 8% remaining seizure free on CLB and LAM respectively. CONCLUSION: Our results demonstrate the efficacy and tolerability of CLB, TOP and LAM in children with difficult to treat epilepsies and a good response in CLB and LAM, and a reasonable response in TOP beyond 12 months.

Piezoelectric driven non-toxic injector for automated cell manipulation.

Stimulated by state-of-the-art robotic and computer technology, Intra Cytoplasmic Sperm Injection (ICSI) automation aims to scale and seamlessly transfer the human hand movements into more precise and fast movements of the micro manipulator. Piezo-drill cell injection, a novel technique using piezo-driven pipettes with a very small mercury column, has significantly improves the survival rates of ICSI process. It is found that complications are due, in large part, to toxicity of mercury and the damage to the cell membrane because of the lateral tip oscillations of injector pipette. In this paper, a new design of piezo-driven cell injector is proposed for automated suspended cell injection. This new piezo-driven cell injector design centralizes the piezo oscillation power on the injector pipette which eliminates the vibration effect on other parts of the micromanipulator. Detrimental lateral tip oscillations of the injector pipette are attenuated to a desirable level even without the help of mercury column. This mercury-free injector can sublime the piezoelectric driven injection technique to completely non-toxic level with great research and commercial application in gene injection, in-vitro fertilization, ICSI and drug development.

The effects of feeding medium-chain triglycerides on the growth, insulin responsiveness, and body composition of Holstein calves from birth to 85 kg of body weight.

The objective of this study was to determine the effects of feeding calves isocaloric, isonitrogenous diets that varied in the amount and type of fatty acids on growth, response to an insulin challenge, and body composition. Thirty-six calves were assigned to a randomized block design with 3 dietary treatments, 10 calves per treatment, and a baseline group of 6 calves. Three different milk-replacer-based diets were designed to deliver less than 2% of the lipid as medium-chain triglycerides (control; diet contained no added medium-chain triglycerides), 32% medium-chain triglycerides primarily as caprylate (CAP oil), and 32% of fatty acids primarily as laurate from coconut oil (CCO). Calves were offered 0.28 Mcal of intake energy/kg of body weight (BW)0.75 from d 1 to 7 and 0.32 Mcal of intake energy/kg of BW0.75 adjusted weekly for BW from d 8 to harvest. Dry matter, intake energy, crude protein, and fat intakes were 53.7 kg, 281.8 Mcal, 14.6 kg, and 13.0 kg; 56.6 kg, 297.2 Mcal, 15.8 kg, and 14.2 kg; and 53.8 kg, 280.4 Mcal, 15.4 kg, and 13.3 kg for the control, CAP oil, and CCO treatments, respectively. Dry matter, energy, protein, and fat intakes did not differ among treatments. At approximately 65 kg of BW, 5 calves per treatment were given an insulin challenge. After the challenge the decrease in plasma glucose concentration was greater for the calves fed the CAP oil diet compared with those fed the control and CCO diets. Calves were harvested at approximately 88 kg of BW. Empty body gains were 0.92, 0.79, and 0.87 kg/d for control-, CAP oil-, and CCO-fed calves, respectively, and the gains of the CAP oil-fed calves were less than those of the control-fed calves. Empty body crude protein, ash, and water were not different among treatments. Empty body retained energy and fat tended to be 5.6 and 8.7% greater for calves consuming the CCO diet than for those fed the control diet. The livers of calves consuming the CCO diet were 330 g heavier and contained 15% more fat than the livers of the control and CAP oil calves. The results of this study demonstrate that the energy demand of the calf to maintain body temperature resulted in increased oxidation of intake energy; thus, overall body composition differences could not be detected. However, the intake of CCO increased the accumulation of lipid in the liver and carcass despite the apparent cold stress conditions.

Congenital myasthenic syndrome with sleep hypoventilation.

We report the case of a 13-year-old boy with acetylcholinesterase deficiency, a congenital myasthenic syndrome, who developed sleep hypoventilation syndrome during a period of rapid growth. His symptoms were insidious and life-threatening despite changes in strength or lung volume measurements that were not marked. He responded well to noninvasive nocturnal ventilation, with reversal of symptoms and normalization of blood gases. His lung volumes, but not motor function, improved after treatment.

Duplications and de novo deletions of the SMNt gene demonstrated by fluorescence-based carrier testing for spinal muscular atrophy.

Approximately 95% of individuals with spinal muscular atrophy (SMA) lack both copies of the SMNt gene at 5q13. The presence of a nearly identical centromeric homolog of the SMNt gene, SMNc, necessitates a quantitative polymerase chain reaction approach to direct carrier testing. Adapting a radioactivity-based method described previously, multiplex polymerase chain reaction was performed using fluorescently labeled primers followed by analysis on an ABI 373a DNA sequencer. The SMNt copy number was calculated from ratios of peak areas using both internal and genomic standards. Samples from 60 presumed carriers (50 parents of affected individuals and 10 relatives implicated by linkage analysis) and 40 normal control individuals were tested. Normalized results (to the mean of five or more control samples harboring two copies of the SMNt gene) were consistently within the ranges of 0.4 to 0.6 for carriers (one copy) and 0.8 to 1.2 for normal controls (two copies), without overlap. Combining linkage analyses with direct carrier test results demonstrated de novo deletions associated with crossovers, unaffected individuals carrying two SMNt gene copies on one chromosome and zero SMNt gene copies on the other chromosome, and unaffected individuals with three copies of the SMNt gene. This report demonstrates that fluorescence-based carrier testing for SMA is accurate, reproducible, and useful for genetic risk assessment, and that carrier testing may need to be combined with linkage analysis in certain circumstances.

Inhibition of the erbB-2 tyrosine kinase receptor in breast cancer cells by phosphoromonothioate and phosphorodithioate antisense oligonucleotides.

Antisense activity against erbB-2 of a variety of sulfur-modified oligonucleotides was examined in a breast cancer cell line which overexpresses this oncogene. Using a 15 base anti-erbB-2 sequence previously shown to be effective, various backbone configurations containing phosphoromonothioate or phosphorodithioate linkages were evaluated for antisense activity by a two-color flow cytometric assay. This sequence was effective in inhibiting the production of erbB-2 protein when it was configured as a monothioate at each linkage and as an alternating dithioate/phosphodiester. Both of these compounds were also able to specifically inhibit erbB-2 mRNA expression, indicative of RNase H-mediated activity. The same sequence protected by either three dithioate or three monothioate linkages at each end was ineffective as an antisense reagent, suggesting that endonuclease activity is a significant determinant of the stability of oligonucleotides. Finally, the erbB-2 sequence target was shifted in an effort to improve antisense activity. A new lead sequence was identified that was significantly more effective in inhibiting erbB-2 protein levels and retained activity at lower concentrations.

Interpersonal dependency in alcoholic and obese men.

While psychological conflict about dependency needs of alcoholic and obese persons has been widely observed, few studies have examined differences in dependency characteristics between these clinical groups. The Interpersonal Dependency Inventory was administered to 22 alcoholic and 8 morbidly obese men in intensive treatment for alcohol and obesity. The original hypothesis that alcoholic and obese men would show similar dependency needs was supported. Dependency correlates of personality may serve as useful predictor variables in the clinical treatment of alcoholic and obese persons.

A note on interpersonal sensitivity and psychotic symptomatology in obese adult outpatients with a history of childhood obesity.

The Symptom Checklist 90-Revised (Derogatis, 1975) was administered to 37 obese adults in outpatient treatment for obesity. Individuals who had become obese during childhood showed greater interpersonal sensitivity and exhibited more psychotic symptoms than those who had become obese later in life. The findings support the belief that morbid obesity is characteristically associated with elevated levels of internal psychological conflict.

Interpersonal dependency correlates and locus of control orientation among obese adults in outpatient treatment for obesity.

Interpersonal dependency and locus of control orientation were studied in obese and nonobese samples. The Interpersonal Dependency Inventory (Hirschfeld, Klerman, Gough, Barrett, & Korchin, 1977) and the I-E Scale (Rotter, 1966) were administered to 106 obese adults in outpatient treatment for obesity. The I-E Scale was also administered to 97 nonobese control subjects. As predicted, the obese subjects were significantly more internally oriented than has been previously identified in obese populations. These findings challenge currently held assumptions about locus of control in obese groups. Although the obese and nonobese subjects maintained similar overall dependency scores, the obese subjects were more likely to show low levels of autonomy in comparison with the nonobese subjects. Interpersonal dependency and locus of control dimensions associated with obese conditions may serve as useful predictor variables influencing obesity treatment approaches and outcome.

Locus of control orientation among obese adults in outpatient treatment for obesity.

We studied locus of control (LOC) in samples of obese and nonobese subjects. We administered Rotter's (1966) I-E Scale to 106 obese adults in outpatient treatment for obesity and to 99 nonobese controls. The obese subjects were significantly more internally oriented than the control group. These findings challenge currently held assumptions on the directionality of LOC in obese groups. The results contradict the consensus among researchers that obese individuals have an external LOC orientation. The finding that internal LOC is associated with the obese condition may be a useful predictor variable influencing obesity treatment, approaches, and outcome.

Perceptions of excessive alcohol consumption in stressful and nonstressful situations among undergraduates.

We devised and administered the Alcohol Perceptions Questionnaire (APQ) to 56 male and 51 female undergraduates in a small midwestern university. Findings yielded statistically significant results between the level of alcohol consumption reported by the subjects and their perceptions of excessive drinking in interpersonal situations, social group situations, and under conditions of subjective stress. The greater the amount of personal alcohol consumption among the sample, the less likely they viewed excessive drinking as a problem. In addition, subjects from lower income environments viewed excessive drinking in general as a problem, whereas subjects from higher income environments viewed excessive drinking as no problem at all.

The relationship between childhood onset obesity and psychopathology in adulthood.

We administered the Symptom Checklist (Derogatis, 1975; SCL-90-R) to 37 obese subjects in outpatient treatment for obesity. Patients with early onset obesity demonstrated a greater frequency and higher levels of emotional distress and psychiatric symptomatology than patients with late onset obesity. Individuals who developed obesity in childhood showed more psychopathology than those who developed obesity later in life. Overall, these findings support the belief that obesity is characteristically associated with greater internal psychological conflict. These findings further suggest that childhood obesity could serve as a predictor variable for possible future psychological disturbance in obese populations.

Locus of control orientation of obese adolescent girls and children of alcoholics in outpatient treatment.

Obese adolescents and children of alcoholics have been reported to exhibit an external locus of control orientation. Due to the perceived loss of control over personal eating behavior versus limited control over environmental circumstances, it was believed that obese adolescent girls would show greater externality than children of alcoholics. Rotter's I-E Scale was administered to 19 moderately obese adolescent girls and 10 girls who were children of alcoholics in outpatient treatment. While both groups scored within the external range of control orientation, there was no statistically significant difference between the samples. Contrary to prediction, obese adolescent girls and adolescents from alcoholic environments have similar world views on control orientation.