Dynamic responses of blood metabolites to nutrient depletion and repletion in broiler chicken nutrition.

A total of 720 male Cobb 500 broiler chicks were used in a 5 treatment and 8 replicate experiment to explore dynamic changes in blood metabolites in response to short-term nutrient depletion and repletion. Day old chicks were offered a corn and soybean meal-based common starter diet from d1 to 14 that was formulated to meet all nutrient requirements of the birds. From d15 to 17, the experimental diets were offered, before returning all groups to a common diet from d18 to 20, at which point the experiment was terminated. A total of 5 experimental diets were designed. A standard grower diet served as a control and was offered to 1 of the 5 groups of chicks. The additional 4 experimental groups comprised diets that were low in digestible phosphorus (P), total calcium (Ca), crude protein and digestible amino acids (AA) or apparent metabolizable energy (AME). The common grower diet that was offered from d18-20 was designed to be nutritionally complete and was intended to explore dynamic response to nutrient repletion. Blood was drawn from 8 chicks per treatment at time 0 (immediately prior to introduction of the experimental diets) and then again 3, 6, 12, 24, and 48h after introduction of the nutrient depleted diets. Additionally, blood was drawn 3, 6, 12, 24, and 48h after the introduction of the nutritionally complete common grower diet. Chicks were not sampled more than once. Feed intake, body weight and feed conversion ratio (FCR) were assessed on d14, 17, and 20. Blood metabolites were analyzed using the iSTAT Alinity V handheld blood analyzer, the Vetscan VS2 Chemistry Analyzer and the iCheck Carotene Photometer. Live performance metrics were not affected by the short-term nutrient depletion and all chicks grew normally throughout the experiment. The diet with low digestible P generated a rapid temporary decrease in plasma P and an increase in plasma Ca, that were returned to baseline following the re-introduction of the common grower feed. Introduction of the diet with low total Ca resulted in a significant increase in plasma P, effects which were also mitigated during the nutrient repletion phase. Total plasma protein, albumin and uric acid (UA) were decreased, and plasma glucose increased, in the chicks that received the diet with low crude protein and digestible AA. There was a delayed increase in aspartate amino transaminase (AST) associated with the diets with low digestible P and low AME. These results demonstrate the capacity of blood biochemistry to adapt to quantitative and qualitative changes in nutrient intake. Point-of-care analysis of blood biomarkers offers nutritionists a valuable opportunity to calibrate nutritional matrices for common dietary ingredients, zootechnical feed additives and to optimize diet phase changes. It can be concluded that many blood biomarkers are plastic to changes in diet nutrient density and offer an objective index for optimization of nutritional programs for commercial broiler production.

The anti-biofilm activity of lemongrass (Cymbopogon flexuosus) and grapefruit (Citrus paradisi) essential oils against five strains of Staphylococcus aureus.

AIMS: To determine the sensitivity of five strains of Staphylococcus aureus to five essential oils (EOs) and to investigate the anti-biofilm activity of lemongrass and grapefruit EOs. METHODS AND RESULTS: Antimicrobial susceptibility screening was carried out using the disk diffusion method. All of the strains tested were susceptible to lemongrass, grapefruit, bergamot and lime EOs with zones of inhibition varying from 2·85 to 8·60 cm although they were resistant to lemon EO. Lemongrass EO inhibited biofilm formation at 0·125% (v/v) as measured by colorimetric assay and at 0·25% (v/v) no metabolic activity was observed as determined by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction. Grapefruit EO did not show any anti-biofilm activity. Following exposure to lemongrass EO extensive disruption to Staph. aureus biofilms was shown under scanning electron microscopy. CONCLUSIONS: In comparison to the other EOs tested, lemongrass exhibited the most effective antimicrobial and anti-biofilm activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of lemongrass EO highlights its potential against antibiotic resistant Staph. aureus in the healthcare environment.

Systemic mastocytosis and probable mast cell leukaemia in a koala (Phascolarctos cinereus).

BACKGROUND: To the authors' knowledge, this is the first report of mast cell neoplasia in a koala (Phascolarctos cinereus). CASE REPORT: An adult female koala was presented for rapidly deteriorating health and death of a pouch young. Significant weight loss was apparent despite supplemental feeding; the abdomen was distended; and the koala was weak and mentally depressed. Haematology revealed a significant mastocytosis with a concurrent population of atypical mononuclear cells. The koala was euthanised and tissues were collected for histology. Bone marrow, lymph node, lung, stomach and spleen exhibited significant infiltration by mast cells. Atypical round cells consistent with those identified in the peripheral blood were also identified in the marrow. A diagnosis of systemic mastocytosis and probable mast cell leukaemia was made. Immunocytochemical and immunohistochemical staining was not able to further characterise the atypical cell population, and the mast cells exhibited only weak staining with CD117. CONCLUSION: The histological diagnosis, in this case, was systemic mastocytosis and myeloproliferative disease of uncertain origin. There was a dominant population of mast cells in the peripheral blood and marrow, and a population of circulating atypical mononuclear cells, appearing similar to mast cell leukaemia-acute myeloid leukaemia in humans.

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Tissue level mechanical properties of cortical bone in skeletally immature and mature dogs.

OBJECTIVES: The purpose of this study was to quantify the tissue level mechanical properties of cortical bone of skeletally immature (~five-month-old) Beagle dogs and compare them to data from mature dogs measured in a previous study. METHODS: Eight femoral cross sectional specimens (two bone sections / dog) were obtained from four skeletally immature dogs. A pair of calcein bone labels were administered intravenously to the dogs to mark sites of active mineralization prior to euthanasia. Prepared bone specimens were placed in a nano- indenter specimen holder and the previously identified calcein labelled osteons were located. Labelled (n = 128) and neighbouring unlabelled (n = 127) osteons in skeletally immature femurs were examined by instrumented indentation testing. Indents were made to a depth of 500 nm at a loading rate of 10 nm/s. Indentation modulus (IM) and hardness (H) were obtained. RESULTS: The overall IM of the cortical bone in the skeletally mature groups was significantly greater than in the immature group (p = 0.0011), however overall H was not significantly different. The differences between the groups in IM were significant for the unlabelled osteons (p = 0.001), but not for the labelled osteons (p = 0.56). CONCLUSION: There are differences in the IM of unlabelled osteons in skeletally immature and mature groups of Beagle dogs. In contrast to whole bone mechanical tests, where there are obvious differences between growing and mature bones, there are only small differences in the micro-mechanical properties.

Generation of MUC1-stimulated mononuclear cells using optimized conditions.

Mucin is a glycoprotein found on the surface of cell membranes of adenocarcinomas. The purpose of these studies was to generate MUC1 multiple tandem repeat (VNTR)-stimulated mononuclear cells (M1SMC). We first determined the optimal conditions to influence the immune response. In these studies, peripheral blood mononuclear cells (PBMC), from patients with adenocarcinomas, were stimulated by different numbers of M1SMC stimulations, various concentrations of MUC1 peptide, washing of PBMC prior to stimulation and days in culture, to determine the optimal conditions to influence the immune response. The results of this study indicate that the mononuclear cells (MC) stimulated twice 1 week apart with MUC1 VNTR1 produced a greater specific killing of the breast cancer cell line MCF-7 than the 0, 1, 3 or 4 weekly stimulations. The optimal molarity for inducing cytotoxicity and cytokines (granulocyte macrophage colony-stimulating factor, gamma-interferon and interleukin-10) was 45 x 10(-8) M (1 microg/ml); except for tumour necrosis factor (TNF)-alpha which was 22 x 10(-8) M (0.5 microg/ml). The unwashed MC were superior to washing them with Ficoll-Hypaque. The optimal number of days in culture for cytotoxicity and cytokine production was after two stimulations (i.e. after day 7). Optimum conditions for generation of M1SMC identified in these studies were two stimulations with peptide, concentration of 45 x 10(-8) M (1 microg/ml) peptide, unwashed cells, and after two stimulations or after 8 days in culture. M1SMC were generated from multiple patients with breast cancer which lysed adenocarcinoma cells.

Baseline morphometric, haematological and plasma biochemical parameters in free-ranging eastern water dragons (Intellagama lesueurii lesueurii).

OBJECTIVE: The aim of this study was to determine baseline reference data for morphometric measurements and haematological and plasma biochemical parameters in clinically healthy eastern water dragons (Intellagama lesueurii lesueurii), accounting for the variables of season and sex. The clinical objective was to provide clinicians and researchers with baseline reference intervals (RIs) in order to assess accurately the health of a population or individual animals. METHODS: The study group comprised 39 free-ranging eastern water dragons. To monitor seasonal changes in data two study periods were assigned: September-October 2009 (spring) and January-March 2010 (summer). The 18 males and 21 females were captured in the grounds of Taronga Zoo, Mosman (33°50'45'' S, 151°15'20'' E) in Sydney, New South Wales, Australia. Basic morphometric, haematological and plasma biochemical parameters were measured. RIs were generated in accordance with the American Society of Veterinary Clinical Pathology guidelines. Data were analysed for the effects of sex, season and sex-season interactions using restricted maximal likelihood modelling. RESULTS: Reference values were established in eastern water dragons for morphometric data and RIs were generated for haematological and plasma biochemical levels. The study showed seasonal variation in uric acid. Weight, total length, snout-vent length and basophil % showed significant differences according to sex. Sex-season interactions were detected for calcium, monocyte % and total protein (refractometry). CONCLUSION: Multiple parameters exhibited normal physiological variation between season and sex and an interaction between the two factors. These variables need to be considered in the clinical context when interpreting results of haematological and biochemical analyses in eastern water dragons. The RIs established from this population will provide a basis for further studies in both free-living and captive eastern water dragons, and for comparison in clinical cases.

Context of MUC1 epitope: immunogenicity.

MUC1 is a glycoprotein found at the secretory poles of normal cells but is hypoglycosylated on the entire surface of cell membranes of adenocarcinomas. In order to determine the influence on the immune response of peptide context for epitope presentation, peripheral blood mononuclear cells (PBMC) from patients with adenocarcinomas, were stimulated with MUC1 peptides derived from the 20 amino acids (aa) long sequence that is characteristic of the MUC1 Variable Number of Tandem Repeats (VNTR). In the seven peptides tested, the T-cell tumor-specific epitope (cTSE) was surrounded by variable numbers of aa and repeated up to 5 times in the same peptide. The results of this study indicate that cultures stimulated with peptide 610 (GSTAPPAHGVTS APDTRPAP) showed the highest specific killing of the MUC1-expressing breast cancer MCF-7 cells. Peptide 610 is also superior to the other peptides in inducing better production of the type 1 cytokines, tissue necrosis factor alpha and interferon gamma. In conclusion, context of the epitope and not sequence alone determines immunogenicity.

Communication education in veterinary in the United Kingdom and Ireland: the NUVACS project coupled to progressive individual school endeavors.

This article reports on the coordination of communications skills training in veterinary schools in the United Kingdom and Ireland and describes the progress and status of training that is occurring in six of these schools.

Sex identification in the eastern blue-tongued lizard (Tiliqua scincoides White, ex Shaw, 1790) using morphometrics.

OBJECTIVE: To establish a repeatable, well-tolerated, non-invasive technique for sex identification in eastern blue-tongued lizards (Tiliqua scincoides). METHODS: Measurements were obtained from 69 free living eastern blue-tongued lizards presented for treatment that were either deceased on arrival or subsequently euthanased on welfare grounds. Weight, head width (H), snout vent length (SVL) and trunk length (T) were recorded. Ratios for head width to snout vent length (H/SVL%) and head width to trunk length (H/T%) were calculated. Sex was identified at necropsy. RESULTS: Evidence of sexual dimorphism in this population was shown. However, the degree of difference was subtle, requiring the use of ratios in order to refine the technique. Ratios of H/SVL% and H/T% showed significant predictability with respect to sex identification in both adult and sub-adult lizards. CONCLUSION: This study established a protocol for a repeatable, non-invasive technique for sex identification in the eastern blue-tongued lizard (Tiliqua scincoides) and provides a normal reference range.

Human T lymphocytes express a protein-tyrosine kinase homologous to p56LSTRA.

We have used oligonucleotide probes, based on a portion of the p60v-src autophosphorylation sequence, Glu-Asp-Asn-Glu-Tyr-Thr, to identify and characterize a cDNA from the human T-leukemia cell line, JURKAT. The JURKAT cDNA (designated ptk-JURKAT) was homologous to but distinct from the src, yes and fgr oncogenes, which encode protein-tyrosine kinases (ATP:protein phosphotransferase, EC 2.7.1.37). The ptk-JURKAT cDNA hybridized with a 2.2 kb RNA transcript from JURKAT cells and the human T-cell lymphoma line, MOLT-4, but failed to identify any transcript in two human B-cell lymphoma lines or a human erythroid-myeloid leukemia line, K562. Recently the nucleotide sequence has been established for the murine lymphocyte protein tyrosine kinase, p56LSTRA. The ptk-JURKAT cDNA appears to encode the human homolog of p56LSTRA.

Effect of megestrol acetate on quality of life in a dose-response trial in women with advanced breast cancer. The Cancer and Leukemia Group B.

PURPOSE: The impact of the side effects of megestrol acetate on the quality of life of noncachectic women with advanced breast cancer was studied in a dose-response clinical trial of the Cancer and Leukemia Group B (CALGB 8741). Side effects of appetite increase and weight gain at higher doses were predicted to have a negative effect on quality of life. PATIENTS AND METHODS: Stage IV breast cancer patients were randomized to receive either 160, 800, or 1,600 mg/d of megestrol acetate. Quality of life was assessed in 131 patients at trial entry and at 1 and 3 months while on treatment, by telephone interview, using the following measures: the Functional Living Index-Cancer (FLIC), Rand Functional Limitations Scale, Rand Mental Health Inventory (MHI), the Body Image Subscale, and linear analog scales of drug side effects. RESULTS: At 3 months, women treated with 160 mg/d reported less severe side effects (P < .0005), better physical functioning (FLS, P < .0005), less psychologic distress (MHI, P = .008), and an improvement in overall quality of life (FLIC, P = .003) from the time of study entry as compared with those treated with 1,600 mg/d. Patients who received the 800-mg/d dose fell between the low- and high-dose arms in reported intensity of drug side effects, but responded similarly to those in the 160-mg/d group in terms of physical functioning, psychologic distress, and overall quality of life. CONCLUSION: Unless additional follow-up data demonstrate a survival advantage at higher doses, the 160-mg/d dose is optimal, achieving maximal treatment effect with the fewest side effects and better quality of life.

Induction in C57BL/KsJ mice of complement-dependent antibody cytotoxic to cultured beta cells.

Despite widespread evidence that autoimmune mechanisms may contribute to the beta cell necrosis associated with type I insulin-dependent diabetes mellitus (IDDM), it has not heretofore been demonstrated that islet cell antibodies (ICAs), directed primarily against cytoplasmic antigens, are capable of specific lysis of beta cells. We utilized a readily accessible source of mouse pancreatic islets [CBA/J mice lacking exocrine pancreas (exocrine pancreatic insufficiency syndrome)] to experimentally induce ICAs inbred mice. Homogenates of these islets were injected weekly for four weeks into syngeneic (CBA/J) and allogeneic (A/J, C57BL/6J, C57BL/KsJ) recipients. Only C57BL/KsJ inbred mice showed the induction of a high titer (greater than or equal to 160) antiserum cytotoxic to 51Cr-labeled CBA/J lymph node target cells. Neither the immunized C57BL/sJ mice with circulating ICAs nor any of the other immunized strains showed any decrease in glucose tolerance as compared with vehicle controls. Moreover, no morphologic evidence of islet necrosis or atrophy was apparent. Thus the ICAs induced were reactive with alloantigenic determinants of the donor and unreactive with antigenic determinants of the recipient strain. The C57BL/KsJ antiserum was further screened for anti-islet cell cytotoxic activity using both a 51Cr release assay from CBA/J islet cell monolayer cultures, and immunocytochemical staining of sections of Bouin's fixed, paraffin-embedded pancreas. This antiserum was cytotoxic to CBA/J beta cells in monolayer culture, but not the other non-beta islet cell types. Immune lysis of the beta cell required rabbit complement. At a concentration of 1% antiserum and 4% complement, beta cell lysis was evident between 3 and 4 h at 37 degrees C. Ultrastructural examination of beta cells exhibiting cytopathic changes revealed cytoplasmic disarray rather than any obvious lytic events at the plasma membrane. Grossly distended, rough, endoplasmic reticulum containing intracisternal type A retrovirus was the most prominent feature distinguishing antiserum and control serum-treated beta cells. This model system suggests that ICAs which recognize beta cell cytoplasmic antigens are capable of specifically lysing beta cells via a complement-dependent mechanism. Immunocytochemical staining revealed that, in addition to islet beta-cells,, the antiserum (1/500 dilution) stained a macrophage-like cell in the spleen and lymph nodes, as well as an epithelial-like cell in the thymus. The possibility is discussed that this multiple specificity may have been due to passenger leukocytes present in the islet homogenates used to immunize.

Separating population structure from population history: a cladistic analysis of the geographical distribution of mitochondrial DNA haplotypes in the tiger salamander, Ambystoma tigrinum.

Nonrandom associations of alleles or haplotypes with geographical location can arise from restricted gene flow, historical events (fragmentation, range expansion, colonization), or any mixture of these factors. In this paper, we show how a nested cladistic analysis of geographical distances can be used to test the null hypothesis of no geographical association of haplotypes, test the hypothesis that significant associations are due to restricted gene flow, and identify patterns of significant association that are due to historical events. In this last case, criteria are given to discriminate among contiguous range expansion, long-distance colonization, and population fragmentation. The ability to make these discriminations depends critically upon an adequate geographical sampling design. These points are illustrated with a worked example: mitochondrial DNA haplotypes in the salamander Ambystoma tigrinum. For this example, prior information exists about restricted gene flow and likely historical events, and the nested cladistic analyses were completely concordant with this prior information. This concordance establishes the plausibility of this nested cladistic approach, but much future work will be necessary to demonstrate robustness and to explore the power and accuracy of this procedure.

Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation.

A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.

Evaluation of methods of administering tyramine to raise systolic blood pressure.

To compare the relative merits of two different administration regimens, tyramine was administered intravenously in ascending doses to 12 healthy subjects to raise systolic blood pressure slightly more than 30 mm Hg. Six subjects received tyramine by bolus injection and six other subjects received tyramine by infusion. The bolus dose of tyramine needed was 4.34 +/- 1.51 mg (X +/- SD) and the infusion rate needed was 1.11 +/- 0.33 mg/min. Four blood pressure response patterns to continuous tyramine infusion were observed. Because different units were measured for the quantity of tyramine administered, the between-subject variance estimate to within-subject variance estimate ratios were calculated. The two techniques had equivalent consistency. With the bolus method, in contrast to the infusion procedure, the dose-response relationship was obvious in most subjects. Therefore the bolus method was judged to be more useful than the infusion method.

A noninvasive protocol for anterior temporal lobectomy.

We report the results of a protocol for choosing candidates for temporal lobectomy using a standard battery of objective tests without intracranial electrodes. We assigned each test a level of importance, and an algorithm was used to determine whether temporal lobectomy could be performed. Fifty-one patients (total pool, 103 patients) met protocol requirements and had an anterior temporal lobectomy with a mean follow-up of 39.4 months (range, 21 to 64 months), most remaining on anticonvulsant therapy. Eighty percent are seizure free, 12% have less than 3 seizures per year or only nocturnal seizures, and 8% have greater than 80% reduction in seizure frequency. One-third of patients who failed protocol criteria did not have temporal lobe seizures when studied with intracranial electrodes. We analyzed and modified the algorithm after comparing these patients with others who were poor candidates for temporal lobectomy. We conclude that this protocol is effective and recommend using such an objective algorithm.

Predictive value of the intracarotid amobarbital test in bihemispheric seizure onset.

The intracarotid amobarbital test (IAT) is used to lateralize language function and assess hemispheric memory. In this study 23 non-lesional epileptic patients were evaluated. The magnitude of the difference in IAT scores between the two hemispheres was significantly greater in the patients whose seizures lateralized on scalp or intracranial EEG than in patients with bihemispheric seizure onset. This suggests that the IAT is useful not only in predicting lateralized seizure focus, but bihemispheric onset as well.

Prostaglandins, thromboxanes and the pregnant rat uterus at term.

1 Prostaglandin and thromboxane release from the term pregnant (Day 22) rat uterus in vitro has been measured by radioimmunoassay and gas chromatography combined with mass spectrometry. 2 Prostacyclin (prostaglandin I2, PGI2) and thromboxane A2 (TXA2) (measured as their metabolites, 6-oxo-PGF1 alpha and TXB2, respectively) were released in large amounts, while PGE2 and PGF2 alpha were released in smaller amounts. PGD2 was released in the largest quantities. 3 Treatment of the term pregnant rat uterus in vitro with the PGI2 synthesis inhibitors, 15-hydroperoxy arachidonic acid (15-OOH AA) and tranylcypromine caused spasm of the tissue. 4 15-OOH AA caused dose-dependent increases in prostaglandin release, while tranylcypromine caused a fall in the release of PGE2 but did not affect the release of other prostaglandins. A possible reason for the effect of 15-OOH AA on prostaglandin release is discussed. 5 Indomethacin prevented spontaneous activity of the term pregnant rat uterus in vitro. Contractions were restored by prostaglandins and their order or potency was PGE2 greater than PGF2 alpha greater than PGI2 greater than PGD2 much greater than 6-oxo-PGF1 alpha = TXB2.

Complete analysis of the presenilin 1 gene in early onset Alzheimer's disease.

The presenilin 1 gene has recently been identified as the locus on chromosome 14 which is responsible for a large proportion of early onset, autosomal dominantly inherited Alzheimer's disease (AD). We have elucidated the intron/exon structure of the gene and designed intronic primers to enable direct sequencing of the entire coding region (10 exons) of the presenilin gene in a large number of families. This strategy has enabled us to find a further two novel mutations in the gene. We discuss the distribution of mutations and the proportions of autosomal dominant AD with a mean age of onset below 60 years caused by mutations in this gene.