Behavioural variability, physical activity, rumination time, and milk characteristics of dairy cattle in response to regrouping.

In the commercial dairy industry worldwide, it is common practice to periodically regroup cows as part of their management strategy within housed systems. While this animal husbandry practice is intended to improve management efficiency, cows may experience social stress as a result of the social environment changes, which may have an impact on their behavioural patterns, performance, and welfare. We investigated whether regrouping altered dairy cows' behaviour and impacted their cortisol concentration (a physiological marker of stress), oxytocin, milk yield, and quality in a robotic milking system. Fifty-two lactating cows (17 primiparous; 35 multiparous) were moved in groups of 3-5 individuals into established pens of approximately 100 cows. Behaviour of the regrouped cows was directly observed continuously for 4 h/day across 4-time blocks (day-prior (d-1), day-of regrouping (d0), day-after (d + 1), and 6-days after (d + 6) regrouping). Cows were categorised as being with others, alone, or feeding every 2.5 min prior to the assessment of behavioural dynamics. Milk yield (MY) and composition, total daily activity, and rumination time (RUM) data were extracted from the Lely T4C management program (Lely Industries, Maassluis, the Netherlands), and milk samples were collected for cortisol and oxytocin concentration analyses; data were analysed using linear mixed-effect modelling. Primiparous cows were less likely to be interacting with others on d + 1 than d-1 compared with multiparous. However, average bout duration (minutes) between being alone and feeding activity states were similar on d-1, d + 1, and d + 6, for both primiparous and multiparous cows. A reduction in the average alone and feeding bout duration was observed on d0. Multiparous cows spent significantly more total time being alone on d0 compared to d-1. Neither regrouping nor parity statistically influenced milk DM content, energy, or cortisol concentration. Primiparous cows produced 3.80 ±â€¯2.42 kg (12.2%) less MY on d + 1 compared to their d-1, whereas multiparous cows did not change MY. A significant decrease of 0.2% fat was found in both parity groups following regrouping and remained low up to d + 6. Daily activity in both parity groups increased significantly and RUM reduced after regrouping. A significant decrease in oxytocin concentration was observed in all cows on d + 1. The results, specifically for primiparous cows, indicated a negative impact of regrouping on social interactions, due to changes in the social environment which may lead to short-term social instability. Multiparous cows may benefit from previous regrouping experiences.

Palaeoproteomics guidelines to identify proteinaceous binders in artworks following the study of a 15th-century painting by Sandro Botticelli's workshop.

Undertaking the conservation of artworks informed by the results of molecular analyses has gained growing importance over the last decades, and today it can take advantage of state-of-the-art analytical techniques, such as mass spectrometry-based proteomics. Protein-based binders are among the most common organic materials used in artworks, having been used in their production for centuries. However, the applications of proteomics to these materials are still limited. In this work, a palaeoproteomic workflow was successfully tested on paint reconstructions, and subsequently applied to micro-samples from a 15th-century panel painting, attributed to the workshop of Sandro Botticelli. This method allowed the confident identification of the protein-based binders and their biological origin, as well as the discrimination of the binder used in the ground and paint layers of the painting. These results show that the approach is accurate, highly sensitive, and broadly applicable in the cultural heritage field, due to the limited amount of starting material required. Accordingly, a set of guidelines are suggested, covering the main steps of the data analysis and interpretation of protein sequencing results, optimised for artworks.

Influence of environmental factors and parity on milk yield dynamics in barn-housed dairy cattle.

We investigated the effects of environmental factors on average daily milk yield and day-to-day variation in milk yield of barn-housed Scottish dairy cows milked with an automated milking system. An incomplete Wood gamma function was fitted to derive parameters describing the milk yield curve including initial milk yield, inclining slope, declining slope, peak milk yield, time of peak, persistency (time in which the cow maintains high yield beyond the peak), and predicted total lactation milk yield (PTLMY). Lactation curves were fitted using generalized linear mixed models incorporating the above parameters (initial milk yield, inclining and declining slopes) and both the indoor and outdoor weather variables (temperature, humidity, and temperature-humidity index) as fixed effects. There was a higher initial milk yield and PTLMY in multiparous cows, but the incline slope parameter and persistency were greatest in primiparous cows. Primiparous cows took 54 d longer to attain a peak yield (mean ± standard error) of 34.25 ± 0.58 kg than multiparous (47.3 ± 0.45 kg); however, multiparous cows yielded 2,209 kg more PTLMY. The best models incorporated 2-d lagged minimum temperature. However, effect of temperature was minimal (primiparous decreased milk yield by 0.006 kg/d and multiparous by 0.001 kg/d for each degree increase in temperature). Both primiparous and multiparous cows significantly decreased in day-to-day variation in milk yield as temperature increased (primiparous cows decreased 0.05 kg/d for every degree increase in 2-d lagged minimum temperature indoors, which was greater than the effect in multiparous cows of 0.008 kg/d). Though the model estimates for both indoor and outdoor were different, a similar pattern of the average daily milk yield and day-to-day variation in milk yield and milk yield's dependence on environmental factors was observed for both primiparous and multiparous cows. In Scotland, primiparous cows were more greatly affected by the 2-d lagged minimum temperature compared with multiparous cows. After peak lactation had been reached, primiparous and multiparous cows decreased milk yield as indoor and outdoor minimum temperature increased.

Direct evidence of milk consumption from ancient human dental calculus.

Milk is a major food of global economic importance, and its consumption is regarded as a classic example of gene-culture evolution. Humans have exploited animal milk as a food resource for at least 8500 years, but the origins, spread, and scale of dairying remain poorly understood. Indirect lines of evidence, such as lipid isotopic ratios of pottery residues, faunal mortality profiles, and lactase persistence allele frequencies, provide a partial picture of this process; however, in order to understand how, where, and when humans consumed milk products, it is necessary to link evidence of consumption directly to individuals and their dairy livestock. Here we report the first direct evidence of milk consumption, the whey protein ß-lactoglobulin (BLG), preserved in human dental calculus from the Bronze Age (ca. 3000 BCE) to the present day. Using protein tandem mass spectrometry, we demonstrate that BLG is a species-specific biomarker of dairy consumption, and we identify individuals consuming cattle, sheep, and goat milk products in the archaeological record. We then apply this method to human dental calculus from Greenland's medieval Norse colonies, and report a decline of this biomarker leading up to the abandonment of the Norse Greenland colonies in the 15(th) century CE.

Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants.

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.

4-Hydroxyproline metabolism and glyoxylate production: A target for substrate depletion in primary hyperoxaluria?

The primary hyperoxalurias are diseases of overproduction of oxalate. The immediate precursor of oxalate is glyoxylate. Metabolism of hydroxyproline, derived from collagen turnover or the diet, appears to be a major source of glyoxylate, and a potential target for a therapeutic strategy of substrate depletion.

Consequences of missense mutations for dimerization and turnover of alanine:glyoxylate aminotransferase: study of a spectrum of mutations.

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme, deficiency of which results in primary hyperoxaluria type 1 (PH1). More than 65 PH1-related mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have generated a spectrum of 15 missense changes including the most common PH1 mutation, G170R, and expressed them on the appropriate background of the major or minor allele, in an Escherichia coli overexpression system and in a rabbit reticulocyte transcription/translation system. We have investigated their effects on enzyme activity, dimerization, aggregation, and turnover. The effect of pyridoxal phosphate (PLP) on dimerization and stability was also investigated. Although all 15 mutant AGTs were expressed as intact proteins in E. coli, only three: G41R and G41V on the major allele, and the common mutation G170R, resulted in significant amounts of enzymatic activity. Dimerization failure was a frequent observation (13/15) except for G41V and D183N. Dimerization was poor with S187F but was substantially improved with PLP. Proteasome-mediated protein degradation was observed for all the mutations except G41R on the major allele, G41V, D183N, G170R, and S218L. Increases in the stability of the mutant enzymes in the presence of PLP were small; however, G41R on the minor allele showed a direct relationship between its half life and the concentration of PLP. The minor allele AGT product and many of the mutants were subject to a limited non-proteasomal proteolytic cleavage when ATP was depleted.

P boutons in lamina IX of the rodent spinal cord express high levels of glutamic acid decarboxylase-65 and originate from cells in deep medial dorsal horn.

Presynaptic inhibition of primary muscle spindle (group Ia) afferent terminals in motor nuclei of the spinal cord plays an important role in regulating motor output and is produced by a population of GABAergic axon terminals known as P boutons. Despite extensive investigation, the cells that mediate this control have not yet been identified. In this work, we use immunocytochemistry with confocal microscopy and EM to demonstrate that P boutons can be distinguished from other GABAergic terminals in the ventral horn of rat and mouse spinal cord by their high level of the glutamic acid decarboxylase (GAD) 65 isoform of GAD. By carrying out retrograde labeling from lamina IX in mice that express green fluorescent protein under the control of the GAD65 promoter, we provide evidence that the cells of origin of the P boutons are located in the medial part of laminae V and VI. Our results suggest that P boutons represent the major output of these cells within the ventral horn and are consistent with the view that presynaptic inhibition of proprioceptive afferents is mediated by specific populations of interneurons. They also provide a means of identifying P boutons that will be important in studies of the organization of presynaptic control of Ia afferents.

The development of targeted chemotherapy for CNS lymphoma-a pilot study of the IDARAM regimen.

PURPOSE: We have developed and evaluated a CNS-targeted chemotherapy regimen based on the pharmacokinetic properties of the individual drugs in the combination. PATIENTS AND METHODS: In a twin-track study, 16 patients with secondary CNS lymphoma (SCNSL) and 8 with primary CNS lymphoma (PCNSL) were treated with IDARAM which comprised idarubicin 10 mg/m(2) i.v., days 1 and 2; dexamethasone 100 mg, 12-h infusion, days 1, 2 and 3; cytosine arabinoside (ARA-C) 1.0 g/m(2), 1-h infusion, days 1 and 2; methotrexate 2.0 g/m(2), 6-h infusion, day 3 (with folinic acid rescue); and cytosine arabinoside 70 mg plus methotrexate 12 mg, intrathecally, days 1 and 8. Two cycles were delivered at 3-weekly intervals. After response assessment, patients received adjuvant cranial radiotherapy (40 Gy over 20 fractions). RESULTS: The series comprised 24 patients, 11 male and 13 female. Their median age was 53 years (range 21 to 73 years). Grade 4 neutropenia and thrombocytopenia occurred in the majority of patients treated. Of the eight PCNSL patients, seven achieved complete remission (CR). Four remained in CR at the time of this report with a median duration of follow-up of 25 months (range 11 to 42 months). Of the 16 SCNSL patients, 12 achieved CR. Seven patients remained in CR at the time of this report with a median duration of follow-up of 24 months (range 18 to 57 months). CONCLUSION: This study suggests that IDARAM is an effective regimen in both PCNSL and SCNSL and is suitable for further development and evaluation.

Total registration of non-Hodgkin's lymphoma and Hodgkin's disease in Scotland: effect of deprivation and caseload on outcome.

All cases S16 years of age with a histological diagnosis of non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) presented in Scotland between 1 January 1994 and 31 December 1996 were registered prospectively in the Scotland and Newcastle Lymphoma Group database by a process of total registration. The census population of Scotland in 1996-1997 was 5.1 million. One thousand seven hundred and sixty three patients were registered with NHL and 350 patients with HD. These patients have been followed up for a median of 47 months in the case of NHL and 51 months for HD cases. Actuarial 5-year survival for adult NHL was 35% and for HD, 75%. Outcome for both NHL and HD was particularly poor in the population over 60 years with median survival of 18 months for NHL and 27 months for HD. When analysis of survival was related to degree of material deprivation using the Carstairs score a significantly poorer survival was seen for NHL with increasing deprivation that could not be explained by a different pattern of age or stage at presentation. Deprivation had no impact on incidence or survival in HD. Analysis of impact of caseload of the physician initiating therapy showed no significant difference in 5-year survival.

Distribution and colocalisation of glutamate decarboxylase isoforms in the rat spinal cord.

The inhibitory neurotransmitter GABA is synthesized by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord have shown that whilst both are present in the dorsal horn, GAD67 is the predominant form in the ventral horn. The present study was carried out to determine the pattern of coexistence of the two GAD isoforms in axonal boutons in different laminae of the cord, and also to examine the relation of the GADs to the glycine transporter GLYT2 (a marker for glycinergic axons), since many spinal neurons are thought to use GABA and glycine as co-transmitters. Virtually all GAD-immunoreactive boutons throughout the spinal grey matter were labelled by both GAD65 and GAD67 antibodies; however, the relative intensity of staining with the two antibodies varied considerably. In the ventral horn, most immunoreactive boutons showed much stronger labelling with the GAD67 antibody, and many of these were also GLYT2 immunoreactive. However, clusters of boutons with high levels of GAD65 immunoreactivity were observed in the motor nuclei, and these were not labelled with the GLYT2 antibody. In the dorsal horn, some GAD-immunoreactive boutons had relatively high levels of labelling with either GAD65 or GAD67 antibody, whilst others showed a similar degree of labelling with both antibodies. GLYT2 immunoreactivity was associated with many GAD-immunoreactive boutons; however, this did not appear to be related to the pattern of GAD expression. It has recently been reported that there is selective depletion of GAD65, accompanied by a loss of GABAergic inhibition, in the ipsilateral dorsal horn in rats that have undergone peripheral nerve injuries [J Neurosci 22 (2002) 6724]. Our finding that some boutons in the superficial laminae showed relatively high levels of GAD65 and low levels of GAD67 immunoreactivity is therefore significant, since a reduction in GABA synthesis in these axons may contribute to neuropathic pain.

The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn.

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid-lumbar spinal cord of the rat. The great majority (94-100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2-immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin- and enkephalin-containing boutons (including somatostatin-immunoreactive boutons that lacked calcitonin gene-related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present. We also investigated VGLUT expression in central terminals of primary afferents. Myelinated afferents were identified with cholera toxin B subunit; most of those in lamina I were VGLUT2-immunoreactive, whereas all those in deeper laminae were VGLUT1-immunoreactive, and some (in laminae III-VI) appeared to contain both transporters. However, peptidergic primary afferents that contained substance P or somatostatin (most of which are unmyelinated), as well as nonpeptidergic C fibres (identified with Bandeiraea simplicifolia isolectin B4) showed low levels of VGLUT2-immunoreactivity, or were not immunoreactive with either VGLUT antibody. As all primary afferents are thought to be glutamatergic, this raises the possibility that unmyelinated afferents, most of which are nociceptors, express a different vesicular glutamate transporter.

Levels of male-specific RNA bacteriophage and Escherichia coli in molluscan bivalve shellfish from commercial harvesting areas.

AIMS: Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator organism, male-specific RNA (FRNA) bacteriophage has been proposed for this role. This study compared the distribution of E. coli and FRNA bacteriophage in shellfish harvesting areas. METHODS AND RESULTS: A total of 608 shellfish samples from 49 shellfish harvesting areas were analysed for E. coli and FRNA bacteriophage using standard published methods. The geometric mean concentration of FRNA bacteriophage in all samples was over three times greater than that of E. coli (1800 and 538 counts/100 g for FRNA bacteriophage and E. coli, respectively). In contrast to E. coli, FRNA bacteriophage concentrations were strongly influenced by season with a geometric mean count of 4503 PFU/100 g in the winter (October-March) compared with 910 PFU/100 g in the summer (April-September). CONCLUSIONS: FRNA bacteriophage were present in shellfish at higher concentrations than E. coli. Elevated levels of FRNA bacteriophage observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in the UK. Levels of FRNA bacteriophage found in many shellfish from category B harvesting areas would not be eliminated by conventional treatment processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study will inform future proposals to introduce FRNA bacteriophage as an indicator of the viral risk associated with shellfish.

A population-based study of intensive multi-agent chemotherapy with or without autotransplant for the highest risk Hodgkin's disease patients identified by the Scotland and Newcastle Lymphoma Group (SNLG) prognostic index. A Scotland and Newcastle Lymphoma Group study (SNLG HD III).

The aim of the study was to identify all patients with poor risk Hodgkin's disease (HD) using a numerical prognostic index in a defined population and to recruit them into a trial of intensive chemotherapy prednisolone, vinblastine, doxorubicin, chlorambucil, etoposide, bleomycin, vincristine, procarbazine (PVACE-BOP)x3+autotransplant (Arm A) versus PVACE-BOPx5 (Arm B) in first remission. In 10 years, the Scotland and Newcastle Lymphoma Group (SNLG) registered 930 patients with HD of whom 178 (19%) were identified as 'poor risk' by the SNLG index and were aged 16-59 years. 126/178 (71%) entered the study. Of the 120 confirmed poor risk HD cases, all completed PVACE-BOPx3 with a 93% Complete Response/unconfirmed Complete Response (CR/CRu) rate. Only 65/107 in CR accepted the randomisation. With a median follow-up of 6 years, both arms of the trial have a similar time to treatment failure (TTF) (Arm A 79%+/-11 versus 85%+/-7 Arm B, P=0.35). Advanced stage 'good risk' patients not included in the trial receiving standard therapy with CLVPP or ABVD had a 75% 5-year survival. The study demonstrates that PVACE-BOP therapy in the poorest risk group (58% had an IPI>/=3) produces excellent CR rates (93%) and overall survival with minimal toxicity, and that the substitution of autotransplant in first CR does not improve outcome. The use of the objective SNLG index accurately helped in the selection of the poorest risk group in this population study. The placing of a randomised control trial within the context of a population-based study of HD enhances the validity of the outcome.

Three novel deletions in the alanine:glyoxylate aminotransferase gene of three patients with type 1 hyperoxaluria.

We describe three novel deletions in the human AGT gene in three patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme, alanine glyoxylate aminotransferase (AGT; EC 2.6.1.44). A deletion of 4 nucleotides in the exon 6/intron 6 splice junction (679-IVS6+2delAAgt) is expected to cause missplicing. It would also code for a K227E missense alteration in any mRNA successfully spliced. A 2-bp deletion in exon 11 (1125-1126del CG, cDNA) results in a frameshift. A deletion of at least 5-6 kb, EX1 EX5del, spanned exons 1-5 and contiguous upstream sequence. All three deletions are heterozygous with previously documented missense mutations; the intron 6 deletion with F152I, the exon 11 deletion with G82E, and EX1 EX5del with the common mistargeting mutation, G170R.

Superposition of macroscopic numbers of atoms and molecules.

We theoretically examine photoassociation of a nonideal Bose-Einstein condensate, focusing on evidence for a macroscopic superposition of atoms and molecules. This problem raises an interest because, rather than two states of a given object, an atom-molecule system is a seemingly impossible macroscopic superposition of different objects. Nevertheless, photoassociation enables coherent intraparticle conversion, and we thereby propose a viable scheme for creating a superposition of a macroscopic number of atoms with a macroscopic number of molecules.

Recurrent mutations in P- and T-proteins of the glycine cleavage complex and a novel T-protein mutation (N145I): a strategy for the molecular investigation of patients with nonketotic hyperglycinemia (NKH).

Screening a DNA bank from 50 patients with enzymatic confirmation of their diagnosis of nonketotic hyperglycinemia gave allele frequencies of 5% for R515S of P-protein (glycine decarboxylase) and 7% for R320H of T-protein (aminomethyltransferase). In a previous report we found that 3% of the same patient alleles were positive for T-protein IVS7-1G>A. In total, testing for these three mutations identified 15% of alleles and positive results (one or two mutations) were found in 11 of the 50 patients. In addition, a novel point mutation in T-protein, N145I, was found in a single case and a PCR/restriction enzyme assay was developed for its detection.

A multicentre, open, non-comparative phase II study of a combination of fludarabine phosphate, cytarabine and granulocyte colony-stimulating factor in relapsed and refractory acute myeloid leukaemia and de novo refractory anaemia with excess of blasts in transformation.

The primary objective of this study was to determine the complete remission (CR) rate achieved with the FLAG (fludarabine phosphate, cytarabine and granulocyte colony-stimulating factor) regimen in patients with relapsed or refractory acute myeloid leukaemia (AML) or de novo refractory anaemia with excess of blasts in transformation (RAEB-t). Secondary objectives were to evaluate survival and toxicity. Induction treatment consisted of between one and two courses of FLAG. Patients achieving CR received between one and two courses of consolidation treatment. Eighty-three of the 89 patients entering the study were eligible for assessment. CR rates were: 17 out of 21 (81%) in late relapse AML (Group 1), 13 out of 44 (30%) in early relapse/refractory AML (Group 2), and 10 out of 18 (56%) in de novo RAEB-t (Group 3). Thirty-four of the 40 responders (85%) achieved CR after one induction course. Median survival times were 1.4 years, 3 months and 1.6 years in Groups 1, 2 and 3 respectively. Other than myelosuppression, the FLAG regimen was not generally associated with clinically significant toxicity and was well tolerated by most patients including the elderly. The FLAG regimen offers a very effective alternative treatment for CR induction in poor prognosis adult patients with either relapsed or refractory AML or de novo RAEB-t. FLAG delivers high-dose treatment without increasing overall toxicity, an approach which is of particular value in older patients, who constitute the majority in these diseases. It is therefore an important advance in developing new treatment options for these patients.

Identification of the first reported splice site mutation (IVS7-1G-->A) in the aminomethyltransferase (T-protein) gene (AMT) of the glycine cleavage complex in 3 unrelated families with nonketotic hyperglycinemia.

A novel splice site mutation (IVS7-1G-->A) in the T-protein gene (aminomethyltransferase, or AMT) of the glycine cleavage enzyme complex was found in a patient with nonketotic hyperglycinemia (NKH). A PCR/restriction enzyme method to detect this mutation was used to screen 100 NKH alleles and identified the mutation in three unrelated families.