Impact of environmental complexity and stocking density on affective states of rainbow trout (Oncorhynchus mykiss).

Environmental condition, such as environmental complexity or stocking density, can directly or indirectly influence animal emotion and ultimately, affective state. Affective states of animals can be assessed through judgement bias tests, evaluating responses to ambiguous situations. In this study, we aimed to determine whether environmental complexity and stocking density impacted rainbow trout affective state. Rainbow trout (n = 108) were housed in recirculating aquaculture systems under commercial conditions while trained at tank-level to discriminate between a positively reinforced chamber (feed) in one location and a negative chamber (positive punishment; chase by net for 1 s) in the opposing location. Fish from successful tanks (two out of five tanks) were then housed in treatment tanks of either high- or low- environmental complexity at either high (165 fish/m3) or low (69 fish/m3) stocking density. Trained fish were tested for latencies to approach three intermediate, ambiguous chambers. Fish housed in high-density tanks were faster to enter all chambers than those housed in low-density tanks (8.5 s vs. 15.2 s; P = 0.001), with faster entries into the positive (7.4 s vs. 15.2 s; P = 0.02) and near-negative chambers (10.2 s vs. 17.4 s; P = 0.006), suggesting that these fish were more optimistic to receive a feed reward. Tank complexity did not affect test outcomes. No differences between treatments were observed between body weight, length, and plasma cortisol. Overall, rainbow trout are capable of discriminating between cues during a judgement bias test and fish housed in high-density environments respond more optimistically in ambiguous situations compared to fish in low-density environments.

Social-pair judgment bias testing in slow-growing broiler chickens raised in low- or high-complexity environments.

Impacts of environmental complexity on affective states in slow-growing broiler chickens (Gallus gallus domesticus) are unknown. Chickens' performance in judgment bias tests (JBT) can be limited as they are tested individually, causing fear and anxiety. The objectives were to apply a social-pair JBT to assess the effect of environmental complexity on slow-growing broiler chickens` affective states, and assess the impact of fearfulness, anxiety, and chronic stress on JBT performance. Six-hundred Hubbard Redbro broilers were housed in six low-complexity (similar to commercial) or six high-complexity (permanent and temporary enrichments) pens. Twelve chicken pairs were trained (1 pair/pen, n = 24 chickens) using a multimodal approach (visual and spatial cues), with reward and neutral cues of opposing color and location. Three ambiguous cues were tested: near-positive, middle, and near-neutral cues. Approach and pecking behavior were recorded. Eighty-three percent of chickens (20/24) were successfully trained in 13 days. Fearfulness, anxiety, and chronic stress did not impact chickens' performance. Chickens successfully discriminated between cues. Low-complexity chickens approached the middle cue faster than high-complexity chickens, indicating that they were in a more positive affective state. The environmental complexity provided in this study did not improve affective states in slow-growing broiler chickens compared to a control. A social-pair JBT resulted in excellent learning and testing outcomes in slow-growing broilers.

Outbreak of abdominal distension and obstipation in a C57BL/6J experimental autoimmune encephalomyelitis study.

Seventy-four 9-week old female C57BL/6J mice housed in a conventional facility were manipulated to induce experimental autoimmune encephalomyelitis, among which 26 developed clinical signs including lethargy, absence of defecation, and abdominal distension. By gross necropsy examination, there was distension of the cecum and colon with fecal impaction. By histologic examination, there was severe ulcerative and proliferative typhlocolitis. Fecal ELISA confirmed the presence of toxins A and B of Clostridium difficile. Alteration in immune status of the immunocompetent mice, due to stress caused by experimental manipulation or autoimmune disease, may have led to intestinal dysbiosis, followed by opportunistic infections resulting in C. difficile-associated disease. This report brings to light the occurrence of the disease in immunocompetent laboratory mice during experimental manipulations associated with alteration in immune status, and it discusses potential hazards associated with conventional housing within a hospital-associated research institute.

Environmental complexity positively impacts affective states of broiler chickens.

Affective state can bias an animal's judgement. Animals in positive affective states can interpret ambiguous cues more positively ("optimistically") than animals in negative affective states. Thus, judgement bias tests can determine an animal's affective state through their responses to ambiguous cues. We tested the effects of environmental complexity and stocking density on affective states of broiler chickens through a multimodal judgement bias test. Broilers were trained to approach reinforced locations signaled by one color and not to approach unreinforced locations signaled by a different color. Trained birds were tested for latencies to approach three ambiguous cues of intermediate color and location. Broilers discriminated between cues, with shorter latencies to approach ambiguous cues closest to the reinforced cue than cues closest to the unreinforced cue, validating the use of the test in this context. Broilers housed in high-complexity pens approached ambiguous cues faster than birds in low-complexity pens-an optimistic judgement bias, suggesting the former were in a more positive affective state. Broilers from high-density pens tended to approach all cues faster than birds from low-density pens, possibly because resource competition in their home pen increased food motivation. Overall, our study suggests that environmental complexity improves broilers' affective states, implying animal welfare benefits of environmental enrichment.

Vacuum-assisted closure of the open abdomen in a resource-limited setting.

AIM: We describe our experience of developing a modified vacuum-assisted closure (VAC) dressing for open abdomens. BACKGROUND: We see a high volume of trauma in our department. Massive delays in presentation of patients with acute abdomen are common. Closure at initial laparotomy is not possible in many cases, either because the patient has or will develop abdominal compartment syndrome, or because several re-look laparotomies will be required. A significant proportion of our patients who have undergone laparotomy therefore spend some of their stay in hospital with an open abdomen. The management of these patients is particularly labour intensive for nursing staff. The Opsite sandwich or Bogota bag invariably leaks, and sometimes needs changing daily. If a patient also has a temporary ileostomy, application can be difficult. The commercial VAC dressing is an improvement on the Opsite sandwich, but is prohibitively expensive. Financial constraints and the volume of abdominal trauma and sepsis we see mean that commercial VAC dressings for laparostomy are not affordable in our setting. METHODS/RESULTS: We describe our adapted VAC dressing. It is inexpensive and easy to apply, has made a big difference in the nursing of patients with an open abdomen, and has enabled us to increase the rate of delayed primary closure (i.e., we have reduced the rate of ventral hernia). CONCLUSION: The modified VAC dressing is now our department's method of choice for temporary abdominal closure.

A simple method to measure proton beam energy in a standard medical cyclotron.

A simple and rapid technique to measure the proton beam energy in the external beam line of a medical cyclotron has been examined. A stack of 0.1 mm thick high purity copper (Cu) foils was bombarded and the relative activity of 65Zn produced in each foil was compared to a computational model that predicted activity, based on proton stopping power, reaction cross-sectional data, and beam energy. In the model, the beam energy was altered iteratively until the best match between computed and measured relative activities of the stack of disks was obtained. The main advantage of this method is that it does not require the comparison of the activities of different isotopes of zinc arising from (p, xn) reactions in the Cu, which would require the gamma photon detector being calibrated for different energy responses. Using this technique the proton beam energy of a nominally 18 MeV standard isochronous medical cyclotron was measured as 17.49 +/- 0.04 (SD) MeV, with a precision of 0.2% CV.

Genome organization in prokaryotes.

Most of the well-characterized prokaryotic genomes consist of double-stranded DNA organized as a single circular chromosome 0.6-10 Mb in length and one or more circular plasmid species of 2 kb-1.7 Mb. The past few years, however, have revealed some major variations in genome organization. In addition, a recent accumulation of data has shown that the location and orientation of the genes and repeated sequences (including prophages and transposons) on and among these elements is not always random. Some of the non-randomness is probably the result of unique historical events; in other cases it reflects selection for the optimization of function.

MOT1 can activate basal transcription in vitro by regulating the distribution of TATA binding protein between promoter and nonpromoter sites.

MOT1 is an ATPase which can dissociate TATA binding protein (TBP)-DNA complexes in a reaction requiring ATP hydrolysis. Consistent with this observation, MOT1 can repress basal transcription in vitro. Paradoxically, however, some genes, such as HIS4, appear to require MOT1 as an activator of transcription in vivo. To further investigate the function of MOT1 in basal transcription, we performed in vitro transcription reactions using yeast nuclear extracts depleted of MOT1. Quantitation of MOT1 revealed that it is an abundant protein, with nuclear extracts from wild-type cells containing a molar excess of MOT1 over TBP. Surprisingly, MOT1 can weakly activate basal transcription in vitro. This activation by MOT1 is detectable with amounts of MOT1 that are approximately stoichiometric to TBP. With amounts of MOT1 similar to those present in wild-type nuclear extracts, MOT1 behaves as a weak repressor of basal transcription. These results suggest that MOT1 might activate transcription via an indirect mechanism in which limiting TBP can be liberated from nonpromoter sites for use at promoters. In support of this idea, excess nonpromoter DNA sequesters TBP and represses transcription, but this effect can be reversed by addition of MOT1. These results help to reconcile previous in vitro and in vivo results and expand the repertoire of transcriptional control strategies to include factor-assisted redistribution of TBP between promoter and nonpromoter sites.

The iso1 gene of Chlamydomonas is involved in sex determination.

Sexual differentiation in the heterothallic alga Chlamydomonas reinhardtii is controlled by two mating-type loci, mt+ and mt-, which behave as a pair of alleles but contain different DNA sequences. A mutation in the mt minus-linked imp11 gene has been shown previously to convert a minus gamete into a pseudo-plus gamete that expresses all the plus gametic traits except the few encoded by the mt+ locus. Here we describe the iso1 mutation which is unlinked to the mt- locus but is expressed only in minus gametes (sex-limited expression). A population of minus gametes carrying the iso1 mutation behaves as a mixture of minus and pseudo-plus gametes: the gametes isoagglutinate but they do not fuse to form zygotes. Further analysis reveals that individual gametes express either plus or minus traits: a given cell displays one type of agglutinin (flagellar glycoprotein used for sexual adhesion) and one type of mating structure. The iso1 mutation identifies a gene unlinked to the mating-type locus that is involved in sex determination and the repression of plus-specific genes.

Properties of deoxyribonuclease 4 from Aspergillus nidulans.

Deoxyribonuclease 4 from Aspergillus nidulans was purified to over 70% homogeneity. It contains a polypeptide of Mr about 30000, and behaves as a dimer, but with some evidence of dissociation on gel filtration and ultracentrifugation. The pH optimum is 7-9. Activity is supported by metal ions in the order (Mn2+ + Ca2+) greater than Mn2+ approximately equal to (Mg2+ + Ca2+) much greater than Mg2+. Mn2+ is optimal at 10-20 mM. DNAase 4 strongly prefers native DNA, for which the Km is about 0.5 mM, and on which it acts as an endonuclease. The specific activity is about 2000 mumol of nucleotide made acid-soluble in 30 min at 37 degrees C per mg of protein. Action on denatured DNA, which has a lower optimal Mn2+ concentration and a different time course from its action on native DNA, may be due to partial renaturation of the DNA used. It has no action on RNA. With native DNA the enzyme gave mainly, or entirely, double-strand cleavages by a single-hit mechanism, with either Mn2+ or Mg2+. The enzyme has no strongly preferred sequences. Action stops, or becomes very slow, when 50-60% acid-solubility has been reached. In a near-limit digest, mononucleotides were absent, dinucleotides to at least heptanucleotides occurred in similar weight yields, there was an excess of chains of 10-11 (or rather longer) and a rapid decline at greater lengths. Products have 3'-OH, 5'-P termini. The products and kinetics can be understood in terms of the enzyme's causing non-staggered double-strand cleavages randomly in DNA but subject to a requirement for at least two base pairs at one side of the cleavage site and at least 10 (or perhaps rather more) base pairs on the other side of the site.

Structure of cryptic lambda prophages.

When Escherichia coli cells lysogenic for bacteriophage lambda are induced with ultraviolet light, cells carrying cryptic lambda prophages are occasionally found among the apparently cured survivors. The lambda variant crypticogen (lambda crg) carries an insertion of the transposable element IS2, which increases the frequency of cryptic lysogens to about 50% of cured cells: 43 of these cryptic prophages have been characterized. They all contain substitutions that replace the early segment of the prophage genome (from the IS2 to near the cos site) with a duplicate copy of a large segment of the host chromosome. The right end of the substitution always results from recombination between the nin-QSR-cos region of the prophage and the homologous incomplete lambdoid prophage Qsr' at 12.5 minutes in the E. coli chromosome. The left end of the substitution is usually a crossover that recombines the IS2 element in the prophage with an E. coli IS2 at 8.5 minutes, near the lac gene, or with a second IS2 located counterclockwise from leu at 2 minutes, generating duplications of at least 200,000 bases. Five cryptic lysogens derived from cells lysogenic for a reference strain of lambda (which lacks the IS2 present in lambda crg) have been characterized. They contain substitutions whose right termini are generated by a crossover with the Qsr' prophage. The left termini of these substitutions are formed either by a crossover between the lambda exo gene and a short exo-homologous segment of Qsr' (2/5), or by a crossover between sequences to the left of attL and an unmapped distant region of the host chromosome (3/5). The large duplications carried by these cryptic lysogens are stable, unlike tandem duplications, and so may significantly influence the cell's evolutionary potential.

Isolation of lambda transducing phage with the bio genes inserted between lambda genes P and Q.

Plaque-forming, biotin-transducing phages were constructed with the bio genes inserted between lambda genes P and Q. These phages were isolated for the eventual aim of fusing the lambda Q gene to the bio operon. The following steps were used to construct these phages: A defective temperature-sensitive lysogen was constructed with the bio genes adjacent to and to the left of lambda genes beta NcI857OPQSRA. Heat-resistant survivors were screened for deletions with endpoints in the bio operon and to the right of lambda P and to the left of lambda A. Five of approximately 1,600 heat-resistant survivors had these properties. Two had the gene order bioAB .... lambda QSRA. When these two strains were lysogenized with lambda cI857b221 and heat induced, the desired transducing phages were obtained. We characterized these phages and studied one in detail. Two-thirds of the plaque-forming transducing phages isolated carried the entire bioB gene and only part of the bioA gene, and one-third carried the entire bioA and bioB genes. The phages isolated lost the bio genes upon propagation, indicating that they contain a partial duplication of phage genes. The duplication was shown not to involve the entire lambda Q gene in one of these phages, lambda bioq1b221. A recombinant of this phage, lambda Nam7am53c17b221, failed to form plaques under biotin-derepression conditions. We conclude that if the lambda Q gene was fused to the bio operon in this phage, not enough lambda Q gene product was made to allow phage propagation.

Xenognosin sensing in virulence: is there a phenol receptor in Agrobacterium tumefaciens?

BACKGROUND: The mechanisms of signal perception and transmission in the 'two-component' autokinase transmitters/response regulators are poorly understood, especially considering the vast number of such systems now known. Virulence induction from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens represents one of the best understood systems with regard to the chemistry of the activating signal, and yet the existing data does not support a receptor-mediated perception event for the xenognostic phenols. RESULTS: Here we provide the first conclusive evidence that a specific receptor must be involved in xenognostic phenol perception, detail structural requirements of the xenognosins necessary for perception by this receptor, and develop a genetic strategy that demonstrates critical components of the phenol recognition system are not encoded on the Ti plasmid. CONCLUSIONS: Although the basic elements of the two-component system required for phenol-mediated induction of virulence gene expression are encoded on the Ti plasmid, they are dependent on the chromosomal background for even the very first stage of signal perception. This discovery suggests a curious evolutionary history, and also provides functional insight into the mechanisms of two-component signal detection and transmission in general.

Cattle and environmental sample-level factors associated with the presence of Salmonella in a multi-state study of conventional and organic dairy farms.

The objective of this study was to evaluate associations between cattle-level factors and environmental samples with the isolation of Salmonella from dairy farms in Minnesota, Wisconsin, Michigan, and New York. The study farms included 129 conventional and organic farms enrolled without regard to previous history of Salmonella infection. Herds were sampled at two-month intervals over a one-year period. Cattle groups more likely to be associated with Salmonella shedding (compared to preweaned calves) were cows designated as sick by farm personnel (OR=2.5, 95% CI: 1.7, 3.7), cows within 14 days of calving (OR=1.8, 95% CI: 1.1, 2.8), and cows due for culling within 14 days (OR=1.9, 95% CI: 1.0, 3.4). State of origin was also associated with the presence of Salmonella in samples from cattle and the farm environment; Midwestern states were more likely to have Salmonella-positive samples compared to New York. Cattle treated with antimicrobials within 14 days of sampling were more likely to be Salmonella-negative compared with nontreated cattle (OR=2.0, 95% CI: 1.1, 3.4). Farms with at least 100 cows were more likely to have Salmonella-positive cattle compared with smaller farms (OR=2.6, 95% CI: 1.4, 4.6). Season was associated with Salmonella shedding in cattle, and compared to the winter period, summer had the highest odds for shedding (OR=2.4, 95% CI: 1.5, 3.7), followed by fall (OR=1.9, 95% CI: 1.2, 3.1) and spring (OR=1.8, 95% CI: 1.2, 2.6). Environmental samples significantly more likely to be Salmonella-positive (compared to bulk tank milk) included, in descending order, samples from sick pens (OR=7.4, 95% CI: 3.4, 15.8), manure storage areas (OR=6.4, 95% CI: 3.5, 11.7), maternity pens (OR=4.2, 95% CI: 2.2, 8.1), haircoats of cows due to be culled (OR=3.9, 95% CI: 2.2, 7.7), milk filters (OR=3.3, 95% CI: 1.8, 6.0), cow waterers (OR=2.8, 95% CI: 1.4, 5.7), calf pens (OR=2.7, 95% CI: 1.3, 5.3), and bird droppings from cow housing (OR=2.4, 95% CI: 1.3, 4.4). Parity, stage of lactation, and calf age were not associated with Salmonella shedding.

Chronic psychosocial stress impairs learning and memory and increases sensitivity to yohimbine in adult rats.

BACKGROUND: It is well known that intense and prolonged stress can produce cognitive impairments and hippocampal damage and increase noradrenergic activity in humans. This study investigated the hypothesis that chronic psychosocial stress would affect behavior, drug sensitivity, and hippocampal-dependent learning and memory in rats. The work provides a novel connection between animal and human studies by evaluating the effects of stress on a rat's response to yohimbine, an alpha(2) adrenergic receptor antagonist. METHODS: Rats were exposed to a cat for 5 weeks and randomly housed with a different group of cohorts each day (psychosocial stress). The effects of the stress manipulations were then assessed on open field behavior, spatial learning and memory in the radial arm water maze and the behavioral response to a low dose of yohimbine (1.5 mg/kg). RESULTS: Stressed rats displayed impaired habituation to a novel environment, heightened anxiety, and increased sensitivity to yohimbine. In addition, the stressed rats exhibited impaired learning and memory. CONCLUSIONS: There are commonalities between the current findings on stressed rats and from studies on traumatized people. Thus, psychosocial stress manipulations in rats may yield insight into the basis of cognitive and neuroendocrine disturbances that commonly occur in people with anxiety disorders.

Co-chairman's remarks: genetic recombination in the molecular era.

The present status of some general questions about DNA recombination is assessed. Topics include the mechanisms of synapsis and strand exchange, and the functions of recombination in nature.

Functional elements of DNA upstream from the integrase operon that are conserved in bacteriophages 434 and lambda.

A 1488-bp restriction fragment of bacteriophage 434 DNA contains the integrase promoter and an adjacent nucleotide sequence (t'I) resembling a Rho-independent terminator. To identify and quantitate transcription termination, DNA segments were cloned into a plasmid between the galactose promoter and assayable galactokinase gene and tested for termination. Whereas the entire fragment effectively terminated transcription, a 331-bp restriction fragment containing the t'I terminator had only weak terminator activity. Random sequential deletions of the 434 DNA segment defined a strong terminator 650-bp upstream from t'I. This proposed Rho-independent terminator called tL4 consists of a 7-bp stem and 6-nt loop followed by a uridine-rich region in the RNA. Phage lambda contains an even stronger tL4 terminator that differs in 4 nt from 434 tL4. Thus, despite some sequence divergence, terminator activity has been conserved in these phages. The 434 DNA segment was also tested for promoter activity. Rightward promoter activity (opposite to pL in the phage) was located about 200 bp to the right of tL4 and was followed by an open reading frame (ORF) capable of encoding a 91 amino acid protein. Promoter activity in the same approximate location was also found in phage lambda. Thus the rightward promoter, the tL4 and t'I terminators, and ORF-55 all are elements in this segment of the genome that are conserved for function despite sequence divergence.

Sequence and properties of operator mutations in the bio operon of Escherichia coli.

The nucleotide-sequence changes occurring in newly isolated operator-constitutive mutations of the divergently transcribed bio operon have been determined. The observed point mutations are single GC leads to AT changes which occur at two symmetrical points in the hyphenated inverted repeat present in the control region. The changes at position -15 (with respect to the center of the inverted repeat) cause constitutivity of leftward operon expression and decreased expression of bioB, due to alteration of the -35 region of the rightward promoter. The change at position +15 is identical to one of the changes that Otsuka and Abelson (1978) detected in the bio p98 bio o34 double mutant. The location of the bio p131::IS1 insertion, which affects both leftward and rightward transcription, is also within the operator region. Both of the operator-constitutive mutations and the bio p131 insertion cause decreased repressor binding in vivo as shown by repressor titration tests on multicopy plasmids which bear them. The operator-constitutive mutations also decrease repressor binding in vitro, where added repressor fails to protect the TaqI site that is protected by repressor in bio o+ DNA. These results confirm several aspects of the model for bio operon regulation proposed by Otsuka and Abelson (1978).

Flow cytometric analysis of tumour-draining lymph nodes in breast cancer patients.

The phenotype and activation status of lymphocytes from the peripheral blood and axillary lymph nodes of 40 patients with breast cancer were analysed using flow cytometry and compared with lymphocytes from the blood and lymph nodes of 7 control subjects. There was little difference in the overall proportions of T and B lymphocytes but there was a much larger population of B cells bearing surface IgG and a greater number of CD4+ helper T cells, particularly in the regional nodes, in the breast cancer patients. Many more T cells in the cancer patients were found to be carrying the HLA DR and Tac antigens. The axillary lymph nodes were the major site of B cells and CD4+ T cells whilst the primary tumour was the source of the CD8+ suppressor/cytotoxic T cells. Any immune response appeared to be largely loco-regional and may therefore destroyed by conventional surgery or radiotherapy.

Aneuploid subpopulations in tumour-invaded lymph nodes from breast cancer patients.

Fresh, paired primary tumours and lymph node metastases from breast cancer patients were compared by DNA flow cytometry. Although 65% of primary tumours were aneuploid, the detection of aneuploid peaks in corresponding nodal metastases was rare (only 6 cases out of 25) in single-parameter DNA analysis. Detection of aneuploid subpopulations in lymph nodes was greatly improved in dual-parameter DNA analysis using an anticytokeratin (CK) antibody which allowed ploidy determination on CK+ epithelial cells alone. Examination of 12 lymph nodes for CK+ cells revealed the presence of both diploid and aneuploid tumour cells in tumour invaded nodes. In patients with multiploid primary tumours, a subpopulation of the primary aneuploid cells was dominant in the nodal metastases. This suggests that aneuploidy is an integral property of metastatic cells and that within a primary tumour a subpopulation may have a higher metastatic potential.