The impact of chronic environmental stressors on growing pigs, Sus scrofa (Part 1): stress physiology, production and play behaviour

Commercially farmed animals are frequently housed in conditions that impose a number of concurrent environmental stressors. For pigs housed indoors, elevated levels of mechanical noise, atmospheric ammonia and low light intensities are commonplace. This experiment examined the effects on growing pigs of chronic exposure to combinations of commercially relevant levels of these potential stressors. Four-week-old hybrid female pigs (n = 224) were housed under experimentally manipulated conditions of nominally either <5 or 20 ppm atmospheric concentration of ammonia (24 h), a light intensity of 40 lux or 200 lux (12 h) and mechanical noise at either ⩽60 or 80 dB(A) (24 h) for 15 weeks in a fully factorial arrangement (2³) of treatments. The response of pigs to these environmental factors was assessed using a suite of physiological, production and behavioural measures. These included indicators of hypothalamic–pituitary–adrenal (HPA) axis activation such as salivary cortisol and adrenal morphometry, as well as body weight, food conversion efficiency and general health scores. Play behaviour was recorded as it is thought to be inversely related to stress. Chronic exposure to ammonia produced the strongest effect, shown by lower concentrations of salivary cortisol and larger adrenal cortices in the pigs reared under 20 ppm ammonia, which may have been indicative of a period of HPA activation leading to a downregulation of cortisol production. The pigs in the ammoniated rooms also performed less play behaviour than pigs in non-ammoniated rooms. There was evidence for an interaction between high noise and ammonia on the health scores of pigs and for brighter light to ameliorate the effect of ammonia on salivary cortisol. However, there was no measurable impact of these potential stressors on the productivity of the pigs or any of the other physiological parameters measured. We conclude that there should be little concern in terms of performance about the physical stressors tested here, within current European Union legal limits. However, 20 ppm ammonia may have had an adverse influence on the well-being of growing pigs. In this study, all other aspects of the pigs’ husbandry were optimal; therefore, it is possible that under less favourable conditions, more pronounced effects of ammonia, noise and dim light would be observed.     

Influence of Von Willebrand Disease (VWD) and pregnancy on the expression of angiogenic factors in the porcine female reproductive tract

Von Willebrand Disease (VWD) is a heritable disorder caused by defects of the Von Willebrand Factor (VWF), leading to deficiencies in coagulation and also angiogenesis. Women affected by VWD frequently show bleeding concerning the reproductive tract and may present with increased rates of miscarriages. We used a porcine model representing VWD type 1 and type 3 as well as the wildtype. Samples were obtained from the reproductive tract of non-pregnant sows and sows pregnant at time of placentation. Relative expression of the genes CALR, CCN2, CXCL8, ECE1, EDN1, F8, IGFBP7, and LGALS3 was analyzed. CCN2 and FVIII proteins were additionally analyzed using immunohistochemistry. In uterus and ovary significant upregulation of CCN2 was seen in non-pregnant pigs affected by VWD. This might be caused by the higher VEGFA-levels in these pigs and could have an influence angiogenesis. During pregnancy, CCN2 expression increased in wildtype pig uteri but hardly changed in those of pregnant pigs affected by VWD, presumably because the expression level in the latter pigs already was significantly increased before pregnancy. F8 expression was significantly reduced in uterus and ovary of VWD-affected pigs. VWF is known to protect FVIII from decomposition and a lack of VWF leads to lower levels of FVIII. Our results suggest that a reduced F8 expression primarily might contribute to those reduced FVIII levels in VWD-affected pigs. Additional significant results involving the pregnant pigs were detected for CALR, EDN1, and LGALS3. These genes are promising candidates for more detailed future studies.     

The impact of chronic environmental stressors on growing pigs, Sus scrofa (Part 2): social behaviour

The effects of common and concurrent environmental stressors on the social behaviour of farm animals are poorly understood. Here, we report the results of a multifactorial experiment designed specifically to examine the individual, additive or interactive effects of elevated ammonia, noise and low light (LL) levels on the social behaviour of growing pigs. Social behaviour was measured in terms of the nature, frequency and duration of both initiated and response behaviours for 4 weeks following mixing of the groups. General activity patterns, group cohesion and social discrimination were also examined as a function of the environmental treatments. Elevated concentrations of atmospheric ammonia (～20 v. <5 ppm) and LL intensity (～40 v. 200 lux) had the most pronounced effects, particularly on the nature of social interactions, with pigs under these conditions showing more aggression in the early stages of the experiment. In addition, pigs exposed to a high level of mechanical noise representative of artificial ventilation (～80 v. 40 dB [A]) were less submissive to aggressive acts, while pigs in ～20 ppm ammonia showed more reciprocated aggression when in coincident LL (<40 lux). The results indicate that atmospheric ammonia at commonly experienced concentrations may undermine social stability, particularly in the presence of low lighting, though the mechanisms are currently unknown. These findings have implications for the welfare of growing pigs and hence policy makers and farmers alike, with respect to the improvement of welfare in intensive pig farming.     

Effects of Organic Substrates on Dechlorination of Aroclor 1242 in Anaerobic Sediments

The effects of different organic substrates on the abilities of anaerobic sediment enrichments to reductively dechlorinate polychlorinated biphenyls (PCBs) were studied. Sediments collected from a site previously contaminated with PCBs were dosed with additional PCBs (Aroclor 1242; approximately 300 ppm [300 μg/g], sediment dry weight) and incubated anaerobically with acetate, acetone, methanol, or glucose. The pattern of dechlorination was similar for each substrate-fed batch; however, the extents and rates of dechlorination were different. Significant dechlorination over time was observed, with the relative rates and extents of dechlorination being greatest for methanol-, glucose-, and acetone-fed batches and least for acetate-fed batches. Dechlorination occurred primarily on the meta- and para- positions of the highly chlorinated congeners, resulting in the accumulation of less-chlorinated, primarily ortho-substituted products. No significant dechlorination was observed in incubation batches receiving no additional organic substrate, even though identical inorganic nutrients were added to all incubation batches. In addition, dechlorination was not observed in autoclaved controls that received substrate and nutrients.     

Effect of pens with an elevated platform on space utilization,skin lesions and growth performance in nursery pigs

Environmental enrichment, adequate space and the ability to separate a living area into suitable functional areas are key elements for pig welfare. In this study, a two-level pen for nursery pigs was explored with the aim of analysing the use of space and the impact on pig behaviour, health and performance. Therefore, per batch, three experimental groups in pens with elevated platforms (two-level pens) and one control group in a standard pen were formed after weaning at the age of 4 weeks. Thereafter, groups were studied for a period of 6 weeks. In a total of eight batches (n = 882 pigs), the occurrence of skin lesions was determined per individual using a lesion score, and in seven batches (n = 761 pigs) individual daily weight gain was analysed. In five batches (n = 450 pigs), the individual use of space and the behaviour of pigs were investigated by direct observation (4 h per day on 2 days in weeks 1, 3 and 6 of the housing period, respectively). Results revealed that 98.9% of pigs (n = 445) were observed on the elevated platform at least once. The probability that an individual pig used the platform was significantly higher than 0.95 (P < 0.0001, confidence interval = 0.977, 1). The use of the platform was not affected by sex (odds ratio (OR) = 1.013, P = 0.937). However, the probability of observing an individual on the elevated platform increased with increasing BW during the experimental period (OR = 1.043, P < 0.0001). The presence of a platform decreased the probability of seeing a pig fighting (P = 0.014) and increased the probability of observing locomotor behaviour as opposed to lying (P < 0.0001). In two-level pens, pigs sustained fewer skin injuries than in standard pens (day 41: OR = 0.731, P < 0.0001). Over 41 days of experiment, pigs in two-level pens had higher daily weight gains than animals in standard pens (416 versus 393 g/day, P = 0.006). We conclude that usable space for nursery pigs can be effectively extended by introducing elevated platforms into the pigs’ pen. Furthermore, pigs may benefit from two-level systems by establishing spaces for activity as well as for retreat from other pen mates, thereby reducing aggressive behaviour and social stress. The installation of two-level pens is therefore encouraged, if they are intended to provide more space than legally stipulated.     

Divergent selection for residual feed intake in group-housed growing pigs: characteristics of physical and behavioural activity according to line and sex

The aim of the study was to assess the impact of selection for residual feed intake (RFI) on the behavioural activity of lines divergently selected for RFI during seven generations. In all, six successive batches from the seventh generation of selection were raised in collective pens equipped with a single-place electronic feeder (SEF) from 10 weeks of age to 100 kg BW. Each batch included four groups of 12 pigs: high RFI (RFI⁺) castrated males, RFI⁺ females, low RFI (RFI⁻) castrated males, RFI⁻ females. At 17 weeks of age, health criteria were evaluated using a gradient scale for increased severity of lameness, body lesions, bursae and tail biting. Individual behavioural activities were recorded by 24-h video tape on the day after health evaluation. The investigative motivation towards unfamiliar objects was quantified at 18 weeks of age. The daily individual feeding patterns were computed from SEF records during the 4 weeks surrounding 12, 17 and 22 weeks of age. All pigs spent significantly most of their time lying in diurnal (80% of total scan) and nocturnal (>89%) periods. The RFI⁻ pigs showed a lower proportion of health problems (P<0.01) than RFI⁺ pigs. The RFI⁻ pigs used the SEF less than the RFI⁺ pigs, in diurnal (5.3% v. 6.4% of video scans, P<0.05) and nocturnal periods (3.6% v. 4.5% of video scans, P<0.05). This was confirmed by a significantly lower daily number and duration of visits to the SEF computed from the SEF data. The feeding activity measured from the video recording was significantly correlated (R>0.34; P<0.05) with feeding patterns computed from the SEF. The RFI⁻ pigs spent less time standing over the 24-h period (9.7% v. 12.2% of scans, i.e. 35 min/day, P<0.05). In terms of energy costs, this amounted to 14% of the line difference in terms of daily metabolizable energy intake. The castrated males used the SEF more than females, especially at night (4.7% v. 3.4% of total scans, P<0.05), whereas females displayed greater investigation of their environment (7.7±0.3% v. 6.6±0.2% of total scans, P<0.05) and the novel objects (10.7% v. 4.9% of total scans, P<0.05). In conclusion, the lower physical activity associated with reduced energy expenditure in RFI⁻ pigs compared with RFI⁺ pigs contributed significantly to their improved efficiency and was not related to worsened health scores.     

CXCL14 enhances insulin-dependent glucose uptake in adipocytes and is related to high-fat diet-induced obesity

Accumulating evidence suggests an association between obesity and adipose tissue inflammation. Chemokines are involved in the regulation of inflammation status. Chemokine (C-X-C motif) ligand 14 (CXCL14) is known to be a chemoattractant for monocyte and dendritic cells. Recently, it was reported that CXCL14-deficient mice show resistance to high-fat diet-induced obesity. In this study, we identified CXCL14 as a growth hormone (GH)-induced gene in HepG2 hepatoma cells. Substantial in vivo expression of CXCL14 was detected in the adipose tissue and liver. Its expression and secretion were strikingly increased by insulin administration and high-fat diet. Intriguingly, incubation of 3T3-L1 adipocytes with CXCL14 stimulated insulin-dependent glucose uptake. Further, this effect was associated with enhanced insulin signaling. CXCL14 enhanced the insulin-induced tyrosine phosphorylation of insulin receptors and insulin receptor substrate-1. These results suggest that CXCL14 plays a causal role in high-fat diet-induced obesity.     

Prevalence and risk factors for gastric ulceration in pigs slaughtered at 170 kg

Oesophago-gastric ulcers (OGU) are a production and welfare problem in pigs. Stomach condition was scored for 22 551 pigs in 228 batches over a 7-month period at an abattoir in Italy processing heavy pigs for ham production. Mild or severe ulceration was observed in 20.7% of pigs, of which 13% had scar tissue. Variation between batches was high (0% to 36% prevalence of severe ulcers) and showed a significant effect of farm of origin (P<0.001). Overnight lairage increased the prevalence of mild ulcers (P<0.001), but not severe or scarred ulcers. Scarred ulcers increased in the hottest summer months. Prevalence of ulcers showed only few and weak correlations at batch level with pathologies of the pleura, lungs and liver, but a strong correlation with on-farm mortality of the batch. Analysis of farm risk factors for OGU was assessed by questionnaire with a response rate of 17% of farms. Risk factors retained in a multivariable model included a protective effect of anthelmintic treatment (risk ratio (RR)=5.1, P=0.03), increased risk in farms using Mycoplasma vaccination (RR=5.6, P=0.04) and a tendency for association with type of flooring (P=0.06). Univariable analyses also highlighted possible influences of other stress-inducing factors including lack of enrichment objects and mixing of pigs during fattening, suggesting that the role of on-farm stressors merits further investigation. It is concluded that abattoir screening of OGU in future programmes for the assessment of well-being on farm should encompass only severe lesions and scarring, and results be returned to farmers to facilitate improvement of production and welfare.     

Effects of Thyroxine Exposure on Osteogenesis in Mouse Calvarial Pre-Osteoblasts

The incidence of craniosynostosis is one in every 1,800–2500 births. The gene-environment model proposes that if a genetic predisposition is coupled with environmental exposures, the effects can be multiplicative resulting in severely abnormal phenotypes. At present, very little is known about the role of gene-environment interactions in modulating craniosynostosis phenotypes, but prior evidence suggests a role for endocrine factors. Here we provide a report of the effects of thyroid hormone exposure on murine calvaria cells. Murine derived calvaria cells were exposed to critical doses of pharmaceutical thyroxine and analyzed after 3 and 7 days of treatment. Endpoint assays were designed to determine the effects of the hormone exposure on markers of osteogenesis and included, proliferation assay, quantitative ALP activity assay, targeted qPCR for mRNA expression of Runx2, Alp, Ocn, and Twist1, genechip array for 28,853 targets, and targeted osteogenic microarray with qPCR confirmations. Exposure to thyroxine stimulated the cells to express ALP in a dose dependent manner. There were no patterns of difference observed for proliferation. Targeted RNA expression data confirmed expression increases for Alp and Ocn at 7 days in culture. The genechip array suggests substantive expression differences for 46 gene targets and the targeted osteogenesis microarray indicated 23 targets with substantive differences. 11 gene targets were chosen for qPCR confirmation because of their known association with bone or craniosynostosis (Col2a1, Dmp1, Fgf1, 2, Igf1, Mmp9, Phex, Tnf, Htra1, Por, and Dcn). We confirmed substantive increases in mRNA for Phex, FGF1, 2, Tnf, Dmp1, Htra1, Por, Igf1 and Mmp9, and substantive decreases for Dcn. It appears thyroid hormone may exert its effects through increasing osteogenesis. Targets isolated suggest a possible interaction for those gene products associated with calvarial suture growth and homeostasis as well as craniosynostosis.     

Lymphotoxin-beta receptor blockade reduces CXCL13 in lacrimal glands and improves corneal integrity in the NOD model of Sjögren's syndrome

Introduction

In Sjögren's syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren's syndrome.

Methods

Male NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögren's syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores.

Results

LTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögren's patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01).

Conclusions

Blockade of LTBR pathways may have therapeutic potential for treatment of Sjögren's syndrome.     

CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.     

Using human microarrays to identify differentially expressed genes associated with increased steroidogenesis in boars

Human microarrays are readily available, and it would be advantageous if they could be used to study gene expression in other species, such as pigs. The objectives of this research were to validate the use of human microarrays in the analysis of porcine gene expression, to assess the variability of the data generated, and to compare gene expression in boars with different levels of steroidogenesis. Cytochrome b5 (CYB5) expression was used to assess array detection sensitivity. Samples having high or low CYB5 RNA levels were hybridized to microarrays to determine if the known expression difference could be detected. Six hybridizations were conducted using human microarrays containing 3840 total spots representing 1718 characterized human ESTs. To analyze gene expression in boars with different levels of steroidogenesis, testis RNA from four boars with high levels of plasma estrone sulphate was hybridized to testis RNA from four boars with lower levels. Eight microarray hybridizations were conducted including fluor-flips. Self-self hybridizations were also conducted to assess the variability of array experiments. The Cy5 and Cy3 intensity values for each array were normalized using a locally weighted linear regression (LOESS). Statistical significance was assessed using a Student's t-test followed by the Benjamini and Hochberg multiple testing correction procedure. Quantitative real-time PCR (Q-RT-PCR) was used to verify select gene expression differences. The results show that CYB5 was significantly overexpressed in the high CYB5 sample by 1.8 fold (P < 0.05), verifying the known expression difference. The average log2 ratio of the majority of genes (1643) falls within one standard deviation of the mean, indicating the data were reproducible. In the high versus low steroidogenesis experiment, seven genes were significantly overexpressed in the high group (P < 0.05). Quantitative real-time PCR was used to validate five genes with the highest fold change, and the results corroborated those found by the microarray experiments. The results of the self-self hybridizations showed that no genes were significantly differentially expressed following the application of the Benjamini and Hochberg multiple testing correction procedure. The results presented in this report show that human arrays can be used for gene expression analysis in pigs.     

Chemokine CXCL14/BRAK transgenic mice suppress growth of carcinoma cell xenografts

We reported previously that the forced expression of the chemokine BRAK, also called CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor xenografts compared with mock-vector treated cells in athymic nude mice or in severe combined immunodeficiency mice. This suppression occurred even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that a high expression level of the gene in tumor cells is important for the suppression of tumor establishment in vivo. The aim of this study was to determine whether CXCL14/BRAK transgenic mice show resistance to tumor cell xenografts or not. CXCL14/BRAK cDNA was introduced into male C57BL/6 J pronuclei, and 10 founder transgenic mice (Tg) were obtained. Two lines of mice expressed over 10 times higher CXCL14/BRAK protein levels (14 and 11 ng/ml plasma, respectively) than normal blood level (0.9 ng/ml plasma), without apparent abnormality. The sizes of Lewis lung carcinoma and B16 melanoma cell xenografts in Tg mice were significantly smaller than those in control wild-type mice, indicating that CXCL14/BRAK, first found as a suppressor of tumor progression of HNSCC, also suppresses the progression of a carcinoma of other tissue origin. Immunohistochemical studies showed that invasion of blood vessels into tumors was suppressed in tumor xenografts of CXCL14/BRAK Tg mice. These results indicate that CXCL14/BRAK suppressed tumor cell xenografts by functioning paracrine or endocrine fashion and that CXCL14/BRAK is a very promising molecular target for tumor suppression without side effects.     

Global hypomethylation and promoter methylation in small intestinal neuroendocrine tumors: An in vivo and in vitro study

Aberrant DNA methylation is a feature of human cancer affecting gene expression and tumor phenotype. Here, we quantified promoter methylation of candidate genes and global methylation in 44 small intestinal-neuroendocrine tumors (SI-NETs) from 33 patients by pyrosequencing. Findings were compared with gene expression, patient outcome and known tumor copy number alterations. Promoter methylation was observed for WIF1, RASSF1A, CTNNB1, CXCL14, NKX2–3, P16, LAMA1, and CDH1. By contrast APC, CDH3, HIC1, P14, SMAD2, and SMAD4 only had low levels of methylation. WIF1 methylation was significantly increased (P = 0.001) and WIF1 expression was reduced in SI-NETs vs. normal references (P = 0.003). WIF1, NKX2–3, and CXCL14 expression was reduced in metastases vs. primary tumors (P < 0.02). Low expression of RASSF1A and P16 were associated with poor overall survival (P = 0.045 and P = 0.011, respectively). Global methylation determined by pyrosequencing of LINE1 repeats was reduced in tumors vs. normal references, and was associated with loss in chromosome 18. The tumors fell into three clusters with enrichment of WIF1 methylation and LINE1 hypomethylation in Cluster I and RASSF1A and CTNNB1 methylation and loss in 16q in Cluster II. In Cluster III, these alterations were low-abundant and NKX2-3 methylation was low. Similar analyses in the SI-NET cell lines HC45 and CNDT2 showed methylation for CDH1 and WIF1 and/or P16, CXCL14, NKX2-3, LAMA1, and CTNNB1. Treatment with the demethylating agent 5-azacytidine reduced DNA methylation and increased expression of these genes in vitro. In conclusion, promoter methylation of tumor suppressor genes is associated with suppressed gene expression and DNA copy number alterations in SI-NETs, and may be restored in vitro.     

Study of Plasma Flow Modes in Imploding Nested Arrays

Results from experimental studies of implosion of nested wire and fiber arrays at currents of up to 4 МА at the Angara-5-1 facility are presented. Depending on the ratio between the radii of the inner and outer arrays, different modes of the plasma flow in the space between the inner and outer arrays were implemented: the sub-Alfvénic (V _r < V _А ) and super-Alfvénic (V _r > V _А ) modes and a mode with the formation of the transition shock wave (SW) region between the cascades. By varying the material of the outer array (tungsten wires or kapron fibers), it is shown that the plasma flow mode between the inner and outer arrays depends on the ratio between the plasma production rates ?_in /?_out in the inner and outer arrays. The obtained experimental results are compared with the results of one-dimensional MHD simulation of the plasma flow between the arrays. Stable implosion of the inner array plasma was observed in experiments with combined nested arrays consisting of a fiber outer array and a tungsten inner array. The growth rates of magnetic Rayleigh?Taylor (MRT) instability in the inner array plasma at different numbers of fibers in the outer array and different ratios between the radii of the inner and outer arrays are compared. Suppression of MRT instability during the implosion of the inner array plasma results in the formation of a stable compact Z-pinch and generation of a soft X-ray pulse. A possible scenario of interaction between the plasmas of the inner and outer arrays is offered. The stability of the inner array plasma in the stage of final compression depends on the character of interaction of plasma jets from the outer array with the magnetic field of the inner array.     

Antimicrobial and anti-inflammatory activities of chemokine CXCL14-derived antimicrobial peptide and its analogs

CXCL14 is a CXC chemokine family that exhibits antimicrobial activity and contains an amphipathic cationic α-helical region in the C-terminus, a characteristic structure of antimicrobial peptides (AMPs). In this study, we designed three analogs of CXCL14_59–75 (named CXCL14-C17) corresponding to the C-terminal α-helix of CXCL14, which displayed potential antimicrobial activity against a wide variety of gram-negative and gram-positive bacteria with minimum inhibitory concentrations of 4?16?μM without mammalian cell toxicity. Furthermore, two CXCL14-C17 analogs (CXCL14-C17-a1 and CXCL14-C17-a3) with improved cell selectivity were engineered by introducing Lys, Arg, or Trp in CXCL14-C17. Additionally, CXCL14-C17 analogs showed much greater synergistic effect (FICI: 0.3125–0.375) with chloramphenicol and ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa (MDRPA) than LL-37 did (FICI: 0.75–1.125). CXCL14-C17 analogs were more active against antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), MDRPA, and vancomycin-resistant Enterococcus faecium (VREF) than LL-37 and melittin. In particular, CXCL14-C17-a2 and CXCL14-C17-a3 completely inhibited the biofilm formation at sub-MIC and all of the peptides were able to eliminate pre-formed biofilm as well. Membrane depolarization, flow cytometry, sytox green uptake, ONPG hydrolysis and confocal microscopy revealed the possible target of the native peptide (CXCL14-C17) to likely be intracellular, and the amphipathic designed analogs targeted the bacterial membrane. CXCL14-C17 also showed DNA binding characteristic activity similar to buforin-2. Interestingly, CXCL14-C17-a2 and CXCL14-C17-a3 effectively inhibited the production and expression of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 from lipopolysaccharide (LPS)-stimulated RAW264.7 cells, suggesting that these peptides could be promising anti-inflammatory and antimicrobial agents.     

Effects of feeding finisher pigs with chicory or lupine feed for one week or two weeks before slaughter with respect to levels of Bifidobacteria and Campylobacter

This study aimed to assess whether inclusion of chicory or lupine (prebiotics) in the diet of pre-slaughter pigs for just 1 or 2 weeks could change the composition of their intestinal microbiota, stimulate the growth of bifidobacteria and help to lower the amount of thermoplilic Campylobacter spp. (mainly Campylobacter jejuni and Campylobacter coli), which are a major cause of food-borne infections in humans. A total of 48 pigs that had an initial live weight of 90 kg were fed with either a lupine (organic concentrate with 25% blue lupine seeds), chicory (organic concentrate with 10% dried chicory roots) or control (100% organic concentrate) diet for 1 week (24 pigs) or 2 weeks (24 pigs) before slaughter. The Campylobacter spp. level in rectal faecal samples after 0, 1 and 2 weeks of feeding and in the luminal content from ileum, caecum and colon at slaughter was determined by direct plating on modified charcoal-cefoperazone-deoxycholate agar plates. DNA extracted from the luminal content of distal ileum and caecum was used for terminal restriction fragment length polymorphism (T-RFLP) analysis of the composition of intestinal microbiota and for measuring the amount of bifidobacterial and total bacterial DNA by quantitative real-time PCR (qPCR). Campylobacter spp. were excreted by all pigs and present in the luminal content from distal ileum to midway colon with particularly high numbers in the caecum, but the excretion was reduced by 10-fold in pigs fed lupines for 1 week as compared with control- and chicory-fed pigs (mean log₁₀ 2.9 v. 4.1 CFU/g; P < 0.05). The qPCR analysis showed that feeding with lupines resulted in higher levels of bifidobacteria in caecum as compared with the other diets (P < 0.05). T-RFLP analysis showed that four of the most abundant bacteria with terminal restriction fragment values >5% relative to the intensity of total abundance differed between the feed treatments (P < 0.05). Therefore, this study showed that even a short-term alternative feeding strategy with prebiotics in the diet of pre-slaughter pigs elicited changes in the composition of the intestinal microbiota, where lupine increased the level of bifidobacteria in caecum and reduced the Campylobacter spp. excretion level after 1 week.