Effect of 1,25 dihydroxyvitamin D3 on intracellular IFN-γ and TNF-α positive T cell subsets in pulmonary tuberculosis

We studied the immunomodulatory effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α cytokines in T cell subsets of pulmonary tuberculosis (PTB) patients (n = 22) and normal healthy subjects (n = 22). Peripheral blood mononuclear cells (PBMCs) were cultured with live Mycobacterium tuberculosis (MTB) with or without 1,25(OH)₂D₃ (10^?7 M) for 48 h. T cell subsets positive for IFN-γ and TNF-α were enumerated by flow cytometry and the culture supernatants were assayed for both the cytokines using ELISA. In both NHS and PTB patients, a significantly reduced percentage of IFN-γ and TNF-α expressing CD3+, CD3+CD4+ and CD3+CD8+ T cells were observed in cultures stimulated with live MTB and treated with 1,25(OH)₂D₃ compared to cultures without 1,25(OH)₂D₃ (NHS; CD3+ IFN-γ+: p < 0.0001; CD3+TNF-α +: p = 0.0292 and PTB; CD3+ IFN-γ+: p = 0.0292; CD3+ TNF-α +: p = 0.0028). The levels of IFN-γ and TNF-α in the culture supernatants of 1,25(OH)₂D₃ treated cultures were also found to be significantly decreased in both groups (NHS; IFN-γ: p = 0.0001; TNF-α: p < 0.0001) and (PTB; IFN-γ: p < 0.0001; TNF-α: p < 0.0001). A positive correlation was observed between IFN-γ and TNF-α expressing CD3+CD8+ T cells in MTB stimulated cultures treated with or without 1,25(OH)₂D₃ in NHS (p = 0.0001; p = 0.001, respectively) and PTB patients (p = 0.002; p = 0.005, respectively). The present study revealed the suppressive effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α by CD3+CD4+ and CD3+CD8+ T cells in pulmonary tuberculosis. This suppressive effect of 1,25(OH)₂D₃ on proinflammatory and Th1 cytokine positive cells might have a role in reducing inflammation at the site of infection.     

Effect of 5-fluoro-2′-deoxyuridine on toxin production and cell cycle regulation in marine dinoflagellate,Alexandrium tamarense

The effect of metabolic inhibitor, 5-fluoro-2′-deoxyuridine (FUdR) on toxin production and the cell cycle of marine dinoflagellate, Alexandrium tamarense, was investigated. Compared to untreated cells, FUdR at 3 μM (p < 0.05) to 300 μM (p < 0.01) inhibited the cell proliferation and toxin production in a dose-dependent manner for A. tamarense cultured in modified T₁ medium. FUdR at 203 μM resulted in cell cycle arrest at the S phase at day 4 and toxigenesis was inhibited after day 2. The toxin profiles of the FUdR-treated cultures were similar to those of the control culture. These results suggest that FUdR inhibits saxitoxin (STX) biosynthesis in the early stage of the pathway. This report is the first to demonstrate the inhibition of toxin production in A. tamarense by a nucleoside analog.     

Neuromuscular fatigue during a prolonged intermittent exercise: Application to tennis

The time course of alteration in neuromuscular function of the knee extensor muscles was characterized during a prolonged intermittent exercise. Maximal voluntary contraction (MVC) and surface EMG activity of both vastii were measured during brief interruptions before (T₀), during (30, 60, 90, 120, 150 and 180 min: T₃₀, T₆₀, T₉₀, T₁₂₀, T₁₅₀, T₁₈₀) and 30 min after (T₊₃₀) a 3 h tennis match in 12 trained players. M-wave and twitch contractile properties were analyzed following single stimuli. Short tetani at 20 Hz and 80 Hz were also applied to six subjects at T₀ and T₁₈₀. Significant reductions in MVC (P < 0.05; −9%) and electromyographic activity normalized to the M wave for both vastii (P < 0.01) occurred with fatigue at T₁₈₀. No significant changes in M-wave duration and amplitude nor in twitch contractile properties were observed. The ratio between the torques evoked by 20 Hz and 80 Hz stimulation declined significantly (P < 0.001; −12%) after exercise. Central activation failure and alterations in excitation–contraction coupling are probable mechanisms contributing to the moderate impairment of the neuromuscular function during prolonged tennis playing.     

Impact of riding in a coercively obtained Rollkur posture on welfare and fear of performance horses

Rollkur, the usually coercively obtained hyperflexion of the horse's neck, is employed as a training method by some dressage riders; however, its use is controversial as it may cause discomfort and adversely affect the horse's welfare. The objectives of this study were to determine: (1) if horses showed differences in stress, discomfort and fear responses as measured by heart rate and behaviour when ridden in Rollkur (R) obtained by pressure on the reins compared to regular poll flexion (i.e. with the nose-line being at or just in front of the vertical; N), and (2) if they showed a preference between the two riding styles when given the choice. Fifteen riding horses were ridden 30 times through a Y-maze randomly alternating between sides. Riding through one arm of the Y-maze was always followed by a short round ridden in R, whereas riding through the other arm was followed by a short round ridden in N. Immediately after the conditioning phase, horses were again repeatedly ridden into the maze; however, riders left it to the horse to decide which arm of the maze to enter. During R, horses moved slower and showed more often behavioural signs of discomfort, such as tail-swishing, head-tossing or attempted bucks (P < 0.05), and 14 of the 15 horses chose significantly (P < 0.05) more often the maze-arm associated with N rather than R. Subsequently, eight of the horses were also subjected to two fear tests following a short ride in N as well as a ride in R. During R, horses tended to react stronger (P = 0.092) to the fear stimuli and to take longer (P = 0.087) to approach them. These findings indicate that a coercively obtained Rollkur position may be uncomfortable for horses and that it makes them more fearful and therefore potentially more dangerous to ride. Further studies need to assess horses’ reaction to gradual training of Rollkur, as opposed to a coercively obtained hyperflexion, in order to decide whether the practice should be banned.     

Influence of forward leaning and incentive spirometry on inspired volumes and inspiratory electromyographic activity during breathing exercises in healthy subjects

Breathing exercises (BE), incentive spirometry and positioning are considered treatment modalities to achieve lung re-expansion. This study evaluated the influence of incentive spirometry and forward leaning on inspired tidal volumes (V_T) and electromyographic activity of inspiratory muscles during BE. Four modalities of exercises were investigated: deep breathing, spirometry using both flow and volume-oriented devices, and volume-oriented spirometry after modified verbal instruction. Twelve healthy subjects aged 22.7 ± 2.1 years were studied. Surface electromyography activity of diaphragm, external intercostals, sternocleidomastoid and scalenes was recorded. Comparisons among the three types of exercises, without considering spirometry after modified instruction, showed that electromyographic activity and V_T were lower during volume-oriented spirometry (p = 0.000, p = 0.054, respectively). Forward leaning resulted in a lower V_T when compared to upright sitting (p = 0.000), but electromyographic activity was not different (p = 0.606). Inspired V_T and electromyographic activity were higher during volume-oriented spirometry performed after modified instruction when compared with the flow-oriented device (p = 0.027, p = 0.052, respectively). In conclusion BE using volume-oriented spirometry before modified instruction resulted in a lower work of breathing as a result of a lower V_T and was not a consequence of the device type used. Forward leaning might not be assumed by healthy subjects during situations of augmented respiratory demand.     

Aluminum chloride impacts dentate gyrus structure in male adult albino Wistar rats

To get better insights into the aluminum neurotoxicity, rats were treated with AlCl₃ for increasing doses and periods. Body and brain weights, plasma and brain AlCl₃ levels were assayed. Light microscopy observation of brain was performed. AlCl₃ exposure showed a significant decrease (p < 0.05) on body and brain weight with the highest dose at 18 months. Statistical analysis confirms no significant interaction during 6 months (ρ = 0.357; p > 0.05) while, significant correlation was observed during 12 (ρ = 0.836; p < 0.001) and 18 months (ρ = 0.769; p < 0.001) between body and brain weight. Plasma and brain AlCl₃ concentration increased significantly (p < 0.05) with dose and period dependent manner. Statistical analysis confirms significant interaction between brain concentrations of AlCl₃ and administrated doses during 6 (ρ = 0.969; p < 0.001), 12 (ρ = 0.971; p < 0.001) and 18 months (ρ = 0.965; p < 0.001). Similar relation was established between plasma AlCl₃ concentration and administrated doses during 6 (ρ = 0.970; p < 0.001), 12 (ρ = 0.971; p < 0.001) and 18 months (ρ = 0.964; p < 0.001). Significant relation was confirmed between plasma and brain AlCl₃ concentration during 6 (ρ = 0.926; p < 0.001), 12 (ρ = 0.983; p < 0.001) and 18 months (ρ = 0.906; p < 0.001). Morphological alterations mainly targeted the subgranular layer with modulation of the dentate gyrus appearance. This study highlights the toxic effect of AlCl₃ on the brain which may affects learning and memory and seems to be different according to dose and duration of exposure.     

Disproportionate thermophysiological strain between intensively- and extensively-managed goats during summer

Goat keeping is feasible for smallholder farmers in many world regions especially those best suited for extensive management. However, summertime grazing in arid zones entails major challenges to animal thermoregulation and well-being. An experiment was conducted to evaluate the thermoregulatory performance and selected hemogramic parameters in intensively (INT) or extensively (EXT) managed goat kids (N = 14). We applied a previously established technique to evaluate body thermal state of freely ranging animals, in which contemporaneous temperatures of the core (T_c) and periphery (T_p) are chronically recorded. Animals were initially kept for 12 days under INT management. Subsequently, seven animals were transferred to a grazing pasture and gradually transitioned over a four-day acclimatization period, then kept for the last 22 days under EXT conditions. Water drinking was limited to twice daily in both groups. Excessive solar radiation-induced heat load – with daytime black globe temperatures (T_bg) often exceeding 40 °C – under EXT was primarily responsible (r² = 0.49; P < 0.05) for 0.57 and 1.72 °C rises in T_c and T_p, respectively, over INT kids. Unlike the typically biphasic pattern noticed for daily temperatures of both body sites in INT goats, that of EXT counterparts became rather polyphasic, whereby water drinking had drastic and prolonged thermolytic effect, inducing 0.40–0.41 and 0.79–1.45 °C declines in T_c and T_p, on midday and afternoon watering bouts, respectively. Despite indication for added daytime heat load, EXT goats displayed lower early morning T_c than INT. All animals exhibited hypohydration, as reflected by rises in hematocrit, serum osmolality, albumin, potassium, and sodium, being more pronounced in EXT conditions. Results emphasize the excessive thermophysiological strain facing grazing animals in arid zones during the summer.     

Evaluation and comparison of four methods of ranking horses based on reactivity

Four methods of ranking horses on reactivity were evaluated and compared: isolation from conspecifics, presentation of a static novel stimulus, traversing a novel stimulus in a runway (isolation, novel stimulus and runway tests, respectively) and assigning subjective emotionality scores. In all tests, horses’ heart rates were recorded and behaviour was videotaped. To be considered a valid test of reactivity, at least one heart rate and one behavioural measurement in the test had to change significantly between treatments (tranquilizer administation versus sham tranquilizer administration), and behavioural measures had to be displayed in at least 75% of the trials. Forty horses performed each of the three tests daily on three different days in a switchback design. Horses were assigned randomly to a daily test sequence, which was maintained throughout the study. In the runway test, no significant difference in heart rate values in tranquilized and non-tranquilized horses was found, and no behavioural attribute was displayed in more than 52% of the trials; therefore it was rejected as a valid test of reactivity. Both isolation and novel stimulus tests produced valid measurements. Mean heart rate was the most precise physiological measure for these tests, and walking and defecation frequency were the most precise behavioural measures for novel stimulus and isolation tests, respectively. Mean heart rates on the novel stimulus and isolation tests were correlated (r_s = 0.79, P < 0.01) indicating that these tests produced similar rankings based on physiological responses. However, behavioural measures ranked horses differently (r_s = 0.27, P < 0.10) on the tests. Rank correlations between mean heart rates and behavioural measures were higher in the novel stimulus (r_s = 0.66, P < 0.01) than the isolation test (r_s = 0.55, P < 0.01), indicating that the novel stimulus test ranked horses based on either physiological or behavioural responses more similarly than did the isolation test. Therefore, the novel stimulus test was considered the more accurate evaluation of reactivity. Subjective emotionality scores were correlated moderately with mean heart rates (r_s > 0.33, P < 0.01) from the novel stimulus and isolation tests and with walking scores (r_s = 0.47, P < 0.01) from the novel stimulus test. Assignment of subjective emotionality scores was not as accurate as the novel stimulus or isolation tests in ranking horses for reactivity. Using physiological data alone, combining physiological and behavioural measurements or using more than one behavioural measurement in reactivity tests may reflect the reactivity of the horse better than a single behavioural measurement.     

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background: Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods: TXS and VEGF metabolite levels were measured in NSCLC serum samples (n = 46) by EIA. TXB₂ levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results: Serum TXB₂ levels were correlated with VEGF (p < 0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p < 0.0001) and microvessel density (CD-31; p < 0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p < 0.0001) and female patients (p < 0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p < 0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion: TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.     

Housing conditions and breed are associated with emotionality and cognitive abilities in riding school horses

Horses’ emotional reactivity is of a major importance in riding schools where a variety of more or less experienced riders are present. Horses’ learning abilities may also be important for work. Previous studies have shown that different intrinsic or extrinsic factors, such as breed, housing conditions, sire, and work may have an influence, and that different facilities present horses with different characteristics. In this study, we tested the reactions of 184 horses from 22 riding schools, all practicing the same type of work, but differing in particular in terms of housing conditions, to 3 emotionality tests and to one instrumental learning task, in order to (1) try and characterize riding schools, and (2) determine how general management could explain some of the potential differences observed between sites.Multivariate analysis conducted on the whole set of data showed that riding schools could be classified into four categories from those where horses showed low emotionality levels and good learning abilities to those where horses showed high emotionality levels and poor learning abilities. Breed, in accordance to previous studies, had an impact on the time to cross a novel obstacle in hand (“Bridge Test”) (Kruskall–Wallis, H (14, N = 184) = 27.08, p < 0.05) while housing conditions (e.g. box housing) influenced emotionality (emotionality index, MW: p < 0.05), when horses were released in the arena. Box housing was associated with more active locomotion patterns (including trot, canter and passage) (MW: p < 0.005). These results underline the importance for riding schools to take into account both the individual characteristics of the horse, and in particular the breed, and to consider the impact of general management including housing conditions, on horses’ reactivity, and its consequences in terms of human security.     

The effect of time left alone at home on dog welfare

The aim of this study was to investigate the effect of time left alone on dog behaviour and cardiac activity. Twelve privately owned dogs, with no history of separation related behaviour problems, were video-recorded on three different occasions when left alone in their home environment. The treatments lasted for 0.5 h (T_0.5); 2 h (T₂) and 4 h (T₄). Video-recording started 10 min before the owner left the house and continued until 10 min after the owner returned, so that interactions between dog and owner as well as behaviour during separation could be studied. Data on heart rate (HR) and heart rate variability (HRV) were collected within the same time period in each treatment. In addition to analysing behaviours separately, behaviours were also grouped together and defined as new variables; physically active, attentive behaviour, vocal, interaction initiated by owner and interaction initiated by dog. There were no differences in behaviour between treatments at equivalent time intervals until the owner returned, although a number of differences were observed at reunion with the owner. Dogs showed a higher frequency of physical activity (P < 0.05) and attentive behaviour (P < 0.01) in T₂ (0.37 ± 0.07; 0.52 ± 0.08, mean frequency of occurrence/15 s ± SE) and T₄ (0.48 ± 0.08; 0.48 ± 0.07) compared to T_0.5 (0.20 ± 0.07; 0.21 ± 0.05). They also showed more tail wagging (P < 0.01) and interacted more with their owners (P < 0.01) in T₂ (0.27 ± 0.08; 0.47 ± 0.09) and T₄ (0.26 ± 0.04; 0.42 ± 0.09) compared to T_0.5 (0.09 ± 0.04; 0.14 ± 0.03). After a longer time of separation, the dogs also showed higher frequencies of lip licking (P < 0.05) and body shaking (P < 0.05) at the owner's return (T_0.5 = 0.09 ± 0.05; T₂ = 0.24 ± 0.08; T₄ = 0.27 ± 0.06 and T_0.5 = 0.03 ± 0.01; T₂ = 0.08 ± 0.03; T₄ = 0.07 ± 0.01, respectively). There was a tendency for higher HR (P < 0.1) during the first and second minute after reunion in T₂ (127.6 ± 1.25, mean bpm ± SE; 111.3 ± 1.24) compared to T_0.5 (106.2 ± 1.06; 87.5 ± 1.02). According to the results of this study, the effect of time left alone was shown by a more intense greeting behaviour by the dog towards their owner as well as by a higher frequency of physical activity and attentive behaviour when the owner returned, already after 2 h of separation. Although this study cannot distinguish between whether dogs were aware of the length of time they were alone (but did not signal it) or whether they were unaware until reminded of it by the return of their owner, it does confirm that dogs are affected by the duration of time at home alone.     

Effects of considering the rate of comminution of particles and microbial contamination on accuracy of in situ studies of feed protein degradability in ruminants

Ruminal degradation of dry matter (DM) and crude protein (CP) in samples of soybean meal (SBM), barley grain (BG) and lucerne hay (LH) were measured by an in situ ruminal technique considering either only the outflow rate of particles from the rumen (k_p) or, both k_p and rate of particle comminution (k_c). The effect of correction for the microbial contamination using an ¹⁵N technique was also determined for LH. Degradation and transit studies were completed on three wethers cannulated in the rumen and duodenum and fed a mixed diet of LH and concentrate (2:1 on DM), at an intake level of 50 g DM/kg BW^0.75. Transit studies of concentrates and forages were completed with SBM and LH samples marked with Yb and Eu, respectively. Mean values of k_c were higher in SBM than in LH (0.357 versus 0.204 h, P < 0.05). The same trend was observed for k_p (0.0587 versus 0.0452/h; P < 0.1). Apparent estimates of the proportion of rumen undegraded CP obtained using both rates were 0.884, 0.745 and 0.825 of those obtained using only k_p (0.535, 0.247 and 0.303 for SBM, BG and LH, respectively). Differences occurred for SBM and LH (P < 0.01) and for BG (P < 0.05). The pattern of microbial contamination of LH, expressed as a proportion of residual DM or CP, fitted exponential curves with asymptotic values of 0.076 and 0.586, respectively. Correction for microbial contamination reduced (P < 0.05) estimates of undegraded CP from 0.303 to 0.238, which was further reduced to 0.178 (P < 0.01) when the effect of k_c was also considered.     

Expression of orexin receptors 1 (OX1R) and 2 (OX2R) in the porcine pituitary during the oestrous cycle

Orexin A and B, also termed hypocretin 1 and 2, are associated with the stimulation of food intake and arousal. The biological actions of the hormones are mediated via two distinct G protein-coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). OX1R is selective for orexin A and OX2R binds orexin A and orexin B with similar affinity. The present study analyzed mRNA and protein expressions of OX1R and OX2R in adenohypophysis (AP) and neurohypophysis (NP) of cycling pigs. The tissue samples were harvested on days 2–3, 10–12, 14–16, and 17–19 of the oestrous cycle. Using quantitative real-time PCR higher OX1R gene expression was detected in AP on days 2–3 relative to days 10–12, 14–16 and 17–19 (p < 0.05). In NP the OX1R mRNA level was elevated on days 10–12 compared to the remaining stages (p < 0.05). OX2R gene expression in AP was the lowest on days 10–12 (p < 0.05 compared to days 2–3 and 17–19) and the expression peak occurred on days 17–19 (p < 0.05 vs. the all studied stages). In NP the highest (p < 0.05) expression of OX2R mRNA was noted on days 17–19 in relation to the remaining periods. OX1R protein content in AP was greatest on days 10–12 (p < 0.05), whereas in NP it was greatest on days 2–3 and 14–16 (p < 0.05 vs. days 10–12 and 17–19). In both cases the lowest OX1R protein expression was observed during follicular phase (p < 0.05 in relation to three remaining studied stages). OX2R protein in AP was lower (p < 0.05) on days 2–3 and 14–16 compared to days 10–12 and 17–19. In NP the lowest (p < 0.05) expression of this protein was on days 17–19 and the highest on days 10–12 (p < 0.05 compared to days 2–3 and 17–19). In summary, the present findings provide the first evidence that OX1R and OX2R mRNAs and proteins occur in the pituitary of the pig and indicate the dependence of orexin receptor expression on the endocrine reproductive state.     

IL28B gene polymorphisms and Th1/Th2 cytokine levels might be associated with HTLV-associated arthropathy

The present study is the first investigation of the association between single nucleotide polymorphisms (SNPs – rs8099917, rs12979860 and rs8103142) of the IL28B gene and the development of human T-lymphotropic virus (HTLV)-associated arthropathy (HAA). Individuals with HAA exhibited low interleukin (IL) 6 (p < 0.05) and high IL-10 (p < 0.05) levels compared with asymptomatic patients. TNF-α/CD4⁺ T cell count, TNF-α/CD8⁺ T cell count and IFN-γ/proviral load positively correlated in asymptomatic patients. The allelic and genotypic frequencies did not differ between patients with HAA and asymptomatic patients. Seven haplotypes were detected in the investigated population, with haplotype CCT (p < 0.05) being the most frequent among the HTLV-infected individuals, while haplotype TTG (p < 0.05) was detected in the group with HAA only. Compared with asymptomatic patients, individuals with HAA and genotype TT (rs8099917) exhibited larger numbers of CD8⁺ T cells (p < 0.05) and higher proviral load levels (p < 0.05). Those patients with HAA and genotypes CC (rs12979860) and TT (rs8103142) exhibited high TNF-β (p < 0.05) and IFN-γ (p < 0.05) levels. Those patients with HAA and genotype CT/TT (rs12979860) exhibited high IL-10 levels (p < 0.05). These results suggest that haplotypes CCT and TTG might be associated with susceptibility to HTLV infection and progression to HAA, respectively. Genotype TT (rs8099917) might be a risk factor for elevation of the proviral load and CD8⁺ T cell count. In addition, genotypes CC (rs12979860) and TT (rs8103142) seem to be associated with increased TNF-β and IFN-γ levels.     

Effects of dietary whole cottonseed and crude protein level on rumen protozoal population and fermentation parameters

In this investigation in vitro and in vivo trials were performed to determine the efficacy of a cottonseed to limit protozoal population and fermentation parameters. The composition of diets given to the different treatments were as follow: (1) control (without whole cottonseed), 16% crude protein (CP), 3.2% ether extract (E.E.); (2) 20% whole cottonseed, 16% CP, 6.5% E.E.; (3) 20% whole cottonseed, 13% CP, 6.4% E.E. and (4) 20% crushed whole cottonseed, 13% CP, 6.4% E.E. DM disappearance (DMD) and fermentation characteristics of the treatments were determined by in vitro incubation studies. In the in vivo trial, ruminal fluid was taken by rumenocentesis (3 h after feeding) on days 1, 3, 5, 7, 9, 11, 14, 21 and 28 from four sheep fed about treatment diets. The pH and protozoal counts were determined in each sample, while ammonia nitrogen and volatile fatty acid (VFA) were determined in samples taken on days 7, 14, 21 and 28. The in vitro DMD after 24 h incubation decreased (p < 0.01) with the addition of cottonseed in diets 3 and 4 and DMD after 72 h incubation was highest (p < 0.01) for the control diet. The fractional rate of gas production (c) for the control and diet 2 was higher (p < 0.05) than for the diets 3 and 4. Feeding crushed whole cottonseed decreased molar proportion of propionate (p < 0.05) and increased molar proportion of butyrate (p < 0.01). Low crude protein level increased the molar proportion of propionate (p < 0.05) and decreased molar proportion of butyrate (p < 0.05) and cellolytic protozoa population (p < 0.05). Feeding cottonseed decreased (p < 0.05) the total protozoa population from approximately 500,000 to 250,000 ml⁻¹ and Holotrich and cellulolytic protozoa disappeared from the rumen of sheep and only Entodinium sp., remained. This was associated with lower concentration of ammonia nitrogen in rumen fluid of sheep fed diets 4 (p < 0.05) and 2 (p < 0.01). It was concluded that cottonseed reduced rumen fauna and ammonia nitrogen, but had no effect on ruminal VFA while the crushed whole cottonseed decreased molar proportion of isovalerate only. In vivo molar proportion of propionate and butyrate and valerate were increase and decrease, respectively, by decreasing CP percentage in treatment diets.     

Angiotensin II-induced activation of c-Ret signaling is critical in ureteric bud branching morphogenesis

The renin-angiotensin system (RAS) plays a critical role in ureteric bud (UB) and kidney morphogenesis. Mutations in the genes encoding components of the RAS cause a spectrum of congenital abnormalities of the kidney and urinary tract (CAKUT). However, the mechanisms by which aberrations in the RAS result in CAKUT are poorly understood. Given that c-Ret receptor tyrosine kinase (RTK) is a major inducer of UB branching, the present study tested the hypothesis that angiotensin (Ang) II-induced activation of c-Ret plays a critical role in UB branching morphogenesis. E12.5 mice metanephroi were grown for 24 h in the presence or absence of Ang II, Ang II AT₁ receptor (AT₁R) antagonist candesartan, phosphatidylinositol 3-kinase (PI3 K) inhibitor LY294002 or ERK1/2 inhibitor PD98059. Ang II increased the number of UB tips (61 ± 2.4 vs. 45 ± 4.3, p < 0.05) compared with control. Quantitative RT-PCR analysis demonstrated that Ang II increased c-Ret mRNA levels in the kidney (1.35 ± 0.05 vs. 1.0 ± 0, p < 0.01) and in the UB cells (1.28 ± 0.04 vs. 1.0 ± 0, p < 0.01) compared to control. This was accompanied by increased Tyr¹⁰⁶²Ret phosphorylation by Ang II (5.5 ± 0.9 vs. 1.8 ± 0.4 relative units, p < 0.05). In addition, treatment of UB cells with Ang II (10^?5 M) increased phosphorylation of Akt compared to control (213 ± 16 vs. 100 ± 20%, p < 0.05). In contrast, treatment of metanephroi or UB cells with candesartan decreased c-Ret mRNA levels (0.72 ± 0.06 vs. 1.0 ± 0, p < 0.01; 0.68 ± 0.07 vs. 1.0 ± 0, p < 0.05, respectively) compared with control. Ang II-induced UB branching was abrogated by LY294002 (24 ± 2.6 vs. 37 ± 3.0, p < 0.05) or PD98059 (33 ± 2.0 vs. 48 ± 2.2, p < 0.01). These data demonstrate that Ang II-induced UB branching depends on activation of Akt and ERK1/2. We conclude that cross-talk between the RAS and c-Ret signaling plays an important role in the development of the renal collecting system.     

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p < 0.001) in MR signal intensity or a significant decrease (p < 0.001) in T₁ relaxation, measured as %T₁ change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p < 0.0001) in T₁ relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p < 0.001) and 3-nitrotyrosine (3-NT) (p < 0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.     

Light thresholds derived from seagrass loss in the coastal zone of the northern Great Barrier Reef,Australia

There is a world-wide trend for deteriorating water quality and light levels in the coastal zone, and this has been linked to declines in seagrass abundance. Localized management of seagrass meadow health requires that water quality guidelines for meeting seagrass growth requirements are available. Tropical seagrass meadows are diverse and can be highly dynamic and we have used this dynamism to identify light thresholds in multi-specific meadows dominated by Halodule uninervis in the northern Great Barrier Reef, Australia. Seagrass cover was measured at ∼3 month intervals from 2008 to 2011 at three sites: Magnetic Island (MI) Dunk Island (DI) and Green Island (GI). Photosynthetically active radiation was continuously measured within the seagrass canopy, and three light metrics were derived. Complete seagrass loss occurred at MI and DI and at these sites changes in seagrass cover were correlated with the three light metrics. Mean daily irradiance (I_d) above 5 and 8.4 mol m⁻² d⁻¹ was associated with gains in seagrass at MI and DI, however a significant correlation (R = 0.649, p < 0.05) only occurred at MI. The second metric, percent of days below 3 mol m⁻² d⁻¹, correlated the most strongly (MI, R = −0.714, p < 0.01 and DI, R = −0.859, p = <0.001) with change in seagrass cover with 16–18% of days below 3 mol m⁻² d⁻¹ being associated with more than 50% seagrass loss. The third metric, the number of hours of light saturated irradiance (H_sat) was calculated using literature-derived data on saturating irradiance (E_k). H_sat correlated well (R = 0.686, p < 0.01; and DI, R = 0.704, p < 0.05) with change in seagrass abundance, and was very consistent between the two sites as 4 H_sat was associated with increases in seagrass abundance at both sites, and less than 4 H_sat with more than 50% loss. At the third site (GI), small seasonal losses of seagrass quickly recovered during the growth season and the light metrics did not correlate (p > 0.05) with change in percent cover, except for I_d which was always high, but correlated with change in seagrass cover. Although distinct light thresholds were observed, the departure from threshold values was also important. For example, light levels that are well below the thresholds resulted in more severe loss of seagrass than those just below the threshold. Environmental managers aiming to achieve optimal seagrass growth conditions can use these threshold light metrics as guidelines; however, other environmental conditions, including seasonally varying temperature and nutrient availability, will influence seagrass responses above and below these thresholds.