Paralogous antirepressors acting on the master regulator for biofilm formation in Bacillus subtilis

Matrix production during biofilm formation by Bacillus subtilis is governed by a gene control circuit at the heart of which are three dedicated regulatory proteins, the antirepressor SinI, the repressor SinR and the downstream regulator SlrR. Matrix production is triggered by the synthesis of SinI, which binds to and inactivates SinR, thereby derepressing genes for matrix production as well as the gene for SlrR. Recently, two additional regulators of matrix genes were identified: SlrA, which was reported to be an activator of SlrR, and YwcC, a repressor of SlrA synthesis (Kobayashi, 2008). We present evidence indicating that SlrA, which is a paralogue of SinI, is like SinI, an antirepressor that binds to, and inactivates, SinR. We also show that SlrA does not activate SlrR for expression of matrix genes. Instead, SlrR binds to, and inhibits the activity of, SlrA. Thus, the YwcC-SlrA-SinR-SlrR pathway is a negative feedback loop in which SlrA indirectly stimulates the synthesis of SlrR, and SlrR, in turn, inhibits the activity of SlrA. Finally, we report that under standard laboratory conditions SlrA makes only a small contribution to the expression of genes for matrix production. We propose that in response to an unknown signal recognized by the YwcC repressor, SlrA transiently boosts matrix production.     

Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis

sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI. Each forms a pair of α-helices that hook together to form an intermolecular four-helix bundle. Here, we are interested in the assembly of the SinR tetramer and its binding to DNA. Size-exclusion chromatography with multi-angle laser light scattering and crystallographic analysis reveal that a DNA-binding fragment of SinR (residues 1-69) is a monomer, while a SinI-binding fragment (residues 74-111) is a tetramer arranged as a dimer of dimers. The SinR(74-111) chain forms two α-helices with the organisation of the dimer similar to that observed in the SinR-SinI complex. The tetramer is formed through interactions of residues at the C-termini of the four chains. A model of the intact SinR tetramer in which the DNA binding domains surround the tetramerisation core was built. Fluorescence anisotropy and surface plasmon resonance experiments showed that SinR binds to an oligonucleotide duplex, 5′-TTTGTTCTCTAAAGAGAACTTA-3′, containing a pair of SinR consensus sequences in inverted orientation with a K_d of 300 nM. The implications of these data for promoter binding and the curious quaternary structural transitions of SinR upon binding to (i) SinI and (ii) the SinR-like protein SlrR, which “repurposes” SinR as a repressor of autolysin and motility genes, are discussed.     

A novel factor controlling bistability in Bacillus subtilis: the YmdB protein affects flagellin expression and biofilm formation

Cells of Bacillus subtilis can either be motile or sessile, depending on the expression of mutually exclusive sets of genes that are required for flagellum or biofilm formation, respectively. Both activities are coordinated by the master regulator SinR. We have analyzed the role of the previously uncharacterized ymdB gene for bistable gene expression in B. subtilis. We observed a strong overexpression of the hag gene encoding flagellin and of other genes of the σ(D)-dependent motility regulon in the ymdB mutant, whereas the two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were not expressed. As a result, the ymdB mutant is unable to form biofilms. An analysis of the individual cells of a population revealed that the ymdB mutant no longer exhibited bistable behavior; instead, all cells are short and motile. The inability of the ymdB mutant to form biofilms is suppressed by the deletion of the sinR gene encoding the master regulator of biofilm formation, indicating that SinR-dependent repression of biofilm genes cannot be relieved in a ymdB mutant. Our studies demonstrate that lack of expression of SlrR, an antagonist of SinR, is responsible for the observed phenotypes. Overexpression of SlrR suppresses the effects of a ymdB mutation.     

Effects of Changes in Water Intake on Mood of High and Low Drinkers

Objective

To evaluate the effects of a change in water intake on mood and sensation in 22 habitual high-volume (HIGH; 2-4 L/d) and 30 low-volume (LOW; <1.2 L/d) drinkers who were asked to respectively decrease and increase their daily water intake.

Method

During baseline HIGH consumed 2.5 L and LOW 1 L of water/day. During 3 controlled intervention days HIGH''s water intake was restricted to 1 L/day whereas LOW''s was increased to 2.5 L water/day. Several mood scales (Bond & Lader Visual Analog Scale (VAS), Profile of Mood States, Karolinska Sleepiness Scale, Thirst & Emotional VAS) were administered at different time points during the study. ANOVA including intervention, time point and intervention by time point as fixed effects on mean values (i.e.; baseline data vs. mean of 3 intervention days) for each mood scale was performed.

Results

At baseline HIGH and LOW were comparable in mood state, except for thirst scores (estimate = 17.16, p<0.001) and POMS depression-dejection scores (estimate = 0.55, p<0.05) which were both higher in the HIGH vs. LOW. In HIGH the restricted water intake resulted in a significant increase in thirst (p<0.001) and a decrease in contentedness (p<0.05), calmness (p<0.01), positive emotions (p<0.05) and vigor/activity (p<0.001). In LOW, increased water consumption resulted in a significant decrease in fatigue/inertia (p<0.001), confusion/bewilderment (p = 0.05) and thirst (p<0.001) and a trend to lower sleepiness (p = 0.07) compared to baseline.

Conclusion

Increasing water intake has beneficial effects in LOW, especially sleep/wake feelings, whereas decreasing water intake has detrimental effects on HIGH''s mood. These deleterious effects in HIGH were observed in some sleep/wake moods as well as calmness, satisfaction and positive emotions.     

Effects of muscle glycogen and plasma FFA availability on human metabolic responses in cold water.

The purpose of this study was to investigate whether simultaneous alterations in the availability of plasma free fatty acids and muscle glycogen would impair the maintenance of thermal balance during cold water immersion in humans. Eight seminude subjects were immersed on two occasions in 18 degrees C water for 90 min or until rectal temperature (Tre) decreased to 35.5 degrees C. Each immersion followed 2.5 days of a specific dietary and exercise regimen designed to elicit low (LOW) or high glycogen levels (HIGH) in large skeletal muscle groups. Nicotinic acid (1.6 mg/kg) was administered for 2 h before and during immersion to inhibit white adipose tissue lipolysis. Biopsies from the vastus lateralis showed that the glycogen concentration before the immersion was significantly lower in LOW than in HIGH (223 +/- 19 vs. 473 +/- 24 mmol glucose units/kg dry muscle). However, the mean rates of glycogen utilization were not significantly different between trials (LOW 0.62 +/- 0.14 vs. HIGH 0.88 +/- 0.15 mmol glucose units.kg-1.min-1). Nicotinic acid dramatically reduced plasma free fatty acid levels in both trials, averaging 127 +/- 21 mumol/l immediately before the immersion. Cold water immersion did not significantly alter those levels. Plasma glucose levels were significantly reduced after cold water immersion to a similar extent in both trials (18 +/- 4%). Mean respiratory exchange ratio at rest and during immersion was greater in HIGH than LOW, whereas there were no intertrial differences in O2 uptake. The calculated average metabolic heat production during immersion tended to be lower (P = 0.054) in LOW than in HIGH (15.3 +/- 1.9 vs. 17.5 +/- 1.9 kJ/min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pre-slaughter handling,exercise and the presence of a dog on lamb welfare and meat quality

Before slaughter, lambs may experience several stressors such as feed and water deprivation, handling and transport that have the potential to negatively impact welfare and meat quality. The objective of this study was to evaluate the effect of pre-slaughter handling, exercise and the presence of a dog on the behaviour and physiology of lambs and meat quality at slaughter. At 6 months of age, 60 lambs (n=20 lambs/replicate; three replicates) were allocated to one of the two treatment groups (n=30 lambs/treatment): low (LOW) intensive handling or high (HIGH) intensive handling. LOW lambs were moved short distances, quietly and without the use of a dog before transport. HIGH lambs were moved quickly, long distances and with a dog present before transport. Lamb behaviour (standing, lying, rumination and panting) was recorded for 1 h before (post-treatment) and after transport (post-transport), and for 30 min before slaughter (pre-slaughter). Blood samples were collected before (baseline), after transport (post-transport) and at exsanguination (at slaughter) to assess cortisol, lactate and non-esterified fatty acid (NEFA) concentrations. At slaughter, lamb carcases (M. longissimus lumborum) were evaluated for pH levels, drip and cook loss, and tenderness. HIGH lambs spent more time standing (P<0.001) and panting (P<0.001) and less time lying (P<0.001) and ruminating (P<0.001) post-treatment than LOW lambs, but more (P<0.001) time ruminating post-transport. All lambs spent more time standing (P<0.001) and less time lying (P<0.001) and panting (P<0.001) post-transport and pre-slaughter than post-treatment. Cortisol concentrations were greater (P<0.001) in lambs post-transport and at slaughter compared with baseline values. Lactate concentrations were lower (P=0.002) in HIGH than LOW lambs. In addition, NEFA concentrations were higher (P<0.001) post-transport and at slaughter in HIGH compared with LOW lambs. Ultimate pH was higher (P<0.001) in HIGH than LOW lambs and pH declined quicker (P=0.012) in LOW than HIGH lambs. Cook loss, drip loss and shear force were lower (P⩽0.05) in HIGH than LOW lambs. The HIGH intensive pre-slaughter handling regime used in the present study caused stress in lambs and increased ultimate pH that could potentially negatively impact welfare, product quality and consistency.     

Detrimental effect of eicosapentaenoic acid supplementation on bone following ovariectomy in rats

Although several studies have reported a positive effect of n-3 essential fatty acids (EFAs) on bone density post-ovariectomy, the role of specific EFAs has yet to be fully elucidated. In this study, ovariectomised (OVX) rats were supplemented with 0.1 g (LOW) or 1.0 g (HIGH) of eicosapentaenoic acid (EPA)/kg body weight for 9 weeks. Bone mineral density (BMD), 25-hydroxyvitamin D(3) and plasma fatty acid profile were compared to those of OVX and sham animals fed a non-supplemented diet. BMD decreased significantly in all OVX (P<0.001) but not sham rats. There was no difference in BMD between the LOW group and OVX controls. BMD was significantly lower in the HIGH group compared to OVX and sham controls. 25-hydroxyvitamin D(3) levels were significantly higher in both the LOW and HIGH groups compared to OVX controls (P=0.0006 and 0.02, respectively). In conclusion, high-dose EPA supplementation exacerbated the effects of ovariectomy on BMD.     

The maximal and submaximal vertical jump: implications for strength and conditioning

The vertical jump is a widely used activity to develop explosive strength, particularly in plyometric and maximal power training programs. It is a multijoint action that requires substantial muscular effort from primarily the ankle, knee, and hip joints. It is not known if submaximal performances of a vertical jump have a proportional or differential training effect on the major lower-limb muscles compared to maximal jump performance. Therefore, the purpose of this study was to investigate the contribution that each of the major lower-limb joints makes to vertical jump performance as jump height increases and to comment on the previously mentioned uncertainty. Adult males (N = 20) were asked to perform a series of submaximal (LOW and HIGH) and maximal (MAX) vertical jumps while using an arm swing. Force, motion, and electromyographical data were recorded during each performance and used to compute a range of kinematic and kinetic data, including ankle, knee, and hip joint torques, powers, and work done. It was found that the contribution to jump height made by the ankle and knee joints remains largely unchanged as jump height increases (work done at the ankle: LOW =1.80, HIGH = 1.97, MAX = 2.06 J.kg(-1), F = 3.596, p = 0.034; knee: LOW = 1.62, HIGH = 1.77, MAX = 1.94 J.kg(-1), F = 1.492, p = 0.234) and that superior performance in the vertical jump is achieved by a greater effort of the hip extensor muscles (work done at the hip: LOW = 1.03, HIGH = 1.84, MAX = 3.24 J.kg(-1), F = 110.143, p < 0.001). It was concluded that the role of submaximal and maximal jumps can be differentiated in terms of their effect on ankle, knee, and hip joint muscles and may be of some importance to training regimens in which these muscles need to be differentially trained.     

Evidence that metabolism and chromosome copy number control mutually exclusive cell fates in Bacillus subtilis

Bacillus subtilis chooses between matrix production and spore formation, which are both controlled by the regulator Spo0A~P. We report that metabolism and chromosome copy number dictate which fate is adopted. Conditions that favour low Spo0A~P levels promote matrix production, whereas conditions favouring high levels trigger sporulation. Spo0A~P directs the synthesis of SinI, an antirepressor for the SinR repressor of matrix genes. The regulatory region of sinI contains an activator site that Spo0A~P binds strongly and operators that bind Spo0A~P weakly. Evidence shows that low Spo0A~P levels turn sinI ON and high levels turn sinI OFF and instead switch sporulation ON. Cells in which sinI and sinR were transplanted from their normal position near the chromosome replication terminus to positions near the origin and cells that harboured an extra copy of the genes were blocked in matrix production. Thus, matrix gene expression is sensitive to the number of copies of sinI and sinR. Because cells at the start of sporulation have two chromosomes and matrix-producing cells one, chromosome copy number could contribute to cell-fate determination.     

Does Anxiety Cause Freezing of Gait in Parkinson's Disease?

Individuals with Parkinson''s disease (PD) commonly experience freezing of gait under time constraints, in narrow spaces, and in the dark. One commonality between these different situations is that they may all provoke anxiety, yet anxiety has never been directly examined as a cause of FOG. In this study, virtual reality was used to induce anxiety and evaluate whether it directly causes FOG. Fourteen patients with PD and freezing of gait (Freezers) and 17 PD without freezing of gait (Non-Freezers) were instructed to walk in two virtual environments: (i) across a plank that was located on the ground (LOW), (ii) across a plank above a deep pit (HIGH). Multiple synchronized motion capture cameras updated participants'' movement through the virtual environment in real-time, while their gait was recorded. Anxiety levels were evaluated after each trial using self-assessment manikins. Freezers performed the experiment on two separate occasions (in their ON and OFF state). Freezers reported higher levels of anxiety compared to Non-Freezers (p<0.001) and all patients reported greater levels of anxiety when walking across the HIGH plank compared to the LOW (p<0.001). Freezers experienced significantly more freezing of gait episodes (p = 0.013) and spent a significantly greater percentage of each trial frozen (p = 0.005) when crossing the HIGH plank. This finding was even more pronounced when comparing Freezers in their OFF state. Freezers also had greater step length variability in the HIGH compared to the LOW condition, while the step length variability in Non-Freezers did not change. In conclusion, this was the first study to directly compare freezing of gait in anxious and non-anxious situations. These results present strong evidence that anxiety is an important mechanism underlying freezing of gait and supports the notion that the limbic system may have a profound contribution to freezing in PD.     

The influence of maternal nutrition on expression of genes responsible for adipogenesis and myogenesis in the bovine fetus

The objective of this study was to determine whether altered maternal energy supply during mid-gestation results in differences in muscle histology or genes regulating fetal adipose and muscle development. In total, 22 Angus cross-bred heifers (BW=527.73±8.3 kg) were assigned randomly to the three dietary treatments providing 146% (HIGH; n=7), 87% (INT; n=7) or 72% (LOW; n=8) of the energy requirements for heifers from day 85 to day 180 of gestation. Fetuses were removed via cesarean section at day 180 of gestation and longissimus muscle (LM) and subcutaneous fat were collected and prepared for analysis of gene expression. Samples from the LM and semitendinosus (ST) were evaluated for muscle fiber diameter, area and number. The right hind limb was dissected and analyzed to determine compositional analysis. Fetal growth and muscle histology characteristics of the LM and ST were similar among treatments. Preadipocyte factor-1 expression was up-regulated in fetal LM (P<0.05) of HIGH fetuses as compared with INT, whereas LOW fetuses showed increased CCAAT/enhancer-binding protein-β (C/EBP-β) expression in LM as compared with INT (P<0.05). Peroxisome proliferator-activated receptor γand C/EBP-α did not differ as a result of dietary treatment in LM or subcutaneous fat samples. There was a tendency for increased expression of fatty acid synthase in LM of LOW fetuses as compared with INT (P<0.10). Myogenin was more highly expressed (P<0.05) in LM of the LOW fetuses, whereas μ-calpain expression was increased in the HIGH treatment compared with INT. A tendency for increased expression of IGF-II was observed for both LOW and HIGH fetuses compared with INT (P<0.10). Expression of stearoyl-CoA desaturase, myoblast determination protein 1, myogenic factor 5, myogenic regulatory factor-4, m-calpain, calpastatin, IGF-I and myostatin was similar between treatments. Collectively, these results suggest that fetal growth characteristics are not affected by the level of maternal nutritional manipulation imposed in this study during mid-gestation. However, differences in expression of fetal genes regulating adipose and muscle tissue growth and development could lead to differences in postnatal composition and warrants further investigation.     

Exploring nitrogen remobilization for seed filling using natural variation in Arabidopsis thaliana

Nineteen Arabidopsis accessions grown at low (LOW N) and high (HIGH N) nitrate supplies were labelled using (15)N to trace nitrogen remobilization to the seeds. Effects of genotype and nutrition were examined. Nitrate availability affected biomass and yield, and highly modified the nitrogen concentration in the dry remains. Surprisingly, variations of one-seed dry weight (DW(1S)) and harvest index (HI) were poorly affected by nutrition. Nitrogen harvest index (NHI) was highly correlated with HI and showed that nitrogen use efficiency (NUE) was increased at LOW N. Nitrogen remobilization efficiency (NRE), as (15)N partitioning in seeds ((15)NHI), was also higher at LOW N. The relative specific abundance (RSA) in seeds and whole plants indicated that the (14)NO(3) absorbed post-labelling was mainly allocated to the seeds (SEEDS) at LOW N, but to the dry remains (DR) at HIGH N. Nitrogen concentration (N%) in the DR was then 4-fold higher at HIGH N compared with LOW N, whilst N% in seeds was poorly modified. Although NHI and (15)NHI were highly correlated to HI, significant variations in NUE and NRE were identified using normalization to HI. New insights provided in this report are helpful for the comprehension of NUE and NRE concepts in Arabidopsis as well as in crops and especially in Brassica napus.     

Effects of a passive exoskeleton on the mechanical loading of the low back in static holding tasks

With mechanical loading as the main risk factor for LBP in mind, exoskeletons are designed to reduce the load on the back by taking over a part of the required moment. The present study assessed the effect of a passive exoskeleton on back and abdominal muscle activation, hip and lumbar flexion and on the contribution of both the human and the exoskeleton to the L5/S1 net moment, during static bending at five different hand heights. Two configurations of the exoskeleton (LOW & HIGH) differing in angle-torque characteristics were tested. L5/S1 moments generated by the subjects were significantly reduced (15–20% for the most effective type) at all hand heights. LOW generated 4–11 Nm more support than HIGH at 50%, 25% and 0% upright stance hand height and HIGH generated 4–5 Nm more support than LOW at 100% and 75%. Significant reductions (11–57%) in back muscle activity were found compared to WITHOUT for both exoskeletons for some conditions. However, EMG reductions compared to WITHOUT were highly variable across subjects and not always significant. The device allowed for substantial lumbar bending (up to 70°) so that a number of participants showed the flexion-relaxation phenomenon, which prevented further reduction of back EMG by the device and even an increase from 2% to 6% MVC in abdominal activity at 25% hand height. These results indicate that flexion relaxation and its interindividual variation should be considered in future exoskeleton developments.     

Patterns of movement by baboons in the Drakensberg mountains: Primary responses to the environment

The movement patterns of two chacma baboon troops in the Drakensberg mountains of South Africa were studied over an 18-month period. One of these troops (HIGH) lived on the escarpment slopes (1920–2980 m), while the other (LOW) lived primarily in the montane belt (1560–2000 m). In this paper we analyze the effects of environmental variables on day journeys and home range use. We found the troops to be similar in the temporal patterning of their daily movement, in the resistance of travel speed to variations in local climatic conditions, and in their response to the nutritional bottleneck at the end of the austral winter. While both troops traveled farther as food became scarcer, HIGH troop undertook longer day journeys than LOW troop. This was probably due to the interactive effects of absolutely lower food availability and lower ambient temperatures. HIGH troop traveled farther when food was scarcer and when minimum temperatures were higher. Neither variable accounted for variance in the day journeys of LOW troop. Both troops used some areas of their home ranges preferentially. For LOW troop this preference followed the spatial distribution of food. While the same was generally true for HIGH troop, its home range use was complicated by the adverse temperatures of winter and spring. It abandoned the higher, more productive part of its range once temperatures became severe, returning only after minimum ground temperatures had risen above 0°C. These usage patterns differ from those described for eastern African baboon populations and reflect both the severity and sesonality of climate and the poor quality and extreme dispersion of food that characterize these southern mountains.