Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPgammaS

The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K(12) of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate or guanosine-5'-(beta,gamma-methyleno)triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state (state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) leads to (31)P NMR spectra that indicate the prevalence of only one conformational state with K(12) > 10. Titration with the Ras-binding domain of Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding state 2. In the GTPgammaS complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affinity states 1a and 1b. From line-shape analysis of the spectra measured at various temperatures an activation energy DeltaH(|) (1a1b) of 61 kJ.mol(-1) and an activation entropy DeltaS(|) (1a1b) of 65 J.K(-1).mol(-1) are derived. Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity K(A) for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild-type with the strongest reduction observed for the GTPgammaS complex.     

Detection of testosterone propionate administration in horse hair samples

A sensitive and specific method has been developed to detect semi-quantitatively testosterone in horse hair samples. The method involved a washing step with sodium dodecylsulfate aqueous solution. The mane and tail hair samples (100mg) were dissolved in 1 mL of sodium hydroxide for 15 min at 95 degrees C in the presence of d3-boldenone used as internal standard. The next three steps involved diethyl ether extraction and a solid phase extraction on Isolute C18 (EC) cartridges eluted with methanol. The residue was derivatized by adding 100 microL of acetonitrile and 30 microL of PFPA then incubating for 15 min at 60 degrees C. After evaporation, 30 microL of hexane was added and 2.5 microL was injected into the column (a bonded phase fused silica capillary column DB5MS, 30 m x 0.25 mm i.d. x 0.25 microm film thickness) of a Trace GC chromatograph. In order to improve the sensitivity of the method, damping gas flow has been optimized. Testosterone was identified in MS(2) full scan mode on the Polaris Q instrument. The assay was capable of detecting less than 1 pg mg(-1). The recovery was close to 90%. The analysis of tail and mane samples collected from a gelding horse having received a single dose of testosterone propionate (1 mg kg(-1)) showed the presence of testosterone in the range of 1-6 pg mg(-1) in hair collected during 5 months after administration.     

Effects of preconception lifestyle intervention in infertile women with obesity: The FIT-PLESE randomized controlled trial

BackgroundWomen with obesity and infertility are counseled to lose weight prior to conception and infertility treatment to improve pregnancy rates and birth outcomes, although confirmatory evidence from randomized trials is lacking. We assessed whether a preconception intensive lifestyle intervention with acute weight loss is superior to a weight neutral intervention at achieving a healthy live birth.Methods and findingsIn this open-label, randomized controlled study (FIT-PLESE), 379 women with obesity (BMI ≥ 30 kg/m²) and unexplained infertility were randomly assigned in a 1:1 ratio to 2 preconception lifestyle modification groups lasting 16 weeks, between July 2015 and July 2018 (final follow-up September 2019) followed by infertility therapy. The primary outcome was the healthy live birth (term infant of normal weight without major anomalies) incidence. This was conducted at 9 academic health centers across the United States. The intensive group underwent increased physical activity and weight loss (target 7%) through meal replacements and medication (Orlistat) compared to a standard group with increased physical activity alone without weight loss. This was followed by standardized empiric infertility treatment consisting of 3 cycles of ovarian stimulation/intrauterine insemination. Outcomes of any resulting pregnancy were tracked. Among 191 women randomized to standard lifestyle group, 40 dropped out of the study before conception; among 188 women randomized to intensive lifestyle group, 31 dropped out of the study before conception. All the randomized women were included in the intent-to-treat analysis for primary outcome of a healthy live birth. There were no significant differences in the incidence of healthy live births [standard 29/191(15.2%), intensive 23/188(12.2%), rate ratio 0.81 (0.48 to 1.34), P = 0.40]. Intensive had significant weight loss compared to standard (−6.6 ± 5.4% versus −0.3 ± 3.2%, P < 0.001). There were improvements in metabolic health, including a marked decrease in incidence of the metabolic syndrome (baseline to 16 weeks: standard: 53.6% to 49.4%, intensive 52.8% to 32.2%, P = 0.003). Gastrointestinal side effects were significantly more common in intensive. There was a higher, but nonsignificant, first trimester pregnancy loss in the intensive group (33.3% versus 23.7% in standard, 95% rate ratio 1.40, 95% confidence interval [CI]: 0.79 to 2.50). The main limitations of the study are the limited power of the study to detect rare complications and the design difficulty in finding an adequate time matched control intervention, as the standard exercise intervention may have potentially been helpful or harmful.ConclusionsA preconception intensive lifestyle intervention for weight loss did not improve fertility or birth outcomes compared to an exercise intervention without targeted weight loss. Improvement in metabolic health may not translate into improved female fecundity.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02432209","term_id":"NCT02432209"}}NCT02432209.

Richard Legro and colleagues investigate the impact of a preconception weight loss intervention on healthy live birth rates in women with obesity and unexplained infertility.     

Context-dependent memory: colour versus odour

An olfactory stimulus and a visual stimulus were employed in a context- dependent memory study using a prose passage as the to-be-remembered item. Ninety-five university students (aged 17-35 years) learned the passage of prose in the presence of one of the stimuli and were then asked to recall the passage with the original context either reinstated or not reinstated. The results revealed a significant context-dependent memory effect for the olfactory cue but not for the visual cue. They demonstrate support for the effectiveness of odours as context cues and it is suggested that context-dependent memory processes may underlie the formation and retrieval of odour-evoked autobiographical memories.      

Effect of shear rate and culture pH on the production of xylanase by Thermomyces lanuginosus

Summary The pH-value and the stirrer speed during cultivation of the thermophilic fungus Thermomyces lanuginosus were found to have a pronounced influence on xylanase production using corn cobs as carbon source. The highest xylanase activity of 32500 nkat/ml was produced in labscale fermentation within 118 hours at a stirrer speed of 50 rpm and a controlled pH-value of 7.5.     

Proteomics: applications in basic and applied biology

The rapid evolution of proteomics has continued during the past year, with a series of innovations in the core technologies of two-dimensional electrophoresis and mass spectrometry, and a diversity of productive research programmes. Well-annotated proteomics databases are now emerging in a number of fields to provide a platform for systematic research, with particularly promising progress in clinical applications such as cardiology and oncology. Large-scale quantitative research, comparable in power and sensitivity to that achieved for gene expression, is thus becoming a reality at the protein level.     

The genetic basis of phenotypic convergence in beach mice: similar pigment patterns but different genes

Convergent evolution is a widespread phenomenon seen in diverseorganisms inhabiting similar selective environments. However,it is unclear if similar phenotypes are produced by the sameor different genes and mutations. Here we analyze the molecularmechanisms underlying convergent pigment pattern among subspeciesof the beach mouse (Peromyscus polionotus) inhabiting the Gulfand Atlantic coasts of Florida. In these two geographic regions,separated by more than 300 km, "beach mice" have lighter coloredcoats than do their mainland counterparts, produced by naturalselection for camouflage against the pale coastal sand dunes.We measured color pattern in eight beach mouse subspecies andshowed that three of the Gulf Coast subspecies are more phenotypicallysimilar to an Atlantic coast subspecies than to their Gulf Coastneighbors. However, light-colored beach mice do not form a monophyleticgroup. Previous results implicated a single derived amino acidchange in the melanocortin-1 receptor (Mc1r) as a major contributorto pigment pattern in the Gulf Coast beach mice; despite phenotypicsimilarities, the derived Mc1r allele was not found in the Atlanticcoast beach mouse populations. Here we show that Atlantic coastbeach mice have high levels of Mc1r polymorphism but they lackunique alleles. Functional assays revealed that single aminoacid mutations segregating in Atlantic coast beach mice do notcause any change in Mc1r activity compared with the activityof Mc1r from dark-colored mice. These joint results show thatconvergent pigment patterns in recently diverged beach mousesubspecies—whose developmental constraints are presumablysimilar—have evolved through a diversity of genetic mechanisms.     

Thinking and doing: the effects of dopamine and oxytocin genes and executive function on mothering behaviours

Animal and human studies suggest that initial expression of maternal behaviour depends on oxytocin and dopamine systems. However, the mechanism by which these systems affect parenting behaviours and the timing of these effects are not well understood. This article explores the role of mothers' executive function in mediating the relation between oxytocin and dopamine gene variants and maternal responsiveness at 48 months post‐partum. Participants (n = 157) were mothers recruited in the Maternal Adversity, Vulnerability and Neurodevelopment Study, which assesses longitudinally two cohorts of mothers and children in Canada. We examined single nucleotide polymorphisms (SNPs) related to the dopamine and oxytocin systems (DRD1 rs686, DRD1 rs265976, OXTR rs237885 and OXTR rs2254298), assessed mothers' decision‐making at 48 months using the Cambridge Neurological Automated Testing Battery (CANTAB) and evaluated maternal responsiveness from videotaped interactions during the Etch‐A‐Sketch co‐operation task. Mediation analyses showed that OXTR rs2254298 A‐carriers had an indirect effect on positive parenting which was mediated by mothers' performance on decision‐making task (estimate = 0.115, P < 0.005), while OXTR rs2254298 A‐carriers had both direct and indirect effects on physically controlling parenting, also mediated through enhanced performance on decision‐making (estimate = ?0.059, P < 0.005). Dopamine SNPs were not associated with any measure of executive function or parenting (all P > 0.05). While oxytocin has previously been associated with only the early onset of maternal behaviour, we show that an OXTR polymorphism is involved in maternal behaviour at 48 months post‐partum through mothers' executive function. This research highlights the importance of the oxytocin system to maternal parenting beyond infancy.     

Analysis of bacterial diversity in the canine duodenum, jejunum, ileum, and colon by comparative 16S rRNA gene analysis

The study aim was to describe the diversity of the intraluminal intestinal microbial community in dogs by direct sequence analysis of the 16S rRNA gene. Intestinal content was collected from the duodenum, jejunum, ileum, and colon from six healthy dogs. Bacterial 16S rRNA gene was amplified with universal bacterial primers. Amplicons were ligated into cloning vectors and near-full-length 16S rRNA gene inserts were analyzed. From a total of 864 clones analyzed, 106 nonredundant 16S rRNA gene sequences were identified. Forty-two (40%) sequences showed<98% sequence similarity to 16S rRNA gene sequences reported previously. Operation taxonomic units were classified into four phyla: Firmicutes, Fusobacteria, Bacteroidetes, and Proteobacteria. Clostridiales predominated in the duodenum (40% of clones) and jejunum (39%), and were highly abundant in the ileum (25%) and colon (26%). Sequences affiliated with Clostridium cluster XI and Clostridium cluster XIVa dominated in the proximal small intestine and colon, respectively. Fusobacteriales and Bacteroidales were the most abundant bacterial order in the ileum (33%) and colon (30%). Enterobacteriales were more commonly observed in the small intestine than in the colon. Lactobacillales occurred commonly in all parts of the intestine.