Exploring the Structural Mechanism of Covalently Bound E3 Ubiquitin Ligase: Catalytic or Allosteric Inhibition?

Covalent inhibition has recently gained a resurgence of interest in several drug discovery areas. The expansion of this approach is based on evidence elucidating the selectivity and potency of covalent inhibitors when bound to particular amino acids of a biological target. The Nedd4-1, an E3 ubiquitin ligase, is characterized by two covalent binding sites, of which catalytic Cys^cat and allosteric Cys^allo are enclosed. This enzyme has demonstrated inhibition at both the above-mentioned binding sites; however, a detailed molecular understanding of the structural mechanism of inhibition upon Cys^cat and Cys^allo binding remains vague. This prompted us to provide the first account of investigating the preferential covalent binding mode and the underlying structural and molecular dynamic implications. Based on the molecular dynamic analyses, it was evident that although both catalytic and allosteric covalent binding led to greater stability of the enzyme, a preferential covalent mechanism of inhibition was seen in the allosteric-targeted system. This was supported by a more favorable binding energy in the allosteric site compared to the catalytic site, in addition to the larger number of residue interactions and stabilizing hydrogen bonds occurring in the allosteric covalent bound complex. The fundamental dynamic analysis presented in this report compliments, as well as adds to previous experimental findings, thus leading to a crucial understanding of the structural mechanism by which Nedd4-1 is inhibited. The findings from this study may assist in the design of more target-specific Nedd4-1 covalent inhibitors exploring the surface-exposed cysteine residues.     

Emerging Evidence on the Effectiveness of Tropical Forest Conservation

The PLOS ONE Collection “Measuring forest conservation effectiveness” brings together a series of studies that evaluate the effectiveness of tropical forest conservation policies and programs with the goal of measuring conservation success and associated co-benefits. This overview piece describes the geographic and methodological scope of these studies, as well as the policy instruments covered in the Collection as of June 2016. Focusing on forest cover change, we systematically compare the conservation effects estimated by the studies and discuss them in the light of previous findings in the literature. Nine studies estimated that annual conservation impacts on forest cover were below one percent, with two exceptions in Mexico and Indonesia. Differences in effect sizes are not only driven by the choice of conservation measures. One key lesson from the studies is the need to move beyond the current scientific focus of estimating average effects of undifferentiated conservation programs. The specific elements of the program design and the implementation context are equally important factors for understanding the effectiveness of conservation programs. Particularly critical will be a better understanding of the causal mechanisms through which conservation programs have impacts. To achieve this understanding we need advances in both theory and methods.     

A stretch of polybasic residues mediates Cdc42 GTPase-activating protein (CdGAP) binding to phosphatidylinositol 3,4,5-trisphosphate and regulates its GAP activity

The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions.     

The effect of strength training at the same time of the day on the diurnal fluctuations of muscular anaerobic performances

The aim of this study was to examine the effects of training at the same time of the day on the diurnal variations of anaerobic performances to provide some recommendations to adjust training hours with the time of the day of competitive events. Thirty participants underwent 8 weeks of lower-extremity progressive resistance training performed 3 times per week designed to promote muscular strength and power. These subjects were randomly assigned to a morning training group (MTG, 07:00-08:00 hours, n = 10), an evening training group (ETG, 17:00-18:00 hours, n = 10), and a control group (CG, completed all tests but did not train, n = 10). Performance in the squat jump, the countermovement jump, the Wingate and 1 repetition maximum (1RM) during leg extension, leg curl, and squat tests was recorded just before and 2 weeks after an 8-week course of regular training. For all the subjects, the morning and evening tests were scheduled at the same time of the day as for the morning and evening training sessions. Before training, the results indicated a significant increase in performance from morning to evening tests (ca. 2.84-17.55% for all tests) for all groups. After training, the diurnal variations in anaerobic performances were blunted in the MTG. In fact, there was no significant difference in muscular power or strength between morning and evening tests. However, these intradaily variations in anaerobic performances persisted in the ETG and CG. From a practical point of view, adaptation to strength training is greater at the time of the day at which training was scheduled than at other times.     

Applying digital technology to promote active and healthy confinement lifestyle during pandemics in the elderly

Although recognized as effective measures to curb the spread of the COVID19 outbreak, social distancing and home confinement have generated a mental health burden with older adults who are considered to be more vulnerable to psychosocial strains. To date, the application of digital technologies in response to COVID-19 pandemic has been narrowed to public-health needs related to containment and mitigation. However, information and communications technology (ICT)-based initiatives directed toward prediction and prevention of psychosocial support are still limited. Given the power of digital health solutions to allow easy and accurate characterization and intervention for health and disease, as well as to flatten the COVID19 incidence curves in many countries, our ECLB-COVID19 consortium is highlighting the importance of providing innovative ICT-based solutions (ICT-COVID-Companion) to improve elderly physical and mental health, thereby preventing/dampening psychosocial strain during pandemics. Based on innovative approaches (e.g., emotional/social computing, open social platform, interactive coaching, gamification, fitness-tracker, internet of things) and smart digital solutions (smartwatch/smartphone), smart companions must provide safe personalised physical, mental and psychosocial health surveillance. Additionally, by delivering personalised multi-dimension crisis-oriented health recommendations, such innovative crisis-oriented solutions would help (i) facilitate a user’s adherence to active and healthy confinement lifestyle (AHCL), (ii) achieve a rapid psychosocial recovery in case of depression issues and (iii) enhance preparedness for eventual future pandemics.     

The effect of post-lunch napping on mood,reaction time,and antioxidant defense during repeated sprint exercice

To compare the effects of two nap opportunities (20 and 90 min) to countermeasure the transient naturally occurring increased sleepiness and decreased performances during the post-lunch dip (PLD). Fourteen highly trained judokas completed in a counterbalanced and randomized order three test sessions (control (No-nap), 20- (N20) and 90-min (N90) nap opportunities). Test sessions consisted of the running-based anaerobic sprint test (RAST), simple and multiple-choice reaction times (MCRT) and the Epworth sleepiness scale (ESS). From the RAST, the maximum (P_max), mean (P_mean) and minimum (P_min) powers were calculated. Blood samples were taken before and after the RAST to measure the effect of pre-exercise napping on energetic and muscle damage biomarkers and antioxidant defense. N20 increased P_max and P_mean compared to No-nap (p < 0.001, d = 0.59; d = 0.66) and N90 (p < 0.001, d = 0.98; d = 0.72), respectively. Besides, plasma lactate and creatinine increased only when the exercise was performed after N20. Both N20 (p < 0.001, d = 1.18) and N90 (p < 0.01, d = 0.78) enhanced post-exercise superoxide dismutase activity compared to No-nap. However, only N20 enhanced post-exercise glutathione peroxidase activity (p < 0.001, d = 1.01) compared to pre-nap. Further, MCRT performance was higher after N20 compared to No-nap and N90 (p < 0.001, d = 1.15; d = 0.81, respectively). Subjective sleepiness was lower after N20 compared to No-nap (p < 0.05, d = 0.92) and N90 (p < 0.01, d = 0.89). The opportunity to nap for 20 min in the PLD enhanced RAST, MCRT performances, and antioxidant defense, and decreased sleepiness. However, the opportunity of 90 min nap was associated with decreased repeated sprint performances and increased sleepiness, probably because of the sleep inertia.     

Conservation of Genomic Localization and Sequence Content of Sau3AI-Like Restriction-Modification Gene Cassettes among Listeria monocytogenes Epidemic Clone I and Selected Strains of Serotype 1/2a

Listeria monocytogenes is a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported among L. monocytogenes isolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.Listeria monocytogenes is a Gram-positive, facultative intracellular food-borne pathogen capable of causing severe disease (listeriosis) in animals and humans. Listeriosis most often affects pregnant women and their fetuses, neonates, the elderly, and immunocompromised individuals. The disease is predominantly transmitted via the consumption of contaminated foods and has a ca. 20% fatality rate (12, 27). Application of numerous genotyping methods has consistently shown that the organism has a clonal population structure with three major phylogenetic lineages: lineage I consists of strains of serotypes 1/2b, 3b, and 4b, while those of serotypes 1/2a, 1/2c, 3a, and 3c are clustered in lineage II; strains of serotypes 4a and 4c, along with certain serotype 4b strains, constitute lineage III (37, 38).Most epidemics of human listeriosis have involved a small number of closely related strains (epidemic clones), predominantly of serotype 4b (7, 35). The earliest identified clone, epidemic clone I (ECI), has been responsible for several major outbreaks in Europe and North America. In addition, strains of this clonal group are frequently encountered in sporadic illness (10, 28, 29). ECI strains have also been found to comprise a significant portion of the serotype 4b strains from foods and from the environments of food processing plants (10, 11, 40).Genomic DNA of ECI strains has been long known to resist digestion with Sau3AI, suggesting methylation of cytosine at GATC sites (41). Genome sequencing of the ECI strain F2365, implicated in the 1985 California outbreak of listeriosis, revealed a putative restriction-modification (RM) gene cassette with specificity for GATC sites (25). This RM cassette was harbored by all tested serotype 4b strains with Sau3AI-resistant DNA and was absent from those with DNA that could be digested with Sau3AI (40). These findings were in agreement with previous evidence that a fragment of the putative methyltransferase gene was specific to ECI and absent from other strains (14).In spite of extensive documentation for the presence of this putative RM cassette in ECI strains, and its apparent absence among other serotype 4b strains, limited information is available about the possible presence of the cassette among other lineages of L. monocytogenes. Furthermore, conclusive evidence for involvement of the cassette in the resistance of the DNA of ECI strains to Sau3AI digestion has been lacking. In this study, we investigated a panel of food-derived serotype 1/2a strains with Sau3AI-resistant DNA and characterized the genetic content and genomic localization of the RM cassette harbored by these strains. Furthermore, we employed deletion mutagenesis to assess the involvement of the RM cassette in Sau3AI resistance of the DNA of the ECI strain F2365, as well as of a serotype 1/2a strain harboring the cassette.     

Effect of the chitin binding domain deletion from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> chitinase Chi255 on its stability in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene, chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed in the native chitinase, and for improving its activity.     

First trimester exposure to paroxetine and risk of cardiac malformations in infants: the importance of dosage

BACKGROUND: Conflicting findings with regard to the teratogenic risks of first trimester use of paroxetine have prompted the FDA, Health Canada, and the manufacturer of the drug to issue warnings against its use during pregnancy. Given that untreated depression during pregnancy can lead to deleterious effect on the mother and her unborn fetus, data on the relationship between the dose and the range of malformations is warranted. This study attempts to quantify the association between first trimester exposure to paroxetine and congenital cardiac malformations, adjusting for possible confounders, and to quantify the dose-response relationship between paroxetine use and cardiac defects. METHODS: The Medication and Pregnancy registry was used. This population-based registry was built by linking three administrative databases (RAMQ, Med-Echo, and ISQ), and includes all pregnancies in Quebec between 01/01/1997 and 06/30/2003. Date of entry in the registry is the date of the first day of the last menstrual period. To be eligible for this study, women had to: 1) be 15-45 years of age at entry; 2) be covered by the RAMQ drug plan >or=12 months before and during pregnancy; 3) be using only one type of antidepressant during the first trimester; and 4) have a live birth. Two nested case-control studies were carried out comparing the prevalence of paroxetine use in the first trimester of pregnancy to the prevalence of other antidepressant exposures during the same time period. Cases were defined as: 1) any major malformations; or 2) any cardiac malformations diagnosed in the first year of life; controls were defined as no major or minor malformations. Multivariate logistic regression techniques were used to analyze data. RESULTS: Among the 1,403 women meeting inclusion criteria, 101 infants with major congenital malformations were identified; 24 had cardiac malformations. Adjusting for possible confounders, the use of paroxetine (odds ratio [OR] = 1.38, 95% confidence interval [CI] = 0.49-3.92), and the use of other SSRIs (OR = 0.89, 95% CI = 0.28-2.84) during the first trimester of pregnancy did not increase the risk of congenital cardiac malformations compared with the use of non-SSRI antidepressants. When considering the dose, however, a dose-response relationship was observed, thus women exposed to >25 mg/day of paroxetine during the first trimester of pregnancy were at increased risk of having an infant with major congenital malformations (adjusted [adj] OR = 2.23, 95% CI = 1.19, 4.17), or major cardiac malformations (adj OR = 3.07, 95% CI = 1.00, 9.42). CONCLUSIONS: Gestational exposure to paroxetine is associated with major congenital malformations and major cardiac malformations for only first trimester exposure above 25 mg/day.