Targeting SxIP-EB1 interaction: An integrated approach to the discovery of small molecule modulators of dynamic binding sites

End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule (MT) growing ends, which has a fundamental role in MT polymerisation. EB1 is an important protein target as it is involved in regulating MT dynamic behaviour, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved four amino acid motif, (serine-X-isoleucine-proline) which exists within an intrinsically disordered region. Here we report the use of a multidisciplinary computational and experimental approach for the discovery of the first small molecule scaffold which targets the EB1 recruiting domain. This approach includes virtual screening (structure- and ligand-based design) and multiparameter compound selection. Subsequent studies on the selected compounds enabled the elucidation of the NMR structures of the C-terminal domain of EB1 in the free form and complexed with a small molecule. These structures show that the binding site is not preformed in solution, and ligand binding is fundamental for the binding site formation. This work is a successful demonstration of the combination of modelling and experimental methods to enable the discovery of compounds which bind to these challenging systems

Identification and optimisation of ligands to target protein-protein interactions: EB1-SxIP proteins

End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule growing ends which has a fundamental role in microtubule polymerisation. EB1 regulates the microtubule dynamic behaviour, through protein recruitment, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved SxIP motif within an intrinsically disordered region enriched with basic, serine and proline residues. Crystal structure of EB1 in complex with a peptide containing the SxIP motif demonstrated that the isoleucine-proline dipeptide is bound into a well‐defined cavity of EB1 that may be suitable for small molecule targeting. The research described herein reports the use of a multidisciplinary approach for the discovery of the first small molecule scaffold to target the EB1 recruiting domain. This approach included virtual screening (structure and ligand based design) and multiparameter compound selection. Solution NMR structures of the C-terminal domain of EB1 in the free form and in complex with the small molecule are also reported. A key finding from these structures is that the hydrophobic binding pocket reported to be essential for recruiting SxIP proteins is not pre-formed but highly dynamic in solution. This brings new insights to the protein recruitment mechanism regulated by EB1 and for the identification of new small molecule inhibitors for the EB1-SxIP protein interactions. The interaction of short length peptides containing the SxIP motif with EB1 was characterised through the use of solution NMR and ITC methods. The contributions for the binding of the SxIP motif and neighbouring residues to EB1 were quantified in terms of binding energy. A structural model shows that the binding pocket of EB1 is largely extended when in complex. This research describes not only the first chemical scaffold that targets EB1, it details important structural features of the interaction of this protein with SxIP containing peptides. This structural information provides fundamental understanding of this interaction that can be exploited in the future to discover higher affinity ligands

Life satisfaction and time-use among full-time and part-time working mothers in Brazil

BACKGROUND: Working part-time or full-time is a persistent dilemma for women, especially mothers, as they strive to manage their time across various roles. However, the existing literature remains unclear on whether part-time work contributes more to life satisfaction than full-time work. OBJECTIVE: This study aims to describe and compare time-use patterns in different occupational roles and levels of life satisfaction among 795 mothers in Brazil who are employed either part-time or full-time. METHODS: Data was collected through an online survey and the snowball technique. Regression and correlation analyses were performed on the data obtained from the following instruments: the Role Checklist for assessing time-use within occupational roles, the Satisfaction with Life Scale, and demographic information. RESULTS: Our analysis revealed no significant differences in life satisfaction between mothers working full-time and those working part-time (t (793)=1.160, p = 0.24). However, life satisfaction scores were positively correlated with the number of occupational roles performed (r = 0.222, p <  0.01), higher family income (P <  0.001), and engagement in social roles such as Friend (r = 0.106, p = 0.003), Hobbyist/Amateur (r = 0.08, p = 0.018), and Caregiver (r = 0.07, p = 0.026). CONCLUSION: While our society places significant emphasis on time spent at work, our findings highlight that life satisfaction extends far beyond the dichotomy of working part-time or full-time. There are deeper dimensions to consider, including the number of occupational roles, family income, and engagement in social roles, which have a more substantial impact on overall life satisfaction

LD Motif Recognition by Talin: Structure of the Talin-DLC1 Complex

Cell migration requires coordination between integrin-mediated cell adhesion to the extracellular matrix and force applied to adhesion sites. Talin plays a key role in coupling integrin receptors to the actomyosin contractile machinery, while deleted in liver cancer 1 (DLC1) is a Rho GAP that binds talin and regulates Rho, and therefore actomyosin contractility. We show that the LD motif of DLC1 forms a helix that binds to the four-helix bundle of the talin R8 domain in a canonical triple-helix arrangement. We demonstrate that the same R8 surface interacts with the paxillin LD1 and LD2 motifs. We identify key charged residues that stabilize the R8 interactions with LD motifs and demonstrate their importance in vitro and in cells. Our results suggest a network of competitive interactions in adhesion complexes that involve LD motifs, and identify mutations that can be used to analyze the biological roles of specific protein-protein interactions in cell migration

Inhibition of CXCR2 Plays a Pivotal Role in Re-Sensitizing Ovarian Cancer to Cisplatin Treatment

cDNA microarray data conducted by our group revealed overexpression of CXCL2 and CXCL8 in ovarian cancer (OC) microenvironment. Herein, we have proven that the chemokine receptor, CXCR2, is a pivotal molecule in re-sensitizing OC to cisplatin, and its inhibition decreases cell proliferation, viability, tumor size in cisplatinresistant cells, as well as reversed the overexpression of mesenchymal epithelium transition markers. Altogether, our study indicates a central effect of CXCR2 in preventing tumor progression, due to acquisition of cisplatin chemoresistant phenotype by tumor cells, and patients’ high lethality rate. We found that the overexpression of CXCR2 by OC cells is persistent and anomalously confined to the cellular nuclei, thus pointing to an urge in developing highly lipophilic molecules that promptly permeate cells, bind to and inhibit nuclear CXCR2 to fight OC, instead of relying on the high-cost genetic engineered cells.acknowledge financial support from CAPES, FAPES and CNPq, as well as the Biotechnology Program/ RENORBIO from the Federal University of Espirito Santo, Espirito Santo, Brazil; Institute of Pathology and Molecular Immunology (IPATIMUP) and the Institute of Innovation and Health Research (I3s), Porto, Portugal

Natural variation in immune responses to neonatal mycobacterium bovis bacillus calmette-guerin (BCG) vaccination in a cohort of Gambian infants

Background There is a need for new vaccines for tuberculosis (TB) that protect against adult pulmonary disease in regions where BCG is not effective. However, BCG could remain integral to TB control programmes because neonatal BCG protects against disseminated forms of childhood TB and many new vaccines rely on BCG to prime immunity or are recombinant strains of BCG. Interferon-gamma (IFN-) is required for immunity to mycobacteria and used as a marker of immunity when new vaccines are tested. Although BCG is widely given to neonates IFN- responses to BCG in this age group are poorly described. Characterisation of IFN- responses to BCG is required for interpretation of vaccine immunogenicity study data where BCG is part of the vaccination strategy. Methodology/Principal Findings 236 healthy Gambian babies were vaccinated with M. bovis BCG at birth. IFN-, interleukin (IL)-5 and IL-13 responses to purified protein derivative (PPD), killed Mycobacterium tuberculosis (KMTB), M. tuberculosis short term culture filtrate (STCF) and M. bovis BCG antigen 85 complex (Ag85) were measured in a whole blood assay two months after vaccination. Cytokine responses varied up to 10 log-fold within this population. The majority of infants (89-98% depending on the antigen) made IFN- responses and there was significant correlation between IFN- responses to the different mycobacterial antigens (Spearman’s coefficient ranged from 0.340 to 0.675, p=10-6-10-22). IL-13 and IL-5 responses were generally low and there were more non-responders (33-75%) for these cytokines. Nonetheless, significant correlations were observed for IL-13 and IL-5 responses to different mycobacterial antigens Conclusions/Significance Cytokine responses to mycobacterial antigens in BCG-vaccinated infants are heterogeneous and there is significant inter-individual variation. Further studies in large populations of infants are required to identify the factors that determine variation in IFN- responses

World health system performance revisited: the impact of varying the relative importance of health system goals

BACKGROUND: In 2002, the World Health Organization published a health system performance ranking for 191 member countries. The ranking was based on five indicators, with fixed weights common to all countries. METHODS: We investigate the feasibility and desirability of using mathematical programming techniques that allow weights to vary across countries to reflect their varying circumstances and objectives. RESULTS: By global distributional measures, scores and ranks are found to be not very sensitive to changes in weights, although differences can be large for individual countries. CONCLUSIONS: Building the flexibility of variable weights into calculation of the performance index is a useful way to respond to the debates and criticisms appearing since publication of the ranking

The dimensions of responsiveness of a health system: a Taiwanese perspective

BACKGROUND: Responsiveness is an indicator used to measure how well a health system performs relative to non-health aspects. This study assessed whether seven dimensions proposed by the World Health Organization (WHO) to measure responsiveness (dignity, autonomy, confidentiality, prompt attention, social support, basic amenities, and choices of providers) are applicable in evaluating the health system of Taiwan. METHODS: A key informant survey and focus group research were used in this study. The translated WHO proposed questionnaire was sent to 205 nominated key informants by mail, and 132 (64.4%) were returned. We used principal component analysis to extract factors. Linear regression analysis was used to assess the relationship between the total score and the extracted factors. A qualitative content analysis was also carried out in focus group research. RESULTS: Principal component analysis produced five factors (respect, access, confidentiality, basic amenities, and social support) that explained 63.5% of the total variances. These five factors demonstrated acceptable internal consistency and four of them (except social support) were significantly correlated with the total responsiveness score. The focus group interviews revealed health providers' communication ability and medical ethics were also highly appraised by Taiwanese. CONCLUSION: When the performance of a health system is to be evaluated, elements of responsiveness proposed by WHO may have to be tailored to fit different cultural backgrounds. Four key features illustrate the uniqueness of Taiwanese perspectives: the idea of autonomy may not be conceptualized, prompt attention and choice of providers are on the same track, social support during care is trivially correlated to the total responsiveness score, and accountability of health providers is deemed essential to a health system