Cancer Mutations in SPOP Put a Stop to Its Inter-compartmental Hops.

In this issue of Molecular Cell, Bouchard et al. (2018) identify liquid-liquid phase separation as a mechanism for substrate-triggered localization of SPOP and ubiquitination machinery to different nuclear bodies and describe how cancer mutations disrupt this process.

Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions.

Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.

Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units.

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains.

Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin-binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.

Short disordered termini and proline-rich domain are major regulators of UBQLN1/2/4 phase separation.

Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2, and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates, and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2, and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2, and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs and the need to apply caution when using epitope tags to prevent experimental artifacts.

Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid-Liquid Phase Separation.

Liquid-liquid phase separation (LLPS) is a process by which biomacromolecules, particularly proteins, condense into a dense phase that resembles liquid droplets. Dysregulation of LLPS is implicated in disease, yet the relationship between protein conformational changes and LLPS remains difficult to discern. This is due to the high flexibility and disordered nature of many proteins that phase separate under physiological conditions and their tendency to oligomerize. Here, we demonstrate that ion mobility mass spectrometry (IM-MS) overcomes these limitations. We used IM-MS to investigate the conformational states of full-length ubiquilin-2 (UBQLN2) protein, LLPS of which is driven by high-salt concentration and reversed by noncovalent interactions with ubiquitin (Ub). IM-MS revealed that UBQLN2 exists as a mixture of monomers and dimers and that increasing salt concentration causes the UBQLN2 dimers to undergo a subtle shift toward extended conformations. UBQLN2 binds to Ub in 2:1 and 2:2 UBQLN2/Ub complexes, which have compact geometries compared to free UBQLN2 dimers. Together, these results suggest that extended conformations of UBQLN2 are correlated with UBQLN2's ability to phase separate. Overall, delineating protein conformations that are implicit in LLPS will greatly increase understanding of the phase separation process, both in normal cell physiology and disease states.

Whole-Exome Sequencing of 21 Families: Candidate Genes for Early-Onset High Myopia.

High myopia is the most severe and pathological form of myopia. It occurs when the spherical refractive error exceeds -6.00 spherical diopters (SDs) or the axial length (AL) of the eye is greater than 26 mm. This article focuses on early-onset high myopia, an increasingly common condition that affects children under 10 years of age and can lead to other serious ocular pathologies. Through the genetic analysis of 21 families with early-onset high myopia, this study seeks to contribute to a better understanding of the role of genetics in this disease and to propose candidate genes. Whole-exome sequencing studies with a panel of genes known to be involved in the pathology were performed in families with inconclusive results: 3% of the variants found were classified as pathogenic, 6% were likely pathogenic and the remaining 91% were variants of uncertain significance. Most of the families in this study were found to have alterations in several of the proposed genes. This suggests a polygenic inheritance of the pathology due to the cumulative effect of the alterations. Further studies are needed to validate and confirm the role of these alterations in the development of early-onset high myopia and its polygenic inheritance.

The STOP Program: a Hybrid Smoking Prevention and Cessation Training for Cancer Care Providers in Colombia and Peru.

We designed and tested the feasibility of the Smoking Cessation Training Program for Oncology Practice (STOP), a hybrid (face-to-face plus web-based) educational intervention to enhance Spanish-speaking cancer care professionals' (CCPs') ability to provide brief smoking prevention and cessation counseling to cancer patients and survivors. Changes in the CCPs' competencies (knowledge, attitude, self-efficacy, and practices toward smoking and smoking cessation services) were assessed post-training. Sixty CCPs from one major cancer center in Colombia (n = 30) and Peru (n = 30) were invited to participate in a 4-module hybrid training program on smoking prevention and cessation. Demographic and pre- and post-test evaluation data were collected. The training's acceptability was measured after each module. Bivariate analysis was conducted using Wilcoxon signed-rank test to compare the CCPs' competencies before and after the delivery of the STOP Program. Effect sizes were computed over time to assess the sustainability of the acquired competencies. Twenty-nine CCPs in Colombia and 24 CCPs in Peru completed the STOP Program (96.6% and 80.0% retention rates, respectively). In both countries, 98.2% of the CCPs reported that the overall structure and organization of the program provided an excellent learning experience. The pre-post-test evaluations indicated that the CCPs significantly improved their knowledge, attitude, self-efficacy, and practices toward smoking, smoking prevention, and cessation services. We found that the CCPs' self-efficacy and practices increased over time (1-, 3-, and 6-month assessments after completing the 4 educational modules). The STOP Program was effective and well-received, demonstrating remarkable changes in CCPs' competencies in providing smoking prevention and cessation services to cancer patients.

Ubiquitin-Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?

Liquid-liquid phase separation (LLPS) has recently emerged as a possible mechanism that enables ubiquitin-binding shuttle proteins to facilitate the degradation of ubiquitinated substrates via distinct protein quality control (PQC) pathways. Shuttle protein LLPS is modulated by multivalent interactions among their various domains as well as heterotypic interactions with polyubiquitin chains. Here, the properties of three different shuttle proteins (hHR23B, p62, and UBQLN2) are closely examined, unifying principles for the molecular determinants of their LLPS are identified, and how LLPS is connected to their functions is discussed. Evidence supporting LLPS of other shuttle proteins is also found. In this review, it is proposed that shuttle protein LLPS leads to spatiotemporal regulation of PQC activities by mediating the recruitment of PQC machinery (including proteasomes or autophagic components) to biomolecular condensates, assembly/disassembly of condensates, selective enrichment of client proteins, and extraction of ubiquitinated proteins from condensates in cells.

ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells.

Mutations in ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells remains unknown. Here, we overexpress mCherry-fused UBQLN2 with five patient-derived ALS-linked mutations and employ live-cell imaging and photokinetic analysis to investigate how each of these mutations impact stress-induced UBQLN2 condensate assembly and condensate material properties. Unlike endogenous UBQLN2, exogenously introduced UBQLN2 forms condensates distinct from stress granules. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, mutant UBQLN2 forms fewer stress-induced UBQLN2 condensates than wild-type UBQLN2. Exogenously expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.

The path to a better biomarker: application of a risk management framework for the implementation of PD-L1 and TILs as immuno-oncology biomarkers in breast cancer clinical trials and daily practice.

Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin-stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD-L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immuno-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk-management framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in BC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Alternative Strategies for the Estimation of a Disease's Basic Reproduction Number: A Model-Agnostic Study.

This work presents a model-agnostic evaluation of four different models that estimate a disease's basic reproduction number. The evaluation presented is twofold: first, the theory behind each of the models is reviewed and compared; then, each model is tested with eight impartial simulations. All scenarios were constructed in an experimental framework that allows each model to fulfill its assumptions and hence, obtain unbiased results for each case. Among these models is the one proposed by Thompson et al. (Epidemics 29:100356, 2019), i.e., a Bayesian estimation method well established in epidemiological practice. The other three models include a novel state-space method and two simulation-based approaches based on a Poisson infection process. The advantages and flaws of each model are discussed from both theoretical and practical standpoints. Finally, we present the evolution of Covid-19 outbreak in Colombia as a case study for computing the basic reproduction number with each one of the reviewed methods.

Structure, dynamics and functions of UBQLNs: at the crossroads of protein quality control machinery.

Cells rely on protein homeostasis to maintain proper biological functions. Dysregulation of protein homeostasis contributes to the pathogenesis of many neurodegenerative diseases and cancers. Ubiquilins (UBQLNs) are versatile proteins that engage with many components of protein quality control (PQC) machinery in cells. Disease-linked mutations of UBQLNs are most commonly associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerative disorders. UBQLNs play well-established roles in PQC processes, including facilitating degradation of substrates through the ubiquitin-proteasome system (UPS), autophagy, and endoplasmic-reticulum-associated protein degradation (ERAD) pathways. In addition, UBQLNs engage with chaperones to sequester, degrade, or assist repair of misfolded client proteins. Furthermore, UBQLNs regulate DNA damage repair mechanisms, interact with RNA-binding proteins (RBPs), and engage with cytoskeletal elements to regulate cell differentiation and development. Important to the myriad functions of UBQLNs are its multidomain architecture and ability to self-associate. UBQLNs are linked to numerous types of cellular puncta, including stress-induced biomolecular condensates, autophagosomes, aggresomes, and aggregates. In this review, we focus on deciphering how UBQLNs function on a molecular level. We examine the properties of oligomerization-driven interactions among the structured and intrinsically disordered segments of UBQLNs. These interactions, together with the knowledge from studies of disease-linked mutations, provide significant insights to UBQLN structure, dynamics and function.

Neoadjuvant letrozole plus taselisib versus letrozole plus placebo in postmenopausal women with oestrogen receptor-positive, HER2-negative, early-stage breast cancer (LORELEI): a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Endocrine therapy-based neoadjuvant treatment for luminal breast cancer allows efficient testing of new combinations before surgery. The activation of the phosphatidylinositol-3-kinase (PI3K) pathway is a known mechanism of resistance to endocrine therapy. Taselisib is an oral, selective PI3K inhibitor with enhanced activity against PIK3CA-mutant cancer cells. The LORELEI trial tested whether taselisib in combination with letrozole would result in an increased proportion of objective responses and pathological complete responses. METHODS: In this multicentre, randomised, double-blind, parallel-cohort, placebo-controlled phase 2, study, we enrolled postmenopausal women (aged ≥18 years) with histologically confirmed, oestrogen receptor (ER)-positive, HER2-negative, stage I-III, operable breast cancer, from 85 hospitals in 22 countries worldwide. To be eligible, patients had have an Eastern Cooperative Oncology Group (ECOG) performance status 0-1, adequate organ function, and had to have evaluable tumour tissue for PIK3CA genotyping. Patients were randomly assigned (1:1) by means of a permuted block algorithm (block size of four) via an interactive voice or web-based response system, to receive letrozole (2·5 mg/day orally, continuously) with either 4 mg of oral taselisib or placebo (on a 5 days-on, 2 days-off schedule) for 16 weeks, followed by surgery. Randomisation was stratified by tumour size and nodal status. Site staff, patients, and the sponsor were masked to treatment assignment. Coprimary endpoints were the proportion of patients who achieved an objective response by centrally assessed breast MRI and a locally assessed pathological complete response in the breast and axilla (ypT0/Tis, ypN0) at surgery in all randomly assigned patients and in patients with PIK3CA-mutant tumours. Analyses were done in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT02273973, and is closed to accrual. FINDINGS: Between Nov 12, 2014, and Aug 12, 2016, 334 participants were enrolled and randomly assigned to receive letrozole and placebo (n=168) or letrozole and taselisib (n=166). Median follow-up was 4·9 months (IQR 4·7-5·1). The study met one of its primary endpoints: the addition of taselisib to letrozole was associated with a higher proportion of patients achieving an objective response in all randomly assigned patients (66 [39%] of 168 patients in the placebo group vs 83 [50%] of 166 in the taselisib group; odds ratio [OR] 1·55, 95% CI 1·00-2·38; p=0·049) and in the PIK3CA-mutant subset (30 [38%] of 79 vs 41 [56%] of 73; OR 2·03, 95% CI 1·06-3·88; p=0·033). No significant differences were observed in pathological complete response between the two groups, either in the overall population (three [2%] of 166 in the taselisib group vs one [1%] of 168 in the placebo group; OR 3·07 [95% CI 0·32-29·85], p=0·37) or in the PIK3CA-mutant cohort (one patient [1%) vs none [0%]; OR not estimable, p=0·48). The most common grade 3-4 adverse events in the taselisib group were gastrointestinal (13 [8%] of 167 patients), infections (eight [5%]), and skin-subcutaneous tissue disorders (eight [5%]). In the placebo group, four (2%) of 167 patients had grade 3 or worse vascular disorders, two (1%) had gastrointestinal disorders, and two (1%) patients had grade 3 or worse infections and infestations. There was no grade 4 hyperglycaemia and grade 3 cases were asymptomatic. Serious adverse events were more common in the taselisib group (eight [5%] patients with infections and seven [4%] with gastrointestinal effects) than in the placebo group (one [1%] patient each with grade 3 postoperative wound and haematoma infection, grade 4 hypertensive encephalopathy, grade 3 acute cardiac failure, and grade 3 breast pain). One death occurred in the taselisib group, which was not considered to be treatment-related. INTERPRETATION: The increase in the proportion of patients who achieved an objective response from the addition of taselisib to endocrine therapy in a neoadjuvant setting is consistent with the clinical benefit observed in hormone receptor-positive, HER2-negative, metastatic breast cancer. FUNDING: Genentech and F Hoffmann-La Roche.

Update on tumor-infiltrating lymphocytes (TILs) in breast cancer, including recommendations to assess TILs in residual disease after neoadjuvant therapy and in carcinoma in situ: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer.

Morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer is gaining momentum as evidence strengthens the clinical relevance of this immunological biomarker. TILs in the post-neoadjuvant residual disease setting are acquiring increasing importance as a stratifying marker in clinical trials, considering the raising interest on immunotherapeutic strategies after neoadjuvant chemotherapy. TILs in ductal carcinoma in situ, with or without invasive carcinoma, represent an emerging area of clinical breast cancer research. The aim of this report is to update pathologists, clinicians and researchers on TIL assessment in both the post-neoadjuvant residual disease and the ductal carcinoma in situ settings. The International Immuno-Oncology Working Group proposes a method for assessing TILs in these settings, based on the previously published International Guidelines on TIL Assessment in Breast Cancer. In this regard, these recommendations represent a consensus guidance for pathologists, aimed to achieve the highest possible consistency among future studies.

A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid.

Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.IMPORTANCE Negative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.

[Programa de detección del alelo APOE-E4 en adultos mayores mexicanos con deterioro cognitivo].

INTRODUCTION: In Mexico, the prevalence of neurocognitive disorders (NCDs) has increased in parallel with the increase in life expectancy. The E4 allele of the gene that encodes apolipoprotein E (APOE) is the main genetic risk factor for cognitive impairment. OBJECTIVE: To replicate the association of APOE-E4 allele with neurocognitive impairment in a Mexican population, as well as to implement a genetic risk-detection program with the APOE-E4 allele. METHOD: A program was structured for the detection of APOE-E4 allele risk in different recruiting centers from the central zone of the Mexican Republic, with three stages: recruitment and selection of candidates for the detection of the risk-allele, genetic risk analysis and delivery of results. RESULTS: In the genetic-association study to replicate the association with neurocognitive disorders by means of multivariate logistic models, the APOE-E4 allele increased the risk for cognitive impairment in the Mexican populations by approximately 6 % (OR: 5.83, p = 0.0025). In addition, 367 genetic risk results were delivered. CONCLUSIONS: The present program is the first one to be implemented in Mexico with the purpose to inform on a genetic risk factor for neurocognitive disorders in several centers of the country.

INTRODUCCIÓN: En México, la prevalencia de los trastornos neurocognitivos (TNC) han aumentado a la par del incremento en la esperanza de vida. El alelo E4 del gen que codifica la apolipoproteína E (APOE) es el principal factor de riesgo genético para deterioro neurocognitivo. OBJETIVO: Reproducir la asociación en población mexicana entre APOE-E4 y el deterioro neurocognitivo, así como implementar un programa de detección de riesgo genético con el alelo APOE-E4. MÉTODO: Se estructuró un programa de detección de riesgo basado en APO-EA en diferentes centros de reclutamiento en la zona centro de la República Mexicana, con tres etapas: reclutamiento y selección de los candidatos para la detección del alelo de riesgo, análisis del riesgo genético y entrega del resultado. RESULTADOS: El análisis de asociación genética para replicar la asociación con trastornos neurocognitivos mediante modelos logísticos multivariados mostró que el alelo E4 de APOE incrementó aproximadamente 6 % el riesgo en población mexicana (RM = 5.83, p = 0.0025). Se entregaron 367 resultados de riesgo genético. CONCLUSIONES: El presente programa es el primero en México implementado para dar a conocer un factor de riesgo genético para trastornos neurocognitivos en varios centros del país.

The implementation of the Plan Esperanza and response to the imPACT Review.

Following the implementation of the National Cancer Prevention and Control Results-based Budget Programme (PpR Cancer-024) in 2011, the Peruvian Government approved the Plan Esperanza-a population-based national cancer control plan-in 2012. Legislation that ensured full government-supported funding for people who were otherwise unable to access or afford care and treatment accompanied the Plan. In 2013, the Ministry of Health requested an integrated mission of the Programme of Action for Cancer Therapy (imPACT) report to strengthen cancer control in Peru. The imPACT Review, which was executed in 2014, assessed Peru's achievements in cancer control, and areas for improvement, including cancer control planning, further development of population-based cancer registration, increased prevention, early diagnosis, treatment and palliative care, and the engagement and participation of civil society in the health-care system. This Series paper gives a brief history of the development of the Plan Esperanza, describes the innovative funding model that supports it, and summarises how funds are disseminated on the basis of disease, geography, and demographics. An overview of the imPACT Review, and the government's response in the context of the Plan Esperanza, is provided. The development and execution of the Plan Esperanza and the execution of and response to the imPACT Review demonstrates the Peruvian Government's commitment to fighting cancer across the country, including in remote and urban areas.

Functional tuning of the catalytic residue pKa in a de novo designed esterase.

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p-nitrophenyl acetate (pNPA) and 4-nitrophenyl-(2-phenyl)-propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single-histidine residue (His144 ) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pKa value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pKa calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pKa value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pKa value of this residue in water. We determined that a short-range favorable interaction with Glu127 contributes to the elevated pKa of His144 . We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pKa of its active nucleophile, His144 , by 0.7 units. As a direct result of the decrease in the His144 pKa value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine-promoted catalysis. Proteins 2017; 85:1656-1665. © 2017 Wiley Periodicals, Inc.

ß-Alanine and N-terminal cationic substituents affect polyamide-DNA binding.

Minor-groove binding hairpin polyamides (PAs) bind specific DNA sequences. Synthetic modifications can improve PA-DNA binding affinity and include flexible modules, such as ß-alanine (ß) motifs to replace pyrroles (Py), and increasing compound charge using N-terminal cationic substituents. To better understand the variations in kinetics and affinities caused by these modifications on PA-DNA interactions, a comprehensive set of PAs with different numbers and positions of ß and different types of N-cationic groups was systematically designed and synthesized to bind their cognate sequence, the λB motif. The λB motif is also a strong binding promoter site of the major groove targeting transcription factor PU.1. The PA binding affinities and kinetics were evaluated using a spectrum of powerful biophysical methods: thermal melting, biosensor surface plasmon resonance and circular dichroism. The results show that ß inserts affect PA-DNA interactions in a number and position dependent manner. Specifically, a ß replacement between two imidazole heterocycles (ImßIm) generally strengthens binding. In addition, N-terminal cationic groups can accelerate the association between PA and DNA, but the bulky size of TMG can cause steric hindrance and unfavourable repulsive electrostatic interactions in some PAs. The future design of stronger binding PA requires careful combination of ßs and cationic substituents.