Identification of potential selective autophagy receptors from protein-content profiling of autophagosomes.

Selective autophagy receptors (SARs) are central to cellular homeostatic and organellar recycling pathways. Over the last two decades, more than 30 SARs have been discovered and validated using a variety of experimental approaches ranging from cell biology to biochemistry, including high-throughput imaging and screening methods. Yet, the extent of selective autophagy pathways operating under various cellular contexts, for example, under basal and starvation conditions, remains unresolved. Currently, our knowledge of all known SARs and their associated cargo components is fragmentary and limited by experimental data with varying degrees of resolution. Here, we use classical predictive and modeling approaches to integrate high-quality autophagosome content profiling data with disparate datasets. We identify a global set of potential SARs and their associated cargo components active under basal autophagy, starvation-induced, and proteasome-inhibition conditions. We provide a detailed account of cellular components, biochemical pathways, and molecular processes that are degraded via autophagy. Our analysis yields a catalog of new potential SARs that satisfy the characteristics of bonafide, well-characterized SARs. We categorize them by the subcellular compartments they emerge from and classify them based on their likely mode of action. Our structural modeling validates a large subset of predicted interactions with the human ATG8 family of proteins and shows characteristic, conserved LC3-interacting region (LIR)-LIR docking site (LDS) and ubiquitin-interacting motif (UIM)-UIM docking site (UDS) binding modes. Our analysis also revealed the most abundant cargo molecules targeted by these new SARs. Our findings expand the repertoire of SARs and provide unprecedented details into the global autophagic state of HeLa cells. Taken together, our findings provide motivation for the design of new experiments, testing the role of these novel factors in selective autophagy.

NS1 from Two Zika Virus Strains Differently Interact with a Membrane: Insights to Understand Their Differential Virulence.

Zika virus (ZIKV) from Uganda (UG) expresses a phenotype related to fetal loss, whereas the variant from Brazil (BR) induces microcephaly in neonates. The differential virulence has a direct relation to biomolecular mechanisms that make one strain more aggressive than the other. The nonstructural protein 1 (NS1) is a key viral toxin to comprehend these viral discrepancies because of its versatility in many processes of the virus life cycle. Here, we aim to examine through coarse-grained models and molecular dynamics simulations the protein-membrane interactions for both NS1ZIKV-UG and NS1ZIKV-BR dimers. A first evaluation allowed us to establish that the NS1 proteins, in the membrane presence, explore new conformational spaces when compared to systems simulated without a lipid bilayer. These events derive from both differential coupling patterns and discrepant binding affinities to the membrane. The N-terminal domain, intertwined loop, and greasy finger proposed previously as binding membrane regions were also computationally confirmed by us. The anchoring sites have aromatic and ionizable residues that manage the assembly of NS1 toward the membrane, especially for the Ugandan variant. Furthermore, in the presence of the membrane, the difference in the dynamic cross-correlation of residues between the two strains is particularly pronounced in the putative epitope regions. This suggests that the protein-membrane interaction induces changes in the distal part related to putative epitopes. Taken together, these results open up new strategies for the treatment of flaviviruses that would specifically target these dynamic differences.

Recurrent and functional regulatory mutations in breast cancer.

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.

How the Strain Origin of Zika Virus NS1 Protein Impacts Its Dynamics and Implications to Their Differential Virulence.

Viruses can impact and affect human populations in a severe way. The appropriate differentiation among several species or strains of viruses is one of the biggest challenges for virology and infectiology studies. The detection of measurables-quantified discrepancies allows for more accurate clinical diagnoses and treatments for viral diseases. In the present study, we have used a computational approach to explore the dynamical properties of the nonstructural protein 1 from two strains of Zika virus. Our results show that despite a high sequence similarity, the two viral proteins from different origins can exhibit significant dissimilar structural dynamics, which complement their reported differential virulence. The present study opens up new ways in the understanding of the infectivity for these biological entities.

Identification of Electrostatic Epitopes in Flavivirus by Computer Simulations: The PROCEEDpKa Method.

Viruses are enthusiastically studied due to the great impact that these organisms can have on human health. Computational approaches can contribute offering tools that can shed light on important molecular mechanisms that help to design new diagnostic procedures. Several cellular processes between the immune-host system and the pathogenic organism are dependent on specific intermolecular interactions. In this study, we evaluated theoretical approaches to understand some properties of the antigen-antibody interactions considering the titratable properties of all ionizable residues of the nonstructural viral protein 1 (NS1) of the West Nile virus (WNV) and the Zika virus (ZIKV). Constant-pH Monte Carlo simulations were performed to estimate electrostatic properties such as the pKa shifts (ΔpKa). We proposed an alternative criterion for the discrimination of antigenic residues based on ΔpKas. Our outcomes were analyzed by an evaluation of the sensitivity and specificity through a receiver operating characteristic (ROC). As a starting point, we used the known crystallographic structure for the complex of NS1WNV(176-352) and the specific antibody 22NS1 (PDB ID 4OII ) to differentiate the residues belonging to that interface. With an optimal threshold for the absolute value of the pKa shifts, we found that is possible to predict antigenic epitopes reproducing the interfaces as defined by the X-ray structure. After this validation, we evaluated theoretical predictions based on protein-protein (PP) complexation simulations. From them, we observe amino acids with an antigenic potential and defined the optimum threshold that was applied for two strains of ZIKV (i.e., Uganda and Brazil). Several ionizable residues with antigenic capacity were identified. This is favorably related to some studies that show the high immunogenicity of secreted NS1. This approach opens up an important discussion about what are termed here "electrostatic epitopes" and how they work as an important reference in the paratope-epitope interaction for viral systems.

Sequence analysis of mutations and translocations across breast cancer subtypes.

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3-AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

Complete sequence of the ATP6 and ND3 mitochondrial genes in breast cancer tissue of postmenopausal women with different body mass indexes.

Due to the fact that mitochondrial defects and oxidative stress have been related with obesity and breast cancer is more aggressive in women with obesity, we investigated if postmenopausal Mexican-Mestizo women with breast cancer presented somatic mutations in the sequence of the ATP6 and/or ND3 genes. Twenty one postmenopausal Mexican-Mestizo women with breast cancer who underwent mastectomy or breast conserving surgery were studied. Height and weight were used to calculate body mass index. DNA from tumor tissue samples and blood leukocytes was amplified by polymerase chain reaction and sequenced the ATP6 and ND3 mitochondrial genes. Ages ranged from 46 to 82. According to World Health Organization criteria among the 21 women, 7 had a normal BMI, 7 were overweight and 7 had obesity. In regard to the molecular study, after sequencing the coding region of ATP6 and ND3 genes of the DNA obtained from both leukocytes and tumor tissue, we did not find somatic mutations. All of the changes that we found in both genes were polymorphisms: in ATP6, we identified in ten patients 3 non-synonymous nucleotide changes and in ND3 we observed that six patients presented polymorphisms, three of them were synonymous and two non-synonymous. To our knowledge, this constitutes the first report where the complete sequence of the ATP6 and ND3 genes has been analyzed in postmenopausal Mexican-Mestizo women with breast cancer and diverse BMI. Our results differ with those reported in Caucasian and Asian populations, possibly due to ethnic differences.

A microRNA signature associated with pathological complete response to novel neoadjuvant therapy regimen in triple-negative breast cancer.

Neoadjuvant chemotherapy aims to improve the outcome of breast cancer patients, but only few would benefit from this treatment. Pathological complete response has been proposed as a surrogate marker for the prediction of long-term clinical benefits; however, 50%-85% patients have an unfavorable pathological complete response to chemotherapy. MicroRNAs are known biomarkers of breast cancer progression; nevertheless, their potential to identify patients with pathological complete response remains poorly understood. Here, we investigated whether a microRNA profile could be associated with pathological complete response in triple-negative breast cancer patients receiving 5-fluorouracil, adriamycin, cyclophosphamide-cisplatin/paclitaxel as a novel neoadjuvant chemotherapy. In the discovery cohort, the expression of 754 microRNAs was examined in tumors from 10 triple-negative breast cancer patients who achieved pathological complete response and 8 without pathological complete response using TaqMan Low-Density Arrays. Unsupervised hierarchical cluster analysis identified 11 microRNAs with significant differences between responder and no-responder patients (fold change ≥ 1.5; p < 0.05). The differential expression of miR-30a, miR-9-3p, miR-770, and miR-143-5p was validated in an independent group of 17 patients with or without pathological complete response. Moreover, Kaplan-Meier analysis showed that expression of these four microRNAs was associated with an increased disease-free survival. Gene ontology classification of predicted microRNA targets indicated that numerous genes are involved in pathways related to chemoresistance, such as vascular endothelial growth factor, focal adhesion kinase, WNT, ERbB, phosphoinositide 3-kinase, and AKT signaling. In summary, we identified a novel microRNA expression signature associated with pathological complete response in breast cancer. We propose that the four validated microRNAs could be used as molecular biomarkers of clinical response in triple-negative breast cancer patients with pathological complete response to neoadjuvant therapy.

Suppression of cell migration is promoted by miR-944 through targeting of SIAH1 and PTP4A1 in breast cancer cells.

BACKGROUND: Aberrant expression of microRNAs has been associated with migration of tumor cells. In this study, we aimed to investigate the biological significance of miR-944 whose function is unknown in breast cancer. METHODS: MiR-944 expression in breast cancer cells and tumors was evaluated by Taqman qRT-PCR assays. Transcriptional profiling of MDA-MB-231 cells expressing miR-944 was performed using DNA microarrays. Cell viability, migration and invasion were assessed by MTT, scratch/wound-healing and transwell chamber assays, respectively. The luciferase reporter assay was used to evaluate targeting of SIAH1, PTP4A1 and PRKCA genes by miR-944. SIAH1 protein levels were measured by Western blot. Silencing of SIAH1 gene was performed by RNA interference using shRNAs. RESULTS: Our data showed that miR-944 expression was severely repressed in clinical specimens and breast cancer cell lines. Suppression of miR-944 levels was independent of hormonal status and metastatic potential of breast cancer cells. Gain-of-function analysis indicated that miR-944 altered the actin cytoskeleton dynamics and impaired cell migration and invasion. Genome-wide transcriptional profiling of MDA-MB-231 cells that ectopically express miR-944 showed that 15 genes involved in migration were significantly repressed. Notably, luciferase reporter assays confirmed the ability of miR-944 to bind the 3´UTR of SIAH1 and PTP4A1 genes, but not PRKCA gene. Congruently, an inverse correlation between miR-944 and SIAH1 protein expression was found in breast cancer cells. Moreover, SIAH1 was upregulated in 75 % of miR-944-deficient breast tumors. Finally, SIAH1 gene silencing by RNA interference significantly impaired cell migration of breast cancer cells. CONCLUSIONS: Our results pointed out that miR-944 is a novel upstream negative regulator of SIAH1 and PTP4A1 genes and provided for the first time evidence for its functional role in migration and invasion of breast cancer cells. They also suggest that miR-944 restoration may represent a potential strategy for breast cancer therapy.

[The burden of cervical cancer in patients with limited access to health services]. / Repercusión del cáncer cervicouterino en pacientes con limitaciones de acceso a los servicios de salud.

BACKGROUND: Cervical cancer is caused by high-risk HPV, a sexually transmitted virus. In Mexico, this disease represents a public health problem. San Luis Potosi is located within ten states with the highest rates in the country. Indigenous women of Mexico will likely to develop cervical cancer due to inequality in access to health services and their determinants. Epidemiological studies can be supported by investigations of diverse geographical nature to undertake the identification and analysis of spatial patterns of disease. OBJECTIVE: To locate by geographical distribution of Huasteca Potosina women high-risk HPV positive to observe the burden of disease in patients with limited access to health services and propose specific primary prevention activities was made with a sample of 605 women. Cervico-vaginal specimens were taken. High-risk HPV infection was determined by hybrid capture. Age and date of the last Papanicolaou were obtained through a structured poll. It was use descriptive statistics and georeference was made in a map using the software ILWIS 3.3. RESULTS: Countyes with the highest and lowest percentages of infection were found. The prevalence of infection with high-risk HPV was 9.9% and age groups with the highest percentages of infection were in 51-60 and 41-50 years. Most women had been made the Papanicolaou at time of the present study. CONCLUSIONS: Georeferenceas like epidemiological tool for generating risk profiles allowed suggest strategies for improve prevention, early detection and control of the cervical cancer.

[Neoadjuvant Chemotherapy (NC) Response in Patients with Breast Cancer According to Immunohistochemical Intrinsic Subtypes (IHC)]. / Respuesta a la quimioterapia neoadyuvante (QTneo) según los subtipos intrínsecos determinados por inmunohistoquímica (IHQ) en pacientes con cáncer de mama.

INTRODUCTION: Breast cancer is heterogeneous, with different responses to NC even within similar histology and stages. OBJECTIVE: To evaluate clinical/pathological response to NC according to different tumor subtypes in Mexican breast cancer patients. PATIENTS AND METHODS: Retrospective study of patients with breast cancer stages II-III, and complete immunohistochemistry (IHC), such as hormonal receptors HER2 and Ki67, treated with NC and surgery. Descriptive and comparative analyses between different intrinsic subtypes were performed. RESULTS: A total of 117 patients were included with 48.6 ± 10.6 years of age, stage II (24%), and III (76%). We identified 20 (17.1%) cases of luminal A, 37 (31.6%) luminal B HER2-, 13 (11.1%) luminal B HER2+, 12 (10.3%) HER2+, and 35 (29.9%) triple negative. Clinical complete response (tumor and lymph nodes) in luminal A was 10%, in luminal B HER2- 10.8%, luminal B HER2+ 15.4%, HER2+ 25%, and in triple negative 14.3%. Conservative surgeries were done in 9 (7.7%) patients. There is a weak positive association between Ki67 expression and tumor clinical response. Pathological complete response occurred in 8 (6.83%) cases, being more frequent in luminal B HER2+ patients (23%). CONCLUSIONS: Pathological complete responses were more often in luminal B HER2+ cases.

[Impact on quality of life with breast reconstructive surgery after mastectomy for breast cancer]. / Impacto en la calidad de vida con cirugía reconstructiva posterior al tratamiento de cáncer de mama.

BACKGROUND: Breast cancer treatment leads mutilation and destruction of breast shape, with negative effects on body image and self-esteem. OBJECTIVES: Assessment on quality of life after breast reconstruction surgery, impact on sexuality, the cosmetic outcome experienced by the patient, and compare result with patients who refused breast reconstruction. MATERIAL AND METHODS: Retrospective, observational, descriptive, analytic study. We included breast cancer patients treated between April 15 2010 to April 15, 2011. Application of "The Survey Questionnaire short form Health 36" (SF-36) with valid use on Mexican population was conducted to measure quality of life, which uses 8 concepts: physical functioning, physical role, body pain, general health, vitality, social function, emotional role and mental health, the results are transferred to a scale 0 (worst health) to 100 (best health). RESULTS: 37 patients whit breast reconstruction had the inclusion criteria, mean age was 48.4 years. The score of SF-36 questionnaire in reconstructed patients was 76.8, in control group was 85.19 and mastectomy patients without reconstruction was 72.6. Among the items studied those with the greatest difference was the mental health, emotional role and social function, this means that patients with breast reconstruction are less affected in their social and sexual interaction. CONCLUSIONS: The reconstructed patients have a positive impact on quality of life slightly higher, sexuality is significantly worse in patients without breast reconstruction, it is important to inform and offer breast reconstruction because many do not require these procedures for fear or lack of information.

Fluid mixing by an electromagnetically driven floating rotor.

We analyze the mixing properties of a floating stirrer driven electromagnetically in a thin layer of electrolyte, consisting of two free-floating magnets with opposite polarities connected by a rigid coupling. The magnetic rotor is set in circular motion using Lorentz forces created due to the interaction of the magnetic field of the rotor with dc currents actuated in logic sequence. We identify a coherent structure similar to a tripolar vortex whose central vortex rotates in the same direction of the rotor promoting chaotic mixing of the fluid in the laminar regime (Re=45). Dyed water visualization and particle image velocimetry were performed to characterize experimentally the mixing and flow dynamics at the surface of the electrolyte layer. A quasitwo-dimensional numerical simulation based on the immersed boundary method, which incorporates the fluid-solid interaction and reproduces the experimental observations satisfactorily, was carried out. Optimal mixing conditions are determined through the exponential growth of the material interfaces, which are established mainly by varying the distance separating the magnets of the rotor.

An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.

MetastamiRs: non-coding MicroRNAs driving cancer invasion and metastasis.

MicroRNAs (miRNAs) are small non-coding RNAs of ~22 nucleotides that function as negative regulators of gene expression by either inhibiting translation or inducing deadenylation-dependent degradation of target transcripts. Notably, deregulation of miRNAs expression is associated with the initiation and progression of human cancers where they act as oncogenes or tumor suppressors contributing to tumorigenesis. Abnormal miRNA expression may provide potential diagnostic and prognostic tumor biomarkers and new therapeutic targets in cancer. Recently, several miRNAs have been shown to initiate invasion and metastasis by targeting multiple proteins that are major players in these cellular events, thus they have been denominated as metastamiRs. Here, we present a review of the current knowledge of miRNAs in cancer with a special focus on metastamiRs. In addition we discuss their potential use as novel specific markers for cancer progression.

Self-association features of NS1 proteins from different flaviviruses.

Flaviviruses comprise a large group of arboviral species that are distributed in several countries of the tropics, neotropics, and some temperate zones. Since they can produce neurological pathologies or vascular damage, there has been intense research seeking better diagnosis and treatments for their infections in the last decades. The flavivirus NS1 protein is a relevant clinical target because it is involved in viral replication, immune evasion, and virulence. Being a key factor in endothelial and tissue-specific modulation, NS1 has been largely studied to understand the molecular mechanisms exploited by the virus to reprogram host cells. A central part of the viral maturation processes is the NS1 oligomerization because many stages rely on these protein-protein assemblies. In the present study, the self-associations of NS1 proteins from Zika, Dengue, and West Nile viruses are examined through constant-pH coarse-grained biophysical simulations. Free energies of interactions were estimated for different oligomeric states and pH conditions. Our results show that these proteins can form both dimers and tetramers under conditions near physiological pH even without the presence of lipids. Moreover, pH plays an important role mainly controlling the regimes where van der Waals interactions govern their association. Finally, despite the similarity at the sequence level, we found that each flavivirus has a well-characteristic protein-protein interaction profile. These specific features can provide new hints for the development of binders both for better diagnostic tools and the formulation of new therapeutic drugs.

[Breast cancer in Mexican women under 40]. / Cancer de mama en mujeres mexicanas menores de 40 años.

BACKGROUND: Breast cancer is the leading cause of death from malignancy in women. The incidence increases with age, but the relationship between age and survival of breast cancer patients is not well defined. It is observed that young women with breast cancer have patterns more aggressive biological. OBJECTIVE: To determine the frequency, sociodemographic, clinical and histopathological features of breast cancer in women under 40 years attending a specialist breast unit in Mexico City. PATIENTS AND METHOD: Transversal, descriptive and retrospective study of patients under 40 years of age with breast cancer treated between 2005 and 2010. RESULTS: 1430 cases were diagnosed with breast cancer five years with a mean age of 53.64 +/- 11.87 years (range 23 to 93 years), 142 cases were women under 40 years of age (10%). The auto-detection of a breast lump was the most frequent clinical manifestation (50%). CONCLUSION: The prevalence of clinical stage III in this age group suggests the difficulty of diagnosis, the high breast density, which is one factor limiting studies of screening with mammography, it diminishes their effectiveness in early detection of breast cancer.

First breast cancer mammography screening program in Mexico: initial results 2005-2006.

Breast cancer is the most frequent malignant neoplasia worldwide. In emergent countries as Mexico, an increase has been shown in frequency and mortality, unfortunately, most cases in advanced loco-regional stages developed in young women. The success of breast screening in mortality reduction has been observed since 1995 in Western Europe and the United States, where as many as 40% mortality reduction has been achieved. Most countries guidelines recommends an annual or biannual mammography for all women >40 years of age. In 2005, FUCAM, a nonlucrative civil foundation in Mexico join with Mexico City government, initiated the first voluntary mammography screening program for women >40 years of age residing in Mexico City's Federal District. Mammographies were carried out with analogical mammographs in specially designed mobile units and were performed in the area of women's domiciles. This report includes data from the first 96,828 mammographies performed between March 2005 and December 2006. There were 1% of mammographies in Breast Imaging Reporting and Data System 0, 4, or 5 and 208 out of 949 women with abnormal mammographies (27.7%) had breast cancer, a rate of 2.1 per thousand, most of them in situ or stage I (29.4%) or stage II (42.2%) nevertheless 21% of those women with abnormal mammography did not present for further clinical and radiologic evaluation despite being personally notified at their home addresses. The breast cancer rate of Mexican women submitted to screening mammography is lower than in European or North American women. Family history of breast cancer, nulliparity, absence of breast feeding, and increasing age are factors that increase the risk of breast cancer. Most cancers were diagnosed in women's age below 60 years (68.5%) with a mean age of 53.55 corroborating previous data published. It is mandatory to sensitize and educate our population with regard to accepting to visit the Specialized Breast Centers.