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Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Relacionadas con la Autofagia , Ubiquitinación , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/química , Proteínas Relacionadas con la Autofagia/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Humanos , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Ubiquitina/metabolismo , Ubiquitina/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Ubiquitinadas/metabolismo , Proteínas Ubiquitinadas/aislamiento & purificación , Proteínas Ubiquitinadas/química , Separación de FasesRESUMEN
Purpose: In Peru, breast cancer (BC) stands as the most predominant malignancy neoplasm among women. Trastuzumab has marked a significant milestone in the management of this disease. It has been shown to improve prognosis in human epidermal growth factor receptor 2 (HER2)-expressing female patients, but its repercussions and efficacy are yet to be analyzed in a context with limited resources. Methods: The study population is made of woman patients aged 18 years and older diagnosed with HER2-positive BC at Instituto Nacional de Enfermedades Neoplásicas (INEN, Lima, Peru) during 2019-2021 and treated with at least one dose of subcutaneous trastuzumab. We reviewed medical records to register treatment characteristics, adverse events (AEs), disease progression, and survival status. We considered a median follow-up time of 36 and 45 months for progression and survival status. Results: The majority of patients were over 50 years old (54.29%). Tumor size averaged 19.7 ± 16.1 mm. Lymph nodes were present in 44.78% of patients. Most patients received adjuvant chemotherapy (63.8%) as first-line treatment. Descriptive analyses of treatment outcomes revealed a 30% toxicity rate, primarily attributed to arthralgia (47.62%), followed by diarrhea, fatigue, and injection site reactions, with relatively lower discontinuation rates compared to larger scale studies. Differences in demographic, clinical, and treatment characteristics were not statistically significant concerning the emergence of AEs (p > 0.05). Progression appeared in nine patients, and the overall survival (OS) rate stood at 98.6% and 92.8%, respectively, during a median follow-up of 36 and 45 months. Conclusion: The research suggests that subcutaneous trastuzumab is comparable in effectiveness and safety to the intravenous administration. Regional-specific studies may provide valuable insights into demographic factors influencing treatment outcomes in Peru or other countries. Furthermore, it could represent a more accessible alternative, potentially enhancing patient adherence and optimizing healthcare resource logistics.
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BACKGROUND: The influence of Helicobacter-pylori (H. pylori) infection and the characteristics of gastric cancer (GC) on tumor-infiltrating lymphocyte (TIL) levels has not been extensively studied. Analysis of infiltrating-immune-cell subtypes as well as survival is necessary to obtain comprehensive information. AIM: To determine the rates of deficient mismatch-repair (dMMR), HER2-status and H. pylori infection and their association with TIL levels in GC. METHODS: Samples from 503 resected GC tumors were included and TIL levels were evaluated following the international-TILs-working-group recommendations with assessment of the intratumoral (IT), stromal (ST) and invasive-border (IB) compartments. The density of CD3, CD8 and CD163 immune cells, and dMMR and HER2-status were determined by immunohistochemistry (IHC). H. pylori infection was evaluated by routine histology and quantitative PCR (qPCR) in a subset of samples. RESULTS: dMMR was found in 34.4%, HER2+ in 5% and H. pylori-positive in 55.7% of samples. High IT-TIL was associated with grade-3 (P = 0.038), while ST-TIL with grade-1 (P < 0.001), intestinal-histology (P < 0.001) and no-recurrence (P = 0.003). dMMR was associated with high TIL levels in the ST (P = 0.019) and IB (P = 0.01) compartments, and ST-CD3 (P = 0.049) and ST-CD8 (P = 0.05) densities. HER2- was associated with high IT-CD8 (P = 0.009). H. pylori-negative was associated with high IT-TIL levels (P = 0.009) when assessed by routine-histology, and with high TIL levels in the 3 compartments (P = 0.002-0.047) and CD8 density in the IT and ST compartments (P = 0.001) when assessed by qPCR. A longer overall survival was associated with low IT-CD163 (P = 0.003) and CD8/CD3 (P = 0.001 in IT and P = 0.002 in ST) and high IT-CD3 (P = 0.021), ST-CD3 (P = 0.003) and CD3/CD163 (P = 0.002). CONCLUSION: TIL levels were related to dMMR and H. pylori-negativity. Low CD8/CD3 and high CD163/CD3 were associated with lower recurrence and longer survival.
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Rapid and uncontrolled urban growth and land use changes in watersheds worldwide have led to increased surface runoff within metropolitan areas, coupled with climate change, creating a risk for residents during the rainy season. The city of San Luis Potosí is no exception to this phenomenon. One affected watercourse is the Garita Stream, which flows inside the city near urbanization. It is essential to analyze the effects of urban sprawl on this stream based on historical precipitation data for the town. Hydrological and topographical information were required to conduct this research. The hydrological study of the basin involved analyzing the region's geomorphology and historical climatological data. For the stream's topography, aerial photogrammetry using an unmanned aerial Vehicle (UAV) and Global Navigation Satellite System (GNSS) equipment was employed to conduct topographic surveys in the area. To find out when the Garita stream would overflow and which areas are most likely to flood, numerical modeling was done using 1D, 2D, and 3D programs like SWMM5 (Storm Water Management Model), HEC-RAS (Hydrologic Engineering Center's River Analysis System), and EDFC Explorer (Environmental Fluid Dynamics Code). These models simulated different return periods and their correlation with current flooding events recorded in the area, thereby further proposing solutions to mitigate overflow issues. By conducting these simulations and analyzing the results, solutions can be suggested to address the overflow problems in the area based on historical flood events at various return periods caused by the Garita Stream.
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The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in concert to transmit cell-generated forces to the extracellular matrix required for cell motility, wound healing, and tissue maintenance. While we know cell-generated forces are driven by actomyosin contractility and balanced by microtubule network resistance, the effect of intermediate filaments on cellular forces is unclear. Using a combination of theoretical modeling and experiments, we show that vimentin intermediate filaments tune cell stress by assisting in both actomyosin-based force transmission and reinforcement of microtubule networks under compression. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. These results reconcile seemingly contradictory results in the literature and provide a unified description of vimentin's effects on the transmission of cell contractile forces to the extracellular matrix.
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Actomiosina , Mecanotransducción Celular , Microtúbulos , Vimentina , Microtúbulos/metabolismo , Actomiosina/metabolismo , Vimentina/metabolismo , Humanos , Matriz Extracelular/metabolismo , AnimalesRESUMEN
Ubiquitination is one of the most common post-translational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity towards K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage dependent manner, thus serving as an interpreter of the ubiquitin code.
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The ubiquitin-adaptor protein UBQLN2 promotes degradation of several aggregate-prone proteins implicated in neurodegenerative diseases. Missense UBQLN2 mutations also cause X-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previously we demonstrated that the liquid-like properties of UBQLN2 molecular assemblies are altered by a specific pathogenic mutation, P506T, and that the propensity of UBQLN2 to aggregate correlated with neurotoxicity. Here, we systematically assess the effects of multiple, spatially distinct ALS/FTD-linked missense mutations on UBQLN2 aggregation propensity, neurotoxicity, phase separation, and autophagic flux. In contrast to what we observed for the P506T mutation, no other tested pathogenic mutant exhibited a clear correlation between aggregation propensity and neurotoxicity. These results emphasize the unique nature of pathogenic UBQLN2 mutations and argue against a generalizable link between aggregation propensity and neurodegeneration in UBQLN2-linked ALS/FTD.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Demencia Frontotemporal/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Mutación , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Para la evaluación del fitness cardiorrespiratorio existen pruebas directas en laboratorio que proporcionan información objetiva respecto a esta cualidad. No obstante, cuando no se cuenta con los recursos se recurre al uso de pruebas de campo como el Shuttle Run Test 20 metros (SRT-20m), el cual busca establecer el VO2max para cada sujeto, relacionando los valores obtenidos con aspectos como la composición corporal, edad y contexto poblacional, siendo una de las pruebas más confiables y con concordancia clínica existente. El propósito fue reconocer los cambios o variaciones presentes en los modelos matemáticos o lineales empleados en esta prueba con diferentes grupos poblacionales. Se realizó una revisión de la literatura bajo el marco básico para revisiones integradoras con algunas precisiones dadas por la guía PRISMA, identificando en su mayoría estudios de corte transversal los cuales evidencian los cambios realizados al planteamiento de la fórmula matemática donde las variables que inciden en el valor resultante para la resistencia aeróbica son: velocidad final, etapa de la prueba, edad y aspectos antropométricos del sujeto. Por lo anterior, se concluye que, para efectos prácticos, la estimación del VO2máx debe considerar factores intrínsecos del sujeto y aquellos relacionados con la ejecución de la prueba (AU)
For the evaluation of cardiorespiratory fitness, there are direct laboratory tests that provide objective information regarding this quality. However, when the resources are not available, the use of field tests such as the Shuttle Run Test 20 meters (SRT-20m) is used, which seeks to establish the VO2max for each subject, relating the values obtained with aspects such as body composition, age, and population context, being one of the most reliable tests with existing clinical agreement. The purpose was to recognize the changes or variations present in the mathematical or linear models used in this test with different population groups. A review of the literature was carried out under the basic framework for integrative reviews with some clarifications given by the PRISMA guide, identifying mostly cross-sectional studies which show the changes made to the approach of the mathematical formula where the variables that affect the resulting value for aerobic endurance are final speed, stage of the test, age, and anthropometric aspects of the subject. Therefore, it is concluded that, for practical purposes, the estimation of VO2max should consider intrinsic factors of the subject and those related to the execution of the test (AU)
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Humanos , Consumo de Oxígeno/fisiología , Ventilación Voluntaria Máxima/fisiología , Pruebas Respiratorias , Modelos BiológicosRESUMEN
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.
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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.
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Miopía , Niño , Humanos , Secuenciación del Exoma , Miopía/genéticaRESUMEN
Introduction: Triple-negative breast cancer (TNBC) is a heterogeneous disease associated with a poor prognosis. Delaying in time to start adjuvant chemotherapy (TTC) has been related to an increased risk of distant recurrence-free survival (DRFS). We aimed to develop a prognostic model to estimate the effects of delayed TTC among TNBC risk subgroups. Materials and methods: We analyzed 687 TNBC patients who received adjuvant chemotherapy at the Instituto Nacional de Enfermedades Neoplasicas (Lima, Peru). Database was randomly divided to create a discovery set (n=344) and a validation set (n=343). Univariate and multivariate Cox regression models were performed to identify prognostic factors for DRFS. Risk stratification was implemented through two models developed based on proportional hazard ratios from significant clinicopathological characteristics. Subpopulation treatment effect pattern plot (STEPP) analysis was performed to determine the best prognostic cut-off points for stratifying TNBC subgroups according to risk scores and estimate Kaplan-Meier differences in 10-year DRFS comparing TTC (≤30 vs.>30 days). Results: In univariate analysis, patients aged ≥70 years (HR=4.65; 95% CI: 2.32-9.34; p=<0.001), those at stages pT3-T4 (HR=3.28; 95% CI: 1.57-6.83; p=0.002), and pN2-N3 (HR=3.00; 95% CI: 1.90-4.76; p=<0.001) were notably associated with higher risk. STEPP analysis defined three risk subgroups for each model. Model N°01 categorized patients into low (score: 0-31), intermediate (score:32-64), and high-risk (score: 65-100) cohorts; meanwhile, Model N°02: low (score: 0-26), intermediate (score: 27-55), and high (score: 56-100). Kaplan-Meier plots showed that in the discovery set, patients with TTC>30 days experienced a 17.5% decrease in 10-year DRFS rate (95%CI=6.7-28.3), and the impact was more remarkable in patients who belong to the high-risk subgroup (53.3% decrease in 10 years-DRFS rate). Similar results were found in the validation set. Conclusions: We developed two prognostic models based on age, pT, and pN to select the best one to classify TNBC. For Model N°02, delayed adjuvant chemotherapy conferred a higher risk of relapse in patients ≥70 years and who were characterized by pT3/T4 and pN2/N3. Thus, more efforts should be considered to avoid delayed TTC in TNBC patients, especially those in high-risk subgroups.
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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 (Pxx) 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.
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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.
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Poliubiquitina , Ubiquitina , Ubiquitina/metabolismo , Poliubiquitina/metabolismo , Ligandos , Ubiquitinación , Sitios de UniónRESUMEN
It is well known that induction motors consume active and reactive energy from the utility grid to operate; additionally, when a power converter drives the motor, a high content of current harmonics is produced, and both circumstances decrease the utility grid power factor, which later requires to be improved. To this end, this paper presents a novel complete solution through a robust control system employed in a back-to-back topology power converter to deliver, instead of consuming, regulated reactive power toward the main grid, which comes from a capacitor bank in a DC-bus. This salient feature of delivering reactive power, and simultaneously, regulating the speed for an induction motor, becomes one of the contributions of this work to enhance the power factor. The robust converter controller is synthesized in a cascade form, by applying the linearization block control and state-feedback techniques. These techniques are combined with the super-twisting strategy for canceling the nonlinearities and the effect of the external disturbances. The complete system consists of a back-to-back converter, an LCL filter coupled to the main grid for mitigating the current harmonic content, and an induction motor under variable load conditions. Experimental tests expose the performance and robustness of the proposed controller, where a robust control for the reactive power acts under sudden changes in the active power produced through abrupt variations in the motor load.
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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.
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Neoplasias , Cese del Hábito de Fumar , Humanos , Prevención del Hábito de Fumar , Colombia , Perú , Fumar , Neoplasias/prevención & controlRESUMEN
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.
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Factores de Transcripción , Ubiquitina , Conformación Proteica , Espectrometría de MasasRESUMEN
Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world's first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5' terminus capping and 3' terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.
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COVID-19 , Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodos , SARS-CoV-2/genética , Vacunas contra la COVID-19 , Oligonucleótidos/genética , COVID-19/prevención & control , Vacunas de ARNm , Mapeo Peptídico/métodos , ARN Mensajero/genéticaRESUMEN
In this issue of Structure, Buel et al. (2023) combined NMR data with AlphaFold2 to map out the interaction between the AZUL domain of ubiquitin ligase E6AP and UBQLN1/2 UBA. The authors demonstrated that this interaction enhances the self-association of the helix neighboring UBA and enables E6AP to localize to UBQLN2 droplets.
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Ubiquitina-Proteína Ligasas , Ubiquitina , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/química , Unión ProteicaRESUMEN
Introduction: Breast cancer is a heterogeneous disease, and the distribution of the different subtypes varies by race/ethnic category in the United States and by country. Established breast cancer-associated factors impact subtype-specific risk; however, these included limited or no representation of Latin American diversity. To address this gap in knowledge, we report a description of demographic, reproductive, and lifestyle breast cancer-associated factors by age at diagnosis and disease subtype for The Peruvian Genetics and Genomics of Breast Cancer (PEGEN-BC) study. Methods: The PEGEN-BC study is a hospital-based breast cancer cohort that includes 1943 patients diagnosed at the Instituto Nacional de Enfermedades Neoplásicas in Lima, Peru. Demographic and reproductive information, as well as lifestyle exposures, were collected with a questionnaire. Clinical data, including tumor Hormone Receptor (HR) status and Human Epidermal Growth Factor Receptor 2 (HER2) status, were abstracted from electronic medical records. Differences in proportions and mean values were tested using Chi-squared and one-way ANOVA tests, respectively. Multinomial logistic regression models were used for multivariate association analyses. Results: The distribution of subtypes was 52% HR+HER2-, 19% HR+HER2+, 16% HR-HER2-, and 13% HR-HER2+. Indigenous American (IA) genetic ancestry was higher, and height was lower among individuals with the HR-HER2+ subtype (80% IA vs. 76% overall, p=0.007; 152 cm vs. 153 cm overall, p=0.032, respectively). In multivariate models, IA ancestry was associated with HR-HER2+ subtype (OR=1.38,95%CI=1.06-1.79, p=0.017) and parous women showed increased risk for HR-HER2+ (OR=2.7,95%CI=1.5-4.8, p<0.001) and HR-HER2- tumors (OR=2.4,95%CI=1.5-4.0, p<0.001) compared to nulliparous women. Multiple patient and tumor characteristics differed by age at diagnosis (<50 vs. >=50), including ancestry, region of residence, family history, height, BMI, breastfeeding, parity, and stage at diagnosis (p<0.02 for all variables). Discussion: The characteristics of the PEGEN-BC study participants do not suggest heterogeneity by tumor subtype except for IA genetic ancestry proportion, which has been previously reported. Differences by age at diagnosis were apparent and concordant with what is known about pre- and post-menopausal-specific disease risk factors. Additional studies in Peru should be developed to further understand the main contributors to the specific age of onset and molecular disease subtypes in this population and develop population-appropriate predictive models for prevention.
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Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. 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 the UBQLN2-binding surface of Ub or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, 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. Importantly, the extent to which polyubiquitin hubs can promote UBQLN2 phase separation are encoded in the spacings between Ub units as found for naturally-occurring 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. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.