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Portuguese and Azorean immigrants make up 0.4% of the United States population, but they comprise over 6% of the population of Rhode Island. A vast number of Azorean immigrants in this region have made immense contributions to our medical community and the local cultural identity. Within Rhode Island Hospital (RIH), the roles held by staff who are Azorean immigrants are critical in maximizing the quality of care and efficiency of a large academic medical center. To properly illustrate their contributions, we highlight the experiences of four Azorean immigrants with decades of experience at RIH. Much like how the keelson is a structural member that provides continuity and connects the bow stem, frames, and transom to hold a whaleboat or wooden vessel together, these stalwart employees have long done similarly for Rhode Island's only tertiary care center.
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Emigrantes e Inmigrantes , Rhode Island , Humanos , Azores , FemeninoRESUMEN
We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms.
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Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Humanos , Espectrometría de Masas/métodos , Unión Proteica , Espectrometría de Masas en Tándem/métodosRESUMEN
Biochemical and biophysical assays using recombinant RAS require the protein to be in either the active or inactive state. Here we describe methods to exchange the nucleotide present in the purified RAS protein with either GDPßS, GppNHp, or GTP depending on the assay requirement. In addition, we also describe the HPLC method used to validate the exchange process and provide information on the efficiency of the nucleotide exchange.
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Proteínas ras , Guanosina Trifosfato/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Guanosina DifosfatoRESUMEN
RAS research has entered the world of translational and clinical science. Progress has been based on our appreciation of the role of RAS mutations in different types of cancer and the effects of these mutations on the biochemical, structural, and biophysical properties of the RAS proteins themselves, particularly KRAS, on which most attention has been focused. This knowledge base, while still growing, has enabled creative chemical approaches to targeting KRAS directly. Our understanding of RAS signaling pathways in normal and cancer cells plays an important role for developing RAS inhibitors but also continues to reveal new approaches to targeting RAS through disruption of signaling complexes and downstream pathways.
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Antineoplásicos , Neoplasias , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Mutación , Neoplasias/metabolismo , Transducción de Señal , Antineoplásicos/farmacologíaRESUMEN
Surface plasmon resonance (SPR) is an optical effect at an electron-rich surface that enables affinity measurements of biomolecules in real time. It is label free and versatile, not limited to proteins, nucleic acids, and small molecules. SPR is a widely accepted method to measure not only affinity of molecular interactions but also association and dissociation rates of such interactions. In this chapter, we describe a general method to measure the affinity of a small molecule drug, MRTX849, to GDP bound HRAS, KRAS, and NRAS.
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Acetonitrilos , Proteínas Proto-Oncogénicas p21(ras) , Pirimidinas , Resonancia por Plasmón de Superficie , Resonancia por Plasmón de Superficie/métodos , Proteínas Proto-Oncogénicas p21(ras)/genética , Piperazinas , Isoformas de Proteínas , MutaciónRESUMEN
Oncogenic mutations in KRAS typically impact the GAP-mediated and intrinsic GTP hydrolysis activity resulting in elevated levels of cellular KRAS-GTP. The development of biochemical assays for GTPase activity provides an opportunity to quantitatively measure the impact of these mutations on GTP hydrolysis. Here we describe a biochemical assay that measures the release of free phosphate upon hydrolysis of the GTP nucleotide and allows the measurement of intrinsic or GAP-stimulated GTP hydrolysis by KRAS. This assay can be used to measure GTPase activity under single turnover conditions.
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Proteínas Activadoras de GTPasa , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/genética , Hidrólisis , Mutación , Cinética , Guanosina Trifosfato , Proteínas Activadoras de GTPasa/metabolismoRESUMEN
Homogenous time-resolved FRET (HTRF) assays have become one of the most popular tools for pharmaceutical drug screening efforts over the last two decades. Large Stokes shifts and long fluorescent lifetimes of lanthanide chelates lead to robust signal to noise, as well as decreased false positive rates compared to traditional assay techniques. In this chapter, we describe an HTRF protein-protein interaction (PPI) assay for the KRAS4b G-domain in the GppNHp-bound state and the RAF-1-RBD currently used for drug screens. Application of this assay contributes to the identification of lead compounds targeting the GTP-bound active state of K-RAS.
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Descubrimiento de Drogas , Transferencia Resonante de Energía de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , QuelantesRESUMEN
BACKGROUND: Pip is a novel digital health platform (DHP) that combines human health coaches (HCs) and technology with patient-facing content. This combination has not been studied in perioperative surgical optimization. OBJECTIVE: This study's aim was to test the feasibility of the Pip platform for deploying perioperative, digital, patient-facing optimization guidelines to elective surgical patients, assisted by an HC, at predefined intervals in the perioperative journey. METHODS: We conducted an institutional review board-approved, descriptive, prospective feasibility study of patients scheduled for elective surgery and invited to enroll in Pip from 2.5 to 4 weeks preoperatively through 4 weeks postoperatively at an academic medical center between November 22, 2022, and March 27, 2023. Descriptive primary end points were patient-reported outcomes, including patient satisfaction and engagement, and Pip HC evaluations. Secondary end points included mean or median length of stay (LOS), readmission at 7 and 30 days, and emergency department use within 30 days. Secondary end points were compared between patients who received Pip versus patients who did not receive Pip using stabilized inverse probability of treatment weighting. RESULTS: A total of 283 patients were invited, of whom 172 (60.8%) enrolled in Pip. Of these, 80.2% (138/172) patients had ≥1 HC session and proceeded to surgery, and 70.3% (97/138) of the enrolled patients engaged with Pip postoperatively. The mean engagement began 27 days before surgery. Pip demonstrated an 82% weekly engagement rate with HCs. Patients attended an average of 6.7 HC sessions. Of those patients that completed surveys (95/138, 68.8%), high satisfaction scores were recorded (mean 4.8/5; n=95). Patients strongly agreed that HCs helped them throughout the perioperative process (mean 4.97/5; n=33). The average net promoter score was 9.7 out of 10. A total of 268 patients in the non-Pip group and 128 patients in the Pip group had appropriate overlapping distributions of stabilized inverse probability of treatment weighting for the analytic sample. The Pip cohort was associated with LOS reduction when compared to the non-Pip cohort (mean 2.4 vs 3.1 days; median 1.9, IQR 1.0-3.1 vs median 3.0, IQR 1.1-3.9 days; mean ratio 0.76; 95% CI 0.62-0.93; P=.009). The Pip cohort experienced a 49% lower risk of 7-day readmission (relative risk [RR] 0.51, 95% CI 0.11-2.31; P=.38) and a 17% lower risk of 30-day readmission (RR 0.83, 95% CI 0.30-2.31; P=.73), though these did not reach statistical significance. Both cohorts had similar 30-day emergency department returns (RR 1.06, 95% CI 0.56-2.01, P=.85). CONCLUSIONS: Pip is a novel mobile DHP combining human HCs and perioperative optimization content that is feasible to engage patients in their perioperative journey and is associated with reduced hospital LOS. Further studies assessing the impact on clinical and patient-reported outcomes from the use of Pip or similar DHPs HC combinations during the perioperative journey are required.
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In this paper, a Monte Carlo (MC)-based extended Kalman filter is proposed for a two-dimensional bearings-only tracking problem (BOT). This problem addresses the processing of noise-corrupted bearing measurements from a sea acoustic source and estimates state vectors including position and velocity. Due to the nonlinearity and complex observability properties in the BOT problem, a wide area of research has been focused on improving its state estimation accuracy. The objective of this research is to present an accurate approach to estimate the relative position and velocity of the source with respect to the maneuvering observer. This approach is implemented using the iterated extended Kalman filter (IEKF) in an MC-based iterative structure (MC-IEKF). Re-linearizing dynamic and measurement equations using the IEKF along with the MC campaign applied to the initial conditions result in significantly improved accuracy in the estimation process. Furthermore, an observability analysis is conducted to show the effectiveness of the designed maneuver of the observer. A comparison with the widely used UKF algorithm is carried out to demonstrate the performance of the proposed method.
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Object detection and tracking are pivotal tasks in machine learning, particularly within the domain of computer vision technologies. Despite significant advancements in object detection frameworks, challenges persist in real-world tracking scenarios, including object interactions, occlusions, and background interference. Many algorithms have been proposed to carry out such tasks; however, most struggle to perform well in the face of disturbances and uncertain environments. This research proposes a novel approach by integrating the You Only Look Once (YOLO) architecture for object detection with a robust filter for target tracking, addressing issues of disturbances and uncertainties. The YOLO architecture, known for its real-time object detection capabilities, is employed for initial object detection and centroid location. In combination with the detection framework, the sliding innovation filter, a novel robust filter, is implemented and postulated to improve tracking reliability in the face of disturbances. Specifically, the sliding innovation filter is implemented to enhance tracking performance by estimating the optimal centroid location in each frame and updating the object's trajectory. Target tracking traditionally relies on estimation theory techniques like the Kalman filter, and the sliding innovation filter is introduced as a robust alternative particularly suitable for scenarios where a priori information about system dynamics and noise is limited. Experimental simulations in a surveillance scenario demonstrate that the sliding innovation filter-based tracking approach outperforms existing Kalman-based methods, especially in the presence of disturbances. In all, this research contributes a practical and effective approach to object detection and tracking, addressing challenges in real-world, dynamic environments. The comparative analysis with traditional filters provides practical insights, laying the groundwork for future work aimed at advancing multi-object detection and tracking capabilities in diverse applications.
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Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.
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Proteómica , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/genética , Mutación , Sitios de UniónRESUMEN
The oncogene RAS, extensively studied for decades, presents persistent gaps in understanding, hindering the development of effective therapeutic strategies due to a lack of precise details on how RAS initiates MAPK signaling with RAF effector proteins at the plasma membrane. Recent advances in X-ray crystallography, cryo-EM, and super-resolution fluorescence microscopy offer structural and spatial insights, yet the molecular mechanisms involving protein-protein and protein-lipid interactions in RAS-mediated signaling require further characterization. This study utilizes single-molecule experimental techniques, nuclear magnetic resonance spectroscopy, and the computational Machine-Learned Modeling Infrastructure (MuMMI) to examine KRAS4b and RAF1 on a biologically relevant lipid bilayer. MuMMI captures long-timescale events while preserving detailed atomic descriptions, providing testable models for experimental validation. Both in vitro and computational studies reveal that RBDCRD binding alters KRAS lateral diffusion on the lipid bilayer, increasing cluster size and decreasing diffusion. RAS and membrane binding cause hydrophobic residues in the CRD region to penetrate the bilayer, stabilizing complexes through ß-strand elongation. These cooperative interactions among lipids, KRAS4b, and RAF1 are proposed as essential for forming nanoclusters, potentially a critical step in MAP kinase signal activation.
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Membrana Dobles de Lípidos , Lípidos de la Membrana , Lípidos de la Membrana/metabolismo , Membrana Dobles de Lípidos/metabolismo , Membrana Celular/metabolismo , Membranas/metabolismo , Transducción de SeñalRESUMEN
Electrospinning is an advanced manufacturing strategy used to create innovative medical devices from continuous nanoscale fibers that is endowed with tunable biological, chemical, and physical properties. Innovative medical patches manufactured entirely by electrospinning are discussed in this paper, using a specific plant-derived formulation "1 Primary Wound Dressing©" (1-PWD) as an active pharmaceutical ingredient (API). 1-PWD is composed of neem oil (Azadirachta indica A. Juss.) and the oily extracts of Hypericum perforatum (L.) flowers, according to the formulation patented by the ENEA of proven therapeutic efficacy as wound dressings. The goal of this work is to encapsulate this API and demonstrate that its slow release from an engineered electrospun patch can increase the therapeutic efficacy for wound healing. The prototyped patch is a three-layer core-shell membrane, with a core made of fibers from a 1-PWD-PEO blend, enveloped within two external layers made of medical-grade polycaprolactone (PCL), ensuring mechanical strength and integrity during manipulation. The system was characterized via electron microscopy (SEM) and chemical and contact angle tests. The encapsulation, release, and efficacy of the API were confirmed by FTIR and LC-HRMS and were validated via in vitro toxicology and scratch assays.
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KRAS, the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4a's thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably, KRAS4a exhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approaching KRAS4b in the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells.
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Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Conformación Molecular , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.
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Proteínas Proto-Oncogénicas p21(ras) , Proteínas ras , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/metabolismo , Guanosina Trifosfato/metabolismo , Espectroscopía de Resonancia Magnética , Transducción de Señal , MutaciónRESUMEN
A large proportion of surgical residents that are applying to surgical critical care(SCC) and acute care surgery(ACS) fellowships are describing cases where they cared for patients with injuries from penetrating trauma in their personal statements. These cases appear to have served as an inspiration for their fellowship and career decision. However a substantial percentage of training in these fellowships occurs in the ICU and there also have been steadily decreasing rates of operative penetrating trauma throughout the United States over the last several decades. This incongruity is explored and suggestions are made for formal mentorship to occur between surgical residents interested in further training in SCC and ACS and attendings practicing within these fields.
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The whale optimization algorithm (WOA) is a widely used metaheuristic optimization approach with applications in various scientific and industrial domains. However, WOA has a limitation of relying solely on the best solution to guide the population in subsequent iterations, overlooking the valuable information embedded in other candidate solutions. To address this limitation, we propose a novel and improved variant called Pbest-guided differential WOA (PDWOA). PDWOA combines the strengths of WOA, particle swarm optimizer (PSO), and differential evolution (DE) algorithms to overcome these shortcomings. In this study, we conduct a comprehensive evaluation of the proposed PDWOA algorithm on both benchmark and real-world optimization problems. The benchmark tests comprise 30-dimensional functions from CEC 2014 Test Functions, while the real-world problems include pressure vessel optimal design, tension/compression spring optimal design, and welded beam optimal design. We present the simulation results, including the outcomes of non-parametric statistical tests including the Wilcoxon signed-rank test and the Friedman test, which validate the performance improvements achieved by PDWOA over other algorithms. The results of our evaluation demonstrate the superiority of PDWOA compared to recent methods, including the original WOA. These findings provide valuable insights into the effectiveness of the proposed hybrid WOA algorithm. Furthermore, we offer recommendations for future research to further enhance its performance and open new avenues for exploration in the field of optimization algorithms. The MATLAB Codes of FISA are publicly available at https://github.com/ebrahimakbary/PDWOA.
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Many important engineering optimization problems require a strong and simple optimization algorithm to achieve the best solutions. In 2020, Rao introduced three non-parametric algorithms, known as Rao algorithms, which have garnered significant attention from researchers worldwide due to their simplicity and effectiveness in solving optimization problems. In our simulation studies, we have developed a new version of the Rao algorithm called the Fully Informed Search Algorithm (FISA), which demonstrates acceptable performance in optimizing real-world problems while maintaining the simplicity and non-parametric nature of the original algorithms. We evaluate the effectiveness of the suggested FISA approach by applying it to optimize the shifted benchmark functions, such as those provided in CEC 2005 and CEC 2014, and by using it to design mechanical system components. We compare the results of FISA to those obtained using the original RAO method. The outcomes obtained indicate the efficacy of the proposed new algorithm, FISA, in achieving optimized solutions for the aforementioned problems. The MATLAB Codes of FISA are publicly available at https://github.com/ebrahimakbary/FISA.