An Atomic Structure of the Human Spliceosome.

Mechanistic understanding of pre-mRNA splicing requires detailed structural information on various states of the spliceosome. Here we report the cryo electron microscopy (cryo-EM) structure of the human spliceosome just before exon ligation (the C∗ complex) at an average resolution of 3.76 Å. The splicing factor Prp17 stabilizes the active site conformation. The step II factor Slu7 adopts an extended conformation, binds Prp8 and Cwc22, and is poised for selection of the 3'-splice site. Remarkably, the intron lariat traverses through a positively charged central channel of RBM22; this unusual organization suggests mechanisms of intron recruitment, confinement, and release. The protein PRKRIP1 forms a 100-Å α helix linking the distant U2 snRNP to the catalytic center. A 35-residue fragment of the ATPase/helicase Prp22 latches onto Prp8, and the quaternary exon junction complex (EJC) recognizes upstream 5'-exon sequences and associates with Cwc22 and the GTPase Snu114. These structural features reveal important mechanistic insights into exon ligation.

The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action.

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1R1074H and PHF5AY36C The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5AR38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.

Reduction in the incidence of pressure ulcers upon implementation of a reminder system for health-care providers.

PURPOSE: To measure the clinical impact of the introduction of a reminder system for healthcare professionals to alert patients who are at risk for pressure ulcers (PU). METHODS: This was a pre- and post-test study of patients who were discharged from 6 medical-surgical units of the University Hospital of Fuenlabrada in 2009 and 2010. Beginning in January 2010, implementation of an on-screen list of reminders was automatically updated daily on the units' computers including patient arrival date, last assessment of ulceration risk and location of any PU. The cumulative incidence of PU was measured for patients discharged in 2009 (group A: healthcare professionals were not exposed to on-screen reminder) and 2010 (group B: healthcare professionals were exposed to on-screen reminder list). The relative risk (RR) was estimated. The study was completed with a stratified analysis and binary logistic regression. RESULTS: In group A, there were 84 cases of PU among 9263 patients discharged (0.9%); whereas in group B, there were 59 cases among 9220 patients discharged (0.6%). The RR of PU for group B/group A was 0.706 (p=0.038). In the logistic regression analysis, after adjusting for study variables, the odds ratio of PU B/A was 0.558. CONCLUSION: A list of on-screen reminders at the beginning of a healthcare professional's shift to inform them of patients at risk for developing a PU was effective at reducing the incidence of these clinical burdens.

Adsorption of linear aliphatic α,ω-dithiols on plasmonic metal nanoparticles: a structural study based on surface-enhanced Raman spectra.

The adsorption mechanism of linear aliphatic α,ω-dithiols with chain lengths of 6, 8 and 10 carbon atoms on silver and gold nanoparticles has been studied by surface-enhanced Raman scattering (SERS) spectroscopy. SERS spectra provided the structural marker bands of these compounds and they were employed to obtain information about the adsorption and coordination mechanism, the orientation, conformational order, and packing of the aliphatic chains of dithiols on the metal nanoparticle surface. The effect of the type of metal (silver or gold) and the extent of surface coverage on all the above mentioned properties is discussed. It was found that the adsorption of dithiols on Au nanoparticles leads to a more disordered structure of the aliphatic chains of dithiols in comparison with the adsorption on Ag nanoparticles. The interaction through both thiol groups makes the adsorption of dithiols on metal surfaces substantially different from that of monothiols; in particular, the orientation of dithiols is perpendicular, while monothiols adopt a tilted orientation. Dithiols may act as linkers between metal nanoparticles and induce the formation of nanogaps with a controllable interparticle distance. The nanogaps thus formed are able to produce hot spots exhibiting a large intensification of electromagnetic field in these points which has been proved by the observation of intense SERS spectra of dithiols until a concentration of 10(-8) M, corresponding to a large Raman enhancement factor of 5 × 10(6).

Heart rate recovery in elite Spanish male athletes.

AIM: During postexercise recovery, heart rate (HR) initially falls rapidly, followed by a period of slower decrease, until resting values are reached. The aim of the present work was to examine the differences in the recovery heart rate (RHR) between athletes engaged in static and dynamic sports. METHODS: The study subjects were 294 federated sportsmen competing at the national and international level in sports classified using the criteria of Mitchell et al. as either prevalently static (N.=89) or prevalently dynamic (N.=205). Within the dynamic group, the subjects who practised the most dynamic sports were assigned to further subgroups: triathlon (N.=20), long distance running (N.=58), cycling (N.=28) and swimming (N.=12). All athletes were subjected to a maximum exertion stress test and their HR recorded at 1, 2, 3 and 4 min (RHR1,2,3,4) into the HR recovery period. The following indices of recovery (IR) were then calculated: IR1=(HRpeak-RHR1,2,3,4)/(HRmax-HRrest)*100, IR2=(HRpeak-RHR1,2,3,4)/(HRmax/HRpeak), and IR3=HRpeak-RHR1,2,3,4. The differences in the RHR and IR for the static and dynamic groups were examined using two way ANOVA. RESULTS: The RHR at minutes 2 (138.7±15.2 vs. 134.8±14.4 beats·min⁻¹) and 3 (128.5±15.2 vs. 123.3±14.4 beats·min⁻¹) were significantly higher for the static group (Group S) than the dynamic group (Group D), respectively. Significant differences were seen between Group D and S with respect to IR1 at minutes 1 (26.4±8.7 vs. 24.8±8.4%), 2 (43.8±8.1 vs. 41.5±7.8%), 3 (52.1±8.3 vs. 49.1±8%) and 4 (56.8±8.6 vs. 55.4±7.4%) of recovery. For IR2, significant differences were seen between the same groups at minutes 2 (59.7±12.5 vs. 55.9±10.8 beats·min⁻¹) and 3 (71.0±13.5 vs. 66.1±11.4 beats·min⁻¹) of recovery. Finally, for IR3, the only significant difference between Group D and S was recorded at minute 3 of recovery (72.2±12.5 vs. 66.2±11.5 beats·min⁻¹). CONCLUSION: This work provides information on RHR of a large population of elite Spanish athletes, and shows marked differences in the way that HR recovers in dynamic and static sports.

Analysis of machine learning algorithmic models for the prediction of vital status at six months after hip fracture in patients older than 74 years. / Análisis de modelos algorítmicos de aprendizaje automático para la predicción del estado vital a los seis meses tras fractura de cadera en pacientes mayores de 74 años.

BACKGROUND AND OBJECTIVE: The objective is to develop a model that predicts vital status six months after fracture as accurately as possible. For this purpose we will use five different data sources obtained through the National Hip Fracture Registry, the Health Management Unit and the Economic Management Department. MATERIAL AND METHODS: The study population is a cohort of patients over 74 years of age who suffered a hip fracture between May 2020 and December 2022. A warehouse is created from five different data sources with the necessary variables. An analysis of missing values and outliers as well as unbalanced classes of the target variable («vital status¼) is performed. Fourteen different algorithmic models are trained with the training. The model with the best performance is selected and a fine tuning is performed. Finally, the performance of the selected model is analyzed with test data. RESULTS: A data warehouse is created with 502 patients and 144 variables. The best performing model is Linear Regression. Sixteen of the 24 cases of deceased patients are classified as live, and 14 live patients are classified as deceased. A sensitivity of 31%, an accuracy of 34% and an area under the curve of 0.65 is achieved. CONCLUSIONS: We have not been able to generate a model for the prediction of six-month survival in the current cohort. However, we believe that the method used for the generation of algorithms based on machine learning can serve as a reference for future works.

Structural dynamics of RAF1-HSP90-CDC37 and HSP90 complexes reveal asymmetric client interactions and key structural elements.

RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 µs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1's unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding.

Collaborating with cancer patients and informal caregivers in a European study on quality of life: protocol to embed patient and public involvement within the EUonQoL project.

BACKGROUND: Patient and public involvement (PPI) has become an essential part of health research. There is a need for genuine involvement in order to ensure that research is relevant to patients. This can then improve the quality, relevance, and impact of health research, while at the same time reducing wasted research and in doing so bringing science and society closer together. Despite the increasing attention for this involvement, it is not yet common practice to report on proposed activities. An article reporting planned PPI could provide guidance and inspiration for the wider academic community in future activities. Therefore, this current article aims to describe the way in which PPI principles are incorporated in the research project called "Quality of Life in Oncology: measuring what matters for cancer patients and survivors in Europe (EUonQoL)." This project aims to develop a new set of questionnaires to enable cancer patients to assess their quality of life, entitled the EUonQoL-Kit. METHODS: The first step is to recruit cancer patients and their informal caregivers as co-researchers in order to train them to collaborate with the researchers. Based on their skills and preferences, they are then assigned to several of the project's work packages. Their individual roles, tasks, and responsibilities regarding the work packages, to which they have been assigned, are evaluated and adapted when necessary. The impact of their involvement is evaluated by both the researchers and co-researchers. DISCUSSION: PPI is a complex and dynamic process. As such, the overall structure of the research may be defined while at the same time leaving room for certain aspects to be filled in later. Our research is, we believe, relevant as co-researcher involvement in such a large European project as EUonQoL is a new development.

Emergency department admissions and economic costs burden related to ambulatory care sensitive conditions in older adults living in care homes.

OBJECTIVES: To assess the frequency of emergency department admissions (EDA) for ambulatory care sensitive conditions (ACSC) and non-ACSC among older adults living in care homes (CH), to describe and compare their demographic and clinical characteristics, the outcomes of the hospitalisation process and the associated costs. METHOD: This multicenter, retrospective and observational study evaluated 2444 EDAs of older adults ≥ 65 years old living in care homes in 5 emergency departments in Catalonia (Spain) by ACSC and non-ACSC, in 2017. Sociodemographic variables, prior functional and cognitive status, and information on diagnosis and hospitalisation were collected. Additionally, the costs related with the EDAs were calculated, as well as a sensitivity analysis using different assumptions of decreased admissions due to ACSC. RESULTS: A total of 2444 ED admissions were analysed. The patients' mean (SD) age was 85.9 (7.2) years. The frequency of ACSC-EDA and non-ACSC-EDA was 56.6% and 43.4%, respectively. Severe dependency and cognitive impairment were present in 56.6% and 78%, respectively, with no differences between the two groups. The three most frequent ACSC were falls/trauma (13.8%), chronic obstructive pulmonary disease/asthma (11.4%) and urinary tract infection (7.4%). The average cost per ACSC-EDA was є1,408.24. Assuming a 60% reduction of ACSC-EDA, the estimated cost savings would be є1.2 million. CONCLUSIONS: Emergency admissions for ACSC from care homes have a significant impact on both frequency and costs. Reducing these conditions through targeted interventions could redirect the avoided costs towards improving care support in residential settings.

Effectiveness of a Weight-Loss Intervention in Preventing Frailty and Functional Decline in Community-Dwelling Obese Older People. A Randomized Controlled Trial.

BACKGROUND: Obesity is a risk factor for frailty and muscle weakness, so weight loss in obese older adults may prevent frailty and functional decline. OBJECTIVE: To assess the safety and efficacy of a multimodal weight-loss intervention in improving functional performance and reducing frailty risk in obese older adults. DESIGN: Randomized controlled trial with 2 parallel arms. SETTING AND PARTICIPANTS: Community-dwelling obese adults aged 65-75 years with body mass index (BMI) 30-39 kg/m2. INTERVENTION: 6-month multimodal intervention based on diet and a physical activity program. CONTROL GROUP: Usual care. Main and secondary outcome measures: Frailty (Fried criteria) rate and functional performance at 6, 12, and 24 months of follow-up, respectively. Intermediate outcome measures: Weight loss, body composition changes, and metabolic and inflammatory biomarker changes. RESULTS: N=305. The study intervention increased gait speed at 12 and 24 months of follow-up, but had no significant effect on frailty prevention. It was effective in reducing weight, BMI, fat mass, interleukin 6, and insulin resistance and improving self-reported quality of life. CONCLUSIONS: The study intervention was not demonstrated to be effective in preventing frailty in obese people aged 65-75 years at 24 months of follow-up. However, it allowed weight loss and a reduction in inflammatory and insulin resistance markers, which could have a long-term effect on frailty that requires further research.

Structure of the SHOC2-MRAS-PP1C complex provides insights into RAF activation and Noonan syndrome.

SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions.

Use of healthcare REsources and associated COsts in controlled versus uncontrolled carcinoid SYndrome in patients with neuroendocrine tumours: the RECOSY study.

PURPOSE: To report healthcare resource use and associated costs in controlled versus uncontrolled carcinoid syndrome (CS) in patients with neuroendocrine tumours. METHODS: A cross-sectional, non-interventional multicentre study was conducted with retrospective data analysis. Resource use was compared between two patient groups: those with controlled CS (> 12 months with no uncontrolled CS episodes) and uncontrolled CS (< 12 months since last uncontrolled episode). Patients were matched for age, sex, and origin and grade of tumour. When no matching patients were available, data from deceased patients were used. Information on healthcare resource use came from review of medical records, patient history and physician reports. Working capacity was assessed using the Work Productivity and Activity Impairment General Health questionnaire. RESULTS: Twenty-six university hospitals in Spain participated, between July 2017 and April 2018. 137 patients were enrolled; 104 were analysed (2 groups of 52). Patients with uncontrolled CS had 10 times more emergency department (ED) visits (mean 1.0 vs 0.10 visits; P = 0.0167), were more likely to have a hospital admission (40.4% vs 19.2%; P = 0.0116) and had longer hospital stays (mean 7.87 vs 2.10 days; P = 0.0178) than those with controlled CS. This corresponded to higher annual hospitalisation costs (mean €5511.59 vs €1457.22; P = 0.028) and ED costs (€161.25 vs €14.85; P = 0.0236). The mean annual total healthcare costs were 60.0% higher in patients with uncontrolled than controlled CS (P = NS). CONCLUSION: This study quantifies higher health resource use, and higher hospitalisation and ED costs in patients with uncontrolled CS. Better control of CS may result 3in lower medical costs.

Spontaneous skin ulceration and defective T cell function in CD18 null mice.

A null mutation was prepared in the mouse for CD18, the beta2 subunit of leukocyte integrins. Homozygous CD18 null mice develop chronic dermatitis with extensive facial and submandibular erosions. The phenotype includes elevated neutrophil counts, increased immunoglobulin levels, lymphadenopathy, splenomegaly, and abundant plasma cells in skin, lymph nodes, gut, and kidney. Very few neutrophils were found in spontaneously occurring skin lesions or with an induced toxic dermatitis. Intravital microscopy in CD18 null mice revealed a lack of firm neutrophil attachment to venules in the cremaster muscle in response to N-formyl- methionyl-leucyl-phenylalanine. A severe defect in T cell proliferation was found in the CD18 null mice when T cell receptors were stimulated either by staphylococcal enterotoxin A or by major histocompatibility complex alloantigens demonstrating a greater role of CD11/CD18 integrins in T cell responses than previously documented. The null mice are useful for delineating the functions of CD18 in vivo.

Infectious susceptibility and severe deficiency of leukocyte rolling and recruitment in E-selectin and P-selectin double mutant mice.

During the initial phase of the inflammatory response, leukocytes marginate and roll along the endothelial surface, a process mediated largely by the selectins and their ligands. Mice with mutations in individual selectins show no spontaneous disease and have mild or negligible deficiencies of inflammatory responses. In contrast, we find that mice with null mutations in both endothelial selectins (P and E) develop a phenotype of leukocyte adhesion deficiency characterized by mucocutaneous infections, plasma cell proliferation, hypergammaglobulinemia, severe deficiencies of leukocyte rolling in cremaster venules with or without addition of TNF-alpha, and an absence of neutrophil emigration at 4 h in response to intraperitoneal Streptococcus pneumoniae peritonitis. These mice provide strong evidence for the functional importance of selectins in vivo.

Adsorption of beta-adrenergic agonists used in sport doping on metal nanoparticles: a detection study based on surface-enhanced Raman scattering.

The adsorption of beta(2)-adrenergic agonist (ßAA) drugs clenbuterol, salbutamol, and terbutaline on metal surfaces has been investigated in this work by means of surface-enhanced Raman scattering (SERS). To assist in this investigation, a previous vibrational (IR and normal Raman) characterization of these drugs was performed, supported by ab initio density functional theory calculations. The application of SERS was aimed to apply this highly sensitive technique, based on localized surface plasmon resonance, in the detection of ßAA at trace concentrations and as a possible alternative method which can be postulated in routine antidoping analysis. The adsorption of these drugs was studied in depth at different experimental conditions: on Au and Ag, at different pHs, and with varying adsorbate concentration. Moreover, plasmon resonance spectroscopy was employed to investigate the adsorption of these drugs on the metal nanoparticles as well as their aggregation. It was found that the adsorption of these molecules is more effective on gold nanoparticles and at acidic pH, based on the direct interaction of the aromatic or aliphatic moieties through ionic or coordination bonds with the metal. These drugs followed a Langmuir adsorption model from which the adsorption constant and the limit of detection can be determined.

Influence of oral anticoagulation on stroke severity and outcomes: A propensity score matching case-control study.

BACKGROUND: Oral anticoagulants (OAC) such as vitamin K antagonists (VKA) and direct-acting OACs (DOAC) remain the mainstay for prevention of cardioembolic stroke. The influence of previous OAC treatment on stroke severity and outcomes is not well stablished. We compared patients with incident cardioembolic strokes according to pre-stroke treatment. METHODS: Retrospective observational study of patients with cardioembolic stroke. Demographic data, vascular risk factors, pre-stroke treatments, reperfusion therapies and outcomes were analyzed. Propensity score matching of baseline characteristics was used to compare case-control samples across different treatment groups: adequate OAC vs no OAC; inadequate VKA vs no OAC; adequate VKA vs inadequate VKA; adequate VKA vs DOAC. RESULTS: 462 patients (76 ±â€¯11.6 years) included. 255 (55%) had a known major cardioembolic source, but only 151 (59%) of them were under OAC upon admission (127 VKA, 24 DOAC). Four patients received VKA for other reasons. Of those taking VKA, 91 (69%) had an inadequate anticoagulation. After propensity score matching, we found no significant differences in stroke severity across the different groups. Patients receiving DOAC had lower mortality at 3 months (8% vs 33%, p = .033) and higher successful recanalization rates after thrombectomy (100% vs 25%, p = .033) compared with adequate VKA anticoagulation. CONCLUSIONS: DOAC treatment significantly reduced mortality at three months compared with adequate VKA anticoagulation. Further studies are needed to confirm its influence on endovascular thrombectomy outcomes.

SEOM clinical guideline on hereditary colorectal cancer (2019).

In the last 2 decades, clinical genetics on hereditary colorectal syndromes has shifted from just a molecular characterization of the different syndromes to the estimation of the individual risk of cancer and appropriate risk reduction strategies. In the last years, new specific therapies for some subgroups of patients have emerged as very effective alternatives. At the same time, germline multigene panel testing by next-generation sequencing (NGS) technology has become the new gold standard for molecular genetics.

Total Body Water and Intracellular Water Relationships with Muscle Strength, Frailty and Functional Performance in an Elderly Population.

BACKGROUND: As a person ages, total body water (TBW), intracellular water (ICW), muscle mass and muscle strength tend to decline. The decline in ICW may reflect losses in the number of muscle cells but may also be responsible for less hydrated muscle cells. AIM: To assess whether TBW and ICW are associated with muscle strength, functional performance and frailty in an aged population, independently of muscle mass. METHODOLOGY: Design: An observational cross-sectional study of community-dwelling individuals aged 75 years and older. TBW, ICW, fat mass, lean mass and muscle mass were assessed by bioelectrical impedance analysis, frailty status was measured according to Fried criteria, handgrip strength was measured using the hand-held JAMAR dynamometer, and functional performance was measured according to the Barthel index and gait speed. RESULTS: A total of 324 subjects were recruited (mean age 80.1 years, 47.5% women). TBW and ICW were closely correlated with muscle mass in both sexes. ICW was also associated with Barthel score, gait speed and frailty in both sexes and with handgrip in men. Considerable variability in ICW was observed for the same muscle mass. Multivariate analysis showed a positive effect of ICW on handgrip, functional performance and gait speed and a protective effect of ICW on frailty, independently of age, sex, body mass index and number of comorbidities. CONCLUSIONS: In elderly individuals with similar muscle mass, those with higher ICW had a better functional performance and a lower frailty risk, suggesting a protective effect of cell hydration, independently of muscle mass.