Validation of the BOADICEA model in a prospective cohort of BRCA1/2 pathogenic variant carriers.

BACKGROUND: No validation has been conducted for the BOADICEA multifactorial breast cancer risk prediction model specifically in BRCA1/2 pathogenic variant (PV) carriers to date. Here, we evaluated the performance of BOADICEA in predicting 5-year breast cancer risks in a prospective cohort of BRCA1/2 PV carriers ascertained through clinical genetic centres. METHODS: We evaluated the model calibration and discriminatory ability in the prospective TRANsIBCCS cohort study comprising 1614 BRCA1 and 1365 BRCA2 PV carriers (209 incident cases). Study participants had lifestyle, reproductive, hormonal, anthropometric risk factor information, a polygenic risk score based on 313 SNPs and family history information. RESULTS: The full multifactorial model considering family history together with all other risk factors was well calibrated overall (E/O=1.07, 95% CI: 0.92 to 1.24) and in quintiles of predicted risk. Discrimination was maximised when all risk factors were considered (Harrell's C-index=0.70, 95% CI: 0.67 to 0.74; area under the curve=0.79, 95% CI: 0.76 to 0.82). The model performance was similar when evaluated separately in BRCA1 or BRCA2 PV carriers. The full model identified 5.8%, 12.9% and 24.0% of BRCA1/2 PV carriers with 5-year breast cancer risks of <1.65%, <3% and <5%, respectively, risk thresholds commonly used for different management and risk-reduction options. CONCLUSION: BOADICEA may be used to aid personalised cancer risk management and decision-making for BRCA1 and BRCA2 PV carriers. It is implemented in the free-access CanRisk tool (https://www.canrisk.org/).

The expanded spectrum of human disease associated with GREB1L likely includes complex congenital heart disease.

OBJECTIVE: GREB1L has been linked prenatally to Potter's sequence, as well as less severe anomalies of the kidney, uterus, inner ear, and heart. The full phenotypic spectrum is unknown. The purpose of this study was to characterize known and novel pre- and postnatal phenotypes associated with GREB1L. METHODS: We solicited cases from the Fetal Sequencing Consortium, screened a population-based genomic database, and conducted a comprehensive literature search to identify disease cases associated with GREB1L. We present a detailed phenotypic spectrum and molecular changes. RESULTS: One hundred twenty-seven individuals with 51 unique pathogenic or likely pathogenic GREB1L variants were identified. 24 (47%) variants were associated with isolated kidney anomalies, 19 (37%) with anomalies of multiple systems, including one case of hypoplastic left heart syndrome, five (10%) with isolated sensorineural hearing loss, two (4%) with isolated uterine agenesis; and one (2%) with isolated tetralogy of Fallot. CONCLUSION: GREB1L may cause complex congenital heart disease (CHD) in humans. Clinicians should consider GREB1L testing in the setting of CHD, and cardiac screening in the setting of GREB1L variants.

ATXN3 deubiquitinates YAP1 to promote tumor growth.

The ubiquitin-specific peptidase Ataxin-3 (ATXN3) has emerged as a potential oncogene in a variety of human cancers. However, the molecular mechanisms underlying how ATXN3 achieves its tumorigenic functions remain largely undefined. Herein, we report that targeted deletion of the ATXN3 gene in cancer cells by the CRISPR-Cas9 system resulted in decreased protein expression of Yes-associated protein 1 (YAP1) without altering its mRNA transcription. Interestingly, genetic ATXN3 suppression selectively inhibited the expression levels of YAP1 target genes including the connective tissue growth factor (Ctgf) and cysteine-rich angiogenic inducer 61 (Cyr61), both of which have important functions in cell adhesion, migration, proliferation and angiogenesis. Consequently, ATXN3 suppression resulted in reduced cancer cell growth and migration, which can also be largely rescued by YAP1 reconstitution. At the molecular level, ATNX3 interacts with the WW domains of YAP1 to protect YAP1 from ubiquitination-mediated degradation. Immunohistology analysis revealed a strong positive correlation between ATXN3 and YAP1 protein expression in human breast and pancreatic cancers. Collectively, our study defines ATXN3 as a previously unknown YAP1 deubiquitinase in tumorigenesis and provides a rationale for ATXN3 targeting in antitumor chemotherapy.

Predicting line of therapy transition via similar patient augmentation.

Analyzing large EHR databases to predict cancer progression and treatments has become a hot trend in recent years. An increasing number of modern deep learning models have been proposed to find the milestones of essential patient medical journey characteristics to predict their disease status and give healthcare professionals valuable insights. However, most of the existing methods are lack of consideration for the inter-relationship among different patients. We believe that more valuable information can be extracted, especially when patients with similar disease statuses visit the same doctors. Towards this end, a similar patient augmentation-based approach named SimPA is proposed to enhance the learning of patient representations and further predict lines of therapy transition. Our experiment results on a real-world multiple myeloma dataset show that our proposed approach outperforms state-of-the-art baseline approaches in terms of standard evaluation metrics for classification tasks.

The chromosome 21 kinase DYRK1A: emerging roles in cancer biology and potential as a therapeutic target.

Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) is a serine/threonine kinase that belongs to the DYRK family of proteins, a subgroup of the evolutionarily conserved CMGC protein kinase superfamily. Due to its localization on chromosome 21, the biological significance of DYRK1A was initially characterized in the pathogenesis of Down syndrome (DS) and related neurodegenerative diseases. However, increasing evidence has demonstrated a prominent role in cancer through its ability to regulate biologic processes including cell cycle progression, DNA damage repair, transcription, ubiquitination, tyrosine kinase activity, and cancer stem cell maintenance. DYRK1A has been identified as both an oncogene and tumor suppressor in different models, underscoring the importance of cellular context in its function. Here, we review mechanistic contributions of DYRK1A to cancer biology and its role as a potential therapeutic target.

Assessing the predictive capabilities of design heuristics and coarse-grain simulation toward understanding and optimizing site-specific covalent immobilization of ß-lactamase.

For industrial applications, covalent immobilization of enzymes provides minimum leakage, recoverability, reusability, and high stability. Yet, the suitability of a given site on the enzyme for immobilization remains a trial-and-error procedure. Here, we investigate the reliability of design heuristics and a coarse-grain molecular simulation in predicting the optimum sites for covalent immobilization of TEM-1 ß-lactamase. We utilized Escherichia coli-lysate-based cell-free protein synthesis (CFPS) to produce variants containing a site-specific incorporated unnatural amino acid with a unique moiety to facilitate site directed covalent immobilization. To constrain the number of potential immobilization sites, we investigated the predictive capability of several design heuristics. The suitability of immobilization sites was determined by analyzing expression yields, specific activity, immobilization efficiency, and stability of variants. These experimental findings are compared with coarse-grain simulation of TEM-1 domain stability and thermal stability and analyzed for a priori predictive capabilities. This work demonstrates that the design heuristics successfully identify a subset of locations for experimental validation. Specifically, the nucleotide following amber stop codon and domain stability correlate well with the expression yield and specific activity of the variants, respectively. Our approach highlights the advantages of combining coarse-grain simulation and high-throughput experimentation using CFPS to identify optimal enzyme immobilization sites.

Assessing site-specific PEGylation of TEM-1 ß-lactamase with cell-free protein synthesis and coarse-grained simulation.

PEGylation is a broadly used strategy to enhance the pharmacokinetic properties of therapeutic proteins. It is well established that the location and extent of PEGylation have a significant impact on protein properties. However, conventional PEGylation techniques have limited control over PEGylation sites. Emerging site-specific PEGylation technology provides control of PEG placement by conjugating PEG polymers via click chemistry reaction to genetically encoded non-canonical amino acids. Unfortunately, a method to rapidly determine the optimal PEGylation location has yet to be established. Here we seek to address this challenge. In this work, coarse-grained molecular dynamic simulations are paired with high-throughput experimental screening utilizing cell-free protein synthesis to investigate the effect of site-specific PEGylation on the two-state folder protein TEM-1 ß-lactamase. Specifically, the conjugation efficiency, thermal stability, and enzymatic activity are studied for the enzyme PEGylated at several different locations. The results of this analysis confirm that the physical properties of the PEGylated protein vary considerably with PEGylation site and that traditional design recommendations are insufficient to predict favorable PEGylation sites. In this study, the best predictor of the most favorable conjugation site is coarse-grained simulation. Thus, we propose a dual combinatorial screening approach in which coarse-grained molecular simulation informs site selection for high-throughput experimental verification.

Towards detection of SARS-CoV-2 RNA in human saliva: A paper-based cell-free toehold switch biosensor with a visual bioluminescent output.

The COVID-19 pandemic has illustrated the global demand for rapid, low-cost, widely distributable and point-of-care nucleic acid diagnostic technologies. Such technologies could help disrupt transmission, sustain economies and preserve health and lives during widespread infection. In contrast, conventional nucleic acid diagnostic procedures require trained personnel, complex laboratories, expensive equipment, and protracted processing times. In this work, lyophilized cell-free protein synthesis (CFPS) and toehold switch riboregulators are employed to develop a promising paper-based nucleic acid diagnostic platform activated simply by the addition of saliva. First, to facilitate distribution and deployment, an economical paper support matrix is identified and a mass-producible test cassette designed with integral saliva sample receptacles. Next, CFPS is optimized in the presence of saliva using murine RNase inhibitor. Finally, original toehold switch riboregulators are engineered to express the bioluminescent reporter NanoLuc in response to SARS-CoV-2 RNA sequences present in saliva samples. The biosensor generates a visible signal in as few as seven minutes following administration of 15 µL saliva enriched with high concentrations of SARS-CoV-2 RNA sequences. The estimated cost of this test is less than 0.50 USD, which could make this platform readily accessible to both the developed and developing world. While additional research is needed to decrease the limit of detection, this work represents important progress toward developing a diagnostic technology that is rapid, low-cost, distributable and deployable at the point-of-care by a layperson.

Mechanistic discoveries and simulation-guided assay optimization of portable hormone biosensors with cell-free protein synthesis.

Nuclear receptors (NRs) influence nearly every system of the body and our lives depend on correct NR signaling. Thus, a key environmental and pharmaceutical quest is to identify and detect chemicals which interact with nuclear hormone receptors, including endocrine disrupting chemicals (EDCs), therapeutic receptor modulators, and natural hormones. Previously reported biosensors of nuclear hormone receptor ligands facilitated rapid detection of NR ligands using cell-free protein synthesis (CFPS). In this work, the advantages of CFPS are further leveraged and combined with kinetic analysis, autoradiography, and western blot to elucidate the molecular mechanism of this biosensor. Additionally, mathematical simulations of enzyme kinetics are used to optimize the biosensor assay, ultimately lengthening its readable window by five-fold and improving sensor signal strength by two-fold. This approach enabled the creation of an on-demand thyroid hormone biosensor with an observable color-change readout. This mathematical and experimental approach provides insight for engineering rapid and field-deployable CFPS biosensors and promises to improve methods for detecting natural hormones, therapeutic receptor modulators, and EDCs.

Subcuticular Barbed Suture and Skin Glue Wound Closure Decreases Reoperation and Length of Stay in Geriatric Hip Fractures When Compared With Staples.

INTRODUCTION: Patients with geriatric hip fracture are notoriously frail and at risk for complications. Persistent postoperative wound drainage can lead to prolonged hospital stay, increased risk for infection, and need for revision surgery. The purpose of this study was to determine the effect of wound closure technique, barbed monofilament subcuticular suture and skin glue versus staples on rates of intervention for wound drainage and length of hospital stay after geriatric hip fracture fixation. METHODS: A retrospective review of isolated hip fractures in patients older than 60 years at a single institution over a 3-year period was done. Hip fractures included femoral neck, intertrochanteric, and subtrochanteric femur fractures treated with internal fixation or arthroplasty. Skin closure technique, at the discretion of the operating surgeon, included either barbed subcuticular monofilament suture and skin glue or staples. Charts and radiographs were reviewed to determine patient characteristics, Charlson Comorbidity Index, type of wound closure, length of stay, and interventions for persistent wound drainage. RESULTS: There were 175 patients in the barbed suture and skin glue group and 211 patients in the staples group. The barbed suture group had an average postsurgical length of stay of 5.0 days which was significantly lower than the staples group (7.0 days, P < 0.00001). In the staples group, 17 patients (8%) required incisional negative pressure wound therapy due to wound drainage with five patients (2.4%) returning to the operating room secondary to persistent wound drainage. No patients were observed in the barbed suture group that required intervention for wound drainage. DISCUSSION: Barbed suture and skin glue closure is associated with markedly shorter hospital stay and fewer interventions for wound drainage when compared with staples after surgical treatment of geriatric hip fractures.

Treatment initiation prediction by EHR mapped PPD tensor based convolutional neural networks boosting algorithm.

Electronic health records contain patient's information that can be used for health analytics tasks such as disease detection, disease progression prediction, patient profiling, etc. Traditional machine learning or deep learning methods treat EHR entities as individual features, and no relationships between them are taken into consideration. We propose to evaluate the relationships between EHR features and map them into Procedures, Prescriptions, and Diagnoses (PPD) tensor data, which can be formatted as images. The mapped images are then fed into deep convolutional networks for local pattern and feature learning. We add this relationship-learning part as a boosting module on a commonly used classical machine learning model. Experiments were performed on a Chronic Lymphocytic Leukemia dataset for treatment initiation prediction. Experimental results show that the proposed approach has better real world modeling performance than the baseline models in terms of prediction precision.

Leveraging graph-based hierarchical medical entity embedding for healthcare applications.

Automatic representation learning of key entities in electronic health record (EHR) data is a critical step for healthcare data mining that turns heterogeneous medical records into structured and actionable information. Here we propose ME2Vec, an algorithmic framework for learning continuous low-dimensional embedding vectors of the most common entities in EHR: medical services, doctors, and patients. ME2Vec features a hierarchical structure that encapsulates different node embedding schemes to cater for the unique characteristic of each medical entity. To embed medical services, we employ a biased-random-walk-based node embedding that leverages the irregular time intervals of medical services in EHR to embody their relative importance. To embed doctors and patients, we adhere to the principle "it's what you do that defines you" and derive their embeddings based on their interactions with other types of entities through graph neural network and proximity-preserving network embedding, respectively. Using real-world clinical data, we demonstrate the efficacy of ME2Vec over competitive baselines on diagnosis prediction, readmission prediction, as well as recommending doctors to patients based on their medical conditions. In addition, medical service embeddings pretrained using ME2Vec can substantially improve the performance of sequential models in predicting patients clinical outcomes. Overall, ME2Vec can serve as a general-purpose representation learning algorithm for EHR data and benefit various downstream tasks in terms of both performance and interpretability.

Coarse-grained simulation of PEGylated and tethered protein devices at all experimentally accessible surface residues on ß-lactamase for stability analysis and comparison.

PEGylated and surface-tethered proteins are used in a variety of biotechnological applications, but traditional methods offer little control over the placement of the functionalization sites on the protein. Fortunately, recent experimental methods functionalize the protein at any location on the amino acid sequence, so the question becomes one of selecting the site that will result in the best protein function. This work shows how molecular simulation can be used to screen potential attachment sites for surface tethering or PEGylation. Previous simulation work has shown promise in this regard for a model protein, but these studies are limited to screening only a few of the surface-accessible sites or only considered surface tethering or PEGylation separately rather than their combined effects. This work is done to overcome these limitations by screening all surface-accessible functionalization sites on a protein of industrial and therapeutic importance (TEM-1) and to evaluate the effects of tethering and PEGylation simultaneously in an effort to create a more accurate screen. The results show that functionalization site effectiveness appears to be a function of super-secondary and tertiary structures rather than the primary structure, as is often currently assumed. Moreover, sites in the middle of secondary structure elements, and not only those in loops regions, are shown to be good options for functionalization-a fact not appreciated in current practice. Taken as a whole, the results show how rigorous molecular simulation can be done to identify candidate amino acids for functionalization on a protein to facilitate the rational design of protein devices.

Mortality risk of loneliness in the oldest old over a 10-year follow-up.

Objective: To investigate the impact of loneliness on all-cause mortality in the oldest old population over a 10-year follow-up.Method: Participants were from the third wave of the Cambridge City over-75s Cohort (CC75C) study, a population-based longitudinal study of older people aged 75 or over. Loneliness was measured two further times. At each wave, participants were asked how often they felt lonely and the answers were divided into three levels: not lonely, slightly lonely and lonely. The relationship between loneliness and all-cause mortality was examined using Cox regression with loneliness as a time-varying predictor. The association was adjusted for socio-demographic factors, number of chronic diseases, functional ability and depression.Results: Seven hundred thirteen participants were seen at wave 3 (out of 2166 at baseline), of whom 665 had data on loneliness. The prevalence of feeling slightly lonely and lonely was 16% and 25%, respectively. Vital status was followed for a further 10 years. A total of 562 participants died during the follow-up. After adjusting for age, sex and other socio-demographic factors, loneliness was associated with a 20% increased risk of mortality (HR: 1.2, 95% CI: 1.0-1.6). The association was disappeared after further adjusting for health conditions and depression (HR: 1.0, 95% CI: 0.8-1.4). Individuals who reported being slightly lonely were not at risk of mortality.Conclusions: The association between loneliness and mortality was fully explained by health conditions, suggesting that in the very old age, health problem is the proximal risk factor for mortality.

Thermostable lyoprotectant-enhanced cell-free protein synthesis for on-demand endotoxin-free therapeutic production.

Cell-free protein synthesis has emerged as a promising platform for the production of therapeutic proteins due to its inherently open reaction environment, flexible reaction conditions and rapid protein synthesis capabilities. In recent years, lyophilized cell-free systems have widened the application space of cell-free technology by improving reagent stability outside of cold-chain storage. Current embodiments of the system, however, demonstrate poor stability at elevated temperatures. Lyoprotectants have long been recognized for the ability to preserve the activity of biological molecules during drying processes, but the application of this technology to lyophilized cell-free systems has been limited and has failed to address the negative effects that such lyoprotectants may have on cell-free systems. Here, several lyoprotected, lyophilized cell-free protein synthesis systems are demonstrated using antiplasticized sugar glasses as lyoprotectants, showing significant improvement over standard lyophilized systems or trehalose-preserved systems. Furthermore, we demonstrate for the first time, preservation and therapeutic expression, specifically of FDA-approved crisantaspase, from a truly single-pot lyophilized, endotoxin-free, cell-free protein synthesis system, exemplifying the potential for on-site therapeutic synthesis.

Is loneliness associated with increased health and social care utilisation in the oldest old? Findings from a population-based longitudinal study.

OBJECTIVES: The present study aimed to examine the impact of loneliness on health and social care service use in the oldest old over a 7-year follow-up. DESIGN: Prospective study. SETTING: UK population-based cohort. PARTICIPANTS: 713 people aged 80 years or older were interviewed at wave 3 of the Cambridge City over-75s Cohort Study. Of these, 665 provided data on loneliness. During 7 years' follow-up, 480 participants left the study, of which 389 due to death. 162 still in the study answered the loneliness question. MAIN OUTCOME MEASURE: Use of health and social care services, assessed at each wave from wave 3 to wave 5. RESULTS: At wave 3, of 665 participants who had data on loneliness, about 60% did not feel lonely, 16% felt slightly lonely and 25% felt lonely. Being slightly lonely at wave 3 was associated with a shorter time since last seeing a general practitioner (ß=-0.5, 95% CI: -0.8 to -0.2); when examining the association between time-varying loneliness and health and social care usage, being lonely was associated with three times greater likelihood of having contact with community nurses and using meals on wheels services (community nurse contact: incidence rate ratio (IRR)=3.4, 95% CI: 1.4 to 8.7; meals on wheels service use: IRR=2.5, 95% CI: 1.1 to 5.6). No associations between loneliness and other health and social care services use were found. CONCLUSION: Loneliness was a significant risk factor for certain types of health and social care utilisations, independently of participants' health conditions, in the oldest old. Study findings have several implications, including the need for awareness-raising and prevention of loneliness to be priorities for public health policy and practice.

The Black Cloud Phenomenon in Hand Surgery.

Background: The term black cloud for a surgeon is generally used to describe someone who is unusually busy compared with his or her counterparts, and it is a superstition that tends to pervade the medical world. The purpose of this study is to investigate whether black clouds exist in hand surgery. Methods: We examined one academic year's worth of hand surgery-specific call at a level I trauma center and tabulated the number of hand-related patient transfers and add-on cases per surgeon. Each surgeon was given a black cloud rating by the fellows who were in training that year. Correlations were made between the black cloud rating and the surgeons' call volume. Results: There were 12 surgeons who shared 365 days of hand call, and 5 of them are hand surgery fellowship trained. Those 5 surgeons tended to be busier on their call days, with more cases added on overnight and the next day, and also had worse black cloud ratings than the 7 non-hand fellowship trained surgeons. Conclusions: In regard to hand surgery, while true emergencies occur and require emergent intervention, how busy hand surgeons may be during call may be influenced by a variety of factors not related to their patients' problems but rather their daily schedules, their hospitals' ability to facilitate add-on cases, and their rapport with their fellow surgeons to share case loads.

Streamlining the preparation of "endotoxin-free" ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase.

An "endotoxin-free" E. coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand. However, preparation of the most complex CFPS reagent - the cell extract - remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3) strain. Here we report a streamlined procedure for preparing E. coli cell extract from ClearColi™ using auto-induction media. In this work, the term auto-induction describes cell culture media which eliminates the need for manual induction of protein expression. Culturing Clearcoli™ cells in autoinduction media significantly reduces the hands-on time required during extract preparation, and the resulting "endotoxin-free" cell extract maintained the same cell-free protein synthesis capability as extract produced with traditional induction as demonstrated by the high-yield expression of crisantaspase, an FDA approved leukemia therapeutic. It is anticipated that this work will lower the barrier for researchers to enter the field and use this technology as the method to produce endotoxin-free E. coli-based extract for CFPS.

The role of biologic in foot and ankle trauma-a review of the literature.

PURPOSE OF REVIEW: The use of biologics in orthopedics is becoming increasingly popular as an adjuvant in healing musculoskeletal injuries. Though many biologics involved in the management of foot and ankle injuries are used based on physician preference, reports of improved outcomes when combined with standard operative treatment has led to further clinical interest especially in foot and ankle trauma. RECENT FINDINGS: The most recent studies have shown benefits for biologic use in patients predisposed to poor bone and soft tissue healing. Biologics have shown benefit in treating soft tissue injuries such as Achilles ruptures as well as the complications of trauma such as non-unions and osteoarthritis. Biologics have shown some benefit in improving functional and pain scores, as well as reducing time to heal in foot and ankle traumatic injuries, with particular success shown with patients that have risk factors for poor healing. As the use of biologics continues to increase, there is a need for high-level studies to confirm early findings of lower level reports.

Accuracy of death certification of dementia in population-based samples of older people: analysis over time.

BACKGROUND: death certification data are routinely collected in most developed countries. Coded causes of death are a readily accessible source and have the potential advantage of providing complete follow-up, but with limitations. OBJECTIVE: to investigate the reliability of using death certificates for surveillance of dementia, the time trend of recording dementia on death certificates and predictive factors of recording of dementia. SUBJECTS: individuals aged 65 and over in six areas across England and Wales were randomly selected for the Medical Research Council Cognitive Function and Ageing Study (CFAS) and CFAS II with mortality follow-up. METHODS: prevalence of dementia recorded on death certificates were calculated by year. Reporting of dementia on death certificates compared with the study diagnosis of dementia, with sensitivity, specificity and Cohen's κ were estimated. Multivariable logistic regression models explored the impact of potential factors on the reporting of dementia on the death certificate. RESULTS: the overall unadjusted prevalence of dementia on death certificates rose from 5.3% to 25.9% over the last 26 years. Dementia reported on death certificates was poor with sensitivity 21.0% in earlier cohort CFAS, but it had increased to 45.2% in CFAS II. Dementia was more likely to be recorded on death certificates in individuals with severe dementia, or those living in an institution, yet less likely reported if individuals died in hospital. CONCLUSION: recording dementia on death certificate has improved significantly in the England and Wales. However, such information is still an underestimate and should be used alongside epidemiological estimations.