A systematic analysis of regression models for protein engineering.

To optimize proteins for particular traits holds great promise for industrial and pharmaceutical purposes. Machine Learning is increasingly applied in this field to predict properties of proteins, thereby guiding the experimental optimization process. A natural question is: How much progress are we making with such predictions, and how important is the choice of regressor and representation? In this paper, we demonstrate that different assessment criteria for regressor performance can lead to dramatically different conclusions, depending on the choice of metric, and how one defines generalization. We highlight the fundamental issues of sample bias in typical regression scenarios and how this can lead to misleading conclusions about regressor performance. Finally, we make the case for the importance of calibrated uncertainty in this domain.

Hematocrit control and thrombotic risk in patients with polycythemia vera treated with ruxolitinib in clinical practice.

Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by unregulated red blood cell production resulting in elevated hemoglobin and/or hematocrit levels. Patients often have symptoms such as fatigue, pruritus, and painful splenomegaly, but are also at risk of thrombosis, both venous and arterial. Ruxolitinib, a selective Janus kinase inhibitor, is approved by the US Food and Drug Administration as second-line cytoreductive treatment after intolerance or inadequate response to hydroxyurea. Although ruxolitinib has been widely used in this setting, limited data exist in the literature on ruxolitinib treatment patterns and outcomes among patients with PV in routine clinical practice. We report a retrospective, observational, cohort study of patients treated for PV with ruxolitinib across three US centers (academic and regional practice) from December 2014-December 2019. The study included 69 patients, with a median follow-up duration of 3.7 years (95% CI, 2.9-4.4). Our data demonstrate very high rates of hematocrit control (88% of patients by three months and 89% by six months); few patients required dose adjustments or suspension. No arterial thromboses were observed; however, the follow-up duration does not allow for the generation of meaningful conclusions from this. Three patients had thrombotic events; one was in the setting of a second malignancy, one post-operative, and a third related to prolonged immobility. We also found that 28% of patients initiated ruxolitinib as a result of poorly controlled platelet counts, second only to hydroxyurea intolerance (46%) as a reason to start therapy. In clinical practice, ruxolitinib continues to be effective in controlling hematocrit levels after three and six months of treatment in patients and is associated with low thrombotic risk.

Characterization of recombinant phytase of Klebsiella sp. and the influence of novel 3-phytase on mineral solubility in broiler diets under an in vitro digestion assay.

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.

Confirming Silent Translocation through Nanopores with Simultaneous Single-Molecule Fluorescence and Single-Channel Electrical Recordings.

Most of what is known concerning the luminal passage of materials through nanopores arises from electrical measurements. Whether nanopores are biological, solid-state, synthetic, hybrid, glass-capillary-based, or protein ion channels in cells and tissues, characteristic signatures embedded in the flow of ionic current are foundational to understanding functional behavior. In contrast, this work describes passage through a nanopore that occurs without producing an electrical signature. We refer to the phenomenon as "silent translocation." By definition, silent translocations are invisible to the standard tools of electrophysiology and fundamentally require a simultaneous ancillary measurement technique for positive identification. As a result, this phenomenon has been largely unexplored in the literature. Here, we report on a derivative of Cyanine 5 (sCy5a) that passes through the α-hemolysin (αHL) nanopore silently. Simultaneously acquired single-molecule fluorescence and single-channel electrical recordings from bilayers formed over a closed microcavity demonstrate that translocation does indeed take place, albeit infrequently. We report observations of silent translocation as a function of time, dye concentration, and nanopore population in the bilayer. Lastly, measurement of the translocation rate as a function of applied potential permits estimation of an effective energy barrier for transport through the pore as well as the effective charge on the dye, all in the absence of an information-containing electrical signature.

An Electrochemical Approach to Directed Fluorination.

Electrosynthesis has made a revival in the field of organic chemistry and, in particular, radical-mediated reactions. Herein, we report a simple directed, electrochemical C-H fluorination method. Employing a dabconium mediator, commercially available Selectfluor, and RVC electrodes, we provide a range of steroid-based substrates with competent regioselective directing groups, including enones, ketones, and hydroxy groups, as well as never reported before lactams, imides, lactones, and esters.

Imaging of pediatric extremity soft tissue tumors: A COG Diagnostic Imaging Committee/SPR Oncology Committee White Paper.

Pediatric soft tissue tumors of the extremity include rhabdomyosarcoma and nonrhabdomyosarcoma neoplasms. This manuscript provides consensus-based imaging recommendations for imaging evaluation at diagnosis, during treatment, and following completion of therapy for patients with a soft tissue tumor of the extremity.

Physical properties and biological effects of ceramic materials emitting infrared radiation for pain, muscular activity, and musculoskeletal conditions.

BACKGROUND: Up to 33% of the general population worldwide suffer musculoskeletal conditions, with low back pain being the single leading cause of disability globally. Multimodal therapeutic options are available to relieve the pain associated with muscular disorders, including physical, complementary, and pharmacological therapies. However, existing interventions are not disease modifying and have several limitations. METHOD: Literature review. RESULTS: In this context, the use of nonthermal infrared light delivered via patches, fabrics, and garments containing infrared-emitting bioceramic minerals have been investigated. Positive effects on muscular cells, muscular recovery, and reduced inflammation and pain have been reported both in preclinical and clinical studies. There are several hypotheses on how infrared may contribute to musculoskeletal pain relief, however, the full mechanism of action remains unclear. This article provides an overview of the physical characteristics of infrared radiation and its biological effects, focusing on those that could potentially explain the mechanism of action responsible for the relief of musculoskeletal pain. CONCLUSIONS: Based on the current evidence, the following pathways have been considered: upregulation of endothelial nitric oxide synthase, increase in nitric oxide bioavailability, anti-inflammatory effects, and reduction in oxidative stress.

C-F Bonds as "Frozen" Nucleophiles: Unconsummated SN2 Reactions.

In this note, we present a series of rigid molecules that show close enforced interactions between Ar-F moieties and -CH2X groups in a "tetrel bond" configuration similar to a nascent SN2 attack. We explore the spectroscopic, crystallographic, and chemical reactivity consequences of these unusual interactions, including significant through-space spin-spin couplings, short C-F···CH2X distances, and differential SN1 and SN2 reaction pathways. We also reveal experimental evidence of carbon-based tetrel bonds influencing chemical reactivity in solution. Finally, density functional theory (DFT) calculations are employed throughout this study to confirm and illuminate our experimental data.

Paediatric Gastrointestinal, Hepatic and Pancreatic Inflammatory Myofibroblastic Tumours, A Single Centre Experience.

ABSTRACT: Inflammatory myofibroblastic tumours (IMTs) are rare soft tissue tumours. Reports of gastrointestinal tract, liver and pancreas tumours are limited. The objective of this study is to identify presenting features, contributing prognostic / etiological factors and any variability in outcomes in the context of different historical treatments. We retrospectively reviewed the records of seven children treated at our hospital between 2006 and 2019 and assessed the demographic, presentation, treatment, immunohistochemistry, and outcomes of their tumours. Age range at presentation was 4 months-15 years with a male predominance. Presentations were typically due to local mass effect or incidental discovery. Systemic symptoms were rare. Outcomes were good with six out of seven stable or in remission irrespective of treatment. Surgical resection where possible is the treatment of choice. Medical therapy had good outcomes with chemotherapy acting as first line treatment when required. The only negative prognostic factor identified was local spread at the time of presentation.

The Close Interaction of a C-F Bond with an Amide Carbonyl: Crystallographic and Spectroscopic Characterization.

The putative interaction of a C-F bond with an amide carbonyl has been an intriguing topic of interest in this century for reasons spanning basic physical organic chemistry to biochemistry. However, to date, there exist no examples of a close, well-defined interaction in which its unique aspects can be identified and exploited. Herein, we finally present an engineered system possessing an exceptionally tight C-F-amide interaction, allowing us to obtain spectroscopic, crystallographic, and kinetic details of a distinctive, biochemically relevant chemical system for the first time. In turn, we also explore Lewis acid coordination, C-F bond promotion of amide isomerization, enantiomerization, and ion protonation processes.

Obesity and revision surgery, mortality, and patient-reported outcomes after primary knee replacement surgery in the National Joint Registry: A UK cohort study.

BACKGROUND: One in 10 people in the United Kingdom will need a total knee replacement (TKR) during their lifetime. Access to this life-changing operation has recently been restricted based on body mass index (BMI) due to belief that high BMI may lead to poorer outcomes. We investigated the associations between BMI and revision surgery, mortality, and pain/function using what we believe to be the world's largest joint replacement registry. METHODS AND FINDINGS: We analysed 493,710 TKRs in the National Joint Registry (NJR) for England, Wales, Northern Ireland, and the Isle of Man from 2005 to 2016 to investigate 90-day mortality and 10-year cumulative revision. Hospital Episodes Statistics (HES) and Patient Reported Outcome Measures (PROMs) databases were linked to the NJR to investigate change in Oxford Knee Score (OKS) 6 months postoperatively. After adjustment for age, sex, American Society of Anaesthesiologists (ASA) grade, indication for operation, year of primary TKR, and fixation type, patients with high BMI were more likely to undergo revision surgery within 10 years compared to those with "normal" BMI (obese class II hazard ratio (HR) 1.21, 95% CI: 1.10, 1.32 (p < 0.001) and obese class III HR 1.13, 95% CI: 1.02, 1.26 (p = 0.026)). All BMI classes had revision estimates within the recognised 10-year benchmark of 5%. Overweight and obese class I patients had lower mortality than patients with "normal" BMI (HR 0.76, 95% CI: 0.65, 0.90 (p = 0.001) and HR 0.69, 95% CI: 0.58, 0.82 (p < 0.001)). All BMI categories saw absolute increases in OKS after 6 months (range 18-20 points). The relative improvement in OKS was lower in overweight and obese patients than those with "normal" BMI, but the difference was below the minimal detectable change (MDC; 4 points). The main limitations were missing BMI particularly in the early years of data collection and a potential selection bias effect of surgeons selecting the fitter patients with raised BMI for surgery. CONCLUSIONS: Given revision estimates in all BMI groups below the recognised threshold, no evidence of increased mortality, and difference in change in OKS below the MDC, this large national registry shows no evidence of poorer outcomes in patients with high BMI. This study does not support rationing of TKR based on increased BMI.

Utility of bedside leucocyte esterase testing to rule out septic arthritis.

INTRODUCTION: Suspected septic arthritis is a common presentation to EDs. The underlying diagnosis is often non-infective pathology. Differentiating between aetiologies is difficult. A bedside test with high negative predictive value (NPV) may allow safe discharge of patients, reduce the time in the ED, hospital admission and associated costs. This study aims to evaluate the NPV of bedside leucocyte esterase (LE) in the assessment of these patients. METHODS: A prospective multicentre observational study of ED adult patients referred to orthopaedics with suspected native joint septic arthritis between October 2015 and April 2016. At three hospital sites in the Bristol region, the results of the LE test exposed to aspirated synovial fluid were recorded along with Gram stain, culture, haematinics and length of stay. A positive LE test was considered 2+ or 3+ leucocytes based on the test strip colour. Data were analysed to establish sensitivity, specificity, NPV and positive predictive value (PPV) against the gold standard 48-hour culture. We determined the potential number of inpatient bed-days that might be avoided using this bedside test. RESULTS: Eighty patients underwent joint aspiration. Five cases had positive 48-hour culture. All (5/5) infected cases showed ≥2+ LE, sensitivity of 100% (95% CI 47.8% to 100%) while the Gram stain was positive in only one case (sensitivity 20%, 95% CI 0.51% to 71.6%). Twenty-three LE were read negative or 1+, all with negative 48-hour culture results, resulting in an NPV of 100% (95% CI 82.1% to 1.00%) for a negative LE test. Specificity of a positive LE test was 30.7% (95% CI 20.5% to 42.45%) with PPV of 8.77% (95% CI 7.64% to 10.1%). It was calculated that 57 orthopaedic bed-days could have potentially been saved by immediately discharging those with a negative LE test. CONCLUSIONS: LE point-of-care testing for suspected septic arthritis of native joints has a high NPV. Implementation of LE may facilitate more rapid discharge of patients with negative results. This test has the potential to reduce diagnostic uncertainty and costs to the healthcare system.

Factors associated with implant survival following total hip replacement surgery: A registry study of data from the National Joint Registry of England, Wales, Northern Ireland and the Isle of Man.

BACKGROUND: Nearly 100,000 people underwent total hip replacement (THR) in the United Kingdom in 2018, and most can expect it to last at least 25 years. However, some THRs fail and require revision surgery, which results in worse outcomes for the patient and is costly to the health service. Variation in the survival of THR implants has been observed between units and reducing this unwarranted variation is one focus of the "Getting it Right First Time" (GIRFT) program in the UK. We aimed to investigate whether the statistically improved implant survival of THRs in a high-performing unit is associated with the implants used or other factors at that unit, such as surgical skill. METHODS AND FINDINGS: We analyzed a national, mandatory, prospective, cohort study (National Joint Registry for England, Wales, Northern Ireland and the Isle of Man [NJR]) of all THRs performed in England and Wales. We included the 664,761 patients with records in the NJR who have received a stemmed primary THR between 1 April 2003 and 31 December 2017 in one of 461 hospitals, with osteoarthritis as the only indication. The exposure was the unit (hospital) in which the THR was implanted. We compared survival of THRs implanted in the "exemplar" unit with THRs implanted anywhere else in the registry. The outcome was revision surgery of any part of the THR construct for any reason. Net failure was calculated using Kaplan-Meier estimates, and adjusted analyses employed flexible parametric survival analysis. The mean age of patients contributing to our analyses was 69.9 years (SD 10.1), and 61.1% were female. Crude analyses including all THRs demonstrated better implant survival at the exemplar unit with an all-cause construct failure of 1.7% (95% CI 1.3-2.3) compared with 2.9% (95% CI 2.8-3.0) in the rest of the country after 13.9 years (log-rank test P < 0.001). The same was seen in analyses adjusted for age, sex, and American Society of Anesthesiology (ASA) score (difference in restricted mean survival time 0.12 years [95% CI 0.07-0.16; P < 0.001]). Adjusted analyses restricted to the same implants as the exemplar unit show no demonstrable difference in restricted mean survival time between groups after 13.9 years (P = 0.34). A limitation is that this study is observational and conclusions regarding causality cannot be inferred. Our outcome is revision surgery, and although important, we recognize it is not the only marker of success of a THR. CONCLUSIONS: Our results suggest that the "better than expected" implant survival results of this exemplar center are associated with implant choice. The survival results may be replicated by adopting key treatment decisions, such as implant selection. These decisions are easier to replicate than technical skills or system factors.

The association between cement type and the subsequent risk of revision surgery in primary total hip replacement.

Background and purpose - To further improve the success of joint replacement surgery, attention needs to be paid to variations associated with improved or worsened outcomes. We investigated the association between the type of bone cement used and the risk of revision surgery after primary total hip replacement. Methods - We conducted a prospective study of data from the National Joint Registry for England and Wales between April 1, 2003 and December 31, 2013. 199,205 primary total hip replacements performed for osteoarthritis where bone cement was used were included. A multilevel over-dispersed piecewise Poisson model was used to estimate differences in the rate of revision by bone cement type adjusted for implant type, head size, age, sex, ASA grade, and surgical approach. Results - The rate of revision was higher in DePuy CMW3 medium viscosity with gentamicin (IRR 2.0, 95% CI 1.5-2.7) and DePuy SmartSet high viscosity plain (IRR 2.7, 95% CI 1.1-5.5), and lower in DePuy CMW1 high viscosity plain (IRR 0.44, 95% CI 0.19-0.89) bone cements compared with Heraeus Palacos high viscosity with gentamicin. Revision rates were similar between plain and antibiotic-loaded bone cement. Interpretation - The majority of bone cements performed similarly well, excluding DePuy SmartSet high viscosity and CMW3 high viscosity with gentamicin, which both had higher revision rates. We found no clear differences by viscosity or antibiotic content.

Low serum magnesium levels are associated with increased risk of fractures: a long-term prospective cohort study.

Magnesium, which is an essential trace element that plays a key role in several cellular processes, is a major component of bone; however, its relationship with risk of major bone fractures is uncertain. We aimed to investigate the association of baseline serum magnesium concentrations with risk of incident fractures. We analyzed data on 2245 men aged 42-61 years in the Kuopio Ischemic Heart Disease prospective cohort study, with the assessment of serum magnesium measurements and dietary intakes made at baseline. Hazard ratios [95% confidence intervals (CI)] for incident total (femoral, humeral, and forearm) and femoral fractures were assessed. During a median follow-up of 25.6 years, 123 total fractures were recorded. Serum magnesium was non-linearly associated with risk of total fractures. In age-adjusted Cox regression analysis, the hazard ratio (HR) (95% CIs) for total fractures in a comparison of the bottom quartile versus top quartile of magnesium concentrations was 2.10 (1.30-3.41), which persisted on adjustment for several established risk factors 1.99 (1.23-3.24). The association remained consistent on further adjustment for renal function, socioeconomic status, total energy intake, and several trace elements 1.80 (1.10-2.94). The corresponding adjusted HRs for femoral fractures were 2.56 (1.38-4.76), 2.43 (1.30-4.53) and 2.13 (1.13-3.99) respectively. There was no evidence of an association of dietary magnesium intake with risk of any fractures. In middle-aged Caucasian men, low serum magnesium is strongly and independently associated with an increased risk of fractures. Further research is needed to assess the potential relevance of serum magnesium in the prevention of fractures.

Algorithm to Diagnose Delayed and Late PJI: Role of Joint Aspiration.

Total Joint Arthroplasty (TJA) continues to gain acceptance as the standard of care for the treatment of severe degenerative joint disease, and is considered one of the most successful surgical interventions in the history of medicine. A devastating complication after TJA is infection. Periprosthetic joint infection (PJI), represents one of the major causes of failure and remains a significant challenge facing orthopaedics today. PJI usually requires additional surgery including revision of the implants, fusion or amputations causing tremendous patient suffering but also a heavy health economics burden. PJI is at the origin of around 20-25 % of total knee arthroplasty (Bozic et al. 2010; de Gorter et al. 2015; Sundberg et al. 2015) and 12-15 % of total hip arthroplasty (Bozic et al. 2009; Garellick et al. 2014; de Gorter et al. 2015) failures.

Lack of uniformity in the investigation and management of suspected ß-lactam allergy in children.

BACKGROUND: Beta-lactam allergy is commonly suspected in childhood with health implications for the individual and wider public. Diagnostic modalities include skin prick tests (SPT), specific immunoglobulin-E (sp-IgE) tests, intradermal tests (IDT) and drug provocation challenges (DPC). The aim of this research was to establish whether variation exists around the world in the investigation and management of beta-lactam allergy. METHODS: Anonymized electronic questionnaire surveys were distributed over 3 months through International Allergy Societies for completion by clinicians who investigate drug allergy in children. RESULTS: Eighty-one clinicians, practising in 16 countries, completed the questionnaire. There is variability in the selection of diagnostic tests used by clinicians around the world and poor agreement on positive cut-off values (sp-IgE, SPT and IDT) and practical techniques used to measure SPT or IDT wheal diameters. DPC were considered the gold standard investigation with 94% of respondents undertaking DPC over the last 12 months; 64% of respondents considered DPC extremely useful for both exclusion and confirmation of beta-lactam allergy. However, there is a lack of consensus on when and how DPC should be performed. Overall, DPC are safe - only 3% of our respondents had patients who required intramuscular adrenaline and none had patients requiring admission to intensive care. CONCLUSIONS: There is lack of consistency amongst clinicians in different countries in the diagnosis and management of suspected beta-lactam allergy. The development of a standardized approach is a priority.