Systematic molecular evolution enables robust biomolecule discovery.

Evolution occurs when selective pressures from the environment shape inherited variation over time. Within the laboratory, evolution is commonly used to engineer proteins and RNA, but experimental constraints have limited the ability to reproducibly and reliably explore factors such as population diversity, the timing of environmental changes and chance on outcomes. We developed a robotic system termed phage- and robotics-assisted near-continuous evolution (PRANCE) to comprehensively explore biomolecular evolution by performing phage-assisted continuous evolution in high-throughput. PRANCE implements an automated feedback control system that adjusts the stringency of selection in response to real-time measurements of each molecular activity. In evolving three distinct types of biomolecule, we find that evolution is reproducibly altered by both random chance and the historical pattern of environmental changes. This work improves the reliability of protein engineering and enables the systematic analysis of the historical, environmental and random factors governing biomolecular evolution.

Circulating Pro-Inflammatory Exosomes Worsen Stroke Outcomes in Aging.

[Figure: see text].

General Synthetic Approach to Diverse Taxane Cores.

Chemical synthesis of natural products is typically inspired by the structure and function of a target molecule. When both factors are of interest, such as in the case of taxane diterpenoids, a synthesis can both serve as a platform for synthetic strategy development and enable new biological exploration. Guided by this paradigm, we present here a unified enantiospecific approach to diverse taxane cores from the feedstock monoterpenoid (S)-carvone. Key to the success of our approach was the use of a skeletal remodeling strategy which began with the divergent reorganization and convergent coupling of two carvone-derived fragments, facilitated by Pd-catalyzed C-C bond cleavage tactics. This coupling was followed by additional restructuring using a Sm(II)-mediated rearrangement and a bioinspired, visible-light induced, transannular [2 + 2] photocycloaddition. Overall, this divergent monoterpenoid remodeling/convergent fragment coupling approach to complex diterpenoid synthesis provides access to structurally disparate taxane cores which have set the stage for the preparation of a wide range of taxanes.

A cost-utility analysis comparing endovascular coiling to neurosurgical clipping in the treatment of aneurysmal subarachnoid haemorrhage.

Endovascular coiling (EC) has been identified in systematic reviews and meta-analyses to produce more favourable clinical outcomes in comparison to neurosurgical clipping (NC) when surgically treating a subarachnoid haemorrhage from a ruptured aneurysm. Cost-effectiveness analyses between both interventions have been done, but no cost-utility analysis has yet been published. This systematic review aims to perform an economic analysis of the relative utility outcomes and costs from both treatments in the UK. A cost-utility analysis was performed from the perspective of the National Health Service (NHS), over a 1-year analytic horizon. Outcomes were obtained from the randomised International Subarachnoid Aneurysm Trial (ISAT) and measured in terms of the patient's modified Rankin scale (mRS) grade, a 6-point disability scale that aims to quantify a patient's functional outcome following a stroke. The mRS score was weighted against the Euro-QoL 5-dimension (EQ-5D), with each state assigned a weighted utility value which was then converted into quality-adjusted life years (QALYs). A sensitivity analysis using different utility dimensions was performed to identify any variation in incremental cost-effectiveness ratio (ICER) if different input variables were used. Costs were measured in pounds sterling (£) and discounted by 3.5% to 2020/2021 prices. The cost-utility analysis showed an ICER of - £144,004 incurred for every QALY gained when EC was utilised over NC. At NICE's upper willingness-to-pay (WTP) threshold of £30,000, EC offered a monetary net benefit (MNB) of £7934.63 and health net benefit (HNB) of 0.264 higher than NC. At NICE's lower WTP threshold of £20,000, EC offered an MNB of £7478.63 and HNB of 0.374 higher than NC. EC was found to be more 'cost-effective' than NC, with an ICER in the bottom right quadrant of the cost-effectiveness plane-indicating that it offers greater benefits at lower costs. This is supported by the ICER being below the NICE's threshold of £20,000-£30,000 per QALY, and both MNB and HNB having positive values (> 0).

Integrins Increase Sarcoplasmic Reticulum Activity for Excitation-Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes.

Engagement of the sarcoplasmic reticulum (SR) Ca2+ stores for excitation-contraction (EC)-coupling is a fundamental feature of cardiac muscle cells. Extracellular matrix (ECM) proteins that form the extracellular scaffolding supporting cardiac contractile activity are thought to play an integral role in the modulation of EC-coupling. At baseline, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show poor utilisation of SR Ca2+ stores, leading to inefficient EC-coupling, like developing or human CMs in cardiac diseases such as heart failure. We hypothesised that integrin ligand-receptor interactions between ECM proteins and CMs recruit the SR to Ca2+ cycling during EC-coupling. hiPSC-CM monolayers were cultured on fibronectin-coated glass before 24 h treatment with fibril-forming peptides containing the integrin-binding tripeptide sequence arginine-glycine-aspartic acid (2 mM). Micropipette application of 40 mM caffeine in standard or Na+/Ca2+-free Tyrode's solutions was used to assess the Ca2+ removal mechanisms. Microelectrode recordings were conducted to analyse action potentials in current-clamp. Confocal images of labelled hiPSC-CMs were analysed to investigate hiPSC-CM morphology and ultrastructural arrangements in Ca2+ release units. This study demonstrates that peptides containing the integrin-binding sequence arginine-glycine-aspartic acid (1) abbreviate hiPSC-CM Ca2+ transient and action potential duration, (2) increase co-localisation between L-type Ca2+ channels and ryanodine receptors involved in EC-coupling, and (3) increase the rate of SR-mediated Ca2+ cycling. We conclude that integrin-binding peptides induce recruitment of the SR for Ca2+ cycling in EC-coupling through functional and structural improvements and demonstrate the importance of the ECM in modulating cardiomyocyte function in physiology.

Cytokine and Growth Factor Response in a Rat Model of Radiation Induced Injury to the Submental Muscles.

Submental muscles (i.e., mylohyoid and geniohyoid) play a vital role during swallowing, protecting the airway from ingested material. To design therapies to reduce the functional deficits associated with radiation treatment relies in part on our understanding of the changes in the cytokine and growth factor response that can impact muscle function. The purpose of this study is to quantify changes in the inflammatory, pro-fibrotic, and pro-angiogenic factors following 48 Gy of fractionated radiation to the mylohyoid muscle. We hypothesized that (1) irradiation will provoke increases in TGF-1ß and MMP-2 mRNA in the mylohyoid muscle; and (2) muscles surrounding the target location (i.e., geniohyoid and digastric muscles) will exhibit similar alterations in their gene expression profiles. Rats were exposed to 6 fractions of 8 Gy using a 6 MeV electron beam on a clinical linear accelerator. The highest dose curve was focused at the mylohyoid muscle. After 2- and 4-weeks post-radiation, the mylohyoid, geniohyoid, and digastric muscles were harvested. Expression of TNF-α, IFNγ, IL-1ß, IL-6, TGF-1ß, VEGF, MMP-2, and MMP-9 mRNA was analyzed via PCR and/or RT-PCR. TGF-1ß, MMP-2, and IL-6 expression was upregulated in the irradiated mylohyoid compared to non-irradiated controls. No notable changes in TNF-α, IFNγ, and IL-1ß mRNA expression were observed in irradiated muscles. Differing expression profiles were found in the surrounding muscles post-radiation. Results demonstrated that irradiation provokes molecular signals involved in the regulation of wound healing, which could lead to fibrosis or atrophy in the swallowing muscle after radiation.

A Case of Novel Coronavirus Disease 19 in a Chronic Hemodialysis Patient Presenting with Gastroenteritis and Developing Severe Pulmonary Disease.

Novel coronavirus disease 2019 (COVID-19) is a highly infectious, rapidly spreading viral disease with an alarming case fatality rate up to 5%. The risk factors for severe presentations are concentrated in patients with chronic kidney disease, particularly patients with end-stage renal disease (ESRD) who are dialysis dependent. We report the first US case of a 56-year-old nondiabetic male with ESRD secondary to IgA nephropathy undergoing thrice-weekly maintenance hemodialysis for 3 years, who developed COVID-19 infection. He has hypertension controlled with angiotensin receptor blocker losartan 100 mg/day and coronary artery disease status-post stent placement. During the first 5 days of his febrile disease, he presented to an urgent care, 3 emergency rooms, 1 cardiology clinic, and 2 dialysis centers in California and Utah. During this interval, he reported nausea, vomiting, diarrhea, and low-grade fevers but was not suspected of COVID-19 infection until he developed respiratory symptoms and was admitted to the hospital. Imaging studies upon admission were consistent with bilateral interstitial pneumonia. He was placed in droplet-eye precautions while awaiting COVID-19 test results. Within the first 24 h, he deteriorated quickly and developed acute respiratory distress syndrome (ARDS), requiring intubation and increasing respiratory support. Losartan was withheld due to hypotension and septic shock. COVID-19 was reported positive on hospital day 3. He remained in critical condition being treated with hydroxychloroquine and tocilizumab in addition to the standard medical management for septic shock and ARDS. Our case is unique in its atypical initial presentation and highlights the importance of early testing.

A Single-Step Synthesis of Azetidine-3-amines.

The azetidine group is frequently encountered within contemporary medicinal chemistry. However, the introduction of an azetidine can be synthetically challenging. Herein, a straightforward synthesis of azetidine-3-amines, starting from a bench stable, commercial material is presented. The reaction tolerates common functionality and proceeds in moderate-to-high yield with secondary amines, and moderate-to-low yield with primary amines. The methodology compares favorably to alternative procedures and can be utilized in "any-stage" functionalization, including late-stage azetidinylation of approved drugs and other compounds with pharmacological activity.

Combined effect of interventions with pure or enriched mixtures of (poly)phenols and anti-diabetic medication in type 2 diabetes management: a meta-analysis of randomized controlled human trials.

PURPOSE: (Poly)phenols have been reported to confer protective effects against type 2 diabetes but the precise association remains elusive. This meta-analysis aimed to assess the effects of (poly)phenol intake on well-established biomarkers in people with type 2 diabetes or at risk of developing diabetes. METHODS: A systematic search was conducted using the following selection criteria: (1) human randomized controlled trials involving individuals with prediabetes and type 2 diabetes; (2) one or more of the following biomarkers: glucose, glycated haemoglobin (HbA1c), insulin, pro-insulin, homeostatic model assessment of insulin resistance (HOMA-IR), islet amyloid polypeptide (IAPP)/amylin, pro-IAPP/pro-amylin, glucagon, C-peptide; (3) chronic intervention with pure or enriched mixtures of (poly)phenols. From 488 references, 88 were assessed for eligibility; data were extracted from 27 studies and 20 were used for meta-analysis. The groups included in the meta-analysis were: (poly)phenol mixtures, isoflavones, flavanols, anthocyanins and resveratrol. RESULTS: Estimated intervention/control mean differences evidenced that, overall, the consumption of (poly)phenols contributed to reduced fasting glucose levels (- 3.32 mg/dL; 95% CI - 5.86, - 0.77; P = 0.011). Hb1Ac was only slightly reduced (- 0.24%; 95% CI - 0.43, - 0.044; P = 0.016) whereas the levels of insulin and HOMA-IR were not altered. Subgroup comparative analyses indicated a stronger effect on blood glucose in individuals with diabetes (- 5.86 mg/dL, 95% CI - 11.34, - 0.39; P = 0.036) and this effect was even stronger in individuals taking anti-diabetic medication (- 10.17 mg/dL, 95% CI - 16.59, - 3.75; P = 0.002). CONCLUSIONS: Our results support that the consumption of (poly)phenols may contribute to lower glucose levels in individuals with type 2 diabetes or at risk of diabetes and that these compounds may also act in combination with anti-diabetic drugs.

Transition Metal-Mediated C-C Single Bond Cleavage: Making the Cut in Total Synthesis.

Transition-metal-mediated cleavage of C-C single bonds can enable entirely new retrosynthetic disconnections in the total synthesis of natural products. Given that C-C bond cleavage inherently alters the carbon framework of a compound, and that, under transition-metal catalysis, the generated organometallic or radical intermediate is primed for further complexity-building reactivity, C-C bond-cleavage events have the potential to drastically and rapidly remodel skeletal frameworks. The recent acceleration of the use of transition-metal-mediated cleavage of C-C single bonds in total synthesis can be ascribed to a communal recognition of this fact. In this Review, we highlight ten selected total syntheses from 2014 to 2019 that illustrate how transition-metal-mediated cleavage of C-C single bonds at either the core or the periphery of synthetic intermediates can streamline synthetic efforts.

Calculation of delivered composite dose from Calypso tracking data for prostate cancer: And subsequent evaluation of reasonable treatment interruption tolerance limits.

PURPOSE: In this study we calculate composite dose delivered to the prostate by using the Calypso tracking -data- stream acquired during patient treatment in our clinic. We evaluate the composite distributions under multiple simulated Calypso tolerance level schemes and then recommend a tolerance level. MATERIALS AND METHODS: Seven Calypso-localized prostate cancer patients treated in our clinic were selected for retrospective analysis. Two different IMRT treatment plans, with prostate PTV margins of 5 and 3 mm respectively, were computed for each patient. A delivered composite dose distribution was computed from Calypso tracking data for each plan. Additionally, we explored the dosimetric implications for "worst case" scenarios by assuming that the prostate position was located at one of the eight extreme corners of a 3 or 5 mm "box." To characterize plan quality under each of the studied scenarios, we recorded the maximum, mean, and minimum doses and volumetric coverage for prostate, PTV, bladder, and rectum. RESULTS AND DISCUSSIONS: Calculated composite dose distributions were very similar to the original plan for all patients. The difference in maximum, mean, and minimum doses as well as volumetric coverage for the prostate, PTV, bladder, and rectum were all < 4.0% of prescription dose. Even for worst scenario cases, the results show acceptable isodose distribution, with the exception for the combination of a 3 mm PTV margin with a 5 mm position tolerance scheme. CONCLUSIONS: Calculated composite dose distributions show that the vast majority of dosimetric metrics agreed well with the planned dose (within 2%). With significant/detrimental deviations from the planned dose only occurring with the combination of a 3 mm PTV margin and 5 mm position tolerance, the 3 mm position tolerance strategy appears reasonable, confirming that further reducing prostate PTV margins to 3 mm is possible when using Calypso with a position tolerance of 3 mm.

Investigation of a source model for a new electronic brachytherapy tandem by film measurement.

PURPOSE: To investigate the accuracy of a vendor-supplied source model for a new Xoft Axxent 0-degree titanium tandem by film measurement. METHODS: We measured the anisotropy factors at varying distances and angles from the tandem in water using radiochromic film (Gafchromic EBT3) and an Epson Perfection v750 desktop flatbed scanner (US Epson, Long Beach, CA). A 0-degree tandem was placed vertically in a water phantom. Four pieces of film, each at varying depths, were positioned orthogonal to the longitudinal axis of the tandem for azimuthal anisotropy measurements. Polar anisotropy measurements were taken with the film aligned parallel to the tandem. An absolute dose calibration for the film was verified with a PTW 34013 Soft X-Ray Chamber. The film measurements were analyzed using different color channels. The measured polar anisotropy for varying source positions was compared to the vendor's data. Azimuthal anisotropy was measured as a function of the radius and angle, and normalized to the mean value over all angles at the specified radius. RESULTS: The azimuthal anisotropy of the tandem and source was found to be consistent for different positions along the tandem's longitudinal axis and at varying distances from the tandem. Absolute dose using a calibrated parallel plate chamber showed agreement to within 2% of expected TPS values. The custom tandem, which has a thicker tip than the wall, was attenuating the 50 kV photons more than expected, at the angles where the photons had more wall material to traverse. This discrepancy was verified at different distances from the tandem and with different measurement techniques. As distance increased, anisotropy values had better agreement. CONCLUSIONS: We quantified the agreement between the measured and provided anisotropy factors for a new Xoft Axxent 0-degree titanium tandem. Radiochromic film response at low kV energy was also investigated. Our results showed that vendor-supplied TG-43 values were appropriate for clinical use at majority of the angles. A rigorous quality assurance method for new electronic brachytherapy sources and applicators, along with complete knowledge of all dosimetric measuring tools, should be implemented for all parts of the verification and commissioning process.

Many Cells Make Life Work-Multicellularity in Stem Cell-Based Cardiac Disease Modelling.

Cardiac disease causes 33% of deaths worldwide but our knowledge of disease progression is still very limited. In vitro models utilising and combining multiple, differentiated cell types have been used to recapitulate the range of myocardial microenvironments in an effort to delineate the mechanical, humoral, and electrical interactions that modulate the cardiac contractile function in health and the pathogenesis of human disease. However, due to limitations in isolating these cell types and changes in their structure and function in vitro, the field is now focused on the development and use of stem cell-derived cell types, most notably, human-induced pluripotent stem cell-derived CMs (hiPSC-CMs), in modelling the CM function in health and patient-specific diseases, allowing us to build on the findings from studies using animal and adult human CMs. It is becoming increasingly appreciated that communications between cardiomyocytes (CMs), the contractile cell of the heart, and the non-myocyte components of the heart not only regulate cardiac development and maintenance of health and adult CM functions, including the contractile state, but they also regulate remodelling in diseases, which may cause the chronic impairment of the contractile function of the myocardium, ultimately leading to heart failure. Within the myocardium, each CM is surrounded by an intricate network of cell types including endothelial cells, fibroblasts, vascular smooth muscle cells, sympathetic neurons, and resident macrophages, and the extracellular matrix (ECM), forming complex interactions, and models utilizing hiPSC-derived cell types offer a great opportunity to investigate these interactions further. In this review, we outline the historical and current state of disease modelling, focusing on the major milestones in the development of stem cell-derived cell types, and how this technology has contributed to our knowledge about the interactions between CMs and key non-myocyte components of the heart in health and disease, in particular, heart failure. Understanding where we stand in the field will be critical for stem cell-based applications, including the modelling of diseases that have complex multicellular dysfunctions.

Negative capacitance in a ferroelectric capacitor.

The Boltzmann distribution of electrons poses a fundamental barrier to lowering energy dissipation in conventional electronics, often termed as Boltzmann Tyranny. Negative capacitance in ferroelectric materials, which stems from the stored energy of a phase transition, could provide a solution, but a direct measurement of negative capacitance has so far been elusive. Here, we report the observation of negative capacitance in a thin, epitaxial ferroelectric film. When a voltage pulse is applied, the voltage across the ferroelectric capacitor is found to be decreasing with time--in exactly the opposite direction to which voltage for a regular capacitor should change. Analysis of this 'inductance'-like behaviour from a capacitor presents an unprecedented insight into the intrinsic energy profile of the ferroelectric material and could pave the way for completely new applications.

Implementation of an in-house visual feedback system for motion management during radiation therapy.

In this Technical Note, we describe an in-house video goggles feedback system assembled using several commercially available products. This goggle video feed-back system is currently being used at University of Louisville and Mayo Clinic for both CT simulation and linac treatment delivery. The setup details, including specific recommendations, are provided, along with an alternative option for using the video goggles system.

Comparison of surface matching and target matching for image-guided pelvic radiation therapy for both supine and prone patient positions.

We investigate the difference between surface matching and target matching for pelvic radiation image guidance. The uniqueness of our study is that all patients have multiple CT-on-rails (CTOR) scans to compare to corresponding AlignRT images. Ten patients receiving pelvic radiation were enrolled in this study. Two simulation CT scans were performed in supine and prone positions for each patient. Body surface contours were generated in treatment planning system and exported to AlignRT to serve as reference images. During treatment day, the patient was aligned to treatment isocenter with room lasers, and then scanned with both CTOR and AlignRT. Image-guidance shifts were calculated for both modalities by com-parison to the simulation CT and the differences between them were analyzed for both supine and prone positions, respectively. These procedures were performed for each patient once per week for five weeks. The difference of patient displace-ment between AlignRT and CTOR was analyzed. For supine position, five patients had an average difference of displacement between AlignRT and CTOR along any direction (vertical, longitudinal, and lateral) greater than 0.5 cm, and one patient greater than 1 cm. Four patients had a maximum difference greater than 1 cm. For prone position, seven patients had an average difference greater than 0.5 cm, and three patients greater than 1 cm. Nine patients had a maximum difference greater than 1 cm. The difference of displacement between AlignRT and CTOR was greater for the prone position than for the supine position. For the patients studied here, surface matching does not appear to be an advisable image-guidance approach for pelvic radiation therapy for patients with either supine or prone position. There appears to be a potential for large alignment discrepancies (up to 2.25 cm) between surface matching and target matching.

Current perspectives on the link between neuroinflammation and neurogenesis.

The link between neuroinflammation and neurogenesis is an area of intensive research in contemporary neuroscience. The burgeoning amount of evidence accumulated over the past decade has been incredible, and now there remains the figuring out of minutia to give us a more complete picture of what individual, synergistic, and antagonistic events are occurring between neurogenesis and neuroinflammation. An intricate study of the inflammatory microenvironment influenced by the presence of the various inflammatory components like cytokines, chemokines, and immune cells is essential for: 1) understanding how neurogenesis can be affected in such a specialized niche and 2) applying the knowledge gained for the treatment of cognitive and/or motor deficits arising from inflammation-associated diseases like stroke, traumatic brain injury, Alzheimer's disease, and Parkinson's disease. This review is written to provide the reader with up-to-date information explaining how these inflammatory components are effecting changes on neurogenesis.

Site-specific tolerance tables and indexing device to improve patient setup reproducibility.

While the implementation of tools such as image-guidance and immobilization devices have helped to prevent geometric misses in radiation therapy, many treatments remain prone to error if these items are not available, not utilized for every fraction, or are misused. The purpose of this project is to design a set of site-specific treatment tolerance tables to be applied to the treatment couch for use in a record and verify (R&V) system that will insure accurate patient setup with minimal workflow interruption. This project also called for the construction of a simple indexing device to help insure reproducible patient setup for patients that could not be indexed with existing equipment. The tolerance tables were created by retrospective analysis on a total of 66 patients and 1,308 treatments, separating them into five categories based on disease site: lung, head and neck (H&N), breast, pelvis, and abdomen. Couch parameter tolerance tables were designed to encompass 95% of treatments, and were generated by calculating the standard deviation of couch vertical, longitudinal, and lateral values using the first day of treatment as a baseline. We also investigated an alternative method for generating the couch tolerances by updating the baseline values when patient position was verified with image guidance. This was done in order to adapt the tolerances to any gradual changes in patient setup that would not correspond with a mistreatment. The tolerance tables and customizable indexing device were then implemented for a trial period in order to determine the feasibility of the system. During this trial period we collected data from 1,054 fractions from 65 patients. We then analyzed the number of treatments that would have been out of tolerance, as well as whether or not the tolerances or setup techniques should be adjusted. When the couch baseline values were updated with every imaging fraction, the average rate of tolerance violations was 10% for the lung, H&N, abdomen, and pelvis treatments. Using the indexing device, tolerances for patients with pelvic disease decreased (e.g., from 5.3 cm to 4.3 cm longitudinally). Unfortunately, the results from breast patients were highly variable due to the complexity of the setup technique, making the couch an inadequate surrogate for measuring setup accuracy. In summary, we have developed a method to turn the treatment couch parameters within the R&V system into a useful alert tool, which can be implemented at other institutions, in order to identify potential errors in patient setup.