Identification of plant transcriptional activation domains.

Gene expression in Arabidopsis is regulated by more than 1,900 transcription factors (TFs), which have been identified genome-wide by the presence of well-conserved DNA-binding domains. Activator TFs contain activation domains (ADs) that recruit coactivator complexes; however, for nearly all Arabidopsis TFs, we lack knowledge about the presence, location and transcriptional strength of their ADs1. To address this gap, here we use a yeast library approach to experimentally identify Arabidopsis ADs on a proteome-wide scale, and find that more than half of the Arabidopsis TFs contain an AD. We annotate 1,553 ADs, the vast majority of which are, to our knowledge, previously unknown. Using the dataset generated, we develop a neural network to accurately predict ADs and to identify sequence features that are necessary to recruit coactivator complexes. We uncover six distinct combinations of sequence features that result in activation activity, providing a framework to interrogate the subfunctionalization of ADs. Furthermore, we identify ADs in the ancient AUXIN RESPONSE FACTOR family of TFs, revealing that AD positioning is conserved in distinct clades. Our findings provide a deep resource for understanding transcriptional activation, a framework for examining function in intrinsically disordered regions and a predictive model of ADs.

Central fixation element type and length affect glenoid baseplate micromotion in reverse shoulder arthroplasty.

BACKGROUND: Reverse shoulder arthroplasty (RSA) is commonly used to treat patients with rotator cuff tear arthropathy. Loosening of the glenoid component remains one of the principal modes of failure and represents a significant complication that requires revision surgery. This study assessed the effects of various factors on glenoid baseplate micromotion for primary fixation of RSA. MATERIALS AND METHODS: A half-fractional factorial design of experiment was used to assess 4 factors: central element type (central peg or screw), central cortical engagement according to length (13.5 or 23.5 mm), anterior-posterior peripheral screw type (nonlocking or locking), and cancellous bone surrogate density (160 or 400 kg/m3, 10 or 25 PCF). Glenoid baseplates were implanted into high- or low-density Sawbones rigid polyurethane foam blocks and cyclically loaded at 60° for 1000 cycles (500-N compressive force range) using a custom-designed loading apparatus. Micromotion at the 4 peripheral screw positions was recorded using linear variable differential transformers. RESULTS: Central peg fixation generated 358% greater micromotion at all peripheral screw positions compared with central screw fixation (P < .001). Baseplates with short central elements that lacked cortical bone engagement generated 328% greater micromotion than those with long central elements (P = .001). No significant effects were observed when varying anterior-posterior peripheral screw type or bone surrogate density. There were significant interactions between central element type and length (P < .001). DISCUSSION: A central screw and a long central element that engaged cortical bone reduced RSA baseplate micromotion. These findings serve to inform surgical decision making regarding baseplate fixation elements to minimize the risk of glenoid loosening and, thus, the need for revision surgery.

The Fading Gloss of Data Science: Towards an Agenda that Faces the Challenges of Big Data for Development and Humanitarian Action.

Different UN and international agencies are busy trying to leverage big data to unlock its value for evidence-based decision-making in development and humanitarian action. But many vulnerable people are invisible to the data infrastructure, while just integrating their data without understanding the consequences can make them even more vulnerable. This article unpacks the challenges presented by data science for development and humanitarianism.

Obesity and occupation in Thailand: using a Bayesian hierarchical model to obtain prevalence estimates from the National Health Examination Survey.

BACKGROUND: Like many developing countries, Thailand has experienced a rapid rise in obesity, accompanied by a rapid change in occupational structure. It is plausible that these two trends are related, with movement into sedentary occupations leading to increases in obesity. National health examination survey data contains information on obesity and socioeconomic conditions that can help untangle the relationship, but analysis is challenging because of small sample sizes. METHODS: This paper explores the relationship between occupation and obesity using data on 10,127 respondents aged 20-59 from the 2009 National Health Examination Survey. Obesity is measured using waist circumference. Modelling is carried out using an approach known as Multiple Regression with Post-Stratification (MRP). We use Bayesian hierarchical models to construct prevalence estimates disaggregated by age, sex, education, urban-rural residence, region, and occupation, and use census population weights to aggregate up. The Bayesian hierarchical model is designed to protect against overfitting and false discovery, which is particularly important in an exploratory study such as this one. RESULTS: There is no clear relationship between the overall sedentary nature of occupations and obesity. Instead, obesity appears to vary occupation by occupation. For instance, women in professional occupations, and men who are agricultural or fishery workers, have relatively low rates of obesity. CONCLUSION: Bayesian hierarchical models plus post-stratification offers new possibilities for using surveys to learn about complex health issues.

Long-term primary results of accelerated partial breast irradiation after breast-conserving surgery for early-stage breast cancer: a randomised, phase 3, equivalence trial.

BACKGROUND: Whole-breast irradiation after breast-conserving surgery for patients with early-stage breast cancer decreases ipsilateral breast-tumour recurrence (IBTR), yielding comparable results to mastectomy. It is unknown whether accelerated partial breast irradiation (APBI) to only the tumour-bearing quadrant, which shortens treatment duration, is equally effective. In our trial, we investigated whether APBI provides equivalent local tumour control after lumpectomy compared with whole-breast irradiation. METHODS: We did this randomised, phase 3, equivalence trial (NSABP B-39/RTOG 0413) in 154 clinical centres in the USA, Canada, Ireland, and Israel. Adult women (>18 years) with early-stage (0, I, or II; no evidence of distant metastases, but up to three axillary nodes could be positive) breast cancer (tumour size ≤3 cm; including all histologies and multifocal breast cancers), who had had lumpectomy with negative (ie, no detectable cancer cells) surgical margins, were randomly assigned (1:1) using a biased-coin-based minimisation algorithm to receive either whole-breast irradiation (whole-breast irradiation group) or APBI (APBI group). Whole-breast irradiation was delivered in 25 daily fractions of 50 Gy over 5 weeks, with or without a supplemental boost to the tumour bed, and APBI was delivered as 34 Gy of brachytherapy or 38·5 Gy of external bream radiation therapy in 10 fractions, over 5 treatment days within an 8-day period. Randomisation was stratified by disease stage, menopausal status, hormone-receptor status, and intention to receive chemotherapy. Patients, investigators, and statisticians could not be masked to treatment allocation. The primary outcome of invasive and non-invasive IBTR as a first recurrence was analysed in the intention-to-treat population, excluding those patients who were lost to follow-up, with an equivalency test on the basis of a 50% margin increase in the hazard ratio (90% CI for the observed HR between 0·667 and 1·5 for equivalence) and a Cox proportional hazard model. Survival was assessed by intention to treat, and sensitivity analyses were done in the per-protocol population. This trial is registered with ClinicalTrials.gov, NCT00103181. FINDINGS: Between March 21, 2005, and April 16, 2013, 4216 women were enrolled. 2109 were assigned to the whole-breast irradiation group and 2107 were assigned to the APBI group. 70 patients from the whole-breast irradiation group and 14 from the APBI group withdrew consent or were lost to follow-up at this stage, so 2039 and 2093 patients respectively were available for survival analysis. Further, three and four patients respectively were lost to clinical follow-up (ie, survival status was assessed by phone but no physical examination was done), leaving 2036 patients in the whole-breast irradiation group and 2089 in the APBI group evaluable for the primary outcome. At a median follow-up of 10·2 years (IQR 7·5-11·5), 90 (4%) of 2089 women eligible for the primary outcome in the APBI group and 71 (3%) of 2036 women in the whole-breast irradiation group had an IBTR (HR 1·22, 90% CI 0·94-1·58). The 10-year cumulative incidence of IBTR was 4·6% (95% CI 3·7-5·7) in the APBI group versus 3·9% (3·1-5·0) in the whole-breast irradiation group. 44 (2%) of 2039 patients in the whole-breast irradiation group and 49 (2%) of 2093 patients in the APBI group died from recurring breast cancer. There were no treatment-related deaths. Second cancers and treatment-related toxicities were similar between the two groups. 2020 patients in the whole-breast irradiation group and 2089 in APBI group had available data on adverse events. The highest toxicity grade reported was: grade 1 in 845 (40%), grade 2 in 921 (44%), and grade 3 in 201 (10%) patients in the APBI group, compared with grade 1 in 626 (31%), grade 2 in 1193 (59%), and grade 3 in 143 (7%) in the whole-breast irradiation group. INTERPRETATION: APBI did not meet the criteria for equivalence to whole-breast irradiation in controlling IBTR for breast-conserving therapy. Our trial had broad eligibility criteria, leading to a large, heterogeneous pool of patients and sufficient power to detect treatment equivalence, but was not designed to test equivalence in patient subgroups or outcomes from different APBI techniques. For patients with early-stage breast cancer, our findings support whole-breast irradiation following lumpectomy; however, with an absolute difference of less than 1% in the 10-year cumulative incidence of IBTR, APBI might be an acceptable alternative for some women. FUNDING: National Cancer Institute, US Department of Health and Human Services.

Minimal acute toxicity from proton beam therapy for major salivary gland cancer.

Introduction: Proton beam therapy (PBT) reduces normal organ dose compared to intensity-modulated radiation therapy (IMRT) for patients with major salivary gland tumors. It is not known whether this dosimetric advantage is clinically meaningful for reducing acute toxicity.Methods: We evaluated treatment parameters and acute toxicity outcomes of patients with major salivary gland cancers enrolled on the Proton Collaborative Group REG001-09 trial (NCT01255748).Results: One-hundred and five patients with a median age of 61 years were included. The majority had parotid (N = 90) versus submandibular gland (N = 15) tumors. The patients were treated across seven institutions in the United States between 2010 and 2017, most commonly in the postoperative setting (70.5%) although a minority were treated definitively (29.5%). Median PBT dose was 66.5 GyE in 33 fractions; only one patient was prescribed less than 50 GyE. Chemotherapy was given concurrently to 20%. Median follow-up was 14.3 months. Acute grade 2 or higher toxicity included nausea (1.5%), dysgeusia (4.8%), xerostomia (7.6%), mucositis (10.5%) and dysphagia (10.5%).Conclusions: PBT should be strongly considered when ipsilateral radiation therapy is indicated for major salivary gland cancer based on a considerably lower incidence of acute grade 2 or higher toxicity in this analysis compared to historical IMRT outcomes.

Regulatory networks controlling the development of the root system and the formation of lateral roots: a comparative analysis of the roles of pericycle and vascular cambium.

Background: The production of a new lateral root from parental root primary tissues has been investigated extensively, and the most important regulatory mechanisms are now well known. A first regulatory mechanism is based on the synthesis of small peptides which interact ectopically with membrane receptors to elicit a modulation of transcription factor target genes. A second mechanism involves a complex cross-talk between plant hormones. It is known that lateral roots are formed even in parental root portions characterized by the presence of secondary tissues, but there is not yet agreement about the putative tissue source providing the cells competent to become founder cells of a new root primordium. Scope: We suggest models of possible regulatory mechanisms for inducing specific root vascular cambium (VC) stem cells to abandon their activity in the production of xylem and phloem elements and to start instead the construction of a new lateral root primordium. Considering the ontogenic nature of the VC, the models which we suggest are the result of a comparative review of mechanisms known to control the activity of stem cells in the root apical meristem, procambium and VC. Stem cells in the root meristems can inherit various competences to play different roles, and their fate could be decided in response to cross-talk between endogenous and exogenous signals. Conclusions: We have found a high degree of relatedness among the regulatory mechanisms controlling the various root meristems. This fact suggests that competence to form new lateral roots can be inherited by some stem cells of the VC lineage. This kind of competence could be represented by a sensitivity of specific stem cells to factors such as those presented in our models.

Fibrinolysis in Trauma: "Myth," "Reality," or "Something in Between".

The emphasis on fibrinolysis as an important contributor to trauma-induced coagulopathy (TIC) has led to a debate regarding the relative clinical significance of fibrinolysis in the setting of trauma. The debate has centered on two camps. The one camp defines fibrinolysis in trauma by standard coagulation tests as well as fibrin split products, D-dimers, and plasmin/antiplasmin levels. This camp favors a more liberal use of tranexamic acid and attributes more significance to hyperfibrinolysis in TIC. The other camp favors a definition of fibrinolysis based on the viscoelastic tests (VET), rotational thromboelastometry (ROTEM), and thromboelastography (TEG). These whole blood assays define hyperfibrinolysis at a higher threshold than plasma-based tests. Therefore, this VET camp reserves antifibrinolytic treatment for patients who demonstrate severe coagulopathy associated with hyperfibrinolysis. This bimodal attribution of the clinical relevance of fibrinolysis in trauma suggests that there may be an underlying "Myth" of the concept of TIC that was historically defined by plasma-based tests and a future "Reality" of the concept of TIC that is grounded on an understanding of TIC based on a VET-defined "fibrinolytic spectrum" of TIC. This narrative review explores this "Myth" and "Reality" of fibrinolysis in TIC and proposes a direction that will allow a "Future" interpretation of TIC that incorporates both the past "Myth" and present "Reality" of fibrinolysis TIC.

Tranexamic Acid for Trauma Resuscitation in the United States of America.

The utilization of tranexamic acid (TXA) for the management of bleeding trauma patients has been a subject of much debate on both sides of the Atlantic and in Australia. As a result of the large randomized controlled study called the Clinical Randomization of an Antifibrinolytic in Severe Hemorrhage (CRASH-2), there was an initial enthusiasm for the use of TXA to treat bleeding patients. However, the adoption of TXA in the United States was delayed by concerns of "knowledge and evidence gaps" of the CRASH-2 study and because of a lack of mechanistic rationale that would explain the survival benefit noted in the study. Subsequent nonrandomized controlled trials questioned the liberal use of TXA in trauma patients. This narrative review explores the historical as well as clinical and theoretical grounds for the more measured use of TXA in the United States and proposes a clinical and point-of-care guided utilization of TXA, blood components, and adjunctive hemostatic agents in bleeding trauma patients.

Global agricultural intensification during climate change: a role for genomics.

Agriculture is now facing the 'perfect storm' of climate change, increasing costs of fertilizer and rising food demands from a larger and wealthier human population. These factors point to a global food deficit unless the efficiency and resilience of crop production is increased. The intensification of agriculture has focused on improving production under optimized conditions, with significant agronomic inputs. Furthermore, the intensive cultivation of a limited number of crops has drastically narrowed the number of plant species humans rely on. A new agricultural paradigm is required, reducing dependence on high inputs and increasing crop diversity, yield stability and environmental resilience. Genomics offers unprecedented opportunities to increase crop yield, quality and stability of production through advanced breeding strategies, enhancing the resilience of major crops to climate variability, and increasing the productivity and range of minor crops to diversify the food supply. Here we review the state of the art of genomic-assisted breeding for the most important staples that feed the world, and how to use and adapt such genomic tools to accelerate development of both major and minor crops with desired traits that enhance adaptation to, or mitigate the effects of climate change.

Biogeography of Alaska paper birch (Betula neoalaskana): latitudinal patterns in chemical defense and plant architecture.

The latitudinal herbivory-defense hypothesis (LHDH) predicts that plants near the equator will be more heavily defended against herbivores than are plants at higher latitudes. Although this idea is widely found in the literature, recent studies have called this biogeographic pattern into question. We sought to evaluate the LHDH in a high-latitude terrestrial ecosystem where fire and mammalian herbivores may contribute to selection for higher levels of defensive chemistry. To address this objective, we collected seeds of Alaska paper birch (Betula neoalaskana) from nine locations along two north-south transects between 55 degrees N and 62 degrees N latitudes in western, interior Canada. The birch seeds were planted in pots in a common garden in Madison, Wisconsin, USA. From the resulting seedlings, we determined levels of chemical defense by assessing the density of resin glands, which have been shown to be negatively correlated with browsing. To assess plant architectural traits such as height, mean individual leaf area, and root-to-shoot ratio, we harvested a subset of the birch seedlings. Further, we used these traits to examine growth-defense trade-offs. Contrary to the LHDH, we found a positive correlation between chemical defense and latitude. Investigating relationships with fire, we found a strong positive correlation between resin gland density and percentage of area annually burned (PAAB) around each collection location and also between PAAB and latitude. Additionally, birch seedlings originating from higher latitudes were shorter, smaller-leaved, and rootier than their lower-latitude counterparts. Growth-defense trade-offs were observed in negative correlations between resin gland density and height and leaf size. Seedlings with higher resin gland densities also allocated less biomass to shoots and more to roots. These results further call into question the LHDH and provide specific information about latitudinal trends in plant defense at high, northern latitudes where fire is a major ecosystem driver and mammals are the main herbivores of deciduous trees such as birches. We propose that these interconnected relationships are the key drivers of the positive correlation between defense and latitude in B. neoalaskana. Understanding patterns of boreal plant defense and growth is especially important because high latitude ecosystems are particularly susceptible to climate change. Key words: Alaska paper birch; Betula neoalaskana; biogeography; chemical defense; grotvth-defense trade-offs; inherent growth rate; latitude; latitudinal herbivory-defense hypothesis; papyr'feric acid; plant ar- chitecture; resin glands; root-to-shoot ratio.

Bayesian demography 250†years after Bayes.

Bayesian statistics offers an alternative to classical (frequentist) statistics. It is distinguished by its use of probability distributions to describe uncertain quantities, which leads to elegant solutions to many difficult statistical problems. Although Bayesian demography, like Bayesian statistics more generally, is around 250 years old, only recently has it begun to flourish. The aim of this paper is to review the achievements of Bayesian demography, address some misconceptions, and make the case for wider use of Bayesian methods in population studies. We focus on three applications: demographic forecasts, limited data, and highly structured or complex models. The key advantages of Bayesian methods are the ability to integrate information from multiple sources and to describe uncertainty coherently. Bayesian methods also allow for including additional (prior) information next to the data sample. As such, Bayesian approaches are complementary to many traditional methods, which can be productively re-expressed in Bayesian terms.

Clinical correlates in an experimental model of repetitive mild brain injury.

OBJECTIVE: Although there is growing awareness of the long-term cognitive effects of repetitive mild traumatic brain injury (rmTBI; eg, sports concussions), whether repeated concussions cause long-term cognitive deficits remains controversial. Moreover, whether cognitive deficits depend on increased amyloid ß deposition and tau phosphorylation or are worsened by the apolipoprotein E4 allele remains unknown. Here, we use an experimental model of rmTBI to address these clinical controversies. METHODS: A weight drop rmTBI model was used that results in cognitive deficits without loss of consciousness, seizures, or gross or microscopic evidence of brain damage. Cognitive function was assessed using a Morris water maze (MWM) paradigm. Immunostaining and enzyme-linked immunosorbent assay (ELISA) were used to assess amyloid ß deposition and tau hyperphosphorylation. Brain volume and white matter integrity were assessed by magnetic resonance imaging (MRI). RESULTS: Mice subjected to rmTBI daily or weekly but not biweekly or monthly had persistent cognitive deficits as long as 1 year after injuries. Long-term cognitive deficits were associated with increased astrocytosis but not tau phosphorylation or amyloid ß (by ELISA); plaques or tangles (by immunohistochemistry); or brain volume loss or changes in white matter integrity (by MRI). APOE4 was not associated with worse MWM performance after rmTBI. INTERPRETATION: Within the vulnerable time period between injuries, rmTBI produces long-term cognitive deficits independent of increased amyloid ß or tau phosphorylation. In this model, cognitive outcome is not influenced by APOE4 status. The data have implications for the long-term mental health of athletes who suffer multiple concussions.

Cell cycling with the SEB: a personal view.

This review, written from a personal perspective, traces firstly the development of plant cell cycle research from the 1970s onwards, with some focus on the work of the author and of Dr Dennis Francis. Secondly there is a discussion of the support for and discussion of plant cell cycle research in the SEB, especially through the activities of the Cell Cycle Group within the Society's Cell Biology Section. In the main part of the review, selected aspects of DNA replication that have of been of special interest to the author are discussed. These are DNA polymerases and associated proteins, pre-replication events, regulation of enzymes and other proteins, nature and activation of DNA replication origins, and DNA endoreduplication. For all these topics, there is mention of the author's own work, followed by a brief synthesis of current understanding and a look to possible future developments.

Pre-hospital ambulance notification and initiation of treatment of ST elevation myocardial infarction is associated with significant reduction in door-to-balloon time for primary PCI.

BACKGROUND: Mortality in ST elevation myocardial infarction (STEMI) is strongly predicted by the time from first medical contact to reperfusion. The aim of this study was to examine the impact of pre-hospital diagnosis by paramedics in the field on the door-to-balloon (DTB) times of patients with ST elevation myocardial infarction undergoing primary percutaneous intervention. METHODS: Paramedics in the field identified patients with ST elevation myocardial infarction on a 12-lead electrocardiograph, activated the cardiac catheter laboratory team from the field and initiated therapy with anticoagulants and antiplatelet agents in the pre-hospital setting. This cohort of patients was compared to a similar group of patients without pre-hospital diagnosis and notification. The primary outcome measure was DTB times. A secondary end point was mortality at 30 days and mortality at six months. RESULTS: A total of 281 patients, mean age of 61.1±12.9 years underwent primary percutaneous intervention with pre-hospital notification occurring in 63 cases. DTB times were lower in those with pre notification than in those without pre-hospital notification (40.4 vs. 75.6 minutes, p<0.001). This represented a 47.6% shorter DTB time. A non-statistically significant mortality reduction at one month and six months was observed in the pre-hospital notification group (1.6 versus 4.3%, p= 0.307 and 1.6 versus 6.4%, p= 0.203, respectively). CONCLUSION: Pre-hospital intervention at our centre had a powerful effect in reducing the time to reperfusion in patients with STEMI undergoing primary percutaneous intervention.

TidyTron: Reducing lab waste using validated wash-and-reuse protocols for common plasticware in Opentrons OT-2 lab robots.

Every year biotechnology labs generate a combined total of ∼5.5 million tons of plastic waste. As the global bioeconomy expands, biofoundries will inevitably increase plastic consumption in-step with synthetic biology scaling. Decontamination and reuse of single-use plastics could increase sustainability and reduce recurring costs of biological research. However, throughput and variable cleaning quality make manual decontamination impractical in most instances. Automating single-use plastic cleaning with liquid handling robots makes decontamination more practical by offering higher throughput and consistent cleaning quality. However, open-source, validated protocols using low-cost lab robotics for effective decontamination of plasticware-facilitating safe reuse-have not yet been developed. Here we introduce and validate TidyTron: a library of protocols for cleaning micropipette tips and microtiter plates that are contaminated with DNA, E. coli, and S. cerevisiae. We tested a variety of cleaning solutions, contact times, and agitation methods with the aim of minimizing time and cost, while maximizing cleaning stringency and sustainability. We tested and validated these cleaning procedures by comparing fresh (first-time usage) versus cleaned tips and plates for contamination with cells, DNA, or cleaning solutions. We assessed contamination by measuring colony forming units by plating, PCR efficiency and DNA concentration by qPCR, and event counts and debris by flow cytometry. Open source cleaning protocols are available at https://github.com/PlantSynBioLab/TidyTron and hosted on a graphical user interface at https://jbryantvt.github.io/TidyTron/.

Novel Postoperative Hypofractionated Accelerated Radiation Dose-Painting Approach for Soft Tissue Sarcoma.

Purpose: Hypofractionated radiation therapy (RT) offers benefits in the treatment of soft tissue sarcomas (STS), including exploitation of the lower α/ß, patient convenience, and cost. This study evaluates the acute toxicity of a hypofractionated accelerated RT dose-painting (HARD) approach for postoperative treatment of STS. Methods and Materials: This is a retrospective review of 53 consecutive patients with STS who underwent resection followed by postoperative RT. Standard postoperative RT dosing for R0/R1/gross disease with sequential boost (50 Gy + 14/16/20 Gy in 32-35 fractions) were replaced with dose-painting, which adapts dose based on risk of disease burden, to 50.4 and 63, 64.4, 70 Gy in 28 fractions, respectively. The first 10 patients were replanned with a sequential boost RT approach and dosimetric indices were compared. Time-to-event outcomes, including local control, regional control, distant control, and overall survival, were estimated with Kaplan-Meier analysis. Results: Median follow-up was 25.2 months. Most patients had high-grade (59%) STS of the extremity (63%) who underwent resection with either R1 (40%) or close (36%) margins. Four patients experienced grade 3 acute dermatitis which resolved by the 3-month follow-up visit. The 2-year local control, regional control, distant control, and overall survival were 100%, 92%, 68%, and 86%, respectively. Compared with the sequential boost plan, HARD had a significantly lower field size (total V50 Gy; P = .002), bone V50 (P = .031), and maximum skin dose (P = .008). Overall treatment time was decreased by 4 to 7 fractions, which translated to a decrease in estimated average treatment cost of $3056 (range, $2651-$4335; P < .001). Conclusions: In addition to benefits in cost, convenience, and improved biologic effect in STS, HARD regimen offers a safe treatment approach with dosimetric advantages compared with conventional sequential boost, which may translate to improved long-term toxicity.

Novel Definitive Hypofractionated Accelerated Radiation Dose-painting (HARD) for Unresected Soft Tissue Sarcomas.

Purpose: Soft tissue sarcomas (STS) are historically radioresistant, with surgery being an integral component of their treatment. With their low α/ß, STS may be more responsive to hypofractionated radiation therapy (RT), which is often limited by long-term toxicity risk to surrounding normal tissue. An isotoxic approach using a hypofractionated accelerated radiation dose-painting (HARD) regimen allows for dosing based on clinical risk while sparing adjacent organs at risk. Methods and Materials: We retrospectively identified patients from 2019 to 2022 with unresected STS who received HARD with dose-painting to high, intermediate, and low-risk regions of 3.0 Gy, 2.5 Gy, and 2.0 to 2.3 Gy, respectively, in 20 to 22 fractions. Clinical endpoints included local control, locoregional control, progression free survival, overall survival, and toxicity outcomes. Results: Twenty-seven consecutive patients were identified and had a median age of 68 years and tumor size of 7.0 cm (range, 1.2-21.0 cm). Tumors were most often high-grade (70%), stage IV (70%), located in the extremities (59%), and locally recurrent (52%). With a median follow-up of 33.4 months, there was a 3-year locoregional control rate of 100%. The 3-year overall and progression-free survival were 44.9% and 23.3%, respectively. There were 5 (19%) acute and 2 (7%) late grade 3 toxicities, and there were no grade 4 or 5 toxicities at any point. Conclusions: The HARD regimen is a safe method of dose-escalating STS, with durable 3-year locoregional control. This approach is a promising alternative for unresected STS, though further follow-up is required to determine long-term control and toxicity.

AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots.

As one of the newest fields of engineering, synthetic biology relies upon a trial-and-error Design-Build-Test-Learn (DBTL) approach to simultaneously learn how a function is encoded in biology and attempt to engineer it. Many software and hardware platforms have been developed to automate, optimize and algorithmically perform each step of the DBTL cycle. However, there are many fewer options for automating the build step. Build typically involves deoxyribonucleic acid (DNA) assembly, which remains manual, low throughput and unreliable in most cases and limits our ability to advance the science and engineering of biology. Here, we present AssemblyTron, an open-source Python package to integrate j5 DNA assembly design software outputs with build implementation in Opentrons liquid handling robotics with minimal human intervention. We demonstrate the versatility of AssemblyTron through several scarless, multipart DNA assemblies, beginning from fragment amplification. We show that AssemblyTron can perform polymerase chain reactions across a range of fragment lengths and annealing temperatures by using an optimal annealing temperature gradient calculation algorithm. We then demonstrate that AssemblyTron can perform Golden Gate and homology-dependent in vivo assemblies (IVAs) with comparable fidelity to manual assemblies by simultaneously building four four-fragment assemblies of chromoprotein reporter expression plasmids. Finally, we used AssemblyTron to perform site-directed mutagenesis reactions via homology-dependent IVA also achieving comparable fidelity to manual assemblies as assessed by sequencing. AssemblyTron can reduce the time, training, costs and wastes associated with synthetic biology, which, along with open-source and affordable automation, will further foster the accessibility of synthetic biology and accelerate biological research and engineering.