Quality improvement approach to reduce patient cycle time at a student-run free healthcare clinical network.

BACKGROUND: The University of Florida (UF) Equal Access Clinic Network (EACN) is the largest student-run free healthcare clinic network in Florida. The UF EACN serves those who are underinsured or uninsured in Alachua County and its surrounding area. Nationally, average total clinic time per medical visit has been established to be 84 min. PROBLEM: Before this project, average patient cycle time at the UF EACN was 125.3 min, and there was no established quality improvement (QI) team to implement changes to address inefficiencies. METHODS: This was a prospective QI study that recorded patient cycle times for patients who received healthcare at any of the four primary care free clinics across the UF EACN from 5 July 2022 to 6 April 2023. INTERVENTIONS: Eighteen Plan-Do-Study-Act cycles were tailored to each of the four primary care clinic's needs with a focus on reducing patient cycle time by addressing the following identified problems: prolonged intake process, translation services, limited numbers of volunteers, and other inefficiencies and bottlenecks in workflow. RESULTS: The median patient cycle time at the EACN shifted from 125.3 min to 112.7 min over a nine month period. This drop of 12.6 min meant patients saw a 10.1% reduction in patient cycle time across the EACN. CONCLUSION: Underserved patients at EACN are experiencing increased value by having shorter patient cycle times.

Hypothesis: 2 Major Environmental and Pharmaceutical Factors-Acetaminophen Exposure and Gastrointestinal Overgrowth of Clostridia Bacteria Induced By Ingestion of Glyphosate-Contaminated Foods-Dysregulate the Developmental Protein Sonic Hedgehog and Are Major Causes of Autism.

Epidemiological studies have found 2 significant factors associated with the increased incidence of autism spectrum disorder (ASD): the increased use of acetaminophen in the 1970s when this drug largely replaced the use of aspirin for many patients because of a fear of Reye syndrome, and the agricultural use in the 1990s of the herbicide glyphosate on crops that were genetically modified (GM) to tolerate glyphosate. The incidence of autism in the United States, where acetaminophen is widely available, is more than 1000 times greater than in Cuba, where acetaminophen is available only by prescription. Metabolites of both glyphosate and acetaminophen likely alter the function of the developmental protein sonic hedgehog (SHH). Glyphosate likely affects SHH indirectly by decreasing the beneficial flora of the gastrointestinal tract and increasing pathogenic Clostridia bacteria, which are resistant to glyphosate. The marked increase of certain Clostridia species caused by glyphosate results in Clostridia production of large amounts of 3-(3-hydroxyphenyl)-3-hydroxypropionate (HPHPA) and 4-cresol (p-cresol). The 4-cresol metabolite 4-methyl-o-hydroquinone and the acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) likely react with the sulfhydryl group of the N-terminal cysteine of SHH, blocking the function of this critical amino acid required for the activation of SHH. HPHPA and 4-cresol also inhibit dopamine ß-hydroxylase, resulting in overproduction of dopamine and its toxic metabolites, such as aminochrome, that cause biochemical damage to mitochondria and structural proteins in brain cells. Elevated amounts of these Clostridia products in body fluids in people with autism and in animals with autistic signs have been documented in laboratories throughout the world. The synthesis of the HPHPA molecule in extremely large quantities depletes the body of free coenzyme A, which is needed for the palmitoylation of SHH. SHH covalently coupled to palmitic acid is 30 times more active than SHH without palmitic acid. These possible modifications of SHH help to explain the significantly altered quantities of SHH in the blood serum of patients with autism. The severity of autism is related to the degree of SHH abnormality. The spread of pathogenic Clostridia worldwide from soil to food animals to humans, which may be promoted by glyphosate use, is a great public health concern, not only for autism but perhaps for all the neuropsychiatric diseases that appear to be related to gastrointestinal Clostridia overgrowth These diseases include seizures, tremors, tic disorders, Parkinson disease, chronic fatigue syndrome, obsessive compulsive disorder, schizophrenia, bipolar and unipolar depression, ADHD, and anorexia nervosa.

The Job Demands and Accommodation Planning Tool (JDAPT): A Nine-Month Evaluation of Use, Changes in Self-efficacy, Presenteeism, and Absenteeism in Workers with Chronic and Episodic Disabilities.

PURPOSE: Enhancing workplace communication and support processes to enable individuals living with disabilities to sustain employment and return to work is a priority for workers, employers, and community stakeholders. The objective of this study was to evaluate a new resource that addresses support challenges, the Job Demands and Accommodation Planning Tool (JDAPT), and assess its use, relevance, and outcomes over a nine-month follow-up period. METHODS: Workers with physical and mental health/cognitive conditions causing limitations at work were recruited using purposive sampling. Online surveys were administered at baseline (prior to using the JDAPT), and at three and nine months post-baseline. Information was collected on demographics (e.g., age, gender) and work characteristics (e.g., job sector, organization size). Outcomes included assessing JDAPT use and relevance, and changes in self-efficacy, work productivity difficulties, employment concerns, difficulties with job demands, and absenteeism. RESULTS: Baseline participants were 269 workers (66% women; mean age 41 years) of whom 188 (69.9%) completed all three waves of data collection. Many workers reported using JDAPT strategies at and outside of work, and held positive perceptions of the tool's usability, relevance, and helpfulness. There were significant improvements (Time 1-2; Time 1-3) in self-efficacy, perceived work productivity, and absenteeism with moderate to large effect sizes in self-efficacy and productivity (0.46 to 0.78). Findings were consistent across gender, age, health condition, and work context variables. CONCLUSIONS: The JDAPT can enhance support provision and provide greater transparency and consistency to workplace disability practices, which is critical to creating more inclusive and accessible employment opportunities.

A tensor decomposition reveals ageing-induced differences in muscle and grip-load force couplings during object lifting.

Do motor patterns of object lifting movements change as a result of ageing? Here we propose a methodology for the characterization of these motor patterns across individuals of different age groups. Specifically, we employ a bimanual grasp-lift-replace protocol with younger and older adults and combine measurements of muscle activity with grip and load forces to provide a window into the motor strategies supporting effective object lifts. We introduce a tensor decomposition to identify patterns of muscle activity and grip-load force ratios while also characterizing their temporal profiles and relative activation across object weights and participants of different age groups. We then probe age-induced changes in these components. A classification analysis reveals three motor components that are differentially recruited between the two age groups. Linear regression analyses further show that advanced age and poorer manual dexterity can be predicted by the coupled activation of forearm and hand muscles which is associated with high levels of grip force. Our findings suggest that ageing may induce stronger muscle couplings in distal aspects of the upper limbs, and a less economic grasping strategy to overcome age-related decline in manual dexterity.

A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces cerevisiae.

Synthetic biology toolkits are one of the core foundations on which the field has been built, facilitating and accelerating efforts to reprogram cells and organisms for diverse biotechnological applications. The yeast Saccharomyces cerevisiae, an important model and industrial organism, has benefited from a wide range of toolkits. In particular, the MoClo Yeast Toolkit (YTK) enables the fast and straightforward construction of multigene plasmids from a library of highly characterized parts for programming new cellular behavior in a more predictable manner. While YTK has cultivated a strong parts ecosystem and excels in plasmid construction, it is limited in the extent and flexibility with which it can create new strains of yeast. Here, we describe a new and improved toolkit, the Multiplex Yeast Toolkit (MYT), that extends the capabilities of YTK and addresses strain engineering limitations. MYT provides a set of new integration vectors and selectable markers usable across common laboratory strains, as well as additional assembly cassettes to increase the number of transcriptional units in multigene constructs, CRISPR-Cas9 tools for highly efficient multiplexed vector integration, and three orthogonal and inducible promoter systems for conditional programming of gene expression. With these tools, we provide yeast synthetic biologists with a powerful platform to take their engineering ambitions to exciting new levels.

Metformin inhibits digestive proteases and impairs protein digestion in mice.

Metformin is among the most prescribed medications worldwide and the first-line therapy for type 2 diabetes. However, gastrointestinal side effects are common and can be dose limiting. The total daily metformin dose frequently reaches several grams, and poor absorption results in high intestinal drug concentrations. Here, we report that metformin inhibits the activity of enteropeptidase and other digestive enzymes at drug concentrations predicted to occur in the human duodenum. Treatment of mouse gastrointestinal tissue with metformin reduces enteropeptidase activity; further, metformin-treated mice exhibit reduced enteropeptidase activity, reduced trypsin activity, and impaired protein digestion within the intestinal lumen. These results indicate that metformin-induced protein maldigestion could contribute to the gastrointestinal side effects and other impacts of this widely used drug.

Gastrointestinal parasites in captive olive baboons in a UK safari park.

From the safety inside vehicles, Knowsley Safari offers visitors a close-up encounter with captive olive baboons. As exiting vehicles may be contaminated with baboon stool, a comprehensive coprological inspection was conducted to address public health concerns. Baboon stools were obtained from vehicles, and sleeping areas, inclusive of video analysis of baboon­vehicle interactions. A purposely selected 4-day sampling period enabled comparative inspections of 2662 vehicles, with a total of 669 baboon stools examined (371 from vehicles and 298 from sleeping areas). As informed by our pilot study, front-line diagnostic methods were: QUIK-CHEK rapid diagnostic test (RDT) (Giardia and Cryptosporidium), Kato­Katz coproscopy (Trichuris) and charcoal culture (Strongyloides). Some 13.9% of vehicles were contaminated with baboon stool. Prevalence of giardiasis was 37.4% while cryptosporidiosis was <0.01%, however, an absence of faecal cysts by quality control coproscopy, alongside lower than the expected levels of Giardia-specific DNA, judged RDT results as misleading, grossly overestimating prevalence. Prevalence of trichuriasis was 48.0% and strongyloidiasis was 13.7%, a first report of Strongyloides fuelleborni in UK. We advise regular blanket administration(s) of anthelminthics to the colony, exploring pour-on formulations, thereafter, smaller-scale indicator surveys would be adequate.

Interplay between nutrient transporters ensures fertility in the malaria mosquito Anopheles gambiae.

Females from many mosquito species feed on blood to acquire nutrients for egg development. The oogenetic cycle has been characterized in the arboviral vector Aedes aegypti, where after a bloodmeal, the lipid transporter lipophorin (Lp) shuttles lipids from the midgut and fat body to the ovaries, and a yolk precursor protein, vitellogenin (Vg), is deposited into the oocyte by receptor-mediated endocytosis. Our understanding of how the roles of these two nutrient transporters are mutually coordinated is however limited in this and other mosquito species. Here, we demonstrate that in the malaria mosquito Anopheles gambiae, Lp and Vg are reciprocally regulated in a timely manner to optimize egg development and ensure fertility. Defective lipid transport via Lp silencing triggers abortive ovarian follicle development, leading to misregulation of Vg and aberrant yolk granules. Conversely, depletion of Vg causes an upregulation of Lp in the fat body in a manner that appears to be at least partially dependent on target of rapamycin (TOR) signaling, resulting in excess lipid accumulation in the developing follicles. Embryos deposited by Vg-depleted mothers are completely infertile, and are arrested early during development, likely due to severely reduced amino acid levels and protein synthesis. Our findings demonstrate that the mutual regulation of these two nutrient transporters is essential to safeguard fertility by ensuring correct nutrient balance in the developing oocyte, and validate Vg and Lp as two potential candidates for mosquito control.

Qualitative Assessment of Perceived Organizational Support for Employed Breast Cancer Survivors.

OBJECTIVE: A more detailed understanding of unmet organizational support needs and workplace-based best practices for supporting cancer survivors is needed. METHODS: Ninety-four working breast cancer survivors responded to an open-ended survey question regarding the desired types of organizational support that were and were not received during early survivorship. We performed content-analysis of qualitative data. RESULTS: Major themes included instrumental support, emotional support, and time-based support. The need for flexible arrangements and reduced workloads was mostly met. Unmet needs included navigation/coordination, understanding/empathy, and time off for treatment and recovery. CONCLUSIONS: Organizational support can help cancer survivors manage their health and work roles, diminishing work-health conflict and turnover intent. Study findings can be used to design targeted interventions to fulfill cancer survivors' unmet organizational support needs, which may also apply to workers with other chronic health conditions.

Inhibition of the Beta-oxidation Pathway of Fatty Acids and Dopamine- Beta-hydroxylase by Phenyl Derivatives of Short- Chain Fatty Acids from Gastrointestinal Clostridia Bacteria is a (the) Major Cause of Autism.

Background: A The author's comprehensive evaluation of the biochemical metabolomic literature over more than 40 years discusses multiple studies documenting abnormal elevations of the neurotransmitter dopamine and its metabolites as well as inhibitors of dopamine beta hydroxylase (DBH) from Clostridia bacteria in urine samples and cerebrospinal fluid samples of children with autism. Aims of Review: The evaluation intends to elucidate the reasons for the elevation of dopamine and its metabolites in urine and their relationship to increased Clostridia colonization of the gastrointestinal tract in children with autism. In addition, to the evaluation of Clostridia metabolism and its effects on abnormal dopamine metabolism in autism, a secondary aim intends to demonstrate as a hypothesis that one particular metabolite of Clostridia bacteria-3-hydroxy-(3-hydroxyphenyl)- 3-hydroxypropionic acid (HPHPA)-may cause even more severe effects on in autism than other metabolites by leading to depletion of free coenzyme A (CoASH). This depletion of free Coenzyme A leads to a deficiency of cholesterol and activated palmitic acid needed for activation of the key brain developmental protein sonic hedgehog, which has recently been research has shown to be severely abnormal in severe autism. Key Scientific Concepts of Review: Laboratories throughout the world have consistently found high quantities of HPHPA and 4-cresol in high percentages of urine samples of children with autism. Those inhibitors, which intestinal Clostridia bacteria produce, cause an elevation in dopamine and its metabolites, which affect the brain's and the sympathetic nervous system's key enzyme dopamine-beta-hydroxylase (DBH). Excessive dopamine and its toxic metabolites due to these DBH inhibitors may cause brain damage due to excessive unstable dopamine quinones, toxic adducts of dopamine disrupting brain mitochondrial energy production, and oxygen superoxide. HPHPA, a short chain phenyl compound, may have additional biochemical effects on the brain in autism, causing a reduction in free CoASH needed to produce the CoA palmitic acid derivative necessary to activate the key brain developmental protein sonic hedgehog. The depletion of CoASH appears to be a new therapeutic target to reverse the adverse effects of the HPHPA metabolite on the beta oxidation of fatty acids and cholesterol synthesis that are prevalent in autism. Conclusions: Variations in the severity of autism could be based on the types and concentrations of the Clostridia markers produced and the extent to which these markers, such as HPHPA, have depleted critical lipids, such as cholesterol and CoA palmitic acid derivative. Patients need those lipids for the activation of the developmental protein sonic hedgehog. In addition, the sequestration of coenzyme A by short chain adducts of Clostridia leads to the depletion of critical free CoASH, needed throughout intermediary metabolism, and creates a biochemical storm that especially affects brain function.

Modular Metabolic Engineering and Synthetic Coculture Strategies for the Production of Aromatic Compounds in Yeast.

Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast Saccharomyces cerevisiae to produce aromatic molecules by exploiting the concept of modularity in synthetic biology. Three different modular approaches were investigated for the production of the valuable fragrance raspberry ketone (RK), found in raspberry fruits and mostly produced from petrochemicals. The first strategy used was modular cloning, which enabled the generation of combinatorial libraries of promoters to optimize the expression level of the genes involved in the synthesis pathway of RK. The second strategy was modular pathway engineering and involved the creation of four modules, one for product formation: RK synthesis module (Mod. RK); and three for precursor synthesis: aromatic amino acid synthesis module (Mod. Aro), p-coumaric acid synthesis module (Mod. p-CA), and malonyl-CoA synthesis module (Mod. M-CoA). The production of RK by combinations of the expression of these modules was studied, and the best engineered strain produced 63.5 mg/L RK from glucose, which is the highest production described in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported in any organism without p-coumaric acid supplementation. The third strategy was the use of modular cocultures to explore the effects of division of labor on RK production. Two two-member communities and one three-member community were created, and their production capacity was highly dependent on the structure of the synthetic community, the inoculation ratio, and the culture media. In certain conditions, the cocultures outperformed their monoculture controls for RK production, although this was not the norm. Interestingly, the cocultures showed up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone, the direct precursor of RK, which can be used for the semi-synthesis of RK. This study illustrates the utility of modularity in synthetic biology tools and their applications to the synthesis of products of industrial interest.

The Job Leeway Scale: Initial Evaluation of a Self-report Measure of Health-Related Flexibility and Latitude at Work.

Purpose Evidence suggests that workers manage health-related challenges at work, in part, by using available leeway to perform work differently. The purpose of this study was to evaluate the reliability and validity of the Job Leeway Scale (JLS), a new 18-item self-report questionnaire designed to assess worker perceptions of available flexibility and latitude to manage health-related challenges at work. Methods Workers seeking assistance for workplace difficulties due to chronic medical conditions (n = 119, 83% female, median age = 49) completed the JLS along with other workplace and health measures. Construct validity was assessed using exploratory factor analysis (EFA), and concurrent validity was assessed by associations with related measures. Results Mean item scores ranged from 2.13 to 4.16 within a possible range of 0-6. The EFA supported three underlying factors: organizational leeway (9 items), task leeway (6 items), and staffing leeway (3 items). Internal consistency (alpha) ranged from 0.78 to 0.91 for subscale scores and 0.94 for the total score. The JLS showed moderate correlations with other work outcome measures including work fatigue, self-efficacy, engagement, and productivity. Conclusion The JLS is a promising new measure with initial support for its reliability and validity to assess worker beliefs of available flexibility to manage health symptoms at work, and this construct may have organizational implications for worker support and accommodation.

A Sensibility Assessment of the Job Demands and Accommodation Planning Tool (JDAPT): A Tool to Help Workers with an Episodic Disability Plan Workplace Support.

Purpose Sensibility refers to a tool's comprehensiveness, understandability, relevance, feasibility, and length. It is used in the early development phase to begin assessing a new tool or intervention. This study examined the sensibility of the job demands and accommodation planning tool (JDAPT). The JDAPT identifies job demands related to physical, cognitive, interpersonal, and working conditions to better target strategies for workplace supports and accommodations aimed at assisting individuals with chronic health conditions. Methods Workers with a chronic health condition and workplace representatives were recruited from health charities, workplaces, and newsletters using convenience sampling. Cognitive interviews assessed the JDAPT's sensibility. A 70% endorsement rate was the minimum level of acceptability for sensibility concepts. A short screening tool also was administered, and answers compared to the complete JDAPT. Results Participants were 46 workers and 23 organizational representatives (n = 69). Endorsements highly exceeded the 70% cut-off for understandability, relevance, and length. Congruence between screening questions and the complete JDAPT suggested both workers and organizational representatives overlooked job demands when completing the screener. Participants provided additional examples and three new items to improve comprehensiveness. The JDAPT was rated highly relevant and useful, although not always easy to complete for someone with an episodic condition. Conclusions This study highlights the need for tools that facilitate accommodations for workers with episodic disabilities and provides early evidence for the sensibility of the JDAPT.

Preventing Pain and Stress-Related Ill-Health in Employees: A 6-Months Follow-Up of a Psychosocial Program in a Cluster Randomized Controlled Trial.

Purpose Pain and stress-related ill-health are major causes of long-term disability and sick leave. This study evaluated the effects of a brief psychosocial program, which previously has been tested for an at-risk population of employees. Methods The Effective Communication within the Organization (ECO) program, where supervisors and employees were trained in communication and problem solving, was compared to an active control consisting of psychoeducative lectures (PE) about pain and stress in a cluster randomized controlled trial. First-line supervisors were randomized to ECO or PE, and a total of 191 mainly female employees with self-reported pain and/or stress-related ill-health were included. The hybrid format programs consisted of 2-3 group sessions. Sick leave data was collected from social insurance registers, before and 6-months after the program. Secondary outcomes (work ability, work limitations, pain-disability risk, exhaustion symptoms, perceived stress, perceived health, quality of life, perceived communication and support from supervisors) were assessed at baseline, post intervention, and at 6-months follow-up. Results No effects were observed on primary or secondary outcome variables. Pain symptoms were common (89%), however a lower proportion (30%) were identified as at risk for long-term pain disability, which might explain the lack of evident effects. The Covid-19 pandemic affected participation rates and delivery of intervention. Conclusion In this study, preventive effects of the ECO program were not supported. Altogether, the findings point at the importance of selecting participants for prevention based on screening of psychosocial risk. Further research on workplace communication and support, and impact on employee health is warranted.

Early Secondary Alveolar Bone Grafting and Facial Growth of Patients with Complete Unilateral Cleft Lip and Palate.

OBJECTIVE: To investigate the craniofacial growth outcomes of early secondary alveolar bone grafting(ABG) around 6 years of age. DESIGN: Retrospective cohort study. SETTING: 1 North-American and 5 Northern-European cleft centers. SUBJECTS: 33 subjects with CUCLP consecutively treated with secondary ABG around 6 years of age were compared to 105 subjects from 4 centers treated with late secondary ABG and 19 subjects from 1 center with primary ABG. METHODS: Preorthodontic standardized lateral cephalometric radiographs taken after 12 years of age were traced and analyzed according to the Eurocleft Study protocol. Fourteen angular and two proportional measurements were performed. Measurement means from the Study Center(SC) were compared to 5 Northern-European centers using analysis of variance and Welch's modified t-tests, and P < .05 was considered statistically significant. RESULTS: For the SC, the mean age ± SD at the time of bone graft was 5.85 ± 0.71 years and the mean age at the time of the lateral cephalogram was 13.4 ± 1.8 years. The sagittal maxillary prominence of the SC was favorably comparable to the 5 Northern-European centers. The mean SNA (78.1 ± 4.3) for the SC was significantly higher compared to 4 of the 5 Northern-European centers(all P < .05), and the mean ANB angle was comparable to 4 of the 5 centers. Similarly, the mean soft tissue ANB angle was not significantly different to the 5 centers. The soft tissue vertical proportions compared favorably to all 5 Northern-European centers(all P < .01). CONCLUSIONS: Craniofacial growth outcomes of early secondary ABG around 6 years compare favorably to the outcomes of late secondary ABG.

Inter-rater reliability in classification of canonical babbling status based on canonical babbling ratio in infants with isolated cleft palate randomised to Timing of Primary Surgery for Cleft Palate (TOPS).

Canonical babbling (CB) is commonly defined as present when at least 15% of all syllables produced are canonical, in other words a canonical babbling ratio (CBR) ≥0.15. However, there is limited knowledge about inter-rater reliability in classification of CB status based on CBR and inter-rater differences in assessment of CBR. We investigated inter-rater reliability of experienced Speech Language Therapists (SLTs) on: classification of CB status based on CBR ≥ 0.15, CBRs and the total number of syllables per infant used to calculate CBR.Each infant (n = 484) was video-recorded at a clinical site in play interaction with their parent as part of the randomised controlled trial Timing of Primary Surgery for Cleft Palate. Each recording was subsequently assessed by three independent SLTs, from a pool of 29 SLTs. They assessed the recordings in real time.The three assessing SLTs agreed in classification of CB status in 423 (87.4%) infants, with higher complete agreement for canonical (91%; 326/358) than non-canonical (77%; 97/126). The average difference in CBR and total number of syllables identified between the SLT assessments of each infant was 0.12 and 95, respectively.This study provided new evidence that one trained SLT can reliably classify CB status (CBR ≥ 0.15) in real time when there is clear distinction between the observed CBR and the boundary (0.15); however, when the observed CBR approaches the boundary multiple SLT assessments are beneficial. Thus, we recommend to include assessment of inter-rater reliability, if the purpose is to compare CBR and total syllable count across infants or studies.Trial registration number here: www.clinicaltrials.gov, identifier NCT00993551.

Screening microbially produced Δ9-tetrahydrocannabinol using a yeast biosensor workflow.

Microbial production of cannabinoids promises to provide a consistent, cheaper, and more sustainable supply of these important therapeutic molecules. However, scaling production to compete with traditional plant-based sources is challenging. Our ability to make strain variants greatly exceeds our capacity to screen and identify high producers, creating a bottleneck in metabolic engineering efforts. Here, we present a yeast-based biosensor for detecting microbially produced Δ9-tetrahydrocannabinol (THC) to increase throughput and lower the cost of screening. We port five human cannabinoid G protein-coupled receptors (GPCRs) into yeast, showing the cannabinoid type 2 receptor, CB2R, can couple to the yeast pheromone response pathway and report on the concentration of a variety of cannabinoids over a wide dynamic and operational range. We demonstrate that our cannabinoid biosensor can detect THC from microbial cell culture and use this as a tool for measuring relative production yields from a library of Δ9-tetrahydrocannabinol acid synthase (THCAS) mutants.

Inducible expression of large gRNA arrays for multiplexed CRISPRai applications.

CRISPR gene activation and inhibition (CRISPRai) has become a powerful synthetic tool for influencing the expression of native genes for foundational studies, cellular reprograming, and metabolic engineering. Here we develop a method for near leak-free, inducible expression of a polycistronic array containing up to 24 gRNAs from two orthogonal CRISPR/Cas systems to increase CRISPRai multiplexing capacity and target gene flexibility. To achieve strong inducibility, we create a technology to silence gRNA expression within the array in the absence of the inducer, since we found that long gRNA arrays for CRISPRai can express themselves even without promoter. Using this method, we create a highly tuned and easy-to-use CRISPRai toolkit in the industrially relevant yeast, Saccharomyces cerevisiae, establishing the first system to combine simultaneous activation and repression, large multiplexing capacity, and inducibility. We demonstrate this toolkit by targeting 11 genes in central metabolism in a single transformation, achieving a 45-fold increase in succinic acid, which could be precisely controlled in an inducible manner. Our method offers a highly effective way to regulate genes and rewire metabolism in yeast, with principles of gRNA array construction and inducibility that should extend to other chassis organisms.

Clinical acceptability of fully automated external beam radiotherapy for cervical cancer with three different beam delivery techniques.

PURPOSE: To fully automate CT-based cervical cancer radiotherapy by automating contouring and planning for three different treatment techniques. METHODS: We automated three different radiotherapy planning techniques for locally advanced cervical cancer: 2D 4-field-box (4-field-box), 3D conformal radiotherapy (3D-CRT), and volumetric modulated arc therapy (VMAT). These auto-planning algorithms were combined with a previously developed auto-contouring system. To improve the quality of the 4-field-box and 3D-CRT plans, we used an in-house, field-in-field (FIF) automation program. Thirty-five plans were generated for each technique on CT scans from multiple institutions and evaluated by five experienced radiation oncologists from three different countries. Every plan was reviewed by two of the five radiation oncologists and scored using a 5-point Likert scale. RESULTS: Overall, 87%, 99%, and 94% of the automatically generated plans were found to be clinically acceptable without modification for the 4-field-box, 3D-CRT, and VMAT plans, respectively. Some customizations of the FIF configuration were necessary on the basis of radiation oncologist preference. Additionally, in some cases, it was necessary to renormalize the plan after it was generated to satisfy radiation oncologist preference. CONCLUSION: Approximately, 90% of the automatically generated plans were clinically acceptable for all three planning techniques. This fully automated planning system has been implemented into the radiation planning assistant for further testing in resource-constrained radiotherapy departments in low- and middle-income countries.