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Obtener híbridos comerciales competitivos de maíz (Zea mays L.) requiere de estrategias de selección eficientes para mejorar poblaciones y formar progenitores élite. El objetivo de esta investigación fue seleccionar líneas y cruzas de alta aptitud combinatoria general (ACG) y específica (ACE) para rendimiento de grano (RG) y características agronómicas, al combinar las pruebas de mestizos (línea × probador) y cruzas dialélicas. En el ciclo agrícola otoño-invierno 2010-2011 se formaron 144 mestizos con líneas S3 de la población subtropical ST C2 F2, y como probador la población subtropical PABGT-CE C9 F2. Los mestizos se evaluaron en secano en el ciclo agrícola primavera-verano (PV) 2012 en Tlajomulco y Tepatitlán, Jalisco, Peña del Panal, Michoacán, y Santa María del Oro, Nayarit, que cubren un intervalo de 1100 a 1900 m de altitud. En esta prueba se seleccionaron, a través de localidades, nueve mestizos por ACG para RG y tolerancia al acame. Con la semilla remanente de las líneas de mestizos seleccionados, en PV 2015 se formaron cruzas dialélicas. Las cruzas se evaluaron en PV 2016 en las localidades anteriores e Iguala, Guerrero. Las líneas ST C2 F2-21-2-1 y ST C2 F2-29-2-1 sobresalieron en ambas pruebas por su alta ACG para RG, pero la última se eliminó porque en cruzas dialélicas la ACG y ACE para acame fueron positivas y significativas (P ≤ 0.05). La ACE de la cruza ST C2 F2-33-1-1 × ST C2 F2-49-2-1 para RG fue significativa (P ≤ 0.05), y en acame negativa, pero no significativa. El asociar las pruebas de mestizos y cruzas dialélicas permitió seleccionar de una forma más rigurosa líneas con alta ACG y ACE para RG y características agronómicas, además de eliminar líneas antes de formar el sintético y obtener cruzas simples que podrían usarse para formar híbridoscon progenitores élite del otro componente heterótico.

Hepatitis C virus (HCV) testing in persons born from 1945 to 1965 has had limited adoption despite guidelines, particularly among racial/ethnic minorities and socioeconomically disadvantaged patients, who have a higher prevalence of disease burden. We examined the effectiveness of a multifaceted intervention to improve HCV screening in a large safety-net health system. We performed a multifaceted intervention that included provider and patient education, an electronic medical record-enabled best practice alert, and increased HCV treatment capacity. We characterized HCV screening completion before and after the intervention. To identify correlates of HCV screening, we performed logistic regression for the preintervention and postintervention groups and used a generalized linear mixed model for patients observed in both preintervention and postintervention time frames. Before the intervention, 10.1% of 48,755 eligible baby boomer patients were screened. After the intervention, 34.6% of the 34,093 eligible baby boomers were screened (P < 0.0001). Prior to the intervention, HCV screening was lower among older baby boomers and providers with large patient panels and higher in high-risk subgroups including those with signs of liver disease (e.g., elevated transaminases, thrombocytopenia), human immunodeficiency virus-positive patients, and homeless patients. Postintervention, we observed increased screening uptake in older baby boomers, providers with larger patient panel size, and patients with more than one prior primary care appointment. Conclusion: Our multifaceted intervention significantly increased HCV screening, particularly among older patients, those engaged in primary care, and providers with large patient panels.

121 Background: As part of a CPRIT prevention services grant led by UTSW, Parkland Health adapted a patient navigation model previously piloted in 2017 to optimize low-dose computed tomography (LDCT) imaging for lung cancer screening and evidence-based tobacco cessation services. Methods: Non-clinical patient navigators (PN) contacted patients with a LDCT order to discuss their lung scan and smoking cessation options via telephone encounters. The PN has three planned touchpoints to assess patient knowledge, identify barriers, and refer to appropriate resources. Program evaluation data has been collected monthly for enrolled patients (n = 460) from August 2021-June 2022 via REDCap and EPIC. Navigation excluded patients who were incarcerated, had no phone number listed in medical record, or patients opted out of the program on intake. Results: Of the LDCT-eligible patients, PNs were able to complete two or more navigation calls with 55.62% of patients assigned. 77% of patients completed their first LDCT, compared to 80% in the initial trial. The most common barriers to screening completion included transportation (n = 38), insurance coverage (n = 22), and cost (n = 9). Of the navigated patients who were active smokers (75%), 33% scheduled at least one smoking cessation-related visit. Some patients unresponsive to conversations about quitting, were responsive to language around “reducing” tobacco use. Conclusions: Disproportionately under- and uninsured, Parkland Health patients face a variety of barriers to screening. As a result, PN staff must balance reaching all patients with a LDCT order and spending additional time navigating patients with complex barriers. To address these barriers, the PN program facilitates coordination of patients between primary care providers, radiology operations, smoking cessation clinics, patient financial services, lung diagnostic clinic, and social work. For example, patients can schedule a smoking cessation visit directly with our PN instead of being referred. As Parkland has a decentralized LCS program model, having embedded non-clinical PNs within existing roles in the cancer center, like medical practice assistants, increases continuity of care. These individuals have the benefit from day-to-day knowledge of downstream services that bolster their ability to connect patients to external services to address barriers.

Key Points Question What are the barriers to coordinating care for underinsured and uninsured patients with cancer and chronic conditions during and after cancer treatment at a safety-net health system? Findings This qualitative study of interviews and observation of 93 health system stakeholders identified system-level challenges (ie, challenges to accessing primary care, lack of communication between oncology and primary care clinicians, leadership awareness of care coordination challenges) and clinician-level challenges (ie, unclear role delineation, lack of clinician knowledge and preparedness to manage cancer and chronic conditions). Meaning These findings suggest that survivorship care delivery strategies that bridge oncology and primary care may be required to meet the care coordination needs of underinsured and uninsured patients with cancer amid the fragmented US health care system., This qualitative study assesses barriers to coordinating care for patients with cancer and chronic conditions during and after cancer treatment., Importance Although a majority of underinsured and uninsured patients with cancer have multiple comorbidities, many lack consistent connections with a primary care team to manage chronic conditions during and after cancer treatment. This presents a major challenge to delivering high-quality comprehensive and coordinated care. Objective To describe challenges and opportunities for coordinating care in an integrated safety-net system for patients with both cancer and other chronic conditions. Design, Setting, and Participants This multimodal qualitative study was conducted from May 2016 to July 2019 at a county-funded, vertically integrated safety-net health system including ambulatory oncology, urgent care, primary care, and specialty care. Participants were 93 health system stakeholders (clinicians, leaders, clinical, and administrative staff) strategically and snowball sampled for semistructured interviews and observation during meetings and daily processes of care. Data collection and analysis were conducted iteratively using a grounded theory approach, followed by systematic thematic analysis to organize data, review, and interpret comprehensive findings. Data were analyzed from March 2019 to March 2020. Main Outcomes and Measures Multilevel factors associated with experiences of coordinating care for patients with cancer and chronic conditions among oncology and primary care stakeholders. Results Among interviews and observation of 93 health system stakeholders, system-level factors identified as being associated with care coordination included challenges to accessing primary care, lack of communication between oncology and primary care clinicians, and leadership awareness of care coordination challenges. Clinician-level factors included unclear role delineation and lack of clinician knowledge and preparedness to manage the effects of cancer and chronic conditions. Conclusions and Relevance Primary care may play a critical role in delivering coordinated care for patients with cancer and chronic diseases. This study’s findings suggest a need for care delivery strategies that bridge oncology and primary care by enhancing communication, better delineating roles and responsibilities across care teams, and improving clinician knowledge and preparedness to care for patients with cancer and chronic conditions. Expanding timely access to primary care is also key, albeit challenging in resource-limited safety-net settings.

Transient expression systems in mesophyll protoplasts have been utilised in many plant species as an indispensable tool for gene function analysis and efficacious genome editing constructs. However, such a system has not been developed inCannabisdue to the recalcitrant nature of the plant to tissue culture as well as its illegal status for many years. In this study, young expanding leaves from asepticin vitro Cannabisexplants were used for protoplast isolation. Factorial designs were used to optimise variables in viable protoplast isolation and transient expression of GFP, with a range analyses performed to determine, and quantify, significantly impacting variables. Viable protoplast yields as high as 5.7 × 106were achieved with 2.5% (w/v) Cellulase R-10, 0.3% (w/v) Macerozyme R-10 and 0.7 M mannitol, incubated for 16 h. As indicated by the transient expression of GFP, efficiency reached 23.2% with 30 μg plasmid, 50% PEG, 1 × 106protoplasts and a transfection duration of 20 min. Application of the optimised protocol for protoplast isolation was successfully evaluated on three subsequent unrelated genotypes to highlight the robustness and broad applicability of the developed technique.

Appropriate design and selection of PCR primers plays a critical role in determining the sensitivity and specificity of a metabarcoding assay. Despite several studies applying metabarcoding to insect pest surveillance, the diagnostic performance of the short “mini-barcodes” required by high-throughput sequencing platforms has not been established across the broader taxonomic diversity of invasive insects. We address this by computationally evaluating the diagnostic sensitivity and predicted amplification bias for 68 published and novel cytochrome c oxidase subunit 1 (COI) primers on a curated database of 110,676 insect species, including 2,625 registered on global invasive species lists. We find that mini-barcodes between 125-257 bp can provide comparable resolution to the full-length barcode for both invasive insect pests and the broader Insecta, conditional upon the subregion of COI targeted and the genetic similarity threshold used to identify species. Taxa that could not be identified by any barcode lengths were phylogenetically clustered within ‘problem groups’, many arising through taxonomic inconsistencies rather than insufficient diagnostic information within the barcode itself. Substantial variation in predicted PCR bias was seen across published primers, with those including 4-5 degenerate nucleotide bases showing almost no mismatch to major insect orders. While not completely universal, a single COI mini-barcode can successfully differentiate the majority of pest and non-pest insects from their congenerics, even at the small amplicon size imposed by 2 × 150 bp sequencing. We provide a ranked summary of high-performing primers and discuss the bioinformatic steps required to curate reliable reference databases for metabarcoding studies.

A FinnishFrom June to October 2019, specimens with discrepant AC2/ACT CT results were submitted to Public Health England and screened for detectable CT DNA using an inhouse real-time (RT)-PCR. When enough DNA was present, partial CT 23S rRNA gene sequencing was performed. Analysis of available relative light units and interpretative data was performed.A total of 317 discordant AC2/ACT specimens were collected from 315 patients. Three hundred were tested on the RT-PCR; 53.3% (n=160) were negative and 46.7% (n=140) were positive. Due to low DNA load in most specimens, sequencing was successful for only 36 specimens. The CT 23S rRNA wild-type sequence was present in 32 specimens, and two variants with C1514T or G1523A mutation were detected in four specimens from three patients. Of the discordant specimens with NG interpretation, 36.6% of NG-negative/CT-negative AC2 specimens had detectable CT DNA on the inhouse RT-PCR vs 53.3% of NG-positive/CT-negative specimens.No widespread dissemination of AC2 diagnostic-escape CT variants has occurred in England. We however identified the impact of NG positivity on the discordant AC2/ACT specimens; a proportion appeared due to NG positivity and the associated NG signal, rather than any diagnostic-escape variants or low DNA load. Several patients with gonorrhoea may therefore receive false-negative AC2 CT results. Single diagnostic targets and multiplex diagnostic assays have their limitations such as providing selection pressure for escape mutants and potentially reduced sensitivity, respectively. These limitations must be considered when establishing diagnostic pathways.

Additional file 2: Figure S1 Prediction equation under various ratios of selection pressure. This figure includes a scatterplot for observed versus predicted Bud Dry Weight (BDW) under different ratios of selection pressure. Predicted values are derived from the multiple regression prediction equation (Equation 1, see manuscript). Dot point colours represent prediction accuracy; black colouring consists of 59 genotypes and marks the prediction within 80%-100% accuracy, brown colouring consists of 41 genotypes and marks the prediction within 60% - 80% accuracy and grey colouring consists of 21 genotypes and marks the prediction that is less than 60% accuracy. Red, green, blue and yellow broken lines indicate the BDW value which defined the top 10%, 15%, 20% and 33% observed and predicted genotypes, respectively. Section I signifies matches between predicted and observed high performing genotypes; Section II contains genotypes identified as false negatives by the prediction equation; Section III signifies matches between predicted and observed low performing genotypes and Section IV contains genotypes identified as false positives by the prediction equation.

Additional file 5: Figure S1 Trial design and spatial arrangement. Black boxes represent rolling benches, red and blue lines represent room rows and room columns, respectively and green numbers indicate the first and last plot on each bench.

Additional file 4: The determination of replicate quantities.

Additional file 3.

Background Cannabis has been used worldwide for centuries for industrial, recreational and medicinal use, however, to date no successful attempts at editing genes involved in cannabinoid biosynthesis have been reported. This study proposes and develops an in silico best practices approach for the design and implementation of genome editing technologies in cannabis to target all genes involved in cannabinoid biosynthesis. Results A large dataset of reference genomes was accessed and mined to determine copy number variation and associated SNP variants for optimum target edit sites for genotype independent editing. Copy number variance and highly polymorphic gene sequences exist in the genome making genome editing using CRISPR, Zinc Fingers and TALENs technically difficult. Evaluation of allele or additional gene copies was determined through nucleotide and amino acid alignments with comparative sequence analysis performed. From determined gene copy number and presence of SNPs, multiple online CRISPR design tools were used to design sgRNA targeting every gene, accompanying allele and homologs throughout all involved pathways to create knockouts for further investigation. Universal sgRNA were designed for highly homologous sequences using MultiTargeter and visualised using Sequencher, creating unique sgRNA avoiding SNP and shared nucleotide locations targeting optimal edit sites. Conclusions Using this framework, the approach has wider applications to all plant species regardless of ploidy number or highly homologous gene sequences. Significance statement Using this framework, a best-practice approach to genome editing is possible in all plant species, including cannabis, delivering a comprehensive in silico evaluation of the cannabinoid pathway diversity from a large set of whole genome sequences. Identification of SNP variants across all genes could improve genome editing potentially leading to novel applications across multiple disciplines, including agriculture and medicine.

Cannabis is a diploid species (2n = 20), the estimated haploid genome sizes of the female and male plants using flow cytometry are 818 and 843 Mb respectively. Although the genome of Cannabis has been sequenced (from hemp, wild and high-THC strains), all assemblies have significant gaps. In addition, there are inconsistencies in the chromosome numbering which limits their use. A new comprehensive draft genome sequence assembly (~900 Mb) has been generated from the medicinal cannabis strain Cannbio-2, that produces a balanced ratio of cannabidiol and delta-9-tetrahydrocannabinol using long-read sequencing. The assembly was subsequently analysed for completeness by ordering the contigs into chromosome-scale pseudomolecules using a reference genome assembly approach, annotated and compared to other existing reference genome assemblies. The Cannbio-2 genome sequence assembly was found to be the most complete genome sequence available based on nucleotides assembled and BUSCO evaluation in Cannabis sativa with a comprehensive genome annotation. The new draft genome sequence is an advancement in Cannabis genomics permitting pan-genome analysis, genomic selection as well as genome editing.