Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification.

CRISPR base editing screens enable analysis of disease-associated variants at scale; however, variable efficiency and precision confounds the assessment of variant-induced phenotypes. Here, we provide an integrated experimental and computational pipeline that improves estimation of variant effects in base editing screens. We use a reporter construct to measure guide RNA (gRNA) editing outcomes alongside their phenotypic consequences and introduce base editor screen analysis with activity normalization (BEAN), a Bayesian network that uses per-guide editing outcomes provided by the reporter and target site chromatin accessibility to estimate variant impacts. BEAN outperforms existing tools in variant effect quantification. We use BEAN to pinpoint common regulatory variants that alter low-density lipoprotein (LDL) uptake, implicating previously unreported genes. Additionally, through saturation base editing of LDLR, we accurately quantify missense variant pathogenicity that is consistent with measurements in UK Biobank patients and identify underlying structural mechanisms. This work provides a widely applicable approach to improve the power of base editing screens for disease-associated variant characterization.

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification.

CRISPR base editing screens are powerful tools for studying disease-associated variants at scale. However, the efficiency and precision of base editing perturbations vary, confounding the assessment of variant-induced phenotypic effects. Here, we provide an integrated pipeline that improves the estimation of variant impact in base editing screens. We perform high-throughput ABE8e-SpRY base editing screens with an integrated reporter construct to measure the editing efficiency and outcomes of each gRNA alongside their phenotypic consequences. We introduce BEAN, a Bayesian network that accounts for per-guide editing outcomes and target site chromatin accessibility to estimate variant impacts. We show this pipeline attains superior performance compared to existing tools in variant classification and effect size quantification. We use BEAN to pinpoint common variants that alter LDL uptake, implicating novel genes. Additionally, through saturation base editing of LDLR, we enable accurate quantitative prediction of the effects of missense variants on LDL-C levels, which aligns with measurements in UK Biobank individuals, and identify structural mechanisms underlying variant pathogenicity. This work provides a widely applicable approach to improve the power of base editor screens for disease-associated variant characterization.

Phytophthora cactorum causing bleeding canker of Acer x freemanii in southern Idaho.

Since 2018, bleeding cankers have been observed on maple trees in multiple home gardens in southwest Idaho. The cankers ooze a dark sap and and are approximately 10 cm to 35 cm in diameter. Cankers typically occur on the main trunk but are also present on scaffold branches in severe infecrions. Symptoms of foliar chlorois, branch dieback, and premature autumn senescence were also associated with the disease. Phytophthora DNA was detected in symptomatic material from five trees using real-time PCR (Miles et al., 2017). In July 2019 recovery of a causal agent from a symptomatic Acer x freemanii tree was attempted. Excisions were made from the interface of healthy and diseased tissue around the cankers using a chisel. The tissue was then placed in sealed plastic ziplock bags at 4°C for 7 days. Hyphae were then removed with forceps and placed onto potato dextrose agar (PDA) amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). Colonies resembling Phytophthora cactorum were consistently observed after 5 days at 21°C. Tentative P. cactorum identification was based on the presence of abundant papillate and caducous sporangia on a short pedicel; sporangia were approximately 30 µm long and 26 µm wide (Bush et al., 2006; Hudler, 2013). Individual hyphal tips were transferred to fresh PDA plates and sequencing of both the rDNA ITS region and Cytochrome c oxidase subunit I (COI) was completed for a representative isolate (D19-130). DNA extraction, PCR and sequencing were as previously described (Woodhall et al. 2013; Robideau et al., 2011). The resulting DNA sequences for rDNA ITS (MW315449) and COI (MW881040) were both 100% identical (723/723 bp and 728/728 bp) with sequences from cultures previously identified as P. cactorum (MH171627 and MH136858). To determine pathogenicity, 14 month-old maple (A. x freemanii) trees in individual containers with potting mix were wounded 15 mm above the soil line with a single 10 mm incision using a sterile razor blade and inoculated by placing a 10 mm2 fully colonized PDA plug of isolate D19-130 on the wound. The inoculum and wound were then covered with a damp cotton ball that was secured loosely with parafilm. Control plants consisted of uninoculated plants and wounded plants inoculated with a PDA agar plug. Each treatment was replicated five times and placed in a controlled environment chamber set at 24ºC and 90% relative humidity. All treatments were sprayed with water daily to ensure the cotton balls remained damp. After 8 weeks, black lesions, up to approximately 25 mm above the soil line, were observed on the stem base of all P. cactorum-inoculated plants. No black lesions were observed on non-inoculated plants or plants inoculated with a PDA agar plug. P. cactorum was isolated from lesions, as described above, except polystyrene foam boxes containing moist paper towels were used instead of bags. This report confirms P. cactorum as a causal agent of bleeding canker of maple in Idaho for the first time. It has been shown that several Phytophthora species can infect maple (Jung and Burgess, 2009; Huddler, 2013). P. cactorum has a wide host range but certain strains have been associated with lethal bleeding stem cankers in maple and other deciduous trees worldwide (Huddler, 2013). Knowledge of the causal agent of bleeding canker on maple will help determine appropriate disease management practices.

Aquatic invertebrate protein sources for long-duration space travel.

During the summer of 2020, NASA returned to launching astronauts to the International Space Station (ISS) from American soil. By 2024, NASA's mission is to return to the Moon, and by 2028 create a sustainable presence. Long duration missions come with obstacles, especially when trying to create a sustainable environment in a location where "living off the land" is impossible. Some resources on the Moon can be recovered or resupplied; however, many resources such as those needed for sustaining life must be recycled or grown to support humans. To achieve sustainability, food and water must be grown and recycled using elements found within the habitat. NASA's current work focuses on food resupply and growing plants as supplemental nutrient content. This paper examines the possibility for using aquaculture systems to purify water while growing nutrient-rich species as food sources, which aquatic food sources would be ideal for a habitat environment, and which species might provide an ideal test case for future studies aboard ISS. The aquatic species should be rapidly grown with high protein content and low launch mass requirements. Although there are numerous challenges and unknown technology gaps for maintaining aquaculture systems in reduced gravity environments, the benefit of employing such systems would be of great advantage towards creating a sustainable presence beyond Earth's orbit for sustainable aquaculture.

First report of rubbery rot of potato caused by Geotrichum candidum in the United States.

Rubbery rot of potato caused by Geotrichum candidum Link is characterized by symptoms of damp, flaccid tubers that feel rubbery when squeezed (Humpreys-Jones 1969), similar in consistency to potato diseases such as pink rot (caused by Phytophthora erythroseptica) and Pythium leak (caused by species of Pythium). In November 2019, several symptomatic tubers of potato variety 'Ciklamen' that had been held in storage since harvest and originated from an over-head irrigated, sandy-loam production field in Bingham county, Idaho were submitted to the University of Idaho for diagnosis. Shipping-point inspection records indicated 4-9% of tubers were affected. External symptoms included irregularly shaped, randomly located sunken black-colored lesions on more severely affected rubbery-textured tubers. When cut, internal affected tissue developed a greyish appearance after several minutes. Lens-shaped cavities were apparent in two of the tubers, indicating an advanced infection. A sour-milk smell accompanied the sample. To isolate the pathogen, pieces of tuber tissue approximately 5 mm in diameter were collected from the margins of symptomatic areas and surface-sanitized in sodium hypochlorite (2%) for two minutes, rinsed twice in sterile water and plated onto tap water agar amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). After three days at 21°C, colonies having distinct creamy white mycelia, a sweet, juniper-like odor, and hyaline hyphae were consistently associated with diseased tissue. Cylindrical to oval-shaped arthroconidia ranged in size from 6.6-11.0 × 3.2-5.9 µm (mean = 8.4 × 4.7, n=21), within dimensions as reported by Carmichael (1957). No other pathogens including species of Pythium and Phytophthora were recovered from the sample. Pure cultures were obtained by transferring hyphal tips to potato dextrose agar plates. Species identity was confirmed via rDNA ITS sequencing using primers ITS5/4 (White et al., 1990). DNA extraction and PCR conditions were as previously described (Woodhall et al., 2013). Resulting sequences (NCBI accession numbers MT893312 and MT893315) shared 99.4% identity with G. candidum Accession KY103453.1 on GenBank. To confirm pathogenicity, ten tubers (cv. Ciklamen) were inoculated by placing a 10mm2 plug of fully colonized PDA of G. candidum on the tuber surface, and ten tubers were mock inoculated with sterile PDA plugs. After 27 days at 21C in a dew chamber, tubers were examined for symptoms. Eight of the 10 inoculated tubers exhibited a rubbery texture and fluid leaking from tubers when cut, with two tubers also exhibiting a grey internal discoloration and the distinctive smell. Control tubers did not exhibit any symptoms. Isolations were attempted from four symptomatic tubers and G. candidum was successfully recovered from three tubers. The disease has been reported sporadically in the United Kingdom (Humphreys-Jones 1969) and Korea (Kim et al., 2011) and the pathogen occurs worldwide (Carmichael 1957). Though the fungus causes a tomato rot in the United States (US) (Pritchard and Porte, 1923; Bourret et al., 2013), and potatoes with rubbery rot originating from Australia were intercepted at a US port (Farr et al., 2020), the disease has not to our knowledge been documented on potato grown in the US. Because symptoms may be confused with pink rot and Pythium leak, it is critical for producers to obtain a correct diagnosis to facilitate appropriate management strategies.

Rhizoctonia solani AG 2-2 IIIB causing root rot of onion in Idaho.

In September of 2018, onion plants (Allium cepa cv. Joaquin) grown in one field in southwest Idaho were observed to have roots with brown discoloration over 10-20% of the total root surface area. Approximately 10% of plants over a 1 ha area were affected and these plants were about visually 50% smaller than the typical bulb size present in the field. To determine the causal agent, 3 mm pieces of symptomatic roots from four plants were placed in sodium hypochlorite (2%) for one minute, followed by two rinses in sterile water and plated on to water agar medium amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). After 3 days at 21°C, fungal colonies with septate hyphae with right-angled branching resembling Rhizoctonia solani were observed in over half of the 16 isolations attempted. Species identity was confirmed through rDNA ITS sequencing, as described previously (Woodhall et al., 2013), with DNA obtained from a single representative hyphal tip culture grown on Potato Dextrose Agar (PDA) which was designated isolate ON3. The resulting sequence (MT672318), was 100% identical (678/678bp) to a sequence previously identified as R. solani AG 2-2 IIIB on GenBank (FJ492137). Pathogenicity of the culture was determined by inoculating ten 20-day-old plants (cv. Joaquin) grown in premium potting compost (Scotts) with a single, fully colonized 10 mm2 plug taken from a 2-week-old PDA culture of isolate ON3. A further nine plants were inoculated with sterile PDA plugs as controls. Plants were grown in the greenhouse at 21C in a 16-hour light regime. After 24 days, each plant was assessed for root rot disease as described previously (Misawa et al. 2017). Root rot was observed on nine of the inoculated plants. Mean diseased root area was 32% of the total root surface, with a minimum of 5% and a maximum of 100% diseased root area and a standard deviation equal to 39.6. No root browning was observed on any of the control plants. Isolations were attempted from nine symptomatic plants and R. solani was successfully isolated from seven plant samples onto water agar. Sequencing was used to confirm identity as AG2-2IIIB. To our knowledge, this is the first report of R. solani AG 2-2 IIIB affecting onions in Idaho. Previous work in the Pacific Northwest recovered R. solani AG2-1, 3, 4 and 8 and also BNR AG A from stunted onions (Patzek et al., 2013). In Japan, Misawa et al. (2017) found AG 2-2 IIIB to be pathogenic to Welsh onion (Allium fistulosum). In Idaho, R. solani AG 2-2 IIIB has was previously reported causing disease in sugar beets (Strausbaugh et al. 2011) and potatoes (Woodhall et al. 2012). Growers should consider crop rotation strategies or soil treatments if R. solani AG2-2IIIB is causing disease in their crops.

Failure to detect equid herpesvirus types 1 and 4 DNA in placentae and healthy new-born Thoroughbred foals.

Equid herpesvirus type 1 is primarily a respiratory tract virus associated with poor athletic performance that can also cause late gestation abortion, neonatal foal death and encephalomyelopathy. Horizontal transmission is well described, whereas evidence of vertical transmission of equid herpesvirus type 1 associated with the birth of a healthy foal has not been demonstrated. This study sampled a population of Thoroughbred mares (n = 71), and their healthy neonatal foals and foetal membranes, to test for the presence of both equid herpesvirus types 1 and 4 using a quantitative polymerase chain reaction assay. Foetal membrane swabs and tissue samples were taken immediately post-partum, and venous blood samples and nasal swabs were obtained from both mare and foal 8 h after birth. Neither equid herpesvirus type 1 nor equid herpesvirus type 4 nucleic acid was detected in any sample, and it was concluded that there was no active shedding of equid herpesvirus types 1 and 4 at the time of sampling. Consequently, no evidence of vertical transmission of these viruses could be found on this stud farm during the sampling period.

Water requirements of canine athletes during multi-day exercise.

BACKGROUND: Exercise increases water requirements, but there is little information regarding water loss in dogs performing multi-day exercise OBJECTIVES: Quantify the daily water turnover of working dogs during multi-day exercise and establish the suitability of SC administration of tracer to determine water turnover. ANIMALS: Fifteen privately owned Labrador retrievers trained for explosive detection duties and 16 privately owned Alaskan Huskies conditioned for mid-distance racing. METHODS: All dogs received 0.3 g D2 O/kg body weight by IV infusion, gavage, or SC injection before the start of a multi-day exercise challenge. Explosive detection dogs conducted 5 days of simulated off-leash explosive detection activity. Alaskan sled dogs completed a mid-distance stage race totaling 222 km in 2 days. Total body water (TBW) and daily water turnover were calculated using both indicator dilution and elimination regression techniques. RESULTS: Total body water (% of body weight) varied from 60% ± 8.6% in minimally conditioned Labrador retrievers to 74% ± 4.5% in highly conditioned Labrador retrievers. Daily water turnover was as high as 45% of TBW during exercise in cold conditions. There was no effect of sex or speed on daily water turnover. There was good agreement between results calculated using the indicator dilution approach and those calculated using a semilog linear regression approach when indicator isotope was administered IV or SC. CONCLUSIONS AND CLINICAL IMPORTANCE: Water requirements are influenced primarily by the amount of work done. SC administration of isotope-labeled water offers a simple and accurate alternative method for metabolic studies.

Childhood disability population-based surveillance: Assessment of the Ages and Stages Questionnaire Third Edition and Washington Group on Disability Statistics/UNICEF module on child functioning in a rural setting in South Africa.

BACKGROUND: Epidemiological information on childhood disability provides the basis for a country to plan, implement and manage the provision of health, educational and social services for these vulnerable children. There is, however, currently no population-based surveillance instrument that is compatible with the International Classification of Functioning, Disability and Health (ICF), internationally comparable, methodologically sound and comprehensively researched, to identify children under 5 years of age who are living with disability in South Africa and internationally. We conducted a descriptive pilot study to investigate the sensitivity and specificity of translated versions of the Ages and Stages Questionnaire Third Edition (ASQ-III) and the Washington Group on Disability Statistics/UNICEF module on child functioning (WG/UNICEF module) as parent-reported measures. The aim of our study was to identify early childhood disabilities in children aged 24-48 months in a rural area of South Africa, to determine the appropriateness of these instruments for population-based surveillance in similar contexts internationally. METHODS: This study was conducted in the Xhariep District of the Free State Province in central South Africa, with 50 carers whose children were registered on the South African Social Security Agency (SASSA) database as recipients of a grant for one of the following: Care Dependency, Child Support or Foster Care. The researchers, assisted by community healthcare workers and SASSA staff members, conducted structured interviews using forward-backward translated versions of the ASQ-III and the WG/UNICEF module. RESULTS: Both measurement instruments had a clinically meaningful sensitivity of 60.0%, high specificity of 95.6% for the ASQ-III and 84.4% for the WG/UNICEF module, and the two instruments agreed moderately (Kappa = 0.6). CONCLUSION: Since the WG/UNICEF module is quicker to administer, easier to understand and based on the ICF, it can be considered as an appropriate parent-reported measure for large-scale, population-based as well as smaller, community-specific contexts. It is, however, recommended that future research and development continues with the WG/UNICEF module to enhance its conceptual equivalence for larger-scale, population-based studies in South Africa and internationally.