Digital Commons Network^™

Protocol For Preclinical Evaluation Of Locoregionally Delivered Car T Cells In Patient-Derived Xenograft Models Of Brain Tumors, Benjamin Draper, Chantelle Bowers, Eleni Vassalou, Ailsa Greppi, John Anderson, Nabil Ahmed, Michael D Taylor, Laura K Donovan

Faculty, Staff and Students Publications

Here, we present a protocol for preclinical evaluation of locoregionally delivered CAR T cells in patient-derived xenograft models of primary, metastatic, and recurrent brain tumors. We provide instructions for isolating peripheral blood mononuclear cells (PBMCs), producing CAR T cells in conjunction with locoregional delivery, and preclinical trial design and analysis involving CAR T cells. Additionally, we describe comprehensive preclinical readouts and guidelines for critical endpoint sample collections. In line with clinical trial procedures, our protocol broadens available treatment modalities for direct clinical translation. For complete details on the use and execution of this protocol, please refer to Donovan et al …

Analyzing Efficiency Of A Lentiviral Shrna Knockdown System In Human Enteroids Using Western Blot And Flow Cytometry, Adam P Wilson, Karni S Moshal, Addison P Franca, Sasirekha Ramani, Randle Gallucci, Hala Chaaban, Kathryn Y Burge

Faculty, Staff and Students Publications

Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes.

Identifying Interventions To Improve Diagnostic Safety In Emergency Departments: Protocol For A Participatory Design Study, Woosuk Seo, Sun Young Park, Zhan Zhang, Hardeep Singh, Kalyan Pasupathy, Prashant Mahajan

Faculty, Staff and Students Publications

Background: Emergency departments (EDs) are complex and fast-paced clinical settings where a diagnosis is made in a time-, information-, and resource-constrained context. Thus, it is predisposed to suboptimal diagnostic outcomes, leading to errors and subsequent patient harm. Arriving at a timely and accurate diagnosis is an activity that occurs after an effective collaboration between the patient or caregiver and the clinical team within the ED. Interventions such as novel sociotechnical solutions are needed to mitigate errors and risks.

Objective: This study aims to identify challenges that frontline ED health care providers and patients face in the ED diagnostic process and …

Initiation Of Hnrnpa1 Low-Complexity Domain Condensation Monitored By Dynamic Light Scattering, Phoebe S Tsoi, Josephine C Ferreon, Allan Chris M Ferreon

Faculty, Staff and Students Publications

Biomolecular condensates (BMCs) exhibit physiological and pathological relevance in biological systems. Both liquid and solid condensates play significant roles in the spatiotemporal regulation and organization of macromolecules and their biological activities. Some pathological solid condensates, such as Lewy Bodies and other fibrillar aggregates, have been hypothesized to originate from liquid condensates. With the prevalence of BMCs having functional and dysfunctional roles, it is imperative to understand the mechanism of biomolecular condensate formation and initiation. Using the low-complexity domain (LCD) of heterogenous ribonuclear protein A1 (hnRNPA1) as our model, we monitored initial assembly events using dynamic light scattering (DLS) while modulating …

Alzheimer's Disease Risk Gene Cd2ap Is A Dose-Sensitive Determinant Of Synaptic Structure And Plasticity, Taeyong Ahn, Gabrielle E Largoza, Julia Younis, Mary E Dickinson, Chih-Wei Hsu, Joshua D Wythe

Faculty, Staff and Students Publications

Tissue clearing is an essential prerequisite for 3D volumetric imaging of larger tissues or organs. Here, we present a detailed protocol for optical, aqueous-based clearing of adult murine tissues using EZ Clear. We describe steps to ensure successful perfusion and fixation of organs from the adult mouse and supply guidelines for optimal lipid removal, refractive index matching, and tissue clearing. Finally, we provide imaging parameters for visualizing both exogenous perfused fluorescent dyes and endogenous fluorescence reporters in the adult mouse. For complete details on the use and execution of this protocol, please refer to Hsu et al.

Comprehensive Single-Cell Atlas Of The Mouse Retina, Jin Li, Jongsu Choi, Xuesen Cheng, Justin Ma, Shahil Pema, Joshua R Sanes, Graeme Mardon, Benjamin J Frankfort, Nicholas M Tran, Yumei Li, Rui Chen

Faculty, Staff and Students Publications

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cellular heterogeneity by characterizing cell types across tissues and species. While several mouse retinal scRNA-seq datasets exist, each dataset is either limited in cell numbers or focused on specific cell classes, thereby hindering comprehensive gene expression analysis across all retina types. To fill the gap, we generated the largest retinal scRNA-seq dataset to date, comprising approximately 190,000 single cells from C57BL/6J mouse retinas, enriched for rare population cells via antibody-based magnetic cell sorting. Integrating this dataset with public datasets, we constructed the Mouse Retina Cell Atlas (MRCA) for wild-type mice, encompassing …

Esr1 And P53 Interactome Alteration Defines Mechanisms Of Tamoxifen Response In Luminal Breast Cancer, Chetan C Oturkar, Spencer R Rosario, Alan D Hutson, Adrianne Groman, Stephen B Edge, Carl D Morrison, Wendy M Swetzig, Jianmin Wang, Jun Hyoung Park, Benny Abraham Kaipparettu, Prashant K Singh, Shicha Kumar, Helen H Cappuccino, Manish Ranjan, Araba Adjei, Mohammad Ghasemi, Andrew K L Goey, Swati Kulkarni, Gokul M Das

Faculty, Staff and Students Publications

The canonical mechanism behind tamoxifen's therapeutic effect on estrogen receptor α/ESR1+ breast cancers is inhibition of ESR1-dependent estrogen signaling. Although ESR1+ tumors expressing wild-type p53 were reported to be more responsive to tamoxifen (Tam) therapy, p53 has not been factored into choice of this therapy and the mechanism underlying the role of p53 in Tam response remains unclear. In a window-of-opportunity trial on patients with newly diagnosed stage I-III ESR1+/HER2/wild-type p53 breast cancer who were randomized to arms with or without Tam prior to surgery, we reveal that the ESR1-p53 interaction in tumors was inhibited by Tam. This resulted in …

Protocol For Optical, Aqueous-Based Clearing Of Murine Tissues Using Ez Clear, Taeyong Ahn, Gabrielle E Largoza, Julia Younis, Mary E Dickinson, Chih-Wei Hsu, Joshua D Wythe

Faculty, Staff and Students Publications

Tissue clearing is an essential prerequisite for 3D volumetric imaging of larger tissues or organs. Here, we present a detailed protocol for optical, aqueous-based clearing of adult murine tissues using EZ Clear. We describe steps to ensure successful perfusion and fixation of organs from the adult mouse and supply guidelines for optimal lipid removal, refractive index matching, and tissue clearing. Finally, we provide imaging parameters for visualizing both exogenous perfused fluorescent dyes and endogenous fluorescence reporters in the adult mouse. For complete details on the use and execution of this protocol, please refer to Hsu et al.

Clinical Prediction For Surgical Versus Nonsurgical Interventions In Patients With Vertebral Osteomyelitis And Discitis, Jennifer Lee, Miguel A. Ruiz-Cardozo, Rujvee P. Patel, Saad Javeed, Raj Swaroop Lavadi, Catherine Newsom-Stewart, Anton Alyakin, Camilo A. Molina, Nitin Agarwal, Wilson Z. Ray, Michele Santacatterina, Brenton H. Pennicooke

2020-Current year OA Pubs

BACKGROUND: Vertebral osteomyelitis and discitis (VOD), an infection of intervertebral discs, often requires spine surgical intervention and timely management to prevent adverse outcomes. Our study aims to develop a machine learning (ML) model to predict the indication for surgical intervention (during the same hospital stay) versus nonsurgical management in patients with VOD.

METHODS: This retrospective study included adult patients (≥18 years) with VOD (ICD-10 diagnosis codes M46.2,3,4,5) treated at a single institution between 01/01/2015 and 12/31/2019. The primary outcome studied was surgery. Candidate predictors were age, sex, race, Elixhauser comorbidity index, first-recorded lab values, first-recorded vital signs, and admit diagnosis. …

Noninvasive Assessment Of The Lung Inflammation-Fibrosis Axis By Targeted Imaging Of Cmklr1, Philip Z Mannes, Taylor S Adams, Samaneh Farsijani, Clayton E Barnes, Joseph D Latoche, Kathryn E Day, Jessie R Nedrow, Farida Ahangari, Naftali Kaminski, Janet S Lee, Sina Tavakoli

2020-Current year OA Pubs

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated

Harmonized Cross-Species Cell Atlases Of Trigeminal And Dorsal Root Ganglia, Shamsuddin A Bhuiyan, Lite Yang, Lisa A Mcilvried, Bryan A Copits, Zachariah Bertels, John S Del Rosario, Allie J Widman, Richard A Slivicki, Jiwon Yi, Robert W Gereau Iv, Et Al.

2020-Current year OA Pubs

Sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli to the central nervous system. Single-cell RNA sequencing has provided insights into the diversity of sensory ganglia cell types in rodents, nonhuman primates, and humans, but it remains difficult to compare cell types across studies and species. We thus constructed harmonized atlases of the DRG and TG that describe and facilitate comparison of 18 neuronal and 11 non-neuronal cell types across six species and 31 datasets. We then performed single-cell/nucleus RNA sequencing of DRG from both human and the …

Symbiotic Behaviour Of Alfalfa Varieties Grown In Different Soils, C Scotti, S Gnocchi, M Carelli, P Rotili

IGC Proceedings (1977-2023)

Six alfalfa (Medicago sativa) varieties were grown in two contrasting soils to study their symbiotic ability as estimated by nodule biomass and dry matter yield in presence of the natural population of rhizobia of soil. Varieties can be discriminated on the basis of total (year) nodule biomass; their ranking for nodule biomass was consistent in the different soil types although soils significantly influenced nodulation and dry matter yield.

Long Term Effects Of Liming A Basalt-Derived Soil, On Lucerene And Phalaris Production, K.M. F. Reed, J F. Chin

IGC Proceedings (1977-2023)

Lime incorporated into a basalt-derived clay loam lowered exchangeable aluminium in soil. Soil pH was still elevated, 9 years after 2 t/ha of lime was incorporated. With lower rates of lime the density of lucerne plants declined. Lucerne yield more than doubled with lime. After six years, the area was sown to a phalaris-clover pasture. A 15% increase in the yield of phalaris was recorded from limed plots over the next three years. Despite this, 11 years after liming, herbage P concentrations increased with lime, from 0.28% to 0.35%.

Can Maize Influence The Dissolution Of Low Water Soluble Phosphorus Fertilizers?, S Sinaj, E Frossard

IGC Proceedings (1977-2023)

The objectives of this study were to evaluate the respective importance of maize (Zea mays) and soil properties on the dissolution of two sparingly soluble P fertilizers. The surface horizon of an acidic and a calcareous soils were amended with Thomas’ slag or phosphate rock and were either incubated or cultivated with maize. Maize cultivation increased the rate of dissolution of both fertilizers in both soils. Its influence remained limited compared to fertilizer dissolution in the incubated acidic soil. In the calcareous soil, however, the presence of maize increased significantly the rate of fertilizer dissolution compared to that observed in …

Phosphorus Availability, Defoliation Tolerance, And Genetic Differentiation In White Clover, D F. Chapman

IGC Proceedings (1977-2023)

The genetic composition of mixed populations of white clover growing in low fertility, grass-dominant hill pastures was estimated four and eight years after populations were established. Populations were based on an adapted ('Grasslands Tahora') or poorly adapted ('Grasslands Huia') cultivar, and received either no fertiliser, or 35kg P /ha /year. The P response curve of surviving plants was also determined, and compared with plants grown from 'standard' seedlines of the respective cultivars. Between years 4 and 8, the proportion of the populations constituted by plants true-to-type for the sown cultivar remained steady at 56 - 58% for Tahora, but fell …

Seasonal Variations Of Labile Soil Organic Carbon And Nitrogen Fertility In Grassland Soil In Tokyo, Japan, T Murata, H Tanaka, Y Kurokawa

IGC Proceedings (1977-2023)

We investigated seasonal variations of soil enzyme activities, soil inorganic N, soil microbial biomass N (BN) and soil total sugar C (TSC) during March to December in cattle-grazed pasture in Japan (Andisols). Dehydrogenase activity had three peaks in a year, on the other hand, phosphomonoesterase activity increased from spring (April) up to summer (August) but then gradually decreased toward winter. BN also showed a seasonal variation, which gradually increased from April to August, but in the end of August once decreased rapidly because of soil becoming dry. Soil inorganic N had three peaks which appeared after the appearance of dehydrogenase …

Reed Canarygrass (Phalaris Arundinacea L.) Response To Liquid Dairy Manure Or Fertilizer N, M P. Russelle, G W. Randall, P D. Clayton, M A. Schmitt, L J. Greub, C C. Sheaffer, R R. Kalton, D H. Taylor

IGC Proceedings (1977-2023)

Livestock farmers face constraints in efficient manure management because of limited land availability, especially during the growing season, but reed canarygrass (Phalaris arundinacea L.) may provide a solution. We measured yield and apparent N removal of established reed canarygrass with surface-applied dairy manure slurry at three locations in the Upper Midwest, USA. Slurry was broadcast or banded at rates up to 375 m3 ha-1 (1190 kg N ha-1) in single or split applications and N fertilizer was applied at rates up to 448 kg N ha- 1 for comparison. Herbage was harvested three times per year. Nitrogen removal in …

The Influence Of Plant Density And Level Of Nitrogen Fertilization On The Production Of Eragrostis Curvula, P A. Pieterse, N F.G. Rethman, J Van Bosch

IGC Proceedings (1977-2023)

The objective of this study was to determine whether plant density would have an influence on dry matter (CM) yield and to determine it’s interaction (if any) with levels of N fertilization. Plants on a broadcast establishment of lovegrass (Eragrostis curvula) were mechanically thinned, to leave 30 cm rows with spacing of 0.5 and 1 m. N fertilizer was applied in spring at levels equivalent to 0, 50, 100 and 150 kg N ha-1. The trial was conducted over three growing seasons. In the second and third years the DM yields with 100 and 150 kg N ha-1 on the …

Potassium Rates For Macrotyloma Axillare Growth, R A. Martim, F A. Monteiro, P.R. L. Portilho

IGC Proceedings (1977-2023)

The response of the tropical forage legume macrotiloma guata (Macrotyloma axillare (E. Mey) Verdc) to potassium rates was studied in a greenhouse experiment. The plants were grown in nutrient solution with seven potassium (K) rates, 0; 3.5; 7; 21; 35; 105 and 245 mg l-1 of solution, in randomized complete block design, with four replications. There was significant response of the legume to potassium rates. Considerations are made on the influence of these levels on plant dry matter yield in two harvests, root dry matter production and potassium and magnesium concentrations in the plant tissue.

Phosphorus Use Efficiency For Biomass Production Of Legume Species, A T. Ayala Torales, V A. Deregibus, P R. Moauro

IGC Proceedings (1977-2023)

The objective of the experiment was to identify traits associated with the production of plant biomass, and hence with P utilization efficiency, when legume species were fertilized. Red clover (Trifolium pratense) and narrowleaf birdsfoot trefoil (Lotus tenuis) were compared in a greenhouse experiment using phosphorus deficient soils. Three plants grew in pots fertilized with 0, 2, 4, 8, 15, 20, 30 and 40 ppm in soil, in a randomized experimental design. Harvest was made at 60 days from the sowing date. Leaf areas, shoot and root biomass, and tissue phosphorus contents were measured. Results showed that …

Phosphogypsum As A Source Of Nutrients For Bahiagrass, J E. Rechcigl, I S. Alcordo

IGC Proceedings (1977-2023)

Phosphogypsum is a by-product of the manufacture of phosphoric acid from phosphate rock and is a potential source of sulfur and calcium for crops. There are currently more than 700 million Mg of phosphogypsum in Florida alone stacked in waste piles and an additional 30 million Mg produced annually. A 3-year study was conducted to determine whether addition of phosphogypsum to bahiagrass would increase production and quality. Results indicate that addition of up to 4.0 Mg/ha phosphogypsum increased bahiagrass yields, protein content, and in vitro digestibility of forage. This study has demonstrated that phosphogypsum can be used as an alternative …

A Review Of Centrosema Pascuorum (Centurion) Cvv. Cavalcade And Bundey As A Pasture Legume In The Ley Farming System Studies In North West Australia, K Thiagalingam, D Zuill, T Price

IGC Proceedings (1977-2023)

Field experiments and commercial experience with the pasture legume Centrosema pascuorum (centurion) in the Katherine and Douglas Daly districts of the Northern Territory over the past ten years were reviewed. Results show that cultivars of this species (Cavalcade and Bundey) have good potential for improving both soil fertility and the quality of dry season forage (as hay or grazing) for cattle in mixed farming systems in the semi-arid tropics of north west Australia.

Influence Of N Fertilization Application And Regrowth On The Yields Of Grassland, J Tasi

IGC Proceedings (1977-2023)

The effect of different levels of fertilization and regrowth periods on the quantity and quality and distribution of yields of eleven grassland species were studied in two plots in Hungary. The effect of the following variables were tested: regrowth period of 20 and 40 days; control, 100 and 200 kg N ha-1 and 11 grass and leguminous species.

Effect Of Plant Age And N And P Fertilization On Mineral Composition Of Andropogon Gayanus, D E. Morillo, A Caraballo, J Faria-Marmoll, L R. Mcdowell

IGC Proceedings (1977-2023)

Mineral concentrations of Andropogon gayanus Kunth, as affected by age of regrowth, 42 and 63 days (six and four cuttings); rate (kg/ ha/year) of N fertilizer, 0, 100 and 200, and of P2O5, 0 and 75, were evaluated on a sandy-loam Aridisol (pH 5.5) in Venezuela, using a split-split-plot experimental design with two replications. Forage mineral concentrations did not differ (P>0.05) among ages. Mean Ca, P, Mg, Na were: 0.20, 0.11, 0.10 and 0.029 %, and regarded as deficient for ruminants. Only K (1.21 %) was considered adequate. Mean Fe, Zn, Cu, Mn and Co were 57, 24, 2.7, …

Seasonality In The Response Of Perennial Grass-Clover Pastures To Phosphorus Fertiliser, A K. Metherell, A C. Mckenzie

IGC Proceedings (1977-2023)

Pasture dry matter yields from individual cuts of multi-year phosphorus (P) fertilizer field trials were analyzed for evidence of consistent seasonal trends in the response to P. The relative response to P was greatest in the late autumn - winter period, with the seasonal variation in relative yield being greater where soil fertility and mean annual relative yields were lowest.

Interaction Of Endomycorrhizal Fungi And Phosphate On Neonotonia Wightii, Lackey, V T. Paulino, O.M. A.A. Ghisi, M.A. C. Lucena, M J. Valarini, J C. Werner

IGC Proceedings (1977-2023)

This experiment was carried out in greenhouse condition, to study the effect of four phosphorus levels (20, 80, 140 and 200 kg of P2O5/ ha) with or without fungi mycorrhizal inoculation (Acaulospora longula + A. morrowae ). Five accessions of Neonotonia wightii (Wightii & Arn) Lackey (perennial soyabean) were studied: Malawi, Tinaroo, NO1264, NO409 and NO 250. The growth of Neonotonia was improved by the phosphorus fertilization.The effect of VAM inoculation was greater in the lower levels of P fertilization reducing its effect in the higher levels of P applied. The presence of soluble P induced higher N accumulation …

Effects Of Agropastoral Systems On Microbially Bound Phosphorus In Low P Acid Soils, A Oberson, D K. Friesen, C Morel, H Tiessen, E Frossard

IGC Proceedings (1977-2023)

The effect of agropastoral systems replacing native savanna on microbially bound phosphorus (Pmic) in low P acid soils was investigated. Chloroform released P (PChl) was measured to estimate Pmic. In a long-term improved pasture experiment, PChl was higher in grass-legume (GL) than grass-only pasture (GO). Although the P balance was slightly higher in GO than in GL, available P contents followed the same trend as PChl suggesting that the presence of legumes enhances the maintenance of P fertility. In a rice-pasture system, PChl was higher than under rice monocrop indicating an effect of the cropping system on PChl that goes …

Potassium Release From Clays By Biosignal Of Grass Root Exudates, S Suzuki

IGC Proceedings (1977-2023)

The objective of this study was to clarify that the root exudates of Italian ryegrass (Lolium multiflorum Lam.) expand the basal spacing of hydroxy potassium vermiculite (K-Vt) in the rhizosphere model with a clay-agar system, and to discuss that the action of the root exudates is regarded as a biosignal to release potassium ions from the interlayers of vermiculite clays.

Effect Of Slurry Application On Cell Wall Components And Silage Intake By Sheep Of Italian Ryegrass And Triticale, H Kawamoto, N Shimizu, R Uegaki, R Kobayashi

IGC Proceedings (1977-2023)

The study was to elucidate the effect of application of slurry manure from dairy cows on chemical components, the rumen degradation characteristics and silage intake by sheep of Italian ryegrass (Lolium multiflorum Lam.) and Triticale ( rye-wheat hybrid). The amounts of application of slurry were 0, 50, 100 and 150 t/ha. The increasing level of slurry application resulted in a decrease of lower digestible cell wall and an increase of degradation rate. The silage of 150 t /ha treatment was significantly more consumed by sheep than the other treatments (p<0.05). The changes in cell wall components in both quantity and quality by the slurry application were advantageous to the increase of voluntary dry matter intake of the forage.

Different Response Of Lucerne (Medicago Sativa L.) Cultivars To P And Zn Fertilization And Susceptibility To Phosphorus-Induced Zinc Deficiency On Calcareous Soils, Gansu, China, Chen Wen, Graham Eagleton, Graeme Blair

IGC Proceedings (1977-2023)

The three pot experiments were carried out at the Qingyang Research Station, Gansu, China to determine the yield response of P and Zn, and examine the effect of soil types on P and Zn requirement of the local and introduced lucerne cultivars in the establishment phase on calcareous soils. The results showed marked growth and dry matter yield benefit at lucerne establishment when P fertiliser was applied on both soils. There were no consistent difference among the three cultivars in response to added P. A significant shoot yield response to Zn was only recorded at high P supply in the …