Response Assay Research Articles

Fluorescence Lifetime Imaging of NAD(P)H in Patients’ Lymphocytes: Evaluation of Efficacy of Immunotherapy

Background: The wide variability in clinical responses to anti-tumor immunotherapy drives the search for personalized strategies. One of the promising approaches is drug screening using patient-derived models composed of tumor and immune cells. In this regard, the selection of an appropriate in vitro model and the choice of cellular response assay are critical for reliable predictions. Fluorescence lifetime imaging microscopy (FLIM) is a powerful, non-destructive tool that enables direct monitoring of cellular metabolism on a label-free basis with a potential to resolve metabolic rearrangements in immune cells associated with their reactivity. Objective: The aim of the study was to develop a patient-derived glioma explant model enriched by autologous peripheral lymphocytes and explore FLIM of the redox-cofactor NAD(P)H in living lymphocytes to measure the responses of the model to immune checkpoint inhibitors. Methods: The light microscopy, FLIM of NAD(P)H and flow cytometry were used. Results: The results demonstrate that the responsive models displayed a significant increase in the free NAD(P)H fraction α1 after treatment, associated with a shift towards glycolysis due to lymphocyte activation. The non-responsive models exhibited no alterations or a decrease in the NAD(P)H α1 after treatment. The FLIM data correlated well with the standard assays of immunotherapy drug response in vitro, including morphological changes, the T-cells activation marker CD69, and the tumor cell proliferation index Ki67. Conclusions: The proposed platform that includes tumor explants co-cultured with lymphocytes and the NAD(P)H FLIM assay represents a promising solution for the patient-specific immunotherapeutic drug screening.

Exploring CAR T-Cell Dynamics: Balancing Potent Cytotoxicity and Controlled Inflammation in CAR T-Cells Derived from Systemic Sclerosis and Myositis Patients.

Systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and idiopathic inflammatory myositis (IIM) are autoimmune diseases managed with long-term immunosuppressive therapies. Hu19-CD828Z, a fully human anti-CD19 chimeric antigen receptor (CAR) with a CD28 costimulatory domain, is engineered to potently deplete B-cells. In this study, we manufactured Hu19-CD828Z CAR T-cells from peripheral blood of SLE, IIM, and SSc patients and healthy donors (HDs). CAR-mediated, CD19-specific activity of these cells was evaluated in vitro by assessing cytotoxicity, cytokine release, and proliferation assays in response to autologous CD19+ B-cells, the CD19+ NALM-6 B-cell line, or a CD19- U937 non-B-cell line as targets. The results demonstrated an increased proliferation of Hu19-CD828Z CAR T-cells and dose-dependent cytotoxicity against primary autologous and NALM-6 B-cells compared to non-transduced controls or co-cultures with non-B-cells. Notably, autoimmune-patient-derived CAR T-cells produced lower levels of inflammatory cytokines than healthy-donor-derived CAR T-cells in response to CD19+ B-cell targets. These data support the potential of Hu19-CD828Z and its therapeutic cell product KYV-101 as a therapeutic strategy to achieve deep B-cell depletion in SLE, IIM, and SSc patients, and highlights its promise for broader application in B-cell-driven autoimmune disorders.

Dimeric guaianolide sesquiterpenoids from the flowers of Chrysanthemum indicum ameliorate hepatic steatosis through mitigating SIRT1-mediated lipid accumulation and ferroptosis.

Non-alcoholic fatty liver disease (NAFLD) acts as the primary contributor to non-alcoholic steatohepatitis, fibrosis, cirrhosis, and potentially hepatocellular carcinoma. The flowers of Chrysanthemum indicum, a traditional edible medicinal herb, have been widely used in China for more than 2000 years. However, the function of C. indicum in managing NAFLD has seldom been investigated. To reveal the novel active components and underlying mechanisms of C. indicum in treating NAFLD. An MS/MS-based molecular networking-guided strategy was used for the chemical investigation. The structure identification of the new compounds involved high resolution electrospray ionization mass spectrometry (HRESIMS), 1D and 2D nuclear magnetic resonance (NMR) spectra, electronic circular dichroism (ECD), and X-ray crystallographic analysis. The biological evaluation was performed using Nile Red staining, flow cytometry, commercial kits, western blotting, co-immunoprecipitation, isothermal titration calorimetry, cellular thermal shift assay, drug affinity responsive target stability assay, molecular docking, and confocal immunofluorescence. A total of 27 new dimeric sesquiterpenoids, chryindicolides A-Z (1-26) and chrysanthemolide C (27), together with seven known compounds, were isolated from the flowers of C. indicum under the guide of MS/MS-based molecular networking. Among them, compounds 1-7 were rare chlorine-containing guaianolide dimers. Chryindicolide O (15) directly bound and activated the deacetylase Sirtuin 1 (SIRT1) to reduce de novo lipogenesis, enhance fatty acid β-oxidation, and inhibit ferroptosis in palmitic acid and oleic acid (P/O)-induced AML12 hepatocytes. In addition, chryindicolide O significantly ameliorated liver steatosis in high-fat diet-fed zebrafish. Novel guaianolide dimers from C. indicum alleviated hepatic steatosis through mitigating SIRT1-mediated lipid accumulation and ferroptosis, suggesting that they could be further developed as candidates against NAFLD.

Two TAL Effectors of Xanthomonas citri pv. malvacearum Induce Water Soaking by Activating GhSWEET14 Genes in Cotton.

Bacterial blight of cotton (BBC) caused by Xanthomonas citri pv. malvacearum (Xcm) is an important and destructive disease affecting cotton plants. Transcription activator-like effectors (TALEs) released by the pathogen regulate cotton resistance to the susceptibility. In this study, we sequenced the whole genome of Xcm Xss-V2-18 and identified eight tal genes: seven on the plasmids and one on the chromosome. Deletion and complementation experiments of Xss-V2-18 tal genes demonstrated that Tal1b is required for full virulence on cotton. Transcriptome profiling coupled with TALE-binding element prediction revealed that Tal1b targets GhSWEET14A04/D04 and GhSWEET14D02 simultaneously. Expression analysis confirmed the independent inducibility of GhSWEET14A04/D04 and GhSWEET14D02 by Tal1b, whereas GhSWEET14A04/D04 is additionally targeted by Tal1. Moreover, β-glucuronidase and Xa10-mediated hypersensitive response assays indicated that the effector-binding element (EBEs) are required for the direct and specific activation of the candidate targets by Tal1 and Ta1b. These insights enhance our understanding of the underlying mechanisms of bacterial blight in cotton and might lead to improved resistance through EBEs disruption or a TALE-trap strategy.

Cytokine and Chemokine Responses of Peripheral Blood Mononuclear Cells from Dogs Infected with Mycobacterium bovis.

Mycobacterial infections are an important emerging zoonosis in companion animals for which diagnostic options remain imperfect, and the canine immunological response to these infections has been poorly investigated. We sought to further define the cellular response of peripheral blood mononuclear cells (PBMCs) from dogs infected with Mycobacterium bovis, as determined using a commercial interferon-gamma response assay (IGRA). To this end, PBMCs from healthy or infected dogs were collected. Serum samples were tested to further classify dogs as seropositive or seronegative for circulating antibodies against M. bovis using the DPP® VetTB Assay, Idexx M. bovis antibody ELISA, and a novel purified protein derivative ELISA. Isolated PBMCs were stimulated with mycobacterial proteins (PPDB or ESAT-6/CFP-10), and 13 cytokines/chemokines were measured in the supernatant. These concentrations were determined using the CYTOMAG-90K MILLIPLEX MAP Canine Cytokine/Chemokine system. PBMCs from infected dogs released IFN-γ in response to stimulation, but this response was reduced in those that had seroconverted. Similarly, cells stimulated with PPDB secreted increased amounts of TNF-α when dogs were seronegative, but cells taken from seropositive dogs did not. Finally, the IL-18 response of seropositive dogs was reduced compared to those that were seronegative in response to PPDB, potentially suggesting that these dogs have a reduced macrophage functionality. This work demonstrates that the inflammatory cytokine response may wane following seroconversion with deleterious consequences for the host response. Overall, combining IFN-γ and TNF-α assessment during diagnosis may increase IGRA sensitivity, whilst further work is needed to better understand the prognostic and diagnostic implications of seroconversion in dogs.

Sex-dependent effects of chronic jet lag on circadian rhythm and metabolism in mice

BackgroundThe circadian clock integrates external environmental changes into the internal physiology of organisms. Perturbed circadian clocks due to misaligned light cycles increase the risk of diseases, including metabolic disorders. However, the effects of sex differences in this context remain unclear.MethodsCircadian misalignment was induced by a chronic jet lag (CJL) shift schedule (light-on time advanced by 6 h every 2 days) in C57BL/6N male and female mice. Core body temperature and activity rhythms were recorded using a nano tag, and the gene expression rhythms of clock and clock-controlled genes in the liver and adrenal glands were analyzed using qPCR. Glucose metabolism and insulin response were evaluated using glucose tolerance, insulin sensitivity, and glucose response assays. Castration and testosterone replacement were performed to assess the fundamental role of testosterone in male phenotypes under CJL.ResultsUnder CJL treatment, male mice exhibited increased weight gain, whereas females exhibited decreased weight gain compared to that of the respective controls. CJL treatment induced a lower robustness of circadian rhythms in core body temperature and a weaker rhythm of clock gene expression in the liver and adrenal glands in females, but not in males. Only male mice exhibited glucose intolerance under CJL conditions, without the development of insulin resistance. Castrated mice without testosterone exhibited decreased weight gain and reduced robustness of body temperature rhythm, as observed in intact females. Testosterone replacement in castrated mice recovered the CJL-induced weight gain, robustness of temperature rhythm, and glucose intolerance observed in intact males.ConclusionsSignificant sex-based differences were observed in circadian clock organization and metabolism under CJL. Testosterone plays a crucial role in maintaining the circadian clock and regulating CJL metabolism in males.

A High-Throughput Neurosphere-Based Colony Formation Assay to Test Drug and Radiation Sensitivity of Different Patient-Derived Glioblastoma Lines.

The gold standard assay for radiation response is the clonogenic assay, a normalized colony formation assay (CFA) that can capture a broad range of radiation-induced cell death mechanisms. Traditionally, this assay relies on two-dimensional (2D) cell culture conditions with colonies counted by fixing and staining protocols. While some groups have converted these to three-dimensional (3D) conditions, these models still utilize 2D-like media compositions containing serum that are incompatible with stem-like cell models such as brain tumor initiating cells (BTICs) that form self-aggregating spheroids in neural stem cell media. BTICs are the preferred patient-derived model system for studying glioblastoma (GBM) as they tend to better retain molecular and phenotypic characteristics of the original tumor tissue. As such, it is important that preclinical radiation studies should be adapted to BTIC conditions. In this study, we describe a series of experimental approaches for performing CFA experiments with BTIC cultures. Our results indicate that serum-free clonogenic assays are feasible for combination drug and radiation testing and may better facilitate translatability of preclinical findings.

Functional Testing of Humoral Immunity in the Prevnar 20 Era.

Humoral immune disorders such as common variable immunodeficiency (CVID) and specific antibody deficiency (SAD) are prevalent in clinical practice and require accurate functional testing of humoral immunity for diagnosis and to guide treatment approach. Traditionally, the 23-valent pneumococcal polysaccharide vaccine (PPSV23) has been used to assess polysaccharide antibody responses by measuring pre- and post-vaccination pneumococcal titers. However, the recent introduction of pneumococcal conjugate vaccines (PCVs), such as PCV13, PCV15, and PCV20, into the childhood and adult vaccine schedules has significantly reduced the number of unique serotypes available for testing and in turn has complicated the evaluation process. We retrospectively analyzed serotype-specific antibody responses in patients aged 2-65 years who received PPSV23 at the University of Virginia Health System to compare diagnostic outcomes using all 23 serotypes versus the limited number of unique serotypes not included in prior PCVs-11 serotypes for PCV13 recipients and 4 for PCV20 recipients. Our findings demonstrate that although prior PCVs mean there is a reduced number of serotypes available for interpretation, PPSV23 testing maintains diagnostic accuracy between 81% and 84%. Despite limitations, the use of PPSV23 remains a valuable tool for identifying patients with clinically significant humoral immune deficiencies. In the future, alternative diagnostic approaches like Salmonella typhi polysaccharide vaccine response and opsonophagocytosis assays may become more commonly utilized as part of the evaluation of humoral immune disorders.

Using Vesicular Stomatitis Virus as a Platform for Directed Protease Evolution.

Antiviral drugs are essential medications to save the lives of infected people. However, they are under constant threat to become ineffective as viruses evolve quickly. Studying the development of resistance is therefore paramount to understand the impact of mutations on pharmacological treatment and to make informed decisions. Yet, such studies are open to scrutiny, as they are considered gain-of-function research, which is especially problematic with viruses of pandemic potential. In this article, we present a protocol that allows for the selection of antiviral resistance mutations safely, without using the actual virus (e.g., SARS-CoV-2, MERS-CoV). Instead, we use vesicular stomatitis virus (VSV) that serves as a surrogate virus; like other RNA viruses, it is prone to mutations due to its polymerase lacking proofreading. By replacing parts of the VSV genome with transgenes from other viruses, VSV becomes dependent on their function. Thus, we can mount a selection pressure with antivirals targeting the transgenes to subsequently sequence selected resistance mutations. This article provides a protocol for this process as well as a sequencing pipeline that we used to collect mutations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Using VSV as a platform for directed protease evolution Alternate Protocol: Dose response assay with TCID50 readout Support Protocol 1: A pipeline for high-throughput VSV sequencing Support Protocol 2: Rescue of VSV.

Induction of CD4 T cell memory responses following BCG vaccination in cattle.

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is a zoonotic pathogen that contributes to economic losses in the cattle industry and poses a public health risk worldwide. Bacillus Calmette-Guerin, or BCG, is a live attenuated strain of M. bovis that is used for human vaccination against tuberculosis and is considered a potential vaccine candidate against bTB. However, BCG affords widely variable levels of protection against challenge and interferes with current diagnostic methods, and as such, it is not currently approved for use as a livestock or wildlife vaccine in the United States. Many efforts have been made to develop bTB vaccines that are reliable and do not interfere with diagnostic testing, but BCG continues to be the most effective option. Previous work has shown that a T helper 1 immune response is essential for protection against virulent M. bovis infection, characterized by CD4+ central and effector memory T cells. In an effort to identify an efficacious bTB intervention strategy, the study presented here used an in vitro recall response assay and concurrent evaluation of CD4+ T cell proliferation and cytokine production to characterize the surface and functional phenotypes of memory responses to BCG vaccination in cattle. Our findings enhance understanding of the bovine immune response to BCG and provide insights into the development of improved vaccines for the control of bTB.

Novel Uveal Melanoma Patient-Derived Organoid Models Recapitulate Human Disease to Support Translational Research.

A lack of representative human disease models has limited the translation of new and more effective treatments in uveal melanoma (UM), the most common primary adult intraocular malignancy. To fill this critical need, we developed and characterized a multicenter biobank of UM patient-derived organoids (PDOs). UM patients requiring enucleation from 2019 to 2024 donated tumor tissue for PDO generation. PDOs were cultured in Cultrex and compared to donor primary tumor using exome sequencing, RNA sequencing, and immunohistochemistry. The ability of PDOs to maintain the transformed phenotype was evaluated in an orthotopic xenograft model and monitored with fundus imaging. ATAC sequencing and drug response assays were done in a subset of PDOs to explore the feasibility of their use for mechanistic and translational studies. PDOs were successfully established in 40 of 44 cases (91%), retained clinically relevant mutations and molecular markers from the primary tumor, and displayed similar gene expression profiles and well-validated clinical prognostic markers of the disease. PDOs retained tumorigenic capacity in an in vivo model resembling human disease progression. Finally, we demonstrated that PDOs were a feasible platform to identify and evaluate novel therapeutic targets and investigate differential, personalized drug response. PDO models offer a new platform with improved representation of human UM to aid in translational research for this dismal condition.

The cell adhesion molecule CD44 acts as a modulator of 5-HT7 receptor functions

BackgroundHomo- and heteromerization of G protein-coupled receptors (GPCRs) plays an important role in the regulation of receptor functions. Recently, we demonstrated an interaction between the serotonin receptor 7 (5-HT7R), a class A GPCR, and the cell adhesion molecule CD44. However, the functional consequences of this interaction on 5-HT7R-mediated signaling remained enigmatic.MethodsUsing a quantitative FRET (Förster resonance energy transfer) approach, we determined the affinities for the formation of homo- and heteromeric complexes of 5-HT7R and CD44. The impact of heteromerization on 5-HT7R-mediated cAMP signaling was assessed using a cAMP responsive luciferase assay and a FRET-based cAMP biosensor under basal conditions as well as upon pharmacological modulation of the 5-HT7R and/or CD44 with specific ligands. We also investigated receptor-mediated G protein activation using BRET (bioluminescence resonance energy transfer)-based biosensors in both, homo- and heteromeric conditions. Finally, we analyzed expression profiles for 5-HT7R and CD44 in the brain during development.ResultsWe found that homo- and heteromerization of the 5-HT7R and CD44 occur at similar extent. Functionally, heteromerization increased 5-HT7R-mediated cAMP production under basal conditions. In contrast, agonist-mediated cAMP production was decreased in the presence of CD44. Mechanistically, this might be explained by increased Gαs and decreased GαoB activation by 5-HT7R/CD44 heteromers. Unexpectedly, treatment of the heteromeric complex with the CD44 ligand hyaluronic acid boosted constitutive 5-HT7R-mediated cAMP signaling and receptor-mediated transcription, suggesting the existence of a transactivation mechanism.ConclusionsInteraction with the hyaluronan receptor CD44 modulates both the constitutive activity of 5-HT7R as well as its agonist-mediated signaling. Heteromerization also results in the transactivation of 5-HT7R-mediated signaling via CD44 ligand.

SeptAsTERS- SeptiCyte® RAPID as assessment tool for early recognition of sepsis - a prospective observational study.

Early recognition of sepsis is critical to patient outcome, with mortality increasing with every hour of delay in treatment. The aim of this study was to investigate the use of a point-of-care molecular host response assay to differentiate sepsis from inflammation after surgery. Three molecular host response assays (SeptiCyte® RAPID) were performed in 61 patients after major abdominal surgery with admission to the intensive care unit and drawn blood cultures. The first (T0) was taken ± 3h around the time of obtaining blood cultures, the second 24h later (T24) and the third at discharge from the intensive care unit (Tex). The primary endpoint was the agreement of SeptiCyte® RAPID results with the diagnosis of sepsis. SeptiScore® indicates sepsis probability (low risk 0 - high risk 15). Patients were retrospectively classified into sepsis and inflammation by three blinded experts. 25 (42.4%) patients were categorized as "inflammation" and 34 (57.6%) patients as "sepsis". At T0 and T24 septic patients showed significantly higher mean SeptiScores® of 8.0 (± 2.2 SD) vs. 6.3 (± 2.1 SD) and 8.5 (± 2.1 SD) vs. 6.2 (± 1.8 SD), respectively. The Receiver Operating Curves (ROC) for the ability to discriminate between sepsis and inflammation had an Area Under the Curve (AUC) of 0.71 (T0) and 0.80 (T24). Embedded in a comprehensive diagnostic algorithm molecular host response analysis could broaden the possibilities for infection diagnostics to differentiate between sepsis and inflammatory response after surgery. But validation in larger cohorts is needed.

Effect of protease inhibitors on the intraerythrocytic development of Babesia microti and Babesia duncani, the causative agents of human babesiosis

AbstractHuman babesiosis is a malaria‐like, tick‐borne infectious disease with a global distribution. Babesiosis is caused by intraerythrocytic, apicomplexan parasites of the genus Babesia. In the United States, human babesiosis is caused by Babesia microti and Babesia duncani. Current treatment for babesiosis includes either the combination of atovaquone and azithromycin or the combination of clindamycin and quinine. However, the side effects of these agents and the resistance posed by these parasites call for alternative approaches for treating human babesiosis. Proteases play several roles in the context of parasitic lifestyle and regulate basic biological processes including cell death, cell progression, and cell migration. Using the SYBR Green‐1 assay, we screened a protease inhibitor library that consisted of 160 compounds against B. duncani in vitro and identified 13 preliminary hits. Dose response assays of hit compounds against B. duncani and B. microti under in vitro conditions identified five effective inhibitors against parasite growth. Of these compounds, we chose ixazomib, a proteasome inhibitor as a potential drug for animal studies based on its lower IC50 and a higher therapeutic index in comparison with other compounds. Our results suggest that Babesia proteasome may be an important drug target and that developing this class of drugs may be important to combat human babesiosis.

Abstract B013: An mDETECT assay for monitoring treatment response in metastatic breast cancer patients

Abstract Metastatic breast cancer treatment response is currently assessed every 3-6 months and this long delay results in many patients undergoing ineffective and toxic treatments for long periods of time. We have developed the methylation DETEction of Circulating Tumour DNA (mDETECT) assay, a targeted DNA methylation-based Next Generation Sequencing liquid biopsy that detects cancer-specific patterns in all subtypes of breast cancer. The assay targets 60 differentially hypermethylated regions in breast cancer across the genome and assesses over 400 individual CpGs with an average of 8 CpGs per target amplicon. The assay has been assessed on DNA from 73 breast cancer tumour samples which on average had 40 regions hypermethylated per patient with similar rates across all subtypes. We will present results from plasma samples from locally advanced and metastatic breast cancer patients along with plasma from age-matched, healthy female controls. A prospective, multi-centre observational cohort study to monitor metastatic breast cancer patients using the mDETECT liquid biopsy as they undergo treatment is active and recruiting patients. Blood is drawn with every standard-of-care blood draw for up to 3 years. Plasma has been assessed using the mDETECT assay to determine the methylation status of the DNA at each time point. 35 patients have been enrolled to date with ongoing recruitment up to 150 patients. Patients are being monitored with the mDETECT assay for earlier detection of disease progression on a treatment. They will also be monitored through treatment changes to assess a patient’s initial response to a given treatment. Changes in mDETECT levels will be correlated with clinical response to determine if the mDETECT breast cancer assay can detect a response to treatment at an earlier timepoint. This assay could allow for improved monitoring of treatment response allowing clinicians to make informed decisions, improving outcomes and prolonging patient’s lives. Citation Format: Keira Frosst, Brooke Wilson, Katrina Cristall, Catherine Crawford-Brown, Christopher R Mueller. An mDETECT assay for monitoring treatment response in metastatic breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B013.

OGC O05 - Utilising patient derived organoids to predict the response to treatment in gastric cancer

Abstract Background Gastric adenocarcinoma remains a significant global health burden. For advanced disease, treatment remains multi-modal with neoadjuvant chemotherapy followed by resectional surgery. Although the FLOT4-AIO trial has established the regimen of 5-fluorauracil, Leucovorin, Oxaliplatin and Docetaxel (FLOT) as the gold standard, some patients demonstrate a poor or no response to treatment, resulting in adverse oncological outcomes. Currently there are no accurate biomarkers to assess or predict the treatment response to neoadjuvant therapy. This study aims to explore the potential of patient-derived organoids as a predictive model to assess the treatment response to neoadjuvant chemotherapy in gastric adenocarcinoma. Method Patient derived organoids are a novel, 3 dimensional in-vitro model which recapitulates the different cells that constitute the tissue of origin. From June 2023 to June 2024, 25 patients with a confirmed diagnosis of gastric adenocarcinoma, were recruited for tissue acquisition for organoid generation. Endoscopic biopsies of cancer tissue taken during OGD and staging laparoscopy were used to generate organoids from each patient. Organoids were subsequently expanded to a sufficient volume to allow cytotoxicity assays in response to different doses of the FLOT regimen. The organoid cytotoxicity response was compared to the patients radiological and pathological response following treatment. Results Organoids were successfully generated from 76% of recruited patients (n=19). Of these, 4 patients and were excluded due to being unfit for neoadjuvant chemotherapy, whilst 2 were excluded due to the confirmation of metastatic disease, precluding neoadjuvant treatment. Finally a single patient was excluded due to mortality during neoadjuvant treatment. Of the remaining organoids, 5 were subjected to FLOT treatment within 2 weeks of tissue acquisition. Organoids demonstrated unique dose response profiles following with statistically significant differences in the IC50 values (p =0.048). The organoid responses corresponded to the clinical responses demonstrated by the patient they were derived from. Conclusion In conclusion, this study underscores the potential of patient-derived cancer organoids as a predictive model for assessing the response to neoadjuvant chemotherapy in gastric adenocarcinoma. The generation of organoids from a significant proportion of patients and the subsequent drug response testing within 2 weeks of tissue acquisition suggests this novel method has the potential to become a valuable tool for predicting patient-specific responses and guiding personalised treatment plans. As such, further research is warranted to further assess the validity of this model, refine the existing techniques and explore its potential integration into clinical practice.

Investigation of resistance mechanisms to flucarbazone-sodium in wild oat (Avena fatua L.) from China

BackgroundWild oat (Avena fatua L.) is a self-pollinating, allohexaploid species in the family Gramineae (grasses), which is a malignant weed that mainly harms crops such as wheat. In recent years, a decline in the control efficiency of flucarbazone-sodium against wild oat has occurred in some regions of China.ResultsWe identified an ALS-resistant A. fatua population (R population). Whole-plant response assays revealed that the R population exhibited a moderate level of resistance (5.9-fold) to flucarbazone-sodium. Pre-treatment with malathion significantly reduced flucarbazone-sodium resistance in the R population. The known mutation sites and ALS gene relative expression that confer resistance to ALS inhibitor herbicides were not found in R population. Following flucarbazone-sodium treatment, the expression of eight genes related to metabolic enzymes was investigated using quantitative real-time PCR (qRT-PCR). CYP92A6 and the Aldo/keto reductase family were highly expressed in the R population after the application of flucarbazone-sodium.ConclusionsThe mechanism of flucarbazone-sodium resistance in A. fatua is mediated by NTSR, nor TSR. Two genes, CYP92A6 and the Aldo/keto reductase family, were discovered to be possibly related in the metabolism of NTSR in the A. fatua population, justifying more functional studies. The results will serve as a data resource for further studies on the molecular mechanisms of A. fatua to flucarbazone-sodium.

Emergence of multiple resistance to EPSPS and ALS herbicides in smooth pigweed (Amaranthus hybridus): a growing concern in Brazil

Abstract Recently, farmers in Brazil have observed a decline in efficacy of glyphosate, chlorimuron, and imazethapyr control of smooth pigweed (Amaranthus hybridus L.). The objectives of this study were to quantify the resistance of Amaranthus in Brazil to glyphosate and acetolactate synthase (ALS)-inhibiting herbicides, elucidate the mechanism of resistance, and assess the frequency of sensitivity shifts to glyphosate and chlorimuron in Brazil. Dose–response assays were conducted in a greenhouse with glyphosate, chlorimuron, and imazethapyr. This was followed by sequencing of the EPSPS and ALS genes. Additionally , 740 Amaranthus populations across several Brazilian states were monitored over 4 yr, subjected to a single discriminatory dose of glyphosate and chlorimuron. The populations BR18Asp051 and BR21Asp205 were resistant to glyphosate, chlorimuron, and imazethapyr. The elevated resistance level to glyphosate in these populations is attributed to multiple amino acid substitutions (TAP-IVS) in the EPSPS gene; and cross-resistance to sulfonylureas and imidazolinones is conferred by the Trp-574-Leu substitution in the ALS gene in both populations. Overall, resistance distribution indicated that 88% of the sampled populations were considered sensitive to glyphosate, while 66% were sensitive to chlorimuron. Furthermore, 10% of the samples demonstrated multiple resistance to both active ingredients. A shift in glyphosate sensitivity was observed in four states in Brazil; however, sensitivity shifts to chlorimuron were more widely dispersed in Brazilian agricultural regions.

Hydroponic Culture of Rice Seedlings for Stress Response Assay.

The major environmental factors limiting rice growth and production are osmotic stresses such as drought and high salinity. High osmotic stresses directly disrupt cellular activities, leading to plant growth retardation or death. Plants have various response mechanisms to survive under such stresses. Understanding rice's stress response mechanisms is necessary to enhance the osmotic stress tolerance of rice. However, assessing specific physiological responses to osmotic stresses is difficult because multiple environmental factors affect rice growth. Here, we describe a simple method for analyzing the osmotic stress responses of rice plants using a hydroponic culture system. This method allows comprehensive gene expression and phenotypic analyses under osmotic stress conditions in rice. Various osmotic stress conditions and samples can be tested simultaneously because this method is small-scale. In addition, the procedure is easy, and highly reproductive results can be obtained.

The Rolled Towel Method for Hormone Response Assays in Maize.

The rolled towel assay (RTA) is a soil-free method to evaluate juvenile phenotypes in crops such as maize and soybean. Here, we provide an updated RTA-based protocol to phenotype maize seedling responses to chemicals of interest. We exemplify the protocol with two synthetic auxin herbicides (2,4-dichlorophenoxyacetic acid and picloram), an auxin precursor (indole-3-butyric acid), and an auxin inhibitor (N-1-naphthylphthalamic acid), but the method can be used with other hormones or plant growth regulators that are soluble in growth media. We also include instructions on how to annotate root traits and analyze primary root length trait data. The protocol can be scaled up for use in genetic screens, preparing tissue for gene expression analyses, carrying out genome-wide association studies (GWASs), and quantitative trait locus (QTL) identification.