Differential Response Research Articles

Differential Responses To Heat Stress On The Regeneration Of Injured Rat Soleus And Plantaris Muscles

Differential Responses To Heat Stress On The Regeneration Of Injured Rat Soleus And Plantaris Muscles

Post-exercise Differential Response Of Central And Brachial Blood Pressure In Patients With Coronary Artery Disease

Post-exercise Differential Response Of Central And Brachial Blood Pressure In Patients With Coronary Artery Disease

Abstract C084: Genetic polymorphism rs3820282 and WNT4-mediated metabolic plasticity contributes to differential therapy response in ovarian cancer clear carcinoma

Abstract C084: Genetic polymorphism rs3820282 and WNT4-mediated metabolic plasticity contributes to differential therapy response in ovarian cancer clear carcinoma

The association between short-term temperature variability and mortality in Virginia

The objective of this study is to determine the relationship between short-term temperature variability on neighboring days and mortality. The change in maximum temperature in Northern Virginia, Richmond, Roanoke, and Norfolk, Virginia, on neighboring days was calculated from airport observations and associated with total mortality over a multi-county area surrounding each weather station. The association between day-to-day temperature change and mortality, lagged over a 28-day period, was analyzed using distributed lag non-linear models that controlled for air quality, temporal trends, and other factors. Days following large temperature declines were associated with an increased risk of mortality in three of the four locations, and temperature increases were linked to higher mortality risk in two cities. For example, the relative risk of mortality for a 12°C daily temperature decline (1st percentile) was 1.74 [0.92, 3.27] in Roanoke and 1.16 [0.70, 1.92] in Richmond. The net effect of short-term temperature increases was smaller, with the largest relative risk of 1.03 [0.58, 1.83] for a 12°C increase (99th percentile) in maximum temperature in Norfolk. In Richmond and Roanoke, there was an observed lagged effect of increased mortality (maximum relative risks varying from 1.08 to 1.10) that extended from 5 to 25 days associated with large temperature declines of 15°C or more. In contrast, there was a strong and immediate (lag 0–3 day) increase in the risk of mortality (1.10 to 1.15) in northern Virginia and Norfolk when the temperature increase exceeded 10°C (short-term warming). In general, consecutive day warming had a more immediate mortality impact than short-term cooling, when the peak mortality is lagged by one week or more. However, cooling of at least 10°C after a hot (summer) day reduced mortality relative to comparable cooling following a cold (winter) day, which is associated with high mortality. This differential mortality response as a function of temperature suggests that there is some relationship between average temperature, temperature variability, and season. The findings of this study may be useful to public health officials in developing mitigation strategies to reduce the adverse health risks associated with short-term temperature variability.

Spillover and competitive exclusion in the crustacean community following the implementation of a marine reserve

Abstract Flamanville marine protected area (MPA) located in Normandy, France, was created in 2000 to sustain the local crustacean fishery. In 1985, an annual survey targeting crustacean populations (e.g. European lobster Homarus gammarus, edible crab Cancer pagurus, and European spider crab Maja brachydactyla) that include the future MPA started. The MPA implementation effects were investigated in this study. The sampling design allowed the integration of spatial and temporal covariances to estimate the MPA effects. With respect to the initial objective, the MPA significantly improves the most economically valuable species, H. gammarus, abundance. Lobster catch per unit effort increased by 597% in the MPA, whereas outside it only increased by 156%. The MPA creation also led to an increase in lobster size inside the protected area. Furthermore, a few years after the MPA implementation, lobster catches showed a significant non-linear decline with distance from the centre of the reserve, suggesting a spillover effect. However, the edible crab catches were halved following the MPA implementation suggesting an opposite effect. Spider crab abundance seemed unaffected. Finally, the MPA implementation had no effect on edible crab and spider crab sizes. These differential responses appear to result from these species’ variable movement and competitive abilities.

A Medicago truncatula HD-ZIP gene MtHB2 is involved in modulation of root development by regulating auxin response

HD-Zip proteins are plant-specific transcription factors known for their diverse functions in regulating plant growth, development, and responses to environmental stresses. Among the Medicago truncatula HD-Zip II genes, MtHB2 has been previously linked to abiotic stress responses. In this study, we conducted a functional characterization of MtHB2 in the regulation of root growth and development. Upon auxin stimulation, expression of MtHB2 was promptly up-regulated. Overexpression of MtHB2 in Arabidopsis thaliana led to reduced primary root growth and inhibited lateral root formation. Interestingly, the transgenic plants expressing MtHB2 exhibited differential responses to three types of auxins (IAA, NAA, and 2,4-D) in terms of root growth and development compared to the wild-type plants. Specifically, primary root growth was less affected, and lateral root formation was enhanced in the transgenic plants when exposed to auxins. This differential response suggests a potential role for MtHB2 in modulating auxin transport and accumulation, as evidenced by the reduced sensitivity of the transgenic plants to the auxin transport inhibitor NPA and lower expression levels of auxin-related reporters such as PIN-FORMED (PIN1)::PIN1-GFP, PIN3::PIN3-GFP, PIN7::PIN7-GFP, and DR5::GFP compared to wild-type plants. Additionally, microarray analysis of the root tissues revealed down-regulation of several auxin-responsive genes in transgenic seedlings compared to wild-type plants. These findings collectively indicate that MtHB2 plays a critical regulatory role in root growth and development by modulating auxin accumulation and response in the roots.

Transcriptional and hormonal profiling uncovers the interactions between plant developmental stages and RNA virus infection.

Arabidopsis thaliana is more susceptible to certain viruses during its later developmental stages. The differential responses and the mechanisms behind this development-dependent susceptibility to infection are still not fully understood. Here we explored the outcome of a viral infection at different host developmental stages by studying the response of A. thaliana to infection with turnip mosaic virus at three developmental stages: juvenile vegetative, bolting, and mature flowering plants. We found that infected plants at later stages downregulate cell wall biosynthetic genes and that this downregulation may be one factor facilitating viral spread and systemic infection. We also found that, despite being more susceptible to infection, infected mature flowering plants were more fertile (i.e. produce more viable seeds) than juvenile vegetative and bolting infected plants; that is, plants infected at the reproductive stage have greater fitness than plants infected at earlier developmental stages. Moreover, treatment of mature plants with salicylic acid increased resistance to infection at the cost of significantly reducing fertility. Together, these observations support a negative trade-off between viral susceptibility and plant fertility. Our findings point towards a development-dependent tolerance to infection.

Genome‑wide analysis of cotton SCAMP genes and functional characterization of GhSCAMP2 and GhSCAMP4 in salt tolerance

BackgroundSecretory carrier membrane proteins (SCAMPs) form a family of integral membrane proteins and play a crucial role in mediating exocytosis in both animals and plants. While SCAMP genes have been studied in several plant species, their functions in cotton, particularly in response to abiotic stress, have not yet been reported.ResultsIn this study, a total of 53 SCAMP genes were identified in G. arboreum, G. raimondii, G. hirsutum, and G. barbadense. These genes were classified into five groups based on a phylogenetic analysis with SCAMPs from Arabidopsis thaliana. The main factor driving the expansion of the SCAMP gene family in G. hirsutum is tandem and segmental duplication events. Using MEME, in addition to the conserved SCAMP domain, we identified 3–13 other domains in each GhSCAMP. The cis-element analysis suggested that GhSCAMPs were widely involved in cotton growth and development, and responses to abiotic stresses. RNA sequencing (RNA-Seq) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results showed that most GhSCAMPs were expressed highly in many tissues and had differential expression responses to drought, cold, and heat stresses. Knock-down of GhSCAMP2 and GhSCAMP4 by virus-induced gene silencing (VIGS) lead to a salt-sensitive phenotype and had a lower content of CAT, POD, and SOD.ConclusionsThis study identified SCAMP genes in four cotton species, enhancing our understanding of the potential biological functions of SCAMPs. Additionally, we demonstrated that GhSCAMP2 and GhSCAMP4 positively regulate cotton tolerance to salt stress.

Differential immunological responses in lamb rumen and colon to alfalfa hay and wheat straw in a concentrate-rich diet: insights into microbe-host interactions.

The impact of a concentrate-rich (CR) diet on the gut microbiome and epithelium homeostasis is well documented. However, it has not been systematically studied whether and how host-microbial interaction contributes to the immune homeostasis in the rumen and colon of lambs fed alfalfa hay and wheat straw, alone or combined, in a CR diet. In all, 63 lambs (initial body weight, 16.69 ± 1.50 kg) were randomly allotted to three dietary groups, each consisting of three pens with seven lambs per pen. Over 14 weeks, the lambs were fed diets as follows: 60% concentrate supplemented with either 40% wheat straw (WG), 20% alfalfa hay combined with 20% wheat straw (MG), or 40% alfalfa hay (AG). The present findings showed that lambs in the AG group had greater (P < 0.05) IgG and lower (P = 0.067) tumor necrosis factor-alpha concentrations relative to those in the MG and WG groups. The 16S rRNA analysis highlighted that various bacterial phyla and genera in the rumen and colon preferentially degrade fiber and starch derived from alfalfa hay and wheat straw. The weighted gene co-expression network analysis revealed that the bacterial genera from the Firmicutes are broadly associated with genes involved in various signaling pathways, underscoring the potential role of Firmicutes as key drivers of host-microbial interactions under the present feeding conditions. These findings shed light on the fact that the rumen and colon immune homeostasis is distinctively influenced by diets of alfalfa hay, wheat straw, or their combination in a CR diet. Further studies should examine the prolonged effects of replacing wheat straw with alfalfa hay in a concentrate-rich diet formulated to provide equivalent neutral detergent fiber levels. This could reveal how various forage fibers influence host-microbial interactions and gut health.IMPORTANCEIn contemporary feedlots, a growing trend is to feed animals a concentrate-rich (CR) diet that could disrupt the synchronized interplay between microbes and host metabolism, leading to altered metabolic functions. Wheat straw and alfalfa hay have different levels of protein and neutral detergent fiber, each with varying rates of digestion. It is unclear how including alfalfa hay and wheat straw, alone or combined in a CR diet, influences the host-microbial consortia and immune homeostasis. Herein, we showed that rumen and colon showed differential immune responses to the alfalfa hay, wheat straw, or both. Bacterial genera preferentially degrade fiber and starch derived from alfalfa hay, wheat straw, or both. Bacterial genera from Firmicutes phylum play a pivotal role in driving the host-microbial interactions, as indicated by their extensive association with genes across various signaling pathways.

Influence of Cultivar-Dependent Alstroemeria Floral Emissions on Frankliniella occidentalis Host Selection Behavior

Frankliniella occidentalis, commonly known as the western flower thrips (WFT), is one of the world’s most significant cosmopolitan pests. This pest infests many ornamental species, including Alstroemeria, leading to substantial economic losses. F. occidentalis damages flowers, transmits viruses, and causes the rejection of shipments for exports. Farmers have observed variations in the occurrence of F. occidentalis among different Alstroemeria cultivars. It is hypothesized that differences in flower color and odor among cultivars may influence the host-choice behavior of this insect, potentially explaining the observed variations in incidence and damage in the field. To test this hypothesis, we analyzed one year’s worth of sampling data for the occurrence of the WFT complex in a commercial Alstroemeria greenhouse crop. This analysis identified cultivars with high and low thrips incidences. The ‘Himalaya’ and ‘Whistler’ cultivars exhibited the highest incidence, while the ‘Shakira’ and ‘Nora’ cultivars showed the lowest incidence values. To investigate the potential role of host odors in these field incidence differences, we conducted behavioral tests (choice, non-choice, and repellency) using glass boxes where visual stimuli were blocked. These tests confirmed a differential attraction response of thrips to the flowers of Alstroemeria cultivars, although all cultivars were viable options for WFT choice. Preferences under laboratory conditions differed from the incidence observed in the greenhouse, ruling out a repellency effect of some cultivars on insects. This study provides evidence that differential emissions from Alstroemeria flowers can influence host selection by WFT, a phenomenon that warrants further in-depth exploration in future studies.

Haemosporidian Infection Is Associated with the Oxidative Status in a Neotropical Bird

Haemosporidians are common blood parasites in wild bird populations, yet their effects on oxidative status remain understudied. Here, we measured the levels of thiobarbituric acid-reactive substances (TBARS) as an indicator of reactive oxygen species (ROS), total antioxidant capacity (TAC) as an indicator of non-enzymatic molecular antioxidants, and TBARS/TAC ratio as an indicator of oxidative status. We also used parasite genus-specific primers and PCR techniques to detect haemosporidians in 117 adults of Rufous-collared Sparrow (Zonotrichia capensis) from four locations in south–central Chile. Mixed-effect models were employed to compare oxidative indicators between infected and uninfected birds. Infected birds showed significantly higher TBARS levels, but no significant differences in TAC, leading to a higher TBARS/TAC ratio, especially in reproductive individuals. This suggests increased oxidative damage in infected birds, irrespective of sex or body condition. A positive relationship between TBARS and TAC was observed in both groups, but the antioxidant response was weaker in infected birds, indicating differential oxidative stress responses based on infection status. Body condition did not differ significantly between infected and uninfected individuals. These results demonstrate that haemosporidian infections impose oxidative costs on birds, potentially compounding the oxidative costs associated with reproduction.

The effect of gradual extinction training on the renewal of electrodermal conditional responses.

Extinction, the repeated presentation of a conditional stimulus (CS) without the unconditional stimulus (US), is the standard paradigm to reduce conditional responding acquired by the repeated pairing of CS and US in acquisition. However, this reduction of conditional responding is prone to relapse. In rodent fear-conditioning, gradual extinction, the fading out of CS-US pairings during extinction, has been shown to reduce the return of fear. The current study replicated the gradual extinction procedure in human fear conditioning and assessed whether it reduced the return of fear due to ABA renewal and reacquisition. During extinction, one group received standard extinction, a second received gradual extinction (increasing the spacing of USs presented after the 1st, 3rd, 6th, 10th, and 15th CS+ trials), and a third received reversed extinction training (decreasing the spacing of USs presented after the 1st, 6th, 10th, 13th, and 15th CS+ trials). Larger renewal and faster reacquisition of differential electrodermal responses to CS+ and CS- were expected after standard and reversed extinction than after gradual extinction training. The results were inconclusive due to the failure to find extinction of differential electrodermal responses and US expectancy ratings in both gradual and reversed extinction groups. Despite successful extinction in group standard, renewal was only observed in US expectancy. Visualization of US expectancy ratings during extinction suggested that potential identification of the US presentation patterns during extinction in the gradual and reversed groups delayed extinction learning.

Modulatory effects of sub-MIC concentrations silver nanoparticles on virulence factor gene expression in Staphylococcus aureus

BackgroundSilver nanoparticles (AgNPs) exhibit a dose-dependent anti-bacterial effect, and it was aimed in this study to investigate the impact of sub-minimum inhibitory concentration (MIC) doses of AgNPs on the expression of virulence genes in Staphylococcus aureus (S. aureus). MethodsMinimum inhibitory concentration (MIC) values for AgNPs were determined for 183 S. aureus isolates. Gene expression was assessed in 14 isolates with sea and seb genes treated with AgNPs at a sub-MIC dose of 1 μg/ml. Accordingly, these strains were exposed to 1 μg/ml doses of AgNPs, and gene expression levels of sea, seb, and agr were assessed using quantitative RT-PCR after 4- and 12-hour post-AgNPs inoculation at 37 °C. The impact of AgNPs on the virulence factors of S. aureus was investigated over different time points, focusing on methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates. ResultsAnalysis revealed significant reductions in gene expression levels of seb and agr after 4 h post-AgNPs treatment in the MSSA group (p < 0.05), with further decreases observed at 12 h for sea, seb, and agr genes (p < 0.0001). MRSA isolates exhibited significant declines in sea and agr gene expression levels at both time points (p < 0.0001). However, no significant changes were observed in seb gene expression among MRSA isolates. Fold-change analysis indicated time-dependent effects of AgNP treatment on gene expression, highlighting substantial alterations in gene expression levels over time, particularly in seb and agr genes. ConclusionThese results show that sub-MIC levels of AgNPs greatly decrease the gene expression of important virulence factors in MSSA and MRSA strains, indicating their promise as treatments for S. aureus infections, particularly at 12 h post-treatment. The differential response between MSSA and MRSA isolates highlights the importance of strain variation in antimicrobial strategies.

Deciphering avian hematopoietic stem cells by surface marker screening and gene expression profiling

BackgroundAvian species have played a pivotal role in developmental hematopoiesis research, leading to numerous critical discoveries. Avian influenza, particularly the H5N1 strain, poses a significant threat to poultry and has zoonotic potential for humans. Infections often result in abnormal hematologic profiles, highlighting the complex interplay between avian diseases and hematopoiesis. Many avian diseases can suppress immune cells in the bone marrow (BM), impacting immune responses. Studying hematopoietic stem cells (HSCs) in avian BM is crucial for understanding these processes and developing effective vaccines and protection strategies for both avian and human health. MethodsThis study adapted methods from mouse studies to isolate avian HSCs as Lineage-negative (Lin-) cells. These isolated cells were further identified as Lin-Sca1+c-Kit+ (LSK) and were found to be more prevalent than in control groups. RT-PCR analyses were conducted, showing that genes like MEIS1 and TSC1 were upregulated, while SIRT1, FOXO1, and AHR were downregulated in these stem cells. Screening for LSK markers revealed ten unique surface antigens in the Sca1+c-Kit+ cell populations, including highly enriched antigens such as CD178, CD227, and CD184. Additionally, studies on quail HSCs demonstrated that similar labeling techniques were effective in quail BM. ResultsThe research demonstrated that the identification of avian HSC-specific surface antigens provides valuable insights into the pathogenesis of avian influenza and other diseases, enhancing our understanding of how these diseases suppress HSC function. Notably, the upregulation of MEIS1 and TSC1 genes in LSK cells underscores their critical roles in regulating hematopoietic processes. Conversely, the downregulation of SIRT1, FOXO1, and AHR genes provides important clues about their roles in differentiation and immune response mechanisms. DiscussionThe findings of this study deepen our understanding of the effects of avian diseases on the immune system by identifying surface markers specific to avian HSCs. The suppression of HSC function by pathogens such as influenza highlights the importance of understanding these cells in developing targeted vaccines. These results represent a significant step towards improving global health security by mitigating risks associated with avian pathogens.

The Immunomodulatory Effects of Lipoteichoic Acid from Lactobacillus reuteri L1 on RAW264.7 Cells and Mice Vary with Dose.

The probiotic properties of Lactobacillus reuteri (L. reuteri) and its impact on immune function are well-documented. Lipoteichoic acid (LTA) is a crucial immune molecule in Gram-positive bacteria. Despite extensive research on LTA's structural diversity, the immunomodulatory mechanisms of L. reuteri LTA remain largely unexplored. This study investigates the immunomodulatory effects of L. reuteri L1 LTA at various concentrations on RAW 264.7 cells and mice under normal and inflammatory conditions. We found that LTA does not significantly affect healthy subjects; however, low-concentration LTA can reduce inflammation induced by LPS in cells and mice, enhancing the abundance of dominant intestinal bacteria. In contrast, high-concentration LTA exacerbates intestinal damage and dysbiosis. Creatinine may play a role in this differential response. In summary, while LTA does not alter immune homeostasis in healthy organisms, low-concentration LTA may mitigate damage from immune imbalance, but high-concentration LTA can worsen it. This suggests a quantitative requirement for probiotic intake. Our study provides critical theoretical support for understanding the immunomodulatory effects of probiotics on the host and paves the way for future research into the immune mechanisms of probiotics.

Temperature differentially regulates estuarine microbial N2O production along a salinity gradient

Nitrous oxide (N2O) is atmospheric trace gas that contributes to climate change and affects stratospheric and ground-level ozone concentrations. Ammonia oxidizers and denitrifiers contribute to N2O emissions in estuarine waters. However, as an important climate factor, how temperature regulates microbial N2O production in estuarine water remains unclear. Here, we have employed stable isotope labeling techniques to demonstrate that the N2O production in estuarine waters exhibited differential thermal response patterns between nearshore and offshore regions. The optimal temperatures (Topt) for N2O production rates (N2OR) were higher at nearshore than offshore sites. 15N-labeled nitrite (15NO2-) experiments revealed that at the nearshore sites dominated by ammonia-oxidizing bacteria (AOB), the thermal tolerance of 15N-N2OR increases with increasing salinity, suggesting that N2O production by AOB-driven nitrifier denitrification may be co-regulated by temperature and salinity. Metatranscriptomic and metagenomic analyses of enriched water samples revealed that the denitrification pathway of AOB is the primary source of N2O, while clade II N2O-reducers dominated N2O consumption. Temperature regulated the expression patterns of nitrite reductase (nirK) and nitrous oxide reductase (nosZ) genes from different sources, thereby influencing N2O emissions in the system. Our findings contribute to understanding the sources of N2O in estuarine waters and their response to global warming.

Abstract A022: Neoadjuvant ablative radiation downstages high-risk features and alters immune response in pancreatic cancer

Abstract Purpose: Increasing evidence shows neoadjuvant (NA) therapy benefits surgical selection and survival in localized pancreatic cancer. However, the optimal NA therapy for resectable, borderline resectable (BRPC), and locally advanced pancreatic cancer (LAPC) remains unclear. We hypothesize that NA stereotactic ablative radiotherapy (SAbR) benefits patients with BRPC and LAPC who do not progress on chemotherapy (CTH), especially with arterial involvement. To evaluate this, we retrospectively reviewed outcomes and molecular features of patients treated with NA CTH and NA CTH followed by SAbR to understand the contribution of radiation. Methods: We retrospectively reviewed clinical data for 133 patients treated with NA CTH and 48 with NA CTH + SAbR. Bulk RNA sequencing was performed on fresh surgical tissue from 29 CTH-treated and 14 CTH + SABR-treated patients. We conducted gene set enrichment analysis (GSEA), CIBERSORTx cellular deconvolution, and Single-Cell Identification of Subpopulations with Bulk Sample Phenotype Correlation (Scissor) analysis to correlate clinical outcomes with transcriptomic features. Results: Molecular subtyping of PDAC transcriptomic signatures from post-treatment tissues revealed similar subtype proportions after both treatments. However, at baseline and before surgery, patients receiving SABR had more advanced clinical T staging, NCCN resectability, and arterial involvement. Despite this, the NA CTH + SAbR cohort showed similar overall survival, local-regional progression-free survival (LRPFS), and distant metastasis progression-free survival (DPFS) compared to NA CTH only. NA CTH + SABR-treated patients had significantly improved post-treatment T and N staging, tumor size, and treatment effect. These improvements correlated with better survival, LRPFS, and DPFS. RNAseq analysis indicated that patients with nodal involvement had significantly increased GSEA of MYC targets and worse DPFS. Tumor-arterial involvement at diagnosis significantly reduced local-regional control for NA CTH only. In contrast, patients with arterial involvement receiving NA CTH + SAbR had significantly improved local control. CIBERSORTx cell fraction deconvolution revealed no significant differences in tumor and immune cell subsets between treatments, but GSEA indicated substantial changes in immune-related gene sets, particularly those related to myeloid and T cell activation, suggesting functional changes correlating with treatment response and local control. Scissor identified gene signatures from activated CD8 T cells correlating with improved local control, while signatures from Treg and naïve T cells negatively impacted LRPFS. Conclusions: Molecular analysis and clinical features showed that patients treated with NA CTH + SAbR experienced downstaging and had similar outcomes, with indications of a differential immune response. Given these findings, the role of SAbR in the NA setting should be further evaluated, especially in the context of improved chemotherapy and novel combination therapies that leverage biological responses. Citation Format: Peter Q. Leung, Eslam A. Elghonaimy, Ahmed M. Elamir, Megan Wachsmann, Song Zhang, Neha Barrows, Rachel von Ebers, Cassandra Hamilton, Grace Josephson, Salwan Al Mutar, Patricio M. Polanco, Nina N. Sanford, Syed M. Ali Kazmi, Matthew R. Porembka, David Hsieh, Adam C. Yopp, Muhammad S. Beg, Herbert J. Zeh, Todd A. Aguilera. Neoadjuvant ablative radiation downstages high-risk features and alters immune response in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A022.

Tumorspheres as In Vitro Model for Identifying Predictive Chemoresistance and Tumor Aggressiveness Biomarkers in Breast and Colorectal Cancer

Chemoresistance remains a major challenge in the treatment of breast and colorectal cancer. For this reason, finding reliable predictive biomarkers of response to chemotherapy has become a significant research focus in recent years. However, validating in vitro results may be problematic due to the outcome heterogeneity. In this study, we evaluate the use of tumorspheres as an in vitro model for validating biomarkers of chemoresistance in breast and colorectal cancer. Our investigation highlights the crucial role of inflammation-related pathways in modulating the response to chemotherapy. Using in silico approaches, we identified specific markers elevated in responders versus non-responders patients. These markers were consistently higher in three-dimensional (3D) tumorsphere models compared to traditional adherent cell culture models. Furthermore, the number of tumorspheres from breast and colorectal cancer cells increased in response to cisplatin and oxaliplatin treatment, respectively, whereas cell viability decreased in adherent cell culture. This differential response underscores the importance of the 3D tumorsphere model in mimicking the tumor microenvironment more accurately than adherent cell culture. The enhanced chemoresistance observed in the 3D tumorspheres model and their correlation of data with the in silico study suggest that 3D culture models are a better option to approach the in vivo model and also to validate in silico data. Our findings indicate that tumorspheres are an ideal model for validating chemoresistance biomarkers and exploring the interplay between inflammation and chemoresistance in breast and colon cancer.

Abstract C023: PDAC tumors from patients treated with combination MEK and autophagy inhibition reveal a treatment sensitive epithelial subtype that is enriched in murine models

Abstract Combining MEK and autophagy inhibitors in preclinical PDAC mouse models resulted in tumor regressions and improved rodent survival. These findings provided the rationale for clinical investigation of this combination. The phase 1 MEKiAUTO trial evaluated combination cobimetinib, HCQ, with or without atezolizumab in 14 patients (pts) with KRAS-mutated pancreatic adenocarcinoma (PDAC). The study was closed due to a lack of preliminary efficacy and poor tolerability; however, three patients experienced progression free survival (PFS) beyond 12 weeks (two with KRAS G12R , one with KRAS G12D ). Correlative analyses on twenty-one biopsies (8-paired FFPE and 6-paired fresh frozen specimens) with multiplex immunofluorescence (mIF) demonstrated decreased phosphorylation of ERK and accumulation of p62 indicating a pharmacodynamic effect. Reverse phase protein array (RPPA) also confirmed MAPK pathway inhibition, with a decrease in pERK, and autophagy inhibition, with decreased autophagy signature score, within the tumor compartment of on-treatment biopsies compared to baseline. Single nucleus RNAseq with protein expression activity extrapolated using ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks) and VIPER (Virtual Inference of Protein-activity by Enriched Regulon) pipelines identified five distinct tumor subpopulations. Comparing prevalence of tumor clusters before and after treatment identified differential responses to treatment. Gene set enrichment analysis revealed that the most sensitive tumor cluster, tumor cluster 2, was enriched in autophagy and MAPK signaling. Pre- treatment frequency of tumor cluster 2 was higher in patients who experienced a clinical benefit and independently correlated with PFS. Among the non-tumor cell clusters identified, patients with clinical benefit also had increased immune cell infiltration. To explain the discordance in efficacy with this combination in preclinical models and humans, we interrogated the preclinical tumor model systems for tumor heterogeneity. Single cell RNA-Seq analysis of tumors from Kras LSL.G12D/+ ; p53 LSL.R172H/+ ; Pdx1-Cre tg/+ (KPC) mice treated with the combination confirmed presence of similar treatment-sensitive and treatment-resistant clusters, with the former being enriched for MAPK and autophagy signatures. Parallel analysis of cell lines previously shown to be sensitive to combination MEK and autophagy inhibition, including MiaPACA2 and PANC1, showed they were mostly enriched in the treatment-sensitive human cluster 2 phenotype but not treatment-resistant clusters. Our results suggest that some preclinical models may overrepresent subtypes which are sensitive to MEK and autophagy inhibitors and thus do not recapitulate the extent of intertumoral heterogeneity in human PDAC. Observed preclinical responses likely represented elimination of treatment-sensitive cluster(s), with the remaining clusters responsible for maintained resistance. Citation Format: Brian W Labadie, Aleksandar Z Obradovic, Sarah Sta Ana, Michael May, Naomi Sender, Isabelle V Ross, Monica Rodriguez-Mendez, Urszula Wasko, Lorenzo Tomassoni, Li Li, Manan Vij, Sinan Abuzaid, Julia Wulfkuhle, Winston Wong, Ilenia Pellicciotta, Benjamin Izar, Emanuel F Petricoin, Aik Choon Tan, Kenneth P Olive, Alex G Raufi, Gulam A Manji. PDAC tumors from patients treated with combination MEK and autophagy inhibition reveal a treatment sensitive epithelial subtype that is enriched in murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C023.

Virulence variation and pathotypes of Zymoseptoria tritici isolates causing wheat leaf blotch in Oromia, Ethiopia

Leaf blotch, caused by Zymoseptoria tritici, is a fungal disease that poses a severe threat to wheat production worldwide. Knowledge of virulence variability is crucial in choosing effective control measures. However, there have only been a few studies of the pathogenic variability and pathotypes within Ethiopian isolates. Hence, the objective of this study was to assess the virulence spectrum and variability of Z. tritici isolates. Forty-three isolates were tested for their virulence and pathotype against 7 wheat differential lines that have different resistance genes. A pathogenicity assay detected 41 differential line-specific virulent isolates among 301 interactions between a host and pathogen based on the percentage coverage of the leaf area by pycnidia. Some isolates were virulent against 50 %–60 % of the resistant genes, but most of them were virulent against some differential lines. Isolates such as EtA-11, EtSh-1, EtSh-2, EtSh-4, and EtA-19 expressed broad-spectrum virulence, highlighting that such isolates are useful for germplasm screening. The isolates were classified into 25 pathotypes, defined by their differential virulence responses. They were also assigned to two clusters according to their mean pycnidia percent. Pathotypes and principal component analysis detected 58.1 % and 62.2 % pathogenic diversity in Ethiopian isolates, respectively. The current findings provide information that breeders can use to identify and select more resistant varieties for farmers.