Wild-type Research Articles

Molecular Characterization of SHV, BLA-OXA and B-Lactamase Resistant Genes Leading to Computational Based Inhibitor Screening Against Bacillus Species

Antibiotic resistance in bacterial pathogen is a great challenge that is connected with excessive morbidity and mortality of living beings. A common motive of antibiotic resistance in bacteria is an increased abundance of Β- lactamases. There chromosomal Β- lactamases do not generally provide effective antibiotic resistance in wild type Bacilli despite evidence that the genes are not completely silenced. Under antibiotic selection pressure however, a number of strains show increased resistance suggesting mutation induced up regulation of Β- lactamases. Computer-assisted drug design has made significant progress in predicting biologically active molecules and their receptor binding conformation, with results that are sometimes more exact than those acquired through traditional methods like as high-throughput screening. The results of computational studies are sometimes more precise than the experimental possibilities, and they contribute to the improvement of the experimental array. In this study, identified pathogenic species of bacillus, and analyzed the antibiotic resistant genes. The Previous study of its characterization showed the presence of BLA-OXA and SHV genes in Bacillus cereus and Bacillus paramycoides. The sequenced antibiotics resistant genes were subjected to computational analysis for the interpretation of the potential inhibitors against BLA-OXA and SHV. The analyzed inhibitor will enable to study the diversity of antibiotic resistant mechanism and to minimize the resistance of bacterial species against antibiotics.

Semaglutide promotes the transition of microglia from M1 to M2 type to reduce brain inflammation in APP/PS1/tau mice.

A growing number of studies show that the diabetes drug Semaglutide is neuroprotective in Alzheimer's disease (AD) animal models, but its mode of action is not fully understood. In order to explore the mechanism of Semaglutide, 7-month-old APP/PS1/tau transgenic (3xTg) mice and wild-type (WT) mice were randomly divided into four groups: control group (WT + PBS), AD model group (3xTg + PBS), Semaglutide control group (WT + Semaglutide) and Semaglutide treatment group (3xTg + Semaglutide). Semaglutide (25 nmol/kg) or PBS was administered intraperitoneally once every two days for 30 days, followed by behavioral and molecular experiments. The results show that Semaglutide can improve working memory and spatial reference memory of 3xTg-AD mice, promote the release of anti-inflammatory factors and inhibit the production of pro-inflammatory factors in the cortex and hippocampus, and reduce Aβ deposition in the hippocampal CA1 region of 3xTg mice. Semaglutide can inhibit the apoptosis of BV2 cells induced by Aβ1-42 in a dose-dependent manner and promote the transformation of microglia from M1 to M2, thereby exerting anti-inflammatory and neuroprotective effects. Therefore, we speculate that Semaglutide shows an anti-inflammatory effect by promoting the transformation of microglia from M1 to M2 type in the brain of 3xTg mice, and thus exerts a neuroprotective effect.

Bacillus quorum quenching shapes the citrus mycobiome through interkingdom signaling

Microbiomes are sustained through infinite yet mutually interacting microbial communities, with bacteria and fungi serving as the major constituents. In recent times, microbial interventions have become popular for microbiome manipulation to achieve sustainable goals. Whether and how the introduced biocontrol agent drives fungal microbial assemblages (mycobiome) and the role of interkingdom signaling in shaping the microbiome structure and function remain poorly understood. Here, we implemented wild-type (WT) Bacillus subtilis L1–21 and its quorum quenching (QQ) mutants (L1–21Δytnp, L1–21Δyxel) individually and as consortia to explore the enrichment patterns of key mycobiome members in Huanglongbing (HLB) infected citrus compartments including leaf endosphere, root endosphere, and rhizosphere soil. The application of WT and its QQ mutants produced differential mycobiome enrichment across citrus compartments. Our findings reveal that application of WT B. subtilis enriched beneficial fungi such as Trichoderma (15.82 %) in leaf endosphere. In contrast, pathogenic fungi Fusarium (47.5 %) and Gibberella (0.47 %) involved in citrus root decline were adundant in the L1–21Δytnp treated root endosphere while Nigrospora (11 %) was predominant in L1–21Δyxel treated leaf endosphere, affirming the role of bacterial quorum sensing (QS) molecules in shaping the fungal community composition. In general, based on the fungal functional prediction, fungal pathogens were highly abundant in mutant-treated plants, particularly in leaf endosphere (L1–21Δytnp: 25 %; L1–21Δyxel: 36.35 %) compared to WT (20.93 %). Additionally, some fungal members exhibited strong compartment specificity and both mutants induced distinct mycobiome shifts in rhizosphere soil, leaf, and root endopshere. In conclusion, B. subtilis QQ modifies bacterial QS networks facilitating beneficial fungi to establish, while loss of QQ leads to enrichment of pathogenic fungal groups. Our study provides a direct link of perception and regulation of mycobiome through bacterial-based QS and QQ system, and its association with disease outcomes.

Development of rapid, efficient and cost effective screening technique for testing arecanut against Phytophthora meadii incitant of fruit rot disease

To accelerate identification of disease resistant arecanut germplasm or hybrids against Phytophthora, it is very much imperative to develop bioassays which could differentiate resistant and susceptible cultivars efficiently. Here, developed a cost effective and rapid technique called “Detached Leaf Assay” to identify resistant germplasm at the seedling stage itself. Standardized zoospore production in highly virulent Phytophthora meadii (P19) by incubating under 12 hours light and dark regime. Zoospore suspension adjusted to 105spores ml-1 in petri plates. Subsequently, surface sterilized arecanut leaves were floated in zoospore suspension and incubated at temperature of 24±1°C. Disease symptoms, including water-soaked lesions, were recorded three days after inoculation. Infection lesion increased from 1 to 7.3 cm2. The pathogen was re-isolated and confirmed with the original culture. The assay was successfully validated to screen arecanut accessions, wild types and hybrids against P. meadii. This technique is the first to be developed, and it is simple, cost-effective, and faster. It also provides consistent infection and could be effectively utilized to screen arecanut germplasm or hybrids against P. meadii in the seedling stage itself.• Developed a cost effective, efficient and rapid screening technique• The technique was validated to identify resistant genotypes against Phytophthora meadii at the seedling stage.

GhBZR15 improved the tolerance of cotton to cold and salt stress revealed by genome-wide characterization of BZR genes family

Brassinazole resistant (BZR) transcription factors play important roles in brassinosteroids (BRs) signaling pathway of plants, which are involved in many developmental processes and the responses to abiotic stress by regulating the expression of related genes. In order to explore the molecular mechanism of BZR transcription factors in cotton in response to abiotic stress, the BZR transcription factors in four cotton genomes were identified and analyzed. A total of 67 BZR transcription factors were identified and divided into three subfamilies according to their genetic relationship. The expression levels of BZR under cold, heat, salt, and drought treatments were analyzed to predict their function under abiotic stress. Promoter analysis of BZR proteins showed that light signaling can enhance the BZR transcriptional activity. The subcellular localization experiment showed that GhBZR15 protein was mainly distributed in the nucleus. Arabidopsis thaliana L. overexpressing GhBZR15 gene showed stronger cold tolerance and salt tolerance than wild type. These results, combined with the GhBZR protein-interaction network, suggest that the interaction of GhBZR with phytochrome interacting factors (PIF4) plays an important role in abiotic stress responses. These findings provide a basis for further understanding the function of BZR in cotton.

Microbiota responses to mutations affecting NO homeostasis in Arabidopsis thaliana.

Interactions between plants and microorganisms are pivotal for plant growth and productivity. Several plant molecular mechanisms that shape these microbial communities have been identified. However, the importance of nitric oxide (NO) produced by plants for the associated microbiota remains elusive. Using Arabidopsis thaliana isogenic mutants overproducing NO (nox1, NO overexpression) or down-producing NO (i.e. nia1nia2 impaired in the expression of both nitrate reductases NR1/NIA1 and NR2/NIA2; the 35s::GSNOR1 line overexpressing nitrosoglutathione reductase (GSNOR) and 35s::AHB1 line overexpressing haemoglobin 1 (AHB1)), we investigated how altered NO homeostasis affects microbial communities in the rhizosphere and in the roots, soil microbial activity and soil metabolites. We show that the rhizosphere microbiome was affected by the mutant genotypes, with the nox1 and nia1nia2 mutants causing opposite shifts in bacterial and fungal communities compared with the wild-type (WT) Col-0 in the rhizosphere and roots, respectively. These mutants also exhibited distinctive soil metabolite profiles than those from the other genotypes while soil microbial activity did not differ between the mutants and the WT Col-0. Our findings support our hypothesis that changes in NO production by plants can influence the plant microbiome composition with differential effects between fungal and bacterial communities.

The sensor protein AaSho1 regulates infection structures differentiation, osmotic stress tolerance and virulence via MAPK module AaSte11-AaPbs2-AaHog1 in Alternaria alternata

The high-osmolarity-sensitive protein Sho1 functions as a key membrane receptor in phytopathogenic fungi, which can sense and respond to external stimuli or stresses, and synergistically regulate diverse fungal biological processes through cellular signaling pathways. In this study, we investigated the biological functions of AaSho1 in Alternaria alternata, the causal agent of pear black spot. Targeted gene deletion revealed that AaSho1 is essential for infection structure differentiation, response to external stresses and synthesis of secondary metabolites. Compared to the wild-type (WT), the ∆AaSho1 mutant strain showed no significant difference in colony growth, morphology, conidial production and biomass accumulation. However, the mutant strain exhibited significantly reduced levels of melanin production, cellulase (CL) and ploygalacturonase (PG) activities, virulence, resistance to various exogenous stresses. Moreover, the appressorium and infection hyphae formation rates of the ∆AaSho1 mutant strain were significantly inhibited. RNA-Seq results showed that there were four branches including pheromone, cell wall stress, high osmolarity and starvation in the Mitogen-activated Protein Kinase (MAPK) cascade pathway. Furthermore, yeast two-hybrid experiments showed that AaSho1 activates the MAPK pathway via AaSte11-AaPbs2-AaHog1. These results suggest that AaSho1 of A. alternata is essential for fungal development, pathogenesis and osmotic stress response by activating the MAPK cascade pathway via Sho1-Ste11-Pbs2-Hog1.

Defects of WFS1-mediated peptide hormones secretion contribute to the manifestations of Wolfram syndrome

AimsThe study aims to investigate whether WFS1 is involved in the regulation of the exportation and secretion of other peptide hormones, as well as to elucidate the precise molecular mechanisms underlying WS caused by pathogenic mutations in the WFS1 gene. Materials and methodsThe plasma proteome from the WS patients (n = 2, male) and WFS1-deficient mice (n = 5, male) were analyzed using liquid-chromatography tandem mass spectrometry (LC-MS/MS), while age- and gender-matched healthy individuals and wildtype (WT) mice serve as controls. WFS1-deficient mice were intraperitoneally injected with IGF1 starting from 4 weeks of age. Body weight was monitored every 2 days, fasting blood glucose and glucose tolerance test were performed on the day 30 and day 40 after injection of IGF1, respectively. BiFC (bimolecular fluorescence complementation) and Co-immunoprecipitation (IP) were used to analyze the interaction between WFS1 and peptide hormones. Confocal microscopy was employed to analyze the colocalization of IGF1 with ER and Golgi. Key findingsPeptide hormones are deficient in both the plasma of WS patients and WFS1-deficient mice. WFS1 binds to and mediates the secretion of these peptide hormones, suggesting that WFS1 serves as a general COPII vesicular receptor for sorting peptide hormones. Interestingly, the WFS1 pathogenic mutations significantly disrupt its interaction with these peptide hormones. Furthermore, intraperitoneal administration of IGF1 partially attenuates high blood glucose levels in WFS1-deficient male mice. SignificanceThis study suggests that WS is characterized by defective peptide hormone secretion and proposes administration of these deficient peptide hormones as a promising treatment regimen for WS.

Determination of T cell response against XBB variants in adults who received either monovalent wild type inactivated whole virus or mRNA vaccine or bivalent WT/BA.4-5 COVID-19 mRNA vaccine as the additional booster

ObjectivesAs the SARS-CoV-2 virus evolves more rapidly than vaccines are updated, T cell immunity potentially confers protection against disease progression and death from new variants. In this study, we aimed to assess whether the current boosting vaccination schemes offer sufficient T cell protection against new SARS-CoV-2 variants. Methods. 292 adults who had received the second booster of either monovalent wild type vaccines (inactivated virus or mRNA) (cohort 1) or the second/third booster of bivalent WT/BA.4-5 mRNA vaccine (cohort 2) were recruited in Hong Kong. All participants showed no serological evidence of recent infection of SARS-CoV-2. Blood samples of each participant were collected before and one-month after receiving the booster. T cell and antibody responses were determined by flow cytometry and neutralization test respectively. Results. Among all vaccination strategies, only the adults who had received the bivalent vaccine as the third booster dose significantly elicited T cell responses to the XBB variant. Either monovalent or bivalent mRNA but not inactivated virus vaccine as the second/third booster induced antibody against different XBB variants. Conclusions. Receiving bivalent mRNA vaccine as the third booster is preferrable to induce both T cell and antibody responses against XBB.

Discovery of a Potential Allosteric Site in the SARS-CoV-2 Spike Protein and Targeting Allosteric Inhibitor to Stabilize the RBD Down State using a Computational Approach.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a worldwide public health crisis. At present, the development of effective drugs and/or related therapeutics is still the most urgent and important task for combating the virus. The viral entry and associated infectivity mainly rely on its envelope spike protein to recognize and bind to the host cell receptor angiotensin-converting enzyme 2 (ACE2) through a conformational switch of the spike receptor binding domain (RBD) from inactive to active state. Thus, it is of great significance to design an allosteric inhibitor targeting spike to lock it in the inactive and ACE2-inaccessible state. This study aims to discover the potential broad-spectrum allosteric inhibitors capable of binding and stabilizing the diverse spike variants, including the wild type, Delta, and Omicron, in the inactive RBD down state. In this work, we first detected a potential allosteric pocket within the SARS-CoV-2 spike protein. Then, we performed large-scale structure-based virtual screening by targeting the putative allosteric pocket to identify allosteric inhibitors that could stabilize the spike inactive state. Molecular dynamics simulations were further carried out to evaluate the effects of compound binding on the stability of spike RBD. Finally, we identified three potential allosteric inhibitors, CPD3, CPD5, and CPD6, against diverse SARS-CoV-2 variants, including Wild-type, Delta, and Omicron variants. Our simulation results showed that the three compounds could stably bind the predicted allosteric site and effectively stabilize the spike in the inactive state. The three compounds provide novel chemical structures for rational drug design targeting spike protein, which is expected to greatly assist in the development of new drugs against SARS-CoV-2.

Enhanced catalytic performance of methanol-tolerant Rhizopus stolonifera immobilized on polyurethane foam with hydrophobic coating for biodiesel production

The limited methanol tolerance and glycerol adsorption tendency of whole-cell catalysts are the main factors contributing to the extended catalytic cycle and limited catalyst reusability in biodiesel preparation. In this study, a 7.5 % methanol-tolerant Rhizopus stolonifera mutant (MTS2) was developed through the atmospheric and room temperature plasma (ARTP), exhibiting a 1.5-fold increase in methanol tolerance compared with the wild type strain (WT-RS), along with a 93.5 % increase in lipases activity. Polyurethane foam PF3 with an average pore size of 0.15 mm and 90 A rigidity is optimal for MTS2 immobilization (MTS2 @PF3), enabling its application in continuous processing for industrial applications. Utilizing soybean oil as a substrate, the catalyst MTS2 @PF3 produced 95.5 % of fatty acid methyl esters (FAME) in 36 hours under optimal conditions (30°C, 180 r/min, catalyst dosage of 15 % g/g oil, 30 % water content, and methanol addition in Scheme 3), achieving over a 50 % reduction in reaction time compared with WT-RS@PF3. To address limitations in catalyst reusability due to glycerol adsorption, various modifiers, including rosin-based modifier (RM), amino silicone oil (ASO), graphene (GRA), betaine and Tween 80 were evaluated. The glycerol adsorption rates experienced substantial reductions (39.3 % with RM and 85.6 % with betaine), and the catalysts maintained above 60 % FAME yield even after 8 reaction cycles. The combination of enhanced methanol tolerance, lipase activity, and reduced glycerol adsorption contribute to enhanced catalytic efficiency and reusability, which can reduce production costs for biodiesel manufacturing. The composite of these attributes makes MTS2 @RM1-PF3 and Betaine-MTS2 @PF3 as promising candidate for industrial biodiesel production, addressing previous limitations in catalyst performance and sustainability. The developed immobilized whole-cell catalysts exhibit promising potential for more sustainable and cost-effective processes in biodiesel manufacturing.

Structure-guided discovery and rational design of a new poly(ethylene terephthalate) hydrolase from AlphaFold protein structure database.

Enzymatic degradation offers a promising eco-friendly solution to plastic pollution, especially for polyethylene terephthalate (PET). Current efforts have focused on screening PET-degrading enzymes from microbial and metagenomic sources and obtaining superior candidates with a limited set of templates. More efficient PET hydrolases are required for PET-waste biorefinery. Here, using a structure-guided bioinformatic workflow, we identified a novel PET hydrolase, LSPET4, from Micromonospora sp. HM5-17, by screening the AlphaFold protein structure database. LSPET4 features a unique carbohydrate-binding module (CBM) and a distinctive linear substrate binding conformation. The intrinsic CBM in LSPET4 exhibited superior binding ability on PET surfaces and enhanced PET hydrolysis performance compared to the previously reported most effective CBM3. Through rational protein engineering focused on stabilizing and modifying the linear substrate binding conformation, we developed LSPET4M6 (D130P, N127F, Y96F, Q209E, A238K, D241S), a variant that achieved a 38.79-fold improvement in activity compared to the wild type, and was comparable to the reported most effective PET hydrolase derived from IsPETase, FAST-PETase at 45℃. This variant also demonstrated effectiveness in degrading various commercial PET materials, including PET food sealing films, PET strawberry boxes, and PET tomato boxes used in the food industry. This study not only provides a new template for protein engineering endeavors to create efficient biocatalysts for PET recycling but also offers an effective enzyme discovery approach to uncover enzymes of interest from the AlphaFold protein structure database.

Pleiotrophin modulates acute and long-term LPS-induced neuroinflammatory responses and hippocampal neurogenesis

The hippocampus is one of the most vulnerable regions affected in disorders characterized by overt neuroinflammation such as neurodegenerative diseases. Pleiotrophin (PTN) is a neurotrophic factor that modulates acute neuroinflammation in different contexts. PTN is found highly upregulated in the brain in different chronic disorders characterized by neuroinflammation, suggesting an important role in the modulation of sustained neuroinflammation. To test this hypothesis, we studied the acute and long-term effects of a single lipopolysaccharide (LPS; 5mg/kg) administration in Ptn+/+ and Ptn-/- mice, and in mice with Ptn-overexpression (Ptn-Tg). Endogenous PTN levels proportionally modulate LPS-induced increase in TNF-α plasma levels one hour after treatment. In the dentate gyrus (DG) of the hippocampus, a lower percentage of DCX+ cells were detected in saline-treated Ptn-/- mice compared to Ptn+/+ mice, suggesting a crucial role of PTN in the maintenance of hippocampal neuronal progenitors. The data show that PTN overexpression tends to potentiate acute microglial responses in the DG 16hours after LPS treatment. Remarkably, a significant increase in the number of neuronal progenitors together with astrogliosis was detected 10 months after a single injection of LPS treatment in wild type mice. However, these LPS-induced long-term effects were prevented in Ptn-/- and Ptn-Tg mice, suggesting that PTN modulates LPS-induced long-term neurogenesis changes and astrocytic response in the hippocampus. The data presented here suggest that endogenous PTN levels are crucial in the regulation of acute LPS-induced systemic and hippocampal microglial responses in young mice. Furthermore, our findings provide evidence of the key role of PTN in the regulation of long-term LPS effects on astrocytic response and neurogenesis in the hippocampus.

The double deficiency of the SNARE proteins vti1a and vti1b affects neurite outgrowth and signaling in N1E-115 neuroblastoma cells.

During intracellular trafficking N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins catalyze the membrane fusion by assembling into a four-helix complex. In the mouse model, loss of the endosomal SNAREs vti1a and vti1b results in a perinatal lethal phenotype and neuronal defects including decreased neurite outgrowth in cultured primary neurons.We used a CRISPR/Cas9 system to generate a Vti1a Vti1b double knockout (DKO) in the neuroblastoma cell line N1E-115. Three different DKO cell lines were obtained and examined at genome and protein level. The double deficiency impaired proper differentiation based on lower levels of synaptic proteins as well as reduced neurite formation and elongation compared to wild type cells in differentiation medium. Neurite elongation can be induced by a variety of extracellular signals via different signaling cascades. Treatment with the Rho kinase inhibitor Y27632, which stimulates enlargeosome exocytosis, or with neurotrophic factors (BDNF, NGF and NT3) resulted in reduced stimulation of all DKO clones and in significantly shorter neurites compared to wild type cells. The loss of vti1a and vti1b disrupted Akt signaling during enlargeosome-mediated and Erk signaling during BDNF-induced neurite outgrowth.

RNAseq and targeted metabolomics implicate RIC8 in regulation of energy homeostasis, amino acid compartmentation, and asexual development in Neurospora crassa

ABSTRACT Heterotrimeric G protein signaling pathways control growth and development in eukaryotes. In the multicellular fungus Neurospora crassa , the guanine nucleotide exchange factor RIC8 regulates heterotrimeric Gα subunits. In this study, we used RNAseq and liquid chromatography-mass spectrometry (LC-MS) to profile the transcriptomes and metabolomes of N. crassa wild type, the Gα subunit mutants Δ gna-1 and Δ gna-3 , and Δ ric8 strains. These strains exhibit defects in growth and asexual development (conidiation), with wild-type and Δ gna-1 mutants producing hyphae in submerged cultures, while Δ gna-3 and Δ ric8 mutants develop conidiophores, particularly in the Δ ric8 mutant. RNAseq analysis showed that the Δ gna-1 mutant possesses 159 mis-regulated genes, while Δ gna-3 and Δ ric8 strains have more than 1,000 each. Many of the mis-regulated genes are involved in energy homeostasis, conidiation, or metabolism. LC-MS revealed changes in levels of primary metabolites in the mutants, with several arginine metabolic intermediates impacted in Δ ric8 strains. The differences in metabolite levels could not be fully explained by the expression or activity of pathway enzymes. However, transcript levels for two predicted vacuolar arginine transporters were affected in Δ ric8 mutants. Analysis of arginine and ornithine levels in transporter mutants yielded support for altered compartmentation of arginine and ornithine between the cytosol and vacuole in Δ ric8 strains. Furthermore, we validated previous reports that arginine and ornithine levels are low in wild-type conidia. Our results suggest that RIC8 regulates asexual sporulation in N. crassa at least in part through altered expression of vacuolar transporter genes and the resultant mis-compartmentation of arginine and ornithine. IMPORTANCE Resistance to inhibitors of cholinesterase-8 (RIC8) is an important regulator of heterotrimeric Gα proteins in eukaryotes. In the filamentous fungus Neurospora crassa , mutants lacking ric8 undergo inappropriate asexual development (macroconidiation) during submerged growth. Our work identifies a role for RIC8 in regulating expression of transporter genes that retain arginine and ornithine in the vacuole (equivalent of the animal lysosome) and relates this function to the developmental defect. Arginine is a critical cellular metabolite, both as an amino acid for protein synthesis and as a precursor for an array of compounds, including proline, ornithine, citrulline, polyamines, creatine phosphate, and nitric oxide. These results have broad relevance to human physiology and disease, as arginine modulates immune, vascular, hormonal, and other functions in humans.

Abstract C009: MK2 knockout and radiation enhances immune cell influx in a mouse model of HPV+ head and neck squamous cell carcinoma

Abstract Introduction: HPV mediated head and neck squamous cell carcinoma (HNSCC) has an immunosuppressive microenvironment which contributes to local and distant failures. We have previously shown that MAPKAPK2 (MK2) inhibition (MK2i) in HNSCC results in improved response to irradiation (RT). MK2i was synergistic with RT resulting in better control than monotherapy. To understand the mechanism behind the improvement of RT in MK2i treated mice we tested the hypothesis that MK2 ablation can enhance the radiotherapy mediated immunogenic response in a solid tumor. Methods: We used CRISPR gene editing to knockout the MK2 gene in the highly metastatic murine cell line, MLM3 (KO). We implanted tumors into c57bl6 and NSG mice and compared tumor growth compared to wild type (WT) with and without radiation. At end point, tumors were excised, digested and immunophenotyped utilizing labelled antibodies and flow cytometry. Before immunophenotyping, 8 mgs of tumor were cut off and incubated in media for 24 hours to assess cytokine release by cytokine array analysis. Results: We found that the differences in tumor growth between WT and KO cells in c57bl6 mice were significantly greater than those seen in NSG mice. Survival studies with c57bl6 mice implanted with WT or KO tumors with and without RT showed that MLM3 KO implanted mice had significantly greater survival (58.5 days) compared to WT (35.5 days). WT tumors treated with RT improved median overall survival with the WT + RT group surviving longer than WT (56 days) and the MK2 KO + RT had the longest survival overall (65 days). Immunophenotyping WT v KO tumors grown in c57bl6 mice showed an increase in CD45 cells in the KO tumors compared to WT. The cells that were increased in number in the KO tumors were labelled by Ly6G (neutrophils), F4/80 (macrophages), CD14+MD11c (dendritic cells), CD4 (Th), CD8 (Tc), CD3+NK1.1 (NKt) and NK1.1 (NK cells). Irradiating the tumors accentuated the influx of immune cells causing a greater influx of CD45 cells at 3 days post-RT which included increases in F4/80+CD80 (M1 macrophages), CD14 (monocytes), CD11c+MHCII, NK1.1, NK1.1+CD3 and CD4 cells. However, the influx of CD8 cells were reduced at 3 days. At 8 days post RT, many cell types remained elevated in the KO tumors compared to wildtype and there was a partial recovery of CD8 cell influx. However, there was also an increase in immune suppressor cells into the tumors including Ly6C+arginase (M-MDSCs), Ly6C+arginase (PMN-MDSCs) and CD4+T-bet (Th1) cells. The cytokine analysis showed that six cytokines were differentially secreted from tumors: Dkk-1, IL-2, IGFBP-5, IL-3, IL-17a and CD14. All these cytokines were suppressed in the KO with respect to the WT tumors. Conclusion: Here we show evidence that genetic ablation of MK2 in a mouse model of HPV mediated HNSCC results in an increase in immune cells infiltrate into tumors. Irradiation enhances this difference by stimulating a greater influx of immune cells. These data suggest that tumoral MK2 may aid in the immunosuppression of tumors in HNSCC. Citation Format: Deri Morgan, Colby Spiess, Alyssa Schmidt, Dakota D.D. Okuwone, Harmony Saunders, Chris E. Lominska, Mary A. Markiewicz, Yelder Tonia, Devin Shrock, Yuting Lin, Hao Gao, Gregory N Gan. MK2 knockout and radiation enhances immune cell influx in a mouse model of HPV+ head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C009.

Abstract B046: Pyk2 and MEK/ERK signaling regulate the extracellular vesicle release and tumor-associated myeloid Cells in glioblastoma

Abstract Glioblastoma (GBM) is often fatal within a year despite treatment, with immune checkpoint blockade therapies benefiting only about 10% of patients. Non-responsive GBMs exhibit an immunosuppressive profile and higher levels of tumor-associated myeloid cells (TAMs). Addressing this immunosuppressive microenvironment is a major challenge in GBM immunotherapy. Our recent studies have identified a positive correlation between Proline-Rich Tyrosine Kinase 2 (Pyk2) activation in GBM tumor cells and the cytokine expression profile of TAMs. This identified Pyk2 as a potential prognostic marker for the immune state in GBM tumor microenvironment. We hypothesize that Pyk2 and MEK/Erk signaling in GBM cells regulate the release of chemokines and cytokines through extracellular vesicles (EVs) by modulating the actin cytoskeleton, thereby impacting TAM activation. The study aimed to investigate the role of Pyk2/MEK/Erk signaling in EV release in GBM cells, potentially revealing new therapeutic targets. Two humans primary GBM cell lines, with and without Pyk2 CRISPR/Cas9 knockout (Pyk2KO), were used. Flow cytometric analysis of EVs, enriched from cell-conditioned medium, identified a shift towards larger in diameter EVs populations in Pyk2 KO cells, compared to wild-type (WT) cells. Analysis using Integrin as a plasma membrane marker showed that 84.70% of Integrin+ and 15.30% of Integrin- EVs were derived from WT cells, whereas Pyk2KO cells produced 89.07% Integrin+ and 10.93% Integrin- EVs. Treatment of WT cells with the Erk/MEK inhibitor Avutometinib (1µM) altered the EV population’s ratio to 79.11% Integrin+ and 20.89% Integrin-. Similarly, Avutometinib treatment of Pyk2KO cells resulted in 78.83% Integrin+ and 21.17% Integrin- EVs, similar to the ratio observed in Avutometinib-treated WT cells. Western blot and PCR analysis of EVs content revealed a significant reduction in CCL2, CCL5, tumor necrosis factor (TNF), and vascular endothelial growth factors (VEGF) in EVs from Pyk2KO GBM cells, compared to WT. In vivo studies using GL261/C57Bl/6 mouse glioma implantation model and flow cytometric analysis of TAMs from tumors generated with WT and Pyk2KO GL261 cells demonstrated increased infiltration of TNF+/IFNg+ CD8+ lymphocytes and Ly6C+/CD206- myeloid cells, alongside a reduction in CD45+/CD11b+/CD33+/MHCII- myeloid-derived suppressor cells. PCR analysis of TAM isolated from Pyk2KO tumors revealed lower gene expression of TNF, CCL5, CCL2, VEGFa, and epidermal growth factor (EGF) compared to WT tumors. The study highlights that Pyk2/MEK/Erk signaling regulates the immune microenvironment in GBM by influencing EV release, which impacts TAM cell activation. Pyk2 primarily regulates the release of Integrin- EVs, while MEK/Erk predominantly regulates EVs shed from the plasma membrane. The findings underscore the interplay between Pyk2 and MEK/Erk signaling in regulating EV biogenesis and composition. Citation Format: Neisha M. Ramirez. Pyk2 and MEK/ERK signaling regulate the extracellular vesicle release and tumor-associated myeloid Cells in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B046.

Abstract C010: Tumor MK2 drives tumor progression and an immunosuppressive microenvironment in head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous carcinoma (HNSCC) results in poor patient outcomes driven primarily by locoregional and distant metastatic tumor spread. We have demonstrated that highly phosphorylated/active MAPKAPK-2 (MK2) is a poor prognostic factor in HNSCC patients. MK2 is associated with the expression of pro-inflammatory cytokines that are linked with creating a pro-tumorigenic microenvironment. Nevertheless, how MK2 contributes to HNSCC development has not been established. In this study, we aimed to identify the effect of MK2 on cancer progression, immune infiltration, and cytokine secretion. Method: To assess the effect of tumor MK2 in vivo, we used CRISPR-Cas9 to knock out (KO) MK2 in a metastatic murine HNSCC cell line, Ly2, and orthotopically implanted wildtype (WT) or MK2 KO cells into the floor-of-mouth of Balb/c mice. At the endpoint of the experiment, the tumors were excised, and myeloid and lymphocyte immune profiling was done via flow cytometry. Mouse lungs and cervical lymph nodes were dissected, paraffin-fixed, sectioned, and H&E stained, and the presence/quantity of metastatic foci was evaluated by a blinded board-certified pathologist. We broadly evaluated the tumor secretome via a 105-cytokine dot blot array using 8 mg of the same resected tumor sample and performed densitometric analysis comparing WT and KO tumors. Results: Loss of MK2 significantly reduced primary tumor growth, as well as lymph node and lung metastasis, compared to WT tumors. Tumor immunophenotyping by flow cytometry revealed that MK2 KO tumors had increased overall immune cell (CD45+) infiltration, but decreased quantities of immunosuppressive neutrophils (CD11b+Ly6G+Arg1+Ly6C-) and a shift in macrophage polarity favoring M1 (anti-tumor) macrophages (CD11b+Ly6G-F4-80+CD80+). In addition, there were increased quantities of anti-tumor immune cells including natural killer (NK)-T, CD4+, and CD8+ T cells in the MK2-KO tumors, while the proportion of CD4+CD25+ Tregs and exhausted CD8 T cells (CD8+PD1+) was decreased compared to WT tumors. Furthermore, cytokine array analysis revealed that secreted levels of several cytokines, including CXCL1, CXCL2, GM-CSF, G-CSF, IL-1alpha, and IL-6, were significantly lower in MK2 KO tumors compared to WT tumors. Conclusion: These results show that tumoral MK2 signaling promotes tumor growth, metastasis, immunosuppressive inflammation, and pro-tumorigenic cytokine release which substantiates future therapeutic targeting of this pathway. Citation Format: Dakota D.D. Okwuone, Deri Morgan, Alyssa Schmidt, Devin Shrock, Yuting Lin, Hao Gao, Sufi M. Thomas, Gregory N. Gan. Tumor MK2 drives tumor progression and an immunosuppressive microenvironment in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C010.

Abstract B045: Requirement of MenaINV for metastatic colonization of the lungs by breast cancer stem cells

Abstract Background: Breast cancer is the most frequently diagnosed cancer, and the second leading cause of cancer-related death in women. Most breast cancer related deaths are due to metastasis to distant organs. The primary tumor and secondary site microenvironments provide cues that activate cancer cell plasticity programs such as those associated with invasion and stemness, which are needed for dissemination and the establishment of metastatic foci. We demonstrated that in the primary tumor >50% of cancer stem cells (CSCs) co-express the invasive phenotype marker, MenaINV. Given that ∼80% of newly arriving circulating tumor cells to the lung are CSCs, we questioned if extravasation of CSCs to the lung is mediated solely by MenaINV or if other compensatory pathways are at play. We describe here our discovery that CSCs require MenaINV for extravasation to the lung parenchyma. Methods: To visualize breast cancer stem cell extravasation into the lung parenchyma, we expressed the stemness biosensor, SORE6, in MDA-MB-231 breast cancer cells. SORE6 is composed of 6 tandem repeats of the Sox2 and Oct4 response elements placed upstream of destabilized Green Fluorescent Protein (GFP). To distinguish CSCs (green) from non-CSCs, all cells were also labeled with red tracker dye. Breast cancer cells transduced with lenti-SORE6 were FACS sorted into low SORE6-GFP cells (non- CSCs) and high SORE6-GFP (CSCs). Activation of the stem program in FACS sorted cells was validated by qRT-PCR analysis of the widely used stem cell markers CD44, CD133, P63, ALDH1, Sox9, Sox2, and Oct4. The extravasation efficiency of non-CSCs vs. CSCs was assessed in vitro by the extravasation directed transendothelial migration (eTEM) assay, which mimics extravasation by allowing migration of cancer cells through endothelial cells cultured on a porous membrane, as well as in vivo, by intravital imaging. The impact of MenaINV on CSC extravasation was evaluated through CRISPR/Cas9 mediated knockout of MenaINV. In in vivo experiments, a 50/50 mixture of non-CSCs and CSCs expressing either wild type or knocked-out MenaINV were injected into the vasculature of Rag2-/- MacBlue mice. in vivo visualization of cancer cell extravasation was accomplished by implantation of an indwelling lung imaging window prior to the injection of cancer cells, followed by high-resolution intravital imaging. Results: CSCs exhibit significantly higher expression of all stem cell markers and express more MenaINV compared to non-CSCs. Moreover, CSCs show significantly better transendothelial migration in eTME assay (p=0.03) and lung extravasation (p<0.0001) in intravital imaging, compared to non-CSCs. Finally, knocking out MenaINV reduced lung extravasation to the levels comparable to non-CSCs. Conclusions: This study highlights the crucial role of MenaINV in facilitating lung metastasis by CSCs. Thus, targeting MenaINV may represent a promising therapeutic strategy to reduce metastatic burden in breast cancer patients. Citation Format: Mohd Nauman, Yookyung Jung, Burcu Karadal-Ferrena, Camille Duran, Madeline Friedman-DeLuca, Nicole Barth, Prachiben Patel, John Condeelis, David Entenberg, Maja H. Oktay. Requirement of MenaINV for metastatic colonization of the lungs by breast cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B045.

The transmission of mutation effects in a multiprotein machine: A comprehensive metadynamics study of the cardiac thin filament.

The binding of Ca2+ ions within the troponin core of the cardiac thin filament (CTF) regulates normal contraction and relaxation. Mutations within the troponin complexes are known to alter normal functions and result in the eventual development of cardiomyopathy. However, despite the importance of the problem, detailed microscopic knowledge of the mechanism of pathogenic effect of point mutations and their effects on the conformational free energy surface of CTF remains elusive. Mutations are known to transmit their effects hundreds of angstroms along this protein complex and between different component proteins. To explore the impact of point mutations on the conformational free energy barrier between the closed and blocked state of CTF, and to understand the transmission of mutation, we have carried out metadynamics simulations for the wild-type (WT) and two mutants (cardiac troponin T Arg92Trp (R92W) and Arg92Leu (R92L)). Specifically, we have investigated the conformational modification of the tropomyosin (Tm) and the troponin (Tn) complex during the closed-to-blocked state transition for both the WT and two hypertrophic cardiomyopathy causing mutations. Our calculations demonstrated that mutations within the cardiac troponin T (cTnT) protein alter conformational properties of the Tm and the other proteins of the Tn complex as well as the Ca2+ binding affinity of the cTnC protein through the indirect mediation of cardiac troponin I (cTnI). Importantly, the data revealed a significant influence of the mutations on the conformational transition free energy barriers for both the Tm and cTnC proteins. Furthermore, we found both mutations independently alter the free energy barrier of transitions of cTnT. Such alteration in the free energy upon mutation of one protein in a complex, allosterically affects the others through structural and dynamical changes, leading to a pathogenic effect on the function of the thin filament.