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Pancreatic ductal adenocarcinoma (PDA) remains resistant to immune therapies, largely owing to robustly fibrotic and immunosuppressive tumor microenvironments. It has been postulated that excessive accumulation of immunosuppressive myeloid cells influences immunotherapy resistance, and recent studies targeting macrophages in combination with checkpoint blockade have demonstrated promising preclinical results. Yet our understanding of tumor-associated macrophage (TAM) function, complexity, and diversity in PDA remains limited. Our analysis reveals significant macrophage heterogeneity, with bone marrow-derived monocytes serving as the primary source for immunosuppressive TAMs. These cells also serve as a primary source of TNF-α, which suppresses expression of the alarmin IL-33 in carcinoma cells. Deletion of Ccr2 in genetically engineered mice decreased monocyte recruitment, resulting in profoundly decreased TNF-α and increased IL-33 expression, decreased metastasis, and increased survival. Moreover, intervention studies targeting CCR2 with a new orthosteric inhibitor (CCX598) rendered PDA susceptible to checkpoint blockade, resulting in reduced metastatic burden and increased survival. Our data indicate that this shift in antitumor immunity is influenced by increased levels of IL-33, which increases dendritic cell and cytotoxic T cell activity. These data demonstrate that interventions to disrupt infiltration of immunosuppressive macrophages, or their signaling, have the potential to overcome barriers to effective immunotherapeutics for PDA.

Over the past decade, immunotherapeutic strategies—mainly targeting the PD-1–PD-L1 immune checkpoint axis—have altered cancer treatment for many solid tumours, but few patients with gastrointestinal forms of cancer have benefited to date. There remains an urgent need to extend immunotherapy efficacy to more patients while addressing resistance to current immune checkpoint inhibitors. The aim of this study was to determine the safety and anti-tumour activity of knockout of CISH, which encodes cytokine-inducible SH2-containing protein, a novel intracellular immune checkpoint target and a founding member of the SOCS family of E3-ligases, using tumour infiltrating lymphocyte (TILs) genetically edited with CRISPR-Cas9 in patients with metastatic gastrointestinal epithelial cancers. For this first-in-human, single-centre, phase 1 trial, patients aged 18–70 years with a diagnosis of metastatic gastrointestinal epithelial cancer with progressive disease following at least one first line standard therapy, measurable disease with at least one lesion identified as resectable for TIL generation and at least one other lesion meeting RECIST criteria as measurable to serve as an indicator of disease response, and an ECOG performance status of 0 or 1 were screened and enrolled if meeting these and all other eligibility criteria. TILs procured from tumour biopsies were expanded on the basis of neoantigen reactivity, subjected to CRISPR-Cas9-mediated CISH knockout, and infused intravenously into 12 patients after non-myeloablative lymphocyte depleting chemotherapy (cyclophosphamide 60 mg/kg per dose on study days –6 and –5, and fludarabine 25 mg/m2 per dose on days –7 to –3) followed by high-dose IL-2 (aldesleukin; 720 000 IU/kg per dose). The primary endpoint was safety of administration of neoantigen-reactive TILs with knockout of the CISH gene, and a key secondary endpoint was anti-tumour activity measured as objective radiographic response and progression-free and overall survival. This study is registered with ClinicalTrials.gov, NCT04426669, and is complete. Between May 12, 2020, and Sept 16, 2022, 22 participants were enrolled in the trial (one patient was enrolled twice owing to lack of TIL outgrowth on the first attempt); ten patients were female, and 11 were male (self-defined). One patient was Asian, the remainder were White (self-defined). We successfully manufactured CISH knockout TIL products for 19 (86%) of the patients, of whom 12 (63%) received autologous CISH knockout TIL infusion. The median follow-up time for the study was 129 days (IQR 15–283). All 12 (100%) patients had treatment-related severe adverse events. The most common grade 3–4 adverse events included haematological events (12 patients [100%]) attributable to the preparative lymphodepleting chemotherapy regimen or expected effects of IL-2, fatigue (four patients [33%]), and anorexia (three patients [25%]). Deaths of any cause for patients on study were attributed to the underlying disease under study (metastatic gastrointestinal cancer) and related complications (10 patients) or infection (grade 5 septicaemia in one patient). There were no severe (≥grade 3) cytokine release or neurotoxicity events. Six (50%) of 12 patients had stable disease by day 28, and four (33%) had stable disease ongoing at 56 days. One young adult patient with microsatellite-instability-high colorectal cancer refractory to anti-PD1/CTLA-4 therapies had a complete and ongoing response (>21 months). These results support the safety and potential antitumour activity of inhibiting the immune checkpoint CISH through the administration of neoantigen-reactive CISH-knockout TILs, with implications for patients with advanced metastatic cancers refractory to checkpoint inhibitor immunotherapies, and provide the first evidence that a novel intracellular checkpoint can be targeted with therapeutic effect. Intima Bioscience.

Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported here. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in V(max) of the enzyme activity by these phospholipids significantly decreased the K(m) indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K(½) = 114 nM) compared to PA (K(½) = 335 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response.

A number of 5′-O-fatty acyl derivatives of 3′-fluoro-2′,3′-dideoxythymidine (FLT, 1) were synthesized. These conjugates were evaluated for their potential as topical microbicides with anti-HIV activity against cell-free (X4 and R5), cell-associated, and multidrug-resistant viruses. Compared to FLT and 3′-azido-2′,3′-dideoxythymidine (AZT), 5′-O-(12-azidododecanoyl) (5), 5′-O-myristoyl (6), and 5′-O-(12-thioethyldodecanoyl) (8) derivatives of FLT were found to be more active against both cell-free viruses (lymphocytotropic and monocytotropic strains) with EC50 values of 0.4 μM, 1.1 μM, and <0.2 μM, respectively, as well as cell-associated virus with EC50 values of 12.6, 6.4, and 2.3 μM, respectively. Conjugates 5, 6, and 8 exhibited >4 and >30 times better antiviral index than FLT and AZT, respectively. Conjugates 5 and 8 were significantly more potent than FLT against many multidrug-resistant strains. A comparison of the anti-HIV activity with the corresponding non-hydrolyzable ether conjugates suggested that ester hydrolysis to FLT and fatty acids is critical to enable anti-HIV activity. Cellular uptake studies were conducted using fluorescent derivatives of FLT attached with 5(6)-carboxyfluorescein through either β-alanine (23) or 12-aminododecanoic acid (24) spacers. The lipophilic fluorescent analog with a long chain (24) showed more than 12 times higher cellular uptake profile than the fluorescent analog with a short chain (23). These studies further confirmed that the attachment of fatty acids improved the cellular uptake of nucleoside conjugates. In addition, 5, 6, and 8 were the least cytotoxic and did not alter vaginal cell and sperm viability compared to the positive control, a commercial topical spermicide (N-9), which significantly decreased sperm and vaginal cell viability inducing the generation of proinflammatory cytokines.

BackgroundOpioids such as morphine are widely used for the management of pain associated with acute pancreatitis. Interestingly, opioids are also known to affect the immune system and modulate inflammatory pathways in non-pancreatic diseases. However, the impact of morphine on the progression of acute pancreatitis has never been evaluated. In the current study, we evaluated the impact of morphine on the progression and severity of acute pancreatitis.MethodsEffect of morphine treatment on acute pancreatitis in caerulein, L-arginine and ethanol–palmitoleic acid models was evaluated after induction of the disease. Inflammatory response, gut permeability and bacterial translocation were compared. Experiments were repeated in mu (µ) opioid receptor knockout mice (MORKO) and in wild-type mice in the presence of opioid receptor antagonist naltrexone to evaluate the role of µ-opioid receptors in morphine’s effect on acute pancreatitis. Effect of morphine treatment on pathways activated during pancreatic regeneration like sonic Hedgehog and activation of embryonic transcription factors like pdx-1 and ptf-1 were measured by immunofluorescence and quantitative PCR.ResultsHistological data show that treatment with morphine after induction of acute pancreatitis exacerbates the disease with increased pancreatic neutrophilic infiltration and necrosis in all three models of acute pancreatitis. Morphine also exacerbated acute pancreatitis-induced gut permeabilisation and bacteraemia. These effects were antagonised in the MORKO mice or in the presence of naltrexone suggesting that morphine’s effect on severity of acute pancreatitis are mediated through the µ-opioid receptors. Morphine treatment delayed macrophage infiltration, sonic Hedgehog pathway activation and expression of pdx-1 and ptf-1.ConclusionMorphine treatment worsens the severity of acute pancreatitis and delays resolution and regeneration. Considering our results, the safety of morphine for analgesia during acute pancreatitis should be re-evaluated in future human studies.

In plants, calcium-dependent protein kinases (CDPKs) are key intermediates in calcium-mediated signaling that couple changes in Ca 2+ levels to a specific response. In the present study, we report the high-level soluble expression of calcium-dependent protein kinase1 from Cicer arietinum ( Ca CDPK1) in Escherichia coli . The expression of soluble Ca CDPK1 was temperature dependent with a yield of 3–4 mg/l of bacterial culture. Ca CDPK1 expressed as histidine-tag fusion protein was purified using Ni–NTA affinity chromatography till homogeneity. The recombinant Ca CDPK1 protein exhibited both calcium-dependent autophosphorylation and substrate phosphorylation activities with a V max and K m value of 13.2 nmol/min/mg and 34.3 μM, respectively, for histone III-S as substrate. Maximum autophosphorylation was seen only in the presence of calcium. Optimum temperature for autophosphorylation was found to be 37 °C. The recombinant protein showed optimum pH range of 6–9. The role of autophosphorylation in substrate phosphorylation was investigated using histone III-S as exogenous substrate. Our results show that autophosphorylation happens before substrate phosphorylation and it happens via intra-molecular mechanism as the activity linearly depends on enzyme concentrations. Autophosphorylation enhances the kinase activity and reduces the lag phase of activation, and Ca CDPK1 can utilize both ATP and GTP as phosphodonor but ATP is preferred than GTP.

Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported here. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in V.sub.max of the enzyme activity by these phospholipids significantly decreased the K.sub.m indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K.sub.½ = 114 nM) compared to PA (K.sub.½ = 335 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response.

The sole purpose of this study is to propose a new concept to fractional derivative as Ujlayan-Dixit derivative in the classical sense using limit approach. The parameter [alpha] of the derivative is confined within a closed unit interval. It is easy to apply and generates a convex combination of function and its derivative. We observe its geometrical behavior and see, how it is different from the previous definitions of the concerned derivatives. It works as an improved form of Conformable fractional derivative and fulfill the meaningful gaps. Some basic properties of calculus like continuity, differentiability, Mean Value theorem, Comparison theorem and some numerical have discussed. Keywords: Fractional derivative, conformable derivative, fractional differential equations, UD derivative, fractional integral.

Chemical warfare agents (CWAs) are extremely lethal substances used in warfare and terrorism, capable of causing permanent damage even in small doses, despite medical intervention. Therefore, detection, protection, and detoxification of CWAs are vital for the safety of first responders, military personnel, and civilians, driving significant research in this area. Herein, we designed and synthesized a poly(diallyldimethylammonium chloride) (PDDA) mediated cupric oxide (CuO) functionalized activated carbon fabric (ACF), termed ACF@PDDA-CuO, as an adsorbent filter material for self-detoxifying chemical protective clothing. PDDA, a positively charged polyelectrolyte, effectively binds in-situ synthesized CuO to the negatively charged ACF surface, serving as a suitable binder. This study demonstrates the synergistic effects of PDDA-CuO functionalization on ACF, where PDDA treatment enhanced mechanical and comfort properties, and CuO crystal growth significantly improved detoxification efficacy against the CWA Nerve Agent Sarin. Comprehensive analyses, including FTIR, BET surface area analysis, SEM, EDS, TEM, STEM, TGA, XPS, and XRD, confirmed the uniform deposition of CuO and PDDA on the ACF surface. The Cu content on ACF@PDDA-CuO samples was measured via iodometric titration. The materials were evaluated for tensile strength, air permeability, water vapor permeability, nerve agent (Sarin) detoxification, and blister agent (Sulfur Mustard) breakthrough time to assess their applicability for protective clothing. The optimized PDDA-CuO on ACF detoxified 82.04% of Sarin within 18 h, compared to 25.22% by ACF alone, and enhanced tensile strength by 23.67%, air permeability by 24.63%, and water vapor permeability by 3.94%, while maintaining protection against Sulfur Mustard for 24 h. These findings indicate that ACF@PDDA-CuO is a promising candidate for CWA protective clothing, offering robust protection with enhanced comfort. [Display omitted]

Background Plants have developed various sophisticated mechanisms to cope up with climate extremes and different stress conditions, especially by involving specific transcription factors (TFs). The members of the WRKY TF family are well known for their role in plant development, phytohormone signaling and developing resistance against biotic or abiotic stresses. In this study, we performed a genome-wide screening to identify and analyze the WRKY TFs in pearl millet ( Pennisetum glaucum; PgWRKY ), which is one of the most widely grown cereal crops in the semi-arid regions. Results A total number of 97 putative PgWRKY proteins were identified and classified into three major Groups (I-III) based on the presence of WRKY DNA binding domain and zinc-finger motif structures. Members of Group II have been further subdivided into five subgroups (IIa-IIe) based on the phylogenetic analysis. In-silico analysis of PgWRKYs revealed the presence of various cis-regulatory elements in their promoter region like ABRE, DRE, ERE, EIRE, Dof, AUXRR, G-box, etc., suggesting their probable involvement in growth, development and stress responses of pearl millet. Chromosomal mapping evidenced uneven distribution of identified 97 PgWRKY genes across all the seven chromosomes of pearl millet. Synteny analysis of PgWRKYs established their orthologous and paralogous relationship among the WRKY gene family of Arabidopsis thaliana, Oryza sativa and Setaria italica . Gene ontology (GO) annotation functionally categorized these PgWRKYs under cellular components, molecular functions and biological processes. Further, the differential expression pattern of PgWRKY s was noticed in different tissues (leaf, stem, root) and under both drought and salt stress conditions. The expression pattern of PgWRKY33 , PgWRKY62 and PgWRKY65 indicates their probable involvement in both dehydration and salinity stress responses in pearl millet. Conclusion Functional characterization of identified PgWRKY s can be useful in delineating their role behind the natural stress tolerance of pearl millet against harsh environmental conditions. Further, these PgWRKY s can be employed in genome editing for millet crop improvement.