Network pharmacology and experimental insights into STAT3 inhibition by novel isoxazole derivatives of piperic acid in triple negative breast cancer.

STAT3 is a crucial member within a family of seven essential transcription factors. Elevated STAT3 levels have been identified in various cancer types, notably in breast cancer (BC). Consequently, inhibiting STAT3 is recognized as a promising and effective strategy for therapeutic intervention against breast cancer. We herein synthesize a library of isoxazole (PAIs) from piperic acid [2E, 4E)-5-(2H-1,3-Benzodioxol-5-yl) penta-2,4-dienoic acid] on treatment with propargyl bromide followed by oxime under prescribed reaction conditions. Piperic acid was obtained by hydrolysis of piperine extracted from Piper nigrum. First, we checked the binding potential of isoxazole derivatives with breast cancer target proteins by network pharmacology, molecular docking, molecular dynamic (MD) simulation and cytotoxicity analysis as potential anti-breast cancer (BC) agents. The multi-source databases were used to identify possible targets for isoxazole derivatives. A network of protein-protein interactions (PPIs) was generated by obtaining 877 target genes that overlapped gene symbols associated with isoxazole derivatives and BC. Molecular docking and MD modelling demonstrated a strong affinity between isoxazole derivatives and essential target genes. Further, the cell viability studies of isoxazole derivatives on the human breast carcinoma cell lines showed toxicity in all breast cancer cell lines. In summary, our study indicated that the isoxazole derivative showed the significant anticancer activity. The results highlight the prospective utility of isoxazole derivatives as new drug candidates for anticancer chemotherapy, suggesting route for the continued exploration and development of drugs suitable for clinical applications.

Preferential differential gene expression within the WC1.1+ γδ T cell compartment in cattle naturally infected with Mycobacterium bovis.

Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis, continues to cause significant issues for the global agriculture industry as well as for human health. An incomplete understanding of the host immune response contributes to the challenges of control and eradication of this zoonotic disease. In this study, high-throughput bulk RNA sequencing (RNA-seq) was used to characterise differential gene expression in γδ T cells - a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. γδ T cell subsets are classified based on expression of a pathogen-recognition receptor known as Workshop Cluster 1 (WC1) and we hypothesised that bTB disease may alter the phenotype and function of specific γδ T cell subsets. Peripheral blood was collected from naturally M. bovis-infected (positive for single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA) and age- and sex-matched, non-infected control Holstein-Friesian cattle. γδ T subsets were isolated using fluorescence activated cell sorting (n = 10-12 per group) and high-quality RNA extracted from each purified lymphocyte subset (WC1.1+, WC1.2+, WC1- and γδ-) was used to generate transcriptomes using bulk RNA-seq (n = 6 per group, representing a total of 48 RNA-seq libraries). Relatively low numbers of differentially expressed genes (DEGs) were observed between most cell subsets; however, 189 genes were significantly differentially expressed in the M. bovis-infected compared to the control groups for the WC1.1+ γδ T cell compartment (absolute log2 FC ≥ 1.5 and FDR P adj. ≤ 0.1). The majority of these DEGs (168) were significantly increased in expression in cells from the bTB+ cattle and included genes encoding transcription factors (TBX21 and EOMES), chemokine receptors (CCR5 and CCR7), granzymes (GZMA, GZMM, and GZMH) and multiple killer cell immunoglobulin-like receptor (KIR) proteins indicating cytotoxic functions. Biological pathway overrepresentation analysis revealed enrichment of genes with multiple immune functions including cell activation, proliferation, chemotaxis, and cytotoxicity of lymphocytes. In conclusion, γδ T cells have important inflammatory and regulatory functions in cattle, and we provide evidence for preferential differential activation of the WC1.1+ specific subset in cattle naturally infected with M. bovis.

Gas chromatographic-mass spectrometric analysis, antioxidant, antiproliferative and antibacterial activities of the essential oil of Prangospabularia.

The essential oil composition of the shoot parts of Prangos pabularia, growing in Drass area of Ladakh, India, along with its antioxidant, antibacterial and anticancer activity, is reported for the first time. Gas chromatography coupled with mass spectrometry (GC-MS) revealed the presence of 31 constituents, representing 97.342% of the total essential oil. The major constituents of essential oil were Durylaldehyde (62.161%), Bicyclo [3.1.1] hept-2-en-4-ol (8.846%), Chrysanthenyl acetate (5.120%) followed by unknown (3.420%), (-)-Spathulenol (3.028%), Mesityl aldehyde (2.402%) and Hexahydro farnesyl acetone (1.683%. Cytotoxic activity of the essential oil by MTT assay against human breast adenocarcinoma (MCF7), human breast (HBL-100), human cervical cancer (HELA) and human lung adenocarcinoma epithelial (A549) cells, at four different concentrations (20, 30, 50 & 100 µg/mL) revealed that the activity of 56.12% against A549 (human lung) cell line at 20 µg/mL concentration was the highest. The Essential oil displayed a significant free radical scavenging activity with DPPH. Antibacterial activity was carried out against 3 g positive and 2-g negative bacteria at four different concentrations using Agar Well Diffusion Method taking streptomycin sulphate as reference. The essential oil displayed significant and broad-spectrum antibacterial activity against different bacteria used. The MIC of the oil ranged from 2.06 to 5.00 µg/mL. The zones of inhibition were lesser for Micrococcus and Escherichia coli compared to other strains of bacteria.

Bacterial Endophyte Community Dynamics in Apple (Malus domestica Borkh.) Germplasm and Their Evaluation for Scab Management Strategies.

The large genetic evolution due to the sexual reproduction-mediated gene assortments and propensities has made Venturia inaequalis (causing apple scab) unique with respect to its management strategies. The resistance in apple germplasm against the scab, being controlled for by more than fifteen genes, has limited gene alteration-based investigations. Therefore, a biological approach of bacterial endophyte community dynamics was envisioned across the apple germplasm in context to the fungistatic behavior against V. inaequalis. A total of 155 colonies of bacterial endophytes were isolated from various plant parts of the apple, comprising 19 varieties, and after screening for antifungal behavior followed by morphological, ARDRA, and sequence analysis, a total of 71 isolates were selected for this study. The alpha diversity indices were seen to fluctuate greatly among the isolation samples in context to microflora with antifungal behavior. As all the isolates were screened for the presence of various metabolites and some relevant genes that directly or indirectly influence the fungistatic behavior of the isolated microflora, a huge variation among the isolated microflora was observed. The outstanding isolates showing highest percentage growth inhibition of V. inaequalis were exploited to raise a bio-formulation, which was tested against the scab prevalence in eight apple varieties under controlled growth conditions. The formulation at all the concentrations caused considerable reductions in both the disease severity and disease incidence in all the tested apple varieties. Red Delicious being most important cultivar of the northwestern Himalayas was further investigated for its biochemical behavior in formulation and the investigation revealed different levels of enzyme production, chlorophyll, and sugars against the non-inoculated control.

Fragile X premutation rCGG repeats impair synaptic growth and synaptic transmission at Drosophila larval neuromuscular junction.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disease that develops in some premutation (PM) carriers of the FMR1 gene with alleles bearing 55-200 CGG repeats. The discovery of a broad spectrum of clinical and cell-developmental abnormalities among PM carriers with or without FXTAS and in model systems suggests that neurodegeneration seen in FXTAS could be the inevitable end-result of pathophysiological processes set during early development. Hence, it is imperative to trace early PM-induced pathological abnormalities. Previous studies have shown that transgenic Drosophila carrying PM-length CGG repeats are sufficient to cause neurodegeneration. Here, we used the same transgenic model to understand the effect of CGG repeats on the structure and function of the developing nervous system. We show that presynaptic expression of CGG repeats restricts synaptic growth, reduces the number of synaptic boutons, leads to aberrant presynaptic varicosities, and impairs synaptic transmission at the larval neuromuscular junctions. The postsynaptic analysis shows that both glutamate receptors and subsynaptic reticulum proteins were normal. However, a high percentage of boutons show a reduced density of Bruchpilot protein, a key component of presynaptic active zones required for vesicle release. The electrophysiological analysis shows a significant reduction in quantal content, a measure of total synaptic vesicles released per excitation potential. Together, these findings suggest that synapse perturbation caused by riboCGG (rCGG) repeats mediates presynaptically during larval neuromuscular junction development. We also suggest that the stress-activated c-Jun N-terminal kinase protein Basket and CIDE-N protein Drep-2 positively mediate Bruchpilot active zone defects caused by rCGG repeats.

Early immune suppression leads to uncontrolled mite proliferation and potent host inflammatory responses in a porcine model of crusted versus ordinary scabies.

Scabies is a neglected tropical disease of global significance. Our understanding of host-parasite interactions has been limited, particularly in crusted scabies (CS), a severe clinical manifestation involving hyper-infestation of Sarcoptes scabiei mites. Susceptibility to CS may be associated with immunosuppressive conditions but CS has also been seen in cases with no identifiable risk factor or immune deficit. Due to ethical and logistical difficulties with undertaking research on clinical patients with CS, we adopted a porcine model which parallels human clinical manifestations. Transcriptomic analysis using microarrays was used to explore scabies pathogenesis, and to identify early events differentiating pigs with ordinary (OS) and crusted scabies. Pigs with OS (n = 4), CS (n = 4) and non-infested controls (n = 4) were compared at pre-infestation, weeks 1, 2, 4 and 8 post-infestation. In CS relative to OS, there were numerous differentially expressed genes including pro-inflammatory cytokines (IL17A, IL8, IL19, IL20 and OSM) and chemokines involved in immune cell activation and recruitment (CCL20, CCL27 and CXCL6). The influence of genes associated with immune regulation (CD274/PD-L1 and IL27), immune signalling (TLR2, TLR8) and antigen presentation (RFX5, HLA-5 and HLA-DOB) were highlighted in the early host response to CS. We observed similarities with gene expression profiles associated with psoriasis and atopic dermatitis and confirmed previous observations of Th2/17 pronounced responses in CS. This is the first comprehensive study describing transcriptional changes associated with the development of CS and significantly, the distinction between OS and CS. This provides a basis for clinical follow-up studies, potentially identifying new control strategies for this severely debilitating disease.

Checkpoint Blockade Rescues the Repressive Effect of Histone Deacetylases Inhibitors on γδ T Cell Function.

Histone deacetylases (HDAC) are one of the key epigenetic modifiers that control chromatin accessibility and gene expression. Their role in tumorigenesis is well established and HDAC inhibitors have emerged as an effective treatment modality. HDAC inhibitors have been investigated for their specific antitumor activities and also clinically evaluated in treatment of various malignancies. In the present study, we have investigated the effect of HDAC inhibitors on the effector functions of human γδ T cells. HDAC inhibitors inhibit the antigen-specific proliferative response of γδ T cells and cell cycle progression. In antigen-activated γδ T cells, the expression of transcription factors (Eomes and Tbet) and effector molecules (perforin and granzyme B) were decreased upon treatment with HDAC inhibitors. Treatment with HDAC inhibitors attenuated the antitumor cytotoxic potential of γδ T cells, which correlated with the enhanced expression of immune checkpoints programmed death-1 (PD-1) and programmed death ligand-1 in γδ T cells. Interestingly, PD-1 blockade improves the antitumor effector functions of HDAC inhibitor-treated γδ T cells, which is reflected in the increased expression of Granzyme B and Lamp-1. This study provides a rationale for designing HDAC inhibitor and immune check point blockade as a combinatorial treatment modality for cancer.

Inhibition of Notch signalling has ability to alter the proximal and distal TCR signalling events in human CD3+ αß T-cells.

The Notch signalling pathway is an important regulator of T cell function and is known to regulate the effector functions of T cells driven by T cell receptor (TCR). However, the mechanism integrating these pathways in human CD3+ αß T cells is not well understood. The present study was carried out to investigate how Notch and TCR driven signalling are synchronized in human αß T cells. Differential expression of Notch receptors, ligands, and target genes is observed on human αß T cells which are upregulated on stimulation with α-CD3/CD28 mAb. Inhibition of Notch signalling by GSI-X inhibited the activation of T cells and affected proximal T cell signalling by regulating CD3-ζ chain expression. Inhibition of Notch signalling decreased the protein expression of CD3-ζ chain and induced expression of E3 ubiquitin ligase (GRAIL) in human αß T cells. Apart from affecting proximal TCR signalling, Notch signalling also regulated the distal TCR signalling events. In the absence of Notch signalling, α-CD3/CD28 mAb induced activation and IFN-γ production by αß T cells was down-modulated. The absence of Notch signalling in human αß T cells inhibited proliferative responses despite strong signalling through TCR and IL-2 receptor. This study shows how Notch signalling cooperates with TCR signalling by regulating CD3-ζ chain expression to support proliferation and activation of human αß T cells.

Host immune responses to the itch mite, Sarcoptes scabiei, in humans.

Scabies is a parasitic disease due to infestation of skin by the burrowing mite Sarcoptes scabiei. Scabies is a major public health problem and endemic in resource poor communities worldwide affecting over 100 million people. Associated bacterial infections cause substantial morbidity, and in severe cases can lead to renal and cardiac diseases. Mite infestation of the skin causes localised cutaneous inflammation, pruritus, skin lesions, and allergic and inflammatory responses are mounted by the host against the mite and its products. Our current understanding of the immune and inflammatory responses associated with the clinical manifestations in scabies is far outweighed by the significant global impact of the disease. This review aims to provide a better understanding of human immune responses to S. scabiei in ordinary and crusted scabies phenotypes.

Coordination of bis(azol-1-yl)methane-based bisphosphines towards RuII, RhI, PdII and PtII: synthesis, structural and catalytic studies.

The coordination chemistry of bisphosphine ligands assembled on the five-membered heterocyclic platform of bis(azol-1-yl)methane viz.: bis(2-diphenylphosphinoimidazol-1-yl)methane (1), bis(5-diphenylphosphinopyrazol-1-yl)methane (2) and bis(5-diphenylphosphino-1,2,4-triazol-1-yl)methane (3) with RuII, RhI, PdII and PtII is described. The bisphosphines 1-3 react with elemental selenium to give the corresponding bis-selenoyl derivatives 4-6. The reactions of 1-3 with transition metal derivatives produce complexes with different coordination modes. Bisphosphine 1 showed a preference for the κ2-P,P mode of coordination, whereas bisphosphines 2 and 3, besides the κ2-P,P mode also showed a head-to-tail κ2-P,N coordination mode resulting in the formation of binuclear complexes [Rh2(COD)2{(CH2(1,2-C3H2N2PPh2)2)-κ2P,N}][BF4]2 (14), [Rh2(COD)2{(CH2(1,2,4-C2HN3PPh2)2)-κ2P,N}][BF4]2 (15), [Pd2(η3-C3H5)2{(CH2(1,2-C3H2N2PPh2)2)-κ2P,N}][BF4]2 (21) and [Pd2(η3-C3H5)2{(CH2(1,2,4-C2HN3PPh2)2)-κ2P,N}][BF4]2 (22). Several of these complexes have also been structurally characterized. The in situ generated RhI complex of bisphosphine 1 showed moderate to good selectivity in the hydroformylation of various styrene derivatives.

Gold(I) complexes of bisphosphines with bis(azol-1-yl)methane backbone: structure of a rare dinuclear gold(I) complex [(Au2Cl){CH2(1,2-C3H2N2PPh2)2}3Cl].

Gold(i) complexes of bisphosphines assembled on the bis(azol-1-yl)methane platform viz.: bis(2-diphenylphosphinoimidazol-1-yl)methane (1), bis(5-diphenylphosphinopyrazol-1-yl)methane (2), bis(5-diphenylphosphino-1,2,4-triazol-1-yl)methane (3) have been described. Reactions of two equivalents of bisphosphines 1-3 with Au(SMe2)Cl produced dinuclear complexes [(AuCl)2(µ-P^P)] (4-6) with AuAu distances around 3.13 Å. Similar reactions of bisphosphines 1 and 3 in 1 : 1 molar ratios yielded chelate complexes 7 and 9, whereas the ligand 2 formed a rare tetracoordinated digold(i) complex [(Au2Cl){CH2(1,2-C3H2N2PPh2)2}3Cl] (8) along with both chelate and binuclear complexes. The complex 8 was also prepared in good yield in 1.5 : 1 reaction. The structures of complexes 4, 5 and 8 have been confirmed by single crystal analysis.

The Jekyll and Hyde story of IL17-Producing γδT Cells.

In comparison to conventional αßT cells, γδT cells are considered as specialized T cells based on their contributions in regulating immune response. γδT cells sense early environmental signals and initiate local immune-surveillance. The development of functional subtypes of γδT cells takes place in the thymus but they also exhibit plasticity in response to the activating signals and cytokines encountered in the extrathymic region. Thymic development of Tγδ1 requires strong TCR, CD27, and Skint-1 signals. However, differentiation of IL17-producing γδT cells (Tγδ17) is independent of Skint-1 or CD27 but requires notch signaling along with IL6 and TGFß cytokines in the presence of weak TCR signal. In response to cytokines like IL23, IL6, and IL1ß, Tγδ17 outshine Th17 cells for early activation and IL17 secretion. Despite expressing similar repertoire of lineage transcriptional factors, cytokines, and chemokine receptors, Tγδ17 cells differ from Th17 in spatial and temporal fashion. There are compelling reasons to consider significant role of Tγδ17 cells in regulating inflammation and thereby disease outcome. Tγδ17 cells regulate mobilization of innate immune cells and induce keratinocytes to secrete anti-microbial peptides thus exhibiting protective functions in anti-microbial immunity. In contrast, dysregulated Tγδ17 cells inhibit Treg cells, exacerbate autoimmunity, and are also known to support carcinogenesis by enhancing angiogenesis. The mechanism associated with this dual behavior of Tγδ17 is not clear. To exploit, Tγδ17 cells for beneficial use requires comprehensive analysis of their biology. Here, we summarize the current understanding on the characteristics, development, and functions of Tγδ17 cells in various pathological scenarios.