Acemannan coated, cobalt-doped biphasic calcium phosphate nanoparticles for immunomodulation regulated bone regeneration.

Biomaterials are used as scaffolds in bone regeneration to facilitate the restoration of bone tissues. The local immune microenvironment affects bone repair but the role of immune response in biomaterial-facilitated osteogenesis has been largely overlooked and it presents a major knowledge gap in the field. Nanomaterials that can modulate M1 to M2 macrophage polarization and, thus, promote bone repair are known. This study investigates a novel approach to accelerate bone healing by using acemannan coated, cobalt-doped biphasic calcium phosphate nanoparticles to promote osteogenesis and modulate macrophage polarization to provide a prohealing microenvironment for bone regeneration. Different concentrations of cobalt were doped in biphasic calcium phosphate nanoparticles, which were further coated with acemannan polymer and characterized. The nanoparticles showed >90% cell viability and enhanced cell proliferation along with osteogenic differentiation as demonstrated by the enhanced alkaline phosphatase activity and osteogenic calcium deposition. The morphology of MC3T3-E1 cells remained unchanged even after treatment with nanoparticles. Acemannan coated nanoparticles were also able to decrease the expression of M1 markers, iNOS, and CD68 and enhance the expression of M2 markers, CD206, CD163, and Arg-1 as indicated by RT-qPCR, flow cytometry, and ICC studies. The findings show that acemannan coated nanoparticles can create a supportive immune milieu by inducing and promoting the release of osteogenic markers, and by causing a reduction in inflammatory markers, thus helping in efficient bone regeneration. As per our knowledge, this is the first study showing the combined effect of acemannan and cobalt for bone regeneration using immunomodulation. The work presents a novel approach for enhancing osteogenesis and macrophage polarization, thus, offering a potent strategy for effective bone regeneration.

Adipose tissue macrophage-derived microRNA-210-3p disrupts systemic insulin sensitivity by silencing GLUT4 in obesity.

Management of chronic obesity-associated metabolic disorders is a key challenge for biomedical researchers. During chronic obesity, visceral adipose tissue (VAT) undergoes substantial transformation characterized by a unique lipid-rich hypoxic AT microenvironment which plays a crucial role in VAT dysfunction, leading to insulin resistance (IR) and type 2 diabetes. Here, we demonstrate that obese AT microenvironment triggers the release of miR-210-3p microRNA-loaded extracellular vesicles from adipose tissue macrophages, which disseminate miR-210-3p to neighboring adipocytes, skeletal muscle cells, and hepatocytes through paracrine and endocrine actions, thereby influencing insulin sensitivity. Moreover, EVs collected from Dicer-silenced miR-210-3p-overexpressed bone marrow-derived macrophages induce glucose intolerance and IR in lean mice. Mechanistically, miR-210-3p interacts with the 3'-UTR of GLUT4 mRNA and silences its expression, compromising cellular glucose uptake and insulin sensitivity. Therapeutic inhibition of miR-210-3p in VAT notably rescues high-fat diet-fed mice from obesity-induced systemic glucose intolerance. Thus, targeting adipose tissue macrophage-specific miR-210-3p during obesity could be a promising strategy for managing IR and type 2 diabetes.

Recent insights of obesity-induced gut and adipose tissue dysbiosis in type 2 diabetes.

An imbalance in microbial homeostasis, referred to as dysbiosis, is critically associated with the progression of obesity-induced metabolic disorders including type 2 diabetes (T2D). Alteration in gut microbial diversity and the abundance of pathogenic bacteria disrupt metabolic homeostasis and potentiate chronic inflammation, due to intestinal leakage or release of a diverse range of microbial metabolites. The obesity-associated shifts in gut microbial diversity worsen the triglyceride and cholesterol level that regulates adipogenesis, lipolysis, and fatty acid oxidation. Moreover, an intricate interaction of the gut-brain axis coupled with the altered microbiome profile and microbiome-derived metabolites disrupt bidirectional communication for instigating insulin resistance. Furthermore, a distinct microbial community within visceral adipose tissue is associated with its dysfunction in obese T2D individuals. The specific bacterial signature was found in the mesenteric adipose tissue of T2D patients. Recently, it has been shown that in Crohn's disease, the gut-derived bacterium Clostridium innocuum translocated to the mesenteric adipose tissue and modulates its function by inducing M2 macrophage polarization, increasing adipogenesis, and promoting microbial surveillance. Considering these facts, modulation of microbiota in the gut and adipose tissue could serve as one of the contemporary approaches to manage T2D by using prebiotics, probiotics, or faecal microbial transplantation. Altogether, this review consolidates the current knowledge on gut and adipose tissue dysbiosis and its role in the development and progression of obesity-induced T2D. It emphasizes the significance of the gut microbiota and its metabolites as well as the alteration of adipose tissue microbiome profile for promoting adipose tissue dysfunction, and identifying novel therapeutic strategies, providing valuable insights and directions for future research and potential clinical interventions.

Leishmania LPG interacts with LRR5/LRR6 of macrophage TLR4 for parasite invasion and impairs the macrophage functions.

Visceral leishmaniasis (VL) is a severe form of leishmaniasis, primarily affecting the poor in developing countries. Although several studies have highlighted the importance of toll-like receptors (TLRs) in the pathophysiology of leishmaniasis, the role of specific TLRs and their binding partners involved in Leishmania donovani uptake are still elusive. To investigate the mechanism of L. donovani entry inside the macrophages, we found that the parasite lipophosphoglycan (LPG) interacted with the macrophage TLR4, leading to parasite uptake without any significant alteration of macrophage cell viability. Increased parasite numbers within macrophages markedly inhibited lipopolysachharide-induced pro-inflammatory cytokines gene expression. Silencing of macrophage-TLR4, or inhibition of parasite-LPG, significantly stemmed parasite infection in macrophages. Interestingly, we observed a significant enhancement of macrophage migration, and generation of reactive oxygen species (ROS) in the parasite-infected TLR4-silenced macrophages, whereas parasite infection in TLR4-overexpressed macrophages exhibited a notable reduction of macrophage migration and ROS generation. Moreover, mutations in the leucine-rich repeats (LRRs), particularly LRR5 and LRR6, significantly prevented TLR4 interaction with LPG, thus inhibiting cellular parasite entry. All these results suggest that parasite LPG recognition by the LRR5 and LRR6 of macrophage-TLR4 facilitated parasite entry, and impaired macrophage functions. Therefore, targeting LRR5/LRR6 interactions with LPG could provide a novel option to prevent VL.

Oxidized pullulan exhibits potent antibacterial activity against S. aureus by disrupting its membrane integrity.

The capability of bacteria to withstand the misuse of antibiotics leads to the generation of multi-drug resistant strains, posing a new challenge to curb wound infections. The biological macromolecules, due to their biocompatibility, biodegradability, and antimicrobial properties, have been explored for a variety of antimicrobial and therapeutic purposes. This work reports that a single-step oxidation of pullulan polymer leads to the formation of oxidized pullulan (o-pullulan), which shows striking antibacterial and antibiofilm activities against the Gram-positive bacteria, Staphylococcus aureus, implicated in wound-related infections. Oxidation of pullulan generates 28 % aldehyde groups (3.462 mmol/g) which exerted 97 % bactericidal activity against S. aureus by targeting cell wall-associated membrane protein SpA (Staphylococcal protein A). The molecular docking, gene silencing, and fluorescence quenching studies revealed a direct binding of o-pullulan with the B and C domains of SpA, which alters the membrane potential and inhibits Ca2+-Mg2+-ATPase pumps. O-pullulan also exhibited scavenging activity against intracellular reactive oxygen species (ROS), and non-immunotoxic activity and was found to be non-toxic to mammalian cells. Thus, o-pullulan shows great promise as an antimicrobial polymer against S. aureus for chronic wound management.

Pharmacological inhibition of DNMT1 restores macrophage autophagy and M2 polarization in Western diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is associated with an increased ratio of classically activated M1 macrophages/Kupffer cells to alternatively activated M2 macrophages, which plays an imperative role in the development and progression of NAFLD. However, little is known about the precise mechanism behind macrophage polarization shift. Here, we provide evidence regarding the relationship between the polarization shift in Kupffer cells and autophagy resulting from lipid exposure. High-fat and high-fructose diet supplementation for 10 weeks significantly increased the abundance of Kupffer cells with an M1-predominant phenotype in mice. Interestingly, at the molecular level, we also observed a concomitant increase in expression of DNA methyltransferases DNMT1 and reduced autophagy in the NAFLD mice. We also observed hypermethylation at the promotor regions of autophagy genes (LC3B, ATG-5, and ATG-7). Furthermore, the pharmacological inhibition of DNMT1 by using DNA hypomethylating agents (azacitidine and zebularine) restored Kupffer cell autophagy, M1/M2 polarization, and therefore prevented the progression of NAFLD. We report the presence of a link between epigenetic regulation of autophagy gene and macrophage polarization switch. We provide the evidence that epigenetic modulators restore the lipid-induced imbalance in macrophage polarization, therefore preventing the development and progression of NAFLD.

Role of macrophages in cancer progression and targeted immunotherapies.

The vast complexity of the tumor microenvironment (TME) aggrandizes the underlying principles responsible for immune escape, therapy resistance, and treatment failure. The stromal and immune cell population circumjacent to the tumor cells affects the cancer cell cycle leading to tumor progression. Tumor-associated macrophages (TAMs) exhibiting a unique M2 polarization state constitute a significant portion of the TME. They serve as tumor suppressors at early stages and tumor promoters at advanced stages by governing various microenvironmental cues. TAMs secreted various pro-tumoral cytokines, chemokines, and matrix metalloproteases are known to regulate the different cell cycle molecules including checkpoint inhibitors in cancer cells leading to cell cycle progression with faulty cellular components. Moreover, TAMs are well-known immunosuppressors and thereby facilitating the tumor cells' evasion from immune recognition. This chapter will describe the interaction between TAMs and tumor cells, the involvement of TAMs in the regulation of cancer cell progression by controlling cell cycle checkpoints or molecular pathways, and current TAM-based therapies, including restriction of TAM recruitment, anti-survival strategies, or switching polarity. Moreover, this chapter will also emphasize recently developed drug targets and CAR-macrophage cell therapy that restricts tumor progression.

Anti-cancer drug molecules targeting cancer cell cycle and proliferation.

Cancer, a vicious clinical burden that potentiates maximum fatality for humankind, arises due to unregulated excessive cell division and proliferation through an eccentric expression of cell cycle regulator proteins. A set of evolutionarily conserved machinery controls the cell cycle in an extremely precise manner so that a cell that went through the cycle can produce a genetically identical copy. To achieve perfection, several checkpoints were placed in the cycle for surveillance; so, errors during the division were rectified by the repair strategies. However, irreparable damage leads to exit from the cell cycle and induces programmed cell death. In comparison to a normal cell, cancer cells facilitate the constitutive activation of many dormant proteins and impede negative regulators of the checkpoint. Extensive studies in the last few decades on cell division and proliferation of cancer cells elucidate the molecular mechanism of the cell-cycle regulators that are often targeted for the development of anti-cancer therapy. Each phase of the cell cycle has been regulated by a unique set of proteins including master regulators Cyclins, and CDKs, along with the accessory proteins such as CKI, Cdc25, error-responsive proteins, and various kinase proteins mainly WEE1 kinases, Polo-like kinases, and Aurora kinases that control cell division. Here in this chapter, we have analytically discussed the role of cell cycle regulators and proliferation factors in cancer progression and the rationale of using various cell cycle-targeting drug molecules as anti-cancer therapy.

Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair.

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.

A small molecule potent IRAK4 inhibitor abrogates lipopolysaccharide-induced macrophage inflammation in-vitro and in-vivo.

Increasing evidence supports vanillin and its analogs as potent toll-like receptor signaling inhibitors that strongly attenuate inflammation, though, the underlying molecular mechanism remains elusive. Here, we report that vanillin inhibits lipopolysaccharide (LPS)-induced toll-like receptor 4 activation in macrophages by targeting the myeloid differentiation primary-response gene 88 (MyD88)-dependent pathway through direct interaction and suppression of interleukin-1 receptor-associated kinase 4 (IRAK4) activity. Moreover, incubation of vanillin in cells expressing constitutively active forms of different toll-like receptor 4 signaling molecules revealed that vanillin could only able to block the ligand-independent constitutively activated IRAK4/1 or its upstream molecules-associated NF-κB activation and NF-κB transactivation along with the expression of various proinflammatory cytokines. A significant inhibition of LPS-induced IRAK4/MyD88, IRAK4/IRAK1, and IRAK1/TRAF6 association was evinced in response to vanillin treatment. Furthermore, mutations at Tyr262 and Asp329 residues in IRAK4 or modifications of 3-OMe and 4-OH side groups in vanillin, significantly reduced IRAK4 activity and vanillin function, respectively. Mice pretreated with vanillin followed by LPS challenge markedly impaired LPS-induced IRAK4 activation and inflammation in peritoneal macrophages. Thus, the present study posits vanillin as a novel and potent IRAK4 inhibitor and thus providing an opportunity for its therapeutic application in managing various inflammatory diseases.

miR-210-3p Promotes Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance by Targeting SOCS1-Mediated NF-κB Pathway.

Under the condition of chronic obesity, an increased level of free fatty acids along with low oxygen tension in the adipose tissue creates a pathophysiological adipose tissue microenvironment (ATenv), leading to the impairment of adipocyte function and insulin resistance. Here, we found the synergistic effect of hypoxia and lipid (H + L) surge in fostering adipose tissue macrophage (ATM) inflammation and polarization. ATenv significantly increased miR-210-3p expression in ATMs which promotes NF-κB activation-dependent proinflammatory cytokine expression along with the downregulation of anti-inflammatory cytokine expression. Interestingly, delivery of miR-210-3p mimic significantly increased macrophage inflammation in the absence of H + L co-stimulation, while miR-210-3p inhibitor notably compromised H + L-induced macrophage inflammation through increased production of suppressor of cytokine signaling 1 (SOCS1), a negative regulator of the NF-κB inflammatory signaling pathway. Mechanistically, miR-210 directly binds to the 3'-UTR of SOCS1 mRNA and silences its expression, thus preventing proteasomal degradation of NF-κB p65. Direct delivery of anti-miR-210-3p LNA in the ATenv markedly rescued mice from obesity-induced adipose tissue inflammation and insulin resistance. Thus, miR-210-3p inhibition in ATMs could serve as a novel therapeutic strategy for managing obesity-induced type 2 diabetes.

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Development of a Multicellular 3D Tumor Model to Study Cellular Heterogeneity and Plasticity in NSCLC Tumor Microenvironment.

Heterogeneity is a characteristic feature of solid tumors. Intra-tumor heterogeneity includes phenotypic diversity, epigenetic abnormalities, cell proliferation, and plasticity that eventually drives disease progression. Studying tumor heterogeneity in 2D culture is challenging as it cannot simulate the microenvironmental features, such as hypoxia, nutrient unavailability, and cell-ECM interactions. We propose the development of multicellular (tri-culture) 3D spheroids using a hanging drop method to study the non-tumorigenic (BEAS-2B) vs. tumorigenic NSCLC (A549/NCI-H460)cells' interaction with lung fibroblasts (MRC-5) and monocytes (THP-1). Unlike the non-tumorigenic model, the tumorigenic 3D spheroids show significant induction of cell proliferation, hypoxia, pluripotency markers, notable activation of cancer-associated fibroblasts, and tumor-associated macrophages. CD68+ macrophages isolated from tumorigenic spheroids exhibited profound induction of phenotypic endothelial characteristics. The results are zebrafish tumor xenograft model and by using human patient samples. This multicellular 3D tumor model is a promising tool to study tumor-stroma interaction and cellular plasticity, targeting tumor heterogeneity, and facilitating cancer therapy success against NSCLC.

Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition.

An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.

Hypoxia-induced miR-210-3p expression in lung adenocarcinoma potentiates tumor development by regulating CCL2-mediated monocyte infiltration.

In most cancers, tumor hypoxia downregulates the expression of C-C motif chemokine 2 (CCL2), and this downregulation has been implicated in monocyte infiltration and tumor progression; however, the molecular mechanism is yet not clear. We compared non-cancerous and lung-adenocarcinoma human samples for hypoxia-inducible factor 1-alpha (HIF-1A), microRNA-210-3p (mir-210-3p) and CCL2 levels. Mechanistic studies were performed on lung adenocarcinoma cell lines and 3D tumor spheroids to understand the role of hypoxia-induced miR-210-3p in the regulation of CCL2 expression and macrophage polarization. HIF-1 A stabilization increases miR-210-3p levels in lung adenocarcinoma and impairs monocyte infiltration by inhibiting CCL2 expression. Mechanistically, miR-210-3p directly binds to the 3'untranslated region (UTR) of CCL2 mRNA and silences it. Suppressing miR-210-3p substantially downregulates the effect of hypoxia on CCL2 expression. Monocyte migration is significantly hampered in miR-210-3p mimic-transfected HIF-1A silenced cancer cells. In contrast, inhibition of miR-210-3p in HIF-1A-overexpressed cells markedly restored monocyte migration, highlighting a direct link between miR-210-3p level and tumor monocyte burden. Moreover, miR-210-3p inhibition in 3D tumor spheroids promotes monocyte recruitment and skewing towards an anti-tumor M1 phenotype. Anti-hsa-miR-210-3p-locked nucleic acid (LNA) delivery in a lung tumor xenograft zebrafish model caused tumor regression, suggesting that miR-210-3p could be a promising target for immunomodulatory therapeutic strategies against lung adenocarcinoma.

Lipid-induced monokine cyclophilin-A promotes adipose tissue dysfunction implementing insulin resistance and type 2 diabetes in zebrafish and mice models of obesity.

Several studies have implicated obesity-induced macrophage-adipocyte cross-talk in adipose tissue dysfunction and insulin resistance. However, the molecular cues involved in the cross-talk of macrophage and adipocyte causing insulin resistance are currently unknown. Here, we found that a lipid-induced monokine cyclophilin-A (CyPA) significantly attenuates adipocyte functions and insulin sensitivity. Targeted inhibition of CyPA in diet-induced obese zebrafish notably reduced adipose tissue inflammation and restored adipocyte function resulting in improvement of insulin sensitivity. Silencing of macrophage CyPA or pharmacological inhibition of CyPA by TMN355 effectively restored adipocytes' functions and insulin sensitivity. Interestingly, CyPA incubation markedly increased adipocyte inflammation along with an impairment of adipogenesis, however, mutation of its cognate receptor CD147 at P309A and G310A significantly waived CyPA's effect on adipocyte inflammation and its differentiation. Mechanistically, CyPA-CD147 interaction activates NF-κB signaling which promotes adipocyte inflammation by upregulating various pro-inflammatory cytokines gene expression and attenuates adipocyte differentiation by inhibiting PPARγ and C/EBPß expression via LZTS2-mediated downregulation of ß-catenin. Moreover, inhibition of CyPA or its receptor CD147 notably restored palmitate or CyPA-induced adipose tissue dysfunctions and insulin sensitivity. All these results indicate that obesity-induced macrophage-adipocyte cross-talk involving CyPA-CD147 could be a novel target for the management of insulin resistance and type 2 diabetes.

Novel imidazo[1,2-a]pyridine derivatives induce apoptosis and cell cycle arrest in non-small cell lung cancer by activating NADPH oxidase mediated oxidative stress.

AIMS: Imidazo[1,2-a]pyridine-based analogues have recently gained significant interest because of their wide spectrum of biological activities including anti-cancer potential, however the development of targeted therapeutic candidates against non-small cell lung cancer (NSCLC) is of utmost need due to its high prevalence and poor prognosis. Herein, we have aimed to synthesized novel imidazo [1,2-a] pyridine derivatives (IMPA) by coupling with 2-amino-4H-pyran to enhance bioactivity against NSCLC. MAIN METHODS: We have designed and synthesized a series of fifteen novel imidazo [1,2-a] pyridine derivatives through molecular hybridization and studied their anti-cancer activity against in-vitro lung adenocarcinoma and 3D multicellular lung tumor spheroids. KEY FINDINGS: IMPA-2, IMPA-5, IMPA-6, IMPA-8, and IMPA-12 markedly induced cytotoxicity by notably increased NADPH oxidase (NOX) activity, which results in the induction of ROS-mediated apoptosis in A549 lung cancer cells. It caused impairment of mitochondrial membrane potential by increasing pro-apoptotic BAX, and BAK1 expressions, and decreasing anti-apoptotic BCL2 expression, along with the induction of caspase-9/3 activation, however, these attributes were compromised in presence of N-acetyl-L-cysteine (NAC), a free radical scavenger. Increased ROS production by IMPAs also promotes p53 mediated cell cycle arrest through the inactivation of p38MAPK. Reduction of tumor size in IMPAs-treated 3D multicellular lung tumor spheroids gave further validation. SIGNIFICANCE: Beside cytotoxicity, IMPAs also inhibit lung cancer cell invasion and migration, suggesting their applicability in metastatic lung cancer. Therefore, IMPA derivatives could be used as potential anti-cancer agents in treating non-small cell lung cancer.

Dexamethasone-loaded, injectable pullulan-poly(ethylene glycol) hydrogels for bone tissue regeneration in chronic inflammatory conditions.

Chronic inflammation, infection, and fixation stability disrupts bone tissue regeneration by implants. The elevated levels of inflammatory markers and reactive oxygen species (ROS) damage tissues, inhibit osteoblastic differentiation, and promote bone resorption. Activation of local and chronic inflammatory responses due to the implantable biomaterial poses a high risk of implant failure and compromised bone repair in several pathological conditions. Not much progress has been made in the development of biomaterials that can counter inflammation and ROS along with inducing osteogenic activities for managing bone defects/injuries. We have developed, for the first time, injectable polymeric hydrogels by crosslinking oxidized pullulan (OP, 1% w/v) and 8-arm PEG hydrazine (PEG-HY, 10% w/v) using pH-sensitive and dynamic hydrazone linkages at 37 °C in buffer. The hydrogels were loaded with dexamethasone (Dex), an anti-inflammatory corticosteroid and osteogenic inducer, by covalently linking it to PEG-HY by hydrazone linkages, and their morphological, injectability, viscoelastic, self-healing, swelling, and drug-release properties were investigated. The hydrogels provided a pH-sensitive sustained release of PEG-Dex conjugate (3.62 wt%, 9.22 × 10-5 mol of Dex/gram) for 28 days, with 74.54 and 55.15% PEG-Dex conjugate being released at pH 6.5 and 7.4. ABTS assay showed that hydrogels inhibited 68% radicals within 1 h, and treatment with hydrogel releasates inhibited the pro-inflammatory markers, IL-6 and IL-1ß, and elevated the anti-inflammatory marker, TGF-ß, in murine osteoblast precursor cells (MC3T3-E1). The hydrogels were found suitable for cell encapsulation and they exhibited 110% viability on treatment with releasates. Finally, the osteogenic activities of hydrogels were ascertained by alkaline phosphatase (ALP) activities, alizarin red S staining, and osteogenic gene expressions- RUNX2, Col-I, OPN, and IBSP. Overall, PEG-Dex conjugate released from hydrogels improved the cell viability and proliferation, and induced the osteoblastic differentiation. The hydrogels with their promising antioxidant and anti-inflammatory properties along with the osteogenic activities show a strong potential as an injectable, extracellular matrix (ECM)-mimicking implantable drug-depot for bone repair applications in chronic inflammatory conditions.

Remodeling of Stromal Cells and Immune Landscape in Microenvironment During Tumor Progression.

The molecular understanding of carcinogenesis and tumor progression rests in intra and inter-tumoral heterogeneity. Solid tumors confined with vast diversity of genetic abnormalities, epigenetic modifications, and environmental cues that differ at each stage from tumor initiation, progression, and metastasis. Complexity within tumors studied by conventional molecular techniques fails to identify different subclasses in stromal and immune cells in individuals and that affects immunotherapies. Here we focus on diversity of stromal cell population and immune inhabitants, whose subtypes create the complexity of tumor microenvironment (TME), leading primary tumors towards advanced-stage cancers. Recent advances in single-cell sequencing (epitope profiling) approach circumscribes phenotypic markers, molecular pathways, and evolutionary trajectories of an individual cell. We discussed the current knowledge of stromal and immune cell subclasses at different stages of cancer development with the regulatory role of non-coding RNAs. Finally, we reported the current therapeutic options in immunotherapies, advances in therapies targeting heterogeneity, and possible outcomes.

A novel small molecule A2A adenosine receptor agonist, indirubin-3'-monoxime, alleviates lipid-induced inflammation and insulin resistance in 3T3-L1 adipocytes.

Saturated free fatty acid-induced adipocyte inflammation plays a pivotal role in implementing insulin resistance and type 2 diabetes. Recent reports suggest A2A adenosine receptor (A2AAR) could be an attractive choice to counteract adipocyte inflammation and insulin resistance. Thus, an effective A2AAR agonist devoid of any toxicity is highly appealing. Here, we report that indirubin-3'-monoxime (I3M), a derivative of the bisindole alkaloid indirubin, efficiently binds and activates A2AAR which leads to the attenuation of lipid-induced adipocyte inflammation and insulin resistance. Using a combination of in silico virtual screening of potential anti-diabetic candidates and in vitro study on insulin-resistant model of 3T3-L1 adipocytes, we determined I3M through A2AAR activation markedly prevents lipid-induced impairment of the insulin signaling pathway in adipocytes without any toxic effects. While I3M restrains lipid-induced adipocyte inflammation by inhibiting NF-κB dependent pro-inflammatory cytokines expression, it also augments cAMP-mediated CREB activation and anti-inflammatory state in adipocytes. However, these attributes were compromised when cells were pretreated with the A2AAR antagonist, SCH 58261 or siRNA mediated knockdown of A2AAR. I3M, therefore, could be a valuable option to intervene adipocyte inflammation and thus showing promise for the management of insulin resistance and type 2 diabetes.