Autophagy in glioblastoma: A mechanistic perspective.

Glioblastoma (GBM) is one of the most lethal malignancies in humans. Even after surgical resection and aggressive radio- or chemotherapies, patients with GBM can survive for less than 14 months. Extreme inter-tumor and intra-tumor heterogeneity of GBM poses a challenge for resolving recalcitrant GBM pathophysiology. GBM tumor microenvironment (TME) exhibits diverse heterogeneity in cellular composition and processes contributing to tumor progression and therapeutic resistance. Autophagy is such a cellular process; that demonstrates a cell-specific and TME context-dependent role in GBM progression, leading to either the promotion or suppression of GBM progression. Autophagy can regulate GBM cell function directly via regulation of survival, migration, and invasion, or indirectly by affecting GBM TME composition such as immune cell population, tumor metabolism, and glioma stem cells. This review comprehensively investigates the role of autophagy in GBM pathophysiology.

The NLR gene family: from discovery to present day.

The mammalian NLR gene family was first reported over 20 years ago, although several genes that were later grouped into the family were already known at that time. Although it is widely known that NLRs include inflammasome receptors and/or sensors that promote the maturation of caspase 1, IL-1ß, IL-18 and gasdermin D to drive inflammation and cell death, the other functions of NLR family members are less well appreciated by the scientific community. Examples include MHC class II transactivator (CIITA), a master transcriptional activator of MHC class II genes, which was the first mammalian NBD-LRR-containing protein to be identified, and NLRC5, which regulates the expression of MHC class I genes. Other NLRs govern key inflammatory signalling pathways or interferon responses, and several NLR family members serve as negative regulators of innate immune responses. Multiple NLRs regulate the balance of cell death, cell survival, autophagy, mitophagy and even cellular metabolism. Perhaps the least discussed group of NLRs are those with functions in the mammalian reproductive system. The focus of this Review is to provide a synopsis of the NLR family, including both the intensively studied and the underappreciated members. We focus on the function, structure and disease relevance of NLRs and highlight issues that have received less attention in the NLR field. We hope this may serve as an impetus for future research on the conventional and non-conventional roles of NLRs within and beyond the immune system.

Increased Incidence of Obesity in Children and Adolescents Post-COVID-19 Pandemic: A Review Article.

The recent coronavirus 2019 (COVID-19) pandemic has immensely impacted all classes of society, but the effects on children and adolescents are much more pronounced than on others. While obesity and its comorbidities in children and adolescents have always been a concern, the COVID-19 pandemic has proven to be one of the leading causes of health problems in children and adolescents worldwide, leading to various complications. Hence, understanding its long-term sequelae is of utmost importance. The role of physicians in family counseling, nutrition counseling, and diet education is vital in maintaining a healthy lifestyle. The BMI (body mass index) measurements and retrospective cohort studies of various individuals are useful for the pertinent research. During the pandemic, social isolation, staying at home, increased screen time due to online classes, reduced outdoor activities, and more snacking are some of the contributing factors that have increased the prevalence of obesity and further morbidities associated with it. Multiple studies and guidelines are available for combating these issues; still, an increasing number of such cases have been encountered in routine outpatient department (OPD) practice. As opposed to specific infectious illnesses, obesity and its comorbidities are non-infectious, and a slow-growing silent risk; hence parents approach the pediatrician quite late in the disease process. With the emergence of the COVID-19 pandemic, every aspect of our life has entered a more virtual domain and is no longer confined to a mere physical sphere. This sudden shift to virtual online classes has significantly impacted children and adolescents by decreasing their physical activities and social interactions in schools. This has even led to increased use of social media and mobile phone games by children and adolescents, a grave concern for parents, pediatricians, and epidemiologists. A more detailed assessment and multidisciplinary approach might benefit in dealing with the management of this emerging issue. Gaining enhanced clarity by establishing more guidelines can help physicians as well as parents in the management of this critical issue.

Glucose-Induced Biofilm Formation in Bacillus thuringiensis KPWP1 is Associated with Increased Cell Surface Hydrophobicity and Increased Production of Exopolymeric Substances.

Bacillus thuringiensis is an agriculturally and medically important bacteria as it produces insecticidal Cry proteins and can form biofilm on different plant surfaces. Previous studies reported that the ubiquitous carbon source glucose could induce restricted motility and fractal pattern formation in the growing colonies of pH, salt and arsenate tolerant Bacillus thuringiensis KPWP1. As bacteria are evolved with the ability to exhibit multicellular behavior and biofilm formation under limiting conditions for survival, the present study was focused on exploring the effect of glucose in biofilm formation by Bacillus thuringiensis KPWP1. A significant rise in biofilm loads was observed with increased glucose concentrations in growth media. Compared to control, six times more biofilm load was marked in presence of 2% of glucose. Interestingly, it was observed that the effect was glucose specific and also not due to any change in the sugar-induced physicochemical property of the growth media as the addition of galactose or arabinose could not induce any significant increase in KPWP1 biofilm load. Scanning electron-, confocal laser scanning-microscopic studies and biochemical tests revealed that increased concentrations of glucose could induce increased production of exopolymeric substances, increased number of densely-packed micro-colonies in KPWP1 biofilm and increased hydrophobicity and adherence properties in KPWP1cells.

Biochemical, molecular and in silico characterization of arsenate reductase from Bacillus thuringiensis KPWP1 tolerant to salt, arsenic and a wide range of pH.

The present study characterized aresenate reductase of Bacillus thuringiensis KPWP1, tolerant to salt, arsenate and a wide range of pH during growth. Interestingly, it was found that arsC, arsB and arsR genes involved in arsenate tolerance are distributed in the genome of strain KPWP1. The inducible arsC gene was cloned, expressed and the purified ArsC protein showed profound enzyme activity with the KM and Kcat values as 25 µM and 0.00119 s-1, respectively. In silico studies revealed that in spite of 19-26% differences in gene sequences, the ArsC proteins of Bacillus thuringiensis, Bacillus subtilis and Bacillus cereus are structurally conserved and ArsC structure of strain KPWP1 is close to nature. Docking and analysis of the binding site showed that arsenate ion interacts with three cysteine residues of ArsC and predicts that the ArsC from B. thuringiensis KPWP1 reduces arsenate by using the triple Cys redox relay mechanism.

Protocol for induction and characterization of microglia extracellular traps in murine and human microglia cells.

Extracellular traps (ETs) are composed of decondensed chromatin and are embedded with various antimicrobial proteins like myeloperoxidase and histones. Recently, we reported that dopamine (DA) induces ETs in BV2 microglia cell line and primary adult human microglia in a manner independent of cell death, reactive oxygen species, and actin polymerization. This protocol details how to characterize DA-induced ETs in BV2 microglia and human microglia. The protocols for characterization of ETs may also be used for other adherent cell lines. For complete details on the use and execution of this protocol, please refer to Agrawal et al. (2021).

Dopamine induces functional extracellular traps in microglia.

Dopamine (DA) plays many roles in the brain, especially in movement, motivation, and reinforcement of behavior; however, its role in regulating innate immunity is not clear. Here, we show that DA can induce DNA-based extracellular traps in primary, adult, human microglia and BV2 microglia cell line. These DNA-based extracellular traps are formed independent of reactive oxygen species, actin polymerization, and cell death. These traps are functional and capture fluorescein (FITC)-tagged Escherichia coli even when reactive oxygen species production or actin polymerization is inhibited. We show that microglial extracellular traps are present in Glioblastoma multiforme. This is crucial because Glioblastoma multiforme cells are known to secrete DA. Our findings demonstrate that DA plays a significant role in sterile neuro-inflammation by inducing microglia extracellular traps.

Mitochondrial Dysfunction and Alzheimer's Disease: Role of Microglia.

In 1907, Alois Alzheimer observed, as he quoted, development of "numerous fibers" and "adipose saccules" in the brain of his diseased patient Auguste Deter. The neurodegenerative disease became known as Alzheimer's disease (AD) and is the most common cause of dementia worldwide. AD normally develops with aging and is mostly initiated because of the imbalance between the formation and clearance of amyloid-ß (Aß). Formation of neurofibrillary tangles (NFTs) of hyperphosphorylated tau is another hallmark of AD. Neuroinflammation plays a significant role in the development and pathology of AD. This chapter explores the role of mitochondrial dysfunction in microglia in case of AD. Mitochondrial oxidative stress in microglia has been linked to the development of AD. Elevated generation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential through various mechanisms have been observed in AD. Aß interacts with microglial receptors, such as triggering receptor expressed in myeloid cells 2 (TREM2), activating downstream pathways causing mitochondrial damage and aggravating inflammation and cytotoxicity. Fibrillar Aß activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in microglia leading to elevated induction of mitochondrial ROS which further causes neurotoxicity. Elevated ROS in microglia causes activation of inflammatory and cell death pathways. Production of ATP, regulation of mitochondrial health, autophagy, and mitophagy in microglia play significant roles in the AD pathology. Understanding microglial physiology and mitochondrial dysfunction will enable better therapeutic interventions.

Obtaining Human Microglia from Adult Human Brain Tissue.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

Amorphous nanosilica induced toxicity, inflammation and innate immune responses: A critical review.

Nanoparticles are promising bioengineering platforms facilitating various consumer product formulations, including packaged food, electrical, biosensor and biomedical tools. The unique surface and physicochemical properties of amorphous nanosilica supports advanced nano-biomolecular applications for various manufacturing, biotechnology, and healthcare industries including cosmetics, packaging, implants, drug delivery systems and cancer diagnostics. The increased technological and economic benefits of amorphous nanosilica, raises concerns regarding their adverse biological effects on humans. The cellular mechanisms underlying amorphous nanosilica internalization, evasion of biological barriers, inadvertent nano-bio interactions and unexpected long term exposure effects must be taken into consideration from the diverse ecosystems and human safety aspects. Recent research studies reveal cytotoxic, inflammatory and immunomodulatory effects of amorphous nanosilica particles. Our review focuses on studies demonstrating hazardous impact of amorphous nanosilica/bio-systems interface on the cellular and biochemical processes. The review further seeks to evaluate amorphous nanosilica-induced cytotoxicity, innate immune responses, inflammation and immune related dysfunctions, and discuss open research questions related to the use of amorphous nanosilica in biomedicine.

Comprehensive review of ASC structure and function in immune homeostasis and disease.

Apoptosis associated speck like protein containing CARD (ASC) is widely researched and recognized as an adaptor protein participating in inflammasome assembly and pyroptosis. It contains a bipartite structure comprising of a pyrin and a caspase recruitment domain (CARD) domain. These two domains help ASC function as an adaptor molecule. ASC is encoded by the gene PYCARD. ASC plays pivotal role in various diseases as well as different homeostatic processes. ASC plays a regulatory role in different cancers showing differential regulation with respect to tissue and stage of disease. Besides cancer, ASC also plays a central role in sensing, regulation, and/or disease progression in bacterial infections, viral infections and in varied inflammatory diseases. ASC is expressed in different types of immune and non-immune cells. Its localization pattern also varies with different kinds of stimuli encountered by cell. This review will summarize the literature on the structure cellular and tissue expression, localization and disease association of ASC.

Differential Expression Profile of NLRs and AIM2 in Glioma and Implications for NLRP12 in Glioblastoma.

Gliomas are the most prevalent primary brain tumors with immense clinical heterogeneity, poor prognosis and survival. The nucleotide-binding domain, and leucine-rich repeat containing receptors (NLRs) and absent-in-melanoma 2 (AIM2) are innate immune receptors crucial for initiation and progression of several cancers. There is a dearth of reports linking NLRs and AIM2 to glioma pathology. NLRs are expressed by cells of innate immunity, including monocytes, macrophages, dendritic cells, endothelial cells, and neutrophils, as well as cells of the adaptive immune system. NLRs are critical regulators of major inflammation, cell death, immune and cancer-associated pathways. We used a data-driven approach to identify NLRs, AIM2 and NLR-associated gene expression and methylation patterns in low grade glioma and glioblastoma, using The Cancer Genome Atlas (TCGA) patient datasets. Since TCGA data is obtained from tumor tissue, comprising of multiple cell populations including glioma cells, endothelial cells and tumor-associated microglia/macrophages we have used multiple cell lines and human brain tissues to identify cell-specific effects. TCGA data mining showed significant differential NLR regulation and strong correlation with survival in different grades of glioma. We report differential expression and methylation of NLRs in glioma, followed by NLRP12 identification as a candidate prognostic marker for glioma progression. We found that Nlrp12 deficient microglia show increased colony formation while Nlrp12 deficient glioma cells show decreased cellular proliferation. Immunohistochemistry of human glioma tissue shows increased NLRP12 expression. Interestingly, microglia show reduced migration towards Nlrp12 deficient glioma cells.

NLRs as Helpline in the Brain: Mechanisms and Therapeutic Implications.

Nucleotide binding domain, leucine-rich repeat containing proteins (NLRs) are a family of pattern recognition receptors involved in major innate immune defense mechanisms. NLRs play a key role in several cancers, autoimmune, and inflammation-associated diseases. Association of NLRP3 has been widely investigated in neurodegenerative diseases, chronic alcoholism, depression, traumatic brain injury, and pathogenic infections. Several research studies have shown possible involvement of various other inflammasome-forming and non-inflammasome-forming NLRs in the brain; however, their mechanisms of action are yet to be defined clearly. Our review provides a comprehensive overview of the expression of NLRs in human brain and their critical association with inflammation and neurodegenerative diseases. The review also summarizes promising NLR-targeted therapeutics and their prospects for brain pathologies.

NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes.

Inflammation in the brain accompanies several high-impact neurological diseases including multiple sclerosis (MS), stroke, and Alzheimer's disease. Neuroinflammation is sterile, as damage-associated molecular patterns rather than microbial pathogens elicit the response. The inflammasome, which leads to caspase-1 activation, is implicated in neuroinflammation. In this study, we reveal that lysophosphatidylcholine (LPC), a molecule associated with neurodegeneration and demyelination, elicits NLRP3 and NLRC4 inflammasome activation in microglia and astrocytes, which are central players in neuroinflammation. LPC-activated inflammasome also requires ASC (apoptotic speck containing protein with a CARD), caspase-1, cathepsin-mediated degradation, calcium mobilization, and potassium efflux but not caspase-11. To study the physiological relevance, Nlrc4-/- and Nlrp3-/- mice are studied in the cuprizone model of neuroinflammation and demyelination. Mice lacking both genes show the most pronounced reduction in astrogliosis and microglial accumulation accompanied by decreased expression of the LPC receptor G2A, whereas MS patient samples show increased G2A. These results reveal that NLRC4 and NLRP3, which normally form distinct inflammasomes, activate an LPC-induced inflammasome and are important in astrogliosis and microgliosis.

Trigonella seed extract ameliorates inflammation via regulation of the inflammasome adaptor protein, ASC.

Trigonella foenum-graecum (fenugreek) is an important medicinal plant, well known for its anti-inflammatory properties. However, the underlying cellular and molecular mechanisms of its action remain largely unknown. The apoptosis associated speck like protein containing a caspase recruitment domain (CARD) (ASC) is central to inflammatory and cell death pathways in innate and adaptive immunity. Here, we show that fenugreek seed extract provides cytoprotection to bacterial lipopolysaccharide (LPS) inflammed and nanosilica-treated fibroblasts via a reactive oxygen species independent pathway. All atom molecular dynamics simulations of ASC-ligand complex reveal that individual phytochemicals in fenugreek can bind to ASC via specific non-covalent interactions. These data show that a synergistic effect of fenugreek phytochemicals with the ASC protein alters its molecular properties resulting in altered cellular function. Such information is crucial to the development of targeted therapeutic interventions for inflammatory diseases.

Role of NOD- like Receptors in Glioma Angiogenesis: Insights into future therapeutic interventions.

Gliomas are the most common solid tumors among central nervous system tumors. Most glioma patients succumb to their disease within two years of the initial diagnosis. The median survival of gliomas is only 14.6 months, even after aggressive therapy with surgery, radiation, and chemotherapy. Gliomas are heavily infiltrated with myeloid- derived cells and endothelial cells. Increasing evidence suggests that these myeloid- derived cells interact with tumor cells promoting their growth and migration. NLRs (nucleotide-binding oligomerization domain (NOD)-containing protein like receptors) are a class of pattern recognition receptors that are critical to sensing pathogen and danger associated molecular patterns. Mutations in some NLRs lead to autoinflammatory diseases in humans. Moreover, dysregulated NLR signaling is central to the pathogenesis of several cancers, autoimmune and neurodegenerative diseases. Our review explores the role of angiogenic factors that contribute to upstream or downstream signaling pathways leading to NLRs. Angiogenesis plays a significant role in the pathogenesis of variety of tumors including gliomas. Though NLRs have been detected in several cancers including gliomas and NLR signaling contributes to angiogenesis, the exact role and mechanism of involvement of NLRs in glioma angiogenesis remain largely unexplored. We discuss cellular, molecular and genetic studies of NLR signaling and convergence of NLR signaling pathways with angiogenesis signaling in gliomas. This may lead to re-appropriation of existing anti-angiogenic therapies or development of future strategies for targeted therapeutics in gliomas.

Inflammasomes in Myeloid Cells: Warriors Within.

The inflammasome is a large multimeric protein complex comprising an effector protein that demonstrates specificity for a variety of activators or ligands; an adaptor molecule; and procaspase-1, which is converted to caspase-1 upon inflammasome activation. Inflammasomes are expressed primarily by myeloid cells and are located within the cell. The macromolecular inflammasome structure can be visualized by cryo-electron microscopy. This complex has been found to play a role in a variety of disease models in mice, and several have been genetically linked to human diseases. In most cases, the effector protein is a member of the NLR (nucleotide-binding domain leucine-rich repeat-containing) or NOD (nucleotide oligomerization domain)-like receptor protein family. However, other effectors have also been described, with the most notable being AIM-2 (absent in melanoma 2), which recognizes DNA to elicit inflammasome function. This review will focus on the role of the inflammasome in myeloid cells and its role in health and disease.

NLR-regulated pathways in cancer: opportunities and obstacles for therapeutic interventions.

NLRs (nucleotide-binding domain, leucine-rich repeat containing receptors) are pattern recognition receptors associated with immunity and inflammation in response to endogenous and exogenous pathogen and damage associated molecular patterns (PAMPs and DAMPs respectively). Dysregulated NLR function is associated with several diseases including cancers, metabolic diseases, autoimmune disorders and autoinflammatory syndromes. In the last decade, distinct cell and organ specific roles for NLRs have been identified however; their roles in cancer initiation, development and progression remain controversial. This review summarizes the emerging role of NLRs in cancer and their possible future as targets for cancer therapeutics.

Holding the inflammatory system in check: NLRs keep it cool.

Inflammation is a double-edged sword. While short-lived, acute inflammation is essential for the repair and resolution of infection and damage, uncontrolled and unresolved chronic inflammation is central to several diseases, including cancer, autoimmune diseases, allergy, metabolic disease, and cardiovascular disease. This report aims to review the literature regarding several members of the nucleotide-binding domain, leucine-rich repeat-containing receptor (NLR) family of pattern recognition sensors/receptors that serve as checkpoints for inflammation. Understanding the negative regulation of inflammation is highly relevant to the development of therapeutics for inflammatory as well as infectious diseases.