Endogenous Functions Research Articles

Tumor-Suppressive Functions of the Aryl Hydrocarbon Receptor (AhR) and AhR as a Therapeutic Target in Cancer.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in regulating a wide range of biological responses. A diverse array of xenobiotics and endogenous small molecules bind to the receptor and drive unique phenotypic responses. Due in part to its role in mediating toxic responses to environmental pollutants, AhR activation has not been traditionally viewed as a viable therapeutic approach. Nonetheless, the expression and activation of AhR can inhibit the proliferation, migration, and survival of cancer cells, and many clinically approved drugs transcriptionally activate AhR. Identification of novel select modulators of AhR-regulated transcription that promote tumor suppression is an active area of investigation. The development of AhR-targeted anticancer agents requires a thorough understanding of the molecular mechanisms driving tumor suppression. Here, we summarized the tumor-suppressive mechanisms regulated by AhR with an emphasis on the endogenous functions of the receptor in opposing carcinogenesis. In multiple different cancer models, the deletion of AhR promotes increased tumorigenesis, but a precise understanding of the molecular cues and the genetic targets of AhR involved in this process is lacking. The intent of this review was to synthesize the evidence supporting AhR-dependent tumor suppression and distill insights for development of AhR-targeted cancer therapeutics.

In situ MUC1-specific CAR engineering of tumor-supportive macrophages stimulates tumoricidal immunity against pancreatic adenocarcinoma

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized intervention strategies for various primary malignancies. However, the implementation of this approach in solid tumors, including fatal gastrointestinal cancer pancreatic adenocarcinoma (PAAD), is limited. Given the endogenous functions of macrophages and their extensive infiltration in the solid tumor microenvironment (TME), liposomalized nanoporters (LNPs) are constructed for in situ genetic engineering of intratumoral macrophages with a mucin 1-specific CAR, which redirect the phagocytic activity of these cells to target cancerous PAAD cells and subsequently prime locoregional anti-PAAD immunity. When systemically administered, any LNPs that escape clearance by the reticuloendothelial system become passively enriched in tumor tissues and unshell upon triggering with legumain intratumorally. The liberated nanoporters in turn introduce the CAR gene into the nucleus of macrophages, thereby locoregionally yielding PAAD-targeted CAR macrophages, which significantly enhances the antigen-specific phagocytosis by macrophages, elevates the antigen presentation specificity, and serially boosts the population of cytotoxic T lymphocytes, thus reducing the tumor burden and prolonging survival in PAAD mouse models. Our technology provides a practical antitumor immunotherapy for PAAD patients and may be broadly applicable for patients suffering from other solid malignancies that dually overexpress mucin 1 and legumain.

Striving toward hyperthermia-free analgesia: lessons from loss-of-function mutations of human TRPV1

Transient receptor potential vanilloid 1 (TRPV1), a receptor for capsaicin and noxious heat, has been one of the most compelling targets for analgesics. However, systemic inhibition of TRPV1 is an impractical approach as a pain killer, since systemic antagonism induces hyperthermia. Two articles in this issue of the JCI report phenotypes from separate, rare missense mutations of human TRPV1. He, Zambelli, and colleagues investigated TRPV1K710N, which showed reduced functionality, while Katz, Zaguri, and co-authors reported on TRPV1N331K, which led to a complete functional knockout. The findings provide insights that will improve our understanding of the endogenous functions of TRPV1 in humans and may facilitate a rational TRPV1-targeting approach to achieve hyperthermia-free analgesia.

Prx5 of Cristaria plicata has antioxidant function and is regulated by Nrf2/ARE signaling pathway

Cristaria plicata is one of the more important freshwater pearl bivalves in China, which is susceptible to pathogen infection, and greatly impacts the ability of breeding pearls. Nrf2/ARE signaling pathway and its downstream target gene Prx5 have endogenous antioxidant functions to protect cells from oxidative damage. The full-length cDNA of Prx5 was cloned from C. Plicata, which was 1420 bp, encoding a total of 189 amino acids and had two conserved cysteine residues (Cys78 and Cys179). The amino acid sequence of CpPrx5 was highly similar to Prx5 of other species. Real-time fluorescence quantitative PCR showed that CpPrx5 was distributed in various tissues of mussels, and the highest expression was in hepatopancreas. The expression of CpPrx5 up-regulated in hepatopancreas and gills after LPS, PGN and Poly:I:C stimulation. The recombinant plasmid DE3-PGEX-4T-1-CpPrx5 was expressed in Escherichia coli BL21 and showed antioxidant activity. With the increase of CpPrx5 protein concentration, the superhelical form of DNA was protected. The expression of CpPrx5 was up-regulated after interference CpKeap1 and down-regulated after interference CpNrf2. Gel block assay showed that CpNrf2 and CpMafK proteins blocked CpPrx5 promoter. Subcellular localization showed that CpPrx5 was located in 293T nucleus and cytoplasm and CpMafK was located in 293T nucleus. GST-Pull down verified that CpMafK and CpPrx5 could bind in vitro. These results indicated that Prx5 had antioxidant function and could protects DNA from oxidative damage, and participated in transcriptional regulation by combining with the transcription factor MafK. In addition, MafK could combine with Nrf2 to regulate the downstream target gene Prx5.

Generation of Knock-In Mouse by Genome Editing.

Knock-in mice are useful for evaluating endogenous gene expressions and functions in vivo. Instead of the conventional gene-targeting method using embryonic stem cells, an exogenous DNA sequence can be inserted into the target locus in the zygote using genome-editing technology. In this chapter, I describe the generation of epitope-tagged mice using engineered endonuclease and single-strand oligodeoxynucleotide through the mouse zygote as an example of how to generate a knock-in mouse by genome editing.

Proper modulation of AHR signaling is necessary for establishing neural connectivity and oligodendrocyte precursor cell development in the embryonic zebrafish brain.

2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD) is a persistent global pollutant that exhibits a high affinity for the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. Epidemiological studies have associated AHR agonist exposure with multiple human neuropathologies. Consistent with the human data, research studies using laboratory models have linked pollutant-induced AHR activation to disruptions in learning and memory as well as motor impairments. Our understanding of endogenous AHR functions in brain development is limited and, correspondingly, scientists are still determining which cell types and brain regions are sensitive to AHR modulation. To identify novel phenotypes resulting from pollutant-induced AHR activation and ahr2 loss of function, we utilized the optically transparent zebrafish model. Early embryonic TCDD exposure impaired embryonic brain morphogenesis, resulted in ventriculomegaly, and disrupted neural connectivity in the optic tectum, habenula, cerebellum, and olfactory bulb. Altered neural network formation was accompanied by reduced expression of synaptic vesicle 2. Loss of ahr2 function also impaired nascent network development, but did not affect gross brain or ventricular morphology. To determine whether neural AHR activation was sufficient to disrupt connectivity, we used the Gal4/UAS system to express a constitutively active AHR specifically in differentiated neurons and observed disruptions only in the cerebellum; thus, suggesting that the phenotypes resulting from global AHR activation likely involve multiple cell types. Consistent with this hypothesis, we found that TCDD exposure reduced the number of oligodendrocyte precursor cells and their derivatives. Together, our findings indicate that proper modulation of AHR signaling is necessary for the growth and maturation of the embryonic zebrafish brain.

Cyberspace Endogenous Safety and Security

Uncertain security threats caused by vulnerabilities and backdoors are the most serious and difficult problem in cyberspace. This paper analyzes the philosophical and technical causes of the existence of so-called “dark functions” such as system vulnerabilities and backdoors, and points out that endogenous security problems cannot be completely eliminated at the theoretical and engineering levels; rather, it is necessary to develop or utilize the endogenous security functions of the system architecture itself. In addition, this paper gives a definition for and lists the main technical characteristics of endogenous safety and security in cyberspace, introduces endogenous safety and security mechanisms and characteristics based on dynamic heterogeneous redundancy (DHR) architecture, and describes the theoretical implications of a coding channel based on DHR.

OX2R-selective orexin agonism is sufficient to ameliorate cataplexy and sleep/wake fragmentation without inducing drug-seeking behavior in mouse model of narcolepsy.

Acquired loss of hypothalamic orexin (hypocretin)-producing neurons causes the chronic sleep disorder narcolepsy-cataplexy. Orexin replacement therapy using orexin receptor agonists is expected as a mechanistic treatment for narcolepsy. Orexins act on two receptor subtypes, OX1R and OX2R, the latter being more strongly implicated in sleep/wake regulation. However, it has been unclear whether the activation of only OX2R, or both OX1R and OX2R, is required to replace the endogenous orexin functions in the brain. In the present study, we examined whether the selective activation of OX2R is sufficient to rescue the phenotype of cataplexy and sleep/wake fragmentation in orexin knockout mice. Intracerebroventricular [Ala11, D-Leu15]-orexin-B, a peptidic OX2R-selective agonist, selectively activated OX2R-expressing histaminergic neurons in vivo, whereas intracerebroventricular orexin-A, an OX1R/OX2R non-selective agonist, additionally activated OX1R-positive noradrenergic neurons in vivo. Administration of [Ala11, D-Leu15]-orexin-B extended wake time, reduced state transition frequency between wake and NREM sleep, and reduced the number of cataplexy-like episodes, to the same degree as compared with orexin-A. Furthermore, intracerebroventricular orexin-A but not [Ala11, D-Leu15]-orexin-B induced drug-seeking behaviors in a dose-dependent manner in wild-type mice, suggesting that OX2R-selective agonism has a lower propensity for reinforcing/drug-seeking effects. Collectively, these findings provide a proof-of-concept for safer mechanistic treatment of narcolepsy-cataplexy through OX2R-selective agonism.

Endogenous Inverse Demand Functions

Endogenous Inverse Demand Functions Buying or selling assets in a financial market impacts the prices upward or downward. Quantifying these price impacts is fundamental to many problems within finance (e.g., optimal liquidation and systemic risk). In “Endogenous Inverse Demand Functions,” Bichuch and Feinstein construct an equilibrium risk sharing problem that results in an endogenous inverse demand function. This is taken in contrast to the often assumed exogenous linear or exponential forms for the inverse demand functions. The authors determine sufficient joint properties for the financial market and assets to replicate these common exogenous forms. The properties of the general endogenous inverse demand functions, found via the equilibrium risk sharing problem, are investigated; the authors deduce that price cross-impacts, which are often assumed to be zero, naturally arise in this equilibrium setting.

Unmet needs in glaucoma therapy: The potential role of hydrogen sulfide and its delivery strategies.

Glaucoma is an optic neuropathy disorder marked by progressive degeneration of the retinal ganglion cells (RGC). It is a leading cause of blindness worldwide, prevailing in around 2.2% of the global population. The hallmark of glaucoma, intraocular pressure (IOP), is governed by the aqueous humor dynamics which plays a crucial role in the pathophysiology of the diesease. Glaucomatous eye has an IOP of more than 22 mmHg as compared to normotensive pressure of 10-21 mmHg. Currently used treatments focus on reducing the elevated IOP through use of classes of drugs that either increase aqueous humor outflow and/or decrease its production. However, effective treatments should not only reduce IOP, but also offer neuroprotection and regeneration of RGCs. Hydrogen Sulfide (H2S), a gasotransmitter with several endogenous functions in mammalian tissues, is being investigated for its potential application in glaucoma. In addition to decreasing IOP by increasing aqueous humor outflow, it scavenges reactive oxygen species, upregulates the cellular antioxidant glutathione and protects RGCs from excitotoxicity. Despite the potential of H2S in glaucoma, its delivery to anterior and posterior regions of the eye is a challenge due to its unique physicochemical properties. Firstly, development of any delivery system should not require an aqueous environment since many H2S donors are susceptible to burst release of the gas in contact with water, causing potential toxicity and adverse effects owing to its inherent toxicity at higher concentrations. Secondly, the release of the gas from the donor needs to be sustained for a prolonged period of time to reduce dosing frequency as per the requirements of regulatory bodies. Lastly, the delivery system should provide adequate bioavailability throughout its period of application. Hence, an ideal delivery system should aim to tackle all the above challenges related to barriers of ocular delivery and physicochemical properties of H2S itself. This review discusses the therapeutic potential of H2S, its delivery challenges and strategies to overcome the associated chalenges.

Synthesis of Indole-Coupled KYNA Derivatives via C-N Bond Cleavage of Mannich Bases.

KYNAs, a compound with endogenous neuroprotective functions and an indole that is a building block of many biologically active compounds, such as a variety of neurotransmitters, are reacted in a transformation building upon Mannich bases. The reaction yields triarylmethane derivatives containing two biologically potent skeletons, and it may contribute to the synthesis of new, specialised neuroprotective compounds. The synthesis has been investigated via two procedures and the results were compared to those of previous studies. A possible alternative reaction route through acid catalysis has been established.

Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis.

Summary Pseudomonas chlororaphis is a non‐pathogenic, plant growth‐promoting rhizobacterium that secretes phenazine compounds with broad‐spectrum antibiotic activity. Currently available genome‐editing methods for P. chlororaphis are based on homologous recombination (HR)‐dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ‐Red–like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time‐consuming, laborious and inefficient. Here, we established a CRISPR‐assisted base‐editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced‐specificity Cas9 nickase (eSpCas9ppD10A) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO‐inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR‐based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non‐traditional bacterial hosts.

Therapeutic Potential and Clinical Evidence of Hesperidin as Neuroprotective Agent.

Neuroprotection is preserving neural function in various neurodegenerative diseases like Alzheimer's, Huntington's, Parkinson's, and multiple sclerosis. Hesperidin, a flavanone glycoside in citrus fruits such as sweet oranges and lemons, possesses many biological effects, including neuroprotection. The study aims to explore the neuropharmacological mechanisms and therapeutic potential of hesperidin in the management of neurodegenerative disorders. It emphasizes comparative and clinical trial studies with a number of targets reviewed from the data available on PubMed, Science Direct, Clinicaltrails.gov, and from many reputed foundations. Escalating clinical evidence has established the inhibitory effect of hesperidin in the management of neurodegenerative disorders. Neuroprotective potential of hesperidin is characterized by endogenous antioxidant defence functions, improvement of neural growth factors, antineuroinflammatory activity, and apoptotic pathways. The present study highlights the beneficial neuropharmacological potential of hesperidin, including anticonvulsant, antidepressant, antioxidant, anti-inflammatory, memory, and locomotor enhancing activities.

Methods for extracting and analyzing carotenoids from bird feathers.

Carotenoid pigments serve many endogenous functions in organisms, but some of the more fascinating are the external displays of carotenoids in the colorful red, orange and yellow plumages of birds. Since Darwin, biologists have been curious about the selective advantages (e.g., mate attraction) of having such ornate features, and, more recently, advances in biochemical methods have permitted researchers to explore the composition and characteristics of carotenoid pigments in feathers. Here we review contemporary methods for extracting and analyzing carotenoids in bird feathers, with special attention to the difficulties of removal from the feather keratin matrix, the possibility of feather carotenoid esterification and the strengths and challenges of different analytical methods like high-performance liquid chromatography and Raman spectroscopy. We also add an experimental test of current common extraction methods (e.g., mechanical, thermochemical) and find significant differences in the recovery of specific classes of carotenoids, suggesting that no single approach is best for all pigment or feather types.

Redirecting meiotic DNA break hotspot determinant proteins alters localized spatial control of DNA break formation and repair.

During meiosis, DNA double-strand breaks (DSBs) are formed at high frequency at special chromosomal sites, called DSB hotspots, to generate crossovers that aid proper chromosome segregation. Multiple chromosomal features affect hotspot formation. In the fission yeast S. pombe the linear element proteins Rec25, Rec27 and Mug20 are hotspot determinants – they bind hotspots with high specificity and are necessary for nearly all DSBs at hotspots. To assess whether they are also sufficient for hotspot determination, we localized each linear element protein to a novel chromosomal site (ade6 with lacO substitutions) by fusion to the Escherichia coli LacI repressor. The Mug20-LacI plus lacO combination, but not the two separate lac elements, produced a strong ade6 DSB hotspot, comparable to strong endogenous DSB hotspots. This hotspot had unexpectedly low ade6 recombinant frequency and negligible DSB hotspot competition, although like endogenous hotspots it manifested DSB interference. We infer that linear element proteins must be properly placed by endogenous functions to impose hotspot competition and proper partner choice for DSB repair. Our results support and expand our previously proposed DSB hotspot-clustering model for local control of meiotic recombination.

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Boundaries of the Machine Body: Violence, Immunity and Media Assemblages in The Last of Us This article examines the portrayal of bodily boundaries in the videogame series The Last of Us. Drawing on theories of media ecology and posthumanism (most notably Deer’s notion of radical animism, Haraway’s theories of the cyborg, and Fuller’s account of media assemblages), three aspects of this portrayal are described: first, the game’s narrativization of bodily violence through an amalgamation of the player’s sensory systems with media technologies; second, the game’s depiction of monstrous corporeality; and third, its representation of immune systems through the mirrored relationship between external tools and endogenous bodily functions. Connecting these three aspects, it is argued that The Last of Us portrays bodily boundaries as precarious, and that it presents violence, technology and infectious disease as callingcards for moving beyond anthropocentric views of corporeality; of conceptualizing the human body as machine-like and inevitably more-than-human.

LEA motifs promote desiccation tolerance in vivo

BackgroundCells and organisms typically cannot survive in the absence of water. However, some animals including nematodes, tardigrades, rotifers, and some arthropods are able to survive near-complete desiccation. One class of proteins known to play a role in desiccation tolerance is the late embryogenesis abundant (LEA) proteins. These largely disordered proteins protect plants and animals from desiccation. A multitude of studies have characterized stress-protective capabilities of LEA proteins in vitro and in heterologous systems. However, the extent to which LEA proteins exhibit such functions in vivo, in their native contexts in animals, is unclear. Furthermore, little is known about the distribution of LEA proteins in multicellular organisms or tissue-specific requirements in conferring stress protection. Here, we used the nematode C. elegans as a model to study the endogenous function of an LEA protein in an animal.ResultsWe created a null mutant of C. elegans LEA-1, as well as endogenous fluorescent reporters of the protein. LEA-1 mutant animals formed defective dauer larvae at high temperature. We confirmed that C. elegans lacking LEA-1 are sensitive to desiccation. LEA-1 mutants were also sensitive to heat and osmotic stress and were prone to protein aggregation. During desiccation, LEA-1 expression increased and became more widespread throughout the body. LEA-1 was required at high levels in body wall muscle for animals to survive desiccation and osmotic stress, but expression in body wall muscle alone was not sufficient for stress resistance, indicating a likely requirement in multiple tissues. We identified minimal motifs within C. elegans LEA-1 that were sufficient to increase desiccation survival of E. coli. To test whether such motifs are central to LEA-1’s in vivo functions, we then replaced the sequence of lea-1 with these minimal motifs and found that C. elegans dauer larvae formed normally and survived osmotic stress and mild desiccation at the same levels as worms with the full-length protein.ConclusionsOur results provide insights into the endogenous functions and expression dynamics of an LEA protein in a multicellular animal. The results show that LEA-1 buffers animals from a broad range of stresses. Our identification of LEA motifs that can function in both bacteria and in a multicellular organism in vivo suggests the possibility of engineering LEA-1-derived peptides for optimized desiccation protection.

Endogenous regulation of the Akt pathway by the aryl hydrocarbon receptor (AhR) in lung fibroblasts

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to mediate toxic responses to dioxin. However, the role of the AhR in the regulation of cellular physiology has only recently been appreciated, including its ability to control cell cycle progression and apoptosis by unknown mechanisms. We hypothesized that the AhR enhances the activation of the AKT serine/threonine kinase (Akt) pathway to promote cell survival. Utilizing AhR knock-out (Ahr−/−) and wild-type (Ahr+/+) mouse lung fibroblasts (MLFs), we found that Ahr−/− MLFs have significantly higher basal Akt phosphorylation but that AhR did not affect Akt phosphorylation in MLFs exposed to growth factors or AhR ligands. Basal Akt phosphorylation was dependent on PI3K but was unaffected by changes in intracellular glutathione (GSH) or p85α. There was no significant decrease in cell viability in Ahr−/− MLFs treated with LY294002—a PI3K inhibitor—although LY294002 did attenuate MTT reduction, indicating an affect on mitochondrial function. Using a mass spectrometry (MS)-based approach, we identified several proteins that were differentially phosphorylated in the Ahr−/− MLFs compared to control cells, including proteins involved in the regulation of extracellular matrix (ECM), focal adhesion, cytoskeleton remodeling and mitochondrial function. In conclusion, Ahr ablation increased basal Akt phosphorylation in MLFs. Our results indicate that AhR may modulate the phosphorylation of a variety of novel proteins not previously identified as AhR targets, findings that help advance our understanding of the endogenous functions of AhR.

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon

BackgroundLong noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown.ResultsWe found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3ʹ-end. Consequently, the secondary structures were changed, the RNA–protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3ʹ-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs.ConclusionsOur study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study.Graphic

Revisiting Secondary Information Related to Pharmacogenetic Testing.

Incidental or secondary findings have been a major part of the discussion of genomic medicine research and clinical applications. For pharmacogenetic (PGx) testing, secondary findings arise due to the pleiotropic effects of pharmacogenes, often related to their endogenous functions. Unlike the guidelines that have been developed for whole exome or genome sequencing applications for management of secondary findings (though slightly different from PGx testing in that these refer to detection of variants in multiple genes, some with clinical significance and actionability), no corresponding guidelines have been developed for PGx clinical laboratories. Nonetheless, patient and provider education will remain key components of any PGx testing program to minimize adverse responses related to secondary findings.