Innate Immune Response to Cytoplasmic DNA: Mechanisms and Diseases.

DNA has been known to be a potent immune stimulus for more than half a century. However, the underlying molecular mechanisms of DNA-triggered immune response have remained elusive until recent years. Cyclic GMP-AMP synthase (cGAS) is a major cytoplasmic DNA sensor in various types of cells that detect either invaded foreign DNA or aberrantly located self-DNA. Upon sensing of DNA, cGAS catalyzes the formation of cyclic GMP-AMP (cGAMP), which in turn activates the ER-localized adaptor protein MITA (also named STING) to elicit the innate immune response. The cGAS-MITA axis not only plays a central role in host defense against pathogen-derived DNA but also acts as a cellular stress response pathway by sensing aberrantly located self-DNA, which is linked to the pathogenesis of various human diseases. In this review, we summarize the spatial and temporal mechanisms of host defense to cytoplasmic DNA mediated by the cGAS-MITA axis and discuss the association of malfunctions of this axis with autoimmune and other diseases.

Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium.

Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.

Cell death in animal development.

Cell death is an important facet of animal development. In some developing tissues, death is the ultimate fate of over 80% of generated cells. Although recent studies have delineated a bewildering number of cell death mechanisms, most have only been observed in pathological contexts, and only a small number drive normal development. This Primer outlines the important roles, different types and molecular players regulating developmental cell death, and discusses recent findings with which the field currently grapples. We also clarify terminology, to distinguish between developmental cell death mechanisms, for which there is evidence for evolutionary selection, and cell death that follows genetic, chemical or physical injury. Finally, we suggest how advances in understanding developmental cell death may provide insights into the molecular basis of developmental abnormalities and pathological cell death in disease.

Comparison of Light Condition-Dependent Differences in the Accumulation and Subcellular Localization of Glutathione in Arabidopsis and Wheat.

This study aimed to clarify whether the light condition-dependent changes in the redox state and subcellular distribution of glutathione were similar in the dicotyledonous model plant Arabidopsis (wild-type, ascorbate- and glutathione-deficient mutants) and the monocotyledonous crop species wheat (Chinese Spring variety). With increasing light intensity, the amount of its reduced (GSH) and oxidized (GSSG) form and the GSSG/GSH ratio increased in the leaf extracts of both species including all genotypes, while far-red light increased these parameters only in wheat except for GSH in the GSH-deficient Arabidopsis mutant. Based on the expression changes of the glutathione metabolism-related genes, light intensity influences the size and redox state of the glutathione pool at the transcriptional level in wheat but not in Arabidopsis. In line with the results in leaf extracts, a similar inducing effect of both light intensity and far-red light was found on the total glutathione content at the subcellular level in wheat. In contrast to the leaf extracts, the inducing influence of light intensity on glutathione level was only found in the cell compartments of the GSH-deficient Arabidopsis mutant, and far-red light increased it in both mutants. The observed general and genotype-specific, light-dependent changes in the accumulation and subcellular distribution of glutathione participate in adjusting the redox-dependent metabolism to the actual environmental conditions.

B subset cells in patients with chronic granulomatous disease in a Mexican population.

INTRODUCTION: Chronic granulomatous disease (CGD) is a disorder of phagocyte function, characterized by pyogenic infections and granuloma formation caused by defects in NADPH oxidase complex activity. Although the effect of CGD mainly reflects the phagocytic compartment, B cell responses are also impaired in patients with CGD. MATERIALS AND METHODS: Flow cytometric analysis was performed on peripheral blood samples from 35 CGD patients age-matched with healthy controls (HC). The target cells of our study were the naive (IgD+/CD27-), memory (IgD-/CD27+), and B1a (CD5+) cells. Immunoglobulins (Igs) were also measured. This study was performed in a Latin American cohort. RESULTS: We found significantly higher levels of naive B cells and B1a cells, but lower levels of memory B cells were found in CGD patients compared to HC. There was no significant difference of cell percentages per inheritance type. DISCUSSION: Our findings suggest that the deficiency of NADPH oxidase components can affect the differentiation of naive B cells to memory B cells. Consequently, memory cells will be low, which also influenced the expression of CD27 in memory B cells and as a result, the percentage of naive cells increases. An altered phenotype of B lymphocytes in CGD patients may contribute to the opportunistic infections and autoimmune disorders that are seen in this disease.

Investigating Subcellular Compartment Targeting Effect of Porous Coordination Cages for Enhancing Cancer Nanotherapy.

Understanding the key factors for successful subcellular compartment targeting for cargo delivery systems is of great interest in a variety of fields such as bionanotechnology, cell biology, and nanotherapies. However, the fundamental basis for intracellular transportation with these systems has thus far rarely been discussed. As a cargo vector, porous coordination cages (PCCs) have great potential for use in cancer nanotherapy and to elucidate fundamental insight regarding subcellular compartment targeting. Herein, it is shown that the transportation of PCC cargo vectors though various subcellular barriers of the mammalian cell can be manipulated by tuning the vector's electronic property and surface affinity. It is found that the PCCs become selectively aggregated at the cell membrane, the cytoplasm, or the nucleus, respectively. When a DNA topoisomerase inhibitor is delivered into the nucleus by a neutral and lipophilic PCC, the anticancer efficacy is dramatically improved. The findings shed light to tune the interactions at the "bio-nano" interface. This study provides a key strategy for future work in targeting specific cell organelles for cell imaging, cargo delivery, and therapy. This research also offers key insight into the engineering of nanoscopic materials for furnishing cell organelle-specificity.

Cytomegalovirus- and Epstein-Barr Virus-Induced T-Cell Expansions in Young Children Do Not Impair Naive T-cell Populations or Vaccination Responses: The Generation R Study.

BACKGROUND: Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) induce effector memory T-cell expansions, which are variable and potentially depend on the age at primary exposure and coinfections. We evaluated the T-cell compartment and herpesvirus infections in 6-year-old children. METHODS: T-cell subsets and immunoglobulin G seropositivity for CMV, EBV, herpes-simplex virus 1, and varicella-zoster virus were studied in 1079 6-year-old children. A random subgroup of 225 children was evaluated for CMV and EBV seropositivity before 2 years of age and for vaccination responses against measles and tetanus. RESULTS: CMV and EBV infections were associated with significant expansions of CD27(-) and CD27(+) effector memory T cells, respectively. These expansions were enhanced in CMV-EBV-coinfected children and were independent of varicella-zoster virus or herpes-simplex virus 1 coinfection. Naive and central memory T-cell numbers were not affected, nor were anti-tetanus and anti-measles immunoglobulin G levels. Children infected before 2 years of age showed smaller effector memory T-cell expansions than those infected between 2 and 6 years of age. CONCLUSIONS: CMV- and EBV-related T-cell expansions do not impair naive T-cell numbers or maintenance of protective responses against nonrelated pathogens. Duration of infection was not directly related to larger expansions of effector memory T cells in children, suggesting that other mechanisms affect these expansions at later age.

Defective B-cell proliferation and maintenance of long-term memory in patients with chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is a primary immune deficiency characterized by a defect in reactive oxygen species production. Although the effect of CGD mainly reflects on the phagocytic compartment, B-cell responses are also impaired in patients with CGD. OBJECTIVE: We sought to investigate how defective gp91(phox) expression in patients with CGD and CGD carriers might affect the B-cell compartment and maintenance of long-term memory. METHODS: We studied the B-cell compartment of patients with CGD in terms of phenotype and ability to produce reactive oxygen species and proliferate on stimuli differently directed to the B-cell receptor and Toll-like receptor 9. We further studied their capacity to maintain long-term memory by measuring cellular and serologic responses to measles. RESULTS: We show that the memory B-cell compartment is impaired among patients with CGD, as indicated by reduced total (CD19(+)CD27(+)) and resting (CD19(+)CD27(+)CD21(+)) memory B cells in parallel to increased naive (CD19(+)CD27(-)IgD(+)) B-cell frequencies. Data on CGD carriers reveal that such alterations are related to gp91(phox) expression. Moreover, proliferative capabilities of B cells on selective in vitro stimulation of B-cell receptor or Toll-like receptor 9 pathways were reduced in patients with CGD compared with those seen in age-matched healthy control subjects. Significantly lower measles-specific antibody levels and antibody-secreting cell numbers were also observed, indicating a poor ability to maintain long-term memory in these patients. CONCLUSION: Altogether, our data suggest that patients with CGD present a defective B-cell compartment in terms of frequencies of memory B cells, response to in vitro stimulation, and maintenance of long-term antigen-specific memory.

Malignant transformation of colonic epithelial cells by a colon-derived long noncoding RNA.

Recent progress has been made in the identification of protein-coding genes and miRNAs that are expressed in and alter the behavior of colonic epithelia. However, the role of long non-coding RNAs (lncRNAs) in colonic homeostasis is just beginning to be explored. By gene expression profiling of post-mitotic, differentiated tops and proliferative, progenitor-compartment bottoms of microdissected adult mouse colonic crypts, we identified several lncRNAs more highly expressed in crypt bottoms. One identified lncRNA, designated non-coding Nras functional RNA (ncNRFR), resides within the Nras locus but appears to be independent of the Nras coding transcript. Stable overexpression of ncNRFR in non-transformed, conditionally immortalized mouse colonocytes results in malignant transformation, as determined by growth in soft agar and formation of highly invasive tumors in nude mice. Moreover, ncNRFR appears to inhibit the function of the tumor suppressor let-7. These results suggest precise regulation of ncNRFR is necessary for proper cell growth in the colonic crypt, and its misregulation results in neoplastic transformation.

Real-time monitoring of subcellular states with genetically encoded redox biosensor system (RBS) in yeast cell factories.

During industrial fermentation, microbial cell factories are usually confronted with environmental or metabolic stresses, leading to the imbalance of intracellular redox and the reduction of cell metabolic capacity. Here, we constructed the genetically encoded redox biosensor system (RBS) based on redox-sensitive fluorescent proteins to detect redox metabolites, including reactive oxygen species (ROS), oxidized glutathione, NADH, and NADPH in Saccharomyces cerevisiae. The functional biosensors were quantitatively characterized and the orthogonal redox biosensor system (oRBS) was designed for detecting multiple redox metabolites. Furthermore, the compartment targeted redox biosensor system (ctRBS) was constructed to detect ROS and NADPH, revealing the distribution and spatiotemporal dynamics of ROS in yeast under various stress conditions. As a proof-of-concept, RBS was applied to evaluate the redox states of engineered yeast with stress resistance and heterogenous triterpene synthesis in vivo, elucidating the redox balance significantly affecting the growth and production phenotypes. The RBS in this study allowed the exploration of the diversity of compartmental redox state and real-time monitoring of the production process of yeast, providing a reliable and effective approach for accurate and in-depth profiling of bottlenecks of yeast cell factories.

Inhibition of ERO1a and IDO1 improves dendritic cell infiltration into pancreatic ductal adenocarcinoma.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal and treatment resistant cancers. Due to its desmoplastic and hypoxic nature along with an abundance of myeloid cell infiltration and scarce T cell infiltration, PDAC is considered a cold tumor. Methods: Here we sought to investigate myeloid cell infiltration and composition in PDAC spheroids by targeting the hypoxia-associated pathways endoplasmic reticulum oxidoreductase 1 alpha (ERO1a) and indoleamine 2,3-dioxygenase 1 (IDO1). Using MiaPaCa2 spheroids with hypoxic core, we assessed the roles of ERO1a and IDO1 inhibition in modulating monocyte infiltration and differentiation, followed by characterizing immunomodulatory factors secreted using LC-MS/MS. Results: Inhibition of ERO1a and IDO1 significantly improved monocyte infiltration and differentiation into dendritic cells. LC-MS/MS analysis of the PDAC spheroid secretome identified downregulation of hypoxia and PDAC pathways, and upregulation of antigen presentation pathways upon inhibition of ERO1a and IDO1. Furthermore, immunomodulatory factors involved in immune infiltration and migration including interleukin-8, lymphocyte cytosolic protein 1, and transgelin-2, were upregulated upon inhibition of ERO1a and IDO1. Discussion: Collectively, our results show that inhibition of ERO1a and IDO1 modulates the tumor microenvironment associated with improved monocyte infiltration and differentiation into dendritic cells to potentially influence therapeutic responses in patients with PDAC.

Cell Compartment-Specific Folding of Ty1 Long Terminal Repeat Retrotransposon RNA Genome.

The structural transitions RNAs undergo during trafficking are not well understood. Here, we used the well-developed yeast Ty1 retrotransposon to provide the first structural model of genome (g) RNA in the nucleus from a retrovirus-like transposon. Through a detailed comparison of nuclear Ty1 gRNA structure with those established in the cytoplasm, virus-like particles (VLPs), and those synthesized in vitro, we detected Ty1 gRNA structural alterations that occur during retrotransposition. Full-length Ty1 gRNA serves as the mRNA for Gag and Gag-Pol proteins and as the genome that is reverse transcribed within VLPs. We show that about 60% of base pairs predicted for the nuclear Ty1 gRNA appear in the cytoplasm, and active translation does not account for such structural differences. Most of the shared base pairs are represented by short-range interactions, whereas the long-distance pairings seem unique for each compartment. Highly structured motifs tend to be preserved after nuclear export of Ty1 gRNA. In addition, our study highlights the important role of Ty1 Gag in mediating critical RNA-RNA interactions required for retrotransposition.

Localization of muscarinic acetylcholine receptor 2 to the intestinal crypt stem cell compartment.

The data presented in this article are related to the research article entitled "Distribution of muscarinic acetylcholine receptor subtypes in the murine small intestine" (E.D. Muise, N. Gandotra, J.J. Tackett, M.C. Bamdad, R.A. Cowles, 2016) [1]. We recently demonstrated that neuronal serotonin stimulates intestinal crypt cell division, and induces villus growth and crypt depth (E.R. Gross, M.D. Gershon, K.G. Margolis, Z.V. Gertsberg, Z. Li, R.A. Cowles, 2012; M.D. Gershon, 2013) [2], [3]. Scopolamine, a nonspecific muscarinic receptor antagonist, inhibited serotonin-induced intestinal mucosal growth [2]. Here we provide data regarding the localization of muscarinic acetylcholine receptor 2 to the intestinal crypt stem cell compartment.

Distribution of muscarinic acetylcholine receptor subtypes in the murine small intestine.

AIMS: Serotonin stimulates enterocyte turnover in the small intestine and studies suggest this is mediated by neuronal signaling via a cholinergic pathway. Distribution of the five known muscarinic receptor subtypes (mAChRs) in the small intestine has not been fully studied, and their role in intestinal growth is unknown. We hypothesized that mAChRs have distinct anatomic distributions within the bowel, and that mAChRs present within intestinal crypts mediate the effects of acetylcholine on the small intestinal mucosa. MAIN METHODS: Small intestine from male C57BL/6 mice ages 2, 4, 6, and 8weeks were harvested. RNA was isolated and cDNA synthesized for PCR-amplification of subtype specific mAChRs. Ileum was fixed with Nakane, embedded in epon, and immunofluorescence microscopy performed using polyclonal antibodies specific to each mAChR1-5. KEY FINDINGS: All five mAChR subtypes were present in the mouse duodenum, jejunum, and ileum at all ages by RT-PCR. Immunofluorescence microscopy suggested the presence of mAChR1-5 in association with mature enterocytes along the villus and within the myenteric plexus. Only mAChR2 clearly localized to the crypt stem cell compartment, specifically co-localizing with Paneth cells at crypt bases. SIGNIFICANCE: Muscarinic receptors are widely distributed along the entire alimentary tract. mAChR2 appears to localize to the crypt stem cell compartment, suggesting it is a plausible regulator of stem cell activity. The location of mAChR2 to the crypt makes it a potential therapeutic target for treatment of intestinal disease such as short bowel syndrome. The exact cellular location and action of each mAChR requires further study.

High-grade dysplasia, restricted to the basal cell layer involving the entire esophagus.

This report presents a case involving a unique observation of a high-grade squamous dysplasia involving the entire esophagus. Dysplastic cells were located exclusively in the basal portion of the esophageal squamous epithelium. The findings were documented using histologic analysis of the step-biopsies from the entire esophagus, histologic examination of the esophagectomy-specimen, immunohistochemical analysis, and molecular pathologic analysis of the p53 gene. A minimally invasive total esophagectomy was performed at the Department of Surgery of the University of Cologne, and histologic analysis of the resection specimen confirmed extensive high-grade dysplasia involving the oral resection margin, but no invasive carcinoma. This case does not fit the current World Health Organization (WHO) definition of high-grade squamous cell dysplasia, which requires full-thickness involvement of the squamous epithelium. Thus, the WHO criteria should probably be reconsidered in order to allow for a diagnosis of high-grade dysplasia in cases where dysplastic cells are exclusively located in the basal layer of the esophageal squamous epithelium.

Developmental and hypoxia-induced cell death share common ultrastructural and biochemical apoptotic features in the central nervous system.

Hypoxic insults during the perinatal period lead to motor and cognitive impairments that later appear during childhood. In the adult brain, hypoxic events often lead to necrotic neuronal death, depending on the region and intensity of the event. During development an active apoptotic cell death occurs and could be an important variable affecting the hypoxic insult outcome. In the present work we performed a comparative study, in a chick embryo model, of the phenotypes and molecular markers exhibited during developmental and hypoxic cell death (HxCD). Ultrastructural analysis of optic tectum cells of embryos subjected to hypoxia (8% O2, 60 min) revealed a clear apoptotic morphology that did not differ from the one exhibited during developmental cell death. Integrity of plasma membrane, condensation of chromatin in round well-defined bodies, and gradual shrinkage of the cell are all hallmarks of the apoptotic process and were present in both control and hypoxic cells. To elucidate if hypoxic and developmental cell deaths share a common mechanism we evaluated the activation of both intrinsic and extrinsic apoptotic pathways. A basal cleavage of caspase-9 and cytochrome c release was observed by co-immunofluorescence in control embryos, but hypoxic insult significantly increased the incidence of this colocalization. Caspase-8 cleavage remained unchanged after the hypoxic insult, suggesting that the extrinsic pathway would not be involved in hypoxic death. We also observed a significant decrease of Akt activation immediately after hypoxia, possibly facilitating the later release of cytochrome c. In addition we analyzed the influence of retinal ganglion cells (RGC) in neuronal survival. Transection of RGC fibers at embryonic day (ED) 3 did not induce any change in developmental and HxCD at ED12. In conclusion, our findings demonstrate that a hypoxic insult in the developing brain triggers the same apoptotic pathway as the active developmental death.

Biosíntesis de alcaloides bencilisoquinolínicos / Benzylisoquinoline alkaloid biosynthesis / Biossíntese de alcalóides benzilisoquinolinas

Los alcaloides bencilisoquinolínicos (ABI) son metabolitos especializados con una distribución filogenética antigua pero conservadatodavía en clados modernos. Varios de ellos, como la morfina, sanguinerina y berberina tienen importancia en la medicina moderna. Enesta revisión se analizan los aspectos más sobresalientes del estado actual de la biosíntesis de ABI. Se han realizado estudios que hanpermitido conocer la biosíntesis de 22 de estos metabolitos nitrogenados. En su formación participan 43 enzimas agrupadas en oxidoreductasas,transferasas y liasas, que en algunos casos representan ejemplos atípicos de la forma en la que se originó la diversificación delmetabolismo secundario, entre ellos proteínas citocromo P450 (CYP450) con actividades catalíticas para la ruta de los ABI, o la enzimanorcoclaurina sintasa (NCS) que esta emparentada con proteínas alergénicas de defensa. Así mismo, hay avances genéticos en los quese ha podido caracterizar 30 enzimas, permitiendo conocer procesos de regulación. Otro aspecto interesante es la compartimentaciónde los sitios de biosíntesis y acumulación de ABI ya que en varios casos están separados espacialmente y en distintas especies o en lamisma pueden participar varios tipos de células. Ello ha sugerido el transporte intra e intercelular de los alcaloides, los precursores yde las enzimas, se ha documentado el transporte de berberina entre el citoplasma y las vacuolas del almacenamiento. El panorama de labiosíntesis de ABI se ha construido con los estudios de ejemplares de importancia farmacológica...

The benzylisoquinoline alkaloids (BIA) are specialized metabolites with an ancient phylogeneticdistribution, but still preserved in modern clades. Some of them, such as morphine, sanguinerine or berberine, are important for modernmedicine. This review discusses the highlights of the current state of the biosynthesis of BIA. There have been studies that show thebiosynthesis of 22 of these nitrogenous metabolites. In their formation there are 43 enzymes grouped into oxidoreductases, transferasesand lyases, which in some cases represent atypical examples of the manner in which the secondary metabolism diversification wasoriginated. Two of these examples are the cytochrome proteins P450 (P450), with catalytic activities for ABI route, or the norcoclaurinesynthase enzyme (NCS), which share substantial identity with defense allergenic proteins. Likewise, there are genetic advances thathave produced the characterization of 30 enzymes, allowing knowledge of regulatory processes. Another interesting aspect is thecompartmentation of the biosynthesis sites and accumulation of BIA, since in several cases they are spatially separated and in differentspecies, or in the same species several types of cells may be involved. This has suggested intra and intercellular transport of alkaloids,precursors and enzymes, and it has been documented berberine transport between the cytoplasm and the vacuoles of storage. The picturefor the biosynthesis of BIA has been constructed with exemplary studies of alkaloids with pharmacological importance...

Os alcalóides benzilisoquinolinas (ABI) são metabólitos especializados com umadistribuição filogenética antiga, mas ainda preservada em clados modernos. Vários deles, como a morfina, sanguinarina e berberina sãoimportantes na medicina moderna. Neste artigo, se analisam os aspectos mais destacados do estado atual da biossíntese de ABI; há estudosque tem permitido conhecer a biossíntese de 22 desses metabólitos nitrogenados. Na sua síntese participam 43 enzimas agrupadas emoxidoreductases, transferases, liases e, em alguns casos, representam exemplos atípicos da forma pela qual se originou a diversificaçãodo metabolismo secundário, incluindo as proteínas do citocromo P450 (CYP450), com atividades catalíticas para a rota dos ABI, ou aenzima norcoclaurina sintase (NCS), que está relacionada com proteínas alergênicas de defesa. Da mesma forma, há avanços genéticosna caracterização de 30 enzimas, permitindo conhecer processos de regulação. Outro aspecto interessante é a compartimentalização dossítios de biossíntese e acumulação de ABI uma vez que em muitos casos estão separados espacialmente e em diferentes espécies, ou namesma podem participar vários tipos de células. Isto há sugerido o transporte intra e intercelular de alcalóides, precursores das enzimas;tem sido documentado o transporte de berberina entre o citoplasma e os vacúolos de armazenamento. A perspectiva na biossíntese deABI foi construída com os estudos de exemplares de importância farmacológica...