Molecular compartmentalization in a syncytium: restricted mobility of proteins within the sea urchin skeletogenic mesenchyme.

Multinucleated cells, or syncytia, are found in diverse taxa. Their biological function is often associated with the compartmentalization of biochemical or cellular activities within the syncytium. How such compartments are generated and maintained is poorly understood. The sea urchin embryonic skeleton is secreted by a syncytium, and local patterns of skeletal growth are associated with distinct sub-domains of gene expression within the syncytium. For such molecular compartments to be maintained and to control local patterns of skeletal growth: (1) the mobility of TFs must be restricted to produce stable differences in the transcriptional states of nuclei within the syncytium; and (2) the mobility of biomineralization proteins must also be restricted to produce regional differences in skeletal growth. To test these predictions, we expressed fluorescently tagged forms of transcription factors and biomineralization proteins in sub-domains of the skeletogenic syncytium. We found that both classes of proteins have restricted mobility within the syncytium and identified motifs that limit their mobility. Our findings have general implications for understanding the functional and molecular compartmentalization of syncytia.

The lipase cofactor CGI58 controls placental lipolysis.

In eutherians, the placenta plays a critical role in the uptake, storage, and metabolism of lipids. These processes govern the availability of fatty acids to the developing fetus, where inadequate supply has been associated with substandard fetal growth. Whereas lipid droplets are essential for the storage of neutral lipids in the placenta and many other tissues, the processes that regulate placental lipid droplet lipolysis remain largely unknown. To assess the role of triglyceride lipases and their cofactors in determining placental lipid droplet and lipid accumulation, we assessed the role of patatin like phospholipase domain containing 2 (PNPLA2) and comparative gene identification-58 (CGI58) in lipid droplet dynamics in the human and mouse placenta. While both proteins are expressed in the placenta, the absence of CGI58, not PNPLA2, markedly increased placental lipid and lipid droplet accumulation. These changes were reversed upon restoration of CGI58 levels selectively in the CGI58-deficient mouse placenta. Using co-immunoprecipitation, we found that, in addition to PNPLA2, PNPLA9 interacts with CGI58. PNPLA9 was dispensable for lipolysis in the mouse placenta yet contributed to lipolysis in human placental trophoblasts. Our findings establish a crucial role for CGI58 in placental lipid droplet dynamics and, by extension, in nutrient supply to the developing fetus.

Low-Dose Lipopolysaccharide Protects from Lethal Paramyxovirus Infection in a Macrophage- and TLR4-Dependent Process.

Respiratory diseases are a major public health burden and a leading cause of death and disability in the world. Understanding antiviral immune responses is crucial to alleviate morbidity and mortality associated with these respiratory viral infections. Previous data from human and animal studies suggested that pre-existing atopy may provide some protection against severe disease from a respiratory viral infection. However, the mechanism(s) of protection is not understood. Low-dose LPS has been shown to drive an atopic phenotype in mice. In addition, LPS has been shown in vitro to have an antiviral effect. We examined the effect of LPS treatment on mortality to the murine parainfluenza virus Sendai virus. Low-dose LPS treatment 24 h before inoculation with a normally lethal dose of Sendai virus greatly reduced death. This protection was associated with a reduced viral titer and reduced inflammatory cytokine production in the airways. The administration of LPS was associated with a marked increase in lung neutrophils and macrophages. Depletion of neutrophils failed to reverse the protective effect of LPS; however, depletion of macrophages reversed the protective effect of LPS. Further, we demonstrate that the protective effect of LPS depends on type I IFN and TLR4-MyD88 signaling. Together, these studies demonstrate pretreatment with low-dose LPS provides a survival advantage against a severe respiratory viral infection through a macrophage-, TLR4-, and MyD88-dependent pathway.

Mouse Model of Sendai Virus-Induced Lung Disease.

Sendai virus (SeV), also known as the murine parainfluenza virus 1, is an enveloped negative-sense RNA paramyxovirus from the family Paramyxoviridae and genus Respirovirus. The virus was named after Sendai, city in Japan, where it was first isolated (Kuroya, Ishida, Yokohama Med Bull 4:217-233, 1953). Antigenically, SeV is closely related to human parainfluenza viruses 1 and 3. SeV is pneumotropic and naturally infects the respiratory tract of rodents. At the proper inoculum (2 × 105 pfu), SeV causes infection that is limited to the airway mucosa and inflammation mainly restricted to bronchiolar tissues as seen in asthma pathogenesis models using C57BL/6 wild-type mice (Walter et al, J Clin Invest 110:165-175, 2002). We utilize SeV to explore the mechanism(s) by which a respiratory viral infection translates into postviral airway disease in mice. This chapter primarily describes the protocols we use to infect mice in vivo, assay viral replication, and assess outcomes in the lungs of the host.

Lessons from a transcription factor: Alx1 provides insights into gene regulatory networks, cellular reprogramming, and cell type evolution.

The skeleton-forming cells of sea urchins and other echinoderms have been studied by developmental biologists as models of cell specification and morphogenesis for many decades. The gene regulatory network (GRN) deployed in the embryonic skeletogenic cells of euechinoid sea urchins is one of the best understood in any developing animal. Recent comparative studies have leveraged the information contained in this GRN, bringing renewed attention to the diverse patterns of skeletogenesis within the phylum and the evolutionary basis for this diversity. The homeodomain-containing transcription factor, Alx1, was originally shown to be a core component of the skeletogenic GRN of the sea urchin embryo. Alx1 has since been found to be key regulator of skeletal cell identity throughout the phylum. As such, Alx1 is currently serving as a lens through which multiple developmental processes are being investigated. These include not only GRN organization and evolution, but also cell reprogramming, cell type evolution, and the gene regulatory control of morphogenesis. This review summarizes our current state of knowledge concerning Alx1 and highlights the insights it is yielding into these important developmental and evolutionary processes.

Atopic Neutrophils Prevent Postviral Airway Disease.

Respiratory syncytial virus (RSV) infection in infancy is associated with increased risk of asthma, except in those with allergic disease at the time of infection. Using house dust mite allergen, we examined the effect of pre-existing atopy on postviral airway disease using Sendai virus in mice, which models RSV infection in humans. Sendai virus drives postviral airway disease in nonatopic mice; however, pre-existing atopy protected against the development of airway disease. This protection depended upon neutrophils, as depletion of neutrophils at the time of infection restored the susceptibility of atopic mice to postviral airway disease. Associated with development of atopy was an increase in polymorphonuclear neutrophil-dendritic cell hybrid cells that develop in Th2 conditions and demonstrated increased viral uptake. Systemic inhibition of IL-4 reversed atopic protection against postviral airway disease, suggesting that increased virus uptake by neutrophils was IL-4 dependent. Finally, human neutrophils from atopic donors were able to reduce RSV infection of human airway epithelial cells in vitro, suggesting these findings could apply to the human. Collectively our data support the idea that pre-existing atopy derives a protective neutrophil response via potential interaction with IL-4, preventing development of postviral airway disease.

Global patterns of enhancer activity during sea urchin embryogenesis assessed by eRNA profiling.

We used capped analysis of gene expression with sequencing (CAGE-seq) to profile eRNA expression and enhancer activity during embryogenesis of a model echinoderm: the sea urchin, Strongylocentrotus purpuratus We identified more than 18,000 enhancers that were active in mature oocytes and developing embryos and documented a burst of enhancer activation during cleavage and early blastula stages. We found that a large fraction (73.8%) of all enhancers active during the first 48 h of embryogenesis were hyperaccessible no later than the 128-cell stage and possibly even earlier. Most enhancers were located near gene bodies, and temporal patterns of eRNA expression tended to parallel those of nearby genes. Furthermore, enhancers near lineage-specific genes contained signatures of inputs from developmental gene regulatory networks deployed in those lineages. A large fraction (60%) of sea urchin enhancers previously shown to be active in transgenic reporter assays was associated with eRNA expression. Moreover, a large fraction (50%) of a representative subset of enhancers identified by eRNA profiling drove tissue-specific gene expression in isolation when tested by reporter assays. Our findings provide an atlas of developmental enhancers in a model sea urchin and support the utility of eRNA profiling as a tool for enhancer discovery and regulatory biology. The data generated in this study are available at Echinobase, the public database of information related to echinoderm genomics.

Analysis of the DNA-binding properties of Alx1, an evolutionarily conserved regulator of skeletogenesis in echinoderms.

Alx1, a homeodomain-containing transcription factor, is a highly conserved regulator of skeletogenesis in echinoderms. In sea urchins, Alx1 plays a central role in the differentiation of embryonic primary mesenchyme cells (PMCs) and positively regulates the transcription of most biomineralization genes expressed by these cells. The alx1 gene arose via duplication and acquired a skeletogenic function distinct from its paralog (alx4) through the exonization of a 41-amino acid motif (the D2 domain). Alx1 and Alx4 contain glutamine-50 paired-type homeodomains, which interact preferentially with palindromic binding sites in vitro. Chromatin immunoprecipitation sequencing (ChIP-seq) studies have shown, however, that Alx1 binds both to palindromic and half sites in vivo. To address this apparent discrepancy and explore the function of the D2 domain, we used an endogenous cis-regulatory module associated with Sp-mtmmpb, a gene that encodes a PMC-specific metalloprotease, to analyze the DNA-binding properties of Alx1. We find that Alx1 forms dimeric complexes on TAAT-containing half sites by a mechanism distinct from the well-known mechanism of dimerization on palindromic sites. We used transgenic reporter assays to analyze the functional roles of half sites in vivo and demonstrate that two sites with partially redundant functions are essential for the PMC-specific activity of the Sp-mtmmpb cis-regulatory module. Finally, we show that the D2 domain influences the DNA-binding properties of Alx1 in vitro, suggesting that the exonization of this motif may have facilitated the acquisition of new transcriptional targets and consequently a novel developmental function.

PLA2G6 guards placental trophoblasts against ferroptotic injury.

The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown. We found that spontaneous preterm birth is associated with ferroptosis and that inhibition of GPX4 causes ferroptotic injury in primary human trophoblasts and during mouse pregnancy. Importantly, we uncovered a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolize Hp-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia/reoxygenation injury in vivo. Together, we identified ferroptosis signaling in the human and mouse placenta, established a role for PLA2G6 in attenuating trophoblastic ferroptosis, and provided mechanistic insights into the ill-defined placental lipotoxicity that may inspire PLA2G6-targeted therapeutic strategies.

Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis.

Alx1 is a conserved regulator of skeletogenesis in echinoderms and evolutionary changes in Alx1 sequence and expression have played a pivotal role in modifying programs of skeletogenesis within the phylum. Alx1 regulates a large suite of effector genes that control the morphogenetic behaviors and biomineral-forming activities of skeletogenic cells. To better understand the gene regulatory control of skeletogenesis by Alx1, we used genome-wide ChIP-seq to identify Alx1-binding sites and direct gene targets. Our analysis revealed that many terminal differentiation genes receive direct transcriptional inputs from Alx1. In addition, we found that intermediate transcription factors previously shown to be downstream of Alx1 all receive direct inputs from Alx1. Thus, Alx1 appears to regulate effector genes by indirect, as well as direct, mechanisms. We tested 23 high-confidence ChIP-seq peaks using GFP reporters and identified 18 active cis-regulatory modules (CRMs); this represents a high success rate for CRM discovery. Detailed analysis of a representative CRM confirmed that a conserved, palindromic Alx1-binding site was essential for expression. Our work significantly advances our understanding of the gene regulatory circuitry that controls skeletogenesis in sea urchins and provides a framework for evolutionary studies.

Intestinal Microbiota Disruption Reduces Regulatory T Cells and Increases Respiratory Viral Infection Mortality Through Increased IFNγ Production.

Alterations in gastrointestinal microbiota indirectly modulate the risk of atopic disease, but effects on respiratory viral infections are less clear. Using the murine paramyxoviral virus type 1, Sendai virus (SeV), we examined the effect of altering gastrointestinal microbiota on the pulmonary antiviral immune response. C57BL6 mice were treated with streptomycin before or during infection with SeV and resulting immune response studied. Ingestion of the non-absorbable antibiotic streptomycin led to a marked reduction in intestinal microbial diversity without a significant effect on lung microbiota. Reduction in diversity in the gastrointestinal tract was followed by greatly increased mortality to respiratory viral infection (p < 0.0001). This increase in mortality was associated with a dysregulated immune response characterized by decreased lung (p = 0.01) and intestinal (p = 0.03) regulatory T cells (Tregs), and increased lung IFNγ (p = 0.049), IL-6 (p = 0.015), and CCL2 (p = 0.037). Adoptive transfer of Treg cells or neutralization of IFNγ prevented increased mortality. Furthermore, Lin-CD4+ cells appeared to be a potential source of the increased IFNγ. Together, these results demonstrate gastrointestinal microbiota modulate immune responses at distant mucosal sites and have the ability to significantly impact mortality in response to a respiratory viral infection.

Cysteinyl leukotriene receptor 1 expression identifies a subset of neutrophils during the antiviral response that contributes to postviral atopic airway disease.

BACKGROUND: Viral respiratory tract infections increase the risk of development and exacerbation of atopic disease. Previously, we demonstrated the requirement for a neutrophil (PMN) subset expressing CD49d to drive development of postviral atopic airway disease in mice. OBJECTIVE: We sought to determine whether human CD49d+ PMNs are present in the nasal mucosa during acute viral respiratory tract infections and further characterize this PMN subset in human subjects and mice. METHODS: Sixty subjects (5-50 years old) were enrolled within 4 days of acute onset of upper respiratory symptoms. Nasal lavage for flow cytometry and nasal swabs for viral PCR were performed at enrollment and during convalescence. The Sendai virus mouse model was used to investigate the phenotype and functional relevance of CD49d+ PMNs. RESULTS: CD49d+ PMN frequency was significantly higher in nasal lavage fluid during acute respiratory symptoms in all subjects (2.9% vs 1.0%, n = 42, P < .001). In mice CD49d+ PMNs represented a "proatopic" neutrophil subset that expressed cysteinyl leukotriene receptor 1 (CysLTR1) and produced TNF, CCL2, and CCL5. Inhibition of CysLTR1 signaling in the first days of a viral respiratory tract infection was sufficient to reduce accumulation of CD49d+ PMNs in the lungs and development of postviral atopic airway disease. Similar to the mouse, human CD49d+ PMNs isolated from nasal lavage fluid during a viral respiratory tract infection expressed CysLTR1. CONCLUSION: CD49d and CysLTR1-coexpressing PMNs are present during symptoms of an acute viral respiratory tract infection in human subjects. Further study is needed to examine selective targeting of proatopic neutrophils as a potential therapeutic strategy to prevent development of postviral atopic airway disease.

Is dexmedetomidine more effective than remifentanil for neurologic outcomes in patients undergoing CEA surgery using regional anesthesia?

Carotid endarterectomy (CEA) surgery is performed using regional anesthesia and intravenous/sedative drugs, such as dexmedetomidine and remifentanil. It is unclear which drug provides the least amount of hemodynamic variability, sedation, and respiratory depression so cognitive function can be continuously monitored intraoperatively. A search of the literature was conducted to identify the evidence of the effects of dexmedetomidine and remifentanil in patients undergoing awake CEA surgery with regional anesthesia. A literature search was conducted using Google Scholar, Cumulative Index to Nursing and Allied Health Literature, PubMed, MEDLINE, and the Cochrane Systematic Review databases. Four randomized control studies and a retrospective study were critically appraised to evaluate the evidence on the effectiveness of dexmedetomidine compared with remifentanil during CEA surgery, using regional anesthesia and dexmedetomidine or remifentanil from 2004 to 2009. These studies found that dexmedetomidine provides adequate sedation with less respiratory depression than remifentanil when used in adjunct with regional anesthesia, allowing the provider to monitor hemodynamic stability and neurologic status continuously during the intraoperative period. Dexmedetomidine was evaluated as the primary agent of choice for sedation when performing an awake CEA with regional anesthesia.

Copy number variation analysis in 98 individuals with PHACE syndrome.

PHACE syndrome is the association of large segmental facial hemangiomas and congenital anomalies, such as posterior fossa malformations, cerebral arterial anomalies, coarctation of the aorta, eye anomalies, and sternal defects. To date, the reported cases of PHACE syndrome have been sporadic, suggesting that PHACE may have a complex pathogenesis. We report here genomic copy number variation (CNV) analysis of 98 individuals with PHACE syndrome as a first step in deciphering a potential genetic basis of PHACE syndrome. A total of 3,772 CNVs (2,507 duplications and 1,265 deletions) were detected in 98 individuals with PHACE syndrome. CNVs were then eliminated if they failed to meet established criteria for quality, spanned centromeres, or did not contain genes. CNVs were defined as "rare" if not documented in the database of genomic variants. Ten rare CNVs were discovered (size range: 134-406 kb), located at 1q32.1, 1q43, 3q26.32-3q26.33, 3p11.1, 7q33, 10q24.32, 12q24.13, 17q11.2, 18p11.31, and Xq28. There were no rare CNV events that occurred in more than one subject. Therefore, further study is needed to determine the significance of these CNVs in the pathogenesis of PHACE syndrome.

A general path for large-scale solubilization of cellular proteins: from membrane receptors to multiprotein complexes.

Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential "druggable" targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format.

Co-localization of DNA repair proteins with UV-induced DNA damage in locally irradiated cells.

This chapter describes a technique in which indirect immunofluorescence is applied to visualize the process of nucleotide excision repair (NER) at the site of locally induced damage in DNA. UV-irradiation of cells through an isopore polycarbonate membrane filter generates cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PP) on a subnuclear area, which corresponds to the size of a pore on the membrane. Specific antibodies to CPD and 6-4PP define the damaged spot. The NER components co-localize at the damaged-DNA subnuclear spot, where the proteins are stained with the appropriate fluorescent antibodies. This relatively simple and affordable method facilitates the examination of the sequential assembly of NER proteins in the chromatin-embedded DNA photoproducts. The method also enhances the identification of repair auxiliary proteins and complexes, such as ubiquitin E3 ligases, involved in the initiation of NER on non-transcribed DNA.

HIV-1 Vpr loads uracil DNA glycosylase-2 onto DCAF1, a substrate recognition subunit of a cullin 4A-ring E3 ubiquitin ligase for proteasome-dependent degradation.

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4(DCAF1)). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1- and CUL4-associated factor 1 (DCAF1). Here we show that recombinant UNG2 specifically interacts with Vpr, but not with Vpx of simian immunodeficiency virus, forming a heterotrimeric complex with DCAF1 and Vpr in vitro as well as in vivo. Using reconstituted CRL4(DCAF1) and CRL4(DCAF1-Vpr) E3 ubiquitin ligases in vitro reveals that UNG2 ubiquitination (ubiquitylation) is facilitated by Vpr. Co-expression of DCAF1 and Vpr causes down-regulation of UNG2 in a proteasome-dependent manner, with Vpr mutants that are defective in UNG2 or DCAF1 binding abrogating this effect. Taken together, our results show that the CRL4(DCAF1) E3 ubiquitin ligase can be subverted by Vpr to target UNG2 for degradation.

A common CNR1 (cannabinoid receptor 1) haplotype attenuates the decrease in HDL cholesterol that typically accompanies weight gain.

We have previously shown that genetic variability in CNR1 is associated with low HDL dyslipidemia in a multigenerational obesity study cohort of Northern European descent (209 families, median  = 10 individuals per pedigree). In order to assess the impact of CNR1 variability on the development of dyslipidemia in the community, we genotyped this locus in all subjects with class III obesity (body mass index >40 kg/m(2)) participating in a population-based biobank of similar ancestry. Twenty-two haplotype tagging SNPs, capturing the entire CNR1 gene locus plus 15 kb upstream and 5 kb downstream, were genotyped and tested for association with clinical lipid data. This biobank contains data from 645 morbidly obese study subjects. In these subjects, a common CNR1 haplotype (H3, frequency 21.1%) is associated with fasting TG and HDL cholesterol levels (p = 0.031 for logTG; p = 0.038 for HDL-C; p = 0.00376 for log[TG/HDL-C]). The strength of this relationship increases when the data are adjusted for age, gender, body mass index, diet and physical activity. Mean TG levels were 160±70, 155±70, and 120±60 mg/dL for subjects with 0, 1, and 2 copies of the H3 haplotype. Mean HDL-C levels were 45±10, 47±10, and 48±9 mg/dL, respectively. The H3 CNR1 haplotype appears to exert a protective effect against development of obesity-related dyslipidemia.

The cullin 4B-based UV-damaged DNA-binding protein ligase binds to UV-damaged chromatin and ubiquitinates histone H2A.

By removing UV-induced lesions from DNA, the nucleotide excision repair (NER) pathway preserves the integrity of the genome. The UV-damaged DNA-binding (UV-DDB) protein complex is involved in the recognition of chromatin-embedded UV-damaged DNA, which is the least understood step of NER. UV-DDB consists of DDB1 and DDB2, and it is a component of the cullin 4A (CUL4A)-based ubiquitin ligase, DDB1-CUL4A(DDB2). We previously showed that DDB1-CUL4A(DDB2) ubiquitinates histone H2A at the sites of UV lesions in a DDB2-dependent manner. Mutations in DDB2 cause a cancer prone syndrome, xeroderma pigmentosum group E (XP-E). CUL4A and its paralog, cullin 4B (CUL4B), copurify with the UV-DDB complex, but it is unclear whether CUL4B has a role in NER as a separate E3 ubiquitin ligase. Here, we present evidence that CUL4A and CUL4B form two individual E3 ligases, DDB1-CUL4A(DDB2) and DDB1-CUL4B(DDB2). To investigate CUL4B's possible role in NER, we examined its subcellular localization in unirradiated and irradiated cells. CUL4B colocalizes with DDB2 at UV-damaged DNA sites. Furthermore, CUL4B binds to UV-damaged chromatin as a part of the DDB1-CUL4B(DDB2) E3 ligase in the presence of functional DDB2. In contrast to CUL4A, CUL4B is localized in the nucleus and facilitates the transfer of DDB1 into the nucleus independently of DDB2. Importantly, DDB1-CUL4B(DDB2) is more efficient than DDB1-CUL4A(DDB2) in monoubiquitinating histone H2A in vitro. Overall, this study suggests that DDB1-CUL4B(DDB2) E3 ligase may have a distinctive function in modifying the chromatin structure at the site of UV lesions to promote efficient NER.