Cell-specific Activity Research Articles

Uptake and Lysosomal Delivery of Recombinant Human Alpha‐N‐Acetylglucosamine‐6‐Sulfatase to Mucopolysaccharidosis IIID Fibroblasts

Sanfilippo syndrome (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal storage disorder of childhood. There is no cure or effective treatment available. The fundamental cause of MPS III is an inherited mutation in one of the 4 enzymes required to catabolize heparan sulfate (HS), a glycosaminoglycan which plays important structural and functional roles in the brain and elsewhere. We now propose to develop an enzyme replacement treatment (ERT) for MPS IIID that will ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease. The symptoms of MPS IIID are largely localized to the brain, therefore, our strategy proposes to deliver recombinant human alpha‐N‐acetylglucosamine‐6‐sulfatase (rhGNS) intrathecally to effectively treat the underlying causes of the neurologic symptoms that dominate MPS III pathology. We have purified ~100 ug rhGNS from 1500 mL media of CHO cells (specific activity >10,000 nmol/hr/mg), demonstrated maximal enzymatic activity at pH 5.6 (low activity at neutral pH), which is closer to lysosomal pH, demonstrated good enzymatic activity at 37°C and showed it was stable for over one month at 4°C in artificial cerebrospinal fluid storage buffer. Additionally we have demonstrated intracellular enzymatic activity of rhGNS in MPS IIID fibroblasts when rhGNS is added to the media, shown it colocalizes with lysosomal markers using confocal microscopy, and confirmed radiolabelled HS is diminished (33–65%) to WT levels in MPS IIID fibroblasts treated with rhGNS. These results demonstrate scale‐up is feasible in preparation for proof of concept studies in the MPS IIID knock‐out mouse. Our ultimate goal is bring this ERT to the clinic which will likely have a transformational effect on patients and families.Support or Funding InformationThis work was funded by Grant R41 NS089061‐01 from NIH NINDS

Sulfur Isotopic Fractionation of Carbonyl Sulfide during Degradation by Soil Bacteria.

We performed laboratory incubation experiments on the degradation of gaseous phase carbonyl sulfide (OCS) by soil bacteria to determine its sulfur isotopic fractionation constants ((34)ε). Incubation experiments were conducted using strains belonging to the genera Mycobacterium, Williamsia, and Cupriavidus isolated from natural soil environments. The (34)ε values determined were -3.67 ± 0.33‰, -3.99 ± 0.19‰, -3.57 ± 0.22‰, and -3.56 ± 0.23‰ for Mycobacterium spp. strains THI401, THI402, THI404, and THI405; -3.74 ± 0.29‰ for Williamsia sp. strain THI410; and -2.09 ± 0.07‰ and -2.38 ± 0.35‰ for Cupriavidus spp. strains THI414 and THI415. Although OCS degradation rates divided by cell numbers (cell-specific activity) were different among strains of the same genus, the (34)ε values for same genus showed no significant differences. Even though the numbers of bacterial species examined were limited, our results suggest that (34)ε values for OCS bacterial degradation depend not on cell-specific activities, but on genus-level biological differences, suggesting that (34)ε values are dependent on enzymatic and/or membrane properties. Taking our (34)ε values as representative for bacterial OCS degradation, the expected atmospheric changes in δ(34)S values of OCS range from 0.5‰ to 0.9‰, based on previously reported decreases in OCS concentrations at Mt. Fuji, Japan. Consequently, tropospheric observation of δ(34)S values for OCS coupled with (34)ε values for OCS bacterial degradation can potentially be used to investigate soil as an OCS sink.

P16Ink4a-induced senescence of pancreatic beta cells enhances insulin secretion

Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16(Ink4a) is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of p16(Ink4a) in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16(Ink4a) in beta cells induces hallmarks of senescence--including cell enlargement, and greater glucose uptake and mitochondrial activity--which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16(Ink4a) activity. We found that islets from human adults contain p16(Ink4a)-expressing senescent beta cells and that senescence induced by p16(Ink4a) in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16(Ink4a) and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs.

The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity.

Single-cell activity of freshwater aerobic anoxygenic phototrophic bacteria and their contribution to biomass production.

Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that despite their low abundances have been hypothesized to play an ecologically and biogeochemically important role in aquatic systems. Characterizing this role requires a better understanding of the in situ dynamics and activity of AAP bacteria. Here we provide the first assessment of the single-cell activity of freshwater AAP bacteria and their contribution to total bacterial production across lakes spanning a wide trophic gradient, and explore the role of light in regulating AAP activity. The proportion of cells that were active in leucine incorporation and the level of activity per cell were consistently higher for AAP than for bulk bacteria across lakes. As a result, AAP bacteria contributed disproportionately more to total bacterial production than to total bacterial abundance. Interestingly, although environmentally driven patterns in activity did not seem to differ largely between AAP and bulk bacteria, their response to light did, and exposure to light resulted in increases in the proportion of active AAP bacteria with no clear effect on their cell-specific activity. This suggests that light may play a role in the activation of AAP bacteria, enabling these photoheterotrophs to contribute more to the carbon cycle than suggested by their abundance.

Abstract LB-B02: Acquisition of stem cell-like properties accompanying with Src family kinase/Focal adhesion kinase activation contributes to acquired resistance to afatinib in lung cancer cells

Abstract [Background] Most NSCLC patients harboring activating EGFR mutations benefit from treatment with EGFR-TKIs, but the final clinical efficacy of EGFR-TKIs varies because of development of tumor resistant to EGFR-TKIs. Multiple kinase-targeted 2nd generation TKIs such as afatinib have been developed to overcome drug resistance to the 1st generation TKIs. Afatinib is an irreversible multitargeted TKI targeting EGFR including T790M, HER2 and HER4. To develop further personalized therapeutics and drug resistance modifiers, we should understand the molecular based mechanism of drug resistance to 2nd as well as 3rd generation TKIs. In our present study, we present a novel finding that acquisition of cancer stem-like cells properties accompanying with activation of residual Src family kinase (SFK)/focal adhesion kinase (FAK) is responsible for the survival of afatinib-resistant lung cancer cells when expression of targeted EGFR, HER2 and HER4 is abraded. [Materials and methods] We have established afatinib-resistant subclones from human lung cancer cell lines, HCC827, harboring activating EGFR mutation after stepwise exposure to afatinib. The afatinib-resistant clones do not harbor T790M, K-ras mutation or PTEN loss. We have characterized biochemical properties of drug-resistant subclones as compared with their drug-sensitive counterparts by Western blot and RT-PCR. ALDEFLUOR kit was used to isolate cell populations with the stem cell specific ALDH enzymatic activity. [Results] We characterized two independent afatinib-resistant subclones, BR1-8 and BR2-3. [1] Afatinib-resistant subclones showed markedly decreased expression of EGFR, HER2, HER3, c-Met and PDGFRβ as compared with their parental cell lines, HCC827; [2] Akt phosphorylation in afatinib-resistant subclones was not suppressed by afatinib; [3] SFK and FAK were activated in resistant subclones, and combination of afatinib with c-Src siRNA or with dasatinib moderately suppressed cell growth and AKT phosphorylation; [4] Combination of afatinib with siFAK moderately blocked cell growth of resistant subclones. [5] Cancer stem like cell marker, ALDH1, positive cell population increased in afatinib-resistant sublines compared with HCC827 cell lines. [Conclusions] Together, our present study presents that increase of cancer stem like cells and SFK/FAK activation could be a mechanism responsible for acquired resistance to afatinib. We would further discuss the association of ALDH1 expression and SFK/FAK activation with afatinib resistance in lung cancer. Citation Format: Yuichi Murakami, Kahori Sonoda, Koichi Azuma, Kosuke Watari, Michihiko Kuwano, Mayumi Ono. Acquisition of stem cell-like properties accompanying with Src family kinase/Focal adhesion kinase activation contributes to acquired resistance to afatinib in lung cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B02.

The emerging role of coagulation proteases in kidney disease.

A role of coagulation proteases in kidney disease beyond their function in normal haemostasis and thrombosis has long been suspected, and studies performed in the past 15 years have provided novel insights into the mechanisms involved. The expression of protease-activated receptors (PARs) in renal cells provides a molecular link between coagulation proteases and renal cell function and revitalizes research evaluating the role of haemostasis regulators in renal disease. Renal cell-specific expression and activity of coagulation proteases, their regulators and their receptors are dynamically altered during disease processes. Furthermore, renal inflammation and tissue remodelling are not only associated, but are causally linked with altered coagulation activation and protease-dependent signalling. Intriguingly, coagulation proteases signal through more than one receptor or induce formation of receptor complexes in a cell-specific manner, emphasizing context specificity. Understanding these cell-specific signalosomes and their regulation in kidney disease is crucial to unravelling the pathophysiological relevance of coagulation regulators in renal disease. In addition, the clinical availability of small molecule targeted anticoagulants as well as the development of PAR antagonists increases the need for in-depth knowledge of the mechanisms through which coagulation proteases might regulate renal physiology.

Abstract 18138: Endothelial PHD2 Loss Causes Pulmonary Arterial Hypertension via HIF-2 (Best of Basic Science Abstract)

Background: Key mediators of cellular adaption to hypoxia are hypoxia-inducible factor (HIF)-1 and -2, which are regulated by the prolyl hydroxylases (PHDs). Although the HIF pathway has been shown to be involved in the pathogenesis of pulmonary arterial hypertension (PAH), little is known about the cell type-specific functions of the PHD/HIF axis in this context. Here, we used a genetic approach to specifically dissect the contribution of the endothelial PHD/HIF axis in PAH. Methods: Endothelial cell specific HIF activation was achieved by crossing Vecadherin (Cdh5)-Cre transgenics to Phd2 floxed mice (ePHD2), while the contribution of each HIF isoform was assessed by generating double mutants lacking PHD2 and HIF-2 (ePHD2HIF2) or PHD2 and HIF-1 (PHD2HIF1). Right ventricular systolic pressure (RVSP) was directly measured via insertion of a 1.4F Mikro-tip catheter transducer into a surgically exposed right internal jugular vein. Results: Mice deficient for endothelial PHD2 showed significant activation of HIF-signaling as shown by immunoblot analysis of lung tissue for HIF-1 and HIF-2. These mice developed spontaneous PAH (ePHD2: 54.3±6.9 vs Cre-: 24.8±2.2 mmHg, n=5-6, P=0.005) which was associated with right ventricular hypertrophy (RVH) (Fulton Index: 0.52 in ePHD2 vs 0.28 in Cre-, P=0.0004) and increased mortality. The PAH phenotype was maintained in ePHD2HIF1 mutants but was reversed in ePHD2HIF2 mutants. To assess the contribution of endothelial HIF-2 in hypoxia induced PAH, endothelial HIF-2 single mutants or Cre- littermates were exposed to normobaric hypoxia (10% O2) for 4 weeks. In contrast to controls, eHIF2 mutants were completely protected from development of PAH and RVH. Bone marrow transplantation studies showed no contribution from hematopoietic HIF-2 in hypoxia induced PAH. Conclusions: Our data establish that endothelial PHD2 loss results in PAH through a HIF-2 dependent mechanism. Our findings identify the PHD2/HIF2 axis as a potential target for PAH therapies.

GLI2 cell-specific activity is controlled at the level of transcription and RNA processing: Consequences to cancer metastasis

High activity of GLI family zinc finger protein 2 (GLI2) promotes tumor progression. Removal of the repressor domain at the N terminus (GLI2∆N) by recombinant methods converts GLI2 into a powerful transcriptional activator. However, molecular mechanisms leading to the formation of GLI2∆N activator proteins have not been established. Herein we report for the first time that the functional activities of GLI2 are parted into different protein isoforms by alternative promoter usage, selection of alternative splicing, transcription initiation and termination sites. Functional studies using melanoma cells revealed that transcriptional regulation of GLI2 is TGFbeta-dependent and supports the predominant production of GLI2∆N and C-terminally truncated GLI2 (GLI2∆C) isoforms in cells with high migratory and invasive phenotype. Taken together, these results highlight the role of transcription and RNA processing as major processes in the regulation of GLI2 activity with severe impacts in cancer development.

Abundance and diversity of ammonia-oxidizing archaea and bacteria on granular activated carbon and their fates during drinking water purification process.

Ammonia is a precursor to trichloramine, which causes an undesirable chlorinous odor. Granular activated carbon (GAC) filtration is used to biologically oxidize ammonia during drinking water purification; however, little information is available regarding the abundance and diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) associated with GAC. In addition, their sources and fates in water purification process remain unknown. In this study, six GAC samples were collected from five full-scale drinking water purification plants in Tokyo during summer and winter, and the abundance and community structure of AOA and AOB associated with GAC were studied in these two seasons. In summer, archaeal and bacterial amoA genes on GACs were present at 3.7 × 10(5)-3.9 × 10(8) gene copies/g-dry and 4.5 × 10(6)-4.2 × 10(8) gene copies/g-dry, respectively. In winter, archaeal amoA genes remained at the same level, while bacterial amoA genes decreased significantly for all GACs. No differences were observed in the community diversity of AOA and AOB from summer to winter. Phylogenetic analysis revealed high AOA diversity in group I.1a and group I.1b in raw water. Terminal-restriction fragment length polymorphism analysis of processed water samples revealed that AOA diversity decreased dramatically to only two OTUs in group I.1a after ozonation, which were identical to those detected on GAC. It suggests that ozonation plays an important role in determining AOA diversity on GAC. Further study on the cell-specific activity of AOA and AOB is necessary to understand their contributions to in situ nitrification performance.

Bacterial abundance, processes and diversity responses to acidification at a coastal CO2 vent.

Shallow CO2 vents are used as natural laboratories to study biological responses to ocean acidification, and so it is important to determine whether pH is the primary driver of bacterial processes and community composition, or whether other variables associated with vent water have a significant influence. Water from a CO2 vent (46 m, Bay of Plenty, New Zealand) was compared to reference water from an upstream control site, and also to control water acidified to the same pH as the vent water. After 84 h, both vent and acidified water exhibited higher potential bulk water and cell-specific glucosidase activity relative to control water, whereas cell-specific protease activities were similar. However, bulk vent water glucosidase activity was double that of the acidified water, as was bacterial secondary production in one experiment, suggesting that pH was not the only factor affecting carbohydrate hydrolysis. In addition, there were significant differences in bacterial community composition in the vent water relative to the control and acidified water after 84 h, including the presence of extremophiles which may influence carbohydrate degradation. This highlights the importance of characterizing microbial processes and community composition in CO2 vent emissions, to confirm that they represent robust analogues for the future acidified ocean.

The histone variant H2A.Z is an important regulator of enhancer activity.

Gene regulatory programs in different cell types are largely defined through cell-specific enhancers activity. The histone variant H2A.Z has been shown to play important roles in transcription mainly by controlling proximal promoters, but its effect on enhancer functions remains unclear. Here, we demonstrate by genome-wide approaches that H2A.Z is present at a subset of active enhancers bound by the estrogen receptor alpha (ERα). We also determine that H2A.Z does not influence the local nucleosome positioning around ERα enhancers using ChIP sequencing at nucleosomal resolution and unsupervised pattern discovery. We further highlight that H2A.Z-enriched enhancers are associated with chromatin accessibility, H3K122ac enrichment and hypomethylated DNA. Moreover, upon estrogen stimulation, the enhancers occupied by H2A.Z produce enhancer RNAs (eRNAs), and recruit RNA polymerase II as well as RAD21, a member of the cohesin complex involved in chromatin interactions between enhancers and promoters. Importantly, their recruitment and eRNAs production are abolished by H2A.Z depletion, thereby revealing a novel functional link between H2A.Z occupancy and enhancer activity. Taken together, our findings suggest that H2A.Z acts as an important player for enhancer functions by establishing and maintaining a chromatin environment required for RNA polymerase II recruitment, eRNAs transcription and enhancer-promoters interactions, all essential attributes of enhancer activity.

Winter Aconite (Eranthis hyemalis) Lectin as a cytotoxic effector in the lifecycle of Caenorhabditis elegans.

The lectin found in the tubers of the Winter Aconite (Eranthis hyemalis) plant is an N-acetyl-D-galactosamine specific Type II Ribosome Inactivating Protein (RIP); Type II RIPs have shown anti-cancer properties, and hence have potential as therapeutic agents. Here we present a modified protocol for the extraction and purification of the E. hyemalis lectin (EHL) using affinity chromatography. De novo amino acid sequencing of EHL confirms its classification as a Type II Ribosome Inactivating Protein. The biocidal properties of EHL have been investigated against the nematode Caenorhabditis elegans. Arrested first stage larvae treated with EHL have shown some direct mortality, with surviving larvae subsequently showing a range of phenotypes including food avoidance, reduced fecundity, developmental delay and constitutive dauer larvae formation. Both inappropriate dauer larvae development and failure to locate to bacterial food source are consistent with the disruption of chemosensory function and the ablation of amphid neurons. Further investigation indicates that mutations that disrupt normal amphid formation can block the EHL-induced dauer larvae formation. In combination, these phenotypes indicate that EHL is cytotoxic and suggest a cell specific activity against the amphid neurons of C. elegans.

T Lymphocyte-Specific Activation of Nrf2 Protects from AKI.

T lymphocytes are established mediators of ischemia reperfusion (IR)-induced AKI, but traditional immune principles do not explain their mechanism of early action in the absence of alloantigen. Nrf2 is a transcription factor that is crucial for cytoprotective gene expression and is generally thought to have a key role in dampening IR-induced AKI through protective effects on epithelial cells. We proposed an alternative hypothesis that augmentation of Nrf2 in T cells is essential to mitigate oxidative stress during IR-induced AKI. We therefore generated mice with genetically amplified levels of Nrf2 specifically in T cells and examined the effect on antioxidant gene expression, T cell activation, cytokine production, and IR-induced AKI. T cell-specific augmentation of Nrf2 significantly increased baseline antioxidant gene expression. These mice had a high frequency of intrarenal CD25(+)Foxp3(+) regulatory T cells and decreased frequencies of CD11b(+)CD11c(+) and F4/80(+) cells. Intracellular levels of TNF-α, IFN-γ, and IL-17 were significantly lower in CD4(+) T cells with high Nrf2 expression. Mice with increased T cell expression of Nrf2 were significantly protected from functional and histologic consequences of AKI. Furthermore, adoptive transfer of high-Nrf2 T cells protected wild-type mice from IR injury and significantly improved their survival. These data demonstrate that T cell-specific activation of Nrf2 protects from IR-induced AKI, revealing a novel mechanism of tissue protection during acute injury responses.

Using six-colour flow cytometry to analyse the activation and interaction of platelets and leukocytes – A new assay suitable for bench and bedside conditions

IntroductionPlatelets are main effector cells in haemostasis and also promote inflammation. Platelet-leukocyte complexes are key mediators in a variety of thromboinflammatory disorders and consecutive organ failure. Cell-specific epitopes and activation markers on platelets and leukocytes can be measured using flow cytometry. However, until recently a major restriction has been a paucity in antibody combinations and lack of detection strategies. We aimed to develop a six-colour flow cytometry method which depicts multiple aspects of platelet and leukocyte interactions in human whole blood. Materials and MethodsPlatelets, including microparticles and aggregates, were detected in flow cytometry using a platelet-specific anti-CD41-FITC antibody and size-defined regions. The morphology of platelet-leukocyte complexes (including granulocyte and monocyte content) were depicted using anti-CD45-PerCP, anti-CD66b-PE-Cy7, and anti-CD14-APC antibodies in a single sample. Expression of platelet and leukocyte activation markers P-selectin and CD11b were detected using anti-CD62P-PE and anti-CD11b-BV421 antibodies, respectively. ResultsThe sensitivity of this assay to detect the effects of various agonists (TRAP-6, ADP, collagen, epinephrine, TNF-α and LPS) is demonstrated. Furthermore, the assay is shown to detect platelet and leukocyte activation induced by extracorporeal circulation in vitro. The suitability of this assay for bedside analysis is demonstrated exemplarily in a patient treated with mechanical circulatory life support. ConclusionsUsing the concurrent assessment of multiple parameters, this method gives detailed insights into the complexity and dynamics of platelet-leukocyte interactions. This assay carries the potential to increase our understanding of the mechanisms and pathophysiology of platelet-leukocyte interaction in the research laboratory and clinical setting.

Unexpected stimulation of soil methane uptake as emergent property of agricultural soils following bio-based residue application.

Intensification of agriculture to meet the global food, feed, and bioenergy demand entail increasing re-investment of carbon compounds (residues) into agro-systems to prevent decline of soil quality and fertility. However, agricultural intensification decreases soil methane uptake, reducing, and even causing the loss of the methane sink function. In contrast to wetland agricultural soils (rice paddies), the methanotrophic potential in well-aerated agricultural soils have received little attention, presumably due to the anticipated low or negligible methane uptake capacity in these soils. Consequently, a detailed study verifying or refuting this assumption is still lacking. Exemplifying a typical agricultural practice, we determined the impact of bio-based residue application on soil methane flux, and determined the methanotrophic potential, including a qualitative (diagnostic microarray) and quantitative (group-specific qPCR assays) analysis of the methanotrophic community after residue amendments over 2months. Unexpectedly, after amendments with specific residues, we detected a significant transient stimulation of methane uptake confirmed by both the methane flux measurements and methane oxidation assay. This stimulation was apparently a result of induced cell-specific activity, rather than growth of the methanotroph population. Although transient, the heightened methane uptake offsets up to 16% of total gaseous CO2 emitted during the incubation. The methanotrophic community, predominantly comprised of Methylosinus may facilitate methane oxidation in the agricultural soils. While agricultural soils are generally regarded as a net methane source or a relatively weak methane sink, our results show that methane oxidation rate can be stimulated, leading to higher soil methane uptake. Hence, even if agriculture exerts an adverse impact on soil methane uptake, implementing carefully designed management strategies (e.g. repeated application of specific residues) may compensate for the loss of the methane sink function following land-use change.

Enhanced particle fluxes and heterotrophic bacterial activities in Gulf of Mexico bottom waters following storm-induced sediment resuspension

Bottom nepheloid layers (BNLs) in the deep sea transport and remobilize considerable amounts of particulate matter, enhancing microbial cycling of organic matter in cold, deep water environments. We measured bacterial abundance, bacterial protein production, and activities of hydrolytic enzymes within and above a BNL that formed in the deep Mississippi Canyon, northern Gulf of Mexico, shortly after Hurricane Isaac had passed over the study area in late August 2012. The BNL was detected via beam attenuation in CTD casts over an area of at least 3.5km2, extending up to 200m above the seafloor at a water depth of ~1500m. A large fraction of the suspended matter in the BNL consisted of resuspended sediments, as indicated by high levels of lithogenic material collected in near-bottom sediment traps shortly before the start of our sampling campaign. Observations of suspended particle abundance and sizes throughout the water column, using a combined camera-CTD system (marine snow camera, MSC), revealed the presence of macroaggregates (>1mm in diameter) within the BNL, indicating resuspension of canyon sediments. A distinct bacterial response to enhanced particle concentrations within the BNL was evident from the observation that the highest enzymatic activities (peptidase, β-glucosidase) and protein production (3H-leucine incorporation) were found within the most particle rich sections of the BNL. To investigate the effects of enhanced particle concentrations on bacterial activities in deep BNLs more directly, we conducted laboratory experiments with roller bottles filled with bottom water and amended with experimentally resuspended sediments from the study area. Macroaggregates formed within 1 day from resuspended sediments; by day 4 of the incubation bacterial cell numbers in treatments with resuspended sediments were more than twice as high as in those lacking sediment suspensions. Cell-specific enzymatic activities were also generally higher in the sediment-amended compared to the unamended treatments. The broader range and higher activities of polysaccharide hydrolases in the presence of resuspended sediments compared to the unamended water reflected enzymatic capabilities typical for benthic bacteria. Our data suggest that the formation of BNLs in the deep Gulf of Mexico can lead to transport of sedimentary organic matter into bottom waters, stimulating bacterial food web interactions. Such storm-induced resuspension may represent a possible mechanism for the redistribution of sedimented oil-fallout from the Deepwater Horizon spill in 2010.

TAF10 Interacts with the GATA1 Transcription Factor and Controls Mouse Erythropoiesis.

The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cells in vivo, we observed a differentiation block accompanied by deregulated GATA1 target genes, including Gata1 itself, suggesting functional cross talk between GATA1 and TAF10. Additionally, we analyzed by mass spectrometry the composition of TFIID and SAGA complexes in mouse and human cells and found that their global integrity is maintained, with minor changes, during erythroid cell differentiation and development. In agreement with our functional data, we show that TAF10 interacts directly with GATA1 and that TAF10 is enriched on the GATA1 locus in human fetal erythroid cells. Thus, our findings demonstrate a cross talk between canonical TFIID and SAGA complexes and cell-specific transcription activators during development and differentiation.

Nrf2 reduces allergic asthma in mice through enhanced airway epithelial cytoprotective function.

Asthma development and pathogenesis are influenced by the interactions of airway epithelial cells and innate and adaptive immune cells in response to allergens. Oxidative stress is an important mediator of asthmatic phenotypes in these cell types. Nuclear erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that is the key regulator of the response to oxidative and environmental stress. We previously demonstrated that Nrf2-deficient mice have heightened susceptibility to asthma, including elevated oxidative stress, inflammation, mucus, and airway hyperresponsiveness (AHR) (Rangasamy T, Guo J, Mitzner WA, Roman J, Singh A, Fryer AD, Yamamoto M, Kensler TW, Tuder RM, Georas SN, Biswal S. J Exp Med 202: 47-59, 2005). Here we dissected the role of Nrf2 in lung epithelial cells and tested whether genetic or pharmacological activation of Nrf2 reduces allergic asthma in mice. Cell-specific activation of Nrf2 in club cells of the airway epithelium significantly reduced allergen-induced AHR, inflammation, mucus, Th2 cytokine secretion, oxidative stress, and airway leakiness and increased airway levels of tight junction proteins zonula occludens-1 and E-cadherin. In isolated airway epithelial cells, Nrf2 enhanced epithelial barrier function and increased localization of zonula occludens-1 to the cell surface. Pharmacological activation of Nrf2 by 2-trifluoromethyl-2'-methoxychalone during the allergen challenge was sufficient to reduce allergic inflammation and AHR. New therapeutic options are needed for asthma, and this study demonstrates that activation of Nrf2 in lung epithelial cells is a novel potential therapeutic target to reduce asthma susceptibility.

Atg7 plays a critical role in the development of iNKT cells with altered PLZF expression. (HEM8P.242)

Abstract Invariant Natural Killer T (iNKT) cell is a distinct subset of T cells that plays a regulatory role in various immune diseases. Although many studies have reported that various factors regulate development of iNKT cells, the role of autophagy in the development is still unknown. Here, we report that autophagy-related protein 7(Atg7) is required for development of iNKT cells in the thymus. iNKT cells of the thymus and liver expressed Atg7 and their development is completely attenuated in the thymus of CD4-specific Atg7 knock-out (CD4-Cre/Atg7f/f) mice compared with WT mice. The levels of PLZF expression were altered in thymocytes from CD4-Cre/Atg7f/f mice as compared with WT mice. CD4-Cre/Atg7f/f mice showed a few iNKT cells in peripheral organs, which led to functional deficient in iNKT cell-specific activation in vitro and in vivo. Accordingly, autoantibody-induced arthritis was attenuated in CD4-Cre/Atg7f/f mice. Therefore, our findings suggest that Atg7 regulates iNKT cell development with altered PLZF expression.