Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors

Th17 cells contribute to host defense on mucosal surfaces but also provoke autoimmune diseases when directed against self-antigens. Identifying therapeutic targets that regulate Th17 cell differentiation and/or cytokine production has considerable value. Here, we study the aryl hydrocarbon receptor (AhR)-dependent transcriptome in human CD4 T cells treated with Th17-inducing cytokines. We show that the AhR reciprocally regulates IL-17 and IL-22 production in human CD4 T cells. Global gene expression analysis revealed that AhR ligation decreased IL21 expression, correlating with delayed upregulation of RORC during culture with Th17-inducing cytokines. Several of the AhR-dependent genes have known roles in cellular assembly, organization, development, growth and proliferation. We further show that expression of GPR15, GPR55 and GPR68 positively correlates with IL-22 production in the presence of the AhR agonist FICZ. Activation of GPR68 with the lorazepam derivative ogerin resulted in suppression of IL-22 and IL-10 secretion by T cells, with no effect on IL-17. Under neutral Th0 conditions, ogerin and the Gq/11 receptor inhibitor YM254890 blunted IL-22 induction by FICZ. These data reveal the AhR-dependent transcriptome in human CD4 T cells and suggest the mechanism through which the AhR regulates T cell function may be partially dependent on Gq-coupled receptors including GPR68

Chemical and physical modifications of polyethylene containing nanostructures

The polyethylene is an interesting polymer with a good mechanical strength, ductility, biocompatibility and chemical inertia. It founds several applications in many fields, such as chemistry, engineering, bio-medicine and micro-electronics. The polyethylene chemical and physical properties can be modified embedding different nanostructures in its bulk. Carbon nanotubes, metallic oxides, coloured pigments and other species can be inserted at different concentrations in the polymer material during the preparation phase of thin films and sheets. Mechanical, optical, thermal and chemical properties can be modified significantly depending on the filler concentration. The absorption coefficient at different radiations can be controlled by the amount of doping structure. In the visible region, for example, the high transparency of pure polyethylene can be strongly reduced by low concentrations of carbon nanotubes. The colour, the mechanical resistance, the wet ability of the polyethylene and other parameters can be changed by the doping species in order to prepare special devices useful for many applications

Life cycle inventory data for the Italian agri-food sector: background, sources and methodological aspects

Purpose: For the development of any life cycle assessment study, the practitioner frequently integrates primary data collected on-field, with background data taken from various life cycle inventory databases which are part of most commercial LCA software packages. However, such data is often not generally applicable to all product systems since, especially concerning the agri-food sector, available datasets may not be fully representative of the site specificity of the food product under examination. In this context, the present work investigates the background, sources and methodological aspects that characterise the most known commercial databases containing agri-food data, with a focus on four agri-food supply chains (olive oil, wine, wheat products and citrus fruit), which represent an important asset for the Italian food sector. Methods: Specifically, the paper entails a review of currently available LCI databases and their datasets with a twofold scope: firstly, to understand how agri-food data is modelled in these databases for a coherent and consistent representation of regional scenarios and to verify whether they are also suitable for the Italian context and, secondly, to identify and analyse useful and relevant methodological approaches implemented in the existing LCI databases when regional data are modelled. Results: Based on the aforementioned review, it is possible to highlight some problems which may arise when developing an LCI pertaining to the four Italian agri-food supply chains, namely: 1. The need for specific inventory datasets to tackle the specificities of agri-food product systems. 2. The lack of datasets, within the existing DBs, related to the Italian context and to the abovementioned supply chains. In fact, at present, in the currently available LCI DBs, there are very few (or in some cases none) datasets related to Italian wine, olive oil, wheat-based products and citrus fruit. The few available datasets often contain some data related to the Italian context but also approximate data with that of product systems representing other countries. Furthermore, the present study allowed to identify and discuss the main aspects to be used as starting elements for modelling regional data to be included in a future Italian LCI database of the abovementioned four supply chains. Conclusions: The results of the present study represent a starting point for the collection of data and its organisation, in order to develop an Italian LCI agri-food database with datasets which are representative of the regional specificities of four agri-food supply chains which play an important role in the Italian economy

Life cycle inventory data for the Italian agri-food sector: background, sources and methodological aspects

AbstractPurposeFor the development of any life cycle assessment study, the practitioner frequently integrates primary data collected on-field, with background data taken from various life cycle inventory databases which are part of most commercial LCA software packages. However, such data is often not generally applicable to all product systems since, especially concerning the agri-food sector, available datasets may not be fully representative of the site specificity of the food product under examination. In this context, the present work investigates the background, sources and methodological aspects that characterise the most known commercial databases containing agri-food data, with a focus on four agri-food supply chains (olive oil, wine, wheat products and citrus fruit), which represent an important asset for the Italian food sector.MethodsSpecifically, the paper entails a review of currently available LCI databases and their datasets with a twofold scope: firstly, to understand how agri-food data is modelled in these databases for a coherent and consistent representation of regional scenarios and to verify whether they are also suitable for the Italian context and, secondly, to identify and analyse useful and relevant methodological approaches implemented in the existing LCI databases when regional data are modelled.ResultsBased on the aforementioned review, it is possible to highlight some problems which may arise when developing an LCI pertaining to the four Italian agri-food supply chains, namely:1. The need for specific inventory datasets to tackle the specificities of agri-food product systems.2. The lack of datasets, within the existing DBs, related to the Italian context and to the abovementioned supply chains. In fact, at present, in the currently available LCI DBs, there are very few (or in some cases none) datasets related to Italian wine, olive oil, wheat-based products and citrus fruit. The few available datasets often contain some data related to the Italian context but also approximate data with that of product systems representing other countries.Furthermore, the present study allowed to identify and discuss the main aspects to be used as starting elements for modelling regional data to be included in a future Italian LCI database of the abovementioned four supply chains.ConclusionsThe results of the present study represent a starting point for the collection of data and its organisation, in order to develop an Italian LCI agri-food database with datasets which are representative of the regional specificities of four agri-food supply chains which play an important role in the Italian economy

The quadruplex r(CGG)n destabilizing cationic porphyrin TMPyP4 cooperates with hnRNPs to increase the translation efficiency of fragile X premutation mRNA

The 5′ untranslated region of the FMR1 gene which normally includes 4–55 d(CGG) repeats expands to > 55–200 repeats in carriers of fragile X syndrome premutation. Although the levels of premutation FMR1 mRNA in carrier cells are 5–10-fold higher than normal, the amount of the product FMR protein is unchanged or reduced. We demonstrated previously that premutation r(CGG)n tracts formed quadruplex structures that impeded translation and lowered the efficiency of protein synthesis. Normal translation could be restored in vivo by the quadruplex r(CGG)n destabilizing action of CBF-A and hnRNP A2 proteins. Here we report that the quadruplex-interacting cationic porphyrin TMPyP4 by itself and in cooperation with CBF-A or hnRNP A2 also unfolded quadruplex r(CGG)n and increased the efficiency of translation of 5′-(CGG)99 containing reporter firefly (FL) mRNA. TMPyP4 destabilized in vitro a (CGG)33 intramolecular quadruplex structure and enhanced the translation of 5′-(CGG)99-FL mRNA in a rabbit reticulocyte lysate and in HEK293 cells. The efficiency of translation of (CGG)99-FL mRNA was additively increased in cells exposed to TMPyP4 together with CBF-A. Whereas low doses of TMPyP4, CBF-A or hnRNP A2 by themselves did not affect the in vivo utilization of (CGG)99-FL mRNA, introduction of TMPyP4 together with either protein synergistically augmented its translation efficiency

Translation of the FMR1 mRNA is not influenced by AGG interruptions

The fragile X mental retardation 1 (FMR1) gene contains a CGG-repeat element within its 5′ untranslated region (5′UTR) which, for alleles with more than ∼40 repeats, increasingly affects both transcription (up-regulation) and translation (inhibition) of the repeat-containing RNA with increasing CGG-repeat length. Translational inhibition is thought to be due to impaired ribosomal scanning through the CGG-repeat region, which is postulated to form highly stable secondary/tertiary structure. One striking difference between alleles in the premutation range (55–200 CGG repeats) and those in the normal range (<∼40 repeats) is the reduced number/absence of ‘expansion stabilizing’ AGG interruptions in the larger alleles. Such interruptions, which generally occur every 9–11 repeats in normal alleles, are thought to disrupt the extended CGG-repeat hairpin structure, thus facilitating translational initiation. To test this hypothesis, we have measured the translational efficiency of CGG-repeat mRNAs with 0–2 AGG interruptions, both in vitro (rabbit reticulocyte lysates) and in cell culture (HEK-293 cells). We demonstrate that the AGG interruptions have no detectable influence on translational efficiency in either a cell-free system or cell culture, indicating that any AGG-repeat-induced alterations in secondary/tertiary structure, if present, do not involve the rate-limiting step(s) in translational initiation

Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles†

5′- and 3′-untranslated regions (UTRs) are important regulators of gene expression and play key roles in disease progression and susceptibility. The 5′-UTR of the fragile X mental retardation 1 (FMR1) gene contains a CGG repeat element that is expanded (>200 CGG repeats; full mutation) and methylated in fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and known cause of autism. Significant phenotypic involvement has also emerged in some individuals with the premutation (55–200 CGG repeats), including fragile X-associated premature ovarian insufficiency (FXPOI) in females, and the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS), in older adult carriers. Here, we show that FMR1 mRNA in human and mouse brain is expressed as a combination of multiple isoforms that use alternative transcriptional start sites and different polyadenylation sites. Furthermore, we have identified a novel human transcription start site used in brain but not in lymphoblastoid cells, and have detected FMR1 isoforms generated through the use of both canonical and non-canonical polyadenylation signals. Importantly, in both human and mouse, a specific regulation of the UTRs is observed in brain of FMR1 premutation alleles, suggesting that the transcript variants may play a role in premutation-related pathologies

Testing the FMR1 Promoter for Mosaicism in DNA Methylation among CpG Sites, Strands, and Cells in FMR1-Expressing Males with Fragile X Syndrome

Variability among individuals in the severity of fragile X syndrome (FXS) is influenced by epigenetic methylation mosaicism, which may also be common in other complex disorders. The epigenetic signal of dense promoter DNA methylation is usually associated with gene silencing, as was initially reported for FMR1 alleles in individuals with FXS. A paradox arose when significant levels of FMR1 mRNA were reported for some males with FXS who had been reported to have predominately methylated alleles. We have used hairpin-bisufite PCR, validated with molecular batch-stamps and barcodes, to collect and assess double-stranded DNA methylation patterns from these previously studied males. These patterns enable us to distinguish among three possible forms of methylation mosaicism, any one of which could explain FMR1 expression in these males. Our data indicate that cryptic inter-cell mosaicism in DNA methylation can account for the presence of FMR1 mRNA in some individuals with FXS

Bone Marrow Osteoblast Damage by Chemotherapeutic Agents

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-β1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-β1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche

Whole genome sequence analysis of the TALLYHO/Jng mouse

Background: The TALLYHO/Jng (TH) mouse is a polygenic model for obesity and type 2 diabetes first described in the literature in 2001. The origin of the TH strain is an outbred colony of the Theiler Original strain and mice derived from this source were selectively bred for male hyperglycemia establishing an inbred strain at The Jackson Laboratory. TH mice manifest many of the disease phenotypes observed in human obesity and type 2 diabetes. Results: We sequenced the whole genome of TH mice maintained at Marshall University to a depth of approximately 64.8X coverage using data from three next generation sequencing runs. Genome-wide, we found approximately 4.31 million homozygous single nucleotide polymorphisms (SNPs) and 1.10 million homozygous small insertions and deletions (indels) of which 98,899 SNPs and 163,720 indels were unique to the TH strain compared to 28 previously sequenced inbred mouse strains. In order to identify potentially clinically-relevant genes, we intersected our list of SNP and indel variants with human orthologous genes in which variants were associated in GWAS studies with obesity, diabetes, and metabolic syndrome, and with genes previously shown to confer a monogenic obesity phenotype in humans, and found several candidate variants that could be functionally tested using TH mice. Further, we filtered our list of variants to those occurring in an obesity quantitative trait locus, tabw2, identified in TH mice and found a missense polymorphism in the Cidec gene and characterized this variant’s effect on protein function. Conclusions: We generated a complete catalog of variants in TH mice using the data from whole genome sequencing. Our findings will facilitate the identification of causal variants that underlie metabolic diseases in TH mice and will enable identification of candidate susceptibility genes for complex human obesity and type 2 diabetes