Examining the efficiency of porcine gastric mucin-coated magnetic beads in extraction of noroviruses from frozen berries.

Human norovirus is the leading cause of foodborne gastroenteritis worldwide. Due to the low infectious dose of noroviruses, sensitive methodologies are required to detect and characterize small numbers of viral particles that are found in contaminated foods. The ISO 15216 method, which is internationally recognized for detection of foodborne viruses from high-risk food commodities, is based on viral precipitation, followed by RNA extraction and identification of the viral genome by RT-PCR. Although the ISO 15216 method is efficient, it is time consuming and tedious, does not report on the viral infectivity, and is sensitive to the presence of RT-PCR inhibitors. Norovirus capture by the porcine gastric mucin conjugated magnetic beads (PGM-MB) was developed as an alternative virus recovery method. It relies on the integrity of the viral capsid being able to bind to PGM. PGM contains a variety of histo-blood group antigens (HBGAs) that act as norovirus receptors. Therefore, the PGM-MB method allows for extraction of noroviruses, with potentially intact viral capsids, from complex food matrices. The viral genome can then be released through heat-shock of the captured virus. For this reason, we performed a parallel comparison between the ISO 15216 method and the PGM-MB method in isolation and quantification of noroviruses from frozen raspberries. We have demonstrated that the efficiency of the PGM-MB method in extraction of murine norovirus (MNV) and human norovirus GII.4 from raspberries is equal or better than the ISO 15216 method, while the PGM-MB has fewer steps and shorter turnaround time. Moreover, the PGM-MB method is more efficient in removing the inhibitors prior to RT-PCR analysis.

Integrative single-nucleus multi-omics analysis prioritizes candidate cis and trans regulatory networks and their target genes in Alzheimer's disease brains.

BACKGROUND: The genetic underpinnings of late-onset Alzheimer's disease (LOAD) are yet to be fully elucidated. Although numerous LOAD-associated loci have been discovered, the causal variants and their target genes remain largely unknown. Since the brain is composed of heterogenous cell subtypes, it is imperative to study the brain on a cell subtype specific level to explore the biological processes underlying LOAD. METHODS: Here, we present the largest parallel single-nucleus (sn) multi-omics study to simultaneously profile gene expression (snRNA-seq) and chromatin accessibility (snATAC-seq) to date, using nuclei from 12 normal and 12 LOAD brains. We identified cell subtype clusters based on gene expression and chromatin accessibility profiles and characterized cell subtype-specific LOAD-associated differentially expressed genes (DEGs), differentially accessible peaks (DAPs) and cis co-accessibility networks (CCANs). RESULTS: Integrative analysis defined disease-relevant CCANs in multiple cell subtypes and discovered LOAD-associated cell subtype-specific candidate cis regulatory elements (cCREs), their candidate target genes, and trans-interacting transcription factors (TFs), some of which, including ELK1, JUN, and SMAD4 in excitatory neurons, were also LOAD-DEGs. Finally, we focused on a subset of cell subtype-specific CCANs that overlap known LOAD-GWAS regions and catalogued putative functional SNPs changing the affinities of TF motifs within LOAD-cCREs linked to LOAD-DEGs, including APOE and MYO1E in a specific subtype of microglia and BIN1 in a subpopulation of oligodendrocytes. CONCLUSIONS: To our knowledge, this study represents the most comprehensive systematic interrogation to date of regulatory networks and the impact of genetic variants on gene dysregulation in LOAD at a cell subtype resolution. Our findings reveal crosstalk between epigenetic, genomic, and transcriptomic determinants of LOAD pathogenesis and define catalogues of candidate genes, cCREs, and variants involved in LOAD genetic etiology and the cell subtypes in which they act to exert their pathogenic effects. Overall, these results suggest that cell subtype-specific cis-trans interactions between regulatory elements and TFs, and the genes dysregulated by these networks contribute to the development of LOAD.

The APOE-TOMM40 Humanized Mouse Model: Characterization of Age, Sex, and PolyT Variant Effects on Gene Expression.

BACKGROUND: The human chromosome 19q13.32 is a gene rich region and has been associated with multiple phenotypes, including late onset Alzheimer's disease (LOAD) and other age-related conditions. OBJECTIVE: Here we developed the first humanized mouse model that contains the entire TOMM40 and APOE genes with all intronic and intergenic sequences including the upstream and downstream regions. Thus, the mouse model carries the human TOMM40 and APOE genes and their intact regulatory sequences. METHODS: We generated the APOE-TOMM40 humanized mouse model in which the entire mouse region was replaced with the human (h)APOE-TOMM40 loci including their upstream and downstream flanking regulatory sequences using recombineering technologies. We then measured the expression of the human TOMM40 and APOE genes in the mice brain, liver, and spleen tissues using TaqMan based mRNA expression assays. RESULTS: We investigated the effects of the '523' polyT genotype (S/S or VL/VL), sex, and age on the human TOMM40- and APOE-mRNAs expression levels using our new humanized mouse model. The analysis revealed tissue specific and shared effects of the '523' polyT genotype, sex, and age on the regulation of the human TOMM40 and APOE genes. Noteworthy, the regulatory effect of the '523' polyT genotype was observed for all studied organs. CONCLUSION: The model offers new opportunities for basic science, translational, and preclinical drug discovery studies focused on the APOE genomic region in relation to LOAD and other conditions in adulthood.

Results of endovascular treatment of venous anastomotic stenosis in prosthetic arteriovenous for hemodialysis PTFE grafts. Comparative analysis between patent and occluded grafts.

OBJECTIVE: To analyze the results of endovascular treatment of venous anastomotic stenosis (VAS) in humero-axillary arteriovenous grafts (HAG), comparing outcomes between patent and thrombosed HAG. MATERIAL AND METHODS: A retrospective cohort study was made of endovascular treated patients because of a VAS in a HAG between January 2009 and December 2019. Group A: Thrombosed HAG secondary to a VAS. Group B: Patent HAG with a VAS detected during follow-up. Technical success was defined as residual stenosis after treatment <30%, and clinical success as satisfactory immediate dialysis after surgery. After ET a biannual clinical and ultrasound follow-up was performed. STATISTICAL ANALYSIS: Survival analysis was performed for time-to-event data to assess patency. RESULTS: Group A: 55 patients. Group B: 22. There were no significative differences in demographic and anatomical factors between groups. Technical and clinical success were 100% in group B and 94.5% and 91% respectively in group A. Primary patency at 1, 6 and 12 months was: Group A: 81.8%, 22.4% y 15.7% respectively. Group B: 100%, 85.9%, 76,4% (p < 0.001). Secondary patency at 1, 6 and 12 months was: Group A: 85.2%, 45.8% y 31.3% respectively. Group B 100%, 95.3%, 95.2% (p < 0.001). Use of non-covered stents was associated with an increased risk of occlusion (HR 2.669 IC 95% 1.146-6.216, p = 0.010). CONCLUSION: A higher patency of EV performed on a patent HAG is expected. It is therefore advisable to develop surveillance programs that are capable to detect VAS before its occlusion.

Resultados del tratamiento endovascular de la estenosis de la anastomosis venosa en las fístulas arteriovenosas protésicas para hemodiálisis de PTFE. Análisis comparativo entre fístulas permeables y trombosadas / Results of endovascular treatment of venous anastomotic stenosis in prosthetic arteriovenous for hemodialysis PTFE grafts. Comparative analysis between patent and occluded grafts

Objetivo: Analizar los resultados del tratamiento endovascular (TEV) de las estenosis en las anastomosis venosas (EAV) de las fístulas arteriovenosas protésicas (FAVp), comparando su utilidad al realizarse sobre FAVp permeables frente a trombosadas.Material y métodosEstudio retrospectivo de pacientes intervenidos mediante TEV por EAV de fístulas humeroaxilares realizadas entre enero de 2009 y diciembre de 2019 en nuestro centro. Grupo A: FAVp trombosada secundaria a EAV; Grupo B: FAVp permeable con EAV detectada en seguimiento. Se definió éxito técnico como estenosis residual ≤ 30% y éxito clínico como diálisis efectiva inmediata. Tras el TEV se realizó un seguimiento clínico y con eco-Doppler semestral. Estudio estadístico: se realizó un análisis de supervivencia mediante el método Kaplan Meier para el estudio de permeabilidades.ResultadosGrupo A: 55 pacientes. Grupo B: 22 pacientes. No existieron diferencias significativas en las características demográficas ni anatómicas entre grupos.El éxito técnico y clínico fueron del 100% en el grupo B frente a un 94,5% y 91%, respectivamente, en el grupo A.La permeabilidad primaria a 1, 6 y 12 meses en el Grupo A fue: 81,8%, 22,4% y 15,7%, respectivamente, frente al Grupo B: 100%, 85,9%, 76,4% (p<0,001). Permeabilidad secundaria a 1, 6 y 12 meses en el Grupo A fue 85,2%, 45,8% y 31,3%, respectivamente, frente al Grupo B 100%, 95,3%, 95,2% (p<0,001). El uso de stents no cubierto se asoció a un mayor riesgo de oclusión (HR 2,669 IC 95% 1,146-6,216, p=0,010).ConclusiónEs esperable una mayor permeabilidad del TEV realizado sobre una FAVp permeable, por lo que es recomendable elaborar programas de seguimiento que sean capaces de detectar la EAV previo a su trombosis. (AU)

Objective: To analyze the results of endovascular treatment of venous anastomotic stenosis (VAS) in humero-axillary arteriovenous grafts (HAG), comparing outcomes between patent and thrombosed HAG.Material and methodsA retrospective cohort study was made of endovascular treated patients because of a VAS in a HAG between January 2009 and December 2019. Group A: Thrombosed HAG secondary to a VAS. Group B: Patent HAG with a VAS detected during follow-up. Technical success was defined as residual stenosis after treatment <30%, and clinical success as satisfactory immediate dialysis after surgery. After ET a biannual clinical and ultrasound follow-up was performed. Statistical analysis: Survival analysis was performed for time-to-event data to assess patency.ResultsGroup A: 55 patients. Group B: 22. There were no significative differences in demographic and anatomical factors between groups.Technical and clinical success were 100% in Group B and 94.5% and 91% respectively in Group A.Primary patency at 1, 6 and 12 months was: Group A: 81.8%, 22.4% and 15.7% respectively. Group B: 100%, 85.9%, 76.4% (p<0.001). Secondary patency at 1, 6 and 12 months was: Group A: 85.2%, 45.8% and 31.3% respectively. Group B 100%, 95.3%, 95.2% (p<0.001). Use of non-covered stents was associated with an increased risk of occlusion (HR 2.669 95% CI 1.146–6.216, p=0.010).ConclusionA higher patency of EV performed on a patent HAG is expected. It is therefore advisable to develop surveillance programs that are capable to detect VAS before its occlusion. (AU)

Results of endovascular treatment of venous anastomotic stenosis in prosthetic arteriovenous for hemodialysis PTFE grafts. Comparative analysis between patent and occluded grafts. / Resultados del tratamiento endovascular de la estenosis de la anastomosis venosa en las fístulas arteriovenosas protésicas para hemodiálisis de PTFE. Análisis comparativo entre fístulas permeables y trombosadas.

OBJECTIVE: To analyze the results of endovascular treatment of venous anastomotic stenosis (VAS) in humero-axillary arteriovenous grafts (HAG), comparing outcomes between patent and thrombosed HAG. MATERIAL AND METHODS: A retrospective cohort study was made of endovascular treated patients because of a VAS in a HAG between January 2009 and December 2019. Group A: Thrombosed HAG secondary to a VAS. Group B: Patent HAG with a VAS detected during follow-up. Technical success was defined as residual stenosis after treatment <30%, and clinical success as satisfactory immediate dialysis after surgery. After ET a biannual clinical and ultrasound follow-up was performed. STATISTICAL ANALYSIS: Survival analysis was performed for time-to-event data to assess patency. RESULTS: Group A: 55 patients. Group B: 22. There were no significative differences in demographic and anatomical factors between groups. Technical and clinical success were 100% in Group B and 94.5% and 91% respectively in Group A. Primary patency at 1, 6 and 12 months was: Group A: 81.8%, 22.4% and 15.7% respectively. Group B: 100%, 85.9%, 76.4% (p<0.001). Secondary patency at 1, 6 and 12 months was: Group A: 85.2%, 45.8% and 31.3% respectively. Group B 100%, 95.3%, 95.2% (p<0.001). Use of non-covered stents was associated with an increased risk of occlusion (HR 2.669 95% CI 1.146-6.216, p=0.010). CONCLUSION: A higher patency of EV performed on a patent HAG is expected. It is therefore advisable to develop surveillance programs that are capable to detect VAS before its occlusion.

Sex dependent glial-specific changes in the chromatin accessibility landscape in late-onset Alzheimer's disease brains.

BACKGROUND: In the post-GWAS era, there is an unmet need to decode the underpinning genetic etiologies of late-onset Alzheimer's disease (LOAD) and translate the associations to causation. METHODS: We conducted ATAC-seq profiling using NeuN sorted-nuclei from 40 frozen brain tissues to determine LOAD-specific changes in chromatin accessibility landscape in a cell-type specific manner. RESULTS: We identified 211 LOAD-specific differential chromatin accessibility sites in neuronal-nuclei, four of which overlapped with LOAD-GWAS regions (±100 kb of SNP). While the non-neuronal nuclei did not show LOAD-specific differences, stratification by sex identified 842 LOAD-specific chromatin accessibility sites in females. Seven of these sex-dependent sites in the non-neuronal samples overlapped LOAD-GWAS regions including APOE. LOAD loci were functionally validated using single-nuclei RNA-seq datasets. CONCLUSIONS: Using brain sorted-nuclei enabled the identification of sex-dependent cell type-specific LOAD alterations in chromatin structure. These findings enhance the interpretation of LOAD-GWAS discoveries, provide potential pathomechanisms, and suggest novel LOAD-loci.

Development of an RNA Extraction Protocol for Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR.

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster's digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

Cell-Type Specific Changes in DNA Methylation of SNCA Intron 1 in Synucleinopathy Brains.

Parkinson's disease (PD) and dementia with Lewy body (DLB) are the most common synucleinopathies. SNCA gene is a major genetic risk factor for these diseases group, and dysregulation of its expression has been implicated in the genetic etiologies of several synucleinopathies. DNA methylation at CpG island (CGI) within SNCA intron 1 has been suggested as a regulatory mechanism of SNCA expression, and changes in methylation levels at this region were associated with PD and DLB. However, the role of DNA methylation in the regulation of SNCA expression in a cell-type specific manner and its contribution to the pathogenesis of PD and DLB remain poorly understood, and the data are conflicting. Here, we employed a bisulfite pyrosequencing technique to profile the DNA methylation across SNCA intron 1 CGI in PD and DLB compared to age- and sex-matched normal control subjects. We analyzed homogenates of bulk post-mortem frozen frontal cortex samples and a subset of neuronal and glia nuclei sorted by the fluorescence-activated nuclei sorting (FANS) method. Bulk brain tissues showed no significant difference in the overall DNA methylation across SNCA intron 1 CGI region between the neuropathological groups. Sorted neuronal nuclei from PD frontal cortex showed significant lower levels of DNA methylation at this region compared to normal controls, but no differences between DLB and control, while sorted glia nuclei exhibited trends of decreased overall DNA methylation in DLB only. In conclusion, our data suggested disease-dependent cell-type specific differential DNA methylation within SNCA intron 1 CGI. These changes may affect SNCA dysregulation that presumably mediates disease-specific risk. Our results can be translated into the development of the SNCA intron 1 CGI region as an attractive therapeutics target for gene therapy in patients who suffer from synucleinopathies due to SNCA dysregulation.

[Tobacco use in Spain during COVID-19 lockdown: an evaluation through social media.] / Consumo de tabaco en España durante el estado de alarma por COVID-19: resultados de una evaluación a través de redes sociales.

OBJECTIVE: During the state of alarm and once the confinement decreed by the COVID-19 pandemic ended, a cross-sectorial study was carried out in Spain between May 4th and 22nd, 2020 by volunteers who completed a self-administered online survey. The objective of this study was to know how the confinement period affected the consumption of tobacco and other related products in the adult Spanish population. METHODS: The survey consisted of 18 questions concerning sociodemographic characteristics, the consumption of tobacco and other related products, exposure to secondhand smoke and perception of COVID-19 risk associated with consumption. Questions about tobacco and other related products were posed in order to compare consumption prior to and during confinement. The survey was completed by 17,017 people. The analysis of association of variables was carried out with T-student. Variable frequency analysis was performed with χ2. RESULTS: There was a reduction in the prevalence of daily tobacco smoking and no changes were observed in the products consumed in either period (6.73%). The prevalence of exposure to secondhand smoke at home during confinement among non-smokers decreased (61.83%). Most of survey respondents reported that tobacco and e-cigarette consumption increased the risk of contracting COVID-19 and suffering severe complications (39.09% and 31.80% respectively). CONCLUSIONS: During the COVID-19 lockdown in Spain, the tobacco consumption decreased. Also, secondhand smoke exposition reduces in Spain during this period.

OBJETIVO: Durante el estado de alarma y una vez finalizado el confinamiento decretado por la pandemia por COVID-19, en España se realizó, entre el 4 y el 22 de mayo de 2020, un estudio transversal en voluntarios aplicando una encuesta autocumplimentada online. El objetivo de este estudio fue conocer cómo afectó el periodo de confinamiento al consumo de tabaco y relacionados en la población adulta española. METODOS: El cuestionario constaba de 18 preguntas e incluía características sociodemográficas, el consumo de tabaco y otros productos relacionados, exposición al humo ambiental de tabaco y percepción del riesgo de enfermedad por COVID-19 asociada a su consumo. La encuesta fue completada por 17.017 personas. El análisis de la asociación entre variables cuantitativas, fue realizado mediante el test de la T de Student y el de frecuencias de las variables categóricas mediante el test de χ2. RESULTADOS: Se observó reducción en la prevalencia de fumadores diarios de tabaco (6,73%) y no se observaron cambios en los productos consumidos. La prevalencia de exposición al humo ambiental en casa durante el confinamiento entre personas no fumadoras disminuyó (61,83%). Los encuestados declararon que el consumo de tabaco y de cigarrillos electrónicos aumentaba el riesgo de contraer la enfermedad del COVID-19 y sufrir complicaciones (39,09% y 31,80% respectivamente). CONCLUSIONES: Durante el periodo de confinamiento en España debido al COVID-19, se produjo una reducción en el consumo de tabaco y similares. Además de observó una reducción a la exposición al humo ambiental.

Shared genetic etiology underlying Alzheimer's disease and major depressive disorder.

Patients with late-onset Alzheimer's disease (LOAD) frequently manifest comorbid neuropsychiatric symptoms with depression and anxiety being most frequent, and individuals with major depressive disorder (MDD) have an increased prevalence of LOAD. This suggests shared etiologies and intersecting pathways between LOAD and MDD. We performed pleiotropy analyses using LOAD and MDD GWAS data sets from the International Genomics of Alzheimer's Project (IGAP) and the Psychiatric Genomics Consortium (PGC), respectively. We found a moderate enrichment for SNPs associated with LOAD across increasingly stringent levels of significance with the MDD GWAS association (LOAD|MDD), of maximum four and eightfolds, including and excluding the APOE-region, respectively. Association analysis excluding the APOE-region identified numerous SNPs corresponding to 40 genes, 9 of which are known LOAD-risk loci primarily in chromosome 11 regions that contain the SPI1 gene and MS4A genes cluster, and others were novel pleiotropic risk-loci for LOAD conditional with MDD. The most significant associated SNPs on chromosome 11 overlapped with eQTLs found in whole-blood and monocytes, suggesting functional roles in gene regulation. The reverse conditional association analysis (MDD|LOAD) showed a moderate level, ~sevenfold, of polygenic overlap, however, no SNP showed significant association. Pathway analyses replicated previously reported LOAD biological pathways related to immune response and regulation of endocytosis. In conclusion, we provide insights into the overlapping genetic signatures underpinning the common phenotypic manifestations and inter-relationship between LOAD and MDD. This knowledge is crucial to the development of actionable targets for novel therapies to treat depression preceding dementia, in an effort to delay or ultimately prevent the onset of LOAD.

Control del tabaquismo: evolución de la política impositiva de las labores del tabaco y su impacto en los precios de venta al público / Control of the smoking habit: evolution of the tax policy of tobacco products and its impact on the retail sales prices

No disponible

The Landscape of SNCA Transcripts Across Synucleinopathies: New Insights From Long Reads Sequencing Analysis.

Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinson's Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here, we apply the first study of using long read sequencing with targeted capture of both the gDNA and cDNA of the SNCA gene in brain tissues of PD, DLB, and control samples using the PacBio Sequel system. The targeted full-length cDNA (Iso-Seq) data confirmed complex usage of known alternative start sites and variable 3' UTR lengths, as well as novel 5' starts and 3' ends not previously described. The targeted gDNA data allowed phasing of up to 81% of the ~114 kb SNCA region, with the longest phased block exceeding 54 kb. We demonstrate that long gDNA and cDNA reads have the potential to reveal long-range information not previously accessible using traditional sequencing methods. This approach has a potential impact in studying disease risk genes such as SNCA, providing new insights into the genetic etiologies, including perturbations to the landscape the gene transcripts, of human complex diseases such as synucleinopathies.

Spontaneous Aneurysmal Subarachnoid Hemorrhage and Related Cortisol and Immunologic Alterations: Impact on Patients' Health-related Quality of Life.

OBJECTIVE: To highlight the impact of aneurysmal subarachnoid hemorrhage (SAH) on surviving patients' health-related quality of life (HRQoL) with respect to cortisol and interleukin (IL)-6 alterations and also to identify possible clinical predictors for a better HRQoL. METHODS: Fifty surviving patients treated in our hospital for aneurysmal SAH in a 2-year period with sufficient HRQoL data were enrolled. A good clinical outcome was represented by the modified Rankin Scale (mRS) 0 to 2. The patient's HRQoL was assessed using the Short Form health survey questionnaire, the Beck Depression Inventory, and the Daily Fatigue Impact Scale at 6 and 12 months. The results were analyzed regarding possible correlation to 24-hour urinary free cortisol, serum, and cerebrospinal fluid IL-6 levels. RESULTS: A reduction of HRQoL in up to 35% of survivors was observed at 6 months and in a high proportion of patients (47.2%) with an assumable good outcome (mRS 0-2). Reduced HRQoL in survivors was found in terms of SF-36 (34.9%), depression (26.8%), and fatigue (14%) at 6 months and 18.4%, 39.4%, and 18.9% at 12 months, respectively. Improvement was recorded at 12 months, mainly in SF-36. Early elevated 24-hour urinary free cortisol and IL-6 levels showed a significant positive impact on HRQoL. CONCLUSIONS: Early cortisol and IL-6 levels may predict patients' HRQoL after SAH. Twelve months after SAH, a considerable percentage of patients with a presumably good outcome (mRS 0-2) had a lower HRQoL compared with the general population. Implementing corresponding tests at discharge and 12-month follow-up is recommended.

Día Mundial sin Tabaco (31 de mayo de 2019) / No disponible

No disponible

Probing the role of PPARγ in the regulation of late-onset Alzheimer's disease-associated genes.

Peroxisome proliferator-activated receptor-γ (PPARγ), is a transcription factor that governs pathways, such as lipid metabolism and immune response, that have been implicated in the etiology of LOAD. Previously, we established HepG2-derived cell-lines with stable knockdown of PPARγ gene, and showed an increase in mRNA levels of genes mapped in the APOE linkage disequilibrium (LD) region on chromosome 19q13.32, with the greatest effect observed for APOE-mRNA. Here, we extended the analysis using our PPARγ knockdown model system and investigated the broader effect on expression changes of genes implicated in LOAD via genome wide association studies (GWAS). We applied the nCounter gene expression assay (NanoString) using a panel of twenty-four LOAD-associated genes inferred by proximity to the top significantly associated SNPs. Two independent PPARγ knockdown cell-lines showed changes in mRNA levels of a total of seven genes compared to a control HepG2 cell-line; six of which, ABCA7, APOE, CASS4, CELF1, PTK2B, and ZCWPW1, were upregulated and one, DSG2, was downregulated upon PPARγ knockdown. Our results propose that PPARγ may act as a master regulator of the transcription of several genes involved in LOAD pathogenesis. Our study provided the premise for further analyses including a larger set of genes positioned within a wider range of linkage disequilibrium (LD) regions tagged by all LOAD significantly associated SNPs.

The ubiquitin-modifying enzyme A20 restricts ubiquitination of the kinase RIPK3 and protects cells from necroptosis.

A20 is an anti-inflammatory protein linked to multiple human diseases; however, the mechanisms by which A20 prevents inflammatory disease are incompletely defined. We found that A20-deficient T cells and fibroblasts were susceptible to caspase-independent and kinase RIPK3-dependent necroptosis. Global deficiency in RIPK3 significantly restored the survival of A20-deficient mice. A20-deficient cells exhibited exaggerated formation of RIPK1-RIPK3 complexes. RIPK3 underwent physiological ubiquitination at Lys5 (K5), and this ubiquitination event supported the formation of RIPK1-RIPK3 complexes. Both the ubiquitination of RIPK3 and formation of the RIPK1-RIPK3 complex required the catalytic cysteine of A20's deubiquitinating motif. Our studies link A20 and the ubiquitination of RIPK3 to necroptotic cell death and suggest additional mechanisms by which A20 might prevent inflammatory disease.

Cutting edge: ABIN-1 protects against psoriasis by restricting MyD88 signals in dendritic cells.

Psoriasis is a chronic, inflammatory skin disease caused by a combination of environmental and genetic factors. The Tnip1 gene encodes A20 binding and inhibitor of NF-κB-1 (ABIN-1) protein and is strongly associated with susceptibility to psoriasis in humans. ABIN-1, a widely expressed ubiquitin-binding protein, restricts TNF- and TLR-induced signals. In this study, we report that mice lacking ABIN-1 specifically in dendritic cells (DCs), ABIN-1(fl) CD11c-Cre mice, exhibit perturbed immune homeostasis. ABIN-1-deficient DCs display exaggerated NF-κB and MAPK signaling and produce more IL-23 than do normal cells in response to TLR ligands. Challenge of ABIN-1(fl) CD11c-Cre mice with topical TLR7 ligand leads to greater numbers of Th17 and TCRγδ T cells and exacerbated development of psoriaform lesions. These phenotypes are reversed by DC-specific deletion of the TLR adaptor MyD88. These studies link ABIN-1 with IL-23 and IL-17, and they provide cellular and molecular mechanisms by which ABIN-1 regulates susceptibility to psoriasis.

A20 restricts wnt signaling in intestinal epithelial cells and suppresses colon carcinogenesis.

Colon carcinogenesis consists of a multistep process during which a series of genetic and epigenetic adaptations occur that lead to malignant transformation. Here, we have studied the role of A20 (also known as TNFAIP3), a ubiquitin-editing enzyme that restricts NFκB and cell death signaling, in intestinal homeostasis and tumorigenesis. We have found that A20 expression is consistently reduced in human colonic adenomas than in normal colonic tissues. To further investigate A20's potential roles in regulating colon carcinogenesis, we have generated mice lacking A20 specifically in intestinal epithelial cells and interbred these with mice harboring a mutation in the adenomatous polyposis coli gene (APC(min)). While A20(FL/FL) villin-Cre mice exhibit uninflamed intestines without polyps, A20(FL/FL) villin-Cre APC(min/+) mice contain far greater numbers and larger colonic polyps than control APC(min) mice. We find that A20 binds to the ß-catenin destruction complex and restricts canonical wnt signaling by supporting ubiquitination and degradation of ß-catenin in intestinal epithelial cells. Moreover, acute deletion of A20 from intestinal epithelial cells in vivo leads to enhanced expression of the ß-catenin dependent genes cyclinD1 and c-myc, known promoters of colon cancer. Taken together, these findings demonstrate new roles for A20 in restricting ß-catenin signaling and preventing colon tumorigenesis.