The RNA binding protein human antigen R is a gatekeeper of liver homeostasis.

BACKGROUND AND AIMS: NAFLD is initiated by steatosis and can progress through fibrosis and cirrhosis to HCC. The RNA binding protein human antigen R (HuR) controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte HuR in NAFLD development and progression to fibrosis and HCC. APPROACH AND RESULTS: Hepatocyte-specific, HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or an NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis, and HCC development were studied by histology, flow cytometry, quantitative PCR, and RNA sequencing. The liver lipidome was characterized by lipidomics analysis, and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation sequencing. Hepatocyte-specific, HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition compared to control HuR-sufficient mice. On an NAFLD-inducing diet, hepatocyte-specific HuR deficiency resulted in exacerbated inflammation, fibrosis, and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics, and RNA immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady state, a triglyceride signature resembling that of NAFLD livers. Moreover, up-regulation of secreted phosphoprotein 1 expression mediated, at least partially, fibrosis development in hepatocyte-specific HuR deficiency on an NAFLD-inducing diet, as shown by experiments using antibody blockade of osteopontin. CONCLUSIONS: HuR is a gatekeeper of liver homeostasis, preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically.

Detection of mycobacterial DNA by a specific and simple lateral flow assay incorporating cadmium selenide quantum dots.

Cadmium selenide quantum dots have been incorporated to a lateral flow assay for the specific and very simple detection of different mycobacterial DNA targets within only a few minutes, bypassing the complexity of conventional DNA hybridization assays. The method extends our previous work on protein detection using an identical procedure.

Evaluation of indirect immunofluorescence antibody test and enzyme-linked immunosorbent assay for the diagnosis of infection by Leishmania infantum in clinically normal and sick cats.

Cats that live in areas where canine and human leishmaniosis due to Leishmania infantum is endemic may become infected and may develop anti-Leishmania antibodies. In this study 50 clinically normal and 50 cats with cutaneous and/or systemic signs that lived in an endemic area and had been previously examined for infection by L. infantum using PCR in four different tissues were serologically tested for the presence of anti-Leishmania IgG (IFAT and ELISA) and IgM (IFAT). The aim was to compare the results of IFAT, ELISA and PCR and to investigate the possible associations between seropositivity to Leishmania spp and signalment, living conditions, season of sampling, health status of the cats, and seropositivity to other infectious agents. Low concentrations of anti-Leishmania IgG were detected by IFAT in 10% of the cats and by ELISA in 1%, whereas anti-Leishmania IgM were detected by IFAT in 1%. There was disagreement between the results of IFAT and ELISA for anti-Leishmania IgG (P = 0.039) and between all serological tests and PCR (P < 0.001). The diagnostic sensitivity of all serological tests, using PCR as the gold standard, was very low, but ELISA and IFAT for anti-Leishmania IgM had 100% specificity. The diagnostic sensitivity of all serological tests could not be improved by changing the cut-off values. Seropositivity for Leishmania spp was not associated with signalment, living conditions, season of sampling and health status of the cats or with seropositivity to feline leukemia virus, feline immunodeficiency virus, feline coronavirus, Toxoplasma gondii and Bartonella henselae. In conclusion, because of their low sensitivity and very high specificity two of the evaluated serological tests (ELISA for anti-Leishmania IgG and IFAT for anti-Leishmania IgM) may be useless as population screening tests but valuable for diagnosing feline infection by L. infantum.

Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples.

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.

Inactivation of AUF1 in Myeloid Cells Protects From Allergic Airway and Tumor Infiltration and Impairs the Adenosine-Induced Polarization of Pro-Angiogenic Macrophages.

The four isoforms of the RNA-binding protein hnRNPD/AUF1 have been proposed to limit the use of inflammatory mRNAs in innate immune cells. Mice engineered to lack AUF1s in all tissues are sensitive to acute inflammatory assaults; however, they also manifest complex degenerations obscuring assessment of AUF1s' roles in innate immune cells. Here, we restricted a debilitating AUF1 mutation to the mouse myeloid lineage and performed disease-oriented phenotypic analyses to assess the requirement of AUF1s in variable contexts of innate immune reactivity. Contrary to the whole-body mutants, the myeloid mutants of AUF1s did not show differences in their susceptibility to cytokine storms occurring during endotoxemia; neither in type-I cell-mediated reactions driving intestinal inflammation by chemical irritants. Instead, they were resistant to allergic airway inflammation and displayed reductions in inflammatory infiltrates and an altered T-helper balance. The ex-vivo analysis of macrophages revealed that the loss of AUF1s had a minimal effect on their proinflammatory gene expression. Moreover, AUF1s were dispensable for the classical polarization of cultured macrophages by LPS & IFNγ correlating with the unchanged response of mutant mice to systemic and intestinal inflammation. Notably, AUF1s were also dispensable for the alternative polarization of macrophages by IL4, TGFß and IL10, known to be engaged in allergic reactions. In contrast, they were required to switch proinflammatory macrophages towards a pro-angiogenic phenotype induced by adenosine receptor signals. Congruent to this, the myeloid mutants of AUF1 displayed lower levels of vascular remodeling factors in exudates from allergen exposed lungs; were unable to support the growth and inflammatory infiltration of transplanted melanoma tumors; and failed to vascularize inert grafts unless supplemented with angiogenic factors. Mechanistically, adenosine receptor signals enhanced the association of AUF1s with the Vegfa, Il12b, and Tnf mRNAs to differentially regulate and facilitate the pro-angiogenic switch. Our data collectively demonstrates that AUF1s do not act as general anti-inflammatory factors in innate immune cells but have more specialized roles in regulons allowing specific innate immune cell transitions to support tissue infiltration and remodeling processes.

The scaffold protein IQGAP1 links heat-induced stress signals to alternative splicing regulation in gastric cancer cells.

In response to oncogenic signals, Alternative Splicing (AS) regulators such as SR and hnRNP proteins show altered expression levels, subnuclear distribution and/or post-translational modification status, but the link between signals and these changes remains unknown. Here, we report that a cytosolic scaffold protein, IQGAP1, performs this task in response to heat-induced signals. We show that in gastric cancer cells, a nuclear pool of IQGAP1 acts as a tethering module for a group of spliceosome components, including hnRNPM, a splicing factor critical for the response of the spliceosome to heat-shock. IQGAP1 controls hnRNPM's sumoylation, subnuclear localisation and the relevant response of the AS machinery to heat-induced stress. Genome-wide analyses reveal that IQGAP1 and hnRNPM co-regulate the AS of a cell cycle-related RNA regulon in gastric cancer cells, thus favouring the accelerated proliferation phenotype of gastric cancer cells. Overall, we reveal a missing link between stress signals and AS regulation.

Evaluation of clinicopathological abnormalities in sick cats naturally infected by Leishmania infantum.

Feline infection by Leishmania infantum (syn. L. chagasi) has been described in areas where canine leishmaniosis is endemic. A wide variety of clinicopathological abnormalities have been reported in cats presenting clinical signs of leishmaniosis but there is a paucity of information regarding cats infected by L. infantum that do not suffer from leishmaniosis but from other diseases. The aim of this study was to compare: a) the frequency of clinicopathological abnormalities and b) the values of hematology, serum biochemistry and urinalysis parameters, between non-infected sick cats and sick cats that were infected by L. infantum. A total of 50 cats with cutaneous, ocular and/or systemic clinical signs that lived in an endemic area and had been tested for infection by L. infantum using PCR from four different tissues, were included. Based on the results of PCR, 20/50 cats were found to be infected and 30/50 non-infected. The only difference between the two groups of cats was that the concentration of inorganic phosphorus (P = 0.043) was higher in infected cats. This finding may suggest an association between infection by L. infantum and feline kidney disease.

Divergent Innate and Epithelial Functions of the RNA-Binding Protein HuR in Intestinal Inflammation.

HuR is an abundant RNA-binding protein acting as a post-transcriptional regulator of many RNAs including mRNAs encoding inflammatory mediators, cytokines, death signalers and cell cycle regulators. In the context of intestinal pathologies, elevated HuR is considered to enhance the stability and the translation of pro-tumorigenic mRNAs providing the rationale for its pharmacological targeting. However, HuR also possesses specific regulatory functions for innate immunity and cytokine mRNA control which can oppose intestinal inflammation and tumor promotion. Here, we aim to identify contexts of intestinal inflammation where the innate immune and the epithelial functions of HuR converge or diverge. To address this, we use a disease-oriented phenotypic approach using mice lacking HuR either in intestinal epithelia or myeloid-derived immune compartments. These mice were compared for their responses to (a) Chemically induced Colitis; (b) Colitis- associated Cancer (CAC); (c) T-cell mediated enterotoxicity; (d) Citrobacter rodentium-induced colitis; and (e) TNF-driven inflammatory bowel disease. Convergent functions of epithelial and myeloid HuR included their requirement for suppressing inflammation in chemically induced colitis and their redundancies in chronic TNF-driven IBD and microbiota control. In the other contexts however, their functions diversified. Epithelial HuR was required to protect the epithelial barrier from acute inflammatory or infectious degeneration but also to promote tumor growth. In contrast, myeloid HuR was required to suppress the beneficial inflammation for pathogen clearance and tumor suppression. This cellular dichotomy in HuR's functions was validated further in mice engineered to express ubiquitously higher levels of HuR which displayed diminished pathologic and beneficial inflammatory responses, resistance to epithelial damage yet a heightened susceptibility to CAC. Our study demonstrates that epithelial and myeloid HuR affect different cellular dynamics in the intestine that need to be carefully considered for its pharmacological exploitation and points toward potential windows for harnessing HuR functions in intestinal inflammation.

Detection of Leishmania-specific DNA and surface antigens using a combination of functionalized magnetic beads and cadmium selenite quantum dots.

Leishmaniosis is a zoonotic disease that affects millions of people especially in resource-poor settings. The development of reliable diagnostic assays that do not require dedicated equipment or highly trained personnel would improve early diagnosis and effective control. For this purpose, a combination of magnetic bead and cadmium selenite quantum dot probes was applied for the detection of Leishmania-specific surface antigens (proteins) and DNA. Both analytes are isolated from the solution using magnetic bead capture probes whereas the presence of the targeted molecules is demonstrated by quantum dot detection probes. The sensitivity and specificity of this method reached 100% based on an assessment performed on 55 cultured isolates of various microbial pathogens. The low limit of detection was 3125 ng/µl and 10(3)cells/ml for Leishmania DNA and protein, respectively. The method shows considerable potential for clinical application in human and veterinary medicine, especially in resource-poor settings.

Cytological and molecular detection of Leishmania infantum in different tissues of clinically normal and sick cats.

Natural infection of domestic cats by Leishmania infantum (synonym: L. chagasi) has been demonstrated in several European, Latin American, and Asian countries, and the estimated prevalence of infection, based mainly on blood PCR, ranges from 0.3% up to 60.6%. In this study we aimed to: (a) estimate the prevalence of the infection by L. infantum in clinically normal cats (group A; n=50) and in cats with various clinical signs (group B; n=50), living in an endemic region, by both cytological examination of four different tissues (lymph node, skin, bone marrow, and conjunctiva) and by PCR in four different tissues (blood, skin biopsies, bone marrow, and conjunctiva); (b) compare the diagnostic sensitivity of the above methods and evaluate for possible associations between their results; and (c) investigate the possible associations between infection by L. infantum and signalment, living conditions, season of sampling, and health status of the cats. The prevalence of the infection in the study population was 41% and did not differ (P=0.839) between group A (42%) and B (40%) cats. Lymph node, skin, bone marrow and conjunctiva cytology was always negative. Therefore, the diagnosis of the infection was based only on PCR in blood, skin biopsy, bone marrow and conjunctiva, which was positive in 13%, 18.2%, 16% and 3.1% of the cats, respectively. PCR was positive in only one of the four tissues in 80.5% of the infected cats. The results differed (P=0.014) among the four tissues and were less frequently positive in conjunctiva compared to skin biopsies and bone marrow (P=0.007 for both comparisons), thus highlighting the need for multiple tissue PCR testing in order to minimize false-negative results. More PCR-positive cats were found when sampling was performed during the period of sandfly activity (odds ratio: 2.44; P=0.022). Also, in group B cats, the likelihood of PCR-positivity was higher (odds ratio: 3.93; P=0.042) among those presenting at least one systemic clinical sign that had been previously reported in cats with leishmaniosis.

A novel non-amplification assay for the detection of Leishmania spp. in clinical samples using gold nanoparticles.

Leishmaniosis is a zoonose caused by protozoans of the genus Leishmania. The need for accurate diagnostic investigation of cases of leishmaniosis has rendered today the use of molecular biology techniques broadly applicable. However, the reliable application of these methods requires highly-specialised personnel, dedicated equipment and space. The aim of this study was the design and construction of functionalized gold nanoparticles (AuNPs) that would be incorporated into an easily applicable DNA detection methodology for the identification of Leishmania spp. in clinical samples. AuNPs 20nm in diameter were conjugated with four oligonucleotide probes, targeting kinetoplastid minicircle DNA of Leishmania spp. In the absence of complimentary DNA, AuNPs-probes precipitate under acid environment causing a change of color from red to purple, which can be detected by visual observation. In the presence of target DNA the color of the solution remains red. The specific methodology was applied to positive and negative control samples and whole blood collected from dogs with suspected canine leishmaniosis. The method's minimum detection limit was defined to 11.5ng of target DNA per µl of sample. Repeatability and reproducibility were 100%. Relative sensitivity and specificity referenced to PCR were calculated to 92% and 100% regarding collectively control and clinical samples. The proposed approach can be considered an appealing diagnostic solution especially for screening purposes in enzootic areas, where detection of very small amounts of the targeted analyte is not top priority.

Evaluation of the microbial safety of child food of animal origin in Greece.

Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity.

Detection of pathogenic mycobacteria based on functionalized quantum dots coupled with immunomagnetic separation.

Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 10(4) bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer. The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples.

Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles.

Mycobacterial infections have a high economic, human and animal health impact. Herein, we present the development of a colorimetric method that relies on the use of gold nanoparticles for fast and specific detection of Mycobacterium spp. dispensing with the need for DNA amplification. The result can be recorded by visual and/or spectrophotometric comparison of solutions before and after acid induced AuNP-probe aggregation. The presence of a complementary target prevents aggregation and the solution remains pink, whereas in the opposite event it turns to purple. The application of the proposed method on isolated bacteria produced positive results with the mycobacterial isolates and negative with the controls. The minimum detection limit of the assay was defined at 18.75 ng of mycobacterial DNA diluted in a sample-volume of 10 microl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium avium subsp. paratuberculosis DNA in faeces, in comparison with real-time PCR. The concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%. The proposed method could be used as a highly specific and sensitive screening tool for the detection of mycobacteria directly from clinical samples in a very simple manner, without the need of high-cost dedicated equipment. The technology described here, may develop into a platform that could accommodate detection of many bacterial species and could be easily adapted for high throughput and expedite screening of samples.