LTA4H expression in canine oral melanomas: methodological set up and preliminary results

Leukotriene A4 hydrolase (LTA4H) is a hydrolytic enzyme which converts leukotriene A4 intoleukotriene B4 inside the arachidonic acid cascade. Besides playing a well-known role in inflammation,it has also been investigated for possible implications in different types of tumors, including canine uvealmelanoma (Chen et al., 2004; Malho et al., 2013). In the present study, we set up RT-PCR andimmunohistochemical protocols to investigate the expression of LTA4H gene and protein, respectively,in formalin fixed paraffin embedded (FFPE) specimens of canine melanomas. 16 samples of canine oralmelanomas were histopathologically evaluated and divided in two subgroups based on morphologicalcriteria of malignancy. RT-PCR protocols for the target gene LTA4H were set up on frozen and FFPEsamples of the same tumor. Immunohistochemical investigation of LTA4H protein was performed witha mouse monoclonal antibody anti-LTA4H (clone 1E9). Preliminary tests were carried out to define theprotocol: with and without bleaching, with different unmaskings, with different serial dilutions of theprimary antibody. RT-PCR set up resulted in comparable good efficiency on both frozen and FFPEsamples. Final immunohistochemical protocol included: hydrogen peroxide bleaching, water bathunmasking and 1:100 dilution of the primary antibody. Positive immunostaining for LTA4H was presentin neoplastic cells of all melanoma samples, with variable localization and intensity. These preliminaryresults encourage future applications of the studied techniques to quantify differential expression ofLTA4H in canine melanomas both at molecular and histological level. Laboratory results will becompared with follow up data, in order to verify if LTA4H can be proposed as a valuable prognosticmarker

Distinct retrograde microtubule motor sets drive early and late endosome transport

Although subcellular positioning of endosomes significantly impacts on their functions, the molecular mechanisms governing the different steady-state distribution of early endosomes (EEs) and late endosomes (LEs)/lysosomes (LYs) in peripheral and perinuclear eukaryotic cell areas, respectively, are still unsolved. We unveil that such differences arise because, while LE retrograde transport depends on the dynein microtubule (MT) motor only, the one of EEs requires the cooperative antagonism of dynein and kinesin-14 KIFC1, a MT minus end-directed motor involved in cancer progression. Mechanistically, the Ser-x-Ile-Pro (SxIP) motif-mediated interaction of the endoplasmic reticulum transmembrane protein stromal interaction molecule 1 (STIM1) with the MT plus end binding protein 1 (EB1) promotes its association with the p150Glued subunit of the dynein activator complex dynactin and the distinct location of EEs and LEs/LYs. The peripheral distribution of EEs requires their p150Glued-mediated simultaneous engagement with dynein and SxIP motif-containing KIFC1, via HOOK1 and HOOK3 adaptors, respectively. In sum, we provide evidence that distinct minus end directed MT motor systems drive the differential transport and subcellular distribution of EEs and LEs in mammalian cells

Comparison between the diagnostic accuracy of clinico-pathological and molecular tests for feline infectious peritonitis (FIP)

The aim of this study was to compare the diagnostic accuracy for feline infectious peritonitis (FIP) of conventional clinic-pathological tests with that of molecular tests such as routine PCR and PCR followed by the sequencing of the Spike (S) gene. Blood, effusion and tissues specimens were collected from 21 FIP suspected cats. In vivo examination consisted of CBC, serum protein electrophoresis, AGP measurement, cytological and biochemical examination and the evaluation of the ΔTNC on effusions, and of molecular tests such the screening PCR (target: 3’UTR region) and the PCR directed towards the S gene followed by the amplification products sequencing in order to detect the aminoacidic substitution recently considered diagnostic for FIP1. These molecular techniques were applied to tissues collected during necropsy, which also allowed forming an FIP group (13 cats) and a non-FIP group (5 cats) based on histology and immunohistochemistry. The best test on tissues was immunohistochemistry (sens: 92.3%; spec: 100%), while the screening PCR suffered of low specificity (spec: 33.3%) and the S gene sequencing showed low sensitivity (sens: 69.2%).On effusions, the best tests resulted screening PCR and cytology (sens and spec: 100%) in comparison with the ΔTNC measurement (sens: 85.7 %; spec: 100%) and the S gene sequencing (sens: 42.8%; spec: 100%).On blood, the best test resulted AGP measurement (sens: 81.8%; spec: 100%), while serum protein electrophoresis showed a surprisingly low sensitivity (sens: 41.7%). Screening PCR (sens: 55.6%; spec: 100%) and S gene sequencing (sens: 33.3%; spec: 100%) proved again low accuracy.

Circulating Tumor Cells Identify Patients with Super-High-Risk Non-Muscle-Invasive Bladder Cancer: Updated Outcome Analysis of a Prospective Single-Center Trial

Clinical behavior of non-muscle-invasive bladder cancer (NMIBC) is largely unpredictable, and even patients treated according to European Association of Urology recommendations have a heterogeneous prognosis. High-grade T1 (HGT1) bladder cancer is the highest-risk subtype of NMIBC, with an almost 40% rate of recurrence and 20% of progression at 5 years. Nomograms predicting risk of recurrence, progression, and cancer-specific survival (CSS) are not available specifically within HGT1 bladder cancer, and the identification of robust prognostic biomarkers to better guide therapeutic strategies in this subgroup of patients is of paramount importance. Strategies to identify putative biomarkers in liquid biopsies from blood and urine collected from patients with bladder cancer have been intensively studied in the last few years

p53-sensitive epileptic behavior and inflammation in Ft1 hypomorphic mice

Epilepsy is a complex clinical condition characterized by repeated spontaneous seizures. Seizures have been linked to multiple drivers including DNA damage accumulation. Investigation of epilepsy physiopathology in humans imposes ethical and practical limitations, for this reason model systems are mostly preferred. Among animal models, mouse mutants are particularly valuable since they allow conjoint behavioral, organismal, and genetic analyses. Along with this, since aging has been associated with higher frequency of seizures, prematurely aging mice, simulating human progeroid diseases, offer a further useful modeling element as they recapitulate aging over a short time-window. Here we report on a mouse mutant with progeroid traits that displays repeated spontaneous seizures. Mutant mice were produced by reducing the expression of the gene Ft1 (AKTIP in humans). In vitro, AKTIP/Ft1 depletion causes telomere aberrations, DNA damage, and cell senescence. AKTIP/Ft1 interacts with lamins, which control nuclear architecture and DNA function. Premature aging defects of Ft1 mutant mice include skeletal alterations and lipodystrophy. The epileptic behavior of Ft1 mutant animals was age and sex linked. Seizures were observed in 18 mutant mice (23.6% of aged ≥ 21 weeks), at an average frequency of 2.33 events/mouse. Time distribution of seizures indicated non-random enrichment of seizures over the follow-up period, with 75% of seizures happening in consecutive weeks. The analysis of epileptic brains did not reveal overt brain morphological alterations or severe neurodegeneration, however, Ft1 reduction induced expression of the inflammatory markers IL-6 and TGF-β. Importantly, Ft1 mutant mice with concomitant genetic reduction of the guardian of the genome, p53, showed no seizures or inflammatory marker activation, implicating the DNA damage response into these phenotypes. This work adds insights into the connection among DNA damage, brain function, and aging. In addition, it further underscores the importance of model organisms for studying specific phenotypes, along with permitting the analysis of genetic interactions at the organismal level

Bleaching melanin in formalin-fixed and paraffin-embedded melanoma specimens using visible light: a pilot study

In fluorescence microscopy, light radiation can be used to bleach fluorescent molecules in formalin-fixed and paraffin-embedded (FFPE) samples, in order to increase the ratio between signal of interest and background autofluorescence. We tested if the same principle can be exploited in bright field microscopy to bleach pigmented melanoma FFPE sections together with cell morphology maintenance. After dewaxing and rehydration, serial FFPE sections of a feline diffuse iris melanoma, a canine dermal melanoma, a gray horse dermal melanoma and a swine cutaneous melanoma were irradiated with visible light for I, 2, 3, 4 and 5 days, prior to Hematoxylin & Eosin staining. Complete bleaching was obtained after 1-day treatment in feline and swine melanomas, while 2 and 3 days were required in canine and equine neoplasms, respectively. In all treated samples, cell morphology was maintained. Photo-induced bleaching combined with immunohistochemistry was tested after a 3-day photo-treatment using five different markers. According to the literature, in all samples neoplastic cells stained positive for vimentin, S100 and PNL2, while negative for FVIII and pancytokeratin. in conclusion, visible light can be effectively exploited to bleach pigmented melanoma FFPE sections prior to perform routine histochemical and immunohistochemical stains

Subacute Ruminal Acidosis and Evaluation of Blood Gas Analysis in Dairy Cow

Subacute Ruminal Acidosis (SARA) corresponds to an imbalance between lactate-producing bacteria and lactate-using bacteria, which results in a change in ruminal pH associated with a prevalent consumption of rapidly fermentable carbohydrates. In our study, 216 primiparus and multiparus dairy cows were selected from 20 Italian intensive dairy herds and were divided into three groups based on the risk of SARA. All the dairy cows had high average milk production. After blood sampling, a complete blood gas analysis was performed. One-way ANOVA was performed to compare the three groups. O2 Cont, PCO2, blood pH, O2Hb, urinary pH, and rumen pH were significantly lower in cows with rumen pH < 5.5. These results indicate that blood gas analysis is a valuable tool to diagnose acidosis in dairy cows because it provides good assessment of acidosis while being less invasive than rumen pH analysis

NPBTs FOR SUSTAINABLE VITICULTURE MANAGEMENT TO BIOTIC AND ABIOTIC STRESS

New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for plant improvement to biotic and abiotic stresses satisfying the European Policies requirements that promote chemical input reduction and a more sustainable agriculture. We decided to apply genome editing (via CRISPR/Cas9) focusing on susceptibility genes to control powdery mildew: we chosen to knock-out two genes belonging to MLO (Mildew Locus O) family: VvMLO7 and VvMLO6. The same approach was used to cope with abiotic stresses, in specifc drought, performing a knock-out of four genes, two belonging to GST (Glutathione S-Transferase) and two to PME (Pectin Methyl Esterase) gene families. In parallel to genome editing, we also applied cisgenesis to move the resistance locus RPV3-1 (Resistance to Plasmopara viticola) into economically important cultivars. This locus is formed by two di\ufb00erent genes that were inserted individually and in combination to evaluate their e\ufb00ects. One of the drawbacks linked to classical Agrobacterium tumefaciens mediated transformation is the insertion of unrelated transgene (e.g., antibiotic resistance). These markers are required for transgenic plants selection, but undesirable to be retained in commercial plants due to possible toxicity or allergenicity to humans and animals, in addition to their potential hazards for the environment. To overcome these limits, we exploit an inducible excision system based on a Cre-lox recombinase technology controlled by a heat-shock inducible promoter that will be activated once the transformation event(s) will be confrmed. Embryogenic calli of Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese, were used in stable transformation with A. tumefaciens carrying the genome editing construct with the MLO-guideRNAs and the cisgenic construct carrying the two RPV3-1 genes. Embryogenic calli of rootstocks 110 Richter and SO4 were transformed with genome editing construct carrying GST and PME guideRNAs in two independent transformations. Regenerated embryos from all the transformation events are now under evaluation

Widespread extrahepatic expression of acute-phase proteins in chicken (Gallus gallus) tissues

Acute Phase Proteins (APP) are plasma proteins that can modify their expression in response toinflammation caused by tissue injury, infections, immunological disorders, or stress. Although APP areproduced mainly in liver, extrahepatic production has been described (Marques et al., 2016; Lecchi etal., 2012). The aim of this work was to study the extrahepatic gene expression of five APP, namely α1-acid glycoprotein (AGP), Serum amyloid A (SAA), Haptoglobin-like protein (PIT54), C-rective protein(CRP) and Ovotransferrin (OVT) (O'Reilly and Eckersall, 2014) in different healthy chicken (Gallus gallus)tissues by quantitative real time PCR (qPCR) and immunohistochemistry to detect the precise locationof the proteins.APP gene expression was higher in liver compared with other tissues. mRNA coding for CRP, OVT andSAA was detected in all tissues involved in this study with a higher expression in gastrointestinal tract,respiratory system and lymphatic system. SAA expression was particularly high in cecal tonsil, lung,spleen and meckel’s diverticulum, whereas OVT showed a high expression in lung, bursa of Fabricius,pancreas, brain and adipose tissue. AGP and PIT54 was also detected in pericardial adipose tissue,spleen, kidney, lung, mucosa of proventriculus, mucosa of gizzard and pancreas but, oppositely to SAA,their mRNA was not detected in meckel’s diverticulum, cecal tonsil and bursa of Fabricius. These resultssuggest that each tissue is able to express different amount of APP even in healthy conditions andmount a local acute phase reaction. Immunohistochemistry to detect the precise location for AGP, OVTand SAA using available antibodies is ongoing