A reverse transcription loop-mediated isothermal amplification (LAMP) assay for the detection of feline Coronavirus

Loop-mediated isothermal amplification (LAMP) is a molecular method that amplifies DNA underisothermal conditions. It relies on the use of 4 different primers recognizing 6 regions of the templatesequence and on the use of a DNA polymerase with strand displacement activity (Notomi et al., 2000).The addition of two loop primers allows the reaction time to be of one hour only (Nagamine et al., 2002).The aim of this study was to develop a reverse transcription LAMP assay for an easy and inexpensivedetection of feline Coronavirus (FCoV). Six primers binding the conserved 3’UTR region of the FCoVwere designed with the Primer Explorer software. Thirty-two samples of RNA (11 feces, 8 effusions, 9blood samples and 4 tissues) on which a reverse transcription polymerase chain reaction (RT-PCR) forthe 3’UTR region was performed were used. The reaction was carried out in 25μL reaction volume andthe mixture was incubated in a thermocycler at 63°C for 1 hour followed by 10 minutes at 80°C. LAMPproducts were visualized under UV after electrophoresis migration on a 1.5% agarose gel stained withethidium bromide, where they produce a ladder-like pattern if positive. Results where compared withthose obtained on standard PCR. Sensitivity and specificity were respectively 60% and 100% on feces,40% and 100% on effusions, 25% and 100% on blood, and 100% and 100% on tissues. The overall sensitivityand specificity of this method were of 57.1% and 100%, thus limiting a clinical application of this method,except for tissues

Preliminary data about Paraoxonase-1 (PON-1) as a maker for Feline Infectious Peritonitis (FIP)

Feline infectious peritonitis (FIP) is a fatal disease in which the definitive diagnosis is achieved by immunohistochemistry (IHC) on post-mortem biopsies. The clinical suspicion is aroused by signalment, clinical signs and several laboratory tests, including alpha-1-acid glycoprotein measurement for which the only validated kit is no longer available. Paraoxonase-1 (PON-1) is a serum enzyme with antioxidant activity, considered as a negative acute phase protein in several species. Since inflammation plays a major role in FIP, and due to the high susceptibility of cats to oxidation, it could be of great interest the evaluation of this enzyme as a diagnostic marker for FIP. The aim of this study was to measure paraoxonase-1 in healthy cats and cats with clinical signs consistent with FIP (both wet or dry form), in order to evaluate the utility of this parameter in the diagnosis of FIP. Sixty-two cats were enrolled and divided into three groups: healthy (n=16), confirmed FIP (n=22) and NON FIP with similar clinical signs (n=24). PON-1 was measured on serum, using a paraoxon-based enzymatic method, already validated in cats. Results showed significantly lower PON-1 activity in FIP cats (mean ± SD: 29.1 ± 16.3 U/mL; median: 24.4; IQR: 16.6-38.3), compared with healthy cats (90.1 ± 24.1 U/mL; median: 86.0; IQR: 76.7-105.7; P<0.001) and with “non-FIP” cats (55.9 ± 28.3 U/mL; median: 51.9; IQR: 35.7-68.8, P<0.001). A significant difference was also found between healthy and “non-FIP” cats (P<0.001). The receiver operating characteristic (ROC) curve demonstrated that PON-1 may discriminate cats with and without FIP (Fig.1). At the cut-off that maximizes the diagnostic power of the test, sensitivity and specificity for FIP were 77% each, suggesting that PON-1 may be a reliable marker in association with other confirmatory tests and with signs consistent with the disease

GLEm-Net: Unified Framework for Data Reduction with Categorical and Numerical Features

In the era of Big Data, effective data reduction through feature selection is of paramount importance for machine learning. This paper presents GLEm-Net (Grouped Lasso with Embeddings Network), a novel neural framework that seamlessly processes both categorical and numerical features to reduce the dimensionality of data while retaining as much information as possible. By integrating embedding layers, GLEm-Net effectively manages categorical features with high cardinality and compresses their information in a less dimensional space. By using a grouped Lasso penalty function in its architecture, GLEm-Net simultaneously processes categorical and numerical data, efficiently reducing high-dimensional data while preserving the essential information. We test GLEm-Net with a real-world application in an industrial environment where 6 million records exist and each is described by a mixture of 19 numerical and 7 categorical features with a strong class imbalance. A comparative analysis using state-of-the-art methods shows that despite the difficulty of building a high-performance model, GLEm-Net outperforms the other methods in both feature selection and classification, with a better balance in the selection of both numerical and categorical features

Novel perspectives in redox biology and pathophysiology of failing myocytes: modulation of the intramyocardial redox milieu for therapeutic interventions - A review article from the Working Group of Cardiac Cell Biology, Italian Society of Cardiology

The prevalence of heart failure (HF) is still increasing worldwide, with enormous human, social, and economic costs, in spite of huge efforts in understanding pathogeneticmechanisms and in developing effective therapies that have transformed this syndrome into a chronic disease. Myocardial redox imbalance is a hallmark of this syndrome, since excessive reactive oxygen and nitrogen species can behave as signaling molecules in the pathogenesis of hypertrophy and heart failure, leading to dysregulation of cellular calcium handling, of the contractile machinery, of myocardial energetics and metabolism, and of extracellular matrix deposition. Recently, following new interesting advances in understanding myocardial ROS and RNS signaling pathways, new promising therapeutical approaches with antioxidant properties are being developed, keeping in mind that scavenging ROS and RNS tout court is detrimental as well, since these molecules also play a role in physiological myocardial homeostasis

The gut microbiome and mucosal defenses in cats with coronaviruses: a pilot study

Feline Infectious Peritonitis (FIP) develops from a mutation of enteric feline coronaviruses (FCoVs) and an imbalance of the host immune response. The wide polymorphism of FCoVs is associated with the viral replication rate (Licitra et al. 2013).  Changes in the composition of the gut microbiota may induce quali-quantitative modifications in FCoVs and/or different immune profiles (Weese et al., 2015). Few information is available on feline gut microbiome and the association between microbiota and the predisposition to pathological conditions (Ramadan et al., 2014).The aim of this study is to provide preliminary data about the composition of gut microbiota in healthy cats compared with FCoV infected cats (with and without  FIP), in order to evaluate whether changes of gut microbiota may induce changes in FCoV, in its genetic polymorphism and in the mucosal immunity.Screening analyses have been performed on 22 cats:- Routine hematology and biochemistry on EDTA and serum (included electrophoresis and alpha-1-acid glycoprotein measurement for cats suspected with FIP)- Nested RT-PCR-3’UTR on frozen faeces- Effusion evaluation- FIV/FeLV serologyDue to strict inclusion criteria (cats younger than 2.5 years old, indoor and not assuming antibiotics in the previous two months) and based on the results obtained from the complete set of analysis, only 15 cats, specifically 5 cats for each of the following 3 groups: FIP- affected, healthy negative and positive for FCoV, have been recruited to perform the following analyses: - microbiota analysis through NGS of 16S rRNA gene (V4 region) amplicons followed by bioinformatic analysis -  evaluation of secretory IgA (ELISA kit)- phylogenetic analysis of FCoVs S gene sequencesInnovative results will be provided on the feline gut microbiota composition. These will be correlated with the presence and genetic polymorphisms of FCoV and mucosal defenses to establish significant correlations between the analysed factors

Rasch validation of the Activities-specific Balance Confidence Scale and its short versions in patients with Parkinson's disease.

Accepted Jan 10, 2014; Epub ahead of print Mar 28, 2014IntroductIonPostural instability is a key feature of advanced Parkinson's dis-ease (PD) (1), often leading to falls with dramatic consequences (2). Balance impairment can also induce psychological reactions, such as reduced balance confidence and increased fear of fall-ing. Balance confidence (a construct exploring fall-related self-efficacy) and fear of falling may be protective if they interfere only with hazardous activity and increase caution in performing daily living tasks. On the other hand, they can be maladaptive if their effect is to restrict mobility, independence and social par -ticipation, leading to further deconditioning, functional decline and poorer quality of life (3). Therefore, balance confidence is a construct that needs to be clearly understood, accurately meas-ured, and requires timely, appropriate management within fall risk assessment and fall prevention programmes (4).Balance confidence is often analysed with the Activities-specific Balance Confidence scale (ABC) (5), which has been shown to have sound psychometric properties in older adults (5, 6) and people with lower-limb amputations (7, 8) and stroke (9). In subjects with PD, the ABC has only recently been validated (10), by means of a Classical Test Theory approach, while the short versions of ABC proposed for PD (11–13) have undergone only preliminary validation.However, the Classical Test Theory approach does not take into account some standard criteria and attributes (concerning both single items and total score) that need to be considered when evaluating the measurement properties of a tool (14). Rasch analysis is being increasingly recommended in the development and evaluation of clinical tools for healthcare to verify if they comply with the theoretical requirements of measurement, including dimensionality analysis and item-level scale evaluation (15).The aim of this study was to analyse the psychometric prop-erties of the ABC and its 3 short versions in subjects with PD, using both Classical Test Theory and Rasch analysis, in order to determine whether fundamental measurement properties are satisfied and to provide insights into the optimal use of these questionnaires. METHOD

Molecular adaptations in Antarctic fish and bacteria

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An attempt to prevent production diseases in dairy cows by intense monitoring and ad hoc treatment

A trial has been performed on 201 dairy cows from two Italian commercial herds in order to verify whether the mitigation of a recognized negative energy balance (NEB) by a therapeutic mean may influence the incidence of peri-partum diseases. All animals were tested for beta-hydroxybutyrate (β-HOB) and non-esterified fatty acids (NEFA) three times a week from 2 weeks before the expected due time to 2 weeks after calving. Animals whose blood levels were above β-HOB>1.2 or NEFA>0.5 mmol/L were declared POSITIVE and then split in two groups. Group T animals (n=57) were treated with a glycogenic treatment (ENERGAN KETOSIS, Virbac). The treatment was repeated daily as long as biochemical values remained abnormal. Group C animals (n=48) served as untreated controls. Animals with values within the physiological range over the study period were said NEGATIVE (n=96). This study confirmed that animals presenting excessive β-HOB or NEFA concentrations show a higher risk to get sick during the study period (P<0.05), the major risk being clinical ketosis (P<0.01) and in a lesser extend retention of the placenta (P=0.09). The application of a glycogenic treatment did not show an impact on blood metabolite levels due to huge individual differences. However, application of the treatment for an average duration of 5 days tends to reduce the incidence of all the diseases related to a NEB. Moreover, untreated control animals were more likely to get dislocation of the abomasum (P<0.05) than NEGATIVE animals whereas treated animals were not

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.

Validation of a Paraoxon-based method for measurement of Paraoxonase (PON-1) activity and establishment of RI in horses

Paraoxonase-1 (PON-1) is an anti-oxidant compound considered as negative acute phase protein in animals (Rossi et al., 2013) and people (Novak et al., 2010). The paraoxon-based method for measurement of PON-1 in equine serum has not yet been validated.The aim of this study is to validate a paraoxon-based method to measure PON-1 and to establish reference intervals (RIs) in healthy horses and foals.120 horses (40 geldings, 40 stallions, 40 mares; median age: 11 years; 57 Warmbloods, 46 Trotters) and 55 foals (27 females, 28 males; median age: 47 days; 22 Warmbloods, 31 Trotters) considered healthy after physical examination and biochemistry were examined. Horses were grouped by breed: Thoroughbreds, Trotters, Warmbloods, Draft horses and Ponies. Serum PON-1 was measured with an automated spectrophotometer and an enzymatic method validated in other species (Giordano et al., 2013). After the analytical validation (precision, accuracy, interference studies), RIs were determined using the Reference Value Advisor software, according to ASCVP guidelines (Friedrichs et al., 2012). The possible gender-, age- and breed-related differences were statistically investigated.The paraoxon-based method was precise (CVs &lt;4.0%) and accurate (P&lt;0.001 in linearity under dilution and spike-recovery testing) but is affected by interference from mild bilirubinemia, severe lipemia or hemoglobinemia. The RIs recorded in the whole population was 38.1-80.8 U/mL. According to the Harris and Boyd test, separate RIs are recommended only for adult females and for Warmblood and Trotter adults (Figure 1).This study demonstrated that analytical performances of the paraoxon-based method for measurement of PON-1 in horses are acceptable. PON-1 is lower in horses than in other species.If future studies will demonstrate that oxidative stress induces a significant decrease of PON-1, this results will be useful to correctly classify healthy and sick horses; PON-1 could be used, as in human medicine, as a marker of oxidative stress