A High Sensitive Nested PCR for Toxoplasma gondii Detection in Animal and Food Samples

Toxoplasma gondii is a major food and waterborne transmitted parasite world-wide. The tissues and meat samples of many warm blooded animals can contain tissues cysts from chronic toxoplasmosis. Water and vegetable can be contaminated by the parasitic oocysts shed through the feces of infected cats, representing the definitive host of the parasite. A sensitive PCR for Toxoplasma gondii detection is described. The first step amplified the region between the 28S and 18S rDNA in the closely related T. gondii and Neospora caninum; RFLP analysis distinguished the DNA from the two morphologically identical parasites. Although N. caninum is not involved in human transmission, so far, it is important for animal health since is a major responsible for abortion in cattle. The nested PCR was used in a dilution assay in pork sausage samples spiked with T. gondii parasitic DNA. The analysis showed that up to 200fg equivalent to two single parasites only, could be detected. Similar detection limit for T. gondii can be obtained with real-time PCRs, but real time methods need special consumables and expensive equipment

Short communication: Fourier-transform mid-infrared spectroscopy to predict coagulation and acidity traits of sheep bulk milk.

Sheep milk is mainly transformed into cheese; thus, the dairy industry seeks more rapid and cost-effective methods of analysis to determine milk coagulation and acidity traits. This study aimed to assess the feasibility of Fourier-transform mid-infrared spectroscopy to determine milk coagulation and acidity traits of sheep bulk milk and to classify milk samples according to their renneting capacity. A total of 465 bulk milk samples collected in 140 single-breed flocks of Comisana (84 samples, 24 flocks) and Sarda (381 samples, 116 flocks) breeds located in Central Italy were analyzed for coagulation properties (rennet coagulation time, curd firming time, and curd firmness) and acidity traits (pH and titratable acidity) using standard laboratory procedures. Fourier-transform mid-infrared spectroscopy prediction models for these traits were built using partial least squares regression analysis and were externally validated by randomly dividing the full data set into a calibration set (75%) and a validation set (25%). The discriminant capacity of the rennet coagulation time prediction model was determined using partial least squares discriminant analysis. Prediction models were more accurate for acidity traits than for milk coagulation properties, and the ratio of prediction to deviation ranged from 1.01 (curd firmness) to 2.14 (pH). Moreover, the discriminant analysis led to an overall accuracy of 74 and 66% for the calibration and validation sets, respectively, with greater sensitivity for samples that coagulated between 10 and 20 min and greater specificity to detect early-coagulating (<10 min) and late-coagulating (20-30 min) samples. Results suggest that Fourier-transform mid-infrared spectroscopy has the potential to help the dairy sheep industry identify milk with better coagulation ability for cheese production and thus improve milk transformation efficiency. However, further research is needed before this information can be exploited at the industry level

Hydroxyapatite Whiskers Based Resin Composite versus Commercial Dental Composites: Mechanical and Biocompatibility Characterization

A systematic evaluation of mechanical and biocompatibility properties of different volume fractions of hydroxyapatite whiskers in comparison with three commercial dental composites filled with micro- and nanosilica particles was carried out. Six groups with different hydroxyapatite whiskers mass fractions were taken into account in order to be compared with the performances of silica particles based composites group. Flexural properties were evaluated via a universal testing machine (2.5 kN Zwick Line) with a 2 kN load-cell (sensitivity 0.001 N). The test was replicated 10 times for the seven experimental groups to better identify statically the significance of the mechanical performances data. MTT quantitative colorimetric assay was performed in order to evaluate the mitochondrial activity of living cells exposed to different resin composites. Data obtained show better interfacial interaction with filler/matrix until 20 wt% of hydroxyapatite whiskers partially replaced silica particles filler. After this threshold, the mechanical performances decrease dramatically due to both the hydroxyapatite agglomerates formation and the low degree of resin conversion. In addition, biocompatibility test showed less cytotoxic effect with the addition of 20 wt% of hydroxyapatite in comparison with higher rates

Glutamate-evoked redox state alterations are involved in tissue transglutaminase upregulation in primary astrocyte cultures

AbstractThe aim of this study was to evaluate the involvement of oxidative stress in glutamate-evoked transglutaminase (TGase) upregulation in astrocyte cultures (14 DIV). A 24 h exposure to glutamate caused a dose-dependent depletion of glutathione intracellular content and increased the ROS production in cell cultures. These effects were receptor-mediated, as demonstrated by inhibition with GYKI 52466. The pre-incubation with glutathione ethyl ester or cysteamine recovered oxidative status and was effective in significantly reducing glutamate-increased tissue TGase. These data suggest that tissue TGase upregulation may be part of a biochemical response to oxidative stress induced by a prolonged exposure of astrocyte cultures to glutamate

Iodine Extravasation Quantification on Dual-Energy CT of the Brain Performed after Mechanical Thrombectomy for Acute Ischemic Stroke Can Predict Hemorrhagic Complications

BACKGROUND AND PURPOSE: Intracerebral hemorrhage represents a potentially severe complication of revascularization of acute ischemic stroke. The aim of our study was to assess the capability of iodine extravasation quantification on dual-energy CT performed immediately after mechanical thrombectomy to predict hemorrhagic complications. MATERIALS AND METHODS: Because this was a retrospective study, the need for informed consent was waived. Eighty-five consecutive patients who underwent brain dual-energy CT immediately after mechanical thrombectomy for acute ischemic stroke between August 2013 and January 2017 were included. Two radiologists independently evaluated dual-energy CT images for the presence of parenchymal hyperdensity, iodine extravasation, and hemorrhage. Maximum iodine concentration was measured. Follow-up CT examinations performed until patient discharge were reviewed for intracerebral hemorrhage development. The correlation between dual-energy CT parameters and intracerebral hemorrhage development was analyzed by the Mann-Whitney U test and Fisher exact test. Receiver operating characteristic curves were generated for continuous variables. RESULTS: Thirteen of 85 patients (15.3%) developed hemorrhage. On postoperative dual-energy CT, parenchymal hyperdensities and iodine extravasation were present in 100% of the patients who developed intracerebral hemorrhage and in 56.3% of the patients who did not ( P = .002 for both). Signs of bleeding were present in 35.7% of the patients who developed intracerebral hemorrhage and in none of the patients who did not ( P P CONCLUSIONS: The presence of parenchymal hyperdensity with a maximum iodine concentration of >1.35 mg/mL may identify patients developing intracerebral hemorrhage with 100% sensitivity and 67.6% specificity

Intracellular fate and impact on gene expression of doxorubicin/cyclodextrin-graphene nanomaterials at sub-toxic concentration

The graphene road in nanomedicine still seems very long and winding because the current knowledge about graphene/cell interactions and the safety issues are not yet sufficiently clarified. Specifically, the impact of graphene exposure on gene expression is a largely unexplored concern. Herein, we investigated the intracellular fate of graphene (G) decorated with cyclodextrins (CD) and loaded with doxorubicin (DOX) and the modulation of genes involved in cancer-associated canonical pathways. Intracellular fate of GCD@DOX, tracked by FLIM, Raman mapping and fluorescence microscopy, evidenced the efficient cellular uptake of GCD@DOX and the presence of DOX in the nucleus, without graphene carrier. The NanoString nCounter™ platform provided evidence for 34 (out of 700) differentially expressed cancer-related genes in HEp-2 cells treated with GCD@DOX (25 µg/mL) compared with untreated cells. Cells treated with GCD alone (25 µg/mL) showed modification for 16 genes. Overall, 14 common genes were differentially expressed in both GCD and GCD@DOX treated cells and 4 of these genes with an opposite trend. The modification of cancer related genes also at sub-cytotoxic G concentration should be taken in consideration for the rational design of safe and effective G-based drug/gene delivery systems. The reliable advantages provided by NanoString® technology, such as sensibility and the direct RNA measurements, could be the cornerstone in this field

Why laparoscopists may opt for three-dimensional view: a summary of the full HTA report on 3D versus 2D laparoscopy by S.I.C.E. (Società Italiana di Chirurgia Endoscopica e Nuove Tecnologie)

Background: Three-dimensional view in laparoscopic general, gynaecologic and urologic surgery is an efficient, safe and sustainable innovation. The present paper is an extract taken from a full health technology assessment report on three-dimensional vision technology compared with standard two-dimensional laparoscopic systems. Methods: A health technology assessment approach was implemented in order to investigate all the economic, social, ethical and organisational implications related to the adoption of the innovative three-dimensional view. With the support of a multi-disciplinary team, composed of eight experts working in Italian hospitals and Universities, qualitative and quantitative data were collected, by means of literature evidence, validated questionnaire and self-reported interviews, applying a final MCDA quantitative approach, and considering the dimensions resulting from the EUnetHTA Core Model. Results: From systematic search of literature, we retrieved the following studies: 9 on general surgery, 35 on gynaecology and urology, both concerning clinical setting. Considering simulated setting we included: 8 studies regarding pitfalls and drawbacks, 44 on teaching, 12 on surgeons’ confidence and comfort and 34 on surgeons’ performances. Three-dimensional laparoscopy was shown to have advantages for both the patients and the surgeons, and is confirmed to be a safe, efficacious and sustainable vision technology. Conclusions: The objective of the present paper, under the patronage of Italian Society of Endoscopic Surgery, was achieved in that there has now been produced a scientific report, based on a HTA approach, that may be placed in the hands of surgeons and used to support the decision-making process of the health providers

Impact of multiple sclerosis risk loci in postinfectious neurological syndromes

Background: The genetic component of multiple sclerosis (MS) is now set to 200 autosomal common variants. However, it is unclear how genetic knowledge be clinically used in the differential diagnosis between MS and other inflammatory conditions like adult-onset postinfectious neurological syndromes (PINS). The aim of this study was to investigate whether PINS and MS have a shared genetic background using an updated polygenic risk scores. / Methods: Eighty-eight PINS patients have been consecutively recruited between 1996 and 2016 at Mondino Foundation of Pavia, diagnosed according to clinical, MRI and CSF findings and followed-up for several years. Patients were typed using Illumina array, and genotypes imputed using the 1000 Genomes Project reference panel. A weighted genetic risk score (wGRS) has been calculated based on autosomal MS risk loci derived from large-scale studies, and an HLA genetic burden (HLAGB) was also calculated on loci associated to MS. / Results: PINS occurred as an episode of myelitis in 44% of patients, encephalomyelitis in 44%, and encephalitis in remaining cases, with an involvement of peripheral nervous system in 41% of patients. Mean age of onset was 50.1 years, and female:male ratio was 1.4. Patients were followed-up for a mean of 7.2 years, and at last visit 55% had a low disability grade (mRS 0–1). Disease was monophasic in 67% of patients, relapsing in 18% and chronic-progressive in 15%. The wGRS of PINS cases was comparable to 370 healthy controls, while significantly lower compared to 907 bout-onset MS (BOMS) cases (wGRS= 20.9 vs 21.2; p<0.0001). The difference was even larger for PINS with peripheral nervous system involvement (wGRS=20.6) vs BOMS. / Conclusion: The distinction between MS and PINS is not easy to make in clinical practice. However, our study shows that the new set of MS risk alleles does not confer increased susceptibility to PINS. These data support the importance to discriminate these cases from MS with pathophysiological and therapeutic implications

Mutations in alpha-B-crystallin cause autosomal dominant axonal Charcot–Marie–Tooth disease with congenital cataracts

Background and purpose: Mutations in the alpha-B-crystallin (CRYAB) gene have initially been associated with myofibrillar myopathy, dilated cardiomyopathy and cataracts. For the first time, peripheral neuropathy is reported here as a novel phenotype associated with CRYAB. // Methods: Whole-exome sequencing was performed in two unrelated families with genetically unsolved axonal Charcot–Marie–Tooth disease (CMT2), assessing clinical, neurophysiological and radiological features. // Results: The pathogenic CRYAB variant c.358A>G;p.Arg120Gly was segregated in all affected patients from two unrelated families. The disease presented as late onset CMT2 (onset over 40 years) with distal sensory and motor impairment and congenital cataracts. Muscle involvement was probably associated in cases showing mild axial and diaphragmatic weakness. In all cases, nerve conduction studies demonstrated the presence of an axonal sensorimotor neuropathy along with chronic neurogenic changes on needle examination. // Discussion: In cases with late onset autosomal dominant CMT2 and congenital cataracts, it is recommended that CRYAB is considered for genetic testing. The identification of CRYAB mutations causing CMT2 further supports a continuous spectrum of expressivity, from myopathic to neuropathic and mixed forms, of a growing number of genes involved in protein degradation and chaperone-assisted autophagy

Can CANVAS due to RFC1 biallelic expansions present with pure ataxia?

Background Biallelic expansion of AAGGG in the replication factor complex subunit 1 (RFC1) was identified as a major cause of cerebellar ataxia, neuropathy (sensory ganglionopathy, or SG) and vestibular areflexia syndrome (CANVAS). We wanted to clarify if RFC1 expansions can present with pure ataxia and if such expansions could be responsible for some cases where an alternative diagnosis had been made. Methods We identified patients with a combination of ataxia and SG and no other cause found, patients where an alternative diagnosis had been made, and patients with pure ataxia. Testing for RFC1 expansions was done using established methodology. Results Among 54 patients with otherwise idiopathic sporadic ataxia without SG, none was found to have RFC1 expansions. Among 38 patients with cerebellar ataxia and SG in which all other causes were excluded, 71% had RFC1 expansions. Among 27 patients with cerebellar ataxia and SG diagnosed with coeliac disease or gluten sensitivity, 15% had RFC1 expansions. Conclusions Isolated cerebellar ataxia without SG makes the diagnosis of CANVAS due to RFC1 expansions highly improbable, but CANVAS is frequently the cause of the combination of idiopathic cerebellar ataxia with SG. It is important to screen patients diagnosed with other causes of acquired ataxia and SG as a small percentage were found to have RFC1 expansions