Search

Abstract Objective No direct comparisons of the effect of natalizumab and ocrelizumab on progression independent of relapse activity (PIRA) and relapse‐associated worsening (RAW) events are currently available. We aimed to compare the risk of achieving first 6 months confirmed PIRA and RAW events and irreversible Expanded Disability Status Scale (EDSS) 4.0 and 6.0 in a cohort of naïve patients treated with natalizumab or ocrelizumab from the Italian Multiple Sclerosis Register. Methods Patients with a first visit within 1 year from onset, treated with natalizumab or ocrelizumab, and ≥3 visits were extracted. Pairwise propensity score‐matched analyses were performed. Risk of reaching the first PIRA, RAW, and EDSS 4.0 and 6.0 events were estimated using multivariable Cox proportional hazards models. Kaplan–Meier curves were used to show cumulative probabilities of reaching outcomes. Results In total, 770 subjects were included (natalizumab = 568; ocrelizumab = 212) and the propensity score‐matching retrieved 195 pairs. No RAW events were found in natalizumab group and only 1 was reported in ocrelizumab group. A first PIRA event was reached by 23 natalizumab and 25 ocrelizumab exposed patients; 7 natalizumab‐ and 10 ocrelizumab‐treated patients obtained an irreversible EDSS 4.0, while 13 natalizumab‐ and 15 ocrelizumab‐treated patients reached an irreversible EDSS 6.0. No differences between the two groups were found in the risk (HR, 95%CI) of reaching a first PIRA (1.04, 0.59–1.84; p = 0.88) event, an irreversible EDSS 4.0 (1.23, 0.57–2.66; p = 0.60) and 6.0 (0.93, 0.32–2.68; p = 0.89). Interpretation Both medications strongly suppress RAW events and, in the short term, the risk of achieving PIRA events, EDSS 4.0 and 6.0 milestones is not significantly different.

Abstract Tectonic plate motions drive the earthquake cycle, as they result in the slow accrual and sudden release of energy along plate boundaries. Steadiness of plate motions over the earthquake cycle is a central tenet of the plate tectonics theory and has long been a main pillar in models of earthquake genesis, or of plate-margins seismic potential inferred from slip-deficit estimates. The advent of geodesy in the geosciences and the availability of multi-year-long series of position measurements permit tracking the motions of tectonic plates from before to after the time of significant seismic events that occur along their margins. Here, we present evidence that large earthquakes are capable of modifying the motions of entire microplates. We use high precision Global Navigation Satellite System (GNSS) position time-series covering the periods 2001–2004 and 2014–2017 to demonstrate that, contrary to the tenet above, the South China microplate motion changed after the 2008 $$M_W$$ M W 7.9 Great Wenchuan earthquake. The GNSS data and associated uncertainties indicate a plate motion slowdown of up to 20% that is beyond the possible impact of data noise and is thus tectonically meaningful. We use quantitative models of torque balance to show that generating this kinematic change requires a force upon the South China microplate compatible with that imparted by the Great Wenchuan earthquake of 2008. The existence of a kinematic signal linked to the earthquake cycle that impacts an entire microplate might offer an additional, novel perspective to assessing the hazards of earthquake-prone tectonic regions.

Specific characteristics of the eggs from nucleus populations of Ancona (AN), Bianca di Saluzzo (BS), Bionda Piemontese (BP), Livorno, Mericanel della Brianza (MB), Mugellese (MU), Siciliana (SI) and Valdarnese (VA) chicken breeds kept in University Research Centres across Italy were evaluated. The physical characteristics considered were whole egg weight and shell, yolk and albumen proportion. The chemical characteristics considered were dry matter (DM), ash and protein content of the yolk and albumen, and the yolk fat. Among the breed groups evaluated in this study, BS and BP produce large eggs that are rich in total crude protein and lipids, with abundant egg whites. MB and MU produce small eggs with a high yolk percentage. AN and SI hens produce medium-small-sized eggs. The heaviest yolks were observed in AN eggs, and the highest shell percentage was found in SI eggs. LB and VA produce medium-large-sized eggs with low yolk lipid content.

Significant changes in the quality and metabolic profile of fresh turkey sperm as a result of both cryopreservation and reproductive age have already been individually confirmed in our previous studies. This new dataset adds a relevant piece to the tangled puzzle of changes in metabolite levels affecting cryopreserved turkey sperm quality, taking into consideration two different reproductive period ages. Fresh semen samples were collected at 44 and 56 weeks of age and exposed to the cryopreservation process. All fresh and frozen-thawed samples were subjected to analysis of mobility, viability and osmotic tolerance as parameters for evaluating the sperm quality, while NMR spectroscopy was used to assess the quantitative changes in water and lipid-soluble metabolites. Our results showed that the cryopreservation process significantly affected all of the measured qualitative parameters both at 44 and 56 weeks. Concerning the metabolic profile, a greater number of quantitative changes for both water and lipid-soluble components were found in frozen semen at 56 weeks than at 44 weeks of age. These data could contribute to identifying new strategies aimed at improving freezing procedures even as reproductive age increases.

Abstract Over million years, convergence between the Nazca and South America tectonic plates results in Andean orogeny. Over decades/centuries, it fuels the earthquake cycle of the Andean megathrust. It is well recognised that, over the geologically-long term of million years, Andean orogeny feeds back onto plate convergence rates, generating temporal changes documented throughout the Neogene. In contrast, no feedback mechanism operated over the geologically-short term by the earthquake cycle is currently contemplated. In fact, it is commonly assumed that the rates of contemporary convergence, which are accurately measured via geodesy, remain steady during the megathrust earthquake cycle. Here we investigate whether the contemporary Nazca/South America plate motion varies over year-/decade-long periods in response to megathrust stress variations associated with the earthquake cycle. We focus on the decade preceding the three largest and most recent $${\varvec{M_w > 8}}$$ M w > 8 earthquakes (2010 $${\varvec{M_w = 8.8}}$$ M w = 8.8 Maule, 2014 $${\varvec{M_w = 8.1}}$$ M w = 8.1 Iquique, 2015 $${\varvec{M_w = 8.3}}$$ M w = 8.3 Illapel), and find slowdowns of both Nazca and South America whole-plate motions that exceed the impact of data uncertainty or noise. We show that the torque variations required upon Nazca and South America to generate the slowdowns are consistent with that arising from the buildup of interseismic stress preceding the earthquakes.

Abstract Tectonic plate motions are a prime constraint on lithosphere dynamics and on the torques acting upon plates. Researchers typically test hypotheses on the controlling torques via forward computer models, which allow accepting or rejecting hypotheses on the basis of the fit of model–output plate velocities to kinematic reconstructions. Such models typically require a significant amount of input information (e.g., tectonic boundaries, structure of the lithosphere, lateral variations of rheology, among others) to appropriately model the tectonic system, and obtain sufficiently–realistic realizations of plate motions. Alternatively, the inverse problem approach takes the difference between the plate torque–balance at two distinct moments in time. This results in an equation in which (a) torques that have not varied through time are canceled out and (b) torques that instead have varied through time are linked to the resulting plate–motion change through a term that accounts for the plate shape and the rheology of the underlying asthenosphere. This approach sacrifices the capability to describe the different individual forces/torques acting upon a plate. Instead, it focuses on determining torque temporal variations, which significantly reduces the amount of input knowledge required. In this technical note we introduce MYRIAM, an open–source software that implements such an inverse approach. MYRIAM takes plate–motion temporal changes, and outputs an estimate of the torque variation required upon a plate to generate the input kinematic change. MYRIAM is released as an open–source repository hosted at GitHub, complete with source code and executable files for Windows and Unix–based operating systems.

Abstract Background Information processing speed is commonly impaired in people with multiple sclerosis (PwMS). However, depression and fatigue can affect the cognitive profile of patients: fatigue has a negative impact from the disease’s earliest stage and a reduced information processing speed is often associated with higher levels of depression. Therefore, the aim of this study was to investigate the correlations between information processing speed and physical fatigue in a cohort of Italian PwMS from a single center, considering the effect of depression. Methods Two hundred (W = 128; mean age = 39.83 years; SD = 11.86) PwMS, from the Bari University Hospital, underwent testing for processing speed (Symbol Digit Modalities Test [SDMT]), fatigue level (Fatigue Severity Scale [FSS]), and depression (Beck’s Depression Inventory [BDI]). Results Statistically significant correlations emerged between SDMT and FSS, SDMT and BDI, FSS and BDI. Mediation analyses revealed that while physical fatigue had no significant direct negative effect on information processing speed (z=-0.891; p > 0.05), depression predicted the relationship between fatigue and information processing speed (z=-2.181; p

Abstract Chimeric antigen receptor T (CAR-T) cell therapy may achieve long-lasting remission in patients with B-cell malignancies not responding to conventional therapies. However, potentially severe and hard-to-manage side effects, including cytokine release syndrome (CRS), neurotoxicity and macrophage activation syndrome, and the lack of pathophysiological experimental models limit the applicability and development of this form of therapy. Here we present a comprehensive humanized mouse model, by which we show that IFNγ neutralization by the clinically approved monoclonal antibody, emapalumab, mitigates severe toxicity related to CAR-T cell therapy. We demonstrate that emapalumab reduces the pro-inflammatory environment in the model, thus allowing control of severe CRS and preventing brain damage, characterized by multifocal hemorrhages. Importantly, our in vitro and in vivo experiments show that IFNγ inhibition does not affect the ability of CD19-targeting CAR-T (CAR.CD19-T) cells to eradicate CD19+ lymphoma cells. Thus, our study provides evidence that anti-IFNγ treatment might reduce immune related adverse effect without compromising therapeutic success and provides rationale for an emapalumab-CAR.CD19-T cell combination therapy in humans.

BackgroundTreatment switching is a common challenge and opportunity in real-world clinical practice. Increasing diversity in disease-modifying treatments (DMTs) has generated interest in the identification of reliable and robust predictors of treatment switching across different countries, DMTs, and time periods.ObjectiveThe objective of this retrospective, observational study was to identify independent predictors of treatment switching in a population of relapsing-remitting MS (RRMS) patients in the Big Multiple Sclerosis Data Network of national clinical registries, including the Italian MS registry, the OFSEP of France, the Danish MS registry, the Swedish national MS registry, and the international MSBase Registry.MethodsIn this cohort study, we merged information on 269,822 treatment episodes in 110,326 patients from 1997 to 2018 from five clinical registries. Patients were included in the final pooled analysis set if they had initiated at least one DMT during the relapsing-remitting MS (RRMS) stage. Patients not diagnosed with RRMS or RRMS patients not initiating DMT therapy during the RRMS phase were excluded from the analysis. The primary study outcome was treatment switching. A multilevel mixed-effects shared frailty time-to-event model was used to identify independent predictors of treatment switching. The contributing MS registry was included in the pooled analysis as a random effect.ResultsEvery one-point increase in the Expanded Disability Status Scale (EDSS) score at treatment start was associated with 1.08 times the rate of subsequent switching, adjusting for age, sex, and calendar year (adjusted hazard ratio [aHR] 1.08; 95% CI 1.07–1.08). Women were associated with 1.11 times the rate of switching relative to men (95% CI 1.08–1.14), whilst older age was also associated with an increased rate of treatment switching. DMTs started between 2007 and 2012 were associated with 2.48 times the rate of switching relative to DMTs that began between 1996 and 2006 (aHR 2.48; 95% CI 2.48–2.56). DMTs started from 2013 onwards were more likely to switch relative to the earlier treatment epoch (aHR 8.09; 95% CI 7.79–8.41; reference = 1996–2006).ConclusionSwitching between DMTs is associated with female sex, age, and disability at baseline and has increased in frequency considerably in recent years as more treatment options have become available. Consideration of a patient's individual risk and tolerance profile needs to be taken into account when selecting the most appropriate switch therapy from an expanding array of treatment choices.

BackgroundThe debate on how to manage women affected by multiple sclerosis (MS) during reproductive age is still open, as is the issue of fertility in such patients. Main issue regard the identification of the optimal window for pregnancy and how to deal with medical therapy before and during conception. The aim of this Delphi consensus was to collect the opinions of a multidisciplinary group, involving reproductive medicine specialists and neurologists with experience in the management of multiple sclerosis women with reproductive desire.MethodsFour experts plus scientific coordinators developed a questionnaire distributed online to 10 neurologists and later discussed the responses and amended a list of statements. The statements were then distributed via an online survey to 23 neurologists (comprising the first 10), who voted on their level of agreement/disagreement with each statement. Consensus was achieved if agreement or disagreement with a statement exceeded 66%.ResultsTwenty-one statements reached consensus after two rounds of voting, leading to the following main recommendations: (1) Fertility evaluation should be suggested to wMS, in case of the need to shorten time to pregnancy and before treatment switch in women on DMTs contraindicated in pregnancy, particularly in case of highly active disease and age > 35 years. (2) ART should not be discouraged in wMS, but the use of DMTs until pregnancy confirmation should be suggested; ART may be considered in order to reduce time to pregnancy in MS women with a reduced ovarian reserve and/or age > 35 years, but in case of an expected poor ART prognosis and the need for more than one ART cycle, a switch to a high-efficacy DMD before ART should be offered. (3) Oocyte cryopreservation may be considered in women with reduced ovarian reserve, with unpredictable time to complete diagnostic workup and achieve disease control; a risk/cost–benefit analysis must be performed in women >35 years, considering the diminished ovarian reserve.ConclusionThis consensus will help MS neurologists to support family planning in wMS, respecting MS therapeutic needs while also taking into account the safety and impact of advancing age on fertility.

Native breed conservation is an important component of poultry biodiversity. The aim of this work is to describe different steps that lead to donor selection for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. The variability within and between breeds was evaluated, and the stored semen reproductive capacity was in vivo tested using artificial insemination. Semen from Bionda Piemontese, Bianca di Saluzzo and Pepoi roosters was collected and processed. Concentration, volume, sperm membrane integrity, total motile sperm, progressive motile sperm and kinetic parameters were analyzed; sperm parameters accounting for bird variability were used to select male donors. Fresh semen quality parameters measured in donor ejaculates showed significant differences between breeds; no differences were found after cryopreservation. Variability in the fertilizing ability of cryopreserved semen was found within a breed (5–16%) and between birds within a breed (BP = 3–7%; BS = 7–31%; PP = 6–22%); only sperm quality parameters measured in fresh ejaculates, not frozen/thawed, may be associated with in vivo fertility results. In conclusion, sperm concentration and progressive motility were successfully used as selection parameters to identify chicken male donors with improved sperm quality for sperm cryobanking. However, new reliable sperm markers to predict cryopreserved semen’s fertilizing ability are required.

Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers. Briefly, LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) were used to modify HEK293 and HEK293T cells. These cell populations were then expanded, and integrated LV vector genomes were rescued by transfection. LV vectors were harvested, and the process of sequential transduction and rescue-transfection was iterated. Through this workflow, guide RNAs (gRNAs) that target genes that may suppress or enhance LV vector production were enriched and identified with Next-Generation Sequencing (NGS). Though more work is needed to test genes identified in this screen, we expect that perturbations of genes we identified here, such as TTLL12, which is an inhibitor of antiviral innate immunity may be introduced and multiplexed to yield cell lines with improved LV vector productivity.

Agrobacterium rhizogenes transformation is a more rapid method of obtaining transgenic and edited rubber dandelion (Taraxacum kok-saghyz) plants than Agrobacterium tumefaciens. The hairy root rol genes are present alongside transgenes after transformation, and they change the morphology of rubber dandelion significantly. Although these rol genes are useful visual markers indicating successful transformation of rubber dandelion, they modify the phenotype induced by the target transgenes and are ultimately detrimental to agronomic traits. Fortunately, the rol genes can be removed by conventional plant breeding because they segregate in progeny separately from the targeted transgenes. However, it is preferable to have preliminary identification of promising effects induced by transgenes or gene edits before rol gene removal so that only the best plants are used for breeding. Therefore, the goal of this research was to characterize rol– and rol+ plant morphology so that, in the future, rol+ transgene+ plants can be easily distinguished from rol+ transgene– plants. This requires that rol gene–induced morphological changes and simply assayed physiological traits are first characterized thoroughly so that transgene changes may be observed. Taproot formation is reduced or eliminated in rubber dandelion by rol genes, and rol-induced hairy roots are identifiable easily because they grow shallowly in potting soil, so only partial unearthing is needed. Both leaf and flower numbers are increased by rol genes, but leaves and flowers are smaller than in rubber dandelion wild type with longer stalks. The rosette doming phenotype caused by the induction of a large number of leaf primordia is obvious in rooted plants as young as 1 month old. Photosynthetic rates are reduced significantly in rol+ plants, although growth is not. An accurate description of the morphology of rubber dandelion after A. rhizogenes transformation may allow for initial selection of promising transformed plants before confirmation with polymerase chain reaction, by phenotypic comparison of plants expressing transgenes and the rol gene, with those only expressing the rol gene.

Abstract Background Paediatric acute myeloid leukaemia (AML) is characterized by poor outcomes in patients with relapsed/refractory disease, despite the improvements in intensive standard therapy. The leukaemic cells of paediatric AML patients show high expression of the CD123 antigen, and this finding provides the biological basis to target CD123 with the chimeric antigen receptor (CAR). However, CAR.CD123 therapy in AML is hampered by on-target off-tumour toxicity and a long “vein-to-vein” time. Methods We developed an off-the-shelf product based on allogeneic natural killer (NK) cells derived from the peripheral blood of healthy donors and engineered them to express a second-generation CAR targeting CD123 (CAR.CD123). Results CAR.CD123-NK cells showed significant anti-leukaemia activity not only in vitro against CD123+ AML cell lines and CD123+ primary blasts but also in two animal models of human AML-bearing immune-deficient mice. Data on anti-leukaemia activity were also corroborated by the quantification of inflammatory cytokines, namely granzyme B (Granz B), interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α), both in vitro and in the plasma of mice treated with CAR.CD123-NK cells. To evaluate and compare the on-target off-tumour effects of CAR.CD123-T and NK cells, we engrafted human haematopoietic cells (hHCs) in an immune-deficient mouse model. All mice infused with CAR.CD123-T cells died by Day 5, developing toxicity against primary human bone marrow (BM) cells with a decreased number of total hCD45+ cells and, in particular, of hCD34+CD38− stem cells. In contrast, treatment with CAR.CD123-NK cells was not associated with toxicity, and all mice were alive at the end of the experiments. Finally, in a mouse model engrafted with human endothelial tissues, we demonstrated that CAR.CD123-NK cells were characterized by negligible endothelial toxicity when compared to CAR.CD123-T cells. Conclusions Our data indicate the feasibility of an innovative off-the-shelf therapeutic strategy based on CAR.CD123-NK cells, characterized by remarkable efficacy and an improved safety profile compared to CAR.CD123-T cells. These findings open a novel intriguing scenario not only for the treatment of refractory/resistant AML patients but also to further investigate the use of CAR-NK cells in other cancers characterized by highly difficult targeting with the most conventional T effector cells.

This study aimed to investigate the impact of sperm concentrations on the in vitro quality of cryopreserved rabbit semen. The semen pools (n = 8, from 80 donors) were split into five aliquots with final sperm concentrations of 15, 25, 35, 55, and 75 × 106 per straw. The sperm motility parameters (CASA system) and membrane integrity (flow cytometric analysis) were both evaluated at various stages of the cryopreservation process: fresh semen dilution, cooling, equilibration, and immediately after and 30 min post-thawing. The results indicated the significant influence of the sperm concentration on the total motility (TM) and progressive motility (PM), with a consistent decline in all sperm variables over the time points. Notably, the semen with a final concentration of 15 × 106 exhibited a higher TM and PM after cooling and equilibration. The post-thawing quality (TM, PM) was higher (p < 0.05) in the mid-range sperm concentrations of 25 × 106 (49.9% and 19.7%) and 35 × 106 (46.2% and 19.7%) compared to the other concentrations. This study demonstrated that the sperm concentration per straw played a significant role in specific phases of the cryopreservation process. These findings contribute valuable insights for refining and standardizing the cryopreservation protocol for rabbit semen, emphasizing the importance of the sperm concentration.

The Mediterranean brown trout is one of the most endangered freshwater species. A complicated network of climate and human influences has severely harmed its biodiversity. The introduction of alien trout is one of the most serious threats to native populations’ intraspecific diversity. In Molise region (south-Italy) an important conservation program (LIFE Nat.Sal.Mo project) has recently been proposed to preserve the genetic integrity of native Mediterranean trout. This project, alongside safeguarding and re-establishing the habitats’ usefulness aims to restore the genetic integrity of the autochthonous population. This is one of the major goals, and it is accomplished by employing frozen wild breeder semen in conjunction with proper fertilization techniques to carry out artificial reproduction to enhance genetic diversity in the progeny and maintain fitness within self-sustaining populations. In this regard, the implementation of the first European semen cryobank has played a strategic role for conserving extant genomic diversity of native population. The goal of this review is to outline the procedures developed and guidelines established for the creation of a Mediterranean trout sperm cryobank. Here, we specifically provide an overview of some of the main challenges associated with the implementation of semen cryobank, the results achieved, the prospects for restoring genetic integrity in native populations, and lastly, future views for hatchery management to preserve the wild biodiversity of native salmonid species. During the project timeframe 1,683 semen doses, from 150 native breeders were stored inside the cryobank. Our results clearly showed the efficiency of the freezing procedure used, both in vitro and in vivo. In fact, we recorded satisfactory values of post-thaw sperm motility and viability that ranged from 40% to 80%, and excellent fertilization rate in vivo, which ranged from 64% to 81%.

ABSTRACT: The recovery, safeguarding, and valorization of autochthonous poultry breeds may contribute toward the preservation of animal biodiversity and utilization of marginal lands that otherwise offer little agricultural or industrial value. A key strategy in promoting local breeds involves the characterization of morphological traits and productive performances, which are influenced by the breed's genetic make-up as well as its environment. The Mugellese breed is an Italian local poultry breed originating in the Mugello area of north-east Tuscany. It is characterized by frugality, resilience and resistance to disease, cold, and heat stress. Moreover, these birds are particularly suitable for free-range farming. The Mugellese chicken is described as a dwarf breed with a medium neck, broad shoulders, fairly long and horizontal wings, wide, and well-developed breast (especially in the hen). Over the course of a 1-yr observation and data collection period, involving 23 breeders and 405 adult chickens, the Mugellese breed showed the following performances: 1) a hen-day egg production characterized by 2 major peaks: the first in the spring time (March–April, 65.75%), and the second in the late summer period (August–September, 51.86%); 2) high true fertility values (94.35%) throughout the entire breeding season; 3) a weight gain of 732.44 ± 117.06 g and a feed conversion ratio of 3.94 ± 2.42 at an age of 140 d; 4) a slaughter yield of 77.80% (± 3.91); v) a respective protein, fat, and mineral content in the yolk and albumen were: 27.21 ± 4.21 g, 57.77 ± 1.03 g and 3.47 ± 0.40 g per 100 g of yolk; and 82.50 ± 0.57 g, 0.12 ± 0.01 g and 5.43 ± 0.34 g per 100 g of albumen. More data are needed to validate the data obtained in this trial.

A high-resolution model of the motion between Nazca and South American plates is presented. The work shows rapid changes that help explaining tectono-magmatic events via a balance between kinematic energy and gravitational potential energy stored in the roots of the Andes.

Although cladribine induces sustained reductions in peripheral T and B lymphocytes, little is known about its effect on axonal damage reduction in multiple sclerosis (MS), which could be demonstrated by assessing the serum neurofilament light chain (sNfL) levels. We investigated the reduction/reconstitution of different lymphocyte subsets (LS) by verifying the correlation with no evidence of disease activity (NEDA) and the variation in sNfL levels during cladribine treatment. We analysed 33 highly active relapsing MS patients and followed them up for 12 ± 3.3 months; blood samples were collected at treatment start (W0) and after 8, 24 and 48 weeks. Seventeen patients (60.7%) showed NEDA during the first treatment. At week 8, we observed a significant decrease in B memory cells, B regulatory 1 CD19+/CD38+ and B regulatory 2 CD19+/CD25+, a significant increase in T regulatory CD4+/CD25+, a slight increase in T cytotoxic CD3+/CD8+ and a non-significant decrease in T helper CD3+/CD4+. Starting from week 24, the B subsets recovered; however, at week 48, CD19+/CD38+ and CD19+/CD25+ reached values near the baseline, while the Bmem were significantly lower. The T cell subsets remained unchanged except for CD4+/CD25+, which increased compared to W0. The LS changes were not predictive of NEDA achievement. The sNfL levels were significantly lower at week 24 (p = 0.046) vs. baseline. These results could demonstrate how cladribine, by inflammatory activity depletion, can also reduce axonal damage, according to the sNfL levels.

The creation of genetic resource cryobanks provides a crucial link between in situ and ex situ techniques to improve the efficiency of conservation programs. Aim of the present review is to describe all the activities developed for the implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. These activities can be classified into three main topics: (1) identification of species-specific semen freezing/thawing reference procedures; (2) drafting Standard Operative Procedures (SOP) for the implementation of the semen cryobank; (3) storage of semen doses from Italian chicken and turkey breeds to establish the cryobank. Several trials have been developed to identify a specie-specific semen cryopreservation protocol for chickens and turkeys. The major results are reviewed and a final reference protocol described. Taking into consideration the FAO guidelines for cryoconservation of animal genetic resources, SOP were drafted with the aim to provide technical guidance and logistical support on the choice of priority breeds, selection of birds for semen production, infrastructures and storage sites, birds and semen management, cryopreservation process and doses traceability. Lastly, the Italian Semen Cryobank was created. A total of 112 semen doses from 22 cockerels of three breeds, and 74 doses from 12 turkey males of three breeds were stored in the Cryobank. Breed specific semen quality parameters assessed before and after cryopreservation are reported. The described activities provide information and tools useful for the implementation of semen cryobanking in avian species and might be transferred also to other species after appropriate adaptations.HIGHLIGHTS Implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds Drafting Standard Operative Procedures provides technical guidance and logistical support on the design and establishment of the cryobank Semen cryobank is a precious genetic reservoir and could be useful to safeguard genetic variability in small population in vivo conserved

In external fertilizer fish, ovarian fluid (OF) seems to play a key role in fertilization success, improving spermatozoa swimming performance. These OF/sperm interaction mechanisms are frequently species-specific and/or population-specific and could decrease the risk of genetic introgression of wild populations from introduced or escaped zootechnical individuals. The Mediterranean brown trout (Salmo cettii) is threatened by genetic introgression with strains of domestic brown trout (Salmo trutta) that were introduced for recreational purposes. The aim of our study was to test if native S. cettii females, rather than zootechnical S. trutta, produce OF with a greater ability to upregulate the sperm motility of conspecific males. Thus, we compared the sperm swimming performances of males inhabiting the Biferno River (Molise region—Southern Italy) activated in native S. cettii vs. zootechnical S. trutta female’s OFs. In our study, native females’ OFs (20% diluted), compared to spring water, has the ability to significantly boost the sperm performance of the autochthonous males, while zootechnical S. trutta fails. These preliminary results suggest that OF-sperm interactions could potentially influence or direct the hybridization mechanisms involving the native Mediterranean trout inhabiting the Biferno River and the domestic lineage of brown trout introduced in the past.

D-532 fertilization solution is generally used to replace the water or ovarian fluid during artificial reproductive practices in salmonids due to its ability to boost sperm motility and increase fertilization rates compared with natural activation media. However, the maintenance of ovarian fluid in a reproductive microenvironment gives it the advantage of protecting the eggs from potential harmful factors from the external environment and simplifying the field operations related to its removal when D-532 is used alone. In light of this, the aim of the present study was to investigate in vitro, for the first time, the effect of ovarian fluid (OF 100%) on post-thaw sperm swimming performance of Mediterranean trout, comparing it with D-532 and a mixed solution of 50% D-532 and 50% ovarian fluid (OF 50%). The percentage of motile spermatozoa and movement duration was significantly increased in OF 100% and OF 50% compared with D-532. Sperm velocity was higher in D-532, but significant differences were recorded only with OF 100%. In conclusion, these results suggest that the presence of ovarian fluid alone or in combination with D-532 in an artificial microenvironment of reproduction represents a key factor in potentially increasing fertilization success when the frozen semen of Mediterranean brown trout is used.

Intensive fish farming is responsible for the occurrence of various stressors that negatively affect the health of cultured animals. Polyphenols are plant-derived compounds with biological activities and positive influences on the performances and immunity of fish. The aim of the present study was to evaluate the effects of dietary inclusion of a polyphenol extract obtained from Nero di Troia red grape on the quality of farmed sea bass (Dicentrarchus labrax) fillets. Three diets were tested: control (n = 90) received a conventional feed, whereas the two experimental groups (n = 90 each) received the control feed supplemented with the red grape polyphenol extract (GPE) at the concentration of 100 (GPE 100) or 200 mg/kg (GPE 200). The two GPE diets lowered (p < 0.05) the red (a *) and yellow (b *) indexes, fillet hardness, and total lipid content. Chewiness, concentration of saturated fatty acids, and the Atherogenic Index were higher (p < 0.05) in the control group; GPE diets increased (p < 0.05) the polyunsaturated fatty acids content. Furthermore, the concentration of malondialdehyde was lower (p < 0.05) in fillets of the GPE groups, thus confirming the antioxidant effect of the red grape extract and its effectiveness in preventing lipid oxidation.

The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. We used CRISPR-Cas9 genome editing to remove the SV40 T antigen-encoding sequences from HEK293T cells by transfecting them with a recombinant plasmid expressing Cas9 and two distinct single guide RNAs (sgRNAs) corresponding to the beginning and end of the T antigen coding region. Cell clones lacking T antigen-encoding sequences were identified using PCR. Whole-genome (WG) and targeted locus amplification (TLA) sequencing of the parental HEK293T cell line revealed multiple SV40 T antigen-encoding sequences replacing cellular sequences on chromosome 3. The putative T antigen null clones demonstrated a loss of sequence reads mapping to T antigen-encoding sequences. Western blot analysis of cell extracts prepared from the T antigen null clones confirmed that the SV40 large and small T antigen proteins were absent. Lentiviral vectors produced using the T antigen null clones exhibited titers up to 1.5 × 107 transducing units (TU)/mL, while the titers obtained from the parent HEK293T cell line were up to 4 × 107 TU/mL. The capacity of the T antigen-negative cells to produce high titer adeno-associated virus (AAV) vectors was also evaluated. The results obtained revealed that the lack of T antigen sequences did not impact AAV vector titers.

SUMMARY: The Milanino is a heavy Italian chicken breed and represents an important genetic resource for alternative production systems. Due to the absence of standard management guidelines for local chicken breeds, this study aims to determine the space requirement to achieve a good equilibrium between growth performance and stress response in Milanino chickens reared according to a separate-sex free-range program. A total of 140 birds (70M:70F) were reared in outdoor pens from 75 to 235 d of life according to the following experimental groups (35 birds/group): (M2) males in 2 m2/bird; (F2) females in 2 m2/bird; (M10) males in 10 m2/bird; (F10) females in 10 m2/bird. Growth performance and stress condition were recorded. Bird density affected male body weight, that was higher at the lower density of 10 m2/bird. In contrast, females showed a consistent growth rate irrespective of the bird density suggesting lower space requirements. An overall good adaptability of the breed to the separate-sex free-range system was found, irrespective of both sex and bird density. Moreover, the stress markers values assessed at the end of the growing period suggest the ability of Milanino birds to respond well to environmental stressors. The results provided in this report will be implemented into free-range management guidelines for meat production in the Milanino breed: the separate-sex rearing is recommended during the growing period in outdoor pens and the bird density has to be planned according to the sex.

SUMMARY: The Milanino is a heavy Italian chicken breed and represents an important genetic resource for alternative production systems. In order to support its promotion in the market according to consumer expectations on healthy nutrition and animal welfare, this trial aims to study the slaughter performance and the meat quality in male and female Milanino chickens kept at different rearing density in a separate-sex free-range system. A total of 140 birds (70M:70F) were reared in outdoor pens from 75 to 235 d of life according to the following experimental groups (35 birds/group): M2) males in 2 m2/bird; F2) females in 2 m2/bird; M10) males in 10 m2/bird; F10) females in 10 m2/bird. At 235 d of age, 6 birds per group were slaughtered. Slaughter performance and meat quality were assessed. The Milanino chicken is characterized by high carcass weight and carcass yield among local chicken breeds, and a rearing density of 10 m2/bird is recommended for male birds to obtain heavier carcasses. Milanino meat appears bright and intensely colored, and it is characterized by high protein and low fat contents compared with the standard broiler meat. Total lipids of Milanino meat are characterized by a healthy fatty acid composition, corresponding to a high PUFA/SFA ratio. Milanino breast meat is a good supply of nutraceutical PUFA with a positive low n-6/n-3 PUFA ratio. The ability of the Milanino breed either to synthesize or to transfer to tissue a high quantity of PUFA relevant for human health could be a key factor for its economic valorization.

BACKGROUND: The aim of this study is to analyze the prognostic factors in patients that underwent induction therapy and surgery for clinical stage III NSCLC. METHODS: Clinical and pathological characteristics of stage III NSCLC patients for N2 involvement that underwent neoadjuvant treatment (NAD) and surgery from 1/01/1998 to 31/12/2017 were collected and retrospectively analyzed. Tumor characteristics, yClinical, yPathological stage and lymph node characteristics were correlated to Overall Survival (OS). RESULTS: The analysis was conducted on 180 patients. Five-year OS (5YOS) was 50.9%. Univariable analysis results revealed old age (p = 0.003), clinical N2 post-NAD (p = 0.01), pneumonectomy (0.005), persistent pathological N2 (p = 0.039, HR 1.9, 95% CI 1.09–2.68) and adjuvant therapy absence (p = 0.049) as significant negative prognostic factors. Multivariable analysis confirmed pN0N1 (p = 0.02, HR 0.29, 95% CI 0.13–0.62) as a favorable independent prognostic factor and adjuvant therapy absence (p = 0.012, HR 2.61, 95% CI 1.23–5.50) as a negative prognostic factor. Patients with persistent N2 presented a 5YOS of 35.3% vs. 55.8% in pN0N1 patients. Regarding lymph node parameters, the lymph node ratio (NR) significantly correlated with OS: 5YOS of 67.6% in patients with NR < 50% vs. 29.5% in NR > 50% (p = 0.029). CONCLUSION: Clinical response aided the stratification of prognosis in patients that underwent multimodal treatment for stage III NSCLC. Adjuvant therapy seemed to be an important option in these patients, while node ratio was a strong prognosticator in patients with persistent nodal involvement.

The preservation of genetic variability of autochthonous poultry breeds is crucial in global biodiversity. A recent report revealed small breed size and potential risk of extinction of all native Italian poultry breeds; therefore, a correct assessment of their genetic diversity is necessary for a suitable management of their preservation. In this work, we provided an overview of the contribution to poultry biodiversity of some Italian autochthonous breeds reared in conservation centers devoted to local biodiversity preservation. The level of genetic diversity, molecular kinship, inbreeding, contribution to overall genetic diversity, and rate of extinction of each breed were analyzed with a set of 14 microsatellite loci in 17 autochthonous chicken breeds. To evaluate genetic variability, total number (Na), and effective number (Ne) of alleles, observed (Ho) and expected (He) heterozygosity, and F (Wright’s inbreeding coefficient) index were surveyed. The contribution of each analyzed breed to genetic diversity of the whole dataset was assessed using MolKin3.0; global genetic diversity and allelic richness contributions were evaluated. All the investigated loci were polymorphic; 209 alleles were identified (94 of which private alleles). The average number of alleles per locus was 3.62, and the effective number of alleles was 2.27. The Ne resulted lower in all breeds due to the presence of low-frequency alleles that can be easily lost by genetic drift, thus reducing the genetic variability of the breeds, and increasing their risk of extinction. The global molecular kinship was 27%, the average breed molecular kinship was 53%, and the mean inbreeding rate 43%, with a self-coancestry of 78%. Wright’s statistical analysis showed a 41% excess of homozygous due to breed genetic differences (34%) and to inbreeding within the breed (9%). Genetic variability analysis showed that 11 breeds were in endangered status. The contribution to Italian poultry genetic diversity, estimated as global genetic diversity, and ranged from 30.2 to 98.5%. In conclusion, the investigated breeds maintain a unique genetic pattern and play an important role in global Italian poultry biodiversity, providing a remarkable contribution to genetic variability.

Sperm freezing is of interest not only for animal breeding. The ability to use sperm in frozen form for AI is a key factor in ensuring the long-term preservation of genetic diversity through the creation of a sperm cryobank. The most widely used freezing method involves using straws as packaging sperm, then freezing them on liquid nitrogen (N2) vapor and finally immersing them in N2. The cryosurvival of sperm cells also varies among different animal species and has been correlated with the freezing rate, i.e., the distance of the straws above the N2 level. Identifying a freezing procedure that will report a fertilization rate as close as possible to that of fresh semen appears to be a challenge. Thus, there is a clear need to standardize the entire freezing process. In this research, a new device for semen cryopreservation is presented. The cryopreservation prototype allows management of the freezing rate by varying the heights of the support straws above the N2 level. A stainless steel box with a suitable floating frame equipped with a support for the straws was designed and fabricated. The holder is connected to the floating frame by means of two threaded rods that allow for height adjustment by screwing in appropriate guides. Two K-type thermocouples were used, one to measure the temperature of the nitrogen vapor at the height of the support and the other to measure the temperature of the semen inside the straw. In this way, different freezing protocols can be compared. By managing the freezing rate, the sperm freezing procedure, which is specific to animal species, could be standardized and thus variability in results could be minimized. In addition, improved cryosurvival and post-freezing sperm fertilization rates are expected.

Brown trout (Salmo trutta), like many other freshwater species, is threated by the release in its natural environment of alien species and the restocking with allochthonous conspecific stocks. Many conservation projects are ongoing and several morphological and genetic tools have been proposed to support activities aimed to restore genetic integrity status of native populations. Nevertheless, due to the complexity of degree of introgression reached up after many generations of crossing, the use of dichotomous key and molecular markers, such as mtDNA, LDH-C1* and microsatellites, are often not sufficient to discriminate native and admixed specimens at individual level. Here we propose a reduced panel of ancestry-informative SNP markers (AIMs) to support on field activities for Mediterranean trout management and conservation purpose. Starting from the genotypes data obtained on specimens sampled in the main two Molise’s rivers (Central-Southern Italy), a 47 AIMs panel was identified and validated on simulated and real hybrid population datasets, mainly through a Machine Learning approach based on Random Forest classifier. The AIMs panel proposed may represent an interesting and cost-effective tool for monitoring the level of introgression between native and allochthonous trout population for conservation purpose and this methodology could be also applied in other species.

Background and aims: No consensus exists on how aggressively to treat relapsing–remitting multiple sclerosis (RRMS) nor on the timing of the treatment. The objective of this study was to evaluate disability trajectories in RRMS patients treated with an early intensive treatment (EIT) or with a moderate-efficacy treatment followed by escalation to higher-efficacy disease modifying therapy (ESC). Methods: RRMS patients with ⩾5-year follow-up and ⩾3 visits after disease modifying therapy (DMT) start were selected from the Italian MS Registry. EIT group included patients who received as first DMT fingolimod, natalizumab, mitoxantrone, alemtuzumab, ocrelizumab, cladribine. ESC group patients received the high efficacy DMT after ⩾1 year of glatiramer acetate, interferons, azathioprine, teriflunomide or dimethylfumarate treatment. Patients were 1:1 propensity score (PS) matched for characteristics at the first DMT. The disability trajectories were evaluated by applying a longitudinal model for repeated measures. The effect of early versus late start of high-efficacy DMT was assessed by the mean annual Expanded Disability Status Scale (EDSS) changes compared with baseline values (delta-EDSS) in EIT and ESC groups. Results: The study cohort included 2702 RRMS patients. The PS matching procedure produced 363 pairs, followed for a median (interquartile range) of 8.5 (6.5–11.7) years. Mean annual delta-EDSS values were all significantly ( p

Semen cryopreservation represents the main tool for preservation of biodiversity; however, in avian species, the freezing–thawing process results in a sharp reduction in sperm quality and consequently fertility. Thus, to gain a first insight into the molecular basis of the cryopreservation of turkey sperm, the NMR-assessed metabolite profiles of fresh and frozen–thawed samples were herein investigated and compared with sperm qualitative parameters. Cryopreservation decreased the sperm viability, mobility, and osmotic tolerance of frozen–thawed samples. This decrease in sperm quality was associated with the variation in the levels of some metabolites in both aqueous and lipid sperm extracts, as investigated by NMR analysis. Higher amounts of the amino acids Ala, Ile, Leu, Phe, Tyr, and Val were found in fresh than in frozen–thawed sperm; on the contrary, Gly content increased after cryopreservation. A positive correlation (p < 0.01) between the amino acid levels and all qualitative parameters was found, except in the case of Gly, the levels of which were negatively correlated (p < 0.01) with sperm quality. Other water-soluble compounds, namely formate, lactate, AMP, creatine, and carnitine, turned out to be present at higher concentrations in fresh sperm, whereas cryopreserved samples showed increased levels of citrate and acetyl-carnitine. Frozen–thawed sperm also showed decreases in cholesterol and polyunsaturated fatty acids, whereas saturated fatty acids were found to be higher in cryopreserved than in fresh sperm. Interestingly, lactate, carnitine (p < 0.01), AMP, creatine, cholesterol, and phosphatidylcholine (p < 0.05) levels were positively correlated with all sperm quality parameters, whereas citrate (p < 0.01), fumarate, acetyl-carnitine, and saturated fatty acids (p < 0.05) showed negative correlations. A detailed discussion aimed at explaining these correlations in the sperm cell context is provided, returning a clearer scenario of metabolic changes occurring in turkey sperm cryopreservation.

Duckweed (Lemna minor) meal was included in the formulation of three experimental feeds (L1, L2, L3) for rainbow trout at 10%, 20%, 28% of the protein source, respectively. Increasing the duckweed inclusion, the other protein sources were adjusted to get isonitrogenous (41%) and isolipidic (20%) diets, as the control diet (LC). 540 fish (mean body weight 124.5 ± 0.7 g) were randomly allocated in 12 tanks divided equally among the four different diets. After 90 days, fish were weighed and the most important productive performances, fillet quality and fatty acid profile were determined. The final body weight in L1 (340.53 g) and L2 (339.42 g) was not different from LC (348.80 g); L3 trout significantly (p < 0.05) exhibited the lowest one (302.16 g). Similar trends were found in final mean length, weight gain, specific growth rate, food conversion rate. Somatic indices were affected by duckweed inclusion. Diets had not significant effects on the proximate composition and fatty acids of the fillet in L1, L2, L3 respect to LC. Based on this study, duckweed meal derived from Lemna minor can be included in the feed for the rainbow trout without negative effects on the growth performances at 20% of the protein substitution.

Background and purpose: Immunoadsorption (IA) is an antibody-depleting therapy used to treat neuromyelitis optica spectrum disorder (NMOSD) associated to antiaquaporin 4 (anti-AQP4-IgG) and antimyelin oligodendrocyte glycoprotein (anti-MOG-IgG) serum autoantibodies. Our aim was to evaluate longitudinal changes of serum MOG-IgG and AQP4-IgG antibody titer and to correlate it with the clinical status.Methods: Autoantibody titer and clinical features of two MOG-IgG+/AQP4-IgG– and two AQP4-IgG+/MOG-IgG– patients with NMOSD were collected at baseline (T0), after 6 IA courses (T1), and then 2 weeks (T2) and 6 months after treatment (T3). A fluorescent ratiometric assay was used for a quantitative detection of MOG and AQP4 antibodies, based on HEK-293 cells transfected with the full-length hMOG fused to GFP or h-AQP4-M23 isoform fused to m-cherry, respectively. We defined the antibody titer as MOG quantitative ratio (MOGqr) and AQP4 quantitative ratio (AQP4qr).Results: In Case 1, the MOGqr dropped from 0.98 at T0 to 0.14 at T3, and in Case 2, it decreased from 0.96 at T0 to undetectable at T3. In Case3, the AQP4qr remained high: 0.90 at T0 and 0.92 at T3. In Case 4, the AQP4qr decreased from 0.50 at T0 to undetectable at T3. Complete recovery was found in Cases 1, 2, and 4.Conclusions: Semiquantitative ratiometric method accurately detects even slight variation of MOG-IgG and AQP4-IgG titer, suggesting it may be useful to monitor the antibody titer during the disease course and maintenance immunotherapy.

Background: Although over a dozen disease modifying treatments (DMTs) are available for relapsing forms of multiple sclerosis (MS), treatment interruption, switching and discontinuation are common challenges. The objective of this study was to describe treatment interruption and discontinuation in the Big MS data network.Methods: We merged information on 269,822 treatment episodes in 110,326 patients from 1997 to 2016 from five clinical registries in this cohort study. Treatment stop was defined as a clinician recorded DMT end for any reason and included treatment interruptions, switching to alternate DMTs and long-term or permanent discontinuations.Results: The incidence of DMT stopping cross the full observation period was lowest in FTY (19.7 per 100 person-years (PY) of treatment; 95% CI 19.2–20.1), followed by NAT (22.6/100 PY; 95% CI 22.2–23.0), IFNβ (23.3/100 PY; 95% CI 23.2–23.5). Of the 184,013 observed DMT stops, 159,309 (86.6%) switched to an alternate DMT within 6 months. Reasons for stopping a drug were stable during the observation period with lack of efficacy being the most common reason followed by lack of tolerance and side effects. The proportion of patients continuing on most DMTs were similarly stable until 2014 and 2015 when drop from 83 to 75% was noted.Conclusions: DMT stopping reasons and rates were mostly stable over time with a slight increase in recent years, with the availability of more DMTs. The overall results suggest that discontinuation of MS DMTs is mostly due to DMT properties and to a lesser extent to risk management and a competitive market.

Introduction: In relapsing Multiple Sclerosis (RMS) patients treated with disease modifying drugs (DMDs), few data are available regarding the biomarkers of treatment response. We aimed to assess the predictive value of lymphocyte count (LC) and Body Mass Index (BMI) for treatment response in a real life setting of dimethyl fumarate (DMF) treated patients.Materials and Methods: We included in our observational analysis 338 patients who were prescribed DMF in an Italian MS Center. We collected clinical and demographic data at the beginning of DMF (T0), and assessed White Blood Cells (WBC) and LC at T0 and at 3 (T3), 6 (T6), 9 (T9), and 12 (T12) months. Gadolinium enhancing (Gd+), new T2 lesions and relapses within the first year of treatment (T12) were recorded in order to evaluate clinical activity at 12 months. Analysis of correlation was performed to correlate WBC, LC and BMI with clinical and radiological responses. We evaluated whether BMI or LC can predict treatment response by using multivariate logistic regression models at each follow-up.Results: Our cohort was followed up for a mean period of 19.8 ± 6.8 months. The mean BMI at baseline was 24.19 ± 4.48. The multivariate models gave as predictive factors for Gd+ lesions at T12, LC at T3 (OR = 1.003, 95% CI = 1.00-1.07; p = 0.046) and baseline BMI (OR = 0.71, 95% CI = 0.52–0.98; p = 0.037). Predictive factors for new T2 lesions at T12 were LC at T3 (OR = 1.01 95%CI = 1.00–1.95; p = 0.005) and baseline BMI (OR = 0.99, 95% CI = 0.98–1.00; p = 0.026).Conclusions: In our real life-experience, BMI and LC may be early biomarkers to predict treatment response during DMF.

Abstract Background The treatment of cognitive deficits is challenging in pediatric onset multiple sclerosis (POMS) and in patients with attention deficit hyperactivity disorder (ADHD). We performed a pilot double-blind RCT to evaluate the efficacy of a home-based computerized-program for retraining attention in two cohorts of POMS and ADHD patients. Methods POMS and ADHD patients failing in at least 2/4 attention tests on a neuropsychological battery were randomized to specific or nonspecific computerized training (ST, nST), performed in one-hour sessions, twice/week for 3 months. The primary outcome was the effect of the training on global neuropsychological performances measured by the cognitive impairment index (CII). The efficacy of the intervention was evaluated in each disease group by using repeated measures ANOVA. Results Sixteen POMS (9 females, age 15.75 ± 1.74 years) and 20 ADHD (2 females, age 11.19 ± 2.49 years) patients were enrolled. In POMS patients the ST exposure was associated to a significantly more pronounced improvement of the CII (p

The sperm of each avian species and breed have unique characteristics that render them more or less susceptible to the freezing–thawing process; therefore, a suitable cryopreservation protocol that is specific for the sperm of each type of bird is needed. In this context, little information about the common pheasant’s sperm is available. Therefore, the aim of this study was to test different parameters at each step of the process of freezing into pellets and thawing to detect the least deleterious parameter settings. Sixteen different protocols were tested by studying two levels in each of the four steps (dilution, equilibration at 5 °C, final dimethylacetamide concentration, and dimethylacetamide equilibration time) comprising the freezing process. The pheasant sperm exhibited a high susceptibility to the damage caused by freezing into pellets; however, the survival of the sperm reached 29%, and the greatest recovered mobility was 22%. The mobility of the sperm was affected by the dilution and the dimethylacetamide concentration, and the viability of the sperm was affected by the equilibration at 5 °C and the dimethylacetamide equilibration. The protocols that caused the least damage to the pheasant sperm were found to be those with higher dilution rates, 10 min of equilibration at 5 °C, and 6% dimethylacetamide equilibrated for 1 or 5 min. In the present study, we individualise some applicable parameters for certain critical steps of the freezing–thawing process; however, further investigations are needed in order to improve upon and complete a suitable protocol for the cryopreservation and thawing of pheasant sperm.

The aim of this study was to evaluate the effects of different cold-storage time intervals between collection and semen-freezing on both fresh and cryopreserved semen motility parameters and the post-thaw fertilizing ability of Mediterranean brown trout semen. The ejaculates were split into six aliquots and stored on ice from 1 to 6 h, until freezing. Fresh and post-thaw sperm motility was evaluated by a Computer-Assisted Sperm Analysis system, whilst the fertilizing ability was assessed by in vivo trials. In fresh semen, at 3 h of storage, a significant decrease of total motility, linear movement (STR, LIN) and beat cross frequency (BCF) was recorded, whilst the amplitude of lateral displacement of the spermatozoon head (ALH) underwent a significant increase. In frozen semen, no significant difference was observed for all the motility parameters evaluated, except for the total motility between 1 and 6 h of storage and the duration of sperm movement between 1 and 5 h. Cold-storage time did not significantly affect the percentage of live embryos following the use of frozen semen. In conclusion, our results showed that, if necessary, the Mediterranean brown trout semen can be frozen even until 6 h post-collection without losing its fertilizing ability.

Knowledge of the composition of the gut microbiota in freshwater fish living in their natural habitat has taxonomic and ecological importance. Few reports have been produced on the composition of the gut microbiota and on the presence of LAB in the intestines of freshwater fish that inhabit river environments. In this study, we investigated the LAB community that was present in the gastrointestinal tract (GIT) of Mediterranean trout (Salmo macrostigma) that colonized the Biferno and Volturno rivers of the Molise region (Italy). The partial 16S rRNA gene sequences of these strains were determined for the species-level taxonomic placement. The phylogenetic analysis revealed that the isolated LABs belonged to seven genera (Carnobacterium, Enterococcus, Lactobacillus, Lactiplantibacillus, Vagococcus, Lactococcus, and Weissella). The study of the enzymatic activities showed that these LABs could contribute to the breakdown of polysaccharides, proteins, and lipids. In future studies, a greater understanding of how the LABs act against pathogens and trigger the fish immune response may provide practical means to engineer the indigenous fish microbiome and enhance disease control and fish health.

Mediterranean trout is a freshwater fish of particular interest with economic significance for fishery management, aquaculture and conservation biology. Unfortunately, native trout populations’ abundance is significantly threatened by anthropogenic disturbance. The introduction of commercial hatchery strains for recreation activities has compromised the genetic integrity status of native populations. This work assessed the fine-scale genetic structure of Mediterranean trout in the two main rivers of Molise region (Italy) to support conservation actions. In total, 288 specimens were caught in 28 different sites (14 per basins) and genotyped using the Affymetrix 57 K rainbow-trout-derived SNP array. Population differentiation was analyzed using pairwise weighted FST and overall F-statistic estimated by locus-by-locus analysis of molecular variance. Furthermore, an SNP data set was processed through principal coordinates analysis, discriminant analysis of principal components and admixture Bayesian clustering analysis. Firstly, our results demonstrated that rainbow trout SNP array can be successfully used for Mediterranean trout genotyping. In fact, despite an overwhelming number of loci that resulted as monomorphic in our populations, it must be emphasized that the resulted number of polymorphic loci (i.e., ~900 SNPs) has been sufficient to reveal a fine-scale genetic structure in the investigated populations, which is useful in supporting conservation and management actions. In particular, our findings allowed us to select candidate sites for the collection of adults, needed for the production of genetically pure juvenile trout, and sites to carry out the eradication of alien trout and successive re-introduction of native trout.

Despite advances, malignant pleural mesothelioma (MPM) remains a challenging disease in terms of diagnosis, treatment, and overall management. Herein, we analyzed, in a large-scale single-center cohort, the characteristics and perioperative course of patients undergoing surgical diagnosis of MPM. We identified a total of 514 consecutive patients, 71.4% male and 28.6% female, with mean age 71.3 +/− 13.6 years. Most exhibited pleural, respiratory, or general symptoms and American Society of Anesthesiologists (ASA) score was ≥3 in 68.3% of cases. Thoracoscopy was the most frequent approach (92.0%) and short open thoracotomy was performed in the remaining patients. Pleurodesis was simultaneously performed in 74.3% of cases. Diagnostic failure led to redo surgery in 3.7% of patients. Non-epithelioid histology was found in 19.5% of MPMs and was significantly more frequent in right-sided MPM (p = 0.04), and in patients without history of cancer (p = 0.03), or pleural nodules at thoracoscopy (p = 0.01). Minor only or major complications occurred in respectively 7.8% and 3.6% of cases. They were more frequent in patients ≥ 70 years (p = 0.05) and Performance Status > 2 (p = 0.05). The mean hospital stay was 7.5 days. The 30-day and 90-day early mortality rates were 2.3% and 6.4%, respectively. Surgical diagnosis of MPM is a reliable procedure but is associated with significant morbidity and hospital-stay duration.

Cognitive impairment (CI) is a remarkable feature in pediatric-onset multiple sclerosis (POMS). The Symbol Digit Modalities Test (SDMT) is increasingly used to explore CI in MS. Recently, a four-point worsening on the SDMT score has been demonstrated to correlate with a clinically meaningful cognitive worsening in adult MS. We conducted a post hoc analysis of a randomized computer-assisted rehabilitation trial for attention impairment in POMS to test the clinical meaningfulness of the changes in SDMT scores at the end of the trial (delta SDMT). A four-point SDMT cut-off was applied. POMS patients exposed to specific computer training (ST) and non-specific training (nST) were compared. Data of 16 POMS (9 females, age 15.75 ± 1.74 years) patients were analyzed. At the end of the trial, 25% of patients reported no clinically significant changes (−3 to 3), 12.5% a clinically significant worsening (≤−4) and 62.5% a clinically significant improvement (≥4) in the delta SDMT. The proportion of patients reporting a clinically meaningful improvement was significantly (p = 0.008) higher (100%) in patients exposed to ST in comparison to those (25%) exposed to nST. The use of the four-point SDMT cut-off may be useful to assess the clinical meaningfulness of results from cognitive rehabilitation trials.

Cognitive impairment (CI) is a common and disabling symptom of Multiple Sclerosis (MS) with a negative impact on daily living. In this pilot study, we applied magnetoencephalography (MEG) and high density (hd) electroencephalography (EEG) study to evaluate acoustic P300 features in a cohort of early MS. Sixteen MS patients (pwMS) and 19 healthy controls (HCs) matched for age and gender underwent an MEG-/(hd)-EEG-co-recording, using 306-channel Vectorview and 64 scalp electrodes. CI was assessed using Rao’s Brief Repeatable Battery (BRB). Moreover, we performed psychometric tests to assess depression and fatigue. In pwMS, we observed a slight latency prolongation of P300 peak compared to HCs, while P300 amplitude and scalp distribution were similar in the two groups. pwMS did not show an amplitude reduction and different scalp distribution of Event-Related Potentials (ERPs) and Event Related Fields (ERFs) related to an acoustic oddball paradigm. We found an inverse correlation between P300 amplitude and fatigue (r Spearman = −0.4; p = 0.019). In pwMS, phenomena of cortical adaptation to early dysfunction could preserve the cognitive performance of the P300 acoustic task, while the development of fatigue could prospectively lead to amplitude decline of P300, suggesting its possible role as a biomarker.

Abstract The directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it has not been possible to efficiently generate Purkinje neuron (PN) progenitors from human or mouse pluripotent stem cells, let alone to develop a methodology for in vivo transplantation in the adult cerebellum. Here, we present a protocol to obtain an expandable population of cerebellar neuron progenitors from mouse embryonic stem cells. Our protocol is characterized by applying factors that promote proliferation of cerebellar progenitors. Cerebellar progenitors isolated in culture from cell aggregates contained a stable subpopulation of PN progenitors that could be expanded for up to 6 passages. When transplanted into the adult cerebellum of either wild-type mice or a strain lacking Purkinje cells (L7cre-ERCC1 knockout), GFP-labeled progenitors differentiated in vivo to establish a population of calbindin-positive cells in the molecular layer with dendritic trees typical of mature PNs. We conclude that this protocol may be useful for the generation and maturation of PNs, highlighting the potential for development of a regenerative medicine approach to the treatment of cerebellar neurodegenerative diseases.

This data article contains supporting information regarding the research article entitled “Combined effect of permeant and non-permeant cryoprotectants on the quality of frozen/thawed chicken sperm”(Mosca et. al., 2016) [1]. The combined effect of the permeant cryoprotectants agent dimethylacetamide and the non-permeant cryoprotectants agent trehalose on the quality of frozen-thawed chicken semen was assessed. In particular, the quantitative dimethylacetamide/trehalose ratio was investigated freezing semen samples according to the following treatments: trehalose 0.1 M+0% dimethylacetamide (DMA-0), trehalose 0.1 M+3% dimethylacetamide (DMA-3), trehalose 0.1 M+6% dimethylacetamide (DMA-6).

Background: Natalizumab (NTZ) is sometimes discontinued in patients with multiple sclerosis, mainly due to concerns about the risk of progressive multifocal leukoencephalopathy. However, NTZ interruption may result in recrudescence of disease activity. Objective: The objective of this study was to summarize the available evidence about NTZ discontinuation and to identify which patients will experience post-NTZ disease reactivation through meta-analysis of existing literature data. Methods: PubMed was searched for articles reporting the effects of NTZ withdrawal in adult patients (⩾18 years) with relapsing–remitting multiple sclerosis (RRMS). Definition of disease activity following NTZ discontinuation, proportion of patients who experienced post-NTZ disease reactivation, and timing to NTZ discontinuation to disease reactivation were systematically reviewed. A generic inverse variance with random effect was used to calculate the weighted effect of patients’ clinical characteristics on the risk of post-NTZ disease reactivation, defined as the occurrence of at least one relapse. Results: The original search identified 205 publications. Thirty-five articles were included in the systematic review. We found a high level of heterogeneity across studies in terms of sample size (10 to 1866 patients), baseline patient characteristics, follow up (1–24 months), outcome measures (clinical and/or radiological), and definition of post-NTZ disease reactivation or rebound. Clinical relapses were observed in 9–80% of patients and peaked at 4–7 months, whereas radiological disease activity was observed in 7–87% of patients starting at 6 weeks following NTZ discontinuation. The meta-analysis of six articles, yielding a total of 1183 patients, revealed that younger age, higher number of relapses and gadolinium-enhanced lesions before treatment start, and fewer NTZ infusions were associated with increased risk for post-NTZ disease reactivation ( p ⩽ 0.05). Conclusions: Results from the present review and meta-analysis can help to profile patients who are at greater risk of post-NTZ disease reactivation. However, potential reporting bias and variability in selected studies should be taken into account when interpreting our data.

The most reared species of farm animal around the world is the chicken. However, the intensification of livestock systems has led to a gradual increase in the concentration of a limited number of breeds, resulting in substantial erosion to the genetic pool. The initial step of an ‘animal conservation program’ entails establishing the actual conservation statuses of the breeds concerned in a defined area; in this case, in Italy. To this end, a survey of breeds was performed by means of a census questionnaire divided into two parts. The first part collected information on breeds, breeders, housing facilities, and management aspects, the results of which are presented here. The second part of the questionnaire regarded chicken products and their markets, and these data will be reported in a second paper. The breed status of six chicken breeds was shown to be exceptionally worrying, with total numbers ranging from just 18 to 186 birds. Population sizes exceeding 1000 birds was identified for just four breeds, the maximum being 3400. Some improvements in status were noted in relation to breeds which had been the subject of conservation efforts in the past. The two most common breeds reported are the Bionda Piemontese, a double-purpose breed, and the Livorno egg-laying hen. Collo Nudo Italiano, Millefiori Piemontese, Pollo Trentino, and Tirolese chicken breeds and the Castano Precoce turkey breed were not listed by breeders at all. The most reported turkey breeds are the Bronzato Comune and the Ermellinato di Rovigo. The population sizes of native Italian poultry breeds were shown to be generally poor. Italian poultry farmers and the population at large are largely ignorant about indigenous poultry breeds. Thus, promoting the virtues of Italian breeds would help their conservation by encouraging breeders to rear these birds and consumers to buy their products. The identification of strategies to facilitate access to pure breed birds is essential, and will require the collaboration of university research centers, public entities, and breeders. The results presented in this paper constitute the initial part of a more complex conservation program.

The intensive use of high-performing strains in poultry production has led to the extinction of several autochthonous chicken breeds and, consequently, loss of genetic variability. Interest in saving biodiversity is growing rapidly and has become a major objective worldwide. The aim of this study was to shed light on the production trends of native Italian poultry breeds and the related market. A questionnaire, which asked about the production cycles, the number of animals and table eggs produced per year and their retail prices was completed by 121 breeders across Italy. The surveyed breeders were divided into two categories: breeders conducting an agrozootechnical farm, referred to as ‘farmers’ (F); and breeders keeping chickens as backyard poultry, referred to as ‘fancy breeders’ (FB). Analysis of the data acquired indicated that animals were mainly slaughtered between 6 and 12 months of age, with F processing more animals per year. The same production trend was observed for table eggs. The recorded retail prices of native chicken products were higher than those for conventional products, but similar to those reported for valuable niche poultry products, such as the Poulet de Bresse in France and organic eggs. Knowledge about these highly valuable markets should be used to encourage the use of local breeds in alternative poultry farming and help protect biodiversity.