Argas ticks (Ixodida: Argasidae) on migratory birds from Africa: first record of a genotype close to Argas africolumbae in Italy.

In spring, migratory birds reach Europe, mainly from sub-Saharan Africa or from northern African countries. Avian species may be implicated in the spread of pathogens, either as reservoirs, hosts or carriers of infected ectoparasites. In 2021, on Ventotene Island (Latium region, Italy) within a project focused on the potential incoming pathogens via migratory birds from Africa, we found two larvae of Argas sp., on the redstart Phoenicurus phoenicurus, that shared morphological features with the African Argas (Argas) africolumbae. Comparison of the tested larval DNA sequences to the adult reference sequences showed the highest identity (> 92%) with homologous sequences of A. africolumbae collected in South Africa and in Spain. This study reports the first detection of Argas africolumbae-like specimens in Italy.

Soil erosion and sediment transport under climate change for Mera River, in Italian Alps of Valchiavenna.

Erosion is a main form of soil degradation, with severe consequences on slope stability and productivity, and erosion studies are required to predict possible variations of such phenomena, also under climate change scenarios. Here we estimated distributed soil erosion within Valchiavenna valley in the Rhaetian Alps, drained by Mera river, and covering Italy, and Switzerland. We used a Dynamic-RUSLE (D-RUSLE) model, which provides spatially distributed estimates of soil erosion explicitly considering snow dynamic (accumulation/melting) and snow cover, and vegetation seasonality. The model was tuned here during 2010-2019, and validation was pursued using river turbidity data, used to assess riverine sediment transport. The model parameter R-factor for rainfall erosivity was estimated using a hydrological model Poli-Hydro, properly set up in the study area. C-factor for land cover was assessed against land cover maps, with seasonally variable Normalized Difference Vegetation Index from satellite images, to account for variable vegetation stage, and large leaf cover in summer. The K-factor related to erosion susceptibility was evaluated through soil texture and organic content. LS-factor depending on slope was assessed using a DTM. Poli-Hydro and D-RUSLE models were then used to project forward potential soil erosion under climate change scenarios until 2100. Climate series (temperature, precipitation) were generated using 4 shared socio-economic pathways (SSPs) of the Sixth Assessment Report of the IPCC, with 3 global circulation models, properly downscaled locally. We analysed expected soil erosion during 2051-2060, and 2091-2100. We found increase of potential soil erosion, with exception of the EC-Earth model for the SSP2.6. Erosion would especially increase in winter, in response to smaller snow accumulation, and larger liquid rainfall share thereby, and decrease in summer, as due to decreased precipitation. Our results suggest the need for adaptation strategies to counteract increasing soil loss in the future, and may highlight most critical areas of intervention.

Effect of microbial inoculation and particle size on fermentation profile, aerobic stability, and ruminal in situ starch degradation of high-moisture corn ensiled for a short period.

Dairy farmers are often challenged with the need to feed high-moisture corn (HMC) after less than 30 d of fermentation. The objective this study was to assess the effects of microbial inoculation and particle size on fermentation profile, aerobic stability, and ruminal in situ starch degradation of HMC ensiled for a short period. High-moisture corn was harvested, coarsely ground (3,798 ± 40 µm, on average) or finely ground (984 ± 42 µm, on average), then ensiled in quadruplicate vacuum pouches untreated (CON) or with the following treatments: Lactobacillus plantarum CH6072 at 5 × 104 cfu/g and Enterococcus faecium CH212 at 5 × 104 cfu/g of fresh forage (LPEF); or Lactobacillus buchneri LB1819 at 7.5 × 104 cfu/g and Lactococcus lactis O224 at 7.5 × 104 cfu/g (LBLL). Silos were allowed to ferment for 14 or 28 d. Ruminal in situ starch degradation increased when HMC was finely ground. In addition, in situ starch degradation was greater and aerobic stability increased approximately 5-fold with LBLL compared with CON and LPEF. An interaction between microbial inoculation and storage length occurred for lactic acid. At 14 d, concentrations of lactic acid were greatest in LPEF and lowest in LBLL. Lactic acid concentrations increased from 14 to 28 d with CON and LPEF, but decreased with LBLL. At 28 d, concentrations of lactic acid were lower in LBLL compared with CON and LPEF. An interaction between particle size, microbial inoculation, and storage length occurred for acetic acid and ammonia-N. At 14 and 28 d, acetic acid concentrations were greatest in finely ground LBLL followed by coarsely ground LBLL. Ammonia-N concentrations increased across all treatments from 0 to 28 d. At 14 and 28 d, concentrations of ammonia-N were greatest in finely ground LBLL and lowest in coarsely ground CON and coarsely ground LPEF. Results from this study suggest that L. buchneri LB1819 can produce acetic acid in as little as 14 d, and that by 28 d, it has the potential to improve the aerobic stability of HMC. Additionally, results indicate that L. buchneri LB1819 has the potential to improve ruminal degradation of starch by 28 d of storage. Finally, results confirm enhanced fermentation and improved ruminal starch degradation with finely ground HMC by 28 d of storage.

Superhydrophobic lab-on-chip measures secretome protonation state and provides a personalized risk assessment of sporadic tumour.

Secretome of primary cultures is an accessible source of biological markers compared to more complex and less decipherable mixtures such as serum or plasma. The protonation state (PS) of secretome reflects the metabolism of cells and can be used for cancer early detection. Here, we demonstrate a superhydrophobic organic electrochemical device that measures PS in a drop of secretome derived from liquid biopsies. Using data from the sensor and principal component analysis (PCA), we developed algorithms able to efficiently discriminate tumour patients from non-tumour patients. We then validated the results using mass spectrometry and biochemical analysis of samples. For the 36 patients across three independent cohorts, the method identified tumour patients with high sensitivity and identification as high as 100% (no false positives) with declared subjects at-risk, for sporadic cancer onset, by intermediate values of PS. This assay could impact on cancer risk management, individual's diagnosis and/or help clarify risk in healthy populations.

NADPH oxidase 2 (NOX2) enzyme activation in patients with chronic inflammatory demyelinating polyneuropathy.

BACKGROUND AND PURPOSE: Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired immunomediated condition affecting the peripheral nervous system where probably macrophages are the primary effector cells for demyelination. Reactive oxygen species (ROS), catalyzed by the NOX family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, can induce peroxidation and are potentially injurious to myelin. Our aim was to assess the activity of NOX2, an isoform of NOX, in a series of CIDP patients and to analyze the effect of intravenous immunoglobulin (IVIg) on NOX2. METHODS: Thirty CIDP patients treated with IVIg and 30 control subjects were enrolled. To evaluate NOX2 activity, neutrophil and monocyte oxidative burst was measured directly in fresh whole blood using the Phagoburst™ assay, a fluorescence-activated cell sorting method. The mean fluorescence intensity, emitted in response to different stimuli, leads to the production of ROS and corresponds to the percentage of oxidizing cells and their enzymatic activity. RESULTS: Mean fluorescence intensity values for granulocyte and monocyte burst in patients (mean 633.3, SD 191; mean 111.8, SD 28.5) were different from those measured in healthy controls (granulocytes, mean 436.6, SD 137.0, P = 0.0003; monocytes, mean 78.2, SD 17.3, P = 0.000001). Moreover, IVIg administration increased both granulocyte (P = 0.005) and monocyte (P = 0.0009) burst. CONCLUSION: Our findings demonstrate that oxidative burst is significantly increased in CIDP patients and that treatment with IVIg enhances oxidative values, thus representing a possible IVIg therapeutic effect linked to a regulatory effect of ROS. Based on this, the development of treatments targeting the specific activation of NOX may be beneficial in autoimmune disorders.

EZH2 is increased in paediatric T-cell acute lymphoblastic leukemia and is a suitable molecular target in combination treatment approaches.

BACKGROUND: T-cell Acute Lymphoblastic Leukemia (ALL) represents about 10-15 % of pediatric ALL cases. EZH2, one of the components of Polycomb group proteins (PRC2) complex, catalyzes the trimethylation of histone H3 lysine 27 that is associated with transcriptional repression and tumor development. METHODS: We examined the expression levels of PRC2 complex in primary samples of T cells ALL at diagnosis by western blotting and real time PCR. We evaluated the effect of 3-deazaneplanocin-A (DZNep), an EZH2 inhibitor, alone and in combination with Daunoblastine on cell viability, apoptotic death and cell cycle distribution of T cell established Jurkat cell line. RESULTS: EZH2 was expressed in 75 % samples at different extents mainly with high expression level. SUZ12 was expressed in 60 % samples and EED in all samples, respectively. The Kaplan-Meier analysis shows that T-ALL expressing EZH2 had a lower probability of disease-free survival (DFS) compared to T-ALL negative for EZH2 (23 % vs 100 %) (p = 0.01). The EZH2 inhibitor DZNep used in combination with Daunoblastine was synergistic in inducing growth inhibition and increasing the apoptosis in T-ALL Jurkat cells at 48 and 72 h paralleled by EZH2 decreased expression. Moreover, the combination decreased the activity of Erk-1/2 proliferation enzymes with no effects on Akt survival pathway. CONCLUSIONS: The evaluation of EZH2 expression in pediatric T-ALL can be useful in predict the clinical outcome of the patients and EZH2 can be a useful target to improve the efficacy of conventional chemotherapy in this subset of patients with bad prognosis.

Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells.

Recent technical developments have enabled the transcriptomes of hundreds of cells to be assayed in an unbiased manner, opening up the possibility that new subpopulations of cells can be found. However, the effects of potential confounding factors, such as the cell cycle, on the heterogeneity of gene expression and therefore on the ability to robustly identify subpopulations remain unclear. We present and validate a computational approach that uses latent variable models to account for such hidden factors. We show that our single-cell latent variable model (scLVM) allows the identification of otherwise undetectable subpopulations of cells that correspond to different stages during the differentiation of naive T cells into T helper 2 cells. Our approach can be used not only to identify cellular subpopulations but also to tease apart different sources of gene expression heterogeneity in single-cell transcriptomes.

Effect of rMnSOD on survival signaling in pediatric high risk T-cell acute lymphoblastic leukaemia.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that defends against oxidative damage due to reactive oxygen species (ROS). A new isoform of MnSOD with cytotoxic activity was recently discovered in liposarcoma cells. Here, we tested the effectiveness of a recombinant form of this isoform (rMnSOD) on leukemic T cells, Jurkat cells, and lymphocytes. Our results confirm that leukemic T cells can internalize rMnSOD and that rMnSOD causes apoptosis of 99% of leukemic cells without showing toxic effects on healthy cells. Using light and electron microscopy, we determined that an rMnSOD concentration of 0.067 µM most effective on apoptosis induction. Western blot analysis showed that treatment with 0.067 µM rMnSOD resulted in high expression of the pro-apoptotic protein Bax and low expression of the anti-apoptotic protein Bcl-2 in leukemia cells. Concerning signal transduction pathway no influence was observed after treatment except for Jurkat cells showing a slightly decreased expression of ERK phosphorylation. These results suggest that rMnSOD may be an effective and non-toxic treatment option for T-cell leukemia.

Olea Europea-derived phenolic products attenuate antinociceptive morphine tolerance: an innovative strategic approach to treat cancer pain.

Morphine and related opioid drugs are currently the major drugs for severe pain. Their clinical utility is limited in the management of severe cancer pain due to the rapid development of tolerance. Restoring opioid efficacy is therefore of great clinical importance. A great body of evidence suggests the key role of free radicals and posttranslational modulation in the development of tolerance to the analgesic activity of morphine. Epidemiological studies have shown a relationship between the Mediterranean diet and a reduced incidence of pathologies such as coronary heart disease and cancer. A central hallmark of this diet is the high consumption of virgin olive oil as the main source of fat which contains antioxidant components in the non-saponifiable fraction, including phenolic compounds absent in seed oils. Here, we show that in a rodent model of opiate tolerance, removal of the free radicals with phenolic compounds of olive oil such as hydroxytyrosol and oleuropein reinstates the analgesic action of morphine. Chronic injection of morphine in mice led to the development of tolerance and this was associated with increased nitrotyrosin and malondialdehyde (MDA) formation together with nitration and deactivation of MnSOD in the spinal cord. Removal of free radicals by hydroxytyrosol and oleuropein blocked morphine tolerance by inhibiting nitration and MDA formation and replacing the MnSOD activity. The phenolic fraction of virgin olive oil exerts antioxidant activities in vivo and free radicals generation occurring during chronic morphine administration play a crucial role in the development of opioid tolerance. Our data suggest novel therapeutic approach in the management of chronic cancer pain, in particular for those patients who require long-term opioid treatment for pain relief without development of tolerance.

Treatment of children over the age of one year with unresectable localised neuroblastoma without MYCN amplification: results of the SIOPEN study.

BACKGROUND: In children older than 1 year with localised unresectable neuroblastoma (NB), treatment strategies are heterogeneous according to the national groups. The objective of this phase III non-randomised study was to evaluate the efficacy of conventional chemotherapy followed by surgery. PATIENTS AND METHODS: In the presence of surgical risk factors (SRF), six courses of chemotherapy alternating Carboplatin-Etoposide and Vincristin-Cyclophosphamide-Doxorubicin were given, and surgical resection was attempted after four. Survival analyses were performed using an intention-to-treat approach. The main objective was to achieve a 5-year survival over 80%. RESULTS: Out of 191 registered children, 160 were evaluable. There were 62.5% older than 18 months and 52.5% had unfavourable histology according to International Neuroblastoma Pathology Classification (INPC). Chemotherapy reduced the number of SRFs by one third. Delayed surgery was attempted in 86.3% of patients and was complete or nearly complete in 74%. The 5-year EFS and OS were 76.4% and 87.6% respectively, with significant better results for patients younger than 18 months or with favourable histology. CONCLUSION: This strategy provides encouraging results in children older than 1 year or 12 months with localised unresectable NB without MYCN amplification. However, in children older than 18 months and with unfavourable histology, additional treatment is recommended.

Infections from CVC in the pediatric neoplastic patient. Single institution experience.

AIM: The present clinical study was carried out in order to evaluate in a perspective way the incidence of the infections caused by CVC, the micro-organisms mostly involved in the infectious process, the condition of aplasia in patients when blood cultures show positiveness and the incidence of removals expressed as number of performed removals/number of positive blood culture. METHODS: Between January 2003 and December 2009 452 blood cultures from CVC were carried out on 120 patients affected by acute lymphoblastyic and myelougenous leukemia (38), Hodgkin and non-Hodgkin lymphoma (17) and solid tumors (65), with an average of 65 blood cultures per year showing an average positiveness of 21 cases/year. The blood cultures were performed, in hyperpyrexia, when there was a clinical suspicion of infection from CVC. RESULTS: On 452 blood cultures from CVC carried out (31.4% positive per Gram +, 53.7% per Gram-, 14.9% per miceti) 128 (28.3%) resulted positive, excluding presumed contaminations. They were divided as follows: 21 of Staphylococcus epidermidis (16%), 10 of Escherichia coli (8%), 10 of Klebsiella pneumoniae (8%), 8 of Pseudomonas aeruginosa (6%), 8 of Staphylococcus aureus (6%), 6 of Enterobacter cloacae (5%), 4 of Candida parapsilosis (3%) and 61 of other micro-organisms (48%). It was necessary to perform 27 CVC removals. The micro-organisms most frequently involved in removals of the CVC were finally analyzed and the resulting frequency percentages are: - 85% for Gram- germs; -8% for Gram + germs; -7% for Mycete. CONCLUSION: Our clinical study has confirmed that in pediatric age neoplastic individuals there is a prevalence of CVC-correlated infections from Gram- and an elevated association of removals of the CVC caused by infections from Pseudomonas and Klebsiella, germs more frequently associated to clinical conditions of marked aplastic anemia.

Gemcitabine and tamoxifen-loaded liposomes as multidrug carriers for the treatment of breast cancer diseases.

The effects of a lipid composition on the physico-chemical and technological properties of a multidrug carrier (MDC) containing both gemcitabine (GEM) and tamoxifen (TMX), as well as its in vitro antitumoral activity on different breast cancer cell lines, were investigated. In particular, the following three different liposomal formulations were prepared: DPPC/Chol/DSPE-mPEG2000 (6:3:1 molar ratio, formulation A), DPPC/Chol/DOTAP (6:3:1 molar ratio, formulation B) and DPPC/Chol/DPPG (6:3:1 molar ratio, formulation C). The colloidal systems were obtained by the TLE technique and the extrusion process allowed us to obtain vesicles having mean sizes of 150-200 nm, while the surface charges varied between 50 mV and -30 mV. Formulation A showed the best encapsulation efficiency between the two compounds and the presence of TMX influenced the release profile of GEM (hydrophilic compound) as a consequence of its effect on the fluidity of the bilayer. An MDC of formulation A was used to effectuate the in vitro cytotoxicity experiments (MTT-test) on MCF-7 and T47D cells. The liposomal MDC provided the best results with respect to the single drug tested in the free form or entrapped in the same liposomal formulation. The CLSM experiments showed a great degree of cell interaction of liposomal MDC after just 6h.

Pharmacokinetic study of a new angiotensin-AT1 antagonist by HPLC.

Recently an innovative novel class angiotensin-AT1 antagonist has been developed by Rottapharm. In this study, we present a validated method for detecting CR 3834 in biological matrices using high-performance liquid chromatography (HPLC) with diode array detection. After oral administration (30mg/kg) to Wistar rats, the plasma and urine concentrations of CR 3834 and its potential metabolic products were determined. Moreover, the plasmatic time course in rats has been determined after intravenous (IV) administration of CR 3834 (5mg/kg). Biological samples (0.5ml of plasma and 1ml of urine) were purified using solid-phase extraction (SPE) of analytes and the internal standard Idebenone, 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1-4-benzoquinone. A chromatographic separation was performed on an Adsorboshere C18 at 25 degrees C, with a pre-column of the same matrix; the eluent was made up of acetonitrile/acidified water with CF3COOH (pH 2.01) in ratio of 75:25 (v/v); the flow rate was 1.0ml/min and a 100microl loop. The lower limit of detection (LOD) was taken as 25ng/ml in plasma and 50ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 0.1 and 0.2microg/ml in plasma and urine samples, respectively. The procedures were validated according to international standards with a good reproducibility and linear response (r=0.9916 in plasma; r=0.9997 in urine). The coefficients of variation inter assay ranged between 2.579 and 4.951% in plasma, and between 0.813 and 2.460% in urine. Mean recovery for CR 3834 was 79% in plasma and 97% in urine samples. The experiments performed demonstrated that the method presented was suitable for determining this new angiotensin-AT1 antagonist in rat plasma and urine.

Detection of GD2-positive cells in bone marrow samples and survival of patients with localised neuroblastoma.

The impact of bone marrow (BM) GD2-positive cells on survival has been evaluated in 145 Italian children with localised neuroblastoma (NB) evaluated at diagnosis by anti-GD2 immunocytochemistry. Nineteen of these (13.1%) were found to be BM GD2-positive, with the number of positive cells ranging between 1 and 155 out of 1 x 10(6) total cells analysed. Seven/19 (38.8%) GD2-positive vs 12/126 (9.5%) GD2-negative patients relapsed. The 5-year event-free survival (EFS) and overall survival of the GD2-positive patients was significantly worse than that of the GD2-negative ones (62.2 vs 89.9%, P<0.001; and 74.9 vs 95.9%, P=0.005, respectively). GD2 positivity was not associated to other known risk factors, and in particular to Myc-N amplification and 1p deletion. Among Myc-N-negative patients, the EFS of those negative for both GD2 and 1p deletion was significantly better than in children positive for either one of these two markers (EFS=96.9 vs 66.0%, P<0.001). In conclusion, GD2 positivity may represent a prognostic marker for patients with non-metastatic NB without Myc-N amplification, and its combination with genetic alterations might help identifying patients that require a more careful follow-up.

Determination of a potent non-competitive AMPA receptor antagonist in rat brain.

N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative (PS3Ac) has been determined in brain tissues by high performance liquid chromatography (HPLC) coupled with a diode array detection. In a previous paper we presented a validation method for detecting PS3Ac and its metabolites in plasma samples after intraperitoneal administration to Wistar rats. In the present paper, we report the results of the determination of PS3Ac and its N-deacetyl (PS3) and O-demethyl (PS3OH) metabolites, in the brain after extraction based on a polymeric matrix with a high hydrophilic-lipophilic balance, using Oasis cartridges. The chromatographic separation was performed in an octadecylsilica stationary phase at 25 degrees C using a mixture of 10 mM potassium dihydrogen orthophosphate (pH 2.24) and acetonitrile in ratio of 30:70 (v/v) as mobile phase, with a flow rate of 0.8 ml/min. The method exhibited a large linear range from 0.05 to 2 microg/ml for all studied compounds (n=6). In the within-day assay (n=4), the accuracy ranged from 87.5% determined with 0.05 microg/ml of PS3 to 110.1% determined with 0.2 microg/ml of PS3OH. In the between-day assay the coefficient of variation ranged from 2.4 determined with 0.05 microg/ml of PS3 to 9.7 determined with 0.2 microg/ml of PS3OH. The extraction efficiency ranged from 77.8% for PS3OH at 0.2 microg/ml to 94.3 for PS3Ac at 0.5 microg/ml. The limit of detection for all the tetrahydroisoquinoline derivatives ranged around 50 ng/ml. The method proved to be highly sensitive and specific to determinate PS3Ac and its metabolites and has been successfully applied to value their concentrations in brain matrix over the time.

P-glycoprotein 170 expression and function as an adverse independent prognostic factor in childhood acute lymphoblastic leukemia.

Little is known about the prognostic role of multidrug resistance (MDR) in newly diagnosed childhood acute lymphoblastic leukemia (ALL). P-glycoprotein 170 (MDR1), a cellular drug efflux pump, is thought to be one of the major causes of MDR. The aim of this retrospective study was to evaluate in 85 children with ALL the impact of the MDR1 product of the mdr-1 gene on the achievement of complete remission (CR) and outcome. MDR1 protein expression was performed by immunocytochemistry (ICC), and flow cytometry (FC). MDR1 functional activity was performed by a rhodamine (Rhd)-123 efflux test with or without verapamil. All patients enrolled in our study were treated with AIEOP ALL 91-95 protocols. At diagnosis, 40 patients (47%) expressed MDR1 protein at significant levels, and 45 (53%) were MDR1 negative. Forty-three of the latter patients were also negative for MDR1 function, while 34/40 (85%) patients MDR1 positive preserved the function. Rhd-123 efflux was inhibited by the MDR modulator verapamil in 12/40 (30%) patients. After induction treatment, CR was achieved in 77/85 children (90.6%). All patients who did not achieve CR were MDR1 positive. Twenty-nine patients relapsed, 17 (58.6%) of whom were MDR1 positive. The 10-year overall survival (OS) rate, and disease-free survival (DFS) for MDR1 negative patients compared to MDR1 positive patients were 75.7% versus 54.8%, and 67.5% versus 46%, respectively. The 10-year event-free survival (EFS) rate was significantly higher (67.5% versus 36.8%) in the MDR1 negative group compared with the MDR1 positive population (p=0.001). Multivariate analysis showed that only EFS was independent of age, WBC count, immunophenotype, FAB subtype and prednisone response (p=0.019). Our results, derived from a monocentric study, demonstrate that MDR1 expression in childhood ALL is an independent adverse prognostic factor on outcome, and could be a useful biological marker of response in these patients. Moreover, MDR1 function was also a predictor of response, but only in univariate analysis.

[Advances in diagnosis and therapy of breast cancer between the 20th and the 21st centuries]. / L'evoluzione della diagnostica e della terapia chirurgica del carcinoma mammario tra XX e XXI secolo.

During the last two decades several new data have been acquired about tumor biology and therapy. Breast cancer is one of the more common cancers occurring, determining life threatening and depression in affected females. By the way a great interest has been developed during the time attempting to reach the earliest diagnosis and the more conservative treatment. However, breast cancer is now considered as a disease which meet the interests of several specialists (oncologists, surgeons, radiologists, and so), all dedicated to reduce the consequences of such pathology. The aim of this review is the resume of the progresses made in the last century about breast cancer knowledge.