A miRNA Signature in Human Cord Blood Stem and Progenitor Cells as Potential Biomarker of Specific Acute Myeloid Leukemia Subtypes.

MicroRNAs (miRNAs) are important regulators of several cellular processes. During hematopoiesis, specific expression signatures have been reported in different blood cell lineages and stages of hematopoietic stem cell (HSC) differentiation. Here we explored the expression of miRNAs in umbilical cord blood stem (HSC) and progenitor cells (HPC) and compared it to unilineage granulocyte and granulo-monocyte differentiation as well as to primary blasts from patients with acute myeloid leukemia (AML). CD34 + CD38- ad CD34 + CD38 + cells were profiled using a global array consisting of about 2000 miRNAs. An approach combining bioinformatic prediction of miRNA targets with mRNA expression profiling was used to search for putative biologically enriched functions and networks. At least 15 miRNAs to be differentially expressed between HSC and HPC cell population, a cluster of 7 miRNAs are located in the q32 region of human chromosome 14 (miR-377-3p, -136-5p, 376a-3p, 495-3p, 654-3p, 376c-3p and 381-3p) whose expression decreased during the early stages of normal myelopoiesis but were markedly increased in a small set of AML. Interestingly, miR-4739 and -4516, two novel microRNA whose function and targets are presently unknown, showed specific and peculiar expression profile during the hematopoietic stem cells differentiation into unilineages and resulted strongly upregulated in almost all AML subsets. miR-181, -126-5p, -29b-3p and -22-3p resulted dis-regulated in specific leukemias phenotypes. This study provides the first evidence of a miRNA signature in human cord blood stem and progenitor cells with a potential role in hematopoietic stemness properties and possibly in leukemogenesis of specific AML subtypes.

Vacuum evaporation treatment of digestate: full exploitation of cogeneration heat to process the whole digestate production.

Vacuum evaporation represents an interesting and innovative solution for managing animal waste surpluses in areas with high livestock density. To reduce operational costs, a key factor is the availability of an inexpensive source of heat, such as that coming from an anaerobic digestion (AD) plant. The aim of this study was to test vacuum evaporation for the treatment of cattle slurry digestate focusing on heat exploitation. Tests were performed with a pilot plant fed with the digestate from a full-scale AD plant. The results were used to evaluate if and how cogeneration heat can support both the AD plant and the subsequent evaporation of the whole daily digestate production in a full-scale plant. The concentrate obtained (12% total solids) represents 40-50% of the influent. The heat requirement is 0.44 kWh/kg condensate. Heat power availability exceeding the needs of the digestor ranges from 325 (in winter) to 585 kW (in summer) versus the 382 kW required for processing the whole digestate production. To by-pass fluctuations, we propose to use the heat coming from the cogenerator directly in the evaporator, tempering the digestor with the latent heat of distillation vapor.

The selection of paired watersheds affects the assessment of wildfire hydrological impacts.

Wildfires strongly alter hydrological processes and surface and groundwater quality in forested environments. The paired-watershed method, consisting of comparing a burnt (altered) watershed with an unburnt (control) watershed, is commonly adopted in studies addressing the hydrological effects of wildfires. This approach requires a calibration period to assess the pre-perturbation differences and relationships between the control and the altered watershed. Unfortunately, in many studies, the calibration phase is lacking due to the unpredictability of wildfires and the large number of processes that should be investigated. So far, no information is available on the possible bias induced by the lack of the calibration period in the paired-watershed method when assessing the hydrological impacts of wildfires. Through a literature review, the consequences of the lack of calibration on the assessment of wildfire hydrological changes were evaluated, along with the most used watershed pairing strategies. The literature analysis showed that if calibration is lacking, misestimation of wildfire impacts is likely, particularly when addressing low-severity or long-term wildfire effects. The Euclidean distance based on physical descriptors (geology, morphology, vegetation) was proposed as a metric of watersheds similarity and tested in mountain watersheds in Central Italy. The Euclidean distance proved to be an effective metric for selecting the most similar watershed pairs. This work raises awareness of biases exerted by lacking calibration in paired-watershed studies and proposes a rigorous and objective methodology for future studies on the hydrological effects of wildfires.

Genomic and epidemiological evidence for the emergence of a L. infantum/L. donovani hybrid with unusual epidemiology in northern Italy.

Leishmania (L.) infantum is one of the main causative agents of animal and human leishmaniasis across many endemic areas in South America, Europe, North Africa, and Asia. Despite its clinical significance, little is known about the genetic diversity of L. infantum circulating in a given endemic area. Here, we investigate this important open question by applying a comparative genomics approach to seven L. infantum isolates from different hosts and Italian regions, including the northern part of the country (Emilia-Romagna, RER), Sicily, and Sardinia, as an initial attempt to explore the breadth of parasite genetic heterogeneity in Italy. Additionally, microsatellite analysis was carried out to compare the isolates from RER with other 70 L. infantum strains from the same region as well as 65 strains belonging to the L. donovani complex from other countries. We revealed important karyotypic instability and identified strain-specific changes in gene dosage, which affected important virulence factors such as amastins and surface antigen-like proteins. Single nucleotide polymorphism-based clustering analysis of these genomes together with over 80 publicly available L. infantum and L. donovani genomes placed the Italian isolates into three geographically distinct clusters within the Mediterranean basin and uncovered three isolates clustering with putative L. infantum/L. donovani hybrids isolated in Cyprus. As judged by microsatellite profiling, these hybrid isolates are representative of a sub-population of parasites circulating in northern Italy that preferentially infect humans but not dogs. Our results place Italy at the crossroads of L. infantum infection in the Mediterranean and call attention to the public health risk represented by the introduction of non-European Leishmania species.IMPORTANCEThis study closes important knowledge gaps with respect to Leishmania (L.) infantum genetic heterogeneity in a given endemic country, as exemplified here for Italy, and reveals genetic hybridization as a main cause for re-emerging human leishmaniasis in northern Italy. The observed high diversity of Leishmania parasites on the Italian peninsula suggests different geographical origins, with genomic adaptation to various ecologies affecting both pathogenicity and transmission potential. This is documented by the discovery of a putative L. infantum/L. donovani hybrid strain, which has been shown to preferentially infect humans but not dogs. Our results provide important information to health authorities, which need to consider the public health risk represented by the introduction of new Leishmania species into EU countries due to population displacement or travel from countries where exotic/allochthonous parasite species are endemic.

HIV impairs CD34+-derived monocytic precursor differentiation into functional dendritic cells.

Dendritic cells (DCs) perform a basic role in the immune system by allowing the initiation of the primary T-cell-dependent immune response. Given previous indirect evidence that DC maturation and function are impaired by HIV, we have developed an in vitro culture system in order to verify the effect of HIV infection on DC function during the development from hematopoietic progenitors. Considering that monocytic (Mo) differentiating cells efficiently replicate monocytotropic HIV, we examined whether HIV-infected monocytic precursors (MoP) were able to generate functional DCs. CD34+ hematopoietic progenitor cells (HPCs) were induced along Mo differentiative pathway in liquid cultures and at an early stage of culture, MoP were infected with M-tropic BaL HIV strain, and after 2 days they were switched to DC differentiation with GM-CSF and IL-4. Derived DCs were actively infected, as detected by HIV-p24 production. HIV did not significantly affect cell viability, but induced a reduction in cell proliferation and an inefficient functional activity in terms of uptake capability and stimulation of allogenic T cells. These results indicate that HIV-infected MoP lost the capacity to generate functional DCs, and this may represent one of the many mechanisms of immunosuppression exploited by HIV.

Cellular mediators in human leishmaniasis: Critical determinants in parasite killing or disease progression.

Data on cellular immunity mediators in the early phase of human leishmaniasis are still limited and controversial. In order to mimic the changes of humoral mediators during the early phase of human natural infection, some Th1, Th2, Treg, and Breg cytokines, MCP-1, and the nitric oxide (NO) from human PBMC, stimulated by Leishmania infantum, Leishmania major, Leishmania donovani and Leishmania tropica infective metacyclic promastigotes, were determined. After 4 h of L. major, L. donovani, and L. tropica challenge, TNFα, IL-1ß, IL-6 levels were significantly higher than negative control cultures with saline (SF) instead of Leishmania promastigotes, unlike L. infantum-stimulated TNFα and L. major-stimulated IL-1ß. We obtained higher levels of IL-4 and IL-10 cytokines after stimulation of human PBMCs by L. infantum and L. donovani, compared to those observed after the challenge of PBMCs by L. major and L. tropica. Regarding IL-35, such cytokine levels were significantly increased following infection with L. infantum and L. donovani, in contrast to L. major and L. tropica. Up to our knowledge, we are the first to study the effect of four different species of Leishmania on IL-35 levels in human cells. Our study highlights how several Leishmania species can up-regulate different groups of cytokines (Th1, Th2, Treg and Breg) and modulate NO release in a different way. This original aspect can be explained by different Leishmania cell products, such as LPG, obtained from different strains/species of live parasites. Our findings would contribute to the development of new therapeutics or vaccination strategies.

New online management software and DICOM viewer for dentistry.

The way we collect, store, and share dental records of our patients is rapidly becoming digital. Many programs have been designed to run on a single computer or local network to handle various tasks, so selecting a system can be complex; it can require high acquisition costs, update fees, and installation difficulties. The purpose of this article is to explain the architecture, characteristics, and advantages related to Web-based Management Software. In particular, this article describes the first Web-based Electronic Medical Record (DocSapiens.com), which is able to view and edit DICOM files directly online.

Effect of traditional check dams (jessour) on soil and olive trees water status in Tunisia.

In the arid regions of south eastern Tunisia, the land use is predominated by olive trees cropping, where two main cultivation strategies can be found: using of water harvesting techniques to overcome the scarcity and variability of rainfall (in the Matmata mountains) and dryland farming (in the Jeffara plain). In these arid areas, soil moisture is the main limiting factor for crop growth and it should be monitored to benchmark different management options. Different conventional methods are available for point soil moisture monitoring, but the increased availability of remotely sensed data offers major opportunities for spatial analyses. The aim of this paper is to perform a comparative study on the soil water status for rainfed olive tree growing in three major landscape areas: in the mountains with traditional water harvesting check dams (called jessour), in the piedmont on floodwater harvesting (called tabias), and in the plain with full dryland farming conditions. Time series of Normalized Difference Infrared Index (NDII), derived from Landsat 7 satellite, were retrieved from the novel Google Earth Engine platform. NDII values were related to measured soil water content, which was taken at non-regular time intervals between 2009 and 2017. The analysis of NDII data, indicating the water content of the vegetation, shows that jessour can adequately ensure water supply for olive trees. Increased soil moisture conditions in the jessour areas are visible both in the dry and the humid seasons, indicating the effectiveness of this traditional water harvesting system. Moreover, our results show that Landsat 7 NDII values are correlated with the root-zone soil moisture in the monitoring sites (r2 ranging from 0.62 to 0.67), allowing the use of NDII to estimate soil water contents in our study area.

[Neonatal group B Streptococcus infection: an integrated approach (of clinical pathologists, gynecologists, midwives, pediatricians-neonatologists) of the functional area of Cuneo (Italy)]. / Infezione neonatale da Streptococco B: un approccio multidisciplinare integrato (di laboratoristi, ginecologi, ostetriche e pediatri-neonatologi) del Quadrante di Cuneo.

The frequency of early-onset neonatal sepsis without prophylaxis is 1-5/1.000 live births. Since year '70 the most frequent causative microorganism is the group B Streptococcus (S. agalactiae, GBS), followed by Escherichia coli. The mortality rate is now reduced to 4% due to the improvement of neonatal intensive care. In the USA, the incidence of GBS early-onset neonatal sepsis has been markedly reduced by the application of the guidelines released by the Centers for Disease Control (CDC). This strategy, however, is not effective on occurrence of late-onset neonatal group B streptococcal disease. In Italy, the application of CDC guidelines is not customary, and different, often complex, protocols of obstetrical-neonatological integrated approach are applied. The frequency of infectious risk has made the GBS a paramount problem for the neonatologist, even for the legal responsibility issues resulting from the multiplicity of possible options. To reach the best level of protection of the newborn against early-onset GBS infection, the working group of providers of prenatal, obstetric, and neonatal care of the functional area of Cuneo issued an integrated protocol, in order to perform the GBS screening with the optimal culture method suggested by CDC guidelines in the highest possible number of pregnant women, and to standardize the obstetrical and neonatal management.

Unilineage monocytopoiesis in hematopoietic progenitor culture: switching cytokine treatment at all Mo developmental stages induces differentiation into dendritic cells.

We have developed a new culture system whereby human hematopoietic progenitors purified from adult peripheral blood extensively proliferate and gradually differentiate into >95% pure monocytic (Mo) cells. At all developmental stages treatment with interleukin (IL)-4+granulocyte-macrophage colony-stimulating factor or IL-4+c-Kit-ligand+FLT-3 ligand switched the Mo precursors into dendritic cells (DCs). The switching capacity decreased only at the end of the culture, when most Mo cells matured to macrophages. Moreover, the Mo precursors were highly susceptible to transduction with lentiviral vectors: once switched to DCs, they maintained the transgene expression, as well as the phenotype and function of the DC lineage. Our results provide new insight into the potential role of the Mo lineage as a reservoir of DCs in vivo. Furthermore, the methodology for transduction of Mo precursors provides a tool to generate genetically modified, normally functioning DCs potentially useful for immunotherapy.

Temperature-induced states of isolated F1-ATPase affect catalysis, enzyme conformation and high-affinity nucleotide binding sites.

Isolated, nucleotide-depleted bovine-heart F1-ATPase exhibits a break in Arrhenius plot with a 2.7-fold increase in activation energy of ATP hydrolysis below 18-19 degrees C. Analysis of intrinsic tyrosine fluorescence and of the circular dichroism of F1-ATPase showed an abrupt and reversible conformational change occurring at the break temperature, characteristic of a structural tightening at low temperature. Analysis of catalytic nucleotide binding sites using fluorescent ADP analog, 3'-O-(1-naphthoyl)adenosine diphosphate did not show any significant change in affinity of nucleotide binding around the transition temperature but the bound fluorophore exerted a more restricted motion and slower rotation at temperature below the break, indicating a change in the mobility of groups in the close neighbourhood. It is concluded that, as a result of temperature, two kinetically distinct states of F1-ATPase are induced, due to a change in enzyme conformation, which influences directly the properties of catalytic nucleotide binding sites.

Lipid protein interactions in mitochondria. VII. A comparison of the effects of lipid removal and lipid perturbation of the kinetic properties of mitochondrial ATPase.

We investigated the kinetics of mitochondrial ATPase in bovine heart mitochondria and submitochondrial particles upon treatment with phospholipase A2, or upon addition of n-butanol to perturb the lipid protein interactions. The changes observed are the following: (1) Lipid removal or perturbation with butanol is accompanied by loss of ATPase activity with decrease of both V and of the KM for ATP. (2) There are changes of activation energy of ATPase activity at temperatures above the discontinuity normally observed for membrane-bound enzymes in mitochondria. In particular, butanol abolishes the discontinuity, and induces a constant activation energy of about 32 kcal/mol in the range 8--37 degrees C. (3) Butanol modifies the pH dependence of ATPase shifting the pH optimum from around 10 to less alkaline values. The optimum for Mg2+ concentrations is increased by the solvent. (4) Treatment with phospholipase A2 results in a removal of oligomycin-sensitive ATPase, whereas butanol addition prevents oligomycin inhibition of ATPase. (5) In beef heart mitochondria, a spin-labelled analog of the inhibitor, dicyclohexyl carbodiimide, did not show any change in environment upon butanol addition, unlike that found in mitochondria from Saccharomyces cerevisiae.

Saturation kinetics of coenzyme Q in NADH oxidation: rate enhancement by incorporation of excess quinone.

In beef heart mitochondria it has been found that the Km for coenzyme Q10 of the NADH oxidation system is in the range of the membrane concentration of the quinone; this is contrary to succinate oxidation which is in Vmax with respect to quinone content. The same proportional difference between the two systems is maintained in their affinities for the exogenous acceptor CoQ1 in non-extracted mitochondria. The Km of succinate- coenzyme Q reductase for CoQ1 is reversibly lowered in CoQ-depleted mitochondria; while in contrast the Km for NADH-coenzyme Q reductase is reversibly increased by CoQ extraction. Incorporation of exogenous quinones by co-sonication with submitochondrial particles, as evidenced by fluorescence quenching of pyrene, enhances NADH-cytochrome c reductase activity in accordance with the lack of saturation of the former system.

Coenzyme Q deficiency in mitochondria: kinetic saturation versus physical saturation.

The coenzyme Q (CoQ) concentration in the inner membrane of beef heart mitochondria is not kinetically saturating for NADH oxidation inasmuch as the K(m) of NADH oxidation for endogenous CoQ10 is in the mM range in membrane lipids. Using CoQ1 as an electron acceptor from complex I, we have found additional evidence that the high Km of NADH oxidase for CoQ is not an artifact due to the use of organic solvents in reconstitution studies. We have also obtained experimental evidence that CoQ concentration may be rendered more rate-limiting for NADH oxidation either by a decrease of CoQ content (as in liver regeneration or under an acute oxidative stress), or by a possible increase of the Km for CoQ, as in some mitochondrial diseases and ageing. The possibility of enhancing the rate of NADH oxidation by CoQ therapy is hindered by the fact that the CoQ concentration in mitochondria appears to be regulated by its mixability with the membrane phospholipids. Nevertheless CoQ10 incorporated into heart submitochondrial particles by sonication enhances NADH oxidation (but not succinate oxidation) up to twofold. Nontoxic CoQ homologs and analogs having shorter side-chains with respect to CoQ10 can be incorporated in the mitochondrial membrane without sonication, supporting an enhancement of NADH oxidation rate above 'physiological' values. It is worth investigating whether this approach can have a therapeutical value in vivo in mitochondrial bioenergetic disorders.

An updating of the biochemical function of coenzyme Q in mitochondria.

The apparent Km for coenzyme Q10 in NADH oxidation by coenzyme Q (CoQ)-extracted beef heart mitochondria is close to their CoQ content, whereas both succinate and glycerol-3-phosphate oxidation (the latter measured in hamster brown adipose tissue mitochondria) are almost saturated at physiological CoQ concentration. Attempts to enhance NADH oxidation rate by excess CoQ incorporation in vitro were only partially successful: the reason is in the limited amount of CoQ10 that can be incorporated in monomeric form, as shown by lack of fluorescence quenching of membrane fluorescent probes; at difference with CoQ10, CoQ5 quenches probe fluorescence and likewise enhances NADH oxidation rate above normal. Attempts to enhance the CoQ content in perfused rat liver and in isolated hepatocytes failed to show uptake in the purified mitochondrial fraction. Nevertheless CoQ cellular uptake is able to protect mitochondrial activities. Incubation of hepatocytes with adriamycin induces loss of respiration and mitochondrial potential measured in whole cells by flow cytometry using rhodamine 123 as a probe: concomitant incubation with CoQ10 completely protects both respiration and potential. An experimental study of aging in the rat has shown some decrease of mitochondrial CoQ content in heart, and less in liver and skeletal muscle. In spite of the little change observed, it is reasoned that CoQ administration may be beneficial in the elderly, owing to the increased demand for antioxidants.

Fluidizing effect of endogenous ubiquinone in bovine heart mitochondrial membranes.

Extraction of endogenous ubiquinone from lyophilized beef heart mitochondria results in increases of both the order parameter of the spin label 5-NS and of the rotational correlation time of 16-NS; reconstitution with the pentane extract results in restoration of the original spectral parameters. On the other hand, addition of purified ubiquinone homologs restores the original spectra only in the case of 16-NS, whereas the order parameter of 5-NS is restored by addition of mixed phospholipids. The same amounts of ubiquinone homologs incorporated in mixed phospholipid vesicles induce much lower effects. It is suggested that ubiquinone in mitochondria is intercalated with the lipid chains of the membrane in such a way to perturb the fluidity of the hydrophobic core.

Measurement of the lateral diffusion coefficients of ubiquinones in lipid vesicles by fluorescence quenching of 12-(9-anthroyl)stearate.

The lateral diffusion coefficients of some ubiquinone homologues have been measured in phospholipid vesicles exploiting the fluorescence quenching of the probe 12-(9-anthroyl)stearate by the quinones. Diffusion coefficients higher than 10(-6) cm2 X s-1 have been found at 25 degrees C, compatible with the localization of the ubiquinones in the low-viscosity midplane region of the bilayer.

The effect of aging and an oxidative stress on peroxide levels and the mitochondrial membrane potential in isolated rat hepatocytes.

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on the mitochondrial membrane potential. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor age. The hydrogen peroxide is significantly increased in both aged and adult rat hepatocytes, however, due to the high basal level in the aged cells, it is higher in aged rat cells not subjected to oxidative stress than that elicited by 50 microM adriamycin in young rat hepatocytes. The results suggest that a hydrogen peroxide increase in hepatocytes of aged rats is unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that the effects of the drug may be due to the concomitant high level of both superoxide and hydrogen peroxide.

Temperature-dependent conformational changes in isolated oligomycin-sensitive ATPase.

Isolated oligomycin-sensitive ATPase undergoes a kinetic change at 20-25 degrees C with a higher activation energy and a lower Km for ATP below this temperature range. This observation has been correlated with temperature-dependent structural changes detected by circular dichroism in the UV region in the isolated enzyme. The negative ellipticities in the 208-225 nm region, which are proportional to the alpha-helix content, increase with rise in temperature to a maximum above 25 degrees C.