IL-32 promotes angiogenesis.

IL-32 is a multifaceted cytokine with a role in infections, autoimmune diseases, and cancer, and it exerts diverse functions, including aggravation of inflammation and inhibition of virus propagation. We previously identified IL-32 as a critical regulator of endothelial cell (EC) functions, and we now reveal that IL-32 also possesses angiogenic properties. The hyperproliferative ECs of human pulmonary arterial hypertension and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and, significantly, the cytokine colocalized with integrin αVß3. Vascular endothelial growth factor (VEGF) receptor blockade, which resulted in EC hyperproliferation, increased IL-32 three-fold. Small interfering RNA-mediated silencing of IL-32 negated the 58% proliferation of ECs that occurred within 24 h in scrambled-transfected controls. Reduction of IL-32 neither affected apoptosis (insignificant changes in Bak-1, Bcl-2, Bcl-xL, lactate dehydrogenase, annexin V, and propidium iodide) nor VEGF or TGF-ß levels, but siIL-32-transfected adult and neonatal ECs produced up to 61% less NO, IL-8, and matrix metalloproteinase-9, and up to 3-fold more activin A and endostatin. In coculture-based angiogenesis assays, IL-32γ dose-dependently increased tube formation up to 3-fold; an αVß3 inhibitor prevented this activity and reduced IL-32γ-induced IL-8 by 85%. In matrigel plugs loaded with IL-32γ, VEGF, or vehicle and injected into live mice, we observed the anticipated VEGF-induced increase in neocapillarization (8-fold versus vehicle), but unexpectedly, IL-32γ was equally angiogenic. A second signal such as IFN-γ was required to render cells responsive to exogenous IL-32γ; importantly, this was confirmed using a completely synthetic preparation of IL-32γ. In summary, we add angiogenic properties that are mediated by integrin αVß3 but VEGF-independent to the portfolio of IL-32, implicating a role for this versatile cytokine in pulmonary arterial hypertension and neoplastic diseases.

Geochemistry, stable isotopes and statistic tools to estimate threshold and source of nitrate in groundwater (Sardinia, Italy).

In the European Union, nitrate vulnerable zone (NVZ) should be designed for the mitigation of nitrate (NO3-) contamination caused by agricultural practices. Before establishing new NVZ, the sources of NO3- must be recognized. A geochemical and multiple stable isotopes approach (hydrogen, oxygen, nitrogen, sulfur and boron) and statistical tools were applied to define the geochemical characteristics of groundwater (60 samples), calculate the local NO3- threshold and assess potential sources of NO3- contamination in two study areas (hereafter Northern and Southern), located in a Mediterranean environment (Sardinia, Italy). Results of the integrated approach applied to two case study, permits to highlight the strengths of integrating geochemical and statistical methods to provide nitrate source identification as a reference by decision makers to remediate and mitigate nitrate contamination in groundwater. Hydrogeochemical features in the two study areas were similar: near neutral to slightly alkaline pH, electrical conductivity in the range of 0.3 to 3.9 mS/cm, and chemical composition ranging from Ca-HCO3- at low salinity to Na-Cl- at high salinity. Concentrations of NO3- in groundwater were in the range of 1 to 165 mg/L, whereas the nitrogen reduced species were negligible, except few samples having NH4+ up to 2 mg/L. Threshold values in the studied groundwater samples were between 4.3 and 6.6 mg/L NO3-, which was in agreement with previous estimates in Sardinian groundwater. Values of δ34S and δ18OSO4 of SO42- in groundwater samples indicated different sources of SO42-. Sulfur isotopic features attributed to marine SO42- were consistent with groundwater circulation in marine-derived sediments. Other source of SO42- were recognize due to the oxidation of sulfide minerals, to fertilizers, manure, sewage fields, and SO42- derived from a mix of different sources. Values of δ15N and δ18ONO3 of NO3- in groundwater samples indicated different biogeochemical processes and NO3- sources. Nitrification and volatilization processes might have occurred at very few sites, and denitrification was likely to occur at specific sites. Mixing among various NO3- sources in different proportions might account for the observed NO3- concentrations and the nitrogen isotopic compositions. The SIAR modeling results showed a prevalent NO3- source from sewage/manure. The δ11B signatures in groundwater indicated the manure to be the predominant NO3- source, whereas NO3- from sewage was recognized at few sites. Geographic areas showing either a predominant process or a defined NO3- source where not recognize in the studied groundwater. Results indicate widespread contamination of NO3- in the cultivated plain of both areas. Point sources of contamination, due to agricultural practices and/or inadequate management of livestock and urban wastes, were likely to occur at specific sites.

[Pulmonary embolism and SARS-CoV-2: analysis of patients hospitalized for pulmonary embolism and COVID-19 in a Northern Italian center]. / Embolia polmonare e SARS-CoV-2: analisi delle caratteristiche dei pazienti ricoverati presso un centro nel nord Italia per embolia polmonare associata a COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has shown high morbidity and mortality and the relationship between pulmonary embolism (PE) and COVID-19 is well established in the literature. METHODS: We describe the characteristics of a cohort of COVID-19 patients (EP-COV) hospitalized at our Centre with PE, investigating how COVID-19 may have influenced their outcomes, as compared to patients without COVID-19 hospitalized for PE in the same months of 2020 (EP-2020) and 2019 (EP-2019). RESULTS: EP-COV patients (n=25) were younger (60.5 ± 8.5 vs 71.4 ± 14.5 vs 70.9 ± 11.8 years, p=0.003), more frequently male (76% vs 48% vs 35%, p=0.016), with a lower history of neoplasia (12% vs 47% vs 40%, p=0.028) and more clinically severe (SOFA score 3.4 ± 1.4 vs 2.2 ± 1.4 vs 1 ± 1.1, p<0.001 and PaO2/FiO2 ratio 223.8 ± 75.5 vs 306.5 ± 49.3 vs 311.8 ± 107.5) than EP-2020 (n=17) and EP-2019 patients (n=20). D-dimer and C-reactive protein were higher in EP-COV (p=0.038 e p<0.001, respectively). The rate of concomitant deep vein thrombosis associated with PE did not differ significantly between the three groups. EP-COV patients developed PE more frequently during in-hospital stay than non-COVID-19 patients (p = 0.016). The mortality rate was higher in EP-COV than in EP-2020 and EP-2019 patients (36% vs 0% vs 5%, p=0.019). CONCLUSIONS: In our study, the risk factors for PE in COVID-19 patients seem to differ from the traditional risk factors for venous thromboembolism; EP-COV patients are clinically more severe and display a higher mortality rate than EP-2020 and EP-2019 patients.

[An unusual presentation of cardiac involvement during the COVID-19 pandemic]. / COVID-19 e coinvolgimento cardiaco: una presentazione inusuale.

Severe acute respiratory syndrome coronavirus 2 may affect the cardiovascular system and cause acute cardiac injury. Other authors described cases of myocarditis with reduced systolic function and/or a life-threatening presentation. We describe the clinical course of an unusual presentation with isolated reversible high degree atrioventricular block in a patient with COVID-19. In this case, a "wait and see approach" avoided an unnecessary permanent pacemaker implantation.

Pulmonary thromboembolism in hospitalised COVID-19 patients at moderate to high risk by Wells score: a report from Lombardy, Italy.

OBJECTIVES: To present a single-centre experience on CT pulmonary angiography (CTPA) for the assessment of hospitalised COVID-19 patients with moderate-to-high risk of pulmonary thromboembolism (PTE). METHODS: We analysed consecutive COVID-19 patients (RT-PCR confirmed) undergoing CTPA in March 2020 for PTE clinical suspicion. Clinical data were retrieved. Two experienced radiologists reviewed CTPAs to assess pulmonary parenchyma and vascular findings. RESULTS: Among 34 patients who underwent CTPA, 26 had PTE (76%, 20 males, median age 61 years, interquartile range 54-70), 20/26 (77%) with comorbidities (mainly hypertension, 44%), and 8 (31%) subsequently dying. Eight PTE patients were under thromboprophylaxis with low-molecular-weight heparin, four PTE patients had lower-limbs deep vein thrombosis at ultrasound examination (performed in 33/34 patients). Bilateral PTE characterised 19/26 cases, with main branches involved in 10/26 cases. Twelve patients had a parenchymal involvement >75%, the predominant pneumonia pattern being consolidation in 10/26 patients, ground glass opacities in 9/26, crazy paving in 5/26, and both ground glass opacities and consolidation in 2/26. CONCLUSION: COVID-19 patients are prone to PTE. ADVANCES IN KNOWLEDGE: PTE, potentially attributable to an underlying thrombophilic status, may be more frequent than expected in COVID-19 patients. Extension of prophylaxis and adaptation of diagnostic criteria should be considered.

Unlocking the full potential of lipid-based formulations using lipophilic salt/ionic liquid forms.

Lipid-based formulations (LBF) are widely used by industry and accepted by the regulatory authorities for oral drug delivery in the pharmaceutical and consumer healthcare market. Innovation in the LBF field is however needed in order to meet the demands of modern drugs, their more challenging problem statements and growing needs for achieving optimal pharmacokinetics (i.e., no food-effects, low variability) on approval. This review describes a new lipophilic salt / ionic liquid approach in combination with LBF, and how this salt strategy can be used to better tailor the properties of a drug to LBFs. The potential advantages of lipophilic salts are discussed in the context of dose escalation studies during toxicological evaluation, reducing the pill burden, increasing drug absorption of new drugs and in life-cycle management. Commentary on lipophilic salt synthesis, scale-up, LBF design and the regulatory aspects are also provided. These topics are discussed in the broad context of bringing the widely recognized advantages of LBFs to a broader spectrum of drugs.

Accumulation, platinum-DNA adduct formation and cytotoxicity of cisplatin, oxaliplatin and satraplatin in sensitive and resistant human osteosarcoma cell lines, characterized by p53 wild-type status.

P53 gene status is implicated in the cytotoxic drug sensitivity and published research has been mostly addressed to cisplatin (CDDP) activity. Previous study in our laboratory considered p53 mutant cell lines A431 (parental) and A431/Pt (CDDP-resistant counterpart, resistance factor R.F.=2.6). For a comparison which contributes to a deeper appreciation of the process that mediates the Pt drug cellular effects, we extended our investigation to the p53 wild-type cell lines U2-OS (human osteosarcoma) and its CDDP-resistant counterpart U2-OS/Pt (R.F.=5). We compared the activity of CDDP, oxaliplatin (L-OHP) and satraplatin (JM216) whose hydrophobicity rank is JM216>L-OHP>CDDP. In U2-OS cells the three drugs accumulated similarly, while in U2-OS/Pt the most hydrophobic drugs were privileged. No significant differences in efflux were observed between sensitive and resistant cell lines. The growing of CDDP resistance seems to be overcome by increasing the hydrophobicity of the Pt agent. An almost linear trend seems to relate R.F. and drug hydrophobicity in U2-OS/Pt and A431/Pt cells. DNA platination in U2-OS as in A431 cells is at the lowest levels for L-OHP. In U2-OS cell line the IC(50) of CDDP (17.6 microM) and JM216 (88.02 microM) do not correlate with their similar levels of Pt-DNA adducts (mean value approximately 0.14 pmol Pt/microg DNA). The presence of a wild-type p53 exalts either CDDP cytotoxicity (two-fold more active in U2-OS than in A431 cells) and CDDP resistance in comparison to a p53 mutant type. The p53 status seems to not improve JM216 or L-OHP cytotoxicity in both cell lines.

Cross-sectional geometrical properties of distal radius and ulna in large, medium and toy breed dogs.

Fractures of the distal radius and the ulna are the third most common fractures in dogs. Toy and miniature breeds have a propensity to develop antebrachial fractures after falling or jumping. Most affect the distal third of both bones involving between 15% and 37% of the radial length. Larger dogs, instead, typically sustain hyperextension injuries to the carpus. The causative mechanisms for this fracture prevalence in toy dogs are unknown. Breed-related changes in bone geometry and/or mineral density have been suggested as possible etiologic factors. In a multifactorial study, the main etiological factors potentially responsible for determining susceptibility to fractures in toy breeds are considered. The aim of this first study is to evaluate the geometric bone features in different dog sizes. The cortical bone cross-sectional properties along the length of the right radius and the ulna of 28 dogs from three different size categories have been quantified by computerized tomographic scanning. Geometrical cross-sectional parameters were measured and normalized to radial length to allow intergroup comparisons. Discriminant analysis was used to classify the observations into different groups. Through statistical analysis of the normalized values, significant differences in cross-sectional properties of different bone sizes were found. The results suggest that, when proportionally loaded, the antibrachii of toy breed dogs are more susceptible to fracture than those of large breed dogs due to morphological differences.

Chemical synthesis of proteins and circular peptides using Nalpha-(1-phenyl-2-mercaptoethyl) auxiliaries.

An overview of the applications of Nalpha-(1-phenyl-2-mercaptoethyl) auxiliary is presented. We describe the on resin preparation (Calpha-carboxy and thioester) of Nalpha-auxiliary derivatives of glycine and the synthesis and incorporation of preformed Nalpha-auxiliary derivatives of glycine and alanine with the protection schemes, including the thiazolidine strategy for SPPS. Such approaches allowed the synthesis of the protein cytochrome b562 as well as native circular peptides after successful removal of the auxiliary.

Native chemical ligation through in situ O to S acyl shift.

[reaction: see text] A novel strategy to generate thioester peptides compatible with Fmoc chemistry is presented. Peptide-C(alpha)oxy-(2-mercapto-1-carboxyamide)ethyl ester undergoes an O to S acyl shift during ligation and the newly formed thioester intermediate reacts with an N-terminal cysteine fragment generating a product with native amide bond at the ligation site.

Semisynthetic analogues of PSC-RANTES, a potent anti-HIV protein.

New HIV prevention methods are needed, and among those currently being explored are "microbicides", substances applied topically to prevent HIV acquisition during sexual intercourse. The chemokine analogue PSC-RANTES (N(alpha)(n-nonanoyl)-des-Ser(1)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)]-RANTES(4-68)) is a highly potent HIV entry inhibitor which has shown promising efficacy in its initial evaluation as a candidate microbicide. However, a way must be found to produce the molecule by cheaper means than total chemical synthesis. Since the only noncoded structures are located at the N-terminus, a possible solution would be to produce a protein fragment representing all but the N-terminal region using low-cost recombinant production methods and then to attach, site specifically, a short synthetic fragment containing the noncoded N-terminal structures. Here, we describe the evaluation of a range of different conjugation chemistries in order to identify those with potential for development as economical routes to production of a PSC-RANTES analogue with antiviral activity as close as possible to that of the parent protein. The strategies tested involved linkage through oxime, hydrazone/hydrazide, and Psi[CH2-NH] bonds, as well as through a peptide bond obtained either by a thiazolidine rearrangement or by direct alpha-amino acylation of a protein fragment in which 4 of the 5 lysine residues of the native sequence were replaced by arginine (the fifth lysine is essential for activity). Where conjugation involved replacement of one or more residues with a linker moiety, the point in the main chain at which the linker was introduced was varied. The resulting panel of 22 PSC-RANTES analogues was evaluated for anti-HIV activity in an entry inhibition assay. The [Arg (25,45,56,57)] PSC-RANTES analogue has comparable potency to PSC-RANTES, and one of the oxime linked analogues, 4L-57, has potency only 5-fold lower, with scope for improvement. Both represent promising leads for development as microbicide compounds that could be produced at low cost via semisynthesis.

Suction due to left ventricular assist: implications for device control and management.

Left ventricular assist device (LVAD) overpumping is associated with hemolysis, thrombus release, and tissue damage at the pump inlet. However, the impact of LVAD suction on pulmonary circulatory function remains unknown. We investigated LVAD suction as induced by pulmonary artery banding and overpumping in experimental animals and in a computer model. In six sheep, a rotary LVAD was implanted. Before inducing suction, partial support (40-60% of cardiac output) was established and characterized by measuring pressures and flows. In the animals, pulmonary artery occlusion (PAOC) elicited LVAD suction (left ventricular pressure was from -10 to -20 mm Hg) within 5-10 heartbeats. During suction, aortic pressure dropped to 50% and LVAD flow decreased significantly. After releasing the occlusion (20 s), the collapsed state persisted for another 20 s. A similar trend was obtained by simulating PAOC in the computer model. Additional simulations showed that pulmonary vascular resistance (PVR), volume status, and right ventricular (RV) contractility are exponentially related to the persistence of collapse after a suction event. Even modest increases in predisposing factors (elevated PVR, RV dysfunction, hypovolemia) caused sustained hemodynamic collapse lasting in excess of 15 min. Both in selected animals and the computer model, comparable suction-induced collapse was obtained by increasing LVAD speed by about 33%. Attempted compensation by simply decreasing speed was not effective, but temporarily shutting down the LVAD caused rapid reversal of collapse. In conclusion, rotary LVAD suction causes unfavorable conditions for effective unloading. The use of pump interventions appears a promising tool to detect suction and to avoid the associated hemodynamic depression.

The chemical synthesis of the GstI protein by NCL on a X-Met site.

The small GstI protein (63 amino acids) of Rhizobium leguminosarum is the endogenous inhibitor of the glnII (glutamine synthetase II) gene expression. It has been suggested that GstI has a predominantly beta-structure and mediates the block of translation and stabilization of glnII mRNA through direct binding to its 5' untranslated region. Because of the unavailability of adequate amounts of purified recombinant protein, the mechanism as well as the protein tridimensional structure remain very poorly understood. To obtain the full-length protein, we have undertaken the chemical synthesis of the protein by different approaches. In a first attempt, the stepwise synthesis was unsuccessful, with strong aggregation experienced on the N-terminal side, after residue 44 from the C-terminus. In a second approach, we set up the conditions to carry out a native chemical ligation (NCL). Albeit the protein contains two Cysteine residues, located at positions 40 and 47, to minimize the size of the N-terminal segment to be synthesized, we have devised an alternative strategy of ligation on Met32, utilizing homoCys as the ligating moiety and then alkylating the resulting polypeptide with methyl iodide. New conditions to quantitatively methylate thiol groups in complex polypeptides have been conceived, obtaining the protein in very good yields and purity. A CD spectroscopy investigation has revealed that the protein does not adopt canonical secondary structures but is very rich in beta-structure (approximately 60%), in agreement with a previous study carried out on samples obtained by recombinant methods.

Synthesis of N alpha-(1-phenyl-2-mercaptoethyl) amino acids, new building blocks for ligation and cyclization at non-cysteine sites: scope and limitations in peptide synthesis.

A new and convenient method for the synthesis and incorporation of N(alpha)-(1-phenyl-2-mercaptoethyl)-derivatized amino acids applicable to chemical ligation at non-cysteine sites is presented. N(alpha)-Auxiliary derivatives of glycine and alanine were easily prepared using reductive amination approaches. Several strategies for the incorporation of these derivatives into peptide chains were investigated: coupling without protection, with acid-labile protection, with base-labile protection, and via a novel protection strategy using the thiazolidine derivative. All amino acid derivatives were successfully coupled to various peptide resins, and with the exception of those incorporating Boc-protected derivatives, all resins yielded the desired peptide fragments. However, the coupling of the two alanine derivative diastereomers generated some epimerization. Finally, N-terminal auxiliary glycine and alanine peptides were cyclized, and the corresponding native circular peptides were obtained upon successful removal of the auxiliary.

Urban wastewater effects on water flow and solute transport in soils.

This article aims mainly to verify the consequences of urban wastewater reuse in irrigation practices on hydrological behavior of soils. Herein the effects on both the hydraulic and dispersive properties of characteristic soils in South Sardinia are illustrated. The study was carried out at the Ente Autonomo del Flumendosa (EAF) laboratory. Ten undisturbed soil monoliths, 120 cm height and 40 cm diameter, were collected from plots, the locations of which were preliminarily individuated through a prior pedological study. An experimental facility was set up and sensors for monitoring both soil water and solute concentrations were inserted horizontally in each column at different depths. A disturbed layer at the soil surface, which expands in depth with time, was observed, characterized by reduced soil porosity, translation of pore size distribution towards narrower pores and consequent drastic decrease of permeability. It will be shown that these changes occurring in the disturbed soil layer, although local by nature, affect the mean hydrological behavior of the whole soil profile.

Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins.

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.5 liter) approach involving sample pooling for the analysis of smaller proteins (M(r) generally < ca. 40 000 and some fragments of very large proteins). Plasma from healthy males was depleted of abundant proteins (albumin and IgG), then smaller proteins and polypeptides were separated into 12 960 fractions by chromatographic techniques. Analysis of proteins and polypeptides was performed by mass spectrometry prior to and after enzymatic digestion. Thousands of peptide identifications were made, permitting the identification of 502 different proteins and polypeptides from a single pool, 405 of which are listed here. The numbers refer to chromatographically separable polypeptide entities present prior to digestion. Combining results from studies with other plasma pools we have identified over 700 different proteins and polypeptides in plasma. Relatively low abundance proteins such as leptin and ghrelin and peptides such as bradykinin, all invisible to two-dimensional gel technology, were clearly identified. Proteins of interest were synthesized by chemical methods for bioassays. We believe that this is the first time that the small proteins in human blood plasma have been separated and analyzed so extensively.

Design and chemical synthesis of a homogeneous polymer-modified erythropoiesis protein.

We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.

In vitro and in silico processes to identify differentially expressed proteins.

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.