Coping with salinity stress: segmental group 7 chromosome introgressions from halophytic Thinopyrum species greatly enhance tolerance of recipient durum wheat.

Increased soil salinization, tightly related to global warming and drought and exacerbated by intensified irrigation supply, implies highly detrimental effects on staple food crops such as wheat. The situation is particularly alarming for durum wheat (DW), better adapted to arid/semi-arid environments yet more sensitive to salt stress than bread wheat (BW). To enhance DW salinity tolerance, we resorted to chromosomally engineered materials with introgressions from allied halophytic Thinopyrum species. "Primary" recombinant lines (RLs), having portions of their 7AL arms distally replaced by 7el1L Th. ponticum segments, and "secondary" RLs, harboring Th. elongatum 7EL insertions "nested" into 7el1L segments, in addition to near-isogenic lines lacking any alien segment (CLs), cv. Om Rabia (OR) as salt tolerant control, and BW introgression lines with either most of 7el1 or the complete 7E chromosome substitution as additional CLs, were subjected to moderate (100 mM) and intense (200 mM) salt (NaCl) stress at early growth stages. The applied stress altered cell cycle progression, determining a general increase of cells in G1 and a reduction in S phase. Assessment of morpho-physiological and biochemical traits overall showed that the presence of Thinopyrum spp. segments was associated with considerably increased salinity tolerance versus its absence. For relative water content, Na+ accumulation and K+ retention in roots and leaves, oxidative stress indicators (malondialdehyde and hydrogen peroxide) and antioxidant enzyme activities, the observed differences between stressed and unstressed RLs versus CLs was of similar magnitude in "primary" and "secondary" types, suggesting that tolerance factors might reside in defined 7el1L shared portion(s). Nonetheless, the incremental contribution of 7EL segments emerged in various instances, greatly mitigating the effects of salt stress on root and leaf growth and on the quantity of photosynthetic pigments, boosting accumulation of compatible solutes and minimizing the decrease of a powerful antioxidant like ascorbate. The seemingly synergistic effect of 7el1L + 7EL segments/genes made "secondary" RLs able to often exceed cv. OR and equal or better perform than BW lines. Thus, transfer of a suite of genes from halophytic germplasm by use of fine chromosome engineering strategies may well be the way forward to enhance salinity tolerance of glycophytes, even the sensitive DW.

Proteomic analysis identifies a signature of disease severity in the plasma of COVID-19 pneumonia patients associated to neutrophil, platelet and complement activation.

Most patients infected with SARS-CoV-2 display mild symptoms with good prognosis, while 20% of patients suffer from severe viral pneumonia and up to 5% may require intensive care unit (ICU) admission due to severe acute respiratory syndrome, which could be accompanied by multiorgan failure.Plasma proteomics provide valuable and unbiased information about disease progression and therapeutic candidates. Recent proteomic studies have identified molecular changes in plasma of COVID-19 patients that implied significant dysregulation of several aspects of the inflammatory response accompanied by a general metabolic suppression. However, which of these plasma alterations are associated with disease severity remains only partly characterized.A known limitation of proteomic studies of plasma samples is the large difference in the macromolecule abundance, with concentration spanning at least 10 orders of magnitude. To improve the coverage of plasma contents, we performed a deep proteomic analysis of plasma from 10 COVID-19 patients with severe/fatal pneumonia compared to 10 COVID-19 patients with pneumonia who did not require ICU admission (non-ICU). To this aim, plasma samples were first depleted of the most abundant proteins, trypsin digested and peptides subjected to a high pH reversed-phase peptide fractionation before LC-MS analysis.These results highlighted an increase of proteins involved in neutrophil and platelet activity and acute phase response, which is significantly higher in severe/fatal COVID-19 patients when compared to non-ICU ones. Importantly, these changes are associated with a selective induction of complement cascade factors in severe/fatal COVID-19 patients. Data are available via ProteomeXchange with identifier PXD036491. Among these alterations, we confirmed by ELISA that higher levels of the neutrophil granule proteins DEFA3 and LCN2 are present in COVID-19 patients requiring ICU admission when compared to non-ICU and healthy donors.Altogether, our study provided an in-depth view of plasma proteome changes that occur in COVID-19 patients in relation to disease severity, which can be helpful to identify therapeutic strategies to improve the disease outcome.

Incidence of Pneumothorax and Pneumomediastinum in 497 COVID-19 Patients with Moderate-Severe ARDS over a Year of the Pandemic: An Observational Study in an Italian Third Level COVID-19 Hospital.

(1) Background: COVID-19 is a novel cause of acute respiratory distress syndrome (ARDS). Indeed, with the increase of ARDS cases due to the COVID-19 pandemic, there has also been an increase in the incidence of cases with pneumothorax (PNX) and pneumomediastinum (PNM). However, the incidence and the predictors of PNX/PMN in these patients are currently unclear and even conflicting. (2) Methods: The present observational study analyzed the incidence of barotrauma (PNX/PNM) in COVID-19 patients with moderate-severe ARDS hospitalized in a year of the pandemic, also focusing on the three waves occurring during the year, and treated with positive-pressure ventilation (PPV). We collected demographic and clinical data. (3) Results: During this period, 40 patients developed PNX/PNM. The overall incidence of barotrauma in all COVID-19 patients hospitalized in a year was 1.6%, and in those with moderate-severe ARDS in PPV was 7.2% and 3.8 events per 1000 positive-pressure ventilator days. The incidence of barotrauma in moderate-severe ARDS COVID-19 patients during the three waves was 7.8%, 7.4%, and 8.7%, respectively. Treatment with noninvasive respiratory support alone was associated with an incidence of barotrauma of 9.1% and 2.6 events per 1000 noninvasive ventilator days, of which 95% were admitted to the ICU after the event, due to a worsening of respiratory parameters. The incidence of barotrauma of ICU COVID-19 patients in invasive ventilation over a year was 5.8% and 2.7 events per 1000 invasive ventilator days. There was no significant difference in demographics and clinical features between the barotrauma and non-barotrauma group. The mortality was higher in the barotrauma group (17 patients died, 47.2%) than in the non-barotrauma group (170 patients died, 37%), although this difference was not statistically significant (p = 0.429). (4) Conclusions: The incidence of PNX/PNM in moderate-severe ARDS COVID-19 patients did not differ significantly between the three waves over a year, and does not appear to be very different from that in ARDS patients in the pre-COVID era. The barotrauma does not appear to significantly increase mortality in COVID-19 patients with moderate-severe ARDS if protective ventilation strategies are applied. Attention should be paid to the risk of barotrauma in COVID-19 patients in noninvasive ventilation because the event increases the probability of admission to the intensive care unit (ICU) and intubation.

16S rRNA gene sequencing of rectal swab in patients affected by COVID-19.

COronaVIrus Disease-2019 (COVID-19) is a pandemic respiratory infection caused by a new betacoronavirus, the Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2). Few data are reported on the gut microbiota in COVID-19 patients. 16S rRNA gene sequencing was performed to reveal an altered composition of the gut microbiota in patients with COVID-19 pneumonia admitted in intensive care unit (ICU) (i-COVID19), or in infectious disease wards (w-COVID19) as compared to controls (CTRL). i-COVID19 patients showed a decrease of Chao1 index as compared to CTRL and w-COVID19 patients indicating that patients in ICU displayed a lower microbial richness while no change was observed as for Shannon Index. At the phylum level, an increase of Proteobacteria was detected in w-COVID19 patients as compared to CTRL. A decrease of Fusobacteria and Spirochetes has been found, with the latter decreased in i-COVID19 patients as compared to CTRL. Significant changes in gut microbial communities in patients with COVID-19 pneumonia with different disease severity compared to CTRL have been identified. Our preliminary data may provide valuable information and promising biomarkers for the diagnosis of the disease and, when validated in larger cohort, it could facilitate the stratification of patients based on the microbial signature.

A new sorbent tube for atmospheric NOx determination by active sampling.

In this paper we used hydrated mayenite as reactive substrate for NOx active sampling in the air, which is novel. The performance of the mayenite-based sorbent for the NOx tubes was evaluated in two different monitoring surveys (autumn 2015 and winter 2016), characterized by different environmental conditions. Sorbent tubes filled with mayenite were exposed simultaneously to triethanolamine (TEA)-based sorbent tubes and to a chemiluminescence detector, as reference. The comparison of the NOx concentration levels measured by active sampling, using mayenite as NOx sorbent, showed a close relationship with the chemiluminescence analyzer. The effect of the environmental conditions on the performance of both mayenite and TEA-based sorbent tubes was evaluated and limitations connected to the use of TEA were discussed.

First detailed karyo-morphological analysis and molecular cytological study of leafy cardoon and globe artichoke, two multi-use Asteraceae crops.

Traditionally globe artichoke and leafy cardoon have been cultivated for use as vegetables but these crops are now finding multiple new roles in applications ranging from paper production to cheese preparation and biofuel use, with interest in their functional food potential. So far, their chromosome complements have been poorly investigated and a well-defined karyotype was not available. In this paper, a detailed karyo-morphological analysis and molecular cytogenetic studies were conducted on globe artichoke (Cynara cardunculus Linnaeus, 1753 var. scolymus Fiori, 1904) and leafy cardoon (Cynara cardunculus Linneaus, 1753 var. altilis De Candolle, 1838). Fluorescent In Situ Hybridization In Suspension (FISHIS) was applied to nuclei suspensions as a fast method for screening of labelling probes, before metaphase spread hybridization. Classic Fluorescent In Situ Hybridization (FISH) on slide, using repetitive telomeric and ribosomal sequences and Simple Sequence Repeats (SSRs) oligonucleotide as probes, identified homologous chromosome relationships and allowed development of molecular karyotypes for both varieties. The close phylogenetic relationship between globe artichoke and cardoon was supported by the very similar karyotypes but clear chromosomal structural variation was detected. In the light of the recent release of the globe artichoke genome sequencing, these results are relevant for future anchoring of the pseudomolecule sequence assemblies to specific chromosomes. In addition, the DNA content of the two crops has been determined by flow cytometry and a fast method for standard FISH on slide and methodological improvements for nuclei isolation are described.

Organic fraction of municipal solid waste from mechanical selection: biological stabilization and recovery options.

Although current trends address towards prevention strategies, the organic fraction of municipal solid waste is greatly produced, especially in high-income contexts. Its recovery-oriented collection is a common practice, but a relevant portion of the biodegradable waste is not source selected. Mechanical and biological treatments (MBT) are the most common option to sort and stabilize the biodegradable matter ending in residual waste stream. Following the changes of the framework around waste management, this paper aimed at analyzing the quality of the mechanically selected organic waste produced in MBT plants, in order to discuss its recovery options. The material performance was obtained by its composition as well as by its main chemical and physical parameters; biological stability was also assessed by both aerobic and anaerobic methods. On this basis, the effectiveness of an aerobic biostabilization process was assessed at pilot scale. After 21 days of treatment, results proved that the biomass had reached an acceptable biostabilization level, with a potential Dynamic Respirometric Index (DRIP) value lower than the limit required for its use as daily or final landfill cover material. However, the final stabilization level was seen to be influenced by scaling factors and the 21 days of treatment turned to be not so adequate when applied in the existing full-scale facility.

FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

Endocrine disruptors compounds, pharmaceuticals and personal care products in urban wastewater: implications for agricultural reuse and their removal by adsorption process.

In the last years, a lot of emerging contaminants, such as, endocrine disruptors compounds (EDCs), pharmaceuticals, and personal care products (PPCPs) have been detected in wastewater. Because of their toxicity and possible adverse effects on the environment and humans, their release from urban wastewater treatment plants (UWWTPs) effluents should be minimized, particularly when a wastewater reuse for crops irrigation is expected. Many processes have been investigated for advanced treatment of UWWTP effluents as well as for emerging contaminant degradation; among these, adsorption process was successfully used to remove EDCs and PPCPs from wastewater. This article shortly reviews EDCs and PPCPs removal from UWWTP effluents by adsorption process using conventional and non-conventional adsorbents. The fate of EDCs and PPCPs in UWWTPs and the implications for agricultural wastewater reuse has been addressed too. In spite of the adsorption process looking to be a valuable alternative to other advanced technologies for the removal of emerging contaminants from wastewater, some gaps still remain to evaluate the actual feasibility at full scale. However, according to a few studies available in scientific literature on the use of both powdered activated carbon and granular activated carbon at full scale, adsorption process by activated carbon is a promising, potentially effective, and economically feasible solution for producing safe wastewater for agricultural reuse.

Flow sorting and molecular cytogenetic identification of individual chromosomes of Dasypyrum villosum L. (H. villosa) by a single DNA probe.

Dasypyrum villosum (L.) Candargy (sin. Haynaldia villosa) is an annual wild diploid grass species (2n = 2x = 14; genome VV) belonging to the Poaceae family, which is considered to be an important source of biotic and abiotic stress resistance genes for wheat breeding. Enhanced characterization of D. villosum chromosomes can facilitate exploitation of its gene pool and its use in wheat breeding programs. Here we present the cytogenetic identification of D. villosum chromosomes on slide by fluorescent in situ hybridization (FISH), with the GAA simple sequence repeat (SSR) as a probe. We also describe the isolation and the flow cytometric analysis of D. villosum chromosomes in suspension, resulting in a distinguished flow karyotype. Chromosomes were flow sorted into three fractions, according their DNA content, one of which was composed of a single type of chromosome, namely 6 V, sorted with over 85% purity. Chromosome 6 V is known to carry genes to code for important resistance and seed storage characteristics, and its isolation represents a new source of genetic traits and specific markers useful for wheat improvement.

The bean polygalacturonase-inhibiting protein 2 (PvPGIP2) is highly conserved in common bean (Phaseolus vulgaris L.) germplasm and related species.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant protein inhibitors of endo-polygalacturonases (PGs) that belong to the leucine-rich repeat (LRR) protein family. In bean, PGIP is encoded by a small gene family of four members among which Pvpgip2 encodes the most wide-spectrum and efficient inhibitor of fungal PGs. In order to evaluate the sequence polymorphism of Pvpgip2 and its functional significance, we have analyzed a number of wild and cultivated bean (P. vulgaris) accessions of Andean and Mesoamerican origin, and some genotypes from the related species P. coccineus, P. acutifolius, and P. lunatus. Our analyses indicate that the protein encoded by Pvpgip2 is highly conserved in the bean germplasm. The few detected polymorphic sites correspond to synonymous substitutions and only two wild genotypes contain a Pvpgip2 with a single non-synonymous replacement. Sequence comparison showed a slightly larger variation in the related bean species P. coccineus, P. acutifolius, and P. lunatus and confirmed the known phylogenetic relationships with P. vulgaris. The majority of the replacements were within the xxLxLxx region of the leucine rich repeat (LRR) domain and none of them affected residues contributing to structural features. The variant PGIPs were expressed in Nicotiana benthamiana using PVX as vector and their inhibitory activity compared to that of PvPPGIP2. All the variants were able to fully inhibit the four fungal PGs tested with minor differences. Taken together these results support the hypothesis that the overall sequence conservation of PGIP2 and minor variation at specific sites is necessary for high-affinity recognition of different fungal PGs.

LC-MS Determination of remifentanil in maternal and neonatal plasma.

An HPLC-MS with electrospray ionization method for the determination of remifentanil in human plasma samples, pre-treated with SPE cartridge, has been developed and validated. Ionisation was performed by positive-ion electrospray and quadrupole filter mass spectrometer operated in the single ion-recording mode. Pre-treatment was performed using Waters Oasis((R)) SPE cartridges. Chromatographic separation was achieved in isocratic elution using a X-Terra C8 5 microm, 150 mm x 2.1 mm i.d. column. The mobile phase consisted of a mixture of water, methanol and acetonitrile (86:10:4, v/v/v) containing 0.1% of formic acid. The method showed to be linear in the range between 0.5 and 48.0 ng/ml, the estimated LOD is 0.18 ng/ml and the LOQ is 0.5 ng/ml. The method can be used to quantify remifentanil in plasma samples taken from adult and newborn patients in a range suitable for clinical studies.

LC-MS determination of MPTP at sub-ppm level in pethidine hydrochloride.

An HPLC-MS with electrospray ionisation method for the determination of MPTP at sub-ppm level in pethidine hydrochloride has been developed and validated. Ionisation is performed by positive-ion electrospray and the quadrupole filter mass spectrometer is operated in the single ion recording mode. Chromatographic separation was achieved in gradient elution using a symmetry C18, 5 microm, 150 mm x 2.1 mm i.d. The mobile phase comprised water containing 0.1% formic acid (v/v) and acetonitrile containing 0.1% formic acid (v/v). The method showed to be linear in the range between 0.2 and 2.2 ng/ml, the estimated LOD was lower than 0.1 ng/ml and the LOQ was lower than 0.2 ng/ml.

Interaction with membrane lipids and heme ligand binding properties of Escherichia coli flavohemoglobin.

Escherichia coli flavohemoglobin has been shown to be able to bind specifically unsaturated and/or cyclopropanated fatty acids with very high affinity. Unsaturated or cyclopropanated fatty acid binding results in a modification of the visible absorption spectrum of the ferric heme, corresponding to a transition from a pentacoordinated (typical of the ligand free protein) to a hexacoordinated, high spin, heme iron. In contrast, no detectable interaction has been observed with saturated fatty acid, saturated phospholipids, linear, cyclic, and aromatic hydrocarbons pointing out that the protein recognizes specifically double bonds in cis conformation within the hydrocarbon chain of the fatty acid molecule. Accordingly, as demonstrated in gel filtration experiments, flavohemoglobin is able to bind liposomes obtained from lipid extracts of E. coli membranes and eventually abstract phospholipids containing cis double bonds and/or cyclopropane rings along the acyl chains. The presence of a protein bound lipid strongly affects the thermodynamic and kinetic properties of imidazole binding to the ferric protein and brings about significant modifications in the reactivity of the ferrous protein with oxygen and carbon monoxide. The effect of the bound lipid has been accounted for by a reaction scheme that involves the presence of two sites for the lipid/ligand recognition, namely, the heme iron and a non-heme site located in a loop region above the heme pocket.

The X-ray structure of ferric Escherichia coli flavohemoglobin reveals an unexpected geometry of the distal heme pocket.

The x-ray structure of ferric unliganded lipid-free Escherichia coli flavohemoglobin has been solved to a resolution of 2.2 A and refined to an R-factor of 19%. The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophus flavohemoglobin with the notable exception of the E helix positioning within the globin domain and a rotation of the NAD binding module with respect to the FAD-binding domain accompanied by a substantial rearrangement of the C-terminal region. An inspection of the heme environment in E. coli flavohemoglobin reveals an unexpected architecture of the distal pocket. In fact, the distal site is occupied by the isopropyl side chain Leu-E11 that shields the heme iron from the residues in the topological positions predicted to interact with heme iron-bound ligands, namely Tyr-B10 and Gln-E7, and stabilizes a pentacoordinate ferric iron species. Ligand binding properties are consistent with the presence of a pentacoordinate species in solution as indicated by a very fast second order combination rates with imidazole and azide. Surprisingly, imidazole, cyanide, and azide binding profiles at equilibrium are not accounted for by a single site titration curve but are biphasic and strongly suggest the presence of two distinct conformers within the liganded species.