Immuno-Assessment of «Pseudomonas syringae» Lipodepsipeptides (Syringomycins and Syringopeptins)

Following a previous work on the immunological detection of syringopeptins (SPs), polyclonal antibodies with a high specificity for syringomycins (SRs) were raised in rabbits and purified. Assayed in a competitive ELISA, the most common forms of SR, i.e. SR-E and SR-G, were recognised with a detection limit of 0.1 mg per well, whereas other structurally related bacterial lipodepsipeptides (LDP), such as SPs, pseudomycins (PSs) and syringotoxins (STs) were not recognised. The immuno-assay (competitive ELISA) method developed in this work is about 100 times more sensitive than the current chromatographic (HPLC) method and requires no previous extraction of the toxin. The production of LDP in culture by strains of three pathovars of Pseudomonas syringae (pv. aptata, pv. lachrymans and pv. syringae) was found to range from 0.026 to 0.055 mg ml-1 for SRs and from 0.02 to 0.06 mg ml-1 for SPs. Both the concentration of LDP in aqueous extracts from zucchini cotyledons infected by P. syringae pv. lachrymans and the severity of symptoms were shown to increase progressively after infection. The immunologically estimated concentration of SRs in the infected cotyledons averaged 0.22 mg g-1 f wt after 12 hours, and 0.39 mg g-1 after 4 days. The corresponding values for SPs were 0.11 and 0.37 mg g-1. In a recovery experiment, solutions of pure toxins (0.22 mg SR-E and 0.14 mg SP25A g-1 f wt) were injected in healthy cotyledons. After 2 days, overestimation due to toxin complexing in planta was of 10% for (SR-E) and 40% for (SP25A). Applying these percentages to the values estimated for infected cotyledons, the net concentrations were as follows: 12 h after inoculation: 0.20 (SRs) and 0.07 (SPs) mg g-1 f wt; four days after inoculation: 0.35 (SRs) and 0.22 (SPs) mg g-1 f wt. The values obtained with aqueous extracts from infected plants are relatively high if compared to the figures of the in vitro experiments. It is assumed that the high reactivity of ELISA to the immune-LDP-related compounds present in the water extracts from infected plants is due to the presence of high molecular weight LDP complexes having a cross-reactivity with antibodies substantially higher than that of free toxins

Fatality rate and predictors of mortality in an Italian cohort of hospitalized COVID-19 patients

Clinical features and natural history of coronavirus disease 2019 (COVID-19) differ widely among different countries and during different phases of the pandemia. Here, we aimed to evaluate the case fatality rate (CFR) and to identify predictors of mortality in a cohort of COVID-19 patients admitted to three hospitals of Northern Italy between March 1 and April 28, 2020. All these patients had a confirmed diagnosis of SARS-CoV-2 infection by molecular methods. During the study period 504/1697 patients died; thus, overall CFR was 29.7%. We looked for predictors of mortality in a subgroup of 486 patients (239 males, 59%; median age 71 years) for whom sufficient clinical data were available at data cut-off. Among the demographic and clinical variables considered, age, a diagnosis of cancer, obesity and current smoking independently predicted mortality. When laboratory data were added to the model in a further subgroup of patients, age, the diagnosis of cancer, and the baseline PaO2/FiO2 ratio were identified as independent predictors of mortality. In conclusion, the CFR of hospitalized patients in Northern Italy during the ascending phase of the COVID-19 pandemic approached 30%. The identification of mortality predictors might contribute to better stratification of individual patient risk

Dynamics of Viral Infection and Evolution of SARS-CoV-2 Variants in the Calabria Area of Southern Italy

In this study, we report on the results of SARS-CoV-2 surveillance performed in an area of Southern Italy for 12 months (from March 2021 to February 2022). To this study, we have sequenced RNA from 609 isolates. We have identified circulating VOCs by Sanger sequencing of the S gene and defined their genotypes by whole-genome NGS sequencing of 157 representative isolates. Our results indicated that B.1 and Alpha were the only circulating lineages in Calabria in March 2021; while Alpha remained the most common variant between April 2021 and May 2021 (90 and 73%, respectively), we observed a concomitant decrease in B.1 cases and appearance of Gamma cases (6 and 21%, respectively); C.36.3 and Delta appeared in June 2021 (6 and 3%, respectively); Delta became dominant in July 2021 while Alpha continued to reduce (46 and 48%, respectively). In August 2021, Delta became the only circulating variant until the end of December 2021. As of January 2022, Omicron emerged and took over Delta (72 and 28%, respectively). No patient carrying Beta, Iota, Mu, or Eta variants was identified in this survey. Among the genomes identified in this study, some were distributed all over Europe (B1_S477N, Alpha_L5F, Delta_T95, Delta_G181V, and Delta_A222V), some were distributed in the majority of Italian regions (B1_S477N, B1_Q675H, Delta_T95I and Delta_A222V), and some were present mainly in Calabria (B1_S477N_T29I, B1_S477N_T29I_E484Q, Alpha_A67S, Alpha_A701S, and Alpha_T724I). Prediction analysis of the effects of mutations on the immune response (i.e., binding to class I MHC and/or recognition of T cells) indicated that T29I in B.1 variant; A701S in Alpha variant; and T19R in Delta variant were predicted to impair binding to class I MHC whereas the mutations A67S identified in Alpha; E484K identified in Gamma; and E156G and ΔF157/R158 identified in Delta were predicted to impair recognition by T cells. In conclusion, we report on the results of SARS-CoV-2 surveillance in Regione Calabria in the period between March 2021 and February 2022, identified variants that were enriched mainly in Calabria, and predicted the effects of identified mutations on host immune response

Daily Fluctuation of Orexin Neuron Activity and Wiring: The Challenge of “Chronoconnectivity”

In the heterogeneous hub represented by the lateral hypothalamus, neurons containing the orexin/hypocretin peptides play a key role in vigilance state transitions and wakefulness stability, energy homeostasis, and other functions relevant for motivated behaviors. Orexin neurons, which project widely to the neuraxis, are innervated by multiple extra- and intra-hypothalamic sources. A key property of the adaptive capacity of orexin neurons is represented by daily variations of activity, which is highest in the period of the animal’s activity and wakefulness. These sets of data are here reviewed. They concern the discharge profile during the sleep/wake cycle, spontaneous Fos induction, peptide synthesis and release reflected by immunostaining intensity and peptide levels in the cerebrospinal fluid as well as postsynaptic effects. At the synaptic level, adaptive capacity of orexin neurons subserved by remodeling of excitatory and inhibitory inputs has been shown in response to changes in the nutritional status and prolonged wakefulness. The present review wishes to highlight that synaptic plasticity in the wiring of orexin neurons also occurs in unperturbed conditions and could account for diurnal variations of orexin neuron activity. Data in zebrafish larvae have shown rhythmic changes in the density of inhibitory innervation of orexin dendrites in relation to vigilance states. Recent findings in mice have indicated a diurnal reorganization of the excitatory/inhibitory balance in the perisomatic innervation of orexin neurons. Taken together these sets of data point to “chronoconnectivity,” i.e., a synaptic rearrangement of inputs to orexin neurons over the course of the day in relation to sleep and wake states. This opens questions on the underlying circadian and homeostatic regulation and on the involved players at synaptic level, which could implicate dual transmitters, cytoskeletal rearrangements, hormonal regulation, as well as surrounding glial cells and extracellular matrix. Furthermore, the question arises of a “chronoconnectivity” in the wiring of other neuronal cell groups of the sleep-wake-regulatory network, many of which are characterized by variations of their firing rate during vigilance states

Immuno-assessment of Pseudomonas syringae lipodepsipeptides (syringomycins and syringopeptins)

Following a previous work on the immunological detection of syringopeptins (SPs), polyclonal antibodies with a high specificity for syringomycins (SRs) were raised in rabbits and purified. Assayed in a competitive ELISA, the most common forms of SR, i.e. SR-E and SR-G, were recognised with a detection limit of 0.1 mg per well, whereas other structurally related bacterial lipodepsipeptides (LDP), such as SPs, pseudomycins (PSs) and syringotoxins (STs) were not recognised. The immuno-assay (competitive ELISA) method developed in this work is about 100 times more sensitive than the current chromatographic (HPLC) method and requires no previous extraction of the toxin. The production of LDP in culture by strains of three pathovars of Pseudomonas syringae (pv. aptata, pv. lachrymans and pv. syringae) was found to range from 0.026 to 0.055 mg ml-1 for SRs and from 0.02 to 0.06 mg ml-1 for SPs. Both the concentration of LDP in aqueous extracts from zucchini cotyledons infected by P. syringae pv. lachrymans and the severity of symptoms were shown to increase progressively after infection. The immunologically estimated concentration of SRs in the infected cotyledons averaged 0.22 mg g-1 f wt after 12 hours, and 0.39 mg g-1 after 4 days. The corresponding values for SPs were 0.11 and 0.37 mg g-1. In a recovery experiment, solutions of pure toxins (0.22 mg SR-E and 0.14 mg SP25A g-1 f wt) were injected in healthy cotyledons. After 2 days, overestimation due to toxin complexing in planta was of 10% for (SR-E) and 40% for (SP25A). Applying these percentages to the values estimated for infected cotyledons, the net concentrations were as follows: 12 h after inoculation: 0.20 (SRs) and 0.07 (SPs) mg g-1 f wt; four days after inoculation: 0.35 (SRs) and 0.22 (SPs) mg g-1 f wt. The values obtained with aqueous extracts from infected plants are relatively high if compared to the figures of the in vitro experiments. It is assumed that the high reactivity of ELISA to the immune-LDP-related compounds present in the water extracts from infected plants is due to the presence of high molecular weight LDP complexes having a cross-reactivity with antibodies substantially higher than that of free toxins

COMPOSIZIONE CORPOREA E DINAMOMETRIA IN PAZIENTI AFFETTE DA CARCINOMA MAMMARIO AFFERENTI ALL\u2019AMBULATORIO DI CONSULENZA NUTRIZIONALE DELLA S.S.D. BREAST UNIT \u2013 ASST SETTE LAGHI, VARESE

Nonostante la diffusione della cultura della prevenzione e l\u2019attivit\ue0 di diagnosi precoce, il cancro colpisce ancora un gran numero di persone e i progressi fatti nella cura, pur avendo allungato la sopravvivenza dei pazienti, non hanno risolto i gravi problemi legati alla qualit\ue0 di vita e ai bisogni degli ammalati. La prevenzione terziaria del cancro della mammella si basa sull\u2019individuazione e sulla correzione di fattori di rischio modificabili e ha come obiettivo una migliore qualit\ue0 di vita, attraverso l\u2019attenuazione di segni e sintomi secondari alla presenza del tumore. Molti studi hanno riscontrato che la dieta di tipo mediterraneo, l\u2019attivit\ue0 fisica e il mantenimento di un peso corporeo salutare possono contribuire a ridurre il rischio di recidiva. Scopo del nostro lavoro \ue8 stato quello di osservare lo stato nutrizionale di donne affette da carcinoma mammario afferenti all\u2019ambulatorio di consulenza nutrizionale del Centro di Ricerca in Senologia

Plant Defense Elicitation by the Hydrophobin Cerato-Ulmin and Correlation with Its Structural Features

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic–hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein’s loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a β-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability

Plant Defense Elicitation by the Hydrophobin Cerato-Ulmin and Correlation with Its Structural Features

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic–hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein’s loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a β-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability

A new syringopeptin produced by bean strains of Pseudomonas syringae pv. syringae

Two strains (B728a and Y37) of the phytopathogenic bacterium Pseudomonas syringae pv. syringae isolated from bean (Phaseolus vulgaris) plants were shown to produce in culture both syringomycin, a lipodepsinonapeptide secreted by the majority of the strains of the bacterium, and a new form of syringopeptin, SP22Phv. The structure of the latter metabolite was elucidated by the combined use of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and chemical procedures. Comparative phytotoxic and antimicrobial assays showed that SP22Phv did not differ substantially from the previously characterized syringopeptin 22 (SP22) as far as toxicity to plants was concerned, but was less active in inhibiting the growth of the test fungi Rhodotorula pilimanae and Geotrichum candidum and of the Gram-positive bacterium Bacillus megaterium