Simultaneous monitoring of SARS-CoV-2 and bacterial profiles from the air of hospital environments with COVID-19-affected patients

Aerobiologia - The SARS-CoV-2 presence and the bacterial community profile in air samples collected at the Intensive Care Unit (ICU) of the Operational Unit of Infectious Diseases of Santa Caterina...     

Chlorophyta microalgae as dietary protein supplement: a comparative analysis of productivity related to photosynthesis

Journal of Applied Phycology - Microalgae are studied as innovative sources of a wide range of highly valuable products, including proteins for the food/feed sectors. However, protein content...     

A Single Mutation in the Gene Responsible for the Mucoid Phenotype of Bifidobacterium animalis subsp. lactis Confers Surface and Functional Characteristics

Exopolysaccharides (EPS) are extracellular carbohydrate polymers synthesized by a large variety of bacteria. Their physiological functions have been extensively studied, but many of their roles have not yet been elucidated. We have sequenced the genomes of two isogenic strains of Bifidobacterium animalis subsp. lactis that differ in their EPS-producing phenotype. The original strain displays a nonmucoid appearance, and the mutant derived thereof has acquired a mucoid phenotype. The sequence analysis of their genomes revealed a nonsynonymous mutation in the gene Balat_1410, putatively involved in the elongation of the EPS chain. By comparing a strain from which this gene had been deleted with strains containing the wild-type and mutated genes, we were able to show that each strain displays different cell surface characteristics. The mucoid EPS synthesized by the strain harboring the mutation in Balat_1410 provided higher resistance to gastrointestinal conditions and increased the capability for adhesion to human enterocytes. In addition, the cytokine profiles of human peripheral blood mononuclear cells and ex vivo colon tissues suggest that the mucoid strain could have higher anti-inflammatory activity. Our findings provide relevant data on the function of Balat_1410 and reveal that the mucoid phenotype is able to alter some of the most relevant functional properties of the cells.     

The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing

MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100_209–217tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the _mutgp100_201–230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the _mutgp100_209–217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8⁺ T cell stimulation in vitro similar to the _wtgp100_209–217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8⁺ T cell response also towards N-terminally extended versions of the minimal epitope.     

Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation

Lipases (EC 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates, such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or “interfacial activation,” with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported, leading to the conclusion that its behavior was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 Å resolution. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. The findings explain the lack of interfacial activation of CALB and offer new elements to elucidate this mechanism, with the consequent implications for the catalytic properties and classification of lipases.     

Trypanosome Infection Establishment in the Tsetse Fly Gut Is Influenced by Microbiome-Regulated Host Immune Barriers

Tsetse flies (Glossina spp.) vector pathogenic African trypanosomes, which cause sleeping sickness in humans and nagana in domesticated animals. Additionally, tsetse harbors 3 maternally transmitted endosymbiotic bacteria that modulate their host''s physiology. Tsetse is highly resistant to infection with trypanosomes, and this phenotype depends on multiple physiological factors at the time of challenge. These factors include host age, density of maternally-derived trypanolytic effector molecules present in the gut, and symbiont status during development. In this study, we investigated the molecular mechanisms that result in tsetse''s resistance to trypanosomes. We found that following parasite challenge, young susceptible tsetse present a highly attenuated immune response. In contrast, mature refractory flies express higher levels of genes associated with humoral (attacin and pgrp-lb) and epithelial (inducible nitric oxide synthase and dual oxidase) immunity. Additionally, we discovered that tsetse must harbor its endogenous microbiome during intrauterine larval development in order to present a parasite refractory phenotype during adulthood. Interestingly, mature aposymbiotic flies (Gmm ^Apo) present a strong immune response earlier in the infection process than do WT flies that harbor symbiotic bacteria throughout their entire lifecycle. However, this early response fails to confer significant resistance to trypanosomes. Gmm ^Apo adults present a structurally compromised peritrophic matrix (PM), which lines the fly midgut and serves as a physical barrier that separates luminal contents from immune responsive epithelial cells. We propose that the early immune response we observe in Gmm ^Apo flies following parasite challenge results from the premature exposure of gut epithelia to parasite-derived immunogens in the absence of a robust PM. Thus, tsetse''s PM appears to regulate the timing of host immune induction following parasite challenge. Our results document a novel finding, which is the existence of a positive correlation between tsetse''s larval microbiome and the integrity of the emerging adult PM gut immune barrier.     

Direct inhibition of hexokinase activity by metformin at least partially impairs glucose metabolism and tumor growth in experimental breast cancer

Emerging evidence suggests that metformin, a widely used anti-diabetic drug, may be useful in the prevention and treatment of different cancers. In the present study, we demonstrate that metformin directly inhibits the enzymatic function of hexokinase (HK) I and II in a cell line of triple-negative breast cancer (MDA-MB-231). The inhibition is selective for these isoforms, as documented by experiments with purified HK I and II as well as with cell lysates. Measurements of ¹⁸F-fluoro-deoxyglycose uptake document that it is dose- and time-dependent and powerful enough to virtually abolish glucose consumption despite unchanged availability of membrane glucose transporters. The profound energetic imbalance activates phosphorylation and is subsequently followed by cell death. More importantly, the “in vivo” relevance of this effect is confirmed by studies of orthotopic xenografts of MDA-MB-231 cells in athymic (nu/nu) mice. Administration of high drug doses after tumor development caused an evident tumor necrosis in a time as short as 48 h. On the other hand, 1 mo metformin treatment markedly reduced cancer glucose consumption and growth. Taken together, our results strongly suggest that HK inhibition contributes to metformin therapeutic and preventive potential in breast cancer.     

The Fifth Domain of Beta 2 Glycoprotein I Protects from Natural IgM Mediated Cardiac Ischaemia Reperfusion Injury

Reperfusion after a period of ischemia results in reperfusion injury (IRI) which involves activation of the inflammatory cascade. In cardiac IRI, IgM natural antibodies (NAb) play a prominent role through binding to altered neoepitopes expressed on damaged cells. Beta 2 Glycoprotein I (β2GPI) is a plasma protein that binds to neoepitopes on damaged cells including anionic phospholipids through its highly conserved Domain V. Domain I of β2GPI binds circulating IgM NAbs and may provide a link between the innate immune system, IgM NAb binding and cardiac IRI. This study was undertaken to investigate the role of Β2GPI and its Domain V in cardiac IRI using wild-type (WT), Rag-1 ^-/- and β2GPI deficient mice. Compared with control, treatment with Domain V prior to cardiac IRI prevented binding of endogenous β2GPI to post-ischemic myocardium and resulted in smaller myocardial infarction size in both WT and β2GPI deficient mice. Domain V treatment in WT mice also resulted in less neutrophil infiltration, less apoptosis and improved ejection fraction at 24 h. Rag-1 -/- antibody deficient mice reconstituted with IgM NAbs confirmed that Domain V prevented IgM NAb induced cardiac IRI. Domain V remained equally effective when delivered at the time of reperfusion which has therapeutic clinical relevance.Based upon this study Domain V may function as a universal inhibitor of IgM NAb binding in the setting of cardiac IRI, which offers promise as a new therapeutic strategy in the treatment of cardiac IRI.     

A new method to measure the haemoglobin oxygen saturation by the oxygen electrode

We describe a new polarographic method to measure the haemoglobin oxygen saturation in whole blood, employing up to 10 μl of sample in a standard case. The measurement is done in an anaerobic staineless-steel cuvette (1 ml) recording three oxygen tension values: (1) that of an air-equilibrated buffer before the addition of the sample; (ii) that after the addition of the sample; and (iii) that after the addition of an oxidant. The haemoglobin oxygen saturation is then calculated from the three oxygen tension values, the volume of the reagents, and solubility coefficient of oxygen. This method is simple, inexpensive and accurate, and correlates well with other standard methods.