A Gamma-adapted subunit vaccine induces broadly neutralizing antibodies against SARS-CoV-2 variants and protects mice from infection.

In the context of continuous emergence of SARS-CoV-2 variants of concern (VOCs), one strategy to prevent the severe outcomes of COVID-19 is developing safe and effective broad-spectrum vaccines. Here, we present preclinical studies of a RBD vaccine derived from the Gamma SARS-CoV-2 variant adjuvanted with Alum. The Gamma-adapted RBD vaccine is more immunogenic than the Ancestral RBD vaccine in terms of inducing broader neutralizing antibodies. The Gamma RBD presents more immunogenic B-cell restricted epitopes and induces a higher proportion of specific-B cells and plasmablasts than the Ancestral RBD version. The Gamma-adapted vaccine induces antigen specific T cell immune responses and confers protection against Ancestral and Omicron BA.5 SARS-CoV-2 challenge in mice. Moreover, the Gamma RBD vaccine induces higher and broader neutralizing antibody activity than homologous booster vaccination in mice previously primed with different SARS-CoV-2 vaccine platforms. Our study indicates that the adjuvanted Gamma RBD vaccine is highly immunogenic and a broad-spectrum vaccine candidate.

Safety and immunogenicity of a SARS-CoV-2 Gamma variant RBD-based protein adjuvanted vaccine used as booster in healthy adults.

A Gamma Variant RBD-based aluminum hydroxide adjuvanted vaccine called ARVAC CG was selected for a first in human clinical trial. Healthy male and female participants (18-55 years old) with a complete COVID-19-primary vaccine scheme were assigned to receive two intramuscular doses of either a low-dose or a high-dose of ARVAC CG. The primary endpoint was safety. The secondary objective was humoral immunogenicity. Cellular immune responses were studied as an exploratory objective. The trial was prospectively registered in PRIISA.BA (Registration Code 6564) and ANMAT and retrospectively registered in ClinicalTrials.gov (NCT05656508). Samples from participants of a surveillance strategy implemented by the Ministry of Health of the Province of Buenos Aires that were boosted with BNT162b2 were also analyzed to compare with the booster effect of ARVAC CG. ARVAC CG exhibits a satisfactory safety profile, a robust and broad booster response of neutralizing antibodies against the Ancestral strain of SARS-CoV-2 and the Gamma, Delta, Omicron BA.1 and Omicron BA.5 variants of concern and a booster effect on T cell immunity in individuals previously immunized with different COVID-19 vaccine platforms.

Bm Delta-pgm, a vaccine for the control of Brucella melitensis with cross-species protective properties.

Brucellosis remains one of the most worldwide distributed zoonosis inflicting serious economical and human health problems in many areas of the world. The disease is caused by different species of the genus Brucella that have different tropisms towards different mammals being the most relevant for human health Brucella abortus, Brucella melitensis and Brucella suis that infect cows, goats/sheep, and swine respectively. For B. melitensis, considered the species with more zoonotic potential and highly aggressive for animals, only one vaccine is available to date in the market: Rev 1. This attenuated strain has the disadvantage that is has a very high residual virulence for animals and humans and, for this reason, it is applied by ocular instillation which is technically challenging in many productive settings. For this reason, the search for new vaccines for caprine and ovine brucellosis is an active topic of research. We describe here the construction of a novel highly attenuated vaccine strain (Bm Delta-pgm) that confers excellent levels of protection against B. melitensis in the mouse model of infection. This strain is a clean deletion of the phosphoglucomutase (pgm) gene that codes for a protein that catalyzes the conversion of glucose-6-P to glucose-1-P, which is used as a precursor for the biosynthesis of many polysaccharides, including the O-antigen of the lipopolysaccharide and cyclic beta glucans. Our results indicate that vaccination with Bm Delta-pgm induces a robust memory cellular immune response but no antibody production against the O-antigen. Cross protection experiments show that this new vaccine protects against B. abortus and B. suis raising the possibility that Bm Delta-pgm could be used as a universal vaccine for the most important Brucella species.

Comparative study between photodynamic therapy and the use of probiotics in the reduction of halitosis in mouth breathing children: Study protocol for a randomized controlled clinical trial.

INTRODUCTION: Halitosis is a term that defines any odor or foul smell the emanates from the oral cavity, the origin of which may be local or systemic. One of the causes of local or oral halitosis is low salivary flow and dry mouth, which is also one of the complaints of individuals with the mouth-breathing habit. The aim of this study is to determine the effectiveness of antimicrobial photodynamic therapy (aPDT) and the use of probiotics for the treatment of halitosis in mouth-breathing children. METHODS: Fifty-two children between 7 and 12 years of age with a diagnosis of mouth breathing and halitosis determined through an interview and clinical examination will be selected. The participants will be divided into 4 groups: Group 1-treatment with brushing, dental floss and tongue scraper; Group 2-brushing, dental floss and aPDT applied to the dorsum and middle third of the tongue; Group 3-brushing, dental floss and probiotics; Group 4-brushing, dental floss, aPDT and probiotics. The use of a breath meter and microbiological analysis of the tongue coating will be performed before, immediately after treatment and 7 days after treatment. The quantitative analysis will involve counts of colony-forming bacteria per milliliter and real-time polymerase chain reaction. The normality of the data will be determined using the Shapiro-Wilk test. Parametric data will be submitted to analysis of variance and nonparametric data will be compared using the Kruskal-Wallis test. The results of each treatment in the different periods of the study will be compared using the Wilcoxon test. DISCUSSION: Due to the low level of evidence, studies are needed to determine whether treatment with aPDT using annatto as the photosensitizer and blue led as the light source is effective at diminishing halitosis in mouth-breathing children.

Biodeterioration of stone monuments: Studies on the influence of bioreceptivity on cyanobacterial biofilm growth and on the biocidal efficacy of essential oils in natural hydrogel.

An important field of research is devoted to the development of innovative, sustainable, and safe methodologies to counteract biodeterioration of stone monuments due to the growth of microbial communities. However, besides the biocide's efficacy, it is crucial to consider the features of substrates on which biocides must be applied, to define the so-called bioreceptivity of the lithic faces. In this research five different lithotypes, namely Lecce stone, Travertine, Peperino, Serena stone, and Granite, have been used as substrates for the growth of cyanobacterial biofilms. Open porosity, hygroscopic properties, and roughness parameters have been investigated for each lithotype and correlated to the photosynthetic yields of the biofilms colonizing the different stones to propose an easy method to estimate stone bioreceptivity. Different levels of coverage of the stone surfaces have been accomplished in relation to the typology of lithotypes. To develop innovative restoration methodologies against biodeterioration of stone monuments, a hydrogel-biocide system has been optimized by using a polysaccharide dispersion as a matrix where to embed T. vulgaris essential oil (at 0.25 % or 0.1 %) or its main component thymol (at 0.18 % or 0.07 %). The efficacy and the effect of the innovative biocide have been evaluated combining microscopy, photosynthetic measurements, and colorimetric analyses and both the biocides (with T. vulgaris EO or thymol) showed to be highly effective against the cyanobacterial biofilms for at least six months from the treatment without inducing any significant alteration to the lithic surfaces. The efficacy of thymol alone allows to treat colonized surfaces with a single active ingredient, or at least a mixture thereof, much cheaper and reproducible. The results obtained in this work pave the way to develop a sustainable cleaning protocol to counteract the biodeterioration of stone monuments or historic buildings induced by the presence of phototrophic biofilms that endangered their conservation.

Fungal outbreak in the Catacombs of SS. Marcellino and Pietro Rome (Italy): From diagnosis to an emergency treatment.

The present study reports a sudden fungal outbreak that occurred in the corridor near the entrance of the Catacombs of SS. Marcellino and Pietro in Rome (Italy) observed after 1 year of a restoration treatment that interested the walls of the entrance of the Catacombs and some artifacts placed in situ. The colonization was observed on the vault at the entrance and in correspondence with the restored marble pieces displayed on the left side of the corridor. No growth was observed on the right side where similarly treated marble slabs were placed. Samples taken in correspondence with fungal biofilm were analyzed through the combined use of microscopical, cultural, and molecular tools and showed that the vault and the left side of the corridor entrance were colonized by a complex fungal biofilm consisting mainly of Coniophora sp. and other genera, such as Hypomyces, Purpureocillium, Acremonium, Penicillium, and Alternaria, many of which are well known as responsible of biodeterioration of stone surfaces. Regarding the brown-rot basidiomycete Coniophora, it was able to form very large colonies on the substrata with a diameter of up to 57 cm. Although the direct observation under a light microscope evidenced the presence of abundant brown fungal conidia, several attempts to cultivate the microorganism failed, therefore only through DNA sequencing analyses, it was possible to identify and characterize this fungus. There is very little literature on the genus Coniophora which is reported as one of the causes of wet-rot decay of wood in buildings. A connection with calcium-containing materials such as bricks and mortars was demonstrated, but no data were available about the possible role of this species in the biodeterioration of stones. This study features the first finding of a strain related to the basidiomycetous genus of Coniophora in the order Boletales in association with evident phenomena of biodeterioration.

Evaluation of intravascular irradiation of blood in children with sleep bruxism: Study protocol for a randomized controlled clinical trial.

BACKGROUND: The objective of this study will be to evaluate the effect of intravascular irradiation of blood (ILIB) combined with myofunctional exercises for sleep bruxism in pediatrics. METHODS: This study will be a randomized controlled clinical trial. A triage of individuals between 4 and 17 years old with a diagnosis of sleep bruxism will be carried out at the clinic of the Catholic University of Uruguay, and in a private office referred by different private care centers. The selected participants will be evaluated before and after treatment by means of questionnaires on bruxism, sleep quality and nocturnal oxygen saturation measurement. For this, 46 patients with sleep bruxism will be recruited, who will be divided into 2 groups: control group (CG), which will undergo an application of placebo ILIB and an orofacial myofunctional therapy (MFT) exercise protocol; and na ILIB group, which will carry out an active application of ILIB and an exercise protocol, this being once a week for 8 weeks. The laser treatment (808 nm) will be performed twice a week for 8 weeks. The values will be tested for normality by the Kolmogorov-Smirnov test. For the comparison between the groups, t test will be carried out, considering a level of significance of 0.5% (P < .05). DISCUSSION: Although local photobiomodulation (PBM), acupuncture PBM and physiotherapy have been studied in the treatment of bruxism, this is the first study to evaluate the effect of ILIB combined with myofunctional exercises for sleep bruxism in pediatrics.

Vascular photobiomodulation in the treatment of children with temporomandibular disorders: Study protocol for a randomized, controlled, blind, clinical trial.

BACKGROUND: This study aims to verify if the use of intravascular laser irradiation of blood (ILIB) influences the reduction of pain and increases the range of motion in opening and closing of the mouth in children and adolescents with temporomandibular disorders (TMD). METHODS: This will be a blind, randomized, and controlled clinical trial, which will be carried out on children between 6 and 12 years of age who enter the Catholic University of Uruguay, Faculty of Health Sciences, Postgraduate School, for treatment. To be included, children must present temporomandibular disorders, based on the diagnostic criteria will be the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD). Forty-five participants will be randomized to three groups: Group 1-ILIB with 2 sessions of 20 minutes for 12 weeks (n = 15); Group 2-Placebo laser application with 2 sessions of 20 minutes for 12 weeks (n = 15); Group 3-Control with no treatment (n = 15). Irradiation will be performed by continuous and direct transcutaneous application to the radial artery, by means of a bracelet that inserts the laser beam. The laser to be used is infrared, power 100 mW ± 20%, wavelength 808 nm ± 10 nm, continuous application. RDC/TMD and pain evaluated through a visual analog scale will be the outcome measures. DISCUSSION: Due to the low level of evidence, new studies are needed on the effect of ILIB in children with TMD.

A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.

U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

A Novel Bacterial Protease Inhibitor Adjuvant in RBD-Based COVID-19 Vaccine Formulations Containing Alum Increases Neutralizing Antibodies, Specific Germinal Center B Cells and Confers Protection Against SARS-CoV-2 Infection in Mice.

In this work, we evaluated recombinant receptor binding domain (RBD)-based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus alum, AddaS03, AddaVax, or the combination of alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and alum as adjuvants has a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus alum, AddaVax, or AddaS03. Antibodies induced with the formulation containing U-Omp19 and alum not only increased their neutralization capacity against the ancestral virus but also cross-neutralized alpha, lambda, and gamma variants with similar potency. Furthermore, the addition of U-Omp19 to alum vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+alum formulation induced RBD-specific Th1 and CD8+ T-cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge of K18-hACE2 mice.

Exploiting the Potential in Water Cleanup from Metals and Nutrients of Desmodesmus sp. and Ampelodesmos mauritanicus.

Increasing levels of freshwater contaminants, mainly due to anthropogenic activities, have resulted in a great deal of interest in finding new eco-friendly, cost-effective and efficient methods for remediating polluted waters. The aim of this work was to assess the feasibility of using a green microalga Desmodesmus sp., a cyanobacterium Nostoc sp. and a hemicryptophyte Ampelodesmos mauritanicus to bioremediate a water polluted with an excess of nutrients (nitrogen and phosphorus) and heavy metals (copper and nickel). We immediately determined that Nostoc sp. was sensitive to metal toxicity, and thus Desmodesmus sp. was chosen for sequential tests with A. mauritanicus. First, A. mauritanicus plants were grown in the 'polluted' culture medium for seven days and were, then, substituted by Desmodesmus sp. for a further seven days (14 days in total). Heavy metals were shown to negatively affect both the growth rates and nutrient removal capacity. The sequential approach resulted in high metal removal rates in the single metal solutions up to 74% for Cu and 85% for Ni, while, in the bi-metal solutions, the removal rates were lower and showed a bias for Cu uptake. Single species controls showed better outcomes; however, further studies are necessary to investigate the behavior of new species.

Investigating the Drought and Salinity Effect on the Redox Components of Sulla Coronaria (L.) Medik.

For the Mediterranean region, climate models predict an acceleration of desertification processes, thus threatening agriculture. The present work aimed to investigate the effect of drought and salinity on Sulla coronaria (L.) Medik., a Mediterranean forage legume, for understanding plant defence systems activated by these stressors. In detail, we focused our attention on the variations on the plant redox status. Plants were subjected to suboptimal watering and irrigation with sodium chloride (NaCl) solutions. The same salt treatment was applied for in vitro tests on seedlings. Water content did not change after treatments. Salt negatively influenced seed germination and seedling development, but it did not affect photosynthesis parameters, contrary to what was observed in adult plants. Proline concentration increased in all samples, while abscisic acid level increased exclusively in seedlings. NaCl caused accumulation of superoxide anion in plants and seedlings and hydrogen peroxide only in seedlings; nevertheless, lipid peroxidation was not detected. Total phenolics, glutathione, expression level, and activity of antioxidant enzymes were assayed, revealing a complex antiradical molecular response, depending on the type of stress and development stage. Our results confirm Sulla as a drought- and salt-tolerant species and highlight its ability to counteract oxidative stress. This evidence suggests a key role for the redox components, as signal transduction messengers, in Sulla acclimation to desertification. Finally, plants and seedlings showed different acclimation capacity to salinity, revealing a greater genomic plasticity for seedlings.

Multimorbidity in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis: Results From a Longitudinal, Multicenter Data Linkage Study.

OBJECTIVE: Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is considered a chronic, relapsing condition. To date, no studies have investigated multimorbidity in AAV nationally. This study was undertaken to characterize temporal trends in multimorbidity and report excess health care expenditures associated with multimorbidities in a national AAV cohort from Scotland. METHODS: Eligible patients with AAV were diagnosed between 1997 and 2017. Each patient was matched with up to 5 general population controls. Linked morbidity and health care expenditure data were retrieved from a Scottish national hospitalization repository and from published national cost data. Multimorbidity was defined as the development of ≥2 disorders. Prespecified morbidities, individually and together, were analyzed for risks and associations over time using modified Poisson regression, discrete interval analysis, and chi-square test for trend. The relationship between multimorbidities and health care expenditure was investigated using multivariate linear regression. RESULTS: In total, 543 patients with AAV (median age 58.7 years [range 48.9-68.0 years]; 53.6% male) and 2,672 general population controls (median age 58.7 years [range 48.9-68.0 years]; 53.7% male) were matched and followed up for a median of 5.1 years. AAV patients were more likely to develop individual morbidities at all time points, but especially <2 years after diagnosis. The highest proportional risk observed was for osteoporosis (adjusted incidence rate ratio 8.0, 95% confidence interval [95% CI] 4.5-14.2). After 1 year, 23.0% of AAV patients and 9.3% of controls had developed multimorbidity (P < 0.0001). After 10 years, 37.0% of AAV patients and 17.3% of controls were reported to have multimorbidity (P < 0.0001). Multimorbidity was associated with disproportionate increases in health care expenditures in AAV patients. Health care expenditure was highest for AAV patients with ≥3 morbidities (3.89-fold increase in costs, 95% CI 2.83-5.31; P < 0.001 versus no morbidities). CONCLUSION: These findings emphasize the importance of holistic care in patients with AAV, and may identify a potentially critical opportunity to consider early screening.

Stability Studies of the Vaccine Adjuvant U-Omp19.

Unlipidated outer membrane protein 19 (U-Omp19) is a novel mucosal adjuvant in preclinical development to be used in vaccine formulations. U-Omp19 holds two main properties, it is capable of inhibiting gastrointestinal and lysosomal peptidases, increasing the amount of co-administered antigen that reaches the immune inductive sites and its half-life inside cells, and it is able to stimulate antigen presenting cells in vivo. These activities enable U-Omp19 to enhance the adaptive immune response to co-administrated antigens. To characterize the stability of U-Omp19 we have performed an extensive analysis of its physicochemical and biological properties in a 3-year long-term stability study, and under potentially damaging freeze-thawing and lyophilization stress processes. Results revealed that U-Omp19 retains its full protease inhibitor activity, its monomeric state and its secondary structure even when stored in solution for 36 months or after multiple freeze-thawing cycles. Non-enzymatic hydrolysis resulted the major degradation pathway for storage in solution at 4 °C or room temperature which can be abrogated by lyophilization yet increasing protein tendency to form aggregates. This information will play a key role in the development of a stable formulation of U-Omp19, allowing an extended shelf-life during manufacturing, storage, and shipping of a future vaccine containing this pioneering adjuvant.

The Influence of Light and Nutrient Starvation on Morphology, Biomass and Lipid Content in Seven Strains of Green Microalgae as a Source of Biodiesel.

The development of clean and renewable energy sources is currently one of the most important challenges facing the world. Although research interests in algae-based energy have been increasing in the last decade, only a small percentage of the bewildering diversity exhibited by microalgae has been investigated for biodiesel production. In this work, seven strains of green microalgae belonging to the genera Scenedesmus, Tetradesmus and Desmodesmus were grown in liquid medium with or without a nitrogen (N) source-at two different irradiances (120 ± 20 and 200 ± 20 µmol photons m-2 s-1)-to evaluate biomass production and FAME (fatty acid methyl esters) content for biodiesel production. The strains of Tetradesmus obliquus and Desmodesmus abundans grown in N-deprived medium showed the highest FAME content (22.0% and 34.6%, respectively); lipid profile characterization highlighted the abundance of saturated FAME (as C16:0 and C18:0) that favors better viscosity (flow properties) and applicability of biodiesel at low temperatures. Light microscopy and confocal laser scanning microscopy observations were employed as a fast method to monitor the vital status of cells and lipid droplet accumulation after Nile red staining in different culture conditions.

Eukaryotic and Prokaryotic Phytochelatin Synthases Differ Less in Functional Terms Than Previously Thought: A Comparative Analysis of Marchantia polymorpha and Geitlerinema sp. PCC 7407.

This paper reports functional studies on the enzyme phytochelatin synthase in the liverwort Marchantia polymorpha and the cyanobacterium Geitlerinema sp. strain PCC 7407. In vitro activity assays in control samples (cadmium-untreated) showed that phytochelatin synthase was constitutively expressed in both organisms. In the presence of 100 µM cadmium, in both the liverwort and the cyanobacterium, the enzyme was promptly activated in vitro, and produced phytochelatins up to the oligomer PC4. Likewise, in vivo exposure to 10-36 µM cadmium for 6-120 h induced in both organisms phytochelatin synthesis up to PC4. Furthermore, the glutathione (GSH) levels in M. polymorpha were constitutively low (compared with the average content in higher plants), but increased considerably under cadmium stress. Conversely, the GSH levels in Geitlerinema sp. PCC 7407 were constitutively high, but were halved under metal treatments. At odds with former papers, our results demonstrate that, as in M. polymorpha and other plants, the cyanobacterial phytochelatin synthase exposed to cadmium possesses manifest transpeptidasic activity, being able to synthesize phytochelatins with a degree of oligomerization higher than PC2. Therefore, prokaryotic and eukaryotic phytochelatin synthases differ less in functional terms than previously thought.

U-Omp19 from Brucella abortus increases dmLT immunogenicity and improves protection against Escherichia coli heat-labile toxin (LT) oral challenge.

Acute diarrhea disease caused by bacterial infections is a major global health problem. Enterotoxigenic Escherichia coli (ETEC) is one of the top causes of diarrhea-associated morbidity and mortality in young children and travelers to low-income countries. There are currently no licensed vaccines for ETEC. Induction of immunity at the site of entry of the bacteria is key to prevent infection. Current approaches to ETEC vaccines include a less toxic mutant form of E. coli heat-labile toxin (double-mutant heat-labile enterotoxin -dmLT-) with both antigenic and immunostimulatory properties. U-Omp19 is a protease inhibitor from Brucella spp. with immunostimulatory properties that has been used as oral adjuvant. In this work, we use U-Omp19 as adjuvant in an oral vaccine formulation against ETEC containing dmLT in outbred and inbred mice. To evaluate antigen dose sparing by U-Omp19 three different immunization protocols with three different doses of dmLT were evaluated. We demonstrated that U-Omp19 co-delivery increases anti-LT IgA in feces using a mid-dose of dmLT following a prime-boost protocol (after one or two boosts). Oral immunization with U-Omp19 induced protection against LT challenge when co-formulated with dmLT in CD-1 and BALB/c mice. Indeed, there was a significant increase in anti-LT IgG and IgA avidity after a single oral administration of dmLT plus U-Omp19 in comparison with dmLT delivered alone. Interestingly, sera from dmLT plus U-Omp19 vaccinated mice significantly neutralize LT effect on intestine inflammation in vivo compared with sera from the group immunized with dmLT alone. These results demonstrate the adjuvant capacity of U-Omp19 to increase dmLT immunogenicity by the oral route and support its use in an oral subunit vaccine formulation against ETEC.

The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins.

In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.

Omp19 Enables Brucella abortus to Evade the Antimicrobial Activity From Host's Proteolytic Defense System.

Pathogenic microorganisms confront several proteolytic events in the molecular interplay with their host, highlighting that proteolysis and its regulation play an important role during infection. Microbial inhibitors, along with their target endogenous/exogenous enzymes, may directly affect the host's defense mechanisms and promote infection. Omp19 is a Brucella spp. conserved lipoprotein anchored by the lipid portion in the Brucella outer membrane. Previous work demonstrated that purified unlipidated Omp19 (U-Omp19) has protease inhibitor activity against gastrointestinal and lysosomal proteases. In this work, we found that a Brucella omp19 deletion mutant is highly attenuated in mice when infecting by the oral route. This attenuation can be explained by bacterial increased susceptibility to host proteases met by the bacteria during establishment of infection. Omp19 deletion mutant has a cell division defect when exposed to pancreatic proteases that is linked to cell-cycle arrest in G1-phase, Omp25 degradation on the cell envelope and CtrA accumulation. Moreover, Omp19 deletion mutant is more susceptible to killing by macrophage derived microsomes than wt strain. Preincubation with gastrointestinal proteases led to an increased susceptibility of Omp19 deletion mutant to macrophage intracellular killing. Thus, in this work, we describe for the first time a physiological function of B. abortus Omp19. This activity enables Brucella to better thrive in the harsh gastrointestinal tract, where protection from proteolytic degradation can be a matter of life or death, and afterwards invade the host and bypass intracellular proteases to establish the chronic infection.

Characterization and quantification of thiol-peptides in Arabidopsis thaliana using combined dilution and high sensitivity HPLC-ESI-MS-MS.

Although thiol-peptide compounds, such as reduced glutathione (GSH), γ-glutamylcysteine (γ-EC), and phytochelatins, play fundamental roles in plants, their analytical determination and characterization is still somewhat problematic, mainly due to their high polarity and oxidation propensity. Thus, in this work a reliable and sensitive HPLC-ESI-MS-MS method was developed, in order to simultaneously assay, within 14-min instrumental runs, γ-EC, GSH, and phytochelatins up to phytochelatin 4. This analytical method was validated in shoot and root extracts of the model plant Arabidopsis thaliana (Brassicaceae) and guaranteed accurate quantification by using specific isotope labelled-internal standards for both GSH and phytochelatins, as well as standards for external calibration. Good linearities in the method performance were observed (R > 0.99), with a dynamic range over three orders of magnitude in thiol-peptide concentrations. In MRM mode, the detection sensitivity of the thiol-peptides was equal to approximately 16, 6, 7, 13, 10 fmol for γ-EC, GSH, phytochelatin 2, phytochelatin 3, and phytochelatin 4, respectively (20 µl injection each). The reproducibility of the method was confirmed by high intra- and inter-day accuracy and precision values. The recovery rates were estimated approximately in the range of 73.8-91.0% and the matrix effect evaluation revealed that all analytes exhibited ionization suppression. The use of stable isotope-labelled analogs of the thiol-peptides as internal standards was particularly worthy of note: it offered the considerable advantage of overcoming the consequences of matrix effect and thiol-peptide loss through sample preparation, by normalizing the analyte signal during the quantification process. Thus, by validating the method's sensitivity, accuracy, precision, reproducibility, stability, recovery, and matrix effect, data reliability and robustness were ensured.