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Objectives: To describe the differences and similarities in perceptions and attitudes regarding COVID-19 vaccination among Black and Latinx Michiganders. Methods: Utilizing a convergent mixed-methods approach, forty interviews were conducted with 24 Black and 16 Latinx community members between December 2020 and June 2021 across four Michigan counties disproportionately affected by COVID-19. Survey data were collected from a representative sample of 1598 individuals living in Detroit between January and March 2021. Results: Vaccine hesitancy was a more prevalent theme among Black interview participants than Latinx participants. Trust in experts and vaccine access were significantly more influential in the decision to vaccinate for Latinx residents compared to Black residents. Latinx individuals reported greater intention to receive a COVID-19 vaccine compared to Black respondents. Multinomial logit models revealed that 30% of Black participants expressed hesitancy about the COVID-19 vaccine compared to 10% of Latinx respondents. Conclusions and Implications: This study provides a deeper understanding of key differences and similarities in vaccine acceptance/hesitancy across race/ethnicity. The findings can enhance health interventions and outcomes by informing the development of culturally responsive practices tailored to specific communities.

Neuroimaging research requires purpose-built analysis software, which is challenging to install and may produce different results across computing environments. The community-oriented, open-source Neurodesk platform (https://www.neurodesk.org/) harnesses a comprehensive and growing suite of neuroimaging software containers. Neurodesk includes a browser-accessible virtual desktop, command-line interface and computational notebook compatibility, allowing for accessible, flexible, portable and fully reproducible neuroimaging analysis on personal workstations, high-performance computers and the cloud.

In this paper, we propose an autoregressive model for time series in which the variable of interest lies in the unit interval and is subject to certain threshold values below or above which the measurements are not quantifiable. The model includes an independent beta regression (Ferrari and Cribari-Neto, J. Appl. Stat., 31, 799–815 2004) as a special case. A Markov chain Monte Carlo (MCMC) algorithm is tailored to obtain Bayesian posterior distributions of unknown quantities of interest. The likelihood function was used to compute Bayesian model selection measures. We discuss the construction of the proposed model and compare it with alternative models by using simulated data. Finally, we illustrate the use of our proposal by modeling a left-censored weekly series of acid rain data.

Background: Minority communities are disproportionately impacted by COVID-19. In Michigan in 2024, 59% of Latinx residents, 46% of Black residents, and 57% of White residents have received at least one dose of the vaccine. However, just 7% of Black residents and 6% of Latinx residents report being up-to-dateper CDC definition, versus 13% of White residents. Drawing from protection motivation theory,we aimed to identify barriers to COVID-19 vaccination. Methods: Interviews with 24 Black and 10 Latinx Michigan residents self-reported as not up-to-date(n= 15) or up-to-date (n= 19) on COVID-19 vaccines were conducted in 2022–2023. We used a community-based participatory approach in collaboration with 16 leaders from 15 organizations to develop research questions, interview protocols, and methods for data collection and analysis. Thematic coding of interviews was conducted. Results: Findings indicate participants’ lack of confidence in the COVID-19 vaccine’s efficacy, with those not up-to-date expressing greater doubt. Participants were also concerned about vaccine benefits versus risks, safety, and side effects. Distrust in medicine, confusion about public health guidelines, and conspiracy theories were often reported. Younger unvaccinated individuals cited low health risk as reason to remain unvaccinated. Many participants felt that health education, especially through medical professionals, was beneficial. Conclusion: There is great need for more data to make informed decisions given ongoing lack of understanding of the public health benefits of COVID-19 vaccination. Identifying drivers of vaccine uptake, particularly boosters, in communities of color and developing age-appropriate and culturally responsive interventions to increase vaccination rates are of utmost importance.

The aim of this study is to investigate the literature to evaluate dental students’ attitudes regarding the treatment of older adults. A scoping review was performed following Preferred Reporting Items for Systematic Reviews and Meta‐Analyses/PRISMA guidelines to identify articles from four electronic databases: MEDLINE via the PubMed interface, Embase, Cumulative Index to Nursing and Allied Health Literature, and AgeLine. Gray literature searches were also performed in Scopus, Web of Science, and ProQuest Dissertations and Theses‐Health and Medicine. Eleven articles were assessed. The majority (72, 72%) were published between 2011 and 2020, evidencing various contexts of dental students, such as different countries and cultures, and levels of education. The most commonly used tool/instrument to survey dental students’ attitudes was the Aging Semantic Differential Scale. Student age, race, and marital status did not seem to interfere with dental students’ attitudes regarding the treatment of older adults. Dental students tend to have a positive attitude toward older people. In this context, female students, students who interact with older people, and clinical students have more positive attitudes than male and nonclinical students.

Background Olfactory dysfunction (OD)—including anosmia and hyposmia—is a common symptom of COVID-19. Previous studies have identified olfactory training (OT) as an important treatment for postinfectious OD; however, little is known about its benefits and optimizations after SARS-CoV-2 infection.Objective This study aimed to assess whether olfactory training performance can be optimized using more fragrances over a shorter period of time in patients with persistent OD after COVID-19. In addition, we determined the presence of other variables related to OD and treatment response in this population.Methods This multicenter randomized clinical trial recruited 80 patients with persistent OD and prior COVID-19 infection for less than 3 months. The patients were divided into 2 groups receiving either 4 or 8 essences over 4 weeks. Subjective assessments and the University of Pennsylvania Smell Identification Test (UPSIT) were performed before and after the treatment.Results Significant olfactory improvement was measured subjectively and using the UPSIT in both groups; however, no significant differences between the groups were observed. Additionally, the presence of olfactory fluctuations was associated with higher UPSIT scores.Conclusion These data suggest that training intensification by increasing the number of essences for 4 weeks does not show superiority over the classical method. Moreover, fluctuant olfaction seems to be related to a higher score on the UPSIT.

The shortage of reliable primary taxonomic data limits the description of biological taxa and the understanding of biodiversity patterns and processes, complicating biogeographical, ecological, and evolutionary studies. This deficit creates a significant taxonomic impediment to biodiversity research and conservation planning. The taxonomic impediment and the biodiversity crisis are widely recognized, highlighting the urgent need for reliable taxonomic data. Over the past decade, numerous countries worldwide have devoted considerable effort to Target 1 of the Global Strategy for Plant Conservation (GSPC), which called for the preparation of a working list of all known plant species by 2010 and an online world Flora by 2020. Brazil is a megadiverse country, home to more of the world's known plant species than any other country. Despite that, Flora Brasiliensis, concluded in 1906, was the last comprehensive treatment of the Brazilian flora. The lack of accurate estimates of the number of species of algae, fungi, and plants occurring in Brazil contributes to the prevailing taxonomic impediment and delays progress towards the GSPC targets. Over the past 12 years, a legion of taxonomists motivated to meet Target 1 of the GSPC, worked together to gather and integrate knowledge on the algal, plant, and fungal diversity of Brazil. Overall, a team of about 980 taxonomists joined efforts in a highly collaborative project that used cybertaxonomy to prepare an updated Flora of Brazil, showing the power of scientific collaboration to reach ambitious goals. This paper presents an overview of the Brazilian Flora 2020 and provides taxonomic and spatial updates on the algae, fungi, and plants found in one of the world's most biodiverse countries. We further identify collection gaps and summarize future goals that extend beyond 2020. Our results show that Brazil is home to 46,975 native species of algae, fungi, and plants, of which 19,669 are endemic to the country. The data compiled to date suggests that the Atlantic Rainforest might be the most diverse Brazilian domain for all plant groups except gymnosperms, which are most diverse in the Amazon. However, scientific knowledge of Brazilian diversity is still unequally distributed, with the Atlantic Rainforest and the Cerrado being the most intensively sampled and studied biomes in the country. In times of “scientific reductionism”, with botanical and mycological sciences suffering pervasive depreciation in recent decades, the first online Flora of Brazil 2020 significantly enhanced the quality and quantity of taxonomic data available for algae, fungi, and plants from Brazil. This project also made all the information freely available online, providing a firm foundation for future research and for the management, conservation, and sustainable use of the Brazilian funga and flora.

Trypanosoma cruzi has three biochemically and morphologically distinct developmental stages that are programmed to rapidly respond to environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, Trypanosomatid genomes contain protein coding genes that are transcribed into polycistronic pre-mRNAs and have their expression controlled by post-transcriptional mechanisms. Transcriptome analyses comparing three stages of the T. cruzi life cycle revealed changes in gene expression that reflect the parasite adaptation to distinct environments. Several genes encoding RNA binding proteins (RBPs), known to act as key post-transcriptional regulatory factors, were also differentially expressed. We characterized one T. cruzi RBP, named TcZH3H12, which contains a zinc finger domain and is up-regulated in epimastigotes compared to trypomastigotes and amastigotes. TcZC3H12 knockout (KO) epimastigotes showed decreased growth rates and increased capacity to differentiate into metacyclic trypomastigotes. Transcriptome analyses comparing wild type and TcZC3H12 KOs revealed a TcZC3H12-dependent expression of epimastigote-specific genes such as genes encoding amino acid transporters and proteins associated with differentiation (PADs). RNA immunoprecipitation assays showed that transcripts from the PAD family interact with TcZC3H12. Taken together, these findings suggest that TcZC3H12 positively regulates the expression of genes involved in epimastigote proliferation and also acts as a negative regulator of metacyclogenesis. [ABSTRACT FROM AUTHOR]

Aim:The inhibitory and antibiofilm effects of Thymus vulgaris(EOTv) and Hyptis spicigeraessential oils (EOHs) on cariogenic microorganisms were evaluated. Materials & methods:The chemical characterization of EOTv was performed by gas chromatography/mass spectrometry. Streptococcus mutans, Streptococcus gordonii, Streptococcus sanguinis, Streptococcus mitis, Streptococcus sobrinus, Lactobacillus acidophilusand Actinomyces naeslundiiwere used for agar diffusion assays and determination of minimal inhibitory and minimal bactericide concentrations. In addition, 20 streptococci and lactobacilli clinical isolates were also tested. The effects of essential oil on microbial initial biofilm formation and on preformed microcosm biofilm formed from human saliva were studied. Results & conclusion:Both essential oils had inhibitory effects on the cariogenic species and reduced the bacterial adherence to dental enamel. Essential oils were able to disrupt preformed microcosm biofilms. Thymus vulgarisand Hyptis spicigeraessential oils have potential to be used in the development of formulations to the control of cariogenic biofilms.

As the coronavirus pandemic has taken over matters of life and death globally, immigrant communities were some of the most deeply impacted. In the United States (U.S.), Latinx immigrants and other minorities have experienced greater economic burden and worse health outcomes, resulting in alarming rates of death from COVID-19. Yet the government’s relief measures to support individuals did not extend to millions of immigrants. This left many immigrants with the cruel choice to either stay home to protect themselves and their loved ones from the virus or go to work to support their families. Disregard for a large segment of the population is further complemented by strict immigration policies, harsher border restrictions, and public health guidelines that failed to account for the realities faced by immigrants. In this brief, we highlight the unequal toll of the COVID-19 pandemic on immigrants and consider social work response. We argue that the pandemic demands more of the social work profession, as the coronavirus crisis exposed more clearly the systemic inequalities toward immigrants and aggravates their vulnerabilities. Insofar as systems are unequal and racist in the context of coronavirus, there is a great need for social work response that is innovative, brave, and deeply connected to communities.

The Antillean manatee Trichechus manatus manatuscan be found along the northern and northeastern coasts of Brazil. Previous studies on the clinical biochemistry of these animals were conducted in North America and the Caribbean, whereas little is known regarding these parameters in South American manatee populations. Accordingly, the objective of the present study was to examine the hematology and clinical biochemistry of Antillean manatees of different sexes and from different environments in northeast Brazil. Whole‐blood and serum samples were obtained from healthy individuals. The hemogram analysis was performed and the levels of blood biochemical components were determined using an automated platform. The only statistically significant difference observed in the hemogram was a higher number of heterophils in manatees that were screened during the dry season of the year. Clinical biochemistry profiling revealed that free‐ranging manatees presented lower levels of creatinine. Albumin was detected in higher concentrations in animals from rehabilitation captivity, and amylase presented higher levels in manatees that were kept in acclimation captivity. Free‐ranging manatees showed higher serum aspartate aminotransferase levels than manatees in rehabilitation captivity. These results can aid veterinarians and conservation professionals in the development of better captive management procedures and in the clinical approach to manatees.

Various species of the intestinal microbiota have been associated with the development of colorectal cancer1,2, but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia colican carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin3. This compound is believed to alkylate DNA on adenine residues4,5and induces double-strand breaks in cultured cells3. Here we expose human intestinal organoids to genotoxic pks+E. coliby repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pkspathogenicity island.

Graphical Abstract

Most cancer drugs enter the US market first. US Food and Drug Administration (FDA) approvals of new cancer drugs may influence regulatory decisions in other settings. The study examined whether characteristics of available evidence at FDA approval influenced time-to-marketing authorisation (MA) in Brazil, and price differences between the two countries.

Bacterial vaginosis (BV) is characterized by depletion of Lactobacillusand overgrowth of anaerobic and facultative bacteria, leading to increased mucosal inflammation, epithelial disruption, and poor reproductive health outcomes. However, the molecular mediators contributing to vaginal epithelial dysfunction are poorly understood. Here we utilize proteomic, transcriptomic, and metabolomic analyses to characterize biological features underlying BV in 405 African women and explore functional mechanisms in vitro. We identify five major vaginal microbiome groups: L. crispatus(21%), L. iners(18%), Lactobacillus(9%), Gardnerella(30%), and polymicrobial (22%). Using multi-omics we show that BV-associated epithelial disruption and mucosal inflammation link to the mammalian target of rapamycin (mTOR) pathway and associate with Gardnerella, M. mulieris, and specific metabolites including imidazole propionate. Experiments in vitroconfirm that type strain G. vaginalisand M. mulierissupernatants and imidazole propionate directly affect epithelial barrier function and activation of mTOR pathways. These results find that the microbiome-mTOR axis is a central feature of epithelial dysfunction in BV.

The use of information modeling and digital data for highway projects has seen a considerable surge over the past decade. These tools have enhanced the predictability and performance of several tasks in the project delivery process. This study explores the implementation of civil integrated management (CIM) practices in four case studies and documents the lessons learned to enhance CIM inclusion in project delivery processes. CIM encompasses the system of interdependent technologies and practices that facilitate collection, management, and utilization of digital information for project delivery and asset management processes. CIM is an emerging practice, and studies concerning integration of CIM technologies with project life cycle are limited. Through case studies of four highway projects, this study emphasizes the standards and processes that played a vital role in utilizing CIM technologies for contract documentation, design coordination, construction automation, and project management. The two small-scale projects investigated as part of this study demonstrate that pilot initiatives could be successfully carried out to harvest best practices in overcoming contract and legal challenges while embracing new technologies in agencies’ workflow. The two large-scale projects indicate that with the owner’s participation and expertise, the role of CIM technologies can be further enhanced toward performing project management functions. The lessons learned from the case studies are organized to provide a synthesis of process and organizational considerations that would enhance the agencywide adoption of CIM technologies.

Myracrodruon urundeuvaAllemão, Anacardiaceae, is a medicinal plant widely found in Brazil, especially in the northern region. In our previous study, the ethanolic extract from leaves of M. urundeuvashowed antiviral activity against simian rotavirus SA-11. Here, the crude extract was subjected to fractionations in order to subsequently work with more concentrated and pure bioactive compounds, which were analyzed by TLC and HPLC methods to support a better understanding of their virucidal effect. The antiviral activity was evaluated using a rotavirus infection model in MA-104 cells treated with the maximum non-cytotoxic concentration of the crude extract and its fractions. Data were expressed as the percentage inhibition of viral replication calculated by the inhibition of cytopathic effect in the treated cells compared to untreated controls after 48h of incubation. First, we conducted a fractionation, generating five fractions (F1–F5) which were submitted to antiviral assay. Then, the fraction that showed the highest virucidal effect (F3, PI=75%) was subjected to a larger partition, yielding eighteen subfractions, which were submitted to new antiviral assays. Terpenes, flavonoids and tannins were the major secondary metabolites detected by TLC analysis in F3. SF1, a flavonoid-enriched fraction, showed the strongest in vitroactivity against rotavirus (PI=92%), preventing cytopathic effect. Chromatographic profiles were obtained by HPLC for the crude extract and SF1, the most potent subfraction. Overall, our data point to the potential anti-rotavirus activity of flavonoid-enriched fraction (SF1) of M. urundeuvaleaves, corroborating the traditional use of this species to treat diarrhea and broadening our perspectives on in vivoassays in mice with SF1 isolated or associated with other fractions.

Chemical profiles of extracts of four species from Laurenciacomplex (Ceramiales, Rhodophyta) from different populations collected along Southeast Brazilian coast were assessed by High Performance Liquid Chromatography coupled with a Diode Array Detector in order to observe geographic chemical variability. Aiming to evaluate the impact of chemical diversity on potential pharmaceutical uses, the extracts were tested against the promastigote form of Leishmania amazonensis.The most active extracts were submitted to anti-amastigote and cytotoxicity assays. Principal Component Analysis of the chromatograms resulted in four major groups of chemical profiles according to the presence of leishmanicidal chamigranes (-)-elatol and obtusol. The existence of chemotypes, displaying variable pharmacological action, is proposed for the differences observed in L. dendroideasamples. Although all extracts were found active against promastigote form of L. amazonensis, their efficacy was remarkably different and not related to the variation of (-)-elatol and obtusol, which indicates the presence of additional compounds with antileishmanial activity. Moreover, the active extracts also displayed anti-amastigote activity and none of them were considered cytotoxic. The results highlight that the knowledge of chemical geographic variability can be valuable in the search of new antileishmanial compounds from marine sources.

•Photo-assisted electrochemical degradation of dimethyl phthalate was achieved.•High dimethyl phthalate mineralization was obtained, increasing the current density.•Photo assistance partially compensates the mass transfer limitations.

ABSTRACTFlippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1? mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1? mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitrogrowth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformansvia processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection.

ABSTRACTIn a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparumapical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivaxmalaria, we expressed the ectodomain of P. vivaxAMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussismonophosphoryl lipid A (MPLA), flagellin FliC from Salmonella entericaserovar Typhimurium, saponin Quil A, or incomplete Freund's adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under “conditions of good laboratory practice” provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastorisis a promising antigen for use in future preclinical and clinical studies.

Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologs in mice; however for granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e.beyond cleavage positions P4-P4′) match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.

ABSTRACTIn prior studies, we demonstrated that glucuronoxylomannan (GXM), the major capsular polysaccharide of the fungal pathogen Cryptococcus neoformans, interacts with chitin oligomers at the cell wall-capsule interface. The structural determinants regulating these carbohydrate-carbohydrate interactions, as well as the functions of these structures, have remained unknown. In this study, we demonstrate that glycan complexes composed of chitooligomers and GXM are formed during fungal growth and macrophage infection by C. neoformans. To investigate the required determinants for the assembly of chitin-GXM complexes, we developed a quantitative scanning electron microscopy-based method using different polysaccharide samples as inhibitors of the interaction of chitin with GXM. This assay revealed that chitin-GXM association involves noncovalent bonds and large GXM fibers and depends on the N-acetyl amino group of chitin. Carboxyl and O-acetyl groups of GXM are not required for polysaccharide-polysaccharide interactions. Glycan complex structures composed of cryptococcal GXM and chitin-derived oligomers were tested for their ability to induce pulmonary cytokines in mice. They were significantly more efficient than either GXM or chitin oligomers alone in inducing the production of lung interleukin 10 (IL-10), IL-17, and tumor necrosis factor alpha (TNF-a). These results indicate that association of chitin-derived structures with GXM through their N-acetyl amino groups generates glycan complexes with previously unknown properties.

The comparison of the efficiencies for boron-doped diamond (BDD) and for DSA-Cl2(r) type electrodes to degrade the reactive orange 16 (RO16) dye is presented. The results for the dye electrochemical degradation were investigated by spectroscopic techniques UV/VIS analysis, Total Organic Carbon (TOC), and High Performance Liquid Chromatography (HPLC). The BDD electrode showed the higher performance in the whole range of current densities studied for the color removal as well as in the mineralization of organic materials present in such dye.

Boron-doped diamond (BDD) electrodes have shown characteristics that differ from other conventional electrodes, and that make them an excellent electrode in the electroanalysis, in the electrosynthesis and also in the treatment of wastewater with organic compounds. In this work, two different electrodes doped with (2,000 and 15,000 ppm) were characterized by Raman Spectroscopy and by Scanning electron Microscopy (SEM). In addition, they were characterized electrochemically by cyclic voltammetry. The electroactive area was estimated according to Cottrell's equation before and after the anodic and the cathodic treatment. From the results, it was observed that the electrodes have a similar behavior in the morphology and in the homogeneity of the films. The large potential window was confirmed for both electrodes (2.5 V vs Ag/AgCl) and for the less doped electrode was obtained a larger area without any treatment while for the more doped electrode, the major area was obtained after the cathodic treatment.

Cryptococcus neoformans is an encapsulated yeast that causes a life-threatening meningoencephalitis in immunocompromised individuals. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this pathogen. This trait is controlled in part by the Ca2+-calcineurin pathway, which senses and utilizes cytosolic calcium for signaling. In the present study, the identification of the C. neoformans gene VCX1, which encodes a vacuolar calcium exchanger, is reported. The VCX1 knockout results in hypersensitivity to the calcineurin inhibitor cyclosporine A at 35°C, but not at 30°C. Furthermore, high concentrations of CaCl2lead to growth inhibition of the vcx1 mutant strain only in the presence of cyclosporine A, indicating that Vcx1 acts in parallel with calcineurin. The loss of VCX1 does not influence cell wall integrity or capsule size but decreases secretion of the major capsular polysaccharide glucuronoxylomannan (GXM) in culture supernatants.Vcx1 also influences C. neoformans phagocytosis by murine macrophages and is required for full virulence in mice. Analysis of cellular distribution by confocal microscopy confirmed the vacuolar localization of Vcx1 in C. neoformans cells.

ABSTRACTGlucuronoxylomannan (GXM), the major capsular component in the Cryptococcuscomplex, interacts with the immune system in multiple ways, which include the activation of Toll-like receptors (TLRs) and the modulation of nitric oxide (NO) production by phagocytes. In this study, we analyzed several structural parameters of GXM samples from Cryptococcus neoformans(serotypes A and D) and Cryptococcus gattii(serotypes B and C) and correlated them with the production of NO by phagocytes and the activation of TLRs. GXM fractions were differentially recognized by TLR2/TLR1 (TLR2/1) and TLR2/6 heterodimers expressed on TLR-transfected HEK293A cells. Higher NF-κB luciferase reporter activity induced by GXM was observed in cells expressing TLR2/1 than in cells transfected with TLR2/6 constructs. A serotype B GXM from C. gattiiwas the most effective polysaccharide fraction activating the TLR-mediated response. This serotype B polysaccharide, which was also highly efficient at eliciting the production of NO by macrophages, was similar to the other GXM samples in monosaccharide composition, zeta potential, and electrophoretic mobility. However, immunofluorescence with four different monoclonal antibodies and dynamic light-scattering analysis revealed that the serotype B GXM showed particularities in serological reactivity and had the smallest effective diameter among the GXM samples analyzed in this study. Fractionation of additional serotype B GXMs, followed by exposure of these fractions to macrophages, revealed a correlation between NO production and reduced effective diameters. Our results demonstrate a great functional diversity in GXM samples from different isolates and establish their abilities to differentially activate cellular responses. We propose that serological properties as well as physical chemical parameters, such as the diameter of polysaccharide molecules, may potentially influence the inflammatory response against Cryptococcusspp. and may contribute to the differences in granulomatous inflammation between cryptococcal species.

ABSTRACTParacoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia colias a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensisenolase [rPbEno]). The P. brasiliensisnative enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensisyeast cells were capable of degrading purified fibronectin, providing in vitroevidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensiswith epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

Glucuronoxylomannan (GXM), the major capsular component in the Cryptococcus complex, interacts with the immune system in multiple ways, which include the activation of Toll-like receptors (TLRs) and the modulation of nitric oxide (NO) production by phagocytes. In this study, we analyzed several structural parameters of GXM samples from Cryptococcus neoformans (serotypes A and D) and Cryptococcus gattii (serotypes B and C) and correlated them with the production of NO by phagocytes and the activation of TLRs. GXM fractions were differentially recognized by TLR2/TLR1 (TLR2/1) and TLR2/6 heterodimers expressed on TLR-transfected HEK293A cells. Higher NF-B luciferase reporter activity induced by GXM was observed in cells expressing TLR2/1 than in cells transfected with TLR2/6 constructs. A serotype B GXM from C. gattii was the most effective polysaccharide fraction activating the TLR-mediated response. This serotype B polysaccharide, which was also highly efficient at eliciting the production of NO by macrophages, was similar to the other GXM samples in monosaccharide composition, zeta potential, and electrophoretic mobility. However, immunofluorescence with four different monoclonal antibodies and dynamic light-scattering analysis revealed that the serotype B GXM showed particularities in serological reactivity and had the smallest effective diameter among the GXM samples analyzed in this study. Fractionation of additional serotype B GXMs, followed by exposure of these fractions to macrophages, revealed a correlation between NO production and reduced effective diameters. Our results demonstrate a great functional diversity in GXM samples from different isolates and establish their abilities to differentially activate cellular responses. We propose that serological properties as well as physical chemical parameters, such as the diameter of polysaccharide molecules, may potentially influence the inflammatory response against Cryptococcus spp. and may contribute to the differences in granulomatous inflammation between cryptococcal species.

ABSTRACTMolecules composed of ß-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformansvaried depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformanscultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformansin the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.

The capsule of Cryptococcus neoformans is a complex structure whose assembly requires intermolecular interactions to connect its components into an organized structure. In this study, we demonstrated that the wheat germ agglutinin (WGA), which binds to sialic acids and β-1,4-N-acetylglucosamine (GlcNAc) oligomers, can also bind to cryptococcal capsular structures. Confocal microscopy demonstrated that these structures form round or hooklike projections linking the capsule to the cell wall, as well as capsule-associated structures during yeast budding. Chemical analysis of capsular extracts by gas chromatography coupled to mass spectrometry and high-pH anion-exchange chromatography suggested that the molecules recognized by WGA were firmly associated with the cell wall. Enzymatic treatment, competition assays, and staining with chemically modified WGA revealed that GlcNAc oligomers, but not sialic acids, were the molecules recognized by the lectin. Accordingly, treatment of C. neoformans cells with chitinase released glucuronoxylomannan (GXM) from the cell surface and reduced the capsule size. Chitinase-treated acapsular cells bound soluble GXM in a modified pattern. These results indicate an association of chitin-derived structures with GXM and budding in C. neoformans, which may represent a new mechanism by which the capsular polysaccharide interacts with the cell wall and is rearranged during replication.

ABSTRACTThe capsule of Cryptococcus neoformansis a complex structure whose assembly requires intermolecular interactions to connect its components into an organized structure. In this study, we demonstrated that the wheat germ agglutinin (WGA), which binds to sialic acids and ß-1,4-N-acetylglucosamine (GlcNAc) oligomers, can also bind to cryptococcal capsular structures. Confocal microscopy demonstrated that these structures form round or hooklike projections linking the capsule to the cell wall, as well as capsule-associated structures during yeast budding. Chemical analysis of capsular extracts by gas chromatography coupled to mass spectrometry and high-pH anion-exchange chromatography suggested that the molecules recognized by WGA were firmly associated with the cell wall. Enzymatic treatment, competition assays, and staining with chemically modified WGA revealed that GlcNAc oligomers, but not sialic acids, were the molecules recognized by the lectin. Accordingly, treatment of C. neoformanscells with chitinase released glucuronoxylomannan (GXM) from the cell surface and reduced the capsule size. Chitinase-treated acapsular cells bound soluble GXM in a modified pattern. These results indicate an association of chitin-derived structures with GXM and budding in C. neoformans, which may represent a new mechanism by which the capsular polysaccharide interacts with the cell wall and is rearranged during replication.

The purpose of this study was to assess the oral health of patients with chronic renal failure (CRF) undergoing hemodialysis in Brazil. A dentist examined 160 subjects aged 40 to 85 years of age.

ABSTRACTGlucuronoxylomannan (GXM) is the major capsular polysaccharide of Cryptococcus neoformans. GXM receptors have been characterized in phagocytes and endothelial cells, but epithelial molecules recognizing the polysaccharide remain unknown. In the current study, we demonstrate that GXM binds to the CD14 receptor in human type II alveolar epithelial cells, resulting in the production of the proinflammatory chemokine interleukin-8.

The intracellular linker LIII-IV of voltage-gated sodium channels is known to be involved in their mechanism of inactivation. Its primary sequence is well conserved in sodium channels from different tissues and species. However, the role of charged residues in this region, first thought to play an important role in inactivation, has not been well identified, whereas the IFM triad (I1488-M1490) has been characterized as the crucial element for inactivation. In this work, we constructed theoretical models and performed molecular dynamics simulations, exploring the role of LIII-IV-charged residues in the presence of a polar/nonpolar planar interface represented by a dielectric discontinuity. From structural predictions, two α-helical segments are proposed. Moreover, from dynamics simulations, a time-conserved motif is detected and shown to play a relevant role in guiding the inactivation particle toward its receptor site.

Nonlinear mixed effects models have received a great deal of attention in the statistical literature in recent years because of their flexibility in handling longitudinal studies, including human immunodeficiency virus viral dynamics, pharmacokinetic analyses, and studies of growth and decay. A standard assumption in nonlinear mixed effects models for continuous responses is that the random effects and the within-subject errors are normally distributed, making the model sensitive to outliers. We present a novel class of asymmetric nonlinear mixed effects models that provides efficient parameters estimation in the analysis of longitudinal data. We assume that, marginally, the random effects follow a multivariate scale mixtures of skew-normal distribution and that the random errors follow a symmetric scale mixtures of normal distribution, providing an appealing robust alternative to the usual normal distribution. We propose an approximate method for maximum likelihood estimation based on an EM-type algorithm that produces approximate maximum likelihood estimates and significantly reduces the numerical difficulties associated with the exact maximum likelihood estimation. Techniques for prediction of future responses under this class of distributions are also briefly discussed. The methodology is illustrated through an application to Theophylline kinetics data and through some simulating studies.

•The water pH does not influence the sex ratio of tambaqui Colossoma macropomum.•pH 6.7 may cause ionic stress in tambaqui (undifferentiated) larvae.

Multiomics analysis reveals the changes that occur in the extracellular matrix in the brains of Zika virus–infected newborns.

Human innate immunity employs cellular and humoral mechanisms to facilitate rapid killing of invading bacteria. The direct killing of bacteria by human serum is attributed mainly to the activity of the complement system, which forms pores in Gram-negative bacteria.

ABSTRACTThe microbiota of the mammalian gastrointestinal tract is a complex ecosystem of bacterial communities that continuously interact with the mucosal immune system. In a healthy host, the mucosal immune system maintains homeostasis in the intestine and prevents invasion of pathogenic bacteria, a phenomenon termed colonization resistance. Antibiotics create dysbiosis of microbiota, thereby decreasing colonization resistance and facilitating infections caused by antibiotic-resistant bacteria. Here we describe how cephalosporin antibiotics create dysbiosis in the mouse large intestine, allowing intestinal outgrowth of antimicrobial-resistant Enterococcus faecium. This is accompanied by a reduction of the mucus-associated gut microbiota layer, colon wall, and Muc-2 mucus layer. E. faeciumagglutinates intraluminally in an extracellular matrix consisting of secretory IgA (sIgA), polymeric immunoglobulin receptor (pIgR), and epithelial cadherin (E-cadherin) proteins, thereby maintaining spatial segregation of E. faeciumfrom the intestinal wall. Addition of recombinant E-cadherin and pIgR proteins or purified IgA to enterococci in vitromimics agglutination of E. faeciumin vivo. Also, the Ca2+levels temporarily increased by 75% in feces of antibiotic-treated mice, which led to deformation of E-cadherin adherens junctions between colonic intestinal epithelial cells and release of E-cadherin as an extracellular matrix entrapping E. faecium. These findings indicate that during antibiotic-induced dysbiosis, the intestinal epithelium stays separated from an invading pathogen through an extracellular matrix in which sIgA, pIgR, and E-cadherin are colocalized. Future mucosal vaccination strategies to control E. faeciumor other opportunistic pathogens may prevent multidrug-resistant infections, hospital transmission, and outbreaks.IMPORTANCEInfections with antibiotic-resistant enterococci are an emerging worldwide problem because enterococci are resistant to most of the antibiotics used in hospitals. During antibiotic treatment, the normal bacteria are replaced by resistant enterococci within the gut, from which they can spread and cause infections. We studied antibiotic-mediated intestinal proliferation of multidrug-resistant Enterococcus faeciumand the effects on intestinal architecture. We demonstrated that antibiotics allow proliferation of E. faeciumin the gut, alter the mucus-associated gut bacterial layer, and reduce the colon wall, mucus thickness, and amount of Muc-2 protein. E. faeciumis agglutinated in the intestine in a matrix consisting of host molecules. We hypothesize that this matrix maintains a segregation of E. faeciumfrom the epithelium. Understanding the processes that occur in the gut during antibiotic treatment may provide clues for future mucosal vaccination strategies to control E. faeciumor other multidrug-resistant opportunistic pathogens, thereby preventing infections, hospital transmission, and outbreaks.

ABSTRACTEnterococcus faeciumis an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faeciumbiofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faeciumE1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162_2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162_2692 was considered the major autolysin in E. faeciumand renamed atlAEfm. In addition, AtlAEfmwas implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faeciumthat contributes to cell-cell interactions in the atlAEfmmutant and is important for cell separation. In conclusion, AtlAEfmis the major autolysin in E. faeciumand contributes to biofilm stability and Acm localization, making AtlAEfma promising target for treatment of E. faeciumbiofilm-mediated infections.IMPORTANCENosocomial infections caused by Enterococcus faeciumhave rapidly increased, and treatment options have become more limited. This is due not only to increasing resistance to antibiotics but also to biofilm-associated infections. DNA is released in biofilm matrix via cell lysis, caused by autolysin, and acts as a matrix stabilizer. In this study, we identified and characterized the major autolysin in E. faecium, which we designated AtlAEfm. atlAEfmdisruption resulted in resistance to lysis, reduced extracellular DNA (eDNA), deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and chaining. Furthermore, AtlAEfmis associated with Acm cell surface localization, resulting in less binding to collagen types I and IV in the atlAEfmmutant. We also identified AtlAEfm-independent eDNA release that contributes to cell-cell interactions in the atlAEfmmutant. These findings indicate that AtlAEfmis important in biofilm and collagen binding in E. faecium, making AtlAEfma promising target for treatment of E. faeciuminfections.

We evaluated the evolutionary conservation of glycine myristoylation within eukaryotic sequences. Our large-scale cross-genome analyses, available as MYRbase, show that the functional spectrum of myristoylated proteins is currently largely underestimated. We give experimental evidence for in vitromyristoylation of selected predictions. Furthermore, we classify five membrane-attachment factors that occur most frequently in combination with, or even replacing, myristoyl anchors, as some protein family examples show.