Morphological and pathogenic investigation of the emerging fungal threat Emergomyces africanus.

Emergomyces africanus is a highly fatal fungal pathogen affecting individuals with advanced HIV disease. Molecular patterns and ultrastructural aspects of E. africanus are unknown, and pathogenic models have not been investigated in detail. Since the cell wall of fungi is a determinant for interaction with the host and antifungal development, we characterized the ultrastructural aspects of E. africanus and the general properties of cell wall components under different conditions of growth in vitro and in vivo. We also tested the pathogenic potential of E. africanus in a Galleria mellonella model of infection. Transmission electron microscopy revealed the common intracellular, ultrastructural features of fungi in association with a thick cell wall. Scanning electron microscopy revealed a smooth cell surface, with no apparent decorative structures. Yeast cultures of E. africanus showed the distribution of chitin, chitooligomers, and mannoproteins commonly observed in fungi. However, in mixed microenvironments containing yeast and filamenting forms of E. africanus, the detection of chitooligomers was increased in comparison with isolated yeast cells, while the detection of these components in filamenting forms was markedly reduced. These observations were suggestive of the ability of E. africanus to change its cell wall composition in response to different microenvironments. Although E. africanus was unable to kill G. mellonella, this infection model allowed us to isolate infected hemocytes for further analysis of mannoproteins, chitin, and chitooligomers. Once again, the detection of E. africanus chitooligomers was markedly increased. These results reveal previously unknown ultrastructural features of E. africanus and suggest a high plasticity in the cell wall of this lethal pathogen. IMPORTANCE: The epidemiology of fungal infections is very dynamic, and novel health emergencies are hard to predict. New fungal pathogens have been continuously emerging for the last few decades, and Emergomyces africanus is one of these threats to human health. This complex scenario points to the need for generating knowledge about emerging pathogens so that new therapeutic strategies can be designed. In this study, we characterized the general cellular and pathogenic properties of the emerging fungal pathogen E. africanus. Our results reveal that E. africanus manifests some of the typical properties of fungal cells but also exhibits some unique characteristics that might be helpful for the future development of therapeutic strategies.

Contribution of microscopy to a better understanding of the anatomy of pathogenic protists.

In this Inaugural Article the author briefly revises its scientific career and how he starts to work with parasitic protozoa. Emphasis is given to his contribution to topics such as a) the structural organization of the surface of protozoa using freeze-fracture and deep-etching; b) the cytoskeleton of protozoa, especially structures such as the subpellicular microtubules of trypanosomatids, the conoid of Toxoplasma gondii, microtubules and inner membrane complex of this protozoan, and the costa of Tritrichomonas foetus; c) the flagellulm of trypanosomatids, that in addition to the axoneme contains a complex network of filaments that constitute the paraflagellar rod; d) special organelles such as the acidocalcisome, hydrogenosome, and glycosome; and e) the highly polarized endocytic pathway found in epimastigote forms of Trypanosoma cruzi.

Phenotypic and Functional Characterization of Memory CD4+ and CD8+ T Cells After Antigenic Stimulation.

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

[Circulating populations of CD4+ CD25+ CD127- regulatory t lymphocytes in peripheral blood of allergic asthmatic children]. / Determinación de poblaciones circulantes en sangre periférica de linfocitos T, reguladores CD4+ CD25+ CD127-, en niños asmáticos alérgicos.

OBJECTIVE: To carry out a preliminary analysis on the Treg lymphocyte counts present in the peripheral blood of allergic asthmatic children from the city of Cartagena, Colombia, compared to healthy controls. METHODS: We compared cytometry counts of ten asthmatic patients (age 7-16 years) and seven healthy controls (6-12 years), recruited in the city of Cartagena. Peripheral blood samples were stained using Cytek's 14-color cFluor Immunoprofiling kit (Cytek® cFluor® Immunoprofiling Kit 14 Color RUO kit), and analyzed on a Northern Lights™ spectral cytometer (Cytek® Biosciences, Fremont, CA, USA), to read 50.000 events per sample. The data obtained were analyzed in SpectroFlo® and FlowJo. The study was approved by the ethics committee of the University of Cartagena (SGR, Grant BPIN2020000100405). RESULTS: The frequency of CD3+, CD4+, CD25+, CD127- Tregs was 11% of all CD4+ T cells, with a range of minimum 8,1% and maximum 17,7%. There was no significant difference in the proportion of Tregs between allergic asthmatic patients and healthy controls (P = 0,2). CONCLUSIONS: With this preliminary sample size, no significant differences were found in the Treg lymphocyte population between allergic asthmatic patients and healthy controls. The 14-color multiplexed panel is a useful tool not only to count CD3+ and CD4+ populations, but also to obtain the percentage of regulatory T cells using cell surface markers.

OBJETIVO: Realizar un análisis preliminar sobre los conteos de linfocitos Tregs presentes en sangre periférica de niños asmáticos alérgicos de la ciudad de Cartagena, comparado con controles sanos. MÉTODOS: Se compararon los conteos de citometría de diez pacientes asmáticos (entre 7 y16 años) y siete controles sanos (entre 6 y12 años), reclutados en la ciudad de Cartagena. La muestra de sangre periférica fue teñida empleando el kit de inmunofenotipo multiplexado de 14 colores de Cytek (Cytek® cFluor® Immunoprofiling Kit 14 Color), y analizada en un citómetro espectral Northern Lights™ (Cytek® Biosciences, Fremont, CA, USA), a lectura de 50.000 eventos por muestra. Los datos obtenidos fueron analizados en SpectroFlo® y FlowJo. El estudio fue aprobado por el Comité de Ética de la Universidad de Cartagena. RESULTADOS: El panel de tinción funcionó apropiadamente y dentro de los parámetros apropiados. Se obtuvo un promedio de células Tregs CD3+, CD4+, CD25+ y CD127- del 11% de todos los CD4+ en las muestras estudiadas, con un rango de mínimo de 8,1% y un máximo de 17,7%. No hubo diferencias significativas en la proporción de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos (P = 0.2). CONCLUSIONES: Con este tamaño de muestra preliminar, no se encontraron diferencias significativas en la población de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos. El panel multiplexado de 14 colores es una herramienta útil no solo para derivar las poblaciones CD3+ y CD4+, sino también para obtener el porcentaje de células T reguladoras empleando marcadores de superficie celular.

Cell surface markers for mesenchymal stem cells related to the skeletal system: A scoping review.

Multipotent mesenchymal stromal cells (MSCs) have been described as bone marrow stromal cells, which can form cartilage, bone or hematopoietic supportive stroma. In 2006, the International Society for Cell Therapy (ISCT) established a set of minimal characteristics to define MSCs. According to their criteria, these cells must express CD73, CD90 and CD105 surface markers; however, it is now known they do not represent true stemness epitopes. The objective of the present work was to determine the surface markers for human MSCs associated with skeletal tissue reported in the literature (1994-2021). To this end, we performed a scoping review for hMSCs in axial and appendicular skeleton. Our findings determined the most widely used markers were CD105 (82.9%), CD90 (75.0%) and CD73 (52.0%) for studies performed in vitro as proposed by the ISCT, followed by CD44 (42.1%), CD166 (30.9%), CD29 (27.6%), STRO-1 (17.7%), CD146 (15.1%) and CD271 (7.9%) in bone marrow and cartilage. On the other hand, only 4% of the articles evaluated in situ cell surface markers. Even though most studies use the ISCT criteria, most publications in adult tissues don't evaluate the characteristics that establish a stem cell (self-renewal and differentiation), which will be necessary to distinguish between a stem cell and progenitor populations. Collectively, MSCs require further understanding of their characteristics if they are intended for clinical use.

Professor Luiz R. Travassos and the study of surface structures of fungal pathogens.

Brazilian medical mycology considerably expanded in the last decades due to the efforts of several pioneers who started and expanded mycology during the twentieth century. In this manuscript, we highlight some of the contributions of one of these pioneers: Professor Luiz R. Travassos, who started his career in the field of microbiology in the 1960s. We will discuss his contributions to the areas of medical mycology and glycobiology, with a focus on glycosphingolipids, sialic acids, and surface enzymes.

Cell surface receptor kinase FERONIA linked to nutrient sensor TORC signaling controls root hair growth at low temperature linked to low nitrate in Arabidopsis thaliana.

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is determined both by intrinsic and environmental signals. Although nutrient availability and temperature are key factors for a sustained plant growth, the molecular mechanisms underlying their sensing and downstream signaling pathways remain unclear. We use genetics to address the roles of the cell surface receptor kinase FERONIA (FER) and the nutrient sensing TOR Complex 1 (TORC) in RH growth. We identified that low temperature (10°C) triggers a strong RH elongation response in Arabidopsis thaliana involving FER and TORC. We found that FER is required to perceive limited nutrient availability caused by low temperature. FERONIA interacts with and activates TORC-downstream components to trigger RH growth. In addition, the small GTPase Rho of plants 2 (ROP2) is also involved in this RH growth response linking FER and TOR. We also found that limited nitrogen nutrient availability can mimic the RH growth response at 10°C in a NRT1.1-dependent manner. These results uncover a molecular mechanism by which a central hub composed by FER-ROP2-TORC is involved in the control of RH elongation under low temperature and nitrogen deficiency.

Dietary polyphenols and their relationship to the modulation of non-communicable chronic diseases and epigenetic mechanisms: A mini-review.

Chronic Non-Communicable Diseases (NCDs) have been considered a global health problem, characterized as diseases of multiple factors, which are developed throughout life, and regardless of genetics as a risk factor of important relevance, the increase in mortality attributed to the disease to environmental factors and the lifestyle one leads. Although the reactive species (ROS/RNS) are necessary for several physiological processes, their overproduction is directly related to the pathogenesis and aggravation of NCDs. In contrast, dietary polyphenols have been widely associated with minimizing oxidative stress and inflammation. In addition to their antioxidant power, polyphenols have also drawn attention for being able to modulate both gene expression and modify epigenetic alterations, suggesting an essential involvement in the prevention and/or development of some pathologies. Therefore, this review briefly explained the mechanisms in the development of some NCDs, followed by a summary of some evidence related to the interaction of polyphenols in oxidative stress, as well as the modulation of epigenetic mechanisms involved in the management of NCDs.

Targeted Therapy for Locally Advanced or Metastatic Urothelial Cancer (mUC): Therapeutic Potential of Sacituzumab Govitecan.

Urothelial carcinoma is the second most frequent genitourinary malignancy. Despite the poor prognosis, new treatment options have emerged and have expanded the therapeutic landscape for the disease. Although major improvements have been achieved, many patients experience rapid disease progression and low responses in subsequent lines of therapy. Sacituzumab govitecan is an ADC that targets Trop-2, which is highly expressed in urothelial cancers. Promising results in early clinical trials have led to further drug development which confirmed encouraging efficacy. Sacituzumab govitecan has been given accelerated approval in 2021 for patients with locally advanced and metastatic urothelial cancer who previously received a platinum containing chemotherapy and either a programmed death receptor-1 or programmed death ligand inhibitor. The results are promising, with encouraging efficacy and safety, however responses are not universal. There is a growing comprehension of mechanisms of resistance and predictive biomarkers that are crucial to improving outcomes. In this review, we summarize the current knowledge on antibody-drug conjugates and the clinical findings that led to the approval of Sacituzumab govitecan and discuss the therapeutic potential of new combinations, mechanisms of resistance and predictive biomarkers.

Challenges for the development of a universal vaccine against leptospirosis revealed by the evaluation of 22 vaccine candidates.

Leptospirosis is a neglected disease of man and animals that affects nearly half a million people annually and causes considerable economic losses. Current human vaccines are inactivated whole-cell preparations (bacterins) of Leptospira spp. that provide strong homologous protection yet fail to induce a cross-protective immune response. Yearly boosters are required, and serious side-effects are frequently reported so the vaccine is licensed for use in humans in only a handful of countries. Novel universal vaccines require identification of conserved surface-exposed epitopes of leptospiral antigens. Outer membrane ß-barrel proteins (ßb-OMPs) meet these requirements and have been successfully used as vaccines for other diseases. We report the evaluation of 22 constructs containing protein fragments from 33 leptospiral ßb-OMPs, previously identified by reverse and structural vaccinology and cell-surface immunoprecipitation. Three-dimensional structures for each leptospiral ßb-OMP were predicted by I-TASSER. The surface-exposed epitopes were predicted using NetMHCII 2.2 and BepiPred 2.0. Recombinant constructs containing regions from one or more ßb-OMPs were cloned and expressed in Escherichia coli. IMAC-purified recombinant proteins were adsorbed to an aluminium hydroxide adjuvant to produce the vaccine formulations. Hamsters (4-6 weeks old) were vaccinated with 2 doses containing 50 - 125 µg of recombinant protein, with a 14-day interval between doses. Immunoprotection was evaluated in the hamster model of leptospirosis against a homologous challenge (10 - 20× ED50) with L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. Of the vaccine formulations, 20/22 were immunogenic and induced significant humoral immune responses (IgG) prior to challenge. Four constructs induced significant protection (100%, P < 0.001) and sterilizing immunity in two independent experiments, however, this was not reproducible in subsequent evaluations (0 - 33.3% protection, P > 0.05). The lack of reproducibility seen in these challenge experiments and in other reports in the literature, together with the lack of immune correlates and commercially available reagents to characterize the immune response, suggest that the hamster may not be the ideal model for evaluation of leptospirosis vaccines and highlight the need for evaluation of alternative models, such as the mouse.

High occurrence of CRLF2 abnormalities in Mexican children with B-cell acute lymphoblastic leukemia.

The P2RY8-CRLF2 and IGH-CRLF2 rearrangements induce the overexpression of cytokine receptor-like factor 2 (CRLF2) and have been associated with relapse and poor prognosis in B-cell acute lymphoblastic leukemia (B-ALL). Additionally, they are frequently documented in high-risk Hispanic populations. To better understand the potential causes of the adverse prognosis of childhood B-ALL in Mexico, we analyzed these rearrangements and the CRLF2 mRNA and protein levels in 133 Mexican children with B-ALL. We collected bone marrow samples at diagnosis and evaluated the CRLF2 gene expression by qRT-PCR and the total CRLF2 protein by flow cytometry. P2RY8-CRLF2 and IGH-CRLF2 were detected by RT-PCR and FISH, respectively. The median time of follow-up to determine the prognostic significance of the CRLF2 abnormalities was three years. In 82% of the participants, the mRNA levels correlated with the cell-surface and intracellular CRLF2 protein levels. The P2RY8-CRLF2 rearrangement was present in 31.5% (42/133) of the patients, while the IGH-CRLF2 rearrangement was detected in 13.5% (9/67) of patients with high expression of CRLF2 (6.8% of the total sample). CRLF2 copy number variations (gain) were also detected in 7.5% (5/67) of patients with high protein levels. The overall survival (OS) presented significantly lower rates in patients with high white blood cell count (≥50x109/L) regardless of CRLF2 expression, but high levels of CRLF2 gene expression appears to contribute to the reduction of OS within this group of patients. In conclusion, in our cohort, a high occurrence of CRLF2 abnormalities was documented, particularly the P2RY8-CRLF2 rearrangement, which might represent a characteristic of the Mexican population. Targeted therapy to treat this group of patients could improve OS.

Quantum dots conjugated to lectins from Schinus terebinthifolia leaves (SteLL) and Punica granatum sarcotesta (PgTeL) as potential fluorescent nanotools for investigating Cryptococcus neoformans.

This study reports the development of conjugates based on quantum dots (QD)s and lectins from Schinus terebinthifolia leaves (SteLL) and Punica granatum sarcotesta (PgTeL). Cryptococcus neoformans cells were chosen to evaluate the efficiency of the conjugates. Lectins were conjugated to QDs via adsorption, and the optical parameters (emission and absorption) were monitored. Lectin stability in the conjugates towards denaturing agents was investigated via fluorometry. The conjugation was evaluated using fluorescence microplate (FMA) and hemagglutination (HA) assays. The labeling of the C. neoformans cell surface was quantified using flow cytometry and observed via fluorescence microscopy. The QDs-SteLL and QDs-PgTeL conjugates, obtained at pH 7.0 and 8.0, respectively, showed the maintenance of colloidal and optical properties. FMA confirmed the conjugation, and the HA assay indicated that the lectin carbohydrate-binding ability was preserved after conjugation. SteLL and PgTeL showed stability towards high urea concentrations and heating. Conjugates labeled over 90% of C. neoformans cells as observed via flow cytometry and confirmed through fluorescence microscopy. C. neoformans labeling by conjugates was inhibited by glycoproteins, suggesting specific interactions through the lectin carbohydrate-binding site. Thus, an effective protocol for the conjugation of SteLL or PgTeL with QDs was proposed, yielding new nanoprobes useful for glycobiological studies.

T follicular helper cells: Their development and importance in the context of helminthiasis.

The development of T follicular helper cells (Tfh) is a multifactorial process that occurs in multiple stages. After their activation the Tfh cells interact with the B cells to complete their differentiation. During this process, the Tfh cells begin to express canonical molecules such as the transcription factor B-cell lymphoma 6 protein, the CXC chemokine receptors type 5, and the inducible T-cell costimulator, as well as secreting other molecules such as IL-21. This whole process is regulated positively and negatively by several factors so that the best response is offered in the face of diseases of various origins, among them helminthiasis. In this context, the role of circulating Tfh, IL-4 and IgG subtypes is essential for an effective response against these pathogens. In this review, the migration process and the differentiation of Tfh, the regulation, their cell subtypes and the role of Tfh in the context of helminth infections will be addressed.

Method-induced variation in the bacterial cell surface hydrophobicity MATH test.

Bacterial cell surface hydrophobicity is a relevant property in determining the ability of bacteria to adhere to inert surfaces. This property has been measured using the microbial adhesion to hydrocarbon (MATH) test. Several reports in the literature establish the percentage of adhesion to hydrocarbons (PoAtH) value produced by the MATH test for a broad variety of bacteria. Discrepancies in PoAtH values reported for the same strain of a specific microorganism suggest that some method-induced variation may exist, as different research teams employ different versions of the assay. The objective of the present study was to compare the performance of different versions of the MATH test as reported in the literature, to quantify the magnitude of the method-induced variation on PoAtH values. The study demonstrated that PoAtH values are influenced twice as much by variations in the employed assay than by actual differences in cell surface composition or architecture. The two L. reuteri strains studied responded differently to changes in assay conditions showing 40 and 70% method-dependent variation for strain ATCC 53609 and 55730, respectively. These results highlight the need to properly standardize the MATH test to enable comparison of PoAtH values produced by independent research teams.

Investigation of EGFR/pi3k/Akt signaling pathway in seminomas.

Activation of the receptor for epidermal growth factor (EGFR) in some testicular tumors activates several signaling pathways. Some components of these pathways are phosphorylated or mutated in testicular germ tumors (TCGT), including EGFR, Kirstein ras oncogen (KRAS) and cell surface protein of the germ cell (KIT). The latter two activate RAF /MEK/ERK and PI3 K/AKT, and interconnect with the EGFR/pI3 k/Akt pathway. We investigated the expression of EGFR/pI3 k/Akt pathway proteins in seminomas and in their precursor lesion, germinal cell neoplasia in situ (GCNIS) and related genetic mutations. We used immunohistochemistry for pEGFR, pI3 k and pAkt expression with a scoring system for 46 seminoma surgical specimens: 36 classical and 10 GCNIS. In 17 samples, the mutations of EGFR (exons 19 - 21), KIT (exons 11, 17) and KRAS (exons 2, 3) were investigated using qPCR and sequencing. Of the 36 seminomas studied, 22 (61%) expressed pEGFR. Ten samples exhibited high scores for pEGFR, pI3 k and pAkt. In 5 of 17 cases (33%) some mutation was exhibited in the exons studied: 21 of EGFR (2), 17 of EGFR (1), 3 of KRAS (1) and 11 of KIT (1). Six cases exhibited nuclear translocation of EGFR; of these, four exhibited mutations of EGFR, KRAS and KIT. Eight of ten of the GCNIS expressed a high pEGFR score (80%). In 2 of 6 cases (33%), mutation was detected in exon 21 of EGFR and one smear showed EGFR translocation to the nucleus. The translocation represents a subpopulation with worse prognosis for TCGT. The EGFR/pI3 k/Akt signaling pathway is linked to TDRG1, which regulates chemosensitivity to cisplatin; this is a mechanism of resistance to treatment. TDRG1 and the EGFR/pI3 k/pAkt pathway could be therapeutic targets for seminomas resistant to cisplatin.

Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications.

Yeast surface display (YSD) is a "whole-cell" platform used for the heterologous expression of proteins immobilized on the yeast's cell surface. YSD combines the advantages eukaryotic systems offer such as post-translational modifications, correct folding and glycosylation of proteins, with ease of cell culturing and genetic manipulation, and allows of protein immobilization and recovery. Additionally, proteins displayed on the surface of yeast cells may show enhanced stability against changes in temperature, pH, organic solvents, and proteases. This platform has been used to study protein-protein interactions, antibody design and protein engineering. Other applications for YSD include library screening, whole-proteome studies, bioremediation, vaccine and antibiotics development, production of biosensors, ethanol production and biocatalysis. YSD is a promising technology that is not yet optimized for biotechnological applications. This mini review is focused on recent strategies to improve the efficiency and selection of displayed proteins. YSD is presented as a cutting-edge technology for the vectorial expression of proteins and peptides. Finally, recent biotechnological applications are summarized. The different approaches described herein could allow for a better strategy cascade for increasing protein/peptide interaction and production.

Enhancing innate immunity against virus in times of COVID-19: Trying to untangle facts from fictions.

INTRODUCTION: In light of the current COVID-19 pandemic, during which the world is confronted with a new, highly contagious virus that suppresses innate immunity as one of its initial virulence mechanisms, thus escaping from first-line human defense mechanisms, enhancing innate immunity seems a good preventive strategy. METHODS: Without the intention to write an official systematic review, but more to give an overview of possible strategies, in this review article we discuss several interventions that might stimulate innate immunity and thus our defense against (viral) respiratory tract infections. Some of these interventions can also stimulate the adaptive T- and B-cell responses, but our main focus is on the innate part of immunity. We divide the reviewed interventions into: 1) lifestyle related (exercise, >7 h sleep, forest walking, meditation/mindfulness, vitamin supplementation); 2) Non-specific immune stimulants (letting fever advance, bacterial vaccines, probiotics, dialyzable leukocyte extract, pidotimod), and 3) specific vaccines with heterologous effect (BCG vaccine, mumps-measles-rubeola vaccine, etc). RESULTS: For each of these interventions we briefly comment on their definition, possible mechanisms and evidence of clinical efficacy or lack of it, especially focusing on respiratory tract infections, viral infections, and eventually a reduced mortality in severe respiratory infections in the intensive care unit. At the end, a summary table demonstrates the best trials supporting (or not) clinical evidence. CONCLUSION: Several interventions have some degree of evidence for enhancing the innate immune response and thus conveying possible benefit, but specific trials in COVID-19 should be conducted to support solid recommendations.

Strategies to display heterologous proteins on the cell surface of lactic acid bacteria using as anchor the C-terminal domain of Lactobacillus acidophilus SlpA.

The surface-layer (S-layer) protein of Lactobacillus acidophilus is a crystalline array of self-assembling subunits, non-covalently bound to the most outer cell wall envelope, which constitutes up to 20% of the total cell protein content. These attributes make S-layer proteins an excellent anchor for the development of microbial cell-surface display systems. In L. acidophilus, the S-layer is formed predominantly by the protein SlpA. We have previously shown that the C-terminal domain of SlpA is responsible for the cell wall anchorage on L. acidophilus ATCC 4356. In the present study, we evaluated the C-terminal domain of SlpA of L. acidophilus ATCC 4356 as a potential anchor domain to display functional proteins on the surface of non-genetically modified lactic acid bacteria (LAB). To this end, green fluorescent protein (GFP)-CTSlpA was firstly produced in Escherichia coli and the recombinant proteins were able to spontaneously bind to the cell wall of LAB in a binding assay. GFP was successfully displayed on the S-layer stripped surface of L. acidophilus. Both the binding stability and cell survival of L. acidophilus decorated with the recombinant protein were then studied in simulated gastrointestinal conditions. Furthermore, NaCl was tested as a safer alternative to LiCl for S-layer removal. This study presents the development of a protein delivery platform involving L. acidophilus, a microorganism generally regarded as safe, which utilizes the contiguous, non-covalently attached S-layer at the cell surface of the bacterium without introducing any genetic modification.

Differential Expression of IgM and IgD Discriminates Two Subpopulations of Human Circulating IgM+IgD+CD27+ B Cells That Differ Phenotypically, Functionally, and Genetically.

The origin and function of blood IgM+IgD+CD27+ B cells is controversial, and they are considered a heterogeneous population. Previous staining of circulating B cells of healthy donors with rotavirus fluorescent virus-like particles allowed us to differentiate two subsets of IgM+IgD+CD27+: IgMhi and IgMlo B cells. Here, we confirmed this finding and compared the phenotype, transcriptome, in vitro function, and Ig gene repertoire of these two subsets. Eleven markers phenotypically discriminated both subsets (CD1c, CD69, IL21R, CD27, MTG, CD45RB, CD5, CD184, CD23, BAFFR, and CD38) with the IgMhi phenotypically resembling previously reported marginal zone B cells and the IgMlo resembling both naïve and memory B cells. Transcriptomic analysis showed that both subpopulations clustered close to germinal center-experienced IgM only B cells with a Principal Component Analysis, but differed in expression of 78 genes. Moreover, IgMhi B cells expressed genes characteristic of previously reported marginal zone B cells. After stimulation with CpG and cytokines, significantly (p < 0.05) higher frequencies (62.5%) of IgMhi B cells proliferated, compared with IgMlo B cells (35.37%), and differentiated to antibody secreting cells (14.22% for IgMhi and 7.19% for IgMlo). IgMhi B cells had significantly (p < 0.0007) higher frequencies of mutations in IGHV and IGKV regions, IgMlo B cells had higher usage of IGHJ6 genes (p < 0.0001), and both subsets differed in their HCDR3 properties. IgMhi B cells shared most of their shared IGH clonotypes with IgM only memory B cells, and IgMlo B cells with IgMhi B cells. These results support the notion that differential expression of IgM and IgD discriminates two subpopulations of human circulating IgM+IgD+CD27+ B cells, with the IgMhi B cells having similarities with previously described marginal zone B cells that passed through germinal centers, and the IgMlo B cells being the least differentiated amongst the IgM+CD27+ subsets.

Identification of CSP Types and Genotypic Variability of Clinical and Environmental Isolates of Aspergillus fumigatus from Different Geographic Origins.

The CSP (cell surface protein) microsatellite marker is useful for typing Aspergillus fumigatus isolates and determining relationships at the subpopulation level because it has shown high discriminatory power. In the present study, 90 A. fumigatus isolates from Mexico (MX), Argentina (AR), France (FR), and Peru (PE) were identified through a phylogenetic analysis using the benA gene fragment and were typed with the CSP microsatellite, and the types were identified using the nomenclature recommended in the literature. Genetic variability was analyzed through haplotype diversity, nucleotide diversity, polymorphic sites, and nucleotide differences between pairs of sequences. The population structure was evaluated using the Tajima's D statistic. No new CSP types were recorded in the MX, FR, and PE isolates, while in the AR isolates, two new CSP types were identified (t25 and t26). The most common CSP types in the studied populations were t01, t02, t03, and t04A; these results are consistent with findings in other countries. In addition, the genetic diversity parameters we obtained revealed that the greatest genetic diversity was found in the MX population, followed by AR and FR. No population structure was identified among the isolates studied.