Microstructural Changes in Vanilla planifolia Beans after Using High-Hydrostatic-Pressure Treatment in the Curing Process.

During vanilla bean curing, the cell arrangement derived from the killing technique applied to start bean ripening is essential to obtain the characteristic aroma and flavor of vanilla. Hence, killing is an important step to release the enzymes and compounds required for vanillin production. In this work, high hydrostatic pressure (HHP) at 100-400 MPa for 5 min, using water at 7 °C as the pressure-transmitting medium, was applied as the killing method, and its effect on the microstructural changes in vanilla beans during different curing cycles (C0-C20) was evaluated and compared with that observed after scalding by using water at 100 °C for 8 s. Microstructural changes in the cross-sectioned beans were analyzed using a stereomicroscope (SM), confocal laser scanning microscopy (CLSM), and environmental scanning electron microscopy (ESEM). The vanilla beans were cross-sectioned and three main sectors were analyzed: the total, annular, and core. The morphometric descriptors, namely, area, Feret's diameter, and circularity, were quantified via digital image analysis (DIA), from which a shrinkage ratio was calculated. The results show that the total area in the beans presented a maximum decrease in the C16 of curing. The core area was most affected by the HHP treatment, mainly at 400 MPa, rather than scalding. CSLM observations revealed the autofluorescence of the compounds inside the beans. In conclusion, the use of microscopy techniques and DIA allowed us to determine the microstructural changes in the HHP-treated pods, which were found to be more numerous than those found in the scalded beans.

Primary explants of the postnatal thymus allow the expansion of clonogenic thymic epithelial cells that constitute thymospheres.

BACKGROUND: Thymic epithelial cells (TECs) are responsible for shaping the repertoires of T cells, where their postnatal regeneration depends on a subset of clonogenic TECs. Despite the implications for regenerative medicine, their cultivation and expansion remain challenging. Primary explant cell culture is a technique that allows the seeding and expansion of difficult-to-culture cells. Here, we report a reliable and simple culture system to obtain functional TECs and thymic interstitial cells (TICs). METHODS: To establish primary thymic explants, we harvested 1 mm cleaned fragments of thymus from 5-week-old C57/BL6 mice. Tissue fragments of a complete thymic lobe were placed in the center of a Petri dish with 1 mL of DMEM/F-12 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin‒streptomycin. To compare, thymic explants were also cultivated by using serum-free DMEM/F-12 medium supplemented with 10% KnockOut™. RESULTS: We obtained high numbers of functional clonogenic TECs and TICs from primary thymic explants cultivated with DMEM/F-12 with 20% FBS. These cells exhibited a highly proliferative and migration profile and were able to constitute thymospheres. Furthermore, all the subtypes of medullary TECs were identified in this system. They express functional markers to shape T-cell and type 2 innate lymphoid cells repertoires, such as Aire, IL25, CCL21 and CD80. Finally, we also found that ≥ 70% of lineage negative TICs expressed high amounts of Aire and IL25. CONCLUSION: Thymic explants are an efficient method to obtain functional clonogenic TECs, all mTEC subsets and different TICs Aire+IL25+ with high regenerative capacity.

HP0953 - hypothetical virulence factor overexpresion and localization during Helicobacter pylori infection of gastric epithelium.

BACKGROUND: The high prevalence and persistence of Helicobacter pylori (H. pylori) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are varied. Moreover, as its proteome contains 340 hypothetical proteins, it is important to investigate them to completely understand the mechanisms of its virulence and survival. We have previously reported that the hypothetical protein HP0953 is overexpressed during the first hours of adhesion to inert surfaces, under stress conditions, suggesting its role in the environmental survival of this bacterium and perhaps as a virulence factor. AIM: To investigate the expression and localization of HP0953 during adhesion to an inert surface and against gastric (AGS) cells. METHODS: Expression analysis was performed for HP0953 during H. pylori adhesion. HP0953 expression at 0, 3, 12, 24, and 48 h was evaluated and compared using the Kruskal-Wallis equality-of-populations rank test. Recombinant protein was produced and used to obtain polyclonal antibodies for immunolocalization. Immunogold technique was performed on bacterial sections during adherence to inert surfaces and AGS cells, which was analyzed by transmission electron microscopy. HP0953 protein sequence was analyzed to predict the presence of a signal peptide and transmembrane helices, both provided by the ExPASy platform, and using the GLYCOPP platform for glycosylation sites. Different programs, via, I-TASSER, RaptorX, and HHalign-Kbest, were used to perform three-dimensional modeling. RESULTS: HP0953 exhibited its maximum expression at 12 h of infection in gastric epithelium cells. Immunogold technique revealed HP0953 localization in the cytoplasm and accumulation in some peripheral areas of the bacterial body, with greater expression when it is close to AGS cells. Bioinformatics analysis revealed the presence of a signal peptide that interacts with the transmembrane region and then allows the release of the protein to the external environment. The programs also showed a similarity with the Tip-alpha protein of H. pylori. Tip-alpha is an exotoxin that penetrates cells and induces tumor necrosis factor alpha production, and HP0953 could have a similar function as posttranslational modification sites were found; modifications in turn require enzymes located in eukaryotic cells. Thus, to be functional, HP0953 may necessarily need to be translocated inside the cell where it can trigger different mechanisms producing cellular damage. CONCLUSION: The location of HP0953 around infected cells, the probable posttranslational modifications, and its similarity to an exotoxin suggest that this protein is a virulence factor.

Mitochondrial Ultrastructure and Activity Are Differentially Regulated by Glycolysis-, Krebs Cycle-, and Microbiota-Derived Metabolites in Monocytes.

Several intermediate metabolites harbour cell-signalling properties, thus, it is likely that specific metabolites enable the communication between neighbouring cells, as well as between host cells with the microbiota, pathogens, and tumour cells. Mitochondria, a source of intermediate metabolites, participate in a wide array of biological processes beyond that of ATP production, such as intracellular calcium homeostasis, cell signalling, apoptosis, regulation of immune responses, and host cell-microbiota crosstalk. In this regard, mitochondria's plasticity allows them to adapt their bioenergetics status to intra- and extra-cellular cues, and the mechanisms driving such plasticity are currently a matter of intensive research. Here, we addressed whether mitochondrial ultrastructure and activity are differentially shaped when human monocytes are exposed to an exogenous source of lactate (derived from glycolysis), succinate, and fumarate (Krebs cycle metabolic intermediates), or butyrate and acetate (short-chain fatty acids produced by intestinal microbiota). It has previously been shown that fumarate induces mitochondrial fusion, increases the mitochondrial membrane potential (Δψm), and reshapes the mitochondrial cristae ultrastructure. Here, we provide evidence that, in contrast to fumarate, lactate, succinate, and butyrate induce mitochondrial fission, while acetate induces mitochondrial swelling. These traits, along with mitochondrial calcium influx kinetics and glycolytic vs. mitochondrial ATP-production rates, suggest that these metabolites differentially shape mitochondrial function, paving the way for the understanding of metabolite-induced metabolic reprogramming of monocytes and its possible use for immune-response intervention.

Differential response of immobile (pneumocytes) and mobile (monocytes) barriers against 2 types of metal oxide nanoparticles.

BACKGROUND: Inhaled nanoparticles (NPs) challenges mobile and immobile barriers in the respiratory tract, which can be represented by type II pneumocytes (immobile) and monocytes (mobile) but what is more important for biological effects, the cell linage, or the type of nanoparticle? Here, we addressed these questions and we demonstrated that the type of NPs exerts a higher influence on biological effects, but cell linages also respond differently against similar type of NPs. DESIGN: Type II pneumocytes and monocytes were exposed to tin dioxide (SnO2) NPs and titanium dioxide (TiO2) NPs (1, 10 and 50 µg/cm2) for 24 h and cell viability, ultrastructure, cell granularity, molecular spectra of lipids, proteins and nucleic acids and cytoskeleton architecture were evaluated. RESULTS: SnO2 NPs and TiO2 NPs are metal oxides with similar physicochemical properties. However, in the absence of cytotoxicity, SnO2 NPs uptake was low in monocytes and higher in type II pneumocytes, while TiO2 NPs were highly internalized by both types of cells. Monocytes exposed to both types of NPs displayed higher number of alterations in the molecular patterns of proteins and nuclei acids analyzed by Fourier-transform infrared spectroscopy (FTIR) than type II pneumocytes. In addition, cells exposed to TiO2 NPs showed more displacements in FTIR spectra of biomolecules than cells exposed to SnO2 NPs. Regarding cell architecture, microtubules were stable in type II pneumocytes exposed to both types of NPs but actin filaments displayed a higher number of alterations in type II pneumocytes and monocytes exposed to SnO2 NPs and TiO2 NPs. NPs exposure induced the formation of large vacuoles only in monocytes, which were not seen in type II pneumocytes. CONCLUSIONS: Most of the cellular effects are influenced by the NPs exposure rather than by the cell type. However, mobile, and immobile barriers in the respiratory tract displayed differential response against SnO2 NPs and TiO2 NPs in absence of cytotoxicity, in which monocytes were more susceptible than type II pneumocytes to NPs exposure.

Autophagosomes accumulation in the vacuoles of the fungus Ustilago maydis and the role of proteases in their digestion.

In the present study we determined whether Ustilago maydis accumulates autophagosomes within vacuoles when the cells are exposed to nutritional stress conditions. We investigated whether proteinase B and proteinase A are involved in their degradation. To this effect, wild type and Δpep4 mutant were incubated in minimal medium lacking a carbon source. It was observed that after incubation in nutrient-deficient media, spherical bodies appeared within the Δpep4 mutant strains vacuoles. In addition, autophagosomes were accumulated in U. maydis WT cells incubated in the presence of the serine protease inhibitor PMSF and accumulation of large autophagosomes and electrodense structures in the Δpep4 mutant cell vacuoles took place. These results demonstrate that the homologues of both, the proteinase B and the protease A, are involved in the autophagosomes degradation process in U. maydis.

Potential use of Bacillus genera for metals removal from spent catalysts.

The aim of the present study was to isolate microorganisms able to tolerate Ni2+ and V5+ from different sites located close to a mineral mine in Guanajuato, Mexico, and then to evaluate their ability to remove metals contained in a spent catalyst. Seventeen isolates were obtained; among them seven presented a minimum inhibitory concentration (MIC) higher than 200 mg/L of Ni2+ and V5+ each. Nickel and Vanadium removal was evaluated in 9 K liquid medium added with spent catalyst at 16% (s/v) pulp density and incubated at 30 °C, 150 rpm for 7 days. Only three isolates which were coded as PRGSd-MS-2, MNSH2-AH-3, and MNSS-AH-4 showed a significant removal at the end of treatment corresponding in mg kg-1 (or percentage metal removal) of 138 (32%), 123 (29%), and 101 (24%) for Ni, respectively; and 557 (26%), 737 (34%), and 456 (21%) mg kg-1 for V, respectively. The same isolates were capable to remove also Al, Fe, As, and Mg at different extent. Cell morphology changes were observed, in comparison to the control system at the end of biological treatment as a higher quantity of spores for MNSH2-AH-3, 2 µm cells in pairs for MNSS-AH-4, also long chain-vegetative cells having inclusions into the cell surface were observed for PRGSd-MS-2. The three isolated microorganisms were identified by sequencing of the 16S gene as Bacillus thuringiensis, Bacillus megaterium, and Bacillus sp, respectively, suggesting its potential use in the treatment of this solid industrial waste.

In Vitro Analyses Reveal the Effect of Synthetic Cytokinin Forchlorfenuron (FCF) on a Septin-Like Protein of Taeniid Cysticerci.

Cytokinin forchlorfenuron (FCF), a synthetic cytokinin, has been used specifically for the characterization of septins. In spite of genomic evidence of their existence, nothing is known about septin filaments in taeniid cestodes. The aim of this work was to determine the presence of a septin-like protein in cysticerci of Taenia crassiceps and Taenia solium using the deduced amino acid sequence of T. solium septin 4 (SEPT4_Tsm), to design and synthesize a derived immunogenic peptide (residues 88 to 103), to prepare a specific rabbit polyclonal antibody, and to examine the effects of FCF at different concentrations and exposure times on an in vitro culture of T. crassiceps cysticerci. In vitro, FCF altered the morphology and motility of T. crassiceps cysticerci, and its effects were reversible under specific concentrations. In addition, we observed by ultrastructural observation that FCF alters the cellular subunit of the protonephridial system of cestodes, where disruption of the axoneme pattern of flame cells was observed. The rabbit polyclonal antibody prepared against the synthetic peptide recognized a major band of 41 kDa in both parasites. Our results establish the importance of SEPT4_Tsm in the dynamics and survival of taeniid cysticerci, as well as their susceptibility to FCF. This is also the first report that a septin is present in the cytoskeleton of taeniids.

Influence of shape and dispersion media of titanium dioxide nanostructures on microvessel network and ossification.

Titanium dioxide nanoparticles (TiO2 NPs) production has been used for pigment, food and cosmetic industry and more recently, shaped as belts for treatment of contaminated water, self-cleaning windows and biomedical applications. However, the toxicological data have demonstrated that TiO2 NPs inhalation induce inflammation in in vivo models and in vitro exposure leads to cytotoxicity and DNA damage. Dermal exposure has limited adverse effects and the possible risks for implants used for tissue regeneration is still under research. Then, it has been difficult to establish a straight statement about TiO2 NPs toxicity since route of exposure and shapes of nanoparticles play an important role in the effects. In this study we aimed to investigate the effect of three different types of TiO2 NPs (industrial, food-grade and belts) dispersed in fetal bovine serum (FBS) and saline solution (SS) on microvessel network, angiogenesis gene expression and femur ossification using a chick embryo model after an acute exposure of NPs on the day 7 after eggs fertilization. Microvascular density of chorioallantoic membrane (CAM) was analyzed after 7days of NPs injection and vehicles induced biological effects per se. NPs dispersed in FBS or SS have slight differences in microvascular density, mainly opposite effect on angiogenesis gene expression and no effects on femur ossification for NPs dispersed in SS. Interestingly, NPs shaped as belts dramatically prevented the alterations in ossification induced by FBS used as vehicle.

Adherence of Ornithobacterium rhinotracheale to chicken embryo lung cells as a pathogenic mechanism.

Ornithobacterium rhinotracheale is a bacterium that causes respiratory disease in birds and it has been isolated in countries with a large poultry production, including Mexico. The pathogenicity mechanisms of this bacterium have not been completely elucidated yet. The capacity of the bacterium to adhere to epithelial cells of chicken in vitro has been evidenced, and since this bacterium has been isolated from the lungs and air sacs of several avian species, the aim of this study was to determine if this bacterium can adhere to chicken lung cells. We used five O. rhinotracheale reference serovars (A-E) that were in contact with primary lung cells cultured from a 19-day-old chicken embryo. O. rhinotracheale adherence was evaluated through optical and transmission electron microscopies. The results revealed that O. rhinotracheale is capable of adhering to chicken embryo lung cells within 3 h of incubation with a diffuse adherence pattern. The adherence percentages of the chicken embryo lung cells were 51-96% according to the serovar of the bacterium. Relative adherence was from 4 to 8 bacteria per cell. Transmission electron microscope data revealed intracellular bacteria inside a vacuole in less than 3 h of incubation.

Corrigendum: Effects of lng Mutations on LngA Expression, Processing, and CS21 Assembly in Enterotoxigenic Escherichia coli E9034A.

[This corrects the article on p. 1201 in vol. 7, PMID: 27536289.].

Effects of lng Mutations on LngA Expression, Processing, and CS21 Assembly in Enterotoxigenic Escherichia coli E9034A.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity in children under 5 years of age in low- and middle-income countries and a leading cause of traveler's diarrhea worldwide. The ability of ETEC to colonize the intestinal epithelium is mediated by fimbrial adhesins, such as CS21 (Longus). This adhesin is a type IVb pilus involved in adherence to intestinal cells in vitro and bacterial self-aggregation. Fourteen open reading frames have been proposed to be involved in CS21 assembly, hitherto only the lngA and lngB genes, coding for the major (LngA) and minor (LngB) structural subunit, have been characterized. In this study, we investigated the role of the LngA, LngB, LngC, LngD, LngH, and LngP proteins in the assembly of CS21 in ETEC strain E9034A. The deletion of the lngA, lngB, lngC, lngD, lngH, or lngP genes, abolished CS21 assembly in ETEC strain E9034A and the adherence to HT-29 cells was reduced 90%, compared to wild-type strain. Subcellular localization prediction of CS21 proteins was similar to other well-known type IV pili homologs. We showed that LngP is the prepilin peptidase of LngA, and that ETEC strain E9034A has another peptidase capable of processing LngA, although with less efficiency. Additionally, we present immuno-electron microscopy images to show that the LngB protein could be localized at the tip of CS21. In conclusion, our results demonstrate that the LngA, LngB, LngC, LngD, LngH, and LngP proteins are essential for CS21 assembly, as well as for bacterial aggregation and adherence to HT-29 cells.

Haemophilus influenzae triggers autophagy in HEp-2 cells.

The MAP-LC3 system regulates the intracellular formation of autophagy-associated vacuoles. These vacuoles contain the LC3 protein; thus it has been utilized as a marker to identify autophagosomes. The aim of our study was to investigate whether Haemophilus influenzae strains and their supernatants could activate autophagy in human larynx carcinoma cell line (HEp-2). We demonstrate that higher expression of the LC3B-II protein was induced, particularly by nontypeable Haemophilus influenzae (NTHi) 49766 and by supernatants, containing <50 kDa proteins, of both strains. Ultrastructural studies demonstrate vacuoles with a double membrane and/or membrane material inside, showing similar features to those of autophagic vacuoles. Together, our findings demonstrate that H. influenzae strains and their supernatants trigger an autophagic process.

Conditions that induce biofilm production by Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale (ORT) is a Gram-negative bacillus that causes respiratory disease in birds, and directly affects the poultry industry. The mechanisms behind these infections are not completely known. Currently, its capacity to form biofilms on inert surfaces has been reported; however, the conditions for biofilm development have not been described yet. The present work was aimed at identifying the conditions that enhance in vitro biofilm formation and development by ORT. For this, serovars A-E were analysed to assess their ability to induce biofilm development on 96-well flat-bottom polystyrene microtitre plates under diverse conditions: temperature, incubation time, and CO2 concentration. The results obtained showed not only that all serovars have the ability to produce in vitro biofilms, but also that the optimal conditions for biofilm density were 40°C after 72 h at an elevated CO2 concentration. In conclusion, ORT biofilm formation depends on the environmental conditions and may contribute to the persistence of this microorganism.

Vancomycin tolerant, methicillin-resistant Staphylococcus aureus reveals the effects of vancomycin on cell wall thickening.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important opportunistic pathogen that causes both healthcare- and community-acquired infections. An increase in the incidence of these infections may lead to a substantial change in the rate of vancomycin usage. Incidence of reduced susceptibility to vancomycin has been increasing worldwide for the last few years, conferring different levels of resistance to vancomycin as well as producing changes in the cell wall structure. The aim of the present study was to determine the effect of vancomycin on cell wall thickening in clinical isolates of vancomycin-tolerant (VT) MRSA obtained from pediatric patients. From a collection of 100 MRSA clinical isolates from pediatric patients, 12% (12/100) were characterized as VT-MRSA, and from them, 41.66% (5/12) exhibited the heterogeneous vancomycin-intermediate S. aureus (hVISA) phenotype. Multiplex-PCR assays revealed 66.66% (8/12), 25% (3/12), and 8.33% (1/12) of the VT-MRSA isolates were associated with agr group II, I, and III polymorphisms, respectively; the II-mec gene was amplified from 83.3% (10/12) of the isolates, and the mecIVa gene was amplified from 16.66% (2/12) of the isolates. Pulsed field electrophoresis (PFGE) fingerprint analysis showed 62% similarity among the VT-MRSA isolates. Thin transverse sections analyzed by transmission electron microscopy (TEM) revealed an average increase of 24 nm (105.55%) in the cell wall thickness of VT-MRSA compared with untreated VT-MRSA isolates. In summary, these data revealed that the thickened cell walls of VT-MRSA clinical isolates with agr type II and SCCmec group II polymorphisms are associated with an adaptive resistance to vancomycin.

Use of antifungal saponin SC-2 of Solanum chrysotrichum for the treatment of vulvovaginal candidiasis: in vitro studies and clinical experiences.

Saponin SC-2 from Solanum chrysotrichum showed antifungal activity, demonstrated in vitro, which inhibited the growth of dermatophytes, and in vivo, to be effective in the treatment against tinea pedis and pityriasis capitis. Fungistatic and fungicidal activity of saponin SC-2 on Candida albicans and other Candida species, fluconazole and ketoconazole resistaent strains was demostrated. SC-2-associated ultrastructural alterations in several Candida species were observed. An exploratory clinical, randomized, double-blind, and controlled ketoconazole study of ketoconazole was conducted with the aim of assessing the effectiveness and tolerability of an herbal medicinal product containing SC-2, on women with Vulvovaginal candidiasis (VVC). The results exhibited a percentage of therapeutic clinical effectiveness similar to that of ketoconazole (X(2), p ≥0.30), but obtained a smaller percentage of mycological effectiveness, and 100% tolerability. In conclusion, saponin SC-2 possesses fungicidale and fungistatic activity on Candida albicans and other multi resistant Candida species, causes morphological changes and fungal death, and it is an alternative therapy for the treatment of VVC.

Characterization of a novel Bacillus thuringiensis phenotype possessing multiple appendages attached to a parasporal body.

Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the Caribbean that produces a spore-crystal complex containing six long appendages, each comprised of numerous thinner filaments approximately 10 nm in diameter and 2.5 µm in length. Each of the multi-filament appendages was attached to a single, small parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages.