Highly Sensitive Whole-Cell Mercury Biosensors for Environmental Monitoring.

Whole-cell biosensors could serve as eco-friendly and cost-effective alternatives for detecting potentially toxic bioavailable heavy metals in aquatic environments. However, they often fail to meet practical requirements due to an insufficient limit of detection (LOD) and high background noise. In this study, we designed a synthetic genetic circuit specifically tailored for detecting ionic mercury, which we applied to environmental samples collected from artisanal gold mining sites in Peru. We developed two distinct versions of the biosensor, each utilizing a different reporter protein: a fluorescent biosensor (Mer-RFP) and a colorimetric biosensor (Mer-Blue). Mer-RFP enabled real-time monitoring of the culture's response to mercury samples using a plate reader, whereas Mer-Blue was analysed for colour accumulation at the endpoint using a specially designed, low-cost camera setup for harvested cell pellets. Both biosensors exhibited negligible baseline expression of their respective reporter proteins and responded specifically to HgBr2 in pure water. Mer-RFP demonstrated a linear detection range from 1 nM to 1 µM, whereas Mer-Blue showed a linear range from 2 nM to 125 nM. Our biosensors successfully detected a high concentration of ionic mercury in the reaction bucket where artisanal miners produce a mercury-gold amalgam. However, they did not detect ionic mercury in the water from active mining ponds, indicating a concentration lower than 3.2 nM Hg2+-a result consistent with chemical analysis quantitation. Furthermore, we discuss the potential of Mer-Blue as a practical and affordable monitoring tool, highlighting its stability, reliance on simple visual colorimetry, and the possibility of sensitivity expansion to organic mercury.

Ouabain's Influence on TRPV4 Channels of Epithelial Cells: An Exploration of TRPV4 Activity, Expression, and Signaling Pathways.

Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However, its precise effects on the body remain largely unknown. Previous studies have shown that ouabain can influence the phenotype of epithelial cells by affecting the expression of cell-cell molecular components and voltage-gated potassium channels. In this study, we conducted whole-cell clamp assays to determine whether ouabain affects the activity and/or expression of TRPV4 channels. Our findings indicate that ouabain has a statistically significant effect on the density of TRPV4 currents (dITRPV4), with an EC50 of 1.89 nM. Regarding treatment duration, dITRPV4 reaches its peak at around 1 h, followed by a subsequent decline and then a resurgence after 6 h, suggesting a short-term modulatory effect related to on TRPV4 channel activity and a long-term effect related to the promotion of synthesis of new TRPV4 channel units. The enhancement of dITRPV4 induced by ouabain was significantly lower in cells seeded at low density than in cells in a confluent monolayer, indicating that the action of ouabain depends on intercellular contacts. Furthermore, the fact that U73122 and neomycin suppress the effect caused by ouabain in the short term suggests that the short-term induced enhancement of dITRPV4 is due to the depletion of PIP2 stores. In contrast, the fact that the long-term effect is inhibited by PP2, wortmannin, PD, FR18, and IKK16 suggests that cSrc, PI3K, Erk1/2, and NF-kB are among the components included in the signaling pathways.

Design of a Whole-Cell Biosensor Based on Bacillus subtilis Spores and the Green Fluorescent Protein To Monitor Arsenic.

A green fluorescent protein (GFP)-based whole-cell biosensor (WCB-GFP) for monitoring arsenic (As) was developed in Bacillus subtilis. To this end, we designed a reporter gene fusion carrying the gfpmut3a gene under the control of the promoter/operator region of the arsenic operon (Pars::gfpmut3a) in the extrachromosomal plasmid pAD123. This construct was transformed into B. subtilis 168, and the resultant strain was used as a whole-cell biosensor (BsWCB-GFP) for the detection of As. The BsWCB-GFP was specifically activated by inorganic As(III) and As(V), but not by dimethylarsinic acid [DMA(V)], and exhibited high tolerance to the noxious effects of arsenic. Accordingly, after 12 h exposure, B. subtilis cells carrying the Pars::gfpmut3a fusion exhibited 50 and 90% lethal doses (LD50 and LD90) to As(III) of 0.89 mM and As 1.71 mM, respectively. Notably, dormant spores from the BsWCB-GFP were able to report the presence of As(III) in a concentration range from 0.1 to 1,000 µM 4 h after the onset of germination. In summary, the specificity and high sensitivity for As, as well as its ability to proliferate under concentrations of the metal that are considered toxic in water and soil, makes the B. subtilis biosensor developed here a potentially important tool for monitoring environmental samples contaminated with this pollutant. IMPORTANCE Arsenic (As) contamination of groundwater is associated with serious worldwide health risks. Detection of this pollutant at concentrations that are established as permissible for water consumption by WHO is a matter of significant interest. Here, we report the generation of a whole-cell biosensor for As detection in the Gram-positive spore former B. subtilis. This biosensor reports the presence of inorganic As, activating the expression of the green fluorescent protein (GFP) under the control of the promoter/operator of the ars operon. The biosensor can proliferate under concentrations of As(III) that are considered toxic in water and soil and detect this ion at concentrations as low as 0.1 µM. Of note, spores of the Pars-GFP biosensor exhibited the ability to detect As(III) following germination and outgrowth. Therefore, this novel tool has the potential to be directly applied to monitor As contamination in environmental samples.

Loud noise-exposure changes the firing frequency of subtypes of layer 5 pyramidal neurons and Martinotti cells in the mouse auditory cortex.

Introduction: Loud noise-exposure can generate noise-induced tinnitus in both humans and animals. Imaging and in vivo studies show that noise exposure affects the auditory cortex; however, cellular mechanisms of tinnitus generation are unclear. Methods: Here we compare membrane properties of layer 5 (L5) pyramidal cells (PCs) and Martinotti cells expressing the cholinergic receptor nicotinic alpha 2 subunit gene (Chrna2) of the primary auditory cortex (A1) from control and noise-exposed (4-18 kHz, 90 dB, 1.5 h, followed by 1.5 h silence) 5-8 week old mice. PCs were furthermore classified in type A or type B based on electrophysiological membrane properties, and a logistic regression model predicting that afterhyperpolarization (AHP) and afterdepolarization (ADP) are sufficient to predict cell type, and these features are preserved after noise trauma. Results: One week after a loud noise-exposure no passive membrane properties of type A or B PCs were altered but principal component analysis showed greater separation between type A PCs from control and noise-exposed mice. When comparing individual firing properties, noise exposure differentially affected type A and B PC firing frequency in response to depolarizing current steps. Specifically, type A PCs decreased initial firing frequency in response to +200 pA steps (p = 0.020) as well as decreased steady state firing frequency (p = 0.050) while type B PCs, on the contrary, significantly increased steady state firing frequency (p = 0.048) in response to a + 150 pA step 1 week after noise exposure. In addition, L5 Martinotti cells showed a more hyperpolarized resting membrane potential (p = 0.04), higher rheobase (p = 0.008) and an increased initial (p = 8.5 × 10-5) and steady state firing frequency (p = 6.3 × 10-5) in slices from noise-exposed mice compared to control. Discussion: These results show that loud noise can cause distinct effects on type A and B L5 PCs and inhibitory Martinotti cells of the primary auditory cortex 1 week following noise exposure. As the L5 comprises PCs that send feedback to other areas, loud noise exposure appears to alter levels of activity of the descending and contralateral auditory system.

Using an Aluminum Hydroxide-Chitosan Matrix Increased the Vaccine Potential and Immune Response of Mice against Multi-Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, immobile, aerobic nosocomial opportunistic coccobacillus that causes pneumonia, septicemia, and urinary tract infections in immunosuppressed patients. There are no commercially available alternative antimicrobials, and multi-drug resistance is an urgent concern that requires emergency measures and new therapeutic strategies. This study evaluated a multi-drug-resistant A. baumannii whole-cell vaccine, inactivated and adsorbed on an aluminum hydroxide-chitosan (mAhC) matrix, in an A. baumannii sepsis model in immunosuppressed mice by cyclophosphamide (CY). CY-treated mice were divided into immunized, non-immunized, and adjuvant-inoculated groups. Three vaccine doses were given at 0D, 14D, and 28D, followed by a lethal dose of 4.0 × 108 CFU/mL of A. baumannii. Immunized CY-treated mice underwent a significant humoral response, with the highest IgG levels and a higher survival rate (85%); this differed from the non-immunized CY-treated mice, none of whom survived (p < 0.001), and from the adjuvant group, with 45% survival (p < 0.05). Histological data revealed the evident expansion of white spleen pulp from immunized CY-treated mice, whereas, in non-immunized and adjuvanted CY-treated mice, there was more significant organ tissue damage. Our results confirmed the proof-of-concept of the immune response and vaccine protection in a sepsis model in CY-treated mice, contributing to the advancement of new alternatives for protection against A. baumannii infections.

Standardization and Key Aspects of the Development of Whole Yeast Cell Vaccines.

In the context of vaccine development, improving antigenic presentation is critical for the activation of specific immune responses and the success of immunization, in addition to selecting an appropriate target. In this sense, different strategies have been developed and improved. Among them is the use of yeast cells as vehicles for the delivery of recombinant antigens. These vaccines, named whole yeast vaccines (WYVs), can induce humoral and cellular immune responses, with the additional advantage of dispensing with the use of adjuvants due to the immunostimulatory properties of their cell wall components. However, there are some gaps in the methodologies for obtaining and validating recombinant strains and vaccine formulations. The standardization of these parameters is an important factor for WYVs approval by regulatory agencies and, consequently, their licensing. This review aimed to provide an overview of the main parameters to consider when developing a yeast-based vaccine, addressing some available tools, and highlighting the main variables that can influence the vaccine production process.

An expert panel opinion on protection against pertussis in Mexico: The role of acellular and whole-cell vaccines.

Pertussis is a highly contagious disease caused by Bordetella pertussis, which may be preventable by vaccination. There are two types of vaccines: whole-cell vaccines and acellular vaccines. Since pertussis control worldwide is heterogeneous, re-emergence of whooping cough has been observed in some countries. This re-emergence has been related to several factors: increased susceptibility to infection, better detection of disease, problems in obtaining adequate vaccination coverage, increase in susceptible subjects (mainly under 6 months of age), loss of immunity in adolescents and young adults, and likely genetic and adaptive B. pertussis changes. This paper discusses whole-cell and acellular vaccines' characteristics, advantages, and disadvantages. International recommendations are presented, and the participants' position is offered regarding the influence of the use of acellular vaccines and the potential disadvantages of reintroducing whole-cell vaccines, mainly due to their reactogenicity. Finally, strategies to achieve better control of pertussis in Mexico are discussed.

La tos ferina es una enfermedad causada por Bordetella pertussis. Aunque es altamente contagiosa, puede ser prevenible por vacunación. Existen dos tipos de vacunas: las de células enteras y las acelulares. La tos ferina ha resurgido en algunos países debido a que su control a escala mundial es heterogéneo. Esta reemergencia se ha relacionado con diversos factores: mayor sensibilidad hacia la infección, mejor detección de la enfermedad, problemas para obtener adecuadas coberturas de vacunación, incremento en los sujetos susceptibles (especialmente menores de 6 meses), pérdida de la inmunidad en los adolescentes y adultos jóvenes, y probables cambios genéticos y adaptativos de B. pertussis. En este documento se analizan las características, las ventajas y las desventajas de las vacunas de células enteras y de las vacunas acelulares. Se presentan las recomendaciones internacionales y se ofrece el posicionamiento de los participantes con respecto a la influencia del uso de vacunas acelulares y las desventajas potenciales de volver a utilizar vacunas de células enteras, en especial por su reactogenicidad. Por último, se analizan las estrategias para lograr un mejor control de la tos ferina en México.

Successive Fermentation of Aguamiel and Molasses by Aspergillus oryzae and Saccharomyces cerevisiae to Obtain High Purity Fructooligosaccharides.

Fructooligosaccharides (FOS) are usually synthesized with pure enzymes using highly concentrated sucrose solutions. In this work, low-cost aguamiel and molasses were explored as sucrose alternatives to produce FOS, via whole-cell fermentation, with an Aspergillus oryzae DIA-MF strain. FOS production process was optimized through a central composite experimental design, with two independent variables: initial sucrose concentration in a medium composed of aguamiel and molasses (AgMe), and inoculum concentration. The optimized process-165 g/L initial sucrose in AgMe (adjusted with concentrated molasses) and 1 × 107 spores/mL inoculum concentration-resulted in an FOS production of 119 ± 12 g/L and a yield of 0.64 ± 0.05 g FOS/g GFi. Among the FOSs produced were kestose, nystose, 1-fructofuranosyl-nystose, and potentially a novel trisaccharide produced by this strain. To reduce the content of mono- and disaccharides in the mixture, run a successive fermentation was run with two Saccharomyces cerevisiae strains. Fermentations run with S. cerevisiae S227 improved FOS purity in the mixture from 39 ± 3% to 61.0 ± 0.6% (w/w) after 16 h of fermentation. This study showed that agro-industrial wastes such as molasses with aguamiel are excellent alternatives as substrate sources for the production of prebiotic FOS, resulting in a lower-cost process.

Opinión de un panel de expertos sobre la protección contra la tos ferina en México: el papel de las vacunas acelulares y de células enteras / An expert panel opinion on protection against pertussis in Mexico: The role of acellular and whole-cell vaccines

Resumen La tos ferina es una enfermedad causada por Bordetella pertussis. Aunque es altamente contagiosa, puede ser prevenible por vacunación. Existen dos tipos de vacunas: las de células enteras y las acelulares. La tos ferina ha resurgido en algunos países debido a que su control a escala mundial es heterogéneo. Esta reemergencia se ha relacionado con diversos factores: mayor sensibilidad hacia la infección, mejor detección de la enfermedad, problemas para obtener adecuadas coberturas de vacunación, incremento en los sujetos susceptibles (especialmente menores de 6 meses), pérdida de la inmunidad en los adolescentes y adultos jóvenes, y probables cambios genéticos y adaptativos de B. pertussis. En este documento se analizan las características, las ventajas y las desventajas de las vacunas de células enteras y de las vacunas acelulares. Se presentan las recomendaciones internacionales y se ofrece el posicionamiento de los participantes con respecto a la influencia del uso de vacunas acelulares y las desventajas potenciales de volver a utilizar vacunas de células enteras, en especial por su reactogenicidad. Por último, se analizan las estrategias para lograr un mejor control de la tos ferina en México.

Abstract Pertussis is a highly contagious disease caused by Bordetella pertussis, which may be preventable by vaccination. There are two types of vaccines: whole-cell vaccines and acellular vaccines. Since pertussis control worldwide is heterogeneous, re-emergence of whooping cough has been observed in some countries. This re-emergence has been related to several factors: increased susceptibility to infection, better detection of disease, problems in obtaining adequate vaccination coverage, increase in susceptible subjects (mainly under 6 months of age), loss of immunity in adolescents and young adults, and likely genetic and adaptive B. pertussis changes. This paper discusses whole-cell and acellular vaccines' characteristics, advantages, and disadvantages. International recommendations are presented, and the participants' position is offered regarding the influence of the use of acellular vaccines and the potential disadvantages of reintroducing whole-cell vaccines, mainly due to their reactogenicity. Finally, strategies to achieve better control of pertussis in Mexico are discussed.

Synthetic Biology Tools for Engineering Microbial Cells to Fight Superbugs.

With the increase in clinical cases of bacterial infections with multiple antibiotic resistance, the world has entered a health crisis. Overuse, inappropriate prescribing, and lack of innovation of antibiotics have contributed to the surge of microorganisms that can overcome traditional antimicrobial treatments. In 2017, the World Health Organization published a list of pathogenic bacteria, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli (ESKAPE). These bacteria can adapt to multiple antibiotics and transfer their resistance to other organisms; therefore, studies to find new therapeutic strategies are needed. One of these strategies is synthetic biology geared toward developing new antimicrobial therapies. Synthetic biology is founded on a solid and well-established theoretical framework that provides tools for conceptualizing, designing, and constructing synthetic biological systems. Recent developments in synthetic biology provide tools for engineering synthetic control systems in microbial cells. Applying protein engineering, DNA synthesis, and in silico design allows building metabolic pathways and biological circuits to control cellular behavior. Thus, synthetic biology advances have permitted the construction of communication systems between microorganisms where exogenous molecules can control specific population behaviors, induce intracellular signaling, and establish co-dependent networks of microorganisms.

Pneumococcal Vaccines: Past Findings, Present Work, and Future Strategies.

The importance of Streptococcus pneumoniae has been well established. These bacteria can colonize infants and adults without symptoms, but in some cases can spread, invade other tissues and cause disease with high morbidity and mortality. The development of pneumococcal conjugate vaccines (PCV) caused an enormous impact in invasive pneumococcal disease and protected unvaccinated people by herd effect. However, serotype replacement is a well-known phenomenon that has occurred after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) and has also been reported for other PCVs. Therefore, it is possible that serotype replacement will continue to occur even with higher valence formulations, but the development of serotype-independent vaccines might overcome this problem. Alternative vaccines are under development in order to improve cost effectiveness, either using proteins or the pneumococcal whole cell. These approaches can be used as a stand-alone strategy or together with polysaccharide vaccines. Looking ahead, the next generation of pneumococcal vaccines can be impacted by the new technologies recently approved for human use, such as mRNA vaccines and viral vectors. In this paper, we will review the advantages and disadvantages of the addition of new polysaccharides in the current PCVs, mainly for low- and middle-income countries, and we will also address future perspectives.

How were DTP-related adverse events reduced after the introduction of an acellular pertussis vaccine in Chile?

OBJECTIVES: To describe the trend in the frequency of adverse events (AE) records associated to pertussis component vaccines between January 1st, 2015 and June 30th, 2020 in infants younger than 2-years-old in Chile, by reviewing the records submitted to the AEFI NIP, stratified by DTP-vaccine type, wP or aP. MATERIALS AND METHODS: This was a retrospective observational study including all AEFI records of DTP (either aP or wP)-containing vaccines in the described sample. A descriptive analysis was performed according to vaccine type and AEFI, using MedDRA terminology. RESULTS: The total number of AEFI reports was 1,697: 815 corresponding to wP vaccines, 417 to aP vaccines, and 465 with unknown type. The reporting rates for the years 2015 to 2020 were 40.1, 56.2, 37.1, 24.7, 19.1, and 12.2 per 100,000 doses administered, respectively. The most reported AEFI were injection site erythema (42.9%), pyrexia (35.7%), and pain at the injection site (29.2%). Among all cases, 5.8% were SAEs (n = 98), 5.9% were SAEs for wP vaccines (n = 48) and 5.3% were for aP vaccines (n = 22). DISCUSSION: A significant decrease in AEFI reports was observed as of 2018, the year that the DTaP-IPV-HepB-Hib was introduced in the NIP.

Autodisplay of an endo-1,4-ß-xylanase from Clostridium cellulovorans in Escherichia coli for xylans degradation.

The goal of this work was the autodisplay of the endo ß-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and ß-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants Vmax and Km were 149 U/gDCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu2+, Zn2+ and Hg2+ as well as EDTA, detergents, and organic acids, and improved by Ca2+, Co2+ and Mn2+ ions. Ca2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl2 were added. Also, Ca2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.

Heightened respiratory-parasympathetic coupling to airways in the spontaneously hypertensive rat.

KEY POINTS: Carotid body (CB) chemoreceptors are hyperactive in hypertension, and their acute activation produces bronchoconstriction. We show that the respiratory-modulated bronchiolar tone, pulmonary parasympathetic efferent activity, and the firing frequency and synaptic excitation of bronchoconstrictor motoneurones in the nucleus ambiguus were all enhanced in spontaneous hypertensive (SH) rats. In SH rats, CB denervation reduced the respiratory-related parasympathetic-mediated bronchoconstrictor tone to levels seen in normotensive rats. Chemoreflex evoked bronchoconstrictor tone was heightened in SH versus normotensive rats. The intrinsic electrophysiological properties and morphology of bronchoconstrictor motoneurones were similar across rat strains. The heightened respiratory modulation of parasympathetic-mediated bronchoconstrictor tone to the airways in SH rats is caused by afferent drive from the CBs. ABSTRACT: Much research has described heightened sympathetic activity in hypertension and diminished parasympathetic tone, especially to the heart. The carotid body (CB) chemoreceptors exhibit hyperreflexia and are hyperactive, providing excitatory drive to sympathetic networks in hypertension. Given that acute CB activation produces reflex evoked bronchoconstriction via activation of parasympathetic vagal efferents, we hypothesised that the parasympathetic bronchoconstrictor activity is enhanced in spontaneously hypertensive (SH) rats and that this is dependent on CB inputs. In situ preparations of Wistar and SH rats were used in which bronchiolar tone, the pulmonary branch of the vagus (pVN) and phrenic nerves were recorded simultaneously; whole cell patch clamp recordings of bronchoconstrictor vagal motoneurones were also made from the nucleus ambiguus. Bronchiolar tone, pVN and bronchoconstrictor motoneurones were respiratory modulated and this modulation was enhanced in SH rats. These differences were all eliminated after CB denervation. Stimulation of the CBs increased the phrenic frequency that caused a summation of the respiratory-related increases in pVN, resulting in the development of bronchoconstrictor tone. This tone was exaggerated in SH rats. The enhanced respiratory-parasympathetic coupling to airways in SH rats was not due to differences in the intrinsic electrophysiological properties of bronchoconstrictor motoneurones but reflected heightened pre-inspiratory- and inspiratory-related synaptic drive. In summary, in SH rats the phasic respiratory modulation of parasympathetic tone to the airways is elevated and the greater development of this bronchoconstrictor tone is caused by the heightened afferent drive originating from the CBs. Thus, targeting the CBs may prove effective for increasing lower airway patency.

Pichia pastoris displaying ZIKV protein epitopes from the Envelope and NS1 induce in vitro immune activation.

The severe consequences of ZIKV infection and its emergence and re-emergence in several countries have boosted vaccines' development. Yeasts such as Pichia pastoris has been widely employed as antigen carriers for immunization against infectious agents. Components of the yeast cell wall have immunostimulatory properties, and recombinant antigens can be anchored to the cell surface to enhance the presentation to the immune system. Here we aimed at producing and anchoring ZIKV proteins in the P. pastoris surface as a vaccine approach. Expression cassettes were designed with epitopes of the Envelope and NS1 proteins. Immunofluorescence microscopy confirmed the anchoring of recombinant proteins. Yeasts' ability to stimulate immune cells was evaluated in vitro by incubation with lymphocytes and monocytes isolated from mouse spleen. P. pastoris expressing EnvNS1 epitopes promoted increased levels of IL-6, IL-10, and TNF-α cytokines and an increase in the number of CD4+, CD8+, and CD16+ lymphocytes, similarly to ZIKV. This profile is indicative of the activation of immunological cells and suggests an immunogenic potential of the proposed yeast vaccines against ZIKV, reinforcing the possibility of P. pastoris as adjuvant and carrier of antigens.

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.

Lactate does not activate the carotid body of Wistar rat.

The carotid body's glomus cells are the primary sensors of hypoxia in mammals. Previous studies suggested that the glomus cells' hypoxia sensitivity is mediated by lactate in mice. This molecule increases the intracellular [Ca2+] and induces exocytosis in glomus cells, activating the carotid sinus nerve (the axons of chemoreceptive petrosal neurons). On the other hand, how lactate affects the activity of carotid body of rats is still unknown. We hypothesized that lactate activates the carotid body of rats. In Wistar rats, we measured the changes in the electrical properties of isolated glomus cells and petrosal chemoreceptive neurons in in situ preparations in response to different concentrations of lactate. Superfusion of both physiological and supraphysiological concentrations of lactate did not affect the membrane conductance and potential of glomus cells. Moreover, lactate injected into the carotid body did not activate the anatomically and physiologically identified chemoreceptive petrosal neurons. We conclude that the carotid body of Wistar rats is not sensitive to lactate.

Pneumococcal vaccines: past findings, present work, and future strategies

The importance of Streptococcus pneumoniae has been well established. These bacteria can colonize infants and adults without symptoms, but in some cases can spread, invade other tissues and cause disease with high morbidity and mortality. The development of pneumococcal conjugate vaccines (PCV) caused an enormous impact in invasive pneumococcal disease and protected unvaccinated people by herd effect. However, serotype replacement is a well-known phenomenon that has occurred after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) and has also been reported for other PCVs. Therefore, it is possible that serotype replacement will continue to occur even with higher valence formulations, but the development of serotype-independent vaccines might overcome this problem. Alternative vaccines are under development in order to improve cost effectiveness, either using proteins or the pneumococcal whole cell. These approaches can be used as a stand-alone strategy or together with polysaccharide vaccines. Looking ahead, the next generation of pneumococcal vaccines can be impacted by the new technologies recently approved for human use, such as mRNA vaccines and viral vectors. In this paper, we will review the advantages and disadvantages of the addition of new polysaccharides in the current PCVs, mainly for low- and middle-income countries, and we will also address future perspectives.

Whole-cell bioconversion of naringenin to high added value hydroxylated compounds using Yarrowia lipolytica 2.2ab in surface and liquid cultures.

The bioconversion process of bioactive naringenin by whole-cells of Yarrowia lipolytica 2.2ab for the production of increased value-added compounds is successfully achieved in surface and liquid cultures. This approach is an alternative to the commercial production of these bioactive compounds from vegetable sources, which are limited due to their low concentrations and the complexity of the purification processes. The experimentation rendered seven value-added compounds in both surface and liquid bioconversion cultures. Some of the compounds produced have not been previously reported as products from the bioconversion processes, such as the case of ampelopsin. Biosynthetic pathways were suggested for the naringenin bioconversion using whole-cells of Y. lipolytica 2.2ab. Finally, the extracts obtained from the naringenin bioconversion in liquid cultures showed higher percentage of inhibition of DPPH· and ABTS· radicals up to 32.88 and 2.08 times, respectively, compared to commercial naringenin.

Process intensification for production of Streptococcus pneumoniae whole-cell vaccine.

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.