Secretion of Bacillus amyloliquefaciens Transglutaminase from Lactococcus lactis and Its Enhancement of Food Gel Properties

(1) Background: Microbial transglutaminases (MTGase) catalyze protein crosslink. This is useful in the food industry to improve gelation, water holding capacity, and emulsifying capacity during foodstuff manufacturing. The production of MTGase in wild-type strains renders low yield and high costs of downstream purification, limiting its industrial applications. (2) Methods: In this work, MTGase from Bacillus amyloliquefaciens BH072 (BaMTGase) has been heterologously expressed in Lactococcus lactis, using the signal peptide Usp45 to direct the secretion of recombinant BaMTGase out of the cell for easier purification. (3) Results: In these conditions, MTGase was purified with a high yield (48.7 +/- 0.2 mg/L) and high enzyme activity (28.6 +/- 0.5 U/mg). Next, BaMTGase was tested for industrial applications. Recombinant BaMTGase and commercial MTGase were used for SPI solution crosslinking. BaMTGase formed a harder gel with higher water-holding capacity and a dense and smooth gel microstructure. (4) Conclusions: This work provides an attractive food-grade cell factory for the food industry and offers a suitable chassis for MTGase production.Major Project of Science and Technology of Anhui Province 201903a06020007 202103b06020009 202003b06020017Natural Science Foundation of Anhui Province 2108085MC123Fundamental Research Funds for the Central Universities PA2021KCPY0048China Postdoctoral Science Foundation 2019M65101

Aplicación del método intervención en la reducción del índice de accidentabilidad en la contratista minera AESA

Determina la influencia del método intervención en la reducción de índice de accidentabilidad en una contratista minera. Se desarrolló en el contexto de la inseguridad de los trabajadores de una contratista minera y las medidas para reducir accidentes y riesgos. El problema de esta investigación surge sobre los altos índices de accidentabilidad en empresas del rubro de la minería ante ello surge la pregunta “Cuál es la influencia del método intervención en la reducción de índice de accidentabilidad en la unidad minera” para el desarrollo de este problema se usó el tipo de investigación correlacional de diseño observacional transversal, la muestra usada fueron los colaboradores que se desarrollan en el área de Operaciones con un total de 24 trabajadores

Feasability of Introducing a Thioether Ring in Vasopressin by nisBTC Co-expression in Lactococcus lactis

Introducing one or more intramolecular thioether bridges in a peptide provides a promising approach to create more stable molecules with improved pharmacodynamic properties and especially to protect peptides against proteolytic degradation. Lanthipeptides are compounds that naturally possess thioether bonds in their structure. The model lanthipeptide, nisin, is produced by Lactococcus lactis as a core peptide fused to a leader peptide. The modification machinery responsible for nisin production, including the Ser/Thr-dehydratase NisB and the cyclase NisC, can be applied for introducing a thioether bridge into peptides fused to the nisin leader peptide, e.g., to replace a disulfide bond. Vasopressin plays a key role in water homeostasis in the human body and helps to constrict blood vessels. There are two cysteine residues in the structure of wild type vasopressin, which form a disulfide bridge in the mature peptide. Here, we show it is possible to direct the biosynthesis of vasopressin variants in such a way that the disulfide bridge is replaced by a thioether bridge using the nisin modification machinery NisBTC, albeit at low efficiency. Vasopressin mutants were fused either to the nisin leader peptide directly (Type A), after the first three rings of nisin (Type B/C), or after full nisin (Type D). The type B strategy was optimal for expression. LC-MS/MS data verified the formation of a thioether bridge, which provides proof of principle for this modification in vasopressin. This is a first step prior to the necessary increase of the production yield and further purification of these peptides to finally test their biological activity in tissue and animal models.Chinese Scholarship Council (No. 201306770012)MM-L was supported by a grant of the EU FW7 Project SynPeptide

Employing the promiscuity of lantibiotic biosynthetic machineries to produce novel antimicrobials

As the number of new antibiotics that reach the market is decreasing and the demand for them is rising, alternative sources of novel antimicrobials are needed. Lantibiotics are potent peptide antimicrobials that are ribosomally synthesized and stabilized by post-translationally introduced lanthionine rings. Their ribosomal synthesis and enzymatic modifications provide excellent opportunities to design and engineer a large variety of novel antimicrobial compounds. The research conducted in this area demonstrates that the modularity present in both the peptidic rings as well as in the combination of promiscuous modification enzymes can be exploited to further increase the diversity of lantibiotics. Various approaches, where the modifying enzymes and corresponding leader peptides are decoupled from their natural core peptide and integrated in designed plug-and-play production systems, enable the production of modified peptides that are either derived from vast genomic data or designed using functional parts from a wide diversity of core peptides. These approaches constitute a powerful discovery platform to develop novel antimicrobials with high therapeutic potential

Posttranslational Peptide-Modification Enzymes in Action:Key Roles for Leaders and Glutamate

In this issue of Cell Chemical Biology, Ortega et al. (2016) determine the structure of another lantibiotic dehydratase with a tRNA(Glu)-dependent mechanism of modification. Moreover, they identify a common recognition motif involved in leader peptide binding in a number of different peptide-modification enzymes. These findings open up new mining possibilities and allow novel approaches in peptide engineering

Bacteriocins of lactic acid bacteria:extending the family

Lactic acid bacteria (LAB) constitute a heterogeneous group of microorganisms that produce lactic acid as the major product during the fermentation process. LAB are Gram-positive bacteria with great biotechnological potential in the food industry. They can produce bacteriocins, which are proteinaceous antimicrobial molecules with a diverse genetic origin, posttranslationally modified or not, that can help the producer organism to outcompete other bacterial species. In this review, we focus on the various types of bacteriocins that can be found in LAB and the organization and regulation of the gene clusters responsible for their production and biosynthesis, and consider the food applications of the prototype bacteriocins from LAB. Furthermore, we propose a revised classification of bacteriocins that can accommodate the increasing number of classes reported over the last years

Método intervención en la reducción del índice de accidentabilidad en la contratista minera Aesa

The implementation of the intervention method reduces the accident rate in the AESA mining contractor and contributes to the creation of a safety culture in both the worker and the company. Security has been improved in many aspects. Now, there are security norms, procedures, formats, standards, security methodologies based on the behavior of people, incidents and other events generated by nature and rock. There are also immediate response programs for emergencies that arise. The influence of the intervention method in reducing the accident rate has been highly positive since workers have been informed and trained in safety issues by implementing this method; The safety culture has improved, since now 87.5% of the participants know what job safety is, staff turnover has improved considerably since workers previously did not know the benefits of having life insurance, to stay safer in their workplace and avoid staff turnover, the reduction in the frequency of disabling accidents has been considerably since workers have been trained using the method on the benefits offered by IPERC for the identification of hazards and assess risks to qualify them according to the dangerousness.La implementación del método intervención reduce el índice de accidentabilidad en la contratista minera AESA y contribuye a que se vaya creando una cultura de seguridad tanto en el trabajador como en la empresa. Se ha mejorado en muchos aspectos en el tema de seguridad. Ahora, se cuenta con normas de seguridad, procedimientos, formatos, estándares, metodologías de seguridad basadas en el comportamiento de las personas, los incidentes y otros eventos generados por la naturaleza y la roca. También se cuenta con programas de respuesta inmediata ante emergencias suscitadas. La influencia del método de intervención en la reducción del índice de accidentabilidad ha sido altamente positiva puesto que los trabajadores se han informado y capacitado en los temas de seguridad mediante la implementación de este método; ha mejorado la cultura de seguridad, puesto que ahora un 87.5% de los participantes sabe lo que es seguridad laboral, la rotación del personal ha mejorado considerablemente puesto que los trabajadores antes no sabían los beneficios de tener un seguro de vida, para mantenerse más seguros en su lugar de trabajo y evitar la rotación del personal, la reducción de la frecuencia de accidentes incapacitantes ha sido considerablemente puesto que los trabajadores han sido capacitados mediante el método sobre los beneficios que ofrece el IPERC para calificarlos según la peligrosidad

Outer-membrane-acting peptides and lipid II-targeting antibiotics cooperatively kill Gram-negative pathogens

The development and dissemination of antibiotic-resistant bacterial pathogens is a growing global threat to public health. Novel compounds and/or therapeutic strategies are required to face the challenge posed, in particular, by Gram-negative bacteria. Here we assess the combined effect of potent cell-wall synthesis inhibitors with either natural or synthetic peptides that can act on the outer-membrane. Thus, several linear peptides, either alone or combined with vancomycin or nisin, were tested against selected Gram-negative pathogens, and the best one was improved by further engineering. Finally, peptide D-11 and vancomycin displayed a potent antimicrobial activity at low μM concentrations against a panel of relevant Gram-negative pathogens. This combination was highly active in biological fluids like blood, but was non-hemolytic and non-toxic against cell lines. We conclude that vancomycin and D- 11 are safe at >50-fold their MICs. Based on the results obtained, and as a proof of concept for the newly observed synergy, a Pseudomonas aeruginosa mouse infection model experiment was also performed, showing a 4 log10 reduction of the pathogen after treatment with the combination. This approach offers a potent alternative strategy to fight (drug-resistant) Gram-negative pathogens in humans and mammals.China Scholarship Council 201306770012European Union (EU)NWO-NACTAR progra

LAB Bacteriocins Controlling the Food Isolated (Drug-Resistant) Staphylococci

Staphylococci are a group of microorganisms that can be often found in processed food and theymight pose a risk for human health. In this study we have determined the content of staphylococci in 7 different fresh goat-milk cheeses. These bacteria were present in all of them, ranging from 103 to 106 CFU/g based on growth on selective media. Thus, a set of 97 colonies was randomly picked for phenotypic and genotypic identification. They could be clustered by RAPD-PCR in 10 genotypes, which were assigned by 16S rDNA sequencing to four Staphylococcus species: Staphylococcus aureus, Staphylococcus chromogenes, S. simulans, and S. xylosus. Representative strains of these species (n = 25) were tested for antibiotic sensitivity, and 11 of them were resistant to at least one of the antibiotics tested, including erythromycin, amoxicillin-clavulanic acid and oxacillin. We also tested two bacteriocins produced by lactic acid bacteria (LAB), namely the circular bacteriocin AS-48 and the lantibiotic nisin. These peptides have different mechanism of action at the membrane level. Nevertheless, both were able to inhibit staphylococci growth at low concentrations ranging between 0.16–0.73μM for AS-48 and 0.02–0.23μM for nisin, including the strains that displayed antibiotic resistance. The combined effect of these bacteriocins were tested and the fractional inhibitory concentration index (FICI) was calculated. Remarkably, upon combination, they were active at the low micromolar range with a significant reduction of the minimal inhibitory concentration. Our data confirms synergistic effect, either total or partial, between AS-48 and nisin for the control of staphylococci and including antibiotic resistant strains. Collectively, these results indicate that the combined use of AS-48 and nisin could help controlling (pathogenic) staphylococci in food processing and preventing antibiotic-resistant strains reaching the consumer in the final products.This research was funded by the Spanish Ministry of Economy and Competitiveness (SAF2013-48971-C2-1-R project), including funds from the European Regional Development Fundings (ERDF) and the Research Group General (BIO160, UGR

Spanish Lymphoma Group (GELTAMO) guidelines for the diagnosis, staging, treatment, and follow-up of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) accounts for approximately 30% of non-Hodgkin lymphoma (NHL) cases in adult series. DLBCL is characterized by marked clinical and biological heterogeneity, encompassing up to 16 distinct clinicopathological entities. While current treatments are effective in 60% to 70% of patients, those who are resistant to treatment continue to die from this disease. An expert panel performed a systematic review of all data on the diagnosis, prognosis, and treatment of DLBCL published in PubMed, EMBASE and MEDLINE up to December 2017. Recommendations were classified in accordance with the Grading of Recommendations Assessment Development and Evaluation (GRADE) framework, and the proposed recommendations incorporated into practical algorithms. Initial discussions between experts began in March 2016, and a final consensus was reached in November 2017. The final document was reviewed by all authors in February 2018 and by the Scientific Committee of the Spanish Lymphoma Group GELTAMO