Study of pH and Thermodynamic Parameters via Circular Dichroism Spectroscopy of a Recombinant Human Lactoferrin.

The production of human recombinant proteins to be used for therapeutic or nutritional purposes must focus on obtaining a molecule that is as close as possible to the native human protein. This biotechnological tool has been documented in various studies published in recent decades, with lactoferrin being one of those that has generated the most interest, being a promising option for recombinant technology. However, stability studies including thermodynamic parameters have not been reported for recombinant lactoferrin (Lf). The objective of this work was to obtain the human recombinant protein using the yeast Komagataella phaffii to study structural changes modifying pH and temperature using circular dichroism spectroscopy (CD). Thermodynamic parameters such as ΔH, ΔS and Tm were calculated and compared with commercial human lactoferrin. We propose the potential use of CD and thermodynamic parameters as a criterion in the production of recombinant proteins to be used in the production of specialized recombinant proteins.

Thrombin Cleaves Prolactin Into a Potent 5.6-kDa Vasoinhibin: Implication for Tissue Repair.

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (< 10 minutes) generates a 5.6-kDa fragment (residues 1-48) with full vasoinhibin activity, that is, it inhibited the proliferation, invasion, and permeability of cultured endothelial cells and promoted the lysis of a fibrin clot in plasma with a similar potency to that of a conventional 14-kDa vasoinhibin (residues 1-123). The R48-G49 cleavage site is highly conserved throughout evolution and precedes the intramolecular disulfide bond (C58-C174), thereby allowing the 5.6-kDa vasoinhibin to be released without a reduction step. Furthermore, the 5.6-kDa vasoinhibin is produced by endogenous thrombin during the clotting process. These findings uncover the smallest vasoinhibin known, add thrombin to the list of PRL-cleaving proteases generating vasoinhibin, and introduce vasoinhibin as a thrombin-activated mechanism for the regulation of hemostasis, vasopermeability, and angiogenesis in response to tissue injury.

A new classification criterion for the biofilm formation index: A study of the biofilm dynamics of pathogenic Vibrio species isolated from seafood and food contact surfaces.

The bacterial biofilm formation index (BFI) is measured by a microtiter plate assay, and it is typically performed at 72 hr. However, the dynamics of biopolymer formation change during this incubation period. The aims of this study were to follow the biofilm formation dynamics of Vibrio strains isolated from samples of seafood and food contact surfaces (FCS) and to propose a new BFI classification criterion. Samples from seafood (136) and FCS (14) were collected from retail markets in Queretaro, Mexico. The presence of Vibrio spp. was determined, the strains were isolated, and the six major pathogenic species (V. cholerae, V. alginolyticus, V. fluvialis, V. parahaemolyticus, V. vulnificus, V. mimicus) were identified by PCR. The BFI of the isolates was determined by the microtiter plate method. Fifty-one strains were isolated and identified as V. alginolytivcus (25), V. vulnificus (12), V. cholerae (7), V. parahaemolyticus (6), and V. mimicus (1). A quantitative classification criterion of biofilm formation was proposed based on the following factors: BFI dynamics (no formation, continuous increase, and increase followed by decrease), time of maximum BFI (early: 24 hr; late: 48 to 72 hr), and degree of BFI (none, weak, moderate, and strong). A numerical value was assigned to each factor to correlate the resulting BFI profile with a risk level. Thirteen BFI profiles were observed, having risk level values from 0 to 10. Vibrio alginolyticus, V. cholerae, and V. vulnificus showed the highest BFI profile diversities, which included the riskiest profiles. The proposed BFI criterion describes the dynamics of bacterial biopolymer formation and associates them with the possible risk implications. PRACTICAL APPLICATION: In food processing environments, the presence of bacterial biofilms that could include foodborne pathogens might favor cross-contamination due to direct contact or biofilm dispersal into food products. The new quantitative classification criterion for biofilm formation considers their production dynamics over time, the biofilm quantity, and the level of biofilm dispersal. These characteristics are represented by a numerical value that reflects the level of risk associated with the presence of a biofilm-producing strain on a food contact surface.

Analysis of tRNA Cys processing under salt stress in Bacillus subtilis spore outgrowth using RNA sequencing data.

Background: In spore-forming bacteria, the molecular mechanisms of accumulation of transfer RNA (tRNA) during sporulation must be a priority as tRNAs play an essential role in protein synthesis during spore germination and outgrowth. However, tRNA processing has not been extensively studied in these conditions, and knowledge of these mechanisms is important to understand long-term stress survival.    Methods:To gain further insight into tRNA processing during spore germination and outgrowth, the expression of the single copy tRNA Cys gene was analyzed in the presence and absence of 1.2 M NaCl in Bacillus subtilis using RNA-Seq data obtained from the Gene Expression Omnibus (GEO) database. The CLC Genomics work bench 12.0.2 (CLC Bio, Aarhus, Denmark, https://www.qiagenbioinformatics.com/) was used to analyze reads from the tRNA Cys gene.  Results:The results show that spores store different populations of tRNA Cys-related molecules.  One such population, representing 60% of total tRNA Cys, was composed of tRNA Cys fragments.  Half of these fragments (3´-tRF) possessed CC, CCA or incorrect additions at the 3´end. tRNA Cys with correct CCA addition at the 3´end represented 23% of total tRNA Cys, while with CC addition represented 9% of the total and with incorrect addition represented 7%. While an accumulation of tRNA Cys precursors was induced by upregulation of the rrnD operon under the control of  σ A -dependent promoters under both conditions investigated, salt stress produced only a modest effect on tRNA Cys expression and the accumulation of tRNA Cys related species. Conclusions:The results demonstrate that tRNA Cys molecules resident in spores undergo dynamic processing to produce functional molecules that may play an essential role during protein synthesis.

Aggressiveness and molecular characterization of Fusarium spp. associated with foot rot and wilt in Tomato in Sinaloa, Mexico.

Fusarium wilt is one of the main limiting factors for tomato production in Mexico. One thousand and fifty isolates were obtained from vascular tissues tomato plants showing wilt and yellowing symptoms in Sinaloa, Mexico. The pathogenic isolates were evaluated through phylogenetic analysis of the TEF-1α gene and ITS region, morphological markers and pathogenicity tests. Within the 15 pathogenic Fusarium isolates, 7 were identified as F. oxysporum and 8 as F. falciforme. Phylogenetic analysis of Fusarium oxysporum f. sp. lycopersici and Fusarium falciforme isolates confirmed that both populations are constituted by distinct phylogenetic lineages. The isolates showed differences in aggressiveness; F. falciforme was the most aggressive. Isolates of both complexes triggered similar aerial symptoms of yellowing and darkening of the vascular tissues in tomato plants. But only F. falciforme isolates triggered necrosis in the plant crowns. Morphological markers allowed differentiating isolates from distinct complexes but not differentiating between lineages.

Analysis of tRNACys processing in the absence of CCAase in Bacillus subtilis.

In Bacillus subtilis, the tRNACys lacks an encoded CCA 3' end. To gain insight into the role of CCAase and RNases in tRNACys processing, several mutant strains were generated. Northern blot and RT-PCR results suggest that enzymes other than CCAase can participate in CCA addition at the 3' end of the immature tRNACys.

Microbial Diversity in Commercial Bee Pollen from Europe, Chile, and Mexico, Based on 16S rRNA Gene Amplicon Metagenome Sequencing.

Bee pollen is a highly nutritive natural foodstuff. Because of its use as a comestible, the association of bacteria with bee pollen is commercially and biologically important. We report here the bacterial diversity of seven bee pollen samples (five from Europe, one from Chile, and one from Mexico) based on 16S rRNA gene amplicon metagenome sequencing.

Plant Hormesis Management with Biostimulants of Biotic Origin in Agriculture.

Over time plants developed complex mechanisms in order to adapt themselves to the environment. Plant innate immunity is one of the most important mechanisms for the environmental adaptation. A myriad of secondary metabolites with nutraceutical features are produced by the plant immune system in order to get adaptation to new environments that provoke stress (stressors). Hormesis is a phenomenon by which a stressor (i.e., toxins, herbicides, etc.) stimulates the cellular stress response, including secondary metabolites production, in order to help organisms to establish adaptive responses. Hormetins of biotic origin (i.e., biostimulants or biological control compounds), in certain doses might enhance plant performance, however, in excessive doses they are commonly deleterious. Biostimulants or biological control compounds of biotic origin are called "elicitors" that have widely been studied as inducers of plant tolerance to biotic and abiotic stresses. The plant response toward elicitors is reminiscent of hormetic responses toward toxins in several organisms. Thus, controlled management of hormetic responses in plants using these types of compounds is expected to be an important tool to increase nutraceutical quality of plant food and trying to minimize negative effects on yields. The aim of this review is to analyze the potential for agriculture that the use of biostimulants and biological control compounds of biotic origin could have in the management of the plant hormesis. The use of homolog DNA as biostimulant or biological control compound in crop production is also discussed.

Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis.

Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW) thiols. Bacillithiol was recently identified as a major low-molecular-weight thiol in Bacillus subtilis and related Firmicutes. Four genes (bshA, bshB1, bshB2, and bshC) are involved in bacillithiol biosynthesis. The bshA and bshB1 genes are part of a seven-gene operon (ypjD), which includes the essential gene cca, encoding CCA-tRNA nucleotidyltransferase. The inclusion of cca in the operon containing bacillithiol biosynthetic genes suggests that the integrity of the 3' terminus of tRNAs may also be important in oxidative stress. The addition of the 3' terminal CCA sequence by CCA-tRNA nucleotidyltransferase to give rise to a mature tRNA and functional molecules ready for aminoacylation plays an essential role during translation and expression of the genetic code. Any defects in these processes, such as the accumulation of shorter and defective tRNAs under oxidative stress, might exert a deleterious effect on cells. This review summarizes the physiological link between tRNACys regulation and oxidative stress in Bacillus.

Draft Genome Sequence of the Mercury-Resistant Bacterium Acinetobacter idrijaensis Strain MII, Isolated from a Mine-Impacted Area, Idrija, Slovenia.

We report here the first draft assembly for the genome of Acinetobacter idrijaensis strain MII, isolated from the Idrija mercury mine area (Slovenia). This strain shows a strikingly high tolerance to mercury, and the genome sequence shows genes involved in the mechanisms for heavy metal tolerance pathways and multidrug efflux pumps.

Exposure of Bacillus subtilis to mercury induces accumulation of shorter tRNA Cys species.

RNA processing is an essential pathway in the regulation of genetic expression in the cell. In this work, Bacillus subtilis was used to understand the effects of mercury on the mechanism of tRNA metabolism. The CVAAS (cold vapor atomic absorption spectroscopy) method revealed that from the addition of HgCl2 (0.75 µg ml(-1)) during the bacterial exponential phase, ca. 48% of the added mercury was taken up by the cells. This led to an immediate reduction in the rate of cell division. During this response, we observed accumulation of species shorter than mature tRNA(Cys) over a 10 h period. We did not observe this accumulation for another five tRNAs analyzed. tRNA processing is largely dependent on RNase R and PNPase in B. subtilis. Thus, when the exonuclease PNPase was absent, we found that the shorter tRNA(Cys) species increased and mature tRNA(Cys) decreased after mercury addition, but this proportion changed during the time analyzed. However, in the absence of RNase R and PNPase the accumulation of the shorter tRNA(Cys) was more pronounced and the mature form was not recovered. In the single rnr mutant strain the shorter tRNA(Cys) was not observed. All together, we provide in vivo evidence that PNPase and RNase R are indispensable in controlling tRNA(Cys) quality in the presence of mercury.