Effect of carbon and nitrogen concentrations on lipid accumulation and regulation of acetyl-CoA carboxylase in Papiliotrema laurentii.

Biodiesel is an interesting alternative to petroleum diesel as it is renewable, biodegradable, and has a low pollutant content. Yeast oils can be used for biodiesel production instead of edible oils, mitigating the use of arable land and water for biodiesel production. Maximum lipid accumulation is reached at 48 h of cultivation by the oleaginous yeast Papiliotrema laurentii UFV-1. Nevertheless, the effects of carbon and nitrogen concentrations on lipid accumulation, as well as the regulation of lipid metabolism in this yeast are still not well-characterised. Therefore, this work evaluated the effects of carbon and nitrogen concentrations on the lipid accumulation in P. laurentti, the expression of the ACC gene, and the activity of the enzyme acetyl-CoA carboxylase (ACCase) in different carbon:nitrogen ratios (C:N) and glucose concentrations. The variation of ammonium sulfate concentration did not affect the growth and lipid accumulation in P. laurentii UFV-1. On the other hand, glucose concentration remarkably influenced biomass and lipid production by this yeast. Therefore, the carbon concentration is more important than the nitrogen concentration for lipid production by P. laurentii UFV-1. Importantly, the levels of both ACC gene expression and ACCase activity were maximum during the late-exponential growth phase and decreased after reaching the highest lipid contents, which was easier evidenced during the accumulation and maximum lipid levels. As such, the reduction of ACCase enzyme activity seems to be related to the decrease in the expression level of the ACC gene.

Chimeric Protein Designed by Genome-Scale Immunoinformatics Enhances Serodiagnosis of Bovine Neosporosis.

Neosporosis has become a concern since it is associated with abortion in cattle. Currently, in situ diagnosis is determined through anamnesis, evaluation of the history, and perception of the clinical signs of the herd. There is no practical and noninvasive test adapted to a large number of samples, which represents a gap for the use of new approaches that provide information about infections and the risks of herds. Here, we performed a search in the Neospora caninum genome by linear B-cell epitopes using immunoinformatic tools aiming to develop a chimeric protein with high potential to bind specifically to antibodies from infected cattle samples. An enzyme-linked immunosorbent assay with the new chimeric antigen was developed and tested with sera from natural field N. caninum-infected bovines. The cross-reactivity of the new antigen was also evaluated using sera from bovines infected by other abortive pathogens, including Trypanosoma vivax, Leptospira sp., Mycobacterium bovis, and Brucella abortus, and enzootic bovine leucosis caused by bovine leukemia virus, as well as with samples of animals infected with Toxoplasma gondii The assay using the chimeric protein showed 96.6% ± 3.4% of sensitivity in comparison to healthy animal sera. Meanwhile, in relation to false-positive results provided by cross-reactivity with others pathogens, the specificity value was 97.0% ± 2.9%. In conclusion, immunoinformatic tools provide an efficient platform to build an accurate protein to diagnose bovine neosporosis based on serum samples.

Comparative analysis of constitutive proteome between resistant and susceptible tomato genotypes regarding to late blight.

Late blight is one of the most destructive diseases of the tomato, resulting in substantial economic losses. There is difficulty in controlling this disease, so the molecular characterization of tomato genotypes may help in the selection of higher resistance tomato plants against Phytophthora infestans, late blight's pathogen. The objective was to analyze the differences with regard to the constitutive proteome between the access Vegetable Germplasm Bank (BGH)-2127, resistant genotype, and Santa Clara-susceptible genotype to late blight. Proteomic analysis of leaf samples by two-dimensional electrophoresis (2-DE) followed by identification by mass spectrometry (MALDI TOF/TOF) was performed. Nineteen proteins were identified, which were then related to metabolism and energy, photosynthesis, transcription, stress, and defenses. Approximately 90% of these proteins were more abundant in Santa Clara, a susceptible cultivar. Acidic 26 kDa endochitinase and ribonuclease T2 proteins were more abundant in BGH-2127 access. The enzymatic activity confirmed a greater abundance of chitinase in the BGH-2127 access as compared to the cultivar Santa Clara. Gene expression analyses by real-time PCR demonstrated that the mRNA levels were not correlated with the respective protein levels. Abundance of the acidic 26 kDa endochitinase and ribonuclease T2 proteins in the constitutive proteomes of BGH-2127 may be associated with the answer to the resistance of this access.

Computer aided identification of a Hevein-like antimicrobial peptide of bell pepper leaves for biotechnological use.

BACKGROUND: Antimicrobial peptides from plants present mechanisms of action that are different from those of conventional defense agents. They are under-explored but have a potential as commercial antimicrobials. Bell pepper leaves ('Magali R') are discarded after harvesting the fruit and are sources of bioactive peptides. This work reports the isolation by peptidomics tools, and the identification and partially characterization by computational tools of an antimicrobial peptide from bell pepper leaves, and evidences the usefulness of records and the in silico analysis for the study of plant peptides aiming biotechnological uses. RESULTS: Aqueous extracts from leaves were enriched in peptide by salt fractionation and ultrafiltration. An antimicrobial peptide was isolated by tandem chromatographic procedures. Mass spectrometry, automated peptide sequencing and bioinformatics tools were used alternately for identification and partial characterization of the Hevein-like peptide, named HEV-CANN. The computational tools that assisted to the identification of the peptide included BlastP, PSI-Blast, ClustalOmega, PeptideCutter, and ProtParam; conventional protein databases (DB) as Mascot, Protein-DB, GenBank-DB, RefSeq, Swiss-Prot, and UniProtKB; specific for peptides DB as Amper, APD2, CAMP, LAMPs, and PhytAMP; other tools included in ExPASy for Proteomics; The Bioactive Peptide Databases, and The Pepper Genome Database. The HEV-CANN sequence presented 40 amino acid residues, 4258.8 Da, theoretical pI-value of 8.78, and four disulfide bonds. It was stable, and it has inhibited the growth of phytopathogenic bacteria and a fungus. HEV-CANN presented a chitin-binding domain in their sequence. There was a high identity and a positive alignment of HEV-CANN sequence in various databases, but there was not a complete identity, suggesting that HEV-CANN may be produced by ribosomal synthesis, which is in accordance with its constitutive nature. CONCLUSIONS: Computational tools for proteomics and databases are not adjusted for short sequences, which hampered HEV-CANN identification. The adjustment of statistical tests in large databases for proteins is an alternative to promote the significant identification of peptides. The development of specific DB for plant antimicrobial peptides, with information about peptide sequences, functional genomic data, structural motifs and domains of molecules, functional domains, and peptide-biomolecule interactions are valuable and necessary.

Co-expression of human sialyltransferase improves N-glycosylation in Leishmania tarentolae and optimizes the production of humanized therapeutic glycoprotein IFN-beta.

The production of therapeutic glycoproteins is primarily expensive due to the necessity of culturing mammalian cells. These systems often require complex and costly culture media and typically yield low amounts of protein. Leishmania tarentolae, a non-pathogenic protozoan to mammals, has emerged as a cost-effective alternative system for heterologous glycoprotein expression due to its suitability for large-scale production using low-cost culture media, and its ability to perform mammalian-like post-translational modifications, including glycosylation. Nevertheless, differences in the carbohydrate residues at the end of N-glycan chains are observed in Leishmania compared to mammalian cells due to the absence of biosynthetic enzymes in Leishmania that are required for the incorporation of terminal sialic acid. In this study, a genetically optimized L. tarentolae cell line was engineered for the production of recombinant interferon-ß (IFN-ß) featuring a complete mammalian N-glycosylation profile. Genomic and metabolomic analyses revealed that heterologous expression of the sialyltransferase enzyme and cultivation in a medium containing sialic acid were sufficient to generate mammalian-like protein N-glycosylation. N-glycan mass spectrometry analysis demonstrated a glycosylation pattern compatible with the incorporation of sialic acid into the glycan structure. In vitro IFN-ß activity indicated that the expressed protein exhibited reduced inflammatory effects compared to IFN-beta produced by other platforms, such as bacteria, non-optimized L. tarentolae, and mammalian cells.

Two novel synthetic xanthenodiones as antimicrobial, anti-adhesive and antibiofilm compounds against methicillin resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is widely recognized as a causative agent for various infections acquired in healthcare settings as well as in the community. Given the limited availability of effective antimicrobial agents to combat MRSA infections, there is an increasing need to explore alternative therapeutic strategies. This study aimed to assess the antimicrobial, anti-adhesive, anti-biofilm properties, and toxicity of 175 newly synthesized compounds, belonging to seven different classes, against MRSA. Initially, the compounds underwent screening for antimicrobial activity using the agar diffusion method. Subsequently, active compounds underwent further evaluation to determine their minimum inhibitory concentrations through microdilution. Anti-biofilm and anti-adhesive properties were assessed using the crystal violet method, while toxicity was tested using the alternative infection model Galleria mellonella. Among the tested compounds, two xanthenodiones exhibited the most promising activities, displaying bactericidal effects along with anti-adhesive and anti-biofilm properties. Moreover, the observed non-toxicity in G. mellonella larvae suggests that these compounds hold significant potential as alternative therapeutic options to address the escalating challenge of MRSA resistance in both hospital and community settings.

Identification of novel small RNAs in extracellular vesicles produced by Actinobacillus pleuropneumoniae.

Extracellular vesicle (EV) production by bacteria is an important mechanism for microbial communication and host-pathogen interaction. EVs of some bacterial species have been reported to contain nucleic acids. However, the role of small RNAs (sRNAs) packaged in EVs is poorly understood. Here, we report on the RNA cargo of EVs produced by the pig pathogen Actinobacillus pleuropneumoniae, the causal agent of porcine pleuropneumonia, a disease which causes substantial economic losses to the swine industry worldwide. The EVs produced by aerobically and anaerobically grown bacteria were only slightly different in size and distribution. Total cell and outer membrane protein profiles and lipid composition of A. pleuropneumoniae whole cell extracts and EVs were similar, although EVs contained rough lipopolysaccharide compared to the smooth form in whole cells. Approximately 50% of Galleria mellonella larvae died after the injection of EVs. RNAseq, RT-PCR, protection from nuclease degradation, and database searching identified previously described and 13 novel A. pleuropneumoniae sRNAs in EVs, some of which were enriched compared to whole cell content. We conclude that A. pleuropneumoniae EVs contain sRNAs, including those known to be involved in virulence, and some with homologs in other Pasteurellaceae and/or non-Pasteurellaceae. Further work will establish whether the novel sRNAs in A. pleuropneumoniae EVs play any role in pathogenesis.

Physiological comparisons among Spathaspora passalidarum, Spathaspora arborariae, and Scheffersomyces stipitis reveal the bottlenecks for their use in the production of second-generation ethanol.

The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44 g/g) and productivity (1.02 g/L.h) were obtained using Sp. passalidarum grown in 100 g/L xylose at 32 °C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells.

Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator in Actinobacillus pleuropneumoniae.

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

Phakopsora pachyrhizi triggers the jasmonate signaling pathway during compatible interaction in soybean and GmbZIP89 plays a role of major component in the pathway.

The biotrophic fungus Phakopsora pachyrhizi is currently the major pathogen affecting soybean production worldwide. It has already been suggested for the non-host interaction between P. pachyrhizi and Arabidopsis thaliana that the fungus in early infection induces jasmonic acid (JA) pathway to the detriment of the salicylic acid (SA) pathway as a mechanism to the establishment of infection. In this study, we verified that this mechanism might also be occurring during the compatible interaction in soybean (Glycine max L. Merril). It was demonstrated that P. pachyrhizi triggers a JA pathway during the early and late stages of infection in a susceptible soybean cultivar. The expression of the GmbZIP89 was induced in a biphasic profile, similarly to other JA responsive genes, which indicates a new marker gene for this signaling pathway. Additionally, plants silenced for GmbZIP89 (iGmZIP89) by the virus-induced gene silencing (VIGS) approach present lower severity of infection and higher expression of pathogenesis related protein 1 (PR1). The lower disease severity showed that the iGmbZIP89 plants became more resistant to infection. These data corroborate the hypothesis that the GmbZIP89 may be a resistance negative regulator. In conclusion, we demonstrated that P. pachyrhizi mimics a necrotrophic fungus and activates the JA/ET pathway in soybean. It is possible to suppose that its direct penetration on epidermal cells or fungal effectors may modulate the expression of target genes aiming the activation of the JA pathway and inhibition of SA defense.

Genome-wide identification and expression analysis of dormancy-associated gene 1/auxin repressed protein (DRM1/ARP) gene family in Glycine max.

Dormancy-Associated gene 1/Auxin Repressed protein (DRM1/ARP) genes are responsive to hormones involved in defense response to biotic stress, such as salicylic acid (SA) and methyl jasmonate (MeJA), as well as to hormones that regulate plant growth and development, including auxins. These characteristics suggest that this gene family may be an important link between the response to pathogens and plant growth and development. In this investigation, the DRM1/ARP genes were identified in the genome of four legume species. The deduced proteins were separated into three distinct groups, according to their sequence conservation. The expression profile of soybean genes from each group was measured in different organs, after treatment with auxin and MeJA and in response to the nematode Meloidogyne javanica. The results demonstrated that this soybean gene family is predominantly expressed in root. The time auxin takes to alter DRM1/ARP expression suggests that these genes can be classified as a late response to auxin. Nevertheless, only the groups 1 and 3 are induced in roots infected by M. javanica and only group 3 is induced by MeJA, which indicates a high level of complexity in expression control mechanisms of DRM1/ARP family in soybean.