Svx Peptidases of Phytopathogenic Pectolytic Bacteria: Structural, Catalytic and Phytoimmune Properties.

Svx proteins are virulence factors secreted by phytopathogenic bacteria of the Pectobacterium genus into the host plant cell wall. Svx-encoding genes are present in almost all species of the soft rot Pectobacteriaceae (Pectobacterium and Dickeya genera). The Svx of P. atrosepticum (Pba) has been shown to be a gluzincin metallopeptidase that presumably targets plant extensins, proteins that contribute to plant cell wall rigidity and participate in cell signaling. However, the particular "output" of the Pba Svx action in terms of plant-pathogen interactions and plant immune responses remained unknown. The Svx proteins are largely unexplored in Dickeya species, even though some of them have genes encoding two Svx homologs. Therefore, our study aims to compare the structural and catalytic properties of the Svx proteins of Pba and D. solani (Dso) and to test the phytoimmune properties of these proteins. Two assayed Dso Svx proteins, similar to Pba Svx, were gluzincin metallopeptidases with conservative tertiary structures. The two domains of the Svx proteins form electronegative clefts where the active centers of the peptidase domains are located. All three assayed Svx proteins possessed phytoimmunosuppressory properties and induced ethylene-mediated plant susceptible responses that play a decisive role in Pba-caused disease.

Exploiting Charge State Distribution To Probe Intramolecular Interactions in Gas-Phase Phosphopeptides and Enhance Proteomics Analyses.

Charging of analytes is a prerequisite for performing mass spectrometry analysis. In proteomics, electrospray ionization is the dominant technique for this process. Although the observation of differences in the peptide charge state distribution (CSD) is well-known among experimentalists, its analytical value remains underexplored. To investigate the utility of this dimension, we analyzed several public data sets, comprising over 250,000 peptide CSD profiles from the human proteome. We found that the dimensions of the CSD demonstrate high reproducibility across multiple laboratories, mass analyzers, and extensive time intervals. The general observation was that the CSD enabled effective partitioning of the peptide property space, resulting in enhanced discrimination between sequence and constitutional peptide isomers. Next, by evaluating the CSD values of phosphorylated peptides, we were able to differentiate between phosphopeptides that indicate the formation of intramolecular structures in the gas phase and those that do not. The reproducibility of the CSD values (mean cosine similarity above 0.97 for most of the experiments) qualified CSD data suitable to train a deep-learning model capable of accurately predicting CSD values (mean cosine similarity - 0.98). When we applied the CSD dimension to MS1- and MS2-based proteomics experiments, we consistently observed around a 5% increase in protein and peptide identification rate. Even though the CSD dimension is not as effective a discriminator as the widely used retention time dimension, it still holds the potential for application in direct infusion proteomics.

RpoS-Regulated Genes and Phenotypes in the Phytopathogenic Bacterium Pectobacterium atrosepticum.

The alternative sigma factor RpoS is considered to be one of the major regulators providing stress resistance and cross-protection in bacteria. In phytopathogenic bacteria, the effects of RpoS have not been analyzed with regard to cross-protection, and genes whose expression is directly or indirectly controlled by RpoS have not been determined at the whole-transcriptome level. Our study aimed to determine RpoS-regulated genes and phenotypes in the phytopathogenic bacterium Pectobacterium atrosepticum. Knockout of the rpoS gene in P. atrosepticum affected the long-term starvation response, cross-protection, and virulence toward plants with enhanced immune status. The whole-transcriptome profiles of the wild-type P. atrosepticum strain and its ΔrpoS mutant were compared under different experimental conditions, and functional gene groups whose expression was affected by RpoS were determined. The RpoS promoter motif was inferred within the promoter regions of the genes affected by rpoS deletion, and the P. atrosepticum RpoS regulon was predicted. Based on RpoS-controlled phenotypes, transcriptome profiles, and RpoS regulon composition, the regulatory role of RpoS in P. atrosepticum is discussed.

A Switch from Latent to Typical Infection during Pectobacterium atrosepticum-Tobacco Interactions: Predicted and True Molecular Players.

Phytopathogenic microorganisms, being able to cause plant diseases, usually interact with hosts asymptomatically, resulting in the development of latent infections. Knowledge of the mechanisms that trigger a switch from latent to typical, symptomatic infection is of great importance from the perspectives of both fundamental science and disease management. No studies to date have compared, at the systemic molecular level, the physiological portraits of plants when different infection types (typical and latent) are developed. The only phytopathogenic bacterium for which latent infections were not only widely described but also at least fluently characterized at the molecular level is Pectobacterium atrosepticum (Pba). The present study aimed at the comparison of plant transcriptome responses during typical and latent infections caused by Pba in order to identify and then experimentally verify the key molecular players that act as switchers, turning peaceful plant-Pba coexistence into a typical infection. Based on RNA-Seq, we predicted plant cell wall-, secondary metabolism-, and phytohormone-related genes whose products contributed to the development of the disease or provided asymptomatic plant-Pba interactions. By treatment tests, we confirmed that a switch from latent to typical Pba-caused infection is determined by the plant susceptible responses mediated by the joint action of ethylene and jasmonates.

Ultra-Fast Mass Spectrometry in Plant Biochemistry: Response of Winter Wheat Proteomics to Pre-Sowing Treatment with Iron Compounds.

In recent years, ultrafast liquid chromatography/mass spectrometry methods have been extensively developed for the use in proteome profiling in biochemical studies. These methods are intended for express monitoring of cell response to biotic stimuli and elucidation of correlation of molecular changes with biological processes and phenotypical changes. New technologies, including the use of nanomaterials, are actively introduced to increase agricultural production. However, this requires complex approbation of new fertilizers and investigation of mechanisms underlying the biotic effects on the germination, growth, and development of plants. The aim of this work was to adapt the method of ultrafast chromatography/mass spectrometry for rapid quantitative profiling of molecular changes in 7-day-old wheat seedlings in response to pre-sowing seed treatment with iron compounds. The used method allows to analyze up to 200 samples per day; its practical value lies in the possibility of express proteomic diagnostics of the biotic action of new treatments, including those intended for agricultural needs. Changes in the regulation of photosynthesis, biosynthesis of chlorophyll and porphyrin- and tetrapyrrole-containing compounds, glycolysis (in shoot tissues), and polysaccharide metabolism (in root tissues) were shown after seed treatment with suspensions containing film-forming polymers (PEG 400, Na-CMC, Na2-EDTA), iron (II, III) nanoparticles, or iron (II) sulfate. Observations at the protein levels were consistent with the results of morphometry, superoxide dismutase activity assay, and microelement analysis of 3-day-old germinated seeds and shoots and roots of 7-day-old seedlings. A characteristic molecular signature involving proteins participating in the regulation of photosynthesis and glycolytic process was suggested as a potential marker of the biotic effects of seed treatment with iron compounds, which will be confirmed in further studies.

Potential of Negative-Ion-Mode Proteomics: An MS1-Only Approach.

Current proteomics approaches rely almost exclusively on using the positive ionization mode, resulting in inefficient ionization of many acidic peptides. This study investigates protein identification efficiency in the negative ionization mode using the DirectMS1 method. DirectMS1 is an ultrafast data acquisition method based on accurate peptide mass measurements and predicted retention times. Our method achieves the highest rate of protein identification in the negative ion mode to date, identifying over 1000 proteins in a human cell line at a 1% false discovery rate. This is accomplished using a single-shot 10 min separation gradient, comparable to lengthy MS/MS-based analyses. Optimizing separation and experimental conditions was achieved by utilizing mobile buffers containing 2.5 mM imidazole and 3% isopropanol. The study emphasized the complementary nature of data obtained in positive and negative ion modes. Combining the results from all replicates in both polarities increased the number of identified proteins to 1774. Additionally, we analyzed the method's efficiency using different proteases for protein digestion. Among the four studied proteases (LysC, GluC, AspN, and trypsin), trypsin and LysC demonstrated the highest protein identification yield. This suggests that digestion procedures utilized in positive-mode proteomics can be effectively applied in the negative ion mode. Data are deposited to ProteomeXchange: PXD040583.

The Role of Intercellular Signaling in the Regulation of Bacterial Adaptive Proliferation.

Bacterial adaptation is regulated at the population level with the involvement of intercellular communication (quorum sensing). When the population density is insufficient for adaptation under starvation, bacteria can adjust it to a quorum level through cell divisions at the expense of endogenous resources. This phenomenon has been described for the phytopathogenic bacterium Pectobacterium atrosepticum (Pba), and it is called, in our study, adaptive proliferation. An important attribute of adaptive proliferation is its timely termination, which is necessary to prevent the waste of endogenous resources when the required level of population density is achieved. However, metabolites that provide the termination of adaptive proliferation remained unidentified. We tested the hypothesis of whether quorum sensing-related autoinducers prime the termination of adaptive proliferation and assessed whether adaptive proliferation is a common phenomenon in the bacterial world. We showed that both known Pba quorum sensing-related autoinducers act synergistically and mutually compensatory to provide the timely termination of adaptive proliferation and formation of cross-protection. We also demonstrated that adaptive proliferation is implemented by bacteria of many genera and that bacteria with similar quorum sensing-related autoinducers have similar signaling backgrounds that prime the termination of adaptive proliferation, enabling the collaborative regulation of this adaptive program in multispecies communities.

Yeast Ribonucleotide Reductase Is a Direct Target of the Proteasome and Provides Hyper Resistance to the Carcinogen 4-NQO.

Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals.

First genome-scale insights into the virulence of the snow mold causal fungus Microdochium nivale.

Pink snow mold, caused by a phytopathogenic and psychrotolerant fungus, Microdochium nivale, is a severe disease of winter cereals and grasses that predominantly occurs under snow cover or shortly after its melt. Snow mold has significantly progressed during the past decade, often reaching epiphytotic levels in northern countries and resulting in dramatic yield losses. In addition, M. nivale gradually adapts to a warmer climate, spreading to less snowy territories and causing different types of plant diseases throughout the growing period. Despite its great economic importance, M. nivale is poorly investigated; its genome has not been sequenced and its crucial virulence determinants have not been identified or even predicted. In our study, we applied a hybrid assembly based on Oxford Nanopore and Illumina reads to obtain the first genome sequence of M. nivale. 11,973 genes (including 11,789 protein-encoding genes) have been revealed in the genome assembly. To better understand the genetic potential of M. nivale and to obtain a convenient reference for transcriptomic studies on this species, the identified genes were annotated and split into hierarchical three-level functional categories. A file with functionally classified M. nivale genes is presented in our study for general use. M. nivale gene products that best meet the criteria for virulence factors have been identified. The genetic potential to synthesize human-dangerous mycotoxins (fumonisin, ochratoxin B, aflatoxin, and gliotoxin) has been revealed for M. nivale. The transcriptome analysis combined with the assays for extracellular enzymatic activities (conventional virulence factors of many phytopathogens) was carried out to assess the effect of host plant (rye) metabolites on the M. nivale phenotype. In addition to disclosing plant-metabolite-upregulated M. nivale functional gene groups (including those related to host plant protein destruction and amino acid metabolism, xenobiotic detoxication (including phytoalexins benzoxazinoids), cellulose destruction (cellulose monooxygenases), iron transport, etc.), the performed analysis pointed to a crucial role of host plant lipid destruction and fungal lipid metabolism modulation in plant-M. nivale interactions.

Host plant physiological transformation and microbial population heterogeneity as important determinants of the Soft Rot Pectobacteriaceae-plant interactions.

Pectobacterium and Dickeya species belonging to the Soft Rot Pectobacteriaceae (SRP) are one of the most devastating phytopathogens. They degrade plant tissues by producing an arsenal of plant cell wall degrading enzymes. However, SRP-plant interactions are not restricted to the production of these "brute force" weapons. Additionally, these bacteria apply stealth behavior related to (1) manipulation of the host plant via induction of susceptible responses and (2) formation of heterogeneous populations with functionally specialized cells. Our review aims to summarize current knowledge on SRP-induced plant susceptible responses and on the heterogeneity of SRP populations. The review shows that SRP are capable of adjusting the host's hormonal balance, inducing host-mediated plant cell wall modification, promoting iron assimilation by the host, stimulating the accumulation of reactive oxygen species and host cell death, and activating the synthesis of secondary metabolites that are ineffective in limiting disease progression. By this means, SRP facilitate host plant susceptibility. During host colonization, SRP populations produce various functionally specialized cells adapted for enhanced virulence, increased resistance, motility, vegetative growth, or colonization of the vascular system. This enables SRP to perform self-contradictory tasks, which benefits a population's overall fitness in various environments, including host plants. Such stealthy tactical actions facilitate plant-SRP interactions and disease progression.

Resistance to Snow Mold as a Target Trait for Rye Breeding.

Winter rye is a versatile crop widely used for food and industry. Although rye is resistant to abiotic stressors and many phytopathogens, it is severely damaged by pink snow mold (SM)-a progressive disease caused by the psychrotolerant fungus Microdochium nivale under the snow cover or during prolonged periods of wet and cool conditions. Due to little use of the SM resistance sources in contemporary breeding, varieties with at least moderate resistance to SM are limited. Our study aimed to integrate field assessment under natural conditions and an artificially enriched infection background with laboratory techniques for testing rye accessions and selecting SM resistant sources for applied breeding programs and genetic research. We revealed valuable sources of SM resistance and split rye accessions, according to the level of the genetic divergence of the SM resistance phenotype. This allowed us to select the most distinct donors of the SM resistance, for their use as parental forms, to include novel variability sources in the breeding program for achieving high genetic variability, as well as enhanced and durable SM resistance, in progeny. The rye accessions analyzed here, and the suggested options for their use in breeding, are valuable tools for rye breeding.

DirectMS1Quant: Ultrafast Quantitative Proteomics with MS/MS-Free Mass Spectrometry.

Recently, we presented the DirectMS1 method of ultrafast proteome-wide analysis based on minute-long LC gradients and MS1-only mass spectra acquisition. Currently, the method provides the depth of human cell proteome coverage of 2500 proteins at a 1% false discovery rate (FDR) when using 5 min LC gradients and 7.3 min runtime in total. While the standard MS/MS approaches provide 4000-5000 protein identifications within a couple of hours of instrumentation time, we advocate here that the higher number of identified proteins does not always translate into better quantitation quality of the proteome analysis. To further elaborate on this issue, we performed a one-on-one comparison of quantitation results obtained using DirectMS1 with three popular MS/MS-based quantitation methods: label-free (LFQ) and tandem mass tag quantitation (TMT), both based on data-dependent acquisition (DDA) and data-independent acquisition (DIA). For comparison, we performed a series of proteome-wide analyses of well-characterized (ground truth) and biologically relevant samples, including a mix of UPS1 proteins spiked at different concentrations into an Echerichia coli digest used as a background and a set of glioblastoma cell lines. MS1-only data was analyzed using a novel quantitation workflow called DirectMS1Quant developed in this work. The results obtained in this study demonstrated comparable quantitation efficiency of 5 min DirectMS1 with both TMT and DIA methods, yet the latter two utilized a 10-20-fold longer instrumentation time.

GIP Affects Hepatic Fat and Brown Adipose Tissue Thermogenesis but Not White Adipose Tissue Transcriptome in Type 1 Diabetes.

CONTEXT: Glucose-dependent insulinotropic polypeptide (GIP) has been proposed to exert insulin-independent effects on lipid and bone metabolism. OBJECTIVE: We investigated the effects of a 6-day subcutaneous GIP infusion on circulating lipids, white adipose tissue (WAT), brown adipose tissue (BAT), hepatic fat content, inflammatory markers, respiratory exchange ratio (RER), and bone homeostasis in patients with type 1 diabetes. METHODS: In a randomized, placebo-controlled, double-blind, crossover study, 20 men with type 1 diabetes underwent a 6-day continuous subcutaneous infusion with GIP (6 pmol/kg/min) and placebo (saline), with an interposed 7-day washout period. RESULTS: During GIP infusion, participants (26 ± 8 years [mean ± SD]; BMI 23.8 ± 1.8 kg/m2; glycated hemoglobin A1c 51 ± 10 mmol/mol [6.8 ± 3.1%]) experienced transiently increased circulating concentrations of nonesterified fatty acid (NEFA) (P = 0.0005), decreased RER (P = 0.009), indication of increased fatty acid ß-oxidation, and decreased levels of the bone resorption marker C-terminal telopeptide (P = 0.000072) compared with placebo. After 6 days of GIP infusion, hepatic fat content was increased by 12.6% (P = 0.007) and supraclavicular skin temperature, a surrogate indicator of BAT activity, was increased by 0.29 °C (P < 0.000001) compared with placebo infusion. WAT transcriptomic profile as well as circulating lipid species, proteome, markers of inflammation, and bone homeostasis were unaffected. CONCLUSION: Six days of subcutaneous GIP infusion in men with type 1 diabetes transiently decreased bone resorption and increased NEFA and ß-oxidation. Further, hepatic fat content, and supraclavicular skin temperature were increased without affecting WAT transcriptomics, the circulating proteome, lipids, or inflammatory markers.

Differential Activity of the Extracellular Phenoloxidases in Different Strains of the Phytopathogenic Fungus, Microdochium nivale.

To cause plant diseases, phytopathogenic fungi use numerous extracellular enzymes, among which, the phenoloxidases (POs) seem underestimated for the pathogens of non-woody plants. Our study aimed to (1) compare extracellular PO activities (lignin peroxidase, Mn peroxidase, laccase, and tyrosinase) in differentially virulent strains (inhabiting winter rye in a single field) of the phytopathogenic species, Microdochium nivale; (2) check whether these activities are responsive to host plant metabolites; and (3) search for correlations between the activities, lignin-decomposing capacity, and virulence. All strains displayed all four enzymatic activities, but their levels and dynamics depended on the particular strain. The activities displayed the hallmarks of co-regulation and responsiveness to the host plant extract. No relationships between the virulence of strains and levels of their extracellular PO activities or lignin-degrading capacity were revealed. We consider that different strains may rely on different POs for plant colonization, and that different POs contribute to the "uniqueness" of the enzymatic cocktails that are delivered into host plant tissues by different virulent strains of M. nivale. Our study supports the hypothesis of the differential behavior of closely related M. nivale strains, and discusses an important role of POs in the interactions of phytopathogens with herbaceous plants.

Structure-Functional Characteristics of the Svx Protein-The Virulence Factor of the Phytopathogenic Bacterium Pectobacterium atrosepticum.

The Svx proteins are virulence factors of phytopathogenic bacteria of the Pectobacterium genus. The specific functions of these proteins are unknown. Here we show that most of the phytopathogenic species of Pectobacterium, Dickeya, and Xanthomonas genera have genes encoding Svx proteins, as well as some plant-non-associated species of different bacterial genera. As such, the Svx-like proteins of phytopathogenic species form a distinct clade, pointing to the directed evolution of these proteins to provide effective interactions with plants. To get a better insight into the structure and functions of the Svx proteins, we analyzed the Svx of Pectobacterium atrosepticum (Pba)-an extracellular virulence factor secreted into the host plant cell wall (PCW). Using in silico analyses and by obtaining and analyzing the recombinant Pba Svx and its mutant forms, we showed that this protein was a gluzincin metallopeptidase. The 3D structure model of the Pba Svx was built and benchmarked against the experimental overall secondary structure content. Structure-based substrate specificity analysis using molecular docking revealed that the Pba Svx substrate-binding pocket might accept α-glycosylated proteins represented in the PCW by extensins-proteins that strengthen the PCW. Thus, these results elucidate the way in which the Pba Svx may contribute to the Pba virulence.

FLASHIda enables intelligent data acquisition for top-down proteomics to boost proteoform identification counts.

The detailed analysis and structural characterization of proteoforms by top-down proteomics (TDP) has gained a lot of interest in biomedical research. Data-dependent acquisition (DDA) of intact proteins is non-trivial due to the diversity and complexity of proteoforms. Dedicated acquisition methods thus have the potential to greatly improve TDP. Here, we present FLASHIda, an intelligent online data acquisition algorithm for TDP that ensures the real-time selection of high-quality precursors of diverse proteoforms. FLASHIda combines fast charge deconvolution algorithms and machine learning-based quality assessment for optimal precursor selection. In an analysis of E. coli lysate, FLASHIda increases the number of unique proteoform level identifications from 800 to 1500 or generates a near-identical number of identifications in one third of the instrument time when compared to standard DDA mode. Furthermore, FLASHIda enables sensitive mapping of post-translational modifications and detection of chemical adducts. As a software extension module to the instrument, FLASHIda can be readily adopted for TDP studies of complex samples to enhance proteoform identification rates.

Proteomic analysis reveals stage-specific reprogramed metabolism for the primary breast cancer cell lines MGSO-3 and MACL-1.

Breast cancer is the most prevalent cancer in women worldwide. Its molecular subtypes are based on the presence/absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). MACL-1 and MGSO-3 are cell lines derived from primary tumor sites of patients diagnosed with luminal A subtype carcinoma (ER+/PR+/HER2-) and ductal carcinoma in situ (ER-/PR-/HER2+), respectively. However, these cell lines lost the expression of these markers over cell culturing, and both have triple-negative phenotypes (ER-/PR-/HER2-), which has the poorest prognosis. Here, we sought to study the proteome signature of MGSO-3 and MACL-1, comparing them with the epithelial cell line MCF-10A and the well-established metastatic-derived breast cancer cell line MDA-MB-231. Our results showed that proteins associated with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) were upregulated in MGSO-3 and MACL-1 cells. These cell lines also showed upregulation of pro-apoptotic proteins when compared with MDA-MB-231. The molecular differences highlighted in this study may clarify the molecular basis behind cancer cells functioning and may reveal novel signatures across the breast cancer cell models.

Phosphoproteomic studies of alamandine signaling in CHO-MrgD and human pancreatic carcinoma cells: An antiproliferative effect is unveiled.

Alamandine is a heptapeptide from the renin-angiotensin system (RAS) with similar structure/function to angiotensin-(1-7) [ang-(1-7)], but they act via different receptors. It remains elusive whether alamandine is an antiproliferative agent like ang-(1-7). The goal of this study was to evaluate the potential antiproliferative activity of alamandine and the underlying cellular signaling. We evaluated alamandine effect in the tumoral cell lines Mia PaCa-2 and A549, and in the nontumoral cell lines HaCaT, CHO and CHO transfected with the alamandine receptor MrgD (CHO-MrgD). Alamandine was able to reduce the proliferation of the tumoral cell lines in a MrgD-dependent fashion. We did not observe any effect in the nontumoral cell lines tested. We also performed proteomics and phosphoproteomics to study the alamandine signaling in Mia PaCa-2 and CHO-MrgD. Data suggest that alamandine induces a shift from anaerobic to aerobic metabolism in the tumoral cells, induces a negative regulation of PI3K/AKT/mTOR pathway and activates the transcriptional factor FoxO1; events that could explain, at least partially, the observed antiproliferative effect of alamandine. This study provides for the first time a comprehensive investigation of the alamandine signaling in tumoral (Mia PaCa-2) and nontumoral (CHO-MrgD) cells, highlighting the antiproliferative activity of alamandine/MrgD and its possible antitumoral effect.

Characterization of Differentially Abundant Proteins Among Leishmania (Viannia) braziliensis Strains Isolated From Atypical or Typical Lesions.

Leishmania (Viannia) braziliensis is the main etiological agent of cutaneous and mucocutaneous leishmaniasis in Latin America. Non-ulcerated atypical tegumentary leishmaniasis cases caused by L. braziliensis have been reported in several regions of the American continent, including the Xacriabá indigenous reserve in São João das Missões/Minas Gerais, Brazil. Parasites isolated from these atypical clinical lesions are resistant to antimony-based therapeutics. In the present study, proteins displaying differential abundance in two strains of L. braziliensis isolated from patients with atypical lesions compared with four strains isolated from patients with typical lesions were identified using a quantitative proteomics approach based on tandem mass tag labeling (TMT) and mass spectrometry. A total of 532 (P<0.05) differentially abundant proteins were identified (298 upregulated and 234 downregulated) in strains from atypical lesions compared to strains from typical lesions. Prominent positively regulated proteins in atypical strains included those that may confer greater survival inside macrophages, proteins related to antimony resistance, and proteins associated with higher peroxidase activity. Additionally, we identified proteins showing potential as new drug and vaccine targets. Our findings contribute to the characterization of these intriguing L. braziliensis strains and provide a novel perspective on Atypical Cutaneous Leishmaniasis (ACL) cases that have been associated with therapeutic failures.

DFT Quantum-Chemical Calculation of Thermodynamic Parameters and DSC Measurement of Thermostability of Novel Benzofuroxan Derivatives Containing Triazidoisobutyl Fragments.

New derivatives of benzofuroxan containing triazidoisobutyl fragments, opening the way for the creation of highly effective compositions with an increased value of energy characteristics, were synthesized for the first time. Such compounds are also an excellent platform for further modification and for the preparation of new biologically-active compounds containing tetrazole and triazole fragments. Calculations of heats of formation performed with the DFT (density functional theory) method showed that the studied compounds are high-energetic density ones, the enthalpies of formation of which are comparable to the enthalpies of formation of similar benzofuroxan derivatives and exceeds experimental enthalpy of formation of CL-14 (5,7-diamino-4,6-dinitrobenzofuroxan). The analysis of DSC indicates a sufficiently high thermal stability of the synthesized azidobenzofuroxans, which are acceptable for their use as components in the creation of highly efficient compositions with an increased value of energy characteristics.