In Vitro Selection of Lactobacillus and Bifidobacterium Probiotic Strains for the Management of Oral Pathobiont Infections Associated to Systemic Diseases

The human oral pathobionts Aggregatibacter actinomycetemcomitans, Streptococcus mitis and Streptococcus mutans, in dysbiosis-promoting conditions, lead to oral infections, which also represent a threat to human systemic health. This scenario may be worsened by antibiotic misuse, which favours multi-drug resistance, making the research on pathogen containment strategies more than crucial. Therefore, we aimed to in vitro select the most promising probiotic strains against oral pathogen growth, viability, biofilm formation, and co-aggregation capacity, employing both the viable probiotics and their cell-free supernatants (CFSs). Interestingly, we also assessed probiotic efficacy against the three-pathogen co-culture, mimicking an environment similar to that in vivo. Overall, the results showed that Lactobacillus CFSs performed better than the Bifidobacterium, highlighting Limosilactobacillus reuteri LRE11, Lacticaseibacillus rhamnosus LR04, Lacticaseibacillus casei LC04, and Limosilactobacillus fermentum LF26 as the most effective strains, opening the chance to deeper investigation of their action and CFS composition. Altogether, the methodologies presented in this study can be used for probiotic efficacy screenings, in order to better focus the research on a viable probiotic, or on its postbiotics, suitable in case of infections.     

Potential Biomarkers in Diagnosis of Renal Acanthamoebiasis

Recent studies indicate that Acanthamoeba spp. may play a significant role in kidney dysfunction. The aim of the study was to examine the levels of kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and monocyte chemotactic protein 1 (MCP-1), as well as an activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9, respectively) in the kidneys of immunocompetent and immunosuppressed mice infected with Acanthamoeba spp. The levels of KIM-1, NGAL, and MCP-1 were analyzed by enzyme-linked immunosorbent assay (ELISA), and the activity of MMPs was determined by gelatin zymography. The elevated KIM-1 level was found in the kidneys of immunocompetent mice at the beginning of Acanthamoeba spp. infection. In the immunosuppressed mice, the KIM-1 level was statistically different. The statistically decreased NGAL level was found in the kidneys of immunocompetent mice compared to the uninfected mice. In the immunocompromised mice, we found statistically significant differences in MCP-1 levels between the uninfected and infected groups. There was an increase in the expression of both MMP-2 and MMP-9 in the kidneys of immunocompetent and immunosuppressed mice infected with Acanthamoeba spp. compared to the uninfected mice. The results indicate that KIM-1, NGAL, MCP-1, MMP-2, MMP-9, and MMP-9/NGAL might be promising biomarkers of renal acanthamoebiasis.     

Genetic Disruption of Toxoplasma gondii peroxiredoxin (TgPrx) 1 and 3 Reveals the Essential Role of TgPrx3 in Protecting Mice from Fatal Consequences of Toxoplasmosis

Toxoplasma gondii is a worldwide protozoan parasite that endangers human health and causes enormous economic losses to the animal production sector. A safe and effective vaccine or treatment is needed to reduce these hazards. In this study, we revealed the cyto-nuclear and mitochondrial localization of TgPrx1 and TgPrx3 proteins, respectively. We knocked out the T. gondii peroxiredoxin (TgPrxKO) 1 and 3 genes using a parental type II Prugniaud strain lacking KU80 and HXGPRT genes (PruΔku80Δhxgprt) via CRISPR-Cas9 technology. The successful KO was confirmed using PCR, IFAT, and Western blotting in two clones of both target genes, named TgPrx1KO and TgPrx3KO. Regarding in vitro assays, no significant variations between any of the knocked-out clones in TgPrx1KO or TgPrx3KO parasite strains, or even PruΔku80Δhxgprt, were obtained in rates of infection, proliferation, or egress. Nevertheless, mice that were infected with tachyzoites of the TgPrx3KO strain showed a marked decrease in survival rate compared with TgPrx1KO- and PruΔku80Δhxgprt-infected mice. This effect was confirmed using different mouse strains (ICR and C57BL/6J mice), sexes (male and female), and immunological backgrounds (ICR and SCID mice). In addition, TgPrx1KO and TgPrx3KO induced high levels of interferon gamma (IFN-γ) in infected mice at 8 days post infection, and increased IL-6 and IL-12p40 production from murine macrophages cultivated in vitro. The results of the present study suggested that TgPrx3 can induce anti-T. gondii immune responses that protect the mice from fatal consequences of toxoplasmosis. The results of our current and previous studies represent TgPrx3 as an excellent candidate for sub-unit vaccines, suggesting it may contribute to the control of toxoplasmosis for susceptible humans and animals.     

Development of the Chromatographic Method for Simultaneous Determination of Azaperone and Azaperol in Animal Kidneys and Livers

A precise and accurate method for the simultaneous determination of azaperone and azaperol in meat tissues has been developed. This paper describes the first method to be so fast, simple, and useful, especially for many laboratories that do not have sophisticated equipment. This method is based on LC separation and UV-Vis detection. During the sample preparation, the meat tissue was homogenized in acetonitrile at a ratio of 1:4 (tissue weight:acetonitrile volume). The homogenate was centrifuged, the supernatant was evaporated in a lyophilizator, and then the evaporation residue was dissolved in 20 µL of ethanol. For deproteinization, 15 µL of perchloric acid was added, and the sample prepared in this way was injected into a chromatographic column and analyzed using reversed-phased HPLC. The mobile phase consisted of 0.05 mol/L phosphate buffer pH 3.00 (component A) and acetonitrile (component B). UV detection was conducted at 245 nm. The experimentally determined LOQs were 0.25 µg/kg for azaperone and 0.12 µg/kg for azaperol. For both analytes, the calibration curves showed linearity in the tested concentration range from 50 to 300 µg/kg of tissue. The accuracy of the presented method did not exceed 15%, and the recovery was in the range of 85–115%. A validated analytical procedure was implemented for the analysis of various animal tissues for their content of azaperone and azaperol.     

Toxoplasma gondii GRA9 Regulates the Activation of NLRP3 Inflammasome to Exert Anti-Septic Effects in Mice

Dense granule proteins (GRAs) are essential components in Toxoplasma gondii, which are suggested to be promising serodiagnostic markers in toxoplasmosis. In this study, we investigated the function of GRA9 in host response and the associated regulatory mechanism, which were unknown. We found that GRA9 interacts with NLR family pyrin domain containing 3 (NLRP3) involved in inflammation by forming the NLRP3 inflammasome. The C-terminal of GRA9 (GRA9C) is essential for GRA9–NLRP3 interaction by disrupting the NLRP3 inflammasome through blocking the binding of apoptotic speck-containing (ASC)-NLRP3. Notably, Q200 of GRA9C is essential for the interaction of NLRP3 and blocking the conjugation of ASC. Recombinant GRA9C (rGRA9C) showed an anti-inflammatory effect and the elimination of bacteria by converting M1 to M2 macrophages. In vivo, rGRA9C increased the anti-inflammatory and bactericidal effects and subsequent anti-septic activity in CLP- and E. coli- or P. aeruginosa-induced sepsis model mice by increasing M2 polarization. Taken together, our findings defined a role of T. gondii GRA9 associated with NLRP3 in host macrophages, suggesting its potential as a new candidate therapeutic agent for sepsis.     

Neurotrophic Factors in Experimental Cerebral Acanthamoebiasis

To date, no studies have addressed the role of neurotrophins (NTs) in Acanthamoeba spp. infections in the brain. Thus, to clarify the role of NTs in the cerebral cortex and hippocampus during experimental acanthamoebiasis in relation to the host immune status, the purpose of this study was to determine whether Acanthamoeba spp. may affect the concentration of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) in brain structures. Our results suggest that at the beginning of infection in immunocompetent hosts, BDNF and NT-3 may reflect an endogenous attempt at neuroprotection against Acanthamoeba spp. infection. We also observed a pro-inflammatory effect of NGF during acanthamoebiasis in immunosuppressed hosts. This may provide important information for understanding the development of cerebral acanthamoebiasis related to the immunological status of the host. However, the pathogenesis of brain acanthamoebiasis is still poorly understood and documented and, therefore, requires further research.     

Evidence of G-Protein-Coupled Receptors (GPCR) in the Parasitic Protozoa Plasmodium falciparum—Sensing the Host Environment and Coupling within Its Molecular Signaling Toolkit

Throughout evolution, the need for single-celled organisms to associate and form a single cluster of cells has had several evolutionary advantages. In complex, multicellular organisms, each tissue or organ has a specialty and function that make life together possible, and the organism as a whole needs to act in balance and adapt to changes in the environment. Sensory organs are essential for connecting external stimuli into a biological response, through the senses: sight, smell, taste, hearing, and touch. The G-protein-coupled receptors (GPCRs) are responsible for many of these senses and therefore play a key role in the perception of the cells’ external environment, enabling interaction and coordinated development between each cell of a multicellular organism. The malaria-causing protozoan parasite, Plasmodium falciparum, has a complex life cycle that is extremely dependent on a finely regulated cellular signaling machinery. In this review, we summarize strong evidence and the main candidates of GPCRs in protozoan parasites. Interestingly, one of these GPCRs is a sensor for K⁺ shift in Plasmodium falciparum, PfSR25. Studying this family of proteins in P. falciparum could have a significant impact, both on understanding the history of the evolution of GPCRs and on finding new targets for antimalarials.     

Metabolomics Analyses Reveal Metabolites Affected by Plant Growth-Promoting Endophytic Bacteria in Roots of the Halophyte Mesembryanthemum crystallinum

Mesembryanthemum crystallinum L. (common ice plant) is an edible halophyte. However, if ice plants are used to phytoremediate salinity soil, there are problems of slow initial growth, and a long period before active NaCl uptake occurs under higher salinity conditions. Application of endophytic bacteria may improve the problem, but there remain gaps in our understanding of how endophytic bacteria affect the growth and the biochemical and physiological characteristics of ice plants. The aims of this study were to identify growth-promoting endophytic bacteria from the roots of ice plants and to document the metabolomic response of ice plants after application of selected endophytic bacteria. Two plant growth-promoting endophytic bacteria were selected on the basis of their ability to promote ice plant growth. The two strains putatively identified as Microbacterium spp. and Streptomyces spp. significantly promoted ice plant growth, at 2-times and 2.5-times, respectively, compared with the control and also affected the metabolome of ice plants. The strain of Microbacterium spp. resulted in increased contents of metabolites related to the tricarboxylic acid cycle and photosynthesis. The effects of salt stress were alleviated in ice plants inoculated with the endobacterial strains, compared with uninoculated plants. A deeper understanding of the complex interplay among plant metabolites will be useful for developing microbe-assisted soil phytoremediation strategies, using Mesembryanthemum species.     

Rhizopus and Fusarium are Selected as Dominant Fungal Genera in Rhizospheres of Brassicaceae

We isolated several strains of Rhizopus and Fusarium spp. as dominant fungi in the rhizospheres of Brassicaceae plants. The Fusarium isolates showed a higher tolerance of the antifungal constituents of "mustard oil," which originates from the glucosinolates that are characteristic secondary metabolites of the Brassicaceae, than other Fusarium isolates from non-Brassicaceae plants. In contrast, the Rhizopus isolates showed a high tolerance regardless of their source. Myrosinase activity was found in Bn-R-1-1 (Rhizopus sp.) isolated from the rhizoplane of Brassica napus and Ls-F-in-4-1 (Fusarium sp.) isolated from a surface-disinfected root of Lepidium sativum (Brassicaceae). Ls-F-in-4-1 was the Fusarium most tolerant of the Brassicaceae antifungal constituents. These results suggest that fungi in the rhizospheres of Brassicaceae plants may be selected because of secondary metabolites exuded from the roots of host plants.     

光谱法在菌液活性检测中的初步应用

[目的]为了明确菌液的理化性质与杀线活性之间的关系.[方法]采用紫外吸收光谱法初步探索了利用物理化学方测定杀线虫活性的可能性.[结果]将菌液紫外光谱图分析结果与该组分的生物测定结果进行初步比较,显示波长在291 nm处的吸光度值与杀线虫活性生物测定的结果之间存在较好的相关性.     

The Role of Plant Hormones in the Interaction of Colletotrichum Species with Their Host Plants

Colletotrichum is a plant pathogenic fungus which is able to infect virtually every economically important plant species. Up to now no common infection mechanism has been identified comparing different plant and Colletotrichum species. Plant hormones play a crucial role in plant-pathogen interactions regardless whether they are symbiotic or pathogenic. In this review we analyze the role of ethylene, abscisic acid, jasmonic acid, auxin and salicylic acid during Colletotrichum infections. Different Colletotrichum strains are capable of auxin production and this might contribute to virulence. In this review the role of different plant hormones in plant—Colletotrichum interactions will be discussed and thereby auxin biosynthetic pathways in Colletotrichum spp. will be proposed.     

Potential of Silver Nanoparticles in Overcoming the Intrinsic Resistance of Pseudomonas aeruginosa to Secondary Metabolites from Carnivorous Plants

Carnivorous plants are exemplary natural sources of secondary metabolites with biological activity. However, the therapeutic antimicrobial potential of these compounds is limited due to intrinsic resistance of selected bacterial pathogens, among which Pseudomonas aeruginosa represents an extreme example. The objective of the study was to overcome the intrinsic resistance of P. aeruginosa by combining silver nanoparticles (AgNPs) with secondary metabolites from selected carnivorous plant species. We employed the broth microdilution method, the checkerboard titration technique and comprehensive phytochemical analyses to define interactions between nanoparticles and active compounds from carnivorous plants. It has been confirmed that P. aeruginosa is resistant to a broad range of secondary metabolites from carnivorous plants, i.e., naphthoquinones, flavonoids, phenolic acids (MBC = 512 µg mL⁻¹) and only weakly sensitive to their mixtures, i.e., extracts and extracts’ fractions. However, it was shown that the antimicrobial activity of extracts and fractions with a significant level of naphthoquinone (plumbagin) was significantly enhanced by AgNPs. Our studies clearly demonstrated a crucial role of naphthoquinones in AgNPs and extract interaction, as well as depicted the potential of AgNPs to restore the bactericidal activity of naphthoquinones towards P. aeruginosa. Our findings indicate the significant potential of nanoparticles to modulate the activity of selected secondary metabolites and revisit their antimicrobial potential towards human pathogenic bacteria.     

Venom chemistry of ants in the genusMonomorium

A comparative analysis of the venomous alkaloids produced by ant species in the subgenusMonomorium of the genusMonomorium has been undertaken. All species produce mixtures of unsymmetricaltrans-2,5-dialkylpyrrolidines, but the proportions of the constituents may vary considerably between species. All alkaloids contain both C₆ and C₉ side chains which are present as C₉-saturated. C₆-monounsaturated, and both C₆-and C₉-monounsaturated dialkylpyrrolidines. The structure of 2-(1-hex-5-enyl)-5-(1-non-8-enyl)pyrrolidine, a previously undescribed alkaloid, was proved by unambiguous synthesis after the location of the double bonds was established by the methoxymercuration-demercuration followed by mass spectrometry. The possible chemotaxonomic significance of the mixtures of venomous alkaloids produced by these species ofMonomorium is discussed.     

Dufour's gland and its role in secretion of nest cell lining in bees of the genusColletes (Hymenoptera: Colletidae)

The liquid secretion of the Dufour's gland in mated females comprises a mixture of macrocyclic lactones, straight chain mono- and dicarboxylic acids, together with trace amounts of di- and trihydroxy monocarboxylic acids and a series ofn-alkanes. Polymerization of the liquid to form the membranous nest cell lining, which also contains alanine and glutamic acid as major constituents, may be mediated by an enzyme from the thoracic salivary gland and voided via the mouth. The composition and structure of the cell linings are similar to those of Wigglesworth's cuticulin.     

Volatile compounds from crabapple (Malus spp.) cultivars differing in susceptibility to the Japanese beetle (Popillia japonica Newman)

The volatile compounds emitted by leaves of four crabapple cultivars susceptible to damage by Japanese beetles and four relatively resistant cultivars were examined. Twelve compounds, mostly terpene hydrocarbons, were identified from intact leaves. The terpenes (E)--ocimene, caryophylene, germacrene D and (E,E)--farnesene occurred in significantly higher levels in susceptible cultivars, whereas resistant cultivars produced greater amounts of (E)-4,8-dimethyl 1,3,7-nonatriene and linalool. The relative attractiveness of the cultivars as determined in a pitfall bioassay, however, was not related to their susceptibility to the Japanese beetle as previously determined by defoliation sustained in the field. The attractiveness of individual cultivars was found to be positiviely correlated with linalool as a percent of the total volatile blend emitted by leaves. This study and previous work suggest that variation in susceptibility of crabapple cultivars to defoliation by Japanese beetles is not due to the attractiveness of the individual cultivars but rather to nonvolatile components of susceptibility and/or resistance. A scenario for host location by the Japanese beetle is presented.     

The triglyceride composition,structure, and presence of estolides in the oils ofLesquerella and related species

Members of the genusLesquerella, native to North America, have oils containing large amounts of hydroxy fatty acids and are under investigation as potential new crops. The triglyceride structure of oils from twenty-fiveLesquerella species in the seed collection at our research center has been examined after being hydrolysis-catalyzed by reverse micellar-encapsulated lipase and alcoholysis-catalyzed by immobilized lipase. These reactions, when coupled with supercritical-fluid chromatographic analysis, provide a powerful, labor-saving method for oil triglyceride analysis. A comprehensive analysis of overall fatty acid composition of these oils has been conducted as well.Lesquerella oils (along with oils from two other Brassicaceae:Physaria floribunda andHeliophilia amplexicaulis) have been grouped into five categories: densipolic acid-rich (Class I); auricolic acid-rich (Class II); lesquerolic acid-rich (Class III); an oil containing a mixture of hydroxy acids (Class IV); and lesquerolic and erucic acid-rich (Class V). The majority of Class I and II triglycerides contain one or two monoestolides at the 1- and 3-glycerol positions and a C₁₈ polyunsaturated acyl group at the 2-position. Most Class III and IV oil triglycerides contain one or two hydroxy acids at the 1- and 3-positions and C₁₈ unsaturated acid at the 2-position. A few of the Class III oils have trace amounts of estolides. The Class V oil triglycerides are mostly pentaacyl triglycerides and contain monestolide and small amounts of diestolide. Our triglyceride structure assignments were supported by¹H nuclear magnetic resonance data and mass balances.     

Response of the clerid predatorThanasimus dubius (F.) to bark beetle pheromones and tree volatiles in a wind tunnel

Tree volatiles and pheromones produced by southern bark beetles were bioassayed for response by the clerid predatorThanasimus dubius (F.). Upwind flights in a laboratory olfactometer, modified from Visser (1976), were used to determine the attractiveness of compounds. Differences in response to a solvent control and pheromone treatment were tested for statistical significance using the Wilcoxon signed ranks test. Both sexes ofT. dubius responded to frontalin, ipsdienol, and -pinene in a dose-dependent manner with different but overlapping concentration ranges. Strong differences between the sexes were observed in response totrans-verbenol, verbenone, andl--pinene. Neither sex responded to ipsenol orendo-brevicomin.Miss. Agric. and For. Exp. Sta. Publication No. 5251.