In vitro and in vivo efficacy of rosmarinic acid on quorum sensing mediated biofilm formation and virulence factor production in Aeromonas hydrophila

Rosmarinic acid (RA) was assessed for its quorum sensing inhibitory (QSI) potential against Aeromonas hydrophila strains AH 1, AH 12 and MTCC 1739. The pathogenic strains of A. hydrophila were isolated from infected zebrafish and identified through biochemical analysis and amplification of a species-specific gene (rpsL). The biofilm inhibitory concentration (BIC) of RA against A. hydrophila strains was found to be 750 μg ml^?1. At this concentration, RA reduced the QS mediated hemolysin, lipase and elastase production in A. hydrophila. In FT-IR analysis, RA treated A. hydrophila cells showed a reduction in cellular components. Gene expression analysis confirmed the down-regulation of virulence genes such as ahh1, aerA, lip and ahyB. A. hydrophila infected zebrafish upon treatment with RA showed increased survival rates. Thus, the present study demonstrates the use of RA as a plausible phytotherapeutic compound to control QS mediated biofilm formation and virulence factor production in A. hydrophila.     

Effect of glucose on Listeria monocytogenes biofilm formation,and assessment of the biofilm’s sanitation tolerance

Listeria monocytogenes is an important cause of human foodborne infections and its ability to form biofilms is a serious concern to the food industry. To reveal the effect of glucose conditions on biofilm formation of L. monocytogenes, 20 strains were investigated under three glucose conditions (0.1, 1.0, and 2.0% w v^–1) by quantifying the number of cells in the biofilm and observing the biofilm structure after incubation for 24, 72, and 168 h. In addition, the biofilms were examined for their sensitivity to sodium hypochlorite. It was found that high concentrations of glucose reduced the number of viable cells in the biofilms and increased extracellular polymeric substance production. Moreover, biofilms formed at a glucose concentration of 1.0 or 2.0% were more resistant to sodium hypochlorite than those formed at a glucose concentration of 0.1%. This knowledge can be used to help design the most appropriate sanitation strategy.     

In vitro effect of pesticides on the germination, vegetative growth, and conidial production of two strains of Metarhizium anisopliae

Entomopathogenic fungi are widely used as biological control agents against a broad range of insect and arachnid pests. However, the control efficacy of entomopathogenic fungi is variable because of unfavourable and fluctuating environmental conditions and intrinsic factors. One strategy to enhance entomopathogenic fungi efficacy is a combined use of entomopathogenic fungi and low dosages of pesticides. These sub-lethal dosages of chemicals can increase the control efficiency of entomopathogenic fungi but only if they do not affect the fungi. Adverse effects could include the inhibition of germination and/or vegetative growth as well as conidiogenesis. The present study investigated the in vitro effects of different concentrations of fipronil, permethrin, imidacloprid, NeemAzal, and amitraz as potential candidates for combined applications on two strains of the entomopathogenic fungus Metarhizium anisopliae (MA). MA was inoculated on a medium amended with five different concentrations (0.32-200 ppm) of the abovementioned pesticides. The germination, vegetative growth, and sporulation were evaluated. The results showed, according to a physiology parameter compatibility classification, that all pesticides were compatible with both tested MA strains. Only fipronil in the higher dose rates of 40 and 200 ppm was close to moderately toxic to MA-7. Furthermore, only higher concentrations of the pesticides caused a slight inhibition (about 15%) of conidial germination and a reduction in colony size. Sporulation was reduced at most by approximately 50% by 40 or 200 ppm of fipronil or amitraz, respectively. Therefore, it is possible to use the tested pesticides in combination with either strain of MA for an integrated pest management approach. Studies on the effect of these combinations on target organisms are in progress.     

In vitro ectomycorrhization of Tricholoma matsutake strains is differentially affected by soil type

The Japanese delicacy Tricholoma matsutake has been conducted in vitro ectomycorrhizal syntheses for more than 20 y. The development of its ectomycorrhizal structures varies among experimental systems. Here, we examined the effects of soil-fungus interactions on the early stage of in vitro T. matsutake ectomycorrhization. Axenic Pinus densiflora seedlings were transplanted into autoclaved natural inorganic soil, inoculated with the cultured mycelium of T. matsutake, and incubated for 90 d in vitro. Both soil type and fungal strain significantly affected host plant growth; host plant growth and mycorrhization levels significantly differed among soil type/fungal strain combinations. Therefore, the selection of T. matsutake strains for optimal mycorrhization must take into account such fungal and soil properties.     

In vitro complex formation between bacteriophage φ29 terminal protein and deoxynucleotide

It has previously been shown that the 5′-terminal deoxyadenosine residue of each φ29 DNA strand is linked covalently to the 30,000 dalton terminal protein. When extracts prepared from φ29-infected$\text{Bacillus}\text{subtilis}$ cells are incubated with [α-³²p]dATP, complexes consisting of the nucleotide covalently linked to a 30,000 dalton protein can be detected. The formation of this complex requires the presence of φ29 DNA containing the bound 30,000 dalton terminal protein and Mg⁺⁺. When uninfected cell extracts were used, there was no complex formation. When [α-³²p]dCTP was used in place of [α-³²p]dATP, no complex was formed. DNA-protein templates prepared from φ29 related phages, φ15, Nf, M2Y and GA-1, also supported the complex formation in various degrees. These results support the hypothesis that the terminal protein serves as a primer for the initiation of φ29 DNA replication.     

In vitro evaluation of single- and multi-strain probiotics: Inter-species inhibition between probiotic strains, and inhibition of pathogens

Many studies comparing the effects of single- and multi-strain probiotics on pathogen inhibition compare treatments with different concentrations. They also do not examine the possibility of inhibition between probiotic strains with a mixture. We tested the ability of 14 single-species probiotics to inhibit each other using a cross-streak assay, and agar spot test. We then tested the ability of 15 single-species probiotics and 5 probiotic mixtures to inhibit Clostridium difficile, Escherichia coli and S. typhimurium, using the agar spot test. Testing was done with mixtures created in two ways: one group contained component species incubated together, the other group of mixtures was made using component species which had been incubated separately, equalised to equal optical density, and then mixed in equal volumes. Inhibition was observed for all combinations of probiotics, suggesting that when used as such there may be inhibition between probiotics, potentially reducing efficacy of the mixture. Significant inter-species variation was seen against each pathogen. When single species were tested against mixtures, the multi-species preparations displayed significantly (p < 0.05 or less) greater inhibition of pathogens in 12 out of 24 cases. Despite evidence that probiotic species will inhibit each other when incubated together in vitro, in many cases a probiotic mixture was more effective at inhibiting pathogens than its component species when tested at approximately equal concentrations of biomass. This suggests that using a probiotic mixture might be more effective at reducing gastrointestinal infections, and that creating a mixture using species with different effects against different pathogens may have a broader spectrum of action that a single provided by a single strain.     

In vitro inhibition of monkeypox virus production and spread by Interferon-β

Background

Despite inducing a sustained increase in CD4+ T cell counts, intermittent recombinant IL-2 (rIL-2) therapy did not confer a better clinical outcome in HIV-infected patients enrolled in large phase III clinical trials ESPRIT and SILCAAT. Several hypotheses were evoked to explain these discrepancies. Here, we investigated the impact of low and high doses of IL-2 in Rhesus macaques of Chinese origin infected with SIVmac251 in the absence of antiretroviral therapy (ART).

Results

We demonstrated that rIL-2 induced a dose dependent expansion of CD4+ and CD8+ T cells without affecting viral load. rIL-2 increased CD4 and CD8 Treg cells as defined by the expression of CD25^highFoxP3⁺CD127^low. We also showed that rIL-2 modulated spontaneous and Fas-mediated CD4⁺ and CD8⁺ T cell apoptosis. The higher dose exhibited a dramatic pro-apoptotic effect on both CD4⁺ and CD8⁺ T cell populations. Finally, all the animals treated with rIL-2 developed a wasting syndrome in the month following treatment simultaneously to a dramatic decrease of circulating effector T cells.

Conclusion

These data contribute to the understanding of the homeostatic and dosage effects of IL-2 in the context of SIV/HIV infection.     

Overview — In vitro inhibition of aldehyde dehydrogenase by disulfiram and metabolites

Disulfiram (DSF) has found extensive use in the aversion therapy treatment of recovering alcoholics. It is known that DSF or a metabolite irreversibly inhibits aldehyde dehydrogenase (ALDH). However, the actual mechanism of inhibition is still not known. In this work we describe the in vitro interactions of DSF, as well as a principal metabolite S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), with both recombinant rat liver mitochondrial monomeric ALDH (rmALDH) and homotetrameric rmALDH. We show that DSF directly inhibits rmALDH (IC₅₀=36.4 μM) by inducing the formation of an intramolecular disulfide bond. We also demonstrate by HPLC-MS analysis of a Glu-C digest of DSF-treated rmALDH that the intramolecular disulfide bridge formed involves two of the three cysteines located at the active site of the enzyme. Using a combination of HPLC-MS and HPLC-MS/MS, we further show that the electrophilic metabolite MeDTC-SO also inhibits rmALDH (IC₅₀=4.62 μM). We isolate and identify a carbamoylated peptide at Cys₃₀₂ with the sequence FNQGQC₃₀₁C₃₀₂C₃₀₃. Hence we show that MeDTC-SO exhibits its inhibitory effect by covalently modifying the -SH side-chain of Cys₃₀₂, present at the active site rmALDH. Finally we show using SEC-MS that both DSF and MeDTC-SO do not prevent formation of the homotetramer of rmALDH, but inhibit the enzyme by acting directly at the active site of specific monomers of rmALDH.     

Hydralazine modifies Aβ fibril formation and prevents modification by lipids in vitro

Lipid oxidative damage and amyloid β (Aβ) misfolding contribute to Alzheimer's disease (AD) pathology. Thus, the prevention of oxidative damage and Aβ misfolding are attractive targets for drug discovery. At present, no AD drugs approved by the Food and Drug Administration (FDA) prevent or halt disease progression. Hydralazine, a smooth muscle relaxant, is a potential drug candidate for AD drug therapy as it reduces Aβ production and prevents oxidative damage via its antioxidant hydrazide group. We evaluated the efficacy of hydralazine, and related hydrazides, in reducing (1) Aβ misfolding and (2) Aβ protein modification by the reactive lipid 4-hydroxy-2-nonenal (HNE) using transmission electron microscopy and Western blotting. While hydralazine did not prevent Aβ aggregation as measured using the protease protection assay, there were more oligomeric species observed by electron microscopy. Hydralazine prevented lipid modification of Aβ, and Aβ was used as a proxy for classes of proteins which either misfold or are modified by HNE. All of the other hydrazides prevented lipid modification of Aβ and also did not prevent Aβ aggregation. Surprisingly, a few of the compounds, carbazochrome and niclosamide, appeared to augment Aβ formation. Thus, hydrazides reduced lipid oxidative damage, and hydralazine additionally reduced Aβ misfolding. While hydralazine would require specific chemical modifications for use as an AD therapeutic itself (to improve blood brain barrier permeability, reduce vasoactive side effects, and optimization for amyloid inhibition), this study suggests its potential merit for further AD drug development.     

The prebiotic effect of human milk oligosaccharides 3′- and 6′-sialyllactose on adhesion and biofilm formation by Clostridioides difficile – pilot study

Bacterial adhesion is the first stage of colonisation and biofilm formation by Clostridioides difficile. Cell wall proteins (Cwp) 84 and 66 play crucial roles in the pathophysiology of C. difficile and may affect bacterial adhesion. Sialylated human milk oligosaccharides (HMOs) have potential to inhibit bacterial adhesion in vitro. The aim of this study was to investigate how 3′-sialyllactose (SL) and 6′-SL affect adhesion and C. difficile biofilm formation. Also, the influence of these substances on cwp84 and cwp66 genes expression by C. difficile was assessed. An adhesion assay was performed using three human colon cells in vitro, and biofilm formation was evaluated using crystal violet staining and confocal laser scanning microscopy. The effect of 3′-SL and 6′SL on cwp expression was measured using real time-PCR. Both tested HMOs decreased expression of the cwp84 gene, adhesion of C. difficile to human colon cells in vitro and biofilm formation.     

In vitro activity of steroidal dendrimers on Trypanosoma cruzi epimastigote form with PAMAM dendrons modified by “click” chemistry

The increasing use of dendrimers shows promise for the treatment of inflammatory diseases, Chagas disease and other conditions such as cancer. In this study, the activity of 1st and 2nd generation dendrimers over T. cruzi in the epimastigote stage was tested. Dendrimers were derived from α-ethynylestradiol (EE) modified with PAMAM-type dendrons through a triazole ring. The activity of each compound was evaluated in five doses (from 1.3 to 20 µmol/mL) by flow cytometry, including benznidazole (Bz) as positive control. The findings show that an equivalent concentration of 14.8 µmol/mL of 2nd generation (G) dendrimer is 8 times more effective than Bz at 24 h, and it maintains its superiority at 48 h with an IC₅₀ = 1.25 ± 0.19 µmol/mL. A TUNEL assay showed that dendrimers induce cell death in T. cruzi epimastigotes mostly via apoptosis, unlike Bz, which induces death via necrosis in more than 50% of cells.     

Inhibitory effects of the essential oils α-longipinene and linalool on biofilm formation and hyphal growth of Candida albicans

Candida albicans is one of the most common fungal pathogens, and causes systemic and invasive infections in humans. C. albicans biofilms are composed of yeast and hyphal and pseudohyphal elements, and the transition of yeast to the hyphal stage could be a virulence factor. In this study, diverse essential oils were initially investigated for anti-biofilm activity against C. albicans strains, and cascarilla bark oil and helichrysum oil and their components α-longipinene (a major constituent of both) and linalool were found to markedly inhibit biofilm formation without affecting planktonic cell growth. Moreover, α-longipinene and linalool were found to synergistically reduce biofilm formation. Notably, treatments with cascarilla bark oil, helichrysum oil, α-longipinene, or linalool clearly inhibited hyphal formation, and this appeared to be largely responsible for their anti-biofilm effect. Furthermore, the two essential oils, α-longipinene and linalool, reduced C. albicans virulence in Caenorhabditis elegans.     

In vitro production of prostaglandins E,F, and 6-KETO prostaglandin F1α by human pregnant uterus,decidua and amnion

The production of prostaglandins (PGs) E and F and 6-keto PGF_1α was investigated by using human decidua, amnion, and pregnant myometrium obtained before and after onset of labor, and human nonpregnant myometrium collected during luteal phase. PGs produced were measured by radioimmunoassay developed in our laboratory. The radioimmunoassay for 6-keto PGF_1α is specific and accurate. PGE was formed at an almost constant rate regardless of the condition under which the three tissues tested were obtained, with the amnion showing the highest activity compared with decidua and myometrium. PGF production increased remarkably during labor in the myometrium and decidua but not in the amnion. In the myometrium during labor, PGF levels were three times higher than before labor. The pregnant myometrium produced more PGs than the nonpregnant myometrium. The production of 6-keto PGF_1α, a stable, nonenzymatically formed derivative of PGI₂, was observed to be much higher in the pregnant myometrium than in decidua, amnion, or nonpregnant myometrium. Contrary to the pattern of PGF production, 6-keto PGF_1α was lower during labor. The physiological significance of PG production by decidua, amnion, and myometrium in relation to parturition is discussed.     

Androgens and estradiol-17β production by porcine uterine cells: In vitro study

Porcine (Sus scrofa domestica) uterine slices harvested during both early pregnancy and luteolysis produce steroid hormones. The aim of the present study was to determine (1) which porcine separated uterine cells secrete androgens: androstenedione (A₄) and testosterone (T), and estradiol-17β (E₂) in culture; (2) if the production of A₄, T and E₂ in the uterine cells is regulated by P4 and OT; (3) if uterine tissues expressed cytochrome P450arom gene (CYP19). Uteri were collected on Days 14 to 16 of early pregnancy and the estrous cycle. Enzymatically separated epithelial cells, stromal cells, and myocytes were cultured in vitro for 2, 6, and 12 h with control medium, progesterone (P₄; 10^-5 M), oxytocin (OT; 10^-7 M), and both hormones (P₄ + OT). The studied cells secreted A₄, T, and E₂ in vitro. Progesterone served as a substrate for steroid synthesis in the uterine cells. Isolated uterine cells, cultured separately, contributed in equal portion to the basal production of androgens (A₄ and T) during both early pregnancy and luteolysis. In pregnant pigs, the epithelial and stromal cells were rich sources of E₂ compared with myocytes. Myocytes produced E₂ mainly during luteolysis. Pregnant porcine endometrium and myometrium expressed the gene CYP19, which encodes for P450 aromatase, a steroidogenic enzyme. The results indicate an active steroidogenic pathway in porcine uterine cells. The epithelial cells, stromal cells, and myocytes participate in steroid production as an alternative source for their action in pigs.     

“In vitro” Antifungal activity of protease inhibitors

In the last five years, as HAART has become standard therapy in HIV seropositive or AIDS patients, changes have been noted in the numbers and types of opportunistic fungal infections in these cohorts of patients. Particularly, oropharyngeal candidiasis have become rare in HIV infected patients since the introduction of new anti-HIV drugs of the protease inhibitors type. At the Immunology Institute of the Universidad Central de Venezuela the most frequent protease inhibitors (PIs) used for the treatment of these patients have been: Nelfinavir (Viracept^TM, Roche),Indinavir (Crixivan^® Merck),Ritonavir (Norvir^®, Abbott),Saquinavir (Fortovase^®, Roche).Recently, we observed that recurrent candidiasis was less frequent and no Candidacould be isolated in our patients. A direct relation to the PIs was suspected. In order to assess the in vitro antifungal activity of the afore mentioned protease inhibitors on Candida sp., we used both the well diffusion test and the NCCLS broth microdilution test to assay 100 Candida sp. isolates from HIV seropositive or AIDS patients with syntomatic oropharyngeal Candida infection. In general, the data obtained with the well diffusion test were in agreement with those obtained by the broth microdilution test. All 100 isolates were susceptible to Saquinavir and 32 were susceptible to Indinavir using the NCCLS microdilution test,while 97 were susceptible to Saquinavir and 52 to Indinavir by the well diffusion test. From 17 C. albicans resistant to fluconazole, all were susceptible to Saquinavir by the NCCLS micro method and 16 by the well diffusion test. Our results showed anticandidal activity in vitro of PIs, mainly Saquinavir.This revised version was published online in October 2005 with corrections to the Cover Date.     

In vitro effects of ascorbic acid and β-glycerophosphate on human gingival fibroblast cells

Ascorbic acid (AA) and β-glycerophosphate (βG) are considered in vitro osteogenic factors important to the differentiation of osteoblastic progenitor and dental pulp cells into mineralized tissue-forming cells. So, the present study investigated in vitro if these mineralizing inducible factors (AA and βG) could influence differentiation of human gingival fibroblasts when compared with human pulp cells and osteogenic cells derived from rat calvaria cultured. The expression of osteopontin (OPN) and osteoadherin (OSAD) was analyzed by indirect immunofluorescence, immunocytochemistry as well as Western-blotting. In addition, the main ultrastructural aspects were also investigated. No mineralized matrix formation occurred on gingival fibroblasts induced with AA + βG. On these cells, no expression of OPN and OSAD was observed when compared with pulp cells, pulp cells induced with AA + βG as well as osteogenic cells. Ultrastructure analysis additionally showed that gingival fibroblasts exhibited typical fibroblast morphology with no nodule formation. The present findings showed that AA and βG could not promote a mineralized cell differentiation of human gingival fibroblasts and confirm that human dental pulp cells, as the osteogenic cells, are capable to form a mineralized extracellular.     

Complementation of Sulfolobus solfataricus PBL2025 with an α-mannosidase: effects on surface attachment and biofilm formation

Compared to Sulfolobus solfataricus P2, the S. solfataricus mutant PBL2025 misses 50 genes (SSO3004-3050), including genes coding for a multitude of enzymes possibly involved in sugar degradation or metabolism. We complemented PBL2025 with two of the missing proteins, the α-mannosidase (SSO3006, Ssα-man) and the β-galactosidase LacS (SSO3019), and performed comparative fluorescence microscopy and confocal laser scanning microscopy to analyze the recombinant strains. We demonstrated that the Ssα-man complemented strain resembled the S. solfataricus P2 behavior with respect to attachment of cells to glass and growth of cells in static biofilms. During expression of the Ssα-man, but not LacS, glucose and mannose-containing extracellular polymeric substance (EPS) levels changed in the recombinant strain during surface attachment and biofilm formation. These results suggest that the Ssα-man might be involved in the modulation of the EPS composition and/or in the de-mannosylation of the glycan tree, which is attached to extracellular glycosylated proteins in S. solfataricus. On the other hand, LacS expression in PBL2025 reduced the carbohydrate content of the isolated total EPS implying a role in the modulation of the produced EPS during static biofilm formation. These are the first enzymes identified as playing a role in archaeal EPS formation.     

Studies on plasmid stability,cell metabolism and superoxide dismutase production by Pgk− strains of Saccharomyces cerevisiae

Summary A double mutant sod1/pgk1 strain of Saccharomyces cerevisiae has been constructed in order to investigate the effects of different environmental conditions on yeast physiology, plasmid stability, and superoxide dismutase (SOD) production. Strains were transformed with yeast episomal plasmids (YEp) containing both PGK1 and SOD1 genes and were grown on fermentable carbon sources and under vigorous aeration. Under these conditions, the presence of the PGK1 gene was made essential for growth and both genes were efficiently expressed. However, plasmid-borne PGK1 was found not to increase the stability of YEp vectors in batch cultures of Pgk^– cells. Paradoxically, plasmid stability increased during the respiratory phase of growth. An investigation of the metabolism of Pgk^– cells demonstrated that these glycolytic pathway mutants do not appreciably metabolize glycerol. Thus Pgk⁺, plasmid-containg, cells have a selective advantage during the respiratory phase of batch growth since they can utilize both glycerol and ethanol. Correspondence to: S. G. Oliver     

In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.