Number Of CFU Research Articles

The IL-17 Receptor IL-17RA Is Expressed By Early Erythroid Progenitors and Stimulates Erythropoiesis

The central regulator of erythropoeitic rate is erythropoietin (Epo). Epo action is augmented by factors that act on early erythroid progenitors upstream of erythroid terminal differentiation: glucocorticoids, Stem Cell Factor and BMP4. Of these, only Epo and glucocorticoids are useful agents clinically, leaving a gap when they are ineffective or contraindicated. The molecular identities of early erythroid progenitors are not well understood, posing an obstacle to the discovery of novel erythropoietic agents. To address this gap, we used single-cell RNA-seq (scRNA-seq) technology to delineate a continuous erythroid trajectory in the mouse, beginning with multi-potential progenitors (MPPs) and ending with the onset of erythroid terminal differentiation (described in Tusi, Wolock et al.). Based on gene expression analysis, we predicted two novel developmental stages within the erythroid trajectory, that we termed Early Erythroid Progenitors (EEP) and Committed Erythroid Progenitors (CEP). We devised a flow cytometric strategy that enriches for these novel progenitor stages, and found that they correspond to cells with BFU-e and CFU-e activity, respectively.Our scRNA-seq work predicted cell surface molecules expressed by EEPs and CEPs, including receptors with known endogenous ligands or drug agonists or antagonists, but no previously documented role in erythropoiesis. The first three such receptors that we tested, Mst1r, Ryk and IL-17RA, all modified adult and/or fetal liver BFU-e and CFU-e colony formation in vitro . In the case of IL-17RA, its IL-17a ligand cooperated synergistically with Epo to expand the number of adult bone marrow CFU-e in vitro, while having no effect on fetal liver CFU-e. Specifically, at the modestly elevated Epo concentration of 0.05 U/ml, addition of IL-17a (10 ng/ml) resulted in a 2.5 fold increase in CFU-e colonies (p<0.001); at levels of Epo that elicit its maximal effect (2 U/ml), addition of IL-17a increased the number of CFU-e colonies by an additional 44% (p=0.002). A dose response analysis showed that EC50 for IL-17a's synergistic action with Epo was 2 ng/ml (60 pM). Furthermore, addition of IL-17a boosted the formation of human bone marrow CFU-e but not BFU-e in vitro .IL-17a had no effect on CFU-e formation in bone marrow derived from IL-17RA-/- mouse, confirming the specificity of this interaction. In work that is ongoing, we found that IL-17a activated intracellular Stat3 and Stat5 signalings in early erythroid progenitors. Taken together, these findings suggest that IL-17a is a novel erythropoietic stimulator, synergizing with Epo to accelerate erythropoietic rate. [Display omitted] DisclosuresKlein:OneCell Bio: Other: Equity Ownership and Membership on an entity's Board of Directors or advisory committees.

High Mobility Group Box 1 Protein (HMGB1) Mediates Anemia of Inflammation through Lineage Skewing and Direct Inhibition of EPO Signaling

Erythropoiesis is sensitive to inflammation and its associated mediators. We previously identified the damage-associated molecular pattern, high mobility group box 1 protein (HMGB1), as a causative factor involved in the development of anemia using a murine model of chronic inflammation, and recently, showed that HMGB1 negatively affects human erythropoiesis, in part, through altered erythropoietin (EPO) signaling and hematopoietic stem and progenitor cell (HSPC) lineage commitment. Despite these findings, a mechanistic understanding of erythroid dysfunction in the presence of HMGB1 is lacking.We first documented a dose-dependent logarithmic difference in erythroid cell growth at Days 7 and 11 of culture of CD34+ HSPCs during erythroid differentiation as well as a relatively smaller loss of cells at Day 14 between HMGB1-treated and vehicle-treated cultures. These results suggest that HMGB1 exerts its effects at distinct stages of erythroid differentiation: at both the progenitor and the precursor stages. Indeed, flow analysis of erythroblast differentiation showed that HMGB1 caused an accumulation of precursors at the basophilic erythroblast stage. The loss of poly- and orthochromatic erythroblasts resulted from increased apoptosis at these stages of erythroid differentiation as assessed by annexin-V staining. Induction of apoptosis was confirmed by a dose-dependent increase in apoptosis and 3-fold increase in cleaved caspase-3 in HUDEP-2 cells, an erythroid cell line. Furthermore, a mutant form of HMGB1 (3S-HMGB1) in which critical cysteine residues were mutated to abolish the activity of the protein failed to alter erythroid proliferation and survival.HMGB1 appears to deprive differentiating erythroid cells of growth and survival signals through attenuated phosphorylation of downstream EPO effectors including pJAK2 and pSTAT5, independent of SHP1/2 activity. As a consequence of perturbed EPO signaling, erythroid cells contained reduced levels of nuclear HSP70 and GATA1, which ultimately led to apoptosis. Importantly, EPO co-administrated with either tumor necrosis factor (TNF), interleukin-1β (IL-1β), or IL-6 led to phosphorylation levels of JAK2-STAT5 comparable to controls, suggesting that direct inhibition of EPO signaling is specific to HMGB1.To document the effect of HMGB1 on EPO-dependent and independent progenitors, erythroid progenitor growth and differentiation were monitored at 24hr intervals in culture. CFU-E but not BFU-E numbers were decreased following treatment with HMGB1. Moreover, HMGB1-treated cells expressed high levels of GATA2 indicating deregulation of GATA transcription factor switching. Myelo-erythroid culture systems demonstrated that HMGB1 strongly inhibited erythroid commitment of CD34+ HSPCs while it favored the generation of increased numbers of CD11b+ and CD13+ expressing myeloid cells. The induced switch in lineage commitment is consistent with increased GATA2 expression and decreased GATA1 expression. Further, we observed that erythroid and hematopoietic progenitor cultures became more acidic in the presence of HMGB1 suggesting that HMGB1 may also influence progenitor metabolic processes.Our data demonstrate that HMGB1 negatively affects erythropoiesis at EPO-dependent and -independent stages of erythroid differentiation through decreased CFU-E numbers and apoptosis of erythroblasts, and also favor increased myeloid differentiation of HSPCs. Contrary to classical inflammatory mediators, HMGB1 relies on a novel mechanism, involving downstream EPO effectors, to mediate erythroid dysfunction. Ongoing pharmacologic and genetic screens of EPO signaling axes, putative HMGB1 receptor(s), and progenitor metabolic profiling are currently underway to better understand how HMGB1 causes erythropoietin refractory anemia in the setting of inflammation. DisclosuresNo relevant conflicts of interest to declare.

Validation of the Osteomyelitis Induced by Methicillin-Resistant Staphylococcus aureus (MRSA) on Rat Model with Calvaria Defect.

Osteomyelitis resulting from bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA) that are resistant to multiple drugs, brings further clinical challenges. There is currently no model of osteomyelitis induced by MRSA using rats with calvaria defects. So, We induced osteomyelitis in rat models with the calvaria bone defect. The rats were randomly divided into six groups according to inoculation dose levels, which ranged from 6 × 100 to 6 × 105CFU/5µl. Bone tissues were retrieved from all rats used in the study and assessed using histology, microbiology, and radiobiology 4 weeks after surgery to evaluate the relationship between inoculation dose and infectivity. In Histological results, high levels of inflammatory responses, bone necrosis, and bacteria were observed in treatment groups G3 to G5. In IHC staining, high levels of cox-2 expression were observed in treatment groups G3. Microbiological observations also indicated that significantly higher numbers of CFUs were found in G3 to G5. In radiography results, the bone mineral density in G3 to G5 was significantly higher than in the control group, G1, and G2. Our results indicate that an inoculating dose of 6 × 103 CFU/5 μl is sufficient to induce the development of osteomyelitis in rat models. This study suggests that the minimum dose (6 × 103 CFU/5 µl) can induce osteomyelitis in calvaria rat model. This can offer information and ability of more accurately modeling osteomyelitis and simulating the challenge of osteomyelitis treat.

ANTIBACTERIAL ACTIVITY OF SOME NANO-ANTIMICROBIAL POLY(AMIDOAMINE) (PAMAM) DENDRIMERIC PRODUCTS

"Dendrimers are synthetic, branched macromolecules. Compared to linear polymers, dendrimers hold multiple advantages as potential carrier molecules: high capacity to incorporate pharmacologically active substances, chemical stability of micelles under physiological conditions, increased bioavailability of active substances covalently or noncovalently bound to dendrimers, due to increased dendrimers capacity to cross biological barriers. The carrier potential of dendrimers has been evaluated in combination with different antibiotics, as an alternative way to address the increasing resistance of Gram-positive and Gram-negative bacteria. This paper presents a study on the intrinsic antibacterial properties of the following poly(amidoamino) (PAMAM) ethylenediamine core dendrimers: PAMAM generation 3.5, PAMAM generation 4.0 and PAMAM-OH generation 4.0. The following bacterial strains were used: Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442, Enterococcus faecalis ATCC 51299 and a multi-drug resistant (MDR) Acinetobacter baumannii clinical isolate. The dendrimers’ concentrations used in the experiment were established within the range of admissibility obtained following cytotoxicity testing on human cell cultures. The dendrimers’ antibacterial activity varied depending on the concentration and the bacterial species. The antibacterial efficacy was quantified by calculating the percentage and logarithmic reduction of the number of viable microorganisms. The logarithmic reduction varied between 0.06 and 1.10, corresponding to percentage reductions between 13% and 92%. The strongest antibacterial activity of all dendrimers used in this study was registered for the D3.5 dendrimer (50 μg/mL) against Acinetobacter baumannii and Pseudomonas aeruginosa ATCC 15442 reference strain. For A. baumannii, a logarithmic reduction of bacterial activity of 1.10 log10 (percentage reduction in the number of CFU of 92%) was registered, while for P. aeruginosa ATCC 15442 the logarithmic reduction was 0.75 log10 (percentage reduction of 82%)."

Histological and microbiological evaluation of surgical wound closure in mouse skin with cyanoacrylate (Histoacryl®) in comparison to poliglecaprone (Monocryl®) traditional suture

Incisional wound closure is a key surgical step to facilitate tissue healing, reduce the risk of infection and obtain esthetic and functional recovery. Cyanoacrylates such as Histoacryl® have become a popular choice in surgical veterinary practice. However, how Histoacryl® is affecting tissue regeneration and bacterial load in the wound in comparison to poliglecaprone (Monocryl®) traditional suture methods remains to be determined. This work aimed to evaluate how wounded tissue responds to traditional suture with Monocryl® (poliglecaprone 25/4-0) and Histoacryl®, as well as provide evidence of their effects on wound healing in mice. Fortyeight hours after the incisional procedure, wound tissue biopsies were prepared for histological and microbiological analysis. Biopsies were fixed and colored with Mallory's trichrome and hematoxylin-eosin stains. For microbiological assays, biopsies were suspended in tryptic soy broth (TSB) and 1/10 diluted to evaluate the number of CFU in nutrient agar plates. Our results show no differences between Histoacryl® and Monocryl® traditional suture suggesting that both methods could be used to treat wounds in small animals such as rodents.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

SummaryAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

Phenotypic changes in group B streptococci grown in the presence of the polyols, erythritol, sorbitol and mannitol

BackgroundGroup B streptococci (GBS) are important neonatal bacterial pathogens that can cause severe invasive disease in the newborn. It is thought that in many cases of invasive neonatal GBS disease, the bacteria ascend the vagina into the uterus and infect the amniotic fluid surrounding the fetus. Important constituents of this environment include the polyols or sugar alcohols of which erythritol, sorbitol and mannitol are examples. The aim of our study was to investigate the effect of polyols on GBS grown in media containing these sugar alcohols.ResultsGBS incubated in varying concentrations of polyols (erythritol, sorbitol or mannitol) did not display any significant enhancement or inhibition of bacterial growth. However, growth of GBS in the presence of erythritol significantly increased the surface expression of GBS-PGK (a plasminogen binding protein) 1.25 to 1.5-fold depending on the erythritol concentration and significantly enhanced the survival in human blood 3X to 18X depending on the concentration of polyol used. Interestingly, GBS grown in 1% erythritol significantly increased invasion by the bacteria of HeLa cells (epithelial cell line) (150% vs 100%) however, at higher concentrations (2% or 4% of polyol) the number of CFUs was significantly reduced (55-75% vs 100%) suggesting higher concentrations of polyols may inhibit invasion. Erythritol also increased GBS hemolytic activity as well as enhancing biofilm formation 1.4X to 3.3X depending on the concentration of polyol used.ConclusionsGBS grown in the presence of polyols alters the bacteria’s phenotype resulting in changes associated with GBS virulence. This effect was greatest for the polyol erythritol.

Laminar airflow decreases microbial air contamination compared with turbulent ventilated operating theatres during live total joint arthroplasty: a nationwide survey

Preventing surgical site infections and prosthetic joint infections is crucial for patient safety after total joint arthroplasty. Microbial air contamination has been suggested as a risk factor. Therefore, the ventilation system that will reduce air contamination most effectively in operating theatres (OTs) has been discussed. To determine whether laminar airflow (LAF) ventilation is superior to turbulent airflow (TAF) ventilation by looking at the colony forming units (cfu) count during live total hip and knee arthroplasties. Furthermore, to explore whether the number of OT personnel, door and cabinet lock openings and technical parameters of the ventilation systems have an impact on the number of cfu. Active air sampling and passive sedimented bacterial load were performed in 17 OTs, equipped with either LAF or TAF ventilation, during 51 live surgeries while observations were noted. LAF OTs reduced cfu counts compared with TAF OTs during live surgery (P<0.001). All LAF OTs provided ultraclean air whereas TAF had nine procedures exceeding the threshold of 10 cfu/m3. Door and cabinet lock openings and number of personnel did not influence the cfu count, while it decreased with increasing volume and total air change per hour (P<0.05). All LAF OTs had cfu counts within recommendations and provided lower cfu counts compared with TAF OTs. The number of OT personnel and total openings did not have an influence on cfu counts. Increased volume of the OT and total air change per hour showed a decrease in active cfu counts.

Measurement of internal Beauveria bassiana to ascertain non-target impacts on arthropods in field environments

ABSTRACT Effects of Beauveria bassiana on non-target arthropods were assessed following field application on rangeland and alfalfa agroecosystems in Canada. Densities and prevalence of B. bassiana within non-target arthropods were measured by two methods, surface sanitation of non-target cadavers followed by homogenisation and plating on selective medium, or incubation in a high humidity environment. From rangeland, numbers of B. bassiana colony-forming units (cfu) in spiders, and carabid and tenebrionid beetles were low (≤2.0 × 103 cfu/ individual) and none of the 2500 arthropods incubated in high humidity were colonised. In alfalfa, increases in numbers of B. bassiana cfu in homogenised specimens were observed only in the first four days in coccinellid beetles (Coccinellidae) while high cfu numbers persisted in some harvestmen (Phalangidae). Laboratory assays with coccinellids indicated that recovered B. bassiana emanated from ingested conidia within the alimentary tracts rather than from hyphal bodies within hemocoels. A relatively low prevalence of non-target arthropods from which B. bassiana was isolated in rangeland and alfalfa coupled with the decrease in numbers of internal cfu as a function of time indicated that the fungus imparted a minor and short-lived impact on non-target arthropods. Although B. bassiana did not affect leafcutting bee larvae, prepupae or adult emergence following overwintering diapause, a small number of foraging bees were infected under field conditions. Measuring internal fungal populations on selective medium or incubation of cadavers in a high humidity environment were found to be convenient and efficient methods for safety assessment to non-target arthropods of a mycopesticide applied under field conditions.

N-chlorotaurine, a potent weapon against multiresistant bacteria.

Aims N‐chlorotaurine (NCT) is a body‐own mild oxidizing antiseptic that can be applied topically as a well‐tolerated anti‐infective at many body sites. The objective of this study was to demonstrate its activity against representative nosocomial multidrug‐resistant bacteria.Methods and ResultsThe bactericidal activity of NCT was tested in quantitative killing assays against a panel of multiresistant Gram‐positive and Gram‐negative clinical isolates. N‐chlorotaurine (1%, 55 mmol l−1) reduced the number of CFU of strains of methicillin‐resistant Staphylococcus aureus, linezolid‐resistant Staphylococcus epidermidis, vancomycin‐resistant, and linezolid‐ and vancomycin‐resistant Enterococcus faecium, 3MRGN and 4MRGN Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae by at least 2 log10 steps after 15 min and completely or nearly to the detection limit after 30 min at pH 7·1 and 37°C.ConclusionThe activity of NCT against these clinical isolates is similar to that against non‐resistant ATCC strains and therefore not influenced by antibiotic resistance. This can be explained by the oxidizing and chlorinating mechanism of action of NCT, which leads to an attack of multiple targets in the microorganisms.Significance and Impact of the StudyThe bactericidal spectrum of NCT is not restricted by resistance against antibiotics. Therefore, it can be used against resistant strains, too.

Antimicrobial activity of experimental chitosan solutions on acrylic resin and cobalt-chromium surfaces.

To evaluate the application of chitosan as a cleanser in the control of biofilm formation on cobalt-chromium (Co-Cr) alloy and acrylic resin surfaces. In total, 172 Co-Cr discs and 172 acrylic resin discs (14 mm x 3 mm) were contaminated with Streptococcus mutans, Staphylococcus aureus, Candida albicans, or Candida glabrata and incubated for 48 hours. Then, specimens were randomly divided into groups and immersed in the following solutions for 15 minutes: WC = solution without chitosan (control); CH = chitosan solution (5 mg/mL); CN = chitosan nanoparticle solution (3.8 mg/mL); and ET = effervescent tablet. Biofilm recovery rates (n = 9) were evaluated by counting the colony-forming units (CFU/mL). Biofilm morphology was evaluated using scanning electron microscopy. Data were compared with Kruskal-Wallis or analysis of variance followed by Tukey post hoc tests. For acrylic resin, ET showed the lowest number of CFU for S aureus and S mutans (P < .001). CH exhibited intermediate values for S mutans, S aureus, and C albicans, and CN exhibited intermediate values for S mutans and S aureus. For C glabrata, there was no statistical difference between the solutions (P = .264). For Co-Cr, ET showed the highest level of antimicrobial action against all microorganisms (P < .001), and CH showed an intermediate level of action against S mutans and S aureus. Against C albicans and C glabrata, there was no significant difference among CH, CN, and WC. Although ET had a broader spectrum of antimicrobial action, the chitosan solution showed promise as a denture cleanser.

Effect of Propolis Nanoparticles against Enterococcus faecalis Biofilm in the Root Canal.

To determine the antibacterial effect of propolis nanoparticles (PNs) as an endodontic irrigant against Enterococcus faecalis biofilm inside the endodontic root canal system. Two-hundred-ten extracted human teeth were sectioned to obtain 6 mm of the middle third of the root. The root canal was enlarged to an internal diameter of 0.9 mm. The specimens were inoculated with E. faecalis for 21 days. Following this, specimens were randomly divided into seven groups, with 30 dentinal blocks in each group including: group I—saline; group II—propolis 100 µg/mL; group III—propolis 300 µg/mL; group IV—propolis nanoparticle 100 µg/mL; group V—propolis nanoparticle 300µg/mL; group VI—6% sodium hypochlorite; group VII—2% chlorhexidine. Dentin shavings were collected at 200 and 400 μm depths, and total numbers of CFUs were determined at the end of one, five, and ten minutes. The non-parametric Kruskal–Wallis and Mann–Whitney tests were used to compare the differences in reduction in CFUs between all groups, and probability values of p < 0.05 were set as the reference for statistically significant results. The antibacterial effect of PNs as an endodontic irrigant was also assessed against E. faecalis isolates from patients with failed root canal treatment. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were also performed after exposure to PNs. A Raman spectroscope, equipped with a Leica microscope and lenses with curve-fitting Raman software, was used for analysis. The molecular interactions between bioactive compounds of propolis (Pinocembrin, Kaempferol, and Quercetin) and the proteins Sortase A and β-galactosidase were also understood by computational molecular docking studies. PN300 was significantly more effective in reducing CFUs compared to all other groups (p < 0.05) except 6% NaOCl and 2% CHX (p > 0.05) at all time intervals and both depths. At five minutes, 6% NaOCl and 2% CHX were the most effective in reducing CFUs (p < 0.05). However, no significant difference was found between PN300, 6% NaOCl, and 2% CHX at 10 min (p > 0.05). SEM images also showed the maximum reduction in E. faecalis with PN300, 6% NaOCl, and 2% CHX at five and ten minutes. CLSM images showed the number of dead cells in dentin were highest with PN300 compared to PN100 and saline. There was a reduction in the 484 cm−1 band and an increase in the 870 cm−1 band in the PN300 group. The detailed observations of the docking poses of bioactive compounds and their interactions with key residues of the binding site in all the three docking protocols revealed that the interactions were consistent with reasonable docking and IFD docking scores. PN300 was equally as effective as 6% NaOCl and 2% CHX in reducing the E. faecalis biofilms.

Cu(II) biosorption by living biofilms: Isothermal, chemical, physical and biological evaluation

Dissolved copper in stormwater runoff is a significant environmental problem. Biosorption of dissolved metals using microorganisms is known as a green, low-cost and efficient method. However, the role of live biological agents in the remediation of dissolved copper in Sustainable Drainage (SuDS) has not been reported. In this study, the effect of pH, initial concentration and temperature, on bacteria in different stages of biofilm development on a geotextile, along with Cu(II) removal efficiencies, were evaluated. Maximum Cu(II) removal efficiency (92%) was observed at pH 6. By decreasing the pH from 6 to 2, a log 5 reduction in bacteria was observed and Carboxyl groups transformed from -COO- to –COOH. The maximum biosorption capacity (119 mg g−1) was detected on day 1 of biofilm development, however, maximum removal efficiency (97%) was measured on day 21 of biofilm incubation. Exteracellular Polymeric Substance (EPS) showed a better protection of CFUs in more mature biofilms (day 21) with less than 0.1 log decrease when exposed to 200 mL−1 Cu(II), whereas, biofilm on day 1 of incubation showed a 2 log reduction in CFUs number. Thermodynamic studies showed that the maximum Cu(II) biosorption capacity of biofilms, incubated for 7 days (117 mg g−1) occurred at 35 °C. Thermodynamic and kinetic modelling of data revealed that a physical, feasible, spontaneous and exothermic process controlled the biosorption, with a diffusion process observed in external layers of the biofilm, fitting a pseudo-second order model. Equilibrium data modelling and high R2 values of Langmuir model indicated that the biosorption took place by a monolayer on the living biofilm surface in all stages of biofilm development.

The role of Toll-like receptor 9 in a murine model of Cryptococcus gattii infection

Toll-like receptor 9 (TLR9) is crucial to the host immune response against fungi, such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, but its importance in Cryptococcus gattii infection is unknown. Our study aimed to understand the role of TLR9 during the course of experimental C. gattii infection in vivo, considering that the cryptococcal DNA interaction with the receptor could contribute to host immunity even in an extremely susceptible model. We inoculated C57BL/6 (WT) and TLR9 knock-out (TLR9−/−) mice intratracheally with 104C. gattii yeast cells. TLR9−/− mice had a higher mortality rate compared to WT mice and more yeast cells that had abnormal size, known as titan cells, in the lungs. TLR9−/− mice also had a greater number of CFUs in the spleen and brain than WT mice, in addition to having lower levels of IFN-γ and IL-17 in the lung. With these markers of aggressive cryptococcosis, we can state that TLR9−/− mice are more susceptible to C. gattii, probably due to a mechanism associated with the decrease of a Th1 and Th17-type immune response that promotes the formation of titan cells in the lungs. Therefore, our results indicate the participation of TLR9 in murine resistance to C. gattii infection.

Electrical stimulation of Cucumis sativus germination and growth using IrO2-Ta2O5|Ti anodes in Vertisol pelic

The effect of the application of electric fields, magnetic fields, and electric currents in plants is still limited. Some research has yielded favorable results on plant growth, but some has found inhibition of plant development. The physiological, microbiological, and edaphological characteristics of plants could be affected by imposing low-intensity electric fields using a 2D array of electrodes with six modified anodes with transition metal oxides such as IrO2-Ta2O5|Ti anodes around a naked Ti cathode. In the case of cucumber (Cucumis sativus) germination, electrofarming showed a higher percentage of germination (50%) than did conventional farming (30%), and changes in the other physiological properties of cucumber plants were also observed. The results showed that after 4 h of exposure to an electric field (0.2 V cm−1), stem thickness (6%), cotyledon length (5%), true leaf size (49%) and generated biomass (53%) were quantitatively improved; properties such as stem elongation (20%) and root length were quantitatively improved, and significantly different effects were obtained after exposure to the electric field (p < 0.05) than without it.In the case of microbiological characterization, after exposure to the electric field, a greater number of CFUs were obtained for bacteria and fungi, with an increase of 2× in both cases, than in the control soil. The electric field significantly increased the CFUs in the area close to the anodes compared to CFUs in the control soil. Other properties were also improved by electrofarming; in the microscopic characterization of bacteria, a higher percentage of Gram (−) bacilli was obtained along with increased percentages of soil organic matter (42%), cationic exchange capacity (6%), and enzymatic activity (47%) close to the anodes compared to results in the control soil.

Concept and principles of development of a new generation symbiotic preparation

The article presents the results of the research on the cultivation of B. bifidum and B. subtillis in a high-carbohydrate nutrient medium composed of beet molasses, obtaining metabolites and scientific and practical substantiation of the resulting culture liquid as a biologically active additive. The material of the laboratory studies was sugar beet molasses of 5%-10%-15%-20%-25% concentrations. The studies used the probiotic microorganism B. bifidum, strain No. 1 and B. subtilis, strain DSM-3242. To obtain bacterial concentrate, a daily culture of microorganisms was used, which was standardized to 1×106 CFU / cm3. The testing process was carried out according to the indicators of the mass fraction of fermented sugars, determined by the calculation method, branch standard OCT 18-395-82. The number of CFU was calculated in the Goryaev chamber using the method of GPhA (General Pharmacopeic Article) 1.7.2.0008.15. The content of amino acids, vitamins, and organic acids in the culture fluid was determined by capillary electrophoresis in the device “Drops”. During the cultivation of B. bifidum and B. subtillis, a certain metabolic activity of their vitality was found, which is expressed in a decrease in the mass fraction of the amount of fermented sugars in the range of 53.7% to 23.7%. The hydrogen index is from 6.70 to 3.77 in experimental samples of molasses. The number of B. bifidum and B. subtillis CFUs cultivated on 5% molasses on the second day reached 7x106 CFU/cm3 and 8.2 × 106 cm3. In other concentrations, a maximum indicator of the number of microorganisms occurred on the third day and did not exceed 6.6 and 7 × 106 CFU/cm3. A significant synthesis of metabolites in the form of indispensable and conditionally dispensable amino acids and organic acids was determined. The dynamics of amino acid synthesis is consistent with the amylolytic and enzymatic activity of microorganisms in the molasses culture medium. In this case the level of enzymes is directly dependent on the number of CFU.

Comparative evaluation of photoactivated disinfection and sonic irrigation as an adjunct to conventional irrigation on Enterococcus faecalis in root canals: An in vitro study

Objective: The aim of the in vitro study was to compare the antibacterial efficacy of photoactivated disinfection (PAD), sonic irrigation as an adjunct to conventional irrigation against Enterococcus faecalis in vitro.Materials and Methods: A total of 75 extracted teeth were selected and prepared followed by inoculation with strains of E. faecalis, and a preirrigation sample was collected using sterile paper points. These teeth were then divided randomly into three groups for irrigation: Group I (Conventional irrigation), Group II (PAD), and Group III (EndoActivator) followed by postirrigation sample collection using the sterile paper points. The samples were swabbed on blood agar plates and incubated followed by the calculation of colony-forming units (CFU's).Results: The results were statistically analyzed using the SPSS software version 18.0. On comparing the mean values among the groups, the reduction in the number of CFU's after the treatment protocol was highly significant for all groups (P < 0.001). With the preirrigation sample, there is a statistically significant difference in the values of Group I and Group II (P = 0.047). However, in case of postirrigation samples, there is nonsignificant difference between Group II and Group III.Conclusion: PAD using 940 nm diode laser and methylene blue and endoactivator were more effective than sodium hypochlorite (NaOCl) in reducing E. faecalis counts.

Association of immune responses of Zebu and Holstein-Friesian cattle and resistance to mycobacteria in a BCG challenge model.

Mycobacterium bovis is the main cause of bovine tuberculosis (BTB) in cattle and can also infect humans. Zebu cattle are considered more resistant to some infectious diseases compared with Holstein‐Friesian (HF) cattle, including BTB. However, epidemiological studies may not take into account usage differences of the two types of cattle. HF cattle may suffer greater metabolic stress due to their more or less exclusive dairy use, whereas Zebu cattle are mainly used for beef production. In experiments conducted so far, the number of animals has been too small to draw statistically robust conclusions on the resistance differences between these cattle breeds. Here, we used a BCG challenge model to compare the ability of naïve and vaccinated Zebu and HF cattle to control/kill mycobacteria. Young cattle of both breeds with similar ages were housed in the same accommodation for the duration of the experiment. After correcting for multiple comparisons, we found no difference between naïve HF and Zebu (ρ = 0.862) cattle. However, there was a trend for vaccinated HF cattle to have lower cfu numbers than non‐vaccinated HF cattle (ρ = 0.057); no such trend was observed between vaccinated and non‐vaccinated Zebu cattle (ρ = 0.560). Evaluation of antigen‐specific IFNγ secretion by PBMC indicated that Zebu and HF cattle differed in their response to mycobacteria. Thus, whilst there may be difference in immune responses, our data indicate that with the number of animals included in the study and under the conditions used in this work, we were unable to measure any differences between Zebu and HF cattle in the overall control of mycobacteria. Whilst determination of different susceptibilities between Zebu and HF cattle using the BCG challenge model will require larger numbers of animals than the number of animals used in this experiment, these data should inform future experiments.

Effectiveness of chitosan-propolis nanoparticle against Enterococcus faecalis biofilms in the root canal

BackgroundThe successful outcome of endodontic treatment depends on controlling the intra-radicular microbial biofilm by effective instrumentation and disinfection using various irrigants and intracanal medicaments. Instrumentation alone cannot effectively debride the root canals specially due to the complex morphology of the root canal system. A number of antibiotics and surfactants are being widely used in the treatment of biofilms however, the current trend is towards identification of natural products in disinfection. The aim of the study was to determine the antibacterial effect of chitosan-propolis nanoparticle (CPN) as an intracanal medicament against Enterococcus faecalis biofilm in root canal.Methods240 extracted human teeth were sectioned to obtain 6 mm of the middle third of the root. The root canal was enlarged to an internal diameter of 0.9 mm. The specimens were inoculated with E. faecalis for 21 days. Following this, specimens were randomly divided into eight groups (n = 30) according to the intracanal medicament placed: group I: saline, group II: chitosan, group III: propolis100 µg/ml (P100), group IV: propolis 250 µg/ml (P250), group V: chitosan-propolis nanoparticle 100 µg/ml (CPN100), group VI: chitosan-propolis nanoparticle 250 µg/ml (CPN250), group VII: calcium hydroxide(CH) and group VIII: 2% chlorhexidine (CHX) gel. Dentine shavings were collected at 200 and 400 μm depths, and total numbers of CFUs were determined at the end of day one, three and seven. The non-parametric Kruskal Wallis and Mann–Whitney tests were used to compare the differences in reduction of CFUs between all groups and probability values of p < 0.05 were set as the reference for statistically significant results. The scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM) were also performed after exposure to CPNs. The effectiveness of CPNs were also evaluated against E. faecalis isolated obtained from patients having failed root canal treatment.ResultsThe treatments of chitosan, P100, P250, CPN100, CPN250, CH and 2% CHX reduced the CFUs significantly compared to saline (p < .05). On day one and three, at 200 and 400-μm, CPN250 showed significant reduction of CFUs compared to all other groups (p < .05), while CPN100 was significantly better than other groups (p < .05) except CPN250 and 2% CHX. On day seven, at 200-μm CPN250 showed significant reduction of CFUs compared to all other groups (p < .05) except CPN100 and CHX, while at 400 μm CPN250 showed similar effectiveness as CPN100, CH and 2% CHX. SEM images showed root canal dentin treated with CPN250 had less coverage with E. faecalis bacteria similarly, CLSM images also showed higher percentage of dead E. faecalis bacteria with CPN250 than to CPN100.ConclusionCPN250 was the most effective in reducing E. faecalis colonies on day one, three at both depths and at day seven CPN250 was equally effective as CPN100 and 2% CHX.

GRP78 Is Expressed on the Cell Surface of Pediatric AML Blasts and Can be Targeted with GRP78-CAR T Cells without Toxicity to Hematopoietic Progenitor Cells

Finding the ideal immunotherapy target for AML has proven challenging and is limited by overlapping expression of antigens on hematopoietic progenitor cells (HPCs) and AML blasts. Intracellular Glucose-regulated-protein 78 (GRP78) is a key UPR regulator, which normally resides in the endoplasmic reticulum (ER). GRP78 is overexpressed and translocated to the cell surface in a broad range of solid tumors and hematological malignancies in response to elevated ER stress, making it an attractive target for immune-based therapies with T cells expressing chimeric antigen receptors (CARs). The goal of this project was to determine the expression of GRP78 on pediatric AML samples, generate GRP78-CAR T cells, and evaluate their effector function against AML blasts in vitro and in vivo. To demonstrate overexpression of GRP78 in AML, we performed gene expression analysis by RNAseq on a cohort of cord blood CD34+ cell samples (N=5) and 74 primary AML samples. Primary AML samples included RUNX1-RUNX1T1 (N=7), CBFB-MYH11(N=17), KMT2A rearrangement (N=28) and NUP98 (N=22). Analysis showed increased GRP78 expression in AML samples, especially in KMT2A- and NUP98-rearranged AML. To demonstrate surface expression of GRP78, we performed flow cytometry of AML (Kg1a, MOLLM13, THP-1, MV4-11) cell lines as well as 11 primary AML samples and 5 PDX samples; non transduced (NT) T cells served as control. All AML samples, including cell lines, primary AML blasts, and PDX samples, showed increased expression of GRP78 on their cell surface in comparison to NT T cells We then designed a retroviral vector encoding a GRP78-CAR using a GRP78-specific peptide as an antigen recognition domain, and generated GRP78-CAR T cells by retroviral transduction of primary human T cells. Median transduction efficiency was 82% (± 5-8%, N=6), and immunophenotypic analysis showed a predominance of naïve and terminal effector memory subsets on day 7 after transduction (N=5). To determine the antigen specificity of GRP78-CAR T cells, we performed coculture assays in vitro with cell surface GRP78+ (AML cell lines: MOLM13, MV-4-11, and THP-1 and 3 AML PDX samples) or cell surface GRP78- (NT T cells) targets. T cells expressing CARs specific for HER2-, CD19-, or a non-functional GRP78 (DGRP78)-CAR served as negative controls. GRP78-CAR T cells secreted significant amounts of IFNg and IL-2 only in the presence of GRP78+ target cells (N=3, p&amp;lt;0.005); while control CAR T cells did not. GRP78-CAR T cells only killed GRP78+ target cells in standard cytotoxicity assays confirming specificity. To test the effects of GRP78-CAR T cells on normal bone marrow derived HPCs, we performed standard colony forming unit (CFU) assays post exposure to GRP78-CAR or NT T cells (effector to target (E:T) ratio 1:1 and 5:1) and determined the number of BFU-E, CFU-E, CFU-GM, and CFU-GEMM. No significant differences between GRP78-CAR and NT T cells were observed except for CFU-Es at an E:T ratio of 5:1 that was not confirmed for BFU-Es. Finally, we evaluated the antitumor activity of GRP78-CAR T cells in an in vivo xenograft AML model (MOLM13). Tumor growth was monitored by serial bioluminescence imaging. A single intravenous dose of GRP78-CAR T cells induced tumor regression, which resulted in a significant (p&amp;lt;0.001) survival advantage in comparison to mice that had received control CAR T cells. In conclusion, GRP78 is expressed on the cell surface of AML. GRP78-CAR T cells have potent anti-AML activity in vitro and in vivo and do not target normal HPCs. Thus, our cell therapy approach warrants further active exploration and has the potential to improve outcomes for patients with AML. Disclosures Hebbar: St. Jude: Patents &amp; Royalties. Epperly:St. Jude: Patents &amp; Royalties. Vaidya:St. Jude: Patents &amp; Royalties. Gottschalk:TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; Patents and patent applications in the fields of T-cell &amp; Gene therapy for cancer: Patents &amp; Royalties. Velasquez:St. Jude: Patents &amp; Royalties; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees.