Reduction In Metabolic Activity Research Articles

Effects of Adper™ Scotchbond™ 1 XT, Clearfil™ SE Bond 2 and Scotchbond™ Universal in Odontoblasts.

This study aimed to assess the cytotoxicity of commercially available adhesive strategies—etch-and-rinse (Adper™ Scotchbond™ 1 XT, 3M ESPE, St. Paul, MN, USA, SB1), self-etch (Clearfil™ SE Bond 2, Kuraray Noritake Dental Inc., Tokyo, Japan, CSE), and universal (Scotchbond™ Universal, 3M Deutschland GmbH, Neuss, Germany, SBU). MDPC-23 cells were exposed to adhesives extracts in different concentrations and exposure times. To access cell metabolic activity, viability, types of cell death, and cell cycle, the MTT assay, SRB assay, double labeling with annexin V and propidium iodide, and labeling with propidium iodide/RNAse were performed, respectively. Cultures were stained with May-Grünwald Giemsa for qualitative cytotoxicity assessment. The SB1, CSE, and SBU extracts determined a significant reduction in cell metabolism and viability. This reduction was higher for prolonged exposures, even for less concentrated extracts. CSE extracts significantly reduced the cell’s metabolic activity at higher concentrations (50% and 100%) from 2 h of exposure. After 24 and 96 h, a metabolic activity reduction was verified for all adhesives, even at lower concentrations. These changes were dependent on the adhesive, its concentration, and the incubation time. Regarding cell viability, SBU extracts were the least cytotoxic, and CSE was significantly more cytotoxic than SB1 and SBU. The adhesives determined a reduction in viable cells and an increase in apoptotic, late apoptosis/necrosis, and necrotic cells. Moreover, on cultures exposed to SB1 and CSE extracts, a decrease in the cells in S and G2/M phases and an increase in the cells in G0/G1 phase was observed. Exposure to SBU led to an increase of cells in the S phase. In general, all adhesives determined cytotoxicity. CSE extracts were the most cytotoxic and were classified as having a higher degree of reactivity, leading to more significant inhibition of cell growth and destruction of the cell’s layers.

Activity of Two Antimicrobial Peptides against Enterococcus faecalis in a Model of Biofilm-Mediated Endodontic Infection.

Enterococcus faecalis is a common cause of biofilm-associated opportunistic infections, which are often difficult to treat. The formation of E. faecalis biofilms on the dentinal walls of the root canal is frequently the cause of endodontic treatment failure and secondary apical periodontitis. In a preliminary work, two recognized antifungal peptides, KP and L18R, showed antibacterial activity against planktonic E. faecalis cells at micromolar concentrations. Moreover, L18R proved to reduce the biomass in the early stage of E. faecalis biofilm development on polystyrene plates, while a qualitative biofilm inhibition was demonstrated on hydroxyapatite disks by confocal laser scanning microscopy (CLSM). The aim of this study was to better characterize the effect of both peptides on E. faecalis biofilm. A reduction in metabolic activity after peptide treatment was detected by Alamar Blue assay, while a remarkable impairment in the architecture of E. faecalis biofilms on hydroxyapatite disks, along with a significant reduction in viable bacteria, was caused mostly by L18R, as assessed by CLSM and scanning electron microscopy. The lack of cytotoxicity of the investigated peptides against L929 murine fibroblasts was also determined. Obtained results suggest L18R as a promising candidate for the development of new strategies for endodontic infection control.

Optimizing the application of plasma functionalised water (PFW) for microbial safety in fresh-cut endive processing

The microbiological profiles and responses of native microflora of endive were investigated using a model process line, to establish where a defined PFW should be optimally applied to retain or improve produce microbiological quality. The PFW processes were compared with tap water and ClO2. The antimicrobial efficacy of PFW was quantified by determining the reduction in microbial load, the microbial viability and vitality. Depending on the stage of application of PFW, up to 5 log10-cycles reduction was achieved, accompanied by a reduction of metabolic activity, but not necessarily with a decrease in metabolic vitality. Multiple application (3-step-PFW-application) was more effective than single application (1-step-PFW-application) and PFW showed stronger antimicrobial effect in pre-cleaned endive. High concentrations of nitrite (315 mg l−1) and nitrate (472 mg l−1) in PFW were the main factors for the antimicrobial efficacy of PFW against bacteria. Furthermore, H2O2 and an acidic pH supported the mechanism of action against the endive microflora. These results identify the pathway to scale up successful industrial application of PFW targeting microbiological quality and safety of fresh leafy products. Industrial relevanceThe safety, quality and shelf life of freshly cut vegetables, e.g. lettuce, are strongly influenced by the microbial load. In addition, the hygienic design of production line, and a good handling/ production practice are indispensable. This study shows that the application of PFW, as a promising non-thermal sanitation technology, enables the inactivation of native microbial contamination on fresh-cut endive depending on the process stage of application. It further describes the impact of PFW on the metabolic activity and metabolic vitality of the lettuce-associated microflora. For higher acceptance, the mechanism of action of PFW was assumed based on previous chemical analyses and compared to the industrial standard of ClO2. The results contribute to the understanding and product-specificity of PFW-induced effects on safety, quality and shelf life of fresh cut lettuce and could be a basis for a possible industrial implementation and complement of common technologies.

Comparison of the efficiency of solar-powered prototype vapour compression cooler with evaporative charcoal cooler in maintaining quality of French beans

The quality and shelf life of French beans is affected by the time of harvesting and storage period. Cooling immediately after harvesting is important. The use of a solar evaporative cooling system is suitable for use in rural areas with no power grid connections. This study compared evaporative charcoal cooler and solar cooling systems on improving the quality and shelf life of French beans. The prototype was made using mathematical modeling equations and was powered by solar power while the evaporative charcoal cooler was made from iron sheets with charcoal walls. Freshly harvested French beans were stored under the two cooling systems. The weight and temperature of the stored produce were checked hourly. At the end of the day, produce was packed into modified atmosphere packaging (MAP) bags and stored for 7 days in a cold room. After seven days, oxygen and carbon dioxide levels were measured using a Bridge gas analyzer. The results show significant differences in weight loss between produce stored in evaporative charcoal shed and solar cooler prototype with those stored in evaporative charcoal cooler having 5% weight reduction while those solar coolers having 3% weight loss. A higher accumulation of CO2 was realized in the evaporative charcoal cooler than in the solar-powered cooler. The solar-powered cooler maintained weight, reduced the temperature of products, and maintained the balance between carbon dioxide and oxygen thus reduction in metabolic activities. This enhanced the shelf life and quality of the produce.

Growth and metabolic responses to methyl viologen (1,1′-dimethyl - 4,4′-bipyridinium dichloride) on Chlorella vulgaris

Aquatic environments are especially susceptible to being contaminated by pesticides used in agricultural fields. Methyl viologen (MV) is an herbicide with high effectiveness for the control of unwanted land plants; however, it also has a high toxicity towards the algae in the aquatic environment. The objective of this work was to describe the effect of MV on photosynthetic metabolism and its relationship with respiration, growth and the content of photosynthetic pigments of Chlorella vulgaris. The cultures of C. vulgaris were exposed for 72 h at different concentrations of methyl viologen. The results show that growth, pigment content and metabolic activity decrease as the concentration of MV increases. Analysis of the photochemical activity indicates that MV produces an inhibition of electron transport between quinone A and quinone B of photosystem II. The inhibition of photosynthetic electron transport is directly related to the reduction of metabolic activity and cell growth. The results found in this research show that methyl viologen can be a toxic pollutant for primary producers in aquatic environments.

Combinatorial therapy of chitosan hydrogel-based zinc oxide nanocomposite attenuates the virulence of Streptococcus mutans

BackgroundBiofilm formation is an important causative factor in the expansion of the carious lesions in the enamel. Hence, new approaches to efficient antibacterial agents are highly demanded. This study was conducted to evaluate the antimicrobial-biofilm activity of chitosan hydrogel (CS gel), zinc oxide/ zeolite nanocomposite (ZnONC) either separately or combined together [ZnONC / CS gel (ZnONC-CS)] against Streptococcus mutans biofilm.ResultsMTT assay demonstrated that the ZnONC-CS exhibits a non-cytotoxic effect (> 90% cell viability) toward human gingival fibroblast cells at different dosages (78.1–625 μg/mL) within 72 h. In comparison with CS gel and ZnONC, ZnONC-CS was superior at biofilm formation and metabolic activity reduction by 33 and 45%, respectively; (P < 0.05). The field emission scanning electron microscopy micrographs of the biofilms grown on the enamel slabs were largely in concordance with the quantitative biofilm assay results. Consistent with the reducing effect of ZnONC-CS on biofilm formation, the expression levels of gtfB, gtfC, and ftf significantly decreased.ConclusionsTaken together, excellent compatibility coupled with an enhanced antimicrobial effect against S. mutans biofilm has equipped ZnONC-CS as a promising candidate for dental biofilm control.

Synthesis, Neuroprotection, and Antioxidant Activity of 1,1'-Biphenylnitrones as α-Phenyl-N-tert-butylnitrone Analogues in In Vitro Ischemia Models.

Herein, we report the neuroprotective and antioxidant activity of 1,1′-biphenyl nitrones (BPNs) 1–5 as α-phenyl-N-tert-butylnitrone analogues prepared from commercially available [1,1′-biphenyl]-4-carbaldehyde and [1,1′-biphenyl]-4,4′-dicarbaldehyde. The neuroprotection of BPNs 1-5 has been measured against oligomycin A/rotenone and in an oxygen–glucose deprivation in vitro ischemia model in human neuroblastoma SH-SY5Y cells. Our results indicate that BPNs 1–5 have better neuroprotective and antioxidant properties than α-phenyl-N-tert-butylnitrone (PBN), and they are quite similar to N-acetyl-L-cysteine (NAC), which is a well-known antioxidant agent. Among the nitrones studied, homo-bis-nitrone BPHBN5, bearing two N-tert-Bu radicals at the nitrone motif, has the best neuroprotective capacity (EC50 = 13.16 ± 1.65 and 25.5 ± 3.93 μM, against the reduction in metabolic activity induced by respiratory chain blockers and oxygen–glucose deprivation in an in vitro ischemia model, respectively) as well as anti-necrotic, anti-apoptotic, and antioxidant activities (EC50 = 11.2 ± 3.94 μM), which were measured by its capacity to reduce superoxide production in human neuroblastoma SH-SY5Y cell cultures, followed by mononitrone BPMN3, with one N-Bn radical, and BPMN2, with only one N-tert-Bu substituent. The antioxidant activity of BPNs 1-5 has also been analyzed for their capacity to scavenge hydroxyl free radicals (82% at 100 μM), lipoxygenase inhibition, and the inhibition of lipid peroxidation (68% at 100 μM). Results showed that although the number of nitrone groups improves the neuroprotection profile of these BPNs, the final effect is also dependent on the substitutent that is being incorporated. Thus, BPNs bearing N-tert-Bu and N-Bn groups show better neuroprotective and antioxidant properties than those substituted with Me. All these results led us to propose homo-bis-nitrone BPHBN5 as the most balanced and interesting nitrone based on its neuroprotective capacity in different neuronal models of oxidative stress and in vitro ischemia as well as its antioxidant activity.

Corynebacterium accolens Has Antimicrobial Activity against Staphylococcus aureus and Methicillin-Resistant S. aureus Pathogens Isolated from the Sinonasal Niche of Chronic Rhinosinusitis Patients.

Corynebacterium accolens is the predominant species of the healthy human nasal microbiota, and its relative abundance is decreased in the context of chronic rhinosinusitis (CRS). This study aimed to evaluate the antimicrobial potential of C. accolens isolated from a healthy human nasal cavity against planktonic and biofilm growth of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) clinical isolates (CIs) from CRS patients. Nasal swabs from twenty non-CRS control subjects were screened for the presence of C. accolens using microbiological and molecular techniques. C. accolens CIs and their culture supernatants were tested for their antimicrobial activity against eight S. aureus and eight MRSA 4CIs and S. aureus ATCC25923. The anti-biofilm potential of C. accolens cell-free culture supernatants (CFCSs) on S. aureus biofilms was also assessed. Of the 20 nasal swabs, 10 C. accolens CIs were identified and confirmed with rpoB gene sequencing. All isolates showed variable antimicrobial activity against eight out of 8 S. aureus and seven out of eight MRSA CIs. Culture supernatants from all C. accolens CIs exhibited a significant dose-dependent antibacterial activity (p < 0.05) against five out of five representative S. aureus and MRSA CIs. This inhibition was abolished after proteinase K treatment. C. accolens supernatants induced a significant reduction in metabolic activity and biofilm biomass of S. aureus and MRSA CIs compared to untreated growth control (p < 0.05). C. accolens exhibited antimicrobial activity against S. aureus and MRSA CIs in both planktonic and biofilm forms and holds promise for the development of innovative probiotic therapies to promote sinus health.

Posthypoxic behavioral impairment and mortality of Drosophila melanogaster are associated with high temperatures, enhanced predeath activity and oxidative stress

Hypoxia is an underlying pathophysiological condition of a variety of devastating diseases, including acute ischemic stroke (AIS). We are faced with limited therapeutic options for AIS patients, and even after successful restoration of cerebral blood flow, the poststroke mortality is still high. More basic research is needed to explain mortality after reperfusion and to develop adjunct neuroprotective therapies. Drosophila melanogaster (D.m.) is a suitable model to analyze hypoxia; however, little is known about the impacts of hypoxia and especially of the subsequent reperfusion injury on the behavior and survival of D.m. To address this knowledge gap, we subjected two wild-type D.m. strains (Canton-S and Oregon-R) to severe hypoxia (<0.3% O2) under standardized environmental conditions in a well-constructed hypoxia chamber. During posthypoxic reperfusion (21% O2), we assessed fly activity (evoked and spontaneous) and analyzed molecular characteristics (oxidative stress marker abundance, reactive oxygen species (ROS) production, and metabolic activity) at various timepoints during reperfusion. First, we established standard conditions to induce hypoxia in D.m. to guarantee stable and reproducible experiments. Exposure to severe hypoxia under defined conditions impaired the climbing ability and reduced the overall activity of both D.m. strains. Furthermore, a majority of the flies died during the early reperfusion phase (up to 24 h). Interestingly, the flies that died early exhibited elevated activity before death compared to that of the flies that survived the entire reperfusion period. Additionally, we detected increases in ROS and stress marker (Catalase, Superoxide Dismutase and Heat Shock Protein 70) levels as well as reductions in metabolic activity in the reperfusion phase. Finally, we found that changes in environmental conditions impacted the mortality rate. In particular, decreasing the temperature during hypoxia or the reperfusion phase displayed a protective effect. In conclusion, our data suggest that reperfusion-dependent death might be associated with elevated temperatures, predeath activity, and oxidative stress.

Cold Atmospheric Plasma, a Novel Approach against Bladder Cancer, with Higher Sensitivity for the High-Grade Cell Line.

Simple SummaryBladder cancer has a high incidence and mortality. Besides this, currently available therapies for this type of cancer have low efficacy and show considerable adverse effects, urging the need of new therapeutic approaches. Cold Atmospheric Plasma treatment presents itself as a promising alternative, having demonstrated antitumor effects against several types of cancer. The present work arises from a multidisciplinary team, namely, medical doctors and researchers, in an attempt to find new therapeutic strategies to fight bladder cancer. Therefore, our main objective is to evaluate Cold Atmospheric Plasma effects against bladder cancer, as well as the mechanisms by which it exerts its effects. The results obtained demonstrate that Cold Atmospheric Plasma treatment has a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line. This new approach using Cold Atmospheric Plasma for the treatment of bladder cancer presents enormous clinical benefits, since it is able to selectively treat the tumor tissue, sparing the normal urothelium, with an additional glaring positive economic impact, since it entails a decrease in the cost of therapy in comparison with conventional therapeutic options.Antitumor therapies based on Cold Atmospheric Plasma (CAP) are an emerging medical field. In this work, we evaluated CAP effects on bladder cancer. Two bladder cancer cell lines were used, HT-1376 (stage III) and TCCSUP (stage IV). Cell proliferation assays were performed evaluating metabolic activity (MTT assay) and protein content (SRB assay). Cell viability, cell cycle, and mitochondrial membrane potential (Δψm) were assessed using flow cytometry. Reactive oxygen and nitrogen species (RONS) and reduced glutathione (GSH) were evaluated by fluorescence. The assays were carried out with different CAP exposure times. For both cell lines, we obtained a significant reduction in metabolic activity and protein content. There was a decrease in cell viability, as well as a cell cycle arrest in S phase. The Δψm was significantly reduced. There was an increase in superoxide and nitric oxide and a decrease in peroxide contents, while GSH content did not change. These results were dependent on the exposure time, with small differences for both cell lines, but overall, they were more pronounced in the TCCSUP cell line. CAP showed to have a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line.

Amphotericin B, fluconazole, and nystatin as development inhibitors of Candida albicans biofilms on a dental prosthesis reline material: Analytical models in vitro

Statement of problemThe use of antifungals has been suggested during the treatment of denture stomatitis associated with Candida albicans biofilms. However, how time, material surface, and substrates present during adhesion and biofilm development can influence clinical treatment is unclear. PurposeThe purpose of this in vitro study was to investigate the growth kinetics of C. albicans biofilms on surfaces of specimens under the influence of adsorbed films and to evaluate the antibiofilm efficacy of antifungal agents: amphotericin B, fluconazole, and nystatin. Material and methodsSpecimens of Silagum-Comfort Soft Relining were submerged in preconditioning systems: phosphate-buffered saline, artificial saliva, fetal bovine serum, and artificial saliva+fetal bovine serum. Planktonic cells were incubated (phosphate-buffered saline+specimens) for 1.5 hours (adhesion phase) and washed with phosphate-buffered saline solution. The specimens were then incubated (YNB+glucose) for 8, 24, and 48 hours (initial, intermediate, and maturation phases). The biofilm sessile minimum inhibitory concentration was determined by the broth microdilution method (7.81 to 500 μg/mL). The metabolic activity of the biofilms was tested by colorimetric assay (cell metabolic activity). Cell viability, relative biomass (μm3), and the thickness of the biofilm (μm) were evaluated by confocal laser scanning microscopy. ResultsThe highest bioactivity was recorded in the presence of fetal bovine serum. Biofilms treated with fluconazole and amphotericin B were partially inhibited in a dose-dependent manner. Nystatin inhibited metabolic activity mainly from ≥15.63 or 62.5 μg/mL. Variations in magnitude parameters (relative biomass and thickness) were observed depending on the development phases of biofilms, whereas biological parameters (percentage of nonviable cells) were constant throughout the formation of C. albicans biofilms. ConclusionsThe data suggest that partial (fluconazole and amphotericin B) or more effective (nystatin) reduction of metabolic activity of C. albicans biofilms occurred depending on the time and the antifungal and its concentrations.

Insights Into Mechanisms of Antimicrobial Photodynamic Action Toward Biofilms Using Phenalen-1-One Derivatives as Photosensitizers.

IntroductionIn view of increasing resistance against antibiotics and antiseptics, antimicrobial photodynamic therapy (aPDT) may be a promising approach for use in dentistry. The aim of this study was to investigate the mechanism of action of aPDT with the phenalene-1-one derivatives SAPYR and SA-PN-05 as photosensitizers by evaluating bacterial ability to replicate, membrane integrity, metabolic activity, and formation of reactive oxygen species (ROS) in biofilms of Actinomyces naeslundii, Streptococcus mutans, and Escherichia coli.Materials and MethodsSingle-species biofilms (A. naeslundii, S. mutans, and E. coli) were cultured under aerobic conditions for 48 h followed by treatment with the photosensitizers SAPYR and SA-PN-05 at various concentrations (0, 50, 100, 500 μM) and different incubation periods of 5, 10, 20, and 30 min and subsequent irradiation for 10 min (Waldmann PIB 3000; λem = 360–600 nm; 50 mW/cm2; 30 J/cm2). Control samples were treated with dH2O and kept in dark for the same periods. Bacterial ability to replicate was evaluated by colony forming unit (CFU) assay. The cytoplasmic membrane integrity was investigated by flow cytometry using SYBR Green and propidium iodide and visualized by scanning and transmission electron microscopy. For SAPYR, metabolic activity and formation of intracellular ROS after irradiation were evaluated via luminescence and fluorometric assays, respectively.ResultsSAPYR showed antimicrobial effects (>3 log10 CFU reduction) on S. mutans after 5 min and on A. naeslundii after 20 min incubation and light activation. For E. coli, CFU reduction was >2 log10 after 30 min of incubation. SA-PN-05 showed an antimicrobial effect after 5 min for all bacteria. Membrane damage upon aPDT with SAPYR was observed for E. coli, but not for S. mutans and A. naeslundii. Following treatment with SA-PN-05, irradiated samples and dark controls of all three species showed loss of membrane integrity. Luminescence and fluorometric assays showed a reduction in metabolic activity and an increase in formation of intracellular ROS in all three species upon aPDT treatment with SAPYR.ConclusionThe observed loss in ability to replicate upon aPDT with SAPYR in single-species biofilms may be due to an increase in formation of intracellular ROS upon photodynamic treatment.

NSAIDs as a Drug Repurposing Strategy for Biofilm Control

Persistent infections, usually associated with biofilm-producing bacteria, are challenging for both medical and scientific communities. The potential interest in drug repurposing for biofilm control is growing due to both disinvestment in antibiotic R&D and reduced efficacy of the available panel of antibiotics. In the present study, the antibacterial and antibiofilm activities of four non-steroidal anti-inflammatory drugs (NSAIDs), piroxicam (PXC), diclofenac sodium (DCF), acetylsalicylic acid (ASA) and naproxen sodium (NPX) were evaluated against Escherichia coli and Staphylococcus aureus. The minimum inhibitory/bactericidal concentrations (MICs and MBCs) and the dose–response curves from exposure to the selected NSAIDs were determined. MICs were found for PXC (800 μg/mL) and ASA (1750 μg/mL) against E. coli, and for DCF (2000 μg/mL) and ASA (2000 μg/mL) against S. aureus. No MBCs were found (>2000 μg/mL). The potential of NSAIDs to eradicate preformed biofilms was characterized in terms of biofilm mass, metabolic activity and cell culturability. Additionally, the NSAIDs were tested in combination with kanamycin (KAN) and tetracycline (TET). ASA, DCF and PXC promoted significant reductions in metabolic activity and culturability. However, only PXC promoted biofilm mass removal. Additive interactions were obtained for most of the combinations between NSAIDs and KAN or TET. In general, NSAIDs appear to be a promising strategy to control biofilms as they demonstrated to be more effective than conventional antibiotics.

Development of Natural-Based Bone Cement for a Controlled Doxorubicin-Drug Release.

Osteosarcoma (OS) accounts for 60% of all global bone cancer diagnoses. Intravenous administration of Doxorubicin Hydrochloride (DOXO) is the current form of OS treatment, however, systemic delivery has been linked to the onset of DOXO induced cardiomyopathy. Biomaterials including calcium phosphate cements (CPCs) and nanoparticles (NPs) have been tested as localized drug delivery scaffolds for OS cells. However, the tumor microenvironment is critical in cancer progression, with mesenchymal stem cells (MSCs) thought to promote OS metastasis and drug resistance. The extent of MSC assisted survival of OS cells in response to DOXO delivered by CPCs is unknown. In this study, we aimed at investigating the effect of DOXO release from a new formulation of calcium phosphate-based bone cement on the viability of OS cells cocultured with hMSC in vitro. NPs made of PLGA were loaded with DOXO and incorporated in the formulated bone cement to achieve local drug release. The inclusion of PLGA-DOXO NPs into CPCs was also proven to increase the levels of cytotoxicity of U2OS cells in mono- and coculture after 24 and 72 h. Our results demonstrate that a more effective localized DOXO delivery can be achieved via the use of CPCs loaded with PLGA-DOXO NPs compared to CPCs loaded with DOXO, by an observed reduction in metabolic activity of U2OS cells in indirect coculture with hMSCs. The presence of hMSCs offer a degree of DOXO resistance in U2OS cells cultured on PLGA-DOXO NP bone cements. The consideration of the tumor microenvironment via the indirect inclusion of hMSCs in this study can act as a starting point for future direct coculture and in vivo investigations.

Plasma-Functionalized Water: from Bench to Prototype for Fresh-Cut Lettuce

Fresh-cut produce like lettuce may contain a very high microbial load, including human pathogens. Therefore, the need for antimicrobial agents at post-harvest stages to mitigate microbial cross-contamination and growth is evident. Sanitation based on non-thermal plasma (NTP) reveals innovative food processing possibilities by application at different points along the food chain, for production, modification, and preservation, as well as in packaging of plant- and animal-based food. The aim of the present study was to evaluate the applicability of plasma-treated water (PTW) as antimicrobial process water additives for washing in fresh-cut lettuce processing. Antibacterial activities of PTW the natural occurring microflora of lettuce were examined. Different process variants of PTW application inside the washing process were investigated. Fresh-cut lettuce were investigated regarding microbiological safety and food quality. Samples were analyzed for antimicrobial and metabolic activity as well as metabolic vitality to prove food safety. The investigations for food quality included color and texture analyses and nitrate concentration detection in fresh tissue as well as microscopic measurements by scanning electron microscopy (SEM) and atomic force microscopy (AFM) for tissue surface structure and transmission electron microscopy (TEM) for cell organelle investigations. The application of PTW allowed up to 5 log10 cycle reduction, depending on the process variant and scale (lab and pilot scale). The increase of antimicrobial activity was accompanied by a reduction of metabolic activity, but not consequently by a decrease in metabolic vitality. Food quality was not affected by the use of PTW in the washing process of the fresh-cut lettuce. The promising results in color and texture were supported by the results of the microscopic assays. These promising results may lead to an industrial application of PTW as process water additive in fresh-cut produce processing to reduce the microbial load on the food surface and in addition in the process water or on food processing surfaces.

Interaction of Candida albicans with Fluconazole/ Clotrimazole: Effect on Hyphae Formation and Expression of Hyphal Wall Protein 1

Background and Aims: Candida albicans (C. albicans) is the most common opportunistic human pathogen. Therapeutic options for Candida infections are limited to available antifungal drugs. The aim of this study was to investigate the effects of fluconazole/clotrimazole (FLU/CLT) on C. albicans hyphae formation. Materials and Methods: We have established the effectiveness of the combination of FLU/CLT on C. albicans hyphae formation. Interaction of C. albicans with combination of FLU/CLT was performed using the CLSI guidelines and time-killing curves. We investigated the anti-hyphal activities of combination of FLU/CLT against C. albicans using XTT and crystal violet assays as well as scanning electron microscopy and expression of HWP1 gene. Results: The interaction of C. albicans with FLU/CLT resulted in synergistic, partial synergistic and indifferent effects. The interaction of FLU/CLT were confirmed by time-killing curves. FLU/CLT combined resulted in the reduction of metabolic activity and hyphae formation in C. albicans. Images taken by scanning electron microscopy indicated the effectiveness on hyphae disruption. According to relative real time polymerase chain reaction analysis, the mean Ct values revealed the significant decrease in expression level of the HWP1 gene. A 2.86- and 2.33-fold decrease in HWP1 gene expression was observed in combination of FLU/CLT treatment at 2× minimum inhibitory concentration and 1× minimum inhibitory concentration, respectively (p=0.002). Conclusions: We confirmed that the hyphae is a target for the combination of FLU/CLT in C. albicans. HWP1 gene is likely to be considered as a probable targets synergistic interaction of FLU/CLT against C. albicans.

Chlorogenic acid acts upon Leishmania donovani arresting cell cycle and modulating cytokines and nitric oxide in vitro.

Visceral leishmaniasis (VL), caused by Leishmania donovani in India, is fatal if untreated, having serious concern of limited chemotherapeutic options. In this study, we evaluated antileishmanial efficacy of purified chlorogenic acid (CGA) against promastigotes and intracellular amastigotes infected into RAW264.7 macrophages. Chlorogenic acid was effective both on promastigotes (IC50 =78.394µmol/L, i.e. 27.75µg/mL) and intracellular amastigotes (ED50 =26.752µmol/L, i.e. 9.47µg/mL). In promastigotes, significant retardation in mitotic growth was caused both by cell-death and reduction of metabolic activity, evidenced by propidium-iodide uptake and MTT assay, respectively. Flow cytometric analysis revealed that retardation of mitotic growth was due to cell-cycle arrest at G1/S checkpoint. Complete clearance of amastigotes from infected RAW264.7 cells, assessed by microscopic counting, was achieved with 60µmol/L (21.24µg/mL) CGA for 24hours, with negligible toxicity to host macrophages. This parasite clearing efficacy was comparable to 1.0µg/mL (1.082µmol/L) Amphotericin B, and 20µmol/L Miltefosine, two standard antileishmanial drugs. Cytokine-ELISA revealed that elevated IL-10 production by infected macrophages was reduced after parasite clearance. Consequently, IL-12, TNF and NO (assayed by Griess test) production by macrophages were significantly increased after successful resolution of infection. Chlorogenic acid might emerge as a potential antileishmanial drug.

Graduated Controlled Atmosphere: A Novel Approach to Increase "Duke" Blueberry Storage Life.

Blueberries (Vaccinium corymbosum L.) are highly valued for their health-promoting potential, yet they are extremely perishable. Controlled atmosphere (CA) strategies reduce blueberry respiratory metabolism, slowing down senescence. However, the sudden change of atmosphere could elicit a physical abiotic stress in the fruit, negatively affecting quality. We propose an innovative approach based on controlled graduation to slowly reach optimum gas storage conditions as an alternative to standard CA. For two consecutive seasons, “Duke” blueberries were subjected to four different storage conditions: control (air); standard CA (sudden exposure to 5 kPa O2 and 10 kPa CO2 across the experiment); GCA3 and GCA7 (gradually reaching 5 kPa O2 and 10 kPa CO2 in 3 and 7 days, respectively). Fruit were stored for 28 days at 0 ± 0.5°C. Real-time respirometry provided an in-depth insight to the respiratory response of blueberries to their gas environment. Blueberries subjected to the graduated application of CA (GCA) treatments had a lower steady-state respiration rate compared to control and standard CA fruit. This indicated a reduction in metabolic activity that positively impacted quality and storage life extension. For example, GCA3 and GCA7 blueberries had a 25% longer storage life when compared to control, based on reduced decay incidence. In addition, GCA fruit were 27% firmer than control and CA fruit after 28 days of cold storage. GCA3 had a positive effect on maintaining individual sugars concentrations throughout the experiment, and both GCA treatments maintained ascorbic acid content close to initial values compared to a decrease of 44% in the control fruit at the end of the experiment. This work provides a paradigm shift in how CA could be applied and a better understanding of blueberry physiology and postharvest behavior.

Reproductive cycle progression arrest and modification of cell morphology (shape and biovolume) in the alga Pseudokirchneriella subcapitata exposed to metolachlor

Metolachlor (MET) is an herbicide widely used and frequently found (at μg L−1) in aquatic systems. This work aimed to study the modes of action of MET on the green microalga Pseudokirchneriella subcapitata. Algae exposed to 115 or 235 μg L−1 MET, for 48 or 72 h, presented a reduction of metabolic activity, chlorophyll a and b content and photosynthetic efficiency. The exposure to 115 or 235 μg L−1 MET also induced growth yield reduction, mean cell biovolume increase and alteration of the typical algae shape (cells lunate or helically twisted) to “French croissant”-type; at these MET concentrations, algal population was mainly composed by multinucleated cells (≥ 4 nuclei), which suggest that MET impairs the normal progression of the reproductive cycle but did not hinder nuclear division. The accumulation of multinucleated cells seems to be the consequence of the incapacity of the parent cell to release the autospores. In conclusion, MET disrupts the physiology of P. subcapitata cells; the disturbance of the progression of the reproductive cycle should be in the origin of growth slowdown (or even its arrest), increase of mean cell biovolume and modification of algal shape. This work contributed to elucidate, in a systematically and integrated way, the toxic mechanism of MET on the non-target organism, the alga P. subcapitata.

Alcohol Intoxication and Cognition: Implications on Mechanisms and Therapeutic Strategies.

Binge alcohol drinking is highly prevalent in young adults and results in 30% deaths per year in young males. Binge alcohol drinking or acute alcohol intoxication is a risk factor for developing alcohol use disorder (AUD). Three FDA approved drugs are currently in use as therapy for AUD; however, all of them have contra-indications and limitations. Structural brain imaging studies in alcoholics have shown defects in the brain regions involved in memory, cognition and emotional processing. Positron emission tomography (PET) using radiotracers (e.g., 18FDG) and measuring brain glucose metabolism have demonstrated diagnostic and prognostic utility in evaluating patients with cognitive impairment. Using PET imaging, only a few exclusive human studies have addressed the relationship between alcohol intoxication and cognition. Those studies indicate that alcohol intoxication causes reduction in brain activity. Consistent with prior findings, a recent study by us showed that acute alcohol intoxication reduced brain activity in the cortical and subcortical regions including the temporal lobe consisting the hippocampus. Additionally, we have observed a strong correlation between reduction in metabolic activity and spatial cognition impairment in the hippocampus after binge alcohol exposure. We have also demonstrated the involvement of a stress response protein, cold inducible RNA binding protein (CIRP), as a potential mechanistic mediator in acute alcohol intoxication. In this review, we will first discuss in detail prior human PET imaging studies on alcohol intoxication as well as our recent study on acute alcohol intoxication, and review the existing literature on potential mechanisms of acute alcohol intoxication-induced cognitive impairment and therapeutic strategies to mitigate these impairments. Finally, we will highlight the importance of studying brain regions as part of a brain network in delineating the mechanism of acute alcohol intoxication-induced cognitive impairment to aid in the development of therapeutics for such indication.