Enhanced photocatalytic and electrochemical performance of hydrothermally prepared NiO-doped Co nanocomposites.

In this study, we synthesize nanostructured nickel oxide (NiO) and doped cobalt (Co) by combining nickel(II) chloride hexahydrate (NiCl2.6H2O) and sodium hydroxide (NaOH) as initial substances. We analyzed the characteristics of the product nanostructures, including their structure, optical properties, and magnetic properties, using various techniques such as x-ray diffraction (XRD), scanning electron microscopy (SEM), ultraviolet absorption spectroscopy (UV-Vis), Fourier transform infrared (FTIR) spectroscopy, and vibrating sample magnetometers (VSM). The NiO nanoparticles doped with Co showed photocatalytic activity in degrading methylene blue (MB) dye in aqueous solutions. We calculated the degradation efficiencies by analyzing the UV-Vis absorption spectra at the dye's absorption wavelength of 664 nm. It was observed that the NiO-doped Co nanoparticles facilitated enhanced recombination and migration of active elements, which led to more effective degradation of organic dyes during photocatalysis. We also assessed the electrochemical properties of the materials using cyclic voltammetry (CV) and impedance spectroscopy in a 1 mol% NaOH solution. The NiO-modified electrode exhibited poor voltammogram performance due to insufficient contact between nanoparticles and the electrolyte solution. In contrast, the uncapped NiO's oxidation and reduction cyclic voltammograms displayed redox peaks at 0.36 and 0.30 V, respectively.

Methylene Blue Reduces Retinal Cell Inflammation, Apoptosis, and Oxidative Stress in a Rat Model of Diabetic Retinopathy via Sirtuin 1 Activation.

Objective: Diabetic retinopathy (DR), characterized by neuronal damage in the retina, is primarily driven by oxidative stress resulting from diabetes (DM). This study investigated the potential effects of methylene blue (MB) on streptozotocin (STZ)-induced DR. Methods: A rat model of DR was established via STZ injection, while a cell model was created using high-glucose (HG) exposure of human retinal microvascular endothelial cells. Evaluation of oxidative stress markers, pro-inflammatory cytokines, and pro-apoptotic proteins was performed based on their expression profiles in human retinal microvascular endothelial cells. Results: MB treatment significantly upregulated the expression of sirtuin 1 (SIRT1), which was found to be downregulated in the retinal tissues of STZ-treated rats and HG-exposed human retinal microvascular endothelial cells, as determined by polymerase chain reaction (PCR). Furthermore, MB therapy effectively suppressed STZ-induced oxidative stress, inflammation, and cell death. Consistent with the in vivo findings, MB activated the expression of SIRT1, thereby protecting HG-treated human retinal microvascular endothelial cells against oxidative stress, inflammation, and apoptosis. Conclusion: These results support the conclusion that MB mitigates DR by activating SIRT1, leading to a reduction of inflammation, apoptosis, and oxidative stress.

Adverse effects of methylene blue in peripheral neurons: An in vitro electrophysiology and cell culture study.

Methylene blue (MB) is an effective treatment for methemoglobinemia, ifosfamide-induced encephalopathy, cyanide poisoning, and refractory vasoplegia. However, clinical case reports and preclinical studies indicate potentially neurotoxic activity of MB at certain concentrations. The exact mechanisms of MB neurotoxicity are not known, and while the effects of MB on neuronal tissue from different brain regions and myenteric ganglia have been examined, its effects on primary afferent neurons from dorsal root ganglia (DRG) have not been studied. Mouse DRG were exposed to MB (0.3-10 µM) in vitro to assess neurite outgrowth. Increasing concentrations of MB (0.3-10 µM) were associated with neurotoxicity as shown by a substantial loss of cells with neurite formation, particularly at 10 µM. In parallel experiments, cultured rat DRG neurons were treated with MB (100 µM) to examine how MB affects electrical membrane properties of small-diameter sensory neurons. MB decreased peak inward and outward current densities, decreased action potential amplitude, overshoot, afterhyperpolarization, increased action potential rise time, and decreased action potential firing in response to current stimulation. MB induced dose-dependent toxicity in peripheral neurons, in vitro. These findings are consistent with studies in brain and myenteric ganglion neurons showing increased neuronal loss and altered membrane electrical properties after MB application. Further research is needed to parse out the toxicity profile for MB to minimize damage to neuronal structures and reduce side effects in clinical settings.

Molecular Mechanisms of the Neuroprotective Effect of Methylene Blue.

Methylene blue (MB) is the first fully synthetic compound that had found its way into medicine over 120 years ago as a treatment against malaria. MB has been approved for the treatment of methemoglobinemia, but there are premises for its repurposing as a neuroprotective agent based on the efficacy of this compound demonstrated in the models of Alzheimer's, Parkinson's, and Huntington's diseases, traumatic brain injury, amyotrophic lateral sclerosis, depressive disorders, etc. However, the goal of this review was not so much to focus on the therapeutic effects of MB in the treatment of various neurodegeneration diseases, but to delve into the mechanisms of direct or indirect effect of this drug on the signaling pathways. MB can act as an alternative electron carrier in the mitochondrial respiratory chain in the case of dysfunctional electron transport chain. It also displays the anti-inflammatory and anti-apoptotic effects, inhibits monoamine oxidase (MAO) and nitric oxide synthase (NOS), activates signaling pathways involved in the mitochondrial pool renewal (mitochondrial biogenesis and autophagy), and prevents aggregation of misfolded proteins. Comprehensive understanding of all aspects of direct and indirect influence of MB, and not just some of its effects, can help in further research of this compound, including its clinical applications.

Statistical modeling of methylene blue degradation by yeast-bacteria consortium; optimization via agro-industrial waste, immobilization and application in real effluents.

The progress in industrialization everyday life has led to the continuous entry of several anthropogenic compounds, including dyes, into surrounding ecosystem causing arduous concerns for human health and biosphere. Therefore, microbial degradation of dyes is considered an eco-efficient and cost-competitive alternative to physicochemical approaches. These degradative biosystems mainly depend on the utilization of nutritive co-substrates such as yeast extract peptone in conjunction with glucose. Herein, a synergestic interaction between strains of mixed-culture consortium consisting of Rhodotorula sp., Raoultella planticola; and Staphylococcus xylosus was recruited in methylene blue (MB) degradation using agro-industrial waste as an economic and nutritive co-substrate. Via statistical means such as Plackett-Burman design and central composite design, the impact of significant nutritional parameters on MB degradation was screened and optimized. Predictive modeling denoted that complete degradation of MB was achieved within 72 h at MB (200 mg/L), NaNO3 (0.525 gm/L), molasses (385 µL/L), pH (7.5) and inoculum size (18%). Assessment of degradative enzymes revealed that intracellular NADH-reductase and DCIP-reductase were key enzymes controlling degradation process by 104.52 ± 1.75 and 274.04 ± 3.37 IU/min/mg protein after 72 h of incubation. In addition, azoreductase, tyrosinase, laccase, nitrate reductase, MnP and LiP also contributed significantly to MB degradation process. Physicochemical monitoring analysis, namely UV-Visible spectrophotometry and FTIR of MB before treatment and degradation byproducts indicated deterioration of azo bond and demethylation. Moreover, the non-toxic nature of degradation byproducts was confirmed by phytotoxicity and cytotoxicity assays. Chlorella vulgaris retained its photosynthetic capability (˃ 85%) as estimated from Chlorophyll-a/b contents compared to ˃ 30% of MB-solution. However, the viability of Wi-38 and Vero cells was estimated to be 90.67% and 99.67%, respectively, upon exposure to MB-metabolites. Furthermore, an eminent employment of consortium either freely-suspended or immobilized in plain distilled water and optimized slurry in a bioaugmentation process was implemented to treat MB in artificially-contaminated municipal wastewater and industrial effluent. The results showed a corporative interaction between the consortium examined and co-existing microbiota; reflecting its compatibility and adaptability with different microbial niches in different effluents with various physicochemical contents.

Increased photoluminescence and photodynamic therapy efficiency of hydroxyapatite-ß-cyclodextrin-methylene blue@carbon powders with the favor of hydrogen bonding effect.

To meet the requirements of theranostics with diagnosis and treatment, photodynamic-based therapy is simultaneously enabled with the incorporation of methylene blue (MB) as imaging agent and photosensitizer in core-shell structured drug vehicles. Citrate-modified hydroxyapatite (HAp) powders are first grafted with ß-cyclodextrin (CD), then combined with MB molecules through electrostatic interactions, and finally encapsulated with carbon shells through hydro-thermal carbonization of glucose to prepare HAp-CD-MB@C powders. Processing parameters of carbonization temperature, glucose addition, reaction time and CD addition are varied to prepare drug carriers with modulated crystallite degrees and photo-physical properties. Increased crystallite sizes of HAp are accompanied with the formation of C=O, C=C and C-OH groups in carbon shell, endowing sustainable release behaviors of MB through carbonous structures. High photoluminescence intensities are fairly related with red-shifted vibration peaks of groups in tightly combined MB molecules through hydrogen bonds. This hydrogen bonding effect is significantly increased for HAp-CD-MB@C140 with the splitting of CH3-involved vibration peaks in infrared spectra, which causes increase in photoluminescence intensity and four-fold increase in generation ratio of singlet oxygen. The present studies shed light on preparation of core-shell structured drug carriers, modulation of aggregate states of MB molecules, enhancement of photo-physical properties and improvement of generation ratio of singlet oxygen during photodynamic-based therapy.

The ameliorative effects of methylene blue on testicular damage induced by cisplatin in rats.

Cisplatin, a common chemotherapeutic drug, can induce testicular toxicity. Methylene blue, a potent antioxidant, can inhibit the generation of free radicals. This research aimed to study the protective effect of methylene blue against the cisplatin-induced toxicity of the reproductive system in rats. 35 male Wistar rats were divided into five groups: the control group, the cisplatin group (a single dose of 5 mg/kg cisplatin), the low-dose and high-dose methylene blue + cisplatin (2 and 4 mg/kg of methylene blue, respectively, for 7 days) and the methylene blue group (4 mg/kg of methylene blue, for 7 days). The treatments were applied through intraperitoneal injection. Cisplatin treatment reduced the sperm parameters and serum testosterone levels significantly. Methylene blue treatment increased the sperm count (p < .001), viability (p < .001) and motility (p < .001) compared to the cisplatin group. The methylene blue group showed a significant increase in the levels of testosterone compared to the cisplatin group (p < .001) and reverted histopathological changes in cisplatin-treated groups. Immunohistochemical evaluation of the caspase-3 protein revealed that the treatment with methylene blue has significant anti-apoptotic effects on testicular tissue damage. In conclusion, methylene blue can attenuate the cisplatin-induced histological damages and improve the sperm parameters.

Determination of Methylene Blue and Its Metabolite Residues in Aquatic Products by High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A sensitive and reliable method was developed to determine methylene blue (MB) and its metabolite residues, including azure A (AZA), azure B (AZB), and azure C (AZC) in aquatic products by HPLC-MS/MS. The samples were extracted by acetonitrile and cleaned up by alumina-neutral (ALN) cartridges. The analytes were separated on a Sunfire C18 column (150 mm × 2.1 mm, 5 µm). The method was validated according to the European criteria of Commission Decision 2002/657/CE. Good linearity between 1-500 µg/L was obtained with correlation coefficients (R2) greater than 0.99. The limit of quantification (LOQ) was 1.0 µg/kg. The average recoveries at three levels of each compound (1, 5, and 10 µg/kg) were demonstrated to be in the range of 71.8-97.5%, with relative standard deviations (RSDs) from 1.05% to 8.63%. This method was suitable for the detection of methylene blue and its metabolite residues in aquatic products.

Low-cost Fluorescein as an Alternative to Radio-colloid for Sentinel Lymph Node Biopsy-a Prospective Validation Study in Early Breast Cancer.

BACKGROUND: Sentinel lymph node biopsy (SLNB) using radio-pharmaceutical (RP) and a blue dye is gold standard for axillary staging in clinically node-negative early breast cancer. High costs and limited availability of RP and/or gamma probe are major deterrents in performing SLNB in developing countries. Fluorescence-guided SLNB can obviate the need for RP and gamma probe. Fluorescein is an inexpensive fluorescent lymphatic tracer. In this study, we compared SLN identification rate (SLN-IR) and false negative rates (FNR) of fluorescein-guided SLNB and radio-guided SLNB using 99mTc-Sulfur-colloid, in isolation, or in combination with methylene blue dye (MBD). METHODS: Sixty-five cN0 early and large operable breast cancer patients underwent validation SLNB using fluorescein (and blue LED light), 99mTc-Sulfur-colloid (and gamma probe) and MBD. Inj Fluorescein 4% was injected, 1 ml each peri-tumoral and sub-areolar five minutes before axillary incision. Axillary dissection was performed irrespective of SLNB histology. The SLN-IR and FNR with various tracers and their combinations were compared. RESULTS: The mean number of SLNs identified was 3.5 ± 1.8 (range 1-6). The SLN-IR using RP alone was 94%, fluorescein alone was 92%, and MBD alone was 82%. The SLN-IR using fluorescein plus MBD combination was 95.4%, compared to 97% using MBD plus RP combination. FNR was 6.3% using fluorescein plus MBD, as well as RP plus MBD combinations. CONCLUSIONS: SLN-IR of > 90% and SLN-FNR of < 10% using fluorescein plus MBD combination are in acceptable range, and are comparable to that of RP plus MBD combination. Fluorescein can replace RP for performing SLNB, in combination with MBD.

Methylene blue-treated plasma, versus quarantine fresh frozen plasma, for acute thrombotic thrombocytopenic purpura treatment: Comparison between centres and critical review on longitudinal data.

INTRODUCTION: Therapeutic plasma exchange (TPE) is the first-line treatment for acute thrombotic thrombocytopenic purpura (TTP). Methylene blue-plasma (MBP) has been used for over 20 years, but its efficacy in this setting remains controversial. PATIENTS AND METHODS: this is a comparative analysis of the experience of two Centres, with different plasma products, to evaluate their efficacy in TTP. One centre used quarantine plasma (QP), and MBP the other. We performed a retrospective longitudinal study, analysing the clinical files of TTP patients of a 13-year data evaluation period. Duration of treatment and transfusion parameters, medical record, laboratory testing, concomitant medication, and survival rate, were assessed for every episode. RESULTS: During the study period, 12 (55.5 %) and 10 (45.5 %) new cases were treated with QP and MBP, respectively. There were no significant differences between the mean numbers of TPE processes, days elapsed from diagnosis to TPE, and plasma volume transfused. The QP TPE episodes of treatment were significantly associated with an increased time to recovery compared with MBP episodes of treatment (p = 0.004). CONCLUSION: MBP was as effective as QP in the treatment of TTP patients. Since recovery was more favourable when MBP was used, we consider MBP remains a suitable alternative to treat TTP patients.

Methylene blue active substances in plaque of Bacillus subtilis subsp. subtilis and enrichment by supplemental calcium in culture media.

A series of experiments was conducted to identify the molecular species responsible for surface active emulsification (surfactant) bioactivity in Bacillus subtilis subsp. subtilis strain ATCC PTA-125135, and to describe culture conditions to support the enriched production of said bioactivity in cultured plaque of the strain. The assay for methylene blue active substances (MBAS) was found to be suitable for describing surfactant activity, where a solvent-extracted molecular fraction from the biofilm was found to retain surfactant activity and positively quantified as MBAS. Furthermore, an HPLC-refined protein fraction was found to quantify as MBAS with approximately 1·36-fold or greater surfactant activity per mol than sodium dodecyl sulphate, and a proteomic analysis of solvent extracted residues confirmed that biofilm surface layer protein BslA was a primary constituent of extracted residues. Surfactant bioactivity, quantified as MBAS, was enriched in cultured plaque by the supplementation of culture media with calcium chloride or calcium nitrate.

Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency.

A likely mechanism of chromosomal rearrangement formation involves joining the ends from two different chromosomal double-strand breaks (DSBs). These events could potentially be mediated by either of two end-joining (EJ) repair pathways [canonical nonhomologous end joining (C-NHEJ) or alternative end joining (ALT-EJ)], which cause distinct rearrangement junction patterns. The relative role of these EJ pathways during rearrangement formation has remained controversial. Along these lines, we have tested whether the DNA damage response mediated by the Ataxia Telangiectasia Mutated (ATM) kinase may affect the relative influence of C-NHEJ vs. ALT-EJ on rearrangement formation. We developed a reporter in mouse cells for a 0.4-Mbp deletion rearrangement that is formed by EJ between two DSBs induced by the Cas9 endonuclease. We found that disruption of the ATM kinase causes an increase in the frequency of the rearrangement as well as a shift toward rearrangement junctions that show hallmarks of C-NHEJ. Furthermore, ATM suppresses rearrangement formation in an experimental condition, in which C-NHEJ is the predominant EJ repair event (i.e., expression of the 3' exonuclease Trex2). Finally, several C-NHEJ factors are required for the increase in rearrangement frequency caused by inhibition of the ATM kinase. We also examined ATM effectors and found that H2AX shows a similar influence as ATM, whereas the influence of ATM on this rearrangement seems independent of 53BP1. We suggest that the contribution of the C-NHEJ pathway to the formation of a 0.4-Mbp deletion rearrangement is enhanced in ATM-deficient cells.

Usefulness of urinary glycosaminoglycans assay for a mucopolysaccharidosis-specific screening.

BACKGROUND: Mucopolysaccharidoses (MPS), a group of inherited metabolic disorders characterized by the accumulation of glycosaminoglycans, can be diagnosed early through newborn screening programs. Establishing newborn screening in Morocco is a challenging task for multiple economic and social reasons. Screening in a Moroccan population using 1,9-dimethylmethylene blue urinary glycosaminoglycan (GAG) assays may allow for an earlier diagnosis of MPS. We studied the feasibility of implementing screening in Moroccan children as an alternative to national newborn screening. We determined the reference ranges for GAGs in the Moroccan population, their stability during transport, the effectiveness of this test as a screening procedure for MPS in patients, and its use as a screening test for MPS in the Imssouane region, where the rate of consanguineous marriage is 38%. METHODS: Using dimethylmethylene blue assays, urine samples of 47 MPS patients were analyzed, together with urine samples from healthy controls (n = 368, age ranging from 1 month to 25 years), and from Imssouane region children (n = 350, age ranging from 6 months to 24 month). Precision, linearity, recovery, limits, and stability were tested. RESULTS: Urinary GAGs reference values are age and ethnicity dependent. The validation parameters established displayed great precision and accuracy leading to recoveries according to internationally accepted values for bioanalytical methods. Urinary GAGs were stable for a maximum of 7 weeks at 40 °C. Screening of Imssouane children resulted in the detection of a 6-month-old child, diagnosed with MPS I. CONCLUSIONS: Our results demonstrate the usefulness of quantifying glycosaminoglycans for early screening of MPS.

Antibacterial, antibiofilm, and photocatalytic activities of metals-substituted spinel cobalt ferrite nanoparticles.

This study reports the photocatalytic degradation of Methylene Blue (MB) dye (a class of dyestuffs that are resistant to biodegradation) under the influence of UV-light irradiation. Antibacterial and antibiofilm activities of ferrite nanoparticles (FO NPs) were examined against some pathogenic bacteria isolated from the medical operating room surfaces. In the same context, metals-substituted spinel cobalt ferrite nanoparticles with nominal composition [MxCo1-xFe2O4 NPs; (M = Zn, Cu, Mn; x = 0.0, 0.25, 0.5 and 0.75)] were synthesized by citrate sol-gel method. Also, the structures of the synthesized FO NPs were characterized by X-ray diffraction, and Williamson-Hall (WH) method was used to determine the crystallite size. The estimated specific surface area is found in the range from 37.99 to 107.05 m2/g, between the synthesized ferrites, Zn0.5Co0.5Fe2O4 NPs have average pore radius 1.84 nm and the pore volume was 0.136 ml/g. SEM images revealed that, the synthesized FO NPs have an unique pores and uniformly distribution, while EDX spectra shows the elemental composition for the synthesized FO NPs. The elastic properties of FO NPs have been estimated using FTIR data, whereas (M - H) hysteresis loops revealed that, by replacing cobalt ions with Zn, Cu, and Mn ions the magnetic behaviour changed from ferromagnetic to paramagnetic. Results obtained from the photocatalysis indicated that Mn0.75Co0.25Fe2O4 NPs (30.0 mg) were a promising photocatalyst achieving 96.0% removal of MB after 100 min of UV-light exposure in the alkaline solution. Antibacterial results showed that the most effective combination was Zn0.75Co0.25Fe2O4 NPs (20.0 ppm) displaying activity against Staphylococcus aureus, Enterococcus columbae, and Aerococcus viridians by 15.0, 13.0, and 12.0 mm ZOI, respectively. Additionally, Zn0.75Co0.25Fe2O4 NPs were active as antibiofilm factors producing activity by 63.7, 57.9, and 45.5% towards S. aureus, A. viridians, and E. columbae, respectively. Accordingly, Zn0.75Co0.25Fe2O4 and Mn0.75Co0.25Fe2O4 NPs can be utilized in industrial, biological and medical applications.

Screening sediment samples used as anolytes in microbial fuel cells for microbial electron transfer activity using DREAM assay.

OBJECTIVE: The dye reduction-based electron-transfer activity monitoring (DREAM) assay was employed to screen sediment and wastewater samples functioning as anolytes in a microbial fuel cell (MFC) for their microbial electron transfer activity. RESULTS: Electron transfer to redox dyes from microbial activity reduced the dyes and the resulting extent of reduction was measured as DREAM assay coefficient. Methylene blue was decolourised, while resazurin underwent florigenic change from blue to pink to colourless upon formation of resorufin and dihydroxyresorufin. DREAM assay coefficient conformed to power density obtained in the MFC. A correlation was observed between chemical oxygen demand of the sample and the DREAM coefficient (+ 0.934) and also between DREAM coefficient and power density generated (+ 0.976). Highest DREAM coefficient and power density was observed for activated sludge. CONCLUSIONS: The DREAM assay is a rapid, sensitive and low-cost method to assess microbial electron transfer activity for inocula used as anolytes in a MFC.

Photodynamic inactivation diminishes quorum sensing-mediated virulence factor production and biofilm formation of Serratia marcescens.

Serratia marcescens is an opportunistic human pathogen causing nosocomial infections and displays expanded resistance towards the conventional antibiotics. In S. marcescens, quorum sensing (QS) mechanism coordinates the population-dependent behaviors and regulates the virulence factors production. Photodynamic inactivation (PDI) is a promising alternative for the treatment of infections caused by drug resistant bacteria. Although PDI should be applied at lethal doses, it is possible that during PDI treatment, pathogens encounter sub-lethal doses of PDI (sPDI). sPDI cannot kill microorganisms, but it can considerably influence the microbial virulence. So, in this study, the effect of methylene blue (MB)-mediated PDI on QS-mediated virulence factor production and biofilm formation of S. marcescens at lethal and sub-lethal doses was evaluated. The biofilm formation and virulence factor production of S. marcescens ATCC 13,880 and S. marcescens Sm2 were assessed before and after PDI treatment. Besides, the effect of lethal and sub-lethal PDI on expression of bsmA and bsmB (Biofilm maturation), fimA and fimC (Major fimbrial protein), flhD (Regulator of flagellar mediated swarming and swimming motility) and swrR (AHL-dependent regulator) genes were evaluated by quantitative real time polymerase chain reaction. Lethal and sub-lethal PDI resulted in a significant decrease in biofilm formation, swimming/swarming motility, and pigment and hemolysin production ability of S. marcescens strains. bsmA, bsmB, flhD and swrR genes were down-regulated after PDI treatments. In conclusion, QS-mediated virulence factor production and biofilm formation ability of the two studied S. marcescens strains decreased after both lethal and sub-lethal PDI.

[Performance of MALDI-TOF MS for the identification of gram-negative bacteria grown on eosin methylene blue (EMB) agar: A simple method for improving the effectiveness of identification]. / MALDI-TOF MS'nin EMB agarda üreyen gram-negatif bakterileri tanimlama performansi: Tani etkinligini arttiran basit bir yöntem.

Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a new method that is increasingly used in microbiology laboratories due to its ability of reliable and rapid identification (ID) of bacteria and fungi. However, some problems emerge in the routine clinical diagnosis since only one gram-negative selective medium has been suggested up to date. Though EMB agar is one of the traditional gram-negative selective media, there is no data in the scientific literature, about the ID performance of MALDI TOF MS with the gram-negative bacteria grown on this medium. In this study, we tested the ID performance of MALDI-TOF MS for gram-negative isolates on EMB agar and aimed to develop a rapid and easy sample preparation method for improving this performance. A total of 468 clinical isolates of gram-negative bacteria, consisting of 37 different species from 20 genera, were included in this study. The isolates were identified using the Vitek MS MALDI-TOF MS (Bio Mérieux, France) both directly from EMB agar, and also through a two-step colony washing (once with physiologic saline, and three times with 70% ethanol) method. The performances of these two IDs were compared. In the direct reading from EMB medium, 382 (81.6%) of 468 studied isolates were correctly identified at species level; no ID was detected for 80 (17%) isolates, and 6 (1.2%) isolates (four at the genus level) were misidentified Performance of MALDI-TOF MS directly from EMB agar was excellent (100%) for 14 species including Stenotrophomonas maltophilia, Klebsiella oxytoca, Salmonella spp., and Proteus mirabilis; and lowest for Providencia spp. (62.5%), Escherichia coli (70.5%) and Acinetobacter spp. (70.7%). Following the washing procedure which was performed about 20 min with simple laboratory equipment, 434 (92.7%) isolates were correctly identified at species level; 30 (6.4%) strains could not be identified, and four (0.85%) isolates (two at the genus level) were misidentified. Statistical analyses indicated that the washing procedure defined here significantly increased the overall ID performance of MALDI-TOF MS with EMB agar (p= 0.001), particularly with improving the IDs of those markedly dye-absorbing genera, such as E.coli and A.baumannii. In this study, EMB agar which has no data until today on its suitability for mass spectrometric identification has been shown to be useful for bacterial identification with MALDI-TOF MS. In addition, the unidentified gram-negative bacteria in the direct reading from the EMB medium have been shown to be identified after the colony washing method as described here. Determination of the different medium alternatives will contribute to effective usage of MALDI-TOF MS in microbiology laboratories.

A Mechanistic Investigation of Methylene Blue and Heparin Interactions and Their Photoacoustic Enhancement.

We recently reported a real-time method to measure heparin in human whole blood based on the photoacoustic change of methylene blue (MB). Intriguingly, the MB behaved unlike other "turn on" photoacoustic probes-the absorbance decreased as the photoacoustic signal increased. The underlying mechanism was not clear and motivated this study. We studied the binding mechanism of MB and heparin in water and phosphate buffer saline (PBS) with both experimental and computational methods. We found that the photoacoustic enhancement of the MB-heparin mixture was a result of MB-heparin aggregation due to charge neutralization and resulting sequestration of MB in these aggregates. The sequestration of MB in the MB-heparin aggregates led to decreased absorbance-there was simply less free dye in solution to absorb light. The highest photoacoustic signal and aggregation occurred when the number of negatively charged sulfate groups on heparin was approximately equal to the number of positively charged MB molecule. The MB-heparin aggregates dissociated when there were more sulfated groups from heparin than MB molecules because of the electrostatic repulsion between negatively charged sulfate groups. PBS facilitated MB dimer formation regardless of heparin concentration and reprecipitated free MB in aggregates due to ionic strength and ionic shielding. Further molecular dynamics experiments found that binding of heparin occurred at the sulfates and glucosamines in heparin. Phosphate ions could interact with the heparin via sodium ions to impair the MB-heparin binding. Finally, our model found 3.7-fold more MB dimerization upon addition of heparin in MB solution confirming that heparin facilitates MB aggregation. We conclude that the addition of heparin in MB decreases the absorbance of the sample because of MB-heparin aggregation leading to fewer MB molecules in solution; however, the aggregation also increases the PA intensity because the MB molecules in the MB-heparin aggregate have reduced degrees of freedom and poor heat transfer to solvent.

Description of an ultrasound-guided thoracic paravertebral block technique and the spread of dye in dog cadavers.

OBJECTIVES: To describe an ultrasound-guided thoracic paravertebral block and determine the distribution after injection of two volumes of methylene blue in dog cadavers. STUDY DESIGN: Prospective experimental cadaveric study. ANIMALS: Twelve dog cadavers weighing 11 ± 3 kg. METHODS: Ultrasound-guided injections aimed at the fifth thoracic (T5) paravertebral space were performed in randomized order using 0.1 or 0.3 mL kg-1 dye solution (six dogs for each volume). Anatomic dissections determined dye spread characteristics, including the presence and degree of staining of spinal nerves, and the presence of intercostal and sympathetic trunk spread. Staining of mediastinum, epidural, intrapleural and contralateral thoracic paravertebral space was recorded. RESULTS: There was no significant difference in dye distribution between groups. The use of anatomic landmarks resulted in the inaccurate identification of the T5 paravertebral space. The T4, T5 and T6 paravertebral spaces were injected in four, five and three of 12 dogs, respectively. Complete staining of the spinal nerve of the thoracic paravertebral space injected was observed in 11 of 12 dogs, and partial staining in one dog in the low-volume group. Multisegmental distribution was demonstrated with staining of contiguous spinal nerves in one dog in the high-volume group, and multiple segments of intercostal (three dogs) and sympathetic trunk (four dogs) spread in both groups. No mediastinal, epidural, intrapleural or contralateral thoracic paravertebral space staining was observed. CONCLUSIONS AND CLINICAL RELEVANCE: Ultrasound-guided injection at the thoracic paravertebral space resulted in staining of the spinal nerve in all dogs. However, T5 paravertebral space was not accurately identified using anatomic landmarks. Dye distribution was not significantly different between the two groups; therefore, the use of the lower-volume and multiple-site injections would be potentially necessary in clinical cases to achieve ipsilateral blockade of the thoracic wall.

Biogenic synthesis of multifunctional silver nanoparticles from Rhodotorula glutinis and Rhodotorula mucilaginosa: antifungal, catalytic and cytotoxicity activities.

Silver nanoparticles (AgNPs) have several technological applications and may be synthetized by chemical, physical and biological methods. Biosynthesis using fungi has a wide enzymatic range and it is easy to handle. However, there are few reports of yeasts with biosynthetic ability to produce stable AgNPs. The purpose of this study was to isolate and identify soil yeasts (Rhodotorula glutinis and Rhodotorula mucilaginosa). After this step, the yeasts were used to obtain AgNPs with catalytic and antifungal activity evaluation. Silver Nanoparticles were characterized by UV-Vis, DLS, FTIR, XRD, EDX, SEM, TEM and AFM. The AgNPs produced by R. glutinis and R. mucilaginosa have 15.45 ± 7.94 nm and 13.70 ± 8.21 nm (average ± SD), respectively, when analyzed by TEM. AgNPs showed high catalytic capacity in the degradation of 4-nitrophenol and methylene blue. In addition, AgNPs showed high antifungal activity against Candida parapsilosis and increase the activity of fluconazole (42.2% for R. glutinis and 29.7% for R. mucilaginosa), while the cytotoxicity of AgNPs was only observed at high concentrations. Finally, two yeasts with the ability to produce AgNPs were described and these particles showed multifunctionality and can represent a technological alternative in many different areas with potential applications.