Shuttle-Shape Carrier-Free Platinum-Coordinated Nanoreactors with O2 Self-Supply and ROS Augment for Enhanced Phototherapy of Hypoxic Tumor.

The synergistic nanotheranostics of reactive oxygen species (ROS) augment or phototherapy has been a promising method within synergistic oncotherapy. However, it is still hindered by sophisticated design and fabrication, lack of a multimodal synergistic effect, and hypoxia-associated poor photodynamic therapy (PDT) efficacy. Herein, a kind of porous shuttle-shape platinum (IV) methylene blue (Mb) coordination polymer nanotheranostics-loaded 10-hydroxycamptothecin (CPT) is fabricated to address the abovementioned limitations. Our nanoreactors possess spatiotemporally controlled O2 self-supply, self-sufficient singlet oxygen (1O2), and outstanding photothermal effect. Once they are taken up by tumor cells, nanoreactors as a cascade catalyst can efficiently catalyze degradation of the endogenous hydrogen peroxide (H2O2) into O2 to alleviate tumor hypoxia. The production of O2 can ensure enhanced PDT. Subsequently, under both stimuli of external red light irradiation and internal lysosomal acidity, nanoreactors can achieve the on-demand release of CPT to augment in situ mitochondrial ROS and highly efficient tumor ablation via phototherapy. Moreover, under the guidance of near-infrared (NIR) fluorescent imaging, our nanoreactors exhibit strongly synergistic potency for treatment of hypoxic tumors while reducing damages against normal tissues and organs. Collectively, shuttle-shape platinum-coordinated nanoreactors with augmented ROS capacity and enhanced phototherapy efficiency can be regarded as a novel tumor theranostic agent and further promote the research of synergistic oncotherapy.

Selective Imaging of HClO in the Liver Tissue In Vivo Using a Near-infrared Hepatocyte-specific Fluorescent Probe.

Liver injury is typified by an inflammatory response. Hypochlorous acid (HClO), an important endogenous reactive oxygen species, is regarded as a biomarker associated with liver injury. Near-infrared (NIR) fluorescent probes with the advantage of deep tissue penetrating and low auto-fluorescence interference are more suitable for bioimaging in vivo. Thus, in this work, we designed and synthesized a novel NIR hepatocyte-specific fluorescent probe named NHF. The probe NHF showed fast response (<3 s), large spectral variation, and good selectivity to trace HClO in buffer solution. By employing N-acetylgalactosamine (GalNAc) as the targeting ligand, probe NHF can be actively delivered to the liver tissue of zebrafish and mice. It is important that probe NHF is the first NIR hepatocyte-specific fluorescent probe, which successfully visualized the up-regulation of endogenous HClO in the oxygen-glucose deprivation/reperfusion (OGD/R) model HepG2 cells and dynamically monitored APAP-induced endogenous HClO in the liver tissue of zebrafish and mice.

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.

aPDT using nanoconcentration of 1,9-dimethylmethylene blue associated to red light is efficacious in killing Enterococcus faecalis ATCC 29212 in vitro.

The Enterococcus faecalis is a microorganism that causes multiple forms of resistance to a wide range of drugs used clinically. aPDT is a technique in which a visible light activates photosensitizer (PS), resulting in generation of reactive oxygen species that kill bacteria unselectively via an oxidative burst. aPDT is an alternative to antibiotics with the advantage of not causing resistance. The search for an alternative treatment of infections caused by E. faecalis, without using antibiotics, is off great clinical importance. The aim of present investigation was to assess the efficacy of using 3.32 ηg/mL of 1,9-dimethylmethylene blue (DMMB) as photosensitizer associated with the use of either Laser (λ660 nm) or LED (λ632 ±â€¯2 nm) using different energy densities (6, 12 and 18 J/cm2) to kill E. faecalis in vitro. Under different experimental conditions, 14 study groups, in triplicate, were used to compare the efficacy of the aPDT carried out with either the laser or LED lights using different energy densities associated to DMMB. The most probable number method (MPN) was used for quantitative analysis. Photodynamic antimicrobial effectiveness was directly proportional to the energy density used, reaching at 18 J/cm2, 99.999998% reduction of the counts of E. faecalis using both light sources. The results of this study showed that the use of 3.32 ηg/mL of DMMB associated with the use 18 J/cm2 of LED light (λ632 ±â€¯2 nm) reduced >7-log counts of planktonic culture of E. faecalis.

Folding Paper-Based Aptasensor Platform Coated with Novel Nanoassemblies for Instant and Highly Sensitive Detection of 17ß-Estradiol.

Owing to its critical role in the development of female reproductive tissues and as a clinical biomarker, there is an urgent need to develop a rapid and cost-effective method to sensitively detect 17ß-estradiol (E2). In this work, a folding aptasensor platform with microfluidic channels for the label-free electrochemical detection of E2 is described. The platform, designed with a delicate folding structure, integrating filter holes, microfluidic channels, reaction chambers, and a three-electrode system, is extremely easy to use. To increase the detection sensitivity and immobilize the aptamer, we synthesized a novel nanoassembly consisting of amine-functionalized single-walled carbon nanotube/new methylene blue/gold nanoparticles (AuNPs) and modified the working electrode with this nanoassembly. The calibration curve obtained from the experimental results exhibited a linear range between 10 pg mL-1 and 500 ng mL-1 (R2 = 0.993), and a detection limit of 5 pg mL-1 was achieved (S/N = 3). Furthermore, experiments to detect E2 in clinical serum were conducted, and the results were highly similar to those obtained using a large electrochemical luminescence apparatus. By integrating multiple functional components, adopting novel nanoassemblies, and using a folding structure, this paper-based platform not only has great potential as a simple and convenient integrated device for point-of-care testing of E2, but also as a portable, low-cost, and highly sensitive aptasensor platform capable of detecting many diagnostic biomarkers with the appropriate aptamers.

A novel technique of antimicrobial photodynamic therapy - aPDT using 1,9-dimethyl-methylene blue zinc chloride double salt-DMMB and polarized light on Staphylococcus aureus.

Antimicrobial Photodynamic Therapy (aPDT) is an alternative to conventional treatments of local infections such as the use of antibiotics, which may lead to the development of resistance. aPDT besides requiring the use of a photosensitiser also needs a light source do be carried out. In the search for efficient and low-cost procedure the use of multispectral polarized light (λ400-2000 nm) emerges as a possibility for the execution of aPDT. The use of a highly effective photosensitizer is also of great importance. 1,9-Dimethyl-Methylene Blue Zinc Chloride Double Salt - DMMB is a potent phenothiazine derivative that presents high photodynamic action due to its high lipophilicity as well as a greater quantum yield of Singlet oxygen and phototoxicity when compared to other Photosensitizers. The aim of this study was to assess, In Vitro, the efficacy of aPDT on Staphylococcus aureus (ATCC 25923) using different concentrations of DMMB associated to a Polarized light source (Bioptron®, 40 mW, ᴓ = 15.8 cm2) using different energy densities. Based on the IC50, 150 and 300 ng/mL of DMMB concentrations were chosen for this study. Twelve experimental groups were used: (Control, PLs, PSs and aPDTs). Serial dilutions (up to 10-8) of the bacterial inoculum were used and the DMMB was added using the two previously determined concentrations. After 5 min of preincubation the dilutions of the inoculum were illuminated by the polarized light source. Subsequently, 100 µL of each dilution, in triplicate, were inoculated into Petri dishes containing TSA medium and incubated in a bacteriological oven at 37 °C for 24-h and quantification of UFCs was done. The results showed significant exponential reduction (p < .0001) of 99.93% (150 ng/mL + LP 10 J/cm2) and 99.97% (300 ng/mL + LP 5 J/cm2) the CFU counts in comparison to non-illuminated control. The results of this study allow to conclude that aPDT carried out with 1,9-Dimethyl-Methylene Blue Zinc Chloride Double Salt-DMMB and a PL souce was efficacious on the reduction (99.97%), in vitro, of the bacterial counts of S. aureus.

A Self-Powered Biosensor with a Flake Electrochromic Display for Electrochemical and Colorimetric Formaldehyde Detection.

The formaldehyde biosensors with the features of cost effectiveness, high specificity, easy operation, and simplicity are urgently desired in routing and field detection of formaldehyde. Here, we report a new design of an enzymatic self-powered biosensor (ESPB) toward formaldehyde detection. The ESPB involves a formaldehyde dehydrogenase/poly-methylene green/buckypaper bioanode as the sensing electrode and a Prussian blue/Au nanoparticles/carbon fiber paper cathode as the electrochromic display. Formaldehyde acts as the fuel to drive the ESPB, relying on that the concentration of formaldehyde can be determined with the ESPB by both directly measuring the variance in short circuit current and observing the color change of the cathode. By measuring the variance in short circuit current, a linear detection range from 0.01 to 0.35 mM and a calculated detection limit of 0.006 mM are obtained, comparable to or better than those reported before. The color change of the cathode can be distinguished easily and exactly via the naked eye after immersing the ESPB in formaldehyde solution for 90 s with the concentration up to 0.35 mM, covering the permissive level of formaldehyde in some standards associated with environmental quality control. Specially, the formaldehyde concentration can be precisely quantified by analyzing the color change of the cathode digitally using the equation of B/(R + G + B). In the following test of real spiked samples of tap water and lake water, the recovery ratios of formaldehyde with the concentrations from 0.010 to 0.045 mM are tested to be between 95 and 100% by both measuring the variance in short circuit current and analyzing the color change of the cathode digitally. In addition, the ESPB exhibits negligible interference from acetaldehyde and ethanol and can be stored at 4 °C for 21 days with a loss of less than 8% in its initial value of short circuit current. Therefore, the ESPB with the capability of working like disposable test paper can be expected as a sensitive, simple, rapid, cost-effective colorimetric method with high selectivity in routing and field formaldehyde detection.

Proximal RUMM block in dogs: preliminary results of cadaveric and clinical studies.

OBJECTIVE: To design and assess the perioperative analgesic efficacy of an ultrasound (US)-guided radial (R), ulnar (U), median (M) and musculocutaneous (Mc) nerve blocks, performed together in the axillary space by a single, in-plane approach. STUDY DESIGN: Anatomical research and prospective clinical study. ANIMALS: A group of three dog cadavers and 15 client-owned dogs undergoing orthopaedic thoracic limb surgery. METHODS: Phase 1: Anatomical dissection and US study of the axillary space were performed to design the US-guided proximal RUMM block. The technique was considered successful if a total volume of 0.15 mL kg-1 new methylene blue solution completely stained the four nerves in two cadavers for ≥2 cm. Phase 2: In 15 client-owned dogs undergoing orthopaedic thoracic limb surgery, the RUMM block designed in phase 1 was performed to provide analgesia using a total volume of 0.15 mL kg-1 of ropivacaine 0.5%. The block was considered effective if the intraoperative fentanyl requirement was <1.2 mcg kg-1 hour-1 and until the postoperative pain score was [short-form Glasgow Composite Measure Pain Scale (SF-GCMPS)] ≤5/20. RESULTS: Phase1: Detection of the four nerves was always feasible in a single US-window. The axillary artery and Mc nerve were used as landmarks. In-plane needling approach was feasible in both cadavers. All the nerves were completely stained for >2 cm. No intrathoracic dye spread was found. Phase 2: In 14/15 anaesthetized dogs, mean intraoperative fentanyl requirement was 0.25 ± 0.05 mcg kg-1 hour-1. Postoperatively, all dogs had SF-GCMPS ≤5/20 up to 8 hours. CONCLUSIONS AND CLINICAL RELEVANCE: The US-guided proximal RUMM block performed at the axillary level with a single, in-plane needling approach using 0.15 mL kg-1 of ropivacaine 0.5% minimized fentanyl requirement during thoracic limb surgery, contributing to postoperative analgesia up to 8 hours after execution of the peripheral nerve block.

In Vitro and in Vivo Effect of MAPK Signal Transduction Pathway Inhibitors on Echinococcus multilocularis.

To evaluate the effect of mitogen-activated protein kinase (MAPK) signal transduction pathway inhibitors against alveolar echinococcosis in vitro and in vivo, Echinococcus multilocularis metacestode cysts and protoscolices were obtained from infected mice. Protein chip technology was utilized to screen for key highly expressed target proteins in the MAPK pathway in this parasite and their corresponding inhibitors. Four-week-old Balb/c female mice used for the in vivo experiment underwent inoculation of E. multilocularis by intraperitoneal injection, as well as intragastric administration of MAPK inhibitors for 6 wk. We included 6 groups of mice: a phosphate-buffered saline (PBS) group (negative control); an albendazole-treated group (positive group); and 4 experimental groups treated with TRx0237 mesylate, GDC-0994, pifithrin-ß hydrobromide, or Selonsertib. Echinococcus multilocularis protoscolices were collected and cultured in 1066 medium with penicillin/streptomycin and 10% fetal bovine serum. The in vitro experiment included a PBS group (negative control), a dimethyl sulfoxide-treated group (solvent group), and 4 inhibitor-treated groups as in the in vivo experiment (experimental groups). Each inhibitor group received 4 drug concentrations (5, 30, 55, and 80 µM), and the experiment was performed in triplicate per sample. Fluorescence microscopy was used to evaluate the survival rate of the protoscolices every 48 hr beginning from the first 24 hr. The same grouping was used to evaluate cytotoxicity on E. multilocularis germinal cells and L02 cells. The average weights of E. multilocularis metacestode cyst tissue from each group of the in vivo experiment were 873 mg (PBS), 335 mg (albendazole), 323 mg (TRx0237 mesylate), 420 mg (GDC-0994), 340 mg (pifithrin-ß hydrobromide), and 642 mg (Selonsertib). Results showed albendazole, TRx0237 mesylate, and pifithrin-ß hydrobromide had significant inhibitory effects on inhibition of E. multilocularis. We found a positive correlation between drug concentrations and the inhibitory effects seen in the in vitro experiment, with the differences in contrast with the control group becoming statistically significant after 72 hr of treatment ( P < 0.05). The inhibition rates of TRx0237 mesylate to germinal cells by drug concentration were 23.73, 46.59, 74.71, and 77.44%. Other drugs had no effect on germinal cells. All the inhibitors had low toxicity on L02 cells. Inhibitors of the MAPK signal transduction pathway showed significant inhibitory effects on E. multilocularis, suggesting these may be potential candidates for the treatment of alveolar echinococcosis.

Parallel damage in mitochondria and lysosomes is an efficient way to photoinduce cell death.

Cells challenged by photosensitized oxidations face strong redox stresses and rely on autophagy to either survive or die. However, the use of macroautophagy/autophagy to improve the efficiency of photosensitizers, in terms of inducing cell death, remains unexplored. Here, we addressed the concept that a parallel damage in the membranes of mitochondria and lysosomes leads to a scenario of autophagy malfunction that can greatly improve the efficiency of the photosensitizer to cause cell death. Specific damage to these organelles was induced by irradiation of cells pretreated with 2 phenothiazinium salts, methylene blue (MB) and 1,9-dimethyl methylene blue (DMMB). At a low concentration level (10 nM), only DMMB could induce mitochondrial damage, leading to mitophagy activation, which did not progress to completion because of the parallel damage in lysosome, triggering cell death. MB-induced photodamage was perceived almost instantaneously after irradiation, in response to a massive and nonspecific oxidative stress at a higher concentration range (2 µM). We showed that the parallel damage in mitochondria and lysosomes activates and inhibits mitophagy, leading to a late and more efficient cell death, offering significant advantage (2 orders of magnitude) over photosensitizers that cause unspecific oxidative stress. We are confident that this concept can be used to develop better light-activated drugs. Abbreviations: ΔΨm: mitochondrial transmembrane inner potential; AAU: autophagy arbitrary units; ATG5, autophagy related 5; ATG7: autophagy related 7; BAF: bafilomycin A1; BSA: bovine serum albumin; CASP3: caspase 3; CF: carboxyfluorescein; CTSB: cathepsin B; CVS: crystal violet staining; DCF: dichlorofluorescein; DCFH2: 2',7'-dichlorodihydrofluorescein; DMMB: 1,9-dimethyl methylene blue; ER: endoplasmic reticulum; HaCaT: non-malignant immortal keratinocyte cell line from adult human skin; HP: hydrogen peroxide; LC3B-II: microtubule associated protein 1 light chain 3 beta-II; LMP: lysosomal membrane permeabilization; LTG: LysoTracker™ Green DND-26; LTR: LysoTracker™ Red DND-99; 3-MA: 3-methyladenine; MB: methylene blue; mtDNA: mitochondrial DNA; MitoSOX™: red mitochondrial superoxide probe; MTDR: MitoTracker™ Deep Red FM; MTO: MitoTracker™ Orange CMTMRos; MT-ND1: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; MTT: methylthiazolyldiphenyl-tetrazolium bromide; 1O2: singlet oxygen; OH. hydroxil radical; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; PBS: phosphate-buffered saline; PI: propidium iodide; PDT: photodynamic therapy; PS: photosensitizer; QPCR: gene-specific quantitative PCR-based; Rh123: rhodamine 123; ROS: reactive oxygen species RTN: rotenone; SQSTM1/p62: sequestosome 1; SUVs: small unilamellar vesicles; TBS: Tris-buffered saline.

Cysteine-Independent Inhibition of Alzheimer's Disease-like Paired Helical Filament Assembly by Leuco-Methylthioninium (LMT).

Alzheimer's disease is a tauopathy characterized by pathological fibrillization of tau protein to form the paired helical filaments (PHFs), which constitute neurofibrillary tangles. The methylthioninium (MT) moiety reverses the proteolytic stability of the PHF core and is in clinical development for treatment of Alzheimer's disease in a stable reduced form as leuco-MT. It has been hypothesized that MT acts via oxidation of cysteine residues, which is incompatible with activity in the predominantly reducing environment of living cells. We have shown recently that the PHF-core tau unit assembles spontaneously in vitro to form PHF-like filaments. Here we describe studies using circular dichroism, SDS-PAGE, transmission electron microscopy and site-directed mutagenesis to elucidate the mechanism of action of the MT moiety. We show that MT inhibitory activity is optimal in reducing conditions, that the active moiety is the reduced leuco-MT form of the molecule and that its mechanism of action is cysteine independent.

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich's ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.

Cost-effective three dimensional Ag/polymer dyes/graphene-carbon spheres hybrids for high performance nonenzymatic sensor and its application in living cell H2O2 detection.

We describe a facile method to synthesize a new type of catalyst by electrodepositing Ag nanocrystals (AgNCs) on the different polymer dyes, Poly (methylene blue) (PMB) or Poly (4-(2-Pyridylazo)-Resorcinol) (PAR) modified graphene­carbon spheres (GS) hybrids. The self-assembled GS take dual advantages of carbon spheres and graphene. Carbon spheres acts as nano-spacers prevent the aggregation of graphene and guarantee the fast electron transfer of GS. Secondly, polymerized dyes used here are beneficial for AgNCs growing as a linker. The effects of dyes on the growth habits, morphologies and catalytic properties for AgNCs were investigated. A novel electrochemical nonenzymatic sensor for hydrogen peroxide (H2O2) detection is fabricated based on the Ag/Polymer dyes/GS ternary composites modified glass carbon electrode (GCE) for the first time. It was found that the proposed electrodes, especially for Ag/PMB/GS/GCE, displayed a peculiar electrocatalytic activity towards H2O2 reduction synergistically as compared to Ag/PAR/GS/GCE or Ag/GS/GCE alone. Ag/PMB/GS/GCE showed a linear response over the H2O2 concentration range of 0.5 to 1112 µM. The detection limit and sensitivity is 0.15 µM and 400 µA mM-1 cm-2, respectively. These outstanding results enable the practical application of Ag/PMB/GS/GCE for the H2O2 tracking released from MCF-7 (human breast cancer cells) with satisfactory results.

Efficacy of photodynamic therapy against Streptococcus mutans biofilm: Role of singlet oxygen.

In photodynamic therapy (PDT), killing is entirely based on the ROS generation and among different types of ROS generated during PDT, singlet oxygen is considered as the most potential as illustrated in many studies and therefore it is predominantly responsible for photodamage and cytotoxic reactions. The aim of this study was to check whether singlet oxygen (Type II photochemistry) is more potential than free radicals (Type I photochemistry) against Streptococcus mutans biofilm. We have taken two phenothiazinium dyes i.e. toluidine blue O (TBO) and new methylene blue (NMB). TBO was found to have better antibacterial as well as antibiofilm effect than NMB. Antibacterial effect was evaluated by colony forming unit while antibiofilm action by crystal violet and congo red binding assays. We have also evaluated the disruption of preformed biofilm by biofilm reduction assay, confocal laser electron and scanning electron microscopy. More singlet oxygen production was detected in case of TBO than NMB while more Free radical (HO) was produced by NMB than TBO. TBO showed better antibacterial as well as antibiofilm effect than NMB so; we conclude that potency of a photosensitizer is correlated with the capability to produce singlet oxygen.

Ultrasensitive Tyrosinase-Activated Turn-On Near-Infrared Fluorescent Probe with a Rationally Designed Urea Bond for Selective Imaging and Photodamage to Melanoma Cells.

Melanoma is a highly aggressive malignancy and early monitoring and diagnosis are challenging at present. Tyrosinase is overexpressed in melanoma and regarded as an important biological marker for diagnosis and treatment. Thus, the selective and sensitive detection of tyrosinase is of great significance. To date, a few fluorescent probes have been reported for the detection of tyrosinase in vitro or in vivo. However, a highly sensitive near-infrared probe for tyrosinase monitoring is still missing. In this study, the Gibbs free energy change of different urea bonds during spontaneous hydrolysis is analyzed with the aid of chemical thermodynamic computation. On the basis of this analysis, we modified the dye methylene blue with a rationally designed urea bond to specifically create a probe, called MB1, for rapid detection of tyrosinase. Our experimental results demonstrated that MB1 can serve as a highly sensitive near-infrared responsive fluorescent probe for the monitoring and bioimaging of tyrosinase. In addition, the activated MB1 probe can effectively kill melanoma cells by photodynamic therapy. Thus, the near-infrared probe has great potential for monitoring and treating melanoma.

Defining local nerve blocks for feline distal pelvic limb surgery: a cadaveric study.

Objectives Anatomical and methodological detail is lacking regarding local anesthetic peripheral nerve block techniques for distal pelvic limb surgery in cats. The aim of this study was to develop, describe and test nerve block methods based on cadaveric dissections and dye injections. Methods Ten pairs of feline pelvic limbs (n = 20) were dissected and the tibial nerve (T n.), common fibular (peroneal) nerve (CF n., and its two branches, the superficial fibular [peroneal] nerve [SpF n.] and the deep fibular [peroneal] nerve [DpF n.]) and the saphenous nerve (Sa n.) were identified. Based on these dissections, a 'distal crus block' (selective blockade of the CF n., T n. and Sa n.) and a 'distal pes block' (selective blockade of the SpF n., DpF n., T n. and Sa n.) were developed for surgical procedures in two different regions of the distal pelvic limb. Techniques were tested using new methylene blue (NMB) dye injections in feline pelvic limbs (n = 12). Using a 25 G × 5/8 inch needle and 1 ml syringe, 0.1 ml/kg of NMB dye solution was injected at the site of the CF n., and 0.05 ml/kg was injected at the sites of the SpF n., DpF n., Sa n. and T n. The length and circumference (fully or partially stained) of each stained nerve were measured. Results Positive staining of nerves was observed in 12/12 limbs. The lengths stained for the CF n., DpF n., SpF n., Sa n. and T n. were 27.19 ± 7.13, 20.39 ± 5.57, 22.82 ± 7.13, 30.89 ± 6.99 and 25.16 ± 8.09 mm, respectively. The nerves were fully stained in 12, 12, 10, 11 and 11 out of 12 limbs, respectively. Conclusions and relevance These two, three-point injection methods may be an effective perioperative analgesia technique for feline distal pelvic limb procedures.

Intraoperative nerve staining in nerve-sparing radical hysterectomy: a pilot study.

OBJECTIVE: To evaluate the feasibility and effectiveness of intraoperative nerve staining by modified leucomethylene blue (MLB). METHODS: Animal experiment was performed to assure whether the tissues dyed blue by MLB were nerves with microscopic examination. Ten patients with cervical cancer were performed by nerve-sparing radical hysterectomy (NSRH) and nerve staining intraoperatively by MLB. The status of staining was recorded. The post-void residual urine volume after removing was measured by ultrasound. The time to post-void residual urine volume of less than 100 ml and the first defecation were recorded. RESULTS: In animal experiment, the tissues dyed blue obviously showed abundant nerve fibers by microscopic examination. The minor nerves were dyed blue clearly in NSRH. The time to post-void residual urine volume of less than 100 ml after removal of the urethral catheter was 10.3 (7-13) days by records. The time to the first defecation was 67.7 (60-82) h. CONCLUSION: Intraoperative nerve staining by MLB provided a new method for nerve location in NSRH. It was safe, effective and convenient.

Screening for mucopolysaccharidoses in the Turkish population: Analytical and clinical performance of an age-range specific, dye-based, urinary glycosaminoglycan assay.

Comprehensive analytical and diagnostic performance of urinary quantitative GAG analysis with dimethylmethylene blue (DMB) and the age-specific reference ranges were determined in Turkish population, which has a high incidence of MPSs. Precision, linearity, recovery and accuracy/trueness, limits, stability, and effect of interferents were tested according to CLSI guideline. Clinical performance was evaluated with ROC analyses including 45 MPS patients. Intra-day and inter-day precisions were <5% and <11% (CV), respectively. LoD was 9.12mg/L and LoQ was 23.3mg/L. The highest reference values for urinary GAG excretion were determined in an age-specific manner. In the 2-13years age cohort, a cut-off of 89.86mg/g creatinine resulted in 98.07% sensitivity and 93.33% specificity. Proteinuria and hematuria interfered with analysis in some instances. Neither leukocyturia nor pH changes affected the assay. Stability analysis indicated that freezing urine samples for transfer is unnecessary. Of the 45 MPS patient samples evaluated, only three tested negative including MPS II, IVA and VI. Despite limitations due to low levels of urinary GAG excretion in some cases, urinary GAG analysis with DMB with its technical simplicity, low cost, and precise quantitative results, is a valuable screening method, particularly in populations with a high rate of MPSs.

The effects of photodynamic treatment with new methylene blue N on the Candida albicans proteome.

Candida albicans is a human pathogenic fungus mainly affecting immunocompromised patients. Resistance to the commonly used fungicides can lead to poor treatment of mucosal infections which, in turn, can result in life-threatening systemic candidiasis. In this scenario, antimicrobial photodynamic treatment (PDT) has emerged as an effective alternative to treat superficial and localized fungal infections. Microbial death in PDT is a consequence of the oxidation of many cellular biomolecules, including proteins. Here, we report a combination of two-dimensional electrophoresis and tandem mass spectrometry to study the protein damage resulting from treating C. albicans with PDT with new methylene blue N and red light. Two-dimensional gels of treated cells showed an increase in acidic spots in a fluence-dependent manner. Amino acid analysis revealed a decrease in the histidine content after PDT, which is one plausible explanation for the observed acidic shift. However, some protein spots remained unchanged. Protein identification by mass spectrometry revealed that both modified and unmodified proteins could be localized to the cytoplasm, ruling out subcellular location as the only explanation for damage selectivity. Therefore, we hypothesize that protein modification by PDT is a consequence of both photosensitizer binding affinity and the degree of exposure of the photooxidizable residues on the protein surface.

In Vitro Antimicrobial Photodynamic Therapy Against Trichophyton mentagrophytes Using New Methylene Blue as the Photosensitizer. / Terapia fotodinámica antimicrobiana in vitro aplicada sobre Trichophyton mentagrophytes con nuevo azul de metileno como fotosensibilizador.

INTRODUCTION AND OBJECTIVES: Antimicrobial photodynamic therapy combines the use of a photosensitizing drug with light and oxygen to eradicate pathogens. Trichophyton mentagrophytes is a dermatophytic fungus able to invade the skin and keratinized tissues. We have investigated the use of new methylene blue as the photosensitizing agent for antimicrobial photodynamic therapy to produce the in vitro inactivation of T mentagrophytes. MATERIAL AND METHODS: A full factorial design was employed to optimize the parameters for photoinactivation of the dermatophyte. The parameters studied were new methylene blue concentration, contact time between the photosensitizing agent and the fungus prior to light treatment, and the fluence of red light (wavelength, 620-645nm) applied. RESULTS: The minimum concentration of new methylene blue necessary to induce the death of all T. mentagrophytes cells in the initial suspension (approximate concentration, 106 colony forming units per milliliter) was 50µM for a fluence of 81J/cm2 after a contact time of 10minutes with the photosensitizing-agent. Increasing the concentration to 100µM allowed the fluence to be decreased to 9J/cm2. CONCLUSIONS: Comparison of our data with other published data shows that the susceptibility of T. mentagrophytes to antimicrobial photodynamic therapy with new methylene blue is strain-dependent. New methylene blue is a photosensitizing agent that should be considered for the treatment of fungal skin infections caused by this dermatophyte.