Adrenergic Modulation With Photochromic Ligands.

Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition, and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability, and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice.

Clinical evaluation of three chromogenic media for the isolation of Staphylococcus aureus in respiratory samples in patients with cystic fibrosis.

We evaluated the performance of three chromogenic media (BBL CHROMagar™ Staph aureus, ChromID™ S. aureus SAID, ChromID™ S. aureus Elite SAIDE) for the isolation of Staphylococcus aureus in respiratory samples in patients with cystic fibrosis in comparison with CNA media. We reported a similar ability of the four media to support the growth of S. aureus and that sensitivity increased when incubation lasted more than 24 h. SAIDE had the higher sensitivity compared to the other media and kept a high specificity even after 72 h.

ß-Lactamase triggered visual detection of bacteria using cephalosporin functionalized biomaterials.

We report the conjugation of a chromogenic cephalosporin ß-lactamase (ßL) substrate to polymers and integration into biomaterials for facile, visual ßL detection. Identification of these bacterial enzymes, which are a leading cause of antibiotic resistance, is critical in the treatment of infectious diseases. The ßL substrate polymer conjugate undergoes a clear to deep yellow color change upon incubation with common pathogenic Gram-positive and Gram-negative bacteria species. We have demonstrated the feasibility of formulating hydrogels with the ßL substrate covalently tethered to a poly(ethylene glycol) (PEG) polymer matrix, exhibiting a visible color change in the presence of ßLs. This approach has the potential to be used in diagnostic biomaterials for point-of-care detection of ßL-producing bacteria, helping combat the spread of drug resistant microbes.

Activity measurements of dalcinonacog alfa.

INTRODUCTION: Many recombinant and modified FIX products have been, and continue to be, developed with the aim of improving treatment for patients with haemophilia B. One such new product is dalcinonacog alfa, a recombinant FIX with modifications to provide improved features such as subcutaneous administration. AIM: In view of previously observed assay discrepancies with modified FIX therapeutics, the aim of this study was to assess potential discrepancies in potency measurement of dalcinonacog alfa between and within different assay methods. METHODS: Potency of dalcinonacog alfa was measured against the 5th International Standard (IS) for FIX Concentrate and the 4th IS for FIX Plasma by One-Stage Clotting Assay, using 9 different APTT reagents and 2 commercially available FIX chromogenic kits. Plasma-derived concentrate and recombinant FIX samples were also included for comparison in every assay. RESULTS: Substantial discrepancies were observed when assaying dalcinonacog alfa using the one-stage clotting assay against both standards. No statistically valid results were obtained when testing dalcinonacog alfa using either chromogenic kit. Increasing the incubation time with the activation reagent in both chromogenic kits resulted in valid assays and increased the potency to become more in line with potencies by one-stage clotting assays. Increasing the incubation time in the chromogenic kits had no effect on the potencies of the plasma-derived or recombinant samples. However, incubation time influenced in the one-stage clotting assay using Dapttin. CONCLUSIONS: Within and between assay method discrepancy was found when assaying dalcinonacog alfa. Methods for potency labelling and clinical monitoring should be given careful consideration.

[Contribution of the Uriselect4® and the CPS ID3® chromogenic media to the isolation and identification of urinary tract infections bacteria]. / Apport des milieux chromogènes Uriselect4® et CPS ID3® dans l'isolement et l'identification des germes responsables d'infections urinaires.

Rapid and accurate identification of pathogens involved in urinary tract infections helps to guide antimicrobial therapy. Chromogenic agars provide presumptive identification directly from primary isolation media. They have been intended to make the bacterial isolation and identification process easier and faster. Our study aimed to compare the performance and the cost of the CPS ID3® and the Uriselect4® chromogenic agars with the conventional method for the isolation and identification of urinary tract infections bacteria. We included 301 urinary samples in a prospective study conducted in May 2018 in the clinical laboratory of the National institute of nutrition and food technology of Tunis. Isolates from routine media were identified using API® system while isolates from chromogenic media were directly identified by colony color with reference to the manufacturer's recommendations. Chromogenic media yielded more pure positive cultures and allowed better isolation of Escherichia coli, Klebsiella pneumoniae, Citrobacter koseri, Morganella morganii and Streptococcus agalactiae. Sensitivity and specificity of the presumptive identification of most commonly isolated uropathogens were higher with the Uriselect4® medium than with the CPS ID3® medium. Chromogenic media yielded the identification of pathogenic organisms 24 hours sooner than the conventional method in approximately 63 % of cases with the CPS ID3® medium and in 77.7% of cases with the Uriselect4® medium. Chromogenic media allowed a much better isolation of bacteria commonly involved in urinary tract infections with a quick, easy and accurate presumptive identification especially with the Uriselect4® medium.

Photochromic peptidic NPY Y4 receptor ligands.

The neuropeptide Y (NPY) Y4 receptor is a G protein coupled receptor, which is targeted by pancreatic polypeptide, a homologue of NPY. Selective Y4R agonists were suggested as potential therapeutics for the treatment of obesity. Highly potent dimeric peptidic Y4R agonists, constituting two pentapeptide moieties connected through an aliphatic linker, represent an interesting class of Y4R ligands. Based on this compound class, photoresponsive Y4R ligands, containing an azobenzene, azopyrazole, diethienylethene or a fulgimide chromophore were prepared to explore structural requirements of such Y4R agonists on Y4R binding. The synthesized Y4R ligands, containing a non-aliphatic rigid photochromic linker, switch reversibly in aqueous buffer and exhibited high Y4R affinity throughout. This demonstrated that the replacement of the highly flexible aliphatic linker by a considerably less flexible photochromic linker was well tolerated with respect to Y4R binding. Differences in Y4R affinity and activity between the individual photoisomers (varying in spatial orientation and flexibility) were marginal suggesting that the linking element in the dimeric ligands is less critical for the adaptation of high-affinity binding modes at the receptor.

Stability, Activity, and Application of Topical Doxycycline Formulations in a Diabetic Wound Case Study.

INTRODUCTION: Tetracycline molecules comprise a group of broad-spectrum antibiotics whose primary mechanism of action is the inhibition of protein synthesis through the binding of the bacterial ribosome. In addition, tetracyclines inhibit matrix metalloproteases (MMPs), a family of zinc-dependent proteases that contribute to tissue remodeling, inflammation, and angiogenesis and are overexpressed in certain pathophysiologies such as diabetic foot ulcers (DFUs). OBJECTIVE: This study aims to develop a liquid chromatography and mass spectrometry (LC-MS/MS) doxycycline quantification methodology to facilitate the development of a stable topical doxycycline hyclate (DOXY) formulation as well as evaluate the topical DOXY formulation for the efficacy in MMP-9 inhibition in vitro and in a clinical application of diabetic lower extremity wounds. MATERIALS AND METHODS: A simple quantification method utilizing LC-MS/MS was used to develop a topical DOXY formulation, a sample of which was analyzed in stability testing. The formulation was evaluated in vitro for MMP-9 activity using a commercial assay and compared with internal kit controls as well as in a clinical setting for wound healing. RESULTS: Two formulations of 2% (w/w) DOXY demonstrated acceptable stability (±10% target concentration) for 70 days when stored at 4°C. Using an in vitro assay of MMP-9 enzyme activity, the 2% DOXY formulation imparted a ~30% decrease in MMP-9 inhibitory potential as compared with the control drug alone (IC50 values 62.92 µM and 48.27 µM, respectively). This topical product was evaluated for clinical utility in a patient with a DFU, and preliminary data suggest this intervention may promote wound healing. CONCLUSIONS: In summary, novel DOXY formulations may be stable and biologically active tools amenable to complex wound care.

Detection of carbapenem-resistant Enterobacteriaceae by chromogenic screening media.

Enterobacteriaceae are a part of the human intestinal flora easily spread by hand carriage, water and food. Treatment of infections due to Enterobacteriaceae is difficult mainly in countries with a low socioeconomic state. Carbapenems are considered as a last-resort antibiotic for the treatment of infections due to multidrug-resistant Enterobacteriaceae. Carbapenem-resistant Enterobacteriaceae (CRE) presents a major threat to public health and are rapidly disseminating globally. The most important resistance mechanism of Enterobacteriaceae to carbapenems is carbapenemase production. The treatment options for CRE are limited; attention is focused here on the rapid detection of CRE. Laboratory surveillance of cultures and screening of patients are essential and desirable in areas where these strains are endemic. Culture-based methods such as chromogenic media are used for the initial detection of these strains. These media offer a sensitive, convenient, and low-cost way of identifying CRE species. This article provides an overview of the current state of culture-based chromogenic screening media for the detection of CRE.

Advanced endoscopic imaging for diagnosis of inflammatory bowel diseases: Present and future perspectives.

Crohn's disease and ulcerative colitis are chronic inflammatory bowel diseases (IBD) causing severe damage of the luminal gastrointestinal tract. Differential diagnosis between both disease entities is sometimes awkward requiring a multifactorial pathway, including clinical and laboratory data, radiological findings, histopathology and endoscopy. Apart from disease diagnosis, endoscopy in IBD plays a major role in prediction of disease severity and extent (i.e. mucosal healing) for tailored patient management and for screening of colitis-associated cancer and its precursor lesions. In this state-of-the-art review, we focus on current applications of endoscopy for diagnosis and surveillance of IBD. Moreover, we will discuss the latest guidelines on surveillance and provide an overview of the most recent developments in the field of endoscopic imaging and IBD.

Isolation and quantitation of Clostridium difficile in aqueous and fecal matter using two types of selective media.

We evaluated the isolation and quantitation of Clostridium difficile from aqueous and fecal samples utilizing ChromID CDIF and cycloserine, cefoxitin, and fructose-containing agar with horse blood and taurocholate media. Growth was similar between the two. ChromID CDIF provided enhanced isolation and required no ethanol pretreatment to inhibit normal flora. ChromID CDIF also improved turn-around time, requiring only 24 hours' incubation.

[Comparison of routine use of two chromogenic media ChromID CPS (bioMérieux) and UriSelect4 (Bio-Rad) for the detection of Escherichia coli and major uropathogenics in urine]. / Comparaison de l'utilisation en routine de deux milieux chromogènes ChromID CPS (bioMérieux) et UriSelect4 (Bio-Rad) pour la détection d'Escherichia coli et des principaux uropathogènes dans les urines.

Escherichia coli is the most common bacterial cause of urinary tract infections. Its rapid and specific identification in urine samples represents a major challenge within the rendering results and optimizing the management of the patient. We aimed to compare the sensitivity and specificity of two commercially available chromogenic media for E. coli: ChromID CPS (Biomérieux) and UriSelect4 (Bio(-)Rad), without carrying out further tests. 99 consecutive and non-redundant urine samples considered to be infected were simultaneously plated onto blood agar and the two chromogenic media. Colony color and bacterial growth quantification were compared 18 and 48 hours after incubation. Bacteria were identified with mass spectrometry. A complementary analysis on 80 bacterial strains known to pose potential identification problems was performed. 43 urines samples grew E. coli, and 42 of them were pink-colored on the two chromogenic mediums, as expected (sensibility=97.7%). Growth quantification was significantly greater on blood agar than on chromogenic media (p<0.001).We noted specificity issues at the complementary analysis with the UriSelect4 medium: Citrobacter freundii and some strains of Citrobacter brakii, Enterobacter cloacae and Hafnia alvei were pink-colored, and could be misidentified as E. coli. ChromID CPS medium did not show such misidentification. In conclusion, the agar ChromID CPS proved to be greater than the UriSelect4 agar in our work in terms of specificity of direct identification of E. Coli, without the use of additional test.

Inhibitory effect of chromogenic culture media on the growth of Rhodotorula: relevance to the diagnosis of Rhodotorula spp. infections.

With the increasing incidence and diverse etiologies of fungal infections, chromogenic yeast culture media are increasingly used for routine diagnosis. Rhodotorula species, which are characterized by the production of carotenoid pigments, are considered as emerging opportunistic pathogens. We recently diagnosed two fungemia due to Rhodotorula spp. and noticed that in both cases, the yeast failed to grow in subculture on the chromogenic yeast culture medium. This study was thus undertaken to investigate more thoroughly the ability (or inability) of Rhodotorula species to grow on different commercially available chromogenic media for yeast. Eighteen Rhodotorula spp. were checked for their ability to grow on four chromogenic yeast culture media: CHROMagar Candida (BD), Candi 4 Select (Biorad), Brilliance Candida (Oxoid), and Candida ID 2 (BioMerieux). All the Rhodotorula spp. strains grew on Brilliance and Candida ID 2, while only six isolates grew on Candi 4, and seven on CHROMagar. Two chromogenic yeast culture media showed a significant inhibitory effect on the growth of Rhodotorula species. As all Rhodotorula species are resistant to echinocandins and fluconazole, it is essential to isolate and identify these yeast quickly to initiate appropriate amphotericin B antifungal treatment as early as possible. The choice of media for routine use should take into account the ability of different media to allow all emerging fungal pathogens to grow.

Quantum dots induced monocyte chemotactic protein-1 expression via MyD88-dependent Toll-like receptor signaling pathways in macrophages.

Quantum dots (QDs) are nano-sized semiconductors. Previously, intratracheal instillation of QD705s induces persistent inflammation in mouse lungs. In our present study, QD705-COOH and QD705-PEG activated NF-κB and increased monocyte chemotactic protein-1 (MCP-1) expression in macrophages RAW264.7 via MyD88 dependent Toll-like receptor (TLR) signaling pathways. MyD88 is an adapter protein for most TLRs to activate NF-κB. Silencing expression of MyD88 or p65 with siRNA or co-treatment with a NF-κB inhibitor tremendously abolished QD705s-induced NF-κB activity and MCP-1 expression. The involved TLRs might locate either on the cell surface or inside of cells. Co-treatment with a TLR4 inhibitor completely prevented MCP-1 induction by QD705-PEG. Nevertheless, QD705-COOH readily entered cells, and co-treatment with either inhibitors of endocytosis or intracellular TLRs prevented MCP-1 induction. These findings indicate that, depending on their surface modification, OD705s activate MyD88 dependent-TLRs at the surface or inside of the cells, which is an important mechanism for nanoparticles-induced inflammatory responses. But other MyD88-independent pathways may also involve in these responses.

A chromogenic cephalosporin for ß-lactamase inhibitor screening assays.

Production of ß-lactamases is the primary mechanism of antibiotic resistance employed by gram-negative pathogens. Chromogenic ß-lactams are important reagents for detection and assay of ß-lactamases, but limited commercial availability and exorbitant pricing of these compounds are prohibitive. Here we describe a straightforward synthesis of a chromogenic cephalosporin for ß-lactamase assay that gives an overall yield of 74%. On hydrolysis, its λ(max) undergoes a bathochromic shift that is easy to see and measure spectrophotometrically with a Δε(442 nm) of 14,500 cm⁻¹ M⁻¹. This compound was shown to be a substrate for a variety of ß-lactamases.

Evaluation of commercial selective agars in screening for methicillin-resistant Staphylococcus aureus.

To evaluate methicillin-resistant S. aureus (MRSA) detection methods, we compared (a) mannitol salt agar with cefoxitin (MSA-FX), (b) MRSASelect agar (Bio-Rad), (c) MRSA ID (bioMerieuex), and (d) CHROMagar MRSA (BD Diagnostics) as selective media for culturing nasal swab specimens collected from intensive care unit (ICU) patients and healthcare personnel. A total of 99 (17.1%) cases of MRSA were recovered from 578 specimens. Four (5.5%) cases were identified from healthcare personnel and 95 (18.8%) were from ICU patients. The sensitivity of MSA-FX, MRSASelect, MRSA ID, and CHROMagar MRSA was 83.8, 87.9, 80.8, and 84.8% after 18 hr; 92.9, 94.9, 90.9, and 91.9% after 24 hr; and 96.0, 100, 99.0, and 99.0% after 48 hr, respectively. The specificity of MSA-FX, MRSASelect, MRSA ID, and CHROMagar MRSA was 97.7, 99.0, 98.7. and 99.8% after 18 hr; 97.1, 98.5, 98.1, and 99.5% after 24 hr; and 95.2, 97.7, 97.9, and 99.0% after 48 hr, respectively. In conclusion, all four media showed good results after the 24 hr readings, but MRSA ID and CHROMagar MRSA media required readings at 48 hr due to increased sensitivity at this time point.

In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi--Rhodnius prolixus interaction using confocal microscopy.

Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals markers that allow long photobleaching and do not destroy the parasites. In this paper, we used fluorescent core shell quantum dots to perform studies of live parasite-vector interaction processes without any observable effect on the vitality of parasites. These nanocrystals were synthesized in aqueous medium and physiological pH, which is very important for monitoring live cells activities, and conjugated with molecules such as lectins to label specific carbohydrates involved on the parasite-vector interaction. These QDs were successfully used for the study of in vitro and in vivo interaction of Trypanosoma cruzi and the triatomine Rhodnius prolixus. These QDs allowed us to acquire real time confocal images sequences of live T. cruzi-R. prolixus interactions for an extended period, causing no damage to the cells. By zooming to the region of interest, we have been able to acquire confocal images at the three to four frames per second rate. Our results show that QDs are physiological fluorescent markers capable to label living parasites and insect vector cells. QDs can be functionalized with lectins to specifically mark surface carbohydrates on perimicrovillar membrane of R. prolixus to follow, visualize, and understand interaction between vectors and its parasites in real-time.

Labelling heparan sulphate saccharides with chromophore, fluorescence and mass tags for HPLC and MS separations.

The analysis of heparin/HS saccharides derived from small amounts of tissue or cells is a considerable technical challenge and the development of methods to characterise these carbohydrates has progressed comparatively slowly. A number of procedures have been devised to tag glycans selectively at the reducing end with a group that will enhance the sensitivity of detection and facilitate chromatographic separations. Outlined in this chapter are two useful strategies designed specifically for the analysis of heparin/HS saccharides. The first involves a fluorophore label, Bodipy-FL-hydrazide, which permits highly sensitive (fmol level) detection of saccharides utilising high performance strong anion exchange chromatography. The second facilitates oligosaccharide separation by gel-permeation chromatography and reverse phase high performance ion-pairing chromatography (RP-HPIPC) through the use of a phenylsemicarbazide tag. The latter also serves as an effective mass tag for electrospray mass spectrometry, permitting enhanced analysis of HS saccharides. These methods provide new opportunities for the development of glycomics approaches to study the structure and function of the heparan sulfate family of glycans.

Purification, crystallization and preliminary X-ray analysis of CMS1MS2: a cysteine proteinase from Carica candamarcensis latex.

Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P2(1)), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 A, beta = 96.81 degrees , and diffract X-rays to 1.8 A resolution.

Synthesis and testing of chromogenic phenoxazinone substrates for beta-alanyl aminopeptidase.

Novel 7-N-(beta-alanyl)aminophenoxazin-3-one salts 27a-d have been synthesized and tested as chromogenic substrates for beta-alanyl aminopeptidase, which is present in Pseudomonas aeruginosa, the most common respiratory pathogen in patients with cystic fibrosis. The biological results show that 7-N-(beta-alanyl)amino-1-pentylphenoxazin-3-one trifluoroacetate salt 27a is a chromogenic substrate for this bacterium, with a low degree of diffusion in nutrient media for growing bacterial cultures and a bright red colour, making it easily distinguishable from the agar background.

Trafficking of QD-conjugated MPO-ANCA in murine systemic vasculitis and glomerulonephritis model mice.

In systemic vasculitis, the serum level of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA) is significantly elevated with the progression of disease. We have established a model of murine systemic vasculitis by administration of MPO-ANCA and fungal mannoprotein to C57BL/6 mice. We examined the role of MPO and MPO-ANCA in the pathogenesis of glomerulonephritis and systemic vasculitis in this model using quantum dots (QDs). We demonstrated that QD-conjugated MPO-ANCA (ANCA-QD) visualized the translocation of MPO on the neutrophil membrane surface after stimulation with proinflammatory cytokines. We also observed that MPO translocation on neutrophils in both patients with rapid progressive glomerulonephritis and these model mice without any stimulation, suggesting that MPO translocation is certain to contribute to the development of glomerular lesion. In addition, blood flow on the kidney surface vessel was significantly decelerated in both SCG/Kj mice and this model, suggesting that ANCA induces the damage of blood vessel. These results indicate that MPO-ANCA and surface-translocated MPO on the activated neutrophils coordinately plays essential roles in the initial steps of the glomerulonephritis.