Carboxylate-Assisted C(sp^3)–H Activation in Olefin Metathesis-Relevant Ruthenium Complexes

The mechanism of C–H activation at metathesis-relevant ruthenium(II) benzylidene complexes was studied both experimentally and computationally. Synthesis of a ruthenium dicarboxylate at a low temperature allowed for direct observation of the C–H activation step, independent of the initial anionic ligand-exchange reactions. A first-order reaction supports an intramolecular concerted metalation–deprotonation mechanism with ΔG^(‡)_(298K) = 22.2 ± 0.1 kcal·mol^(–1) for the parent N-adamantyl-N′-mesityl complex. An experimentally determined ΔS^(‡) = −5.2 ± 2.6 eu supports a highly ordered transition state for carboxylate-assisted C(sp^3)–H activation. Experimental results, including measurement of a large primary kinetic isotope effect (k_(H)/k_(D) = 8.1 ± 1.7), agree closely with a computed six-membered carboxylate-assisted C–H activation mechanism where the deprotonating carboxylate adopts a pseudo-apical geometry, displacing the aryl ether chelate. The rate of cyclometalation was found to be influenced by both the electronics of the assisting carboxylate and the ruthenium ligand environment

Gradient elution LC-ESI-MS determination of cilostazol in rat plasma and its application

A sensitive and simple liquid chromatography/electrospray mass spectrometry (LC-ESI-MS) method for determination of cilostazol in rat plasma using one-step protein precipitation was developed. After addition of midazolam as internal standard (IS), protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on an SB-C18 (2.1 × 50 mm, 5.0 μm) column with acetonitrile-0.1 % formic acid as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selected ion monitoring (SIM) mode was used to quantification using target fragment ions m/z 370.0 for cilostazol and m/z 325.9 for the IS. Calibration plots were linear over the range of 10-2000 ng/mL for cilostazol in rat plasma. Lower limit of quantification (LLOQ) for cilostazol was 10 ng/mL. Mean recovery of cilostazol from plasma was in the range 90.14- 95.10 %. RSD of intra-day and inter-day precision were both less than 15 %. This method is simple and sensitive enough to be used in pharmacokinetic research for determination of cilostazol in rat plasma.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Gastric Subserous Vaccination With Helicobacter pylori Vaccine: An Attempt to Establish Tissue-Resident CD4+ Memory T Cells and Induce Prolonged Protection

Tissue-resident memory T (Trm) cells are enriched at the sites of previous infection and required for enhanced protective immunity. However, the emergence of Trm cells and their roles in providing protection are unclear in the field of Helicobacter pylori (H. pylori) vaccinology. Here, our results suggest that conventional vaccine strategies are unable to establish a measurable antigen (Ag)-specific memory cell pool in stomach; in comparison, gastric subserous injection of mice with micro-dose of Alum-based H. pylori vaccine can induce a pool of local CD4+ Trm cells. Regional recruitment of Ag-specific CD4+ T cells depends on the engagement of Ag and adjuvant-induced inflammation. Prior subcutaneous vaccination enhanced this recruitment. A stable pool of Ag-specific CD4+ T cells can be detected for 240 days. Two weeks of FTY720 administration in immune mice suggests that these cells do not experience the recirculation. Immunohistochemistry results show that close to the vaccination site, abundant CD4+T cells locate on epithelial niches, independent of lymphocyte cluster. Paradigmatically, Ag-specific CD4+ T cells with a phenotype of CD69+CD103- are preferential on lymphocytes isolated from epithelium. Upon Helicobacter infection, CD4+ Trm cells orchestrate a swift recall response with the recruitment of circulating antigen-specific Th1/Th17 cells to trigger a tissue-wide pathogen clearance. This study investigates the vaccine-induced gastric CD4+ Trm cells in a mice model, and highlights the need for designing a vaccine strategy against H. pylori by establishing the protective CD4+ Trm cells

Determination of free cyclosporine A with a LC-MS/MS method: Application to C2 monitoring in rabbits

Cyclosporine A (CsA) is a cyclic peptide widely used as an immunosuppressant. Therapeutic drug monitoring (TDM) of CsA is becoming mandatory for transplant patients who received CsA therapy in the routine clinical practice because of large individual variability, dose-related toxicity and the risk of acute rejection. In this study, a rapid, sensitive, and selective LC-MS/MS method was developed and validated for the quantitative analysis of free CsA (fCsA), a better indicator for the prediction of efficacy and safety of CsA-based therapy. Following ultrafiltration for fCsA, chromatographic separation was performed on an Agilent Zorbax SB-C18 column (100 mm x 2.1 mm, 3.5 μm ) with acetonitrile and 0.1 % ammonium hydroxide in water (85:15, v/v) as the mobile phases. The compounds were quantified by positive electrospray ionization tandem mass spectrometry. Selectivity, linearity, accuracy, precision, recovery, and stability were evaluated during method validation. The validated method was applied to a single blood concentration measurement 2 h after CsA administration (C2) measurement study of fCsA after an oral administration of a single 15 mg/kg intravenous dose of CsA to six rabbits.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Complete genome sequence of biocontrol strain Bacillus velezensis YC89 and its biocontrol potential against sugarcane red rot

IntroductionSugarcane is one of the most important sugar crops worldwide, however, sugarcane production is seriously limited by sugarcane red rot, a soil-borne disease caused by Colletotrichum falcatum. Bacillus velezensis YC89 was isolated from sugarcane leaves and can significantly inhibited red rot disease caused by C. falcatum.MethodsIn this study, the genome of YC89 strain was sequenced, its genome structure and function were analyzed using various bioinformatics software, and its genome was compared with those of other homologous strains. In addition, the effectiveness of YC89 against sugarcane red rot and the evaluation of sugarcane plant growth promotion were also investigated by pot experiments.ResultsHere, we present the complete genome sequence of YC89, which consists of a 3.95 Mb circular chromosome with an average GC content of 46.62%. The phylogenetic tree indicated that YC89 is closely related to B. velezensis GS-1. Comparative genome analysis of YC89 with other published strains (B. velezensis FZB42, B. velezensis CC09, B. velezensis SQR9, B. velezensis GS-1, and B. amyloliquefaciens DSM7) revealed that the strains had a part common coding sequences (CDS) in whereas 42 coding were unique of strain YC89. Whole-genome sequencing revealed 547 carbohydrate-active enzymes and identified 12 gene clusters encoding secondary metabolites. Additionally, functional analysis of the genome revealed numerous gene/gene clusters involved in plant growth promotion, antibiotic resistance, and resistance inducer synthesis. In vitro pot tests indicated that YC89 strain controlled sugarcane red rot and promoted the growth of sugarcane plants. Additionally, it increased the activity of enzymes involved in plant defense, such as superoxide dismutase, peroxidase, polyphenol oxidase, chitinase, and β-1,3-glucanase.DiscussionThese findings will be helpful for further studies on the mechanisms of plant growth promotion and biocontrol by B. velezensis and provide an effective strategy for controlling red rot in sugarcane plants