ABSTRACT
Label-free assays, and particularly those based on the combination of mass spectroscopy with surface chemistries, enable high-throughput experiments of a broad range of reactions. However, these methods can still require the incorporation of functional groups that allow immobilization of reactants and products to surfaces prior to analysis. In this paper, we report a traceless method for attaching molecules to a self-assembled monolayer for matrix-assisted laser desorption and ionization (SAMDI) mass spectrometry. This method uses monolayers that are functionalized with a 3-trifluoromethyl-3-phenyl-diazirine group that liberates nitrogen when irradiated and gives a carbene that inserts into a wide range of bonds to covalently immobilize molecules. Analysis of the monolayer with SAMDI then reveals peaks for each of the adducts formed from molecules in the sample. This method is applied to characterize a P450 drug metabolizing enzyme and to monitor a Suzuki-Miyaura coupling chemical reaction and is important because modification of the substrates with a functional group would alter their activities. This method will be important for high-throughput experiments in many areas, including reaction discovery and optimization.
Subject(s)
Azirines/metabolism , Cytochrome P-450 Enzyme System/metabolism , High-Throughput Screening Assays , Azirines/chemistry , Cytochrome P-450 Enzyme System/chemistry , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Molecular Structure , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Surface PropertiesABSTRACT
OBJECTIVES: To investigate the aetiology of persistent cough among vaccinated children as suspected cases of pertussis in Khairpur District, Sindh, Pakistan. Pertussis or whooping cough, caused by Bordetella pertussis, is re-appearing in many countries despite vaccination coverage. In Khairpur, persistent cough and symptoms similar to pertussis among vaccinated children are common but the aetiology has not been investigated previously. STUDY DESIGN: B. pertussis was isolated from cough samples of suspected pertussis patients (n = 700) using the cough plate method with charcoal agar. METHODS: Isolation and confirmation of the clinical isolates of B. pertussis was performed by culture on Bordet-Gengou medium, biochemical tests and polymerase chain reaction. RESULTS: In total, 22 strains of B. pertussis were isolated from clinical cough samples. CONCLUSION: To the authors' knowledge, this is the first report of the presence of pertussis in vaccinated children in Khairpur. There is a need for continuous monitoring of pertussis after immunization programmes in order to assess the efficacy of pertussis vaccination.
Subject(s)
Bordetella pertussis/isolation & purification , Disease Outbreaks , Pertussis Vaccine/administration & dosage , Whooping Cough/epidemiology , Antigens, Bacterial , Bordetella pertussis/classification , Child , Child, Preschool , Cough/etiology , Female , Humans , Infant , Male , Pakistan/epidemiology , Pertussis Vaccine/immunology , Vaccination/statistics & numerical data , Whooping Cough/complications , Whooping Cough/diagnosis , Whooping Cough/immunology , Whooping Cough/prevention & controlABSTRACT
The aim of this study was to device a simple, sensitive and convenient assay for the detection of toxicity of the Adenylate cyclase toxin (ACT), a newly recognized toxin of Bordetella pertussis. Haemolytic activity of ACT was analysed as an assay and sheep RBCs were found to be suitable to detect the toxic effect down to the dose of 6.25 nmol cAMP/min/ml enzymic activity applied. The haemolytic activity was found to be dose-dependent.
ABSTRACT
A total 72 drinking water sample were collected and analyzed by membrane filtration method during 1 year study from various points in Khairpur City. Out of these 58 (80.55%) samples were found to be contaminated with thermo-tolerant Escherichia coli 2. The susceptibility of these isolates to 35 antibiotics was studied by disc diffusion method and the organism was highly sensitive to levoflaxin, cefipime, enoxobid, noroxin, tarivid, ciproxin, avelox, amikacin, kanamycin, rocifin, pipenedic acid and slightly sensitive to cravit, naladixic acid, neomycin, cefizox, fortum cefotaxime, cefizox, fortum, tobramycin and cefoperoxone. The resistance against 16 antibiotics such as meropenem, linkomycin, fusidic acid, orbenin, penicillin, streptomycin, bacitracin, minocin, zinacef, amoxil, ceclor, claracid, cephalexin, augmentin, cephradin and dalacin was shown by these isolates. We report the presence of multi-drug resistance in thermo-tolerant Escherichia coli isolated in municipal water with different levels of prevalence in Khairpur City. In this study a higher number of positive results were obtained in all sampling points indicating the more fecally polluted municipal water.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli , Water Microbiology , Water Supply , Drug Resistance, Multiple, Bacterial/drug effects , Escherichia coli/drug effects , Escherichia coli/isolation & purification , Humans , Microbial Sensitivity TestsABSTRACT
The incidence of internal hernia through a defect in the falciform ligament, mostly congenital, is very rare. In this era of minimally invasive laparoscopic surgeries, a few cases of internal hernia through an iatrogenic defect in the falciform ligament have also been reported. Here, we present a case of a 65-year-old patient who presented with acute small-bowel obstruction. The patient had undergone a laparoscopic fundoplication 4 years ago. On diagnostic laparoscopy, it was found that the cause of the intestinal obstruction was herniation of the small bowel through a window in the falciform ligament (which was probably created due to port insertion during the previous surgery of laparoscopic fundoplication). The obstruction was relieved by the division of the falciform ligament.
Subject(s)
Hernia, Abdominal/complications , Intestinal Obstruction/etiology , Intestine, Small , Ligaments/injuries , Aged , Female , Hernia, Abdominal/surgery , Humans , Iatrogenic Disease , Intestinal Obstruction/surgery , Ligaments/surgeryABSTRACT
When baby hamster kidney (BHK) cells are allowed to spread on fibronectin-coated substrata in the absence of serum and the presence of agents which elevate intracellular 3':5'-cyclic AMP (cAMP) levels they adopt an abnormal, stellated morphology. To determine whether the invasive adenylate cyclase (AC) toxin of Bordetella pertussis induced the same response, cell extracts were prepared from several B. pertussis strains. They were characterized for AC toxin production by enzymic assay and by immunoblotting with an AC-toxin-specific monoclonal antibody. Extracts of strains producing AC toxin induced elevated levels of intracellular cAMP in BHK cells and promoted a stellation response during cell spreading. Extracts prepared from strains defective in AC toxin production showed no effect. Using image analysis to quantify the morphological change, we have demonstrated that the effect of AC toxin on cell spreading is dose dependent. This technique is a rapid and sensitive assay for the invasive AC toxin.