188 research outputs found
Expression and Purification of Diphtheria Toxin Variant CRM197 in Escherichia coli
No full textThe cross reacting material 197 (CRM197) is a nontoxic variant of the diphtheria toxin (DTx) featuring identical immunological properties and similar ability to bind the heparin-binding epidermal growth factor (HB-EGF). The only difference between CRM197 and DTx is a single amino acid substitution at position 52 (glutamic acid instead of glycine). Due to the absence of toxicity, and to its strong inflammatory-immunological property, the CRM197 protein is currently used in several conjugate vaccines. Diphtheria toxin and other related CRM proteins are generally produced using cultures of Corynebacterium diphteriae infected by specific β-phages carrying the tox gene (wt or mutated, respectively).
Here we propose a new and alternative procedure for the production of CRM197 using Escherichia coli as host strain. This process presents several advantages: a reduced time for the coltivation of bacteria; a simple culture medium; the safety of E. coli bacteria compared to C. diptheriae. To this aim, a synthetic gene coding for CRM197 and optimized for E. coli codon usage, was cloned into a specific vector (pET9a) based on the T7 RNA polymerase system. The over-expression was induced in BL21AI E. coli strain simply by adding arabinose to the culture medium. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilized using urea as denaturant. After the expression and solubilization steps, the refolding and purification conditions were experimentally assayed to define a simple procedure for the production of CRM197 in a pure and active form. In particular, the recombinant protein was purified by two different chromatographic steps (affinity and gel-filtration chromatography) and the purity of the final preparation reached up to 95%
Clinical management of rivaroxaban-treated patients
No full textIntroduction: Until recently, only vitamin K antagonists (VKAs) were used for long-term anticoagulation. New oral anticoagulants, with pharmacokinetic and pharmacodynamic characteristics different to VKAs, are now available for some indications. Rivaroxaban (Xarelto (R)) is an oral Factor Xa inhibitor approved in many countries for long-term treatment of patients with atrial fibrillation or venous thromboembolism. This article is addressed to all professionals involved in the management of treated patients to highlight the characteristics of rivaroxaban and provide practical guidance on management of treated patients. Areas covered: This article is based on a consensus of specialists involved in the management of anticoagulant treatment, including thrombosis experts, cardiologists, neurologists, emergency medicine specialists, and general practitioners. The authors performed a nonsystematic review of the literature, and expressed guidance statements based on the results of the review as well as personal experience. Expert opinion: Availability of new anticoagulant drugs, including rivaroxaban, is an important step forward to allow easier, more effective, and safer long-term anticoagulation in patients in whom adequate anticoagulation is currently denied due to the limitations of VKAs. However, given their totally new properties, associated risks, and expected broad clinical use, expert professionals and manufacturers must urgently tackle a series of issues
Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli
No full textThe expression of the recombinant diphtheria toxin mutant CRM197 in bacteria other than Corynebacterium diphtheriae has proven to be difficult. Here we propose a new and alternative procedure for the production of full-length CRM197 in Escherichia coli. The present study relates specifically to the expression of an artificial sequence and to a method for the isolation and purification of the corresponding protein. In particular, a synthetic gene coding for CRM197, bearing a short histidine tag and optimized for E. coli codon usage, was cloned in the pET9a vector. Accordingly, the over-expression of the protein was simply induced with arabinose in E. coli BL21AI. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilised using urea. Surprisingly, the expression of CRM197, devoid of the short tag, always failed. Following a refolding step, the his-tagged CRM197 was purified by affinity and gel-filtration chromatography and the purity of the final preparation reached 95%. Interestingly, the recombinant protein features DNase activity, indicating that the presence of the tag is not affecting its biochemical properties. However, the removal of the synthetic tag could be easily obtained by incubating the target protein with a proper quantity of a commercial enterokinase
Selettività di emamectina benzoato (Affirm®) nei confronti degli acari fitoseidi su vite e melo
No full textChromosomal localization and physical linkage of the genes encoding the human alpha 3, alpha 5, and beta 4 neuronal nicotinic receptor subunits
No full textAnswer to:Genetic paroxysmal neurological disorders featuring episodic ataxia and epilepsy (Amadori E et al., 2022). EJMG-D-22-00384
No full textAnswer to: Genetic paroxysmal neurological disorders featuring episodic ataxia and epilepsy
(Amadori E et al., 2022). EJMG-D-22-0038
Clinical management of rivaroxaban-treated patients
No full textUntil recently, only vitamin K antagonists (VKAs) were used for long-term anticoagulation. New oral anticoagulants, with pharmacokinetic and pharmacodynamic characteristics different to VKAs, are now available for some indications. Rivaroxaban (Xarelto®) is an oral Factor Xa inhibitor approved in many countries for long-term treatment of patients with atrial fibrillation or venous thromboembolism. This article is addressed to all professionals involved in the management of treated patients to highlight the characteristics of rivaroxaban and provide practical guidance on management of treated patients. AREAS COVERED: This article is based on a consensus of specialists involved in the management of anticoagulant treatment, including thrombosis experts, cardiologists, neurologists, emergency medicine specialists, and general practitioners. The authors performed a nonsystematic review of the literature, and expressed guidance statements based on the results of the review as well as personal experience. EXPERT OPINION: Availability of new anticoagulant drugs, including rivaroxaban, is an important step forward to allow easier, more effective, and safer long-term anticoagulation in patients in whom adequate anticoagulation is currently denied due to the limitations of VKAs. However, given their totally new properties, associated risks, and expected broad clinical use, expert professionals and manufacturers must urgently tackle a series of issues
Differential diagnosis of pulmonary embolism in outpatients with non-specific cardiopulmonary symptoms
No full textMost cardiopulmonary diseases share at least one symptom with pulmonary embolism (PE). The aim of this study was to identify the most common acute causes of dyspnea, chest pain, fainting or palpitations, which diagnostic procedures were performed and whether clinicians investigate them appropriately. An Italian multicenter collaboration gathered 17,497 Emergency Department (ED) records of patients admitted from January 2007 to June 2007 in six hospitals. A block random sampling procedure was applied to select 800 hospitalised patients. Results of the overall 17,497 patients were obtained by weighting sampled cases according to the probability of the randomisation block variables in the whole population. The case-mix of enrolled patients was assessed in terms of cardiopulmonary symptoms, and the prevalence of acute disorders. The actual performance of procedures was compared with a measure of their accuracy as expected in the most common clinical presentations. PE occurred in less than 4% of patients with cardiopulmonary symptoms. Acute heart failure, pneumonia and chronic obstructive pulmonary disease exacerbation were the most likely diagnoses in patients with dyspnea. Acute myocardial infarction was present in roughly 10% of patients with chest pain. Atrial fibrillation was the prevalent diagnosis in patients with palpitations. Echocardiography, computed tomographic pulmonary angiography, perfusion lung scan, D-dimer test and B-type natriuretic peptide were performed less than expected from their accuracy. Diagnostic strategies, starting from non-specific symptoms and coping with the eventuality of PE, are likely to benefit from an increased awareness of the examination's accuracy in discriminating among several competing hypotheses, rather than in testing the single PE suspicion
Differential diagnosis of pulmonary embolism in outpatients with non-specific cardiopulmonary symptoms
No full textMost cardiopulmonary diseases share at least one symptom with pulmonary embolism (PE). The aim of this study was to identify the most common acute causes of dyspnea, chest pain, fainting or palpitations, which diagnostic procedures were performed and whether clinicians investigate them appropriately. An Italian multicenter collaboration gathered 17,497 Emergency Department (ED) records of patients admitted from January 2007 to June 2007 in six hospitals. A block random sampling procedure was applied to select 800 hospitalised patients. Results of the overall 17,497 patients were obtained by weighting sampled cases according to the probability of the randomisation block variables in the whole population. The case-mix of enrolled patients was assessed in terms of cardiopulmonary symptoms, and the prevalence of acute disorders. The actual performance of procedures was compared with a measure of their accuracy as expected in the most common clinical presentations. PE occurred in less than 4% of patients with cardiopulmonary symptoms. Acute heart failure, pneumonia and chronic obstructive pulmonary disease exacerbation were the most likely diagnoses in patients with dyspnea. Acute myocardial infarction was present in roughly 10% of patients with chest pain. Atrial fibrillation was the prevalent diagnosis in patients with palpitations. Echocardiography, computed tomographic pulmonary angiography, perfusion lung scan, D-dimer test and B-type natriuretic peptide were performed less than expected from their accuracy. Diagnostic strategies, starting from non-specific symptoms and coping with the eventuality of PE, are likely to benefit from an increased awareness of the examination's accuracy in discriminating among several competing hypotheses, rather than in testing the single PE suspicion
Chromosomal localization and physical linkage of the genes encoding the human alpha 3, alpha 5, and beta 4 neuronal nicotinic receptor subunits.
No full textThe genes coding alpha 3, alpha 5, and beta 4 neuronal nicotinic receptor subunits have been localized on human metaphase chromosomes by in situ hybridization
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