Actinoplanes teichomyceticus ATCC 31121 as a cell factory for producing teicoplanin

<p>Abstract</p> <p>Background</p> <p>Teicoplanin is a glycopeptide antibiotic used clinically in Europe and in Japan for the treatment of multi-resistant Gram-positive infections. It is produced by fermenting <it>Actinoplanes teichomyceticus</it>. The pharmaceutically active principle is teicoplanin A₂, a complex of compounds designated T-A_2-1-A_2-5differing in the length and branching of the fatty acid moiety linked to the glucosamine residue on the heptapeptide scaffold. According to European and Japanese Pharmacopoeia, components of the drug must be reproduced in fixed amounts to be authorized for clinical use.</p> <p>Results</p> <p>We report our studies on optimizing the fermentation process to produce teicoplanin A₂in <it>A. teichomyceticus </it>ATCC 31121. Robustness of the process was assessed on scales from a miniaturized deep-well microtiter system to flasks and 3-L bioreactor fermenters. The production of individual factors T-A_2-1-A_2-5was modulated by adding suitable precursors to the cultivation medium. Specific production of T-A_2-1, characterized by a linear C10:1 acyl moiety, is enhanced by adding methyl linoleate, trilinoleate, and crude oils such as corn and cottonseed oils. Accumulation of T-A_2-3, characterized by a linear C10:0 acyl chain, is stimulated by adding methyl oleate, trioleate, and oils such as olive and lard oils. Percentages of T-A_2-2, T-A_2-4, and, T-A_2-5bearing the iso-C10:0, anteiso-C11:0, and iso-C11:0 acyl moieties, respectively, are significantly increased by adding precursor amino acids <smcaps>L</smcaps>-valine, <smcaps>L</smcaps>-isoleucine, and <smcaps>L</smcaps>-leucine. Along with the stimulatory effect on specific complex components, fatty acid esters, oils, and amino acids (with the exception of <smcaps>L</smcaps>-valine) inhibit total antibiotic productivity overall. By adding industrial oils to medium containing <smcaps>L</smcaps>-valine the total production is comparable, giving unusual complex compositions.</p> <p>Conclusions</p> <p>Since the cost and the quality of teicoplanin production depend mainly on the fermentation process, we developed a robust and scalable fermentation process by using an industrial medium in which a complex composition can be modulated by the combined addition of suitable precursors. This work was performed in the wild-type strain ATCC 31121, which has a clear genetic background. This is important for starting a rational improvement program and also helps to better control teicoplanin production during process and strain development.</p

Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in <it>Escherichia coli </it>of type II cholesterol oxidase from <it>Brevibacterium sterolicum </it>(BCO).</p> <p>Results</p> <p>Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant <it>E. coli </it>cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from ~500 up to ~25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies.</p> <p>Conclusions</p> <p>Comparison of our results with those published on non-covalent (type I) COs expressed in recombinant form (either in <it>E. coli </it>or <it>Streptomyces </it>spp.), shows that the fully active type II BCO can be produced in <it>E. coli </it>at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.</p

Early career researchers want Open Science : Comment

Creative Commons Attribution License (CC BY 4.0)Open Science is encouraged by the European Union and many other political and scientific institutions.However, scientific practice is proving slow to change.We propose, as early career researchers, that it is our task to change scientific research into open scientific research and commit to Open Science principles.Non peer reviewe

Presynaptic GABAergic inhibition regulated by BDNF contributes to neuropathic pain induction

The gate control theory proposes the importance of both pre- and post-synaptic inhibition in processing pain signal in the spinal cord. However, although postsynaptic disinhibition caused by brain-derived neurotrophic factor (BDNF) has been proved as a crucial mechanism underlying neuropathic pain, the function of presynaptic inhibition in acute and neuropathic pain remains elusive. Here we show that a transient shift in the reversal potential (E GABA) together with a decline in the conductance of presynaptic GABA A receptor result in a reduction of presynaptic inhibition after nerve injury. BDNF mimics, whereas blockade of BDNF signalling reverses, the alteration in GABA A receptor function and the neuropathic pain syndrome. Finally, genetic disruption of presynaptic inhibition leads to spontaneous development of behavioural hypersensitivity, which cannot be further sensitized by nerve lesions or BDNF. Our results reveal a novel effect of BDNF on presynaptic GABAergic inhibition after nerve injury and may represent new strategy for treating neuropathic pain

Manic and Depressive Symptoms in Children Diagnosed with Noonan Syndrome

Noonan syndrome (NS) is a dominant clinically variable and genetically heterogeneous developmental disorder caused by germ-line mutations encoding components of the Ras–MAPK signaling pathway. A few studies have investigated psychopathological features occurring in individuals with NS, although they were poorly analyzed. The aim of the present work is to investigate the psychopathological features in children and adolescents with NS focusing on depressive and hypo-manic symptoms. Thirty-seven subjects with molecularly confirmed diagnosis were systematically evaluated through a psychopathological assessment. In addition, an evaluation of the cognitive level was performed. Our analyses showed a high recurrence of attention deficit and hyperactivity disorder symptoms, emotional dysregulation, irritability, and anxiety symptomatology. The mean cognitive level was on the average. The present study provides new relevant information on psychopathological features in individuals with NS. The implications for clinicians are discussed including the monitoring of mood disorders in a clinical evolution

SARS-CoV-2 in myelodysplastic syndromes: a snapshot from early Italian experience

Myelodysplastic syndrome patients are subjects of advanced age, vulnerable and frail, whose outcome is heavily influenced by pre-existing comorbidities worsening the hematologic condition. Infections are a rather common cause of death (around 30%), especially, but not only, for IPSS-R higher risk patients.1–3 In MDS there is a significant impairment of lymphopoiesis, resulting in lymphopenia (ALC&lt;1.0109 /l) in around 38% of MDS patients and poor prognosis.4 Data on innate and adoptive immune systems (either disease related or due to immunosenescence) and the subsequent supposed susceptibility and incidence of viral infections in MDS are scarce.