Psychotropic medications sales during COVID-19 outbreak in Italy changed according to the pandemic phases and related lockdowns.

OBJECTIVES: We have investigated the psychotropic medications sales (i.e. benzodiazepines, mood stabilisers and selective serotonin reuptake inhibitors) during the COVID-19 pandemic in the period from March 2020 to February 2021 compared with the same period in the preceding year. STUDY DESIGN: This was a retrospective and observational study. METHODS: Data were obtained from five pharmacies located in a working-class zone populated by approximately 150,000 people in the urban area of Rome (Italy). RESULTS: A general slight increase in psychotropic medications sales was observed during the whole pandemic period compared with the previous year. CONCLUSION: Our data showed that (1) the percentage of sales seems to vary according to the pandemic phases and related lockdowns and (2) the sales differ between the classes of medications considered.

Cloning and sequencing of the ilvBNC gene cluster from Mycobacterium avium.

Disseminated Mycobacterium avium (Ma)-M. intracellulare disease is a prevalent opportunistic infection in patients with Acquired Immune Deficiency Syndrome. Mycobacteria produce a variety of fatty acids which provide the first line of defence against potentially lethal environmental conditions. The metabolism of the branched-chain amino acids (BCAA) could be correlated to the production of branched-chain fatty acids in mycobacteria. In order to develop a better understanding of Mycobacterium BCAA biosynthesis, three genes, ilvBN and ilvC, encoding acetohydroxy acid synthase (AHS) and acetohydroxy acid isomeroreductase (IR), respectively, were cloned from Ma. The genes were isolated by screening a Ma genomic library with a heterologous probe. The deduced amino acid sequences revealed significant homology to the AHS and IR proteins from other bacterial species.

Sequence of the Bacillus stearothermophilus gene encoding aspartokinase II.

The complete nucleotide sequence of the gene encoding aspartokinase (Ask) II from thermophilic Bacillus stearothermophilus has been determined. Degenerate oligodeoxyribonucleotides primed the amplification of a 932-bp gene. This sequence was successively used for constructing new primers applied in inverse polymerase chain reaction using, as template, self-ligating DNA fragments. The deduced amino-acid sequence is 68.7% identical with the sequence of the Bacillus sp. strain MGA3 Ask II.

Cloning, sequencing and expression of the ilvBNC gene cluster from Streptomyces avermitilis.

The metabolism of the branched-chain amino acids (BCAA) isoleucine, leucine and valine is correlated to the production of polyketide antibiotics in many streptomycetes. Despite its significance, this biosynthetic pathway is poorly understood in Streptomyces. In order to develop a better understanding of Streptomyces BCCA biosynthesis, two genes, ilvBN and ilvC, encoding acetohydroxy acid synthase (AHS) and acetohydroxy acid isomeroreductase (IR), respectively, were cloned from Streptomyces avermitilis, a strain producing avermectins, potent antiparasitic compounds. The genes were isolated by applying a combination of PCR and genomic library screening. The deduced amino-acid sequences revealed significant homology to the AHS and IR proteins from other bacterial species. The ilvBN gene, expressed in Escherichia coli (Ec) by using the expression vector pGEX-4T-1, complemented the ilv- mutation of Ec PS1283. Ec transformants produced high levels of AHS, whose activity was feedback inhibited by valine.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Characterization of gram-positive broad host-range plasmids carrying a thermophilic replicon.

The cryptic plasmid pBC1 (1.6 kb) isolated from Bacillus coagulans Zu1961 was genetically marked with the genes for chloramphenicol and ampicillin resistance (CmR and ApR) from the Escherichia coli plasmid pJH101. The recombinant vector obtained (pCP49, 7.0 kb) replicated and expressed CmR in B. subtilis and CmR and ApR in E. coli. Different shuttle vectors for Gram+ bacteria were also constructed by inserting pBC1 into the Staphylococcus aureus plasmid pC194. The smallest of these, pLM6 (2.8 kb), containing essentially pBC1 and the chloramphenicol acetyl transferase gene from pC194, replicated in B. subtilis at a copy number of 60. By electroporation, these plasmids were introduced and stably maintained in B. subtilis, B. amyloliquefaciens, S. aureus, S. carnosus and Lactobacillus reuteri.

New shuttle vector for cloning in Bacillus stearothermophilus.

Cloning vector plasmid pRP9 was constructed on the basis of the broad host-range plasmid pLM6. pRP9 was a small plasmid (2.9 kb), possessed a convenient polyrestriction site sequence and efficiently transformed Bacillus subtilis, Bacillus stearothermophilus and Escherichia coli. Furthermore, pRP9 presented a very high segregational stability in Bacillus hosts. Also, the structural stability in Bacillus strains, grown under selective pressure, of pRP9 carrying a 3-kb fragment, was high. No single-stranded and high-molecular weight pRP9 DNA was found in B. stearothermophilus. The host/vector systems described possessed all the properties required for efficient gene cloning.

Genetic transformation of intact cells of Bacillus subtilis by electroporation.

Plasmid DNAs were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation. The maximum electroporation efficiency was 10(4) transformants/microgram DNA. Parameters including growth phase of cells, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformation efficiency.

Detection and characterization of naturally occurring plasmids in Bacillus licheniformis.

Twenty-two Bacillus licheniformis strains, freshly isolated from pasture-land, were studied for the presence of plasmid DNA. Among these strains, 14 were shown to harbor one or more plasmids of different size. Southern-hybridization experiments showed a high homology between all plasmids investigated and a 2.2-kb PvuII/HindIII fragment of pBL1, a B. licheniformis plasmid previously isolated. Three fragments of pBL1, including the 2.2-kb PvuII/HindIII region, were cloned into pJH101 vector. The resulting chimeras were able to transform Bacillus subtilis. The fragment with high homology probably contains the region with the replicative functions of plasmids from B. licheniformis species.

Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance.

Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.

Modelos anatómicos personalizados impresos en 3D como herramientas para el aprendizaje y la preparación de intervenciones / Custom 3D printed anatomical models as tools for learning and preparation of Interventions / Modelos anatômicos personalizados impressos em 3D como ferramentas para aprendizagem e preparação de intervenções

Objetivo: presentar un método de desarrollo basado en dos casos de modelos anatómicos personalizados impresos en 3D, el\r\nprimero una arteria cerebral y el segundo una estructura ósea del húmero humano, a fin de ejemplificar el uso de herramientas\r\nde visualización tridimensionales para planificar intervenciones quirúrgicas. Método: se seleccionaron imágenes médicas de\r\ntomografías computarizadas o imágenes de resonancia magnética de pacientes anónimos y la sección específica del órgano se\r\nsegmentó con el software 3D Slicer. El modelo se convirtió en mallas poligonales en tres dimensiones, se optimizó y se imprimió\r\nen 3D. La morfología del órgano representada en el modelo anatómico se validó con especialistas para determinar si son oportunas\r\npara planificar procedimientos médicos. Resultados: diversos modelos anatómicos de los mismos casos se elaboraron en dos laboratorios de fabricación digital, uno en la Universidad El Bosque y otro en el FabLab Valencia, con diferentes variables en su\r\nproceso técnico y características, dada la dificultad de morfologías y delicadeza de las estructuras presentes en el cuerpo humano.\r\nConclusiones: con el método presentado sí es posible realizar modelos anatómicos personalizados en 3D para visualizar y simular\r\nestructuras anatómicas de pacientes útiles en la planeación de cirugías y la enseñanza de anatomía, que podrían mejorar el éxito\r\nen las intervenciones y el entrenamiento de profesionales en áreas de la salud.

Objective: To present a method of development based on\r\ntwo cases of custom anatomical models printed in 3D; the\r\nfirst one a cerebral artery and the second a bone structure\r\nof the human humerus, to exemplify the use of three-dimensional\r\nvisualization tools to perform planning of surgical\r\noperations. Method: It consisted of: a) Searching CT or MRI\r\nimages of anonymous patients, b) Segmenting with the 3D\r\nSlicer software the specific section of the organ, c) Converting\r\nthe model into polygonal meshes in three dimensions, d)\r\nOptimizing and printing in 3D, e) Validating with specialists\r\nthe organ morphology to determine if they are pertinent to\r\nplanning medical procedures. Results: Models were made in\r\ntwo different manufacturing laboratories; El Bosque University\r\nand FabLab in Valencia, with various anatomical models\r\nmanufactured of the same case with different variables in their\r\nprocess and characteristics given the difficulty of morphologies\r\nand delicacy of the structures present in the human body.\r\nConclusion: The method presented does provide useful results\r\nas an example for the planning of surgeries and anatomy\r\nteaching of anatomical structures in different scenarios that\r\ncould improve the success in interventions and the training of\r\nprofessionals in health areas.

Objetivo: apresentar um método de desenvolvimento de\r\nmodelos anatômicos personalizados em 3D para exemplificar\r\no uso de ferramentas de visualização para realizar planificação\r\nde operações cirúrgicas. Realizaram-se dois modelos como\r\nexemplo; o primeiro uma artéria cerebral e o segundo uma\r\nestrutura óssea do úmero humano. Método: o método de\r\ntrabalho consistiu em a) Buscar imagens médicas realizadas\r\ncom CT ou MRI de pacientes anônimos, b) Segmentar com o\r\nsoftware 3D Slicer a seção específica do órgão, c) Converter o\r\nmodelo em malhas poligonais em três dimensões, d) Otimizar\r\ne imprimir em 3D, e) Validar com especialistas a morfologia do\r\nórgão para determinar se são pertinentes para planejar procedimentos\r\nmédicos. Resultados: fabricou-se em dois laboratórios\r\nde fabricação diferentes, um na Universidad El Bosque e outro\r\nno FabLab Valencia, diversos modelos anatômicos do mesmo\r\ncaso com diferentes variáveis em seu processo e características\r\ndada a dificuldade de morfologias e delicadeza das estruturas\r\npresentes no corpo humano. Conclusão: o método apresentado\r\nproporciona resultados úteis para o planejamento de\r\ncirurgias e ensino de anatomia de estruturas anatômicas em\r\ndiferentes cenários que poderiam melhorar o êxito das intervenções\r\ne o treinamento de profissionais na área de saúde.

Decaprenylphosphoryl-ß-D-ribose 2'-epimerase from Mycobacterium tuberculosis is a magic drug target.

Tuberculosis is still a leading cause of death in developing countries and a resurgent disease in developed countries. The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need of new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Several enzymes involved in various metabolic processes have been described as potential targets for the development of new drugs. Recently, two different classes of most promising drugs, the benzothiazinones (BTZ) and the dinitrobenzamide derivatives (DNB), have been found to be highly active against M. tuberculosis, including XDR-TB strains. Interestingly, both drugs have the same target: the heteromeric decaprenylphosphoryl-ß-D-ribose 2'-epimerase encoded by dprE1 (Rv3790) and dprE2 (Rv3791) genes, respectively. DprE1 and DprE2 are involved in the biosynthesis of D-arabinose and, in particular, they are essential to perform the transformation of decaprenylphosphoryl-D-ribose to decaprenylphosphoryl-D-arabinose, which is a substrate for arabinosyltransferases in the synthesis of the cell-envelope arabinogalactan and liporabinomannan polysaccharides of mycobacteria. Arabinogalactan is a fundamental component of the mycobacterial cell wall, which covalently binds the outer layer of mycolic acids to peptidoglycan. The heteromeric decaprenylphosphoryl-ß-D-ribose 2'-epimerase thus represents a valid vulnerable antimycobacterial drug target which could result in "magic" for tuberculosis treatment.

Molecular cloning and sequencing of the beta-isopropylmalate dehydrogenase gene from the cyanobacterium Spirulina platensis.

The gene for beta-isopropylmalate dehydrogenase (EC 1.1.1.85) of Spirulina platensis (leuB) was cloned from a lambda EMBL3 genomic library by heterologous hybridization using the Nostoc UCD 7801 leuB gene as a probe. The sequence of the entire leuB coding region was determined as well as 645 bp of 5' flanking region and 956 bp of 3' flanking region. DNA sequencing revealed an open reading frame of 1065 nucleotides capable of encoding a polypeptide of 355 amino acids. Homologies between the amino acid sequence deduced from the nucleotide sequence of the S. platensis leuB gene and the amino acid sequences published for corresponding proteins either from bacteria or yeasts are 45% or more. Northern hybridization analysis indicated that the S. platensis leuB gene is transcribed as a single monocistronic RNA of approximately 1200 bases.

Cloning of the glutamine synthetase gene fromspirulina platensis.

An 8 Kilobase-pair (Kbp) HindIII fragment containing the coding sequence forSpirulina platensis glutamine synthetase [EC 6.3.1.1.] has been identified utilizing a probe derived fromAnabaena 7120 and cloned in the vector pAT153.

Molecular characterization of the genes encoding acetohydroxy acid synthase in the cyanobacterium Spirulina platensis.

The enzyme acetohydroxy acid synthase (AHS), which catalyses the first common step in the biosynthesis of isoleucine, leucine and valine, has been demonstrated to be present in Spirulina platensis in two isoenzymic forms. The complete nucleotide sequences of the genes ilvX and ilvW encoding these two enzymes have been determined. Sequence analysis revealed the presence of two open reading frames, of 1836 and 1737 nucleotides for ilvX and ilvW, respectively. The predicted amino acid sequences of the two isoenzymes, compared with the Synechococcus PCC 7942 AHS enzyme and the large subunits of the Escherichia coli AHSI, II, III isoenzymes, revealed a notable degree of similarity. A small subunit has not been identified for either of the S. platensis AHS isoenzymes. Analysis by Northern blot hybridization demonstrated that the ilvX and ilvW genes are transcribed to give mRNA species of approximately 2.15 kb and 1.95 kb, respectively.

The katE gene, which encodes the catalase HPII of Mycobacterium avium.

Disseminated Mycobacterium avium-Mycobacterium intracellulare disease is a prevalent opportunistic infection in patients with acquired immune deficiency syndrome (AIDS). These pathogens are generally resistant to isoniazid (INH), a powerful antituberculosis drug. It is now generally accepted that the INH susceptibility of Mycobacterium tuberculosis results from the transformation of the drug into a toxic derivative, as a result of the action of the enzyme catalase-peroxidase (HPI), encoded by the katG gene. It has been speculated that the presence of a second catalase (HPII) in some mycobacterial species, but lacking in M. tuberculosis, may impair the action of INH. In this report, the nucleotide sequence of the M. avium katE gene, encoding catalase HPII, is described. This enzyme shows strong similarity to Escherichia coli catalase HPII and eukaryotic catalases. All amino acids previously postulated as participating directly in catalysis by liver catalase and most of the amino acids binding the prosthetic group are conserved in M. avium catalase HPII. The enzyme is expressed in E. coli and is inhibited by 3-amino-1,2,4-triazole (AT). Furthermore, Southern blot hybridizations and polymerase chain reaction experiments demonstrate the distribution of katE gene in several mycobacterial species. To evaluate the potentially antagonistic effect of HPII catalase on INH susceptibility, the katE gene was transformed into M. tuberculosis H37Rv and the minimum inhibitory concentration (MIC) for INH was determined. Despite strong expression of the katE gene, no change in MIC was observed, thus ruling out a possible contribution of this enzyme to the natural resistance of M. avium to the drug. The availability of the gene probe, encoding the second mycobacterial catalase HPII, should open the way for the development of new drugs and diagnostic tests to combat drug-resistant pathogen strains.

Molecular cloning and expression of Spirulina platensis acetohydroxy acid synthase genes in Escherichia coli.

The coding sequence for Spirulina platensis acetohydroxy acid synthase (AHAS, EC 4.1.3.18) is shown to be contained within a 4.2 Kb ClaI fragment (ilvX) that has been cloned from a recombinant lambda library. This fragment was able to complement a suitable mutant of Escherichia coli when inserted into the ClaI site of plasmid pAT153 in either orientation, demonstrating that transcription of ilvX originated within the cloned fragment. The probe used for hybridization experiments was the corresponding gene from Anabaena sp. PCC7120. The same probe allowed us to identify a second putative gene encoding AHAS in the S. platensis genomic library.

mmr, a Mycobacterium tuberculosis gene conferring resistance to small cationic dyes and inhibitors.

The mmr gene, cloned from Mycobacterium tuberculosis, was shown to confer to Mycobacterium smegmatis resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [3H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP.

Structural organization of pBC1, a cryptic plasmid from Bacillus coagulans.

The complete nucleotide sequence of the Bacillus coagulans plasmid pBC1 was determined. The sequence revealed an open reading frame encoding a polypeptide of 259 amino acids. This open reading frame shows sequence similarity to genes coding for replication-associated proteins in a group of gram-positive bacterial plasmids known to replicate via single-stranded intermediates. A region required for replication in cis, when the intact replicon is supplied in trans, was identified as well.

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis.

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.