Interplay between carbon, nitrogen and phosphate utilization in the control of secondary metabolite production in Streptomyces.

Streptomyces species are a wide and diverse source of many therapeutic agents (antimicrobials, antineoplastic and antioxidants, to name a few) and represent an important source of compounds with potential applications in medicine. The effect of nitrogen, phosphate and carbon on the production of secondary metabolites has long been observed, but it was not until recently that the molecular mechanisms on which these effects rely were ascertained. In addition to the specific macronutrient regulatory mechanisms, there is a complex network of interactions between these mechanisms influencing secondary metabolism. In this article, we review the recent advances in our understanding of the molecular mechanisms of regulation exerted by nitrogen, phosphate and carbon sources, as well as the effects of their interconnections, on the synthesis of secondary metabolites by members of the genus Streptomyces.

Carbon catabolite regulation in Streptomyces: new insights and lessons learned.

One of the most significant control mechanisms of the physiological processes in the genus Streptomyces is carbon catabolite repression (CCR). This mechanism controls the expression of genes involved in the uptake and utilization of alternative carbon sources in Streptomyces and is mostly independent of the phosphoenolpyruvate phosphotransferase system (PTS). CCR also affects morphological differentiation and the synthesis of secondary metabolites, although not all secondary metabolite genes are equally sensitive to the control by the carbon source. Even when the outcome effect of CCR in bacteria is the same, their essential mechanisms can be rather different. Although usually, glucose elicits this phenomenon, other rapidly metabolized carbon sources can also cause CCR. Multiple efforts have been put through to the understanding of the mechanism of CCR in this genus. However, a reasonable mechanism to explain the nature of this process in Streptomyces does not yet exist. Several examples of primary and secondary metabolites subject to CCR will be examined in this review. Additionally, recent advances in the metabolites and protein factors involved in the Streptomyces CCR, as well as their mechanisms will be described and discussed in this review.

Transcriptomic analysis of a classical model of carbon catabolite regulation in Streptomyces coelicolor.

BACKGROUND: In the genus Streptomyces, one of the most remarkable control mechanisms of physiological processes is carbon catabolite repression (CCR). This mechanism regulates the expression of genes involved in the uptake and utilization of alternative carbon sources. CCR also affects the synthesis of secondary metabolites and morphological differentiation. Even when the outcome effect of CCR in different bacteria is the same, their essential mechanisms can be quite different. In several streptomycetes glucose kinase (Glk) represents the main glucose phosphorylating enzyme and has been regarded as a regulatory protein in CCR. To evaluate the paradigmatic model proposed for CCR in Streptomyces, a high-density microarray approach was applied to Streptomyces coelicolor M145, under repressed and non-repressed conditions. The transcriptomic study was extended to assess the ScGlk role in this model by comparing the transcriptomic profile of S. coelicolor M145 with that of a ∆glk mutant derived from the wild-type strain, complemented with a heterologous glk gene from Zymomonas mobilis (Zmglk), insensitive to CCR but able to grow in glucose (ScoZm strain). RESULTS: Microarray experiments revealed that glucose influenced the expression of 651 genes. Interestingly, even when the ScGlk protein does not have DNA binding domains and the glycolytic flux was restored by a heterologous glucokinase, the ScGlk replacement modified the expression of 134 genes. From these, 91 were also affected by glucose while 43 appeared to be under the control of ScGlk. This work identified the expression of S. coelicolor genes involved in primary metabolism that were influenced by glucose and/or ScGlk. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. CONCLUSIONS: The transcriptome of a classical model of CCR was studied in S. coelicolor to differentiate between the effects due to glucose or ScGlk in this regulatory mechanism. Glucose elicited important metabolic and transcriptional changes in this microorganism. While its entry and flow through glycolysis and pentose phosphate pathway were stimulated, the gluconeogenesis was inhibited. Glucose also triggered the CCR by repressing transporter systems and the transcription of enzymes required for secondary carbon sources utilization. Our results confirm and update the agar model of the CCR in Streptomyces and its dependence on the ScGlk per se. Surprisingly, the expected regulatory function of ScGlk was not found to be as global as thought before (only 43 out of 779 genes were affected), although may be accompanied or coordinated by other transcriptional regulators. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. These findings offer new opportunities to study and understand the CCR in S. coelicolor by increasing the number of known glucose and ScGlk -regulated pathways and a new set of putative regulatory proteins possibly involved or controlling the CCR.

Anti-interleukin-6 receptor antibody for the treatment of remitting seronegative symmetrical synovitis with pitting oedema: a new outlook?

We present the case of an elderly man with a small-joint polyarthritis, accompanied by pitting oedema, involving hands and feet, raising clinical suspicion of remitting seronegative symmetrical synovitis with pitting oedema (RS3PE). Treatment with corticosteroids was initiated with significant improvement, but unacceptable iatrogeny ensued, and tapering was not possible without disease flare-up. A trial of tocilizumab allowed disease activity control, slow weaning of corticosteroids and, ultimately, its suspension. RS3PE is a rare rheumatological entity, initially thought to be a variant of rheumatoid arthritis (RA), with shared traits with polymyalgia rheumatica (PMR), and other seronegative spondyloarthropathies, thereby implying a shared pathophysiological background. Elevated levels of interleukin 6 (IL-6) are found in patients with RA, have shown to mirror disease activity in PMR and have also been described in the serum and synovial fluid of patients with RS3PE. Tocilizumab, an anti-IL-6 receptor antibody, shows auspicious results in several other rare rheumatic diseases other than RA.

Self-Poisoning With Household Bleach in an Elderly Man.

Caustic self-poisoning is a major health hazard, which affects any age, but is particularly serious among the elderly. Household bleach is a caustic that contains 3% to 6% sodium hypochlorite solution, one of the most common agents in unintentional caustic poisoning. In this clinical case, we present a household bleach self-poisoning by an older man with no relevant medical history. He presented with extensive burns on the oral cavity mucosa and tongue, requiring orotracheal intubation by video laryngoscopy. He was then admitted to the intensive care unit, where he evolved poorly. Given the poor prognosis and the lack of physiological reserve for the invasiveness required for a surgical approach, a conservative strategy was chosen after a multidisciplinary team discussion. With the conservative strategy, the patient survived, being discharged to the general ward after one month, where he underwent a Stamm gastrostomy and placement of a percutaneous endoscopic gastrostomy. In the follow-up consultation three months later, the patient was found to be weakened, with high frailty status, presenting anxiety, depression, and causing high family burden.

Lipase-Catalysed Polymerization of Eutectic Mixtures.

Aiming to reduce the toxicity and operational costs often associated to chemical processes, the enzymatic synthesis is applied herein as a sustainable route for producing polyesters. The use of NADES' (Natural Deep Eutectic Solvents) components as a source of monomers for the synthesis of polymers through lipase-catalyzed esterification in an anhydrous medium is detailed for the first time. Three NADES composed by glycerol and an organic base, or acid, were used to produce polyesters, through polymerization reactions catalyzed by Aspergillus oryzae lipase. High polyester conversion rates (above 70 %), containing at least 20 monomeric units (glycerol:organic acid/base (1 : 1)), were observed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis. The NADES monomers' capacity for polymerization, along with their non-toxicity, cheap cost, and simplicity of production, sets up these solvents as a greener and cleaner approach for the synthesis of high value-added products.

Venous Thromboembolism Prophylaxis in Medical and Surgical Patients - What's Our Reality?

INTRODUCTION: Venous thromboembolism (VTE) includes pulmonary embolism (PE), deep vein thrombosis (DVT) in lower limbs, and thrombosis in rare locations. VTE is a common cardiovascular disease, being the leading preventable cause of in-hospital death. Both surgical and acute medical patients have an elevated risk of developing VTE. VTE risk assessment is essential to identify patients who might benefit from VTE prophylaxis accurately. Clinical data on risk factors and prophylaxis in Portugal are scarce. We aimed to determine the proportion of at-risk patients who received prophylaxis and the incidence of bleeding events. We also intended to study the rate of VTE in a cohort of medical and surgical patients during the hospitalization period and three months after discharge. METHODS: During one week in 2020, adults admitted for more than 72hr to a medical or surgical ward were included. The study excluded patients with a diagnosis of VTE three months before hospitalization and who were either chronically receiving anticoagulation therapy or had started it 48 hours after admission. Risk assessments were based on the Padua Prediction Score (PPS) for medical patients and the Caprini Risk Assessment Model (CRAM) for surgical patients. We used CHEST guidelines, 9th edition, to determine the adequacy of the prophylactic method. RESULTS: A total of 123 patients were analyzed, 18.7% of which tested positive for SARS-CoV-2. VTE risk in surgical patients was categorized as very low or low (16.6%), moderate (37.5%), and high (43.8%), according to the CRAM. Risk in medical patients was categorized as low (60.0%) or high (40.0%) according to the PPS. We estimated that VTE chemoprophylaxis was overused in about 30.0% of patients vs. 7.0% who were at risk and did not receive appropriate chemoprophylaxis. The rate of thromboembolic events was 4.1% (n=5), 2 of which happened after discharge. Two of these patients were under VTE prophylaxis during hospitalization. Major bleeding occurred in 2.4% of patients (n=3). DISCUSSION: A significant number of hospitalized patients are deemed to be at risk for VTE, making appropriate prophylaxis essential. The results emphasize the insufficient management of VTE prophylaxis.

Spontaneous Pneumomediastinum: A Rare Cause of Chest Pain.

Spontaneous pneumomediastinum is a rare medical condition characterized by the presence of free air in the mediastinum, not preceded by trauma, surgery, or another medical procedure. It predominantly affects young adult males and usually has a benign course, and in most cases, it is not possible to identify the precipitating factor. There are some conditions that predispose to its occurrence, namely those that lead to an increase in intrapleural pressure, such as coughing, vomiting, or vigorous exercise. We report a case of a 21-year-old male who presented with acute-onset shortness of breath after an episode of coughing and was found to have mediastinal and subcutaneous emphysema. Clinical, laboratory, and radiological studies did not demonstrate any predisposing factor, and the case was classified as spontaneous pneumomediastinum.

Review of Acute Leukemia as a New Cause of Dual Thrombosis (Pulmonary Vein Thrombosis and Pulmonary Embolism).

Pulmonary vein thrombosis is rare in surgical situations and medical pathologies. The coexistence of pulmonary thrombosis with pulmonary vein thrombosis has been called dual thrombosis and has been reported in a scarce number of post-surgical patients undergoing lung transplantation. We describe a patient with acute leukemia who attended an evaluation for dyspnea. CT angiography of the chest reveals the presence of bilateral pulmonary thromboembolism, as well as pulmonary vein thrombosis. The review of the medical literature in the English language does not report similar cases in medical pathology. It would be the first case of dual thrombosis secondary to acute leukemia.

Review of the Myelodysplastic Syndrome as a Cause of Group 5 Pulmonary Arterial Hypertension: An Orphan Disease in an Orphan Pulmonary Hypertension Group.

The coexistence of MDS and pulmonary hypertension (PH) is not a common finding and often goes unnoticed because symptoms such as dyspnea can be confused with the underlying pathology. The annual incidence of idiopathic pulmonary arterial hypertension (PAH) is only around 0.2 cases per 100,000 inhabitants, while MDS is 1 to 8 cases per 100,000 inhabitants. This review summarizes the clinical manifestations, functional respiratory tests, hemodynamic parameters using right heart catheterization, and imaging findings using echocardiography and tomography of pulmonary hypertension in myelodysplastic syndrome. We centered our discussion on the diagnosis of these patients within the hematologic disorders, especially in patients with the detriment of the functional class, as we were not used to looking for this diagnosis as a first choice. Several specialties dealing with patients with hematologic disorders (internists, hematologists, family physicians, geriatrics, oncologists) will find helpful the contents of this review.

Atrial fibrillation as a new prognosis factor in chronic patients after hospitalization: the CHRONIBERIA index.

A collaborative project in different areas of Spain and Portugal was designed to find out the variables that influence the mortality after discharge and develop a prognostic model adapted to the current healthcare needs of chronic patients in an internal medicine ward. Inclusion criteria were being admitted to an Internal Medicine department and at least one chronic disease. Patients' physical dependence was measured through Barthel index (BI). Pfeiffer test (PT) was used to establish cognitive status. We conducted logistic regression and Cox proportional hazard models to analyze the influence of those variables on one-year mortality. We also developed an external validation once decided the variables included in the index. We enrolled 1406 patients. Mean age was 79.5 (SD = 11.5) and females were 56.5%. After the follow-up period, 514 patients (36.6%) died. Five variables were identified as significantly associated with 1 year mortality: age, being male, lower BI punctuation, neoplasia and atrial fibrillation. A model with such variables was created to estimate one-year mortality risk, leading to the CHRONIBERIA. A ROC curve was made to determine the reliability of this index when applied to the global sample. An AUC of 0.72 (0.7-0.75) was obtained. The external validation of the index was successful and showed an AUC of 0.73 (0.67-0.79). Atrial fibrillation along with an advanced age, being male, low BI score, or an active neoplasia in chronic patients could be critical to identify high risk multiple chronic conditions patients. Together, these variables constitute the new CHRONIBERIA index.

Measuring adherence to inhaled control medication in patients with asthma: Comparison among an asthma app, patient self-report and physician assessment.

BACKGROUND: Previous studies have demonstrated the feasibility of using an asthma app to support medication management and adherence but failed to compare with other measures currently used in clinical practice. However, in a clinical setting, any additional adherence measurement must be evaluated in the context of both the patient and physician perspectives so that it can also help improve the process of shared decision making. Thus, we aimed to compare different measures of adherence to asthma control inhalers in clinical practice, namely through an app, patient self-report and physician assessment. METHODS: This study is a secondary analysis of three prospective multicentre observational studies with patients (≥13 years old) with persistent asthma recruited from 61 primary and secondary care centres in Portugal. Patients were invited to use the InspirerMundi app and register their inhaled medication. Adherence was measured by the app as the number of doses taken divided by the number of doses scheduled each day and two time points were considered for analysis: 1-week and 1-month. At baseline, patients and physicians independently assessed adherence to asthma control inhalers during the previous week using a Visual Analogue Scale (VAS 0-100). RESULTS: A total of 193 patients (72% female; median [P25-P75] age 28 [19-41] years old) were included in the analysis. Adherence measured by the app was lower (1 week: 31 [0-71]%; 1 month: 18 [0-48]%) than patient self-report (80 [60-95]) and physician assessment (82 [51-94]) (p < 0.001). A negligible non-significant correlation was found between the app and subjective measurements (ρ 0.118-0.156, p > 0.05). There was a moderate correlation between patient self-report and physician assessment (ρ = 0.596, p < 0.001). CONCLUSIONS: Adherence measured by the app was lower than that reported by the patient or the physician. This was expected as objective measurements are commonly lower than subjective evaluations, which tend to overestimate adherence. Nevertheless, the low adherence measured by the app may also be influenced by the use of the app itself and this needs to be considered in future studies.

Mortars for Conservation of Late 19th and Early 20th Century Buildings-Combination of Natural Cements with Air Lime.

With the availability of commercial Natural cements (NC) for the conservation purposes raises a fundamental question about the compatibility between historic and repair mortars. The properties of Natural cements are dependent on the geological location of the raw material extraction and also on the production parameters, both having an impact on the final properties of the mortars produced from each distinct. Therefore, the significance of preservation of 19th and 20th century heritage and selection of the proper binder compatible with the original materials necessitate the study of existing NCs, that nowadays are produced by several manufacturers. This work provides a complex study of the mortars prepared from three NCs available in the market: Groupe Prompt Vicat, France (NCPV); Cemento Collet Marfil (NCM) and Cemento Natural Tigre (NCT), both from Spain. Various mortar sets based on individual NC containing different binder/aggregate ratios and air lime additions were analyzed after 28, 60, and 90 days of curing with the focus on their mineralogical composition (XRD), morphology (SEM), mechanical (flexural and compressive strength), and physical properties such as water absorption by capillarity, water vapor permeability, and water vapor diffusion resistance. Mortars prepared from NCPV, NCM, and NCT show distinct physical-mechanical properties with varying binder/aggregate ratio and air lime addition. This study shows that the NC variability should be taken into consideration when selecting materials for the conservation and rehabilitation of historic renders and plasters. Based on the comparison with original NC mortars, several NC mortars developed in this study show adequate properties for conservation of the buildings from late 19th and early 20th century in terms of compressive strength (>12 MPa), water absorption by capillarity (<20 kg·m−2·h−0.5), water vapor permeability (<4 × 10−10 kg·s−1·m−1·Pa−1), and water vapor diffusion resistance (<28) values.

[Occupational Dermatoses in Healthcare Workers during the COVID-19 Pandemic: A Narrative Review]. / Dermatoses Ocupacionais em Profissionais de Saúde durante a Pandemia de COVID-19: Revisão Narrativa.

INTRODUCTION: On the 11th March 2020, the World Health Organization declared the novel coronavirus outbreak as a pandemic. Preventive measures such as the appropriate use of personal protective equipment and proper hand hygiene are essential in decreasing transmissibility. Healthcare workers have been required to wear personal protective equipment and to wash their hands even more frequently. These preventive measures are associated with an increased risk of skin conditions, namely, contact dermatitis and exacerbation of pre-existing skin lesions such as acne and seborrheic dermatitis. The aim of this article is to provide an overview of the main skin lesions, and highlighting the role in occupational dermatology and preventive measures. METHODS: A search was conducted on PubMed and some guidelines from organizations like World Health Organization and Portuguese General Directorate of Health were included. RESULTS: Most skin conditions seen in healthcare professionals throughout the pandemic were associated with hand hygiene and the use of personal protective equipment and comprised hand eczema, mask-induced contact dermatitis, personal protective equipment related irritant contact dermatitis, cheilitis and exacerbation of pre-existing conditions such as acne and seborrheic dermatitis. Prevention and early recognition of dermatitis associated with personal protective equipment and hand hygiene are essential to maximize work performance, healthcare workers' safety and the adherence to preventive measures. CONCLUSION: Protection of healthcare workers' health and safety is an essential measure in the fight against the pandemic. Occupational Health has a fundamental role, acting in terms of disease prevention and health promotion, for example, through educational sessions and orientation of skin conditions associated with work, thus maximizing workers' well-being.

Introdução: A 11 de março de 2020, a Organização Mundial da Saúde declarou a doença provocada pelo novo coronavírus uma pandemia. As medidas de prevenção, nomeadamente, o uso apropriado de equipamento de proteção individual e a higienização das mãos, são essenciais na diminuição da transmissibilidade. Os profissionais de saúde têm particular indicação para o seu uso e para o aumento da frequência e duração da lavagem e desinfeção das mãos. Estas medidas estão associadas a um maior risco de desenvolvimento de patologias cutâneas como a dermatite de contacto e a exacerbação de lesões cutâneas pré-existentes, tais como a acne e a dermatite seborreica. O objetivo deste artigo é apresentar uma descrição das principais lesões cutâneas, e salientar o papel da dermatologia ocupacional e medidas preventivas.Métodos: Foi realizada uma pesquisa na PubMed e incluídas orientações de organizações como a Direção-Geral da Saúde e a Organização Mundial da Saúde.Resultados: As principais patologias cutâneas observadas nos profissionais de saúde ao longo da pandemia associam-se à higienização das mãos e à utilização de equipamentos de proteção individual, consistindo, sucintamente, em eczema das mãos, dermatite de contacto induzida por máscaras, dermatite de contacto irritativa por traumatismo do equipamento de proteção individual, queilite e exacerbação de condições pré-existentes como acne e dermatite seborreica. A prevenção e o reconhecimento atempado das dermatites associadas ao uso de equipamento de proteção individual e ao reforço da higiene das mãos são fundamentais para otimizar o desempenho laboral e a segurança dos profissionais de saúde e maximizar a adesão às medidas preventivas.Conclusão: A proteção da saúde e da segurança dos profissionais de saúde constitui uma medida essencial no combate da pandemia, pelo que a Medicina do Trabalho assume um papel fundamental, atuando a nível da prevenção da doença e promoção da saúde, por exemplo, através da realização de sessões de educação e orientação das várias patologias cutâneas associadas ao trabalho, maximizando o bem-estar do trabalhador.

Widespread elevated concentrations of gaseous elemental mercury in Guanajuato, Mexico, centuries after historical silver refining by mercury amalgamation.

Silver (Ag) production in Hispanic America between the 16th and 19th centuries is thought to be one of the largest sources of anthropogenic mercury (Hg) emissions in history. Recent reviews of the chemistry behind the patio process, which used Hg amalgamation to extract Ag from ore, reveal that a large amount of the Hg may not have been immediately released to the atmosphere; instead, it may have been captured in the form of calomel (Hg2Cl2, in which Hg exists as monovalent HgI) and remained in the local environment. Here we show that Hg used in the patio process centuries ago in the Guanajuato Mining District of Mexico continues to elevate present-day concentrations of gaseous elemental mercury (GEM) throughout the region. In the ground-level air, GEM ranged from 8 to 454 ng m-3, exceeding the Northern Hemispheric average (~1.4 ng m-3) by up to two orders of magnitude. Much higher concentrations, up to 44,700 ng m-3, were found in the interstitial air of reprocessed mineral wastes, sediment, and soil. These highly elevated present-day GEM values are due, at least in part, to the disproportionation of legacy calomel, as supported by the presence of HgI in the reprocessed wastes and by the GEM release pattern from calomel disproportionation. Our results imply that the contribution of historical Ag refining to atmospheric Hg emissions must be re-evaluated to account for calomel and its subsequent disproportionation and releases of GEM to the present-day.

Interaction of SCO2127 with BldKB and its possible connection to carbon catabolite regulation of morphological differentiation in Streptomyces coelicolor.

In Streptomyces coelicolor, the sco2127 gene is located upstream of the gene encoding for glucose kinase. This region restores sensitivity to carbon catabolite repression (CCR) of Streptomyces peucetius var. caesius mutants, resistant to 2-deoxyglucose (Dog(R)). In order to search for the possible mechanisms behind this effect, sco2127 was overexpressed and purified for protein-protein interaction studies. SCO2127 was detected during the late growth phase of S. coelicolor grown in a complex media supplemented with 100 mM glucose. Pull-down assays using crude extracts from S. coelicolor grown in the same media, followed by far-western blotting, allowed detection of two proteins bound to SCO2127. The proteins were identified by MALDI-TOF mass spectrometry as SCO5113 and SCO2582. SCO5113 (BldKB) is a lipoprotein ABC-type permease (∼66 kDa) involved in mycelium differentiation by allowing the transport of the morphogenic oligopeptide Bld261. SCO2582, is a putative membrane metalloendopeptidase (∼44 kDa) of unknown function. In agreement with the possible role of SCO2127 in mycelium differentiation, delayed aerial mycelium septation and sporulation was observed when S. coelicolor A3(2) was grown in the presence of elevated glucose concentrations (100 mM), an effect not seen in a Δ-sco2127 mutant derived from it. We speculate that SCO2127 might represent a key factor in CCR of mycelium differentiation by interacting with BldKB.

Production of microbial secondary metabolites: regulation by the carbon source.

Microbial secondary metabolites are low molecular mass products, not essential for growth of the producing cultures, but very important for human health. They include antibiotics, antitumor agents, cholesterol-lowering drugs, and others. They have unusual structures and are usually formed during the late growth phase of the producing microorganisms. Its synthesis can be influenced greatly by manipulating the type and concentration of the nutrients formulating the culture media. Among these nutrients, the effect of the carbon sources has been the subject of continuous studies for both, industry and research groups. Different mechanisms have been described in bacteria and fungi to explain the negative carbon catabolite effects on secondary metabolite production. Their knowledge and manipulation have been useful either for setting fermentation conditions or for strain improvement. During the last years, important advances have been reported on these mechanisms at the biochemical and molecular levels. The aim of the present review is to describe these advances, giving special emphasis to those reported for the genus Streptomyces.

Benzo[d]thiazol-2(3H)-ones as new potent selective CB2 agonists with anti-inflammatory properties.

The high distribution of CB2 receptors in immune cells suggests their important role in the control of inflammation. Growing evidence offers this receptor as an attractive therapeutic target: selective CB2 agonists are able to modulate inflammation without triggering psychotropic effects. In this work, we report a new series of selective CB2 agonists based on a benzo[d]thiazol-2(3H)-one scaffold. This drug design project led to the discovery of compound 9, as a very potent CB2 agonist (Ki = 13.5 nM) with a good selectivity versus CB1. This compound showed no cytotoxicity, acceptable ADME-Tox parameters and demonstrates the ability to counteract colon inflammatory process in vivo.

Development of an efficient conjugal DNA transfer system between Escherichia coli and a non-sporulating Streptomyces strain.

Bacterial conjugation is a powerful tool used for DNA transfer from Escherichia coli into various bacteria including streptomycetes. In this methodology, spores are usually employed as recipient cells of the genetic information. However, some industrially important Streptomyces do not produce spores making difficult their genetic manipulation. In these strains, the use of mechanically fragmented mycelia allows DNA transfer with low efficiency. Streptomyces peucetius var. caesius is a non-sporulating bacteria which produces the antitumor compound doxorubicin. The use of aerial mycelia of this microorganism, failed to get intergeneric conjugation with E. coli. In the present work, by using young aerial mycelia of this microorganism and an excess of E. coli cells (~7×108cellsmL-1) in soybean-mannitol medium (MS) supplemented with 20mMMgCl2 resulted in a high number of exconjugant colonies (5×10-4) when compared to other reports from this genus (1.1×10-5 to 2.5×10-8). The effectiveness of these conditions was confirmed by isolating null mutants of two different glucokinases from S. peucetius var. caesius. The novelty in using young aerial mycelia as receptor cells, allowed an efficient conjugative process and opened the way for genetic manipulation of additional non-spore forming actinobacteria exhibiting natural resistance to be genetically manipulated.

S-allylcysteine scavenges singlet oxygen and hypochlorous acid and protects LLC-PK(1) cells of potassium dichromate-induced toxicity.

It has been found that S-allylcysteine (SAC), a garlic-derived compound, has in vivo and in vitro antioxidant properties. In addition, it is known that SAC is able to scavenge different reactive oxygen or nitrogen species including superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radical (OH()), and peroxynitrite anion (ONOO(-)) although the IC(5O) values for each reactive species has not been calculated and the potential ability of SAC to scavenge singlet oxygen ((1)O(2)) and hypochlorous acid (HOCl) has not been explored. The purposes of this work was (a) to explore the potential ability of SAC to scavenge (1)O(2) and HOCl, (b) to further characterize the O(2)(-), H(2)O(2), OH(), and ONOO(-) scavenging ability of SAC by measuring the IC(50) values using in vitro assays, and (c) to explore the potential ability of SAC to ameliorate the potassium dichromate (K(2)Cr(2)O(7))-induced cytotoxicity in LLC-PK1 cells in which oxidative stress is involved. The scavenging activity was compared against the following reference compounds: N-acetylcysteine for O(2)(-), sodium pyruvate for H(2)O(2), dimethylthiourea for OH(), lipoic acid and glutathione for (1)O(2), lipoic acid for HOCl, and penicillamine for ONOO(-). It was found that SAC was able to scavenge concentration-dependently all the species assayed with the following IC(5O) (mean+/-SEM, mM): O(2)(-) (14.49+/-1.67), H(2)O(2) (68+/-1.92), OH() (0.68+/-0.06), (1)O(2) (1.93+/-0.27), HOCl (2.86+/-0.15), and ONOO(-) (0.80+/-0.05). When the ability of SAC to scavenge these species was compared to those of the reference compounds it was found that the efficacy of SAC (a) to scavenge O(2)(-), H(2)O(2), OH(), and ONOO(-) was lower, (b) to scavenge HOCl was similar, and (c) to scavenge (1)O(2) was higher. In addition, it was found that SAC was able to prevent K(2)Cr(2)O(7)-induced toxicity in LLC-PK1 cells in culture. It was showed for the first time that SAC is able to scavenge (1)O(2) and HOCl and to ameliorate the K(2)Cr(2)O(7)-induced toxicity.