MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters.

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.

Expression in CHO cells of a bacterial biosynthetic pathway producing a small non-ribosomal peptide aldehyde prevents proteolysis of recombinant proteins.

A significant problem during recombinant protein production is proteolysis. One of the most common preventive strategies is the addition of protease inhibitors, which has drawbacks, such as their short half-life and high cost, and their limited prevention of extracellular proteolysis. Actinomycetes produce the most commonly used inhibitors, which are non-ribosomal small aldehydic peptides. Previously, an unprecedented biosynthetic route involving a condensation-minus non-ribosomal peptide synthetase (NRPSs) and a tRNA utilizing enzyme (tRUE) was shown to direct the synthesis of one of these inhibitor peptides, livipeptin. Here, we show that expression of the livipeptin biosynthetic pathway encoded by the lvp genes in CHO cells resulted in the production of this metabolite with cysteine protease inhibitory activity, implying that mammalian tRNAs were recruited by the lvp system. CHO cells transiently expressing the biosynthetic pathway produced livipeptin without affecting cell growth or viability. Expression of the lvp system in CHO cells producing two model proteins, secreted alkaline phosphatase (hSeAP) and a monoclonal antibody, resulted in higher specific productivity with reduced proteolysis. We show for the first time that the expression of a bacterial biosynthetic pathway is functional in CHO cells, resulting in the efficient, low-cost synthesis of a protease inhibitor without adverse effects on CHO cells. This expands the field of metabolic engineering of mammalian cells by expressing the overwhelming diversity of actinomycetes biosynthetic pathways and opens a new option for proteolysis inhibition in bioprocess engineering.

Discovery of Streptomyces species CS-62, a novel producer of the Acinetobacter baumannii selective antibiotic factumycin.

Narrow-spectrum antibiotics are of great interest given their ability to spare the microbiome and decrease widespread antibiotic resistance compared to broad-spectrum antibiotics. Herein, we screened an in-house library of Actinobacteria strains for selective activity against Acinetobacter baumannii and successfully identified Streptomyces sp. CS-62 as a producer of a natural product with this valuable activity. Analysis of the cultures via high-resolution mass spectrometry and tandem mass spectrometry, followed by comparison with molecules in the Natural Product Atlas and the Global Natural Products Social Molecular Networking platform, suggested a novel natural product. Genome mining analysis initially supported the production of a novel kirromycin derivative. Isolation and structure elucidation via mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses revealed that the active natural product was the known natural product factumycin, exposing omissions and errors in the consulted databases. While public databases are generally very useful for avoiding rediscovery of known molecules, rediscovery remains a problem due to public databases either being incomplete or having errors that result in failed dereplication. Overall, the work describes the ongoing problem of dereplication and the continued need for public database curation.

Streptomyces lividans 66 produces a protease inhibitor via a tRNA-utilizing enzyme interacting with a C-minus NRPS.

Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by non-ribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster (BGC) in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-utilizing enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here, we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize. SIGNIFICANCE: The discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, for example of their evolution, activity, and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data.

Phylogeny of Telmatobius marmoratus complex (Anura, Telmatobiidae) reveals high cryptic diversity in the Andean Altiplano.

Telmatobius is the most diverse group of anurans in the Andean Altiplano (highlands) Morphologically, these amphibians have a generally conserved morphology but in turn present large intraspecific variation, which has led to a complex taxonomy and systematics. T. marmoratus has the widest distribution of the genus and forms a complex composed of at least two Telmatobius species. Partial systematic studies based on molecular evidence reveal the existence of three lineages with a complex spatial distribution. However, these studies did not include the entire distribution of T. marmoratus. Our study aims to reassess the current systematic scenario including the complete distribution of the complex. For this, we used a multilocus approach based on mitochondrial (16S, Cytb) and nuclear (RAG1-1, BFIB) DNA sequences to build a phylogenetic hypothesis based on Bayesian inference, maximum likelihood and maximum parsimony. Subsequently, we performed single-locus (ABGD and PTP) and multilocus (STACEY) species delimitation analyses to verify the diversity of nominal species within the complex. The analyses suggest seven non-sibling lineages and 6-10 candidate species within the marmoratus complex. Only one of the two lineages restricted to the central northern plateau correspond to T. marmoratus sensu stricto. South-central marbled water frogs belong to completely new lineages closer to T. gigas and T. culeus, evidencing the polyphyletic condition of the marmoratus complex. The findings of several sympatric lineages in some localities reveal a complex history of ancient water connections in south-central Altiplano.

Morphine and Fentanyl Repeated Administration Induces Different Levels of NLRP3-Dependent Pyroptosis in the Dorsal Raphe Nucleus of Male Rats via Cell-Specific Activation of TLR4 and Opioid Receptors.

Morphine promotes neuroinflammation after NOD-like receptor protein 3 (NLRP3) oligomerization in glial cells, but the capacity of other opioids to induce neuroinflammation and its relationship to the development of analgesic tolerance is unknown. We studied the effects of morphine and fentanyl on NLRP3 inflammasome activation in glial and neuronal cells in the dorsal raphe nucleus (DRN), a region involved in pain regulation. Male Wistar rats received i.p. injections of morphine (10 mg/kg) or fentanyl (0.1 mg/kg) 3 × daily for 7 days and were tested for nociception. Two hours after the last (19th) administration, we analyzed NLRP3 oligomerization, caspase-1 activation and gasdermin D-N (GSDMD-N) expression in microglia (CD11b positive cells), astrocytes (GFAP-positive cells) and neurons (NeuN-positive cells). Tolerance developed to both opioids, but only fentanyl produced hyperalgesia. Morphine and fentanyl activated NLRP3 inflammasome in astrocytes and serotonergic (TPH-2-positive) neurons, but fentanyl effects were more pronounced. Both opioids increased GFAP and CD11b immunoreactivity, caspase-1 and GSDMD activation, indicating pyroptotic cell death. The opioid receptor antagonist (-)-naloxone, but not the TLR4 receptor antagonist (+)-naloxone, prevented microglia activation and NLRP3 oligomerization. Only (+)-naloxone prevented astrocytes' activation. The anti-inflammatory agent minocycline and the NLRP3 inhibitor MCC950 delayed tolerance to morphine and fentanyl antinociception and prevented fentanyl-induced hyperalgesia. MCC950 also prevented opioid-induced NLRP3 oligomerization. In conclusion, morphine and fentanyl differentially induce cell-specific activation of NLRP3 inflammasome and pyroptosis in the DRN through TLR4 receptors in astrocytes and through opioid receptors in neurons, indicating that neuroinflammation is involved in opioid-induced analgesia and fentanyl-induced hyperalgesia after repeated administrations.

Co-administration of morphine and levamisole increases death risk, produces neutropenia and modifies antinociception in mice.

Levamisole is a veterinary anthelmintic drug and a common adulterant of misused drugs. This study analyses the lethal, antinociceptive and haematological effects produced by acute or repeated levamisole administration by itself or combined with morphine. Independent groups of male Swiss Webster mice were i.p. injected with 100 mg/kg morphine, 31.6 mg/kg levamisole (lethal doses at 10%, LD10 ) or the same doses combined. Naloxone pretreatment (10 mg/kg, i.p.) prevented morphine-induced death, as did 2.5 mg/kg, i.p. mecamylamine with levamisole. Co-administration of levamisole and morphine (Lvm + Mor) increased lethality from 10% to 80%. This augmented effect was prevented by 30 mg/kg, i.p. naloxone and reduced with 10 mg/kg naloxone plus 2.5 mg/kg, i.p. mecamylamine. In independent groups of mice, 17.7 mg/kg, i.p. levamisole antagonized the acute morphine's antinociceptive effect evaluated in the tail-flick test. Repeated 17.7 mg/kg levamisole administration (2×/day/3 weeks) did not affect tolerance development to morphine (10 mg/kg, 3×/day/1 week). Blood samples obtained from mice repeatedly treated with levamisole showed leukopenia and neutropenia. Morphine also produced neutropenia, increased erythrocyte count and other related parameters (e.g. haemoglobin). Lvm + Mor had similar effects on leukocyte and neutrophil counts to those seen with levamisole only, but no erythrocyte-related alterations were evident. Blood chemistry analysis did not indicate liver damage but suggested some degree of electrolyte balance impairment. In conclusion, Lvm + Mor increased death risk, altered morphine-induced antinociceptive effects and produced haematologic abnormalities. The importance of studying combinations of drugs of abuse lies in the fact that drug users frequently combine drugs, which are commonly adulterated.

[Relationship between occupational commitment and teaching practices among healthcare teachers]. / ¿Enseña diferente un docente comprometido? Relación entre compromiso docente y prácticas pedagógicas en académicos de carreras de la salud.

BACKGROUND: A central aspect of professional identity is occupational commitment, which has a significant relationship with motivation and positive mental states in healthcare environments. However, it is not clear how occupational commitment is related to teaching practices in universities. AIM: To apply the Occupational Commitment Questionnaire (OCQ) to university teachers and relate its results with the Teaching Practices Questionnaire. MATERIAL AND METHODS: OCQ and Teaching Practices Questionnaire were answered by 319 university teachers from 13 Chilean regions, who were selected through a non-probabilistic volunteer sampling. Data collection was done after informed consent. An exploratory factor analysis was performed for OCQ. The internal consistency of each factor was calculated with Cronbach's Alpha. Spearman correlations were used to explore the relationship between the two questionnaires. RESULTS: Two factors were identified in OCQ, namely Teacher Commitment and Ethical-Professional Commitment. Cronbach's Alpha was 0.85 for both factors. A moderate direct relationship between teacher commitment, ethical-professional commitment, and the Teaching Practices Questionnaire was found. CONCLUSIONS: OCQ showed an adequate internal consistency, and it was directly related with teaching practices. Committed teachers had better teaching practices. This confirms the importance of teachers' values and perceptions on their professional performance.

Minocycline prevents neuronal hyperexcitability and neuroinflammation in medial prefrontal cortex, as well as memory impairment caused by repeated toluene inhalation in adolescent rats.

Toluene can be intentionally misused by adolescents to experience psychoactive effects. Toluene has a complex mechanism of action and broad behavioral effects, among which memory impairment is reported consistently. We have previously reported that repeated toluene inhalation (8000 ppm) increases layer 5 prelimbic pyramidal cells' excitability in the medial prefrontal cortex (mPFC) of adolescent rats. Toluene also produces reactive oxygen species (ROS), which activate glial cells. Here, we tested the hypothesis that the anti-inflammatory agent minocycline would decrease toluene's effects because it inhibits NF-κB (nuclear factor enhancer of the kappa light chains of activated B cells) and reduces pro-inflammatory cytokine and ROS production. Our results show that minocycline (50 mg/kg, ip, for 10 days) prevents the hyperexcitability of mPFC neurons observed after repeated 8000 ppm toluene exposure (30 min/day, 2×/day for 10 days). Minocycline prevents toluene-induced hyperexcitability by a mechanism that averts the loss of the slow calcium-dependent potassium current, and normalizes mPFC neurons' firing frequency. These effects are accompanied by significant decreased expression of astrocytes and activated microglia in the mPFC, reduced NLRP3 inflammasome activation and mRNA expression levels of the pro-inflammatory cytokine interleukin 1ß (IL-1ß), as well as increased mRNA expression of the anti-inflammatory cytokine transforming growth factor ß (TGF-ß). Minocycline also prevents toluene-induced memory impairment in adolescent rats in the passive avoidance task and the temporal order memory test in which the mPFC plays a central role. These results show that neuroinflammation produces several effects of repeated toluene administration at high concentrations, and minocycline can significantly prevent them.

Comparison between simulated SO2 concentrations using satellite emission data and Pemex emission inventories in Tabasco, Mexico.

This study presents a comparison of SO2 concentrations simulated by a dispersion model, using as input both emission data inventories of the Mexican petroleum company Petroleos Mexicanos and emissions estimated by the Ozone Monitoring Instrument on board of the Aura satellite, contrasting results with data measured by an on-ground sensor located in the city of Villahermosa, Tabasco, Mexico, during the month of February 2013. The emissions used came from both oil extraction facilities located in the Gulf of Mexico and oil installations located on land. The days selected to be modeled were those when the winds had a northern component, and there was an observable emission detected by the Aura satellite coming from the emission sources. Evaluation analysis of the results showed that satellite data overestimate SO2 concentration, while the inventories of Petroleos Mexicanos underestimate it. Good correlations (r, r2, and concordance index) were observed between simulated and measured concentrations when the winds had a high frequency of directions of northern component and not-so weak speeds. It is recommended that Mexican oil company calculates more precise and updated emission inventories, considering not only a higher sulfur content of the gas emitted to the atmosphere and its combustion process but also from other existing sources of SO2 emission in this region.

Questionnaire on conceptions about teaching: Factorial structure and reliability in academics of health careers in Chile. Questionnaire on conceptions about teaching.

OBJECTIVE: To evaluate the factorial structure of the questionnaire on conceptions about teaching in the domain of health careers in Chile. DESIGN: The cross-sectional analytical study was conducted from July 2016 to December 2016 at different cities in Chile, and comprised healthcare academics in Chile. The questionnaire on conceptions about teaching was used for data collection in addition to a socio-demographic proforma. Exploratory factorial analysis of the questionnaire was performed, and the reliability of the factors located was evaluated through Cronbach's alpha. Descriptive analysis of the data was subsequently performed. RESULTS: Of the 312 subjects, 196(63%) were women and 116(37%) were men. The overall mean age was 45.70}12.04 years (range: 24-73 years). A total of three factors were identified: student as an autonomous learner, professor as a content transmitter, and professor as a trained mediator. These presented reliabilities with Cronbach's alpha between 0.74 and 0.84 and had direct and statistically significant correlations among them (p<0.05). CONCLUSIONS: There was psychometric evidence supporting both validity and reliability of the questionnaire on conceptions about teaching.

Analysis of differentially upregulated proteins in ptsHIcrr- and rppH- mutants in Escherichia coli during an adaptive laboratory evolution experiment.

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS- derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH-) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH- mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS- and rppH- mutations in the deep of a proteomic approach to understanding the relevance of rppH- phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH-, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH- were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS- strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS- derivatives during the ALE experiment. This result suggests that the evolving PTS- population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH- mutation in the PTS- background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.

[Psychometric properties of a newly developed occupational identity questionnaire for university teachers]. / Identidad ocupacional en docentes de carreras de la salud y su relación con las prácticas pedagógicas.

BACKGROUND: Occupational identity is defined as the perception of occupational interests, abilities, goals, and values, and the structure of the meanings that link these self-perceptions to career role. We developed an occupational identity questionnaire suitable to be used with university teachers. AIM: To evaluate the psychometric properties of the Occupational Identity Questionnaire (OIQ) and relate its results with those of the Teaching Practices questionnaire. MATERIAL AND METHODS: The OIQ and Teaching Practices Questionnaire were answered by 311 university teachers along Chile, who were selected through a non-probabilistic volunteer sampling. An exploratory factor analysis was performed and the internal consistency of each factor was calculated with Cronbach's Alpha. Spearman correlations were used to explore the relationship between the two questionnaires. RESULTS: Two factors were identified in OIQ: Teaching Vocation and Sense of Competence. Cronbach's Alpha was 0.75 for teaching vocation and 0.82 for the sense of competence. There was a moderate direct relationship between teaching vocation, sense of competence and the results of the Teaching Practices Questionnaire. CONCLUSIONS: OIS has an adequate internal consistency, its factorial structure showed two different types of identities and it is directly related to the results of the Teaching Practices Questionnaire.

[Porphyria cutanea tarda as extrahepatic manifestation of chronic hepatitis C: a case report]. / Porfiria cutánea tarda como manifestación extrahepática de hepatitis Ccrónica: reporte de un caso.

The present case is a 56 year old male who present hyperpigmented and hypopigmented scars in both hands, associated with the presence of milia cysts. It was studied the metabolism of porphyrins and skin biopsy of the lesions which were compatible with porphyria cutanea tarda. In the initial laboratory, elevated transaminases values were found and subsequently identified chronic infection of hepatitis C virus. In order to treat viral infection and resolve the dermal commitment; considered extrahepatic manifestation of hepatitis C virus, treatment was started with pegylated interferon and ribavirin, with favorably development and rapid viral response, with undetectable viral load until now (24 weeks of treatment), decreased level of serum transaminases and improvement of skin lesions.

[Factors associated with delay to reperfusion therapy in patients with ST-Segment Elevation Myocardial Infarction in a hospital in the southeast of Mexico]. / Factores asociados con retraso en la terapia de reperfusión en infarto agudo de miocardio con elevación del segmento ST (IMCEST) en un hospital del sureste mexicano.

UNLABELLED: Introduction and subject: The aim of the study was to determine the factors involved in the delayed medical care of patients with ST-Segment Elevation Myocardial Infarction. METHODS: A prospective observational study was conducted in patients admitted to the coronary care unit at Dr. Juan Graham Casasús hospital with a diagnosis of ST-Segment Elevation Acute Myocardial Infarction. In all patients, clinical data, type and time of reperfusion treatment, and factors associated with delay were identified. RESULTS: Between November 2012 and January 2015 we included 213 patients with ST-Segment Elevation Myocardial Infarction. Age, diabetes, atypical chest angina and patient arrival period (night or weekend), were more frequent in patients presenting after 12 hours of onset of symptoms of myocardial infarction. Of these, hospital admission at night or weekend was the only independent predictor for delay to the emergency room. CONCLUSIONS: This study shows that in a referral hospital in southeast of Mexico, the delay attributable to the patient was the most common factor associated with care in patients with ST-Segment Elevation Myocardial Infarction. Patient arrival period was associated with delay to medical care.

Deletion of the 2-acyl-glycerophosphoethanolamine cycle improve glucose metabolism in Escherichia coli strains employed for overproduction of aromatic compounds.

BACKGROUND: As a metabolic engineering tool, an adaptive laboratory evolution (ALE) experiment was performed to increase the specific growth rate (µ) in an Escherichia coli strain lacking PTS, originally engineered to increase the availability of intracellular phosphoenolpyruvate and redirect to the aromatic biosynthesis pathway. As result, several evolved strains increased their growth fitness on glucose as the only carbon source. Two of these clones isolated at 120 and 200 h during the experiment, increased their µ by 338 and 373 %, respectively, compared to the predecessor PB11 strain. The genome sequence and analysis of the genetic changes of these two strains (PB12 and PB13) allowed for the identification of a novel strategy to enhance carbon utilization to overcome the absence of the major glucose transport system. RESULTS: Genome sequencing data of evolved strains revealed the deletion of chromosomal region of 10,328 pb and two punctual non-synonymous mutations in the dhaM and glpT genes, which occurred prior to their divergence during the early stages of the evolutionary process. Deleted genes related to increased fitness in the evolved strains are rppH, aas, lplT and galR. Furthermore, the loss of mutH, which was also lost during the deletion event, caused a 200-fold increase in the mutation rate. CONCLUSIONS: During the ALE experiment, both PB12 and PB13 strains lost the galR and rppH genes, allowing the utilization of an alternative glucose transport system and allowed enhanced mRNA half-life of many genes involved in the glycolytic pathway resulting in an increment in the µ of these derivatives. Finally, we demonstrated the deletion of the aas-lplT operon, which codes for the main components of the phosphatidylethanolamine turnover metabolism increased the further fitness and glucose uptake in these evolved strains by stimulating the phospholipid degradation pathway. This is an alternative mechanism to its regeneration from 2-acyl-glycerophosphoethanolamine, whose utilization improved carbon metabolism likely by the elimination of a futile cycle under certain metabolic conditions. The origin and widespread occurrence of a mutated population during the ALE indicates a strong stress condition present in strains lacking PTS and the plasticity of this bacterium that allows it to overcome hostile conditions.

Actinomycetota bioprospecting from ore-forming environments.

Natural products from Actinomycetota have served as inspiration for many clinically relevant therapeutics. Despite early triumphs in natural product discovery, the rate of unearthing new compounds has decreased, necessitating inventive approaches. One promising strategy is to explore environments where survival is challenging. These harsh environments are hypothesized to lead to bacteria developing chemical adaptations (e.g. natural products) to enable their survival. This investigation focuses on ore-forming environments, particularly fluoride mines, which typically have extreme pH, salinity and nutrient scarcity. Herein, we have utilized metagenomics, metabolomics and evolutionary genome mining to dissect the biodiversity and metabolism in these harsh environments. This work has unveiled the promising biosynthetic potential of these bacteria and has demonstrated their ability to produce bioactive secondary metabolites. This research constitutes a pioneering endeavour in bioprospection within fluoride mining regions, providing insights into uncharted microbial ecosystems and their previously unexplored natural products.

Atypical Seropositive Striated Muscle Antibody Myasthenia Gravis Presentation With Metastatic B1 Thymoma: A Rare Case.

The association between myasthenia gravis (MG) and thymomas is well-documented. Thymomas are rare epithelial cell tumors that arise from the thymus gland and occur in the mediastinum. Myasthenia gravis is a neuromuscular disorder that causes skeletal muscle weakness due to the presence of anti-acetylcholinesterase antibodies. Roughly 60% of thymomas are associated with MG, while only 10% of MG patients have thymomas. We present an atypical presentation of myasthenia gravis with an associated unusual metastatic thymoma. This case is of a young, previously healthy 26-year-old male with no previous medical history who presented with non-specific symptoms of fatigue, diarrhea, abdominal pain, back pain, blurry vision, and unintended weight loss. He underwent treatment with intravenous immunoglobulins (IVIG), had two surgical resections of the thymoma, and ultimately received radiotherapy. Based on our experience with this case, diagnosing myasthenia gravis by testing for specific muscle antibodies for patients with ptosis in the setting of non-specific complaints, including fatigue, vomiting, diarrhea, and abdominal or back pain, should be considered. Routine imaging should follow with a chest computed tomography to screen for thymomas if the specific anti-titin and anti-ryanodine receptor (anti-RyR) muscle antibodies are positive and myasthenia gravis is suspected. If a thymoma is confirmed, it is best to confirm; and mass characterizes with chest magnetic resonance imaging. A treatment approach of IVIG followed by surgical resection and possible debulking if the lesion is deemed metastatic could also be considered thereafter, especially in young patients with few comorbidities. Treatment with Pyridostigmine 30 mg twice daily for 25 days post-surgically and radiation for treatment of any remaining unresectable tumor should also be considered.

Genetic changes during a laboratory adaptive evolution process that allowed fast growth in glucose to an Escherichia coli strain lacking the major glucose transport system.

BACKGROUND: Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), which is the major bacterial component involved in glucose transport and its phosphorylation, accumulate high amounts of phosphoenolpyruvate that can be diverted to the synthesis of commercially relevant products. However, these strains grow slowly in glucose as sole carbon source due to its inefficient transport and metabolism. Strain PB12, with 400% increased growth rate, was isolated after a 120 hours adaptive laboratory evolution process for the selection of faster growing derivatives in glucose. Analysis of the genetic changes that occurred in the PB12 strain that lacks PTS will allow a better understanding of the basis of its growth adaptation and, therefore, in the design of improved metabolic engineering strategies for enhancing carbon diversion into the aromatic pathways. RESULTS: Whole genome analyses using two different sequencing methodologies: the Roche NimbleGen Inc. comparative genome sequencing technique, and high throughput sequencing with Illumina Inc. GAIIx, allowed the identification of the genetic changes that occurred in the PB12 strain. Both methods detected 23 non-synonymous and 22 synonymous point mutations. Several non-synonymous mutations mapped in regulatory genes (arcB, barA, rpoD, rna) and in other putative regulatory loci (yjjU, rssA and ypdA). In addition, a chromosomal deletion of 10,328 bp was detected that removed 12 genes, among them, the rppH, mutH and galR genes. Characterization of some of these mutated and deleted genes with their functions and possible functions, are presented. CONCLUSIONS: The deletion of the contiguous rppH, mutH and galR genes that occurred simultaneously, is apparently the main reason for the faster growth of the evolved PB12 strain. In support of this interpretation is the fact that inactivation of the rppH gene in the parental PB11 strain substantially increased its growth rate, very likely by increasing glycolytic mRNA genes stability. Furthermore, galR inactivation allowed glucose transport by GalP into the cell. The deletion of mutH in an already stressed strain that lacks PTS is apparently responsible for the very high mutation rate observed.

Evolutionary Genome Mining for the Discovery and Engineering of Natural Product Biosynthesis.

Genome mining has become an invaluable tool in natural products research to quickly identify and characterize the biosynthetic pathways that assemble secondary or specialized metabolites. Recently, evolutionary principles have been incorporated into genome mining strategies in an effort to better assess and prioritize novelty and understand their chemical diversification for engineering purposes. Here, we provide an introduction to the principles underlying evolutionary genome mining, including bioinformatic strategies and natural product biosynthetic databases. We introduce workflows for traditional genome mining, focusing on the popular pipeline antiSMASH, and methods to predict enzyme substrate specificity from genomic information. We then provide an in-depth discussion of evolutionary genome mining workflows, including EvoMining, CORASON, ARTS, and others, as adopted by our group for the discovery and prioritization of natural products biosynthetic gene clusters and their products.