Constitutive expression of selected genes from the pentose phosphate and aromatic pathways increases the shikimic acid yield in high-glucose batch cultures of an Escherichia coli strain lacking PTS and pykF

BACKGROUND: During the last two decades many efforts have been directed towards obtaining efficient microbial processes for the production of shikimic acid (SA); however, feeding high amounts of substrate to increase the titer of this compound has invariably rendered low conversion yields, leaving room for improvement of the producing strains. In this work we report an alternative platform to overproduce SA in a laboratory-evolved Escherichia coli strain, based on plasmid-driven constitutive expression of six genes selected from the pentose phosphate and aromatic amino acid pathways, artificially arranged as an operon. Production strains also carried inactivated genes coding for phosphotransferase system components (ptsHIcrr), shikimate kinases I and II (aroK and aroL), pyruvate kinase I (pykF) and the lactose operon repressor (lacI). RESULTS: The strong and constitutive expression of the constructed operon permitted SA production from the beginning of the cultures, as evidenced in 1 L batch-mode fermentors starting with high concentrations of glucose and yeast extract. Inactivation of the pykF gene improved SA production under the evaluated conditions by increasing the titer, yield and productivity of this metabolite compared to the isogenic pykF(+) strain. The best producing strain accumulated up to 43 g/L of SA in 30 h and relatively low concentrations of acetate and aromatic byproducts were detected, with SA accounting for 80% of the produced aromatic compounds. These results were consistent with high expression levels of the glycolytic pathway and synthetic operon genes from the beginning of fermentations, as revealed by transcriptomic analysis. Despite the consumption of 100 g/L of glucose, the yields on glucose of SA and of total aromatic compounds were about 50% and 60% of the theoretical maximum, respectively. The obtained yields and specific production and consumption rates proved to be constant with three different substrate concentrations. CONCLUSIONS: The developed production system allowed continuous SA accumulation until glucose exhaustion and eliminated the requirement for culture inducers. The obtained SA titers and yields represent the highest reported values for a high-substrate batch process, postulating the strategy described in this report as an interesting alternative to the traditionally employed fed-batch processes for SA production

RNA-Seq quantification of the human small airway epithelium transcriptome

<p>Abstract</p> <p>Background</p> <p>The small airway epithelium (SAE), the cell population that covers the human airway surface from the 6^thgeneration of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq).</p> <p>Results</p> <p>The data demonstrate that 48% of SAE expressed genes are ubiquitous, shared with many tissues, with 52% enriched in this cell population. The most highly expressed gene, SCGB1A1, is characteristic of Clara cells, the cell type unique to the human SAE. Among other genes expressed by the SAE are those related to Clara cell differentiation, secretory mucosal defense, and mucociliary differentiation. The high sensitivity of RNA-Seq permitted quantification of gene expression related to infrequent cell populations such as neuroendocrine cells and epithelial stem/progenitor cells. Quantification of the absolute smoking-induced changes in SAE gene expression revealed that, compared to ubiquitous genes, more SAE-enriched genes responded to smoking with up-regulation, and those with the highest basal expression levels showed most dramatic changes. Smoking had no effect on SAE gene splicing, but was associated with a shift in molecular pattern from Clara cell-associated towards the mucus-secreting cell differentiation pathway with multiple features of cancer-associated molecular phenotype.</p> <p>Conclusions</p> <p>These observations provide insights into the unique biology of human SAE by providing quantit-ative assessment of the global transcriptome under physiological conditions and in response to the stress of chronic cigarette smoking.</p

Reconstructing native American migrations from whole-genome and whole-exome data.

There is great scientific and popular interest in understanding the genetic history of populations in the Americas. We wish to understand when different regions of the continent were inhabited, where settlers came from, and how current inhabitants relate genetically to earlier populations. Recent studies unraveled parts of the genetic history of the continent using genotyping arrays and uniparental markers. The 1000 Genomes Project provides a unique opportunity for improving our understanding of population genetic history by providing over a hundred sequenced low coverage genomes and exomes from Colombian (CLM), Mexican-American (MXL), and Puerto Rican (PUR) populations. Here, we explore the genomic contributions of African, European, and especially Native American ancestry to these populations. Estimated Native American ancestry is 48% in MXL, 25% in CLM, and 13% in PUR. Native American ancestry in PUR is most closely related to populations surrounding the Orinoco River basin, confirming the Southern American ancestry of the Taíno people of the Caribbean. We present new methods to estimate the allele frequencies in the Native American fraction of the populations, and model their distribution using a demographic model for three ancestral Native American populations. These ancestral populations likely split in close succession: the most likely scenario, based on a peopling of the Americas 16 thousand years ago (kya), supports that the MXL Ancestors split 12.2kya, with a subsequent split of the ancestors to CLM and PUR 11.7kya. The model also features effective populations of 62,000 in Mexico, 8,700 in Colombia, and 1,900 in Puerto Rico. Modeling Identity-by-descent (IBD) and ancestry tract length, we show that post-contact populations also differ markedly in their effective sizes and migration patterns, with Puerto Rico showing the smallest effective size and the earlier migration from Europe. Finally, we compare IBD and ancestry assignments to find evidence for relatedness among European founders to the three populations

Human Tyrosine Hydroxylase Natural Allelic Variation: Influence on Autonomic Function and Hypertension

The catecholamine biosynthetic pathway consists of several enzymatic steps in series, beginning with the amino acids phenylalanine and tyrosine, and eventuating in the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). Since the enzyme tyrosine hydroxylase (TH; tyrosine 3-mono-oxygenase; EC 1.14.16.2; chromosome 11p15.5) is generally considered to be rate-limiting in this pathway, probed as to whether common genetic variation at the TH gene occurred, and whether such variants contributed to inter-individual alterations in autonomic function, either biochemical or physiological. We began with sequencing a tetranucleotide (TCAT) repeat in the first intron, and found that the two most common versions, (TCAT)6 and (TCAT)10i, predicted heritable autonomic traits in twin pairs. We then conducted systematic polymorphism discovery across the ~8 kbp locus, and discovered numerous variants, principally non-coding. The proximal promoter block contained four common variants, and its haplotypes and SNPs (especially C-824T, rs10770141) predicted catecholamine secretion, environmental stress-induced BP increments, and hypertension. Finally, we found that two of the common promoter variants, C-824T (rs10770141) and A-581G (rs10770140), were functional in that they differentially affected transcriptional activity of the isolated promoter, disrupted recognition motifs for specific transcription factor binding, altered the promoter responses to the co-transfected (exogenous) factors, and bound the endogenous factors in the chromatin fraction of the nucleus. We concluded that common variation in the proximal TH promoter is functional, giving rise to changes in autonomic function and consequently cardiovascular risk

Reconstructing Native American Migrations from Whole-Genome and Whole-Exome Data

There is great scientific and popular interest in understanding the genetic history of populations in the Americas. We wish to understand when different regions of the continent were inhabited, where settlers came from, and how current inhabitants relate genetically to earlier populations. Recent studies unraveled parts of the genetic history of the continent using genotyping arrays and uniparental markers. The 1000 Genomes Project provides a unique opportunity for improving our understanding of population genetic history by providing over a hundred sequenced low coverage genomes and exomes from Colombian (CLM), Mexican-American (MXL), and Puerto Rican (PUR) populations. Here, we explore the genomic contributions of African, European, and especially Native American ancestry to these populations. Estimated Native American ancestry is 48% in MXL, 25% in CLM, and 13% in PUR. Native American ancestry in PUR is most closely related to populations surrounding the Orinoco River basin, confirming the Southern America ancestry of the Taíno people of the Caribbean. We present new methods to estimate the allele frequencies in the Native American fraction of the populations, and model their distribution using a demographic model for three ancestral Native American populations. These ancestral populations likely split in close succession: the most likely scenario, based on a peopling of the Americas 16 thousand years ago (kya), supports that the MXL Ancestors split 12.2kya, with a subsequent split of the ancestors to CLM and PUR 11.7kya. The model also features effective populations of 62,000 in Mexico, 8,700 in Colombia, and 1,900 in Puerto Rico. Modeling Identity-by-descent (IBD) and ancestry tract length, we show that post-contact populations also differ markedly in their effective sizes and migration patterns, with Puerto Rico showing the smallest effective size and the earlier migration from Europe. Finally, we compare IBD and ancestry assignments to find evidence for relatedness among European founders to the three populations.Facultad de Ciencias Naturales y MuseoInstituto Multidisciplinario de Biología Celula

Reconstructing Native American Migrations from Whole-Genome and Whole-Exome Data

There is great scientific and popular interest in understanding the genetic history of populations in the Americas. We wish to understand when different regions of the continent were inhabited, where settlers came from, and how current inhabitants relate genetically to earlier populations. Recent studies unraveled parts of the genetic history of the continent using genotyping arrays and uniparental markers. The 1000 Genomes Project provides a unique opportunity for improving our understanding of population genetic history by providing over a hundred sequenced low coverage genomes and exomes from Colombian (CLM), Mexican-American (MXL), and Puerto Rican (PUR) populations. Here, we explore the genomic contributions of African, European, and especially Native American ancestry to these populations. Estimated Native American ancestry is 48% in MXL, 25% in CLM, and 13% in PUR. Native American ancestry in PUR is most closely related to populations surrounding the Orinoco River basin, confirming the Southern America ancestry of the Taíno people of the Caribbean. We present new methods to estimate the allele frequencies in the Native American fraction of the populations, and model their distribution using a demographic model for three ancestral Native American populations. These ancestral populations likely split in close succession: the most likely scenario, based on a peopling of the Americas 16 thousand years ago (kya), supports that the MXL Ancestors split 12.2kya, with a subsequent split of the ancestors to CLM and PUR 11.7kya. The model also features effective populations of 62,000 in Mexico, 8,700 in Colombia, and 1,900 in Puerto Rico. Modeling Identity-by-descent (IBD) and ancestry tract length, we show that post-contact populations also differ markedly in their effective sizes and migration patterns, with Puerto Rico showing the smallest effective size and the earlier migration from Europe. Finally, we compare IBD and ancestry assignments to find evidence for relatedness among European founders to the three populations.Facultad de Ciencias Naturales y MuseoInstituto Multidisciplinario de Biología Celula

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Next generation flow for minimally-invasive blood characterization of MGUS and multiple myeloma at diagnosis based on circulating tumor plasma cells (CTPC)

Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p < 0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p < 0.0001) and a shorter survival in MM patients with active disease requiring treatment (p <= 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM