Gene regulatory networks during muscle development

Congenital myopathies are caused by heritable mutations in particular genes. Genes mutated in congenital muscular dystrophies often encode cytoskeletal proteins, which contribute to the shape and movement of cells. We would like to know how such molecular defects lead to the muscle weakness in patients. It is therefore important to understand the molecular and cellular mechanisms by which muscle cells form, grow, and assemble into functional muscles. The bicoid–related homeobox gene Pitx2 is expressed in the developing embryo, regulates organogenesis, and is associated with several human congenital diseases. Pitx2 specifies and maintains several cell populations as they form specific organs by acting as a node within individual distinct network kernels. We hypothesize that lack of functional Pitx2 results in major changes in the gene regulatory networks involved in skeletal myogenesis. Pitx2 is expressed in all muscles during development, and thus the Pitx2-null mouse is an informative model to study myogenesis. Absence of Pitx2 results in an open abdominal wall followed by absence of abdominal musculature. Gene expression microarrays of E10.5 wild type and mutant Pitx2 mouse body wall have shown that Pitx2 can act as an inhibitor of protein transport and cell apoptosis. Loss of Pitx2 results in the altered specification of the abdominal Hox network that is involved in patterning, of which the Hox genes are normally occupied, positively regulated, and stabilized by Pitx2 function in the abdomen. The loss of proper Hox patterning results in the loss of the somatopleure and thus loss of somite-derived musculature. In the somites, the initiation and maintenance of the myogenic program is independent of Pitx2 function, in lieu of Pitx2 occupancy on the myogenic regulatory factors (MRFs). Absence of functional Pitx2 in the limbs results in muscle malformation with defects in the higher order assembly, including adhesion molecules and cytoskeletal proteins. The chromatin state of normal and Pitx2 mutant limbs as a whole at E12.5 indicated no significant difference between genes involved in transcription, transcriptional regulation, or organ development. However, genes involved in myogenic networks are bivalently marked and thus likely being poised for activation or repression. Future work will focus on identifying differences in the chromatin state of myogenic cells as they progress to make a functional muscle

Parietal epithelial cell differentiation to a podocyte fate in the aged mouse kidney

Healthy aging is typified by a progressive and absolute loss of podocytes over the lifespan of animals and humans. To test the hypothesis that a subset of glomerular parietal epithelial cell (PEC) progenitors transition to a podocyte fate with aging, dual reporte

Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid

<p>Abstract</p> <p>Background</p> <p>Due to the global occurrence of multi-drug-resistant malarial parasites (<it>Plasmodium falciparum</it>), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (<it>Artemisia annua</it>). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from <it>A. annua</it>. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required.</p> <p>Results</p> <p>Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (<it>AMO </it>or <it>CYP71AV1</it>), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 μg mL^-1in shake-flask cultures and 1 g L^-1in bio-reactors with the use of <it>Leu2d </it>selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (<it>PDR</it>) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast.</p> <p>Conclusion</p> <p>The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.</p

High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli

BACKGROUND: Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step. METHODOLOGY/ PRINCIPAL FINDINGS: By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene. CONCLUSIONS/ SIGNIFICANCE: Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs

Genome-wide mapping of chromatin state of mouse forelimbs

BACKGROUND: Cell types are defined at the molecular level during embryogenesis by a process called pattern formation and created by the selective utilization of combinations of sequence-specific transcription factors. Developmental programs define the sets of genes that are available to each particular cell type, and real-time biochemical signaling interactions define the extent to which these sets are used at any given time and place. Gene expression is regulated through the integrated action of many cis-regulatory elements, including core promoters, enhancers, silencers, and insulators. The chromatin state in developing body parts provides a code to cellular populations that directs their cell fates. Chromatin profiling has been a method of choice for mapping regulatory sequences in cells that go through developmental transitions. RESULTS: We used antibodies against histone H3 lysine 4 trimethylations, a modification associated with promoters and open/active chromatin, histone H3 lysine 27 trimethylations associated with Polycomb-repressed regions, and ribonucleic acid polymerase II associated with transcriptional initiation to identify the chromatin state signature of the mouse forelimb during mid-gestation at embryonic day 12. The families of genes marked included those related to transcriptional regulation and embryogenesis. One-third of the marked genes were transcriptionally active, whereas only a small fraction were bivalent marked. Sequence-specific transcription factors that were activated were involved in cell specification, including bone and muscle formation. CONCLUSION: Our results demonstrate that embryonic limb cells do not exhibit the plasticity of the embryonic stem cells but rather are programmed for a finer tuning for cell lineage specification.Keywords: chromatin, sequence-specific transcription factors, mouse genome, forelimbKeywords: chromatin, sequence-specific transcription factors, mouse genome, forelim

Loss of Abdominal Muscle in Pitx2 Mutants Associated with Altered Axial Specification of Lateral Plate Mesoderm

Correction 17 Oct 2012: Eng D, Ma HY, Xu J, Shih HP, Gross MK, et al. (2012) Correction: Loss of Abdominal Muscle in Pitx2 Mutants Associated with Altered Axial Specification of Lateral Plate Mesoderm. PLOS ONE 7(10): 10.1371/annotation/c980d5a1-1899-4d30-af7b-573d4049cf4bSequence specific transcription factors (SSTFs) combinatorially define cell types during development by forming recursively linked network kernels. Pitx2 expression begins during gastrulation, together with Hox genes, and becomes localized to the abdominal lateral plate mesoderm (LPM) before the onset of myogenesis in somites. The somatopleure of Pitx2 null embryos begins to grow abnormally outward before muscle regulatory factors (MRFs) or Pitx2 begin expression in the dermomyotome/myotome. Abdominal somites become deformed and stunted as they elongate into the mutant body wall, but maintain normal MRF expression domains. Subsequent loss of abdominal muscles is therefore not due to defects in specification, determination, or commitment of the myogenic lineage. Microarray analysis was used to identify SSTF families whose expression levels change in E10.5 interlimb body wall biopsies. All Hox9-11 paralogs had lower RNA levels in mutants, whereas genes expressed selectively in the hypaxial dermomyotome/myotome and sclerotome had higher RNA levels in mutants. In situ hybridization analyses indicate that Hox gene expression was reduced in parts of the LPM and intermediate mesoderm of mutants. Chromatin occupancy studies conducted on E10.5 interlimb body wall biopsies showed that Pitx2 protein occupied chromatin sites containing conserved bicoid core motifs in the vicinity of Hox 9-11 and MRF genes. Taken together, the data indicate that Pitx2 protein in LPM cells acts, presumably in combination with other SSTFs, to repress gene expression, that are normally expressed in physically adjoining cell types. Pitx2 thereby prevents cells in the interlimb LPM from adopting the stable network kernels that define sclerotomal, dermomyotomal, or myotomal mesenchymal cell types. This mechanism may be viewed either as lineage restriction or specification

Molecular determinants of plaque size as an indicator of dengue virus attenuation

The development of live viral vaccines relies on empirically derived phenotypic criteria, especially small plaque sizes, to indicate attenuation. However, while some candidate vaccines successfully translated into licensed applications, others have failed safety trials, placing vaccine development on a hit-or-miss trajectory. We examined the determinants of small plaque phenotype in two dengue virus (DENV) vaccine candidates, DENV-3 PGMK30FRhL3, which produced acute febrile illness in vaccine recipients, and DENV-2 PDK53, which has a good clinical safety profile. The reasons behind the failure of PGMK30FRhL3 during phase 1 clinical trial, despite meeting the empirically derived criteria of attenuation, have never been systematically investigated. Using in vitro, in vivo and functional genomics approaches, we examined infections by the vaccine and wild-type DENVs, in order to ascertain the different determinants of plaque size. We show that PGMK30FRhL3 produces small plaques on BHK-21 cells due to its slow in vitro growth rate. In contrast, PDK53 replicates rapidly, but is unable to evade antiviral responses that constrain its spread hence also giving rise to small plaques. Therefore, at least two different molecular mechanisms govern the plaque phenotype; determining which mechanism operates to constrain plaque size may be more informative on the safety of live-attenuated vaccines

Support for UNRWA's survival

The United Nations Relief and Works Agency for Palestine Refugees in the Near East (UNRWA) provides life-saving humanitarian aid for 5·4 million Palestine refugees now entering their eighth decade of statelessness and conflict. About a third of Palestine refugees still live in 58 recognised camps. UNRWA operates 702 schools and 144 health centres, some of which are affected by the ongoing humanitarian disasters in Syria and the Gaza Strip. It has dramatically reduced the prevalence of infectious diseases, mortality, and illiteracy. Its social services include rebuilding infrastructure and homes that have been destroyed by conflict and providing cash assistance and micro-finance loans for Palestinians whose rights are curtailed and who are denied the right of return to their homeland

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

Characteristics and outcomes of over 300,000 patients with COVID-19 and history of cancer in the United States and Spain

Background: We described the demographics, cancer subtypes, comorbidities, and outcomes of patients with a history of cancer and coronavirus disease 2019 (COVID-19). Second, we compared patients hospitalized with COVID-19 to patients diagnosed with COVID-19 and patients hospitalized with influenza. Methods: We conducted a cohort study using eight routinely collected health care databases from Spain and the United States, standardized to the Observational Medical Outcome Partnership common data model. Three cohorts of patients with a history of cancer were included: (i) diagnosed with COVID-19, (ii) hospitalized with COVID-19, and (iii) hospitalized with influenza in 2017 to 2018. Patients were followed from index date to 30 days or death. We reported demographics, cancer subtypes, comorbidities, and 30-day outcomes. Results: We included 366,050 and 119,597 patients diagnosed and hospitalized with COVID-19, respectively. Prostate and breast cancers were the most frequent cancers (range: 5%–18% and 1%–14% in the diagnosed cohort, respectively). Hematologic malignancies were also frequent, with non-Hodgkin’s lymphoma being among the five most common cancer subtypes in the diagnosed cohort. Overall, patients were aged above 65 years and had multiple comorbidities. Occurrence of death ranged from 2% to 14% and from 6% to 26% in the diagnosed and hospitalized COVID-19 cohorts, respectively. Patients hospitalized with influenza (n ¼ 67,743) had a similar distribution of cancer subtypes, sex, age, and comorbidities but lower occurrence of adverse events. Conclusions: Patients with a history of cancer and COVID-19 had multiple comorbidities and a high occurrence of COVID-19-related events. Hematologic malignancies were frequent. Impact: This study provides epidemiologic characteristics that can inform clinical care and etiologic studies.</p