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Various fire emission estimates for southern Africa during 2019, derived with multiple burned area data sets with resolutions ranging from 500 to 20 m, are evaluated using satellite carbon monoxide (CO) observations. Southern African emissions derived from burned area generated by 20 m Sentinel‐2 satellite imagery are up to 120% higher than other estimates because small fires are better detected with a higher‐resolution satellite instrument. A comprehensive comparison between simulated and observed atmospheric CO indicates that the Sentinel‐2 burned area data significantly improves emission estimates, with up to 15% reduction in CO concentration biases in comparison to emissions based on coarser resolution burned area data. We also found that the temporal lag between emissions and atmospheric CO concentrations during the peak fire month was related to atmospheric transport. These findings emphasize the importance of utilizing higher‐resolution satellite instruments in accurately estimating emissions and understanding the impact of small fires on global climate. Plain Language Summary: We studied how much carbon monoxide (CO) was released into the air in southern Africa due to fires. To derive CO emissions we used different remotely sensed data of area burned, with varying levels of detail, from 500 to 20 m, and used these emissions in a model to mimic atmospheric transport and chemistry of CO. We found that emissions derived from the very detailed 20‐m satellite images from Sentinel‐2 produced a similar large amount of CO in the atmosphere over southern Africa as seen by CO satellite observations. This is because the smaller fires, which are harder to spot, were detected with the 20‐m burned area satellite observations. We also found that the air movement and inflow of polluted air from outside Africa impacted the peak of air pollution. Key Points: Sentinel‐2 burned area detects small fires, resulting in up to 120% higher fire emissions compared to conventional resolution estimatesImproved fuel load modeling and new emission factors yield more trustworthy fire emissions that can be evaluated with top‐down constraintsAtmospheric modeling and TROPOMI CO observations indicate a 15% bias reduction using Sentinel‐2 burned area compared with coarser fire data [ABSTRACT FROM AUTHOR]

Boreal Eurasia is a region where the interaction between droughts and the carbon cycle may have significant impacts on the global carbon cycle. Yet the region is extremely data sparse with respect to meteorology, soil moisture and carbon fluxes as compared to e.g. Europe. To better constrain our vegetation model SiBCASA, we increase data usage by assimilating two streams of satellite derived soil moisture. We study if the assimilation improved SiBCASA's soil moisture and its effect on the simulated carbon fluxes. By comparing to unique in situ soil moisture observations, we show that the passive microwave soil moisture product did not improve the soil moisture simulated by SiBCASA, but the active data seem promising in some aspects. The match between SiBCASA and ASCAT soil moisture is best in the summer months over low vegetation. Nevertheless, ASCAT failed to detect the major droughts occurring between 2007 and 2013. The performance of ASCAT soil moisture seems to be particularly sensitive to ponding, rather than to biomass. The effect on the simulated carbon fluxes is large, 5-10% on annual GPP and TER, and tens of percent on local NEE, and 2% on area-integrated NEE, which is the same order of magnitude as the inter-annual variations. Consequently, this study shows that assimilation of satellite derived soil moisture has potentially large impacts, while at the same time further research is needed to understand under which conditions the satellite derived soil moisture improves the simulated soil moisture. [ABSTRACT FROM AUTHOR]

We present an enhanced version of the SiBCASA terrestrial biosphere model that is extended with (a) biomass burning emissions from the SiBCASA carbon pools using remotely sensed burned area from the Global Fire Emissions Database (GFED), (b) an isotopic discrimination scheme that calculates 13C signatures of photosynthesis and autotrophic respiration, and (c) a separate set of 13C pools to carry isotope ratios into heterotrophic respiration. We quantify in this study the terrestrial exchange of CO2 and 13CO2 as a function of environmental changes in humidity and biomass burning. The implementation of biomass burning yields similar fluxes as CASA-GFED both in magnitude and spatial patterns. The implementation of isotope exchange gives a global mean discrimination value of 15.2‰, ranges between 4 and 20‰ depending on the photosynthetic pathway in the plant, and compares favorably (annually and seasonally) with other published values. Similarly, the isotopic disequilibrium is similar to other studies that include a small effect of biomass burning as it shortens the turnover of carbon. In comparison to measurements, a newly modified starch/sugar storage pool propagates the isotopic discrimination anomalies to respiration much better. In addition, the amplitude of the drought response by SiBCASA is lower than suggested by the measured isotope ratios. We show that a slight increase in the stomatal closure for large vapor pressure deficit would amplify the respired isotope ratio variability. Our study highlights the importance of isotope ratio observations of 13C to assess and improve biochemical models like SiBCASA, especially with regard to the allocation and turnover of carbon and the responses to drought. [ABSTRACT FROM AUTHOR]

Radiocarbon dioxide (14CO2, reported in Δ14CO2) can be used to determine the fossil fuel CO2 addition to the atmosphere, since fossil fuel CO2 no longer contains any 14C. After the release of CO2 at the source, atmospheric transport causes dilution of strong local signals into the background and detectable gradients of Δ14CO2 only remain in areas with high fossil fuel emissions. This fossil fuel signal can moreover be partially masked by the enriching effect that anthropogenic emissions of 14CO2 from the nuclear industry have on the atmospheric Δ14CO2 signature. In this paper, we investigate the regional gradients in 14CO2 over the European continent and quantify the effect of the emissions from nuclear industry. We simulate the emissions and transport of fossil fuel CO2 and nuclear 14CO2 for Western Europe using the Weather Research and Forecast model (WRF-Chem) for a period covering 6 summer months in 2008. We evaluate the expected CO2 gradients and the resulting Δ14CO2 in simulated integrated air samples over this period, as well as in simulated plant samples. We find that the average gradients of fossil fuel CO2 in the lower 1200m of the atmosphere are close to 15 ppm at a 12 km x 12 km horizontal resolution. The nuclear influence on Δ14CO2 signatures varies considerably over the domain and for large areas in France and the UK it can range from 20 to more than 500% of the influence of fossil fuel emissions. Our simulations suggest that the resulting gradients in Δ14CO2 are well captured in plant samples, but due to their time-varying uptake of CO2, their signature can be different with over 3‰ from the atmospheric samples in some regions. We conclude that the framework presented will be well-suited for the interpretation of actual air and plant 14CO2 samples. [ABSTRACT FROM AUTHOR]

Previous studies suggest that a large part of the variability in the atmospheric ratio of 13CO2/12CO2originates from carbon exchange with the terrestrial biosphere rather than with the oceans. Since this variability is used to quantitatively partition the total carbon sink, we here investigate the contribution of interannual variability (IAV) in biospheric exchange to the observed atmospheric 13C variations. We use the Simple Biosphere - Carnegie-Ames-Stanford Approach biogeochemical model, including a detailed isotopic fractionation scheme, separate 12C and 13C biogeochemical pools, and satellite-observed fire disturbances. This model of 12CO2 and 13CO2 thus also produces return fluxes of 13CO2from its differently aged pools, contributing to the so-called disequilibrium flux. Our simulated terrestrial 13C budget closely resembles previously published model results for plant discrimination and disequilibrium fluxes and similarly suggests that variations in C3 discrimination and year-to-year variations in C3and C4 productivity are the main drivers of their IAV. But the year-to-year variability in the isotopic disequilibrium flux is much lower (1 σ=±1.5 PgC ‰ yr−1) than required (±12.5 PgC ‰ yr−1) to match atmospheric observations, under the common assumption of low variability in net ocean CO2 fluxes. This contrasts with earlier published results. It is currently unclear how to increase IAV in these drivers suggesting that SiBCASA still misses processes that enhance variability in plant discrimination and relative C3/C4productivity. Alternatively, 13C budget terms other than terrestrial disequilibrium fluxes, including possibly the atmospheric growth rate, must have significantly different IAV in order to close the atmospheric 13C budget on a year-to-year basis. [ABSTRACT FROM AUTHOR]

Objective: Amlodipine, a long-acting dihydropyridine calcium channel blocker, is frequently prescribed to pediatric patients. To date, no suitable pediatric formulation has been available. In this study, an amlodipine oral solution was developed and tested for bioequivalence to tablets in healthy adult volunteers., Methods: This study was designed as an open-label, single-dose, twosequence, two-period, crossover trial to assess the bioequivalence of a newly developed amlodipine besylate oral solution 0.5 mg/mL compared to Norvasc® 5 mg tablets. 13 adult subjects (mean [standard deviation] age of 23.2 [3.6] years, weight 71.5 [7.7] kg) were included and blood samples were collected for 72 hours. Amlodipine plasma levels were determined using a validated UPLC-MS/MS assay. Non-compartmental pharmacokinetic parameters were compared between the formulations according to European Medicines Agency (EMA) bioequivalence guidelines., Results: The 90% confidence intervals of the test/reference ratios of the geometric means for the primary pharmacokinetic parameters AUC(0-72) (88.24 - 104.37%) and C(max) (99.00 - 121.40%) were within the acceptance range of 80.00 - 125.00% for bioequivalence. Mean (SD) AUC(0-72) was 102.7 (26.8) (26.8) μg × h/L for the solution and 108.2 (30.6) μg × h/L for the tablet. Mean (SD) Cmax of the solution was 3.11(1.06) μg/L with a median (IQR) t(max) of 4.0 (2.6 - 7.5) hours. Mean (SD) C(max) of the tablet was 2.91 (0.84) μg/L with a median (IQR) tmax of 6.0 (4.0 - 14.0) hours. Intrasubject coefficients of variation were 10.2% (AUC(0-72)) and 12.4% (C(max))., Conclusions: The formulations are bioequivalent according to EMA guidelines. This warrants further study of our novel amlodipine oral solution in pediatric patients.

Mesenchymal stromal cells (MSCs) are attractive for cellular therapy of muscular dystrophies as they are easy to procure, can be greatly expanded ex vivo, and contribute to skeletal muscle repair in vivo. However, detailed information about the contribution of bone marrow (BM)-derived human MSCs (BM-hMSCs) to skeletal muscle regeneration in vivo is very limited. Here, we present the results of a comprehensive study of the fate of LacZ-tagged BM-hMSCs following implantation in cardiotoxin (CTX)-injured tibialis anterior muscles (TAMs) of immunodeficient mice. β-Galactosidase-positive (β-gal(+)) human-mouse hybrid myofibers (HMs) were counted in serial cross sections over the full length of the treated TAMs of groups of mice at monthly intervals. The number of human cells was estimated using chemiluminescence assays. While the number of human cells declined gradually to about 10% of the injected cells at 60 days after transplantation, the number of HMs increased from day 10 onwards, reaching 104 ± 39.1 per TAM at 4 months postinjection. β-gal(+) cells and HMs were distributed over the entire muscle, indicating migration of the former from the central injection site to the ends of the TAMs. The identification of HMs that stained positive for human spectrin suggests myogenic reprogramming of hMSC nuclei. In summary, our findings reveal that BM-hMSCs continue to participate in the regeneration/remodeling of CTX-injured TAMs, resulting in ±5% HMs at 4 months after damage induction. Moreover, donor-derived cells were shown to express genetic information, both endogenous and transgenic, in recipient myofibers., (© 2011 Cognizant Comm. Corp.)

Background: Late onset disorders are often associated with cerebral disfunctioning and cognitive impairment in elderly patients. It is unknown whether the age of onset affects cognition in patients with bipolar disorder. The authors compare cognition and clinical characteristics of early- and late-onset bipolar patients in a stable and euthymic condition., Method: One hundred and nineteen older patients (age >60) with an early- (<40 years) or late-onset bipolar disorder and a group of 78 comparison subjects were extensively tested for cognitive functioning., Results: Bipolar subjects scored lower on most cognitive measures. The late-onset patients were more impaired in psychomotor performance and mental flexibility than the early-onset patients. These differences could not be explained by differences in exposure to cerebrovascular risk factors., Conclusions: Older patients with bipolar disorder have substantial cognitive impairments. Late onset bipolar disorder is associated with more severe cognitive impairment than early-onset bipolar disorder. For clinical practice, it is important to develop treatment strategies which take this into account.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), making it amenable to gene- or cell-based therapies. Another possible treatment entails the combination of both principles by transplantation of autologous myogenic cells after their genetic complementation. This approach requires efficient and stable transduction of these cells with recombinant DMD. Recently, we generated a dual high-capacity (hc) adenovirus (Ad)-adeno-associated virus (AAV) hybrid vector (HV) that can deliver two full-length dystrophin-encoding modules into target cells. We showed that HV transduction of human cells containing AAV Rep proteins leads to the insertion of foreign DNA into the AAVS1 locus. Here, we improved HV entry into muscle cells from DMD patients. After having verified that these cells barely express the coxsackie B virus and Ad receptor (CAR), which constitutes the attachment molecule for Ad serotype 5 (Ad5) fibers, we equipped dual hcAd/AAV HV particles with Ad serotype 50 fiber domains to achieve CAR-independent uptake. These retargeted vectors complemented much more efficiently the genetic defect of dystrophin-defective myoblasts and myotubes than their isogenic counterparts with conventional Ad5 fibers. Importantly, the accumulation of beta-dystroglycan along the membranes of vector-treated DMD myotubes indicated proper assembly of dystrophin-associated glycoprotein complexes.

Duchenne muscular dystrophy (DMD) is the most prevalent inheritable muscle disease. It is caused by mutations in the approximately 2.5-megabase dystrophin (Dys) encoding gene. Therapeutic attempts at DMD have relied on injection of allogeneic Dys-positive myoblasts. The immune rejection of these cells and their limited availability have prompted the search for alternative therapies and sources of myogenic cells. Stem cell-based gene therapy aims to restore tissue function by the transplantation of gene-corrected autologous cells. It depends on (i) the capacity of stem cells to participate in tissue regeneration and (ii) the efficient genetic correction of defective autologous stem cells. We explored the potential of bone marrow-derived human mesenchymal stem cells (hMSCs) genetically modified with the full-length Dys-coding sequence to engage in myogenesis. By tagging hMSCs with enhanced green fluorescent protein (EGFP) or the membrane dye PKH26, we demonstrated that they could participate in myotube formation when cultured together with differentiating human myoblasts. Experiments performed with EGFP-marked hMSCs and DsRed-labeled DMD myoblasts revealed that the EGFP-positive DMD myotubes were also DsRed-positive indicating that hMSCs participate in human myogenesis through cellular fusion. Finally, we showed that hMSCs transduced with a tropism-modified high-capacity hybrid viral vector encoding full-length Dys could complement the genetic defect of DMD myotubes.

Bone marrow-derived human mesenchymal stem cells (hMSCs) lack the Coxsackie-adenovirus (Ad) receptor and thus are poorly transduced by vectors based on human Ad serotype 5 (Ad5). We investigated whether this problem could be overcome by using tropism-modified Ad5 vectors carrying fiber shaft domains and knobs of different human species B Ads (Ad5FBs). To allow quantitative analyses, these vectors coded for the enhanced green fluorescent protein (eGFP). Transgene expression analysis showed superior transduction of hMSCs by all Ad5FBs tested as compared with conventional Ad5 vectors. This was evident both by the frequency of eGFP-positive cells and by the eGFP level per cell. Highly efficient transduction of hMSCs, with limited variability between cells from different donors, was achieved with vectors displaying fiber domains of Ad serotypes 50, 35, and 16. These findings could not be reconciled with the very low levels of CD46, a recently identified receptor for species B Ads, on hMSCs, suggesting that AdFBs probably use receptors other than CD46 to enter these cells. We further observed that high eGFP levels were maintained in replication-restricted hMSCs for more than 30 days. In dividing hMSCs, foreign DNA delivered by Ad5FBs was expressed in a large fraction of the cells for approximately 3 weeks without compromising their replication capacity. Importantly, the transduced hMSCs retained their capacity to differentiate into adipocytes and osteoblasts when exposed to the appropriate stimuli.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.

Human hematopoietic stem cells (HSCs) are poorly transduced by vectors based on adenovirus serotype 5 (Ad5). This is primarily due to the paucity of the coxsackievirus-Ad receptor on these cells. In an attempt to change the tropism of Ad5, we constructed a series of chimeric E1-deleted Ad5 vectors in which the shaft and knob of the capsid fibers were exchanged with those of other Ad serotypes. In all these vectors, the Ad E1 region was replaced by an expression cassette containing the cytomegalovirus immediate-early promoter and the gene for enhanced green fluorescent protein (GFP). Experiments performed in vitro showed an efficient transduction of umbilical cord blood (UCB) monocytes, granulocytes, and their precursors as well as the undifferentiated CD34(+) CD33(-) CD38(-) CD71(-) cells by Ad5 vectors carrying Ad subgroup B-specific fiber chimeras (Ad5FBs). In the latter subpopulation, which comprises less than 1% of the CD34(+) cells and is highly enriched with cells repopulating immunodeficient mice, more than 90% of the cells were GFP(+). Transduction by Ad5FBs of the less primitive fraction within UCB CD34(+) cells was significant lower. Actually, the transduction frequency and GFP level declined gradually with increased expression of the CD33, CD38, and CD71 antigens. Flow cytometric analysis of transduced UCB CD34(+) cells that were cultured for 5 days on an allogeneic human bone marrow stroma layer showed maintenance of the phenotypically defined HSCs at levels similar to those of control cultures. The latter finding indicates that neither the transduction procedure nor the high levels of GFP were toxic for these cells.

The presence of replication-competent adenoviruses (RCAs) in batches of replication-defective adenovirus (Ad) vectors is a major problem for the application of these vectors in gene therapy. RCAs are generated by recombination between sequences in the Ad vector and homologous Ad sequences in the helper cells, resulting in the acquisition by the vector of early region 1. To prevent the formation of RCAs, we have developed helper cell lines, which we named PER, and matched Ad vectors that do not have sequence overlap. PER cells contain the Ad serotype 5 (Ad5) E1A- and E1B-encoding sequences (Ad5 nucleotides 459-3510) under the control of the human phosphoglycerate kinase (PGK) promoter. We demonstrate that PER cells synthesize high levels of the Ad5 E1A and E1B proteins. The yields from PER cells of E1-deleted Ads are similar to those obtained from earlier helper cells, such as 911 and 293 cells. Propagation of matched Ad vectors, which lack Ad5 nucleotides 459-3510, in one of the PER clones, PER.C6, does not result in the generation of RCAs, in contrast to propagation in 293 cells. We conclude that the combination of PER.C6 cells and nonoverlapping E1-deleted adenoviral vectors eliminates the problem of RCA generation by homologous recombination, and allows cost-effective production of safe, clinical-grade batches of recombinant Ad vectors.

In the present study, we describe the cloning and sequence analysis of rat IL-3. Two different mRNA isoforms were isolated after transfection of COS cells with the cytokine genomic sequences. One of the isoforms has been predicted before by Cohen et al. (1986), and the other one is identical except that it encodes a protein with an insertion of three amino acids at position 56. As names for the two isoforms, we propose IL-3alpha for the predicted and IL-3beta for the novel molecule. IL-3beta mRNA was detected as the predominant isoform in rat lymphocytes in vivo. High levels of the cytokine were obtained after infection of human cells (A549) with a recombinant adenovirus harboring rIL-3beta cDNA (IG.Ad.CMV.IL-3beta). The biological properties of the IL-3beta protein were tested in a FDC-P1 proliferation assay and in a hematopoietic progenitor colony forming assay. To assess in-vivo bioactivity, lysed 293 cells containing IG.Ad.CMV.rIL-3beta virus were injected subcutaneously into F344 rats. Stimulation of hematopoiesis and leucocytosis were observed during the treatment. After subcutaneous injections of the lysed adeno-producer cells in mice, the only effect observed was a cellular infiltration at the site of injection, confirming the poor cross-reactivity between the two species. The biological properties in vitro and in vivo demonstrate that the cDNA sequences of IL-3beta presented here encode active rat IL-3 protein.