Explore the vast range of titles available.

Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies 1 , 2 , 3 , 4 . The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, we have previously introduced a technology—linear amplification–mediated (LAM) PCR—for detecting and sequencing unknown DNA flanking sequences down to the single cell level 5 ( Supplementary Note online). LAM-PCR analyses have enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies, and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies 4 , 6 , 7 , 8 . The reliability and robustness of this method results from the initial preamplification of the vector-genome junctions preceding nontarget DNA removal via magnetic selection. Subsequent steps are carried out on a semisolid streptavidin phase, including synthesis of double complementary strands, restriction digest, ligation of a linker cassette onto the genomic end of the fragment and exponential PCR(s) with vector- and linker cassette–specific primers. LAM-PCR can be adjusted to all unknown DNA sequences adjacent to a known DNA sequence. Here we describe the use of LAM-PCR analyses to identify 5′ long terminal repeat (LTR) retroviral vector adjacent genomic sequences (Fig. 1 and Box 1 ).

Nanocarrier-based drug delivery is a promising therapeutic approach that offers unique possibilities for the treatment of various diseases. However, inside the blood stream, nanocarriers’ properties may change significantly due to interactions with proteins, aggregation, decomposition or premature loss of cargo. Thus, a method for precise, in situ characterization of drug nanocarriers in blood is needed. Here we show how the fluorescence correlation spectroscopy that is a well-established method for measuring the size, loading efficiency and stability of drug nanocarriers in aqueous solutions can be used to directly characterize drug nanocarriers in flowing blood. As the blood is not transparent for visible light and densely crowded with cells, we label the nanocarriers or their cargo with near-infrared fluorescent dyes and fit the experimental autocorrelation functions with an analytical model accounting for the presence of blood cells. The developed methodology contributes towards quantitative understanding of the in vivo behavior of nanocarrier-based therapeutics. While nanocarrier-based drug delivery is a promising therapeutic approach, in situ characterization of drug nanocarriers in blood remains difficult. Here, the authors demonstrate how the fluorescence correlation spectroscopy can be used to directly characterize drug nanocarriers in flowing blood.

The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has reached over five million confirmed cases worldwide, and numbers are still growing at a fast rate. Despite the wide outbreak of the infection, a remarkable asymmetry is observed in the number of cases and in the distribution of the severity of the COVID-19 symptoms in patients with respect to the countries/regions. In the early stages of a new pathogen outbreak, it is critical to understand the dynamics of the infection transmission, in order to follow contagion over time and project the epidemiological situation in the near future. While it is possible to reason that observed variation in the number and severity of cases stems from the initial number of infected individuals, the difference in the testing policies and social aspects of community transmissions, the factors that could explain high discrepancy in areas with a similar level of healthcare still remain unknown. Here, we introduce a binary classifier based on an artificial neural network that can help in explaining those differences and that can be used to support the design of containment policies. We found that SARS-CoV-2 infection frequency positively correlates with particulate air pollutants, and specifically with particulate matter 2.5 (PM2.5), while ozone gas is oppositely related with the number of infected individuals. We propose that atmospheric air pollutants could thus serve as surrogate markers to complement the infection outbreak anticipation.

Landscape fragmentation is increasingly considered an important environmental indicator in the fields of sustainable land use and biodiversity. To set goals for future development and to plan appropriate measures, suitable empirical data on the degree of landscape fragmentation are needed to identify trends and compare different regions. However, there is still a significant lack of data on landscape fragmentation as an indicator, despite the substantial scientific literature on this topic, likely because of confusion over the definition of “fragmentation,” questions associated with scale and data issues, and lack of general agreement on a fragmentation measure. This study presents a state-wide quantitative analysis of landscape fragmentation in Baden-Württemberg, Germany, by means of the “effective mesh size” (m eff), which characterizes the anthropogenic penetration of landscapes from a geometric point of view and is based on the probability that two randomly chosen points in a landscape are connected, i.e., not separated by barriers such as roads, railroads, or urban areas. Baden-Württemberg is fragmented to a far greater extent than indicated by previous studies. Them effhas decreased by 40% since 1930. This development is strongly related to the growing number of inhabitants, the increased use of motorized vehicles, and the hierarchical regional planning system based on the central place theory. To illustrate the suitability of them effmethod for environmental monitoring, as a planning instrument and as an assessment instrument for impact assessment studies, we explored several variations of applying the method with regard to choice of fragmenting elements, consideration of noise bands, spatial differentiation (e.g., administrative districts vs. ecoregions), and way of dealing with patches at the boundaries of the reporting units. Depending on the objectives of the investigation (e.g., recreational quality vs. suitability for wildlife habitat), different variations may be most appropriate. The insights and quantitative results from Baden-Württemberg provide a yardstick for analyzing and assessing landscape fragmentation in other countries.

HIV-1 recurrently targets active genes and integrates in the proximity of the nuclear pore compartment in CD4 + T cells. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are proximal to super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results provide evidence of the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection. HIV-1 usually targets active genes and integrates near the nuclear pore compartment. Here the authors show that recurrently targeted genes are proximal to super-enhancer genomic elements, which cluster in specific spatial compartments of the T cell nucleus, suggesting a role for nuclear organisation in viral infection.

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.

An adeno-associated virus (AAV) vector encoding a variant of human lipoprotein lipase was recently approved in Europe as the first gene therapy for the treatment of LPL deficiency. Here Manfred Schmidt and his colleagues report their analysis of AAV integration sites after injection of the gene therapy construct in LPL-deficient patients and in mice. The clinical application of adeno-associated virus vectors (AAVs) is limited because of concerns about AAV integration–mediated tumorigenicity. We performed integration-site analysis after AAV1-LPL S447X intramuscular injection in five lipoprotein lipase–deficient subjects, revealing random nuclear integration and hotspots in mitochondria. We conclude that AAV integration is potentially safe and that vector breakage and integration may occur from each position of the vector genome. Future viral integration-site analyses should include the mitochondrial genome.

Crustaceans express genes for at least three classes of putative chemosensory proteins. These are: Ionotropic Receptors (IRs), derived from the heterotetrameric ionotropic glutamate receptors (iGluRs); Transient Receptor Potential (TRP) channels, a diverse set of sensor-channels that include several families of chemoreceptor channels; and Gustatory Receptor Like receptors (GRLs), ionotropic receptors that are homologues of Gustatory Receptors (GRs) of insects and are expressed sparingly in most crustaceans so far studied. IRs are typically numerically the most dominant of these receptor proteins in crustaceans and include two classes: co-receptor IRs, which are necessary for making a functional receptor-channel; and tuning IRs, whose specific combination in the IR subunits in the heterotetramer confers chemical specificity. Previous work showed that the transcriptomes from two major chemosensory organs-the lateral flagellum of the antennule (LF) and the tips of the legs (dactyls)-of the Caribbean spiny lobster Panulirus argus express four co-receptor IRs and over 100 tuning IRs. In this paper, we examined and compared the transcriptomes from the LF and dactyls of P. argus and three other decapod crustaceans-the clawed lobster Homarus americanus, red swamp crayfish Procambarus clarkii, and the blue crab Callinectes sapidus. Each species has at least ca. 100 to 250 IRs, 1 to 4 GRLs, and ca. 15 TRP channels including those shown to be involved in chemoreception in other species. The IRs show different degrees of phylogenetic conservation: some are arthropod-conserved, others are pancrustacean-conserved, others appear to be crustacean-conserved, and some appear to be species-specific. Many IRs appear to be more highly expressed in the LF than dactyl. Our results show that decapod crustaceans express an abundance of genes for chemoreceptor proteins of different types, phylogenetic conservation, and expression patterns. An understanding of their functional roles awaits determining their expression patterns in individual chemosensory neurons and the central projections of those neurons.

We consider matching, rewriting, critical pairs and the Knuth–Bendix confluence test on rewrite rules in a nominal setting extended by atom-variables. We utilize atom-variables instead of atoms to formulate and rewrite rules on constrained expressions, which is an improvement of expressiveness over previous approaches. Nominal unification and nominal matching are correspondingly extended. Rewriting is performed using nominal matching, and computing critical pairs is done using nominal unification. We determine the complexity of several problems in a quantified freshness logic. In particular we show that nominal matching is $$\\prod _2^p$$ -complete. We prove that the adapted Knuth–Bendix confluence test is applicable to a nominal rewrite system with atom-variables, and thus that there is a decidable test whether confluence of the ground instance of the abstract rewrite system holds. We apply the nominal Knuth–Bendix confluence criterion to the theory of monads and compute a convergent nominal rewrite system modulo alpha-equivalence.

The spiny lobster, Panulirus argus, has two classes of chemosensilla representing \"olfaction\" and \"distributed chemoreception,\" as is typical for decapod crustaceans. Olfactory sensilla are found exclusively on antennular lateral flagella and are innervated only by olfactory receptor neurons (ORNs) that project into olfactory lobes organized into glomeruli in the brain. Distributed chemoreceptor sensilla are found on all body surfaces including the antennular lateral flagella (LF) and walking leg dactyls (dactyls), and are innervated by both chemoreceptor neurons (CRNs) and mechanoreceptor neurons that project into somatotopically organized neuropils. Here, we examined expression of three classes of chemosensory genes in transcriptomes of the LF (with ORNs and CRNs), dactyls (with only CRNs), and brain of P. argus: Ionotropic Receptors (IRs), which are related to ionotropic glutamate receptors and found in all protostomes including crustaceans; Gustatory Receptors (GRs), which are ionotropic receptors that are abundantly expressed in insects but more restricted in crustaceans; and Transient Receptor Potential (TRP) channels, a diverse set of sensor-channels that include several chemosensors in diverse animals. We identified 108 IRs, one GR, and 18 homologues representing all seven subfamilies of TRP channels. The number of IRs expressed in the LF is far greater than in dactyls, possibly reflecting the contribution of receptor proteins associated with the ORNs beyond those associated with CRNs. We found co-receptor IRs (IR8a, IR25a, IR76b, IR93a) and conserved IRs (IR21a, IR40a) in addition to the numerous divergent IRs in the LF, dactyl, and brain. Immunocytochemistry showed that IR25a is expressed in ORNs, CRNs, and a specific type of cell located in the brain near the olfactory lobes. While the function of IRs, TRP channels, and the GR was not explored, our results suggest that P. argus has an abundance of diverse putative chemoreceptor proteins that it may use in chemoreception.