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Abstract Introduction The distal radius fracture is considered the most common fracture in humans. For fractures classified as Arbeitsgemeinschaft für Osteosynthese (AO) 23-C1 + C2, there is no consensus on treatment in older patients due to inconsistent study results. The aim of this study was to compare conservative and surgical treatment in relation to wrist function and satisfaction in patients older than 65 years. Methods In this prospective randomized clinical trial, patients aged older than 65 years who suffered an isolated AO-classified C1 or C2 distal radius fracture were randomized to surgical treatment using palmar plate osteosynthesis or conservative treatment. Patient-rated wrist evaluation (PRWE) score and disabilities of arm, shoulder, and hand (DASH) was assessed 3, 6 and 12 months post-interventionally. Satisfaction, range of motion (ROM) and pain scores were evaluated at 6 weeks and 3, 6 and 12 months post-interventionally. Results A total of 80 patients with a mean age of 77.3 years (± 6.1 years) in the conservative group and 72.5 years (± 5.3 years) in the surgery group were included. Both the PRWE score, and the DASH score showed a statistically significant difference between the two groups after 3 months, 6 months and 12 months (p

Abstract Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 (LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient’s specific factors.

Background: Due to the high frequency of postoperative complications after body-contouring surgeries, the need for a unifying postoperative complication grading system that correlates with outcomes is of key importance. Here we therefore consider the application of the Clavien–Dindo classification to evaluate postoperative complications after body-contouring surgeries. Methods: A retrospective study on 602 patients who underwent body-contouring surgery between 2009 and 2015 at our institution was performed. The length of hospital stays, age, sex, follow-up visits, and postoperative complications were evaluated and classified using the Clavien–Dindo classification. Results: We raised a total of 672 body-contouring procedures on 602 patients (563 female, 39 male). According to the Clavien–Dindo System, the severity of postoperative complications following body-contouring procedures was significantly correlated with the duration of hospitalization (mean 5.8 ± 2.7 days) and the number of follow-up visits (mean 4.4 ± 4.7). Conclusions: The Clavien–Dindo classification offers a valid prediction for postoperative hospital stay and the number of follow-up visits after body-reshaping surgery. By becoming a validated and reliable grading system that correlates patients’ outcomes after body-contouring procedures, this classification has the potential to significantly improve patients’ healthcare and quality of life.

AbstractIntroduction Neoadjuvant chemotherapy and radiotherapy for the management of soft tissue sarcomas (STS) are still preferably delivered sequentially, with or without concurrent hyperthermia. Concurrent delivery of chemo-, radio- and thermotherapy may produce synergistic effects and reduce chemotherapy-free intervals. The few available studies suggest that concurrent chemoradiation (CRT) has a greater local effect. Data on the efficacy and toxicity of adding hyperthermia to CRT (CRTH) are sparse.Materials and methods A cohort of 101 patients with STS of the extremities and trunk who received CRT (n = 33) or CRTH (n = 68) before resection of macroscopic tumor (CRT: n = 19, CRTH: n = 49) or re-resection following a non-oncological resection, so called ‘whoops procedure’, (CRT: n = 14, CRTH: n = 19) were included in this retrospective study. CRT consisted of two cycles of doxorubicine (50 mg/m2 on d2) plus ifosfamide (1500 mg/m2 on d1-5, q28) plus radiation doses of up to 60 Gy. Hyperthermia was delivered in two sessions per week.Results All patients received the minimum dose of 50 Gy. Median doses of ifosfamide and doxorubicin were comparable between CRT (75%/95%) and CRTH (78%/97%). The median number of hyperthermia sessions was seven. There were no differences in acute toxicities. Major wound complications occurred in 15% (CRT) vs. 25% (CRTH) (p = 0.19). In patients with macroscopic disease, the addition of hyperthermia resulted in a tendency toward improved remission: regression ≥90% occurred in 21/48 (CRTH) vs. 4/18 (CRT) patients (p = 0.197). With a median postoperative follow-up of 72 months, 6-year local control and overall survival rates for CRTH vs. CRT alone were 85 vs. 78% (p = 0.938) and 79 vs. 71% (p = 0.215).Conclusions Both CRT and CRTH are well tolerated with an expected rate of wound complications. The results suggest that adding hyperthermia may improve tumor response.

Undiagnosed disorders or gene mutations can lead to life-threatening events even in cosmetic surgery. Herein, the authors present a case of a young and supposedly healthy 36-year-old woman who survived an episode of bilateral pulmonary embolism and cryptogenic stroke after cosmetic breast augmentation-mastopexy. Two days after cosmetic surgery, the patient presented at the emergency stroke unit after she collapsed at home, following frequent generalized seizures. After she was transferred to the intensive care unit, it was revealed that the patient had an undiagnosed patent foramen ovale and several thrombophilic gene mutations. During the stay, two episodes of active bleeding and beginning nipple-areola complex hypoperfusion led to bilateral implant removal. As a final result, the patient lost her breast implants and experienced persistent hypoesthesia of the entire left hemi body. However, this case might highlight deficits in current venous thromboembolism risk assessment and prophylaxis due to the missing considerations of undiagnosed disorders or gene mutations. Further, recommendations on dealing with implants or aesthetic outcome in crucial episodes should be introduced.

The mechanisms that shape the regulatory T cell repertoire in patients with cancer are not completely understood. Here, the authors observe that, in breast cancer patients, tumor-resident regulatory T cells do not show clonal relationship with their circulating counterpart, but share a common origin with intratumoral antigen-experienced conventional T cells.

Viral vector characterization and analysis are important components for the development of safe gene therapeutic products, elucidating the potential genotoxic and immunogenic effects of vectors and establishing their safety profiles. Here, we present VSeq-Toolkit, which offers varying analysis modes for viral gene therapy data. The first mode determines the undesirable known contaminants and their frequency in viral preparations or other sequencing data. The second mode is designed for the analysis of intra-vector fusion breakpoints and the third mode for unraveling the viral-host fusion events distribution. Analysis modes of our toolkit can be executed independently or together and allow the analysis of multiple viral vectors concurrently. It has been designed and evaluated for the analysis of short read high-throughput sequencing data, including whole-genome or targeted sequencing. VSeq-Toolkit is developed in Perl and Bash programming languages and is available at https://github.com/CompMeth/VSeq-Toolkit.

Introduction Within the hyperthermia community, consensus exists that clinical outcome of the treatment radiotherapy and/or chemotherapy plus hyperthermia (i.e. elevating tumor temperature to 40 − 44 °C) is related to the applied thermal dose; hence, treatment quality is crucial for the success of prospective multi-institution clinical trials. Currently, applicator quality assurance (QA) measurements are implemented independently at each institution using basic cylindrical phantoms. A multi-institution comparison of heating quality using magnetic resonance thermometry (MRT) and anatomical representative anthropomorphic phantoms provides a unique opportunity to obtain novel QA insights to facilitate multi-institution trial evaluation. Objective Perform a systematic QA procedure to compare the performance of MR-compatible hyperthermia systems in five institutions. Methods and materials Anthropomorphic phantoms, including pelvic and spinal bones, were produced. Clinically relevant power of 600 watts was applied for ∼12 min to allow for 8 sequential MR-scans. The 3D-heating distribution, steering capabilities, and presence of off-target heating were analyzed. Results The evaluated devices show comparable heating profiles for centric and eccentric targets. The differences observed in the 3D-heating profiles are the result of variations in the exact phantom positioning and applicator characteristics, whereby positioning of the phantom followed current ESHO-QA guidelines. Conclusion Anthropomorphic phantoms were used to perform QA-measurements of MR-guided hyperthermia systems operating in MR-scanners of different brands. Comparable heating profiles are shown for the five evaluated institutions. Subcentimeter differences in position substantially affected the results when evaluating the heating patterns. Integration of advanced phantoms and precise positioning in QA-guidelines should be evaluated to guarantee the best quality patient care.

Abstract Background Individual radiosensitivity is influencing the outcome of radiation therapy. A general ex vivo testing is very work-intensive. It is of interest to see if a significant prediction concerning the sensitivity can be made by in vivo irradiation during radiation treatment. Methods Blood samples of 274 patients with rectal cancer and 43 lung cancer patients receiving radiotherapy were examined after 2 Gy ex vivo and in vivo ionizing radiation. Chromosomes # 1, 2 and 4 were stained by the 3-color-fluorescence in situ hybridization. Chromosomal aberrations were analyzed as breaks per metaphase (B/M). The deposited energy per session was calculated for each patient. Results Weak correlation could be found between the chromosomal aberrations ex and in vivo. Though receiving significantly smaller deposited energy during radiation therapy (RT) the lung cancer cohort displayed B/M values similar to the rectal cancer cohort. Considering the individual deposit energy differences improved slightly the correlation. Conclusions As various factors influence the induction of chromosomal aberrations it seems not feasible to estimate individual radiosensitivity via in vivo irradiation. An ex vivo estimation of individual radiosensitivity should be preferred.

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.

This paper is a contribution to exploring and analyzing space-improvements in concurrent programming languages, in particular in the functional process-calculus CHF. Space-improvements are defined as a generalization of the corresponding notion in deterministic pure functional languages. The main part of the paper is the O(n*log n) algorithm SpOptN for offline space optimization of several parallel independent processes. Applications of this algorithm are: (i) affirmation of space improving transformations for particular classes of program transformations; (ii) support of an interpreter-based method for refuting space-improvements; and (iii) as a stand-alone offline-optimizer for space (or similar resources) of parallel processes.

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.

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.

Although breast implants have been in clinical use for almost 6 decades and have undergone considerable development during this time, implant rupture is still a dreaded long-term complication. Some obvious external factors, such as trauma, can lead to implant rupture, but many studies have reported a high rate of “spontaneous” implant rupture. Herein, we present two cases with the aim of raising awareness of a new possible cause of “spontaneous” implant rupture: mechanical irritation by bony protrusions.

In vivo reprogramming of somatic cells is hampered by the need for vectors to express the OKSM factors in selected organs. Here the authors report new AAV-based vectors capable of in vivo reprogramming at low doses.

We explore space improvements in LRP, a polymorphically typed call-by-need functional core language. A relaxed space measure is chosen for the maximal size usage during an evaluation. It abstracts from the details of the implementation via abstract machines, but it takes garbage collection into account and thus can be seen as a realistic approximation of space usage. The results are: a context lemma for space improving translations and for space equivalences; all but one reduction rule of the calculus are shown to be space improvements, and for the exceptional one we show bounds on the space increase. Several further program transformations are shown to be space improvements or space equivalences in particular the translation into machine expressions is a space equivalence. We also classify certain space-worsening transformations as space-leaks or as space-safe. These results are a step forward in making predictions about the change in runtime space behavior of optimizing transformations in call-by-need functional languages.

Abstract Although tumor‐initiating cell (TIC) self‐renewal has been postulated to be essential in progression and metastasis formation of human pancreatic adenocarcinoma (PDAC), clonal dynamics of TICs within PDAC tumors are yet unknown. Here, we show that long‐term progression of PDAC in serial xenotransplantation is driven by a succession of transiently active TICs producing tumor cells in temporally restricted bursts. Clonal tracking of individual, genetically marked TICs revealed that individual tumors are generated by distinct sets of TICs with very little overlap between subsequent xenograft generations. An unexpected functional and phenotypic plasticity of pancreatic TICs in vivo underlies the recruitment of inactive TIC clones in serial xenografts. The observed clonal succession of TIC activity in serial xenotransplantation is in stark contrast to the continuous activity of limited numbers of self‐renewing TICs within a fixed cellular hierarchy observed in other epithelial cancers and emphasizes the need to target TIC activation, rather than a fixed TIC population, in PDAC.

In T cells with transgenic high-avidity T cell receptors (TCRs), endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) from the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells. Additionally, we were able to verify targeted gene addition of a GFP gene by homology-directed repair at the TALEN target site, using a donor suitable for replacement of the reporter transgene with therapeutic TCR chains. Remarkably, analysis of TALEN and CRISPR/Cas9 specificity using integrase-defective lentiviral vector capture revealed only one off-target site for one of the gRNAs and three off-target sites for both of the TALENs, indicating a high level of specificity. Collectively, our work shows highly efficient and specific nucleases for T cell engineering.

Integration site profiling and clonality analysis of viral vector distribution in gene therapy is a key factor to monitor the fate of gene-corrected cells, assess the risk of malignant transformation, and establish vector biosafety. We developed the Genome Integration Site Analysis Pipeline (GENE-IS) for highly time-efficient and accurate detection of next-generation sequencing (NGS)-based viral vector integration sites (ISs) in gene therapy data. It is the first available tool with dual analysis mode that allows IS analysis both in data generated by PCR-based methods, such as linear amplification method PCR (LAM-PCR), and by rapidly evolving targeted sequencing (e.g., Agilent SureSelect) technologies. GENE-IS makes use of trimming strategies, customized reference genome, and soft-clipped information with sequential filtering steps to provide annotated IS with clonality information. It is a scalable, robust, precise, and reliable tool for large-scale pre-clinical and clinical data analysis that provides users complete flexibility and control over analysis with a broad range of configurable parameters. GENE-IS is available at https://github.com/G100DKFZ/gene-is. Keywords: gene therapy, bioinformatical analysis, next-generation sequencing, viral integration sites, LAM-PCR, targeted sequencing

We present an implementation of an interpreter LRPi for the call-by-need calculus LRP, based on a variant of Sestoft's abstract machine Mark 1, extended with an eager garbage collector. It is used as a tool for exact space usage analyses as a support for our investigations into space improvements of call-by-need calculi.

With next generation sequencing thousands of virus and viral vector integration genome targets are now under investigation to uncover specific integration preferences and to define clusters of integration, termed common integration sites (CIS), that may allow to assess gene therapy safety or to detect disease related genomic features such as oncogenes. Here, we addressed the challenge to: 1) define the notion of CIS on graph models, 2) demonstrate that the structure of CIS enters in the category of scale-free networks and 3) show that our network approach analyzes CIS dynamically in an integrated systems biology framework using the Retroviral Transposon Tagged Cancer Gene Database (RTCGD) as a testing dataset.

With next-generation sequencing, the genomic data available for the characterization of integration sites (IS) has dramatically increased. At present, in a single experiment, several thousand viral integration genome targets can be investigated to define genomic hot spots. In a previous article, we renovated a formal CIS analysis based on a rigid fixed window demarcation into a more stretchy definition grounded on graphs. Here, we present a selection of supporting data related to the graph-based framework (GBF) from our previous article, in which a collection of common integration sites (CIS) was identified on six published datasets. In this work, we will focus on two datasets, ISRTCGD and ISHIV, which have been previously discussed. Moreover, we show in more detail the workflow design that originates the datasets.

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.

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.

The inverted terminal repeats (ITRs) of adeno-associated virus (AAV) are notoriously difficult to sequence owing to their high GC-content (70%) and palindromic sequences that result in the formation of a very stable, 125 bp long, T-shaped hairpin structure. Here we evaluate the performance of two widely used next-generation sequencing platforms, 454 GS FLX (Roche) and MiSeq Benchtop Sequencer (Illumina), in analyzing ITRs in comparatively sequencing linear amplification-meditated PCR (LAM-PCR) amplicons derived from AAV-concatemeric structures. While our data indicate that both platforms can sequence complete ITRs, efficiencies (MiSeq: 0.11% of sequence reads; 454: 0.02% of reads), frequencies (MiSeq: 171 full ITRs, 454: 3 full ITRs), and rates of deviation from the derived ITR consensus sequence (MiSeq: 0.8%–1.3%; 454: 0.5%) did differ. These results suggest that next-generation sequencing platforms can be used to specifically detect ITR mutations and sequence complete ITRs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

This paper is an extended abstract of an analysis of term rewriting where the terms in the rewrite rules as well as the term to be rewritten are compressed by a singleton tree grammar (STG). This form of compression is more general than node sharing or representing terms as dags since also partial trees (contexts) can be shared in the compression. In the first part efficient but complex algorithms for detecting applicability of a rewrite rule under STG-compression are constructed and analyzed. The second part applies these results to term rewriting sequences. The main result for submatching is that finding a redex of a left-linear rule can be performed in polynomial time under STG-compression. The main implications for rewriting and (single-position or parallel) rewriting steps are: (i) under STG-compression, n rewriting steps can be performed in nondeterministic polynomial time. (ii) under STG-compression and for left-linear rewrite rules a sequence of n rewriting steps can be performed in polynomial time, and (iii) for compressed rewrite rules where the left hand sides are either DAG-compressed or ground and STG-compressed, and an STG-compressed target term, n rewriting steps can be performed in polynomial time.

The curative potential of retroviral vectors for somatic gene therapy has been demonstrated impressively in several clinical trials leading to sustained long-term correction of the underlying genetic defect. Preclinical studies and clinical monitoring of gene modified hematopoietic stem and progenitor cells in patients have shown that biologically relevant vector induced side effects, ranging from in vitro immortalization to clonal dominance and oncogenesis in vivo, accompany therapeutic efficiency of integrating retroviral gene transfer systems. Most importantly, it has been demonstrated that the genotoxic potential is not identical among all retroviral vector systems designed for clinical application. Large scale viral integration site determination has uncovered significant differences in the target site selection of retrovirus subfamilies influencing the propensity for inducing genetic alterations in the host genome. In this review we will summarize recent insights gained on the mechanisms of insertional mutagenesis based on intrinsic target site selection of different retrovirus families. We will also discuss examples of side effects occurring in ongoing human gene therapy trials and future prospectives in the field.

Correctness of program transformations in extended lambda calculi with a contextual semantics is usually based on reasoning about the operational semantics which is a rewrite semantics. A successful approach to proving correctness is the combination of a context lemma with the computation of overlaps between program transformations and the reduction rules, and then of so-called complete sets of diagrams. The method is similar to the computation of critical pairs for the completion of term rewriting systems. We explore cases where the computation of these overlaps can be done in a first order way by variants of critical pair computation that use unification algorithms. As a case study we apply the method to a lambda calculus with recursive let-expressions and describe an effective unification algorithm to determine all overlaps of a set of transformations with all reduction rules. The unification algorithm employs many-sorted terms, the equational theory of left-commutativity modelling multi-sets, context variables of different kinds and a mechanism for compactly representing binding chains in recursive let-expressions.

This paper shows equivalence of several versions of applicative similarity and contextual approximation, and hence also of applicative bisimilarity and contextual equivalence, in LR, the deterministic call-by-need lambda calculus with letrec extended by data constructors, case-expressions and Haskell's seq-operator. LR models an untyped version of the core language of Haskell. The use of bisimilarities simplifies equivalence proofs in calculi and opens a way for more convenient correctness proofs for program transformations. The proof is by a fully abstract and surjective transfer into a call-by-name calculus, which is an extension of Abramsky's lazy lambda calculus. In the latter calculus equivalence of our similarities and contextual approximation can be shown by Howe's method. Similarity is transferred back to LR on the basis of an inductively defined similarity. The translation from the call-by-need letrec calculus into the extended call-by-name lambda calculus is the composition of two translations. The first translation replaces the call-by-need strategy by a call-by-name strategy and its correctness is shown by exploiting infinite trees which emerge by unfolding the letrec expressions. The second translation encodes letrec-expressions by using multi-fixpoint combinators and its correctness is shown syntactically by comparing reductions of both calculi. A further result of this paper is an isomorphism between the mentioned calculi, which is also an identity on letrec-free expressions.

Human cytomegalovirus (HCMV) harmfully impacts survival after peripheral blood hematopoietic stem cell transplantation (PB-HSCT). Delayed immune reconstitution after cord blood (CB)-HSCT leads to even higher HCMV-related morbidity and mortality. Towards a feasible dendritic cell therapy to accelerate de novo immunity against HCMV, we validated a tricistronic integrase-defective lentiviral vector (coexpressing GM-CSF, IFN-α, and HCMV pp65 antigen) capable to directly induce self-differentiation of PB and CB monocytes into dendritic cells processing pp65 (âSmyleDCpp65â). In vitro, SmyleDCpp65 resisted HCMV infection, activated CD4+ and CD8+ T cells and expanded functional pp65-specific memory cytotoxic T lymphocytes (CTLs). CD34+ cells obtained from PB and CB were transplanted into irradiated NOD.Rag1â/â.IL2γcâ/â mice. Donor-derived SmyleDCpp65 administration after PB-HSCT stimulated peripheral immune effects: lymph node remodeling, expansion of polyclonal effector memory CD8+ T cells in blood, spleen and bone marrow, and pp65-reactive CTL and IgG responses. SmyleDCpp65 administration after CB-HSCT significantly stimulated thymopoiesis. Expanded frequencies of CD4+/CD8+ T cell precursors containing increased levels of T-cell receptor excision circles in thymus correlated with peripheral expansion of effector memory CTL responses against pp65. The comparative in vivo modeling for PB and CB-HSCT provided dynamic and spatial information regarding human T and B cell reconstitution. In vivo potency supports future clinical development of SmyleDCpp65.

Gene transfer to hematopoietic stem cells with integrating vectors not only allows sustained correction of monogenic diseases but also tracking of individual clones in vivo. Quantitative real-time PCR (qPCR) has been shown to be an accurate method to quantify individual stem cell clones, yet due to frequently limited amounts of target material (especially in clinical studies), it is not useful for large-scale analyses. To explore whether vector integration site (IS) recovery techniques may be suitable to describe clonal contributions if combined with next-generation sequencing techniques, we designed artificial ISs of different sizes which were mixed to simulate defined clonal situations in clinical settings. We subjected all mixes to either linear amplificationâmediated PCR (LAM-PCR) or nonrestrictive LAM-PCR (nrLAM-PCR), both combined with 454 sequencing. We showed that nrLAM-PCR/454-detected clonality allows estimating qPCR-detected clonality in vitro. We then followed the kinetics of two clones detected in a patient enrolled in a clinical gene therapy trial using both, nrLAM-PCR/454 and qPCR and also saw nrLAM-PCR/454 to correlate to qPCR-measured clonal contributions. The method presented here displays a feasible high-throughput strategy to monitor clonality in clinical gene therapy trials is at hand.

HIV-1 derived vectors are among the most efficient for gene transduction in mammalian tissues. As the parent virus, they carry out vector genome insertion into the host cell chromatin. Consequently, their preferential integration in transcribed genes raises several conceptual and safety issues. To address part of these questions, HIV-derived vectors have been engineered to be nonintegrating. This was mainly achieved by mutating HIV-1 integrase at functional hotspots of the enzyme enabling the development of streamlined nuclear DNA circles functional for transgene expression. Few integrase mutant vectors have been successfully tested so far for gene transfer. They are cleared with time in mitotic cells, but stable within nondividing retina cells or neurons. Here, we compared six HIV vectors carrying different integrases, either wild type or with different mutations (D64V, D167H, Q168A, K186Q+Q214L+Q216L, and RRK262-264AAH) shown to modify integrase enzymatic activity, oligomerization, or interaction with key cellular cofactor of HIV DNA integration as LEDGF/p75 or TNPO3. We show that these mutations differently affect the transduction efficiency as well as rates and patterns of integration of HIV-derived vectors suggesting their different processing in the nucleus. Surprisingly and most interestingly, we report that an integrase carrying the D167H substitution improves vector transduction efficiency and integration in both HEK-293T and primary CD34+ cells.

Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.

Dendritic cells (DCs) constitute an attractive target for specific delivery of nanovaccines for immunotherapeutic applications. Here we tested nano-sized dextran (DEX) particles to serve as a DC-addressing nanocarrier platform. Non-functionalized DEX particles had no immunomodulatory effect on bone marrow (BM)-derived murine DCs in vitro. However, when adsorbed with ovalbumine (OVA), DEX particles were efficiently engulfed by BM-DCs in a mannose receptor-dependent manner. A DEX-based nanovaccine containing OVA and lipopolysaccharide (LPS) as a DC stimulus induced strong OVA peptide-specific CD4(+) and CD8(+) T cell proliferation both in vitro and upon systemic application in mice, as well as a robust OVA-specific humoral immune response (IgG1>IgG2a) in vivo. Accordingly, this nanovaccine also raised both a more pronounced delayed-type hypersensitivity response and a stronger induction of cytotoxic CD8(+) T cells than obtained upon administration of OVA and LPS in soluble form. Therefore, DEX-based nanoparticles constitute a potent, versatile and easy to prepare nanovaccine platform for immunotherapeutic approaches.

Vectors based on γ-retroviruses or lentiviruses have been shown to stably express therapeutical transgenes and effectively cure different hematological diseases. Molecular follow up of the insertional repertoire of gene corrected cells in patients and preclinical animal models revealed different integration preferences in the host genome including clusters of integrations in small genomic areas (CIS; common integrations sites). In the majority, these CIS were found in or near genes, with the potential to influence the clonal fate of the affected cell. To determine whether the observed degree of clustering is statistically compatible with an assumed standard model of spatial distribution of integrants, we have developed various methods and computer programs for γ-retroviral and lentiviral integration site distribution. In particular, we have devised and implemented mathematical and statistical approaches for comparing two experimental samples with different numbers of integration sites with respect to the propensity to form CIS as well as for the analysis of coincidences of integration sites obtained from different blood compartments. The programs and statistical tools described here are available as workspaces in R code and allow the fast detection of excessive clustering of integration sites from any retrovirally transduced sample and thus contribute to the assessment of potential treatment-related risks in preclinical and clinical retroviral gene therapy studies.

The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

Compositionally and structurally abrupt Si/SiO2 interfaces were prepared under ultrahigh vacuum conditions by RF plasma oxidation of Si(111) substrates with thermalized neutral oxygen atoms. The chemical, structural and electronic properties of the interface were analyzed and discussed with respect to a possible application in photovoltaic Si/SiO2 quantum well structures. The benefits of using neutral atomic oxygen were explored and turned out to be manifold: ultrathin SiO2 layers (thickness: 1–2 nm) can be precisely grown, the formation of suboxides is mostly suppressed and abrupt Si/SiO2 interfaces are obtained even at moderate substrate temperatures of 300 to 600 °C. Due to the perfect Si/SiO2 interfaces, the SiO2 layers allow thermal post-oxidation treatment up to 1000 °C without significant change in oxide thickness or stoichiometry. This is an essential prerequisite for the envisaged realization of Si/SiO2 superlattices with high crystallinity and low strain. It was shown that a post-oxidation annealing step lowers the strain and disorder at the interface resulting in lower intrinsic density of interface states. A further decrease of the density of interface states was achieved by hydrogen passivation as a result of saturation of dangling bonds.

Features such as mutations or structural characteristics can be non-randomly or non-uniformly distributed within a genome. So far, computer simulations were required for statistical inferences on the distribution of sequence motifs. Here, we show that these analyses are possible using an analytical, mathematical approach. For the assessment of non-randomness, our calculations only require information including genome size, number of (sampled) sequence motifs and distance parameters. We have developed computer programs evaluating our analytical formulas for the real-time determination of expected values and p-values. This approach permits a flexible cluster definition that can be applied to most effectively identify non-random or non-uniform sequence motif distribution. As an example, we show the effectivity and reliability of our mathematical approach in clinical retroviral vector integration site distribution.

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34(+)cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

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" (meff), 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. The meff has 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 the meff method 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.