Search

Abstract Background The precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA—despite being successful in E. coli and animal models—still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies. Methods Previously, we have demonstrated the utility of our phage λ-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a ‘gene of interest’. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII. Results The two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of ~ 10 kb and of a DUX4 reporter cassette of ~ 7 kb in hESCs. Conclusion We present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications.

Abstract Apex carnivores are wide‐ranging, low‐density, hard to detect, and declining throughout most of their range, making population monitoring both critical and challenging. Rapid and inexpensive index calibration survey (ICS) methods have been developed to monitor large African carnivores. ICS methods assume constant detection probability and a predictable relationship between the index and the actual population of interest. The precision and utility of the resulting estimates from ICS methods have been questioned. We assessed the performance of one ICS method for large carnivores—track counts—with data from two long‐term studies of African lion populations. We conducted Monte Carlo simulation of intersections between transects (road segments) and lion movement paths (from GPS collar data) at varying survey intensities. Then, using the track count method we estimated population size and its confidence limits. We found that estimates either overstate precision or are too imprecise to be meaningful. Overstated precision stemmed from discarding the variance from population estimates when developing the method and from treating the conversion from tracks counts to population density as a back‐transformation, rather than applying the equation for the variance of a linear function. To effectively assess the status of species, the IUCN has set guidelines, and these should be integrated in survey designs. We propose reporting the half relative confidence interval width (HRCIW) as an easily calculable and interpretable measure of precision. We show that track counts do not adhere to IUCN criteria, and we argue that ICS methods for wide‐ranging low‐density species are unlikely to meet those criteria. Established, intensive methods lead to precise estimates, but some new approaches, like short, intensive, (spatial) capture–mark–recapture (CMR/SECR) studies, aided by camera trapping and/or genetic identification of individuals, hold promise. A handbook of best practices in monitoring populations of apex carnivores is strongly recommended.

Abstract Background Biomass contains cellulose (C6-sugars), hemicellulose (C5-sugars) and lignin. Biomass ranks amongst the most abundant hydrocarbon resources on earth. However, biomass is recalcitrant to enzymatic digestion by cellulases. Physicochemical pretreatment methods make cellulose accessible but partially destroy hemicellulose, producing a C5-sugar-rich liquor. Typically, digestion of pretreated LCB is performed with commercial cellulase preparations, but C5-sugars could in principle be used for “on site” production of cellulases by genetically engineered microorganism, thereby reducing costs. Results Here we report a succession of genetic interventions in Aspergillus nidulans that redesign the natural regulatory circuitry of cellulase genes in such a way that recombinant strains use C5-sugar liquors (xylose) to grow a vegetative tissue and simultaneously accumulate large amounts of cellulases. Overexpression of XlnR showed that under xylose-induction conditions only xylanase C was produced. XlnR overexpression strains were constructed that use the xynCp promoter to drive the production of cellobiohydrolases, endoglucanases and β-glucosidase. All five cellulases accumulated at high levels when grown on xylose. Production of cellulases in the presence of pretreated-biomass C5-sugar liquors was investigated, and cellulases accumulated to much higher enzyme titers than those obtained for traditional fungal cell factories with cellulase-inducing substrates. Conclusions By replacing expensive substrates with a cheap by-product carbon source, the use of C5-sugar liquors directly derived from LCB pretreatment processes not only reduces enzyme production costs, but also lowers operational costs by eliminating the need for off-site enzyme production, purification, concentration, transport and dilution.

Rapidly dividing cells maintain chromatin supercoiling homeostasis via two specialized classes of enzymes, DNA topoisomerase type 1 and 2 (TOP1/2). Several important anticancer drugs perturb this homeostasis by targeting TOP1/2, thereby generating genotoxic DNA damage. Our recent studies indicated that the oncofetal chromatin structuring high‐mobility group AT‐hook 2 (HMGA2) protein plays an important role as a DNA replication fork chaperone in coping with DNA topological ramifications that occur during replication stress, both genomewide and at fragile sites such as subtelomeres. Intriguingly, a recent large‐scale clinical study identified HMGA2 expression as a sole predicting marker for relapse and poor clinical outcomes in 350 acute myeloid leukemia (AML) patients receiving combinatorial treatments that targeted TOP2 and replicative DNA synthesis. Here, we demonstrate that HMGA2 significantly enhanced the DNA supercoil relaxation activity of the drug target TOP2A and that this activator function is mechanistically linked to HMGA2's known ability to constrain DNA supercoils within highly compacted ternary complexes. Furthermore, we show that HMGA2 significantly reduced genotoxic DNA damage in each tested cancer cell model during treatment with the TOP2A poison etoposide or the catalytic TOP2A inhibitor merbarone. Taken together with the recent clinical data obtained with AML patients targeted with TOP2 poisons, our study suggests a novel mechanism of cancer chemoresistance toward combination therapies administering TOP2 poisons or inhibitors. We therefore strongly argue for the future implementation of trials of HMGA2 expression profiling to stratify patients before finalizing clinical treatment regimes.

European natural history collections are a critical infrastructure for meeting the most important challenge humans face over the next 30 years – creating a sustainable future for ourselves and the natural systems on which we depend – and for answering fundamental scientific questions about ecological, evolutionary, and geological processes. Since 2004 SYNTHESYS has been an essential instrument supporting this community, underpinning new ways to access and exploit collections, harmonising policy and providing significant new insights for thousands of researchers, while fostering the development of new approaches to face urgent societal challenges. SYNTHESYS+ is a fourth iteration of this programme, and represents a step change in the evolution of this community. For the first time SYNTHESYS+ brings together the European branches of the global natural science organisations (GBIF https://www.gbif.org/, TDWG https://www.tdwg.org/, GGBN http://www.ggbn.org/ggbn_portal/ and CETAF https://cetaf.org/) with an unprecedented number of collections, to integrate, innovate and internationalise our efforts within the global scientific collections community. Major new developments addressed by SYNTHESYS+ include the delivery of a new virtual access programme, providing digitisation on demand services to a significantly expanded user community; the construction of a European Loans and Visits System (ELViS) providing, for the first time, a unified gateway to accessing digital, physical and molecular collections; and a new data processing platform (the Specimen Data Refinery), applying cutting edge artificial intelligence to dramatically speed up the digital mobilisation of natural history collections. The activities of SYNTHESYS+ form a critical dependency for DiSSCo - the Distributed System of Scientific Collections (https://dissco.eu/), which is the European Research Infrastructure for natural science collections, under the ESFRI umbrella. DiSSCo will undertake the maintenance and sustainability of SYNTHESYS+ products at the end of the programme.

Abstract Background Hospital wastewaters contain fecal material from a large number of individuals, of which many are undergoing antibiotic therapy. It is, thus, plausible that hospital wastewaters could provide opportunities to find novel carbapenemases and other resistance genes not yet described in clinical strains. Our aim was therefore to investigate the microbiota and antibiotic resistome of hospital effluent collected from the city of Mumbai, India, with a special focus on identifying novel carbapenemases. Results Shotgun metagenomics revealed a total of 112 different mobile antibiotic resistance gene types, conferring resistance against almost all classes of antibiotics. Beta-lactamase genes, including encoding clinically important carbapenemases, such as NDM, VIM, IMP, KPC, and OXA-48, were abundant. NDM (0.9% relative abundance to 16S rRNA genes) was the most common carbapenemase gene, followed by OXA-58 (0.84% relative abundance to 16S rRNA genes). Among the investigated mobile genetic elements, class 1 integrons (11% relative abundance to 16S rRNA genes) were the most abundant. The genus Acinetobacter accounted for as many as 30% of the total 16S rRNA reads, with A. baumannii accounting for an estimated 2.5%. High throughput sequencing of amplified integron gene cassettes identified a novel functional variant of an IMP-type (proposed IMP-81) carbapenemase gene (eight aa substitutions) along with recently described novel resistance genes like sul4 and bla RSA1. Using a computational hidden Markov model, we detected 27 unique metallo-beta-lactamase (MBL) genes in the shotgun data, of which nine were novel subclass B1 genes, one novel subclass B2, and 10 novel subclass B3 genes. Six of the seven novel MBL genes were functional when expressed in Escherichia coli. Conclusion By exploring hospital wastewater from India, our understanding of the diversity of carbapenemases has been extended. The study also demonstrates that the microbiota of hospital wastewater can serve as a reservoir of novel resistance genes, including previously uncharacterized carbapenemases with the potential to spread further.

The ongoing softwarization of networks creates a big need for automated testing solutions to ensure service quality. This becomes even more important if agile environments with short time to market and high demands, in terms of service performance and availability, are considered. In this paper, we introduce a novel testing solution for virtualized, microservice-based network functions and services, which we base on TTCN-3, a well known testing language defined by the European standards institute (ETSI). We use TTCN-3 not only for functional testing but also answer the question whether TTCN-3 can be used for performance profiling tasks as well. Finally, we demonstrate the proposed concepts and solutions in a case study using our open-source prototype to test and profile a chained network service. Keywords: Network function virtualization, Service chains, Automated testing, Performance profiling, Test definition

Abstract Background Shotgun metagenome data sets of microbial communities are highly diverse, not only due to the natural variation of the underlying biological systems, but also due to differences in laboratory protocols, replicate numbers, and sequencing technologies. Accordingly, to effectively assess the performance of metagenomic analysis software, a wide range of benchmark data sets are required. Results We describe the CAMISIM microbial community and metagenome simulator. The software can model different microbial abundance profiles, multi-sample time series, and differential abundance studies, includes real and simulated strain-level diversity, and generates second- and third-generation sequencing data from taxonomic profiles or de novo. Gold standards are created for sequence assembly, genome binning, taxonomic binning, and taxonomic profiling. CAMSIM generated the benchmark data sets of the first CAMI challenge. For two simulated multi-sample data sets of the human and mouse gut microbiomes, we observed high functional congruence to the real data. As further applications, we investigated the effect of varying evolutionary genome divergence, sequencing depth, and read error profiles on two popular metagenome assemblers, MEGAHIT, and metaSPAdes, on several thousand small data sets generated with CAMISIM. Conclusions CAMISIM can simulate a wide variety of microbial communities and metagenome data sets together with standards of truth for method evaluation. All data sets and the software are freely available at https://github.com/CAMI-challenge/CAMISIM

New technologies like ecoacoustic surveys promise time and cost efficiency for biodiversity assessments, serve as a basis for effective conservation policies, and are particularly appealing for remote and highly diverse tropical areas. Acoustic indices facilitate the analysis of large acoustic datasets but no consensus on their performance has been reached yet. We evaluated the efficacy of four acoustic indices (Acoustic Complexity Index, Acoustic Diversity Index, Acoustic Evenness Index, Acoustic Entropy) for sound data analysis and biodiversity assessments inside a national park and the agricultural mosaic landscape of north-eastern Madagascar, a global biodiversity hotspot. We used self-built sound recorders to continuously record soundscapes on 80 plots across seven land-use types (old-growth forest, forest fragment, forest–derived and fallow-derived vanilla agroforest, herbaceous and woody fallow, rice paddy) and compared index values between land–use types, assessed the correlation with bird species richness as measured by point counts, and related the acoustic indices to plot- and landscape-scale parameters. The Acoustic Diversity Index, Acoustic Evenness Index (inverse) and Acoustic Entropy were highest in old-growth forest and lowest for rice paddies and fallow land. Index values for structurally similar land-use types did not differ significantly from each other. The correlation of the three acoustic indices with bird species richness was strongest during daytime (R2≥0.30). Differences in the index values were best explained by land-use type and vegetation density. Our results showed that all investigated indices except the Acoustic Complexity Index were suitable biodiversity indicators for a tropical, agricultural landscape. Soundscape diversity was positively affected by plot-scale vegetation structure, emphasizing the importance of forests and particularly old-growth forest for conservation. We demonstrated that acoustic indices and sound recordings are a useful tool for assessing biodiversity in tropical agricultural mosaic landscapes. To realize the full potential of ecoacoustics in conservation, sampling guidelines and user-friendly analysis packages will be key to facilitate a wider implementation.

The aim of the ArDM project is the development and operation of a one ton double-phase liquid argon detector for direct Dark Matter searches. The detector measures both the scintillation light and the ionization charge from ionizing radiation using two independent readout systems. This paper briefly describes the detector concept and presents preliminary results from the ArDM R&D program, including a 3 l prototype developed to test the charge readout system.

Aim: To examine the stroke risks associated with aircraft, road traffic, and railway noise exposure in a large case–control study. Materials and Methods: All people aged ≥40 years living around the Frankfurt airport that were insured by one of three large statutory health insurance funds between 2005 and 2010 were included in the study (n = 1,026,670). Address-specific exposure to aircraft, road, and railway traffic noise was estimated for 2005. We used insurance claim data to identify 25,495 newly diagnosed cases of stroke between 2006 and 2010 and compared them with 827,601 control participants. Logistic regression analysis was used to calculate the odds ratios adjusted for age, sex, local proportion of people receiving unemployment benefits, and if available individual indicators of socioeconomic status (education, occupation). Results: For 24-h continuous aircraft noise exposure, neither increased risk estimates nor a positive linear exposure–risk relation was found. However, stroke risk was statistically significantly increased by 7% [95% confidence intervals (95%CI): 2–13%] for people who were exposed to

Background: Acute myocardial infarction (AMI) and cancer are common and serious diseases. As the prognosis and treatment of both diseases has improved, more cancer patients will suffer an AMI. Unfortunately, data on these “double hit” patients is scarce. Methods: From the largest public German health insurance, anonymized data of all patients with pre-existing cancer who were hospitalized due to ST-elevation MI (STEMI) between 2010 and 2017 were analyzed and followed-up until 2018. Results: Of 175,262 STEMI patients, 27,213 had pre-existing cancer (15.5%). Most frequent were skin (24.9%), prostate (17.0%), colon (11.0%), breast (10.9%), urinary tract (10.6%), and lung cancer (5.2%). STEMI patients with malignancies were older and presented more often with coronary three-vessel disease, atrial arrhythmias, chronic kidney disease, chronic heart failure, cerebrovascular and peripheral artery disease (PAD, each p < 0.001). They showed more often previous AMI, percutaneous coronary interventions (PCI), cardiac surgery, and stroke (all p < 0.001). Acute PCIs were applied between 2 and 6% less frequently compared to those without cancer. In-hospital adverse events occurred more frequently in cancer. Eight-year survival was 57.3% (95% CI 57.0–57.7%) without cancer and ranged between 41.2% and 19.2% in distinct cancer types. Multivariable Cox regression for all-cause mortality found, e.g., lung cancer (HR 2.04), PAD stage 4–6 (HR 1.78), metastasis (HR 1.72), and previous stroke (HR 1.44) to have the strongest impact (all p < 0.001). Conclusion: In this large “real world” data, prognosis after STEMI in cancer patients was markedly reduced but differed widely between cancer types. Of note, no withholding of interventional treatments in cancer patients could be observed.

Genetic variability in transforming growth factor beta pathway (TGFB) was suggested to affect adverse events of radiotherapy. We investigated comprehensive variability in TGFB1 (gene coding for TGFβ1 ligand) and TGFBR1 (TGFβ receptor-1) in relation to radiotoxicity. Prostate cancer patients treated with primary radiotherapy (n = 240) were surveyed for acute and late toxicity. Germline polymorphisms (n = 40) selected to cover the common genetic variability in TGFB1 and TGFBR1 were analyzed in peripheral blood cells. Human lymphoblastoid cell lines (LCLs) were used to evaluate a possible impact of TGFB1 and TGFBR1 genetic polymorphisms to DNA repair capacity following single irradiation with 3 Gy. Upon adjustment for multiplicity testing, rs10512263 in TGFBR1 showed a statistically significant association with acute radiation toxicity. Carriers of the Cytosine (C)-variant allele (n = 35) featured a risk ratio of 2.17 (95%-CI 1.41–3.31) for acute toxicity ≥ °2 compared to Thymine/Thymine (TT)-wild type individuals (n = 205). Reduced DNA repair capacity in the presence of the C-allele of rs10512263 might be a mechanistic explanation as demonstrated in LCLs following irradiation. The risk for late radiotoxicity was increased by carrying at least two risk genotypes at three polymorphic sites, including Leu10Pro in TGFB1. Via comprehensive genotyping of TGFB1 and TGFBR1, promising biomarkers for radiotoxicity in prostate cancer were identified.

Analyzing and understanding the movement of the mitral valve is of vital importance in cardiology, as the treatment and prevention of several serious heart diseases depend on it. Unfortunately, large amounts of noise as well as a highly varying image quality make the automatic tracking and segmentation of the mitral valve in two-dimensional echocardiographic videos challenging. In this paper, we present a fully automatic and unsupervised method for segmentation of the mitral valve in two-dimensional echocardiographic videos, independently of the echocardiographic view. We propose a bias-free variant of the robust non-negative matrix factorization (RNMF) along with a window-based localization approach, that is able to identify the mitral valve in several challenging situations. We improve the average f1-score on our dataset of 10 echocardiographic videos by 0.18 to a f1-score of 0.56.

OBJECTIVES: Aircraft, road, and rail traffic noise can cause sleep disturbances. Since night work and shorter sleep durations have been linked to increased risks of breast cancer, we examined if 24-hour, or day- or night-time traffic noise exposure may also increase the risk of breast cancer. METHODS: To investigate the noise-related risks of breast cancer, the pseudonymized insurance records of three large statutory health companies (2005–2010) for women aged ≥40 years living in the region surrounding the Frankfurt international airport were analyzed with address-specific acoustic data representing aircraft, road, and rail-traffic noise. Noise exposure among women with incident breast cancer (N=6643) were compared with that of control subjects (N=471 596) using logistic regression and adjusting for age, hormone replacement therapy, education and occupation (only available for 27.9%), and a regional proportion of persons receiving long-term unemployment benefits as an ecological indicator of socioeconomic level. Analyses were also stratified according to estrogen receptor (ER) status. RESULTS: An increased odds ratio (OR) was observed for ER negative (ER-) tumors at 24-hour aircraft noise levels 55–59 dB [OR 55–59 dB 1.41, 95% confidence interval (CI) 1.04–1.90] but not for ER positive (ER+) breast cancers (OR 55–59 dB 0.95, 95% CI 0.75–1.20). Clear associations between road and rail traffic noise were not observed. CONCLUSIONS: The results indicate increased aircraft noise may be an etiologic factor for ER- breast cancers. However, information regarding potential confounding factors was largely unattainable. Further research is required to understand how environmental noise may be involved in the pathogenesis of ER- breast cancers.

Locally advanced head and neck squamous cell carcinomas (HNSCC) are often managed with surgery followed by postoperative radiochemotherapy (RCT). With the general increase in life expectancy, the proportion of elderly patients with HNSCC is expected to grow rapidly. Until now, a deeper understanding of specific management strategies for these patients in clinical routine was lacking. In the present study, we compared elderly patients (≥70 years, n = 52) and younger patients (n = 245) treated with postoperative RCT for HNSCC at our tertiary cancer center. All patients were irradiated with modern radiotherapy techniques (IMRT/VMAT). Patients ≥70 years of age had more comorbidities. Additionally, elderly patients less frequently received concomitant systemic treatment. The rates of mucositis and dermatitis were lower in patients ≥70 years. Elderly patients had significantly worse overall and progression-free survival. Locoregional and distant control were comparable in elderly and younger patients. In conclusion, postoperative RCT is a safe and effective treatment option in patients ≥70 years. In light of comorbidities and poor overall survival rates, benefits and harms of radiotherapy and concomitant systemic treatment should be weighed carefully. When exclusively applying up-to-date radiotherapy techniques with, at the same time, careful use of concomitant systemic therapy, favorable acute toxicity profiles are achieved.

Chronic limb threatening ischaemia (CLTI) has a devastating prognosis with high rates of lower limb amputation (LLA) and deaths. This is an illustration of contemporary management and the long term fate of patients after ischaemic LLA, particularly with respect to sex, using real world data. This was a multisectoral cross sectional and longitudinal analysis of health claims data from the largest German health insurance database (AOK). Data of 39 796 propensity score matched patients hospitalised for ischaemic LLA between 2010 and 2018 were analysed for cardiovascular comorbidities, treatment, and for subsequent cardiovascular and limb events, with a distinct focus on sex. Matching was performed, to ensure that the rate of major amputations and the age distribution were equal in both groups (in both sexes). An observation period of two years before index and a follow up (FU) period until 2019 were included. Before index amputation, 68% of patients had received any kind of peripheral revascularisation. The use of statins (37.0% vs. 42.6%) and antithrombotic substances (54.9% vs. 61.8%) was lower in women than in men (p <.001). During two year FU, cardiovascular and limb events occurred among women and men as follows: limb re-amputation (26.7% vs. 31.2%), myocardial infarction (10.9% vs. 14.5%), stroke (20.8% vs. 20.7%), and death from any cause (51.0% vs. 53.3%, p <.001 except for stroke). After adjustment for cardiovascular comorbidities and vascular procedures, female sex was associated with a higher probability of death (HR 1.04, 95% CI 1.04 – 1.04). Patients undergoing ischaemic LLA still have a poor prognosis marked by high rates of recurrent cardiovascular and limb events resulting in a > 50% mortality rate within two years. The continuous lack of guideline recommended therapies, particularly in women, may be associated with the persisting poor outcome, necessitating urgent further investigation. [ABSTRACT FROM AUTHOR]

Despite excellent loco-regional control by multimodal treatment of locally advanced rectal cancer, a substantial portion of patients succumb to this disease. As many treatment effects are mediated via reactive oxygen species (ROS), we evaluated the effect of single nucleotide polymorphisms (SNPs) in ROS-related genes on clinical outcome. Based on the literature, eight SNPs in seven ROS-related genes were assayed. Eligible patients (n = 287) diagnosed with UICC stage II/III rectal cancer were treated multimodally starting with neoadjuvant radiochemotherapy (N-RCT) according to the clinical trial protocols of CAO/ARO/AIO-94, CAO/ARO/AIO-04, TransValid-A, and TransValid-B. The median follow-up was 64.4 months. The Ser326Cys polymorphism in the human OGG1 gene affected clinical outcome, in particular cancer-specific survival (CSS). This effect was comparable in extent to the ypN status, an already established strong prognosticator for patient outcome. Homozygous and heterozygous carriers of the Cys326 variant (n = 105) encountered a significantly worse CSS (p = 0.0004 according to the log-rank test, p = 0.01 upon multiple testing adjustment). Cox regression elicited a hazard ratio for CSS of 3.64 (95% confidence interval 1.70–7.78) for patients harboring the Cys326 allele. In a multivariable analysis, the effect of Cys326 on CSS was preserved. We propose the genetic polymorphism Ser326Cys as a promising biomarker for outcome in rectal cancer.

Background: Intensity-modulated radiotherapy (IMRT) is the standard of care in chemoradiotherapy (CRT) for anal cancer. Until now, only a limited number of studies have analyzed the results with VMAT (volumetric modulated arc therapy). We conducted a retrospective study on patients treated at our institution. Patients and Methods: We included patients who received curative CRT for anal cancer. We compared VMAT-treated and 3DCRT (3D conformal radiotherapy)-treated patients. We analyzed toxicities (acute: CTCAE criteria; late: LENT/SOMA criteria), treatment compliance, overall survival, cancer-specific survival (CSS), distant control (DC), and locoregional control. Results: A total of 149 patients (3DCRT: n = 87, VMAT: n = 62) were included. The median follow-up was longer in 3DCRT-treated patients (3DCRT: 61.3 months; VMAT: 39.1 months; p < 0.05). VMAT-treated patients had more G3 tumors (3DCRT: 12/87 (13.8%); VMAT: 18/62 (29.0%), p < 0.001). VMAT reduced acute toxicities ≥grade 3 (3DCRT: n = 48/87 (55.2%); VMAT: n = 11/62 (17.7%), p < 0.001). VMAT improved treatment compliance (less interruptions/delays) (3DCRT: 37/87, 42.5%; VMAT: 4/62, 6.5%; p < 0.001), provided a shorter median overall treatment time (3DCRT: 41 days; VMAT: 38 days; p = 0.02), and gave a higher median absolute 5-fluorouracil dose (3DCRT: 13,700 mg; VMAT: 14,400 mg; p = 0.001). Finally, we found improved CSS (p = 0.02; 3DCRT: 81.9% at 3 years; VMAT: 94.1% at 3 years) and DC (p = 0.01; 3DCRT: 89.4% at 3 years; VMAT: 100.0% at 3 years) with VMAT. Summary: Our study is the first to demonstrate improved treatment compliance and outcomes with VMAT for anal cancer. Previous studies have indicated that organs at risk sparing might be more improved with the use of VMAT vs. with conventional IMRT. Future studies should address whether these advantages lead to a further reduction in CRT-associated morbidity.

We retrospectively studied outcomes in patients treated with preoperative radiochemotherapy and surgery for esophageal squamous cell cancer. We put special focus on the comparison of patients treated with 5-fluorouracil/cisplatin (‘Walsh’) or carboplatin/paclitaxel (‘CROSS’). We compared characteristics between patients treated according to ‘Walsh’ vs. ‘CROSS’. Cox regression was performed to test for an association of parameters with outcomes. Study eligibility was met by 90 patients. First, the higher age and more comorbidities of the ‘CROSS’ patients, along with a shorter intensive care/intermediate care stay, might reflect an improvement in supportive and surgical/perioperative procedures over the periods. Second, the ‘CROSS’ patients experienced more hematologic toxicity and were less likely to complete chemotherapy as per protocol. This indicates that efforts should be taken to guide patients through a toxic treatment regimen by supportive measures. Third, the negative prognostic impact of radiochemotherapy-related toxicities (i.e., dysphagia and hematologic toxicities) and the duration of the intensive care/intermediate care unit stay underlines that further optimization of treatment procedures remains an important goal. We found no differences in tumor downstaging and survival between treatment regimen. Toxicity profiles could be improved by tailoring the regimen to individual patients (e.g., careful use of the taxane-based regimen in elderly patients).

Abstract HMGA2 belongs to the family of the high mobility group (HMG) proteins. It binds DNA via three AT-hook domains to the minor groove of adenine-thymine (AT) rich DNA. Recently, a new function of HMGA2 as a replication fork chaperone that protects stem and cancer cells from replication fork collapse induced by chemotherapeutic agents was uncovered, suggesting a previously uncharacterized binding at replication forks. In this study, we examined HMGA2 binding to four DNA structures relevant to replication forks, namely ds DNA, ss DNA, forked DNA and supercoiled DNA plectonemes. We detected HMGA2 binding to supercoiled DNA at the lowest concentration and this binding mode transiently stabilizes the supercoiled plectonemes against relaxation by type I topoisomerase. Together, these findings suggest a plausible mechanism how fork regression and collapse are attenuated by HMGA2 during replication stress, i.e. through transient stabilization of positively supercoiled plectonemes in the parental duplex.

Abstract Within a large carnivore guild, subordinate competitors (African wild dog, Lycaon pictus, and cheetah, Acinonyx jubatus) might reduce the limiting effects of dominant competitors (lion, Panthera leo, and spotted hyena, Crocuta crocuta) by avoiding them in space, in time, or through patterns of prey selection. Understanding how these competitors cope with one other can inform strategies for their conservation. We tested how mechanisms of niche partitioning promote coexistence by quantifying patterns of prey selection and the use of space and time by all members of the large carnivore guild within Liuwa Plain National Park in western Zambia. Lions and hyenas specialized on wildebeest, whereas wild dogs and cheetahs selected broader diets including smaller and less abundant prey. Spatially, cheetahs showed no detectable avoidance of areas heavily used by dominant competitors, but wild dogs avoided areas heavily used by lions. Temporally, the proportion of kills by lions and hyenas did not detectably differ across four time periods (day, crepuscular, early night, and late night), but wild dogs and especially cheetahs concentrated on time windows that avoided nighttime hunting by lions and hyenas. Our results provide new insight into the conditions under which partitioning may not allow for coexistence for one subordinate species, the African wild dog, while it does for cheetah. Because of differences in responses to dominant competitors, African wild dogs may be more prone to competitive exclusion (local extirpation), particularly in open, uniform ecosystems with simple (often wildebeest dominated) prey communities, where spatial avoidance is difficult.

The transient build-up of DNA supercoiling during the translocation of replication forks threatens genome stability and is controlled by DNA topoisomerases (TOPs). This crucial process has been exploited with TOP poisons for cancer chemotherapy. However, pinpointing cellular determinants of the best clinical response to TOP poisons still remains enigmatic. Here, we present an integrated approach and demonstrate that endogenous and exogenous expression of the oncofetal high-mobility group AT-hook 2 (HMGA2) protein exhibited broad protection against the formation of hydroxyurea-induced DNA breaks in various cancer cells, thus corroborating our previously proposed model in which HMGA2 functions as a replication fork chaperone that forms a protective DNA scaffold at or close to stalled replication forks. We now further demonstrate that high levels of HMGA2 also protected cancer cells against DNA breaks triggered by the clinically important TOP1 poison irinotecan. This protection is most likely due to the recently identified DNA supercoil constraining function of HMGA2 in combination with exclusion of TOP1 from binding to supercoiled substrate DNA. In contrast, low to moderate HMGA2 protein levels surprisingly potentiated the formation of irinotecan-induced genotoxic covalent TOP1-DNA cleavage complexes. Our data from cell-based and several in vitro assays indicate that, mechanistically, this potentiating role involves enhanced drug-target interactions mediated by HMGA2 in ternary complexes with supercoiled DNA. Subtelomeric regions were found to be extraordinarily vulnerable to these genotoxic challenges induced by TOP1 poisoning, pointing at strong DNA topological barriers located at human telomeres. These findings were corroborated by an increased irinotecan sensitivity of patient-derived xenografts of colorectal cancers exhibiting low to moderate HMGA2 levels. Collectively, we uncovered a therapeutically important control mechanism of transient changes in chromosomal DNA topology that ultimately leads to enhanced human subtelomere stability.

Ungulate populations face declines across the globe, and populations are commonly conserved by using protected areas. However, assessing the effectiveness of protected areas in conserving ungulate populations has remained difficult. Using herd size data from four years of line transect surveys and distance sampling models, we modeled population densities of four important herbivore species across a gradient of protection on the edge of Zambia's South Luangwa National Park (SLNP) while accounting for the role of various ecological and anthropogenic variables. Our goal was to test whether protection was responsible for density dynamics in this protection gradient, and whether a hunting moratorium impacted herbivore densities during the studies. For all four species, we estimated lower densities in partially protected buffer areas adjacent to SLNP (ranging from 4.5-fold to 13.2-fold lower) compared to protected parklands. Density trends through the study period were species-specific, with some species increasing, decreasing, or remaining stable in all or some regions of the protection gradient. Surprisingly, when controlling for other covariates, we found that these observed differences were not always detectably related to the level of protection or year. Our findings highlight the importance of accounting for variables beyond strata of interest in evaluating the effectiveness of a protected area. This study highlights the importance of comprehensively modeling ungulate population density across protection gradients, identifies lands within an important protection gradient for targeted conservation and monitoring, documents prey depletion and expands our understanding on the drivers in a critical buffer area in Zambia.

Telomerase RNA (TR) provides the template for DNA repeat synthesis at telomeres and is essential for genome stability in continuously dividing cells. We mapped the RNA interactome of human TR (hTR) and identified a set of non-coding and coding hTR-interacting RNAs, including the histone 1C mRNA (HIST1H1C). Disruption of the hTR-HIST1H1C RNA association resulted in markedly increased telomere elongation without affecting telomerase enzymatic activity. Conversely, over-expression of HIST1H1C led to telomere attrition. By using a combination of mutations to disentangle the effects of histone 1 RNA synthesis, protein expression, and hTR interaction, we show that HIST1H1C RNA negatively regulates telomere length independently of its protein coding potential. Taken together, our data provide important insights into a surprisingly complex hTR-RNA interaction network and define an unexpected non-coding RNA role for HIST1H1C in regulating telomere length homeostasis, thus offering a glimpse into the mostly uncharted, vast space of non-canonical messenger RNA functions.

Numerous clinical trials sought to improve outcomes in endometrial cancer patients with multimodal treatment strategies. We tested the hypothesis that specific histopathological and clinical parameters are prognosticators for outcomes at our Gynecological Cancer Center. A total of 203 patients (median age, 69.5 years) was included. They were irradiated postoperatively (n = 184: Brachytherapy, n = 19: Teletherapy) between 05/2007 and 03/2020. The median follow-up was 37.2 months. As statistical methods, we used the univariable Cox proportional hazards regression, and log-rank statistics. First, we found a significant influence of grading and nodal stage on outcomes. These findings underline the recommendations of more intense treatment in these patient groups, as already reflected in current guidelines. Secondly, we found that patient age had a significant influence on survival be it due to comorbidities and/or due to too hesitant treatment regimen in the elderly. Thus, it should be aimed at particular strategies in treatment of these patients. Lastly, we found very low rates of treatment-related side effects in patients treated with brachytherapy and moderate rates of side effects in patients treated with teletherapy. Overall, our study serves as basis for further improvement of treatment strategies and for conceptualization of clinical trials.