Culture expansion of CAR T cells results in aberrant DNA methylation that is associated with adverse clinical outcome.

Chimeric antigen receptor (CAR) T cells provide new perspectives for treatment of hematological malignancies. Manufacturing of these cellular products includes culture expansion procedures, which may affect cellular integrity and therapeutic outcome. In this study, we investigated culture-associated epigenetic changes in CAR T cells and found continuous gain of DNAm, particularly within genes that are relevant for T cell function. Hypermethylation in many genes, such as TCF7, RUNX1, and TOX, was reflected by transcriptional downregulation. 332 CG dinucleotides (CpGs) showed an almost linear gain in methylation with cell culture time, albeit neighboring CpGs were not coherently regulated on the same DNA strands. An epigenetic signature based on 14 of these culture-associated CpGs predicted cell culture time across various culture conditions. Notably, even in CAR T cell products of similar culture time higher DNAm levels at these CpGs were associated with significantly reduced long-term survival post transfusion. Our data demonstrate that cell culture expansion of CAR T cells evokes DNA hypermethylation at specific sites in the genome and the signature may also reflect loss of potential in CAR T cell products. Hence, reduced cultivation periods are beneficial to avoid dysfunctional methylation programs that seem to be associated with worse therapeutic outcome.

Site-Selective Acylation of Pyranosides with Immobilized Oligopeptide Catalysts in Flow.

We report the site-selective acetylation of partially protected monosaccharides using immobilized oligopeptide catalysts, which are readily accessible via solid-phase peptide synthesis. The catalysts are able to invert the intrinsic selectivity, which was determined using N-methylimidazole, for a variety of pyranosides. We demonstrate that the catalysts are stable for multiple reaction cycles and can be easily reused after separation from the reaction solution. The catalysts can also be used in flow without loss of reactivity and selectivity.

Site-Selective Acylation of Pyranosides with Oligopeptide Catalysts.

Herein, we report the oligopeptide-catalyzed site-selective acylation of partially protected monosaccharides. We identified catalysts that invert site-selectivity compared to N-methylimidazole, which was used to determine the intrinsic reactivity, for 4,6-O-protected glucopyranosides (trans-diols) as well as 4,6-O-protected mannopyranosides (cis-diols). The reaction yields up to 81% of the inherently unfavored 2-O-acetylated products with selectivities up to 15:1 using mild reaction conditions. We also determined the influence of protecting groups on the reaction and demonstrate that our protocol is suitable for one-pot reactions with multiple consecutive protection steps.

2000-year-old pathogen genomes reconstructed from metagenomic analysis of Egyptian mummified individuals.

BACKGROUND: Recent advances in sequencing have facilitated large-scale analyses of the metagenomic composition of different samples, including the environmental microbiome of air, water, and soil, as well as the microbiome of living humans and other animals. Analyses of the microbiome of ancient human samples may provide insights into human health and disease, as well as pathogen evolution, but the field is still in its very early stages and considered highly challenging. RESULTS: The metagenomic and pathogen content of Egyptian mummified individuals from different time periods was investigated via genetic analysis of the microbial composition of various tissues. The analysis of the dental calculus' microbiome identified Red Complex bacteria, which are correlated with periodontal diseases. From bone and soft tissue, genomes of two ancient pathogens, a 2200-year-old Mycobacterium leprae strain and a 2000-year-old human hepatitis B virus, were successfully reconstructed. CONCLUSIONS: The results show the reliability of metagenomic studies on Egyptian mummified individuals and the potential to use them as a source for the extraction of ancient pathogen DNA.

In Situ Switching of Site-Selectivity with Light in the Acetylation of Sugars with Azopeptide Catalysts.

We present a novel concept for the in situ control of site-selectivity of catalytic acetylations of partially protected sugars using light as external stimulus and oligopeptide catalysts equipped with an azobenzene moiety. The isomerizable azobenzene-peptide backbone defines the size and shape of the catalytic pocket, while the π-methyl-l-histidine (Pmh) moiety transfers the electrophile. Photoisomerization of the E- to the Z-azobenzene catalyst (monitored via NMR) with an LED (λ = 365 nm) drastically changes the chemical environment around the catalytically active Pmh moiety, so that the light-induced change in the catalyst shape alters site-selectivity. As a proof of principle, we employed (4,6-O-benzylidene)methyl-α-d-pyranosides, which provide a change in regioselectivity from 2:1 (E) to 1:5 (Z) for the monoacetylated products at room temperature. The validity of this new catalyst-design concept is further demonstrated with the regioselective acetylation of the natural product quercetin. In situ irradiation NMR spectroscopy was used to quantify photostationary states under continuous irradiation with UV light.

DACCOR-Detection, characterization, and reconstruction of repetitive regions in bacterial genomes.

The reconstruction of genomes using mapping-based approaches with short reads experiences difficulties when resolving repetitive regions. These repetitive regions in genomes result in low mapping qualities of the respective reads, which in turn lead to many unresolved bases. Currently, the reconstruction of these regions is often based on modified references in which the repetitive regions are masked. However, for many references, such masked genomes are not available or are based on repetitive regions of other genomes. Our idea is to identify repetitive regions in the reference genome de novo. These regions can then be used to reconstruct them separately using short read sequencing data. Afterward, the reconstructed repetitive sequence can be inserted into the reconstructed genome. We present the program detection, characterization, and reconstruction of repetitive regions, which performs these steps automatically. Our results show an increased base pair resolution of the repetitive regions in the reconstruction of Treponema pallidum samples, resulting in fewer unresolved bases.

Ancient genomes reveal a high diversity of Mycobacterium leprae in medieval Europe.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom-a skeleton from the Great Chesterford cemetery with a calibrated age of 415-545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide.

Alzheimer's brains show inter-related changes in RNA and lipid metabolism.

Alzheimer's disease (AD) involves changes in both lipid and RNA metabolism, but it remained unknown if these differences associate with AD's cognition and/or post-mortem neuropathology indices. Here, we report RNA-sequencing evidence of inter-related associations between lipid processing, cognition level, and AD neuropathology. In two unrelated cohorts, we identified pathway-enriched facilitation of lipid processing and alternative splicing genes, including the neuronal-enriched NOVA1 and hnRNPA1. Specifically, this association emerged in temporal lobe tissue samples from donors where postmortem evidence demonstrated AD neuropathology, but who presented normal cognition proximate to death. The observed changes further associated with modified ATP synthesis and mitochondrial transcripts, indicating metabolic relevance; accordingly, mass-spectrometry-derived lipidomic profiles distinguished between individuals with and without cognitive impairment prior to death. In spite of the limited group sizes, tissues from persons with both cognitive impairment and AD pathology showed elevation in several drug-targeted genes of other brain, vascular and autoimmune disorders, accompanied by pathology-related increases in distinct lipid processing transcripts, and in the RNA metabolism genes hnRNPH2, TARDBP, CLP1 and EWSR1. To further detect 3'-polyadenylation variants, we employed multiple cDNA primer pairs. This identified variants that showed limited differences in scope and length between the tested cohorts, yet enabled superior clustering of demented and non-demented AD brains versus controls compared to total mRNA expression values. Our findings indicate inter-related cognition-associated differences in AD's lipid processing, alternative splicing and 3'-polyadenylation, calling for pursuing the underlying psychological and therapeutics implications.

Improving ancient DNA genome assembly.

Most reconstruction methods for genomes of ancient origin that are used today require a closely related reference. In order to identify genomic rearrangements or the deletion of whole genes, de novo assembly has to be used. However, because of inherent problems with ancient DNA, its de novo assembly is highly complicated. In order to tackle the diversity in the length of the input reads, we propose a two-layer approach, where multiple assemblies are generated in the first layer, which are then combined in the second layer. We used this two-layer assembly to generate assemblies for two different ancient samples and compared the results to current de novo assembly approaches. We are able to improve the assembly with respect to the length of the contigs and can resolve more repetitive regions.

InFusion: Advancing Discovery of Fusion Genes and Chimeric Transcripts from Deep RNA-Sequencing Data.

Analysis of fusion transcripts has become increasingly important due to their link with cancer development. Since high-throughput sequencing approaches survey fusion events exhaustively, several computational methods for the detection of gene fusions from RNA-seq data have been developed. This kind of analysis, however, is complicated by native trans-splicing events, the splicing-induced complexity of the transcriptome and biases and artefacts introduced in experiments and data analysis. There are a number of tools available for the detection of fusions from RNA-seq data; however, certain differences in specificity and sensitivity between commonly used approaches have been found. The ability to detect gene fusions of different types, including isoform fusions and fusions involving non-coding regions, has not been thoroughly studied yet. Here, we propose a novel computational toolkit called InFusion for fusion gene detection from RNA-seq data. InFusion introduces several unique features, such as discovery of fusions involving intergenic regions, and detection of anti-sense transcription in chimeric RNAs based on strand-specificity. Our approach demonstrates superior detection accuracy on simulated data and several public RNA-seq datasets. This improved performance was also evident when evaluating data from RNA deep-sequencing of two well-established prostate cancer cell lines. InFusion identified 26 novel fusion events that were validated in vitro, including alternatively spliced gene fusion isoforms and chimeric transcripts that include intergenic regions. The toolkit is freely available to download from http:/bitbucket.org/kokonech/infusion.

Origin of modern syphilis and emergence of a pandemic Treponema pallidum cluster.

The abrupt onslaught of the syphilis pandemic that started in the late fifteenth century established this devastating infectious disease as one of the most feared in human history1. Surprisingly, despite the availability of effective antibiotic treatment since the mid-twentieth century, this bacterial infection, which is caused by Treponema pallidum subsp. pallidum (TPA), has been re-emerging globally in the last few decades with an estimated 10.6 million cases in 2008 (ref. 2). Although resistance to penicillin has not yet been identified, an increasing number of strains fail to respond to the second-line antibiotic azithromycin3. Little is known about the genetic patterns in current infections or the evolutionary origins of the disease due to the low quantities of treponemal DNA in clinical samples and difficulties in cultivating the pathogen4. Here, we used DNA capture and whole-genome sequencing to successfully interrogate genome-wide variation from syphilis patient specimens, combined with laboratory samples of TPA and two other subspecies. Phylogenetic comparisons based on the sequenced genomes indicate that the TPA strains examined share a common ancestor after the fifteenth century, within the early modern era. Moreover, most contemporary strains are azithromycin-resistant and are members of a globally dominant cluster, named here as SS14-Ω. The cluster diversified from a common ancestor in the mid-twentieth century subsequent to the discovery of antibiotics. Its recent phylogenetic divergence and global presence point to the emergence of a pandemic strain cluster.

EAGER: efficient ancient genome reconstruction.

BACKGROUND: The automated reconstruction of genome sequences in ancient genome analysis is a multifaceted process. RESULTS: Here we introduce EAGER, a time-efficient pipeline, which greatly simplifies the analysis of large-scale genomic data sets. EAGER provides features to preprocess, map, authenticate, and assess the quality of ancient DNA samples. Additionally, EAGER comprises tools to genotype samples to discover, filter, and analyze variants. CONCLUSIONS: EAGER encompasses both state-of-the-art tools for each step as well as new complementary tools tailored for ancient DNA data within a single integrated solution in an easily accessible format.

The Enantioselective Dakin-West Reaction.

Here we report the development of the first enantioselective Dakin-West reaction, yielding α-acetamido methylketones with up to 58 % ee with good yields. Two of the obtained products were recrystallized once to achieve up to 84 % ee. The employed methylimidazole-containing oligopeptides catalyze both the acetylation of the azlactone intermediate and the terminal enantioselective decarboxylative protonation. We propose a dispersion-controlled reaction path that determines the asymmetric reprotonation of the intermediate enolate after the decarboxylation.

Child sexual abuse in religiously affiliated and secular institutions: a retrospective descriptive analysis of data provided by victims in a government-sponsored reappraisal program in Germany.

BACKGROUND: The disclosure of widespread sexual abuse committed by professional educators and clergymen in institutions in Germany ignited a national political debate, in which special attention was paid to church-run institutions. We wanted to find out whether the nature of the abuse and its effect on victims differed depending on whether the abuse had been experienced in religiously affiliated versus secular institutions. METHODS: In 2010, the German government established a hotline that victims could contact anonymously to describe their experiences of sexual abuse. The information provided by callers was documented and categorized. Our analysis looked at a subset of the data collected, in order to compare the nature of the abuse experienced at three types of institutions: Roman Catholic, Protestant, and non-religiously affiliated. Non-parametric tests were used to compare frequency distributions, and qualitative data were analyzed descriptively. RESULTS: Of the 1050 victims in our sample, 404 had been in Roman Catholic, 130 in Protestant, and 516 in non-religious institutions. The overall mean age at the time of reporting was 52.2 years. Males (59.8%) outnumbered females. Victims who had been in religiously affiliated institutions were significantly older than those who had been in secular institutions. Almost half the victims had been abused physically as well as sexually, and most victims reported that the abuse had occurred repeatedly and that the assaults had been committed by males. Patterns of abuse (time, type, and extent), and the gender of the offenders did not differ between the three groups. Intercourse was more frequently reported by older victims and by females. Similar percentages of victims in all groups reported current psychiatric diagnoses (depression, anxiety disorders, PTSD). Significantly more victims from Protestant institutions reported having current psychosocial problems. CONCLUSION: The results suggest that child sexual abuse in institutions is attributable to the nature of institutional structures and to societal assumptions about the rights of children more than to the attitudes towards sexuality of a specific religion. The exploratory data arising from this study may serve as a starting point for building hypotheses, and may point the way toward improvements in prevention and intervention strategies.

Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours.

We have shown recently that downregulation of the androgen receptor (AR), one of the key players in prostate tumor cells, with short antisense oligodeoxynucleotides (ODNs) results in inhibition of prostate tumor growth. Particularly with regard to an application of these antisense drugs in vivo, we now investigated the usefulness of microbubble-enhanced ultrasound to deliver these ODNs into prostate cancer cells. Our short antisense AR ODNs were loaded onto the lipid surface of cationic gas-filled microbubbles by ion charge binding, and delivered into the cells by bursting the loaded microbubbles with ultrasound. In vitro experiments were initially performed to show that this kind of delivery system works in principle. In fact, transfection of prostate tumor cells with antisense AR ODNs using microbubble-enhanced ultrasound resulted in 49% transfected cells, associated with a decrease in AR expression compared to untreated controls. In vivo, uptake of a digoxigenin-labelled ODN was found in prostate tumour xenografts in nude mice following intratumoral or intravenous injection of loaded microbubbles and subsequent exposure of the tumour to ultrasound, respectively. Our results show that ultrasound seems to be the driving force of this delivery system. Uptake of the ODN was also observed in tumors after treatment with ultrasound alone, with only minor differences compared to the combined use of microbubbles and ultrasound.

Sense and antisense transcripts of the apolipoprotein E gene in normal and ApoE knockout mice, their expression after spinal cord injury and corresponding human transcripts.

The apolipoprotein E (ApoE) gene has been linked to maladies such as hypercholesterolemia, CNS injury and disease. In this study, we present evidence that, in addition to the known transcript (ApoE S1) that translates into ApoE, there are three additional transcripts in mice. Two of these transcripts, ApoE S2 and ApoE S3, which are predicted to be transmembrane proteins, are transcribed from the sense strand. ApoE AS1 is transcribed from the antisense strand and is complementary to exon 4 of ApoE S1. The open reading frame of ApoE AS1 is conserved between human and mouse. The antisense transcript falls within the region of the human epsilon 4 allele that has been linked to the familial onset form of Alzheimer's disease. We also demonstrate the expression of ApoE S3 and ApoE AS1 in ApoE knockout mice, and ApoE S1 and ApoE S2 do not get transcribed. We had previously identified ApoE S1 as being upregulated in mice after spinal cord injury. In this study, we show that in spinal cord-injured C57BL/6 mice, both ApoE S1 and ApoE S3 transcripts are 10-fold upregulated and the antisense ApoE AS1 is 100-fold upregulated compared with normal levels. Such data suggest that these alternate transcripts are involved in the molecular pathogenesis of CNS disease and perhaps in ApoE expression in general, as we show that ApoE S2 and AS1 are also transcribed in human.

Matrix metalloproteinase activity correlates with blastema formation in the regenerating MRL mouse ear hole model.

The MRL mouse was proposed as a model of mammalian regeneration because it can close ear holes completely with the restoration of normal tissue. This regeneration process involves the formation of a blastema during healing, the re-appearance of cartilage and hair follicles, and healing without scarring. Such a process requires extensive tissue remodeling. To characterize differences in ear wounding responses between regenerating and nonregenerating mice, we examined and compared the extracellular matrix remodeling and the matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) response in the MRL and C57BL/6 mouse strains after injury. We found a correlation between the MRL's ability to break down the basement membrane, form a blastema, and close ear hole wounds and an inflammatory response with neutrophils and macrophages seen in the ear after injury. These cells were positive for MMP-2 and MMP-9 as well as TIMP-2 and TIMP-3. Clear differences between the MRL and B6 response to injury were seen that could explain the differences in healing and blastema formation in the MRL and lack of it in the B6 mice. This finding was further supported by enzyme activity as determined by gelatin zymography.

Apolipoprotein E expression after spinal cord injury in the mouse.

Apolipoprotein E (apo-E), a protein involved in lipid metabolism and cholesterol transport, has been found to be up-regulated in CNS injury and is associated with Alzheimer's disease in humans. In this study, we show that apo-E is also up-regulated after complete spinal cord transection in the C57BL/6 mouse. In the uninjured cord, the cellular localization of apo-E protein is in astrocytes, in individual neurons throughout the laminae except for the dorsal horn, and in endothelial cells of capillaries in the immediate vicinity of those neurons. After injury, RNA levels are elevated as early as 4 days and reach a maximal level between 1 and 2 weeks. Protein levels follow closely but remain up-regulated beyond 3 weeks. Early on, the protein can be found in neutrophils and macrophages at the injury site and only at later times in astrocytes during the remodeling of white matter tracts, most prominently in degenerating parts of the fasciculus gracilis.

Expression of preadipocyte factor-1(Pref-1), a delta-like protein, in healing mouse ears.

Preadipocyte factor-1 (Pref-1), a delta-like protein containing epidermal growth factor-repeats, is expressed in proliferating cells in a variety of tissues and is believed to be involved in maintaining the undifferentiated state of these cells. Using microarray analysis, reverse transcriptase-polymerase chain reaction, in-situ hybridization, and immunohistochemistry, we have identified Pref-1 expression in the healing ears of two strains of mice, MRL and C57BL/6. MRL is unusual in that ear punches completely regenerate the ear tissue along with new cartilage with no scarring. Pref-1 is more highly expressed in the MRL wounds, is uniquely found in a condensation of cells within the regenerating tissue of the blastema, and may contribute to the regenerative capacity of the MRL ear wound.