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Chronic lymphocytic leukemia (CLL) is a B-cell malignancy mainly occurring at an advanced age with no single major genetic driver. Transgenic expression of TCL1 in B cells leads after a long latency to a CLL-like disease in aged Eµ-TCL1 mice suggesting that TCL1 overexpression is not sufficient for full leukemic transformation. In search for secondary genetic events and to elucidate the clonal evolution of CLL, we performed whole exome and B-cell receptor sequencing of longitudinal leukemia samples of Eµ-TCL1 mice. We observed a B-cell receptor stereotypy, as described in patients, confirming that CLL is an antigen-driven disease. Deep sequencing showed that leukemia in Eµ-TCL1 mice is mostly monoclonal. Rare oligoclonality was associated with inability of tumors to develop disease upon adoptive transfer in mice. In addition, we identified clonal changes and a sequential acquisition of mutations with known relevance in CLL, which highlights the genetic similarities and therefore, suitability of the Eµ-TCL1 mouse model for progressive CLL. Among them, a recurrent gain of chromosome 15, where Myc is located, was identified in almost all tumors in Eµ-TCL1 mice. Interestingly, amplification of 8q24, the chromosomal region containing MYC in humans, was associated with worse outcome of patients with CLL., Clonal evolution and genomic alterations in the Eµ-TCL1 mouse model mirror human CLL and identify Myc as oncogenic hit associated with disease progression in patients.

Synthesis of a novel series of hydrazine clubbed 1,3-thiazoles (5a–m) has been described by reacting hydrazine-1-carbothioamides (3a–k) with α-chloro- or bromo-acetophenones (4a–d) in refluxing ethanol in good to excellent yields (65–86%). Structural confirmation was based upon spectroscopic techniques such as 1H-NMR, 13C-NMR, FT-IR and mass spectrometry. The biological application of these motifs has been demonstrated in terms of their strong urease inhibition activity. The results of in vitro study revealed that all the compounds are the potent inhibitors of urease. The IC50 (ranging in between 110 and 440 nM) values were higher as compared to that of standard, i.e., thiourea (IC50 = 490 ± 10 nM). The synthesized compounds were docked at the active sites of the Jack bean urease enzyme in order to explore the possible binding interactions of enzyme–ligand complexes; the results reinforced the in vitro biological activity results.

In vivo reprogramming of somatic cells into induced pluripotent stem cells (iPSC) holds vast potential for basic research and regenerative medicine. However, it remains hampered by a need for vectors to express reprogramming factors (Oct-3/4, Klf4, Sox2, c-Myc; OKSM) in selected organs. Here, we report OKSM delivery vectors based on pseudotyped Adeno-associated virus (AAV). Using the AAV-DJ capsid, we could robustly reprogram mouse embryonic fibroblasts with low vector doses. Swapping to AAV8 permitted to efficiently reprogram somatic cells in adult mice by intravenous vector delivery, evidenced by hepatic or extra-hepatic teratomas and iPSC in the blood. Notably, we accomplished full in vivo reprogramming without c-Myc. Most iPSC generated in vitro or in vivo showed transcriptionally silent, intronic or intergenic vector integration, likely reflecting the increased host genome accessibility during reprogramming. Our approach crucially advances in vivo reprogramming technology, and concurrently facilitates investigations into the mechanisms and consequences of AAV persistence., 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.

Tuberculosis (TB) is an infectious chronic bacterial disease caused by Mycobacterium tuberculosis (MTB). More than 95% of cases and deaths are reported in developing countries. Tuberculosis affects all age groups but adults are mostly affected in their most productive age. However, all age groups are at risks. In theworld, TB is one of the major killer infectious diseases, which place it in one of the top 5 causes of death for females aged 15 to 44. The death rate due to this isease has declined by 47% from 1990 to 2015.Objective: This study was conducted to determine the demographic parameters of patients of Tuberculosis at public sector health facilities Lahore, Pakistan.Methods: A cross sectional study was conducted in outpatient Department of chest Medicine in Public sector health facilities Lahore, Pakistan from 1st January 2017 to 30th June 2017 after informed consent from Tuberculosis patients. After simple random sampling 1120 patients coming from different parts of the Punjabprovince were included in this study. Data were collected and analyzed by SPSS version 22. Results: A total of 1120 patients were included in this study; the mean age of cases was 33 ± 16.76 years with minimum and maximum age of 15 and 100 years. The median and mode of ages was 27 and 15 years, respectively. The most common age group was 15-24 years that comprised of 473(42.23%) of the cases. A total of 529(47.23%) patients were male and 591(52.77%) were female in this study. In male and female cases, frequency of cases was decreased as age increased. Both male and female cases had almost similar distribution of age. There were 830(74.11%) cases those belonged to Lahore while 25.895% were from other cities. Approximately 48.96% males and 38.92% females were educated, 37.24% males and 52.79% females had monthly income 10,000 or less whereas 34.82% males and 25.04% females had monthly income between ten thousands and thirty thousands.38.75% males and 44.84% females belonged to lower class.1004 (89.64%) cases were relapsed and 116(10.36%) were new cases. 366(32.68%) cases had pulmonary tuberculosis (PTB) and 754(67.32%) cases had extra pulmonary tuberculosis.Conclusion: There were 830(74.11%) cases that belonged to Lahore while 25.895 were from other cities. Majority of the cases were relapsed after treatment. The extra pulmonary lymph node was the most frequent site for involvement.

Members of the ectonucleoside triphosphate diphosphohydrolases (NTPDases) constitute the major family of enzymes responsible for the maintenance of extracellular levels of nucleotides and nucleosides by catalyzing the hydrolysis of nucleoside triphosphate (NTP) and nucleoside diphosphates (NDP) to nucleoside monophosphate (NMP). Although, NTPDase inhibitors can act as potential drug candidates for the treatment of various diseases, there is lack of potent as well as selective inhibitors of NTPDases. The current study describes the synthesis of a number of carboxamide derivatives that were tested on recombinant human (h) NTPDases. The most promising inhibitors were 2h (h-NTPDase1, IC50: 0.12 ± 0.03 µM), 2d (h-NTPDase2, IC50: 0.15 ± 0.01 µM) and 2a (h-NTPDase3, IC50: 0.30 ± 0.04 µM; h-NTPDase8, IC50: 0.16 ± 0.02 µM). Four compounds (2e, 2f, 2g and 2h) were associated with the selective inhibition of h-NTPDase1 while 2b was identified as a selective h-NTPDase3 inhibitor. Considering the importance of NTPDase3 in the regulation of insulin release, the NTPDase3 inhibitors were further investigated to elucidate their role in the insulin release. The obtained data suggested that compound 2a was actively participating in regulating the insulin release without producing any effect on NTPDase3 mRNA. Moreover, the most potent inhibitors were docked within the active site of respective enzyme and the observed interactions were in compliance with in vitro results. Hence, these compounds can be used as pharmacological tool to further investigate the role of NTPDase3 coupled to insulin release.

Overexpression of NTPDases leads to a number of pathological situations such as thrombosis, and cancer. Thus, effective inhibitors are required to combat these pathological situations. Different classes of NTPDase inhibitors are reported so far including nucleotides and their derivatives, sulfonated dyes such as reactive blue 2, suramin and its derivatives, and polyoxomatalates (POMs). Suramin is a well-known and potent NTPDase inhibitor, nonetheless, a range of side effects are also associated with it. Reactive blue 2 also had non-specific side effects that become apparent at high concentrations. In addition, most of the NTPDase inhibitors are high molecular weight compounds, always required tedious chemical steps to synthesize. Hence, there is still need to explore novel, low molecular weight, easy to synthesize, and potent NTPDase inhibitors. Keeping in mind the known NTPDase inhibitors with imine functionality and nitrogen heterocycles, Schiff bases of tryptamine, 1–26, were synthesized and characterized by spectroscopic techniques such as EI-MS, HREI-MS, 1H-, and 13C NMR. All the synthetic compounds were evaluated for the inhibitory avidity against activities of three major isoforms of NTPDases: NTPDase-1, NTPDase-3, and NTPDase-8. Cumulatively, eighteen compounds were found to show potent inhibition (Ki = 0.0200–0.350 μM) of NTPDase-1, twelve (Ki = 0.071–1.060 μM) of NTPDase-3, and fifteen compounds inhibited (Ki = 0.0700–4.03 μM) NTPDase-8 activity. As a comparison, the Kis of the standard inhibitor suramin were 1.260 ± 0.007, 6.39 ± 0.89 and 1.180 ± 0.002 μM, respectively. Kinetic studies were performed on lead compounds (6, 5, and 21) with human (h-) NTPDase-1, -3, and -8, and Lineweaver-Burk plot analysis showed that they were all competitive inhibitors. In silico study was conducted on compound 6 that showed the highest level of inhibition of NTPDase-1 to understand the binding mode in the active site of the enzyme.

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site ( trs )-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

As tools for quantitative label-free mass spectrometry (MS) rapidly develop, a consensus about the best practices is not apparent. In the work described here we compared popular statistical methods for detecting differential protein expression from quantitative MS data using both controlled experiments with known quantitative differences for specific proteins used as standards as well as "real" experiments where differences in protein abundance are not known a priori. Our results suggest that data-driven reproducibility-optimization can consistently produce reliable differential expression rankings for label-free proteome tools and are straightforward in their application.

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

For biosafety assessment and the understanding of viral integration mechanisms the determination of the exact position and distribution of viral integration sites in the host genome is crucial. Current methods for mapping of vector insertion sites are based on primer binding within the vector genome, its elongation into the host genome, ligation of a common adapter on the host part, and subsequent PCRs. Thus, these methods depend on the presence of the primer binding site in the viral genome and biases are introduced during restriction digest (if applied), ligation, and PCR. Here, we present results from a targeted sequencing approach (SureSelect, Agilent) in which we enriched for genomic regions including vector sequences. This approach has major advantages over primer based approaches like LAM PCR: •As less PCR cycles are needed, a more quantitative estimation of cell clonality (clones with the sample integration site) should be possible.•Genomic regions that are captured together with the vector allow for relative quantification of vector copies per genome.•Integrations of incomplete vectors (common for adeno-associated vectors) can be detected without the need of specific primers.•As the entire vector is sequenced, mutations within the vector (and transgene) can be detected.Using control samples we defined the sensitivity, background, and dynamic quantitative range of this method. As the entire vector is sequenced, mutations within the vector (and transgene) can be detected. In summary, targeted sequencing provides an important complementary tool to primer based approaches (like LAM-PCR) for mapping of vector/viral integration sites.

Over the last two decades, gene therapy has shown rapid advancements as a promising approach to treat genetic diseases by introducing corrected genes into patient cells. Viruses are the most common carriers in the vector-mediated gene therapy. However, integration of viral vectors at undesirable genomic locations can lead to deleterious effects, e.g. insertional mutagenesis. Therefore, an efficient, stable and safe vector system is the major prerequisite for a successful gene therapy. Long term monitoring of the distribution pattern of vector integration sites (IS) is the most feasible strategy to address vector safety and stability concerns.Recent advancements in next generation sequencing technologies have dramatically increased the possibility to generate substantial amount of vector-genome sequencing data for comprehensive IS analysis. An efficient downstream analysis of this data requires automated and fast computational methods. Here, we present Genome Insertion Site (GENIS) pipeline, a suite for time-efficient and reliable analysis of vector-genome junctions. GENIS has been designed to analyze the sequencing data generated from traditional linear amplification mediated PCR (LAM-PCR) based methods and also from new targeted DNA single and paired end sequencing technologies (e.g., Agilent SureSelect). Our suite consists of six basic modules including barcode sorting, quality filtering and adapter trimming, mapping of sequence reads to the reference genome, extraction of soft-clip reads and clustering of IS for subsequent annotation.GENIS is implemented on Linux platform with minimum external software dependencies. Users can adjust the required parameters in the provided configuration file. It takes about 30 minutes for complete processing, starting from raw reads till annotation, of 10 million paired end reads generated by targeted sequencing. In case of LAM-PCR data, 30 million reads are sorted in about 30 minutes (50 different PCR) and time required for rest of processing to obtain annotated IS is also approximately 30 minutes for 15 million reads. Three final files present the conclusion of the analysis process and contain: 1) the information about read ID, chromosome position (genomic IS), vector position (vector IS), sequence, genomic and vector orientation and sequence span; 2) all the clustered IS with their respective sequence count and 3) the annotated IS with respect to nearby genomic features, including gene identifier and gene name, transcription start site, coding region start and end sites etc. Our tool is highly appropriate for in-depth quantitative analysis of biosafety and transduction efficiency of viral vectors.

A subpopulation of tumor-initiating cells (TIC) drives colorectal cancer (CRC) progression in serial xenotransplantation. Strikingly, the CRC TIC compartment itself is heterogeneous and comprised of a hierarchy of long-term (LT-) TIC, tumor transient amplifying cells (T-TAC) and delayed contributing (DC-) TIC. Whether this functionally heterogeneous TIC compartment is genetically homogenous or whether distinct genetic subclones drive the functional heterogeneity of the TIC compartment is yet unknown. To address this question, we performed high coverage (91-126 fold) whole genome sequencing on primary CRC patient tumors (n = 3), corresponding serially passaged TIC enriched spheroids as well as spheroid derived serial xenografts. Sequenced samples harbored between 22.800 and 232.000 synonymous or non-synonymous single nucleotide variants (SNVs). In addition, all samples contained multiple focal or large-scale somatic copy number alterations (CNAs). Clustering of SNVs as well as subclonal copy numbers from serial xenografts and spheroids were used to define SNV- and CNA-based subpopulations. Next, cellular fractions of identified subpopulations were determined and combined applying maximal parsimony to create models of the minimal number of subclones present in each sample. Using this strategy, we found that multiple subclones were present in each sample analyzed. Subclone heterogeneity was maintained during serial in vitro passaging and serial xenografting. Importantly, tumor initiation in xenografts was driven by at least 3 distinct genetic subclones whose relative contribution dynamically changed over time. Strikingly, in serial samples from the same patients, different genetic subclones grew out in vivo and in vitro. To test whether functional heterogeneity of the TIC compartment is related to the presence of genetic subclones, we assessed the contribution of different TIC subtypes - LT-TIC, T-TAC and DC-TIC - at early and late time points of xenografting using secondary genetic marking. Therefore, 1×10⁁5 cells derived from early and late generation xenografts were transduced with an integrating lentiviral vector, thereby generating a stable barcode-like genetic mark which differs in each transduced cell. Following serial transplantation of transduced cells for 3 mouse generations, tumors were harvested and lentiviral integration sites were determined using highly sensitive LAM-PCR and high-throughput sequencing. Assessing the relative contribution of LT-TIC, T-TAC and DC-TIC revealed that the functional heterogeneity of the TIC compartment was preserved despite profound genetic subclone dynamics in serial xenotransplantation. These results strongly indicate that multiple genetic subclones drive long-term tumor formation and that functional heterogeneity of the CRC TIC compartment is not based on specific genetic subclones. Citation Format: Klara M. Giessler, Kortine Kleinheinz, Gnana Prakash Balasubramanian, Daniel Hübschmann, Taronish D. Dubash Rai, Sebastian D. Dieter, Christine Siegl, Christopher M. Hoffmann, Sarah Weber, Raffaele Fronza, Saira Afzal, Manfred Schmidt, Martin Schneider, Alexis Ulrich, Juergen Weitz, Wilko Weichert, Matthias Schlesner, Benedikt Brors, Claudia R. Ball, Hanno Glimm. Genetic subclone heterogeneity of the human colon cancer initiating cell compartment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 910.

In depth study of T-cell receptor (TCR) repertoire, specifically clonal expansion of T cells and diversity of repertoire, can provide invaluable information in various health and diseases states including infection, cancer and genetic disorders etc. Currently, Shannon and Simpson diversity indices used to estimate TCR clonality have certain limitations. These indices are biased either towards total number of different TCR receptor types (richness) or distribution of each clonal type (evenness). We introduced here a new framework for accurate and precise quantification of clonal diversity of TCR cell population that overcomes shortcomings of already available indices. Our concept is based on the generalized form of entropy known as Renyi numbers and two components of diversity, richness and evenness. Based on the Renyi equation, we can prove that at a specific richness value the highest possible evenness obtained cannot go beyond the stated richness value. Plotting richness versus evenness helps to define maximal theoretical polyclonality and monoclonality regions and diversity of sample can be measured by estimating distances of sample from these theoretical bounds. We have validated our method on in-silico data sets and TCR repertoire study. We conclude that our idea of clonal plane and diversity index measurement provides more reliable and robust estimation of immune population diversity. It can prove to be a useful tool for quantitative characterization of TCR repertoire in immunological studies.

Comprehensive analysis of deep sequencing data originating from the newly introduced Targeted Enrichment Sequencing (TES) indicates so far undescribed recombinations within the Inverted Terminal Repeats of recombinant Adeno-Associated Viruses (rAAVs). For the detection of vector integration sites into the host genome we routinely apply LAM-PCR. However, TES, in which we enrich for genomic regions that include vector sequences, has major advantages over LAM-PCR, as it neither depends on the existence of a vector-specific primer binding site (often lost during rAAV integration) nor on a restriction site in the vicinity of the vector insertion site. Furthermore, regions that are captured together with the vector allow for relative quantification of vector copies per genome. As the entire vector can be sequenced by TES, mutations within the vector and transgene can be detected. In summary, we here provide new and so far unpublished information about rAAV integration patterns and show how we optimized TES to become an important complementary tool to primer-based approaches (like LAM-PCR) for mapping of vector/viral integration sites.

Purpose: To synthesise a new series of 5-substituted-1,3,4-Oxadiazol-2yl-4-(morpholin-4yl sulfonyl)benzyl sulfide and evaluate their antibacterial activity. Methods: Different organic acids were converted consecutively into corresponding esters, hydrazides and 5-substituted-1,3,4-Oxadiazol-2-thiols ( 4a-e ). The targets, 6a-e were synthesized by stirring 4a-e with 4-(4-(bromomethyl)phenylsulfonyl) morpholine ( 5 ) in the presence of N,N-dimethylformamide (DMF) and sodium hydride (NaH). All the structures were elucidated by modern spectroscopic techniques and screened against bacteria using standard procedure and ciprofloxacin drug as positive control. Results: The yield of the synthesized compounds ( 4a-e and 6a-e ) were moderate (65 - 90 %). Compounds 6a-e had antibacterial activity against Pseudomonas aeruginosa, Bacillis subtilis and Staphylococcus aureus while some had activity against the other bacteria used. One of the compounds, 6b , exhibited significant activity against all the bacterial strains, i.e., S. typhi (-), E. coli (-), K. pneumoniae (-), P. aeruginosa (-), B. subtilis (+) and S. aureus (+) with MIC (μM) values of 11.01 ± 0.31, 15.37 ± 3.33, 16.11 ± 1.14, 9.70 ± 1.96, 10.01 ± 2.70 and 9.15 ± 0.29, respectively. However, none of the compounds had any inhibitory activity against any bacteria as high as that of ciprofloxacin. Conclusion: Five new compounds with antibacterial activities have been synthesized. Their potential as therapeutic agents is, however, yet to be evaluated. Keywords: 1,3,4-Oxadiazole, Benzyl sulfide, 4-(4-(bromomethyl)phenylsulfonyl)morpholine, Spectral analysis, Antibacterial activity

To establish latency, wildtype adeno-associated virus (wtAAV) is able to integrate its DNA into a specific region on chromosome 19 of the human genome, named AAVS1. This targeted integration mechanism relies on the function of the two larger versions (Rep78/68) of the Rep protein family, expressed by wtAAV. Rep is able to bind a 12 bp long sequence, called Rep binding site (RBS). The RBS can be found both in the viral ITRs and in AAVS1. By the formation of a trimeric complex between wtAAV, Rep and the genomic locus, targeted integration can be achieved. However, the exact mechanisms of Rep-mediated integration still remain unknown. To analyze wtAAV IS distribution genome-wide, large-scale integration analyses using high-throughput next generation sequencing have been performed. These studies revealed that Rep-mediated wtAAV integration preferentially occurs in AAVS1, but that additional integration hotspots associated with RBS can be found as well throughout the genome. Up to this point most integration sites (IS) reported for wtAAV were derived from vector-genome fusion sequences, where the viral sequence fragment was mapped near the ITRs or near the p5 region of the viral genome. Most currently available methods for the analysis of viral IS rely on amplification of the vector-genome junction with primers that bind in the vicinity of the viral ends. Therefore, knowledge about the nature and structure of the internal wtAAV genome remains limited. To address this question, we investigated ~3e6 wtAAV concatemeric structures generated by IS analyses using linear amplification-mediated (LAM) PCR to identify potential alternative breakpoints throughout the wtAAV genome. We discovered that breakpoints occur at any position of the wtAAV genome. Apart from the ITRs and the p5 promoter, there are a number of other preferred breakpoints throughout the wtAAV genome. Using a novel approach involving targeted enrichment of AAV and AAVS1 sequences, we also were able to consider viral IS at any position of the AAV genome. Targeted HiSeq sequencing of wtAAV and AAVS1 determined several hundreds of exact IS and confirmed that wtAAV genome junctions form at any position of its genome. Those cryptic integration events, not picked up by the majority of methods currently available, shed new light onto wtAAV persistence and may further improve current AAV persistence analyses in gene therapy.