SHT-based public auditing protocol with error tolerance in FDL-empowered IoVs

Peer reviewe

Addressing intrinsic challenges for next generation sequencing of immunoglobulin repertoires.

textAntibodies are essential molecules that help to provide immunity against a vast population of environmental pathogens. This antibody conferred protection is dependent upon genetic diversification mechanisms that produce an impressive repertoire of lymphocytes expressing unique B-cell receptors. The advent of high throughput sequencing has enabled researchers to sequence populations of B-cell receptors at an unprecedented depth. Such investigations can be used to expand our understanding of mechanistic processes governing adaptive immunity, characterization of immunity related disorders, and the discovery of antibodies specific to antigens of interest. However, next generation sequencing of immunological repertoires is not without its challenges. For example, it is especially difficult to identify biologically relevant features within large datasets. Additionally, within the immunology community, there is a severe lack of standardized and easily accessible bioinformatics analysis pipelines. In this work, we present methods which address many of these concerns. First, we present robust statistical methods for the comparison of immunoglobulin repertoires. Specifically, we quantified the overlap between the antibody heavy chain variable domain (V H ) repertoire of antibody secreting plasma cells isolated from the bone marrow, lymph nodes, and spleen lymphoid tissues of immunized mice. Statistical analysis showed significantly more overlap between the bone marrow and spleen VH repertoires as compared to the lymph node repertoires. Moreover, we identified and synthesized antigen-specific antibodies from the repertoire of a mouse that showed a convergence of highly frequent VH sequences in all three tissues. Second, we introduce a novel algorithm for the rapid and accurate alignment of VH sequences to their respective germline genes. Our tests show that gene assignments reported from this algorithm were more than 99% identical to assignments determined using the well-validated IMGT software, and yet the algorithm is five times faster than an IgBlast based analysis. Finally, in an effort to introduce methods for the standardization, transparency, and replication of future repertoire studies, we have built a cloud-based pipeline of bioinformatics tools specific to immunoglobulin repertoire studies. These tools provide solutions for data curation and long-term storage of immunological sequencing data in a database, annotation of sequences with biologically relevant features, and analysis of repertoire experiments.Chemical Engineerin

In silico prediction of regulators of neuronal identity through phylogenetic footprinting

How individual neurons in a nervous system give rise to complex function, behavior and consciousness in higher animals has been studied for over a century, yet scientist have only begun to understand how brains work at the molecular level. This level of study is made possible through technological advances, especially transgenic analysis of the cells that make up nervous systems. To date, no other system has been used as extensively as the nematode Caenorhabditis elegans in this pursuit. With just 302 neurons in the adult hermaphrodite, extensive neuronal maps at the anatomical, functional, and molecular level have been built over the past 30 years. One way to understand how nervous systems develop and differentiate into diverse cell types such as sensory or motor neurons that make higher level behaviors possible, is to unravel the underlying gene regulatory programs that control development. Throughout my PhD I investigated neuron type identity regulators to understand how nervous system diversity is generated and maintained using several bioinformatic approaches. First, I developed a software program and community resource tool, TargetOrtho, useful for identifying novel regulatory targets of transcription factors such as the cell type selector proteins termed terminal selectors evidenced to control terminal cell identity of 74 of the 118 neuron types in C. elegans. Analysis of terminal selector candidate target genes led to the further discovery that predicted target genes with cis-regulatory binding sites are enriched for neuron type specific genes suggesting an overarching theme of direct regulation by terminal selectors to specify cell type. Using this knowledge, I make predictions for novel regulators of neuronal identity to further elucidate how the C. elegans nervous system diversifies into 118 neuron types

Investigation of Factors Influencing var Gene Expression in Plasmodium falciparum Parasites from Acute and Chronic Infections

Im Jahre 2015 gab es weltweit 214 Millionen neue Malariafälle und 438.000 Todesfälle durch Malaria. Der einzellige Parasit Plasmodium falciparum (P. falciparum) verursacht die schwerste Form der Malaria und ist verantwortlich für die Mehrheit der auftretenden Todesfälle. Durch die Expression des Plasmodium falciparum erythrocyte membrane proteins 1 (PfEMP1) auf der Oberfläche von infizierten Erythrozyten können diese an Endothelrezeptoren des Wirts adhärieren, was zu den Symptomen der Malaria führt. PfEMP1 wird von der Familie der hypervariablen var-Gene kodiert. Jeder Parasit besitzt etwa 60 verschiedene var-Gene, von denen jeweils nur eins pro Parasit exprimiert wird. Ein ständiger Wechsel des aktiv transkribierten var-Lokus führt zur Antigenvariation, die es dem Parasit ermöglicht, der Immunantwort des Wirtes zu entgehen. Im ersten Projekt dieser Dissertation konnte gezeigt werden, dass die Moskito- und Humanpassage bei malaria-naiven Individuen die var-Gen Transkription grundlegend verändert. Die in vitro var-Gen Transkription wird maßgeblich durch die Replikationsdauer von P. falciparum im Wirt beeinflusst. Je länger eine Parasitenpopulation der Rezeptorselektion im Wirt ausgesetzt ist, umso mehr verschiebt sich die var-Gen Transkription zugunsten von wenigen, sehr stark transkribierten var-Genen. Des weiteren wurde herausgefunden, dass die var-Gen Transkription in Abwesenheit eines Selektionsdrucks scheinbar vor allem durch ein festgelegtes genetisches Programm definiert wird. Die Daten aus dem zweiten Projekt dieser Dissertation weisen darauf hin, dass PfEMP1 nicht allein für das variable Oberflächensignal von Parasiten in chronischen Infektionen verantwortlich ist, sondern andere variable Oberflächenproteine, wie z.B. die STEVOR und RIFIN Proteinfamilien, am variablen Obeflächensignal beteiligt sind. Zudem scheint die Rezeptorselektion in naiven Wirten und die Antikörperantwort in semi-immunen Wirten die var-Gen Expression zu beeinflussen. Im dritten Projekt dieser Dissertation konnte in zwei von 10 Feldisolaten aus verschiedenen afrikanischen Regionen ein var-Gen gefunden werden, welches ebenfalls konserviert ist. Die Feldisolate wiesen ansonsten eine hohe Diversität in den nichtkodierenden Regionen auf. Dies weist auf eine Selektion gegen die Diversität dieses spezifischen var-Genes hin. In zukünftigen Untersuchungen mit kontrollierten Malariainfektionen (controlled human malaria infections (CHMI)) von malaria-naiven und semi-immunen Individuen könnte der Einfluss der Selektionsdrücke des Wirtes auf die var-Gen-Expression und die Rolle der anderen variable Oberflächenprotein bei der Antigenvariation untersucht werden

Proceedings of the 21st Conference on Formal Methods in Computer-Aided Design – FMCAD 2021

The Conference on Formal Methods in Computer-Aided Design (FMCAD) is an annual conference on the theory and applications of formal methods in hardware and system verification. FMCAD provides a leading forum to researchers in academia and industry for presenting and discussing groundbreaking methods, technologies, theoretical results, and tools for reasoning formally about computing systems. FMCAD covers formal aspects of computer-aided system design including verification, specification, synthesis, and testing

Quantum Cloning Machines and the Applications

No-cloning theorem is fundamental for quantum mechanics and for quantum information science that states an unknown quantum state cannot be cloned perfectly. However, we can try to clone a quantum state approximately with the optimal fidelity, or instead, we can try to clone it perfectly with the largest probability. Thus various quantum cloning machines have been designed for different quantum information protocols. Specifically, quantum cloning machines can be designed to analyze the security of quantum key distribution protocols such as BB84 protocol, six-state protocol, B92 protocol and their generalizations. Some well-known quantum cloning machines include universal quantum cloning machine, phase-covariant cloning machine, the asymmetric quantum cloning machine and the probabilistic quantum cloning machine etc. In the past years, much progress has been made in studying quantum cloning machines and their applications and implementations, both theoretically and experimentally. In this review, we will give a complete description of those important developments about quantum cloning and some related topics. On the other hand, this review is self-consistent, and in particular, we try to present some detailed formulations so that further study can be taken based on those results.Comment: 98 pages, 12 figures, 400+ references. Physics Reports (published online

Nanopore sequencing and assembly of a human genome with ultra-long reads

We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ~30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ~3 Mb). Next, we developed a protocol to generate ultra-long reads (N50 > 100kb, up to 882 kb). Incorporating an additional 5×-coverage of these data more than doubled the assembly contiguity (NG50 ~6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4 Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length and closure of gaps in the reference human genome assembly GRCh38

EVALUATION OF GENE EDITING APPROACHES IN HIGHER PLANTS

Advances in gene editing have facilitated new approaches to improve traits of interest in plants. Specifically, CRISPR-Cas9 and associated endonuclease systems allow for the targeting of agriculturally important genes such as disease resistance, improving yield and shelf life, and changing specific traits related to a final high-quality product. SpCas9 uses a tracrRNA: crRNA (gRNA) sequence to target the DNA and induce double-stranded breaks at targeted locations. SpCas9 is widely used for genome editing in mammals and plants; however, other promising endonucleases have been discovered with the potential for greater editing efficiency. Particularly, the nucleases Cas12a (also known as Cpf1) and Cms1 are promising for plant transformation and genome editing approaches. Cas12a, discovered in the bacteria Francisella novicida U112, and Cms1, discovered in the genera Microgenomates and Smithella, are smaller enzymes than SpCas9. Both utilize only a gRNA formed by a mature crRNA to target a T-rich PAM sequence and generate cohesive DNA ends. Although successful gene editing has been achieved in several plants, a single approach that is useful, economically efficient, and reliable to use across different crops has been challenging to develop. The first aim of this work was to answer this question among our candidates: Cas12a, Cms1, and SpCas9. We explore efficiency by targeting the GFP gene in N. benthamiana protoplast as an evaluation system.The generation of gene-edited plants requires identifying the most suitable method to deliver the CRISPR system into the plant cell. Therefore, recalcitrant crops to transformation is a challenge for gene editing in certain crops. Moreover obtaining lines without transgenic elements relies on outcrossing the transformed-edited obtained plant to non-transgenic plants. This outcrossing approach is incompatible with several high-value, obligated outcrossing crops. The second aim of this work is to present a novel method of delivering gene editing reagents across graft junctions using transgenic tobacco as the donor and wild-type tobacco as a scion. This innovation expands the capabilities of plant gene editing to generate non-transgenic edited plants.Implementing gene editing in agriculturally relevant cases is a promising tool for addressing challenges (such as generating climate-resilient crops and creating the next generation of elite cultivars). Grapes were reported to produce 74 million tonnes in 2021 (FAO); 70% of the grape production is used for the wine industry. A promising target for editing in wine grapes, an economically significant crop, is the o-methyltransferase-encoding gene VvOMT3. The VvOMT3 protein is involved in the synthesis of methoxypyrazine, which imparts an undesirable green pepper flavor in wines and produces low anthocyanin content, weak color, and poor tannin quality. Removal of this compound could result in important improvements to flavor. Therefore, the third aim of this work is to target the OMT3 gene, using gene editing, for expression knock-out in organogenic cultures from Vitis vinifera (cv. ‘Cabernet Sauvignon’).Knowledge of the efficacy of multiple nuclease systems, plant tissues in which they are most efficient, and their utility in both fundamental and applied systems, is expected to greatly improve the success of future gene editing projects. As new editing technologies improve and broaden to employ additional endonucleases, the success of inducing changes in genes of interest, such as the proposed OMT modification in grapes, becomes increasingly feasible.

Understanding the Imprinting Mechanism of UBE3A for Therapeutic Intervention

Human chromosome 15q11-q13 contains a cluster of imprinted genes that are associated with a number of neurological disorders that exhibit non-Mendelian patterns of inheritance, such as Angelman syndrome (AS) and Prader-Willi syndrome. Angelman syndrome is caused by the loss-of-expression of maternally inherited ubiquitin E3A protein ligase gene (UBE3A). Prader-Willi syndrome is caused by loss-of-function of paternally inherited SNORD116 snoRNAs (small nucleolar RNAs), which are expressed as part of a long polycistronic transcriptional unit (PTU) comprised of SNURF-SNRPN, additional orphan C/D box snoRNA clusters, and the UBE3A antisense transcript (UBE3A-AS). The full-length transcript of PTU, including UBE3A-AS, is only expressed in neurons causing the imprinting of paternal UBE3A. Why this occurs in only neurons remains largely unknown. Furthermore, this neuron-specific imprinting adds additional difficulty for therapeutic intervention. In this dissertation, the imprinting mechanism of UBE3A is examined in detail, while an alternative high-throughput screening (HTS) method for drug discovery in neurons is developed. A combination of bioinformatic and molecular analysis of the human and mouse PTU revealed that UBE3A-AS/Ube3a-AS is extensively processed via 5’ capping 3’polyadenyation and alternative splicing, suggesting that the antisense may have regulatory functions apart from imprinting UBE3A in neurons. Following this discovery, the transcriptional profiles and processing of mouse paternal Ube3a was investigated as literature suggested that imprinted paternal Ube3a, unlike other imprinted genes, was transcribed up to intron 4. This analysis unveiled a fourth Ube3a isoform that terminates within intron 4. Moreover, expression of this isoform correlated with Ube3a-AS expression, suggesting alternative reasons for the imprinting of Ube3a. In addition to the analysis of the imprinting of Ube3a, an alternative solution for drug discovery for central nervous system disorders was developed and validated. Here, an embryonic stem cell-derived neuronal culture system was developed for HTS and tested using the paternal Ube3a^Y FP reporter cell-line. Using a known reactivator of paternal Ube3a, Topotecan - a topoisomerase inhibitor, as a positive control a proof-of-concept study demonstrated the utility of this method for HTS drug discovery. Collectively, these results advance the field and understanding of antisense lncRNAs and provide a versatile tool for drug discovery for neurological disorders