1,721,147 research outputs found
Phage lytic enzymes
Full text linkPhage lytic enzymes are enzymes produced by bacterial viruses, either as part of their virion to facilitate bacterial infection through local peptidoglycan degradation, or as soluble proteins to induce massive cell lysis at the end of the lytic replication cycle [...
The preclinical and clinical progress of bacteriophages and their lytic enzymes : the parts are easier than the whole
Full text linkThe therapeutic potential of phages has been considered since their first identification more than a century ago. The evident concept of using a natural predator to treat bacterial infections has, however, since then been challenged considerably. Initially, the vast success of antibiotics almost eliminated the study of phages for therapy. Upon the renaissance of phage therapy research, the most provocative and unique properties of phages such as high specificity, self-replication and co-evolution prohibited a rapid preclinical and clinical development. On the one hand, the typical trajectory followed by small molecule antibiotics could not be simply translated into the preclinical analysis of phages, exemplified by the need for complex broad spectrum or personalized phage cocktails of high purity and the more complex pharmacokinetics. On the other hand, there was no fitting regulatory framework to deal with flexible and sustainable phage therapy approaches, including the setup and approval of adequate clinical trials. While significant advances are incrementally made to eliminate these hurdles, phage-inspired antibacterials have progressed in the slipstream of phage therapy, benefiting from the lack of hurdles that are typically associated with phage therapy. Most advanced are phage lytic enzymes that kill bacteria through peptidoglycan degradation and osmotic lysis. Both phages and their lytic enzymes are now widely considered as safe and have now progressed to clinical phase II to show clinical efficacy as pharmaceutical. Yet, more initiatives are needed to fill the clinical pipeline to beat the typical attrition rates of clinical evaluation and to come to a true evaluation of phages and phage lytic enzymes in the clinic
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Accelerating the lysin hit-to-lead pipeline through metagenomic lysin identification, modular engineering and ultrahigh-throughput screening
No full textThe escalating global antimicrobial resistance crisis necessitates the exploration of alternative antimicrobial strategies. For nearly a century, antibiotics have been the cornerstone of antimicrobial therapy, effectively treating a wide range of bacterial infections. However, widespread misuse and overuse of antibiotics have driven the emergence and spread of resistant bacterial strains, rendering even last-resort antibiotics ineffective in the case of pandrug-resistant strains. This crisis has been exacerbated by the lack of success in developing new conventional antibiotic classes, highlighting the urgent need for innovative approaches.
Bacteriophage-encoded lysins offer a promising alternative due to their targeted mode of action by degrading the bacterial peptidoglycan, their reduced potential for resistance development, and their modular structure, which facilitates customization of drug properties through modular engineering. While initial research focused on naturally occurring lysins, subsequent engineering efforts have expanded their therapeutic potential. However, translating these promising candidates into clinically approved treatments remains a significant challenge.
To overcome this, an efficient hit-to-lead pipeline for the discovery, engineering, and screening of lysin variants are essential to accelerate progress. This dissertation specifically addressed these three critical aspects of the lysin hit-to-lead pipeline. First, metagenomic databases and approaches were used to uncover endolysins present in the untapped source of uncultivable bacteriophages. Secondly, the new VersaTile technique was used as an engineering method to assess the impact of different lysin domain architectures on activity, and thirdly, an ultrahigh-throughput, microfluidic-based method for the screening of lysin activity was investigated.
Metagenomics is the study of genetic material (metagenomes) directly extracted from microorganisms, in this case bacteriophages, collected from environmental samples. This approach provides a comprehensive view of the natural, mainly unexplored diversity among bacteriophages and their lysins. This dissertation focused on two primary metagenomic strategies: sequence-based metagenomics, which involves analysing the extracted and sequenced DNA sequences of the bacteriophages to predict their functions, and functional metagenomics, which directly evaluates the activity of the bacteriophage DNA without relying on prior DNA sequence information.
The sequence-based metagenomic approach began with the selection of six lysin candidates from a metagenomic dataset comprising predicted endolysins for detailed characterization. These endolysins were selected for their unannotated domains, interesting ecosystems and predicted bacterial hosts, and underwent in-depth identification through structural and sequence distance-based analyses and functional characterization. Functional characterization through turbidity reduction assays elucidated the host ranges and the putative activity mechanisms. Additionally, a Random Forest model was developed to predict endolysins within protein sequence datasets or phage genomes. This model was applied to a metagenomic dataset, revealing valuable insights into the physicochemical properties of unpredicted metagenomic endolysins. It was also used on a phage genome with an unknown endolysin, generating a list of putative candidates, thereby expediting the annotation process.
The functional metagenomic approach bypasses the reliance on sequence homology when functionally characterizing the phage-derived lysins. Typically, the extracted metagenomic DNA is first fragmented into smaller sequences to capture genes within these fragments. Subsequently, these fragments are inserted into expression vectors, generating libraries of metagenomic sequences. Each fragment is then individually expressed and screened for the presence of enzymatic activity. In this dissertation, a workflow for constructing these libraries was successfully developed using two control phage genomes. Specifically, libraries derived from a Gram-positive-infecting and a Gram-negative-infecting bacteriophage were created and screened using a halo-based assay, where halo formation indicates lysin activity. Halos were observed in the libraries for a model phage infecting Gram-positive Staphylococcus aureus, indicating the presence of complete endolysin genes within the plasmid inserts. Additionally, near-complete coverage of the phage genomes was achieved. These results highlight the feasibility and potential of this approach for metagenomic applications, specifically for discovering novel lysins in environmental samples.
To further enhance the engineering capacities of the lysin hit-to-lead pipeline, a screening project targeting Klebsiella pneumoniae with an unprecedented throughput was performed. Specifically, the VersaTile technique was used to create five combinatorial libraries with varying lysin architectures, which were subsequently tested. A total of 940 variants were evaluated using a microtiter plate-based growth inhibition assay, with 53 variants from the library with the most active configuration sequenced for further analysis. This led to the identification of certain design rules under the tested conditions, which could guide future engineering efforts. Notably, some enzymatically active domains (EADs) and outer membrane-permeabilizing peptides (OMPs) were found to be overrepresented, and introducing a linker when fusing an OMP to the lysin structure was observed to have a positive effect.
To increase the screening throughput, an ultrahigh-throughput screening assay was explored and optimized using a microdroplet platform. This approach involved pico-injection steps for adding necessary reaction components and droplet sorting based on fluorogenic substrate conversion after bacterial lysis. Lysin expression was accomplished through a cell-free system. Although further optimizations are required, the integration of the respective assay miniaturization, fluorescence detection method, and cell-free expression technique showed considerable promise for future ultrahigh-throughput screening of novel lysins from both metagenomic libraries and engineered combinatorial variant libraries.
This dissertation successfully explored, investigated, and highlighted the potential of the methodologies applied to each key aspect of this specific lysin hit-to-lead pipeline, demonstrating its promising and valuable contribution to the field of lysin research
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Enzyme colocalisation on synthetic protein scaffolds : a VersaTile approach
No full textIn nature, enzymes are often colocalised to promote substrate channeling and to enhance synergism.
Synthetic protein scaffolds are state-of-the-art multimodular proteins that can be employed to mimic
this natural phenomenon and bring multiple enzymes in close proximity in a laboratory environment.
A synthetic protein scaffold is composed of a range of cohesins, which are separated by linkers.
Enzymes can be recruited to the scaffold by fusing them to dockerins that have a strong affinity for
cohesins. Many different parameters have an influence on the efficiency of these multi-enzyme
complexes. On the one hand, the selection of cohesin, dockerin, enzyme and linker domains has a
major impact. On the other hand, protein engineers are also able to control the spatial organisation
and enzyme stoichiometry within the scaffold. The optimisation of these multi-modular protein
complexes is therefore a complex and often empirical process. Due to the high modularity of the
interacting proteins and the extensive DNA work to prepare all components, this is a particularly
tedious job.
In this work, we describe the extension of the VersaTile technique, a rapid DNA assembly method for
modular proteins, to allow the efficient construction of synthetic protein scaffolds and their
corresponding docking enzymes. As a proof of concept, we illustrate the multiparametric optimisation
of three distinct multi-enzyme complexes. Two of the constructed complexes are catabolic, allowing
the complete degradation of hemicelluloses galactomannan and xyloglucan, respectively. The final
complex is anabolic, incorporating three pathway enzymes to convert a simple glucose monomer to
fructose-1,6-biphosphate. With VersaTile, we have removed a major hurdle in the field. However, the
in vitro production and optimisation process remains labour intensive. Future research should
therefore focus on the parallel production and characterisation of synthetic protein scaffolds. Tackling
this last hurdle will allow us to fully exploit both the combinatorial power offered by VersaTile and the
opulence provided by nature
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