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https://www.readbyqxmd.com/read/28820969/a-disulfide-stabilized-human-vl-single-domain-antibody-library-is-a-source-of-soluble-and-highly-thermostable-binders
#1
Kevin A Henry, Hiba Kandalaft, Michael J Lowden, Martin A Rossotti, Henk van Faassen, Greg Hussack, Yves Durocher, Dae Young Kim, Jamshid Tanha
We have previously shown that incorporation of a second intradomain disulfide linkage into camelid VHH and human VH/VL single-domain antibodies confers increased thermostability. Here, we explored the effects of introducing an additional disulfide linkage, formed between Cys48 and Cys64 (Kabat numbering), into a phage-displayed synthetic human VL library. In comparison to an identical library bearing only the highly conserved Cys23-Cys88 disulfide linkage, the disulfide-stabilized VL library tolerated a similar degree of randomization but retained a higher level of functional diversity after selection with protein L...
August 15, 2017: Molecular Immunology
https://www.readbyqxmd.com/read/28820325/small-alarmone-synthetases-as-novel-bacterial-rna-binding-proteins
#2
Vasili Hauryliuk, Gemma C Atkinson
The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, antibiotic tolerance and pathogenicity. We have recently shown that the Small Alarmone Synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis has an RNA-binding activity (Beljantseva et al. 2017). RelQ's activities as an enzyme and as a RNA-binding protein are mutually incompatible: binding of single-stranded RNA potently inhibits (p)ppGpp synthesis in a sequence-specific manner, and RelQ's enzymatic activity destabilizes the RNA:RelQ complex...
August 18, 2017: RNA Biology
https://www.readbyqxmd.com/read/28818634/mining-na%C3%A3-ve-rabbit-antibody-repertoires-by-phage-display-for-monoclonal-antibodies-of-therapeutic-utility
#3
Haiyong Peng, Thomas Nerreter, Jing Chang, Junpeng Qi, Xiuling Li, Pabalu Karunadharma, Gustavo Martinez, Mohammad Fallahi, Jo Soden, Jim Freeth, Roger R Beerli, Ulf Grawunder, Michael Hudecek, Christoph Rader
Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but in some cases also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity...
August 14, 2017: Journal of Molecular Biology
https://www.readbyqxmd.com/read/28815907/t7-rna-polymerase-driven-inducible-cell-lysis-for-dna-transfer-from-escherichia-coli-to-bacillus-subtilis
#4
Mario Juhas, James W Ajioka
The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E...
August 16, 2017: Microbial Biotechnology
https://www.readbyqxmd.com/read/28815494/use-of-the-dice-dual-integrase-cassette-exchange-system
#5
Alfonso P Farruggio, Mital S Bhakta, Michele P Calos
When constructing transgenic cell lines via plasmid DNA integration, precise targeting to a desired genomic location is advantageous. It is also often advantageous to remove the bacterial backbone, since bacterial elements can lead to the epigenetic silencing of neighboring DNA. The least cumbersome method to remove the plasmid backbone is recombinase-mediated cassette exchange (RMCE). RMCE is accomplished by arranging recombinase sites in the genome and in a donor plasmid such that a recombinase can both integrate the donor plasmid and excise its bacterial backbone...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28813669/the-phage-nucleus-and-tubulin-spindle-are-conserved-among-large-pseudomonas-phages
#6
Vorrapon Chaikeeratisak, Katrina Nguyen, MacKennon E Egan, Marcella L Erb, Anastasia Vavilina, Joe Pogliano
We recently demonstrated that the large Pseudomonas chlororaphis bacteriophage 201φ2-1 assembles a nucleus-like structure that encloses phage DNA and segregates proteins according to function, with DNA processing proteins inside and metabolic enzymes and ribosomes outside the nucleus. Here, we investigate the replication pathway of the Pseudomonas aeruginosa bacteriophages φKZ and φPA3. Bacteriophages φKZ and φPA3 encode a proteinaceous shell that assembles a nucleus-like structure that compartmentalizes proteins and DNA during viral infection...
August 15, 2017: Cell Reports
https://www.readbyqxmd.com/read/28812171/complete-genome-sequence-of-a-novel-virulent-ahjdlikevirus-bacteriophage-that-infects-enterococcus-faecium
#7
Shaozhen Xing, Xianglilan Zhang, Qiang Sun, Jian Wang, Zhiqiang Mi, Guangqian Pei, Yong Huang, Xiaoping An, Kaifei Fu, Lijun Zhou, Baohua Zhao, Yigang Tong
A novel virulent bacteriophage named vB_EfaP_IME199 that specifically infects Enterococcus faecium was isolated and characterized. Its optimal multiplicity of infection was 0.01, and it had a 30 minute outbreak period. High-throughput sequencing revealed that the phage has a dsDNA genome of 18,838 bp with 22 open reading frames. The genome has very low homology to all other bacteriophage sequences in the GenBank database. Run-off sequencing experiments confirmed that vB_EfaP_IME199 has short inverted terminal repeats...
August 16, 2017: Archives of Virology
https://www.readbyqxmd.com/read/28811841/bacteriophages-in-the-gastrointestinal-tract-and-their-implications
#8
Marzanna Łusiak-Szelachowska, Beata Weber-Dąbrowska, Ewa Jończyk-Matysiak, Renata Wojciechowska, Andrzej Górski
The gut microbiota plays an essential role in health and disease of humans. Bacteriophages are the most abundant members of the gut virobiota and display great diversity. Phages can translocate through the mucosa to lymph and internal organs and play a role as regulators of the bacterial population in the gut. Increasing abundance of phages in the gut mucosa may reduce colonization by bacteria. Moreover, phages may have an immunomodulatory role in the immune response in the human gut. The role of phages in inflammatory bowel disease (IBD) remains unknown...
2017: Gut Pathogens
https://www.readbyqxmd.com/read/28811656/phageterm-a-tool-for-fast-and-accurate-determination-of-phage-termini-and-packaging-mechanism-using-next-generation-sequencing-data
#9
Julian R Garneau, Florence Depardieu, Louis-Charles Fortier, David Bikard, Marc Monot
The worrying rise of antibiotic resistance in pathogenic bacteria is leading to a renewed interest in bacteriophages as a treatment option. Novel sequencing technologies enable description of an increasing number of phage genomes, a critical piece of information to understand their life cycle, phage-host interactions, and evolution. In this work, we demonstrate how it is possible to recover more information from sequencing data than just the phage genome. We developed a theoretical and statistical framework to determine DNA termini and phage packaging mechanisms using NGS data...
August 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28811374/recruitment-of-crispr-cas-systems-by-tn7-like-transposons
#10
Joseph E Peters, Kira S Makarova, Sergey Shmakov, Eugene V Koonin
A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference...
August 15, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28811342/bacteriophages-of-gordonia-spp-display-a-spectrum-of-diversity-and-genetic-relationships
#11
Welkin H Pope, Travis N Mavrich, Rebecca A Garlena, Carlos A Guerrero-Bustamante, Deborah Jacobs-Sera, Matthew T Montgomery, Daniel A Russell, Marcie H Warner, Graham F Hatfull
The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are "singletons" with no closely related genomes...
August 15, 2017: MBio
https://www.readbyqxmd.com/read/28808331/pro-and-anti-inflammatory-responses-of-peripheral-blood-mononuclear-cells-induced-by-staphylococcus-aureus-and-pseudomonas-aeruginosa-phages
#12
Jonas D Van Belleghem, Frédéric Clement, Maya Merabishvili, Rob Lavigne, Mario Vaneechoutte
The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile of peripheral blood monocytes from six donors for twelve immunity-related genes (i.e. CD14, CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, IL10, LYZ, SOCS3, TGFBI and TNFA) induced by Staphylococcus aureus phage ISP and four Pseudomonas aeruginosa phages (i...
August 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28808028/immune-evasion-by-a-staphylococcal-inhibitor-of-myeloperoxidase
#13
Nienke W M de Jong, Kasra X Ramyar, Fermin E Guerra, Reindert Nijland, Cindy Fevre, Jovanka M Voyich, Alex J McCarthy, Brandon L Garcia, Kok P M van Kessel, Jos A G van Strijp, Brian V Geisbrecht, Pieter-Jan A Haas
Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN)...
August 14, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28807928/role-of-translesion-synthesis-dna-polymerases-in-dna-replication-in-the-presence-of-a-weak-dna-polymerase-%C3%AE-in-saccharomyces-cerevisiae
#14
Likui Zhang, Yanchao Huang, Xinyuan Zhu, Yuxiao Wang, Haoqiang Shi, Min Chen, Kunming Dong, Xiaojian Zhou
Translesion synthesis (TLS) DNA polymerases (pols) are often mutagenic for lesion bypass due to their low fidelity. Here, we isolated a weak yeast DNA pol δ mutant that possessed amino acid substitution V592G by genetic selection. The pol3-V592G cells were sensitive to hydroxyurea (HU), which increases the requirement for dNTPs, and their HU sensitivity was suppressed by L612M substitution. We also demonstrated that V592G substitution suppressed the phosphonoacetic acid (PAA) sensitivity of pol3-L612M cells, suggesting a cycle of mutual suppression in the HU- and PAA-sensitive yeast DNA pol δ mutants as similarly observed in the optA1- and PAA-sensitive T4 DNA pol mutants...
August 14, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28807826/structural-plasticity-of-t4-transcription-co-activator-gp33-revealed-by-a-protease-resistant-unfolded-state
#15
Radhakrishnan Mahalakshmi, Svetlana Rajkumar Maurya, Bhawna Burdak, Parini Surti, Manoj S Patel, Vikas Jain
Gene 33 protein (gp33) is a transcriptional coactivator for late genes of the T4 bacteriophage. gp33 possesses a 5-helix bundle core, with unstructured N- and C-terminal regions that account for >50% of the protein sequence. It plays a unique role of interacting with host RNA polymerase, couples transcription with DNA replication, and plays the dual function as repressor and co-activator in phage transcription. Here, we identify protein structural plasticity as the molecular basis of the dual nature in gp33...
August 12, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28807301/a-candidate-recombinant-antigen-for-diagnosis-of-schistosomiasis-japonica-in-domestic-animals
#16
Jintao Feng, Rui Xu, Xin Zhang, Yu Han, Chuanchuan He, Chao Lu, Yang Hong, Ke Lu, Hao Li, Yamei Jin, Jiaojiao Lin, Jinming Liu
Domestic animals infected with Schistosoma japonicum are a major source of infection and play an important role in transmission to humans. A key strategy for the elimination of schistosomiasis is to control the sources of infection. In the present study, we identified a candidate diagnostic antigen-encoding gene, SjMRP1, the putative multidrug resistance protein 1 gene, by screening a cDNA phage display library from 44-day-old S. japonicum worms using IgGs from goat, cattle, and buffalo infected with S. japonicum...
August 30, 2017: Veterinary Parasitology
https://www.readbyqxmd.com/read/28805554/apitherapeutics-and-phage-loaded-nanofibers-as-wound-dressings-with-enhanced-wound-healing-and-antibacterial-activity
#17
Wessam A Sarhan, Hassan Me Azzazy
AIM: Develop green wound dressings which exhibit enhanced wound-healing ability and potent antibacterial effects. METHODS: Honey, polyvinyl alcohol, chitosan nanofibers were electrospun and loaded with bee venom, propolis and/or bacteriophage against the multidrug-resistant Pseudomonas aeruginosa and examined for their antibacterial, wound-healing ability and cytotoxicity. RESULTS: Among different formulations of nanofibers, honey, polyvinyl alcohol, chitosan-bee venom/bacteriophage exhibited the most potent antibacterial activity against all tested bacterial strains (Gram-positive and -negative strains) and achieved nearly complete killing of multidrug-resistant P...
September 2017: Nanomedicine
https://www.readbyqxmd.com/read/28804739/selection-of-genetically-modified-bacteriophages-using-the-crispr-cas-system
#18
Miriam Manor, Udi Qimron
We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages...
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28803933/phage-in-urban-wastewater-have-the-potential-to-disseminate-antibiotic-resistance
#19
Gayathri Upeksha Gunathilaka, Varun Tahlan, Abdullah Ibn Mafiz, Manisha Polur, Yifan Zhang
A total of 29 Escherichia coli phage were isolated from wastewater samples collected from an urban wastewater treatment plant (WWTP) and characterized by host range determination, Transmission Electron Microscopy (TEM), antibiotic resistance gene identification, and phage transduction. β-lactam resistance genes, blaCMY, blaTEM, blaSHV, blaCTXM, and blaOXA, were amplified on phage DNA by PCR. Out of nine host range patterns observed, six were able to multiply in three or more indicator strains, including Shiga-toxin producing E...
August 10, 2017: International Journal of Antimicrobial Agents
https://www.readbyqxmd.com/read/28802409/exploring-the-potential-of-t7-bacteriophage-protein-gp2-as-a-novel-inhibitor-of-mycobacterial-rna-polymerase
#20
J du Plessis, R Cloete, L Burchell, P Sarkar, R M Warren, A Christoffels, S Wigneshweraraj, S L Sampson
Over the past six decades, there has been a decline in novel therapies to treat tuberculosis, while the causative agent of this disease has become increasingly resistant to current treatment regimens. Bacteriophages (phages) are able to kill bacterial cells and understanding this process could lead to novel insights for the treatment of mycobacterial infections. Phages inhibit bacterial gene transcription through phage-encoded proteins which bind to RNA polymerase (RNAP), thereby preventing bacterial transcription...
September 2017: Tuberculosis
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