Tag: MOI

Structure-guided discovery of anti-CRISPR and anti-phage defense proteins

Identification of putative anti-crispr proteins using structural features To identify Acrs in phage genomes, we began by retrieving ~66.5 million proteins from Integrated Microbial Genomes Virus database (IMG/VR)37. We excluded large proteins because over 90% of known Acrs contain less than 200 amino acids38 (Fig. 1A, Supplementary Data 1). To reduce computational…

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Proteomic analysis of SARS-CoV-2 particles unveils a key role of G3BP proteins in viral assembly

Capturing proteins associated with SARS-CoV-2 virions To identify host cell proteins potentially present in extracellular SARS-CoV-2 virions, viral particles were produced from two lung epithelial cell models, A549 cells overexpressing ACE2 receptor (A549-ACE2) and Calu-3 cells that naturally express ACE2 receptor and TMPRSS2. Following infection with the D614G SARS-CoV-2 strain32,…

Continue Reading Proteomic analysis of SARS-CoV-2 particles unveils a key role of G3BP proteins in viral assembly

Crispr Library Download

CRISPR guide RNA libraries have been iteratively improved to provide increasingly efficient reagents, although their large size is a barrier for many applications. We design an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9) by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared…

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Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein | Virology Journal

Design of the recombinant virus MVA-HBZ The multiepitope protein design was based on HBZ sequence deposited in the GenBank database [17, 23, 24], and in silico analyses performed using the epitope prediction tools: NetMHCpan 4.0 (Threshold: weak binders—2.0; strong binders—0.5), NetCTL 1.2 (Threshold of 0.75) and IEDB—MHC-I Binding Predictions. Epitopes…

Continue Reading Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein | Virology Journal

Generation of 17C-2 iCas9 hiPSCs containing a custom CRISPR library (17C-2-CC) by lentiviral transduction, associated with Fig 2.

(A) (left) Phase-contrast images of lenti-viral transduced 17C-2 hiPSCs before (day 1) and after (day 3) puromycin selection. Volumes of virus-containing supernatant added per well (0 μl, 8 μl and 16 μl; virus titer) are indicated. (+), with puromycin; (-), without puromycin. (right) The number of cells counted at day…

Continue Reading Generation of 17C-2 iCas9 hiPSCs containing a custom CRISPR library (17C-2-CC) by lentiviral transduction, associated with Fig 2.

An ncRNA transcriptomics-based approach to design siRNA molecules against SARS-CoV-2 double membrane vesicle formation and accessory genes | BMC Infectious Diseases

When compared to the genomes of other RNA viruses, coronaviruses have been found to possess largest genome sizes. They are capable of establishing reservoirs in both human and zoonotic populations, enabling their transmission and circulation among a range of animal hosts, including bats, pangolins, civets, cats, mice, pigs, whales, dogs,…

Continue Reading An ncRNA transcriptomics-based approach to design siRNA molecules against SARS-CoV-2 double membrane vesicle formation and accessory genes | BMC Infectious Diseases

CRISPRi gene modulation and all-optical electrophysiology in post-differentiated human iPSC-cardiomyocytes

A feasibility CRISPRi study was performed in post-differentiated iPSC-CMs targeting key genes important in cardiac electrophysiology. Comprehensive analysis using all-optical electrophysiology and a pipeline enabling correlative analysis of functional and molecular data in the same samples helped quantify the CRISPRi gene modulation in this in vitro model. Time-dependent control of…

Continue Reading CRISPRi gene modulation and all-optical electrophysiology in post-differentiated human iPSC-cardiomyocytes

Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells

Abstract A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing,…

Continue Reading Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells

Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs

Introduction Oxford Nanopore Technologies (ONT) direct RNA sequencing (Fig 1A) enables detection of RNA modifications. A modified base produces an altered electrical current and/or dwell time relative to a canonical base that can be detected with algorithms (Garalde et al, 2018; Smith et al, 2019; Workman et al, 2019). Figure…

Continue Reading Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs

Release Testing Of AAV Gene Therapies For Clinical Trials

By Mark Haydock, biologics CMC consultant, Agile Biologics Consulting LLC Adeno-associated virus (AAV) gene therapies that are designed to deliver a therapeutic transgene to patients are complex products that can be challenging to manufacture. Because of their complexity, a variety of analytical methods are required to ensure that these viral…

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Genome sequence and characterization of Streptomyces phages Vanseggelen and Verabelle, representing two new species within the genus Camvirus

Bacteriophage isolation Streptomyces phages Vanseggelen and Verabelle were isolated from a soil sample obtained from the National Park Zuid-Kennemerland in the Netherlands (N52° 23′ 31″, E4° 34′ 49″) using Streptomyces coelicolor as the host. Details on isolation procedures are reported previously in detail12. The collected phage stock solutions were stored…

Continue Reading Genome sequence and characterization of Streptomyces phages Vanseggelen and Verabelle, representing two new species within the genus Camvirus

Ginsenoside Rg3 enriches SCFA-producing commensal bacteria to confer protection against enteric viral infection via the cGAS-STING-type I IFN axis

Rg3 elicits protection against local and systemic enteric virus infection by regulating the intestinal microbiome To assess the antiviral effects of Rg3 in vivo, groups of C57BL/6J mice (referred to as WT B6 hereafter) were orally administered Rg3 for 3 consecutive days, inoculated with MNV-1 by the peroral route, and…

Continue Reading Ginsenoside Rg3 enriches SCFA-producing commensal bacteria to confer protection against enteric viral infection via the cGAS-STING-type I IFN axis

FAT10 is phosphorylated by IKK{beta} to inhibit the antiviral type-I interferon response

Introduction The innate immune system represents the host´s first-line defense against viral infections (Koyama et al, 2008). In this context, IFN-I are produced by all nucleated cells and represent the principal cytokines that counteract viral replication (Ivashkiv & Donlin, 2014). In fact, several receptors of the infected cell can recognize…

Continue Reading FAT10 is phosphorylated by IKK{beta} to inhibit the antiviral type-I interferon response

The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3

Statistics and reproducibility Anti-AMP IP for LC-MS/MS analysis was performed in three independent biological replicates (n = 3). TSA assay data represents technical triplicates. Time-resolved (de)AMPylation analyzed by LC-MS was performed as biological triplicates. CD measurements were performed in technical triplicates. Anisotropy data is shown as technical triplicates. Anisotropy measurements were repeated…

Continue Reading The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3

Correction of a homoplasmic mitochondrial tRNA mutation in patient-derived iPSCs via a mitochondrial base editor

Human induced pluripotent stem cells (iPSCs) Reprogramming and Culture This study was ethically approved by the Medical Ethics Committee of Nanjing Maternal and Child Health Care Hospital (2021KY-131), and informed consents were obtained from the patient’s legal guardian as well as the healthy donors, in accordance with the Declaration of…

Continue Reading Correction of a homoplasmic mitochondrial tRNA mutation in patient-derived iPSCs via a mitochondrial base editor

mRNA vaccine encoding Gn provides protection against severe fever with thrombocytopenia syndrome virus in mice

mRNA preparation The DNA template for the mRNA vaccine comprised a DNA fragment encoding the SFTSV Gn protein. The DNA templates were cloned into a plasmid vector with backbone sequence elements (T7 promoter, 5′- and 3′-UTR, 100 nucleotide poly(A) tail) interrupted by a linker (A50LA50, 20 nucleotides) to improve RNA…

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Identification of CCZ1 as an essential lysosomal trafficking regulator in Marburg and Ebola virus infections

Cells and viruses Haploid mSCs AN3–12 are a feeder independent clonal derivative of HMSc2 isolated from mice oocyte and maintained at IMBA52. The AN3–12 library and knocked out cells used for the haploid screening was obtained from IMBA (Austria)14,52. AN3–12 cells were validated by STR analysis. Haploid mES cells were…

Continue Reading Identification of CCZ1 as an essential lysosomal trafficking regulator in Marburg and Ebola virus infections

Solved Fig. 3. GIGVF2/AEHP complex formation is critical for

Transcribed image text: Fig. 3. GIGVF2/AEHP complex formation is critical for repression of 1/nbf mRNA translation and enabling viral replication. (A) ELSA measurement of ifN-1) production in WT, 4EHP.KO, and GIGVF2-KO HEK 93 cels transiently expressing TLR3 folowing 6 h of treatment with 1 pgimL poly(:C. Data are presented KO…

Continue Reading Solved Fig. 3. GIGVF2/AEHP complex formation is critical for

Subcellular distribution of the rAAV genome depends on genome structure

Optimization of transfection condition for viral genome staining The transfection was prepared by various concentrations of plasmids expressing DD-dSpyCas9-mCherry-APEX2 (pAPEX2) and sgITR (psgITR) with the transfection agent to stain the rAAV genome inside the cells for TEM. To investigate the target viral genome, a specific staining technique is necessary for…

Continue Reading Subcellular distribution of the rAAV genome depends on genome structure

Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells | Parasites & Vectors

Cell lines Hsu and CT cells originally derived from Culex quinquefasciatus and Culex tarsalis, respectively, were used as experimental material (gifts from R. van Rij, Radboud University, Nijmegen, the Netherlands). These cell lines are known to harbor several MSV infections [7]. Aag2 and C6/36 cells were received from A. Kohl…

Continue Reading Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells | Parasites & Vectors

Biological characterization of coronavirus noncanonical transcripts in vitro and in vivo | Virology Journal

Noncanonical coronavirus transcripts were robustly synthesized and could be categorized into seven populations based on differences in sequence elements To determine the amounts and structures of the noncanonical bovine coronavirus (BCoV) transcripts in HRT-18 cells, nanopore direct RNA sequencing was employed. Quantification by read counts revealed that ~ 30% of the total…

Continue Reading Biological characterization of coronavirus noncanonical transcripts in vitro and in vivo | Virology Journal

Cells | Free Full-Text | CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy

Author Contributions Conceptualization, M.G. and R.F.; Design and experimental work, M.G., L.P., V.B., M.C. and R.F.; Data analysis, M.G., L.P., V.B., M.C. and R.F.; Writing—Original Draft Preparation, M.G. and R.F.; Writing—Review and Editing, M.G., L.P., V.B., M.C., A.B., E.G., G.R., I.R. and R.F.; Supervision, M.G. and R.F.; Project Administration, R.F.;…

Continue Reading Cells | Free Full-Text | CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy

Genome Engineering Oxford (GEO) Facility

We also are actively involved in developing new gene editing methods and provide opportunities for collaborations on more extensive or challenging uses of CRISPR techniques such as development of new construct designs, application to whole organisms, development and use of pooled genome-wide CRISPR libraries. Services/Equipment available Engineered mammalian and bacterial…

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Unveiling the biology of defective viral genomes in vitro and in vivo: implications for gene expression and pathogenesis of coronavirus | Virology Journal

The classification, structure, abundance and reproducibility of coronavirus DVGs RNA transcripts other than those encoded by the coronavirus genome are also synthesized during infection [27]. Based on whether they are relevant to transcription regulatory sequence (TRS) (Figure S1A), a sequence motif from which subgenomic mRNAs (sgmRNAs) are synthesized, coronavirus RNA…

Continue Reading Unveiling the biology of defective viral genomes in vitro and in vivo: implications for gene expression and pathogenesis of coronavirus | Virology Journal

The effect of oleic acid supplementation on lipid droplet production, betaoxidation and rotavirus replication

Abstract Rotavirus (RV) is the most common cause of severe acute gastroenteritis in infants and young children globally. Rotavirus infections induce cytoplasmic inclusion bodies called viroplasms serving as a site of RV genome replication and assembly. Viroplasms recruit lipid droplets (LDs), which play major roles in energy homeostasis and is…

Continue Reading The effect of oleic acid supplementation on lipid droplet production, betaoxidation and rotavirus replication

A computationally designed antigen eliciting broad humoral responses against SARS-CoV-2 and related sarbecoviruses

Phylogenetic analysis Protein sequences of spike proteins were downloaded from the NCBI virus database for all the known sarbecoviruses (June 2020). A multiple sequence alignment was generated using MUSCLE36. The resulting multiple sequence alignment was pruned to the RBD region, filtered at 95% sequence identity and used as input for…

Continue Reading A computationally designed antigen eliciting broad humoral responses against SARS-CoV-2 and related sarbecoviruses

Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K+ during entry

An improved STA of the BUNV GP permits modelling of the Gc and Gn hetero-hexameric arrangement To determine the arrangement of Gn and Gc in the BUNV spike by cryo-EM, WT BUNV was concentrated and purified as previously described5,25 (Supplementary Fig. 1). Purified virions (in a pH 7.3/no K+ buffer) were…

Continue Reading Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K+ during entry

Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer

Sample collection Previously, we collected nasal samples from WTD in northeastern Ohio focusing on metropolitan area parks11. In the present study, we expanded geographical study area by approximately 1000-fold to target the entire state of Ohio. We collected 1522 nasal swabs from WTD across 83 of Ohio’s 88 counties from…

Continue Reading Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer

An IgM-like inhalable ACE2 fusion protein broadly neutralizes SARS-CoV-2 variants

Protein expression hACE2-hIgG1 was constructed by fusing the extracellular domain of human ACE2 (amino acid 19–615) (UniProt Q9BYF1) to the human IgG1 hinge and Fc (UniProt P01857). H374N and H378N mutations were introduced to inactivate the Zinc metallo-enzyme activity17. HH-120 was constructed by fusing hACE2-hIgG1 with an IgM tailpiece (PTLYNVSLVMSDTAGTCY)33….

Continue Reading An IgM-like inhalable ACE2 fusion protein broadly neutralizes SARS-CoV-2 variants

CircRNA-CIRH1A Promotes the Development of Osteosarcoma by Regulating PI3K/AKT and JAK2/STAT3 Signaling Pathways

Tissue Collection The study was approved by the Medical Ethics Committee at the Fuyang Traditional Chinese Medicine Orthopedics Hospital (2019-F027). OS tumor tissues and adjacent normal tissues were collected from 40 patients diagnosed with OS at the Fuyang Traditional Chinese Medicine Orthopedics Hospital from June 2018 to Dec 2019. Patient…

Continue Reading CircRNA-CIRH1A Promotes the Development of Osteosarcoma by Regulating PI3K/AKT and JAK2/STAT3 Signaling Pathways

The non-classical major histocompatibility complex II protein SLA-DM is crucial for African swine fever virus replication

Cell lines and viruses Cell lines were received from the cell culture collection for veterinary medicine (CCVM) of the Friedrich-Loeffler-Institut (FLI). The highly passaged wild boar lung cell line (WSL-R-HP, #1346; abbreviated as WSL) was maintained in Ham’s F12 cell culture medium (Ham’s F-12, 5.32 g/L; IMDM, 8.80 g/L; NaHCO3, 2.45 g/L; pH 7.2)…

Continue Reading The non-classical major histocompatibility complex II protein SLA-DM is crucial for African swine fever virus replication

CRISPR library screening to develop HEK293-derived cell lines with improved lentiviral vector titers

doi: 10.3389/fgeed.2023.1218328. eCollection 2023. Affiliations Expand Affiliation 1 Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States. Free PMC article Item in Clipboard Brian J Iaffaldano et al. Front Genome Ed. 2023. Free PMC article Show details Display…

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Viruses | Free Full-Text | Astragaloside IV Regulates cGAS-STING Signaling Pathway to Alleviate Immunosuppression Caused by PRRSV Infection

Received: 12 June 2023 / Revised: 1 July 2023 / Accepted: 13 July 2023 / Published: 20 July 2023 Round 1 Reviewer 1 Report This manuscript examines the effects of AS-IV treatment of PAMs infected with a strain of PRRSV-2. Specifically, aspects of immune regulation responses are investigated. Specific comments:…

Continue Reading Viruses | Free Full-Text | Astragaloside IV Regulates cGAS-STING Signaling Pathway to Alleviate Immunosuppression Caused by PRRSV Infection

Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry

Constructs and expression of human SERINC proteins SERINC3 The gene that encodes human SERINC3 (Genscript-OHu02717D) was inserted upstream of a thrombin protease cleavable linker (LVPRGS) and Strep II epitope (WSHPQFEK) in a modified pFastBac vector by In-Fusion cloning (Clontech). Mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Agilent) was performed to…

Continue Reading Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry

Optimized minimal genome-wide human sgRNA library

sgRNA collection and enzyme cut site annotation All sgRNAs corresponding to potential NGG-containing target sites on the human (GRCh38/hg38) genome were calculated by a customized Perl script. The enzyme cut site was set at the 17 position from 5’ to 3’ of each sgRNA sequence. These sgRNAs have more than…

Continue Reading Optimized minimal genome-wide human sgRNA library

In vitro and in vivo characterization of SARS-CoV-2 resistance to ensitrelvir

Ethics All animal experiments were conducted in accordance with the University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. The committee acknowledged and accepted both the legal and ethical responsibility for the…

Continue Reading In vitro and in vivo characterization of SARS-CoV-2 resistance to ensitrelvir

Characterization of bacteriophage vB_KleM_KB2 possessing high control ability to pathogenic Klebsiella pneumoniae

Electron microscopy observation of phage virions Using the sewage collected from the Diankun River in Haidian island (Haikou, Hainan, China), one phage, named vB_KleM_KB2, was isolated (CCTCC No. M 20221832). This phage forms a plaque with a clear center with a diameter of 1 mm and a turbid rim with a…

Continue Reading Characterization of bacteriophage vB_KleM_KB2 possessing high control ability to pathogenic Klebsiella pneumoniae

Inhalable CAR-T cell-derived exosomes as paclitaxel carriers for treating lung cancer | Journal of Translational Medicine

Cells and plasmids HEK 293 T and LLC cells (ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific) containing 10% FBS (Gibco, USA) and 1% penicillin–streptomycin (Invitrogen) at 37 °C in a 5% CO2 environment. Stbl3 and DH5α competent cells were used for plasmid transformation. The pCDH-CMV-MCS-EF1-GFP-Puro vector (cat….

Continue Reading Inhalable CAR-T cell-derived exosomes as paclitaxel carriers for treating lung cancer | Journal of Translational Medicine

A viral pan-end RNA element and host complex define a SARS-CoV-2 regulon

Cell lines and culture A549-hACE2 [a gift from Dr. Ben tenOever3], 293 T, HEK293, A549, and 3T3-L1 pre-adipocyte cell lines (refer to Supplementary Table 1) were cultured in DMEM and 10% FBS. U937, Calu-3, and HCT116 cells were cultured in RPMI, EMEM, and McCoy’s 5 A media, respectively, with 10% FBS. Caco-2 cells…

Continue Reading A viral pan-end RNA element and host complex define a SARS-CoV-2 regulon

Nuclear permeability during cell division

In late May I had the honor of speaking with Sucharit Bhakdi, MD regarding dsDNA contamination in the vaccines. First, we need to do some numbers. The 100ug Moderna shots deliver over 40 trillion mRNA molecules (14T for Pfizer) and the number of LNPs per shot is reported to be…

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Selection and validation of reference genes for RT-qPCR normalization of porcine alveolar macrophages (PAMs) for PRRSV studies

Sample collection and cDNA synthesis In this study, we used porcine alveolar macrophages (PAMs) that were isolated from 16 different animals between the years 2011 and 2022, and subsequently stored. The PAMs were isolated by bronchoalveolar lavage (BAL) from lungs of euthanized 3-week-old healthy conventional piglets from a PRRSV negative…

Continue Reading Selection and validation of reference genes for RT-qPCR normalization of porcine alveolar macrophages (PAMs) for PRRSV studies

Design of bacteriophage T4-based artificial viral vectors for human genome remodeling

Assembly of T4 artificial viral vectors T4-AVVs were assembled by sequential incorporation of purified biomaterials to generate a virus structural mimic (Fig. 2a and Supplementary Movie 1). Starting with an empty capsid shell purified from E. coli infected by the neck-minus and tail-minus T4 phage mutant (10-amber.13-amber.HocΔ.SocΔ T4) (Supplementary Fig. 1a), a pentameric…

Continue Reading Design of bacteriophage T4-based artificial viral vectors for human genome remodeling

Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs

Recombinant ASFVs with mosaic genomes of genotype I and II viruses detected from field samples in China During our surveillance in China, we isolated three ASFVs from pig samples collected in Jiangsu province, Henan province, and Inner Mongolia Autonomous Region, respectively, and designated them Pig/Jiangsu/LG/2021 (JS/LG/21), Pig/Henan/123014/2022 (HeN/123014/22), and Pig/Inner…

Continue Reading Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs

Vaccinia E5 is a major inhibitor of the DNA sensor cGAS

Screening strategy for identifying viral inhibitors of the cGAS/STING pathway Vaccinia virus is a large cytoplasmic DNA virus with a 190 kilobase pairs (kbp) genome that encodes over 200 proteins21,22. MVA has an approximately 30-kbp deletion from its parental vaccinia genome, resulting in the loss of many immune-modulatory viral genes15….

Continue Reading Vaccinia E5 is a major inhibitor of the DNA sensor cGAS

Identification of age-specific gene regulators of La Crosse virus neuroinvasion and pathogenesis

RNA-seq analyses of adult and weanling brain microvessels identify candidate gene regulators of LACV infection We first sought to identify candidate gene regulators that contribute to age-dependent susceptibility to LACV. Weanling (W) and adult (A) mice were treated with either poly I:C (PI) for 3 h (as a surrogate for a…

Continue Reading Identification of age-specific gene regulators of La Crosse virus neuroinvasion and pathogenesis

Mosquito densovirus significantly reduces the vector susceptibility to dengue virus serotype 2 in Aedes albopictus mosquitoes (Diptera: Culicidae) | Infectious Diseases of Poverty

Identification of MDV presence using open access sequencing data Metagenomic next-generation sequencing (NGS) data for field mosquitoes in China were searched from the NCBI SRA database (www.ncbi.nlm.nih.gov/sra) and CNGB Sequence Archive database (db.cngb.org/cnsa/) using the keywords “mosquito OR Anopheles OR Aedes OR Culex”. Followed by removing the datasets with less…

Continue Reading Mosquito densovirus significantly reduces the vector susceptibility to dengue virus serotype 2 in Aedes albopictus mosquitoes (Diptera: Culicidae) | Infectious Diseases of Poverty

Optimized metrics for orthogonal combinatorial CRISPR screens

Plasmid design The information on each plasmid for combinatorial libraries is indicated in Supplementary Table S1. Plasmids used in this study to generate combinatorial libraries for SpCas9, enAsCas12a, CHyMErA, enAsCas12a (dual-gRNA), CHyMErA.v2, multiSPAS were deposited to Addgene (IDs: 189632, 189633, 189634, 189635, 189636, 189637, respectively). 3Cs oligonucleotide design The protocol…

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Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus

Ethics statement All experiments in this study comply with guidelines of the U.S. Public Health Service and were approved by the Institutional Biosafety Committee at the University of Pittsburgh. All animal husbandry and experimental procedures were conducted in accordance with U.S. Public Health Service policy and approved by the Institutional…

Continue Reading Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus

Metagenomics-enabled reverse-genetics assembly and characterization of myotis bat morbillivirus

Ethics declaration Animal study was performed following the Guide for the Care and Use of Laboratory Animals. Animal experiment was approved by the Institutional Animal Care and Use Committee of Colorado State University (protocol number 1090) in advance and conducted in compliance with the Association for the Assessment and Accreditation…

Continue Reading Metagenomics-enabled reverse-genetics assembly and characterization of myotis bat morbillivirus

Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus

CLIP enables high-efficiency knock-in into an essential locus To create CLIP, we leverage integrase-deficient lentiviruses (IDLV) to deliver an HDR donor template as a viral RNA genome to cells25,26. IDLV consists of lentiviral components but with a D64V mutation in the integrase, maintaining this enzyme’s ability to package and nuclear…

Continue Reading Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus

Lipid-coated mesoporous silica nanoparticles for anti-viral applications via delivery of CRISPR-Cas9 ribonucleoproteins

All methods were performed in accordance with institutional guidelines and regulations. CRISPR-Cas9 protein and guide RNA reagents All Cas9 proteins used in this study were derived from the prototypic Streptococcus pyogenes species. Cas9 protein was produced as described in Seamon et al.33 and contained a N-terminal nuclear localization signal and…

Continue Reading Lipid-coated mesoporous silica nanoparticles for anti-viral applications via delivery of CRISPR-Cas9 ribonucleoproteins

TUT4/7-mediated uridylation of a coronavirus subgenomic RNAs delays viral replication

Cell lines and tissue culture The murine 17-CL1 cell line (derived from 3T3 cells, BEI Resources, Cat. No. NR-53719) was maintained in Dulbecco Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% Fetal Bovine Serum (FBS, Gemini Bio-Products), L-glutamine (Invitrogen), and Sodium pyruvate (Invitrogen). The NCTC clone 1469 cell line…

Continue Reading TUT4/7-mediated uridylation of a coronavirus subgenomic RNAs delays viral replication

Human CEACAM1 is targeted by a Streptococcus pyogenes adhesin implicated in puerperal sepsis pathogenesis

Ethics Human blood was obtained from healthy donors, approved by the Regional Ethics Committee and Imperial College Healthcare NHS Trust Tissue Bank (Regional Ethics Committee approval no. 17/WA/0161, Imperial College Healthcare Tissue Bank Human Tissue Authority license no. 12275, and Imperial College Research Ethics Committee no. 19IC5166). All samples were…

Continue Reading Human CEACAM1 is targeted by a Streptococcus pyogenes adhesin implicated in puerperal sepsis pathogenesis

In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants

Cell culture and reagents HEK293 and HeLa cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM GlutaMAX‐1, 100 μg/mL streptomycin and 100 U/mL penicillin (complete growth medium) at 37 °C under 5% CO2. HeLa cells stably expressing GFP-TIFA were previously described8. Cells…

Continue Reading In vitro kinase assay reveals ADP-heptose-dependent ALPK1 autophosphorylation and altered kinase activity of disease-associated ALPK1 mutants

New carbohydrate binding domains identified by phage display based functional metagenomic screens of human gut microbiota

Purification of T7Select 10-3b phages The T7Select 10-3b phage vector (Novagen, Merck) was used in this study. Non-recombinant/recombinant T7Select 10-3b phages were purified from the lysate of infected E. coli strain BL5403 by PEG-8000 precipitation as per the method described in T7 select system manual. The pellet was suspended in…

Continue Reading New carbohydrate binding domains identified by phage display based functional metagenomic screens of human gut microbiota

A Vaccinia-based system for directed evolution of GPCRs in mammalian cells

Poxvirus vectors for directed evolution of GPCRs in mammalian cells Monoclonal and homogenous expression of the gene of interest are critical prerequisites for selections in directed evolution that are difficult to achieve in mammalian cells with common expression strategies such as transient transfection. To reach both equal gene dosage and…

Continue Reading A Vaccinia-based system for directed evolution of GPCRs in mammalian cells

Genetic association analysis of 77,539 genomes reveals rare disease etiologies

Motivation for developing a sparse RDB Computational approaches for discovering the etiologies of rare diseases typically depend on the analysis of a heterogeneous set of files, each of which can be very large and follow a distinct convention. Genotypes, for example, are ordinarily stored in VCFs containing data for one…

Continue Reading Genetic association analysis of 77,539 genomes reveals rare disease etiologies

An introduction to cell engineering in picodroplets

With an easy-to-use instrument to encapsulate single cells in picolitre-volume water droplets (picodroplets) at high throughput rates of thousands of droplets per second, picodroplet microfluidics is quickly emerging as the technology of choice for single-cell analysis. This highly effective and delicate method has important benefits in terms of cost and…

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From a genome-wide screen of RNAi molecules against SARS-CoV-2 to a validated broad-spectrum and potent prophylaxis

Screening for hyper-potent shRNA against SARS-CoV-2 We parsed the SARS-CoV-2 genome into a series of potential short hairpin RNA (shRNA) targets (Supplemental Fig. 1). This process was conducted by tiling the genome with overlapping 50 nucleotide-long sequences, each shifted by a single nucleotide from the other. The region targeted by each…

Continue Reading From a genome-wide screen of RNAi molecules against SARS-CoV-2 to a validated broad-spectrum and potent prophylaxis

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

Genetic screen identifies modulators of Tun-mediated neuronal death To identify genes that ameliorate neuronal cell death caused by Tun-induced ER stress, we performed a loss-of-function pooled genetic screen using our druggable genome sgRNAs library. We first generated a clonal iPSC line that expresses Cas9 under the GAPDH promoter (Fig. 1A). We…

Continue Reading CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

BdLT-Seq as a barcode decay-based method to unravel lineage-linked transcriptome plasticity

Cell lines Immortalised HA1E (hTERT and SV40ER) and tumourigenic HA1ER cells (hTERT, SV40ER and HRAS-G12V) from stepwise tumourigenesis models generated from normal human embryonic kidney cells were obtained from Dr. Hahn (Broad Institute of MIT and Harvard, Cambridge, USA). Cells were cultured in DMEM (1 g/L glucose) (Gibco, cat. #10567014) supplemented…

Continue Reading BdLT-Seq as a barcode decay-based method to unravel lineage-linked transcriptome plasticity

Rescue and in vitro characterization of a divergent TBEV-Eu strain from the Netherlands

Phylogenetic inference To analyze the phylogenetic relationship of the TBEV sequences obtained from ticks in the Netherlands (GenBank accession numbers LC171402 and MH021184)21,22 with known TBEV-Eu strains, all TBEV-Eu sequences encompassing the complete coding sequence were selected from GenBank (n = 88, retrieved on September 18, 2021). Multiplicities, synthetic and clonal sequences…

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Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains

This research complies with all relevant ethical regulations approved by the Human Investigation Review Board of Albert Szent-Györgyi Clinical Centre of the University of Szeged and the National Biodiversity Authority (NBA) of India. Permission for the faecal sample collection was obtained from the Human Investigation Review Board of Albert Szent-Györgyi…

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CRISPR-Cas13d effectively targets SARS-CoV-2 variants, including Delta and Omicron, and inhibits viral infection

doi: 10.1002/mco2.208. eCollection 2023 Feb. Zongzhi Liu  1   2   3 , Xiang Gao  4 , Chuanwen Kan  2   5 , Lingyu Li  1   2   3 , Yuan Zhang  6 , Yibo Gao  7 , Shengyuan Zhang  4 , Liangji Zhou  6 , Hui Zhao  6   8   9 , Mingkun Li  2  …

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Screening of lymphoma radiotherapy-resistant genes

Introduction Lymphoma, a cancer characterized by a malignant tumor of the immune system that originates in the lymph nodes or lymphoid tissue, is one of the most common cancers worldwide. According to GLOBOCAN, the incidence of non-Hodgkin lymphoma (NHL) in both males and females ranked top ten among all cancers…

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Evolution of giant pandoravirus revealed by CRISPR/Cas9

Viral strains utilized in this work The following viral strains have been used in this study: Pandoravirus neocaledonia3, Pandoravirus macleodensis3, Pandoravirus kuranda (this study, ON887157), Mollivirus kamchatka26, Pithovirus sibericum27 and Mimivirus reunion28. Cloning of DNA constructs All primers used in this study are listed in Table S1. All vectors used in…

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SARS-CoV-2 hijacks cellular kinase CDK2 to promote viral RNA synthesis

CDKs support SARS-CoV-2 RdRp-mediated gene expression To identify cellular proteins that modulate the SARS-CoV-2 RNA synthesis, we first performed immunoprecipitation coupled with mass spectrometry (MS) to determine cellular proteins that associate with SARS-CoV-2 RdRp. Briefly, Flag-tagged nsp7, Flag-tagged nsp8, and Flag-tagged nsp12 plasmids were co-transfected into HEK293T cells, and the…

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Evasion of cGAS and TRIM5 defines pandemic HIV

Cells and reagents HEK293T and U87 cells were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Labtech) and 100 U ml−1 penicillin plus 100 μg ml−1 streptomycin (Pen/Strep; Gibco). THP-1-IFIT1 cells that had been modified to express Gaussia luciferase under the control of the IFIT1 promoter were described previously62. THP-1…

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A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model

Lai, L. et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089–1092. doi.org/10.1126/science.1068228 (2002). ADS  CAS  Article  PubMed  Google Scholar  Kuwaki, K. et al. Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat. Med. 11, 29–31. doi.org/10.1038/nm1171 (2005). CAS  Article  PubMed  Google…

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RNA interference triggers that target SARS-CoV-2 genome

Coronavirus disease 2019 (COVID-19) vaccines have played a critical role in reducing transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. However, with emerging reports of waning vaccine efficacy, there remains an urgent need to develop prophylactic measures against COVID-19. In a recent study published on the bioRxiv*…

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Virology: Module 2 Flashcards | Quizlet

Virology: Module 2 Flashcards | Quizlet   attachment/entry, translation of viral mRNA into viral proteins, replication, assembly, release of viruses from cell steps of infection for viruses? a cell possesses the functional receptor for a virus – virus can bind, possibly enter, but replication isn’t guaranteed a cell does not…

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Cell Strain-Derived Induced Pluripotent Stem Cells as an Isogenic Approach To Investigate Age-Related Host Response to Flaviviral Infection

INTRODUCTION Dengue is the most common mosquito-borne viral disease globally (1). This acute disease, which can be life-threatening, is caused by four different dengue viruses (DENVs) (DENV-1, DENV-2, DENV-3, and DENV-4). An estimated 390 million people are infected with these DENVs annually (2), and populations throughout the tropics face frequent…

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Phospholipase A2 inhibitor and LY6/PLAUR domain-containing protein PINLYP regulates type I interferon innate immunity

Significance Interferon (IFN)-mediated antiviral responses serve as the first line of the host innate immune defense against viral infection. Here we identify a previously uncharacterized protein designated phospholipase A2 inhibitor and LY6/PLAUR domain-containing protein (PINLYP), which is essential for embryonic development and plays an important role in type I IFN…

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Kenya Targets Easing Sickle Cell Disease Pain Through Quality and Affordable Care

Health experts in Kenya aim to reduce the number of children born with sickle cell disease — an inherited blood disorder — by more than 12 times through public education and awareness, as the country premiered a guideline for controlling and managing the disease. The guideline titled Sickle Cell Disease…

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