Table S6. Original pdf outputs from mirDeep2. Data used in Table 1 on IsomiRs (5⠲ modification... more Table S6. Original pdf outputs from mirDeep2. Data used in Table 1 on IsomiRs (5⠲ modifications, 3⠲ modification, and nucleotide substitution) of the miR-34 family activated by p53. (DOCX 68 kb)
Table S5. Highly comparable RPKMs resulted from other small noncoding RNA-Seq using standard Illu... more Table S5. Highly comparable RPKMs resulted from other small noncoding RNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 2013Â kb)
Table S4. Highly comparable RPKMs resulted from MicroRNA-Seq using standard Illumina protocol and... more Table S4. Highly comparable RPKMs resulted from MicroRNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 95Â kb)
Table S3. Highly comparable RPKMs resulted from RNA-Seq using standard Illumina protocol and the ... more Table S3. Highly comparable RPKMs resulted from RNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 1126Â kb)
Table S2. Data of variant calling for the three Bordetella bronchiseptica RB50 strains and one Bo... more Table S2. Data of variant calling for the three Bordetella bronchiseptica RB50 strains and one Bordetella pertussis Tohama I strain. (XLSX 122Â kb)
Table S1. A summary of run metrics of the Illumina protocol and the sub-nanomolar protocol. (DOCX... more Table S1. A summary of run metrics of the Illumina protocol and the sub-nanomolar protocol. (DOCX 24Â kb)
Table S7. Highly similar taxonomic hierarchy and relative abundance of classification (sheet 1) a... more Table S7. Highly similar taxonomic hierarchy and relative abundance of classification (sheet 1) and strong correlation of number of hits (sheet 2) resulted form 16S rRNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 40Â kb)
The four-residue insert (PRRA) at the boundary between the S1 and S2 subunits of SARS-CoV-2 has b... more The four-residue insert (PRRA) at the boundary between the S1 and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2.
Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of... more Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of protein contents per activity unit (or IU/mg, specific activity), available from previous studies (Lin et al, 2004; Butenas et al, 2009) and the prescribing information. The wide range of the specific activities seems to be more pronounced in recombinant FVIII products (rFVIII) compared to plasma-derived FVIII, implying presence of protein with altered structure and biochemical properties. In particular, rFVIII products were reported to contain a protein fraction (FVIII*), unable to bind von Willebrand factor (VWF) without functional activity (Lin et al, 2004;Ofosu et al 2012). Furthermore, FVIII* may represent a risk factor for development of FVIII inhibitors in Hemophilia A patients, as the binding to VWF was shown to reduce FVIII immunogenicity in a tissue culture and mice model systems (Gangadharan et al, 2017; Muczynski et al, 2018). Due to these reasons, FVIII* can be defined as a...
DNA base editors have enabled genome editing without generating DNA double strand breaks. The app... more DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in uninten...
Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors... more Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors (PRRs) on antigen-presenting cells, including monocytes and macrophages, which can cause prostaglandin E2 (PGE2) release and consequently undesired inflammatory responses and fever in vaccine recipients. Here, we studied the mechanism of PGE2 production by human monocytes activated with muramyl dipeptide (MDP) adjuvant, which activates cytosolic nucleotide-binding oligomerization domain 2 (NOD2). In rabbits, administration of MDP elicited an early increase in PGE2 followed by fever. In human monocytes, MDP alone did not induce PGE2 production. However, high amounts of PGE2 and the proinflammatory cytokines IL-1β and IL-6 were secreted by monocytes activated with MDP in the presence of conditioned medium obtained from CD3 bead–isolated T cells (Tc CM) but not from those isolated without CD3 beads. Mass spectrometry and immunoblotting revealed that the costimulatory factor in Tc CM was gl...
This work is supported by Food and Drug Administration intramural funds. This project was support... more This work is supported by Food and Drug Administration intramural funds. This project was supported in part by an appointment to the Internship/ Research Participation Program at the Office of Biotechnology Products, Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the Department of Energy and Food and Drug Administration. The authors declare that they have no conflicts of interest with the contents of this article. The views expressed in this article are those of the authors, and do not necessarily reflect the official policy or position of the U.S. Food and Drug Administration and the Dept. of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This article contains Figs. S1-S14 and Tables S1-S3.
Naturally-occurring chalcones and synthetic chalcone analogues have been demonstrated to have man... more Naturally-occurring chalcones and synthetic chalcone analogues have been demonstrated to have many biological effects, including anti-inflammatory, anti-malarial, anti-fungal, and anti-oxidant/anti-cancerous activities. Compared to other chalcones, trans-chalcone exhibits superior inhibitory activity in cancer cell growth as shown via in vitro assays, and exerts anti-cancerous effects via the activation of the p53 tumor suppressor protein. Thus, characterization of the specific mechanisms, by which trans-chalcone activates p53, can aid development of new chemotherapeutic drugs that can be used individually or synergistically with other drugs. In this report, we found that trans-chalcone modulates many p53 target genes, HSP40 being the most induced gene in the RNA-Seq data using trans-chalcone-treated cells. CRM1 is also inhibited by trans-chalcone, resulting in the accumulation of p53 and other tumor suppressor proteins in the nucleus. Similar effects were seen using trans-chalcone ...
Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM i... more Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other s...
Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, te... more Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, tetanus neurotoxin and botulinum neurotoxin, respectively. Extensive biochemical and genetic investigation has been devoted to identifying and characterizing various C. botulinum strains. Less effort has been focused on studying C. tetani likely because recently sequenced strains of C. tetani show much less genetic diversity than C. botulinum strains and because widespread vaccination efforts have reduced the public health threat from tetanus. Our aim was to acquire genomic data on the U.S. vaccine strain of C. tetani to better understand its genetic relationship to previously published genomic data from European vaccine strains. We performed high throughput genomic sequence analysis on two wild-type and two vaccine C. tetani strains. Comparative genomic analysis was performed using these and previously published genomic data for seven other C. tetani strains. Our analysis focused on singl...
The type 1 taste receptor member 3 (T1R3) is a G protein-coupled receptor (GPCR) involved in swee... more The type 1 taste receptor member 3 (T1R3) is a G protein-coupled receptor (GPCR) involved in sweet taste perception. Besides the tongue, the T1R3 receptor is highly expressed in brain areas implicated in cognition, including the hippocampus and cortex. As cognitive decline is often preceded by significant metabolic or endocrinological dysfunctions, regulated by the sweet taste perception system, we hypothesized that a disruption of the sweet taste perception in the brain could have a key role in the development of cognitive dysfunction. To assess the importance of the sweet taste receptors in the brain, we conducted transcriptomic and proteomic analyses of cortical and hippocampal tissues isolated from T1R3 knockout (T1R3KO) mice. The effect of an impaired sweet taste perception system on cognition functions were examined by analyzing synaptic integrity and performing animal behavior on T1R3KO mice. While T1R3 knockout (T1R3KO) mice did not present a metabolically-disrupted phenotyp...
Table S6. Original pdf outputs from mirDeep2. Data used in Table 1 on IsomiRs (5⠲ modification... more Table S6. Original pdf outputs from mirDeep2. Data used in Table 1 on IsomiRs (5⠲ modifications, 3⠲ modification, and nucleotide substitution) of the miR-34 family activated by p53. (DOCX 68 kb)
Table S5. Highly comparable RPKMs resulted from other small noncoding RNA-Seq using standard Illu... more Table S5. Highly comparable RPKMs resulted from other small noncoding RNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 2013Â kb)
Table S4. Highly comparable RPKMs resulted from MicroRNA-Seq using standard Illumina protocol and... more Table S4. Highly comparable RPKMs resulted from MicroRNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 95Â kb)
Table S3. Highly comparable RPKMs resulted from RNA-Seq using standard Illumina protocol and the ... more Table S3. Highly comparable RPKMs resulted from RNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 1126Â kb)
Table S2. Data of variant calling for the three Bordetella bronchiseptica RB50 strains and one Bo... more Table S2. Data of variant calling for the three Bordetella bronchiseptica RB50 strains and one Bordetella pertussis Tohama I strain. (XLSX 122Â kb)
Table S1. A summary of run metrics of the Illumina protocol and the sub-nanomolar protocol. (DOCX... more Table S1. A summary of run metrics of the Illumina protocol and the sub-nanomolar protocol. (DOCX 24Â kb)
Table S7. Highly similar taxonomic hierarchy and relative abundance of classification (sheet 1) a... more Table S7. Highly similar taxonomic hierarchy and relative abundance of classification (sheet 1) and strong correlation of number of hits (sheet 2) resulted form 16S rRNA-Seq using standard Illumina protocol and the sub-nanomolar protocol. (XLSX 40Â kb)
The four-residue insert (PRRA) at the boundary between the S1 and S2 subunits of SARS-CoV-2 has b... more The four-residue insert (PRRA) at the boundary between the S1 and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2.
Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of... more Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of protein contents per activity unit (or IU/mg, specific activity), available from previous studies (Lin et al, 2004; Butenas et al, 2009) and the prescribing information. The wide range of the specific activities seems to be more pronounced in recombinant FVIII products (rFVIII) compared to plasma-derived FVIII, implying presence of protein with altered structure and biochemical properties. In particular, rFVIII products were reported to contain a protein fraction (FVIII*), unable to bind von Willebrand factor (VWF) without functional activity (Lin et al, 2004;Ofosu et al 2012). Furthermore, FVIII* may represent a risk factor for development of FVIII inhibitors in Hemophilia A patients, as the binding to VWF was shown to reduce FVIII immunogenicity in a tissue culture and mice model systems (Gangadharan et al, 2017; Muczynski et al, 2018). Due to these reasons, FVIII* can be defined as a...
DNA base editors have enabled genome editing without generating DNA double strand breaks. The app... more DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in uninten...
Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors... more Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors (PRRs) on antigen-presenting cells, including monocytes and macrophages, which can cause prostaglandin E2 (PGE2) release and consequently undesired inflammatory responses and fever in vaccine recipients. Here, we studied the mechanism of PGE2 production by human monocytes activated with muramyl dipeptide (MDP) adjuvant, which activates cytosolic nucleotide-binding oligomerization domain 2 (NOD2). In rabbits, administration of MDP elicited an early increase in PGE2 followed by fever. In human monocytes, MDP alone did not induce PGE2 production. However, high amounts of PGE2 and the proinflammatory cytokines IL-1β and IL-6 were secreted by monocytes activated with MDP in the presence of conditioned medium obtained from CD3 bead–isolated T cells (Tc CM) but not from those isolated without CD3 beads. Mass spectrometry and immunoblotting revealed that the costimulatory factor in Tc CM was gl...
This work is supported by Food and Drug Administration intramural funds. This project was support... more This work is supported by Food and Drug Administration intramural funds. This project was supported in part by an appointment to the Internship/ Research Participation Program at the Office of Biotechnology Products, Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the Department of Energy and Food and Drug Administration. The authors declare that they have no conflicts of interest with the contents of this article. The views expressed in this article are those of the authors, and do not necessarily reflect the official policy or position of the U.S. Food and Drug Administration and the Dept. of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This article contains Figs. S1-S14 and Tables S1-S3.
Naturally-occurring chalcones and synthetic chalcone analogues have been demonstrated to have man... more Naturally-occurring chalcones and synthetic chalcone analogues have been demonstrated to have many biological effects, including anti-inflammatory, anti-malarial, anti-fungal, and anti-oxidant/anti-cancerous activities. Compared to other chalcones, trans-chalcone exhibits superior inhibitory activity in cancer cell growth as shown via in vitro assays, and exerts anti-cancerous effects via the activation of the p53 tumor suppressor protein. Thus, characterization of the specific mechanisms, by which trans-chalcone activates p53, can aid development of new chemotherapeutic drugs that can be used individually or synergistically with other drugs. In this report, we found that trans-chalcone modulates many p53 target genes, HSP40 being the most induced gene in the RNA-Seq data using trans-chalcone-treated cells. CRM1 is also inhibited by trans-chalcone, resulting in the accumulation of p53 and other tumor suppressor proteins in the nucleus. Similar effects were seen using trans-chalcone ...
Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM i... more Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other s...
Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, te... more Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, tetanus neurotoxin and botulinum neurotoxin, respectively. Extensive biochemical and genetic investigation has been devoted to identifying and characterizing various C. botulinum strains. Less effort has been focused on studying C. tetani likely because recently sequenced strains of C. tetani show much less genetic diversity than C. botulinum strains and because widespread vaccination efforts have reduced the public health threat from tetanus. Our aim was to acquire genomic data on the U.S. vaccine strain of C. tetani to better understand its genetic relationship to previously published genomic data from European vaccine strains. We performed high throughput genomic sequence analysis on two wild-type and two vaccine C. tetani strains. Comparative genomic analysis was performed using these and previously published genomic data for seven other C. tetani strains. Our analysis focused on singl...
The type 1 taste receptor member 3 (T1R3) is a G protein-coupled receptor (GPCR) involved in swee... more The type 1 taste receptor member 3 (T1R3) is a G protein-coupled receptor (GPCR) involved in sweet taste perception. Besides the tongue, the T1R3 receptor is highly expressed in brain areas implicated in cognition, including the hippocampus and cortex. As cognitive decline is often preceded by significant metabolic or endocrinological dysfunctions, regulated by the sweet taste perception system, we hypothesized that a disruption of the sweet taste perception in the brain could have a key role in the development of cognitive dysfunction. To assess the importance of the sweet taste receptors in the brain, we conducted transcriptomic and proteomic analyses of cortical and hippocampal tissues isolated from T1R3 knockout (T1R3KO) mice. The effect of an impaired sweet taste perception system on cognition functions were examined by analyzing synaptic integrity and performing animal behavior on T1R3KO mice. While T1R3 knockout (T1R3KO) mice did not present a metabolically-disrupted phenotyp...
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