Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Programmable polyproteams built using twin peptide superglues

doi: 10.1073/pnas.1519214113

Figure Lengend Snippet: Establishing the covalently reactive peptide/protein pair SnoopTag and SnoopCatcher. (A) Spontaneous isopeptide bond formation between Lys and Asn, releasing ammonia. (B) Cartoon of splitting RrgA D4 domain [based on Protein Data Bank (PDB) ID code 2WW8] to make SnoopTag and SnoopCatcher. Reactive residues are in cyan. (C) SnoopTag-MBP reaction with SnoopCatcher, each at 10 µM, after 2 h at 25 °C analyzed by SDS/PAGE with Coomassie staining, alongside controls with Ala mutation of SnoopTag’s reactive Lys (KA) or SnoopCatcher’s reactive Asn (NA). (D) Isopeptide bond formation between SnoopTag peptide and SnoopCatcher shown by mass spectrometry. (E) Time course of SnoopTag reaction with a 1:1 or 2:1 ratio of SnoopCatcher to SnoopTag-MBP, tested as in C. (F) Time course of SnoopCatcher reaction with a 1:1, 2:1, or 4:1 ratio of SnoopTag-MBP to SnoopCatcher, tested as in C. Error bars are mean ±1 SD; n = 3. Some error bars are too small to be visible.

Article Snippet: Constructs were initially cloned into chemically competent E. coli DH5α (Life Technologies). pET28a SpyTag-MBP (Addgene plasmid ID 35050), GST-BirA, and pDEST14-SpyCatcher (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JQ478411","term_id":"380294102","term_text":"JQ478411"}} JQ478411 , Addgene plasmid ID 35044) have been described ( 26 ). pET28a SnoopCatcher (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU500646","term_id":"985768551","term_text":"KU500646"}} KU500646 , Addgene plasmid ID 72322) was generated by DNAWorks primer-mediated assembly from residues 749–860 of S. pneumoniae adhesin RrgA (numbering based on PDB ID code 2WW8) ( 28 ), digested with HindIII and NdeI and subcloned into pET28a.

Techniques: SDS Page, Staining, Mutagenesis, Mass Spectrometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Programmable polyproteams built using twin peptide superglues

doi: 10.1073/pnas.1519214113

Figure Lengend Snippet: SnoopTag and SnoopCatcher reaction conditions. (A) Point mutations to make SnoopCatcher. From the C-terminal domain of RrgA in cartoon format (based on PDB ID code 2WW8), residues mutated are shown in space-fill with carbons in cyan (G842 was changed to T and D848 to G). The Lys, Asn, and Glu involved in isopeptide bond formation are shown in stick format in yellow and the rest of SnoopTag colored magenta. (B) Sample gel to test for quantitative reaction of SnoopTag. Shown are 10 µM SnoopTag-MBP and 20 µM SnoopCatcher alone or mixed for 32 min (triplicate samples) before SDS/PAGE with Coomassie staining. (C) Sample gel to test for quantitative reaction of SnoopCatcher. Shown are 10 µM SnoopCatcher and 40 µM SnoopTag-MBP alone or mixed for the indicated times (triplicate samples) before SDS/PAGE with Coomassie staining. (D) Buffer dependence of SnoopTag/SnoopCatcher reaction. We incubated 10 µM SnoopTag-MBP with 10 µM SnoopCatcher at pH 8.0 for 15 min at 25 °C in the indicated buffer and analyzed the samples by SDS/PAGE with Coomassie staining. (E) TMAO dependence of SnoopTag/SnoopCatcher reaction tested as in D. Error bars are all mean ±1 SD; n = 3. Some error bars are too small to be visible.

Article Snippet: Constructs were initially cloned into chemically competent E. coli DH5α (Life Technologies). pET28a SpyTag-MBP (Addgene plasmid ID 35050), GST-BirA, and pDEST14-SpyCatcher (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JQ478411","term_id":"380294102","term_text":"JQ478411"}} JQ478411 , Addgene plasmid ID 35044) have been described ( 26 ). pET28a SnoopCatcher (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU500646","term_id":"985768551","term_text":"KU500646"}} KU500646 , Addgene plasmid ID 72322) was generated by DNAWorks primer-mediated assembly from residues 749–860 of S. pneumoniae adhesin RrgA (numbering based on PDB ID code 2WW8) ( 28 ), digested with HindIII and NdeI and subcloned into pET28a.

Techniques: SDS Page, Staining, Incubation

Journal: Biology

Article Title: A Novel Vitronectin Peptide Facilitates Differentiation of Oligodendrocytes from Human Pluripotent Stem Cells (Synthetic ECM for Oligodendrocyte Differentiation)

doi: 10.3390/biology10121254

Figure Lengend Snippet: Induction and expansion of hPSC-derived OPCs. ( A ) A bright-field image of OPCs at day 12 of differentiation. Scale bar: 20 µm. (Inset) A representative image of NESTIN-positive cells in the culture on day 12. ( B , C ) Immunofluorescent images of OLIG2, A2B5 and NG2. ( D ) Quantification of cells positive for OLIG2, A2B5, and NG2. ( E ) Quantitative gene expression of OPC markers ( OLIG2 and PDGFRα ) and a pluripotency marker ( NANOG ). ( F , G ) Representative images of OLIG2, A2B5- and NG2-positive cells after multiple passages (at passage 11) accompanied with a freeze-thawing cycle. Scale bar: 20 µm. ( H ) Quantification of marker-positive cells at passage 11. ( I ) Quantitative gene expression of OPC markers ( PDGFRα and SOX10 ). Scale bars: 10 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, Student t -test.

Article Snippet: One day later, the cells were infected with a lentivirus encoding SOX10 (Addgene, clone ID number 45843, Watertown, MA, USA) and a lentivirus of reverse tetracycline-controlled transactivator ( rtTA ) (Addgene, clone ID number 20342) for doxycycline (DOX)-induced SOX10 expression and incubated for 14–16 h. Viral vectors and packaging were constructed as described elsewhere [ ].

Techniques: Derivative Assay, Gene Expression, Marker

Journal: Biology

Article Title: A Novel Vitronectin Peptide Facilitates Differentiation of Oligodendrocytes from Human Pluripotent Stem Cells (Synthetic ECM for Oligodendrocyte Differentiation)

doi: 10.3390/biology10121254

Figure Lengend Snippet: Differentiation of oligodendrocytes from hPSC-derived OPCs via SOX10 overexpression. ( A ) A schematic diagram for oligodendrocyte differentiation from hPSC-derived OPCs. ( B – D ) Time-course expression of OPC markers during differentiation. As differentiation proceeded, the transcript levels of OLIG2 and PDGFR-α gradually decreased ( C , D ) whereas that of SOX10 was maintained due to continued exogenous expression ( B ). ( E – G ) Time-course expression of OD markers during differentiation. Expression levels of all marker genes tested were upregulated by SOX10-induced differentiation and plateaued after day 8. ( H , I ) Representative image of O4- and MBP-positive cells induced by the overexpression of SOX10. Scale bars: 12.5 µm. ** p < 0.01, **** p < 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: One day later, the cells were infected with a lentivirus encoding SOX10 (Addgene, clone ID number 45843, Watertown, MA, USA) and a lentivirus of reverse tetracycline-controlled transactivator ( rtTA ) (Addgene, clone ID number 20342) for doxycycline (DOX)-induced SOX10 expression and incubated for 14–16 h. Viral vectors and packaging were constructed as described elsewhere [ ].

Techniques: Derivative Assay, Over Expression, Expressing, Marker

Journal: Biology

Article Title: A Novel Vitronectin Peptide Facilitates Differentiation of Oligodendrocytes from Human Pluripotent Stem Cells (Synthetic ECM for Oligodendrocyte Differentiation)

doi: 10.3390/biology10121254

Figure Lengend Snippet: Screening for ECM materials facilitating oligodendrocyte differentiation induced by SOX10. ( A – F ) Representative images of O4- and MBP-positive cells generated on the indicated matrix. White arrows are pointing to double-positive cells for O4 and MBP. ( G , H ) Quantification of the number of O4- or MBP-positive cells on day 12 after initiation of oligodendrocyte maturation on the indicated substrates. * p < 0.05, ** p < 0.01, one-way ANOVA with Tukey’s post-hoc test.

Article Snippet: One day later, the cells were infected with a lentivirus encoding SOX10 (Addgene, clone ID number 45843, Watertown, MA, USA) and a lentivirus of reverse tetracycline-controlled transactivator ( rtTA ) (Addgene, clone ID number 20342) for doxycycline (DOX)-induced SOX10 expression and incubated for 14–16 h. Viral vectors and packaging were constructed as described elsewhere [ ].

Techniques: Generated

Journal: Viruses

Article Title: Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections

doi: 10.3390/v13071387

Figure Lengend Snippet: Retrograde transgene expression via HiRet and NeuRet vectors after vector injections into M2 in rats. ( a ) GFP immunostaining in the cortex (contralateral M2). Right panels denote higher-power magnifications of the square areas in left panels. Scale bars, 500 μm (left) and 50 μm (right). ( b ) Density (cells per mm 2 ) of GFP-positive cells in the contralateral M2. Mean ± SEM ( n = 4). * p < 0.05, significant differences from the value for the HiRet vector (Student’s t test). ( c ) GFP immunostaining in the ipsilateral thalamus (left, VL; right, CL). Right panels denote higher-power magnifications of the square areas in left panels. HC, hippocampus; R, reticular nucleus of the thalamus; 3V, third ventricle. Scale bars, 500 μm (left) and 50 μm (right). ( d ) Number (cells per section) of GFP-positive cells in the ipsilateral VL and CL. Data are expressed as the mean ± SEM ( n = 4).

Article Snippet: Together with the FuG-B2 type of HiRet vector (Addgene plasmid ID number 67513) and the FuG-E type of NeuRet vector (Addgene plasmid ID number 67509), the FuG-E (P440E) variant of NeuRet vector (Addgene plasmid ID number 119978) was used for the present purpose.

Techniques: Expressing, Plasmid Preparation, Immunostaining

Journal: Viruses

Article Title: Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections

doi: 10.3390/v13071387

Figure Lengend Snippet: Retrograde transgene expression via HiRet and NeuRet vectors after vector injections into A6m in marmosets. ( a ) GFP immunostaining in the cortex (left, contralateral A6m; right, ipsilateral PE and PCC). A6DC, dorsocaudal part of area 6; A24, area 24. Scale bars, 500 µm (left) and 100 μm (right). ( b ) Density (cells per mm 2 ) of GFP-positive cells in contralateral A6m and the ipsilateral PE and PCC. Data obtained in two animals are indicated with hollow and solid symbols (circles, triangles, and squares). ( c ) GFP immunostaining in the ipsilateral thalamus (left, VL; right, CL). Cd, caudate nucleus; LGN, lateral geniculate nucleus of the thalamus; MD, mediodorsal nucleus of the thalamus; VP, ventroposterior nucleus of the thalamus. Other abbreviations are as in . Scale bars, 500 µm (left) and 100 μm (right). ( d ) Number (cells per section) of GFP-positive cells in the ipsilateral VL and CL. Data obtained in two animals are indicated with hollow and solid symbols (circles, triangles, and squares). All other conventions are as in .

Article Snippet: Together with the FuG-B2 type of HiRet vector (Addgene plasmid ID number 67513) and the FuG-E type of NeuRet vector (Addgene plasmid ID number 67509), the FuG-E (P440E) variant of NeuRet vector (Addgene plasmid ID number 119978) was used for the present purpose.

Techniques: Expressing, Plasmid Preparation, Immunostaining

Journal: Viruses

Article Title: Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections

doi: 10.3390/v13071387

Figure Lengend Snippet: Retrograde transgene expression via HiRet and NeuRet vectors after vector injections into SMA in macaques. ( a ) GFP immunostaining in the cortex (ipsilateral ventral premotor cortex, PMv). Cd, caudate nucleus; PMd, dorsal premotor cortex; Put, putamen; sAS, spur of the arcuate sulcus. Scale bars, 1 mm (left) and 100 μm (right). ( b ) Density (cells per mm 2 ) of GFP-positive cells in the contralateral SMA and the ipsilateral PMv. ( c ) GFP immunostaining in the ipsilateral thalamus (VL). All abbreviations are as in and . ( d ) Number (cells per section) of GFP-positive cells in the ipsilateral VL and CL. Scale bars, 1 mm (left) and 100 μm (right). All other conventions are as in .

Article Snippet: Together with the FuG-B2 type of HiRet vector (Addgene plasmid ID number 67513) and the FuG-E type of NeuRet vector (Addgene plasmid ID number 67509), the FuG-E (P440E) variant of NeuRet vector (Addgene plasmid ID number 119978) was used for the present purpose.

Techniques: Expressing, Plasmid Preparation, Immunostaining

Journal: Viruses

Article Title: Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections

doi: 10.3390/v13071387

Figure Lengend Snippet: Inflammatory/immune responses of HiRet and NeuRet vectors. ( a ) GFP (green) and double immunofluorescence staining for Iba1 (red) and CD8 (blue) at the injection sites of the HiRet (upper) and NeuRet (FuG-E; lower) vectors in rats. Insets: higher-power magnifications of the square areas. Scale bars: 500 μm and 100 μm for insets. ( b ) Data obtained for marmosets. Scale bars: 500 μm and 200 μm for insets. ( c ) Data obtained for macaques. HiRet vector (top), NeuRet vector (middle), and higher-titer NeuRet vector (bottom). Scale bars: 2 mm and 0.2 mm for insets.

Article Snippet: Together with the FuG-B2 type of HiRet vector (Addgene plasmid ID number 67513) and the FuG-E type of NeuRet vector (Addgene plasmid ID number 67509), the FuG-E (P440E) variant of NeuRet vector (Addgene plasmid ID number 119978) was used for the present purpose.

Techniques: Double Immunofluorescence Staining, Injection, Plasmid Preparation

Journal: Viruses

Article Title: Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections

doi: 10.3390/v13071387

Figure Lengend Snippet: Neuron specificity of HiRet and NeuRet vectors. ( a ) Double immunofluorescence histochemistry for GFP and NeuN or GFP and GFAP at the injection sites of the HiRet (upper) and NeuRet (FuG-E; lower) vectors in marmosets. Scale bar, 50 μm. ( b ) Ratios of double-labeled cells (GFP + NeuN + , GFP + GFAP + ) to the total GFP-positive cells (GFP + ). ( c , d ) Data obtained for macaques. Scale bar, 50 μm.

Article Snippet: Together with the FuG-B2 type of HiRet vector (Addgene plasmid ID number 67513) and the FuG-E type of NeuRet vector (Addgene plasmid ID number 67509), the FuG-E (P440E) variant of NeuRet vector (Addgene plasmid ID number 119978) was used for the present purpose.

Techniques: Immunofluorescence, Injection, Labeling