Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Expression, cytotoxic activity, and cytokine secretion of T cells expressing altered version of FcγRI. A, Illustration of the modified FcγRI structure with fused signaling domains: A2G (left), FcγRI composed of α-chain and homodimer of FcRγ chain; AG2G (middle), FcγRI α-chain with fused domain of FcRγ chain and homodimer of FcRγ; AGO2GO (right), FcγRI α-chain with fused FcRγ and OX40 domains and homodimer of FcRγ with fused OX40 domain. B, Flow cytometry analysis of A2G, AG2G, and AGO2GO expression following retroviral transduction of healthy donor T cells. C, Mean numbers of HT29 target cells expressing HER2 calculated by incuCyte software through 48-hour incubation with the three FcγRI-based receptor expressing T cells, with 1:1 E:T ratios with or without 6 μg/mL trastuzumab (data shows one of two experiments repeats, n = 4). Each sample is normalized to its cell number at time zero. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. D, Representative incuCyte images showing HER2-expressing HT29 cells, imaged and counted in live imaging system after 48 hours of incubation with different construct-expressing T cells with 1:1 E:T ratio. Scale bar is 100 μm. E, Human Luminex Discovery Assay measurement of cytokine levels in supernatants from 48 hours of culture of T cells 4:1 E:T ratio with HT29 cells expressing HER2 ( n = 4). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Expressing, Activity Assay, Modification, Flow Cytometry, Retroviral, Transduction, Software, Incubation, Imaging, Construct, Luminex

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Characterization of AG2G-expressing T cells, activation, and cytotoxicity. A, ELISA measurement of cytokine levels of AG2G-expressing T cells after 48-hour incubation with HT29 cells expressing HER2. B, IFNγ and TNFα levels measured by ELISA in supernatants from 48-hour incubation of AG2G-expressing T cells and HT29 cells expressing HER2 in different E:T ratios. C, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with 0, 2.5, 5, 10 mg/mL IVIG, with or without 30 μg/mL trastuzumab ( n = 4). D, ELISA measurement of IFNγ levels of AG2G-expressing cells cocultured with HT29 cells expressing HER2 described in B . E, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with different concentrations of trastuzumab over 96 hours ( n = 2). F, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with different E:T ratios of AG2G-expressing T cells that were isolated for their CD4 (left) or CD8 (middle) population or with no isolation (right). G, ELISA measurement of cytokine levels from supernatants of 48-hour culture of 4:1 E:T ratios of isolated CD4, isolated CD8, or AG2G-expressing T cells with HT29 cells expressing HER2 and 30 μg/mL trastuzumab. Graphs show mean ±SD. Statistical significance was calculated using Student t test for a, two-way ANOVA with Sidak correction for multiple comparisons for B and C , two-way ANOVA with Tukey's correction for multiple comparisons for E and F . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Software, Isolation

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: AG2G-expressing T cells differentiate between cells expressing high and low antigen levels, are more specific, and less exhausted, compared with classic CAR T cells. A, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with transduced T cells in combination with antibodies (60 μg/mL each, n = 4). B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-transduced T cells incubated with HER2-expressing HT29 target cells, and with tumor-binding trastuzumab, or irrelevant antibodies cetuximab and rituximab (60 μg/mL). C, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. D, Normalized confluence of kidney epithelial cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell confluence at time zero. E, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D ( n = 4). F, ELISA measurement of IFNγ, granzyme B and TNFα levels in 24-hour supernatants of 10 5 AG2G-expressing T cells or CAR T cells incubated on immobilized trastuzumab or rHER2, in rising concentrations (mmol/mL) respectively. G, Confocal microscope imaging of internalization kinetics of PE-labeled IgG by AG2G-expressing cells (top), or PE-labeled HER2 by CAR T cells (bottom). H, PD-1, LAG-3, and TIM3 flow cytometry analysis of AG2G-expressing cells or CAR T cells after 48-hour incubation with or without 12 mmol/mL immobilized trastuzumab or HER2, respectively. Graphs show mean ± SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for A , C , D , and H . Two-way ANOVA with Sidak correction for multiple comparisons for E and F . One-way ANOVA with Dunnett correction for multiple comparisons for B . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Expressing, Software, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay, Microscopy, Imaging, Labeling, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Human T cells expressing AG2G exert specific tumor cytotoxicity while sparing normal cells. A, Flow cytometry expression analysis of HER2 on different tumor cell lines and primary human normal cells. B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-expressing T cells with different tumor cells or primary normal cells in 4:1 E:T ratio and 60 μg/mL trastuzumab (results shown are from 3 different donors combined, n = 12). C, Quantification of HER2 receptor numbers using DAKO QIFIKIT (Agilent) beads by flow cytometer. Individual populations of the calibration are gated, a linear regression is calculated from the MFI and the ABC values of the five calibration bead populations using MS Excel. Using the linear regression, the ABCs of the beads coated with αHER2 is calculated from their MFI values. SDs are also retrieved from gated populations and transformed using linear regression. D, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. E, Normalized confluence of kidney epithelial (epithel) cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab. Each sample is normalized to its cell confluence at time zero. Graphs are identical and described in . and but with the comparison to ACTR707 instead of CAR T. F, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D and E ( n = 4). G, ELISA measurement of IFNγ levels in 24 hours supernatants of AG2G-expressing T cells or ACTR707 incubated with medium containing different concentration of IVIG ( n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. ****, P < 0.0001. Error bars represent standard error. Endothel, endothelial cells.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Transformation Assay, Software, Incubation, Comparison, Concentration Assay

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Retroviral transduction of γδ-T cells with AG2G endows them with antitumor ADCC. A, Flow cytometry analysis of sham or AG2G-transduced αβ-T cells (two left panels, activated with IL2 and anti-CD3); sham or AG2G-transduced γδ-T cells (two right panels, activated with IL2 and zoledronic acid). B, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with AG2G-expressing γδ-T cells or AG2G-expressing αβ-T cells, with or without trastuzumab ( n = 3). C, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with sham untransduced or AG2G-expressing γδ-T cells with or without trastuzumab, in E:T ratios of 1:1, 2:1 or 4:1 ( n = 4). D, ELISA measurement of IFNγ levels in supernatants of 48-hour sham or AG2G-expressing γδ-T cells cocultured with HT29-HER2 expressing cells, with or without trastuzumab (E:T ratio 4:1, n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for B and C . Two-way ANOVA with Sidak correction for multiple comparisons for D . *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Retroviral, Transduction, Flow Cytometry, Expressing, Software, Enzyme-linked Immunosorbent Assay

Journal: Microbial cell factories

Article Title: Engineering osmolysis susceptibility in Cupriavidus necator and Escherichia coli for recovery of intracellular products.

doi: 10.1186/s12934-023-02064-8

Figure Lengend Snippet: Fig. 5 Application of mechanosensitive knockout for osmolysis in E. coli BL21. A Percent cell lysis following growth in LB with 4% NaCl of BL21 (blue), BL21 ΔmscL (red), and BL21 ΔmscL ΔmscS (yellow) in three different media: commercial B-PER™ Bacterial Protein Extraction Reagent; a 4% NaCl(aq) isotonic solution; and distilled water (diH2O). Cell lysis is determined as the fraction of RFP recovered in supernatant compared to total RFP content, as it was in C. necator, with concentration measured by fluorescence intensity (n = 6). B Growth rate (red diamonds) and osmolysis efficiency (blue circles) of BL21 ΔmscL ΔmscS in LB with various (final) NaCl concentrations (n = 3). C SDS-PAGE gel of various fractions of RFP-expressing BL21 and BL21 ΔmscL ΔmscS. NE: cells not expressing RFP, WC: whole-cell fraction, S: post-osmolysis supernatant fraction, P: post-osmolysis cell pellet. For each data point in (A) and (B), mean ± SD is displayed

Article Snippet: All C. necator strains listed in Table 1 were transformed with the red fluorescent protein (RFP) expression plasmid pBADTrfp via conjugation using E. coli WM3064 as a donor, following the protocol from Windhorst et al. [31]. pBADTrfp was a gift from Nathan Hillson (Addgene plasmid # 99382; http:// n2t. net/ addge ne: 99382; RRID:Addgene_99382) [29]. pBADTrfp was transformed into chemically competent WM3064 cells using heat shock and positive clones were selected for on LB-Agar plates containing DAP and kanamycin (50 μg/ mL) following a 1-h outgrowth in SOC medium.

Techniques: Knock-Out, Lysis, Protein Extraction, Concentration Assay, Fluorescence, SDS Page, Expressing

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: srn_3610_sprC expression increases in the absence of SarA. ( A ) Srn_3610_sprC expression in different Staphylococcus aureus strains lacking transcription factors (TFs). Northern blot analysis of the Srn_3610_sprC and transfer-messenger RNA (tmRNA) transcripts in the S. aureus HG003 strain and in isogenic mutants for the SarA protein family. All samples were harvested after 5 h of growth. A total of 10 μg of total RNA were analyzed, and the Digoxigenin method (Roche) was used to reveal Srn_3610_sprC and tmRNA (loading control). ( B ) Northern blot analysis of Srn_3610_SprC and tmRNA transcript expression in S. aureus HG003 and isogenic HG003Δ sarA . Samples were harvested at the specified time point, and tmRNA was used as a control. In a graph showing optical density as a function of time, the growths of HG003 (diamonds) and HG003Δ sarA (triangles) are represented in a semi-logarithmic scale.

Article Snippet: Briefly, binding reaction medium (10 fmol DNA template, 20 mM HEPES-KOH, pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, 0.1 M MgCl 2 , 5 mM DTT and 0.2 μg poly(dI-dC)), was incubated when necessary with purified SarA or the E. coli RNA polymerase holoenzyme (New England Biolabs) for 30 min at 30°C.

Techniques: Expressing, Northern Blot

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: SarA represses srn_3610_sprC expression. ( A ) Staphylococcus aureus HG003 and HG003Δ sarA strains were co-transformed with: pCN36/pCN41c empty vectors (lanes 1 and 5); pCN36/pCN41c - P sprC (lanes 2 and 6); pCN36-SarA/pCN41c (lanes 3 and 7); and pCN36-SarA/pCN41- P sprC (lanes 4 and 8). Srn_3610_SprC and SarA transcripts levels were assessed by northern blot. A 16S rRNA probe was used to reflect total RNA loading for each lane. In parallel, SarA protein levels were checked by western blot (middle panel) and SYPRO Ruby staining of total protein extracts was done to compare loaded protein levels (bottom). ( B ) Effects of SarA on transcriptional activity of the srn_3610_sprC promoter (P sprC ). S. aureus HG003 wild-type strain (HG003) and a strain lacking sarA (HG003Δ sarA ) were co-transformed with pCN41c or pCN41c-P sprC and either pCN36 or pCN36-SarA. P sprC activity was estimated by measuring ß-lactamase substrate hydrolysis. For each lane, indicated ß-lactamase activity was normalized by subtracting the background signal from the same strain where pCN41c-P sprC was replaced by pCN41c empty vector (not shown). Three independent experiments were done, error bars show ± standard deviation. (Mann–Whitney test; * P < 0.05; *** P < 0.001.).

Article Snippet: Briefly, binding reaction medium (10 fmol DNA template, 20 mM HEPES-KOH, pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, 0.1 M MgCl 2 , 5 mM DTT and 0.2 μg poly(dI-dC)), was incubated when necessary with purified SarA or the E. coli RNA polymerase holoenzyme (New England Biolabs) for 30 min at 30°C.

Techniques: Expressing, Transformation Assay, Northern Blot, Western Blot, Staining, Activity Assay, Plasmid Preparation, Standard Deviation, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: srn _ 9340 possesses a putative binding site for SarA. Srn_3610_sprC belongs to the pathogenicity island SaPIn3. SprC 47 , which can interact with SarA, possesses a 22-nt palindromic sequence (underlined). Blast analysis using sprC 47 as the query led to the discovery of another putative SarA binding site within SaPIn3. This sequence was identified as part of the promoter of another small RNA, Srn_9340.

Article Snippet: Briefly, binding reaction medium (10 fmol DNA template, 20 mM HEPES-KOH, pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, 0.1 M MgCl 2 , 5 mM DTT and 0.2 μg poly(dI-dC)), was incubated when necessary with purified SarA or the E. coli RNA polymerase holoenzyme (New England Biolabs) for 30 min at 30°C.

Techniques: Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: SarA binds srn_9340 on its promoter and represses transcription. ( A ) SarA represses srn_9340 expression. After 5 h of growth, 10 μg of total RNA were obtained from Staphylococcus aureus HG003 and HG003Δ sarA . Srn_9340 expression levels were monitored by northern blot. ( B ) Schematic representation of both the 5′ and 3′ ends of the Srn_9340 transcripts (long and short small RNAs), as determined by rapid amplification of cDNA ends (RACE). ( C ) SarA binds the srn_9340 promoter in vitro . An EMSA was done using 10 fmol of a 32 P-labeled srn_9340 promoter fragment ( srn_9340 P225 ) as a probe in the presence of increasing amounts (0.01–1 pmol) of 6His-tagged SarA. Srn_9340 P225 forms an initial complex with 0.15 pmol of SarA, and a second one at 0.75 pmol. ( D ) SarA specifically binds srn_9340 P225 in vitro . EMSA was done with 10 fmol of 32 P-labeled srn_9340 P225 , 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled srn_9340 P225 ) or non-specific competitors (unlabeled 16S 225 ). ( E ) DNase I footprinting assays were performed in the presence of 10 fmol srn_9340 P225 , 7.5.10 −2 U DNase I and increasing amounts (0.1–0.5 pmol) of 6His-SarA. The region protected by SarA is indicated with a vertical dotted arrow, and the numbers indicate relative positions to the previously determined transcription start site (TSS). Lanes G, A, T and C correspond to sequencing. The nucleotides from −51 to −1 ( srn_9340 51 ) are protected by SarA against DNase I degradation. ( F ) Schematic representation of the DNA probes used for EMSA studies. ( G ) Deletion of the protected SarA sequence from the srn_9340 promoter region abolishes SarA's capacity to bind the srn_9340 promoter. ( H ) A 51 bp protected sequence is sufficient for SarA binding. EMSA were done using 10 fmol srn_9340 51 in the presence of increasing amounts of 6His-tagged SarA (0.25–2 pmol). ( I ) Srn_9340 51 competes with srn_9340 225 for SarA binding. EMSA was performed with 10 fmol of 32 P-labeled srn_9340 P225 , 0.4 or 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled srn_9340 51 ) or non-specific competitors (unlabeled Random 51 ). ( J ) The 23 bp SarA binding site on srn_9340 225 was confirmed by EMSA done in the presence of 0.5 pmol sarA and with increasing amounts of unlabeled srn_9340 225 or unlabeled srn_9340 225 Δ23 .

Article Snippet: Briefly, binding reaction medium (10 fmol DNA template, 20 mM HEPES-KOH, pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, 0.1 M MgCl 2 , 5 mM DTT and 0.2 μg poly(dI-dC)), was incubated when necessary with purified SarA or the E. coli RNA polymerase holoenzyme (New England Biolabs) for 30 min at 30°C.

Techniques: Expressing, Northern Blot, Rapid Amplification of cDNA Ends, In Vitro, Labeling, Footprinting, Sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: In vivo analysis of the srn_3610_sprC and srn_9340 promoters: SarA versus σ A binding. The Staphylococcus aureus HG003Δ sarA strain was transformed either with an empty pCN38 vector (dark gray) or with pCN38-SarA6His (light gray). Chromatin immunoprecipitation (ChIP) experiments were conducted using antibodies for 6His-tag ( A ) or for the σ 70 /σ A RNA polymerase subunit ( B ). DNA enrichment was assessed by qPCR analysis using specific primers for the promoters of srn_3610_sprC (left panel), srn_9340 (middle) or hla (right). Values beneath the bars indicate fold change between specific and non-specific interactions. (Mann–Whitney U test; three independent experiments; * P < 0.05). (A) srn_3610_sprC, srn_9340 and hla promoters co-immunoprecipitate with SarA6His. (B) SarA6His disturbs σ A binding on the srn_3610_sprC promoter.

Article Snippet: Briefly, binding reaction medium (10 fmol DNA template, 20 mM HEPES-KOH, pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, 0.1 M MgCl 2 , 5 mM DTT and 0.2 μg poly(dI-dC)), was incubated when necessary with purified SarA or the E. coli RNA polymerase holoenzyme (New England Biolabs) for 30 min at 30°C.

Techniques: In Vivo, Binding Assay, Transformation Assay, Plasmid Preparation, Chromatin Immunoprecipitation, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: SarA and bacterial RNA polymerase holoenzyme cannot simultaneously bind sprC P267 and srn_9340 P225 . EMSA was done using 10 fmol 32 P-labeled sprC P267 or srn_9340 P225 probes with increasing amounts (2–100 fmol) of purified RNA polymerase. The binding capacity of Escherichia coli RNA polymerase was confirmed by EMSA experiments using sprC P267 ( A ) or srn_9340 P225 ( B ). To demonstrate their binding exclusivity on sprC P267 ( C ) or srn_9340 P225 ( D ), 0.5 pmol purified SarA and 50–100 fmol E. coli RNA polymerase were simultaneously added to EMSA experiments. In vitro transcription was realized using 10 fmol of sprC 456 ( E ) or srn_9340 337 ( F ) and 100 fmol of E. coli RNA polymerase. A total of 5 pmol of the purified TF SarA were added, when indicated.

Article Snippet: Briefly, binding reaction medium (10 fmol DNA template, 20 mM HEPES-KOH, pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.1 M KCl, 0.1 M MgCl 2 , 5 mM DTT and 0.2 μg poly(dI-dC)), was incubated when necessary with purified SarA or the E. coli RNA polymerase holoenzyme (New England Biolabs) for 30 min at 30°C.

Techniques: Labeling, Purification, Binding Assay, In Vitro

Journal: Molecules

Article Title: The Effect of Ethanol on Gelation, Nanoscopic, and Macroscopic Properties of Serum Albumin Hydrogels

doi: 10.3390/molecules25081927

Figure Lengend Snippet: Figure 3. (A) Amide 1 band of 2.6 mM BSA solution with 12.1 wt % EtOH at 37 ◦C on ATR-IR crystal (BioATRCell), the same condition as Figure 2C, shows homogenous changes across all the amide 1 band and no gel formation. (B) Amide 1 band of 2.4 mM BSA solution with 19.9 wt % EtOH at 37 ◦C on ATR-IR crystal, the same condition as that in Figure 2B, shows an increase in the intermolecular β-sheet peak, whereas native α-helices of BSA are lost. In this case, gel formed on the spectrometer crystal. (C) PC1 scores vs. time regarding Figure 3A, (D) PC1 scores vs. time regarding Figure 3B. For more detailed information about PCA analysis, see ref. [6].

Article Snippet: This process is detectable by recording the spectra during gel formation on the crystal of an attenuatated total reflection infrared (ATR-IR) spectrometer (A Bruker Tensor 27 FT-IR spectrometer equipped with a BioATRCell II and an LN-MCT photovoltaic detector and the To elucidate the mechanical properties and the rate of gelation of the gels obtained from ethanol to a more robust and more rapid gelation compared to the pH-induced method.

Techniques: