Journal: Journal of Synchrotron Radiation

Article Title: High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner

doi: 10.1107/S1600577518010354

Figure Lengend Snippet: Crystallography data collection and processing at FMX. Raster scans of ( a ) a bovine trypsin rod-shaped crystal and ( b ) 5–10 µm-sized proteinase K crystals loaded on MiTeGen loops. The raster-scan areas are shown as red-dashed rectangles; the corresponding heat maps acquired after the raster scans are shown in the insets. The grid coloring is the diffraction pattern spot count. ( c ) Clustering of the serial crystallography partial datasets based on unit-cell size. The data shown were acquired from raster scans of proteinase K microcrystals with a 200 Hz detector frame rate. Of these 279 partial datasets, 234 (blue dots) were indexed to c = 106.58 ± 0.41 Å, with 99.6% completeness (Table 1 ▸ ). The remaining 45 partial datasets (orange dots) with c = 110.06 ± 0.84 Å did not yield a complete dataset and were excluded from refinement. The unit-cell size was converted to Cartesian coordinates for better visualization. The data were clustered into two groups using a K -mean clustering algorithm. ( d ) Hierarchical cluster-analysis dendrogram for the proteinase K partial datasets acquired at a 750 Hz detector frame rate. The cutoff limit was set to 0.6 (dashed line).

Article Snippet: Proteinase K crystals were grown on siliconized glass cover slips (hanging drop) by equilibrating 25 mg ml−1 proteinase K (Worthington Biochemical; LS004224) in 40 mM calcium chloride, 400 mM sodium nitrate and 50 mM BisTris (pH 6.5) over a reservoir containing 160 mM calcium chloride, 1.6 M sodium nitrate and 200 mM BisTris (pH 6.5).

Techniques:

Journal: Cell host & microbe

Article Title: Coagulase-Negative Staphylococcal Strain Prevents Staphylococcus aureus Colonization and Skin Infection by Blocking Quorum Sensing

doi: 10.1016/j.chom.2017.11.001

Figure Lengend Snippet: Initial characterization and identification of the S. caprae quorum-sensing inhibitor A . The S. caprae spent media was treated with 65 °C heat, proteinase K (1 mg/mL final), EGTA (5 mM final), or NaOH (to a pH of 11, then neutralized). Each sample was added to MRSA agr type I reporter to 10% final and compared to no treatment control. Florescence was measure at 24 hr, and the results are pooled from two experiments, each with N=3 replicates per condition and normalized to the media control. One-way ANOVA with multiple comparisons (Dunnett’s correction) was performed. **** indicates p

Article Snippet: For proteinase K treatment, 5 μL of a 20 mg/mL proteinase K solution (Fermentas) were added to 100 μL of spent media and incubated in a 37 °C heat block for one hour.

Techniques:

Journal: bioRxiv

Article Title: A novel method to isolate free-floating extracellular DNA from wastewater for quantitation and metagenomic profiling of mobile genetic elements and antibiotic resistance genes

doi: 10.1101/2020.05.01.072397

Figure Lengend Snippet: The impact of enzymatic post-treatment of raw eDNA extracts with proteinase K to release the eDNA from extracellular protein mesh: (a) Agarose gel from three different biological replicates showing the fragment size distributions of intracellular DNA (iDNA) obtained without any preliminary treatment, of iDNA obtained after enzymatic pre-treatment of cells with DNase I prior to cell lysis and DNA extraction, and ofthe raw extract of free-floating extracellular DNA (eDNA) obtained from from centrifuged and filtered activated sludge supernatant samples. (b) Agarose gel of free-floating eDNA after either pre-treatment of cells with DNase I, post-treatment of eDNA extract with proteinase K, or a combination of both pre-and post-treatments.

Article Snippet: The precipitated raw eDNA was incubated with proteinase K (Sigma-Aldrich, UK) at 0.85 g L−1 during 2 h in order to digest remaining co-extracted proteins (e.g. , DNA-bound proteins).

Techniques: Agarose Gel Electrophoresis, Lysis, DNA Extraction

Journal: Nucleic Acids Research

Article Title: Co-transcriptional formation of DNA:RNA hybrid G-quadruplex and potential function as constitutional cis element for transcription control

doi: 10.1093/nar/gkt264

Figure Lengend Snippet: HQ formation during transcription of linear dsDNA that contained a G-core motif from the NRAS gene on the non-template strand. ( A, B ) HQ detection by DMS footprinting in DNA that carried (A) four or (B) three and two G-tracts. DNA was labeled at the 5′-end of the non-template strand with a FAM (asterisk), then subjected either to heat denaturation/renaturation (H) or transcription (T) with ATP and GTP (AG) or all four NTPs (NTP) and followed by RNase A and protease K digestion. Footprinting cleavage fragments were resolved by denaturing gel electrophoresis (left) and digitized (right) for comparison. Arrowheads indicate protected guanines. ( C ) HQ detection by native gel electrophoresis. DNA carrying four (top), three (middle) or two (bottom) G-tracts was heated or transcribed with either GTP or dzGTP and the other three NTPs as in (A, B). After RNase A and protease K digestion, the DNA was resolved on a native gel. Filled and half-filled arrowheads indicate intramolecular DNA G-quadruplexes and intermolecular DNA:RNA hybrid G-quadruplexes (HQ), respectively, and their amounts as the percentage of total DNA. ( D ) Primer extension detection of photo-cross-linking at the non-template strand by 4-thio-uridine incorporated into the RNA transcript. The 5′-FAM labeled primer was annealed to the 3′-end of the non-template of non-transcribed (N, lane 1) or transcribed (T, lane 2) DNA, followed by extension with DNA sequenase. Extension products were resolved by denaturing gel electrophoresis. G and A ladders were obtained by primer extension with ddCTP and ddTTP, respectively. Symbols on the right sides of the lanes and non-template sequence indicate the cross-linking sites (gray heart), G-tracts (black bar), guanines (filled triangle) and adenines (open triangle). Arrows on the left side of the non-template sequence indicate the sites of 4-thio-uridine incorporation. ( E ) HQ formation requires four G-tracts. Transcription and HQ detection were carried out as in (C) using dsDNA carrying the indicated G/C-tracts without or with a single mutation (nucleotide underlined) at the middle of a single G- or C-tract. Transcriptions in (A–E) were all conducted in solution containing 50 mM K + and 40% (w/v) PEG 200.

Article Snippet: For dimethyl sulfate (DMS) footprinting, native gel electrophoresis and photocleavage footprinting, the samples were further treated with 0.4 mg/ml of RNase A (Fermentas, MBI), then 0.6 mg/ml proteinase K (TAKARA, Dalian) at 37°C for 1 h each.

Techniques: Footprinting, Labeling, Nucleic Acid Electrophoresis, Sequencing, Mutagenesis

Journal: Human Molecular Genetics

Article Title: Extensive enteric nervous system abnormalities in mice transgenic for artificial chromosomes containing Parkinson disease-associated ?-synuclein gene mutations precede central nervous system changes

doi: 10.1093/hmg/ddq038

Figure Lengend Snippet: Aggregates of α-synuclein immunoreactivity that resist digestion with proteinase K are found in enteric neurons of Dbl-PAC-Tg( SNCA A53T ); Snca −/− mice. ( A – C ) Myenteric plexus from the ileum of a Dbl-PAC-Tg( SNCA A53T ); Snca −/− mouse. (A) Aggregates of α-synuclein immunoreactivity (arrow) are found in neurons of the myenteric plexus. (B) Bisbenzamide fluorescence of the field illustrated in A. There is some diffusion of bisbenzamide fluorescence due to the digestion of tissue with proteinase K; however, the locations of large neuronal nuclei are evident. (C) Merged image of Alexa 594 and bisbenzamide fluorescence. ( D – F ) Myenteric plexus from the ileum of a PAC-Tg( SNCA WT ); Snca −/− mouse. (D) There is no remaining α-synuclein immunoreactivity after digestion of tissue with proteinase K. (E) Bisbenzamide fluorescence. (F) Merged image of Alexa 594 and bisbenzamide fluorescence. ( G – I ) Submucosal plexus from the ileum of a Dbl-PAC-Tg( SNCA A53T ); Snca −/− mouse. (G) Aggregates of α-synuclein immunoreactivity (arrows) are found in neurons of the submucosal plexus. (H) Bisbenzamide fluorescence of the field illustrated in A. (I) Merged image of Alexa 594 and bisbenzamide fluorescence. ( J – L ) Submucosal plexus from the ileum of a PAC-Tg( SNCA WT ); Snca −/− mouse. (J) There is no remaining α-synuclein immunoreactivity after proteinase K digestion of tissue. (K) Bisbenzamide fluorescence. (L) Merged image of Alexa 594 and bisbenzamide fluorescence. The marker = 25 µm.

Article Snippet: The preparations were permeabilized with Triton X-100 (1% in PBS) for 1 h at room temperature and then digested with proteinase K (50 µg/ml in PBS) at 55° C for 1 h. Following digestion, non-specific staining was blocked with 10% normal horse serum and the specimens were incubated with sheep antibodies to human α-synuclein (diluted 1:250; Abcam, ab6162) for 48 h at room temperature.

Techniques: Mouse Assay, Fluorescence, Diffusion-based Assay, Marker

Journal: Genetics

Article Title: Cross-Talk Between Mitochondrial Fusion and the Hippo Pathway in Controlling Cell Proliferation During Drosophila Development

doi: 10.1534/genetics.115.186445

Figure Lengend Snippet: ChChd3 localizes to the inner mitochondrial membrane and is required for mitochondrial fusion and crista formation. (A) Schematic diagram of Drosophila ChChd3 protein domain structure and sequence comparison of Drosophila ChChd3 with human ChChd3 and mouse ChChd3 within the CHCH domain. (B–B′′′) S2 cells showing colocalization of ChChd3 and ATP5A. Samples were stained with anti-ChChd3 and anti-ATP5A antibodies. (C) Western blot analysis of cytosolic and mitochondrial fractions separated by centrifugation. Samples were probed with anti-ChChd3, anti-ATP5A (inner membrane), and anti-tubulin antibodies. ChChd3 is enriched in the mitochondrial fraction. (D) Western blot analysis of ATP5A (detected by anti-Total OXPHOS), ChChd3, and Tom20 proteins from S2 cell mitochondria. Isolated mitochondria were treated with the indicated concentrations of digitonin followed by Proteinase K digestion. (E–F′′′) Wing imaginal discs containing control (E–E′′′) or ChChd3 D1 homozygous mutant (F–F′′′) clones stained with anti-β-Gal and anti-ATP5A antibodies. Clones are marked by the absence of β-Gal and the dashed white line. ChChd3 mutant clone cells display punctate ATP5A staining. (G) Larval body wall cells from the control larvae expressing UAS-mito-GFP with Mef2-Gal4 and showing mitochondria with tubular morphology. (H) Knockdown of ChChd3 in the larval body wall cells results in shorter mitochondria. (I and J) TEM images of mitochondria from wild-type (I) or ChChd3 D1 mutant larvae. Crista content was reduced in ChChd3 D1 mutant mitochondria. (K and L) TEM images of adult indirect flight muscle from the Mhc-Gal4 control (K) and ChChd3 knockdown flies (L). Knockdown of ChChd3 leads to fragmented mitochondria and reduced crista content. Bars, 10 μm in B; 40 μm in E; 10 μm in G; 0.1 μm in I; and 2 μm in K.

Article Snippet: The samples were then subjected to proteolysis with Proteinase K. Proteins were then precipitated with 10% trichloroacetic acid (TCA) and analyzed by Western blotting using rabbit anti-ChChd3 (1:2000), mouse anti-total OXPHOS (1:4000; Abcam, ab110413) and rabbit anti-Tom20 (1:1000; Proteintech, 11802-1-AP) antibodies.

Techniques: Sequencing, Staining, Western Blot, Centrifugation, Isolation, Mutagenesis, Clone Assay, Expressing, Transmission Electron Microscopy

Journal: Frontiers in Neuroscience

Article Title: A PCR-Based Method for RNA Probes and Applications in Neuroscience

doi: 10.3389/fnins.2018.00266

Figure Lengend Snippet: Preparation and visualization of SST probe in mouse brain sections. Two-step polymerase chain reaction (PCR) amplifications were performed and PCR products were examined by agarose gel electrophoresis. (A) Products of SST from the first PCR. (B) Products of SST containing the T7 promoter from the second PCR. (C) In vitro -transcribed RNA probe of SST. (D) Staining for SST mRNA expression revealed the effect of tissue permeability on signal intensity. The left images show treatment with 1 × PBST (1% Tween-20 in 0.01 M PBS) for 20 min at room temperature (RT); the middle images show treatment with 2 μg/ml proteinase K at RT; and the right images show treatment with 2 μg/ml proteinase K at 37°C. The upper images were captured using 10 × magnification (scale bar = 500 μm), and the bottom images were captured using 20 × magnification (scale bar = 100 μm). (E) Sections were stained with different concentrations of the SST probe at 0, 0.5, 2, and 4 μg/ml, respectively. Images were captured using 10 × magnification (scale bar = 200 μm). SST, somatostatin.

Article Snippet: The tissue sections were given a penetrating treatment with 2 μg/ml proteinase K (diluted in 1 × PBST, AR0056, Bosterbio, USA) solution for 20 min in room temperature (RT), followed by post-fixing with 4% PFA for 10 min. Then the sections were washed 3 times for 10 min each time with 1 × PBST and transferred to 2-ml RNase-free round-bottom tubes.

Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, In Vitro, Staining, Expressing, Permeability

Journal: mBio

Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

doi: 10.1128/mBio.01376-18

Figure Lengend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

Article Snippet: For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed.

Techniques: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

Journal: Cell Death & Disease

Article Title: Uncoupling FoxO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy

doi: 10.1038/s41419-018-0336-0

Figure Lengend Snippet: FoxO3A accumulates into the mitochondria in metabolically stressed cell lines and normal tissues. a Immunogold labeling of HeLa cells cultured in high glucose (HG) or switched to low glucose (LG, 0.75 mM glucose) for 24 h. Black dots represent gold particles recognizing FoxO3A immunocomplexes. b , c Immunoblot analysis of mitochondria isolated from HCT116 cells upon b LG (24 h) and c 2-deoxy-glucose (2-DG) treatment (1 mM, 6 h). Mitochondrial fractions were treated with proteinase K (PK) to degrade outer mitochondrial membrane proteins. PDH: loading control. d , e Immunoblot analysis of mitochondrial fractions isolated from Caco2, HT29, SW-480 and HEK293 ( d ) and from NIH3T3 and MEF murine fibroblasts ( e ) cultured in LG (24 h). Mitochondrial fractions were treated with PK alone or with PK and Triton X-100 to permeabilize mitochondria and degrade all mitochondrial proteins. BCL2: outer membrane control; PDH: mitochondrial matrix control. f Immunoblot analysis of mitochondria-enriched fractions isolated from murine kidney and liver and subjected to PK or combined PK and Triton X-100 treatment. PDH: mitochondrial matrix control. The presented results are representative of at least three independent experiments

Article Snippet: The obtained mitochondria were treated with proteinase K 4 U/ml (#17916, Ambion, Austin, TX) and/or Triton X-100 (0.25%) and analyzed by immunoblotting, as previously described .

Techniques: Metabolic Labelling, Labeling, Cell Culture, Isolation

Journal: Autophagy

Article Title: GORASP2/GRASP55 collaborates with the PtdIns3K UVRAG complex to facilitate autophagosome-lysosome fusion

doi: 10.1080/15548627.2019.1596480

Figure Lengend Snippet: GORASP2 is localized to autophagosomes and lysosomes upon starvation. ( a ) GORASP2 colocalizes with GFP-LC3 upon starvation. GFP-LC3 HeLa cells were treated with growth medium (ctr), BafA1, EBSS or EBSS and 400 nM BafA1 (E + B) for 4 h, stained for GORASP2, GOLGA2 and DNA. The bottom row shows higher magnifications of the indicated area in the above row. Scale bar: 10 µm in the upper four rows, 3 µm in the bottom row. ( b ) Quantification of ( a ) for the percentage of GORASP2 puncta that colocalized with GFP-LC3. ( c ) GORASP2 colocalizes with LC3 and LAMP2 but not EEA1 or LAMP1 upon starvation. HeLa cells were treated with EBSS and 400 nM BafA1 (E + B) for 4 h, stained for GORASP2, LC3, EEA1, LAMP1, or LAMP2 and DNA as indicated. The three rows on the right are higher magnifications of the boxed area in the three rows on the left. Scale bars: 10 µm in the left rows, 3 µm in the right rows. ( d ) Quantification of ( c ) for the percentage of GORASP2 puncta that colocalized with LC3, EEA1, LAMP1 or LAMP2. ( e ) Western blot of the proteinase K protection assay. HeLa cells were treated with growth medium (ctr) or EBSS and 400 nM BafA1 (E + B) for 4 h, then the collected PNS were equally divided into three tubes, one left untreated, one was incubated with 2.5 μg/ml protease K (PK) only, and one was treated with both PK and 1% TritonX-100 (TX-100) for 10 min.

Article Snippet: Each PNS was equally divided into three tubes, one was left untreated, one was incubated with 2.5 μg/ml Protease K (Thermo Fisher Scientific, AM2542), and the other was treated with both protease K and 1% Triton X-100 (from 20% stock) for 10 min on ice.

Techniques: Staining, Western Blot, Incubation