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Patients were referred to the analytic and accurate aggregation of Professor Christoph Klein for added investigations. Informed consent/assent for the abiogenetic and immunological studies, as able-bodied as their advertisement was acquired from all acknowledged assembly and patients. Abiogenetic and anatomic studies on biosamples from patients and their ancestors were performed beneath the framework of a accurate activity advantaged “Genetic characterisation of complete cartilage basal abortion and immunodeficiency syndromes”. This abstraction was accustomed in 2011 by the belief board at LMU (438-11) and includes permission to broadcast the results.
For ancestors A we performed whole-exome sequencing (WES) for accommodating and ancestor application Agilent V4 UTR library alertness and a SOLiD sequencing platform. WES for the accommodating from the ancestors B, the accommodating from ancestors C, as able-bodied as for patient, mother and ancestors (from ancestors D) was performed afterwards Agilent library alertness (V5 UTR or V6 UTR (SY265)) application an IlluminaNextSeq 500 platform. BWA (version 0.7.15) was acclimated to adjust abbreviate reads to the animal advertence genome Grch37.p13. Variants were alleged and recalibrated according to the best convenance activity by GATK (version 3.6). The final variants were again annotated with VEP absolution 85. A custom centralized database was acclimated to clarify variants to be attenuate (not appear in gnomAD or ExAC), as able-bodied as astringent furnishings according to the Ensembl guidelines. Furnishings of filtered variants on protein were predicted with SIFT52 and PolyPhen-253. The actual variants were aggregate and filtered for attenuate homozygous and admixture heterozygous mutations afterward a arrangement of autosomal backward inheritance. Whole-exome sequencing of genomic DNA of affiliated F patients was conducted application Illumina sequencing platforms. Bioinformatics appraisal for apprehension of attenuate arrangement variants afterward Mendelian bequest patterns were performed as declared previously54
FCHO1 Sanger sequencing was performed to affirm WES-detected variants and their allegory with the analytic phenotype beyond the ancestors members. Genomic DNA was PCR-amplified application OneTaq Polymerase (NEB), specific primers are provided in Supplementary Table 3. Amplicons were sequenced either centralized or by application the bartering account of Eurofins Genomics.
Crystal structures of FCHO1 area were modelled application PyMol software with the mutalyzer wizard55. Mu affinity area of Danio rerio with apprenticed Eps15 peptide (5JP2; https://doi.org/10.2210/pdb5JP2/pdb) and animal FCHO2 F-Bar area (2v0O; https://doi.org/10.2210/pdb2V0O/pdb) were called as arrangement for anatomy modelling. To account aftereffect of point mutations on the structures, rotamer configurations of the accomplished anticipation were chosen.
The SK-MEL-2 corpuscle line, engineered application zinc feel nucleases (ZNF) genome alteration to durably accurate RFP beneath the autogenous animal clathrin ablaze alternation A locus (CLTA-RFP), was attentive provided by David G. Drubin, University of Berkeley15. The corpuscle band with alone one RFP-tagged CLTA locus was acclimated to minimise accepted ancillary effects. Beef were maintained in Dulbecco’s adapted Eagle average (DMEM)/F12 average (ThermoFisher Scientific) supplemented with 10% fetal dogie serum (FCS) (ThermoFisher Scientific), 100 U/ml of both penicillin and streptomycin (Gibco). Jurkat beef were purchased from ATCC, USA. They were maintained in RPMI1640 average (ThermoFisher Scientific) supplemented with 10% FCS (ThermoFisher Scientific), 100 U/ml of both penicillin and streptomycin (Gibco) and 2 mM glutamine (Gibco). In all, 100 U/ml of penicillin and streptomycin (Gibco) HEK293T beef and NIH-3T3 beef (both from DSMZ–German Collection of Microorganisms and Corpuscle Cultures) were maintained in DMEM (ThermoFisher Scientific) supplemented with 10% FCS (ThermoFisher Scientific), 100 U/ml anniversary of penicillin and streptomycin (Gibco). Accommodating fibroblasts were developed in Iscove’s Adapted Dulbecco’s Average supplemented with 10% FCS. EBV-LCL were maintained in RPMI with 10% FCS. All corpuscle curve were commonly activated for mycoplasma and were mycoplasma-negative throughout the study.
Full-length cDNA of the animal FCHO1 isoform b (IMAGE 5757146), cloned into the pEGFP-C3 agent with adapted MCS (EcoRI/SalI) was attentive provided by Emmanuel Boucrot (UCL London) and Harvey McMahon (MRC-LMB, Cambridge, UK)5. Accommodating mutations were alien by site-directed PCR mutagenesis application Q5 site-directed mutagenesis kit (NEB) according to manufacturer’s apprenticeship with specific primers advised with NEBase Changer (NEB). Wt and mutated FCHO1 cDNAs were cloned as either GFP admixture proteins or as IRES-containing bicistronic lentiviral pRRL vectors with or afterwards GFP as a anchorman gene, respectively. Full-length cDNA of animal clathrin ablaze alternation tagged with mRFP was attentive provided by Klemens Rottner from Abstruse University in Braunschweig56. It was cloned to lentiviral pRRL agent for assembly of virus particles. The definiteness of the sequences was commonly monitored by Sanger sequencing.
The genomic locus of FCHO1 (transcript ENST00000594202.1) was appointed for gene disruption by inducing double-strand break in exon 7 (T1: 5′-GGACGTTCTCCGCTACGGCG AGG-3′) and intron 7 (T2: 5′-GTGTCGTGGGCGCCGCCCAG CGG-3′). Genome alteration of SK-MEL-2 and Jurkat beef was done beef application the Alt-R CRISPR-Cas9 technology (IDT technology, Belgium). Upon coincubation of crRNA and ATTOTM 550 (ATTO-TEC, Germany) beaming dye-labelled tracRNA, the RNA duplexes were electroporated into ambition beef forth with Cas9 nuclease (SG Corpuscle Band 4D-Nucleofector X Kit and 4D-NucleofectorTM System Lonza, Switzerland). Red beaming protein (RFP)-positive beef were single-sorted application a BD FACSAria breeze sorter (BD Bioscience, USA).
As autogenous announcement of FCHO1 is too low for affectionate appraisal by western blot, abatement of FCHO1 was accepted by PCR. The afterward album brace was acclimated for validation: (F: 5′-GTGAGCCTGATGAACCCTGGGTGTG-3′, R: 5′-TGCATGTGGGTGACAGAGTGAGAC-3′). Beef accustomed homozygous mutations are accepted to accept a connected deletion, spanning amid exon 7 and intron 7. A minimum of bristles clones accustomed homozygous mutations consistent in frameshift were acclimated for consecutive abstracts (Supplementary Fig. 12). Unmodified clones were acclimated as a FCHO1-positive control. To ensure abatement specificity, the ten best apparent astray sites were activated application absolute Sanger sequencing, assuming no signs of ambiguous cuts.
FCHO1 ko SK-MEL-2 beef and FCHO1 ko Jurkat beef were transfected application calcium phosphate transfection kit (Sigma, USA) and 0.1–1 μg of the assorted lentiviral plasmids accustomed either wt or aberrant FCHO1 cDNAs. Beef were about incubated 24 h to accurate the constructs afore imaging.
For assembly of vesicular stomatitis virus G glycoprotein (VSV-g)-pseudotyped lentiviral particles HEK293T beef were transfected application calcium phosphate transfection kit (Sigma, USA) and 5 μg corresponding lentiviral vector, 12 μg pcDNA3.GP.4xCTE (which expresses HIV-1 gag- pol), 5 μg pRSV-Rev and 1.5 μg pMD.G (which encodes VSV-g) in the attendance of 25 μM chloroquine (Sigma, USA). Eight hours afterwards transfection, average was exchanged and afloat absolute lentiviral particles was calm afterwards 24, 48 and 72 h column transfection. Viral titre were bent on 3T3 cells.
FCHO1 ko SK-MEL-2 beef and FCHO1 ko Jurkat beef were transduced with lentiviral particles through centrifugation at 900 × g for 4 h at 32 °C in the attendance of polybrene (8 μg/ml) (Sigma, USA).
The HIV-1NL4-3wt plasmid was acquired from Nathaniel Landau (Alexandria Centre for Life Science, NYU, USA), the HIV-1NL4-3ΔEnv plasmid was a affectionate allowance of Oliver T. Fackler (Universitätsklinikum Heidelberg, Germany) and the BlaM-Vpr plasmid was a allowance from Thomas J. Hope (Northwestern University, Chicago, USA). HIV-1NL4-3ΔEnv and HIV-1NL4-3ΔEnv (BlaM-Vpr), both VSV-G pseudotyped, or HIV-1NL4-3wt (BlaM-Vpr) stocks were produced by PEI co-transfection of HEK293T cells. Forty-eight hours later, supernatants were calm and filtered through a 0.45 µm Stericup (Millipore). Afterwards sucrose beanbag (25% in 1x phosphate-buffered acrid (PBS_) ablution at 24,000 rpm at 4 °C for 1.5 h (Sorvall WX Ultra series; rotor: SW32, Beckmann Coulter), virus pellets were resuspended in PBS and stored at −80 °C until use.
To appraisal for protein–protein interactions, FCHO1-deficient corpuscle curve were used. SK-MEL-2 beef overexpressing altered variants of GFP-FCHO1 admixture proteins were fatigued in serum-free average for 1 h at 37 °C above-mentioned the assay. Jurkat curve were fatigued for 1 h and afterwards angry with an α-CD3 antibiotic (OKT3, 1 mg/ml, BD Biosciences) cross-linked by a dupe α-mouse polyclonal antibiotic (0.5 mg/ml, Jackson ImmunoResearch) for 2 to 20 min at 37 °C. Dispatch was concluded by accession of ice-cold PBS. Corpuscle pellets were lysed in RIPA absorber absolute phenylmethylsulfonyl fluoride protease inhibitors for 30 min in cold. Next, the supernatants were calm for added analysis. GFP-tagged FCHO1 proteins were pulled-down by GFP-Trap alluring chaplet (ChromoTek GmbH) and N’ or C’ Flag-tagged FCHo1 by anti-Flag M2 Affinity Gel (Sigma). Afterwards incubation, chaplet were calm and done two times in RIPA absorber and the pellet was aloft in sample absorber absolute SDS for 10 min at 95 °C. Equal amounts of protein were afar by SDS polyacrylamide gel electrophoresis and blotted assimilate polyvinylidene difluoride (PVDF) membranes application the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 1 h at allowance temperature in 5% non-fat milk afore staining. Afterward primary antibodies were used: FCHO1–rabbit, polyclonal, PA5-31603, lot Q12081994A, Thermo Accurate or rabbit, polyclonal, 84740, lot GR214150−4, Abcam; EPS15–rabbit, carbon D3K8R, Corpuscle Signalling; EPS15R–rabbit, carbon EP1146Y, Abcam; adaptin–mouse, carbon AP6, Abcam; CD3epsilon–rat, carbon OKT3, ThermoFisher Scientific; CD3delta–rabbit, polyclonal, ThermoFisher Scientific; CD3 gamma–rabbit, polyclonal, ThermoFisher Scientific; GAPDH–mouse, carbon 6C5, Santa Cruz. All antibodies acclimated are summarised in Supplementary Table 4.
After abrasion in PBS-T, the PVDF membranes were apparent to horseradish peroxidase-conjugated accessory anti-mouse (BD), anti-rat (CS), or anti-rabbit (CST) Ig antibodies for 1 h at allowance temperature (RT). Western blots were detected application a chemiluminescent substrate (Pierce Technology) and images were captured on a Chemidoc XRS Imaging System (Bio-Rad Laboratories). Blots were bare amid exposures to altered antibodies application a Restore Western Blemish Stripping Absorber (Thermo Scientific). Abstracts appraisal was performed application Quantity One or Image Lab software (Bio-Rad Laboratories). Uncropped immunoblots are apparent in Supplementary Figures.
To minimise dominant-negative furnishings on CCP dynamics consistent from abiding overexpression of FCHO1 protein, alone FCHO1-deficient SK-MEL-2 corpuscle band briefly transduced with N-terminally tagged versions of GFP-FCHO1 were acclimated for experiments. Beef with a low announcement of FCHO1 were analysed about 16 to 20 h column transfection. Beef were argent on bottle coverslips (Karl Hecht, 0.13–0.16 mm thickness, bore 20 mm) in 24-well tissue ability plates and able in complete DMEM/F12 average for min 12 h to facilitate attachment. Coverslips were rinsed with PBS and anchored with 3% formaldehyde (Electron Microscopy Sciences) for 10 min at RT. Next, beef were done in PBS and autofluorescence was quenched for 15 min application 50 mM NH4Cl at RT. Coverslips were done already in PBS and incubated for 30 min at RT in PBS absolute 0.1% (w/v) bovine serum albumin (BSA; Sigma Aldrich), 0.05% (w/v) saponin (Sigma Aldrich). Added blocking band-aid was removed and beef were decrepit with the afterward primary antibodies: rabbit-α-Eps15 (clone D3K8R, Corpuscle Signalling) or mouse-α-Adaptin (AP6, Abcam) at 4 oC, overnight. Subsequently, beef were done in PBS absolute 0.05% (w/v) saponin (Sigma Aldrich), three times for 5 min each. The afterward accessory antibodies accompanying to fluorochromes were used: goat-α-rabbit IgG, AlexaFluor 405 (Invitrogen) and goat-α-mouse IgG, AlexaFluor 633 (Invitrogen).
WGA (wheat antibiotic agglutinin) staining was performed on transfected beef anchored with 3% formaldehyde (Electron Microscopy Sciences) for 10 min at RT. Afterwards all-encompassing abrasion with phenol red chargeless 1xHBSS, WGA conjugated to AlexaFluor 633 (Invitrogen) was added to the beef at absorption 1.25 μg/ml (in phenol red chargeless 1xHBSS) and incubated for 5 min.
Some samples were co-stained with 300 nM DAPI (Invitrogen) for 2 min at RT. Beef were army on bottle slides application Fluoromount-G (SouthernBiotech) and broiled at RT in black for minimum 12 h afore imaging. Samples were analysed by confocal beaming microscopy application the Zeiss LSM880 and Zeiss LSM800 astern microscopes. Images were calm application 63×/1.4 NA or 40×/1.4 NA oil objectives (Zeiss). Four solid-state 5 and 10 mW laser (405, 488, 561, 640 nm) were acclimated as ablaze source; scanner abundance was 400 Hz; line-averaging 2. All images were acquired with GaAsP high-sensitivity detectors. Pearson alternation or co-localisation accessory was adjourned on 16-bits raw agenda files on Zen Blue software (Zeiss). Representative beef from two to three absolute abstracts were called for analysis. Two to four 25 µm2 aboveboard regions were called and minimum ten beef per action were analysed. To abstain bent during analysis, alone one beaming approach was alive while selecting regions.
For live-cell imaging abstracts durably transduced SK-MEL-2 corpuscle curve were used. Beef were seeded on μ-dish 35 mm, aerial Bottle Basal (IBIDI, bottle coverslip no.1.5 H, called quality, 170 μm /− 5 μm) in complete DMEM/F1224h afore imaging. Average was exchanged anon afore alpha of the experiment. During the agreement dishes were placed into a temperature-controlled alcove on the microscope date with 95% air, 5% CO2 and 100% humidity. Live-cell imaging abstracts were acquired application a absolutely motorised astern confocal microscope (Zeiss LSM800) application either 40×/1.4 NA or 63 × /1.4 NA oil objectives (Zeiss) beneath ascendancy of Definite Focus for Axio Observer Z1 (Zeiss). Sixteen-bits agenda images were acquired with GaAsP high-sensitivity detectors, confocal bore and 488 and 561 nm laser curve and a bifold (525/50; 605/70) BP filters. Corpuscle regions were called to acquiesce for 500–950 ms-lasting intervals. Time assurance of the beaming acuteness of FCHo1 and autogenous clathrin were adjourned on movies from three absolute experiments, application Zen Blue software (Zeiss). Up to three 25 µm2 regions per beef were quantified. Both GFP and RFP channels were normalised to the accomplishments and antecedent fluorescence was set to 1.
The virion admixture appraisal was performed in assumption as reported29 employing HIV-1 particles accustomed the HIV-1 Vpr protein alloyed to β-lactamase (BlaM-Vpr). In brief, afterwards admixture of virus particles to ambition cells, the congenital BlaM-Vpr protein is appear into the cytoplasm and is able to carve the CCF2 dye. This leads to a about-face of the dye’s discharge best from 520 to 447 nm, which can be detected and quantified by breeze cytometry. Jurkat T beef were argent at a body of 2 × 105 beef per able-bodied (96-well conical plate, Corning, New York, USA). Where indicated, the HIV-1 admixture inhibitor T20 (50 µM, enfuvirtide, Roche, Rotkreuz, Switzerland) was added 1 h above-mentioned to virus challenge. Serial dilutions of VSV-G HIV-1NL4-3ΔEnv (BlaM-Vpr) were performed in PBS. 4 h afterward claiming with either VSV-G HIV-1NL4-3ΔEnv (BlaM-Vpr) or HIV-1NL4-3 (BlaM-Vpr), Jurkat T beef were done and incubated with the CCF2 dye as ahead reported30,31. The afterward day, beef were anchored for 90 min with 4% PFA/PBS and analysed by breeze cytometry.
Jurkat T beef were argent at a body of 2 × 105 beef per well. T20 (50 µM), the V-ATPase inhibitor bafilomycin A1 (100 nM, Sigma Aldrich, St. Louis, USA) or PBS were added 1 h above-mentioned to virus challenge. Beef were challenged with VSV-G HIV-1NL4-3ΔEnv and 4 h later, 200 µl of beginning ability average were added. 48 h later, beef were anchored for 90 min in 4% PFA/PBS and HIV infection was monitored application an intracellular p24 staining (anti-p24 antibody, carbon KC57-FITC, Beckmann Coulter, Brea, USA) in assumption as reported32.
Fibroblasts from advantageous donor (HD) and accommodating (kindred E) were alone application PBS absolute 10 mM EDTA and serum-starved for 30 min in DMEM at 37 °C. Beef and transferrin conjugated to AlexaFluor 633 (ThermoFisher Scientific) were done in glucose absorber (PBS supplemented with 20 mM Glucose and 1% BSA) and cooled bottomward on ice. 25 µg/ml AlexaFluor 633-conjugated transferrin were added to beef and incubated on ice for 10 min. Beef were again transferred to 37 °C for adumbrated periods of time. Upon adumbrated time, surface-bound transferrin was bare by acerbic ablution (PBS supplemented with 0.1 M glycine and 150 mM NaCl at pH 3) and uptake of beaming transferrin was bent by breeze cytometry. Abstracts are affiliated from four absolute experiments. Error confined announce mean ± SD. Statistical appraisal application two-way ANOVA followed by Sidak’s assorted comparisons appraisal appear no cogent aberration in transferrin uptake amid HD and accommodating fibroblasts.
Blood samples were done with PBS and decrepit with the afterward antibodies for 20 min at RT: α-CD45 BV711 or APC (HI30), α-CD33 PE-Cy7 (P67.6), α-CD3 PE (HIT3a), α-CD19 FITC (HIB19), α-CD8α APC (RPA-T8), α-CD4 PE-Cy7 (A161A1) all from Biolegend. All antibodies acclimated are summarised in Supplementary Table 4. Red claret beef were lysed application 1 × BD FACS Lysing Band-aid (BD Biosciences) according to the manufacturer’s instructions. The samples were acquired application a LSRFortessa (BD Bioscience) cytometer. Abstracts were analysed application FlowJo Software (TreeStar), v9 and v10. Gating action to appraise abundance of claret leucocytes is apparent in Supplementary Fig. 13. Virion admixture and HIV infection of Jurkat T beef were recorded on a BD FACSLyric (BD Biosciences, Franklin Lakes, USA).
PBMC were abandoned from claret samples by Ficoll-Hypaque (Pharmacia) body acclivity centrifugation. Beef were labeled with 1 μM CFSE (eBioscience) according to the manufacturer’s protocol. They were resuspended in complete RPMI1640, argent on 96-well collapsed basal plates and angry with α-CD3-coupled chaplet (bio-α-CD3, carbon OKT3 (eBioscience) accompanying with α-biotin MACSiBeads (MiltenyiBiotec)) at a arrangement 10:1, in the attendance of 1 mg/ml of acrid α-CD28, carbon CD28.2 (eBioscience). Proliferative acknowledgment was abstinent afterwards 3 and 5 days. Gating action to appraise T-cell admeasurement is apparent in Supplementary Fig. 14. Supernatants (four abstruse replicates) were activated for the attendance of IL-2, IL-4, IFN-γ and TNF-α application animal FlowCytomix chaplet (eBioscience) according to the manufacturer’s instructions.
Up to bristles altered FCHO1-sufficient or -deficient Jurkat clones were incubated for 1 h in Ca2 – and Mg2 -free Dulbecco’s serum-free average (Invitrogen) at allowance temperature at a body of 107 cells/ml. Beef were again loaded with Ca2 -sensitive dyes, either Indo-1 or Fluo-4 (3 µM) and FuraRed (6 µM) for 45 min at 37 °C. Further, beef were adequate for 30–45 min at 37 °C. Afterwards establishing of a baseline for 30 s, beef were angry with α-CD3 antibiotic (OKT3, 1 mg/ml, BD Biosciences) and goat-α-mouse polyclonal antibiotic (0.5 mg/ml, Jackson ImmunoResearch) to acquiesce cross-linking and abstracts accretion was connected for four added minutes. To ensure corpuscle viability, 1 min afore the end of acquisition, 2 µg/ml ionomycin (Sigma) was added as absolute Ca2 -flux control. Gating action to appraise Ca2 absolution is apparent in Supplementary Fig. 15.
Confocal microscopy: FCHO1-sufficient or -deficient, or durably transduced (with wt or aberrant FCHO1 viruses) Jurkat FCHO1−/− corpuscle curve were acclimated to visualise TCR internalisation. Beef were argent on poly-d-lysine (0.1 mg/ml, Sigma Aldrich) and α-CD3 (clone OKT3, 0.5 mg/ml, eBioscience) coated bottle coverslips (Karl Hecht, 0.13-0.16 mm thickness, bore 20 mm) in 24-well tissue ability plates and incubated for adumbrated time points. Coverslips were rinsed with PBS and beef were anchored in 3% formaldehyde (Electron Microscopy Sciences) for 10 min at RT. Next, beef were done in PBS and autofluorescence was quenched for 15 min at RT application 50 mM NH4Cl. Coverslips were done already in PBS and incubated for 30 min at RT in PBS absolute 0.1% (w/v) BSA (Sigma Aldrich), 0.05% (w/v) saponin (Sigma Aldrich). Further, blocking band-aid was removed and beef were decrepit with mouse-α-CD3 (OKT3, eBioscience) and goat-α-mouse IgG, AlexaFluor 633 (Invitrogen). Samples were co-stained with 300 nM DAPI (Invitrogen) for 2 min at RT. Beef were army on bottle slides application Fluoromount-G (SouthernBiotech) and broiled at RT in black for at atomic 12 h afore imaging. Samples were analysed by confocal beaming microscopy application the ZEISS LSM800 astern microscopes (ZEISS) as declared above. We acclimated Fiji software57 to quantify the cardinal of CD3 puncta/cell. The cardinal of puncta/cell was averaged on several accidental fields of view.
Flow cytometry: FCHO1-sufficient or -deficient Jurkat beef were decrepit with α-CD3 Ab (OKT3) in algid and TCR internalisation was adjourned over time at 37 °C in the attendance of anti-mouse F(ab’)2 bits labelled with Ax647. Afterwards 2, 5, 15, 45 and 60 min of dispatch the actual apparent TCRs were stripped, appropriately beaming arresting corresponds alone to the internalised TCR. Gating action to appraise TCR internalisation is apparent in Supplementary Fig. 16.
Statistical appraisal was performed application GraphPadPrism software v.6. Pearson alternation and colocalization accessory on called corpuscle bits were adjourned on raw files application Zen Blue software (Zeiss). Corpuscle regions or absolute beef are referred to as n unless adumbrated otherwise. No adjustment of randomisation was used, and no samples were afar from analysis. To abstain bent during analysis, corpuscle regions were called based on the arresting from alone one beaming channel. No statistical adjustment was acclimated to destine sample admeasurement for analyses. Two-way appraisal of about-face (ANOVA) or ANOVA appraisal followed by Sidak’s assorted allegory appraisal were acclimated to appraise differences amid groups. p-values < 0.05 were advised to be statistically significant.
Further advice on analysis architecture is accessible in the Nature Analysis Reporting Summary affiliated to this article.
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