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

Article Title: BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

doi: 10.1073/pnas.1603668113

Figure Lengend Snippet: Increased efficiency of hiPSC generation from FOP HDFs via the BMP-SMAD signaling pathway. (A and B) Number of TRA-1-60–positive colonies (A) or the ratio of TRA-1-60–positive cells (B) from reprogrammed FOP HDFs deposited in the Coriell Institute and collected at UCSF and four different normal HDF lines. TRA1-60–positive cells were analyzed 25 d after transfection from 10,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05 (t test). (C and D) Effects of BMP-SMAD signal inhibitors, Dorsomorphin (1 µM), LDN-193189 (1 µM), P38 MAPK inhibitor, SB203580 (10 µM), and vehicle (0.1% DMSO) on hiPSC generation from HDF-FOP1 (C) and HDF-FOP2 (D) with OSKM retroviral transduction. ESC-like colonies were counted 25 d after the transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with vehicle conditions). (E and F) Effects of the overexpression of inhibitory SMADs SMAD6 or SMAD7 on hiPSC generation from HDF-FOP1 (E) and HDF-FOP2 (F) with OSKM retroviral transduction. ESC-like colonies were counted 25 d after transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with GFP conditions). (G and H) Effects of ACVR1 knockdown on hiPSC generation from HDF-FOP1 (G) and HDF-FOP2 (H) with episomal plasmids. TRA-1-60–positive colonies were counted 25 d after transduction from 5,000 cells per 35-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with nontarget siRNA conditions).

Article Snippet: We obtained two FOP HDF lines (FOP1 and FOP2) from the Coriell Institute (GM00513 and GM00783, respectively) and confirmed that both carry a heterozygous mutation in ACVR1 (617G > A) that is typical in FOP patients ( 14 ) ( ).

Techniques: Transfection, Retroviral, Transduction, Over Expression, Knockdown

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

Article Title: BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

doi: 10.1073/pnas.1603668113

Figure Lengend Snippet: FOP HDFs showed hyperactive BMP-SMAD signaling and increased efficiency of iPSC generation. (A) Direct DNA sequence analysis of 617 G (or A) in ACVR1 from genomic DNA of HDF-1323, HDF-FOP1, and HDF-FOP2. (B) Protein expression of phosphorylated SMAD1/5/8, total SMAD1/5/8, and α-tubulin in normal or FOP fibroblasts treated with 100 ng/mL BMP4 for 1 h and analyzed by Western blotting. (C) Protein expression levels of phosphorylated SMAD1/5/8 (normalized to the amount of α-tubulin expression) in normal or FOP fibroblasts. The results are mean and SE, n = 4. *P < 0.05 (Dunnett’s test with HDF-1323). (D) iPSC colonies have an ESC-like morphology with a flat, round shape and a distinct edge (Upper). Non–ESC-like colonies are epithelial with irregular edges (Lower). (Scale bars: 200 µm.) (E–H) The efficiency of ESC-like colonies from normal or FOP HDFs with retroviral transduction of OSKM in (E and G) or OSK in (F and H) at different seeding densities. Transduced cells were replated at 50,000 (E and F), 150,000, or 500,000 cells per 100-mm dish. Colony numbers were counted 25 or 30 d after transduction in OSKM or OSK conditions, respectively. Results are mean and SE, n = 4. *P < 0.05, **P < 0.01 (Dunnett’s test with HDF-1323). Hashtags (#) indicate that cells became confluent before ESC-like colonies appeared. (I) ALP staining of ESC-like colonies from FOP HDFs of three Japanese patients (FOP-J1, -J2, and -J3) and normal fibroblasts (TIG-120). The images were taken 25 d after OSKM retroviral transduction. (J and K) Numbers of ESC-like colonies from FOP HDFs of three Japanese patients with OSKM (J) and OSK (K) retroviral transduction. Colony numbers are automatically counted by ALP staining in every 5 d after transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 4.

Article Snippet: We obtained two FOP HDF lines (FOP1 and FOP2) from the Coriell Institute (GM00513 and GM00783, respectively) and confirmed that both carry a heterozygous mutation in ACVR1 (617G > A) that is typical in FOP patients ( 14 ) ( ).

Techniques: Sequencing, Expressing, Western Blot, Retroviral, Transduction, Staining

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

Article Title: BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

doi: 10.1073/pnas.1603668113

Figure Lengend Snippet: Expression of BMP-SMAD pathway genes during iPSC generation. (A) Expression levels of ID1-3, SMAD1, and ACVR1 in normal or FOP HDFs stimulated by BMP4 analyzed by RT-qPCR. Normal HDF (HDF-1323) were starved for serum overnight and then treated with 100 ng/mL BMP4 for 1 h. Results are mean and SE, n = 5. *P < 0.05, ***P < 0.001 (Dunnett’s test with HDF-1323). (B) Expression levels of ID1–3, SMAD1, EPCAM, OCLN, SNAI1, SNAI2, and ZEB1 in normal or FOP HDFs untransfected or after 5 d of transfection by epiY4 detected by RT-qPCR. Results are mean and SE, n = 3. *P < 0.05, ***P < 0.001 (t test). (C) Changes in expression levels of ID1, ID2, ID3, EPCAM, P16/INK4A, and NANOG by retroviral transduction. Normal HDFs (HDF-WTc) uninfected or after 7 d of retroviral transduction carrying O(OCT4), S(SOX2), K(KLF4), M(MYC), or their combinations were analyzed by RT-qPCR. Expression level of uninfected HDFs is defined as 1.0. Results are mean and SE, n = 3.

Article Snippet: We obtained two FOP HDF lines (FOP1 and FOP2) from the Coriell Institute (GM00513 and GM00783, respectively) and confirmed that both carry a heterozygous mutation in ACVR1 (617G > A) that is typical in FOP patients ( 14 ) ( ).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Retroviral, Transduction

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

Article Title: BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

doi: 10.1073/pnas.1603668113

Figure Lengend Snippet: Expression of ID genes and p16/INK4A during iPSC generation. (A) Expression levels of ID genes in HDFs transduced by retroviral ID genes. Normal HDFs (HDF-1323 transduced with lentiviral Slc7a1) were transduced with retroviral GFP, ID1, ID2, ID3, or ID4 and cultured for 5 d. Results are mean and SE, n = 3. (B) Expression levels of ID genes in HDFs transfected with siRNAs against ID genes. Normal HDF (HDF-1323) were transfected with nontarget, ID1 no. 1, ID1 no. 2, ID2 no. 1, ID2 no. 2, ID3 no. 1, or ID3 no. 2 siRNA and cultured for 3 d. Results are mean and SE, n = 3. (C) Expression levels of Id genes in Nanog-GFP MEF transduced with retroviral Id genes. Nanog-GFP MEFs were transduced with retroviral DsRed, Id1, Id2, Id3, or Id4 and cultured for 5 d. For Id1–3 expression, results are mean and SE, n = 3. For Id4 expression, a DNA gel image of electrophoresis after 40 cycles of PCR is shown. (D) Expression levels of p16/INK4A, p15/INK4B, and p14/ARF in HDFs carrying siRNA against p16/INK4A analyzed by RT-qPCR. HDF-1323 were transfected with nontarget, p16 no. 1, p16 no. 2, p16 no. 3, or p16 no. 4 siRNA and cultured for 3 d. Results show mean and SE, n = 3. (E) Protein expression of p16/INK4A and GAPDH in HDFs carrying siRNA against P16/INK4A. OSKM-transduced HDF-1323 (at day 2) were treated with nontarget, P16 no. 1, P16 no. 2, p16 no. 3, or P16 no. 4 siRNA and cultured for 3 d and then analyzed with Western blotting. Secondary antibodies for p16/INK4A or GAPDH were labeled with IRdye680LT (red) or IRdye800CW (green), respectively. (F) Protein expression levels of p16/INK4A (normalized to the amount of GAPDH expression) in HDF-1323. Results are mean and SE, n = 4. (G and H) Cell proliferation rates of normal and FOP HDFs transduced with retroviral OSKM (G) and of normal HDFs transduced with retroviral OSKM and ID genes (H). At day 0, transduced cells (3 d after transduction) were replated at 10,000 cells per 35-mm dish and then counted daily. At day 1, the medium was changed to SNL-conditioned hESC medium. Results are mean and SE, n = 4. (I–L) p16/INK4A protein expression of FOP and normal HDFs transduced with retroviral OSKM or untransduced and HiPSCs (I and J) or of normal HDFs transduced with retroviral OSKM and ID genes or transfected with siRNA against ID genes or P16/INK4A (K and L). At day 0, transduced HDFs (3 d after transduction) were replated at 10,000 cells per 35-mm dish. At day 1, the medium was changed to SNL-conditioned hESC medium. The cells were collected for Western blot analysis at day 5. Secondary antibodies for p16/INK4A or GAPDH were labeled with IRdye680LT (red) or IRdye800CW (green), respectively. Results are mean and SE, n = 3. *P < 0.05 (t test in B), *P < 0.05, **P < 0.01 (Dunnett’s test with 4F + GFP conditions in D).

Article Snippet: We obtained two FOP HDF lines (FOP1 and FOP2) from the Coriell Institute (GM00513 and GM00783, respectively) and confirmed that both carry a heterozygous mutation in ACVR1 (617G > A) that is typical in FOP patients ( 14 ) ( ).

Techniques: Expressing, Retroviral, Transduction, Cell Culture, Transfection, Electrophoresis, Quantitative RT-PCR, Western Blot, Labeling

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

Article Title: BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

doi: 10.1073/pnas.1603668113

Figure Lengend Snippet: BMP-SMAD-ID signaling axis is functionally epistatic to p16/INK4A in iPSC generation. (A–C) Effects of p16/INK4A knockdown during reprogramming on hiPSC generation from FOP and normal HDFs (A), normal HDFs (HDF-WTc) transduced with retroviral OSKM and ID1-4 (B), or normal fibroblasts (HDF-WTc) transfected with ID2 or ID1/3 siRNA (C). TRA-1-60–positive colonies were counted 25 d after electroporation of epiY4 from 10,000 cells per 100-mm dish at replating (A and C) or after transduction with retroviral OSKM and ID1-4 from 50,000 cells per 100-mm dish at replating (B). Results are mean and SE, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (Dunnett’s test with each siRNA NT condition). (D) Effects of overexpressed Id genes on generating miPSCs from p16 (+/+), p16 (+/−) MEF, or p16 (−/−) MEFs (crossed with Nanog-GFP) with OSK retroviral transduction. Nanog-GFP–positive colonies were counted at 16 d after transduction from 10,000 cells per 35-mm dish at replating. Results are mean and SE, n = 4. **P < 0.01 (Dunnett’s test with each +DsRed condition).

Article Snippet: We obtained two FOP HDF lines (FOP1 and FOP2) from the Coriell Institute (GM00513 and GM00783, respectively) and confirmed that both carry a heterozygous mutation in ACVR1 (617G > A) that is typical in FOP patients ( 14 ) ( ).

Techniques: Knockdown, Transduction, Retroviral, Transfection, Electroporation

Journal: Chaos, solitons, and fractals

Article Title: Control and Anticontrol of chaos in Fractional-order models of Diabetes, HIV, Dengue, Migraine, Parkinson’s and Ebola Virus diseases

doi: 10.1016/j.chaos.2021.111419

Figure Lengend Snippet: Proposed anticontrollers to introduce chaos in the biological models of diseases

Article Snippet: FOPI , SMAC , 1 , 0.20504 , Initially periodic trajectories turn chaotic after addition of controller at 100s ( Fig. 8 (a) ) , The EEG signals extracted from the central and temporal lobes of the cerebral cortex display periodic or stable dynamics on the onset of FOPI that destroys the normal process of the brain. The positive MLEs show that the anticontrollers designed lead to generation of chaos in the brain signals to bring them to normal healthy functioning..

Techniques: Introduce

Journal: Chaos, solitons, and fractals

Article Title: Control and Anticontrol of chaos in Fractional-order models of Diabetes, HIV, Dengue, Migraine, Parkinson’s and Ebola Virus diseases

doi: 10.1016/j.chaos.2021.111419

Figure Lengend Snippet: Comparative analysis of the designed anticontrollers to induce chaos

Article Snippet: FOPI , SMAC , 1 , 0.20504 , Initially periodic trajectories turn chaotic after addition of controller at 100s ( Fig. 8 (a) ) , The EEG signals extracted from the central and temporal lobes of the cerebral cortex display periodic or stable dynamics on the onset of FOPI that destroys the normal process of the brain. The positive MLEs show that the anticontrollers designed lead to generation of chaos in the brain signals to bring them to normal healthy functioning..

Techniques: Generated

Journal: Molecular Plant Pathology

Article Title: Evidence for horizontal gene transfer and separation of effector recognition from effector function revealed by analysis of effector genes shared between cape gooseberry‐ and tomato‐infecting formae speciales of Fusarium oxysporum

doi: 10.1111/mpp.12700

Figure Lengend Snippet: Pathogenicity tests on M82 tomato plants with Fol‐WT (WT), Fol‐ΔSIX1 (ΔSIX1) and 10 Fol‐ΔSIX1:SIX1a (SIX1a) transformants. (A, C) Photographs taken at 21 days post‐inoculation (dpi) of infected M82 plants from two experiments testing all 10 transformants. (B, D) Top panel in (B) shows the distribution of disease scores for plants shown in (A). Bottom panel in (B) shows the distribution of disease scores at 21 dpi for plants infected with wild‐type (WT), ΔSIX1 or SIX1a transformants 3, 16 or 17 (the transformants showing the highest disease scores from A) pooled from four replicate experiments (n = 38–40; results of individual replicates are shown in Fig. S11, see Supporting Information). (D) shows the distribution of disease scores at 21 dpi for plants infected with WT, ΔSIX1 or SIX1a transformants 22, 25, 28 or 29 pooled from two replicate experiments (n = 18–20; results of individual replicates are shown in Fig. S11). Treatments with different letters are significantly different at P = 0.05.

Article Snippet: The coding sequences of Foph SIX1a and SIX1b were synthesized and cloned into pUC57 by GenScript (Piscataway, NJ, USA).

Techniques: Infection

Journal: Molecular Plant Pathology

Article Title: Evidence for horizontal gene transfer and separation of effector recognition from effector function revealed by analysis of effector genes shared between cape gooseberry‐ and tomato‐infecting formae speciales of Fusarium oxysporum

doi: 10.1111/mpp.12700

Figure Lengend Snippet: Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis showing the expression of Fusarium oxysporumf. sp. physali (Foph) SIX1a or Foph SIX1btransgenes in tomato roots infected with Fol‐ΔSIX1:SIX1a/b transformants at 3 and 6 days post‐inoculation (dpi). Top gel images show bands (expected size of 250 bp) consistent with SIX1a and SIX1bexpression in Fol‐ΔSIX1:SIX1a/b‐infected roots, compared with mock‐, Fol‐WT‐ or Fol‐ΔSIX1‐inoculated controls. Bottom gel images show bands (expected size of 201 bp with RT_Fem1 primers in the SIX1a experiment and 250 bp with q_Fem1 primers in the SIX1b experiment) consistent with FEM1 expression in Fusarium oxysporumf. sp. lycopersici (Fol)‐infected tomato roots.

Article Snippet: The coding sequences of Foph SIX1a and SIX1b were synthesized and cloned into pUC57 by GenScript (Piscataway, NJ, USA).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection

Journal: Molecular Plant Pathology

Article Title: Evidence for horizontal gene transfer and separation of effector recognition from effector function revealed by analysis of effector genes shared between cape gooseberry‐ and tomato‐infecting formae speciales of Fusarium oxysporum

doi: 10.1111/mpp.12700

Figure Lengend Snippet: Pathogenicity tests on IL7‐3 tomato plants with Fol‐WT (WT), Fol‐ΔSIX1 (ΔSIX1) or Fol‐ΔSIX1:SIX1a/btransformants. (A) Photographs taken at 21 days post‐inoculation (dpi) of IL7‐3 plants infected with Fol‐WT (WT), Fol‐ΔSIX1 (ΔSIX1), Fol‐ΔSIX1:SIX1a transformants 3 or 16 or Fol‐ΔSIX1:SIX1b transformants 3 or 4 from one of three replicate experiments. (B) Distribution of disease scores at 21 dpi for plants shown in (A) and two additional replicates (n = 28–30). Treatments with different letters are significantly different at P = 0.05.

Article Snippet: The coding sequences of Foph SIX1a and SIX1b were synthesized and cloned into pUC57 by GenScript (Piscataway, NJ, USA).

Techniques: Infection

Journal: Molecular Plant Pathology

Article Title: Evidence for horizontal gene transfer and separation of effector recognition from effector function revealed by analysis of effector genes shared between cape gooseberry‐ and tomato‐infecting formae speciales of Fusarium oxysporum

doi: 10.1111/mpp.12700

Figure Lengend Snippet: Sequence alignment of Fusarium oxysporumf. sp. lycopersici (Fol) SIX1, Fusarium oxysporumf. sp. physali (Foph) SIX1a and FophSIX1b highlighting positions showing evidence for diversifying and purifying selection among 18 different SIX1 sequences from Fusarium oxysporum.Positions showing evidence for diversifying selection detected using FUBAR, FEL or MEME are highlighted in blue, red and green, respectively.Positions showing evidence for purifying selection detected using FUBAR or FEL are highlighted in dark brown and olive green, respectively.Numbers above the sequence alignment indicate hypervariable positions showing four or more different residues among the 18 sequences analysed. Regions highlighted in grey indicate positions excluded from analysis owing to deletions of three or more amino acid residues in a number of sequences. The predicted signal peptide is boxed and the predicted pro‐peptide region delimited by a Kex2 cleavage site (highlighted in purple) is underlined. Asparagine residues in predicted N‐glycosylation sites are shown in italics and are highlighted in yellow unless located at a position showing purifying selection. Cysteine residues predicted to be involved in disulfide bond formation are highlighted in black unless located at a position showing purifying selection. A hypervariable region showing a high proportion of positions undergoing diversifying selection is underlined with a dotted line. Residues shared between FolSIX1 and FophSIX1b are shown in bold font. Residues unique to Fol SIX1 (relative to FophSIX1a and SIX1b) are double underlined.

Article Snippet: The coding sequences of Foph SIX1a and SIX1b were synthesized and cloned into pUC57 by GenScript (Piscataway, NJ, USA).

Techniques: Sequencing, Selection, Glycoproteomics

Journal: Applied and Environmental Microbiology

Article Title: Use of Comparative Genomics-Based Markers for Discrimination of Host Specificity in Fusarium oxysporum

doi: 10.1128/AEM.01868-17

Figure Lengend Snippet: Overview of fungal strains used in this study and their NCBI genome accession numbers

Article Snippet: Fopis HDV247 , NRRL37622 , pisi , , , , Broad Institute , GCA_000260075.2.

Techniques:

Journal: World Journal of Microbiology & Biotechnology

Article Title: Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered β-fructofuranosidase enzyme in Pichia pastoris

doi: 10.1007/s11274-024-04027-6

Figure Lengend Snippet: Microbial strains in this study

Article Snippet: pJ905- fopA_V1 , Sh ble GAP P -xln2 S -fopA_V1-AOX1 T , This study; ATUM (Menlo Park, USA).

Techniques: Construct