EMBO/sd-character-level-ner huggingface dataset:
SCFFbxw5 ubiquitylates MCAK in a highly efficient and specific manner A.Figure 3A. Coomassie-stained SDS-PAGE of different proteins used in the in vitro ubiquitylation assay. Cul1 was obtained as a full-length protein (and therefore runs at around 85 kDa in contrast to the split version of Fig 1) from Sf21 cells and in vitro neddylated (see Fig EV2A and B). SCFFbxw5 complexes for all ubiquitylation experiments were prepared by mixing equimolar amounts of Fbxw5/Skp1 and Cul1~Nedd8/Rbx1 sub-complexes. B. Drop-out ubiquitylation experiment. 0.2 µM MCAK was incubated with 75 µM ubiquitin (untagged), 170 nM E1, 0.25 µM of UbcH5b and/or Cdc34 and 0.05 µM SCFFbxw5 for 15 min at 30°C followed by western blotting using anti-MCAK antibodies for detection. Top labelling indicates component(s) that have been included (+) or omitted (-) from the reaction. C. in vitro ubiquitylation experiment as in B using Cdc34 as E2, stopped at the indicated time points by adding SDS sample buffer. D. In vitro ubiquitylation experiment as in B. Blots 1&2: Plk1 or RanGAP1 used as substrates instead of MCAK. Blot 3: Left: Experiment as in B. Asterisks indicate signals from Plk1 antibody (same species as anti-Fbxw5). Right: Same reaction except that Cul1~Nedd8/Rbx1 complexes were replaced by Cul4A~Nedd8/DDB1/Rbx1 complexes Bottom blots: Same blots as above incubated with anti-Fbxw5 antibodies. Autoubiquitylation of Fbxw5 indicates activity of the E3 ligase complex. E. in vitro ubiquitylation experiment as in B except that different F-box/Skp1 sub-complexes (Fbxw5/Skp1, Fbxl2/Skp1 or Fbxw7/Skp1) were used F. In vitro ubiquitylation experiment as in B using E.coli-derived MCAK, Kif2a or Kif2b as substrates and Cdc34 as E2. G. In vitro ubiquitylation experiment as in B using either wild type, K48R, K63R, KK48,63RR or methylated ubiquitin (Kmethyl, see Fig EV2K). Top: Cdc34 used as E2. Bottom: UbcH5b used as E2.
Tap-1 encodes a putative recombination site.Figure 7Alignments of the nucleotide and amino acid sequences of the Tap-1 C-termini. Identity in 36 amino acid sequences of Tap-1 or nucleotide sequences of tap-1 are shown (sliding window = 1). Green bars indicate 100% identity, yellow bars indicate 30-99%, and red bars indicate < 30%. Average percent identity is shown over the regions indicated with arrows. Analysis of the GC content (sliding window = 50) of tap-1 and the effector-encoding gene in V. cholerae strain 1587. Average percent GC in regions indicated with arrows is shown. Model for the acquisition of a new 3′ end for tap-1 and an effector-encoding gene as a result of the recombination site within tap-1, and an additional downstream recombination event (not shown).
Figure 4A. Summary of observed phenotypes on SCRN1 recruitment and ER morphology in cells co-expressing indicated SCRN1 and VAP constructs. B. Live hippocampal neurons (DIV17-18) co-expressing TagRFP-ER with GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Scale bars: 5 µm (full size) and 2 µm (zoom). C. Time-lapse of TagRFP-ER dynamics in hippocampal neurons (DIV16-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Intact ER structures (dark arrowheads), intact and dynamic ER structures (light arrowheads) and impaired an non-dynamic ER structures (arrows) are indicated. Scale bars: 5 µm (full size) and 2 µm (zoom). D. Schematic illustration of the effect of VAP-SCRN1 interactions on ER structure and dynamics. E. ER nanostructures visualized with GFP-Sec61β in axons of hippocampal neurons (DIV18) immunostained for α-Tubulin and co-expressed with pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs, and subjected to expansion microscopy. Scale bars: 2 µm (full size) and 500 nm (zoom). F. FRAP experiment of TagRFP-ER in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. Scale bar: 2 µm. G. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. H. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test. I. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. J. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.
Figure 4A, Expression of the artificial mini-nesprin-2G construct results in significant nesprin accumulation (orange arrow) in cells migrating through narrow constrictions. B, Schematic depicting the endogenous protein, and the artificial mini-nesprin-2G and MUT-mini-nesprin-2G constructs. Both constructs contain the first two and last two spectrin repeats of the endogenous protein; the mutant construct presents two mutations on the actin-binding domain that prevents it from binding to actin. C, Quantification of the mini-nesprin-2G accumulation of cells migrating through narrow constrictions. Note that the mutant construct does not accumulate at the front of the nucleus. Error bars correspond to the standard deviation. Mini-nesprin-2G: n=10 cells over 4 biological replicates; Mutant: n=11 cells over 4 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars(*P< 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.
Physical interaction between DAPK and beclin 1.Figure 3(Aa) Protein extracts from COS7 cells, HEK293T cells, or no extracts were added to bacterially produced GST or GST-beclin‐1. The pulled‐down proteins, as well as the total cell extracts, were blotted with the indicated antibodies. (Ab) Ponceau S staining of GST and GST-beclin‐1 to which the extracts were added. (B) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 or with Flag‐tagged beclin 1 lacking the Bcl‐2‐binding domain (ΔBD) together with Bcl‐XL and HA‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted with DAPK, Bcl‐XL and beclin 1 antibodies. (C) HEK293 cells were transfected with Flag-beclin‐1 or Flag-GFP, and the Flag‐tagged proteins were immunoprecipitated using Flag antibodies, and eluted with an excess of Flag peptides. The blot was reacted with DAPK antibodies or with Flag antibodies at different time exposures
1,25D3 induces autophagy in human macrophages co-infected with HIV and M.Figure 2HIV and/or M. tuberculosis (TB) infected MDM were treated for 7 days with 100 pmol/L 1,25D3. (A) Cells were lysed and immunoblots of LC3B isoforms using antibody to LC3B or β-actin performed. B) Cells were incubated with 10 µg/mL pepstatin A for 4 h on day 7 prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin. C) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown. (D) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d followed by 10 µg/mL pepstatin A for a further 4 h. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown. (E) Immunoblots of p62 using antibody to p62 or β-actin 7 d after macrophages treated with 1,25D3. (F) At 7 d post-infection, aliquots of supernatant taken before the addition of WST-1 were tested for lactate dehydrogenase (LDH) spectrophotometrically using the LDHPLUS assay. For the last hour cells were incubated with WST-1, and the reduction of the WST-1 reagent to its formazan product was monitored spectrophotometrically.
Systematic investigation of the impact of fusion-tag orientation A Domain overview of VCP and 10 UBX-domain containing proteins.Figure 5A Domain overview of VCP and 10 UBX-domain containing proteins. The UBX proteins bind with their C-terminally located UBX-domains to the N domain in VCP, with the exception of UBXD1 that binds to the C-terminus of VCP via a PUB domain. B,C,D VCP and the UBX domain-containing proteins were co-expressed as N- and C-terminal NL and PA-mCit fusion proteins in eight different orientations for which cBRET (B) and cLuC (C) ratios were generated. cBRET and cLuC values over the threshold of ≥0.01 and ≥0.03, respectively, are colored in cyan. D The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value.
Figure.Figure 4A, B RT-qPCR analysis of KDM6A (A) and KDM6B (B) mRNA levels in SCNT 2-cell embryo. Data shown are mean expression values relative to Gapdh. The value in ICSI control was set as 1. Error bars, s.e.m., n ≥ 3. **P < 0.01 by Student"s t C. The sketch of KDM6A and KDM6B in vitro transcription vector (right), and the integrity of in vitro transcripted mRNA was confirmed by electrophoresis with formaldehyde gels (left). M, marker; T7, in vitro transcription promoter; HA, hemagglutinin epitope tag D. Immunostaining of SCNT embryo for H3K27me3 and HA epitope tag after injection of different mRNA as indicated. Representative images from ≥ 187 embryos analyzed in four independent micromanipulations for each condition are shown. Scale bar, 20 μm E. Schematic illustration of mRNA injection into oocytes and SCNT F. Representative images of SCNT embryos at 115 h after injection of different mRNA as indicated. Scale bar, 50 μm G. The bar chart showing the efficiency of blastocyst formation. Injection of KDM6A mRNA improved the preimplantation development rate of SCNT embryos. Error bars, s.e.m., n ≥ 3. **P < 0.01, ***P < 0.001 by two-tailed Student"s  t-test. n.s., not signif I. The retrieved rate of embryo assessed at E8.5. Five independent experiment replicates were performed. The numbers at  the bottom of the bars indicated the total number of transferred embryos I. The retrieved rate of embryo assessed at E8.5. Five independent experiment replicates were performed. The numbers at  the bottom of the bars indicated the total number of transferred embryos J. Schematic diagram depicts the position of highly conserved TPR and JmjC domain. The KDM6A are broadly expressed proteins characterized by N-terminal TPRs and C-terminal JmjC domain. In KDM6B the only clearly identifiable domain is the C-terminal JmjC domain. White asterisk indicates the mutation point K. Preimplantation development rates in the KDM6A-HA, KDM6B-HA, or KDM6A-cM/-nM/-ncM-HA mRNA-injected and non-injected control SCNT groups. The efficiency was calculated based on the number of cleavage embryo. Error bars, s.d., the total numbers of cleavage embryos in each condition (KDM6A-HA, KDM6B-HA, KDM6A-cM/-nM/-ncM-HA, and Control) from three independent experiments were 275, 199, 290, 221, 244, and 286, respectively.
Figure 3A - Toeprinting showing inhibition of initiation complex formation on a truncated dgcM mRNA (210 nt from +1) by OmrA and OmrB. Inclusion of 30S ribosomes, tRNAfmet, OmrA, and OmrB is indicated. The unrelated IstR-1 sRNA is a negative control. The 30S/ tRNAfmet-dependent reverse transcription block (toeprint), is indicated by an arrow. Sequencing reactions were loaded next to the primer extension products (UCGA). The AUG start codon is indicated by asterisks. B - In vitro translation of dgcM::3xflag mRNA in the presence or absence of OmrA/ OmrB, with or without purified Hfq protein, was carried out As an internal control, the non-target ompA::3xflag mRNA was included. The Western blot shows DgcM-3xFLAG and OmpA-3xFLAG protein levels.
ALP assay.Figure 1ALP assay. The cells expressing Pho8Δ60p were cultured in SD+CA medium (open bars) and shifted to SD(−N) medium for three hours (filled bars). The ALP activity of the lysates was measured to estimate autophagic activity. Wild-type cells, TN124; Δapg8 mutant harboring vector, TK303; Δapg8 mutant harboring APG8 on a centromeric vector, TK301; Δapg8 mutant harboring 3 × HA-tagged APG8 plasmid, TK307.
Expression and localization of Coi12p A Proteins extracted from wild‐type cells at the indicated time point of conjugation (hpm) were analyzed by Western blot with the newly established anti‐Coi12p antibody (top) and with an anti‐alpha‐tubulin antibody (bottom).Figure 2A Proteins extracted from wild‐type cells at the indicated time point of conjugation (hpm) were analyzed by Western blot with the newly established anti‐Coi12p antibody (top) and with an anti‐alpha‐tubulin antibody (bottom). The positions of the protein molecular weight markers are indicated at the left. B Proteins extracted from wild‐type cells at 2 hpm and from the COI12 KO cells at the indicated time of conjugation (hpm) were analyzed by Western blot as described in (A). C Proteins extracted from wild‐type cells at 4 hpm and recombinantly expressed Coi12p from E. coli were analyzed by Western blot as described in (A). D-F Immunofluorescence microscopy staining of wild‐type cells using the anti‐Coi12p antibody. Coi12p localizes to the cytoplasm and the MAC in early stage (D, meiotic prophase), mid‐stage (E, nuclear exchange), and late stage (F, nuclear alignment) of conjugation. The MICs (i), parental MACs (a) and newly developed MACs (na) are marked with arrowheads. Vegetative (V) and conjugating (C) cells are circled with dotted lines in (D).
Figure 1(A) Detection of Fstl1 protein in ischemic myocardium of WT mice. After MI induction, the heart was harvested at the indicated time points, and separated to ischemic and remote (non-ischemic) areas. Fstl1 protein was detected by western blotting (n=3, each time point). Statistical analysis was performed by ordinary one way ANOVA. Post Hoc test was performed by Dunnett test. (B) Serum Fstl1 was detected by western blotting at the indicated time points. Ponceau S staining of serum reveals equivalent amount of loaded protein (n=4, each time point). Error bars represent mean ± SEM. Statistical analysis was performed by ordinary one way ANOVA. Post Hoc test was performed by Dunnett test.
Figure 2Relative mRNA levels of immune related genes in Vgll4-knockdown LLC or MB49 cells by qRT-PCR analysis Knockdown of Vgll4 in MB49 cells decreased the surface expression of PD-L1 protein on control or shVgll4 MB49 cells measured by flow cytometry analysis with anti-mPD-L1 antibody Relative mRNA levels of immune related genes in Vgll4-knockdown A549 cells by qRT-PCR analysis Immunoblot analysis revealed the reduced expression of PD-L1 protein in VGLL4 knockdown A549, 16HBE and H358 cells compared with control cells qRT-PCR analysis revealed a reduction of PD-L1 mRNA levels in VGLL4-knockdown 16HBE and H358 cells by siRNA Flow cytometry analysis of surface PD-L1 levels in control and VGLL4 knockdown A549 cells treated with IFNγ for the indicated times Immunoblot analysis revealed the reduced IFNγ-inducible PD-L1 expression in VGLL4 knockdown A549 and 16HBE cells Immunoblot analysis of total cell lysates with the indicated antibodies revealed the attenuated IFNγ-inducible PD-L1 expression in VGLL4 knockout (KO) A549 cells generated by CRISPR/Cas9 Loss of VGLL4 dampened the activity of PD-L1 promoter reporter in a luciferase assay. A549 cells were transfected with human PD-L1-promoter luciferase plasmid and indicated siRNA, treated with IFNγ for 12 h, and subjected to a luciferase assay Overexpression of mouse PD-L1 in Vgll4 knockdown LLC cells restored the tumor growth in C57BL/6 mice. Tumor weights of shVgll4 or shVgll4-lenti-mPD-L1 LLC cells were determined 21 days after tumor cell transplantation into C57BL/6 mice (left panel). Expression of mPD-L1 was shown in the right panel by immunoblot analysis with anti-mPD-L1 antibody. n=10 tumors for each group Knockdown of VGLL4 in A549 cells enhances T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and then surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensities are shown in right panel
Fig.Figure 9A-C, Quantitative and morphometric analyses of microglia in the SN area of SNCA-PD mice. Shown in A are representative views of Iba1+ microglia neighboring TH+ mDA neurons in the SNs of PD mice treated with GSK-650394 and vehicle. Insets, enlarged views for the boxed areas. Data are represented as mean ± SEM. n=3 mice per group. Significantly different at p=1.49E-11* in graph B and p=0.000003* in graph C. D and E, The M1 marker CD16/32 expression in Iba1+ microglia. Data are represented as mean ± SEM. n=3 mice per group. Significantly different at p=0.0016* in graph E. F and G, mRNA expression of NLRP3-inflammasome components and pro-inflammatory cytokines in MPTP (F) and SNCA (G) PD mice injected with the SGK1 inhibitor or vehicle (control). Each bar represents the mean ± SEM of 3 PCR values from each animal, n=3 mice per group. Significantly different at p=0.0372*, 0.0173**, 0.0003*** in graph F and p=0.002#, 0.0113##, 0.0053###, 0.0001#### in graph G. H-J, ASC expression in the SNs and SN microglia of SNCA-PD mice. n=5 mice per group. Data are represented as mean ± SEM. Significantly different at p=1.91E-09* in graph I and p=0.0079* in graph J. K and L, β-gal-stained cell senescence in SNCA-PD mouse SN. Data are represented as mean ± SEM. n=5 mice per group. Significantly different at p=0.0001*
Figure 5MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (A) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test). B MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (B) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test). MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the E-cadherin (C) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test). D MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the vimentin (D) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test). MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (E) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test). F MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (F) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).
Physical interaction between Bcl‐XL/Bcl‐2 and the BH3‐like domain of Beclin‐1.Figure 1(A) Selection of Beclin‐1 fragments that interact with Bcl‐XL/Bcl‐2 in the yeast‐two‐hybrid system. Black lines indicate the fragments of Beclin‐1 (grey) that interact with Bcl‐XL (upper part of the graph) or with Bcl‐2 (lower part). ▪, CEMC7 library; •, human thymocyte library; ▴, placenta library and ★, human brain library. The minimal interacting domain required for interaction with Bcl‐XL/Bcl‐2 is situated between aa 112 and 159. (B) Alignment of the BH3‐like domain of Beclin‐1 with established BH3 domains. L116A Beclin‐1 and F123A Beclin‐1 describe two Beclin‐1 mutant peptides. (C) Fluorescence polarization assays demonstrating that a peptide containing the BH3‐like domain of Beclin‐1 interacts with Bcl‐XL. Recombinant Bcl‐XL ΔTM was incubated with carboxyfluorescein‐labeled Bak BH3 peptide, in the absence or presence of wild‐type (WT) Beclin‐1 BH3 or either of the two mutant peptides described in B. (D) Affinity determination of the interaction between recombinant Bcl‐XLΔTM and fluorescent Beclin‐1 BH3, by fluorescence polarization. A comparison between WT Bcl‐XL(squares) and G138A Bcl‐XL (triangles) is shown (means±s.d., n=3 separate experiments). (E) Co‐immunoprecipitation of Bcl‐XL and WT or mutant Beclin‐1. HeLa cells were transfected with the indicated constructs, followed by immunoprecipitation of Flag‐tagged Bcl‐XL and immunochemical detection of Beclin‐1. (F) Co‐immunoprecipitation of Beclin‐1 with‐WT and mutant (G138A) Bcl‐XL. Results are representative of at least three experiments yielding similar results.
Figure 4A) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex, anterior cingulate cortex, premotor cortex, lateral temporal cortex, and hippocampus. The heatmap indicates the associated T-statistic for each voxel, with light blue representing the highest value. B) Magnitude of motor cortex neuronal loss in ALS cases from the UPenn Biobank is associated with higher wPRS. The central bands indicate the median, the box indicates the interquartile range (IQR) between the first and third quartiles, and the upper and lower whiskers indicate the largest or smallest value no further than the IQR multiplied by 1.5. Each data point in the boxplot represents a unique case.
Figure 6After secretion, Bap is proteolytically processed, and the N-terminal containing regions A and B is released. Module 3 acts as a pH and Ca2+ sensor by significant conformational switches. When the pHs become acidic and [Ca2+] is low, modules 1 and 2 of BSP act as scaffold and aggregate into amyloid fibrils over time through LLPS and maturation, contributing to subsequent staphylococcal biofilm formation, which process can be promoted by intrinsically disordered module 3. When [Ca2+] reaches millimolar in the ECM, Ca2+-binding to module 3 stabilizes its ordered folding and the interaction networks among modules 2, 3 and 4, resulting in reduced conformational dynamics across BSP, hindering its self-assembly and aggregation.
Model for TFG function at the ER/ERGIC interfaceA model depicting the assembly of TFG at the ER/ERGIC interface, which facilitates the transient, local retention of COPII vesicles prior to vesicle uncoating and maintains the juxtaposed organization of ER and ERGIC membranes.Figure 8A model depicting the assembly of TFG at the ER/ERGIC interface, which facilitates the transient, local retention of COPII vesicles prior to vesicle uncoating and maintains the juxtaposed organization of ER and ERGIC membranes.
Neonates were obtained by caesarean delivery.figf3Neonates were obtained by caesarean delivery. a, b, Successfully resuscitated pups were monitored in a humidified chamber without milk feeding (a) or with artificial milk feeding provided every 3-6 h through a tube inserted into the stomach (b). Neonates were obtained by caesarean delivery. a, b, Successfully resuscitated pups were monitored in a humidified chamber without milk feeding (a) or with artificial milk feeding provided every 3-6 h through a tube inserted into the stomach (b).