Pengaruh proporsi tepung labu kuning dan tepung terigu terhadap sifat fisikokimia dan organoleptik cake kukus

Labu kuning kaya akan β-karoten, gula, dan serat. Upaya diversifikasi pengolahan labu kuning adalah tepung labu kuning. Tepung labu kuning dapat dimanfaatkan dalam pembuatan cake kukus sebagai bahan pensubstitusi tepung terigu. Penggunaan tepung labu kuning diharapkan dapat meningkatkan kadar β-karoten dalam cake kukus. Penelitian ini bertujuan untuk menentukan proporsi tepung labu kuning dan tepung terigu yang tepat untuk menghasilkan cake kukus dengan sifat fisikokimia dan organoleptik yang dapat diterima oleh konsumen. Rancangan penelitian yang digunakan adalah Rancangan Acak Kelompok dengan satu faktor, yaitu faktor proporsi tepung labu kuning dan tepung terigu dengan lima level, yaitu: 0%:100%; 10%:90%; 20%:80%; 30%:70%; 40%:60%, yang diulang sebanyak lima kali. Parameter pengujian meliputi volume spesifik, firmness, dan organoleptik kesukaan warna, kemudahan digigit, moistness, dan rasa. Data dianalisis dengan ANOVA (Analysis of Variance) pada α = 5% untuk mengetahui ada tidaknya pengaruh proporsi tepung labu kuning dan tepung terigu terhadap sifat fisikokimia dan organoleptik cake kukus. Apabila terdapat pengaruh nyata maka dilanjutkan dengan uji DMRT (Duncan's Multiple Range Test) pada α = 5%. Penentuan perlakuan terbaik dilakukan dengan uji pembobotan. Cake kukus dengan perlakuan terbaik diuji kadar β-karoten. Hasil penelitian menunjukkan bahwa perlakuan proporsi tepung labu kuning : tepung terigu memberikan pengaruh nyata (α = 5%) terhadap volume spesifik, firmness, dan tingkat kesukaan terhadap warna, kemudahan digigit, moistness, dan rasa. Semakin besar proporsi tepung labu kuning, semakin kecil nilai volume spesifik, firmness, dan kesukaan warna cake, namun semakin besar nilai kesukaan kemudahan digigit, moistness, dan rasa cake. Proporsi tepung labu kuning : tepung terigu 20%:80% merupakan perlakuan terbaik dengan kadar β-karoten sebesar 8,569 µg β- karoten/g cake kukus

Molecular characterization of the <i>Sleeping Beauty</i>-mediated integration events in GABEB cell clones.

<p>(A) Southern blot analysis of genomic DNA from GABEB cell clones digested with <i>NheI</i> (SA clones) or <i>AflII</i> (T2 clones), single cutter in the transposon cassette, and hybridized to a Venus probe. A single band higher than 4.2 kb (SA clones) and 3.4 kb (T2 clones) indicates integration of one copy of the transposon into the genome. Multiple Venus-specific bands correspond to repeated integration events. (B) Southern Blot analysis of genomic DNA from 8 (SA) and 9 (T2) clones digested with <i>NcoI</i> (SA clones) or <i>MfeI</i> and <i>NdeI</i> (T2 clones). The expected Venus-specific band corresponding to 6 kb for SA and 8.9 kb for T2 transposon indicates the correct integration of the transposons into the genome. C, mock-transfected cells; red bars, Venus-specific probe. Clone showing rearrangement of the transposon cassette is highlighted by black asterisk. (<b>C</b>) Bi-directional mapping of the junctions between transposon and genomic DNA. The table summarizes 27 integrations belonging to 10 single clones. For each integrant, the underlined sequence represents a portion of the transposon IRs, left (CAGTT) and right (AACTG) separated by dots; TA dinucleotide (in bold) is the target site correctly duplicated after transposition. Hit chromosomes and positions are reported. UnK, unknown region of the human genome based on UCSC hg19 assembly.</p

Characterization of GB/hTERT MSCs.

<p>(A) Schematic representation of a Glucose Biosensor (GB) gene used for the genetic modification of MSCs. The gene encodes a chimeric protein consisting of a signal peptide allowing protein secretion followed by Cyan Fluorescent Protein (CFP), a glucose binding domain and Yellow Fluorescent Protein (YFP). (B) Optical and YFP fluorescence microscopy of GB/hTERT MSCs (scale bar: 100μm). (C) Flow cytometry of GB/hTERT MSCs for mesenchymal CD29, 73, 90, 105 markers. Unstained cells were used as control. (D) Staining of control or differentiated GB/hTERT cells with Alizarine Red/Oil Red O indicative of osteogenic/adipogenic differentiation, respectively (scale bar: 100μm).</p

Correlation between copy number and expression of the integrated cassette.

<p>Mean fluorescence intensity (M.F.I.) of 62 GABEB clones positive for Venus expressing SB transposons are represented as circles; triangles indicate the M.F.I. of 70 GFP⁺ HaCaT clones transduced with a lentiviral vector (LV). Standard deviation bars are present for those clones carrying the same copy number. R² coefficients of determination were extracted from the linear regression plot, green line for SA and T2 transposons and red line for LV.</p

Integration pattern analysis.

<p>(A) Integration sites were annotated as “TSS-proximal” when occurring within a distance of ±2.5 kb from the gene's TSS, as “Intragenic” when occurring in a gene body and as “Intergenic” in all other cases. Black bars represent exons of a schematic gene, arrowhead indicates the direction of transcription. Distribution of SA, T<i>neo</i> and random integration sites in the genome is plotted accordingly to defined annotations. (B) Distribution of Repetitive Elements in SB SA and T<i>neo</i> libraries, in MLV and HIV libraries. Relative weighted random libraries were reported: TA and <i>MseI</i>-weighted for SA, TA-weighted for T<i>neo</i> and <i>MseI</i>-weighted for MLV and HIV libraries. **p≤10⁻³, *p≤10⁻².</p

Self-renewal potential of genetically modified MSCs.

<p>(A) Immunoblots for hTERT in control, GB and GB/hTERT MSC extract using an anti-hTERT antibody. GAPDH was used as a loading control (B) Relative telomerase activity in GB and GB/hTERT MSCs at passage 20. (C) Cumulative population doublings of GB and GB/hTERT MSCs over time. GB MSCs reached replicative senescence at passage 28. (D) mRNA levels of Oct-4, Sox-2 and Nanog pluripotency markers in GB and GB/hTERT MSCs (***: p<0.001, **: p<0.01). Results in (B) and (D) are shown as mean ± SD of 3 independent experiments.</p

Transposition efficiency.

<p>HeLa (A) and GABEB (B) cells were co-transfected with the T2 and SA transposons- and transposase-carrying plasmids. The transposition rate, on the Y axis, is derived by the ratio between the percentage of Venus⁺ cells at about 20 and 2 days post transfection. Data are representative of three independent experiments (mean ± SEM; <i>n</i> = 3).</p

Bimolecular transposition events generated by <i>PB</i>.

<p>Transposition assay was performed by using ‘solo’ transposon substrates, either alone or mixed in equimolar ratios, in the present of the mPB transposase. The statistical significance of differences is shown by asterisk above the bars, *P<0.05. Molecular analysis identified no transposase-mediated integration events in the resistant colonies using <i>PBΔright</i> (background). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen-1004103-t001" target="_blank">Table 1</a>.</p

Both <i>SB</i> and <i>PB</i> transposons are sensitive to the size of the transposon.

<p>A. Excision, autointegration and transposition profiles of <i>SB</i> (left panel) and <i>PB</i> (right panel) transposons. The name and the size of the various constructs are shown below the plots. The values using the smallest constructs (SB2K or PB2K) were set to 100% (n = 3). B. Transposition assay performed by using <i>SB</i> (upper panel) and <i>PB</i> (lower panel) transposon constructs of various sizes.</p

Suicidal Autointegration of <i>Sleeping Beauty</i> and <i>piggyBac</i> Transposons in Eukaryotic Cells

<div><p>Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. ‘Cut and paste’ DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, <i>Sleeping Beauty</i> (<i>SB</i>) and <i>piggyBac (PB)</i> that are widely used for genome manipulation in vertebrate species. <i>SB</i> originates from fish and <i>PB</i> from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither <i>SB</i> nor <i>PB</i> is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the <i>SB</i> transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both <i>SB</i> and <i>PB</i> were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, <i>SB</i> is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for <i>PB</i>.</p></div