The effect of incubation temperature on extracellular (A+C) and intracellular (B+D) lactate concentration.

<p>The concentration of lactate in media (A,C) and lysed cells (B,D) for CHOK1 (A+B) and CHOS (C+D) is plotted as percentage of the maximum of each metabolite recorded within the data set. Shift in culture temperatures is denoted by the wide dashed line, and recovered temperature with the short dashed line. Legend: • 37 °C, ■ 27 °C shift, ▲ 10 °C shift, □ 27 °C recover, △ 10 °C recover.</p

¹H NMR spectra of cell culture media.

<p>The extracellular metabolite profile of (A) CHOK1 and (B) CHOS cultures 1D ¹H NMR spectra. Samples taken at 0 h are shown in black, and 216 h in red. All spectra are shown on the same intensity scale.</p

The effect of incubation temperature on extracellular (A+C) and intracellular (B+D) Isoleucine concentration.

<p>The concentration of isoleucine in media (A,C) and lysed cells (B,D) for CHOK1 (A+B) and CHOS (C+D) is plotted as percentage of the maximum of each metabolite recorded within the data set. Shift in culture temperatures is denoted by the wide dashed line, and recovered temperature with the short dashed line. Legend: • 37 °C, ■ 27 °C shift, ▲ 10 °C shift, □ 27 °C recover, △ 10 °C recover.</p

Principal component analysis of extracellular metabolites of CHOK1 (A+B) and CHOS (C+D) cultures.

<p>The analysis of the identified extracellular metabolites by PCA is plotted by either culturing conditions (A+C) or timepoint (B+D). </p

The effect of incubation temperature on extracellular (A+C) and intracellular (B+D) glucose concentration.

<p>The concentration of glucose in media (A,C) and lysed cells (B,D) for CHOK1 (A+B) and CHOS (C+D) is plotted as percentage of the maximum of each metabolite recorded within the data set. Shift in culture temperatures is denoted by the wide dashed line, and recovered temperature with the short dashed line. Legend: • 37 °C, ■ 27 °C shift, ▲ 10 °C shift, □ 27 °C recover, △ 10 °C recover.</p

The High Performance of Choline Arginate for Biomass Pretreatment Is Due to Remarkably Strong Hydrogen Bonding by the Anion

The ionic liquid choline arginate [Ch]­[Arg] is more effective for biomass pretreatment than other choline based amino acid ILs, but the underlying mechanism has been unclear. In the present work we use the high-level CCSD­(T)/CBS­(MP2) and G4­(MP2) thermochemical protocols to probe the H-bonding interactions of [Ch]­[Arg] with water, and organic functional groups commonly found in biomass. We show that the [Ch]­[Arg] IL forms unusually strong H-bonding interactions with prototypical H-bond donors. For example, we obtain H-bonding interactions of 76.6, 80.0, and 103.6 kJ mol^–1 with water, methanol, and phenol, respectively. Our theoretical results shed light on the capacity of [Ch]­[Arg] to dissolve biomass, and they demonstrate the importance of ion conformation, in addition to speciation, for IL performance more generally. As a point of reference, we compare the H-bonding interactions of [Ch]­[Arg] with those of a related IL, choline glycinate ([Ch]­[Gly]), which does not dissolve biomass as effectively as [Ch]­[Arg]

Internalisation of D25scFv, D34scFv, D25p and D34p in αvβ6-expressing cells.

<p>Internalisation of bound D25scFv (A,B) and D34scFv (D, E) was assessed at 0 mins (A,D) and 45 mins (B,C,E,F) in αvβ6-expressing cells (A,B,D,E) and detected using anti-myc antibody. In control cells (C,F) only anti-myc antibody was used. The scFvs both were internalised by αvβ6-expressing cells and some located to the nucleus. The absence of nuclear staining with anti-myc antibody alone suggests this was a true nuclear localisation. Internalisation of D25p (G, H, I) and D34p (J, K, L) was assessed in αvβ6-expressing cells ((G,H,J,K) and αvβ6-negative cells (I, L) at the times indicated. Both peptides were internalised only by αvβ6-expressing cells. The scale bar shown represents 20 µm.</p

Oligonucleotides used to build the structural guided library.

<p>Oligonucleotides used to build the structural guided library.</p

Screening of phage clones isolated from the α-helix & 3₁₀-helix libraries.

<p>A) Monoclonal scFv screening ELISA testing 96 clones in each library. Bacterial supernatants were added to 5 µg/ml recombinant αvβ6 immobilized onto ELISA plate and then probed with mouse anti-Myc antibody followed by anti-mouse-HRP. B) Clones with unique sequences were screened for binding cellular αvβ6 by flow cytometry. Figure shows examples where scFv was tested at 100 (red histogram), 10 (orange histogram) and 1 (green histogram) µg/ml. For clarity the relevant αvβ6-specific mouse monoclonal antibody 10D5 (grey) and the negative control IgG (black) histograms are also shown in each plot. C) Binding to cellular αvβ6 by D25 and D34 verified by flow cytometry at 100 (red histogram), 10 (orange histogram) and 1 (green histogram) µg/ml. Size-exclusion chromatography profile of purified of D25scFv and D34scFv showed a major peaks at 30 kDa.</p

NMR solution Structure of D25p and D34p.

<p>Figure shows the 3D-rendering model for each peptide based on the mean of 20 NMR structures. Both peptides exhibited the RGD-helix motif. D34 which has 22 amino-acids has a shorter helix than peptide D25, which has 29 amino-acids. Helices for both peptides were defined as standard α-helix with the D34 α-helix running from Leu6-Leu17 and D25 α-helix running from Leu6-Gln25.</p