The Need for the Retina’s Surface “Drying” during Macular Hole Surgery

Purpose: to determine the relevance of retina’s surface “drying” during vitrectomy at the stage of exchange of infusion solution for air based on an experimental study and calculation of the geometric dimensions of a drop of moisture formed in the macular region.Patients and Methods. There were 10 patients (10 eyes), who had a vitrectomy for a macular tear with air injection in one eye. Their age was from 50 to 78 (64.0 ± 3.1), the size of the macular tear 250–631 (431.6 ± 44.3) mkm. After 27G subtotal vitrectomy intake of fluid formed after fluid-air exchange was performed and its volume was measured in equal time intervals 3 times. The next step is to determine the shape of a drop of intraocular fluid (IOF) that forms on the surface of the retina during BSS exchange. For example, in the cadaveric eye, the wetting of the retinal surface was studied with the volume of liquid that was obtained during the operation, and its edge wetting angle was measured. Given the fact that INFLOW during surgery formed from two sources: the result of the production of the ciliary body (CB) (2,5–4,0 μl/min) and dehydration of the vitreous body (VB) due to the pressure of the air supplied in the vitreal cavity was calculated drop diameter INFLOW first, when the product of TST and dehydration VB (CB + VB); second, only if the production CT. This made it possible to understand how a drop of HGH formed during the operation will be projected onto the surface of the macula. Results. Volume of intraocular fluid taken during surgery was 60–80 microliters (68.1 ± 2.8) for 3 minutes, or 22.7 microliters per minute. Rate of fluid formation decreased by 18–25 % for 9 minutes. Contact angle of wetting made 14.5°. Drop diameter calculated for ciliary body secretion and vitreous remnants dehydration equals 17.98 mm. Drop diameter calculated for ciliary body secretion alone equals 2.6 mm. Conclusions. “Drying” of the retina during macular hole surgery is impractical as intraocular fluid is constantly formed on the retina surface. Its volume is sufficient to cause opening of the hole. Refusal from this manipulation would not influence anatomic efficacy of the operation and would reduce surgical trauma

Counterbalance of Stability and Activity Observed for Thermostable Transaminase from Thermobaculum terrenum in the Presence of Organic Solvents

Pyridoxal-5’-phosphate-dependent transaminases catalyze stereoselective amination of organic compounds and are highly important for industrial applications. Catalysis by transaminases often requires organic solvents to increase the solubility of reactants. However, natural transaminases are prone to inactivation in the presence of water-miscible organic solvents. Here, we present the solvent tolerant thermostable transaminase from Thermobaculum terrenum (TaTT) that catalyzes transamination between L-leucine and alpha-ketoglutarate with an optimum at 75 °C and increases the activity ~1.8-fold upon addition of 15% dimethyl sulfoxide or 15% methanol at high but suboptimal temperature, 50 °C. The enhancement of the activity correlates with a decrease in the thermal denaturation midpoint temperature. The blue-shift of tryptophan fluorescence suggested that solvent molecules penetrate the hydration shell of the enzyme. Analysis of hydrogen bonds in the TaTT dimer revealed a high number of salt bridges and surface hydrogen bonds formed by backbone atoms. The latter are sensitive to the presence of organic solvents; they rearrange, conferring the relaxation of some constraints inherent to a thermostable enzyme at low temperatures. Our data support the idea that the counterbalance of stability and activity is crucial for the catalysis under given conditions; the obtained results may be useful for fine-tuning biocatalyst efficiency

Structural basis for the ligand promiscuity of the neofunctionalized, carotenoid-binding fasciclin domain protein AstaP

Abstract Fasciclins (FAS1) are ancient adhesion protein domains with no common small ligand binding reported. A unique microalgal FAS1-containing astaxanthin (AXT)-binding protein (AstaP) binds a broad repertoire of carotenoids by a largely unknown mechanism. Here, we explain the ligand promiscuity of AstaP-orange1 (AstaPo1) by determining its NMR structure in complex with AXT and validating this structure by SAXS, calorimetry, optical spectroscopy and mutagenesis. α1-α2 helices of the AstaPo1 FAS1 domain embrace the carotenoid polyene like a jaw, forming a hydrophobic tunnel, too short to cap the AXT β-ionone rings and dictate specificity. AXT-contacting AstaPo1 residues exhibit different conservation in AstaPs with the tentative carotenoid-binding function and in FAS1 proteins generally, which supports the idea of AstaP neofunctionalization within green algae. Intriguingly, a cyanobacterial homolog with a similar domain structure cannot bind carotenoids under identical conditions. These structure-activity relationships provide the first step towards the sequence-based prediction of the carotenoid-binding FAS1 members

A thermal after-effect of UV irradiation of muscle glycogen phosphorylase <i>b</i>

<div><p>Different test systems are used to characterize the anti-aggregation efficiency of molecular chaperone proteins and of low-molecular-weight chemical chaperones. Test systems based on aggregation of UV-irradiated protein are of special interest because they allow studying the protective action of different agents at physiological temperatures. The kinetics of UV-irradiated glycogen phosphorylase <i>b</i> (UV-Ph<i>b</i>) from rabbit skeletal muscle was studied at 37°C using dynamic light scattering in a wide range of protein concentrations. It has been shown that the order of aggregation with respect to the protein is equal to unity. A conclusion has been made that the rate-limiting stage of the overall process of aggregation is heat-induced structural reorganization of a UV-Ph<i>b</i> molecule, which contains concealed damage.</p></div

Scheme illustrating thermal after-effect of UV irradiation of Ph<i>b</i>.

<p>Heating of UV-Ph<i>b</i> with concealed damage (P⁰) at 37°C results in a structural reorganization of UV-irradiated protein to a state with manifested damage (P^a). Slow transformation of P⁰ into P^a is followed by the nucleation stage and fast stage of aggregate growth with formation of amorphous aggregates. The growth of aggregates proceeds by attachment of P^a to the existing aggregates (basic aggregation pathway). Sticking of protein aggregates can be observed in the course of accumulation of large-sized aggregates (additional aggregation stage).</p

The comparison of the kinetics of aggregation of UV-Ph<i>b</i> (0.26 mg/ml) at 37°C followed by an increase in the concentration of aggregated UV-Ph<i>b</i> and increase in the light scattering intensity.

<p>(A) The time dependences of the concentration of aggregated UV-Ph<i>b</i> irradiated with the following doses: 7.5 (1), 9.4 (2) and 12.5 J/cm² (3). The points are the experimental data. The solid curve was calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#pone.0189125.e009" target="_blank">Eq 6</a> at the following values of parameters: <i>t</i>₀ = 6 min, <i>k</i>_I = 0.074 min^-1 and [UV-Ph<i>b</i>_agg]_lim = 0.16 mg/ml for curve 1; <i>t</i>₀ = 1.71 min, <i>k</i>_I = 0.107 min^-1 and [UV-Ph<i>b</i>_agg]_lim = 0.19 mg/ml for curve 2; <i>t</i>₀ = 0.67 min, <i>k</i>_I = 0.139 min^-1 and [UV-Ph<i>b</i>_agg]_lim = 0.21 mg/ml for curve 3. (B) The relationship between increment of the light scattering intensity (<i>I</i>–<i>I</i>₀) and the concentration of aggregated UV-Ph<i>b</i>. The radiation doses were the following: 7.5 (1), 9.4 (2) and 12.5 J/cm² (3). The concentration of aggregated UV-Ph<i>b</i> was determined from measurements of optical density of supernatant at 280 nm after precipitation of protein aggregates by centrifugation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#sec002" target="_blank">Methods</a>). The error bars were calculated using three independent measurements.</p

The kinetics of aggregation of UV-Ph<i>b</i> followed by the increase in the light scattering intensity at 37°C (radiation dose was 9.4 J/cm²).

<p>(A) The dependences of the light scattering intensity (<i>I–I</i>₀) on time obtained at the following concentrations of UV-Ph<i>b</i>: 0.2 (1), 0.4 (2), 0.6 (3), 0.9 (4) 1.2 (5) and 1.5 mg/ml (6). <i>I</i> and <i>I</i>₀ are the current and initial values of the light scattering intensity, respectively. (B) Fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#pone.0189125.e005" target="_blank">Eq 4</a> to the experimental data obtained at [UV-Ph<i>b</i>] = 0.4 mg/ml. Points are the experimental data. The solid curve was calculated at <i>k</i>_I = 0.115 min^-1 and <i>t</i>₀ = 5.54 min. The horizontal dashes correspond to <i>I</i>₀ and <i>I</i>_lim values. (C, D and E) The dependences of <i>k</i>_I, (<i>I</i>_lim−<i>I</i>₀) and <i>k</i>_I(<i>I</i>_lim−<i>I</i>₀) values on the concentration of UV-Ph<i>b</i>. (F) The kinetic curves represented in coordinates {[d(<i>I—I</i>₀)/d<i>t</i>]/[P]₀; (<i>I—I</i>₀)/[P]₀}, where [P]₀ is the concentration of UV-Ph<i>b</i>. The dimension of [d(<i>I—I</i>₀)/d<i>t</i>]/[P]₀ is [min^-1·(counts/s)]/(mg/ml); the dimension of (<i>I—I</i>₀)/[P]₀ is (counts/s)/(mg/ml). The concentrations of UV-Phb were the following: 0.2 (1), 0.4 (2), 0.6 (3), 0.9 mg/ml (4). The solid line was calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#pone.0189125.e006" target="_blank">Eq 5</a> at <i>k</i>_I = 0.122 min^-1 and ε = 1.67·10⁶ (counts/s)/(mg/ml). Three independent measurements were used to determine the error bars shown in panels C, D and E.</p

A comparison of the properties of intact Ph<i>b</i> and UV-Ph<i>b</i> using CD.

<p>(A) The CD spectra for intact Ph<i>b</i> (1 mg/ml, curve 1) and Ph<i>b</i> irradiated with the dose of 9.4 J/cm² (1 mg/ml, curve 2) at 10°C. (B) The dependence of ellipticity at 220 nm and (C) the portion of unfolded protein (γ_U) on temperature for intact Ph<i>b</i> (0.17 mg/ml, curve 1) and UV-Ph<i>b</i> (0.17 mg/ml, curve 2). The values of γ_U were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189125#pone.0189125.e011" target="_blank">Eq 8</a>. The dashed horizontal lines on panel B correspond to the asymptotes for the regions of relatively low and relatively high temperatures. The dashed horizontal lines on panel C correspond to the γ_U values equal to 0, 0.5 and 1.0.</p

Sedimentation behaviour of UV-Ph<i>b</i> (0.75 mg/ml) at 25°C.

<p>The samples were heated at 37°C for 90 min and cooled to 25°C after heating. (A) Sedimentation profiles for Ph<i>b</i> irradiated with dose of 9.4 (sample 1) and 12.5 J/cm² (sample 2) before heating. <i>A</i>₂₈₈ is absorbance at 288 nm, <i>r</i> is radial distance. Control curves (control 1 and control 2) correspond to non-heated UV-Ph<i>b</i> (radiation doses were 9.4 and 12.5 J/cm², respectively). Sedimentation runs were carried out at 25°C. Rotor speed was 15,000 rpm. (B) Sedimentation profiles for sample 1 (Ph<i>b</i> irradiated with dose of 9.4 J/cm² before heating) at 25°C. Rotor speed was 48,000 rpm. Every 4th scan was taken for presentation. (C) The <i>c</i>(<i>s</i>) sedimentation coefficient distributions obtained at 25°C were transformed to standard <i>s</i>_20,w distributions for samples 1 and 2. Sedimentation runs were carried out at 25°C. Rotor speed was 48,000 rpm.</p

Enzymatic activity and heat stability of UV-Ph<i>b</i>.

<p>(A) Dependence of the relative enzymatic activity <i>A</i>/<i>A</i>₀. (B) Dependences of the excess heat capacity () on temperature for intact Ph<i>b</i> and Ph<i>b</i> irradiated by different UV doses. The initial concentration of Ph<i>b</i> was 1.5 mg/ml. was calculated per Ph<i>b</i> dimer (<i>M</i>_r = 194800 Da). (C) Position of the maximum <i>T</i>_max on the DSC profiles. (D) Relative denaturation heat <i>Q</i>/<i>Q</i>₀. Conditions of the experiments: 0.03 M Hepes buffer, pH 6.8, containing 0.1 M NaCl. <i>A</i>₀ and <i>A</i> are the values of enzymatic activity for intact Ph<i>b</i> and UV-Ph<i>b</i>, respectively. <i>Q</i>₀ and <i>Q</i> are the values of the area under DSC profile for intact Ph<i>b</i> and UV-Ph<i>b</i>, respectively. Three independent measurements were used to determine the arrow bars shown in this figure.</p