Direct Measurement of the Membrane Dipole Field in Bicelles Using Vibrational Stark Effect Spectroscopy

Electrostatic fields in lipid bilayer membranes influence the structure and function of membrane-associated proteins. We present here the first direct measurement of the membrane dipole electrostatic field in lipid bicelles using vibrational Stark effect spectroscopy, in which a nitrile oscillator’s vibrational frequency changes in response to its local electrostatic environment. We synthesized α-helical peptides containing the unnatural amino acid <i>p</i>-cyanophenylalanine (CN-Phe) at four locations along the helix. This peptide was intercalated into bicelles 5 and 15 nm in radius composed of mixtures of 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-<i>sn</i>-glycero-3-phosphocholine (DHPC). Changes in the vibrational absorption energy of the nitrile probe at each position along the helical axis were used to determine changes in the local electrostatic field of the probe. We measured the magnitude of the membrane dipole electrostatic field to be −6 MV/cm, changing rapidly near the membrane surface and more slowly in the low dielectric membrane interior

Human viral targets of CRISPR/Cas9.

<p>Human viral targets of CRISPR/Cas9.</p

Exceptionally Robust CuInS₂/ZnS Nanoparticles as Single Component Photocatalysts for H₂ Evolution

In this work, we report water-soluble CuInS₂/ZnS nanoparticles (NPs) as intrinsic single-component photocatlaysts for light-driven hydrogen generation. In the presence of ascorbic acid (AA), this catalyst can efficiently generate H₂ from water with an exceptionally long lifetime (≫84 h). Mechanistic insight into the catalysis revealed by transient absorption spectroscopy demonstrates that the catalytic cycle initiates by a reductive quenching process through electron transfer from AA to CuInS₂/ZnS NPs, which is followed by a rate limiting step, i.e., proton reduction from electrons in the conduction band/defect states of the CuInS₂/ZnS NPs

<i>In vivo</i> protein transduction of TAT-14-3-3ε in rat brain.

<p>(A) 14-3-3ε protein levels in brain tissues of sham, 2 h ischemia or I/R group. I/R, 2 h ischemia followed by 24 h reperfusion. Lower panel shows the quantification of 14-3-3ε levels in upper panel. Each bar denotes mean ± SD. *<i>p</i><0.05 compared with sham group. (B) Representative Western blot analysis of TAT-14-3-3ε transduction in the rat brain after intravenous infusion of protein using anti-14-3-3ε or anti-His-tag antibody. In the TAT-14-3-3ε treated rat brain, both the TAT-14-3-3ε proteins (as indicated by the solid arrow) and 14-3-3ε proteins (as indicated by the dashed arrow) were detectable in the brain. In the 14-3-3ε-treated rats, only the 14-3-3ε proteins were detectable in the brain (n = 3 per group). Purified TAT-14-3-3ε fusion protein was provided as a positive control. (C) Representative Western blotting with anti-14-3-3ε antibody shows the 14-3-3ε protein levels (including endogenous 14-3-3ε and exogenous TAT-14-3-3ε proteins) in the brains of rats treated with vehicle, 14-3-3ε, TAT-14-3-3ε 2 h before ischemia, 4 h after intravenous administration. Similar results were obtained in 2 other experiments. (D) Western blotting with the anti-14-3-3ε antibody detects the transduction of TAT-14-3-3ε into brain 4 h after intravenous injection, either before ischemia or after ischemia. In all blots, β-actin was used as an internal loading control. Each lane represents an individual animal. The blots are representative of two independent experiments with similar results.</p

TAT-14-3-3ε reduces ischemia-induced neuronal cell apoptosis and caspase-3 activation.

<p>Rats were subjected to MCAO for 2-3-3ε, TAT-14-3-3ε 2 h before ischemia or TAT-14-3-3ε at the end of ischemia, respectively. (A) Representative photographs shows TUNEL staining cells in the striatum of rats. (B) Quantification of the densities of TUNEL-positive cells shown in <i>A</i> (n = 3 per group). Note that TAT-14-3-3ε significantly reduces the density. (C) Colorimetric detection of caspase-3 inhibition by TAT-14-3-3ε. Values given are mean ± SD (n = 4 per group). ##<i>p</i><0.01 compared with sham group. *<i>p</i><0.05 and **<i>p</i><0.01 compared with vehicle and 14-3-3ε treated group. §<i>p</i><0.05 compared with TAT-14-3-3ε pre-treated group. Scale bar  = 50 μm.</p

Rapamycin pretreatment attenuated the neuroprotective effects of TAT-14-3-3ε.

<p>Rats were treated with a single i.c.v. injection of saline or 600-MA at the onset of reperfusion, or RAP (35 pmol, i.c.v.) combined with TAT-14-3-3ε (10 mg/kg, i.v.) 2 h before ischemia, and then followed by 24 h reperfusion. (A) Representative TTC staining from rat brains in the sham, vehicle, 3-MA and RAP + TAT-14-3-3ε groups. (B) Quantitative analysis of brain infarct volume from each group. Note that 3-MA decreases the infarct volume, and RAP weakens the neuroprotective effects of TAT-14-3-3ε. Bar represents mean ± SD, ##<i>p</i><0.01 compared with sham group. *<i>p</i><0.05 compared with vehicle group. §<i>p</i><0.05 compared with RAP + TAT-14-3-3ε group.</p

Protection of TAT-14-3-3ε on infarct volumes and neurological deficit scores of rats subjected to 2 h MCAO followed by 24 h reperfusion.

<p>(A) Representative TTC-stained brain coronal sections of rats treated with vehicle, 14-3-3ε, TAT-14-3-3ε 2 h before ischemia and TAT-14-3-3ε at the end of ischemia. Cerebral infarct volumes (B) and neurological deficit scores (C) of rats treated with vehicle, 14-3-3ε, TAT-14-3-3ε 2 h before ischemia or at the end of ischemia. Note that TAT-14-3-3ε treatment significantly reduced infarct volume and ameliorated neurological performance. ##<i>p</i><0.01 compared with sham group. *<i>p</i><0.05 and **<i>p</i><0.01 compared with vehicle and 14-3-3ε treated group. §<i>p</i><0.05 compared with TAT-14-3-3ε (10 and 20 mg/kg) pre-treated group.</p

Generation of the fusion proteins TAT-14-3-3ε and 14-3-3ε.

<p>(A) The strategy for the synthesis and assembly of the <i>14-3-3ε</i> gene using two step PCR. (B) Diagram of TAT-14-3-3ε and control 14-3-3ε fusion proteins. (C) Expression and purification of TAT-14-3-3ε and 14-3-3ε proteins from bacteria. <i>E. coli</i> BL21(DE3) cells with TAT-14-3-3ε or 14-3-3ε plasmid were induced as described in Materials and Methods. Protein samples were separated by SDS/PAGE and the protein bands are shown after Coomassie blue staining. Lane M, prestained protein markers; Lane 1, total cell proteins before induction; Lane 2-4, whole cell lysate, inclusion body and cellular lysate supernatant of induced BL21 (DE3)/pET-14-3-3ε induced at 28°C, respectively; Lane 5, Purified His-tagged 14-3-3ε proteins. Lane 6-8, whole cell lysate, inclusion body and cellular lysate supernatant of induced BL21 (DE3)/pET-TAT-14-3-3ε induced at 28°C, respectively; Lane 9, Purified His-tagged TAT-14-3-3ε proteins. (D) Western blot analysis of purified His-tagged 14-3-3ε and TAT-14-3-3ε with anti-His-tag antibody and anti-14-3-3ε antibody.</p

Inhibition of autophagy contributes to the neuroprotective effects of TAT-14-3-3ε.

<p>Rats were treated with an i.c.v of saline or 3-MA (600 nmol) at the end of 2h ischemia, or RAP (35 pmol, i.c.v.) combined with TAT-14-3-3ε (10 mg/kg, i.v.) 2 h before 2 h MCAO. Rats were then subjected to 24 h reperfusion, after which all animals were sacrificed. (A) Western blot analysis of the LC3B, p62 and Beclin-1 protein expression in the ischemic cerebral hemisphere. (B, C and D) Quantitation of LC3B-II, p62 and Beclin-1 expression, respectively, normalized to the loading control (β-actin). Bar represents mean ± SD (n = 4 per group). ##<i>p</i><0.01 compared with sham group. *<i>p</i><0.05 compared with vehicle group. §<i>p</i><0.05 compared with RAP + TAT-14-3-3ε group.</p

TAT-14-3-3ε inhibits I/R-induced autophagy in brain.

<p>(A) Representative electron microphotographs showing autophagosomes in the ischemic penumbra of cerebral cortex of sham, vehicle or TAT-14-3-3ε pre-treated animals 24 h after reperfusion. Autophagosomes are indicated by arrows. Scale bar  = 1 μm. (B) Western blot analysis for the expression of LC3B, p62 and Beclin-1 in cerebral hemisphere. (C, D and E) Quantitation of LC3B-II, p62 and Beclin-1 expression, respectively, normalized to β-actin. The data are presented as the mean ± SD (n = 5 per group). #<i>p</i><0.05 and ##<i>p</i><0.01 versus sham group. *<i>p</i><0.05 versus vehicle group.</p