Protein-Protein Affinity Determination by Quantitative FRET Quenching.

The molecular dissociation constant, Kd, is a well-established parameter to quantitate the affinity of protein-protein or other molecular interactions. Recently, we reported the theoretical basis and experimental procedure for Kd determination using a quantitative FRET method. Here we report a new development of Kd determination by measuring the reduction in donor fluorescence due to acceptor quenching in FRET. A new method of Kd determination was developed from the quantitative measurement of donor fluorescence quenching. The estimated Kd values of SUMO1-Ubc9 interaction based on this method are in good agreement with those determined by other technologies, including FRET acceptor emission. Thus, the acceptor-quenched approach can be used as a complement to the previously developed acceptor excitation method. The new methodology has more general applications regardless whether the acceptor is an excitable fluorophore or a quencher. Thus, these developments provide a complete methodology for protein or other molecule interaction affinity determinations in solution

Highly conserved evolution of aquaporin PIPs and TIPs confers their crucial contribution to flowering process in plants

Flowering is the key process for the sexual reproduction in seed plants. In gramineous crops, the process of flowering, which includes the actions of both glume opening and glume closing, is directly driven by the swelling and withering of lodicules due to the water flow into and out of lodicule cells. All these processes are considered to be controlled by aquaporins, which are the essential transmembrane proteins that facilitate the transport of water and other small molecules across the biological membranes. In the present study, the evolution of aquaporins and their contribution to flowering process in plants were investigated via an integration of genome-wide analysis and gene expression profiling. Across the barley genome, we found that HvTIP1;1, HvTIP1;2, HvTIP2;3, and HvPIP2;1 were the predominant aquaporin genes in lodicules and significantly upregulated in responding to glume opening and closing, suggesting the importance of them in the flowering process of barley. Likewise, the putative homologs of the above four aquaporin genes were also abundantly expressed in lodicules of the other monocots like rice and maize and in petals of eudicots like cotton, tobacco, and tomato. Furthermore, all of them were mostly upregulated in responding to the process of floret opening, indicating a conserved function of these aquaporin proteins in plant flowering. The phylogenetic analysis based on the OneKP database revealed that the homologs of TIP1;1, TIP1;2, TIP2;3, and PIP2;1 were highly conserved during the evolution, especially in the angiosperm species, in line with their conserved function in controlling the flowering process. Taken together, it could be concluded that the highly evolutionary conservation of TIP1;1, TIP1;2, TIP2;3 and PIP2;1 plays important roles in the flowering process for both monocots and eudicots

Typestate-Guided Fuzzer for Discovering Use-after-Free Vulnerabilities

© 2020 Association for Computing Machinery. Existing coverage-based fuzzers usually use the individual control flow graph (CFG) edge coverage to guide the fuzzing process, which has shown great potential in finding vulnerabilities. However, CFG edge coverage is not effective in discovering vulnerabilities such as use-after-free (UaF). This is because, to trigger UaF vulnerabilities, one needs not only to cover individual edges, but also to traverse some (long) sequence of edges in a particular order, which is challenging for existing fuzzers. To this end, we propose to model UaF vulnerabilities as typestate properties, and develop a typestateguided fuzzer, named UAFL, for discovering vulnerabilities violating typestate properties. Given a typestate property, we first perform a static typestate analysis to find operation sequences potentially violating the property. Our fuzzing process is then guided by the operation sequences in order to progressively generate test cases triggering property violations. In addition, we also employ an information flow analysis to improve the efficiency of the fuzzing process. We have performed a thorough evaluation of UAFL on 14 widely-used real-world programs. The experiment results show that UAFL substantially outperforms the state-of-the-art fuzzers, including AFL, AFLFast, FairFuzz, MOpt, Angora and QSYM, in terms of the time taken to discover vulnerabilities. We have discovered 10 previously unknown vulnerabilities, and received 5 new CVEs

Platinum composition dependence of spin-orbit torque in (Fe0.8Mn0.2)1−xPtx single-layer ferromagnet

We have investigated the effect of the Pt composition on the spin–orbit torque in a (Fe0.8Mn0.2)1xPtx single-layer ferromagnet. We observed that while the field-like torque decreases and even reverses sign with increasing the Pt composition, the damping-like torque increases monotonically and reaches 0.99 Oe=ð1010 A=m2Þ in a single-layer (Fe0.8Mn0.2)0.52Pt0.48 film. The results corroborate the anomalous Hall effect and surface spin rotation model presented previously, and the relative ratio between the damping-like and field-like torques can be qualitatively understood as the relative phase change in spin-conserving and spin-flip scattering

Photoacoustic quantification of the optical absorption cross-sections of gold nanostructures

This study demonstrates a method for measuring the optical absorption cross-sections (σ_a) of Au-Ag nanocages and Au nanorods using photoacoustic (PA) sensing. PA signals are directly proportional to the absorption coefficient (μ_a) of the nanostructure. For each type of nanostructure, we first obtained μa from the PA signal by benchmarking against a linear calibration curve (PA signal vs. μ_a) derived from a set of methylene blue solutions with different concentrations. We then calculated σ_a by dividing the μ_a by the corresponding concentration of the Au nanostructure. Additionally, we obtained the extinction cross-section (σ_e, sum of absorption and scattering cross-sections) from the extinction spectrum recorded using a conventional UV-vis-NIR spectrometer. From the measurements of σ_a and σ_e, we were able to easily derive both the absorption and scattering cross-sections for each type of gold nanostructure. This method can potentially provide the optical absorption and scattering properties of gold nanostructures and other types of nanomaterials

Gold nanocages covered with thermally-responsive polymers for controlled release by high-intensity focused ultrasound

This paper describes the use of Au nanocages covered with smart, thermally-responsive polymers for controlled release with high-intensity focused ultrasound (HIFU). HIFU is a highly precise medical procedure that uses focused ultrasound to heat and destroy pathogenic tissue rapidly and locally in a non-invasive or minimally invasive manner. The released dosage could be remotely controlled by manipulating the power of HIFU and/or the duration of exposure. We demonstrated localized release within the focal volume of HIFU by using gelatin phantom samples containing dye-loaded Au nanocages. By placing chicken breast tissues on top of the phantoms, we further demonstrated the feasibility of this system for controlled release at depths up to 30 mm. Because it can penetrate more deeply into soft tissues than near-infrared light, HIFU is a potentially more effective external stimulus for rapid, on-demand drug release

Measuring the Optical Absorption Cross Sections of Au−Ag Nanocages and Au Nanorods by Photoacoustic Imaging

This paper presents a method for measuring the optical absorption cross sections (σ_a) of Au−Ag nanocages and Au nanorods. The method is based on photoacoustic (PA) imaging, where the detected signal is directly proportional to the absorption coefficient (μ_a) of the nanostructure. For each type of nanostructure, we first obtained μ_a from the PA signal by benchmarking against a linear calibration curve (PA signal versus μ_a) derived from a set of methylene blue solutions with different concentrations. We then calculated σ_a by dividing the μ_a by the corresponding concentration of the Au nanostructure. Additionally, we obtained the extinction cross section (σ_e, sum of absorption and scattering) from the extinction spectrum recorded using a conventional UV−vis−NIR spectrometer. From the measurements of σ_a and σ_e, we were able to easily derive both the absorption and scattering cross sections for each type of gold nanostructure. The ratios of absorption to extinction obtained from experimental and theoretical approaches agreed well, demonstrating the potential use of this method in determining the optical absorption and scattering properties of gold nanostructures and other types of nanomaterials

Prion protein stabilizes amyloid-β (Aβ) oligomers and enhances Aβ neurotoxicity in a Drosophila model of Alzheimer's disease

The cellular prion protein (PrPC) can act as a cell-surface receptor for β-amyloid (Aβ) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aβ isoforms and PrPC in the Drosophila brain. We found that co-expression of Aβ and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Aβ deposition in the fly brain. In contrast, under the same conditions, expression of Aβ or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Aβ as oligomeric assemblies and fragment-preformed Aβ fibers. The ability of membrane-anchored PrPC to trap Aβ as cytotoxic oligomers at the membrane surface and fragment inert Aβ fibers suggests a mechanism by which PrPC exacerbates Aβ deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Aβ toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Aβ and PrPC represents a potential therapeutic strategy

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics.

We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 microL of crude lysate in under 30 min for the entire process