Water permeation drives tumor cell migration in confined microenvironments.

Cell migration is a critical process for diverse (patho)physiological phenomena. Intriguingly, cell migration through physically confined spaces can persist even when typical hallmarks of 2D planar migration, such as actin polymerization and myosin II-mediated contractility, are inhibited. Here, we present an integrated experimental and theoretical approach ("Osmotic Engine Model") and demonstrate that directed water permeation is a major mechanism of cell migration in confined microenvironments. Using microfluidic and imaging techniques along with mathematical modeling, we show that tumor cells confined in a narrow channel establish a polarized distribution of Na+/H+ pumps and aquaporins in the cell membrane, which creates a net inflow of water and ions at the cell leading edge and a net outflow of water and ions at the trailing edge, leading to net cell displacement. Collectively, this study presents an alternate mechanism of cell migration in confinement that depends on cell-volume regulation via water permeation.

Poly(ADP-ribosyl)ation mediates early phase histone eviction at DNA lesions.

Nucleosomal histones are barriers to the DNA repair process particularly at DNA double-strand breaks (DSBs). However, the molecular mechanism by which these histone barriers are removed from the sites of DNA damage remains elusive. Here, we have generated a single specific inducible DSB in the cells and systematically examined the histone removal process at the DNA lesion. We found that histone removal occurred immediately following DNA damage and could extend up to a range of few kilobases from the lesion. To examine the molecular mechanism underlying DNA damage-induced histone removal, we screened histone modifications and found that histone ADP-ribosylation was associated with histone removal at DNA lesions. PARP inhibitor treatment suppressed the immediate histone eviction at DNA lesions. Moreover, we examined histone chaperones and found that the FACT complex recognized ADP-ribosylated histones and mediated the removal of histones in response to DNA damage. Taken together, our results reveal a pathway that regulates early histone barrier removal at DNA lesions. It may also explain the mechanism by which PARP inhibitor regulates early DNA damage repair.

AI26 inhibits the ADP-ribosylhydrolase ARH3 and suppresses DNA damage repair.

The ADP-ribosylhydrolase ARH3 plays a key role in DNA damage repair, digesting poly(ADP-ribose) and removing ADP-ribose from serine residues of the substrates. Specific inhibitors that selectively target ARH3 would be a useful tool to examine DNA damage repair, as well as a possible strategy for tumor suppression. However, efforts to date have not identified any suitable compounds. Here, we used in silico and biochemistry screening to search for ARH3 inhibitors. We discovered a small molecule compound named ARH3 inhibitor 26 (AI26) as, to our knowledge, the first ARH3 inhibitor. AI26 binds to the catalytic pocket of ARH3 and inhibits the enzymatic activity of ARH3 with an estimated IC50 of ∼2.41 µm in vitro Moreover, hydrolysis of DNA damage-induced ADP-ribosylation was clearly inhibited when cells were pretreated with AI26, leading to defects in DNA damage repair. In addition, tumor cells with DNA damage repair defects were hypersensitive to AI26 treatment, as well as combinations of AI26 and other DNA-damaging agents such as camptothecin and doxorubicin. Collectively, these results reveal not only a chemical probe to study ARH3-mediated DNA damage repair but also a chemotherapeutic strategy for tumor suppression.

Fabrication and characterization of well-ordered PbS nanowires in aluminum oxide template by sulfurization and vacuum injection molding processes.

Lead (Pb) nanowire arrays were fabricated with anodic aluminum oxide (AAO) templates of 30, 100 and 300 nm in pore diameters. Through vacuum injection molding process, Pb/AAO composite was obtained, and lead sulfide (PbS) could further be synthesized after exposing to sulfur gas. AAO templates with different pore sizes were fabricated by using pure aluminum in a two-step anodization. Three types of solutions, which are 10 vol% sulfuric acid, 3 wt% oxalic acid and 1 vol% phosphoric acid, were adopted to achieve AAO of various pore sizes. Different sulfurization temperatures and time spans were applied for studying on the formation mechanism of PbS. Finally, the morphology, composition, structure and elements distribution of the as-prepared Pb and PbS nanowires were confirmed through the use of scanning electron microscopy, energy dispersive x-ray spectroscopy, element-mapping, x-ray diffraction and transmission electron microscopy analysis. The results indicated that Pb nanowires were successfully obtained after applying vacuum injection molding process with 50 kgf cm-2hydraulic pressure, and PbS nano arrays can be formed by sulfurization at 500 °C for 5 h. Furthermore, an optical property, ultraviolet-visible (UV-Vis) absorption, was also measured. The measurement of the PbS nanowires showed that a significant quantum confinement effect made the energy gap produce a blue shift from 0.41 eV to 1.65 eV or 1.72 eV.

Human DNA ligase IV is able to use NAD+ as an alternative adenylation donor for DNA ends ligation.

All the eukaryotic DNA ligases are known to use adenosine triphosphate (ATP) for DNA ligation. Here, we report that human DNA ligase IV, a key enzyme in DNA double-strand break (DSB) repair, is able to use NAD+ as a substrate for double-stranded DNA ligation. In the in vitro ligation assays, we show that the recombinant Ligase IV can use both ATP and NAD+ for DNA ligation. For NAD+-mediated ligation, the BRCA1 C-terminal (BRCT) domain of Ligase IV recognizes NAD+ and facilitates the adenylation of Ligase IV, the first step of ligation. Although XRCC4, the functional partner of Ligase IV, is not required for the NAD+-mediated adenylation, it regulates the transfer of AMP moiety from Ligase IV to the DNA end. Moreover, cancer-associated mutation in the BRCT domain of Ligase IV disrupts the interaction with NAD+, thus abolishes the NAD+-mediated adenylation of Ligase IV and DSB ligation. Disrupting the NAD+ recognition site in the BRCT domain impairs non-homologous end joining (NHEJ) in cell. Taken together, our study reveals that in addition to ATP, Ligase IV may use NAD+ as an alternative adenylation donor for NHEJ repair and maintaining genomic stability.

Super-resolution imaging identifies PARP1 and the Ku complex acting as DNA double-strand break sensors.

DNA double-strand breaks (DSBs) are fatal DNA lesions and activate a rapid DNA damage response. However, the earliest stage of DSB sensing remains elusive. Here, we report that PARP1 and the Ku70/80 complex localize to DNA lesions considerably earlier than other DSB sensors. Using super-resolved fluorescent particle tracking, we further examine the relocation kinetics of PARP1 and the Ku70/80 complex to a single DSB, and find that PARP1 and the Ku70/80 complex are recruited to the DSB almost at the same time. Notably, only the Ku70/80 complex occupies the DSB exclusively in the G1 phase; whereas PARP1 competes with the Ku70/80 complex at the DSB in the S/G2 phase. Moreover, in the S/G2 phase, PARP1 removes the Ku70/80 complex through its enzymatic activity, which is further confirmed by in vitro DSB-binding assays. Taken together, our results reveal PARP1 and the Ku70/80 complex as critical DSB sensors, and suggest that PARP1 may function as an important regulator of the Ku70/80 complex at the DSBs in the S/G2 phase.

The fabrication and thermal properties of bismuth-aluminum oxide nanothermometers.

Bismuth (Bi) nanowires, well controlled in length and diameter, were prepared by using an anodic aluminum oxide (AAO) template-assisted molding injection process with a high cooling rate. A high performance atomic layer deposition (ALD)-capped bismuth-aluminum oxide (Bi-Al2O3) nanothermometer is demonstrated that was fabricated via a facile, low-cost and low-temperature method, including AAO templated-assisted molding injection and low-temperature ALD-capped processes. The thermal behaviors of Bi nanowires and Bi-Al2O3 nanocables were studied by in situ heating transmission electron microscopy. Linear thermal expansion of liquid Bi within native bismuth oxide nanotubes and ALD-capped Bi-Al2O3 nanocables were evaluated from 275 °C to 700 °C and 300 °C to 1000 °C, respectively. The results showed that the ALD-capped Bi-Al2O3 nanocable possesses the highest working temperature, 1000 °C, and the broadest operation window, 300 °C-1000 °C, of a thermal-expanding type nanothermometer. Our innovative approach provides another way of fabricating core-shell nanocables and to further achieve sensing local temperature under an extreme high vacuum environment.

Fabrication of device with poly(N-isopropylacrylamide)-b-ssDNA copolymer brush for resistivity study.

In this study, we grafted bromo-terminated poly(N-isopropylacrylamide) (PNIPAAm) brushes onto thin gold films deposited on silicon, and then reacted with NaN3 to produce azido-terminated PNIPAAm brushes. A probe sequence of single-stranded DNA (ssDNA) with a 4-pentynoic acid succinimidyl ester unit was grafted onto the azido-terminated PNIPAAm brushes through a click reaction, resulting in the formation of block copolymer brushes. The PNIPAAm-b-ssDNA copolymer brushes formed supramolecular complexes stabilized by bio-multiple hydrogen bonds (BMHBs), which enhanced the proton transfer and thereby decreased the resistivity of the structures. In addition, the optimal operation window for DNA detection ranges from 0 to 0.2 M of NaCl concentration. Therefore, the specimens were prepared in the PBS solution at 150 mM NaCl concentration for target hybridization. The supramolecular complex state of the PNIPAAm-b-ssDNA copolymer brushes transformed into the phase-separated state after the hybridization with 0.5 ng/µL of its target DNA sequence owing to the competition between BMHBs and complementary hydrogen bonds. This phase transformation of the PNIPAAm and probe segments inhibited the proton transfer and significantly increased the resistivity at 25 °C. Moreover, there were no significant changes in the resistivity of the copolymer brushes after hybridization with the target sequence at 45 °C. These results indicated that the phase-separated state of the PNIPAAm-b-ssDNA copolymer brushes, which was generally occurred above the LCST, can be substantially generated after hybridization with its target DNA sequence. By performing the controlled experiments, in the same manner, using another sequence with lengths similar to that of the target sequence without complementarity. In addition, the sequences featuring various degrees of complementarity were exploited to verify the phase separation behavior inside the PNIPAAm-b-ssDNA copolymer thin film.

The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response.

Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response.

New Flexible Silicone-Based EEG Dry Sensor Material Compositions Exhibiting Improvements in Lifespan, Conductivity, and Reliability.

This study investigates alternative material compositions for flexible silicone-based dry electroencephalography (EEG) electrodes to improve the performance lifespan while maintaining high-fidelity transmission of EEG signals. Electrode materials were fabricated with varying concentrations of silver-coated silica and silver flakes to evaluate their electrical, mechanical, and EEG transmission performance. Scanning electron microscope (SEM) analysis of the initial electrode development identified some weak points in the sensors' construction, including particle pull-out and ablation of the silver coating on the silica filler. The newly-developed sensor materials achieved significant improvement in EEG measurements while maintaining the advantages of previous silicone-based electrodes, including flexibility and non-toxicity. The experimental results indicated that the proposed electrodes maintained suitable performance even after exposure to temperature fluctuations, 85% relative humidity, and enhanced corrosion conditions demonstrating improvements in the environmental stability. Fabricated flat (forehead) and acicular (hairy sites) electrodes composed of the optimum identified formulation exhibited low impedance and reliable EEG measurement; some initial human experiments demonstrate the feasibility of using these silicone-based electrodes for typical lab data collection applications.