Dna2 removes toxic ssDNA-RPA filaments generated from meiotic recombination-associated DNA synthesis.

During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strands can displace the parental strand to generate single-strand DNAs (ssDNAs). Many programmed DSBs and thus many ssDNAs occur during meiosis. However, it is unclear how these ssDNAs are removed for the complete repair of meiotic DSBs. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) results in an abundant accumulation of RPA and an expansion of RPA from DSBs to broader regions in Saccharomyces cerevisiae. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, Dna2 induction at pachytene is highly effective in removing accumulated RPA and restoring spore viability. Moreover, the depletion of Pif1, an activator of polymerase δ required for meiotic recombination-associated DNA synthesis, and Pif1 inhibitor Mlh2 decreases and increases RPA accumulation in dna2-md, respectively. In addition, blocking DNA synthesis during meiotic recombination dramatically decreases RPA accumulation in dna2-md. Together, our findings show that meiotic DSB repair requires Dna2 to remove ssDNA-RPA filaments generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.

Evaluation of the dual-frequency transducer for controlling thermal ablation morphology using frequency shift keying signal.

PURPOSE: The catheter-based ultrasound (CBUS) can reach the target tissue directly and achieve rapid treatment. The frequency shift keying (FSK) signal is proposed to regulate and evaluate tumor ablation by a miniaturized dual-frequency transducer. METHODS: A dual-frequency transducer prototype (3 × 7 × 0.4 mm) was designed and fabricated for the CBUS applicator (OD: 3.8 mm) based on the fundamental frequency of 5.21 MHz and the third harmonic frequency of 16.88 MHz. Then, the acoustic fields and temperature field distributions using the FSK signals (with 0, 25, 50, 75, and 100% third harmonic frequency duty ratios) were simulated by finite element analysis. Finally, tissue ablation and temperature monitoring were performed in phantom and ex vivo tissue, respectively. RESULTS: At the same input electrical power (20 W), the output acoustic power of the fundamental frequency of the transducer was 10.03 W (electroacoustic efficiencies: 50.1%), and that of the third harmonic frequency was 6.19 W (30.6%). As the third harmonic frequency duty ratios increased, the shape of thermal lesions varied from strip to droplet in simulated and phantom experimental results. The same trend was observed in ex vivo tests. CONCLUSION: Dual-frequency transducers excited by the FSK signal can control the morphology of lesions. SIGNIFICANCE: The acoustic power deposition of CBUS was optimized to achieve precise ablation.

Engineering cell de-adhesion dynamics on thermoresponsive poly(N-isopropylacrylamide).

Poly(N-isopropylacrylamide) (PIPAAm) has been demonstrated as an effective thermoresponsive polymer for non-invasive cell regeneration/recovery. However, little is known about the intricate relationship between the biophysical response of cells and physiochemical properties of PIPAAm during cell recovery. In this study, the de-adhesion kinetics of smooth muscle cell (SMC) on PIPAAm surfaces is probed with unique biophysical techniques. Water-immersion atomic force microscope (AFM) first showed that the nanotopology of PIPAAm surfaces is dependent on the polymerization time and collagen coating. It is found that the initial rate of cell de-adhesion increases with the increase in polymerization time. Moreover, the degree of cell deformation (a/R) and average adhesion energy are reduced with the increase of grafted PIPAAm density during 40min of cell de-adhesion. It has been shown that collagen coating regulates cell adhesion on biomaterial surface. Interestingly, lower collagen density on PIPAAm leads to higher adhesion energy per cell during the initial 20min compared with as-prepared PIPAAm, while the initial rate of cell de-adhesion remains unchanged. In contrast, higher collagen density leads to 50% reduction in the initial rate of cell de-adhesion and higher adhesion energy per cell during the entire 90min. Furthermore, immunostaining of actin provides supporting evidence that the de-adhesion kinetics is correlated with the cytoskeleton transformation during cell de-adhesion below the lower solution critical temperature (LCST).

Dynamics of smooth muscle cell deadhesion from thermosensitive hydroxybutyl chitosan.

Thermoresponsive polymer (TRP) enables the enzyme-free harvesting of cells through an acute increase in surface hydrophilicity of TRP across its lower critical solution temperature (LCST), rendering feasible the generation of polymer-free cell sheets for regenerative medicine applications. To date, the intricate mechanisms of cell deadhesion/detachment on TRP surface remain obscure. Elucidation of such biophysical responses would be valuable for the cell sheet technology. In this study, integrative biophysical techniques are applied to probe the thermal-induced deadhesion kinetics of smooth muscle cell (SMC) on thermoresponsive hydroxybutyl chitosan (HBC29) against different periods of pre-culture time at 37 degrees C. Atomic force microscopy demonstrates that both the surface topography and mechanical property of HBC29 film in water are acutely modulated across its LCST. Firstly, cells show negligible changes in adhesion contact area during low-temperature incubation on unmodified tissue culture polystyrene (TCPS). Secondly, the recession of adhesion contact and retraction of cell body for cells with different pre-culture times are triggered by HBC29 coating on TCPS. Interestingly, the initial rate of reduction in the normalized adhesion contact area of SMC is negatively correlated with the pre-culture time. Thirdly, the degree of cell deformation and average adhesion energy are reducing functions of time only for SMCs with the lowest pre-culture time. In contrast, adhesion energy per cell is a reducing function of time irrespective of the change of pre-culture time. Lastly, the temporal dynamics of cytoskeleton organization and beta-actin/smoothelin-B mRNA expression for SMCs is strongly dependent on the pre-culture time. Overall, this study demonstrates that the thermal-induced deadhesion of SMC on TRP is characterized by the evolution of its contractile phenotypes.

Epigallocatechin-3-gallate induced modulation of cell deadhesion and migration on thermosensitive poly(N-isopropylacrylamide).

Epigallocatechin-3-gallate (EGCG), which is the main polyphenolic constituent of green tea, has emerged as a promising candidate for potential applications in selected anticancer therapeutics. Generally, tumor metastasis is known to be correlated with the alterations in cell adhesion and migration of normal cells. Nevertheless, the effect of EGCG on the biophysical responses of tumor cell adhering on extracellular matrix remains obscure. In this study, a thermosenstive poly(N-isopropylacrylamide) (PIPAAm) system was developed to elucidate the potential anti-tumor effect of EGCG on the deadhesion and migration of HepG2 cells. First, both XPS and ELISA validated the coating of laminin (LA) on PIPAAm. Second, a change of nanotopology of LA layer on PIPAAm across the lower solution critical temperature (LCST) was detected with AFM. HepG2 cells seeded on LA-coated PIPAAm surface was shown to go through deadhesion by lowering the temperature below the LCST. Interestingly, EGCG was shown to decelerate the thermally triggered deadhesion of HepG2 cell on LA coated PIPAAm. Moreover, the inhibition of cell deadhesion in EGCG treated cells was shown to be driven by actin remodeling. Interestingly, the modulation of cell deadhesion on LA coated PIPAAm by EGCG leads to the reduction of cell motility as shown by real-time cell migration assay. Overall, the use of PIPAAm system demonstrated the promise of EGCG as anticancer therapy through the suppression of cell deadhesion and migration.

Effect of cytoskeleton inhibitors on deadhesion kinetics of HepG2 cells on biomimetic surface.

Cytochalasin-D (Cyto-D) and latrunculin-A (Lat-A) are known inhibitors of actin microfilaments and adversely affect the physiological functions of anchorage-dependent cells. Alternatively, doxorubicin (Dox), a chemotherapeutic drug is known to induce apoptosis and cell detachment of tumor cells. However, the intricate interplay between drug administration, cytoskeletal rearrangement and biophysical responses of live cells on immobilized layer of extracellular matrix (ECM) protein remains unknown. In this study, the deadhesion kinetics and actin remodeling of live HepG2 cells following the addition of the three drugs are probed with confocal reflectance interference contrast microscopy (C-RICM) and fluorescence confocal microscopy. First, it is shown that the reduction in two-dimensional spread area of HepG2 cells is 10.5%, 15.4% and 21.9% under the influence of 5 microM of Lat-A, Cyto-D and Dox, respectively. Secondly, C-RICM demonstrates the recession of strong adhesion contact against time of cell seeding upon the addition of the three drugs. Thirdly, the initial cell detachment rate and extent of reduction in the degree of cell deformation (a/R) are dependent on both the drug types and concentration. Lastly, oscillation-like responses of a/R and adhesion energy are uniquely found in Lat-A induced cell detachment. Overall, our biophysical approaches have been proven as a highly quantitative platform for elucidating the interfacial properties of adherent cells on biomimetic surfaces under cytoskeleton disruption.