Po-Yen Lin

DNA and RNA topoisomerase activities of Top3β are promoted by mediator protein Tudor domain-containing protein 3

Significance Tudor domain-containing protein 3 (TDRD3), a multidomain scaffold protein functions as an epigenetic reader on nuclear chromatin and binds with fragile X mental retardation protein on mRNA. It forms a conserved complex with topoisomerase 3β (Top3β), a type IA topoisomerase, and participates in both transcription and translation. The mechanism of how TDRD3 acts as Top3β’s partner in regulating these cellular processes is unknown. Here, we demonstrated that TDRD3 is able to stimulate Top3β’s DNA and RNA topoisomerase catalytic activities, through binding and stabilizing single-stranded regions in DNA and RNA substrates. Because these regions are the preferred site for Top3β, TDRD3 therefore acts as a regulator to provide access of the enzyme to these nucleic acid substrates and to act upon them. Topoisomerase 3β (Top3β) can associate with the mediator protein Tudor domain-containing protein 3 (TDRD3) to participate in two gene expression processes of transcription and translation. Despite the apparent importance of TDRD3 in binding with Top3β and directing it to cellular compartments critical for gene expression, the biochemical mechanism of how TDRD3 can affect the functions of Top3β is not known. We report here sensitive biochemical assays for the activities of Top3β on DNA and RNA substrates in resolving topological entanglements and for the analysis of TDRD3 functions. TDRD3 stimulates the relaxation activity of Top3β on hypernegatively supercoiled DNA and changes the reaction from a distributive to a processive mode. Both supercoil retention assays and binding measurement by fluorescence anisotropy reveal a heretofore unknown preference for binding single-stranded nucleic acids over duplex. Whereas TDRD3 has a structure-specific binding preference, it does not discriminate between DNA and RNA. This unique property for binding with nucleic acids can have an important function in serving as a hub to form nucleoprotein complexes on DNA and RNA. To gain insight into the roles of Top3β on RNA metabolism, we designed an assay by annealing two single-stranded RNA circles with complementary sequences. Top3β is capable of converting two such single-stranded RNA circles into a double-stranded RNA circle, and this strand-annealing activity is enhanced by TDRD3. These results demonstrate that TDRD3 can enhance the biochemical activities of Top3β on both DNA and RNA substrates, in addition to its function of targeting Top3β to critical sites in subcellular compartments.

Long working distance fluorescence lifetime imaging with stimulated emission and electronic time delay

In this work, long working distance fluorescence lifetime imaging is realized with stimulated emission in combination with electronic time delay control. Spatial coherence, as a result of stimulated emission, supports unattenuated fluorescence detection at extended distance, using low NA optics. An electronic time delayed trigger provides an advantageous way in adjusting the pulse separation and probing the fluorescence lifetime in the nanosecond ranges. The fluorescence lifetime of selected fluorophores is accurately determined through the pump-probe configuration. The characteristics and applications in fluorescence lifetime measurement of stimulated emission are investigated and summarized succinctly here.

Fluorescence Lifetime Imaging Microscopy with Subdiffraction-Limited Resolution

In this study, we demonstrate subdiffraction-limited fluorescence lifetime imaging microscopy (FLIM) by engineering the point spread function (PSF) with stimulated emission depletion (STED). The enhanced spatial resolution allows the number of fluorophores in the PSF to reduce in turn the associated heterogeneity in lifetime analysis. Moreover, time gating can be performed using time-correlated single photon counting (TCSPC) to carefully select detected fluorescence photons so as to optimize the spatial resolution and the signal-to-noise ratio in STED imaging. This flexibility also supports the removal of the unintended effects of lifetime reduction that is caused by STED pulses.

A new saw-like self-recovery of interface states in nitride-based memory cell

A new saw-like self-recovery Self-Aligned Nitride (SAN) memory cell is proposed and fabricated in 28nm high-k metal gate (HKMG) CMOS process for high-density logic NVM applications. The cell is operated with Source-Side Injection (SSI) for programming and band-to-band hot holes (BBHH) for erasing. Two effective self-heating recovery mechanisms are proposed and performed to maintain a stable On/Off read window after cycling stresses. Besides, the characteristic and reliability comparison of the SAN cell in other technology nodes, 90nm/45nm/32nm, are characterized to further verify the saw-like self-detrapping and self-recovery operation. The new 28nm HKMG SAN memory cell with the self-detrapping recovery results excellent and superior endurance performance and can provide a very promising solution for logic NVM in advanced technologies.

FOLDING AND STRUCTURAL CHARACTERIZATION OF RECOMBINANT CYCLIN-DEPENDENT KINASE INHIBITOR p21(Cip1, Waf1, Sdi1)

p21Cip1, Waf1, Sdi1 (p21) is a member of the cyclin-dependent kinase (CDK) family of inhibitors in eukaryotes. We report the refolding of an inclusion body of a recombinant p21 (rp21) to its native form, under an alkaline to neutral environment, via an over-critical process describable by a first-order state transition model. The secondary structure of the refolded rp21 possesses a helical-major structure as determined by circular dichroism (CD) analysis, and its diameter is around 3 nm, as measured by dynamic light scattering studies (DLS) and atomic force microscopy (AFM). The differential scanning calorimeter (DSC) measurement indicates that the folded rp21 possesses unique but weak tertiary interactions. That the function of rp21 is reinstated upon refolding under our experimental conditions is evidenced by its binding to proliferating cell nuclear antigen (PCNA) in an immuno-co-precitptation analysis. The conformational changes of the folding intermediates of rp21 are consistent with the framework of a sequential model proposed earlier. The lack of a definitive structure of p21 in acidic condition will be discussed.

Correction to A Mesoporous Silica Biomaterial for Dental Biomimetic Crystallization

Supplementary Figure S8. Dental pulp cell morphology response to (a) CCMS, (b) CCMS-HP through TDD model. ALP staining in (c) DMEM control group, (d) CCMS-HP of TDD model. Pulp cells appeared to have normal morphology when immersion in the elution of CCMS-HP in the TDD model. CCMS-HP group demonstrated blue staining of dental pulp cells under light microscope observations. The result showed that elution of CCMS-HP through a dentin disc did not reduce the activity of ALPase.

Effect of three-dimensional current distribution on characterizing parasitic resistance of FinFETs

A thorough analysis of the FinFET resistance and current distribution is presented. By combining multiple conventional and novel measurement techniques, the key components that contribute to the parasitic resistance of FinFETs can be quantified. Through a three-dimensional (3D) FinFET resistance network model, the impact of the FinFET 3D structure on the current distribution and parasitic resistance is analyzed. The heterogeneous current distribution, known as the current crowding effect, is also discussed with possible alleviation methods through 3D simulation.

Oral Nonviral Gene Delivery for Chronic Protein Replacement Therapy

Abstract Efficient nonviral oral gene delivery offers an attractive modality for chronic protein replacement therapy. Herein, the oral delivery of insulin gene is reported by a nonviral vector comprising a copolymer with a high degree of substitution of branched polyethylenimine on chitosan (CS‐g‐bPEI). Protecting the plasmid from gastric acidic degradation and facilitating transport across the gut epithelium, the CS‐g‐bPEI/insulin plasmid DNA nanoparticles (NPs) can achieve systemic transgene expression for days. A single dose of orally administered NPs (600 µg plasmid insulin (pINS)) to diabetic mice can protect the animals from hyperglycemia for more than 10 d. Three repeated administrations spaced over a 10 d interval produce similar glucose‐lowering results with no hepatotoxicity detected. Positron‐emission‐tomography and computed‐tomography images also confirm the glucose utilization by muscle cells. While this work suggests the feasibility of basal therapy for diabetes mellitus, its significance lies in the demonstration of a nonviral oral gene delivery system that can impact chronic protein replacement therapy and DNA vaccination.

Circulating current reduction for a D-Σ digital controlled transformerless UPS

This paper presents a three-phase transformerless uninterruptible power supply (UPS) with sinusoidal pulse width modulation (SPWM) based division-summation (D-Σ) digital control. A transformerless UPS controls the power flow between dc link and utility grid, as well as tracks the ac reference voltage. The proposed control law derived with D-Σ digital approach takes into account the effects of dc-link voltage fluctuation, grid-voltage distortion and inductance variation due to different current levels. Thus, distortion of input current and filter inductor core size can be reduced significantly. However, circulating current may flow through the common ground between the input power factor corrector (PFC) and the output three-phase four-wire inverter. The derived control law based on SPWM can suppress this circulating current and regulate output voltages tightly. Experimental results measured from a three-phase transformerless UPS have confirmed the analysis and discussion of the proposed control approach.

Improvement of control-law derivation for D-Σ digital controlled three-phase four-wire inverter

A division-summation (D-Σ) digital control can track sinusoidal reference current in four operation modes: grid-connection, rectification, PF leading and PF lagging mode. It can overcome the limitation of abc to α-β-γ frame transformation and cover wide filter inductance variation. However, the switching sequences for the four modes are different from each other, increasing complexity of firmware programming. In this paper, improvement of control-law derivation for D-Σ digital controlled three-phase four-wire inverter is presented. By selecting the zero crossing points of phase voltages as region transitions, the control laws and the related parameter tables for the four modes can be unified to a general form. The three-phase four-wire inverter topology can be also adopted as an uninterruptible power supply (UPS) application and it can supply unbalanced, linear and rectified loads. With the proposed load impendence estimation, the D-Σ digital control laws can be applied for voltage tracking. The switching sequences of the four modes and UPS mode are all unified to the switching pattern of the grid-connection mode. Additionally, a D-Σ transformation matrix is identified to simplify the derivation procedure of the division (D) and summation (Σ), which can obtain the control law directly. Experimental results measured from a 10 kVA inverter have verified the analysis and discussion.