Memory Effect on the Survival of Deinococcus radiodurans after Exposure in Near Space.

Near space (20 to 100 km in altitude) is an extreme environment with high radiation and extreme cold, making it difficult for organisms to survive. However, many studies had shown that there were still microbes living in this extremely harsh environment. It was particularly important to study which factors affected the survival of microorganisms living in near space after exposure to irradiation, as this was related to many studies, such as studies of radioresistance mechanisms, panspermia hypothesis, long-distance microbial transfer, and developing extraterrestrial habitats. Survival after radiation was probably influenced by the growth condition before radiation, which is called the memory effect. In this research, we used different growth conditions to affect the growth of Deinococcus radiodurans and lyophilized bacteria in exponential phase to maintain the physiological state at this stage. Then high-altitude scientific balloon exposure experiments were carried out by using the Chinese Academy of Sciences Balloon-Borne Astrobiology Platform (CAS-BAP) at Dachaidan, Qinghai, China (37°44'N, 95°21'E). The aim was to investigate which factors influence survival after near-space exposure. The results suggested that there was a memory effect on the survival of D. radiodurans after exposure. If the differences in growth rate were caused by differences in nutrition, the survival rate and growth rate were positively correlated. Moreover, the addition of paraquat and Mn2+ during the growth phase can also increase survival. This finding may help to deepen the understanding of the mechanics of radiation protection and provide relevant evidence for many studies, such as of long-distance transfer of microorganisms in near space. IMPORTANCE Earth's near space is an extreme environment with high radiation and extreme cold. Which factors affect the survival of microbes in near space is related to many studies, such as studies of radioresistance mechanisms, panspermia hypothesis, long-distance microbial transfer, and developing extraterrestrial habitats. We performed several exposure experiments with Deinococcus radiodurans in near space to investigate which factors influence the survival rate after near-space exposure; that is, there was a relationship between survival after radiation and the growth condition before radiation. The results suggested that there was a memory effect on the survival of D. radiodurans after exposure. This finding may help to deepen the understanding of the mechanism of radiation protection and provide relevant evidence for many studies, such as of long-distance transfer of microorganisms in near space.

Biomotors, viral assembly, and RNA nanobiotechnology: Current achievements and future directions.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) serves to further the development of a wide variety of functional nucleic acids and other related nanotechnology platforms. To aid in the dissemination of the most recent advancements, a biennial discussion focused on biomotors, viral assembly, and RNA nanobiotechnology has been established where international experts in interdisciplinary fields such as structural biology, biophysical chemistry, nanotechnology, cell and cancer biology, and pharmacology share their latest accomplishments and future perspectives. The results summarized here highlight advancements in our understanding of viral biology and the structure-function relationship of frame-shifting elements in genomic viral RNA, improvements in the predictions of SHAPE analysis of 3D RNA structures, and the understanding of dynamic RNA structures through a variety of experimental and computational means. Additionally, recent advances in the drug delivery, vaccine design, nanopore technologies, biomotor and biomachine development, DNA packaging, RNA nanotechnology, and drug delivery are included in this critical review. We emphasize some of the novel accomplishments, major discussion topics, and present current challenges and perspectives of these emerging fields.

Kinetic assays of DNA polymerase fidelity: A theoretical perspective beyond Michaelis-Menten kinetics.

The high fidelity of DNA polymerase (DNAP) is critical for the faithful replication of DNA. There are several quantitative approaches to measure DNAP fidelity. Directly counting the error frequency in the replication products gives the true fidelity but it turns out very hard to implement in practice. Two biochemical kinetic approaches, the steady-state assay and the transient-state assay, were then suggested and widely adopted. In these assays, the error frequency is indirectly estimated by using kinetic theories combined with the measured apparent kinetic rates. However, whether it is equivalent to the true fidelity has never been clarified theoretically, and in particular there are different strategies using these assays to quantify the proofreading efficiency of DNAP but often lead to inconsistent results. In this paper, we make a comprehensive examination on the theoretical foundation of the two kinetic assays, based on the theory of DNAP fidelity recently proposed by us. Our studies show that while the conventional kinetic assays are generally valid to quantify the discrimination efficiency of DNAP, they are valid to quantify the proofreading efficiency of DNAP only when the kinetic parameters satisfy some constraints which will be given explicitly in this paper. These results may inspire more carefully-designed experiments to quantify DNAP fidelity.

Template-specific fidelity of DNA replication with high-order neighbor effects: A first-passage approach.

DNA replication fidelity is a critical issue in molecular biology. Biochemical experiments have provided key insights on the mechanism of fidelity control by DNA polymerases in the past decades, whereas systematic theoretical studies on this issue began only recently. Because of the underlying difficulties of mathematical treatment, comprehensive surveys on the template-specific replication kinetics are still rare. Here we propose a first-passage approach to address this problem, in particular the positional fidelity, for complicated processes with high-order neighbor effects. Under biologically relevant conditions, we derived approximate analytical expressions of the positional fidelity which show intuitively how some key kinetic pathways are coordinated to guarantee the high fidelity, as well as the high velocity, of the replication processes. It is also shown that the fidelity at any template position is dominantly determined by the nearest-neighbor template sequences, which is consistent with the idea that replication mutations are randomly distributed in the genome.

A New Method for Blood NT-proBNP Determination Based on a Near-infrared Point of Care Testing Device with High Sensitivity and Wide Scope.

OBJECTIVE: To develop a rapid, highly sensitive, and quantitative method for the detection of NT-proBNP levels based on a near-infrared point-of-care diagnostic (POCT) device with wide scope. METHODS: The lateral flow assay (LFA) strip of NT-proBNP was first prepared to achieve rapid detection. Then, the antibody pairs for NT-proBNP were screened and labeled with the near-infrared fluorescent dye Dylight-800. The capture antibody was fixed on a nitrocellulose membrane by a scribing device. Serial dilutions of serum samples were prepared using NT-proBNP-free serum series. The prepared test strips, combined with a near-infrared POCT device, were validated by known concentrations of clinical samples. The POCT device gave the output of the ratio of the intensity of the fluorescence signal of the detection line to that of the quality control line. The relationship between the ratio value and the concentration of the specimen was plotted as a work curve. The results of 62 clinical specimens obtained from our method were compared in parallel with those obtained from the Roche E411 kit. RESULTS: Based on the log-log plot, the new method demonstrated that there was a good linear relationship between the ratio value and NT-proBNP concentrations ranging from 20 pg/mL to 10 ng/mL. The results of the 62 clinical specimens measured by our method showed a good linear correlation with those measured by the Roche E411 kit. CONCLUSION: The new LFA detection method of NT-proBNP levels based on the near-infrared POCT device was rapid and highly sensitive with wide scope and was thus suitable for rapid and early clinical diagnosis of cardiac impairment.

Highly sensitive detection of NT-proBNP by molecular motor.

FoF1-ATPase is an active rotary motor, and generates three-ATP for each rotation. At saturated substrate concentration, the motor can achieve about 103 r.p.m, which means one motor can generate about 105 ATP molecules during 30 min. Here, we constituted a novel nanodevice with a molecular rotary motor and a "battery", FoF1-ATPase and chromatophore, and presented a novel method of sandwich type rotary biosensor based on ε subunit with one target-to-one motor, in which one target corresponds 105 ATP molecules as detection signals during 30 min. The target such as NT-proBNP detection demonstrated that this novel nanodevice has potential to be developed into an ultrasensitive biosensor to detect low expressed targets.

Proofreading of DNA polymerase: a new kinetic model with higher-order terminal effects.

The fidelity of DNA replication by DNA polymerase (DNAP) has long been an important issue in biology. While numerous experiments have revealed details of the molecular structure and working mechanism of DNAP which consists of both a polymerase site and an exonuclease (proofreading) site, there were quite a few theoretical studies on the fidelity issue. The first model which explicitly considered both sites was proposed in the 1970s and the basic idea was widely accepted by later models. However, all these models did not systematically investigate the dominant factor on DNAP fidelity, i.e. the higher-order terminal effects through which the polymerization pathway and the proofreading pathway coordinate to achieve high fidelity. In this paper, we propose a new and comprehensive kinetic model of DNAP based on some recent experimental observations, which includes previous models as special cases. We present a rigorous and unified treatment of the corresponding steady-state kinetic equations of any-order terminal effects, and derive analytical expressions for fidelity in terms of kinetic parameters under bio-relevant conditions. These expressions offer new insights on how the higher-order terminal effects contribute substantially to the fidelity in an order-by-order way, and also show that the polymerization-and-proofreading mechanism is dominated only by very few key parameters. We then apply these results to calculate the fidelity of some real DNAPs, which are in good agreements with previous intuitive estimates given by experimentalists.

A general theory of kinetics and thermodynamics of steady-state copolymerization.

Kinetics of steady-state copolymerization has been investigated since the 1940s. Irreversible terminal and penultimate models were successfully applied to a number of comonomer systems, but failed for systems where depropagation is significant. Although a general mathematical treatment of the terminal model with depropagation was established in the 1980s, a penultimate model and higher-order terminal models with depropagation have not been systematically studied, since depropagation leads to hierarchically-coupled and unclosed kinetic equations which are hard to solve analytically. In this work, we propose a truncation method to solve the steady-state kinetic equations of any-order terminal models with depropagation in a unified way, by reducing them into closed steady-state equations which give the exact solution of the original kinetic equations. Based on the steady-state equations, we also derive a general thermodynamic equality in which the Shannon entropy of the copolymer sequence is explicitly introduced as part of the free energy dissipation of the whole copolymerization system.

Master equation approach for a cross-bridge power-stroke model with a finite number of motors.

The cross-bridge power-stroke model has been widely used to describe the motion of large motor assemblies connected to a common rigid filament. In this paper, we go beyond the original velocity-ensemble approach and propose a master equation approach to account for the cooperative motion of a finite number of motors based on the cross-bridge model. By studying the force-velocity relationship for motors with strain-independent detachment rate, we show the convergence of our approach to the velocity-ensemble approach in the limit of large motor numbers. In the case that the detachment rate of motors is strain dependent, based on two assumptions for the strain distribution among motors, we show the occurrence of the bimodal distribution of the number of motors bound to the filament. This provides a new perspective to look at the instability of a multimotor system, which is essential for all the experimentally observed complex motions displayed by a group of motors, such as hysteresis, bidirectional motion, and spontaneous oscillation. By comparing the velocities calculated using the two assumptions with the stochastic simulation, it suggests that the coupling between motors via the common connection to the filament might facilitate the fast movement of filaments at small loading forces.

The neck linker of kinesin 1 seems optimally designed to approach the largest stepping velocity: a simulation study of an ideal model.

The neck linker is widely believed to play a critical role in the hand-over-hand walking of conventional kinesin 1. Experiments have shown that change of the neck linker length will significantly change the stepping velocity of the motor. In this paper, we studied this length effect based on a highly simplified chemically powered ratchet model. In this model, we assume that the chemical steps (ATP hydrolysis, ADP and P(i) release, ATP binding, neck linker docking) are fast enough under conditions far from equilibrium and the mechanical steps (detachment, diffusional search and re-attachment of the free head) are rate-limiting in kinesin walking. According to this model, and regarding the neck linker as a worm-like-chain polypeptide, we can calculate the steady state stepping velocity of the motor for different neck linker lengths. Our results show, under the actual values of binding energy between kinesin head and microtubule (~15k(B)T) and the persistence length of neck linker (~0.5 nm), that there is an optimal neck linker length (~14-16 a.a.) corresponding to the maximal velocity, which implies that the length of the wild-type neck linker (~15 a.a.) might be optimally designed for kinesin 1 to approach the largest stepping velocity.

FoF1-ATPase, rotary motor and biosensor.

F(o)F(1)-ATPase is an amazing molecular rotary motor at the nanoscale. Single molecule technologies have contributed much to the understanding of the motor. For example, fluorescence imaging and spectroscopy revealed the physical rotation of isolated F(1) and F(o), or F(o)F(1) holoenzyme. Magnetic tweezers were employed to manipulate the ATP synthesis/hydrolysis in F(1), and proton translation in F(o). Here, we briefly review our recent works including a systematic kinetics study of the holoenzyme, the mechanochemical coupling mechanism, reconstituting the delta-free F(o)F(1)-ATPase, direct observation of F(o) rotation at single molecule level and activity regulation through external links on the stator.

F0F1-ATPase activity regulated by external links on beta subunits.

F(o)F(1)-ATPase activity is regulated by external links on beta subunits with different molecular weight. It is inhibited when anti-beta subunit antibody, streptavidin and H9 antibody link on the beta subunits successively, but is activated when virus was binded. Western blotting indicated that the employed anti-beta antibody target was on the non-catalytic site of the beta subunit. Furthermore, an ESR study of spin-labeled ATP (SL-ATP) showed that the affinity of ATP to the holoenzyme increases with increasing external links on the beta subunits. This simple regulation method may have great potential in the design of rapid, free labeled, sensitive and selective biosensors.

Systematic kinetics study of FoF1-ATPase: analytic results and comparison with experiments.

The systematic kinetics of the holoenzyme F oF 1-ATPase has been investigated by a tri-site filled, random binding order, and stochastic mechanochemical tight coupling model. The connection between the mechanical rotation speed and the chemical quantities such as the concentrations of substrates, the proton motive force, and the mechanical damping coefficient, has been analytically derived. The enzymatics based on ensemble experiments and single-molecule assays can be discussed systematically. Our model predictions agree well with both ensemble and single-molecule experimental results. Furthermore, this model can be used to study the dynamics of F oF 1-ATPase in a vesicle system.