Probing the modulation in facilitated diffusion guided by DNA-protein interactions in target search processes.

Many fundamental biophysical processes involving gene regulation and gene editing rely, at the molecular level, on an intricate methodology of searching and locating the precise target base pair sequence on the genome by specific binding proteins. A unique mechanism, known as 'facilitated diffusion', which is a combination of 1D sliding along with 3D movement, is considered to be the key step for such events. This also explains the relatively much shorter timescale of the target searching process, compared to other diffusion-controlled biophysical processes. In this work, we aim to probe the modulation of target search dynamics of a protein moiety by estimating the rate of the target search process, and the statistics of the search rounds and timescales accomplished by the 1D and 3D motions, based on first passage time (FPT) calculations. This is studied with its characteristics getting influenced by various given conditions such as, when the DNA is rigid or flexible, and when the target is placed at different locations on the DNA. The current theoretical framework includes a Brownian dynamics simulation setup adopting a straightforward coarse-grained model for a diffusing protein on DNA. Moreover, this theoretical analysis provides insights into the complex target search dynamics by highlighting the significance of the chain dynamics in the mechanistic details of the facilitated diffusion process.

CRISPR-Cas9 Genome Interrogation: A Facilitated Subdiffusive Target Search Strategy.

The functional application of RNA-guided CRISPR-associated Cas9 protein, a bacterial immune system-based protein complex, via which in vivo, highly specific, and well-regulated, gene-editing processes are being monitored at an unprecedented level, has led to remarkable progress in genetic engineering and technology. The complicated in vivo process of genome interrogation followed by gene editing by the Cas9 complex was recently reported by Knight et al. (Science, 2015, 350, 823-826) using an elegant single-particle tracking method, aided by the two-photon fluorescence correlation spectroscopic technique. In contrast to the usually observed fast target-searching and protein-binding events in biophysical systems, an interesting slow genome-interrogation process by the RNA-guided CRISPR-Cas9 system through a crowded chromatin environment of a mammalian cell has been revealed in Knight et al.'s study. Motivated by this experiment, in this paper, we provide a generalized theoretical framework to capture this particular target-searching mechanism of the CRISPR-Cas9 protein complex. We show that an analysis on the basis of 3D subdiffusion under a cylindrical volume, created by several nonspecific off-target interactions from the DNA strands, can capture the essential details of the process. Moreover, on the basis of this model, we quantify the dynamics of this process and estimate the survival probability, first passage time, and the intensity correlation function of the tagged proteins of the experiment. The results from our theoretical predictions are found to be consistent with the experimental observations, and hence, seem to provide a plausible microscopic picture of the process.

Potential role of deer tick virus in Powassan encephalitis cases in Lyme disease-endemic areas of New York, U.S.A.

Powassan virus, a member of the tick-borne encephalitis group of flaviviruses, encompasses 2 lineages with separate enzootic cycles. The prototype lineage of Powassan virus (POWV) is principally maintained between Ixodes cookei ticks and the groundhog (Marmota momax) or striped skunk (Mephitis mephitis), whereas the deer tick virus (DTV) lineage is believed to be maintained between Ixodes scapularis ticks and the white-footed mouse (Peromyscus leucopus). We report 14 cases of Powassan encephalitis from New York during 2004-2012. Ten (72%) of the patients were residents of the Lower Hudson Valley, a Lyme disease-endemic area in which I. scapularis ticks account for most human tick bites. This finding suggests that many of these cases were caused by DTV rather than POWV. In 2 patients, DTV infection was confirmed by genetic sequencing. As molecular testing becomes increasingly available, more cases of Powassan encephalitis may be determined to be attributable to the DTV lineage.

Subdiffusion as a model of transport through the nuclear pore complex.

Cargo transport through the nuclear pore complex continues to be a subject of considerable interest to experimentalists and theorists alike. Several recent studies have revealed details of the process that have still to be fully understood, among them the apparent nonlinearity between cargo size and the pore crossing time, the skewed, asymmetric nature of the distribution of such crossing times, and the non-exponentiality in the decay profile of the dynamic autocorrelation function of cargo positions. In this paper, we show that a model of pore transport based on subdiffusive particle motion is in qualitative agreement with many of these observations. The model corresponds to a process of stochastic binding and release of the particle as it moves through the channel. It suggests that the phenylalanine-glycine repeat units that form an entangled polymer mesh across the channel may be involved in translocation, since these units have the potential to intermittently bind to hydrophobic receptor sites on the transporter protein.

Emergence of unique variants and inter-genotype recombinants of human astroviruses infecting infants, children and adults in Kolkata, India.

Two conserved genomic fragments viz. 289bp of ORF1a and 449bp of ORF2 amplified by RT-PCR showed emergence of interesting recombinant strains representing new and novel genetic variants (n=5) within eight different genotypes of astroviruses known to date. HAstV-positive cases with ORF1a [HAstV genotype G2 or G8] and ORF2 [HAstV genotype G1, G2, or G3] were detected as sole or mixed infection among infants, children and adults in Kolkata with severe illness owing to acute gastroenteritis that required hospitalization for treatment between 2007 and 2009. The twelve interesting recombinants were of type HAstV _ ORF1a _ ORF2 as HAstV _ G8_ G2 (n=1), HAstV _ G8_ G1 (n=10) and HAstV _ G2_ G3 (n=1).

The stretching of single poly-ubiquitin molecules: static versus dynamic disorder in the non-exponential kinetics of chain unfolding.

Static disorder has recently been implicated in the non-exponential kinetics of the unfolding of single molecules of poly-ubiquitin under a constant force [Kuo, Garcia-Manyes, Li, Barel, Lu, Berne, Urbakh, Klafter, and Fernández, Proc. Natl. Acad. Sci. U.S.A. 107, 11336 (2010)]. In the present paper, it is suggested that dynamic disorder may provide a plausible, alternative description of the experimental observations. This suggestion is made on the basis of a model in which the barrier to chain unfolding is assumed to be modulated by a control parameter r that evolves in a parabolic potential under the action of fractional Gaussian noise according to a generalized Langevin equation. The treatment of dynamic disorder within this model is pursued using Zwanzig's indirect approach to noise averaging [Acc. Chem. Res. 23, 148 (1990)]. In conjunction with a self-consistent closure scheme developed by Wilemski and Fixman [J. Chem. Phys. 58, 4009 (1973); ibid. 60, 866 (1974)], this approach eventually leads to an expression for the chain unfolding probability that can be made to fit the corresponding experimental data very closely.

A model of anomalous enzyme-catalyzed gel degradation kinetics.

We show that a model of target location involving n noninteracting particles moving subdiffusively along a line segment (a generalization of a model introduced by Sokolov et al. [Biophys. J. 2005, 89, 895.]) provides a basis for understanding recent experiments by Pelta et al. [Phys. Rev. Lett. 2007, 98, 228302.] on the kinetics of diffusion-limited gel degradation. These experiments find that the time t(c) taken by the enzyme thermolysin to completely hydrolyze a gel varies inversely as roughly the 3/2 power of the initial enzyme concentration [E]. In general, however, this time would be expected to vary either as [E](-1) or as [E](-2), depending on whether the brownian diffusion of the enzyme to the site of cleavage took place along the network chains (1-d diffusion) or through the pore spaces (3-d diffusion). In our model, the unusual dependence of t(c) on [E] is explained in terms of a reaction-diffusion equation that is formulated in terms of fractional rather than ordinary time derivatives.

Anomalous reaction-diffusion as a model of nonexponential DNA escape kinetics.

We show that data from recent experiments carried out on the kinetics of DNA escape from alpha-hemolysin nanopores [M. Wiggin, C. Tropini, C. T. Cossa, N. N. Jetha, and A. Marziali, Biophys. J. 95, 5317 (2008)] may be rationalized by a model of chain dynamics based on the anomalous diffusion of a particle moving in a harmonic well in the presence of a delta function sink. The experiments of Wiggin et al. found, among other things, that the occasional occurrence of unusually long escape times in the distribution of chain trapping events led to nonexponential decays in the survival probability, S(t), of the DNA molecules within the nanopore. Wiggin et al. ascribed this nonexponentiality to the existence of a distribution of trapping potentials, which they suggested was the result of stochastic interactions between the bases of the DNA and the amino acids located on the surface of the nanopore. Based on this idea, they showed that the experimentally determined S(t) could be well fit in both the short and long time regimes by a function of the form (1+t/tau)(-alpha) (the so called Becquerel function). In our model, S(t) is found to be given by a Mittag-Leffler function at short times and by a generalized Mittag-Leffler function at long times. By suitable choice of certain parameter values, these functions are found to fit the experimental S(t) even better than the Becquerel function. Anomalous diffusion of DNA within the trap prior to escape over a barrier of fixed height may therefore provide a second, plausible explanation of the data, and may offer fresh perspectives on similar trapping and escape problems.

Exact path-integral evaluation of the heat distribution function of a trapped brownian oscillator.

Using path integrals, we derive an exact expression--valid at all times t--for the distribution P(Q,t) of the heat fluctuations Q of a brownian particle trapped in a stationary harmonic well. We find that P(Q,t) can be expressed in terms of a modified Bessel function of zeroth order that in the limit t→∞ exactly recovers the heat distribution function obtained recently by Imparato [Phys. Rev. E 76, 050101(R) (2007)] from the approximate solution to a Fokker-Planck equation. This long-time result is in very good agreement with experimental measurements carried out by the same group on the heat effects produced by single micron-sized polystyrene beads in a stationary optical trap. An earlier exact calculation of the heat distribution function of a trapped particle moving at a constant speed v was carried out by van Zon and Cohen [Phys. Rev. E 69, 056121 (2004)]; however, this calculation does not provide an expression for P(Q,t) itself, but only its Fourier transform (which cannot be analytically inverted), nor can it be used to obtain P(Q,t) for the case v=0 .

Brownian particles in stationary and moving traps: the mean and variance of the heat distribution function.

A recent theoretical model developed by Imparato [Phys. Rev. E 76, 050101(R) (2007)] of the experimentally measured heat and work effects produced by the thermal fluctuations of single micron-sized polystyrene beads in stationary and moving optical traps has proved to be quite successful in rationalizing the observed experimental data. The model, based on the overdamped Brownian dynamics of a particle in a harmonic potential that moves at a constant speed under a time-dependent force, is used to obtain an approximate expression for the distribution of the heat dissipated by the particle at long times. In this paper, we generalize the above model to consider particle dynamics in the presence of colored noise, without passing to the overdamped limit, as a way of modeling experimental situations in which the fluctuations of the medium exhibit long-lived temporal correlations, of the kind characteristic of polymeric solutions, for instance, or of similar viscoelastic fluids. Although we have not been able to find an expression for the heat distribution itself, we do obtain exact expressions for its mean and variance, both for the static and for the moving trap cases. These moments are valid for arbitrary times and they also hold in the inertial regime, but they reduce exactly to the results of Imparato in appropriate limits.

A new variant of Norovirus GII.4/2007 and inter-genotype recombinant strains of NVGII causing acute watery diarrhoea among children in Kolkata, India.

BACKGROUND: Noroviruses (NoVs) are one of the major causal agents of acute gastroenteritis among different age groups. Some of the recent studies reveal that NoV genome is highly prone to mutation and recombination which often leads to emergence of new strains. OBJECTIVES: To explore the genetic diversity of human Caliciviruses (HuCVs) among diarrhoeic children in Kolkata. STUDY DESIGN: The HuCVs were detected by reverse transcription-polymerase chain reaction (RT-PCR) of the partial RNA dependent RNA polymerase gene (RdRp) and capsid gene and confirmed by sequencing. The sequences were analyzed and the recombination point was detected. RESULTS: Faecal specimens of children (n=111) visiting outpatient department of Dr B. C. Roy Memorial Hospital for Children with acute gastroenteritis were studied: 22 cases were HuCV positive with 21 NoVs. Of these, 12 NoV cases (54.5%) were GII.4 and six cases showed 99% identity with the new variant Japanese strain Hu/NoV/GII.4/OC07138/JP. Three novel NoV GII inter-genotype recombinant strains V1628/IND, V1656/IND and V1737/IND were also detected. The RdRp region of V1628 showed 96% identity with Pont de Roide 673/FRN whereas capsid region resembled GII.7/Osaka F140/JPN strain (98%); the strain V1656 showed 98% identity with RdRp region of GII.4/Monastir 375/TUN but capsid region resembled GII.8/Leverkusen 267/DE (91%); the strain V1737 showed 88% identity with RdRp of GII.5/Minato 6/N1/6/JPN whereas capsid region resembled the GII.12/Gifu 96/JPN (93%). During characterization of Caliciviruses two strains of NoV GII.b and one strain of each NoV GI.1/V1622/06/IND, GI.3/V1707/07/IND, GII.3/V1668/IND, GII.16/V1729/IND, Sapovirus GII.1/V1716/IND were also detected. CONCLUSIONS: The emergence of new variant of GII.4/2007, three novel NoV GII inter-genotype recombinant strains and various other NoVs, indicates the remarkable genetic diversity of the HuCVs as diarrhoeagenic viruses circulating in Kolkata, India.

The dynamics of single enzyme reactions: A reconsideration of Kramers' model for colored noise processes.

The utility of an approximate heuristic version of Kramers' theory of reaction rates that was earlier used [Chaudhury and Cherayil, J. Chem. Phys. 125, 024904 (2006)] to successfully describe the nonexponential waiting time distributions of the enzyme beta-galactosidase is reassessed. The original model, based on the Smoluchowski equation, is reformulated in terms of the phase space variables of the reaction coordinate, without neglecting inertial contributions. A new derivation of the Fokker-Planck equation (FPE) that describes the dynamics of this coordinate is presented. This derivation, based on functional methods, provides a more direct alternative to the existing distribution function approach used by Hanggi and Mojtabai [Phys. Rev. A 26, 1168 (1982)]. The time-dependent coefficients in the FPE, when incorporated into the exact expression for the transmission coefficient obtained from a reactive-flux formalism [Kohen and Tannor, J. Chem. Phys. 103, 6013 (1995)], are found to yield virtually the same results as the earlier heuristic model.

The dynamics of intermittent strand separation in double-stranded DNA.

The transient rupture and reformation of hydrogen bonds between base pairs on distinct chains of double-stranded DNA ("bubble" dynamics) is modeled in terms of the fluctuating distance between the bases. The fluctuations in the distance are assumed to be governed by a simple Langevin equation with a quadratic potential under conditions of high friction. A critical distance of separation L must be achieved before a bubble defect is considered to have been formed. The decay of the dynamic correlations between states of the DNA that have such defects and those that do not has been calculated from the above model and has been found to reproduce the trends in experimental measurements of the same quantity.