Live cell imaging of DNA and RNA with fluorescent signal amplification and background reduction techniques

Illuminating DNA and RNA dynamics in live cell can elucidate their life cycle and related biochemical activities. Various protocols have been developed for labeling the regions of interest in DNA and RNA molecules with different types of fluorescent probes. For example, CRISPR-based techniques have been extensively used for imaging genomic loci. However, some DNA and RNA molecules can still be difficult to tag and observe dynamically, such as genomic loci in non-repetitive regions. In this review, we will discuss the toolbox of techniques and methodologies that have been developed for imaging DNA and RNA. We will also introduce optimized systems that provide enhanced signal intensity or low background fluorescence for those difficult-to-tag molecules. These strategies can provide new insights for researchers when designing and using techniques to visualize DNA or RNA molecules

steve bAccumulation of nerve growth factor and its receptors in the uterus and dorsal root ganglia in a mouse model of adenomyosis

<p>Abstract</p> <p>Background</p> <p>Adenomyosis is a common gynecological disease, which is accompanied by a series of immunological and neuroendocrinological changes. Nerve growth factor (NGF) plays a critical role in producing pain, neural plasticity, immunocyte aggregation and release of inflammatory factors. This study aimed to investigate the expression of NGF and its two receptors in uteri and dorsal root ganglia (DRG) in an adenomyosis mouse model, as well as their relationship with the severity of adenomyosis.</p> <p>Methods</p> <p>Forty newborn ICR mice were randomly divided into the adenomyosis model group and control group (n = 20 in each group). Mice in the adenomyosis model group were orally dosed with 2.7 μmol/kg tamoxifen on days 2-5 after birth. Experiments were conducted to identify the expression of NGF- beta and its receptors, tyrosine kinase receptor (trkA) and p75 neurotrophin receptor (p75NTR), in the uterus and DRG in four age groups (90+/-5 d, 140+/-5 d, 190+/-5 d and 240+/-5 d; n = 5 mice in each group) by western bolt, immunochemistry and real time reverse transcription-polymerase chain reaction.</p> <p>Results</p> <p>Adenomyosis, which became more serious as age increased, was successfully induced in dosed ICR mice. NGF-beta, trkA and p75NTR protein levels in the uterus and trkA mRNA levels in DRG were higher in the older aged adenomyosis model group than those in controls (190+/-5 d and 240+/-5 d groups, P < 0.05). The expression of NGF-beta and its receptors in the uterus increased gradually as age increased for adenomyosis mice (190+/-5 d and 240+/-5 d, P < 0.05, compared with 90+/-5 d) but it showed little change in control mice. The mRNA level of trkA in DRG also increased as age increased in the adenomyosis model group (190+/-5 d and 240+/-5 d, P < 0.05, compared with 90+/-5 d) but was unchanged in controls. The mRNA level of p75NTR in DRG was not different between the adenomyosis and control groups and was stable from young to old mice.</p> <p>Conclusions</p> <p>NGF- beta can be used as an indicator for the severity of adenomyosis. The gradually increasing level of NGF- beta and its receptors while the disease becomes more severe suggests an effect of NGF- beta on pathogenic mechanisms of adenomyosis.</p

Enhancement of baryon-to-meson ratios around jets as a signature of medium response

We present a unique signal of jet-induced medium excitations: the enhancement of baryon-to-meson ratios around the quenched jets. To illustrate this, we study jet-particle correlations and the distributions of jet-induced identified particles with respect to the jet direction in Pb+Pb collisions at the LHC via a multi-phase transport model. We find a strong enhancement of baryon-to-meson ratios for associated particles at intermediate transverse momentum around the triggered jets in Pb+Pb collisions relative to p+p collisions, due to the coalescence of jet-excited medium partons. Since the lost energy from jets can diffuse to large angles, such baryon-to-meson-ratio enhancement is more pronounced for larger relative distance from the jet axis. We argue that the experimental confirmation of the enhancement of jet-induced baryon-to-meson ratios around the jets will provide an unambiguous evidence for the medium response to jet quenching in heavy-ion collisions.Comment: 6 pages, 3 figure

Visualizing the dynamic behavior of poliovirus plus-strand RNA in living host cells

Dynamic analysis of viral nucleic acids in host cells is important for understanding virus–host interaction. By labeling endogenous RNA with molecular beacon, we have realized the direct visualization of viral nucleic acids in living host cells and have studied the dynamic behavior of poliovirus plus-strand RNA. Poliovirus plus-strand RNA was observed to display different distribution patterns in living Vero cells at different post-infection time points. Real-time imaging suggested that the translocation of poliovirus plus-strand RNA is a characteristic rearrangement process requiring intact microtubule network of host cells. Confocal-FRAP measurements showed that 49.4 ± 3.2% of the poliovirus plus-strand RNA molecules diffused freely (with a D-value of 9.6 ± 1.6 × 10(−10) cm(2)/s) within their distribution region, while the remaining (50.5 ± 2.9%) were almost immobile and moved very slowly only with change of the RNA distribution region. Under the electron microscope, it was found that virus-induced membrane rearrangement is microtubule-associated in poliovirus-infected Vero cells. These results reveal an entrapment and diffusion mechanism for the movement of poliovirus plus-strand RNA in living mammalian cells, and demonstrate that the mechanism is mainly associated with microtubules and virus-induced membrane structures

Rapid Colorimetric Testing for Pyrazinamide Susceptibility of M. tuberculosis by a PCR-Based In-Vitro Synthesized Pyrazinamidase Method

Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug. But PZA susceptibility test is challenging because PZA activity is optimal only in an acid environment that inhibits the growth of M. tuberculosis. For current phenotypic methods, inconsistent results between different labs have been reported. Direct sequencing of pncA gene is being considered as an accurate predictor for PZA susceptibility, but this approach needs expensive sequencers and a mutation database to report the results. An in-vitro synthesized Pyrazinamidase (PZase) assay was developed based on PCR amplification of pncA gene and an in vitro wheat germ system to express the pncA gene into PZase. The activity of the synthesized PZase was used as an indicator for PZA susceptibility. Fifty-one clinical isolates were tested along with pncA sequencing and the BACTEC MGIT 960 methods. The in-vitro synthesized PZase assay was able to detect PZA susceptibility of M. tuberculosis within 24 h through observing the color difference either by a spectrometer or naked eyes. This method showed agreements of 100% (33/33) and 88% (14/16) with the pncA sequencing method, and agreements of 96% (27/28) and 65% (15/23) with the BACTEC MGIT 960 method, for susceptible and resistant strains, respectively. The novel in-vitro synthesized PZase assay has significant advantages over current methods, such as its fast speed, simplicity, no need for expensive equipment, and the potentials of being a direct test, predicting resistance level and easy reading results by naked eyes. After confirmation by more clinical tests, this method may provide a radical change to the current PZA susceptibility assays

Kernel-based nonlinear discriminant analysis using minimum squared errors criterion for multiclass and undersampled problems

It is well known that there exist two fundamental limitations in the linear discriminant analysis (LDA). One is that it cannot be applied when the within-class scatter matrix is singular, which is caused by the undersampled problem. The other is that it lacks the capability to capture the nonlinearly clustered structure of the data due to its linear nature. In this paper, a new kernel-based nonlinear discriminant analysis algorithm using minimum squared errors criterion (KDA-MSE) is proposed to solve these two problems. After mapping the original data into a higher-dimensional feature space using kernel function, the MSE criterion is used as the discriminant rule and the corresponding dimension reducing transformation is derived. Since the MSE solution does not require the scatter matrices to be nonsingular, the proposed KDA-MSE algorithm is applicable to the undersampled problem. Moreover, the new KDA-MSE algorithm can be applied to multiclass problem, whereas the existing MSE-based kernel discriminant methods are limited to handle twoclass data only. Extensive experiments, including object recognition and face recognition on three benchmark databases, are performed and the results demonstrate that our algorithm is competitive in comparison with other kernel-based discriminant techniques in terms of recognition accuracy. (C) 2009 Elsevier B.V. All rights reserved.National Natural Science Foundation of China [60672046, 60675002]; Fujian Province Science and Technology Foundation [2008H0036]; Specialized Research Fund for the Doctorol Program of Higher Educatio

Measuring the Hubble Constant Using Strongly Lensed Gravitational Wave Signals

The measurement of the Hubble constant $H_0$ plays an important role in the study of cosmology. In this letter, we propose a new method to constrain the Hubble constant using the strongly lensed gravitational wave (GW) signals. By reparameterizing the waveform, we find that the lensed waveform is sensitive to the $H_0$. Assuming the scenario that no electromagnetic counterpart of the GW source can be identified, our method can still give meaningful constraints on the $H_0$ with the information of the lens redshift. We then apply Fisher information matrix and Markov Chain Monte Carlo to evaluate the potential of this method. For the space-based GW detector, TianQin, the $H_0$ can be constrained within a relative error of $\sim$ 0.3-2\%, using a single strongly lensed GW event. Precision varies according to different levels of electromagnetic information.Comment: 8 pages, 4 figure

A Sir2-Like Protein Participates in Mycobacterial NHEJ

In eukaryotic cells, repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway is critical for genome stability. In contrast to the complex eukaryotic repair system, bacterial NHEJ apparatus consists of only two proteins, Ku and a multifunctional DNA ligase (LigD), whose functional mechanism has not been fully clarified. We show here for the first time that Sir2 is involved in the mycobacterial NHEJ repair pathway. Here, using tandem affinity purification (TAP) screening, we have identified an NAD-dependent deacetylase in mycobacteria which is a homologue of the eukaryotic Sir2 protein and interacts directly with Ku. Results from an in vitro glutathione S-transferase (GST) pull-down assay suggest that Sir2 interacts directly with LigD. Plasmid-based end-joining assays revealed that the efficiency of DSB repair in a sir2 deletion mutant was reduced 2-fold. Moreover, the Δsir2 strain was about 10-fold more sensitive to ionizing radiation (IR) in the stationary phase than the wild-type. Our results suggest that Sir2 may function closely together with Ku and LigD in the nonhomologous end-joining pathway in mycobacteria

Phage display mediated immuno-PCR

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10 000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications