Search

Medical devices provide critical care and diagnostic applications through patient contact. Sterility assurance level (SAL) may be defined as the probability of a single viable micro-organism occurring on an item after a sterilization process. Sterilization microbiology often relies upon using an overkill validation method where a 12-log reduction in recalcitrant bacterial endospore population occurs during the process that exploits conventional laboratory-based culture media for enumeration. This timely review explores key assumptions underpinning use of conventional culture-based methods in sterilization microbiology. Consideration is given to how such methods may limit the ability to fully appreciate the inactivation kinetics of a sterilization process such as vaporized hydrogen peroxide (VH2O2) sterilization, and consequently design efficient sterilization processes. Specific use of the real-time flow cytometry (FCM) is described by way of elucidating the practical relevance of these limitation factors with implications and opportunities for the sterilization industry discussed. Application of FCM to address these culture-based limitation factors will inform real-time kinetic inactivation modelling and unlock potential to embrace emerging opportunities for pharma, medical device and sterilization industries including potentially disruptive applications that may involve reduced usage of sterilant., (© 2020 The Society for Applied Microbiology.)

Objective: A standardized procedure of fused REarranged during Transfection (RET) gene detection using fluorescence in situ hybridization (FISH) remains to be established in papillary thyroid carcinoma (PTC). Our purpose was to investigate false-negative and false-positive events and their FISH signal characteristics., Methods: A total of 111 PTC cases were analyzed using break-apart FISH probes for RET status evaluation. All FISH results were validated using fusion-induced asymmetric transcription assay (FIATA)-based reverse transcription droplet digital PCR (RT-ddPCR). Then, suspected RET-positive cases were tested using quantitative reverse transcription-PCR (RT-qPCR), followed by next-generation sequencing (NGS) for recognizing fusion variants., Results: Thirty RET+ cases were revealed, including 20 CCDC6 (exon 1)-RET (exon 12), 6 NCOA4 (exon 8)-RET (exon 12), 1 NCOA4 (exon 7)-RET (exon 12), 1 CCDC186 (exon 7)-RET (exon 12), 1 ERC1 (exon 12)-RET (exon 12) and 1 SPECC1L (exon 9)-RET (exon 12) tumors. All RET fusion cases occurred in the BRAF- population, with a prevalence of 41.7% (30/72). Four cases of 8% to 13% (cutoff was 7.6%) dominant isolated 3' green (IG) FISH signals were RET-. One FISH- case with isolated 5' red (IR) signals with 94% abnormal tumor cells was demonstrated to be positive, harboring the NCOA4 (exon 7)-RET (exon 12) variant. Compared with RET fusions characterized by dominant break-apart signals with 29% to 100% aberrant cells, RET + with dominant IG-signal patterns all showed more frequent FISH+ cells (84%-92%). RET+ PTC with a break-apart signal pattern was more frequently found in unifocal lesions than in multifocal/bilateral tumors (P = 0.049)., Conclusions: A false-positive or false-negative event may exist for RET status detection in PTCs using the traditional FISH scoring method with break-apart probes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here, we describe a procedure to fluorescently contrast the nuclear boundary using the lipophilic carbocyanine dye DiI in cultured human cells. Our procedure is simple and is applicable to detect nuclear boundary defects, which may be relevant to studies on nuclear envelope dynamics, micronuclei formation and cancer biology., Abbreviations: DiI: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiO: 3,3'-dioctadecyloxacarbocyanine perchlorate; NE: nuclear envelope; RanBP2: Ran-binding protein 2/Nucleoporin 358.

Aims: To provide information on the time-dependent behaviour of microbe staining by fluorescent dyes in the order of seconds, which is important in terms of the recent rapid and online techniques for microbe measurements and/or environmental microbe analysis., Methods and Results: For combinations of yeast (Saccharomyces cerevisiae) and typical dyes, including DAPI (4',6-diamidino-2-phenylindole) and Auramine-O, a suspension of yeast cells in ultrapure water was injected into a dye solution in a micro cuvette placed inside a spectrofluorometer and the fluorescence intensity of the resulting solution was measured at 1 s intervals, starting immediately after the mixing and continued until the time for the maximum intensity using various concentrations of yeast and dyes. The relaxation time τ, which corresponds to ~63·2% of the maximum fluorescence intensity, was shown to decrease to below 1 s with increasing DAPI concentration, whereas it remained constant for 2-3 s with increasing Auramine-O concentration, for example at a yeast concentration of 100 µg ml -1 ., Conclusions: For the conditions of yeast >10 µg ml -1 , DAPI >1 µg ml -1 and Auramine-O >0·1 µg ml -1 , τ could be adjusted to below 5 s to achieve a rapid and stable staining., Significance and Impact of the Study: Design and operating conditions for rapid and online measurements of microbes can be optimized., (© 2020 The Society for Applied Microbiology.)

RNA:5-methylcytosine (m5C) methyltransferases are currently the focus of intense research following a series of high-profile reports documenting their physiological links to several diseases. However, no methods exist which permit the specific analysis of RNA:m5C methyltransferases in cells. Herein, we described how a combination of biophysical studies led us to identify distinct duplex-remodelling effects of m5C on RNA and DNA duplexes. Specifically, m5C induces a C3'-endo to C2'-endo sugar-pucker switch in CpG RNA duplex but triggers a B-to-Z transformation in CpG DNA duplex. Inspired by these different 'structural signatures', we developed a m5C-sensitive probe which fluoresces spontaneously in response to m5C-induced sugar-pucker switch, hence useful for sensing RNA:m5C methyltransferase activity. Through the use of this probe, we achieved real-time imaging and flow cytometry analysis of NOP2/Sun RNA methyltransferase 2 (NSUN2) activity in HeLa cells. We further applied the probe to the cell-based screening of NSUN2 inhibitors. The developed strategy could also be adapted for the detection of DNA:m5C methyltransferases. This was demonstrated by the development of DNA m5C-probe which permits the screening of DNA methyltransferase 3A inhibitors. To our knowledge, this study represents not only the first examples of m5C-responsive probes, but also a new strategy for discriminating RNA and DNA m5C methyltransferase activity in cells., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The use of insect markers, such as fluorescent powders, is a useful tool for studying ecological and epidemiological questions. Evaluating their effect on vectors of human disease agents, such as the invasive mosquito vector Aedes aegypti (Linnaeus), is crucial for their practical and reliable use, especially in parameters linked to the risk of disease transmission such as adult survival, dispersal, and host-seeking. Seven fluorescent powders (Hercules Radiant, DayGlo (DG), Risk Reactor (RR), and Angstrom Technologies), applied externally on cohorts of Ae. aegypti female mosquitoes, were tested to determine their impact on survival and recapture by baited mosquito traps, and their detectability after being exposed to controlled laboratory and semifield environments. There were no significant differences in survival among marked and unmarked females across all powders. Marked females were significantly less likely to be captured in baited traps relative to unmarked females, except for one of the DG powders. All females remained visibly marked on five parts of their body for 30 d (under both environments), except for one of the RR powders. The tested powders and application method are suitable for tracking mosquitoes throughout most of their lives under different environments, without significantly affecting their survival, but with potential impact on recapture by baited traps, possibly due to effects on senses or other physiological traits., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Regions of genomic instability are not random and often co-localize with DNA sequences that can adopt alternative DNA structures (i.e. non-B DNA, such as H-DNA). Non-B DNA-forming sequences are highly enriched at translocation breakpoints in human cancer genomes, representing an endogenous source of genetic instability. However, a further understanding of the mechanisms involved in non-B DNA-induced genetic instability is needed. Small molecules that can modulate the formation/stability of non-B DNA structures, and therefore the subsequent mutagenic outcome, represent valuable tools to study DNA structure-induced genetic instability. To this end, we have developed a tunable Förster resonance energy transfer (FRET)-based assay to detect triplex/H-DNA-destabilizing and -stabilizing ligands. The assay was designed by incorporating a fluorophore-quencher pair in a naturally-occurring H-DNA-forming sequence from a chromosomal breakpoint hotspot in the human c-MYC oncogene. By tuning triplex stability via buffer composition, the assay functions as a dual-reporter that can identify stabilizers and destabilizers, simultaneously. The assay principle was demonstrated using known triplex stabilizers, BePI and coralyne, and a complementary oligonucleotide to mimic a destabilizer, MCRa2. The potential of the assay was validated in a 384-well plate with 320 custom-assembled compounds. The discovery of novel triplex stabilizers/destabilizers may allow the regulation of genetic instability in human genomes., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nucleic acid mimics of fluorescent proteins can be valuable tools to locate and image functional biomolecules in cells. Stacking between the internal G-quartet, formed in the mimics, and the exogenous fluorophore probes constitutes the basis for fluorescence emission. The precision of recognition depends upon probes selectively targeting the specific G-quadruplex in the mimics. However, the design of probes recognizing a G-quadruplex with high selectivity in vitro and in vivo remains a challenge. Through structure-based screening and optimization, we identified a light-up fluorescent probe, 9CI that selectively recognizes c-MYC Pu22 G-quadruplex both in vitro and ex vivo. Upon binding, the biocompatible probe emits both blue and green fluorescence with the excitation at 405 nm. With 9CI and c-MYC Pu22 G-quadruplex complex as the fluorescent response core, a DNA mimic of fluorescent proteins was constructed, which succeeded in locating a functional aptamer on the cellular periphery. The recognition mechanism analysis suggested the high selectivity and strong fluorescence response was attributed to the entire recognition process consisting of the kinetic match, dynamic interaction, and the final stacking. This study implies both the single stacking state and the dynamic recognition process are crucial for designing fluorescent probes or ligands with high selectivity for a specific G-quadruplex structure., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ferroptosis is an emerging type of cell death mode that is dependent on iron. Unfortunately, the detailed analysis of the function of organelle labile Fe(ii) in oxidative damage and lethality of the cells has not been demonstrated so far, mainly due to the lack of efficient methods to visualize labile Fe(ii) at the targeted organelles. We have recently reported a series of Fe(ii)-selective fluorescent probes, i.e., Ac-MtFluNox, Lyso-RhoNox, and ER-SiRhoNox, which can detect Fe(ii) specifically in the mitochondria, lysosomes, and endoplasmic reticulum (ER), respectively. These probes demonstrate similar reaction rates and off/on contrasts with various colours and intracellular distributions, enabling simultaneous multi-colour imaging that allows the monitoring of labile Fe(ii) levels at each targeted organelle. In this paper, by using a cocktail of these probes, we successfully visualised the aberrant elevation of labile Fe(ii) in the lysosomes and ER prior to HT1080 cell death induced by erastin, which is an inducer of ferroptosis.

It has been uncovered that chemical dyes are illegally used in traditional Chinese medicines to brighten color and cover up inferiority, which threaten the safety of patients. In the present study, an HPTLC-MS method was developed for the effective screening of 11 chemical dyes (Sudan I, II, III, and IV; 808 Scarlet; Sudan Red 7B; malachite green; Basic Orange 2; auramine; Orange II; and erythrosine) in traditional Chinese medicine (TCM) raw materials and Chinese patent medicines. Firstly, unwashed HPTLC plates were chosen by comparing the background signals of the TLC plates used directly and prewashed with analytical grade and HPLC grade solvents. Twice developments were conducted to isolate chemical dyes of different polarity. Possible adulterants were preliminarily identified by comparing Rf values and in situ UV-Vis spectra with those of the references. Further confirmation was conducted by tandem MS analysis via an elution head-based TLC-MS interface. Sudan I and IV, 808 Scarlet, and Orange II were successfully detected in eight batches of TCM. The proposed method could be applied as a reliable technology for the screening of chemical dyes in TCM.

We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein's turnover time from such data., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Nitric oxide (NO) is both an important regulatory molecule in biological systems and a toxic xenobiotic. Its oxidation products react with sulfhydryl groups and either nitrosylate or oxidize them. The aerobic reaction of NO supplied by diethylamine NONOate (DEA-NO) with pig kidney LLC-PK 1 cells and Zn-proteins within the isolated proteome was examined with three fluorescent zinc sensors, zinquin (ZQ), TSQ, and FluoZin-3 (FZ-3). Observations of Zn 2+ labilization from Zn-proteins depended on the specific sensor used. Upon cellular exposure to DEA-NO, ZQ sequestered about 13% of the proteomic Zn 2+ as Zn(ZQ) 2 and additional Zn 2+ as proteome·Zn-ZQ ternary complexes. TSQ, a sensor structurally related to ZQ with lower affinity for Zn 2+ , did not form Zn(TSQ) 2 . Instead, Zn 2+ mobilized by DEA-NO was exclusively bound as proteome·Zn-TSQ adducts. Analogous reactions of proteome with ZQ or TSQ in vitro displayed qualitatively similar products. Titration of native proteome with Zn 2+ in the presence of ZQ resulted in the sole formation of proteome·Zn-ZQ species. This result suggested that sulfhydryl groups are involved in non-specific proteomic binding of mobile Zn 2+ and that the appearance of Zn(ZQ) 2 after exposure of cells and proteome to DEA-NO resulted from a reduction in proteomic sulfhydryl ligands, favoring the formation of Zn(ZQ) 2 instead of proteome·Zn-ZQ. With the third sensor, FluoZin-3, neither Zn-FZ-3 nor proteome·Zn-FZ-3 was detected during the reaction of proteome with DEA-NO. Instead, it reacted independently with DEA-NO with a modest enhancement of fluorescence.

Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2'OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Zn(2+)-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn(2+) from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn(2+) release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn(2+) concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn(2+), eZinCh-2 (Kd = 1 nM at pH 7.1) and eCALWY-4 (Kd = 0.63 nM at pH 7.1), were expressed in both the cytosol and the ER of wild-type MCF-7 and TamR cells. Treatment of MCF-7 and TamR cells with external Zn(2+) and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn(2+) in both cytosol and ER, suggesting that Zn(2+) was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn(2+) levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn(2+), nor in experiments in which cytosolic and ER Zn(2+) were monitored separately. In contrast to previous work using small-molecule fluorescent dyes, these results indicate that EGF-ionomycin treatment does not result in significant changes in cytosolic Zn(2+) levels as a result from Zn(2+) release from the ER. These results underline the importance of using genetically encoded fluorescent sensors to complement and verify intracellular imaging experiments with synthetic fluorescent Zn(2+) dyes.

Newport Green (NPG) is a recognized sensor of cellular Zn(2+) that displays fluorescence enhancement upon binding to Zn(2+). Because of its modest affinity for Zn(2+), the extent of its capacity to bind cellular Zn(2+) is unclear. The present study investigated the range of reactivity of NPG(ESTER) with cells, isolated (Zn)-proteome, and model Zn-proteins. The sensor accumulated in pig kidney LLC-PK1 cells and was slowly (>40 min) hydrolyzed to the fluorescent, acid form, NPG(ACID). The powerful, cell permeant Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethane-1,2-diamine (TPEN) failed to quench the growing fluorescence emission, indicating that Zn-NPG(ACID) had not formed and NPG-Zn-protein adduct species probably were not present. Furthermore, NPG(ACID) did not bind to Zn-carbonic anhydrase or Zn-alcohol dehydrogenase, two proteins that form adducts with some other sensors. Strikingly, most of the NPG(ACID) that had been converted from NPG(ESTER) was detected in the extracellular medium not the cells. As a result, after cells were incubated with NPG(ESTER) and then Zn-pyrithione to raise the internal concentration of mobile Zn(2+), Zn-NPG(ACID) was only observed in the external medium. Residual cellular NPG(ACID) was unable to bind extra intracellular Zn(2+) delivered by pyrithione. Proteome isolated from the sonicated cell supernatant was also unreactive with NPG(ACID). Titration of proteome or glutathione with Zn(2+) in the presence of NPG(ACID) revealed that NPG(ACID) only weakly competes for mobile Zn(2+) in the presence of these cellular components. In addition, when proteomic Zn(2+) was released by a nitric oxide donor or N-ethyl-maleimide, little Zn(2+) was detected by NPG(ACID). However, exposure to nitric oxide independently enhanced the fluorescence properties of NPG(ACID). Thus, the biochemical properties of NPG related to cellular Zn(2+) chelation deepen the question of how it functions as a Zn(2+) sensor.

Lineage tracing is a method that delineates all progeny produced by a single cell or a group of cells. The possibility of performing lineage tracing initiated the field of Developmental Biology and continues to revolutionize Stem Cell Biology. Here, I introduce the principles behind a successful lineage-tracing experiment. In addition, I summarize and compare different methods for conducting lineage tracing and provide examples of how these strategies can be implemented to answer fundamental questions in development and regeneration. The advantages and limitations of each method are also discussed., (© 2015 AlphaMed Press.)

To explore the effects of X-ray irradiation on mammalian cell cycle dynamics, single cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci) technique were tracked. HeLa cells expressing Fucci were used to visualise cell cycle modifications induced by irradiation. After cultured HeLa-Fucci cells were exposed to 5 Gy X-rays, fluorescent cell images were captured every 20 min for 48 h using a fluorescent microscope. Time dependence of the fluorescence intensity of S/G2 cells was analysed to examine the cell cycle dynamics of irradiated and non-irradiated control cells. The results showed that irradiated cells could be divided into two populations: one with similar cell cycle dynamics to that of non-irradiated cells, and another displaying a prolonged G2 phase. Based on these findings, it is proposed in this article that an underlying switch mechanism is involved in cell cycle regulation and the G2/M checkpoint of HeLa cells., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Fluorescent ubiquitination-based cell-cycle indicator (FUCCI) human cancer (HeLa) cells (red indicates G1; green, S/G2) were exposed to a synchrotron X-ray microbeam. Cells in either G1 or S/G2 were irradiated selectively according to their colour in the same microscopic field. Time-lapse micrographs of the irradiated cells were acquired for 24 h after irradiation. For fluorescent immunostaining, phosphorylated histone proteins (γ-H2AX) indicated the induction of DNA double-strand breaks. The cell cycle was arrested by irradiation at S/G2. In contrast, cells irradiated at G1 progressed to S/G2. The foci were induced in cells irradiated at both G1 and S/G2, suggesting that the G1-S (or S) checkpoint pathway does not function in HeLa cells due to the fact that the cells are functionally p53 deficient, even though X-ray microbeam irradiation significantly induces double-strand breaks. These results demonstrate that single FUCCI cell exposure and live cell imaging are powerful methods for studying the effects of radiation on the cell cycle., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Morphological changes in mitochondria induced by X-irradiation in normal murine mammary gland cells were studied with a live-cell microscopic imaging technique. Mitochondria were visualised by staining with a specific fluorescent probe in the cells, which express fluorescent ubiquitination-based cell-cycle indicator 2 (Fucci2) probes to visualise cell cycle. In unirradiated cells, the number of cells with fragmented mitochondria was about 20 % of the total cells through observation period (96 h). In irradiated cells, the population with fragmented mitochondria significantly increased depending on the absorbed dose. Particularly, for 8 Gy irradiation, the accumulation of fragmentation persists even in the cells whose cell cycle came to a stand (80 % in G1 (G0-like) phase). The fraction reached to a maximum at 96 h after irradiation. The kinetics of the fraction with fragmented mitochondria was similar to that for cells in S/G2/M phase (20 %) through the observation period (120 h). The evidences show that, in irradiated cells, some signals are continually released from a nucleus or cytoplasm even in the G0-like cells to operate some sort of protein machineries involved in mitochondrial fission. It is inferred that this delayed mitochondrial fragmentation is strongly related to their dysfunction, and hence might modulate radiobiological effects such as mutation or cell death., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Artemisinin-induced dormancy is a proposed mechanism for failures of monotherapy and is linked with artemisinin resistance in Plasmodium falciparum. The biological characterization and dynamics of dormant parasites are not well understood. Here we report that after dihydroartemisinin treatment in vitro, a small subset of morphologically dormant parasites was stained with rhodamine 123 (RH), a mitochondrial membrane potential marker, and persisted to recovery. RH-positive parasites sorted with fluorescence-activated cell sorting resumed growth at 10,000/well whereas RH-negative parasites failed to recover at 5 million/well. Furthermore, transcriptional activity for mitochondrial enzymes was detected only in RH-positive dormant parasites. Importantly, after treatment of dormant parasites with different concentrations of atovaquone, a mitochondrial inhibitor, the recovery of dormant parasites was delayed or stopped. This demonstrates that mitochondrial activity is critical for survival and regrowth of dormant parasites and that RH staining provides a means of identifying these parasites. These findings provide novel paths for studying and eradicating this dormant stage., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Hef is an archaeal member of the DNA repair endonuclease XPF (XPF)/Crossover junction endonuclease MUS81 (MUS81)/Fanconi anemia, complementation group M (FANCM) protein family that in eukaryotes participates in the restart of stalled DNA replication forks. To investigate the physiological roles of Hef in maintaining genome stability in living archaeal cells, we studied the localization of Hef-green fluorescent protein fusions by fluorescence microscopy. Our studies revealed that Haloferax volcanii Hef proteins formed specific localization foci under regular growth conditions, the number of which specifically increased in response to replication arrest. Purification of the full-length Hef protein from its native host revealed that it forms a stable homodimer in solution, with a peculiar elongated configuration. Altogether our data indicate that the shape of Hef, significant physicochemical constraints and/or interactions with DNA limit the apparent cytosolic diffusion of halophilic DNA replication/repair complexes, and demonstrate that Hef proteins are dynamically recruited to archaeal eukaryotic-like chromatin to counteract DNA replication stress. We suggest that the evolutionary conserved function of Hef/FANCM proteins is to enhance replication fork stability by directly interacting with collapsed replication forks.

Inteins are protein elements that covalently reassemble proteins from two precursor fragments in a process known as protein splicing. They are commonly used to reassemble a single target protein by protein splicing, but a second target protein can potentially reassemble by intein dimerization. Here, we use the naturally occurring split DnaE intein from Nostoc punctiforme (NpuDnaE) to demonstrate the simultaneous assembly of two target proteins in several examples studied with live cell imaging: yellow fluorescent protein (YFP) with monomeric red fluorescent protein (mRFP), dominant positive mutant of RhoA GTPase with YFP and GCaMP2 Ca(2+) indicator with mRFP. These examples showed the versatility of the strategy along with some interesting attributes: first, the two target proteins are in equal stoichiometry; second, the extent of protein splicing can be reported by a fluorescent protein. In particular, the split GCaMP2 with mRFP could find applications in tissue-specific Ca(2+) imaging in transgenic organisms, where mRFP could control for motion-related intensity changes.

During a 10-day culture ageing, cells of the wild-type Saccharomyces cerevisiae strain JC 482 retain their viability, while mitochondrial function and morphology change. Cell routine and uncoupled respiration rates increase to a maximum on day 4 and then decline to near zero. The decline, which occurs also in mitochondria isolated from cells of different age, is not due to increasing proportion of petites. Rhodamine 123 fluorescence intensity reporting on mitochondrial membrane potential appears to drop slightly for 4 days and then more sharply at the time when respiration rate also decreases. The MitoTracker Green fluorescent signal related to the mitochondrial content per cell also decreases. The branched tubular mitochondrial network of 1-day-old cells dissolves into short fragments; during the first 4 days, this fragmentation is associated with increasing function of mitochondria, while later on, it accompanies functional decline, which is also indicated by the decreasing ratio of Rhodamine 123 fluorescence to MitoTracker Green fluorescence. As shown by cell counting, microscopy and flow cytometry, the cell size distribution in the population broadens, and the population thus becomes more heterogeneous. The changes in respiration rate, mitochondrial membrane potential, mass and structure point to changes in the mitochondrial status during ageing., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Hypersensitive cell death is known to involve dynamic remodeling of intracellular structures that uses energy released during ATP hydrolysis. However, the relationship between intracellular structural changes and ATP levels during hypersensitive cell death remains unclear. Here, to visualize ATP dynamics directly in real time in individual living plant cells, we applied a genetically encoded Förster resonance energy transfer (FRET)-based fluorescent ATP indicator, ATeam1.03-nD/nA, for plant cells. Intracellular ATP levels increased approximately 3 h after inoculation with the avirulent strain DC3000/avrRpm1 of Pseudomonas syringae pv. tomato (Pst), which was accompanied by the simultaneous disappearance of transvacuolar strands and appearance of bulb-like structures within the vacuolar lumen. Approximately 5 h after bacterial inoculation, the bulb-like structures disappeared and ATP levels drastically decreased. After another 2 h, the large central vacuole was disrupted. In contrast, no apparent changes in intracellular ATP levels were observed in the leaves inoculated with the virulent strain Pst DC3000. The Pst DC3000/avrRpm1-induced hypersensitive cell death was strongly suppressed by inhibiting ATP synthesis after oligomycin A application within 4 h after bacterial inoculation. When the inhibitor was applied 7 h after bacterial inoculation, cell death was unaffected. These observations show that changes in intracellular ATP levels correlate with intracellular morphological changes during hypersensitive cell death, and that ATP is required just before vacuolar rupture in response to bacterial infection.

The impact of the thiol binding reagent N-ethylmaleimide (NEM) on proteomic Zn(2+) availability was investigated in rat glioma cells. Zinquin (ZQ) or TSQ, two related fluorescent sensors, were used to observe reactive Zn(2+). Control cells contained proteomic Zn(2+) but no detectable low molecular weight (LMW) Zn(2+). With either sensor, basal cellular fluorescence emission centered near 470 nm, indicative of sensor-Zn-proteins. ZQ sequestered 13% of proteomic Zn(2+) as Zn(ZQ)(2); TSQ reacted only with the Zn-proteome. NEM (100 μM) abolished LMW thiols, including glutathione (GSH) and lowered proteomic sulfhydryl content about 30%. In ZQ-treated cells, NEM exposure enhanced fluorescent intensity and the formation of Zn(ZQ)(2) (λ(MAX), 492 nm). Cells incubated with TSQ and NEM also displayed increased fluorescence without a spectral shift in wavelength maximum, consistent with increased formation of TSQ-Zn-protein adducts but not Zn(TSQ)(2). In neither experiment was Zn(2+) lost from cells. NEM altered Zn(2+) accessibility to sensors in membrane-nuclear and cytosolic fractions, but Zn(ZQ)(2) was only generated in the cytosol. Similar results were obtained when cell supernatant replaced cells. In contrast, when isolated proteome was reacted with ZQ and 100 μM NEM in the absence of GSH, 70% of the proteomic thiols underwent reaction. As a consequence, most of the ZQ-Zn-protein adducts were converted to Zn(ZQ)(2). Substituting TSQ for ZQ, only increased TSQ-Zn-proteins were observed. Evidently, the results of imaging cells with Zn(2+) sensors are dependent upon the specific chemical properties of the sensors and can only be understood after detailed chemical analysis.

Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects.

We report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (∼120 μm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.

Programmed -1 ribosomal frameshifting (-1 PRF) is a mechanism that directs elongating ribosomes to shift-reading frame by 1 base in the 5' direction that is utilized by many RNA viruses. Importantly, rates of -1 PRF are fine-tuned by viruses, including Retroviruses, Coronaviruses, Flavivriuses and in two endogenous viruses of the yeast Saccharomyces cerevisiae, to deliver the correct ratios of different viral proteins for efficient replication. Thus, -1 PRF presents a novel target for antiviral therapeutics. The underlying molecular mechanism of -1 PRF is conserved from yeast to mammals, enabling yeast to be used as a logical platform for high-throughput screens. Our understanding of the strengths and pitfalls of assays to monitor -1 PRF have evolved since the initial discovery of -1 PRF. These include controlling for the effects of drugs on protein expression and mRNA stability, as well as minimizing costs and the requirement for multiple processing steps. Here we describe the development of an automated yeast-based dual fluorescence assay of -1 PRF that provides a rapid, inexpensive automated pipeline to screen for compounds that alter rates of -1 PRF which will help to pave the way toward the discovery and development of novel antiviral therapeutics.

A novel proton-fueled molecular gate-like delivery system has been constructed for controlled cargo release using i-motif quadruplex DNA as caps onto pore outlets of mesoporous silica nanoparticles. Start from simple conformation changes, the i-motif DNA cap can open and close the pore system in smart response to pH stimulus. Importantly, the opening/closing and delivery protocol is highly reversible and a partial cargo delivery can be easily controlled at will. A pH-switchable nanoreactor has also been developed to validate the potential of our system for on-demand molecular transport. This proof of concept might open the door to a new generation of carrier materials and could also provide a general route to use other functional nucleic acids/peptide nucleic acids as capping agents in the fields of versatile controlled delivery nanodevices.

Although micronuclei (MNi) have been extensively used to evaluate genotoxic effects and chromosome instability, the most basic issue regarding their formation was not completely addressed until recently, due to limitations of traditional experimental methods. The development of live-cell imaging, combined with genetically engineered chromosome labelling techniques makes it possible to investigate the origin of a micronucleus in a single cell in a real-time and high-throughput manner. Here, we review all the available studies on the origins of MNi in live cells and discuss novel findings based on this recently emerged methodology. Some unsolved questions on MNi formation and limitations of live-cell imaging in the investigation of MNi have also been discussed.

O(6)-Methylguanine (O(6)-meG), which is produced in DNA following exposure to methylating agents, instructs human RNA polymerase II to mis-insert bases opposite the lesion during transcription. In this study, we examined the effect of O(6)-meG on transcription in human cells and investigated the subsequent effects on protein function following translation of the resulting mRNA. In HEK293 cells, O(6)-meG induced incorporation of uridine or cytidine in nascent RNA opposite the adduct. In cells containing active O(6)-alkylguanine-DNA alkyltransferase (AGT), which repairs O(6)-meG, 3% misincorporation of uridine was observed opposite the lesion. In cells where AGT function was compromised by addition of the AGT inhibitor O(6)-benzylguanine, ∼ 58% of the transcripts contained a uridine misincorporation opposite the lesion. Furthermore, the altered mRNA induced changes to protein function as demonstrated through recovery of functional red fluorescent protein (RFP) from DNA coding for a non-fluorescent variant of RFP. These data show that O(6)-meG is highly mutagenic at the level of transcription in human cells, leading to an altered protein load, especially when AGT is inhibited.

An array of nano-channels was fabricated from silicon based semiconductor materials to stretch long, native dsDNA. Here we present a labeling scheme in which it is possible to identify the location of specific sequences along the stretched DNA molecules. The scheme proceeds by first using the strand displacement activity of the Vent (exo-) polymerase to generate single strand flaps on nicked dsDNA. These single strand flaps are hybridized with sequence specific fluorophore-labeled probes. Subsequent imaging of the DNA molecules inside a nano-channel array device allows for quantitative identification of the location of probes. The highly efficient DNA hybridization on the ss-DNA flaps is an excellent method to identify the sequence motifs of dsDNA as it gives us unique ability to control the length of the probe sequence and thus the frequency of hybridization sites on the DNA. We have also shown that this technique can be extended to a multi color labeling scheme by using different dye labeled probes or by combining with a DNA- polymerase-mediated incorporation of fluorophore-labeled nucleotides on nicking sites. Thus this labeling chemistry in conjunction with the nano-channel platform can be a powerful tool to solve complex structural variations in DNA which is of importance for both research and clinical diagnostics of genetic diseases.

Background: Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential., Methods: In the first experimental series thin slices of cryopreserved and fresh ovine cortical tissue were incubated in 50 μg/ml NR and assessed for the presence of red colouration. Slices were then used for follicular structure isolation and viability evaluation using 5-(and 6)-carboxyfluoresceindiacetate succinimidylester (CFDA-SE), or prepared histologically for follicle counting or evaluation of apoptosis via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL). An additional subset of slices were cultured for 8 days, followed by re-evaluation of follicle viability. NR staining was further assessed in a pilot study using thin slices of cryopreserved human ovarian tissue donated by 17 patients undergoing laparoscopic sterilisation or elective Caesarean section., Results: In both ovine and human ovarian cortex NR concentrated in follicular structures within weakly stained stroma. NR colouration was observed in 41.7 ± 4.6% of cryopreserved and 49.3 ± 6.5% of the fresh ovine tissue slices, and NR staining was consistently predictive of the presence of follicles. Non-stained ovine slices contained highly apoptotic follicles, while lower levels of apoptosis were observed in NR positive slices, indicating preferential detection of viable follicles by NR. Following culture the majority of ovine slices re-stained with NR, no significant increases in the levels of apoptosis were observed and 94.6 ± 3.1% of follicles were viable by CFDA-SE. In the human study, NR identified follicles in 19.3 ± 3.7% of tissue slices, and follicle density tended to decrease with advancing patient age., Conclusions: NR predicts viable follicle density in situ in slices of ovine and human ovarian cortex. Furthermore incubation of tissue in NR prior to culture does not compromise subsequent follicle survival in vitro, indicating the potential suitability of this approach in fertility preservation regimes.

The mechanism responsible for proteinuria in non-genetic idiopathic nephrotic syndrome (iNS) is unknown. Animal models suggest an effect of free radicals on podocytes, and indirect evidence in humans confirm this implication. We determined the oxidative burst by blood CD15+ polymorphonucleates (PMN) utilizing the 5-(and-6)-carboxy-2',7'-dichlorofluorescin diacetate (DCF-DA) fluorescence assay in 38 children with iNS. Results were compared with PMN from normal subjects and patients with renal pathologies considered traditionally to be models of oxidative stress [six anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis, seven post-infectious glomerulonephritis]. Radicals of oxygen (ROS) production was finally determined in a patient with immunodeficiency, polyendocrinopathy, enteropathy X-linked (IPEX) and in seven iNS children after treatment with Rituximab. Results demonstrated a 10-fold increase of ROS production by resting PMN in iNS compared to normal PMN. When PMN were separated from other cells, ROS increased significantly in all conditions while a near-normal production was restored by adding autologous cells and/or supernatants in controls, vasculitis and post-infectious glomerulonephritis but not in iNS. Results indicated that the oxidative burst was regulated by soluble factors and that this regulatory circuit was altered in iNS. PMN obtained from a child with IPEX produced 100 times more ROS during exacerbation of clinical symptoms and restored to a near normal-level in remission. Rituximab decreased ROS production by 60%. In conclusion, our study shows that oxidant production is increased in iNS for an imbalance between PMN and other blood cells. Regulatory T cells (Tregs) and CD20 are probably involved in this regulation. Overall, our observations reinforce the concept that oxidants deriving from PMN are implicated in iNS.

Motivation: Proteins exhibit complex subcellular distributions, which may include localizing in more than one organelle and varying in location depending on the cell physiology. Estimating the amount of protein distributed in each subcellular location is essential for quantitative understanding and modeling of protein dynamics and how they affect cell behaviors. We have previously described automated methods using fluorescent microscope images to determine the fractions of protein fluorescence in various subcellular locations when the basic locations in which a protein can be present are known. As this set of basic locations may be unknown (especially for studies on a proteome-wide scale), we here describe unsupervised methods to identify the fundamental patterns from images of mixed patterns and estimate the fractional composition of them., Methods: We developed two approaches to the problem, both based on identifying types of objects present in images and representing patterns by frequencies of those object types. One is a basis pursuit method (which is based on a linear mixture model), and the other is based on latent Dirichlet allocation (LDA). For testing both approaches, we used images previously acquired for testing supervised unmixing methods. These images were of cells labeled with various combinations of two organelle-specific probes that had the same fluorescent properties to simulate mixed patterns of subcellular location., Results: We achieved 0.80 and 0.91 correlation between estimated and underlying fractions of the two probes (fundamental patterns) with basis pursuit and LDA approaches, respectively, indicating that our methods can unmix the complex subcellular distribution with reasonably high accuracy., Availability: http://murphylab.web.cmu.edu/software.

Cyanobacteria are suitable for sustainable, solar-powered biotechnological applications. Synthetic biology connects biology with computational design and an engineering perspective, but requires efficient tools and information about the function of biological parts and systems. To enable the development of cyanobacterial Synthetic Biology, several molecular tools were developed and characterized: (i) a broad-host-range BioBrick shuttle vector, pPMQAK1, was constructed and confirmed to replicate in Escherichia coli and three different cyanobacterial strains. (ii) The fluorescent proteins Cerulean, GFPmut3B and EYFP have been demonstrated to work as reporter proteins in cyanobacteria, in spite of the strong background of photosynthetic pigments. (iii) Several promoters, like P(rnpB) and variants of P(rbcL), and a version of the promoter P(trc) with two operators for enhanced repression, were developed and characterized in Synechocystis sp. strain PCC6803. (iv) It was shown that a system for targeted protein degradation, which is needed to enable dynamic expression studies, is working in Synechocystis sp. strain PCC6803. The pPMQAK1 shuttle vector allows the use of the growing numbers of BioBrick parts in many prokaryotes, and the other tools herein implemented facilitate the development of new parts and systems in cyanobacteria.

Circadian clocks have long been known to be essential for the maintenance of physiological and behavioral processes in a variety of organisms ranging from plants to humans. Dysfunctions that subvert gene expression of oscillatory circadian-clock components may result in severe pathologies, including tumors and metabolic disorders. While the underlying molecular mechanisms and dynamics of complex gene behavior are not fully understood, synthetic approaches have provided substantial insight into the operation of complex control circuits, including that of oscillatory networks. Using iterative cycles of mathematical model-guided design and experimental analyses, we have developed a novel low-frequency mammalian oscillator. It incorporates intronically encoded siRNA-based silencing of the tetracycline-dependent transactivator to enable the autonomous and robust expression of a fluorescent transgene with periods of 26 h, a circadian clock-like oscillatory behavior. Using fluorescence-based time-lapse microscopy of engineered CHO-K1 cells, we profiled expression dynamics of a destabilized yellow fluorescent protein variant in single cells and real time. The novel oscillator design may enable further insights into the system dynamics of natural periodic processes as well as into siRNA-mediated transcription silencing. It may foster advances in design, analysis and application of complex synthetic systems in future gene therapy initiatives.

Computational modeling of biological systems has become an effective tool for analyzing cellular behavior and for elucidating key properties of the intricate networks that underlie experimental observations. While most modeling techniques rely heavily on the concentrations of intracellular molecules, little attention has been paid to tracking and simulating the significant volume fluctuations that occur over each cell division cycle. Here, we use fluorescence microscopy to acquire single cell volume trajectories for a large population of Saccharomyces cerevisiae cells. Using this data, we generate a comprehensive set of statistics that govern the growth and division of these cells over many generations, and we discover several interesting trends in their size, growth and protein production characteristics. We use these statistics to develop an accurate model of cell cycle volume dynamics, starting at cell birth. Finally, we demonstrate the importance of tracking volume fluctuations by combining cell division dynamics with a minimal gene expression model for a constitutively expressed fluorescent protein. The significant oscillations in the cellular concentration of a stable, highly expressed protein mimic the observed experimental trajectories and demonstrate the fundamental impact that the cell cycle has on cellular functions.

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments.

The purpose of this study was to evaluate the use of rhodamine B as an orally delivered biomarker for rodents and a feed-through transtadial biomarker for phlebotomine sand flies (Diptera: Psychodidae). Rhodamine B-treated hamsters were visibly marked for up to 8 wk, and their feces were fluorescent when examined under a fluorescence microscope. The development and survival of sand fly larvae fed feces of rhodamine B-treated hamsters were not significantly different from control sand flies. Adult male and female sand flies, that had been fed as larvae the feces of rhodamine B-treated hamsters, were fluorescent when examined using fluorescent microscopy and could be distinguished from control sand flies. Adult female sand flies that took bloodmeals from rhodamine B-treated hamsters were fluorescent when examined immediately after feeding. Rhodamine B incorporated rodent baits could be used to detect adult male and female sand flies that fed on the feces of baited rodents as larvae, or adult female sand flies that have taken a bloodmeal from bait-fed rodents. This would allow the delineation of specific foci with rodent-sand fly associations that would be susceptible to control by using feed-through or systemic insecticides.

Background: The inherent fluorescent properties of nucleosides, nucleotides, and nucleic acids are limited, and thus the need has arisen for fluorescent labeling of these molecules for a variety of analytical applications., Content: This review traces the analytical ancestry of fluorescent labeling of nucleosides, nucleotides, and nucleic acids, with an emphasis on the first to publish or patent. The scope of labeling includes (a) direct labeling by covalent labeling of nucleic acids with a fluorescent label or noncovalent binding or intercalation of a fluorescent dye to nucleic acids and (b) indirect labeling via covalent attachment of a secondary label to a nucleic acid, and then binding this to a fluorescently labeled ligand binder. An alternative indirect strategy involves binding of a nucleic acid to a nucleic acid binder molecule (e.g., antibody, antibiotic, histone, antibody, nuclease) that is labeled with a fluorophore. Fluorescent labels for nucleic acids include organic fluorescent dyes, metal chelates, carbon nanotubes, quantum dots, gold particles, and fluorescent minerals., Summary: Fluorescently labeled nucleosides, nucleotides, and nucleic acids are important types of reagents for biological assay methods and underpin current methods of chromosome analysis, gel staining, DNA sequencing and quantitative PCR. Although these methods use predominantly organic fluorophores, new types of particulate fluorophores in the form of nanoparticles, nanorods, and nanotubes may provide the basis of a new generation of fluorescent labels and nucleic acid detection methods.

The extent to which a carrier powder was taken up and horizontally transferred by contaminated Indianmeal moth, Plodia interpunctella (Hübner) individuals to conspecifics was evaluated. Using a marker dye, which was shown to be colocalized with the carrier powder, the amount of powder on the Indianmeal moth, as determined by spectrophotometry, was correlated with quantification based on a novel image analysis approach that enabled differentiation of powder uptake by body region. Over a 48-h period, more powder was retained on the ventral surface than the dorsal side, with the head region showing the greatest amount of powder uptake and retention. During courtship, powder was horizontally transferred by treated males to 1.6% of the head and 0.06% of the untreated females' body. To inoculate moths, a pit-fall style autodissemination station was determined as being more effective than a powder tray, as significantly more material was taken up to the key areas of the moth's body where powder is more effectively retained and more likely to be horizontally transferred. Additionally, a pit-fall station prevented moths from exhibiting avoidance behavior from the powder, which was frequently encountered if they had to walk into a powder tray. This study shows that different regions of the Indianmeal moth body vary in capacity to carry powder and that future research efforts should target these specific regions. Although the proposed autodissemination system was optimized for management strategies of Indianmeal moth, we believe the results presented here can be used to develop novel management strategies for other insect pests.

Tetraalkylammonium (TAA) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (PCR) detected by end-point analysis. In this study, we compared the ability of various TAA derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time PCR based on fluorescence detection from intercalating dye SYBR Green I (SGI). Our data indicate that TAA chlorides and some other TAA derivatives serve as potent enhancers of SGI-monitored real-time PCR. Optimal results were obtained with 10-16 mM tetrapropylammonium chloride. The effect of TAA compounds was dependent on the nature of counter ions present and composition of the reaction mixtures used. Based on measurements of SGI-generated fluorescence signal in the presence of PCR-amplified DNA fragments, oligonucleotide primers and/or various additives, we propose that TAA-derivatives reduce the binding of SGI to oligonucleotide primers and thus enhance primer-template interactions during annealing phase. Furthermore, these compounds serve as stabilizers of SGI-containing PCR mixtures. The combined data indicate that TAA derivatives might be a new class of additives contributing to robustness of real-time PCR monitored by asymmetrical cyanine dye SGI.

Fluorescent microscopy experiments show that when 2'-O-methyl-modified molecular beacons (MBs) are introduced into NIH/3T3 cells, they elicit a nonspecific signal in the nucleus. This false-positive signal can be avoided by conjugating MBs to macromolecules (e.g. NeutrAvidin) that prevent nuclear sequestration, but the presence of a macromolecule makes efficient cytosolic delivery of these probes challenging. In this study, we explored various methods including TAT peptide, Streptolysin O and microporation for delivering NeutrAvidin-conjugates into the cytosol of living cells. Surprisingly, all of these strategies led to entrapment of the conjugates within lysosomes within 24 h. When the conjugates were pegylated, to help prevent intracellular recognition, only microporation led to a uniform cytosolic distribution. Microporation also yielded a transfection efficiency of 93% and an average viability of 86%. When cells microporated with MB-NeutrAvidin conjugates were examined via flow cytometry, the signal-to-background was found to be more than 3 times higher and the sensitivity nearly five times higher than unconjugated MBs. Overall, the present study introduces an improved methodology for the high-throughput detection of RNA at the single cell level.

Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the associated [RFP] expressed from an inducible plasmid. We find that the FCS method allows us to reliably monitor in real-time [mRNA] down to approximately 40 nM (i.e. approximately two transcripts per volume of detection). To validate these measurements, we show that [mRNA] is proportional to the associated expression of the RFP protein. This FCS-based technique establishes a framework for minimally invasive measurements of mRNA concentration in individual living bacteria.

How folding of proteins is coupled to their synthesis remains poorly understood. Here, we apply single-molecule fluorescence imaging to full protein synthesis in vitro. Ribosomes were specifically immobilized onto glass surfaces and synthesis of green fluorescent protein (GFP) was achieved using modified commercial Protein Synthesis using Recombinant Elements that lacked ribosomes but contained purified factors and enzyme that are required for translation in Escherichia coli. Translation was monitored using a GFP mutant (F64L/S65T/F99S/M153T/V163A) that has a high fluorophore maturation rate and that contained the Secretion Monitor arrest sequence to prevent dissociation from the ribosome. Immobilized ribosomal subunits were labeled with Cy3 and GFP synthesis was measured by colocalization of GFP fluorescence with the ribosome position. The rate of appearance of colocalized ribosome GFP was equivalent to the rates of fluorescence appearance coupled with translation measured in bulk, and the ribosome-polypeptide complexes were stable for hours. The methods presented here are applicable to single-molecule investigation of translational initiation, elongation and cotranslational folding.

Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.