Rapid RNA detection through intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction without amplification

The fluorescent dye 1-anilinonapthalene-8-sulfonic acid (ANS) has been used as a probe of changes in membrane conformation accompanying excitation of the electroplax ofElectrophorus electricus. ANS binds reversibly to the excitable membrane at rest. During generation of an action potential, an increase in ANS-fluorescence intensity is observed which resembles but does not strictly follow the membrane potential. Experiments using the current-clamp technique have demonstrated a linear relationship between the change in membrane potential and the change in ANS fluorescence intensity. The change in fluorescence intensity is not a consequence of binding to membrane sites of increased affinity nor of an electrophoretic concentration of ANS molecules at the membrane surface.It is not known whether the change in fluorescence intensity is due to a change in quantum yield of bound ANS or to an increase in the amount of bound ANS. In either case, the change in fluorescence intensity may be interpreted as a change in membrane conformation.

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.

Although the pathophysiology of diabetes is characterized by elevated levels of glucose and long-chain fatty acids (LCFA), nuclear mechanisms linking glucose and LCFA metabolism are poorly understood. As the liver fatty acid binding protein (L-FABP) shuttles LCFA to the nucleus, where L-FABP directly interacts with peroxisome proliferator-activated receptor-α (PPARα), the effect of glucose on these processes was examined. In vitro studies showed that L-FABP strongly bound glucose and glucose-1-phosphate (K(d) = 103 ± 19 nM and K(d) = 20 ± 3 nM, respectively), resulting in altered L-FABP conformation, increased affinity for lipid ligands, and enhanced interaction with PPARα. In living cells, glucose stimulated cellular uptake and nuclear localization of a nonmetabolizable fluorescent fatty acid analog (BODIPY C-16), particularly in the presence of L-FABP. These data suggest for the first time a direct role of glucose in facilitating L-FABP-mediated uptake and distribution of lipidic ligands to the nucleus for regulation of PPARα transcriptional activity.

MQAE is a 'non-ratiometric' chloride ion (Cl-)-quenched fluorescent indicator that is used to determine intracellular Cl- concentration ([Cl-]i). MQAE-based two-photon microscopy is reported to be a useful method to measure [Cl-]i, but it is still controversial because a change in cell volume may alter the MQAE concentration, leading to a change in the fluorescence intensity without any change in [Cl-]i. In an attempt to elucidate the effect or lack of effect of cell volume on MQAE concentration, we studied the effects of changes in cell volume, achieved by applying different levels of osmotic stress, on the intensity of MQAE fluorescence in airway ciliary cells. To study solely the effect of changes in cell volume on MQAE fluorescence intensity, i.e., excluding the effect of any change in [Cl-]i, we first conducted the experiments in a Cl--free nitrate (NO3-) solution to substitute NO3- (non-quenching anion for MQAE fluorescence) for Cl- in the intracellular fluid. Hypo- (-30mM NaNO3) or hyper-osmotic stress (+30mM NaNO3) effected changes in cell volume, but the stress did not result in any significant change in MQAE fluorescence intensity. The experiments were also carried out in Cl--containing solution. Hypo-osmotic stress (-30mM NaCl) increased both MQAE fluorescence intensity and cell volume, while hyper-osmotic stress (+30mM NaCl) decreased both of these properties. These results suggest that the osmotic stress-induced change in MQAE fluorescence intensity was caused by the change in [Cl-]i and not by the MQAE concentration. Moreover, the intracellular distribution of MQAEs was heterogeneous and not affected by the changes in osmotic stress-induced cell volume, suggesting that MQAEs are bound to un-identified subcellular structures. These bound MQAEs appear to have enabled the measurement of [Cl-]i in airway ciliary cells, even under conditions of cell volume change.

Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions.

Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions.

Short communication: Changes in fluorescence intensity induced by soybean soluble polysaccharide–milk protein interactions during acidification

Quantitative measurement of regional signal intensity changes of high grade gliomas following radiotherapy using T1-weighted contrast enhanced MRI imaging

The PilB protein of the Neisseria genus comprises three domains. Two forms have been recently reported to be produced in vivo. One form, containing the three domains, is secreted from the bacterial cytoplasm to the outer membrane, whereas the second form, which is cytoplasmic, only contains the central and the C-terminal domains. The secreted form was shown to be involved in survival under oxidative conditions. Although previous studies indicated that the central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively, no function was described so far for the N-terminal domain. In the present study, the N-terminal domain of the PilB of Neisseria meningitidis was produced as a folded entity, and its biochemical and enzymatic properties have been determined. The data show that the N-terminal domain possesses a disulfide redox-active site with a redox potential in the range of that of thioredoxin. Moreover, the N-terminal domain, either as an isolated form or included in PilB, recycles the oxidized forms of the methionine sulfoxide reductases like thioredoxin. These results, which show that the N-terminal domain exhibits a disulfide reductase activity and probably has a thioredoxin-fold, are discussed in relation to its possible functional role in Neisseria.

The photolysis-induced changes in the protein fluorescence intensity (at 320 nm) during the proton-pumping cycle of bacteriorhodopsin were examined by a delayed two-pulse technique in the time range 1 microsecond-20 msec at room temperature. No detectable change in the protein fluorescence intensity was observed on the earliest time scale within the lifetime of the intermediate K590, when retinal apparently undergoes the largest structural changes. The time dependence of the relative changes in fluorescence intensity did, however, display a close correlation with the population of the L550 and M412 intermediates. From a computer numerical fit of the data, with available published kinetic parameters, the protein fluorescence quantum yields of the K590, L550, and M412 intermediates are found to be 1.0, 0.92, and 0.80 of that for native bR570, respectively. The probable mechanisms of the observed fluorescence quenching during the photochemical cycle are qualitatively discussed.

A common method to evaluate turnover rate in the stratum corneum is to measure the change in fluorescence intensity with time after dyeing the stratum corneum with fluorescent pigments. If these changes in fluorescence over time are carefully observed, the rate of decline in fluorescence intensity differs among different small areas on the skin surface. A possible relationship between these differences and dry skin has been reported. The purpose of this research was to develop a method for analyzing turnover rate in the stratum corneum in each small area on the surface of the skin as well as to investigate the variations in the inconsistencies of turnover rate. The stratum corneum at six body regions (forehead, cheek, forearm, opisthenar, back and lower leg) was dyed with dansyl chloride (DC), and the change in fluorescence intensity over time was imaged with a highly sensitive television camera through special filters. Then, the fluorescent distribution in the images was analyzed to measure the change in fluorescence intensity with time among the small areas. Also, the decline in fluorescence intensity observed was categorized using specific characteristics into six different types. By attaching a filter to an ultraviolet (UV) light source in order to transmit light at the excitation wavelength and a filter to the camera lens to transmit light at the wavelength of DC fluorescence, we could image the low intensity fluorescent light from the DC without interference from the UV light exciting the DC. The characteristics of the variation in the decline in fluorescence intensity were categorized into six patterns. Type I: pattern showing a uniform decline in fluorescence intensity. Type II: pattern showing sporadic areas where fluorescence intensity declines quickly. Type III: pattern showing relatively large areas where fluorescence intensity declines slowly. Type IV: pattern showing sporadic areas of fluorescence intensity, matched with locations of keratotic plugs. Type V: pattern showing sporadic fluorescent areas, not matched with locations of keratotic plugs. Type VI: pattern showing a partial, drastic decline in fluorescence intensity occurring on inflamed skin after sunburn. By analyzing the image generated from a highly sensitive television camera equipped with special filters, we could measure turnover rate of the stratum corneum at any small area. The variations in Types IV and V were believed to be derived from keratotic plugs and closed comedo. Except for Type VI, observed on significant skin inflammation, Type II and Type III were believed to be the patterns that reflected variations in turnover rate in stratum corneum itself.

In recent years, intense research has been carried out worldwide with the goal of developing simple, sensitive, and specific detection tools for biomedical applications. Along these lines, we reported in 2002 on cationic polythiophene derivatives able to provide ultrasensitive detection levels and the capability to distinguish perfect matches from oligonucleotides having as little as a single base mismatch. It was shown that the intrinsic fluorescence of the random-coil polymers quenches as a result of the planar, highly conjugated conformation adopted by the polymers when complexed with a single-strand DNA (ssDNA) capture probe but increases again after hybridization with the perfectly matched complementary strand. This change in fluorescence intensity is mainly due to a modification in the delocalization of pi electrons along the carbon chain backbone that occurs when switching between the two conformations. Thus, by monitoring, via the change in fluorescence intensity, the hybridization of the complementary ssDNA target with the "duplex", one could detect as little as 220 complementary target molecules in a 150 microL sample volume (0.36 zmol) in less than 1 hour. Building on this initial concept, we then reported that tagging the DNA probe with a suitable fluorophore dramatically increases the detection sensitivity. This novel molecular system involves the self-assembly of aggregates of duplexes in solution, prior to the introduction of the target, which allows a highly efficient resonance energy transfer (RET) between a "donor" (being the complex formed of the DNA double helix and the polymer chain wrapped around it) and a large number of neighboring "acceptors" (the fluorophores attached to the DNA probes). The massive intrinsic signal amplification (fluorescence chain reaction or FCR) provided by this novel integrated molecular system allows the specific detection of as little as five dsDNA copies in a 3 mL sample volume in only 5 minutes, without the need for prior amplification of the target. Clearly, direct and reliable detection of DNA hybridization without prior PCR amplification or chemical tagging of the genetic target is now possible with this methodology. We have also shown that proteins can be detected following a similar strategy. Impressive results have also been reported by direct and specific staining of targeted proteins. All these features have recently allowed the development of responsive polymeric supports for the detection of DNA and proteins. All these assays that do not require any chemical manipulation of the biological targets or sophisticated experimental procedures should soon lead to major advances in genomics and proteomics.

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.

Chemical modification of the internal surfaces of cylindrical pores with submicrometer pore diameter in a poly(ethylene terephthalate) (PET) film was examined. The modification involved the alkylation of the carboxylic acid on the surfaces with the alkylation reagent containing a fluorescent probe, and it was monitored by observing the change in fluorescent emission intensity. When the N,N-dimethylformamide solution of 4-(bromomethyl)-6,7-dimethoxycoumarin (BrCU), which bore a coumarin fluorophore, was introduced into the pores, the emission and excitation intensities of the membranes increased proportionally with increases of the pore surface areas. Fluorescent spots about 300 nm in diameter, which were located at the positions of the pores, can be observed in the fluorescence microscope image of the membranes, indicating that highly concentrated fluorescent probes are chemically incorporated on the internal surfaces of the cylindrical pores with 210 nm diameter in the membranes. In the reactions of the PET surfaces with BrCU, the fluorescent intensities increased with increases of the contact angles. This result indicates that the hydrophilicity of the internal pore surfaces can be qualitatively modified by controlling the change in the fluorescent intensities.

Highly advanced phase-change hybrids (PCHs), which consist of a phase-change material and conjugated polymer, were developed for new sensor and actuator applications. PCH films with excellent characteristics were obtained simply by depositing various molten paraffin waxes (PWs) in situ onto poly(diphenylacetylene) (PDPA) films with extremely large fractional free volumes. The phase-change enthalpy of the PWs in the hybrid films was quite high and remained constant over prolonged use. The PCH films underwent critical changes in both fluorescence (FL) intensity and color during the phase change of the PWs, which facilitated various sensor applications such as highly reversible writing/erasing, fingerprinting and array-type thermometer usage. In addition, a biaxially oriented polypropylene (BOPP)-supported PCH film exhibited extremely fast and highly reproducible thermomechanical actuation with reversible curling/uncurling during the phase change of the PWs. These findings will be useful for developing novel PCH materials with highly advanced functions and applications. South Korean scientists have formed a touch-responsive material that can record or remove bright, fluorescent images of human fingerprints. Giseop Kwak from Kyungpook National Univeristy and colleagues improved the optical capabilities of parrafin wax — a ‘phase change’ material that easily melts and releases thermal energy — by mixing it into the numerous microvoids within poly(diphenylacetylene) (PDPA), a fluorescent polymer. When the researchers touched the hybrid polymer with objects (a stamp or fingertip, for example) hotter than 25 °C, fluorescent emission ceased at the contact points due to melted wax disrupting the PDPA structure. This produced high-resolution, photonegative images, which disappeared on cooling the film. By combining their new material with a tough, polypropylene film, the team also built a thermal actuator that twists or uncurls in response to different temperatures. Multifunctional phase-change hybrid materials containing paraffin waxes (PWs) within a microporous conjugated polymer (CP) film were developed for new sensor and actuator applications. The hybrid films were prepared simply by depositing various PWs onto CP films to show critical changes in FL intensity and color during the phase change of PWs. This fascinating FL response behavior to external heat facilitated various applications such as reversible writing/erasing, fingerprinting and thermometer sensors. An appropriate layer-supported hybrid film showed extremely fast and highly reproducible thermomechanical actuation.