Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation.

Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.

Optogenetically engineered Ca2+ oscillation-mediated DRP1 activation promotes mitochondrial fission and cell death.

Mitochondrial dynamics regulate the quality and morphology of mitochondria. Calcium (Ca2+) plays an important role in regulating mitochondrial function. Here, we investigated the effects of optogenetically engineered Ca2+ signaling on mitochondrial dynamics. More specifically, customized illumination conditions could trigger unique Ca2+ oscillation waves to trigger specific signaling pathways. In this study, we found that modulating Ca2+ oscillations by increasing the light frequency, intensity and exposure time could drive mitochondria toward the fission state, mitochondrial dysfunction, autophagy and cell death. Moreover, illumination triggered phosphorylation at the Ser616 residue but not the Ser637 residue of the mitochondrial fission protein, dynamin-related protein 1 (DRP1, encoded by DNM1L), via the activation of Ca2+-dependent kinases CaMKII, ERK and CDK1. However, optogenetically engineered Ca2+ signaling did not activate calcineurin phosphatase to dephosphorylate DRP1 at Ser637. In addition, light illumination had no effect on the expression levels of the mitochondrial fusion proteins mitofusin 1 (MFN1) and 2 (MFN2). Overall, this study provides an effective and innovative approach to altering Ca2+ signaling for controlling mitochondrial fission with a more precise resolution than pharmacological approaches in the temporal dimension.

The efficient induction of human retinal ganglion-like cells provides a platform for studying optic neuropathies.

Retinal ganglion cells (RGCs) are essential for vision perception. In glaucoma and other optic neuropathies, RGCs and their optic axons undergo degenerative change and cell death; this can result in irreversible vision loss. Here we developed a rapid protocol for directly inducing RGC differentiation from human induced pluripotent stem cells (hiPSCs) by the overexpression of ATOH7, BRN3B, and SOX4. The hiPSC-derived RGC-like cells (iRGCs) show robust expression of various RGC-specific markers by whole transcriptome profiling. A functional assessment was also carried out and this demonstrated that these iRGCs display stimulus-induced neuronal activity, as well as spontaneous neuronal activity. Ethambutol (EMB), an effective first-line anti-tuberculosis agent, is known to cause serious visual impairment and irreversible vision loss due to the RGC degeneration in a significant number of treated patients. Using our iRGCs, EMB was found to induce significant dose-dependent and time-dependent increases in cell death and neurite degeneration. Western blot analysis revealed that the expression levels of p62 and LC3-II were upregulated, and further investigations revealed that EMB caused a blockade of lysosome-autophagosome fusion; this indicates that impairment of autophagic flux is one of the adverse effects of that EMB has on iRGCs. In addition, EMB was found to elevate intracellular reactive oxygen species (ROS) levels increasing apoptotic cell death. This could be partially rescued by the co-treatment with the ROS scavenger NAC. Taken together, our findings suggest that this iRGC model, which achieves both high yield and high purity, is suitable for investigating optic neuropathies, as well as being useful when searching for potential drugs for therapeutic treatment and/or disease prevention.

Comparing the effects of chemical Ca2+ dyes and R-GECO on contractility and Ca2+ transients in adult and human iPSC cardiomyocytes.

We compared commonly used BAPTA-derived chemical Ca2+ dyes (fura2, Fluo-4, and Rhod-2) with a newer genetically encoded indicator (R-GECO) in single cell models of the heart. We assessed their performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca2+ dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca2+ handling caused by small molecule modulators of contractility and intracellular ionic homeostasis (mavacamten, levosimendan, and flecainide), but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca2+ handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca2+ transients derived from BAPTA-like chemical Ca2+ dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.

Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging.

Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.

Measurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations.

RATIONALE: Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here, we develop and characterize genetically encoded Ca2+ indicators restricted to the myofilament to directly visualize Ca2+ changes in the sarcomere. OBJECTIVE: To produce and validate myofilament-restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil, and levosimendan) or following cotransduction of 2 established hypertrophic cardiomyopathy disease-causing mutants (cTnT [Troponin T] R92Q and cTnI [Troponin I] R145G) that alter myofilament Ca2+ handling. METHODS AND RESULTS: When expressed in adult ventricular cardiomyocytes RGECO-TnT (Troponin T)/TnI (Troponin I) sensors localize correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Coadenoviral transduction of RGECO-TnT/TnI with hypertrophic cardiomyopathy causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the 2 mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+. CONCLUSIONS: RGECO-TnT/TnI are functionally equivalent. They visualize Ca2+ within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.

Soil Buffering Capacity Can Be Used To Optimize Biostimulation of Psychrotrophic Hydrocarbon Remediation.

Effective bioremediation of hydrocarbons requires innovative approaches to minimize phosphate precipitation in soils of different buffering capacities. Understanding the mechanisms underlying sustained stimulation of bacterial activity remains a key challenge for optimizing bioremediation-particularly in northern regions. Positron emission tomography (PET) can trace microbial activity within the naturally occurring soil structure of intact soils. Here, we use PET to test two hypotheses: (1) optimizing phosphate bioavailability in soil will outperform a generic biostimulatory solution in promoting hydrocarbon remediation and (2) oligotrophic biostimulation will be more effective than eutrophic approaches. In so doing, we highlight the key bacterial taxa that underlie aerobic and anaerobic hydrocarbon degradation in subarctic soils. In particular, we showed that (i) optimized phosphate bioavailability outperformed generic biostimulatory solutions in promoting hydrocarbon degradation, (ii) oligotrophic biostimulation is more effective than eutrophic approaches, and (iii) optimized biostimulatory solutions stimulated specific soil regions and bacterial consortia. The knowledge gleaned from this study will be crucial in developing field-scale biodegradation treatments for sustained stimulation of bacterial activity in northern regions.

Correction to: A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.

Monitoring the Heavy Metal Lead Inside Living Drosophila with a FRET-Based Biosensor.

The harmful impact of the heavy metal lead on human health has been known for years. However, materials that contain lead remain in the environment. Measuring the blood lead level (BLL) is the only way to officially evaluate the degree of exposure to lead. The so-called "safe value" of the BLL seems to unreliably represent the secure threshold for children. In general, lead's underlying toxicological mechanism remains unclear and needs to be elucidated. Therefore, we developed a novel genetically encoded fluorescence resonance energy transfer (FRET)-based lead biosensor, Met-lead, and applied it to transgenic Drosophila to perform further investigations. We combined Met-lead with the UAS-GAL4 system to the sensor protein specifically expressed within certain regions of fly brains. Using a suitable imaging platform, including a fast epifluorescent or confocal laser-scanning/two-photon microscope with high resolution, we recorded the changes in lead content inside fly brains ex vivo and in vivo and at different life stages. The blood-brain barrier was found to play an important role in the protection of neurons in the brain against damage due to the heavy metal lead, either through food or microinjection into the abdomen. Met-lead has the potential to be a powerful tool for the sensing of lead within living organisms by employing either a fast epi-FRET microscope or high-resolution brain imaging.

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

BACKGROUND: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.

Molecular viral epidemiology and clinical characterization of acute febrile respiratory infections in hospitalized children in Taiwan.

Acute respiratory infection (ARI) is a leading cause of morbidity and hospitalization in children. To profile the viruses causing ARI in children admitted to a community-based hospital in central Taiwan, a cross-sectional study was conducted on children under 14 years of age that were hospitalized with febrile ARI. Viral etiology was determined using conventional cell culture and a commercial respiratory virus panel fast assay (xTAG RVP), capable of detecting 19 different respiratory viruses and subtype targets. Demographic, clinical, and laboratory data were recorded and analyzed. The RVP fast assay identified at least one respiratory virus in 130 of the 216 specimens examined (60.2%) and rose to 137 (63.4%) by combining the results of cell culture and RVP fast assay. In order of frequency, the etiological agents identified were, rhinovirus/enterovirus (24.6%), respiratory syncytial virus (13.8%), adenovirus (11.5%), parainfluenza virus (9.2%), influenza B (8.4%), influenza A (5.4%), human metapneumovirus (4.6%), human coronavirus (2%), and human bocavirus (2%). Co-infection did not result in an increase in clinical severity. The RVP assay detected more positive specimens, but failed to detect 6 viruses identified by culture. The viral detection rate for the RVP assay was affected by how many days after admission the samples were taken (P = 0.03). In conclusion, Rhinovirus/enterovirus, respiratory syncytial virus, and adenovirus were prevalent in this study by adopting RVP assay. The viral detection rate is influenced by sampling time, especially if the tests are performed during the first three days of hospitalization.

Evaluation and characterization of a high-resolution melting analysis kit for rapid carrier-screening test of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans, caused by the homozygous absence of the survival motor neuron gene 1 (SMN1). SMN2, a copy gene, influences the severity of SMA. Several assays have been described for molecular diagnosis or carrier screening of SMA. A newly developed tool based on a high-resolution melting analysis (HRMA) that enables high-throughput screening without sophisticated protocols but low costs reveals itself to be powerful. We evaluate the performance of an HRMA-based kit for a carrier-screening test of SMA that was designed to detect the substitution of a single nucleotide in SMN1 exon 7. Carriers were identified in 453 participants by quantifying the SMN1 gene and compared with denaturing high-performance liquid chromatography (DHPLC) assay. An HRMA-based kit had a higher sensitivity (100%) for carrier testing than the DHPLC assay (93%), with the added advantage that some homozygous sequence alterations could be identified. The HRMA kit is a new, fast, and highly reliable quantitative test for the SMA molecular carrier test.

Portable sensing devices for smart healthcare and prevention of lead poisoning.

Lead (Pb) poisoning can damage human bodies silently, without specific symptoms or conspicuous warning signs. To provide safe and user-friendly tools for detecting heavy metals at low concentrations, scientists have developed and optimized versatile biosensors. To practically employ the developed biosensors specific for Pb (eg, the optimized Met-lead 1.44 M1), smartphone applications designed for user convenience and are easily operable for the on-site detection of Pb in environmental water, drinking water, food, and blood/urine are urgently needed. To establish a monitoring system for home health maintenance, a portable device and useful apps installed on a smartphone can be integrated, and the data acquired can be sent to and stored in the cloud for further analysis and evidence preservation. With the high transmissions speeds for 4G and 4G wireless Internet, such a system can be applied for health protection; water-quality data can be provided by anyone and publicly shared for display on smartphone interfaces, alerting individuals of heavy metal contamination. In this review, we describe recent developments in heavy metal-sensing devices, including home health maintenance systems, which have been successfully and practically applied to prevent heavy metal Pb poisoning.

Portable FRET-Based Biosensor Device for On-Site Lead Detection.

Most methods for measuring environmental lead (Pb) content are time consuming, expensive, hazardous, and restricted to specific analytical systems. To provide a facile, safe tool to detect Pb, we created pMet-lead, a portable fluorescence resonance energy transfer (FRET)-based Pb-biosensor. The pMet-lead device comprises a 3D-printed frame housing a 405-nm laser diode-an excitation source for fluorescence emission images (YFP and CFP)-accompanied by optical filters, a customized sample holder with a Met-lead 1.44 M1 (the most recent version)-embedded biochip, and an optical lens aligned for smartphone compatibility. Measuring the emission ratios (Y/C) of the FRET components enabled Pb detection with a dynamic range of nearly 2 (1.96), a pMet-lead/Pb dissociation constant (Kd) 45.62 nM, and a limit of detection 24 nM (0.474 µg/dL, 4.74 ppb). To mitigate earlier problems with a lack of selectivity for Pb vs. zinc, we preincubated samples with tricine, a low-affinity zinc chelator. We validated the pMet-lead measurements of the characterized laboratory samples and unknown samples from six regions in Taiwan by inductively coupled plasma mass spectrometry (ICP-MS). Notably, two unknown samples had Y/C ratios significantly higher than that of the control (3.48 ± 0.08 and 3.74 ± 0.12 vs. 2.79 ± 0.02), along with Pb concentrations (10.6 ppb and 15.24 ppb) above the WHO-permitted level of 10 ppb in tap water, while the remaining four unknowns showed no detectable Pb upon ICP-MS. These results demonstrate that pMet-lead provides a rapid, sensitive means for on-site Pb detection in water from the environment and in living/drinking supply systems to prevent potential Pb poisoning.

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

Virofree, an Herbal Medicine-Based Formula, Interrupts the Viral Infection of Delta and Omicron Variants of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) remains a threat with the emergence of new variants, especially Delta and Omicron, without specific effective therapeutic drugs. The infection causes dysregulation of the immune system with a cytokine storm that eventually leads to fatal acute respiratory distress syndrome (ARDS) and further irreversible pulmonary fibrosis. Therefore, the promising way to inhibit infection is to disrupt the binding and fusion between the viral spike and the host ACE2 receptor. A transcriptome-based drug screening platform has been developed for COVID-19 to explore the possibility and potential of the long-established drugs or herbal medicines to reverse the unique genetic signature of COVID-19. In silico analysis showed that Virofree, an herbal medicine, reversed the genetic signature of COVID-19 and ARDS. Biochemical validations showed that Virofree could disrupt the binding of wild-type and Delta-variant spike proteins to ACE2 and its syncytial formation via cell-based pseudo-typed viral assays, as well as suppress binding between several variant recombinant spikes to ACE2, especially Delta and Omicron. Additionally, Virofree elevated miR-148b-5p levels, inhibited the main protease of SARS-CoV-2 (Mpro), and reduced LPS-induced TNF-α release. Virofree also prevented cellular iron accumulation leading to ferroptosis which occurs in SARS-CoV-2 patients. Furthermore, Virofree was able to reduce pulmonary fibrosis-related protein expression levels in vitro. In conclusion, Virofree was repurposed as a potential herbal medicine to combat COVID-19. This study highlights the inhibitory effect of Virofree on the entry of Delta and Omicron variants of SARS-CoV-2, which have not had any effective treatments during the emergence of the new variants spreading.

Versatile Cell and Animal Models for Advanced Investigation of Lead Poisoning.

The heavy metal, lead (Pb) can irreversibly damage the human nervous system. To help understand Pb-induced damage, we applied a genetically encoded Förster resonance energy transfer (FRET)-based Pb biosensor Met-lead 1.44 M1 to two living systems to monitor the concentration of Pb: induced pluripotent stem cell (iPSC)-derived cardiomyocytes as a semi-tissue platform and Drosophila melanogaster fruit flies as an in vivo animal model. Different FRET imaging modalities were used to obtain FRET signals, which represented the presence of Pb in the tested samples in different spatial dimensions. Using iPSC-derived cardiomyocytes, the relationship between beating activity (20-24 beats per minute, bpm) determined from the fluctuation of fluorescent signals and the concentrations of Pb represented by the FRET emission ratio values of Met-lead 1.44 M1 was revealed from simultaneous measurements. Pb (50 µM) affected the beating activity of cardiomyocytes, whereas two drugs that stop the entry of Pb differentially affected this beating activity: verapamil (2 µM) did not reverse the cessation of beating, whereas 2-APB (50 µM) partially restored this activity (16 bpm). The results clearly demonstrate the potential of this biosensor system as an anti-Pb drug screening application. In the Drosophila model, Pb was detected within the adult brain or larval central nervous system (Cha-gal4 > UAS-Met-lead 1.44 M1) using fast epifluorescence and high-resolution two-photon 3D FRET ratio image systems. The tissue-specific expression of Pb biosensors provides an excellent opportunity to explore the possible Pb-specific populations within living organisms. We believe that this integrated Pb biosensor system can be applied to the prevention of Pb poisoning and advanced research on Pb neurotoxicology.

Development and optimization of heavy metal lead biosensors in biomedical and environmental applications.

The detrimental impact of the heavy metal lead (Pb) on human health has been studied for years. The fact that Pb impairs human body has been established from countless painful and sad historical events. Nowadays, World Health Organization and many developmental countries have established regulations concerning the use of Pb. Measuring the blood lead level (BLL) is so far the only way to officially evaluate the degree of Pb exposure, but the so-called safety value (10 µg/dL in adults and 5 µg/dL in children) seems unreliable to represent the security checkpoint for children through daily intake of drinking water or physical contact with a lower contaminated level of Pb contents. In general, unsolved mysteries about the Pb toxicological mechanisms still remain. In this review article, we report on the methods to prevent Pb poison for further Pb toxicological research. We establish high-sensitivity Pb monitoring, and also report on the use of fluorescent biosensors such as genetically-encoded fluorescence resonance energy transfer-based biosensors built for various large demands such as the detection of severe acute respiratory syndrome coronavirus 2. We also contribute to the development and optimization of the FRET-based Pb biosensors. Our well-performed version of Met-lead 1.44 M1 has achieved a limit of detection of 10 nM (2 ppb; 0.2 µg/dL) and almost 5-fold in dynamic range (DR) supported for the real practical applications-that is, the in-cell Pb sensing device for blood and blood-related samples, and the Pb environmental detections in vitro. The perspective of our powerful Pb biosensor incorporated with a highly sensitive bio-chip of the portable device for quick Pb measurements will be addressed for further manipulation.

Combinatorial roles of mitochondria and cGMP/PKG pathway in the generation of neuronal free Zn2+ under the presence of nitric oxide.

BACKGROUND: Nitric oxide (NO), which possesses both protective and toxic properties, has been observed to have a complicated biphasic character within various types of tissues, including neuronal cells. NO was also found to cause the increase of another important signaling molecular Zn (termed as NZR). The molecular mechanism of NZR has been extensively investigated, but the source of Zn is present of a major candidate that is yet to be answered. The NO-protein kinase G (PKG) pathway, mitochondria, and metallothioneins (MTs), are all proposed to be the individual source of NZR. However, this hypothesis remains inconclusive. In this study, we examined the function of PKG signaling cascades, the mitochondria storage, and MT-1 during NZR of living PC12 cells. METHODS: We applied live-cell imaging in combination with pharmacological inhibitors and activators as well as in vitro Zn assay to dissect the functions of the above candidates in NZR. RESULTS: Two mechanisms, namely, mitochondria as the only Zn source and the opening of NO-PKG-dependent mitochondrial ATP-sensitive potassium channels (mKATP) as the key to releasing NO-induced increase in mitochondrial Zn, were proven to be the two critical paths of NZR in neuronal-related cells. CONCLUSION: This new finding provides a reasonable explanation to previously existing and contradictory conclusions regarding the function of mitochondria/mKATP and PKG signaling on the molecular mechanism of NZR.

Upconversion nanoparticle-mOrange protein FRET nanoprobes for self-ratiometric/ratiometric determination of intracellular pH, and single cell pH imaging.

Fluorescence based intracellular pH nanoprobes have been developed that overcomes the limitations imposed by shallow penetration depth of ultraviolet excitation, photostability, phototoxicity, and interference from background autofluorescence. In this study, we have constructed a Förster Resonance Energy Transfer (FRET) based pH nanoprobe using upconversion nanoparticle (UCNP) as a donor (excitation/emission @ 980/540 nm, green channel), and mOrange fluorescent protein (excitation/emission @ 548/566 nm, red channel) as acceptor. The UCNP-mOrange nanoprobe could be fluorescently imaged with 980 nm excitation, having deep penetration depth, by a fluorescence microscope on a coverslip, or uptaken in a single HeLa cell. The cellular upatake of these nanoparticles were confirmed by transmission electron microscope study. The FRET probes, with a FRET efficiency of ~20% at physiological pH of 7.0, have simultaneous self-ratiometric and ratiometric features varying linearly with local pH. The probe exhibits high accuracy, sensitivity, reversibility, and stability over a wide range of pH (3.0-8.0). The fluorescence intensity ratio from individual green, and red channels in fluorescence microscopic images could be used to estimate the pH of the intracellular compartments of HeLa cell from the pH dependent ratiometric calibration. Nigericin mediated intracellular pH (3.0, 5.0, and 7.0) could be accurately estimated from the CLSM derived FRET ratio. The pH probes demonstrate high stability and reversibility when switched between pH 3, and 8 for at least 5 cycles.