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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

High-performance FRET biosensors for single-cell and in vivo lead detection.

Forms of lead (Pb) have been insidiously invading human life for thousands of years without obvious signs of their considerable danger to human health. Blood lead level (BLL) is the routine measure used for diagnosing the degree of lead intoxication, although it is unclear whether there is any safe range of BLL. To develop a practical detection tool for living organisms, we engineered a genetically encoded fluorescence resonance energy transfer (FRET)-based Pb2+ biosensor, 'Met-lead 1.44 M1', with excellent performance. Met-lead 1.44 M1 has an apparent dissociation constant (Kd) of 25.97 nM, a detection limit (LOD) of 10 nM (2.0 ppb/0.2 µg/dL), and an enhancement dynamic ratio of nearly ~ 5-fold upon Pb2+ binding. The 10 nM sensitivity of Met-lead 1.44 M1 is five times below the World Health Organization-permitted level of lead in tap water (10 ppb; WHO, 2017), and fifteen times lower than the maximum BLL for children (3 µg/dL). We deployed Met-lead 1.44 M1 to measure Pb2+ concentrations in different living models, including two general human cell lines and one specific line, induced pluripotent stem cell (iPSC)-derived cardiomyocytes, as well as in widely used model species in plant (Arabidopsis thaliana) and animal (Drosophila melanogaster) research. Our results suggest that this new biosensor is suitable for lead toxicological research in vitro and in vivo, and will pave the way toward potential applications for both low BLL measures and rapid detection of environmental lead in its divalent form.

Bright and High-Performance Genetically Encoded Ca2+ Indicator Based on mNeonGreen Fluorescent Protein.

Genetically encodable calcium ion (Ca2+) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost 2 decades of steady improvements in the Aequorea victoria GFP-based GCaMP series of GECIs, the performance of the most recent generation (i.e., jGCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression toward ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

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.

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.

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.

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.

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.

Comparison of molecular epidemiology of bloodstream methicillin-resistant Staphylococcus aureus isolates between a new and an old hospital in central Taiwan.

OBJECTIVE: To compare the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolates between an old, urban hospital and a new, rural hospital over the same time period. METHODS: The molecular characteristics of 398 MRSA bloodstream isolates collected between 2007 and 2013 from two hospitals in Taiwan were analyzed retrospectively; 202 isolates were from the old hospital and 196 from the new hospital (opened in 2007). RESULTS: The rate of resistance to multiple antibiotics was significantly higher in the old hospital (93%) than in the new hospital (81%) (p<0.001). Genetic community-associated MRSA carrying staphylococcal cassette chromosome (SCC) type IV or V accounted for 58% of all MRSA isolates in the new hospital, significantly higher than the rate in the old hospital (p=0.018). The rate of spa t037-SCCmec III MRSA was significantly lower in the new hospital than in the old hospital (p=0.02). A significant decreasing trend in spa t002-SCCmec II MRSA isolates was observed in the old hospital (p=0.006), while the proportion of spa t037-SCCmec III MRSA decreased significantly in the new hospital (41.7% to 26.1%, p=0.022). CONCLUSIONS: The rate of multiple antibiotic resistance and the molecular characteristics of MRSA differed significantly between the old and new hospitals and changed over time.