Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission.

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator.

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Fast and sensitive GCaMP calcium indicators for imaging neural populations.

Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

Combined antibacterial effect of essential oils from three most commonly used Ethiopian traditional medicinal plants on multidrug resistant bacteria.

BACKGROUND: An alarm increase the rate of emerging and re-emerging of multidrug resistant bacteria have been caused great public health concern in the worldwide. They have been resisting for most or majority of currently available and affordable antibiotics and imposed socioeconomic catastrophe at global scale. As a result, there is utmost important to discover new or modify currently available antibiotics. The aim of this study was to evaluate combined antibacterial effect of essential oils obtained from Blepharis cuspidata, Boswellia ogadensis and Thymus schimper against multidrug resistance (MDR) Escherichia coli, Klebsiella pneumoniae and Methicillin resistant S. aureus. METHODS: Essential oil (EO) was extracted from the aerial part of B. cuspidata, B.ogadensis and T. schimper by steam distillation and stored in brown bottles at 4 °C. There were mixed in 1:1 ratio and adsorbed to disc and placed on MHA and measured their minimum inhibitory zone seeded with E. coli, K. pneumoniae and MRAS after 18-24 H. minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were measured by broth micro-dilution method. The interaction between EOs was determined by fractional inhibitory concentration index. RESULTS: The antibacterial potential of mixed oil depends on the doses and type of the EOs and bacteria species. The combined EOs of B.cuspidata and T.schimperi had inhibition zone (39 mm), its MIC and MBC value was 0.39 µl/ml against MRSA. It had inhibition zone (28-35 mm), MIC value 0.39-6.25 µl/ml and MBC (0.78-12.5 µl/ml) against MDR E. coli and K. pneumoniae. Whereas, combined effects of B. cuspidata and B. ogadensis had MIC values ranges from 0.78-6.25 µl/ml for E.coli and K. pneumoniae and 1.56 µl/ml for MRSA. There was strong synergistic effect between the combination of B.cuspidata and T.schimperi. This study revealed that gram negative bacteria were slightly less susceptible than gram positive. CONCLUSIONS: This in vitro study of combined EOs has significant antibacterial effect than using each of them and even it was more potent antibacterial effect on MDR as compare to modern antibiotics. Hence, it can be applied to a pharmaceutical composition as modulator or adjuvant or precursor for synthesis of new antibiotic in future activities.

Early impact of rotavirus vaccine in under 5year old children hospitalized due to diarrhea, Swaziland.

BACKGROUND: Swaziland introduced rotavirus vaccine in the National Immunization Program, in May 2015, with the objective of reducing the burden of rotavirus diarrheal disease. We monitored the early impact of the vaccine in reducing rotavirus diarrhea. METHODS: We conducted sentinel rotavirus surveillance from January 2013 to December 2016 in children under five years of age admitted due to diarrhea attending Mbabane Government Referral Hospital in the Hhohho Region and Raleigh Fitkin Memorial Hospital in the Manzini Region. All cases had stool samples collected and tested for rotavirus antigen by enzyme immunoassay. RESULTS: Between 2013 and 2016, 596 samples were collected and tested. Rotavirus positivity reduced from average of 50.8% (172/338) (in 2013-2014 (pre vaccine period)) to 29% (24/82) in 2016, post-vaccine introduction. The median age of children with rotavirus infection increased from average of 10months in 2013-2014 to 13.7months in 2016. The peak season for all-cause diarrhea and rotavirus-specific hospitalizations among children under five years of age was June-August in all years with a blunting of the peak season in 2016. Rotavirus positivity among children 0-11months reduced from an average of 49% in 2013-2014 (116/236) to 33% (15/45) in 2016, a 33% reduction following rotavirus vaccine introduction. CONCLUSION: There has been a rapid reduction of all-cause diarrhea and rotavirus hospitalizations in Swaziland, particularly in young children and during the rotavirus season, after the introduction rotavirus vaccine. Continued surveillance is needed to monitor the long-term impact of rotavirus vaccine introduction.

Sensitive red protein calcium indicators for imaging neural activity.

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.

A neuron-based screening platform for optimizing genetically-encoded calcium indicators.

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.

Comparison of the genome of the oral pathogen Treponema denticola with other spirochete genomes.

We present the complete 2,843,201-bp genome sequence of Treponema denticola (ATCC 35405) an oral spirochete associated with periodontal disease. Analysis of the T. denticola genome reveals factors mediating coaggregation, cell signaling, stress protection, and other competitive and cooperative measures, consistent with its pathogenic nature and lifestyle within the mixed-species environment of subgingival dental plaque. Comparisons with previously sequenced spirochete genomes revealed specific factors contributing to differences and similarities in spirochete physiology as well as pathogenic potential. The T. denticola genome is considerably larger in size than the genome of the related syphilis-causing spirochete Treponema pallidum. The differences in gene content appear to be attributable to a combination of three phenomena: genome reduction, lineage-specific expansions, and horizontal gene transfer. Genes lost due to reductive evolution appear to be largely involved in metabolism and transport, whereas some of the genes that have arisen due to lineage-specific expansions are implicated in various pathogenic interactions, and genes acquired via horizontal gene transfer are largely phage-related or of unknown function.

Identification of competence pheromone responsive genes in Streptococcus pneumoniae by use of DNA microarrays.

Natural genetic transformation in Streptococcus pneumoniae is controlled in part by a quorum-sensing system mediated by a peptide pheromone called competence-stimulating peptide (CSP), which acts to coordinate transient activation of genes required for competence. To characterize the transcriptional response and regulatory events occurring when cells are exposed to competence pheromone, we constructed DNA microarrays and analysed the temporal expression profiles of 1817 among the 2129 unique predicted open reading frames present in the S. pneumoniae TIGR4 genome (84%). After CSP stimulation, responsive genes exhibited four temporally distinct expression profiles: early, late and delayed gene induction, and gene repression. At least eight early genes participate in competence regulation including comX, which encodes an alternative sigma factor. Late genes were dependent on ComX for CSP-induced expression, many playing important roles in transformation. Genes in the delayed class (third temporal wave) appear to be stress related. Genes repressed during the CSP response include ribosomal protein loci and other genes involved in protein synthesis. This study increased the number of identified CSP-responsive genes from approximately 40 to 188. Given the relatively large number of induced genes (6% of the genome), it was of interest to determine which genes provide functions essential to transformation. Many of the induced loci were subjected to gene disruption mutagenesis, allowing us to establish that among 124 CSP-inducible genes, 67 were individually dispensable for transformation, whereas 23 were required for transformation.