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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. Neurons encode information with brief electrical pulses called spikes. Monitoring spikes in large populations of neurons is a powerful method for studying how networks of neurons process information and produce behavior. This activity can be detected using fluorescent protein indicators, or “probes”, which light up when neurons are active. The best existing probes produce green fluorescence. However, red fluorescent probes would allow us to see deeper into the brain, and could also be used with green probes to image the activity and interactions of different neuron types simultaneously. However, existing red fluorescent probes are not as good at detecting neural activity as green probes. By optimizing two existing red fluorescent proteins, Dana et al. have now produced two new red fluorescent probes, each with different advantages. The new protein indicators detect neural activity with high sensitivity and allow researchers to image previously unseen brain activity. Tests showed that the probes work in cultured neurons and allow imaging of the activity of neurons in mice, flies, fish and worms. History has shown that enhancing the techniques used to study biological processes can lead to fundamentally new insights. In the future, Dana et al. would therefore like to make even more sensitive protein indicators that will allow larger networks of neurons deeper in the brain to be imaged.

Calcium imaging with protein-based indicators 1 , 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 indicators 3 – 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. Using large-scale screening and structure-guided mutagenesis, fast and sensitive GCaMP sensors are developed and optimized with improved kinetics without compromising sensitivity or brightness.

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.

Sweet sorghum [ Sorghum bicolor (L.) Moench] is one of the varieties of sorghum having stalk with high concentration of soluble sugar. Biochemical evaluations of sweet sorghum enable it to determine the potential of the crop to be utilized for sugar production at industrial level. This study was designed to evaluate the Ethiopian sweet sorghum germplasms for production of crystalized sugar. In this study, 91 sweet sorghum accessions were evaluated based on total soluble sugar content. As a result, 8 accessions having high total soluble sugar scores were selected and evaluated for further biochemical quality traits. In addition, three agronomic parameters were used to evaluate their effect on the biochemical features of the studied sweet sorghum and sugarcane accessions. These traits include total soluble sugar (degree brix), polarization, sucrose content, sugar purity, days to maturity, stem height and stalk diameter. Three high quality sugarcane ( Saccharum officinarum ) genotypes collected from Kessem sugar industry of Ethiopia were used as standard checks. Analysis of variance (ANOVA); mean separation and correlation were analyzed using R-software. ANOVA revealed significant variations for polarization value and purity of sweet sorghum juice at p ≤ 0.001. The mean separation analysis revealed that maximum and minimum degree brix were obtained from T-11 (20.23%) and T-28 (16.88%), respectively. In the present study, the polarization values ranged from 41.57 (T-28) to 69.30 0 Z (C-86/12).The three sugarcane standard cheeks showed relatively higher values of polarization in comparison with the sweet sorghum accessions. Among the sweet sorghum accessions, T-13 showed relatively higher polarization value whereas the remaining sweet sorghum accessions didn’t show significant variation. The average sucrose content (pol percentage) was ranged from 11.98% (T-28) to 16.77% (C-86/12). C-86/12 sugarcane accession showed the highest (16.77%) mean value for sucrose content whereas T-28 sweet sorghum accession scored the lowest sucrose content (11.98%). Maximum purity of sugar was recorded from SP-70 (90.77%) sugarcane accession whereas; the minimum value was recorded from T-11 (63.62%) sweet sorghum accession. In addition, the correlation analysis revealed that there were both positive and negative correlation among biochemical and agro morphological traits of sweet sorghum and sugarcane. Brix value showed positive correlation with polarization (0.56**) and sucrose content (0.71**) while it was negatively correlated with sugar purity (-0.06), days to maturity (-0.06), stem height (-0.19) and stalk diameter (-0.16). On the other hand, polarization showed strong positive correlation with sucrose content (0.95**), purity (0.76**) and days to maturity (0.78**). In comparison with sugarcane, the studied sweet sorghum accessions revealed quite lower performance in polarization, sucrose content and purity. Nevertheless, this study confirmed the existence of a climate smart and promising sweet sorghum genotypes used for the production of sugar and syrup as an alternative sweetener.

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. jYCaMP1, a yellow variant of the calcium indicator jGCaMP7, enables fast multicolor two-photon imaging at excitation wavelengths above 1,000 nm for use with popular ytterbium-doped fiber and modelocked semiconductor lasers.

Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we present a general approach to engineer electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with positive-going fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters. Genetically encoded voltage indicators (GEVIs) allow visualisation of fast action potentials in neurons but most are bright at rest and dimmer during an action potential. Here, the authors engineer electrochromic FRET GEVIs with fast, bright and positive-going fluorescence signals for in vivo imaging.

Sweet sorghum is a cereal crop in the grass family belonging to the genus Sorghum bicolor L. Moench. It is known in its sugary juice that is accumulated in its stalk and efficient C4 photosynthetic pathway. Only few molecular genetic diversity studies of Ethiopian sweet sorghum have been carried out. Understanding the genetic diversity of plants is the basis for genetic improvement, effective conservation and efficient utilization of genetic resources. Therefore, the objective of this study was to evaluate the genetic diversity and population structure of Ethiopian sweet sorghum genotypes collected from major growing areas of Ethiopia. In the present study, thirteen SSR markers produced a total of 136 alleles across all the 91 sweet sorghum accessions with an average of 10.46 alleles per marker. The major allele frequency per marker ranged from 0.16 to 0.41 with an average of 0.25. The number of alleles per marker ranged from 6 to 15. The mean PIC value was 0.80. The pair-wise genetic differentiation among the five studied sweet sorghum populations ranged from 0.07 to 0.19. The highest Fst (0.19) and the lowest Fst (0.07) population differentiation were observed between sweet sorghum population of South Wollo and Oromia Liyu Zone, and North Shewa and East Gojam sweet sorghum populations, respectively. The analysis of gene flow across populations showed that the highest gene flow was recorded between North Shewa and East Gojam (2.879), whereas the least gene flow was observed between South Wollo and Oromia Liyu Zone (0.618). The analysis of molecular variance revealed that 16% variation was observed among populations and 84% variation has been observed within populations. Meanwhile, the STRUCTURE and UPGMA methods of clustering suggested that the sampled sweet sorghum populations were clustered into two main groups (K =  2). This comprehensive study of genetic diversity and population structure of sweet sorghum (Sorghum bicolor) in Ethiopia suggests that future sweet sorghum improvement and utilization strategies should take the magnitude and pattern of genetic diversity into consideration.

Wheat is one of the most important cereal crops and extensively cultivated in wide ranges of altitudes in Ethiopia. With an alarming population growth in the era of climatic change, there is a need for further crop improvement for sustainable production. In this regard, the study was carried out at the Kulumsa Agricultural Research Center (KARC) in a rainout shelter to investigate the responses of durum and bread wheat varieties to soil water stress in terms of selected morphological, physiological, and biochemical parameters. The 2 factors were combined factorially and arranged in a randomized complete block design with 3 replications. The 12 wheat varieties, 6 bread wheat and other 6 durum wheat, were sown in pots under well-watered (100% field capacity) and water-stressed (30% field capacity) conditions. Results revealed that water stress resulted in 26%, 9%, 23%, 16%, and 11% reductions in plant height, spike length, number of spikelets spike−1, relative water, and chlorophyll contents, respectively. The tested wheat varieties under water stress produced 28% and 6% more proline content and total soluble sugar, respectively, as mitigation strategies against drought. Results further exhibited that wheat varieties significantly differed in all of the measured traits except for the plant height and relative water content. The present study verified that the biochemical parameters needs to be considered as better traits to select wheat (Triticum spp.) varieties for drought tolerance under water stress conditions.

AbstractBackgroundSwaziland 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. MethodsWe 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. ResultsBetween 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 10 months in 2013–2014 to 13.7 months 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 0–11 months reduced from an average of 49% in 2013–2014 (116/236) to 33% (15/45) in 2016, a 33% reduction following rotavirus vaccine introduction. ConclusionThere 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.

Chickpea (Cicer arietinum L.) is a cheap source of protein and rich in minerals for people living in developing countries. In order to assess the existing molecular genetic diversity and determine population structures in selected Ethiopian chickpea germplasm accessions (118), a set of 46 simple sequence repeat (SSR) markers equally distributed on the chickpea genome were genotyped. A total of 572 alleles were detected from 46 SSR markers, and the number of alleles per locus varied from 2 (ICCM0289) to 28 (TA22). The average number of alleles per locus, polymorphism information content, and expected heterozygosity were 12, 0.684, and 0.699, respectively. Phylogenetic analysis grouped the 118 chickpea genotypes from diverse sources into three evolutionary and/or biological groups (improved desi, improved kabuli, and landraces). The population structure analysis revealed six sub-populations from 118 chickpea genotypes studied. AMOVA revealed that 57%, 29%, and 14% of the total genetic variations were observed among individuals, within populations, and among populations. The insights into the genetic diversity at molecular levels in the Ethiopian germplasm lines can be used for designing conservation strategies as well as the diverse germplasm lines identified in this study can be used for trait dissection and trait improvement.