Person:

Patel, Ronak Bhailal

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca(^{2+})-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca(^{2+})]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca(^{2+}) affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

Objectives: Rapid urbanisation has often meant that public infrastructure has not kept pace with growth leading to urban slums with poor access to water and sanitation and high rates of diarrhoea with greater household costs due to illness. This study sought to determine the monetary cost of diarrhoea to urban slum households in Kaula Bandar slum in Mumbai, India. The study also tested the hypotheses that the cost of water and sanitation infrastructure may be surpassed by the cumulative costs of diarrhoea for households in an urban slum community. Design: A cohort study using a baseline survey of a random sample followed by a systematic longitudinal household survey. The baseline survey was administered to a random sample of households. The systematic longitudinal survey was administered to every available household in the community with a case of diarrhoea for a period of 5 weeks. Participants: Every household in Kaula Bandar was approached for the longitudinal survey and all available and consenting adults were included. Results: The direct cost of medical care for having at least one person in the household with diarrhoea was 205 rupees. Other direct costs brought total expenses to 291 rupees. Adding an average loss of 55 rupees per household from lost wages and monetising lost productivity from homemakers gave a total loss of 409 rupees per household. During the 5-week study period, this community lost an estimated 163 600 rupees or 3635 US dollars due to diarrhoeal illness. Conclusions: The lack of basic water and sanitation infrastructure is expensive for urban slum households in this community. Financing approaches that transfer that cost to infrastructure development to prevent illness may be feasible. These findings along with the myriad of unmeasured benefits of preventing diarrhoeal illness add to pressing arguments for investment in basic water and sanitation infrastructure.