The functional characteristics of optogenetic gene therapy for vision restoration

Optogenetic strategies to restore vision in patients blind from end-stage retinal degenerations aim to render remaining retinal neurons light-sensitive. We present an innovative combination of multi-electrode array recordings together with a complex pattern-generating light source as a toolset to determine the extent to which neural retinal responses to complex light stimuli can be restored following viral delivery of red-shifted channelrhodopsin in the retinally degenerated mouse. Our data indicate that retinal output level spatiotemporal response characteristics achieved by optogenetic gene therapy closely parallel those observed for normal mice but equally reveal important limitations, some of which could be mitigated using bipolar-cell targeted gene-delivery approaches. As clinical trials are commencing, these data provide important new information on the capacity and limitations of channelrhodopsin-based gene therapies. The toolset we established enables comparing optogenetic constructs and stem-cell-based techniques, thereby providing an efficient and sensitive starting point to identify future approaches for vision restoration

Reaeration in Supercritical Open Channel Flows: An Experimental Study

Reaeration is a primary path of reoxygenation in streams, fundamental to environmental and ecological integrity. Previous laboratory studies of reaeration rates in subcritical flows showed large scatter in results, with differences in mass transfer coefficients of more than one order of magnitude between comparable flow conditions. Although supercritical flow is commonly observed in natural streams and engineered channels, systematic measurements of supercritical flow reaeration rates have been unavailable. Experiments in a laboratory open channel flume encompassing sub-and supercritical flows have been undertaken. The subcritical data were consistent with a large body of previous studies. Supercritical flows showed 6-10 times stronger reaeration rates than comparable subcritical conditions, while local rates at hydraulic jumps systematically exceed those in the supercritical flows upstream by a factor up to three. A close relationship between reaeration rates and turbulent dissipation rate is observed, and a systematic Froude number dependency is demonstrated for both sub-and supercritical flows. Observed mass transfer coefficients do not correlate as well with flow Reynolds number and shear Reynolds number. The higher reaeration rates associated with supercritical flows indicates that a change in open channel flow regime for the same Reynolds number may be used to improve water quality

A systematic comparison of optogenetic approaches to visual restoration

During inherited retinal degenerations (IRDs), vision is lost due to photoreceptor cell death; however, a range of optogenetic tools have been shown to restore light responses in animal models. Restored response characteristics vary between tools and the neuronal cell population to which they are delivered: the interplay between these is complex, but targeting upstream neurons (such as retinal bipolar cells) may provide functional benefit by retaining intraretinal signal processing. In this study, our aim was to compare two optogenetic tools: mammalian melanopsin (hOPN4) and microbial red-shifted channelrhodopsin (ReaChR) expressed within two subpopulations of surviving cells in a degenerate retina. Intravitreal adeno-associated viral vectors and mouse models utilising the Cre/lox system restricted expression to populations dominated by bipolar cells or retinal ganglion cells and was compared with non-targeted delivery using the chicken beta actin (CBA) promoter. In summary, we found bipolar-targeted optogenetic tools produced faster kinetics and flatter intensity-response relationships compared with non-targeted or retinal-ganglion-cell-targeted hOPN4. Hence, optogenetic tools of both mammalian and microbial origins show advantages when targeted to bipolar cells. This demonstrates the advantage of bipolar-cell-targeted optogenetics for vision restoration in IRDs. We therefore developed a bipolar-cell-specific gene delivery system employing a compressed promoter with the potential for clinical translation

Analyzing the footprints of near-surface aqueous turbulence: An image processing-based approach

In this contribution, a detailed investigation of surface thermal patterns on the water surface is presented, with wind speeds ranging from 1 to 7 m s  − 1 and various surface conditions. Distinct structures can be observed on the surface—small-scale short-lived structures termed fish scales and larger-scale cold streaks that are consistent with the footprints of Langmuir circulations. The structure of the surface heat pattern depends strongly on wind-induced stress. Consistent behavior regarding the spacing of cold streaks can be observed in a range of laboratory facilities when expressed as a function of water-sided friction velocity, u * . This behavior systematically decreased until a point of saturation at u *  = 0.7 cm/s. We present a new image processing-based approach to the analysis of the spacing of cold streaks based on a machine learning approach to classify the thermal footprints of near-surface turbulence. Comparison is made with studies of Langmuir circulation and the following key points are found. Results suggest a saturation in the tangential stress, anticipating that similar behavior will be observed in the open ocean. A relation to Langmuir numbers shows that thermal footprints in infrared images are consistent with Langmuir circulations and depend strongly on wind wave conditions

ON-bipolar cell gene expression during retinal degeneration: Implications for optogenetic visual restoration.

PURPOSE: Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter. METHODS: Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wildtype retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration. RESULTS: A total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration. CONCLUSION: Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies

TRESK is a key regulator of nocturnal suprachiasmatic nucleus dynamics and light adaptive responses

The suprachiasmatic nucleus (SCN) is a complex structure dependent upon multiple mechanisms to ensure rhythmic electrical activity that varies between day and night, to determine circadian adaptation and behaviours. SCN neurons are exposed to glutamate from multiple sources including from the retino-hypothalamic tract and from astrocytes. However, the mechanism preventing inappropriate post-synaptic glutamatergic effects is unexplored and unknown. Unexpectedly we discovered that TRESK, a calcium regulated two-pore potassium channel, plays a crucial role in this system. We propose that glutamate activates TRESK through NMDA and AMPA mediated calcium influx and calcineurin activation to then oppose further membrane depolarisation and rising intracellular calcium. Hence, in the absence of TRESK, glutamatergic activity is unregulated leading to membrane depolarisation, increased nocturnal SCN firing, inverted basal calcium levels and impaired sensitivity in light induced phase delays. Our data reveals TRESK plays an essential part in SCN regulatory mechanisms and light induced adaptive behaviours

Read counts from environmental DNA (eDNA) metabarcoding reflect fish abundance and biomass in drained ponds.

The sampling of environmental DNA (eDNA) coupled with cost-efficient and ever-advancing sequencing technology is propelling changes in biodiversity monitoring within aquatic ecosystems. Despite the increasing number of eDNA metabarcoding approaches, the ability to quantify species biomass and abundance in natural systems is still not fully understood. Previous studies have shown positive but sometimes weak correlations between abundance estimates from eDNA metabarcoding data and from conventional capture methods. As both methods have independent biases a lack of concordance is difficult to interpret. Here we tested whether read counts from eDNA metabarcoding provide accurate quantitative estimates of the absolute abundance of fish in holding ponds with known fish biomass and number of individuals. Environmental DNA samples were collected from two fishery ponds with high fish density and broad species diversity. In one pond, two different DNA capture strategies (on-site filtration with enclosed filters and three different preservation buffers versus lab filtration using open filters) were used to evaluate their performance in relation to fish community composition and biomass/abundance estimates. Fish species read counts were significantly correlated with both biomass and abundance, and this result, together with information on fish diversity, was repeatable when open or enclosed filters with different preservation buffers were used. This research demonstrates that eDNA metabarcoding provides accurate qualitative and quantitative information on fish communities in small ponds, and results are consistent between different methods of DNA capture. This method flexibility will be beneficial for future eDNA-based fish monitoring and their integration into fisheries management

Continuous and non-invasive thermography of mouse skin accurately describes core body temperature patterns, but not absolute core temperature

Body temperature is an important physiological parameter in many studies of laboratory mice. Continuous assessment of body temperature has traditionally required surgical implantation of a telemeter, but this invasive procedure adversely impacts animal welfare. Near-infrared thermography provides a non-invasive alternative by continuously measuring the highest temperature on the outside of the body (Tskin), but the reliability of these recordings as a proxy for continuous core body temperature (Tcore) measurements has not been assessed. Here, Tcore (30 s resolution) and Tskin (1 s resolution) were continuously measured for three days in mice exposed to ad libitum and restricted feeding conditions. We subsequently developed an algorithm that optimised the reliability of a Tskin-derived estimate of Tcore. This identified the average of the maximum Tskin per minute over a 30-min interval as the optimal way to estimate Tcore. Subsequent validation analyses did however demonstrate that this Tskin-derived proxy did not provide a reliable estimate of the absolute Tcore due to the high between-animal variability in the relationship between Tskin and Tcore. Conversely, validation showed that Tskin-derived estimates of Tcore reliably describe temporal patterns in physiologically-relevant Tcore changes and provide an excellent measure to perform within-animal comparisons of relative changes in Tcore

New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR

We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead