Bioluminescent Multi-Characteristic Opsin for Simultaneous Optical Stimulation and Continuous Monitoring of Cortical Activities.

Stimulation and continuous monitoring of neural activities at cellular resolution are required for the understanding of the sensory processing of stimuli and development of effective neuromodulation therapies. We present bioluminescence multi-characteristic opsin (bMCOII), a hybrid optogenetic actuator, and a bioluminescence Ca2+ sensor for excitation-free, continuous monitoring of neural activities in the visual cortex, with high spatiotemporal resolution. An exceptionally low intensity (10 µW/mm2) of light could elicit neural activation that could be detected by Ca2+ bioluminescence imaging. An uninterrupted (>14 h) recording of visually evoked neural activities in the cortex of mice enabled the determination of strength of sensory activation. Furthermore, an artificial intelligence-based neural activation parameter transformed Ca2+ bioluminescence signals to network activity patterns. During continuous Ca2+-bioluminescence recordings, visual cortical activity peaked at the seventh to eighth hour of anesthesia, coinciding with circadian rhythm. For both direct optogenetic stimulation in cortical slices and visually evoked activities in the visual cortex, we observed secondary delayed Ca2+-bioluminescence responses, suggesting the involvement of neuron-astrocyte-neuron pathway. Our approach will enable the development of a modular and scalable interface system capable of serving a multiplicity of applications to modulate and monitor large-scale activities in the brain.

Biodistribution of adeno-associated virus type 2 carrying multi-characteristic opsin in dogs following intravitreal injection.

Gene therapy of retinal diseases using recombinant adeno-associated virus (rAAV) vector-based delivery has shown clinical success, and clinical trials based on rAAV-based optogenetic therapies are currently in progress. Recently, we have developed multi-characteristic opsin (MCO), which has been shown to effectively re-photosensitize photoreceptor-degenerated retina in mice leading to vision restoration at ambient light environment. Here, we report the biodistribution of the rAAV2 carried MCO (vMCO-I) in live samples and post-mortem organs following intraocular delivery in wild-type dogs. Immunohistochemistry showed that the intravitreal injection of vMCO-I resulted in gene transduction in the inner nuclear layer (INL) but did not induce detectable inflammatory or immune reaction in the dog retina. Vector DNA analysis of live body wastes and body fluids such as saliva and nasal secretions using quantitative polymerase chain reaction (qPCR) showed no correlative increase of vector copy in nasal secretions or saliva, minimal increase of vector copy in urine in the low-dose group 13 weeks after injection and in the faeces of the high-dose group at 3-13 weeks after injection suggesting clearance of the virus vector via urine and faeces. Further analysis of vector DNA extracted from faeces using PCR showed no transgene after 3 weeks post-injection. Intravitreal injection of vMCO-I resulted in few sporadic off-target presences of the vector in the mesenteric lymph node, liver, spleen and testis. This study showed that intravitreal rAAV2-based delivery of MCO-I for retinal gene therapy is safe.

The impact of edema and fiber crossing on diffusion MRI metrics assessed in an ex vivo nerve phantom: Multi-tensor model vs. diffusion orientation distribution function.

Diffusion tensor imaging (DTI) has been employed for over 2 decades to noninvasively quantify central nervous system diseases/injuries. However, DTI is an inadequate simplification of diffusion modeling in the presence of coexisting inflammation, edema and crossing nerve fibers. We employed a tissue phantom using fixed mouse trigeminal nerves coated with various amounts of agarose gel to mimic crossing fibers in the presence of vasogenic edema. Diffusivity measures derived by DTI and diffusion basis spectrum imaging (DBSI) were compared at increasing levels of simulated edema and degrees of fiber crossing. Furthermore, we assessed the ability of DBSI, diffusion kurtosis imaging (DKI), generalized q-sampling imaging (GQI), q-ball imaging (QBI) and neurite orientation dispersion and density imaging to resolve fiber crossing, in reference to the gold standard angles measured from structural images. DTI-computed diffusivities and fractional anisotropy were significantly confounded by gel-mimicked edema and crossing fibers. Conversely, DBSI calculated accurate diffusivities of individual fibers regardless of the extent of simulated edema and degrees of fiber crossing angles. Additionally, DBSI accurately and consistently estimated crossing angles in various conditions of gel-mimicked edema when compared with the gold standard (r2 = 0.92, P = 1.9 × 10-9 , bias = 3.9°). Small crossing angles and edema significantly impact the diffusion orientation distribution function, making DKI, GQI and QBI less accurate in detecting and estimating fiber crossing angles. Lastly, we used diffusion tensor ellipsoids to demonstrate that DBSI resolves the confounds of edema and crossing fibers in the peritumoral edema region from a patient with lung cancer metastasis, while DTI failed. In summary, DBSI is able to separate two crossing fibers and accurately recover their diffusivities in a complex environment characterized by increasing crossing angles and amounts of gel-mimicked edema. DBSI also indicated better angular resolution compared with DKI, QBI and GQI.

Acute White-Matter Abnormalities in Sports-Related Concussion: A Diffusion Tensor Imaging Study from the NCAA-DoD CARE Consortium.

Sports-related concussion (SRC) is an important public health issue. Although standardized assessment tools are useful in the clinical management of acute concussion, the underlying pathophysiology of SRC and the time course of physiological recovery after injury remain unclear. In this study, we used diffusion tensor imaging (DTI) to detect white matter alterations in football players within 48 h after SRC. As part of the NCAA-DoD CARE Consortium study of SRC, 30 American football players diagnosed with acute concussion and 28 matched controls received clinical assessments and underwent advanced magnetic resonance imaging scans. To avoid selection bias and partial volume effects, whole-brain skeletonized white matter was examined by tract-based spatial statistics to investigate between-group differences in DTI metrics and their associations with clinical outcome measures. Mean diffusivity was significantly higher in brain white matter of concussed athletes, particularly in frontal and subfrontal long white matter tracts. In the concussed group, axial diffusivity was significantly correlated with the Brief Symptom Inventory and there was a similar trend with the symptom severity score of the Sport Concussion Assessment Tool. In addition, concussed athletes with higher fractional anisotropy performed better on the cognitive component of the Standardized Assessment of Concussion. Overall, the results of this study are consistent with the hypothesis that SRC is associated with changes in white matter tracts shortly after injury, and these differences are correlated clinically with acute symptoms and functional impairments.

Structural characterization of a novel Ca2+-binding protein from Entamoeba histolytica: structural basis for the observed functional differences with its isoform.

A novel Ca(2+)-binding protein (EhCaBP2) was identified from the protozoan parasite Entamoeba histolytica. EhCaBP2 has 79% sequence identity with calcium-binding protein EhCaBP1. The 3D structure of EhCaBP2 was determined using multidimensional nuclear magnetic resonance spectroscopic techniques. The study reveals that the protein consists of two globular domains connected by a short flexible linker region of four residues. On comparison of the 3D structure and dynamics of EhCaBP2 with those of EhCaBP1, it is found that they vary significantly in their N-terminal domains and interdomain linker. Immunofluorescence localization experiments revealed that EhCaBP1 and EhCaBP2 may not carry out similar functions, as their cellular distribution patterns are not the same. The functional differences between the two isoforms are explained on the basis of results obtained from the structural studies. The structural variation in the interdomain linker region and the formation of functionally important hydrophobic clefts in different regions of EhCaBP1 and EhCaBP2 provide interesting insights into the differences in the functionality of these two isoforms.