Sustained neurotrophic factor cotreatment enhances donor and host retinal ganglion cell survival in mice.

Retinal ganglion cells (RGCs) lack regenerative capacity in mammals, and their degeneration in glaucoma leads to irreversible blindness. The transplantation of stem cell-derived RGCs lacks clinically relevant effect due to insufficient survival and integration of donor cells. We hypothesize that the retinal microenvironment plays a critical role in this process, and we can engineer a more acceptable setting for transplantation. Since the adult mammalian retina does not have regenerative capacity, we turned to the developing human retina to reconstruct cell-cell interactions at a single-cell level. We established a human fetal retina atlas by integrating currently available single-cell RNA sequencing datasets of human fetal retinas into a unified resource. We align RGC transcriptomes in pseudotime to map RGC developmental fate trajectories against the broader timeline of retinal development. Through this analysis, we identified brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) as key factors in RGC survival, highly expressed during fetal development but significantly reduced in adulthood despite the persistence of their receptors. To demonstrate the practical application of these findings, we show that using a slow-release formulation of BDNF and GDNF enhances RGC differentiation, survival, and function in vitro and improves RGC transplantation outcomes in a mouse model. BNDF/GDNF co-treatment not only increased survival and coverage of donor RGCs within the retina but also showed neuroprotective effects on host RGCs, preserving retinal function in a model of optic neuropathy. Altogether, our findings suggest that manipulating the retinal microenvironment with slow-release neurotrophic factors holds promise in regenerative medicine for treating glaucoma and other optic neuropathies. This approach not only improves donor cell survival and integration but also provides a neuroprotective benefit to host cells, indicating a significant advancement for glaucoma therapies.

Raman spectroscopy insight into Norovirus encapsulation in Bombyx mori cypovirus cubic microcrystals.

Protein and amino acid structures of Norovirus-like particles (NoVLP) have been investigated by Raman spectroscopy before and after encapsulation into Bombyx mori cypovirus (BmCPV) cubic microcrystals, which are usually referred to as cubes or polyhedra. Two different types of tag were used in co-expression, namely VP3 and H1 tags. VP3 tag is derived from a capsid protein VP4 from BmCPV and H1 tag is N-terminal α-helix of BmCPV polyhedrin, respectively. A major capsid protein VP1 of NoVLP G11.4 was fused with H1 or VP3 tags, and then encapsulated into BmCPV polyhedra. Analyses of the spectroscopic data permitted the assignment of conformation-sensitive Raman bands to viral amino acid constituents and the observation of structural similarities or differences between differently tagged samples. Three separate Raman zones were attentioned, namely, the ring-mode structure region (1000-1500 cm-1), the CO and CC double-bond region and its surroundings (1500-1750 cm-1), and the high-frequency CH stretching region (2800-3100 cm-1). Structural fingerprints could be found in specific spectral zones for differently co-expressed samples. One clear characteristic of the H1-tagged VP1 polyhedra was the increase in tyrosine fraction, which played a critical role in binding neighboring strands through its unpaired negatively charged COO- sites. This feature could consistently be detected in different regions, but it was best represented by Raman signals associated with negatively charged COO- sites and H1 helices in the double-bond region. Such peculiar chemical features were revealed by two relatively broad bands at 1570 and 1630 cm-1, which were assigned to COO- anti-symmetric stretching and amide I in 310-helix extensions to α-helices at N-termini, respectively. These specific features did not display in the spectrum of the VP3-tagged VP1 polyhedra. Concurrently, a strong reduction of CH bond Raman signal was noticed in the high frequency stretching region of the Raman spectrum upon H1-tagged VP1 polyhedra. The Raman activity most strikingly also represented fingerprints of tagged NoVLP VP1 after its encapsulation into BmCPV polyhedra, opening thus the possibility to in situ advanced experiments in the fields of drug delivery and regenerative medicine.