Discoveries in Retina Physiology and Disease Biology Using Single-Cell RNA Sequencing.

The retina, a component of the central nervous system, is composed of six distinct neuronal types and various types of glial cells. A technique for single-cell transcriptome analysis called single-cell RNA sequencing (scRNA-seq) can be employed to study the complicated dynamics of several types of retinal cells. It meticulously examines how various cell types express their genes, shedding light on all biological processes. scRNA-seq is an alternative to regular RNA-seq, which cannot identify cellular heterogeneity. Understanding retinal diseases requires research on retinal cell heterogeneity. The identification of novel cell subpopulations can provide information about disease occurrence and progression as well as the specific biological functions of particular cells. We currently have a better understanding of the interactions among the brain, the retina, and its visual pathways thanks to the use of scRNA-seq to examine retinal development and disease pathogenesis. Additionally, this technology offers fresh perspectives on the sensitivity and molecular basis of cell subtypes linked to retinal diseases. Thanks to scRNA-seq technology, we now have a better understanding of the most recent developments and difficulties in retinal development and disorders. We believe that scRNA-seq is an important tool for developing cutting-edge treatments for retinal diseases. This paper presents a systematic review of the history of sRNA-seq technology development and provides an overview of the unique subtypes of retinal cells and the specific gene markers this technology identifies.

Identification and subcellular localization of NbIAP in the microsporidian Nosema bombycis.

Many organisms go through a process of programmed cell death called apoptosis while newer cells are created. This has the effect of protecting the organism from cellular parasites and is a major line of defense against invading organisms. Apoptosis inhibitors, then, play an important role in aiding infectious agents by inhibiting caspase protease and thus the apoptopic pathway. In this study, we identified an inhibitor of apoptosis protein (IAP) in the microsporidian Nosema bombycis (NbIAP). NbIAP a composed of 218 amino acids containing two overlapping domains; the BIR domain and a zf-C3HC domain. We show, through indirect immunofluorescence, that NbIAP is present throughout the life cycle of N. bombycis and is localized in the nucleus of the parasite and therefor does not act on caspase protease directly. qRT-PCR analysis shows that the expression of the NbIAP gene was the highest on the first day of infection, then decreased to a relatively stable level. In addition, we show that the downregulation of the NbIAP gene directly inhibits the proliferation of N. bombycis. These findings suggest that NbIAP plays an important role in the N. bombycis life cycle.

Blue Light from Cell Phones Can Cause Chronic Retinal Light Injury: The Evidence from a Clinical Observational Study and a SD Rat Model.

BACKGROUND: To investigate the chronic photodamage induced by the low-intensity blue light of phones, we carried out a clinical pilot study and established an animal model by irradiating SD rats with a homemade illuminator. METHODS: Clinical investigation: A total of 25 clinical medical workers in our hospital were selected and divided into a control group and an observation group according to the daily video terminal use time. Multifocal electrophysiological system (Mf-ERG) was used for retinal functional examination. Animal experiment: A total of sixty SD rats were randomly divided into a control group (n = 6) and an experimental group (n = 54). The experimental rats were divided into nine groups, which were exposed to the blue light illuminator of the simulated cell phone array for different time. The visual electrophysiology of the rats was tested, and changes in structure were observed by H&E staining and transmission electron microscopy. RESULTS: In clinical investigation, macular centers near the concave area retinal photoreceptor cells have reduced amplitude. In animal experiments, the amplitude of photoreceptor cells decreased, the peak time was delayed, and the amplitudes were lower in the experimental groups. H&E staining and transmission electron microscope showed retinal tissue structure and functional damage in experimental groups. CONCLUSIONS: Long-term exposure to low-illuminance blue light can cause retinal tissue structure and functional damage, and the chronic damage due to low-illuminance light warrants attention. The clinical registration number is 2018-KY-KS-LHL.

The role of Bombyx mori Bmtutl-519 protein in the infection of BmN cells by Nosema bombycis.

Bmtutl-519 is an isoform of the Bombyx Turtle protein and a member of the immunoglobulin superfamily (IgSF). The relative expression level of Bmtutl-519 was significantly upregulated when BmN cells were infected by Nosema bombycis. The subcellular localization of Bmtutl-519 was studied using an indirect immunoinfluscent assay (IFA), Co-localization assay, Western blotting, and enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bmtutl-519 expression plasmid. The results indicate that Bmtutl-519 is distributed in both the cytoplasm and the cell membrane of BmN cells. Bmtutl-519 may be involved in the infection process of N. bombycis as a cell surface receptor or regulatory factor. Interaction analysis of Bmtutl-519 with NbSWP26, a spore wall protein of N. bombycis involved in host cell adherence and infection, showed that the C-terminal heparin-binding motif (HBM) of NbSWP26 mediates the interaction between these two proteins. Mutation of the NbSWP26 HBM at K208G, K209G, K210G, and K213G led to a loss of the ability to bind the Bmtutl-519 protein. Spore adherence and infection assays showed that Bmtutl-519 enhances the binding ability of N. bombycis to the host cell surface, but this did not enhance host cell infection by N. bombycis. In contrast, the sustained high expression of Bmtutl-519 in BmN cells inhibited the proliferation of N. bombycis spores.

Molecular characterization and expression analysis of Turtle protein in silkworm that is associated with Nosema bombycis infection.

In this report, we describe the cloning and characterization of a member of the immunoglobulin superfamily (IgSF); i.e., Turtle. The cDNA of Turtle was cloned from the silkworm Bombyx mori using the rapid amplification of cDNA ends (RACE) technique. Three isoforms of Bombyx Turtle were obtained, including Bmtutl-464, Bmtutl-519, and Bmtutl-810. The three isoforms had identical 27-amino acid signal peptides and four extracellular immunoglobulin (Ig) domains (IgI-IgIV). Sequence similarity and phylogenic analysis indicated that Bmtutl-810 belongs to the group of insect Turtle isoforms and shares 76.2% identity with Drosophila Turtle. Quantitative real-time PCR analysis revealed that the Bombyx Turtle isoforms were expressed throughout the entire development period, the highest levels of expression of Bmtutl-464 and Bmtutl-519 were observed at the second instar larvae stage, whereas that of Bmtutl-810 peaked at the embryonic stage. The ubiquitous expression of Bmtutl-464, Bmtutl-519, and Bmtutl-810 were observed in all studied tissues, except for Bmtutl-519 in the silk gland. The expression level of Bmtutl-464 was highest in the ovary, whereas that of Bmtutl-519 and Bmtutl-810 was highest in the hemolymph. Bmtutl-519 was upregulated in BmN cells infected by Nosema bombycis, We speculated that Bombyx Turtle was not only involved in neural development in silkworm, as well as Drosophila Turtle, but was also involved in the regulation of other biological functions. For example, Bmtutl-519 might be involved in N. bombycis infection and may play an important role in the immune response of silkworms to N. bombycis infection.

Early responses of silkworm midgut to microsporidium infection--A Digital Gene Expression analysis.

Host-pathogen interactions are complex processes, which have been studied extensively in recent years. In insects, the midgut is a vital organ of digestion and nutrient absorption, and also serves as the first physiological and immune barrier against invading pathogenic microorganisms. Our focus is on Nosema bombycis, which is a pathogen of silkworm pebrine and causes great economic losses to the silk industry. A complete understanding of the host response to infection by N. bombycis and the interaction between them is necessary to prevent this disease. Silkworm midgut infected with N. bombycis is a good model to investigate the early host responses to microsporidia infection and the interaction between the silkworm and the microsporidium. Using Digital Gene Expression analysis, we investigated the midgut transcriptome profile of P50 silkworm larvae orally inoculated with N. bombycis. At 6, 12, 18, 24, 48, 72, and 96 h post-infection (hpi), 247, 95, 168, 450, 89, 80, and 773 DEGs were identified, respectively. KEGG pathway analysis showed the influence of N. bombycis infection on many biological processes including folate biosynthesis, spliceosome, nicotinate and nicotinamide metabolism, protein export, protein processing in endoplasmic reticulum, lysosome, biosynthesis of amino acids, ribosome, and RNA degradation. In addition, a number of differentially expressed genes involved in the immune response were identified. Overall, the results of this study provide an understanding of the strategy used by silkworm as a defense against the invasion by N. bombycis. Similar interactions between hosts and pathogens infection may exist in other species.

Identification of sumoylated proteins in the silkworm Bombyx mori.

Small ubiquitin-like modifier (SUMO) modification (SUMOylation) is an important and widely used reversible modification system in eukaryotic cells. It regulates various cell processes, including protein targeting, transcriptional regulation, signal transduction, and cell division. To understand its role in the model lepidoptera insect Bombyx mori, a recombinant baculovirus was constructed to express an enhanced green fluorescent protein (eGFP)-SUMO fusion protein along with ubiquitin carrier protein 9 of Bombyx mori (BmUBC9). SUMOylation substrates from Bombyx mori cells infected with this baculovirus were isolated by immunoprecipitation and identified by LC-ESI-MS/MS. A total of 68 candidate SUMOylated proteins were identified, of which 59 proteins were functionally categorized to gene ontology (GO) terms. Analysis of kyoto encyclopedia of genes and genomes (KEGG) pathways showed that 46 of the identified proteins were involved in 76 pathways that mainly play a role in metabolism, spliceosome and ribosome functions, and in RNA transport. Furthermore, SUMOylation of four candidates (polyubiquitin-C-like isoform X1, 3-hydroxyacyl-CoA dehydrogenase, cyclin-related protein FAM58A-like and GTP-binding nuclear protein Ran) were verified by co-immunoprecipitation in Drosophila schneide 2 cells. In addition, 74% of the identified proteins were predicted to have at least one SUMOylation site. The data presented here shed light on the crucial process of protein sumoylation in Bombyx mori.

Detection of Nosema bombycis by FTA cards and loop-mediated isothermal amplification (LAMP).

We successfully established a detection method which exhibited a markedly higher sensitivity than previously developed detection methods for Nosema bombycis by combining glass beads, FTA card, and LAMP. Spores of N. bombycis were first broken by acid-washed glass beads; the DNA was subsequently extracted and purified with the FTA card, and LAMP was performed using primers (LSU296) designed based on the sequence of the LSU rRNA of N. bombycis. The minimum detection concentration was 10 spores/mL. When this method was used to detect pebrine disease in silkworm egg, the detection rate for 500 silkworm eggs, in which only one egg was infected with N. bombycis, was 100 % under our optimized conditions. If the number of eggs in the sample increased to 800 or 1,000, the sample was divided into two equal portions, and the eggs were smashed with glass beads after the addition of 1 mL of TE buffer. The liquid in two tubes was later mixed and applied to the FTA card, and the detection rates were 100 %. Furthermore, the LAMP method established in our study could detect N. bombycis infection in silkworm 24 h earlier than microscopy.