Adeno-associated virus as a delivery vector for gene therapy of human diseases.

Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.

Phillygenin Alleviates Arthritis through the Inhibition of the NLRP3 Inflammasome and Ferroptosis by AMPK.

We investigated the potential arthritis-inducing effects of Phillygenin and its underlying mechanisms. RAW264.7 cells were stimulated with lipopolysaccharide to induce inflammation. Phillygenin was found to reduce arthritis score, histopathological changes, paw edema, spleen index, and ALP levels in a dose-dependent manner in a model of arthritis. Additionally, Phillygenin was able to decrease levels of inflammation markers in serum samples of mice with arthritis and also inhibited inflammation markers in the cell supernatant of an in vitro model of arthritis. Phillygenin increased cell viability and JC-1 disaggregation, enhanced calcien-AM/CoCl2, reduced LDH activity levels and IL-1a levels, and inhibited Calcein/PI levels and iron concentration in an in vitro model. Phillygenin was also found to reduce ROS-induced oxidative stress and Ferroptosis, and suppress the NLRP3 inflammasome in both in vivo and in vitro models through AMPK. In the in vivo model, Phillygenin was observed to interact with AMPK protein. These findings suggest that Phillygenin may be a potential therapeutic target for preventing arthritis by inhibiting NLRP3 inflammasome and Ferroptosis through AMPK. This indicates that Phillygenin could have disease-modifying effects on arthritis.

A sleep staging model on wavelet-based adaptive spectrogram reconstruction and light weight CNN.

Effective methods for automatic sleep staging are important for diagnosis and treatment of sleep disorders. EEG has weak signal properties and complex frequency components during the transition of sleep stages. Wavelet-based adaptive spectrogram reconstruction (WASR) by seed growth is utilized to capture dominant time-frequency patterns of sleep EEG. We introduced variant energy from Teager operator in WASR to capture hidden dynamic patterns of EEG, which produced additional spectrograms. These spectrograms enabled a light weight CNN to detect and extract finer details of different sleep stages, which improved the feature representation of EEG. With specially designed depthwise separable convolution, the light weight CNN achieved more robust sleep stage classification. Experimental results on Sleep-EDF 20 dataset showed that our proposed model yielded overall accuracy of 87.6%, F1-score of 82.1%, and Cohen kappa of 0.83, which is competitive compared with baselines with reduced computation cost.

miR-138-5p ameliorates intestinal barrier disruption caused by acute superior mesenteric vein thrombosis injury by inhibiting the NLRP3/HMGB1 axis.

Background: Acute superior mesenteric venous thrombosis (ASMVT) decreases junction-associated protein expression and intestinal epithelial cell numbers, leading to intestinal epithelial barrier disruption. Pyroptosis has also recently been found to be one of the important causes of mucosal barrier defects. However, the role and mechanism of pyroptosis in ASMVT are not fully understood. Methods: Differentially expressed microRNAs (miRNAs) in the intestinal tissues of ASMVT mice were detected by transcriptome sequencing (RNA-Seq). Gene expression levels were determined by RNA extraction and reverse transcription-quantitative PCR (RT-qPCR). Western blot and immunofluorescence staining analysis were used to analyze protein expression. H&E staining was used to observe the intestinal tissue structure. Cell Counting Kit-8 (CCK-8) and fluorescein isothiocyanate/propidine iodide (FITC/PI) were used to detect cell viability and apoptosis, respectively. Dual-luciferase reporter assays prove that miR-138-5p targets NLRP3. Results: miR-138-5p expression was downregulated in ASMVT-induced intestinal tissues. Inhibition of miR-138-5p promoted NLRP3-related pyroptosis and destroyed tight junctions between IEC-6 cells, ameliorating ASMVT injury. miR-138-5p targeted to downregulate NLRP3. Knockdown of NLRP3 reversed the inhibition of proliferation, apoptosis, and pyroptosis and the decrease in tight junction proteins caused by suppression of miR-138-5p; however, this effect was later inhibited by overexpressing HMGB1. miR-138-5p inhibited pyroptosis, promoted intestinal epithelial tight junctions and alleviated ASMVT injury-induced intestinal barrier disruption via the NLRP3/HMGB1 axis.

Relationship between sheep feces scores and gastrointestinal microorganisms and their effects on growth traits and blood indicators.

Fecal scores are crucial for assessing the digestive and gastrointestinal status of animals. The Bristol fecal scoring system is a commonly used method for the subjective evaluation of host feces, there is limited research on fecal scoring standards for fattening Hu sheep. In this study, Hu sheep were collected for rumen, rectum, and colon contents for 16S rDNA sequencing. 514 Hu sheep feces were scored based on the Bristol fecal scoring system, and production performance at each stage was measured. Finally, we developed the scoring standard of the manure of Hu sheep in the fattening period (a total of five grades). The result shows that moisture content significantly increased with higher grades (p < 0.05). We analyzed the relationship between fecal scores and production traits, blood indices, muscle nutrients, and digestive tract microorganisms. The growth traits (body weight, body height, body length, average daily gain (ADG), and average daily feed intake (ADFI) during 80-180 days), body composition traits of the F3 group, and the carcass traits were found to be significantly higher (p < 0.05) than those of the F1 and F2 groups. There was no significant difference in gastrointestinal microflora diversity among all groups (p > 0.05). Significant differences were observed in Aspartate aminotransferase, Glucose, Total bilirubin, and Red Blood Cell Count between groups (p < 0.05). The mutton moisture content in group F4 was significantly higher than in the other groups, and the protein content was also the lowest (p < 0.05). The results of the correlation analysis demonstrated that Actinobacteria, Peptostreptococcaceae, Acidaminococcales, Gammaproteobacteria, and Proteobacteria were the significant bacteria affecting fecal scores. In addition, Muribaculaceae and Oscillospiraceae were identified as the noteworthy flora affecting growth performance and immunity. This study highlights the differences in production traits and blood indicators between fecal assessment groups and the complex relationship between intestinal microbiota and fecal characteristics in Hu sheep, suggesting potential impacts on animal performance and health, which suggest strategies for improved management.

Polymorphism in IGFALS gene and its association with scrotal circumference in Hu lambs.

Scrotal circumference is an important reproductive index of breeding rams, which has a high genetic correlation with ejaculation volume and semen quality. In this study, the scrotal circumference of 1353 male Hu sheep at different stages of development was measured and descriptive statistical analysis was performed. The results showed that the coefficient of variation of scrotal circumference at each stage was greater than 10%, and its heritability were moderately to high, ranging from 0.318 to 0.719. We used PCR amplification and Sanger sequencing to scan the polymorphisms of the IGFALS gene, and performed association analysis with the circumference of the scrotum at different stages. We identified a synonymous mutation g.918 G > C in exon 1 of the IGFALS gene, and this mutation was significantly associated with scrotal circumference at 100, 120, 140, 160 and 180 days (p < 0.05). Therefore, IGFALS gene polymorphism can be used as a molecular marker affecting scrotal circumference of Hu sheep, which can provide a reference for future molecular marker-assisted selection of scrotal circumference in sheep.

Creating Efficient Red Thermally Activated Delayed Fluorescence Materials with Cyano-Substituted 11,12-Diphenyldipyrido[3,2-a:2',3'-c]phenazine Acceptors.

Red luminescent materials are essential components for full color display and white lightening based on organic light-emitting diode (OLED) technology, but the extension of emission color towards red or deep red region generally leads to decreased photoluminescence and electroluminescence efficiencies. Herein, we wish to report two new luminescent molecules (2CNDPBPPr-TPA and 4CNDPBPPr-TPA) consisting of cyano-substituted 11,12-diphenyldipyrido[3,2-a:2',3'-c]phenazine acceptors and triphenylamine donors. As the increase of cyano substituents, the emission wavelength is greatly red-shifted and the reverse intersystem crossing process is promoted, resulting in strong red delayed fluorescence. Meanwhile, due to the formation of intramolecular hydrogen bonds, the molecular structures become rigidified and planarized, which brings about large horizontal dipole ratios. As a result, 2CNDPBPPr-TPA and 4CNDPBPPr-TPA can perform as emitters efficiently in OLEDs, furnishing excellent external quantum efficiencies of 28.8 % at 616 nm and 20.2 % at 648 nm, which are significantly improved in comparison with that of the control molecule without cyano substituents. The findings in this work demonstrate that the introduction of cyano substituents to the acceptors of delayed fluorescence molecules could be a facile and effective approach to explore high-efficiency red or deep red delayed fluorescence materials.

Analysis of liver miRNA in Hu sheep with different residual feed intake.

Feed efficiency (FE), an important economic trait in sheep production, is indirectly assessed by residual feed intake (RFI). However, RFI in sheep is varied, and the molecular processes that regulate RFI are unclear. It is thus vital to investigate the molecular mechanism of RFI to developing a feed-efficient sheep. The miRNA-sequencing (RNA-Seq) was utilized to investigate miRNAs in liver tissue of 6 out of 137 sheep with extreme RFI phenotypic values. In these animals, as a typical metric of FE, RFI was used to distinguish differentially expressed miRNAs (DE_miRNAs) between animals with high (n = 3) and low (n = 3) phenotypic values. A total of 247 miRNAs were discovered in sheep, with four differentially expressed miRNAs (DE_miRNAs) detected. Among these DE_miRNAs, three were found to be upregulated and one was downregulated in animals with low residual feed intake (Low_RFI) compared to those with high residual feed intake (High_RFI). The target genes of DE_miRNAs were primarily associated with metabolic processes and biosynthetic process regulation. Furthermore, they were also considerably enriched in the FE related to glycolysis, protein synthesis and degradation, and amino acid biosynthesis pathways. Six genes were identified by co-expression analysis of DE_miRNAs target with DE_mRNAs. These results provide a theoretical basis for us to understand the sheep liver miRNAs in RFI molecular regulation.

Relationship between hindgut microbes and feed conversion ratio in Hu sheep and microbial longitudinal development.

Feed efficiency is an important indicator in the sheep production process, which plays an important role in improving economic benefits and strengthening energy conservation and emission reduction. Compared with the rumen, the fermentation of the hindgut microorganisms can also provide part of the energy for the host, and the composition of the hindgut microorganisms will affect the feed efficiency. Therefore, we hope to find new ways to regulate sheep feed efficiency by studying the sheep gut microbes. In this study, male Hu sheep with the same birth date were raised under the same conditions until 180 d old. The sheep were divided into high and low groups according to the feed conversion ratio (FCR) at 80 to 180 d old, and the differences in rectal microorganisms between the two groups were compared. The permutational multivariate analysis (PERMANOVA) test showed that there were differences in microorganisms between the two groups (P < 0.05). Combined with linear fitting analysis, a total of six biomarkers were identified, including Ruminobacter, Eubacterium_xylanophilum_group, Romboutsia, etc. Functional enrichment analysis showed that microorganisms may affect FCR through volatile fatty acids synthesis and inflammatory response. At the same time, we conducted a longitudinal analysis of the hindgut microbes, sampling nine-time points throughout the sheep birth to market stages. The microbiota is clearly divided into two parts: before weaning and after weaning, and after weaning microbes are less affected by before weaning microbial composition.

The level of feed efficiency determines the input of sheep production costs and the income of economic benefits. Improving sheep feed efficiency can effectively save energy and reduce emissions. Gut microbes play an important role in the process of feed fermentation. In this study, biomarkers associated with feed efficiency were identified by exploring the relationship between microbes and feed conversion ratio. At the same time, the longitudinal development of microorganisms was explored. It provides a basis for the regulation of intestinal microbes in sheep.

Allele specific binding of histone modifications and a transcription factor does not predict allele specific expression in correlated ChIP-seq peak-exon pairs.

Allele specific expression (ASE) is widespread in many species including cows. Therefore, regulatory regions which control gene expression should show cis-regulatory variation which mirrors this differential expression within the animal. ChIP-seq peaks for histone modifications and transcription factors measure activity at functional regions and the height of some peaks have been shown to correlate across tissues with the expression of particular genes, suggesting these peaks are putative regulatory regions. In this study we identified ASE in the bovine genome in multiple tissues and investigated whether ChIP-seq peaks for four histone modifications and the transcription factor CTCF show allele specific binding (ASB) differences in the same tissues. We then investigate whether peak height and gene expression, which correlates across tissues, also correlates within the animal by investigating whether the direction of ASB in putative regulatory regions, mirrors that of the ASE in the genes they are putatively regulating. We found that ASE and ASB were widespread in the bovine genome but vary in extent between tissues. However, even when the height of a peak was positively correlated across tissues with expression of an exon, ASE of the exon and ASB of the peak were in the same direction only half the time. A likely explanation for this finding is that the correlations between peak height and exon expression do not indicate that the height of the peak causes the extent of exon expression, at least in some cases.

Heritability and recursive influence of host genetics on the rumen microbiota drive body weight variance in male Hu sheep lambs.

BACKGROUND: Heritable rumen microbiota is an important modulator of ruminant growth performance. However, no information exists to date on host genetics-rumen microbiota interactions and their association with phenotype in sheep. To solve this, we curated and analyzed whole-genome resequencing genotypes, 16S rumen-microbiota data, and longitudinal body weight (BW) phenotypes from 1150 sheep. RESULTS: A variance component model indicated significant heritability of rumen microbial community diversity. Genome-wide association studies (GWAS) using microbial features as traits identified 411 loci-taxon significant associations (P < 10-8). We found a heritability of 39% for 180-day-old BW, while also the rumen microbiota likely played a significant role, explaining that 20% of the phenotypic variation. Microbiota-wide association studies (MWAS) and GWAS identified four marker genera (Bonferroni corrected P < 0.05) and five novel genetic variants (P < 10-8) that were significantly associated with BW. Integrative analysis identified the mediating role of marker genera in genotype influencing phenotype and unravelled that the same genetic markers have direct and indirect effects on sheep weight. CONCLUSIONS: This study reveals a reciprocal interplay among host genetic variations, the rumen microbiota and the body weight traits of sheep. The information obtained provide insights into the diverse microbiota characteristics of rumen and may help in designing precision microbiota management strategies for controlling and manipulating sheep rumen microbiota to increase productivity. Video Abstract.

Bioinformatics Tools for Bulk Gene Expression Deconvolution in Diabetic Retinopathy.

Retinal neovascularization is one of the leading causes of vision loss and a hallmark of proliferative diabetic retinopathy (PDR). The immune system is observed to be involved in the pathogenesis of diabetic retinopathy (DR). The specific immune cell type that contributes to retinal neovascularization can be identified via a bioinformatics analysis of RNA sequencing (RNA-seq) data, known as deconvolution analysis. Previous study has identified the infiltration of macrophages in the retina of rats with hypoxia-induced retinal neovascularization and patients with PDR through a deconvolution algorithm, known as CIBERSORTx. Here, we describe the protocols of using CIBERSORTx to perform the deconvolution analysis and downstream analysis of RNA-seq data.

Methods for Ex Vivo AAV Transduction in Rodent and Human Retinal Explants.

Translational research is heavily dependent on animal models, and reliable disease models are essential for the development of novel therapies. Here, we outline the methods for culturing mouse and human retinal explants. In addition, we show efficient adeno-associated virus (AAV) transduction of the mouse retinal explants to aid the study and development of AAV-based therapeutics against ocular diseases.

Polymorphism and expression level of the FADS3 gene and associated with the growth traits in Hu sheep.

Growth traits are the economically important traits of sheep, and screening for genes related to growth and development is helpful for the genetic improvement of ovine growth traits. The fatty acid desaturase 3 (FADS3) is one of the important genes affecting the synthesis and accumulation of polyunsaturated fatty acids in animals. In this study, the expression levels of the FADS3 gene and polymorphism of the FADS3 gene associated with growth traits in Hu sheep were detected using quantitative real-time PCR (qRT-PCR), Sanger sequencing, and KAspar assay. The result showed that the expression levels of the FADS3 gene were widely expressed in all tissues, and the expression level of FADS3 in the lung was significantly higher than in other tissues (p < .05). Then, the polymorphism locus g. 2918 A > C was detected in intron 2 of the FADS3 gene, and associated analysis showed that the mutation in the FADS3 gene was associated significantly with growth traits (including body weight, body height, body length, and chest circumference, p < .05). Therefore, individuals with AA genotype showed significantly better growth traits than those with CC genotype, and FADS3 gene could be a candidate gene for improving growth traits in Hu sheep.

Retinal Aging Transcriptome and Cellular Landscape in Association With the Progression of Age-Related Macular Degeneration.

Purpose: Age is the main risk factor for age-related macular degeneration (AMD), a leading cause of blindness in the elderly, with limited therapeutic options. Methods: Here, we analyze the transcriptomic characteristics and cellular landscape of the aging retinas from controls and patients with AMD. Results: We identify the aging genes in the neural retina, which are associated with innate immune response and inflammation. Deconvolution analysis reveals that the estimated proportions of M2 macrophages are significantly increased with both age and AMD severity. Moreover, we find that proportions of Müller glia are significantly increased only with age but not with AMD severity. Several genes associated with both age and AMD severity, particularly C1s and MR1, are strong positively correlated with the proportions of Müller glia. Conclusions: Our studies expand the genetic and cellular landscape of AMD and provide avenues for further studies on the relationship between age and AMD.

Expression profiles of the CD274 and PLEKHH2 gene and association of its polymorphism with hematologic parameters in sheep.

CD274 and PLEKHH2 genes have been identified as immune- and multiple diseases-related genes, and have recently garnered significant interest. However, their role in regulating immune functions in sheep remains largely unexplored. In this study, we aimed to investigate the effects of polymorphisms in CD274 and PLEKHH2 on hematologic parameters in 915 sheep. Our results showed that the CD274 and PLEKHH2 genes were most highly expressed in the spleen and tail fat, respectively, as determined by qRT-PCR. We also identified a G to A mutation (g 0.11858 G > A) in the exon 4 region of CD274, and a C to G mutation (g 0.38384 C > G) in the intron 8 region of PLEKH2. Association analysis revealed that CD274 g 0.11858 G > A was significantly associated with RBC, HCT, MCHC, and MCV (P < 0.05), while PLEKHH2 g 0.38384 C > G was significantly associated with HCT, MPV, MCHC, and MCV (P < 0.05). These results suggest that CD274 and PLEKHH2 genes may play a role in regulating blood physiological indicators and could be potential functional candidates for influencing immune traits in sheep breeding programs.

Efficient Cyan Delayed Fluorescence Luminogen as Sensitizing Host for OLEDs with High Efficiencies and Extremely Low Roll-Offs.

Organic light-emitting diodes (OLEDs) using thermally activated delayed fluorescence (TADF) materials to sensitizing conventional fluorescence dopants (CFDs) have attracted intensive research interest due to the extraordinary device performances. Herein, a cyan luminogen (TCz-BP-SFAC) with TADF and aggregation-enhanced emission (AEE) character is developed as a sensitizing host for CFDs. TCz-BP-SFAC owns excellent thermal and electrochemical stabilities, prefers horizontal dipole orientation and demonstrates violent delayed fluorescence with high photoluminescence quantum yields in neat and doped films. It exhibits preeminent electroluminescence (EL) performances with maximum external quantum efficiencies (ηext s) of 25.1% and 30.0% and very low efficiency roll-offs. TCz-BP-SFAC also performs well as a sensitizing host for CFDs of different colors. When TCz-BP-SFAC sensitizing green emitter TTPA and orange emitter TBRB, the devices achieve high ηext s of 16.9% and 17.1% as well as very low efficiency roll-offs of 2.4% and 1.2%, respectively. Moreover, TCz-BP-SFAC can serve as a sensitizing host for two-color all-fluorescence white OLEDs, resulting in high ηext s of ∼18% and very low efficiency roll-offs of ∼5%. The outstanding EL performances predict the great potential of TCz-BP-SFAC as emitter and host in practical display and lighting devices.

In it for the long run: perspectives on exploiting long-read sequencing in livestock for population scale studies of structural variants.

Studies have demonstrated that structural variants (SV) play a substantial role in the evolution of species and have an impact on Mendelian traits in the genome. However, unlike small variants (< 50 bp), it has been challenging to accurately identify and genotype SV at the population scale using short-read sequencing. Long-read sequencing technologies are becoming competitively priced and can address several of the disadvantages of short-read sequencing for the discovery and genotyping of SV. In livestock species, analysis of SV at the population scale still faces challenges due to the lack of resources, high costs, technological barriers, and computational limitations. In this review, we summarize recent progress in the characterization of SV in the major livestock species, the obstacles that still need to be overcome, as well as the future directions in this growing field. It seems timely that research communities pool resources to build global population-scale long-read sequencing consortiums for the major livestock species for which the application of genomic tools has become cost-effective.

Knockout of AMD-associated gene POLDIP2 reduces mitochondrial superoxide in human retinal pigment epithelial cells.

Genetic and epidemiologic studies have significantly advanced our understanding of the genetic factors contributing to age-related macular degeneration (AMD). In particular, recent expression quantitative trait loci (eQTL) studies have highlighted POLDIP2 as a significant gene that confers risk of developing AMD. However, the role of POLDIP2 in retinal cells such as retinal pigment epithelium (RPE) and how it contributes to AMD pathology are unknown. Here we report the generation of a stable human RPE cell line ARPE-19 with POLDIP2 knockout using CRISPR/Cas, providing an in vitro model to investigate the functions of POLDIP2. We conducted functional studies on the POLDIP2 knockout cell line and showed that it retained normal levels of cell proliferation, cell viability, phagocytosis and autophagy. Also, we performed RNA sequencing to profile the transcriptome of POLDIP2 knockout cells. Our results highlighted significant changes in genes involved in immune response, complement activation, oxidative damage and vascular development. We showed that loss of POLDIP2 caused a reduction in mitochondrial superoxide levels, which is consistent with the upregulation of the mitochondrial superoxide dismutase SOD2. In conclusion, this study demonstrates a novel link between POLDIP2 and SOD2 in ARPE-19, which supports a potential role of POLDIP2 in regulating oxidative stress in AMD pathology.