Twinning and 9R Phase Transition Mediated Extraordinary Cold-drawn Deformability in NiCoCrFeMo High-Entropy Alloy.

The production and application of materials are evolving towards the low-dimensional micro-nano scale. Nevertheless, the fabrication of micron-scale alloy fibers remains a challenge. Herein, a novel Ni-Co-Cr-Fe-Mo high-entropy alloy (HEA) fiber with a cold-drawn reduction rate of 99.9995% and a strain (ɛ) of 12.19 is presented without requiring intermediate annealing. The exceptional deformation strain of 11.62 within the fiber leads to extraordinary tensile strengths of 2.8 GPa at room temperature and 3.6 GPa at 123 K. The in-depth investigation of the microstructure of fibers has revealed the cold drawing deformation mechanisms mediated by the synergistic effects of plane defects. Specifically, various geometrically necessary dislocation interfaces, such as dislocation walls and microbands, along with deformation twins and long-period 9R structures, form in response to external stress when ɛ≤2.7. As the strain increases, the saturated layered structure emerges and progressively evolves into a 3D equiaxed crystal. Moreover, the formation and evolution of the 9R structure (i.e., the migration of incoherent twin boundaries), coupled with the interaction of partial dislocations and the role of deformation twins, are crucial factors determining the fiber's plastic response. This work provides a novel approach to discovering new high-strength metallic fibers with excellent deformability through plane defects engineering.

A comparison of perceptual-cognitive skills in expert and non-expert sports officials: a systematic review and meta-analysis.

Introduction: The purpose of this study is to systematically compare and assess the differences in perceptual-cognitive skills between expert and non-expert sports officials, and further explore the potential differences caused by different types of sports officials, in order to provide a more comprehensive understanding of the perceptual-cognitive skills of sports officials. Methods: Relevant literature published before 31 December 2022 was searched in four English databases. Review Manager 5.4 and Stata 12.0 software were used for meta-analysis and bias test. Results: Expert sports officials are significantly more accurate in their decision-making than non-expert sports officials, and exhibit a large amount of effect size (SMD = 1.09; 95%CI: 0.52, 1.66; P < 0.05). Expert sports officials had significantly fewer number of fixations than non-expert sports officials and showed a moderate amount of effect size (SMD = 0.71; 95%CI: 1.25, 0.17; P < 0.05). Expert sports officials' duration of fixation (SMD = 0.23; 95%CI: 0.25, 0.71; P = 0.35) were not significantly different from non-expert sports officials. Discussion: It can be seen that there are differences in the Perceptual-cognitive skills of expert and non-expert sports officials. Decision-making accuracy can serve as an important indicator for distinguishing the perceptual-cognitive skills of expert and non-expert sports officials. Number of fixations can serve as important indicators to differentiate the perceptual-cognitive skills of monitors. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=418594, identifier: CRD42023418594.

Epidemiological study of post-pandemic pediatric common respiratory pathogens using multiplex detection.

BACKGROUND: The burden and characteristics of respiratory viral infections in children hospitalized for acute respiratory tract infections (ARTIs) during the post-COVID-19 pandemic era are unclear. We analyzed the epidemiological and clinical characteristics of pediatric patients hospitalized with common respiratory virus infections before and after relaxation of non-pharmaceutical interventions in Hangzhou, China and evaluated the diagnostic value of the six-panel respiratory pathogen detection system. METHODS: Six types of respiratory viruses were detected in respiratory samples from children with suspected ARTIs by multiplex real-time quantitative polymerase chain reaction (RT-qPCR). Changes in virus detection rates and epidemiological and clinical characteristics, obtained from electronic health records, were analyzed. Binary logistic regression was used to identify respiratory tract infections risk factors. Multiplex RT-qPCR and targeted next-generation sequencing results were compared in random samples. RESULTS: Among the 11,056 pediatric samples, 3228 tested positive for one or more of six common respiratory pathogens. RSV and PIV-3 detection rates differed significantly across age groups (both P < 0.001), and were more common in younger children. PIV-1 was more common in infants, toddlers, and preschoolers than in school-age children (P < 0.001). FluB was predominantly detected in school-age children (P < 0.001). RSV-, ADV-, and PIV-1-positivity rates were higher in 2022 than in 2023. Seasonal viral patterns differed across years. RSV (OR 9.156. 95% CI 5.905-14.195) and PIV-3 (OR 1.683, 95% CI 1.133-2.501) were risk factors for lower respiratory tract infections. RSV-positivity was associated with severe pneumonia (P = 0.044). PIV-3 (OR 0.391, 95% CI 0.170-0.899), summer season (OR 1.982, 95% CI 1.117-3.519), and younger age (OR 0.938, 95% CI 0.893-0.986) influenced pneumonia severity. Multiplex RT-qPCR showed good diagnostic performance. CONCLUSION: After changes in COVID-19 prevention and control strategies, six common respiratory viruses in children were prevalent in 2022-2023, with different seasonal epidemic characteristics and age proclivities. RSV and PIV-3 cause lower, and FluA, FluB, and ADV more typically cause upper respiratory tract infections. Infancy and summer season influence severe pneumonia risk. Multiplex RT-qPCR is valuable for accurate and timely detection of respiratory viruses in children, which facilitates management, treatment, and prevention of ARTIs.

Differentiation in detoxification gene complements, including neofunctionalization of duplicated cytochrome P450 genes, between lineages of cotton bollworm, Helicoverpa armigera.

Here we investigate the evolutionary dynamics of five enzyme superfamilies (CYPs, GSTs, UGTs, CCEs and ABCs) involved in detoxification in Helicoverpa armigera. The reference assembly for an African isolate of the major lineages, H. a. armigera, has 373 genes in the five superfamilies. Most of its CYPs, GSTs, UGTs and CCEs and a few of its ABCs occur in blocks and most of the clustered genes are in subfamilies specifically implicated in detoxification. Most of the genes have orthologues in the reference genome for the Oceania lineage, H. a. conferta. However, clustered orthologues and subfamilies specifically implicated in detoxification show greater sequence divergence and less constraint on non-synonymous differences between the two assemblies than do other members of the five superfamilies. Two duplicated CYPs, which were found in the H. a. armigera but not H. a. conferta reference genome, were also missing in 16 Chinese populations spanning two different lineages of H. a. armigera. The enzyme produced by one of these duplicates has higher activity against esfenvalerate than a previously described chimeric CYP mutant conferring pyrethroid resistance. Various transposable elements were found in the introns of most detoxification genes, generating diverse gene structures. Extensive resequencing data for the Chinese H. a. armigera and H. a. conferta lineages also revealed complex copy number polymorphisms in 17 CCE001s in a cluster also implicated in pyrethroid metabolism, with substantial haplotype differences between all three lineages. Our results suggest that cotton bollworm has a versatile complement of detoxification genes which are evolving in diverse ways across its range.

Genome-wide identification and phylogenetic analysis of the tetraspanin gene family in lepidopteran insects and expression profiling analysis in Helicoverpa armigera.

The tetraspanin gene family encodes cell-surface proteins that span the membrane 4 times and play critical roles in a wide range of biological processes across numerous organisms. Recent findings highlight the involvement of a tetraspanin of the lepidopteran pest Helicoverpa armigera in resistance to Bacillus thuringiensis Cry insecticidal proteins, which are extensively used in transgenic crops. Thus, a better understanding of lepidopteran tetraspanins is urgently needed. In the current study, genome scanning in 10 lepidopteran species identified a total of 283 sequences encoding potential tetraspanins. Based on conserved cysteine patterns in the large extracellular loop and their phylogenetic relationships, these tetraspanins were classified into 8 subfamilies (TspA to TspH). Six ancestral introns were identified within lepidopteran tetraspanin genes. Tetraspanins in TspA, TspB, TspC, and TspD subfamilies exhibit highly similar gene organization, while tetraspanins in the remaining 4 subfamilies exhibited variation in intron loss and/or gain during evolution. Analysis of chromosomal distribution revealed a lepidopteran-specific cluster of 10 to 11 tetraspanins, likely formed by tandem duplication events. Selective pressure analysis indicated negative selection across all orthologous groups, with ω values ranging between 0.004 and 0.362. However, positive selection was identified at 18 sites within TspB5, TspC5, TspE3, and TspF10. Furthermore, spatiotemporal expression analysis of H. armigera tetraspanins demonstrated variable expression levels across different developmental stages and tissues, suggesting diverse functions of tetraspanin members in this globally important insect pest. Our findings establish a solid foundation for subsequent functional investigations of tetraspanins in lepidopteran species.

The central retinal thickness and its related genotype in ABCA4-related retinopathy.

PURPOSE: To further explore the influence of genotype, including mutation type and structural domain, on the severity of macular atrophy, we measured the central retinal thickness (CRT) in patients with ABCA4-related retinopathy. METHODS: A total of 66 patients were included in the cohort. This was a retrospective investigation. The patients were tested using whole exon sequencing and ophthalmic exams, including slip lamp exams, best-corrected visual acuity, optical coherence tomography, fundus photo, and fundus autofluorescence. RESULTS: In this study, we discovered that mutations on nucleotide binding domains (NBD) lead to less CRT (45.00 ± 25.25µm, 95% CI: 31.54-58.46) had significantly less CRT than the others (89.75 ± 71.17µm, 95% CI: 30.25-149.25, p = 0.032), and could accelerate the rate of CRT decrease. CONCLUSIONS: Our study provides new perspectives in the understanding of ABCA4-related retinopathy.

GTSE1 promotes nasopharyngeal carcinoma proliferation and angiogenesis by upregulating STMN1.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor with poor survival rate. G2 and S phase-expressed-1 (GTSE1) takes part in the progression of diverse tumors as an oncogene, but its role and potential mechanism in NPC remain unknown. METHODS: The GTSE1 expression was analyzed by western blot in NPC tissues and cells. Knock-down experiments were conducted to determine the function of GTSE1 in NPC by cell counting kit-8, the 5-ethynyl-2'-deoxyuridine (EdU) incorporation experiment, cell scratch wound-healing experiment, transwell assays, tube forming experiment and western blot. In addition, the in vivo role of GTSE1 was addressed in tumor-bearing mice. RESULTS: The expression of was increased in NPC. Silencing of GTSE1 suppressed cell viability, the percent of EdU positive cells, and the number of invasion cells and tubes, but enhanced the scratch ratio in NPC cells. Mechanically, downregulation of GTSE1 decreased the expressions of FOXM1 and STMN1, which were restored with the upregulation of FOXM1. Increased expression of STMN1 reversed the effects of the GTSE1 silencing on proliferation, migration, invasion and angiogenesis of NPC cells. Furthermore, knockdown of GTSE1 repressed the tumor volume and tumor weight of xenografted mice. CONCLUSION: GTSE1 was highly expressed in NPC, and silencing of GTSE1 ameliorated the malignant processes of NPC cells by upregulating STMN1, suggesting a possible therapeutical target for NPC.

Key role of the ryanodine receptor I4790K mutation in mediating diamide resistance in Plutella xylostella.

The diamondback moth Plutella xylostella, a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017-0.917) of the 4790K allele, but not with either 4790M (0-0.183) or 4946E (0.017-0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella, and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest.

Establishment of the induced pluripotent stem cell line SJTUGHi002-A from a CNGA1-related recessive retinitis pigmentosa patient.

Retinitis pigmentosa (RP) is the most common inherited retinal diseases, characterized by photoreceptor cell death and retinal pigment epithelial atrophy. Mutations in cyclic nucleotide gated channel subunit alpha 1 (CNGA1) have been reported to cause retinitis pigmentosa. Here, we established the human induced pluripotent stem cell line (iPSC) SJTUGHi002-A, generated from peripheral blood mononuclear cells of a 36-year-old male RP patient, who carried a homozygous frameshift variant in CNGA1 gene (c.265delC; p.L89Ffs*4). The cell line can serve as a patient-derived disease model for exploring the pathogenesis and drug development of CNGA1-RP.

Cross-resistance and genetics of field-evolved resistance to chlorfenapyr in Plutella xylostella.

Chlorfenapyr is a broad-spectrum halogenated pyrrole insecticide with a unique mode of action. Due to the misuse and overuse of this chemical, resistance has been reported in several arthropods, including Plutella xylostella, which is one of the most destructive insect pests afflicting crucifers worldwide. A better understanding of the cross-resistance and genetics of field-evolved chlorfenapyr resistance could effectively guide resistance management practices. Here, the chlorfenapyr resistance of a field-derived population of P. xylostella was introgressed into the susceptible IPP-S strain using a selection-assisted multigenerational backcrossing approach. The constructed near-isogenic strain, TH-BC5F2, shared 98.4% genetic background with the recurrent parent IPP-S strain. The TH-BC5F2 strain showed 275-fold resistance to chlorfenapyr, but no significant cross-resistance to spinosad, abamectin, chlorpyrifos, ß-cypermethrin, indoxacarb, chlorantraniliprole, or broflanilide (no more than 4.2-fold). Genetic analysis revealed that resistance was autosomal, incompletely dominant, and conferred by 1 major gene or a few tightly linked loci. The synergism of metabolic inhibitors (PBO, DEM, and DEF) to chlorfenapyr was very weak (<1.7-fold), and the metabolic enzyme activities in the TH-BC5F2 strain were not significantly elevated compared with the IPP-S strain. The results enhances our understanding of the genetic traits of chlorfenapyr resistance, and provides essential information for improving resistance management strategies.

GENETIC FACTORS AND CHARACTERISTICS ON SPECTRAL-DOMAIN OPTICAL COHERENCE TOMOGRAPHY ARE ASSOCIATED WITH CHOROIDAL THICKNESS IN ABCA4 -RELATED RETINOPATHY.

PURPOSE: To investigate the possible correlation factors of choroidal thickness in ABCA4 -related retinopathy. METHODS: A total of 66 patients were included in the cohort. It is a retrospective, cross-sectional laboratory investigation. The patients were tested using whole-exon sequencing and ophthalmic examinations, including slit-lamp examinations, best-corrected visual acuity, spectral-domain optical coherence tomography, fundus photograph, and fundus autofluorescence. RESULTS: Besides demographic characteristics (age, onset age, duration), we selected genetic factors and ocular characteristics on spectral-domain optical coherence tomography as the candidates related to choroidal thickness. Mutation type (inframe mutation or premature termination codon), epiretinal membrane, retinal pigment epithelium- Bruch membrane integrity, and macular curvature changes were identified as related factors to choroidal thickness in ABCA4 -related retinopathy after the adjustment of Logistic LASSO regression. CONCLUSION: Mutation type, epiretinal membrane, retinal pigment epithelium-Bruch membrane integrity, and macular curvature changes are related factors to choroidal thinning. These findings could provide us a further understanding for the pathological process and clinical features of ABCA4 mutation.

Helicoverpa armigera ATP-binding cassette transporter ABCA2 is a functional receptor of Bacillus thuringiensis Cry2Ab toxin.

Crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are widely used in transgenic crops to control important insect pests. Bt crops have many benefits compared with traditional broad-spectrum insecticides, including improved pest control with reduced negative impacts on off-target organisms and fewer environmental consequences. Transgenic corn and cotton producing Cry2Ab Bt toxin are used globally to control several major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Resistance to the Cry2Ab toxin and to Bt crops producing Cry2Ab is associated with mutations in the midgut ATP-binding cassette transporter ABCA2 gene in several lepidopterans. Gene-editing knockout has further shown that ABCA2 plays an important functional role in Cry2Ab intoxication. However, the precise role of ABCA2 in the mode of action of Cry2Ab has yet to be reported. Here, we used two in vitro expression systems to study the roles of the H. armigera ABCA2 (HaABCA2) protein in Cry2Ab intoxication. Cry2Ab bound to cultured Sf9 insect cells producing HaABCA2, resulting in specific and dose-dependent susceptibility to Cry2Ab. In contrast, Sf9 cells expressing recombinant mutant proteins missing at least one of the extracellular loop regions 1, 3, 4, and 6 or the intracellular loop containing nucleotide-binding domain 1 lost susceptibility to Cry2Ab, indicating these regions are important for receptor function. Consistent with these results, Xenopus laevis oocytes expressing recombinant HaABCA2 showed strong ion membrane flux in the presence of Cry2Ab, suggesting that HaABCA2 is involved in promoting pore formation during Cry2Ab intoxication. Together with previously published data, our results support HaABCA2 being an important receptor of Cry2Ab where it functions to promote intoxication in H. armigera.

Overexpression of the F116V allele of CYP9A186 in transgenic Helicoverpa armigera confers high-level resistance to emamectin benzoate.

Insect cytochrome P450s play important roles in the detoxification of xenobiotics and the metabolic resistance to insecticides. However, the approach for in vivo validation of the contribution of specific candidate P450s to resistance is still limited in most non-model insect species. Previous studies with heterologous expression and in vitro functional assays have confirmed that a natural substitution (F116V) in the substrate recognition site 1 (SRS1) of the CYP9A186 of Spodoptera exigua is a gain-of-function mutation, which results in detoxification capability of and thus high-level resistance to both emamectin benzoate (EB) and abamectin. In this study, we established an effective piggyBac-based transformation system in the serious agricultural pest Helicoverpa armigera and overexpressed in vivo a resistance P450 allele, CYP9A186-F116V, from another lepidopteran pest Spodoptera exigua. Bioassays showed that transgenic H. armigera larvae expressing CYP9A186-F116V obtained 358-fold and 38.6-fold resistance to EB and abamectin, respectively. In contrast, a transgenic line of Drosophila melanogaster overexpressing this P450 variant only confers ∼20-fold resistance to the two insecticides. This bias towards the resistance level revealed that closely related species might provide a more appropriate cellular environment for gene expression and subsequent toxicokinetics of insecticides. These results not only present an alternative method for in vivo functional characterization of P450s in H. armigera and other phylogenetically close species but also provide a valuable genetic engineering toolkit for the genetic manipulation of H. armigera.

Divergent amplifications of CYP9A cytochrome P450 genes provide two noctuid pests with differential protection against xenobiotics.

Here, we provide mechanistic support for the involvement of the CYP9A subfamily of cytochrome P450 monooxygenases in the detoxification of host plant defense compounds and chemical insecticides in Spodoptera exigua and Spodoptera frugiperda. Our comparative genomics shows that a large cluster of CYP9A genes occurs in the two species but with significant differences in its contents, including several species-specific duplicates and substantial sequence divergence, both between orthologs and between duplicates. Bioassays of CRISPR-Cas9 knockouts of the clusters show that, collectively, the CYP9As can detoxify two furanocoumarin plant defense compounds (imperatorin and xanthotoxin) and insecticides representing three different chemotypes (pyrethroids, avermectins, and oxadiazines). However, in vitro metabolic assays of heterologously expressed products of individual genes show several differences between the species in the particular CYP9As with activities against these compounds. We also find that the clusters show tight genetic linkage with high levels of pyrethroid resistance in field strains of the two species. We propose that their divergent amplifications of the CYP9A subfamily have not only contributed to the development of the broad host ranges of these species over long evolutionary timeframes but also supplied them with diverse genetic options for evolving resistance to chemical insecticides in the very recent past.

Susceptibility and diagnostic concentration for Bacillus thuringiensis toxins and newer chemical insecticides in Spodoptera frugiperda (Lepidoptera: Noctuidae) from China.

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is a cosmopolitan pest that exploits more than 350 host plants, including economically important crops such as corn, cotton and rice. Control of S. frugiperda largely relies on transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) and spraying synthetic insecticides. Here, we established the susceptibility and diagnostic concentration for 2 Bt toxins and 5 newer insecticides in invasive populations of S. frugiperda from southeastern China. Concentrations causing 50% mortality (LC50) in ten field populations sampled in 2022 ranged from 2.13 to 19.29 and 22.43 to 71.12 ng/cm2 for Cry1Fa and Vip3Aa, and 0.83 to 5.30, 2.83 to 9.94, 0.04 to 0.23, 4.59 to 8.40, and 1.49 to 6.79 mg/liter for chlorantraniliprole, chlorfenapyr, emamectin benzoate, indoxacarb, and spinosad, respectively. Relative to the susceptible strain YJ-19, the largest resistance ratio in the field populations was 5.1, 1.6, 6.2, 3.9, 4.6, 2.2, and 3.6 for Cry1Fa, Vip3Aa, chlorantraniliprole, chlorfenapyr, emamectin benzoate, indoxacarb, and spinosad, respectively, indicating that the field populations were generally susceptible to these Bt toxins and insecticides. Based on the pooled response of the field populations, the diagnostic concentration for resistance monitoring, estimated as ca. twice the LC99, was 400 and 1,500 ng/cm2 for Cry1Fa and Vip3Aa, and 2, 40, 60, 60, and 100 mg/liter for emamectin benzoate, chlorantraniliprole, chlorfenapyr, spinosad, and indoxacarb, respectively. These results provide useful information for monitoring resistance to key Bt toxins and insecticides for the control of S. frugiperda in China.

Mechanisms of feeding cessation in Helicoverpa armigera larvae exposed to Bacillus thuringiensis Cry1Ac toxin.

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been applied in sprayable formulations and expressed in transgenic crops for the control of pests in the field. When exposed to Bt proteins insect larvae display feeding cessation, yet the mechanism for this phenomenon remains unknown. In this study, we investigated the feeding behavior and underlying mechanisms of cotton bollworm (Helicoverpa armigera) larvae after exposure to the Cry1Ac protein from Bt. Three H. armigera strains were studied: the susceptible SCD strain, the C2/3-KO strain with HaABCC2 and HaABCC3 knocked out and high-level resistance to Cry1Ac (>15,000-fold), and the SCD-KI strain with a T92C point mutation in tetraspanin (HaTSPAN1) and medium-level resistance to Cry1Ac (125-fold). When determining the percentage of insects that continued feeding after various exposure times to Cry1Ac, we observed quick cessation of feeding in larvae from the susceptible SCD strain, whereas larvae from the C2/3-KO strain did not display feeding cessation. In contrast, larvae from the SCD-KI strain rapidly recovered from the initial feeding cessation. Histopathological analyses and qRT-PCR in midguts of SCD larvae after Cry1Ac exposure detected serious epithelial damage and significantly reduced expression of the neuropeptide F gene (NPF) and its potential receptor gene NPFR, which are reported to promote insect feeding. Neither epithelial damage nor altered NPF and NPFR expression appeared in midguts of C2/3-KO larvae after Cry1Ac treatment. The same treatment in SCD-KI larvae resulted in milder epithelial damage and subsequent repair, and a decrease followed by an initial increase in NPF and NPFR expression. These results demonstrate that the feeding cessation response to Cry1Ac in cotton bollworm larvae is closely associated with midgut epithelial damage and downregulation of NPF and NPFR expression. This information provides clues to the mechanism of feeding cessation in response to Bt intoxication and contributes to the mode of action of the Cry1Ac toxin in target pests.

AAV-mediated base-editing therapy ameliorates the disease phenotypes in a mouse model of retinitis pigmentosa.

Base editing technology is an ideal solution for treating pathogenic single-nucleotide variations (SNVs). No gene editing therapy has yet been approved for eye diseases, such as retinitis pigmentosa (RP). Here, we show, in the rd10 mouse model, which carries an SNV identified as an RP-causing mutation in human patients, that subretinal delivery of an optimized dual adeno-associated virus system containing the adenine base editor corrects the pathogenic SNV in the neuroretina with up to 49% efficiency. Light microscopy showed that a thick and robust outer nuclear layer (photoreceptors) was preserved in the treated area compared with the thin, degenerated outer nuclear layer without treatment. Substantial electroretinogram signals were detected in treated rd10 eyes, whereas control treated eyes showed minimal signals. The water maze experiment showed that the treatment substantially improved vision-guided behavior. Together, we construct and validate a translational therapeutic solution for the treatment of RP in humans. Our findings might accelerate the development of base-editing based gene therapies.

Macrophage Sult2b1 promotes pathological neovascularization in age-related macular degeneration.

Disordered immune responses and cholesterol metabolism have been implicated in age-related macular degeneration (AMD), the leading cause of blindness in elderly individuals. SULT2B1, the key enzyme of sterol sulfonation, plays important roles in inflammation and cholesterol metabolism. However, the role and underlying mechanism of SULT2B1 in AMD have not been investigated thus far. Here, we report that SULT2B1 is specifically expressed in macrophages in choroidal neovascularization lesions. Sutl2b1 deficiency significantly reduced leakage areas and inhibited pathological angiogenesis by inhibiting M2 macrophage activation in vivo and in vitro. Mechanistically, loss of Sult2b1 activated LXRs and subsequently increased ABCA1 and ABCG1 (ABCA1/G1)-mediated cholesterol efflux from M2 macrophages. LXR inhibition (GSK2033 treatment) in Sult2b1 -/- macrophages reversed M2 polarization and decreased intracellular cholesterol capacity to promote pathological angiogenesis. In contrast to SULT2B1, STS, an enzyme of sterol desulfonation, protected against choroidal neovascularization development by activating LXR-ABCA1/G1 signalling to block M2 polarization. Collectively, these data reveal a cholesterol metabolism axis related to macrophage polarization in neovascular AMD.

SYVN1 Promotes STAT3 Protein Ubiquitination and Exerts Antiangiogenesis Effects in Retinopathy of Prematurity Development.

Purpose: SYVN1, a gene involved in endoplasmic reticulum-associated degradation, has been found to exert a protective effect by inhibiting inflammation in retinopathy. This study aimed to clarify whether SYVN1 is involved in the pathogenesis of retinopathy of prematurity (ROP) and its potential as a candidate for target therapy. Methods: Human retinal microvascular endothelial cells (hRMECs) and a mouse model of oxygen-induced retinopathy (OIR) were used to reveal the retinopathy development-associated protein expression and molecular mechanism. An adenovirus overexpressing SYVN1 or vehicle control was injected intravitreally at postnatal day 12 (P12), and the neovascular lesions were evaluated in retinal flatmounts with immunofluorescence staining, and hematoxylin and eosin staining at P17. Visual function was assessed by using electroretinogram (ERG). Results: Endogenous SYVN1 expression dramatically decreased in hRMECs under hypoxia and in ROP mouse retinas. SYVN1 regulated the signal transducer and activator of transcription 3 (STAT3)/vascular endothelial growth factor (VEGF) axis. SYVN1 overexpression promoted ubiquitination and degradation of STAT3, decreased the levels of phospho-STAT3, secretion of VEGF, and formation of neovascularization in hRMECs, which could be rescued by STAT3 activator treatment. In addition, SYVN1 overexpression prevented neovascularization and extended physiologic retinal vascular development in the retinal tissues of OIR mice without affecting retinal function. Conclusions: SYVN1 has a protective effect against OIR, and the molecular mechanisms are partly through SYVN1-mediated ubiquitination of STAT3 and the subsequent downregulation of VEGF. These findings strongly support our assumption that SYVN1 confers ROP resistance and may be a potentially novel pharmaceutical target against proliferative retinopathy.

Rapid and Sensitive Diagnosis of Leber Hereditary Optic Neuropathy Variants Using CRISPR/Cas12a Detection.

Leber hereditary optic neuropathy (LHON) is the most common maternally inherited mitochondrial disease, with >90% of cases harboring one of three point variants (m.3460G>A, m.11778G>A, and m.14484T>C). Rapid and sensitive diagnosis of LHON variants is urgently needed for early diagnosis and timely treatment after onset, which is currently limited. Herein, we adapted the Cas12a-based DNA detection platform for LHON mitochondrial variant diagnosis. Single-strand guide CRISPR RNAs and enzymatic recombinase amplification primers were first screened, the CRISPR/Cas12a system was then optimized with restriction enzymes, and finally compared with Sanger sequencing and next-generation sequencing (NGS) in multicenter clinical samples. This approach can be completed within 30 minutes using only one drop of blood and could reach a sensitivity of 1% of heteroplasmy. Among the 182 multicenter clinical samples, the CRISPR/Cas12a detection system showed high consistency with Sanger sequencing and NGS in both specificity and sensitivity. Notably, a sample harboring a de novo 3.78% m.11778G>A variant detected by NGS, but not by Sanger sequencing, was successfully confirmed using the CRISPR/Cas12a assay, which proved the effectiveness of our method. Overall, our CRISPR/Cas12a detection system provides an alternative for rapid, convenient, and sensitive detection of LHON variants, exhibiting great potential for clinical practice.