Long-Read Sequencing Reveals Alternative Splicing-Driven, Shared Immunogenic Neoepitopes Regardless of SF3B1 Status in Uveal Melanoma.

Tumor-specific neoepitopes are promising targets in cancer immunotherapy. However, the identification of functional tumor-specific neoepitopes remains challenging. In addition to the most common source, single-nucleotide variants (SNV), alternative splicing (AS) represents another rich source of neoepitopes and can be utilized in cancers with low SNVs such as uveal melanoma (UM). UM, the most prevalent adult ocular malignancy, has poor clinical outcomes due to a lack of effective therapies. Recent studies have revealed the promise of harnessing tumor neoepitopes to treat UM. Previous studies have focused on neoepitope targets associated with mutations in splicing factor 3b subunit 1 (SF3B1), a key splicing factor; however, little is known about the neoepitopes that are commonly shared by patients independent of SF3B1 status. To identify the AS-derived neoepitopes regardless of SF3B1 status, we herein used a comprehensive nanopore long-read-sequencing approach to elucidate the landscape of AS and novel isoforms in UM. We also performed high-resolution mass spectrometry to further validate the presence of neoepitope candidates and analyzed their structures using the AlphaFold2 algorithm. We experimentally evaluated the antitumor effects of these neoepitopes and found they induced robust immune responses by stimulating interferon (IFN)γ production and activating T cell-based UM tumor killing. These results provide novel insights into UM-specific neoepitopes independent of SF3B1 and lay the foundation for developing therapies by targeting these actionable neoepitopes.

Insight into the mechanisms of coronaviruses evading host innate immunity.

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) induced coronavirus disease 2019 (COVID-19) has recently caused a pandemic. Patients with COVID-19 presented with a wide spectrum of symptoms for the disease, from entirely asymptomatic disease to full-blown pneumonia and multiorgan failures. More evidence emerged, showing the production of interferons (IFNs) in the severe cases were significantly lower than their milder counterparts, suggesting linkage of COVID-19 to impaired innate immunity. This review presents a brief overview of how coronaviruses evade innate immunity, according to the current studies about SARS-CoV and middle-east respiratory syndrome-coronavirus (MERS-CoV). The coronaviruses manage to block, escape, or dampen the innate immune response by antagonizing double-stranded RNA (dsRNA) sensor, mitochondrial antiviral-signaling protein (MAVS) and stimulator of IFN genes (STING) pathways, epigenetic modification, posttranslational modifications, and host mRNA translation. We provide novel insights into a comprehensive therapy to combat SARS-CoV-2 infection.

The immune-related role of beta-2-microglobulin in melanoma.

Despite the remarkable success of immunotherapy in the treatment of melanoma, resistance to these agents still affects patient prognosis and response to therapies. Beta-2-microglobulin (ß2M), an important subunit of major histocompatibility complex (MHC) class I, has important biological functions and roles in tumor immunity. In recent years, increasing studies have shown that B2M gene deficiency can inhibit MHC class I antigen presentation and lead to cancer immune evasion by affecting ß2M expression. Based on this, B2M gene defect and T cell-based immunotherapy can interact to affect the efficacy of melanoma treatment. Taking into account the many recent advances in B2M-related melanoma immunity, here we discuss the immune function of the B2M gene in tumors, its common genetic alteration in melanoma, and its impact on and related improvements in melanoma immunotherapy. Our comprehensive review of ß2M biology and its role in tumor immunotherapy contributes to understanding the potential of B2M gene as a promising melanoma therapeutic target.

Label-Free Delineation of Human Uveal Melanoma Infiltration With Pump-Probe Microscopy.

Uveal melanoma (UM) is the most frequent primary intraocular malignancy in adults, characterized by melanin depositions in melanocytes located in the uveal tract in the eyes. Differentiation of melanin species (eumelanin and pheomelanin) is crucial in the diagnosis and management of UM, yet it remains inaccessible for conventional histology. Here, we report that femtosecond time-resolved pump-probe microscopy could provide label-free and chemical-specific detection of melanin species in human UM based on their distinct transient relaxation dynamics at the subpicosecond timescale. The method is capable of delineating the interface between melanoma and paracancerous regions on various tissue conditions, including frozen sections, paraffin sections, and fresh tissues. Moreover, transcriptome sequencing was conducted to confirm the active eumelanin synthesis in UM. Our results may hold potential for sensitive detection of tumor boundaries and biomedical research on melanin metabolism in UM.

Benefits of Zebrafish Xenograft Models in Cancer Research.

As a promising in vivo tool for cancer research, zebrafish have been widely applied in various tumor studies. The zebrafish xenograft model is a low-cost, high-throughput tool for cancer research that can be established quickly and requires only a small sample size, which makes it favorite among researchers. Zebrafish patient-derived xenograft (zPDX) models provide promising evidence for short-term clinical treatment. In this review, we discuss the characteristics and advantages of zebrafish, such as their transparent and translucent features, the use of vascular fluorescence imaging, the establishment of metastatic and intracranial orthotopic models, individual pharmacokinetics measurements, and tumor microenvironment. Furthermore, we introduce how these characteristics and advantages are applied other in tumor studies. Finally, we discuss the future direction of the use of zebrafish in tumor studies and provide new ideas for the application of it.

Novel p.G1344E mutation in FBN1 is associated with ectopia lentis.

BACKGROUND: Ectopia lentis refers to dislocation or subluxation of the crystalline lens. Fibrillin-1, encoded by FBN1, is an important microfibrillar structural component that is specifically required for the suspensory ligament of the lens. FBN1 mutations may cause abnormal structure of microfibrils and has been associated with a broad spectrum of clinical phenotypes. In this study, we characterised a Chinese dominant family with late-onset isolated ectopia lentis caused by a novel missense FBN1 mutation. METHODS: Eight family members, including four patients with suspected isolated ectopia lentis, were recruited from Shanghai. Clinical data and family history of the proband and other affected family members were collected. Ophthalmic examination, systemic examination and echocardiography were performed. Whole exome sequencing and Sanger sequencing were used to detect potential pathogenic variants. RESULTS: A novel heterozygous missense mutation c.4031 G>A/p.Gly1344Glu in exon 33 of FBN1 was identified. This mutation was detected in all affected family members and led to specific ocular system phenotypes (ectopia lentis, microspherophakia and secondary glaucoma) with minor skeletal involvement (hallux valgus). CONCLUSION: The novel c.4031G>A mutation in FBN1 is a likely pathogenic mutation for isolated ectopia lentis. Our study expands the spectrum of FBN1 mutations and contributes to better comprehension of genotype-phenotype correlations of ectopia lentis disease.

Traumatic endophthalmitis and the outcome after vitrectomy in young children.

AIM: To explore the traumatic endophthalmitis in young children and the outcome of pars plana vitrectomy (PPV). METHODS: Twenty-two eyes of 22 cases of young children consecutive pediatric traumatic endophthalmitis treated and followed up between September 2014 and May 2018 were included. Aqueous humor or vitreous samples were taken for bacterial culture and sensitivity tests. Intravitreal antibiotics (norvancomycin and ceftazidime) injection, combined with 23-gauge PPV, were administered in 22 eyes. Silicone oil (SO; 5000 centistoke) tamponade or perfluoropropane gas (C3F8) was used in all patients. Main outcome measures were best-corrected visual acuity (BCVA) and retinal attachment, the ratio of penetrating injury, and the existence of intraocular foreign body. RESULTS: The mean age of patients was 6.9±2.2 (range, 3-10)y. All injured eyes suffered from penetrating ocular injury with retained intraocular foreign body in one eye. Bacterial culture was positive in only 2 eyes. The mean follow-up time was 21.1±4.7 (range, 12-30)mo. In the primary PPV, intravitreal antibiotics was administrated in all eyes, SO in 18 eyes, and C3F8 in 4 eyes. The secondary operation of SO removal and C3F8 endotamponade was performed in 16 eyes and a second SO endotamponade due to emulsification of the oil and retinal detachment (RD) was operated in 7 eyes underwent 3 to 11.5mo after primary PPV. A third operation was done in 7 eyes. The final intraocular pressure (IOP) was 8.9±1.8 (range, 6.9-11.4) mm Hg. The final BCVAs were 20/200 or better in 5, counting fingers in 2, and light perception to hand movement in 8 eyes. Whose (66.7%) had retinal injury exhibited worse BCVA (P=0.019, Fisher's exact test). Eyes underwent SO tamponade exhibited worse final BCVA than that with C3F8 in the primary PPV (P=0.026, Fisher's exact test). CONCLUSION: Traumatic endophthalmitis in children is generally more severe and associated with more complicated surgical procedures. Most patients have retinal injury need multiple operations and the final BCVA is poor. Prevention of ocular trauma, especially in children, is still critical.

Intraocular Pharmacokinetics and Safety of Subretinal Injection Compared with Intravitreal Application of Conbercept in Vitrectomized Rabbit Eyes.

PURPOSE: To evaluate the ocular pharmacokinetic properties of subretinal conbercept injection in vitrectomized rabbit eyes and to compare them with those by intravitreal injection. METHODS: The following groups of New Zealand white rabbits received conbercept injections (0.5 mg/0.05 ml): a subretinal group (subretinal injection in vitrectomized eyes), an intravitreal group (intravitreal injection in vitrectomized eyes), and a control group (intravitreal injection in nonvitrectomized eyes). Drug concentrations in the aqueous humor (AH), the vitreous humor (VH), and the retina were measured by the enzyme-linked immunosorbent assay (ELISA), and pharmacokinetic parameters were calculated. Ophthalmic B-ultrasonography, electroretinogram (ERG), and hematoxylin and eosin (H&E) staining were performed to evaluate the safety of subretinal injection. RESULTS: On the 28th day after injection, the drug level in the subretinal group was significantly higher than that in the intravitreal group in the AH (0.90 ± 0.25 µg/ml and 0.11 ± 0.07 µg/ml and 0.11 ± 0.07 P < 0.001, respectively) and the VH (5.00 ± 3.86 µg/ml and 0.11 ± 0.07 µg/ml and 0.11 ± 0.07 P < 0.001, respectively) and the VH (5.00 ± 3.86 P < 0.001, respectively) and the VH (5.00 ± 3.86 P < 0.001, respectively) and the VH (5.00 ± 3.86 . CONCLUSIONS: Our study indicates that applying conbercept by subretinal injection can reduce the drug clearance rate and sustain a long maintenance period in ocular tissue, which suggests that subretinal conbercept injection may be a potentially valuable treatment option.

Intraocular pharmacokinetics of anti-vascular endothelial growth factor agents by intraoperative subretinal versus intravitreal injection in silicone oil-filled eyes of proliferative diabetic retinopathy: a randomized controlled pilot study.

PURPOSE: Intraoperative subretinal anti-vascular endothelial growth factor (VEGF) injections have been used clinically in some case, but the pharmacokinetic characteristics have not yet been determined. In this pilot study, we investigate the pharmacokinetic parameters of anti-VEGF agents by intraoperative subretinal or intravitreal injection in silicone oil (SiO)-filled eyes of patients with proliferative diabetic retinopathy (PDR). METHODS: Randomized controlled trial including 13 patients (16 eyes) with PDR underwent pars plana vitrectomy (PPV) with SiO tamponade and randomly received a subretinal (8 eyes) or intravitreal (8 eyes) conbercept injection (0.5 mg/0.05 ml) intraoperatively. Aqueous humour (AH) was obtained on the 1st, 3rd, 7th, 10th, 14th, 21st and 28th day after the injection. Drug concentrations in the AH were determined by enzyme-linked immunosorbent assay (ELISA). The last best-corrected visual acuity (BCVA) was examined 6 months postoperatively. RESULTS: The clearance rate of anti-VEGF agents by subretinal injection was reduced in vitrectomized eyes with SiO tamponade (p < 0.05). With the same drug dose, subretinal injection (5.49 ± 6.11 µg/ml) resulted in higher drug concentrations in the AH when compared with intravitreal injection (0.42 ± 0.46 µg/ml, p = 0.001) 4 weeks after the treatment. The mean residence time last (MRT0-t ) by subretinal injection (11.57 ± 0.83 days) was significantly longer than the mean MRT0-t by intravitreal injection (7.10 ± 1.00 days, p < 0.001). A self-paired analysis showed that subretinal injection led to the BCVA improvement by +28.59 letters 6 months postoperatively (p = 0.028) while the BCVA did not improve significantly by intravitreal injection (p = 0.715). CONCLUSIONS: The drug maintenance phase was prolonged by intraoperative subretinal injection in SiO-filled eyes of PDR. The results suggest that subretinal injection might be a valuable treatment option for the management of PDR.

MYCN and PRC1 cooperatively repress docosahexaenoic acid synthesis in neuroblastoma via ELOVL2.

BACKGROUND: The MYCN amplification is a defining hallmark of high-risk neuroblastoma. Due to irregular oncogenes orchestration, tumor cells exhibit distinct fatty acid metabolic features from non-tumor cells. However, the function of MYCN in neuroblastoma fatty acid metabolism reprogramming remains unknown. METHODS: Gas Chromatography-Mass Spectrometer (GC-MS) was used to find the potential target fatty acid metabolites of MYCN. Real-time PCR (RT-PCR) and clinical bioinformatics analysis was used to find the related target genes. The function of the identified target gene ELOVL2 on cell growth was detected through CCK-8 assay, Soft agar colony formation assay, flow Cytometry assay and mouse xenograft. Chromatin immunoprecipitation (ChIP) and Immunoprecipitation-Mass Spectrometer (IP-MS) further identified the target gene and the co-repressor of MYCN. RESULTS: The fatty acid profile of MYCN-depleted neuroblastoma cells identified docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid with anti-tumor activity, significantly increased after MYCN depletion. Compared with MYCN single-copy neuroblastoma cells, DHA level was significantly lower in MYCN-amplified neuroblastoma cells. RT-PCR and clinical bioinformatics analysis discovered that MYCN interfered DHA accumulation via ELOVL fatty acid elongase 2 (ELOVL2) which is a rate-limiting enzyme of cellular DHA synthesis. Enforced ELOVL2 expression in MYCN-amplified neuroblastoma cells led to decreased cell growth and counteracted the growth-promoting effect of MYCN overexpression both in vitro and vivo. ELOVL2 Knockdown showed the opposite effect in MYCN single-copy neuroblastoma cells. In primary neuroblastoma, high ELOVL2 transcription correlated with favorable clinical tumor biology and patient survival. The mechanism of MYCN-mediated ELOVL2 inhibition contributed to epigenetic regulation. MYCN recruited PRC1 (Polycomb repressive complex 1), catalysed H2AK119ub (histone 2A lysine 119 monoubiquitination) and inhibited subsequent ELOVL2 transcription. CONCLUSIONS: The tumor suppressive properties of DHA and ELOVL2 are repressed by the MYCN and PRC1 jointly, which suggests a new epigenetic mechanism of MYCN-mediated fatty acid regulation and indicates PRC1 inhibition as a potential novel strategy to activate ELOVL2 suppressive functions.

A modified intrascleral intraocular lens fixation technique with fewer anterior segment manipulations: 27-gauge needle-guided procedure with built-in 8-0 absorbable sutures.

BACKGROUND: To report a modified surgical technique for intrascleral intraocular lens (IOL) fixation with fewer anterior segment manipulations in eyes lacking sufficient capsular support. METHODS: Eyes from 14 patients who underwent 27-gauge needle-guided intrascleral IOL fixation with built-in 8-0 absorbable sutures were studied. The 8-0 absorbable sutures were inserted into 27-gauge round needles and used to create sclerotomies at the 4 o'clock and 10 o'clock positions under the scleral flap. The sutures were used to tie knots at the end of each haptic and guide haptic externalization through the sclerotomy. After externalization, a sufficient flange was created at the end of each haptic and fixed under the scleral flaps. The best corrected visual acuity (BCVA), corneal endothelial cell density (ECD), IOL tilt and decentration, previous surgery history, and complications were determined. RESULTS: Fourteen cases were analyzed. The majority of eyes exhibited an improvement in the BCVA after surgery. When comparing the last follow-up to preoperative visual acuity, the mean change in BCVA was + 26.32 letters (p = 0.011). Postoperative complications included postoperative hypotony in 3 eyes, ocular hypertension in 2 eyes. No cases of postoperative cystoid macular edema (CME), vitreous hemorrhage (VH), IOL dislocation, or endophthalmitis were observed. CONCLUSIONS: The 27-gauge needle-guided intrascleral IOL fixation technique with built-in 8-0 absorbable sutures is easy to perform with fewer anterior chamber manipulations and achieves both anatomical and optical stability.