Immuno-targeting the ectopic phosphorylation sites of PDGFRA generated by MAN2A1-FER fusion in HCC.

BACKGROUND: HCC is one of the most lethal cancers for humans. Mannosidase alpha class 2A member 1 (MAN2A1)-FER is one of the most frequent oncogenic fusion genes in HCC. In this report, we showed that MAN2A1-FER ectopically phosphorylated the extracellular domains of PDGFRA, MET, AXL, and N-cadherin. The ectopic phosphorylation of these transmembrane proteins led to the activation of their kinase activities and initiated the activation cascades of their downstream signaling molecules. METHODS: A panel of mouse monoclonal antibodies was developed to recognize the ectopic phosphorylation sites of PDGFRA. RESULTS AND CONCLUSIONS: The analyses showed that these antibodies bound to the specific phosphotyrosine epitopes in the extracellular domain of PDGFRA with high affinity and specificity. The treatment of MAN2A1-FER-positive cancer HUH7 with one of the antibodies called 2-3B-G8 led to the deactivation of cell growth signaling pathways and cell growth arrest while having minimal impact on HUH7ko cells where MAN2A1-FER expression was disrupted. The treatment of 2-3B-G8 antibody also led to a large number of cell deaths of MAN2A1-FER-positive cancer cells such as HUH7, HEPG2, SNU449, etc., while the same treatment had no impact on HUH7ko cells. When severe combined immunodeficiency mice xenografted with HEPG2 or HUH7 were treated with monomethyl auristatin E-conjugated 2-3B-G8 antibody, it slowed the progression of tumor growth, eliminated the metastasis, and reduced the mortality, in comparison with the controls. Targeting the cancer-specific ectopic phosphorylation sites of PDGFRA induced by MAN2A1-FER may hold promise as an effective treatment for liver cancer.

Association of urinary calcium excretion with chronic kidney disease in patients with type 2 diabetes.

PURPOSE: Herein, we investigated the correlation between urinary calcium excretion (UCaE) and chronic kidney disease (CKD) in patients with type 2 diabetes mellitus (T2DM). METHODS: From August 2018 to January 2023, a total of 2031 T2DM patients providing 24-h urine samples were included in the final analyses. Patients were separated into four cohorts, based on the UCaE quartiles. We then analyzed renal functional indicators like estimated glomerular filtration rate (eGFR) and urinary albumin excretion (UAE) among the four groups. Lastly, we utilized multivariable logistic regression models to investigate the correlation between UCaE and CKD. RESULTS: After adjusting for confounding factors, we observed a decreasing trend in CKD prevalence (36.3%, 13.0%, 7.5%, and 6.6%, respectively, P < 0.001) across the UCaE quartiles. Albuminuria (55.5% vs. 40.0%, 36.5%, 37.4%) and macroalbuminuria prevalence (20.0% vs. 9.3%, 5.2%, 5.7%) in the lowest quartile were markedly elevated, compared to the remaining three quartiles (P < 0.001). Meanwhile, the eGFR level (P < 0.001) showed a clearly increasing trend across the UCaE quartiles, and patients with moderate-to-severe decreases in eGFR levels (with cutoff limits at 30-59, 15-30, and < 15 mL/min/1.73m2) were mostly found in the lowest quartile (P < 0.001). Logistic regression analysis revealed that patients in the lowest quartile experienced an enhanced prevalence of CKD, relative to those in the highest quartile (odds ratio: 5.90, 95% confidence interval: 3.60-9.67, P < 0.001). CONCLUSION: Decreased UCaE was independently associated with the CKD prevalence in T2DM patients.

Serum Fusion Transcripts to Assess the Risk of Hepatocellular Carcinoma and the Impact of Cancer Treatment through Machine Learning.

Hepatocellular carcinoma (HCC) is one of the most fatal malignancies. Early diagnosis of HCC is crucial in reducing the risk for mortality. This study analyzed a panel of nine fusion transcripts in serum samples from 61 patients with HCC and 75 patients with non-HCC conditions, using TaqMan real-time quantitative RT-PCR. Seven of the nine fusions frequently detected in patients with HCC included: MAN2A1-FER (100%), SLC45A2-AMACR (62.3%), ZMPSTE24-ZMYM4 (62.3%), PTEN-NOLC1 (57.4%), CCNH-C5orf30 (55.7%), STAMBPL1-FAS (26.2%), and PCMTD1-SNTG1 (16.4%). Machine-learning models were constructed based on serum fusion-gene levels to predict HCC in the training cohort, using the leave-one-out cross-validation approach. One machine-learning model, called the four fusion genes logistic regression model (MAN2A1-FER≤40, CCNH-C5orf30≤38, SLC45A2-AMACR≤41, and PTEN-NOLC1≤40), produced accuracies of 91.5% and 83.3% in the training and testing cohorts, respectively. When serum α-fetal protein level was incorporated into the machine-learning model, a two fusion gene (MAN2A1-FER≤40, CCNH-C5orf30≤38) + α-fetal protein logistic regression model was found to generate an accuracy of 94.8% in the training cohort. The same model resulted in 95% accuracy in both the testing and combined cohorts. Cancer treatment was associated with reduced levels of most of the serum fusion transcripts. Serum fusion-gene machine-learning models may serve as important tools in screening for HCC and in monitoring the impact of HCC treatment.

Therapeutic targeting at genome mutations of liver cancer by the insertion of HSV1 thymidine kinase through Cas9-mediated editing.

BACKGROUND: Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease. METHODS: In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1- thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers. RESULTS: We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups. CONCLUSIONS: Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.

Long-read single-cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells.

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPSeq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPSeq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

Foveal Microstructure and Visual Outcomes after Pars Plana Vitrectomy in Patients with Different Types of Epiretinal Membrane Foveoschisis.

INTRODUCTION: The aim of this study was to evaluate the clinical characteristics and surgical outcomes of the epiretinal membrane foveoschisis (ERM-FS) with different morphological types. METHODS: This retrospective observational study reviewed 44 consecutive ERM-FS patients who underwent ERM surgery. According to the optical coherence tomography images, ERM-FS was classified into three groups: group A, FS crossed the fovea with the foveola elevated; group B, FS located at the foveal edges with a near-normal central foveal point thickness; and group C, FS with undermined foveal edges with a near-normal central foveal point thickness. RESULTS: There were 10 eyes in group A, 20 eyes in group B, and 14 eyes in group C. Preoperatively, eyes in group A had the best best-corrected visual acuity (BCVA), the thickest central foveal point thickness, and the highest ellipsoid zone (EZ) intact rate among the three groups. After surgery, a resolution of foveoschisis was observed in 40.0%, 45.0%, and 50.0% of the eyes in group A, group B, and group C (p = 0.928), respectively. BCVA was significantly improved postoperatively. Although there was no significant difference in BCVA among the three groups at 1 month postoperatively, BCVA of group A was the best at 4 and 10 months. Correlation analysis indicated that the type of ERM-FS, baseline BCVA, central foveal point thickness, and postoperative EZ continuity (all p < 0.05) were important factors for the final BCVA. CONCLUSIONS: The damage to the retinal structure and visual function was milder in group A ERM-FS. Our study emphasized the necessity of OCT-based subtyping in patients with ERM-FS.

PREDICTIVE FACTORS ASSOCIATED WITH THE VISUAL PROGNOSIS AFTER SURGERY FOR IDIOPATHIC VITREOMACULAR TRACTION.

PURPOSE: To investigate the predictive factors for postsurgical visual prognosis in patients with vitreomacular traction (VMT). METHODS: This retrospective study enrolled 31 eyes from 29 patients who underwent vitrectomy for idiopathic VMT with a follow-up period of ≥3 months. The VMT was divided into three grades based on optical coherence tomography images: Grade 1 denoted partial vitreomacular separation with foveal attachment; Grade 2 exhibited intraretinal cysts or cleft with grade 1 findings; and Grade 3 was Grade 2 plus the subretinal fluid. RESULTS: Three eyes developed a full-thickness macular hole after surgery, all of which were Grade 3 patients. In the rest 28 eyes, the mean postoperative follow-up period was 23.3 ± 25.8 months. The postoperative central foveal thickness ( P = 0.001) and final best-corrected visual acuity (BCVA; P < 0.001) were both significantly improved from baseline. Fifteen eyes (53.8%) gained ≥ two Snellen lines. Multilinear regression analysis showed that the worse the baseline BCVA ( P = 0.004), or the more advanced the VMT grade ( P = 0.049), the worse the final BCVA. Baseline BCVA was negatively associated with the postoperative visual improvement ( P < 0.001). Those Grade 3 patients with baseline Snellen BCVA of ≥20/40 were more likely to achieve a final Snellen BCVA of ≥20/25 ( P = 0.035). CONCLUSION: The VMT grade is an important predictive factor for the postsurgical visual prognosis. Surgical intervention should be performed as early as possible for Grade 3 patients to prevent further disease progression and maximize the postsurgical visual benefit.

Photoreceptor Outer Segment Loss at the Macular Hole Border: Clinical Characteristics and Surgical Prognosis.

INTRODUCTION: During macular hole (MH) formation, the vitreofoveal traction may lead to foveal neural tissue avulsion and consequent photoreceptor loss. However, the clinical significance of the photoreceptor outer segment disruption at the MH border remains unclear. This study aims to investigate the clinical features and surgical outcomes of MHs with photoreceptor outer segment disruption at the MH border. METHODS: This study retrospectively reviewed 86 eyes from 85 patients who underwent vitrectomy combined with internal limiting membrane peeling for idiopathic MHs. Baseline and postoperative best-corrected visual acuity (BCVA) and foveal microstructure on optical coherence tomography images were compared between eyes with smooth and bumpy morphology, the latter of which was defined as those with an uneven and irregular surface with evident lumps and indentations at the MH border. RESULTS: The bumpy morphology was identified in 59 of 86 eyes (68.9%). At baseline, eyes with a bumpy MH morphology had a significantly larger minimum linear diameter (p = 0.043), a longer external limiting membrane defect (p = 0.013), and a worse baseline BCVA (p = 0.017) than those with a smooth morphology. Postoperatively, in a multivariate regression model adjusting preoperative confounding effects, eyes with bumpy borders were associated with a longer ellipsoid zone defect (p = 0.006) and a thinner central fovea (p = 0.002) at 1 month, and a thinner central fovea (p = 0.005) and a worse BCVA (p = 0.015) at 10 months. CONCLUSION: A severe photoreceptor outer segment loss is common at the MH border, which represents a chronic pathogenic process and may be an independent predictor for an incomplete photoreceptor recovery and worse long-term visual outcomes after surgery.

The Evolution and Visual Prognosis of Glial Proliferation with Different Grades after Macular Hole Surgery: An Optical Coherence Tomography-Based Study.

INTRODUCTION: The glial proliferation after macular hole (MH) surgery was divided into two types previously: those replacing the entire intraretinal layer and those involving only the inner foveal layers. The evolution and prognosis of the former type were elaborated on in previous studies, but the latter one has received limited attention. Therefore, this study aims to investigate the evolution of glial proliferation with varying grades after MH surgery and its effects on foveal microstructure and best-corrected visual acuity (BCVA). METHODS: In this retrospective research, we reviewed 202 eyes from 196 consecutive patients who underwent a successful idiopathic MHs repair. Based on optical coherence tomography images, glial proliferation was classified into three types: A-type, which replaced the entire intraretinal layer; B-type, located at the level of and above the external limiting membrane (ELM); and C-type, situated above the ELM. RESULTS: Of the 67 eyes that attended the 1-, 4-, and 10-month follow-up, A-type, B-type, C-type, and no glial proliferation were identified in 27 (40.3%), 17 (25.4%), 20 (29.8%), and 3 eyes (4.5%), respectively, at 1 month. Within 10 months, the prevalence of A-type glial proliferation significantly decreased (p < 0.001), but the changes in B-type (p = 0.261), C-type (p = 0.151), and no glial proliferation (p = 0.492) were not significant. In 32 of the 67 eyes, the grade of glial proliferation gradually improved, with A-type transforming into B- or C-type in 19 of 27 eyes (70.4%), B-type into C-type or no glial proliferation in 11 out of 17 eyes (64.7%), and C-type gradually disappearing in 2 out of 20 eyes (10.0%). Among the eyes that attended at least one follow-up (1 M, 202 eyes; 4 M, 161 eyes; 10 M, 97 eyes), those with A-type glial proliferation showed the most defective outer retinal layers, worst BCVA, and thinnest central fovea compared with the other two types at all follow-up time points (p < 0.001). Eyes with C-type glial proliferation exhibited significantly better photoreceptor layer status and BCVA compared with those with B-type glial proliferation. A-type glial proliferation at 1 month, which showed significant association with BCVA at 10 months, could be accurately predicted by the minimum linear diameter with a cut-off >547.5 µm (p < 0.001). CONCLUSION: Within 10 months, A-type glial proliferation substantially resolves but the prevalence of B- and C-type remains unchanged. B-type glial proliferation hinders the restoration of photoreceptors and impairs visual recovery despite being located within the inner retina.

A novel missense variant expands the phenotype and genotype of PAX6-associated foveal hypoplasia accompanied by various manifestations of anterior segment dysgenesis.

BACKGROUND: According to previous reports, PAX6-associated foveal hypoplasia (FH) could usually be accompanied by various anterior segment anomalies including variable iris changes. This study aims to exhibit unusual phenotypes of a novel missense variant of PAX6 from a Chinese pedigree. METHODS: Ophthalmic examinations including slit-lamp biomicroscopy, gonioscopy, ophthalmic ultrasound, ultrasonic biomicroscopy, optical coherence tomography, wide-field fundus imaging, and visual field test were performed to evaluate the clinical manifestations. Whole-exome sequencing (WES) and bioinformatics analysis were conducted in eight members from this pedigree to identify the causative mutation. RESULTS: WES revealed a novel heterozygous substitution of PAX6 (NM_000280.5:c.157G > A, p.(Val53Met) (chr11:31823309 C > T, hg19)), which cosegregated with the phenotype of this pedigree. All the three patients (a pair of fraternal twins and their mother) exhibited bilateral FH and anterior segment dysgenesis (ASD) including microcornea, sclerocornea, obvious symmetrical corectopia, iris stromal dysplasia, goniodysgenesis, and abnormal distribution of fundus blood vessels. The girl of the fraternal twins also demonstrated bilateral temporal deviation of lenses and abnormal tissue membrane connecting anterior chamber angle and lens anterior capsule in the right eye. The mother additionally showed apparent cataract bilaterally and cupping of the optic disc in her left eye. CONCLUSION: A novel missense variant in PAX6 gene was detected in a Chinese pedigree demonstrating bilateral FH and ASD. It is really distinctive that the ASD involves almost all parts of the anterior segment, and bilateral symmetrical corectopia is the most perceptible sign. This study expands the phenotypic and genotypic spectrum of PAX6-associated ocular diseases, and facilitates the understanding of the crucial role that PAX6 plays in the development of the eye. Meanwhile, PAX6 could be considered as a candidate pathogenic gene of bilateral symmetrical corectopia.

Clinical features of idiopathic epiretinal membrane in children and the factors influencing postoperative visual acuity.

PURPOSE: To describe the characteristics and surgical outcomes of idiopathic epiretinal membrane (iERM) in children and to determine the factors associated with postoperative visual acuity (VA). METHODS: We retrospectively reviewed the medical records of 17 patients with iERM (age, < 18 years) who had undergone ERM surgery from 2009 to 2021. Spectral-domain optical coherence tomography features were documented. The eyes with iERMs involving the fovea were assigned to the localized and diffused groups depending on the morphological description of the membrane. Multiple linear regression analysis was used to explore the factors associated with the final VA. RESULTS: The mean age was 9.2 ± 3.8 years. The mean follow-up period was 38.9 ± 45.4 months. After surgery, the central foveal thickness and the best-corrected VA (BCVA) improved significantly (all, P < 0.05). Fourteen eyes with iERMs showed involvement of the foveal area (localized group, six eyes; diffused group, eight eyes). There were no significant differences in the preoperative BCVA between the two groups (P = 0.064). However, the final BCVA was better in the diffused group than in the localized group (P = 0.043). Multiple regression analysis indicated that the localized membrane (P = 0.042) and lower preoperative BCVA (P = 0.043) were factors associated with a worse final VA in pediatric iERMs. CONCLUSIONS: Surgical removal of ERM showed a high anatomical and functional success rate in children. In pediatric patients with iERMs involving the fovea, a good VA was more common when the membrane was diffused than when it was localized.

Microstructural and microperimetric comparison of internal limiting membrane peeling and insertion in large idiopathic macular hole.

BACKGROUND: The internal limiting membrane (ILM) insertion technique was widely used to treat large macular hole (MH) for the high closure rate. However, the prognosis of closed MH after ILM insertion compared to ILM peeling remains controversial. This study aimed to compare foveal microstructure and microperimeter in large idiopathic MH surgically closed by ILM peeling and ILM insertion. METHODS: This retrospective, non-randomized, comparative study included patients with idiopathic MH (minimum diameter ≥ 650 µm) who underwent primary pars plana vitrectomy (PPV) with ILM peeling or ILM insertion. The initial closure rate was recorded. Patients with initially closed MHs were divided into two groups according to the surgery methods. The best-corrected visual acuity (BCVA), optical coherence tomography (OCT) and microperimeter-3 (MP-3) outcomes of two groups were compared at baseline, 1 and 4 months postoperatively. RESULTS: For idiopathic MH (minimum diameter ≥ 650 µm), ILM insertion had a significantly higher initial closure rate than ILM peeling (71.19% vs. 97.62%, P = 0.001). Among 39 patients with initially closed MHs who were on regular follow-up, twenty-one were assigned to the ILM peeling group and 18 to the ILM insertion group. Postoperative BCVA improved significantly in both groups. The final BCVA (logMAR) (0.40 vs. 0.88, P < 0.001), macular hole sensitivity (19.66 dB vs. 14.14 dB, P < 0.001), peripheral sensitivity of macular hole (24.63 dB vs. 21.95 dB, P = 0.005), and fixation stability (FS) within 2 degrees (82.42% vs. 70.57%, P = 0.031) were significantly better and external limiting membrane (ELM) defect (330.14 µm vs. 788.28 µm, P < 0.001) and ellipsoid zone (EZ) defect (746.95 µm vs. 1105.11 µm, P = 0.010) were significantly smaller in the ILM peeling group than in the ILM insertion group. CONCLUSION: For initially closed MHs (minimum diameter ≥ 650 µm), both ILM peeling and ILM insertion significantly improved the microstructure and microperimeter in the fovea. However, ILM insertion was less efficient at microstructural and functional recovery after surgery.

Bimetallic Ions Functionalized Metal-Organic-Framework Nanozyme for Tumor Microenvironment Regulating and Enhanced Photodynamic Therapy for Hypoxic Tumor.

Photodynamic therapy (PDT), as a light irradiation inducing reactive oxygen species (ROS) generation for cancer treatment, offers facile and promising solutions with respect to spatiotemporal control of ROS generation, and minimizes the systemic toxicity and side effects for highly precise tumor therapy. However, the PDT efficiency is often severely compromised by the complex tumor microenvironment (TME), such as the hypoxic condition and overexpressed antioxidants. Here, for the first time, a bimetallic ion-modified metal-organic framework nanozyme (Zr4+ -MOF-Ru3+ /Pt4+ -Ce6@HA, ZMRPC@HA) is designed. ZMRPC@HA with catalase (CAT) and glutathione oxidase (GSHOx) mimetic activities, can efficiently regulate TME by generation of O2 and deplete the GSH synergistically for enhancing the long-term PDT efficacy toward the hypoxic tumor. The in vitro cell inhibition and in vivo on tumor xenograft evaluations demonstrate the PDT strategy by using ZMRPC@HA can successfully inhibit the differentiation and proliferation of tumor cells under a 660 nm laser irradiation in deep tissues. These findings open a new avenue for the design of multimetallic ions functionalized MOF-based nanozymes with multienzyme mimetic activities toward the antitumor and various other biological applications.

Stage or size? The identity of anatomical and visual outcomes in stage 3 and stage 4 idiopathic macular holes after vitrectomy.

BACKGROUND: Several previous reports suggested that stage 4 idiopathic macular holes (IMHs) may exhibit lower rate of anatomical success and poorer functional results comparing with stage 3 IMHs, while some others showed no differences. Actually, few studies focused on comparison of prognosis between stage 3 and stage 4 IMHs. Our previous study found that IMHs of these two stages demonstrate similar preoperative characteristics, and this study aims to compare anatomical and visual outcomes of IMHs between stage 3 and stage 4, and tries to figure out the outcome-associated factors. METHODS: This retrospective consecutive case series reviewed 317 eyes with IMHs of stage 3 and stage 4 from 296 patients who underwent vitrectomy with internal limiting membrane peeling. Preoperative characteristics like age, gender, and hole size, and intraoperative interventions such as combined cataract surgery were evaluated. Outcome measures included the primary closure rate (type 1), best-corrected visual acuity (BCVA), foveal retinal thickness (FRT) and prevalence of outer retinal defect (ORD) at the last visit. The pre-, intra-, and post-operative information were respectively compared between stage 3 and stage 4. RESULTS: The preoperative characteristics and intraoperative interventions exhibited no significant differences between stages. With comparable follow-up durations (6.6 vs. 6.7 months, P = 0.79), IMHs of the two stages exhibited similar primary closure rate (91.2% vs. 91.8%, P = 0.85), BCVA (0.51 ± 0.12 vs. 0.53 ± 0.11, P = 0.78), FRT (134.8 ± 55.5 µm vs. 138.8 ± 60.7 µm, P = 0.58), and prevalence of ORD (55.1% vs. 52.6%, P = 0.39). IMHs, either < 650 µm or larger, exhibited no significant difference in outcomes between the two stages. However, smaller IMHs (< 650 µm) demonstrated higher rate of primary closure (97.6% vs. 80.8%, P < 0.001), better postoperative BCVA (0.58 ± 0.26 vs. 0.37 ± 0.24, P < 0.001), and thicker postoperative FRT (150.2 ± 54.0 vs. 104.3 ± 52.0, P < 0.001) comparing with larger ones regardless of stage. CONCLUSION: IMHs of stage 3 and stage 4 exhibited considerable identity of anatomical and visual outcomes. In large IMHs, the hole size, instead of stage, may be more important for prediction of surgical outcomes and choice of surgical techniques.

Long-read single-cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells.

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate Long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPseq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection (UMAP) analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen (HLA) molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPseq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

CLINICAL CHARACTERISTICS AND SURGICAL PROGNOSIS OF IDIOPATHIC MACULAR HOLES WITH EPIRETINAL PROLIFERATION.

PURPOSE: To investigate the clinical features and surgical outcomes of idiopathic macular holes with epiretinal proliferation (EP). METHODS: This retrospective study included 282 eyes of 269 consecutive patients who underwent vitrectomy for idiopathic macular holes. Clinical demographic data, baseline and postoperative retinal microstructure features on optical coherence tomography images, best-corrected visual acuity, and postoperative closure rate were compared between eyes with and without EP. RESULTS: EP was identified in 38 of 282 eyes (13.5%). Eyes with EP had a significantly longer symptom duration ( P = 0.019), higher frequency of pseudophakic conditions ( P = 0.034), presence of epiretinal membrane ( P < 0.001), and a larger base diameter ( P = 0.014) than those without EP. In addition, compared with eyes without EP, those with EP had a higher percentage of U-type closure ( P = 0.047) and a thicker central fovea ( P = 0.012) at 1 month postoperatively and a significantly lower frequency of ellipsoid zone disruption ( P = 0.033) and outer foveolar lucency ( P = 0.021) and a thicker central fovea ( P = 0.004) at 10 months postoperatively. CONCLUSION: The presence of EP represents a more chronic pathogenic process but has no negative effect on postoperative outcomes and can even facilitate anatomical recovery after surgery.

Targeting the E3 ligase NEDD4 as a novel therapeutic strategy for IGF1 signal pathway-driven gastric cancer.

The IGF1 signal pathway is highly activated in some subtype of gastric cancer(GC) that exhibits poor survival and chemotherapy resistance. Although the results of clinical trials of anti-IGF1R monoclonal antibodies and IGF-1R inhibitors have been mostly disappointing in unselected cancer patients, some patients benefit from anti-IGF1R therapy in these failed studies. Therefore, it is necessary to characterize the complex IGF signaling in GC and help refine the strategies targeting the IGF1 pathway. We found that GC cell lines exhibit differential responses to the specific IGF1R inhibitor OSI906. According to the phosphorylation status of Akt upon the OSI906 treatment, we divided the GC cell lines into IGF1R-dependent and IGF1R-independent cells. Both in vitro and in vivo experiments indicate that Dox-induced knockdown of NEDD4 significantly suppresses tumor growth of IGF1R-dependent GC cells and NEDD4 overexpression promotes tumor growth of IGF1R-dependent GC cells. In contrast, the proliferation of IGF1R-independent GC cells is not affected by NEDD4 silencing and overexpression. The rescue experiments show that a PTEN-IRS1 axis is required for NEDD4-mediated regulation of Akt activation and tumor growth in GC cells. Clinically, NEDD4 is expressed higher in IGF1-high GC tissues compared with IGF1-low GC tissues and normal tissues, and the co-high expression of NEDD4 and IGF1 predicts a worse prognosis in GC patients. Taken together, our study demonstrated that NEDD4 specifically promotes proliferation of GC cells dependent on IGF1/IGF1R signaling by antagonizing the protein phosphatase activity of PTEN to IRS1, and targeting NEDD4 may be a promising therapeutic strategy for IGF1 signal pathway-driven gastric cancer.

Fusion Gene Detection in Prostate Cancer Samples Enhances the Prediction of Prostate Cancer Clinical Outcomes from Radical Prostatectomy through Machine Learning in a Multi-Institutional Analysis.

Prostate cancer remains one of the most fatal malignancies in men in the United States. Predicting the course of prostate cancer is challenging given that only a fraction of prostate cancer patients experience cancer recurrence after radical prostatectomy or radiation therapy. This study examined the expressions of 14 fusion genes in 607 prostate cancer samples from the University of Pittsburgh, Stanford University, and the University of Wisconsin-Madison. The profiling of 14 fusion genes was integrated with Gleason score of the primary prostate cancer and serum prostate-specific antigen level to develop machine-learning models to predict the recurrence of prostate cancer after radical prostatectomy. Machine-learning algorithms were developed by analysis of the data from the University of Pittsburgh cohort as a training set using the leave-one-out cross-validation method. These algorithms were then applied to the data set from the combined Stanford/Wisconsin cohort (testing set). The results showed that the addition of fusion gene profiling consistently improved the prediction accuracy rate of prostate cancer recurrence by Gleason score, serum prostate-specific antigen level, or a combination of both. These improvements occurred in both the training and testing cohorts and were corroborated by multiple models.

Morphological Characteristics of Foveal Pit Base and Its Impact on Visual Acuity in Macular Pseudoholes.

INTRODUCTION: To investigate the characteristics of macular pseudoholes (MPHs) with different foveal profiles and their impact on preoperative and postoperative visual acuity (VA). METHODS: A retrospective review of 47 eyes from 46 consecutive patients with MPH who had undergone vitrectomy was conducted. The MPHs were classified into u-shape and v-shape according to the morphological description of the foveola base. The best-corrected visual acuity (BCVA), central foveal point thickness, parafoveal thickness, parafoveal inner and outer retinal thickness, stretched lamellar cleavage, microcystic macular edema (MME), disorganization of retinal inner layers (DRIL), and the integrity of outer retinal layers were recorded. RESULTS: The eyes in the v-shaped group (n = 31) had lower BCVA, thicker retinal thickness, more intraretinal cleavage, MME, and DRIL than the u-shaped (n = 16) group (all p < 0.05). Multiple regression analysis revealed that the morphology of the foveola base was significantly related to the preoperative BCVA (p = 0.025). The VA was significantly improved in both groups, and the improvement was greater in the v-shaped group (p = 0.024). No significant difference was found in the postoperative BCVA between the two groups (all p > 0.05). CONCLUSION: The v-shape, reflecting the stretch in the foveola, had a significant impact on preoperative BCVA. However, the VA was improved after surgery whatever their initial foveal profile.

COMPARISON OF AIR VERSUS PERFLUOROPROPANE INTRAOCULAR TAMPONADE COMBINED WITH VITRECTOMY FOR THE TREATMENT OF MACULAR HOLE IN HIGH MYOPIA.

PURPOSE: To compare the efficacy of air and perfluoropropane (C3F8) with pars plana vitrectomy (PPV) in highly myopic macular holes (HMMHs) and explore the factors related to surgical prognosis. METHODS: A retrospective comparison of a consecutive series of HMMHs undergone vitrectomy and internal limiting membrane peeling. According to tamponade type, they were divided into air group and C3F8 group, which were further divided into subgroups based on hole diameter or axial length (AXL). Anatomical and functional outcomes were compared between tamponades. Related factors of initial closure rate and postoperative best-corrected visual acuity (BCVA) were analyzed by logistic regression. RESULTS: The baseline characteristics in air group (n = 63) and C3F8 group (n = 37) were similar except the age of air was older ( P = 0.019). The mean follow-up period was 17.16 ± 17.97 months. After surgery, the initial closure rate of air group was 85.7% and that of C3F8 group was 83.7% ( P = 0.780). And the initial closure rate showed no difference between tamponades in the same subgroup classified by hole diameter or AXL (all P > 0.05). Postoperative BCVA improved significantly in both groups ( P < 0.001), but no difference between them ( P = 0.793). Logistic regression showed that age, minimum linear diameter, and AXL were risk factors of initial closure rate, and preoperative BCVA was the only factor associated with postoperative BCVA (all P < 0.05). CONCLUSION: With a long-term follow-up of HMMH, we found air had a similar tamponade effect anatomically and functionally compared with C3F8. Air may also be a good choice for patients with HMMH.