Vision transformer with masked autoencoders for referable diabetic retinopathy classification based on large-size retina image.

Computer-aided diagnosis systems based on deep learning algorithms have shown potential applications in rapid diagnosis of diabetic retinopathy (DR). Due to the superior performance of Transformer over convolutional neural networks (CNN) on natural images, we attempted to develop a new model to classify referable DR based on a limited number of large-size retinal images by using Transformer. Vision Transformer (ViT) with Masked Autoencoders (MAE) was applied in this study to improve the classification performance of referable DR. We collected over 100,000 publicly fundus retinal images larger than 224×224, and then pre-trained ViT on these retinal images using MAE. The pre-trained ViT was applied to classify referable DR, the performance was also compared with that of ViT pre-trained using ImageNet. The improvement in model classification performance by pre-training with over 100,000 retinal images using MAE is superior to that pre-trained with ImageNet. The accuracy, area under curve (AUC), highest sensitivity and highest specificity of the present model are 93.42%, 0.9853, 0.973 and 0.9539, respectively. This study shows that MAE can provide more flexibility to the input image and substantially reduce the number of images required. Meanwhile, the pretraining dataset scale in this study is much smaller than ImageNet, and the pre-trained weights from ImageNet are not required also.

CircPOSTN/miR-361-5p/TPX2 axis regulates cell growth, apoptosis and aerobic glycolysis in glioma cells.

BACKGROUND: Glioma is the most primary central nervous system tumor in adults. The 5 year survival rate for glioma patients remains poor, although treatment strategies had improved in the past few decades. The cumulative studies have shown that circular RNA (circRNA) is associated with glioma process, so the purpose of this study is to clarify the function of circPOSTN in glioma. METHODS: The expression levels of circPOSTN, miR-361-5p, and targeting protein for Xenopus kinesin-like protein 2 (TPX2) were assessed with real-time quantitative polymerase chain reaction (RT-qPCR). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) and flow cytometry assays were executed to examine proliferation and apoptosis of glioma cells, respectively. Western blot was applied to assess protein expression. The glucose metabolism of glioma cells was analyzed by testing the glucose consumption, lactate production, ATP level, reactive oxygen species (ROS) accumulation and performing Seahorse XF assay. The interaction relationship between miR-361-5p and circPOSTN or TPX2 was analyzed by bioinformatics database and dual-luciferase reporter assay. The influences of circPOSTN silencing in vivo were observed by a xenograft experiment. RESULTS: CircPOSTN was overexpressed in glioma tissues and cells. Absence of circPOSTN in glioma cells promoted apoptosis while impeded proliferation and aerobic glycolysis, which were mitigated by silencing miR-361-5p. What's more, loss-of-functional experiment suggested that knockdown of TPX2 repressed proliferation and aerobic glycolysis, while induced apoptosis in glioma cells. In addition, circPOSTN targetedly regulated TPX2 expression in glioma cells via sponging miR-361-5p. In vivo study revealed that deficiency of circPOSTN restrained tumor growth. CONCLUSION: Mechanistically, circPOSTN regulated cell growth, apoptosis, and aerobic glycolysis in glioma through miR-361-5p/TPX2 axis.

USP2 regulates the intracellular localization of PER1 and circadian gene expression.

Endogenous 24-h rhythms in physiology are driven by a network of circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Posttranslational modifications, including ubiquitination, are important for regulating the clock feedback mechanism. Recently, we showed that the deubiquitinating enzyme ubiquitin-specific peptidase 2 (USP2) associates with clock proteins and deubiquitinates PERIOD1 (PER1) but does not affect its overall stability. Mice devoid of USP2 display defects in clock function. Here, we show that USP2 regulates nucleocytoplasmic shuttling and nuclear retention of PER1 and its repressive role on the clock transcription factors CLOCK and BMAL1. The rhythm of nuclear entry of PER1 in Usp2 knockout mouse embryonic fibroblasts (MEFs) was advanced but with reduced nuclear accumulation of PER1. Although Per1 mRNA expression rhythm remained intact in the Usp2 KO MEFs, the expression profiles of other core clock genes were altered. This was also true for the expression of clock-controlled genes (e.g., Dbp, Tef, Hlf, E4bp4). A similar phase advance of PER1 nuclear localization rhythm and alteration of clock gene expression profiles were also observed in livers of Usp2 KO mice. Taken together, our results demonstrate a novel function of USP2 in the molecular clock in which it regulates PER1 function by gating its nuclear entry and accumulation.

Regulation of behavioral circadian rhythms and clock protein PER1 by the deubiquitinating enzyme USP2.

Endogenous 24-hour rhythms are generated by circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Post-translational modifications, including ubiquitination, are important for regulating the clock feedback mechanism. Previous work has focused on the role of ubiquitin ligases in the clock mechanism. Here we show a role for the rhythmically-expressed deubiquitinating enzyme ubiquitin specific peptidase 2 (USP2) in clock function. Mice with a deletion of the Usp2 gene (Usp2 KO) display a longer free-running period of locomotor activity rhythms and altered responses of the clock to light. This was associated with altered expression of clock genes in synchronized Usp2 KO mouse embryonic fibroblasts and increased levels of clock protein PERIOD1 (PER1). USP2 can be coimmunoprecipitated with several clock proteins but directly interacts specifically with PER1 and deubiquitinates it. Interestingly, this deubiquitination does not alter PER1 stability. Taken together, our results identify USP2 as a new core component of the clock machinery and demonstrate a role for deubiquitination in the regulation of the circadian clock, both at the level of the core pacemaker and its response to external cues.

Mice lacking the USP2 deubiquitinating enzyme have severe male subfertility associated with defects in fertilization and sperm motility.

The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.

RET Gly691Ser mutation is associated with primary vesicoureteral reflux in the French-Canadian population from Quebec.

Primary vesicoureteral reflux (pVUR) is a common, genetically heterogeneous congenital urinary tract abnormality in children. It causes urine to flow backward from the bladder to the ureter due to a developmental defect at the vesicoureteral junction, whose formation requires rearrangement during transformation (Ret)-mediated signaling pathways. To study the genetic causes of pVUR in Quebec patients, we used a sequencing-based candidate gene approach to screen the RET gene and found that 83 out of 118 pVUR patients are carriers of the rare A allele of single nucleotide polymorphism (SNP) rs1799939:G>A that results in a Gly691Ser mutation, a statistically significant increase in allelic frequency, that is absent at six flanking RET SNPs tested. Ser691 is a predicted phosphorylation site and our analysis of transfected cells showed that the Gly691Ser Ret mutant can efficiently interact and associate with a 75-80-kD tyrosine phosphorylated cellular protein, an event not seen with wild-type Ret. This interaction and/or the steric or electric hindrance created by phospho-Ser691 may interfere with the known regulatory functions of the normally phosphorylated phospho-Tyr687 and phospho-Ser696 on the cytoskeleton actin reorganization that are responsible for cell motility and morphology, which consequently may lead to the deficiency in ureteral development observed in pVUR. Our study demonstrates that the Ret Gly691Ser mutation is associated with pVUR and may be one of the genetic causes of this condition in the French-Canadian population in Quebec.

Critical role of Ser-520 phosphorylation for membrane recruitment and activation of the ZAP-70 tyrosine kinase in T cells.

Regulation of protein tyrosine kinases (PTKs) by tyrosine phosphorylation is well recognized; in fact, nearly all PTKs require phosphorylation of tyrosine residues in their "activation loop" for catalytic activity. In contrast, the phosphorylation of PTKs on serine and threonine residues has not been studied nearly as much. We report that the ZAP-70 PTK contains predominately phosphoserine in normal T lymphocytes as well as in Jurkat T leukemia cells. We have identified one site of phosphorylation as Ser-520 and find this site to be important for the recruitment and activation of ZAP-70 in T cells. Mutant ZAP-70-S520A had reduced ability to autophosphorylate and to mediate antigen receptor-induced interleukin 2 gene activation and was not enriched at the plasma membrane. These defects were rescued by addition of a myristylation signal to the N terminus of ZAP-70-S520A to force its plasma membrane and lipid raft localization. We conclude that phosphorylation of ZAP-70 at Ser-520 plays an important role in the correct localization of ZAP-70 and in priming ZAP-70 for its acute recruitment and activation upon antigen receptor ligation.