Dimerization of Hepatitis E Virus Capsid Protein E2s Domain Is Essential for Virus–Host Interaction

Hepatitis E virus (HEV), a non-enveloped, positive-stranded RNA virus, is transmitted in a faecal-oral manner, and causes acute liver diseases in humans. The HEV capsid is made up of capsomeres consisting of homodimers of a single structural capsid protein forming the virus shell. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. To date, no structural information is available for any of the HEV proteins. Here, we report for the first time the crystal structure of the HEV capsid protein domain E2s, a protruding domain, together with functional studies to illustrate that this domain forms a tight homodimer and that this dimerization is essential for HEV–host interactions. In addition, we also show that the neutralizing antibody recognition site of HEV is located on the E2s domain. Our study will aid in the development of vaccines and, subsequently, specific inhibitors for HEV

Sprouty2 Suppresses Epithelial-Mesenchymal Transition of Human Lens Epithelial Cells through Blockade of Smad2 and ERK1/2 Pathways.

Transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) plays a key role in the pathogenesis of anterior subcapsular cataract (ASC) and capsule opacification. In mouse lens, Sprouty2 (Spry2) has a negative regulatory role on TGFβ signaling. However, the regulation of Spry2 during ASC development and how Spry2 modulates TGFβ signaling pathway in human LECs have not been characterized. Here, we demonstrate that Spry2 expression level is decreased in anterior capsule LECs of ASC patients. Spry2 negatively regulates TGFβ2-induced EMT and migration of LECs through inhibition of Smad2 and ERK1/2 phosphorylation. Also, blockade of Smad2 or ERK1/2 activation suppresses EMT caused by Spry2 downregulation. Collectively, our results for the first time show in human LECs that Spry2 has an inhibitory role in TGFβ signaling pathway. Our findings in human lens tissue and epithelial cells suggest that Spry2 may become a novel therapeutic target for the prevention and treatment of ASC and capsule opacification

Objective quantification of lens nuclear opacities using swept-source anterior segment optical coherence tomography

BACKGROUND/AIMS: The primary objective is to quantify the lens nuclear opacity using swept-source anterior segment optical coherence tomography (SS-ASOCT) and to evaluate its correlations with Lens Opacities Classification System III (LOCS-III) system and surgical parameters. The secondary objective is to assess the diagnostic performance for hard nuclear cataract. METHODS: This cross-sectional study included 1222 patients eligible for cataract surgery (1222 eyes). The latest SS-ASOCT (CASIA-2) was used to obtain high-resolution lens images, and the average nuclear density (AND) and maximum nuclear density (MND) were measured by a custom ImageJ software. Spearman's correlations analysis was used to assess associations of AND/MND with LOCS-III nuclear scores, visual acuity and surgical parameters. The subjects were then split randomly (9:1) into the training dataset and validating dataset. Receiver operating characteristic curves and calibration curves were constructed for the classification on hard nuclear cataract. RESULTS: The AND and MND from SS-ASOCT images were significantly correlated with nuclear colour scores (AND: r=0.716; MND: r=0.660; p<0.001) and nuclear opalescence scores (AND: r=0.712; MND: r=0.655; p<0.001). The AND by SS-ASOCT images had the highest values of Spearman's r for preoperative corrected distance visual acuity (r=0.3131), total ultrasonic time (r=0.3481) and cumulative dissipated energy (r=0.4265). The nuclear density had good performance in classifying hard nuclear cataract, with area under the curves of 0.859 (0.831-0.886) for AND and 0.796 (0.768-0.823) for MND. CONCLUSION: Objective and quantitative evaluation of the lens nuclear density using SS-ASOCT images enable accurate diagnosis of hard nuclear cataract

Spry2 expression level is decreased in anterior capsule LECs of ASC patients.

<p>(<b>A</b>) Representative slit-lamp microscope photo of an ASC patient (60 years old, Female). The white arrow indicates the irregular fibrotic opacity beneath the anterior capsule. (<b>B</b>) Total RNA was extracted from the anterior capsules of ASC patients, age-matched cortical cataract patients and postmortem human lens (control). The mRNA level of Spry2 was determined using real-time PCR and normalized to GAPDH. ***<i>P</i><0.001, NS: not significant, n = 6. Fold change relative to the level of the control groups is displayed. (<b>C</b>) Lens anterior capsule whole-mounts from ASC patients, age-matched cortical cataract patients, and postmortem human lens (control) were probed for Spry2 (green), α-SMA (red) and DAPI (blue). Images were acquired from the central area of each sample. Scale bar: 10μm.</p

Spry2 inhibits TGFβ-induced EMT in human lens epithelial cells.

<p><b>(A)</b> Cultured human lens epithelial cells were transfected with Spry2 siRNA or Spry2 plasmid and treated with or without TGFβ for 48h. The mRNA levels of α-SMA, Fn and Vim were determined by real-time PCR and normalized to GAPDH. Fold change relative to the level of the untransfected groups is displayed. **<i>P</i><0.01, ***<i>P</i><0.001, n = 3 <b>(B)</b> Cells were transfected with Spry2 siRNA or Spry2 plasmid and treated with or without TGFβ for 48h. Immunofluorescence was performed by probing Spry2, Fn, Col I, Col IV and DAPI. Scale bar: 50μm <b>(C-D)</b> Cells were transfected with Spry2 siRNA or Spry2 plasmid and treated with or without TGFβ for 48h. Untransfected cells, cells transfected with scrambled siRNA or empty vector were used as controls. Proteins were extracted and probed for Spry2, α-SMA, Fn, Col IV. β-actin was used as a loading control. <b>(E-F)</b> Quantification of the protein expression levels in C and D, respectively. Fold change relative to the level of the untransfected groups is displayed. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, NS: not significant, n = 3.</p

Primers used for real-time quantitative PCR.

<p>Primers used for real-time quantitative PCR.</p

EMT induced by Spry2 downregulation was inhibited by Smad and ERK1/2 specific inhibitors.

<p><b>(A)</b> Cultured human lens epithelial cells were transfected with scrambled siRNA or Spry2 siRNA and treated with Smad inhibitor SB431542 (10.0μM) or ERK1/2 inhibitor U1026 (10.0μM) or both for 24h. Proteins were extracted and probed for pSmad, total Smad, pERK1/2, total ERK1/2, α-SMA and Fn. β-actin was used as a loading control. <b>(B-D)</b> Quantification of the protein expression levels in A. Fold change relative to the level of the scrambled siRNA transfected group is displayed. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, NS: not significant, n = 3.</p