In Silico Screening of Potential Spike Glycoprotein Inhibitors of SARS-CoV-2 with Drug Repurposing Strategy

Objective: To select potential molecules that can target viral spike proteins, which may potentially interrupt the interaction between the human angiotension-converting enzyme 2(ACE2) receptor and viral spike protein by virtual screening. Methods: The three-dimensional(3 D)-coordinate file of the receptor-binding domain(RBD)-ACE2 complex for searching a suitable docking pocket was firstly downloaded and prepared. Secondly, approximately 15,000 molecular candidates were prepared, including US Food and Drug Administration(FDA)-approved drugs from Drug Bank and natural compounds from Traditional Chinese Medicine Systems Pharmacology(TCMSP), for the docking process. Then, virtual screening was performed and the binding energy in Autodock Vina was calculated. Finally, the top 20 molecules with high binding energy and their Chinese medicine(CM) herb sources were listed in this paper. Results: It was found that digitoxin, a cardiac glycoside in Drug Bank and bisindigotin in TCMSP had the highest docking scores. Interestingly, two of the CM herbs containing the natural compounds that had relatively high binding scores, Forsythiae fructus and Isatidis radix, are components of Lianhua Qingwen(莲花清瘟), a CM formula reportedly exerting activity against severe acute respiratory syndrome(SARS)-Cov-2. Moreover, raltegravir, an HIV integrase inhibitor, was found to have a relatively high binding score. Conclusions: A class of compounds, which are from FDA-approved drugs and CM natural compounds, that had high binding energy with RBD of the viral spike protein. Our work provides potential candidates for other researchers to identify inhibitors to prevent SARS-Co V-2 infection, and highlights the importance of CM and integrative application of CM and Western medicine on treating COVID-19.     

Superoxide flashes: elemental events of mitochondrial ROS signaling in the heart

The role of mitochondrial reactive oxygen species (mitoROS) in cellular function remains obscure. By synthesizing recent data, we propose here that local dynamic mitoROS in the form of "superoxide flashes" serve as "signaling ROS" rather than "homeostatic ROS", distinguishable from basal mitoROS due to constitutive leakage of the electron transfer chain (ETC). Individual superoxide flashes are 10-s mitoROS bursts that are compartmentalized to a single mitochondrion or local mitochondrial networks. As a highly-conserved universal mitochondrial activity, it occurs in intact cells, in ex vivo beating hearts, and even in living animals. Unlike basal mitoROS, superoxide flashes are ignited by transient openings of a type of mitochondrial permeability transition pore (mPTP), and their incidence is richly regulated by an array of factors that converge on either the mPTP or ETC. Emerging evidence has shown that superoxide flashes decode dietary and metabolic status or exercise, gauge oxidative stress (e.g., during reoxygenation after hypoxia or anoxia), and constitute early mitochondrial signals that initiate oxidative stress-related apoptosis in a context-dependent manner. That they make only a miniscule contribution to global ROS attests to the high efficiency of local ROS signaling. However, the exact mechanisms underlying superoxide flash formation, regulation and function remain uncertain. Future investigation is warranted to uncover the cellular logic and molecular pathways of local dynamic mitoROS signaling in heart muscle cells and many other cell types.     

Degenerative alterations in noradrenergic neurons of the locus coeruleus in Alzheimer’s disease

Mice carrying mutant amyloid-β precursor protein and presenilin-1 genes (APP/PS1 double transgenic mice) have frequently been used in studies of Alzheimer’s disease; however, such studies have focused mainly on hippocampal and cortical changes. The severity of Alzheimer’s disease is known to correlate with the amount of amyloid-β protein deposition and the number of dead neurons in the locus coeruleus. In the present study, we assigned APP/PS1 double transgenic mice to two groups according to age: young mice (5-6 months old) and aged mice (16-17 months old). Age-matched wild-type mice were used as controls. Immunohistochemistry for tyrosine hydroxylase (a marker of catecholaminergic neurons in the locus coeruleus) revealed that APP/PS1 mice had 23% fewer cells in the locus coeruleus compared with aged wild-type mice. APP/PS1 mice also had increased numbers of cell bodies of neurons positive for tyrosine hydroxylase, but fewer tyrosine hydroxylase-positive fibers, which were also short, thick and broken. Quantitative analysis using unbiased stereology showed a significant age-related increase in the mean volume of tyrosine hydroxylase-positive neurons in aged APP/PS1 mice compared with young APP/PS1 mice. Moreover, the mean volume of tyrosine hydroxylase-positive neurons was positively correlated with the total volume of the locus coeruleus. These findings indicate that noradrenergic neurons and fibers in the locus coeruleus are predisposed to degenerative alterations in APP/PS1 double transgenic mice.     

Efficacy of wet-lab training versus surgical-simulator training on performance of ophthalmology residents during chopping in cataract surgery

AIM: To analyze whether wet-lab training(WLT) or surgical-simulator training(SST) is better for ophthalmology residents to master the chopping technique.METHODS: Sixty ophthalmology residents(in their second year) and three cataract surgeons participated in the study. The residents were randomly separated into two groups, WLT group and SST group. The residents in WLT group were asked to perform 10 trials of chopping using pig eyes and scored by the surgeons, and then they performed and scored using simulator for one time. The residents in SST group underwent 10 trials of chopping using simulator, and the simulator scored each trail. Then, this group were asked to perform the chopping using pig eyes and scored by the surgeons. At last, we investigated the residents’ satisfaction about the training.RESULTS: The demographic characteristics had no significant differences between the two groups. Recorded by the simulator, the residents in SST group got significantly higher overall score(83.90±1.31) than WLT group(78.73±1.92, P=0.03). And the residents in SST group got less corner area injured, and they spend less time than WLT group(P<0.05). Moreover, the residents in WLT group used more ultrasonic energy value than SST group(P=0.03). However, scored by the surgeons, the residents in two groups got nearly the same overall score. The residents in WLT group performed better on the frequencies of posterior capsule torn and incisional stress(P=0.03, 0.008, respectively). In the survey, the residents in two groups held the same opinion that the training was helpful and they strongly recommended this training. And all of them enjoyed the training, and enjoyed being randomized in their own group. However, with respect to the realistic character, the residents thought that WLT was better than SST(P<0.001).CONCLUSION: Both of the Eyesi surgical-stimulator and the wet-lab improve the residents’ chopping ability and each has its own advantages. The combination of the two training ways could be considered to be a part of the training curriculum for new residents.     

Mesenchymal stem cell-derived exosomes inhibit the VEGF-A expression in human retinal vascular endothelial cells induced by high glucose

AIM: To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor A (VEGF-A) in human retinal vascular endothelial cells (HRECs). METHODS: Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy, Western blotting and nanoparticle tracking analysis. HRECs were randomly divided into a normal control group (group A), a high glucose model group (group B), a high glucose group with 25 μg/mL (group C), 50 μg/mL (group D), and 100 μg/mL exosomes (group E). Twenty-four hours after coculture, the cell proliferation rate was detected using flow cytometry, and the VEGF-A level was detected using immunofluorescence. After coculture 8, 16, and 24h, the expression levels of VEGF-A in each group were detected using PCR and Western blots. RESULTS: The characteristic morphology (membrane structured vesicles) and size (diameter between 50 and 200 nm) were observed under transmission electron microscopy. The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis (NTA). The exosomal markers CD9, CD63, and HSP70 were strongly detected. The proliferation rate of the cells in group B increased after 24h of coculture. Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes (F=39.03, P<0.01). The upregulation of VEGF-A protein (group C: F=7.96; group D: F=17.29; group E: F=11.89; 8h: F=9.45; 16h: F=12.86; 24h: F=42.28, P<0.05) and mRNA (group C: F=4.137; group D: F=13.64; group E: F=22.19; 8h: F=7.253; 16h: F=16.98; 24h: F=22.62, P<0.05) in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes (P<0.05). CONCLUSION: hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.     

Analysis of APC/beta-catenin genes mutations and Wnt signalling pathway in desmoid-type fibromatosis

OBJECTIVE: The abnormalities of the Wnt signalling pathway in desmoid-type fibromatosis were analysed, with the purpose of exploring the mechanism of tumorigenesis and progression. METHODS: The clinical and histopathological features of 96 cases were analysed. Beta-catenin, cyclin-D1, c-myc, and Ki-67 proteins were detected in 69 cases using formalin-fixed, paraffin-embedded tissues. Using the same materials, apoptosis of the tumour cells was investigated by terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling (TUNEL) testing. Polymerase chain reaction (PCR), denaturing high performance liquid chromatography (DHPLC) assay, and sequencing were performed to detect abnormalities of the adenomatous polyposis coli (APC) and beta-catenin genes. RESULTS: APC gene mutations were found in 18 cases (26.1%, 18/69). Somatic mutations of codon 41 in exon 3 of beta-catenin were detected in 13 cases (18.8%, 13/69). No correlation of beta-catenin abnormal expression with the mutations of APC gene or beta-catenin gene was identified (p>0.05). The cases with abnormal beta-catenin expression showed a higher level of c-myc protein expression (69.7%, 23/33) than those without (22.2%, 8/36, p = 0.001). The apoptotic indices (AIs) were significantly lower in cyclin-D1 positive cases and c-myc positive cases (p = 0.015, p = 0.007). CONCLUSIONS: There are somatic mutations of the APC and beta-catenin gene in desmoid-type fibromatosis, and there are abnormalities in the Wnt signalling pathway. These abnormalities may result in aberrant cell proliferation and apoptosis, which are likely to be important factors in tumorigenesis and progression.     

CD226 is expressed on the megakaryocytic lineage from hematopoietic stem cells/progenitor cells and involved in its polyploidization

OBJECTIVES: In this study, we examined the expression of CD226 on megakaryocytic, granulocytic and erythroid lineage from hematopoietic stem cells/progenitor cells in adult and fetus and its potential role in megakaryocytic maturation. METHODS: CD34(+) cells from adult and fetus were induced to differentiate toward the megakaryocytic lineage by thrombopoietin (TPO) and the granulocytic lineage by granulocyte colony-stimulating factor (G-CSF), respectively. Mononuclear cells from fetal liver and CD34(+) cells from adult were induced to differentiate toward erythroid-lineage by erythropoiesis (EPO). We investigated the expression of CD226 and lymphocyte function associated antigen-1 (LFA-1) (CD11a) during hemopoiesis. We also studied the effect of CD226 monoclonal antibody (MoAb) and LFA-1 MoAb on megakaryocyte with antibody cross-liking technique. RESULTS: CD34(+) cells from adult and fetus and TPO-induced CD41(+) cells all expressed CD226 molecule. CD226 was not expressed on erythroid progenitor cells and erythroblasts and most cells of granulocytic lineage although G-CSF induced a significant increase of the expression of CD226 on CD34(+) cells in early period of time. CD226 MoAb acts on megakaryocytes by inducing intracellular calcium mobilization. The expression of LFA-1 decreased significantly at late stage of differentiation and maturation of fetal megakaryocytes whereas the expression of LFA-1 on adult megakaryocytes retained at a high level. CD226 MoAb in combination with LFA-1 MoAb shifted the ploidy of generated megakaryocytes from adult-derived CD34(+) cells to higher classes significantly although CD226 and LFA-1 MoAb slightly increased the ploidy of the generated megakaryocytes individually. CD226 MoAb or LFA-1 MoAb or CD226 MoAb plus LFA-1 MoAbs did not increase the ploidy of the generated megakaryocytes from fetus-derived CD34(+) cells. CONCLUSION: CD226 molecules play an important role in maturation of the megakaryocytes in combination with LFA-1.     

Spinal Cord Contusion

Spinal cord injury is a major cause of disability with devastating neurological outcomes and lim-ited therapeutic opportunities, even though there are thousands of publications on spinal cord injury annually. There are two major types of spinal cord injury, transaction of the spinal cord and spinal cord contusion. Both can theoretically be treated, but there is no well documented treatment in human being. As for spinal cord contusion, we have developed an operation with fabulous result.