Expression levels of GLUTs and SIRTs in diabetic liver lesions / 中国病理生理杂志

AIM:To study the expression of glucose transporters(GLUTs)and silent information regulators(SIRTs/sirtuins)in the liver of diabetic rats and human hepatocytes(LO2 cells)treated with high glucose.METHODS:(1)Twenty male SD rats were randomly divided into normal control(NC)group and diabetes mellitus(DM)group.The rats in DM group were given single intraperitoneal injection of streptozotocin(STZ,60 mg/kg)to establish the DM model,while the rats in NC group were intraperitoneally injected with equal volume of solvent once.Fasting blood glucose(FBG)and body mass were measured every 2 weeks.After 12 weeks of rearing,the blood and liver tissues of the rats were ob-tained after anesthesia with 1%sodium pentobarbitone,the biochemical indicators of blood were detected,and the liver in-dex was calculated.Hematoxylin-eosin(HE)staining and periodic acid-Schiff(PAS)staining were used to observe liver histopathological changes.Lipid accumulation in liver tissues was detected by oil red O staining.The expression levels of GLUTs and SIRTs family member proteins were detected in rat liver tissues.(2)The LO2 cells were treated with different concentrations of glucose for 48 h.The viability of the cells in each group was measured by CCK-8 assay,and Western blot was used to detected the protein expression levels of GLUTs and SIRTs in the cells.RESULTS:(1)Compared with NC group,the rats in DM group were depressed,lost weight,and the FBG and liver index were significantly increased(P＜0.05).The results of HE staining showed that the hepatic sinuses were dilatated and congested near the central vein in DM rats,and mild edema and scattered infiltration of inflammatory cells were found in liver cells.The results of oil red O staining showed the red fat droplets were diffusely scattered within liver cells in DM group.The results of PAS staining showed that there were numerous diffuse light purple circular droplets in the cytoplasm of the liver cells in the central ve-nous area of the DM rats.Western blot showed that the protein levels of GLUTs were higher and the protein levels of SIRTs were lower than those in NC group(P＜0.01).(2)The results of CCK-8 assay showed that the viability of LO2 cells was increased in 50 mmol/L glucose group(P＜0.01),without significant difference in 75,100 and 125 mmol/L glucose groups(all P＞0.05),and decreased in 150,175 and 200 mmol/L glucose groups(all P＜0.01).Later,150 mmol/L glu-cose was used as the high-glucose intervention condition.Western blot showed that the protein levels of GLUTs and SIRTs in LO2 cells under high glucose intervention were consistented with the results in animal experiments.CONCLUSION:High concentration of glucose can cause liver damage in SD rats and reduce the viability of human hepatocytes(LO2 cells).It can also increase the expression of GLUTs and decrease the expression of SIRTs in rat liver tissues and LO2 cells.Therefore,GLUTs and SIRTs family members may be the target proteins of diabetes-induced liver injury.

A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA.1 and BA.2

Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC50) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 g of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.

Ultrapotent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD

The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies1-3. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic4,5. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S6. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.

A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

Neutralizing antibodies (Abs) have been considered as promising therapeutics for the prevention and treatment of pathogens. After the outbreak of COVID-19, potent neutralizing Abs to SARS-CoV-2 were promptly developed, and a few of those neutralizing Abs are being tested in clinical studies. However, there were few methodologies detailly reported on how to rapidly and efficiently generate neutralizing Abs of interest. Here, we present a strategically optimized method for precisive screening of neutralizing monoclonal antibodies (mAbs), which enabled us to identify SARS-CoV-2 receptor-binding domain (RBD) specific Abs within 4 days, followed by another 2 days for neutralization activity evaluation. By applying the screening system, we obtained 198 Abs against the RBD of SARS-CoV-2. Excitingly, we found that approximately 50% (96/198) of them were candidate neutralizing Abs in a preliminary screening of SARS-CoV-2 pseudovirus and 20 of these 96 neutralizing Abs were confirmed with high potency. Furthermore, 2 mAbs with the highest neutralizing potency were identified to block authentic SARS-CoV-2 with the half-maximal inhibitory concentration (IC50) at concentrations of 9.88 ng/ml and 11.13 ng/ml. In this report, we demonstrated that the optimized neutralizing Abs screening system is useful for the rapid and efficient discovery of potent neutralizing Abs against SARS-CoV-2. Our study provides a methodology for the generation of preventive and therapeutic antibody drugs for emerging infectious diseases.