Hyperglycemia decreases the expression of ATP synthase ß subunit and enolase 2 in glomerular epithelial cells.

Glomerular epithelial cells (GECs) are known to play a key role in maintaining the structure and function of the glomerulus. GEC injury induced by hyperglycemia is present in early-stage diabetic nephropathy (DN), which is the most common cause of renal failure. In an attempt to identify target proteins involved in the pathogenesis of GEC injury at early DN, we performed the proteomic analysis using primary cultures of GECs, prepared from the dissected rat glomeruli. The protein expression profiles in the two-dimensional electrophoresis gels were compared between GECs treated for three days with normal glucose (5 mM) and those with high glucose (30 mM) concentrations. These concentrations correspond to blood glucose concentrations under normoglycemia and hyperglycemia, respectively. Proteins with differential expression levels were identified using ESI-Q-TOF tandem mass spectrometry. The primary GECs cultured in hyperglycemic conditions showed cellular hypertrophy and increased production of reactive oxygen species, both of which reflect the GEC injury. Our proteomic analysis identified eight proteins with differential expression profiles, depending on glucose concentrations. Among them, we selected ATP synthase ß subunit and enolase 2 that are related to energy metabolism and are down-regulated under hyperglycemia, and confirmed that hyperglycemia decreased the expression levels of ATP synthase ß subunit and enolase 2 proteins by western blotting analysis. Hyperglycemia may impair mitochondrial function and alter glycolysis in GECs by down-regulating the expression of ATP synthase ß subunit and enolase 2. The present study may provide a better understanding of the pathogenic mechanisms of GEC injury in early DN.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

Broader neutralization of CT-P27 against influenza A subtypes by combining two human monoclonal antibodies.

There are several broadly neutralizing monoclonal antibodies that neutralize influenza viruses with different mechanisms from traditional polyclonal antibodies induced by vaccination. CT149, which is one of the broadly neutralizing antibodies, was also previously reported to neutralize group 2 and some of group 1 influenza viruses (13 out of 13 tested group 2 viruses and 5 out of 11 group 1 viruses). In this study, we developed another antibody with the aim of compensating partial coverage of CT149 against group 1 influenza viruses. CT120 was screened among different antibody candidates and mixed with CT149. Importantly, although the binding sites of CT120 and CT149 are close to each other, the two antibodies do not interfere. The mixture of CT120 and CT149, which we named as CT-P27, showed broad efficacy by neutralizing 37 viruses from 11 different subtypes, of both group 1 and 2 influenza A viruses. Moreover, CT-P27 showed in vivo therapeutic efficacy, long prophylactic potency, and synergistic effect with oseltamivir in influenza virus-challenged mouse models. Our findings provide a novel therapeutic opportunity for more efficient treatment of influenza.

Sample preparation method for plasma membrane proteome analysis.

The preparation of plasma membrane (PM) proteome samples is seriously difficult and time-consuming, owing to their profound hydrophobicity and low abundance. We have developed an efficient PM sample preparation method using Ultracentrifugation with Percoll and an aqueous two-phase extraction. The developed method was rapid (3 h) and provided high purities (26-fold of cell lysate) with a high yield (2.6% of whole cell lysate proteins). This method is especially useful for PM proteome studies using 2D gel electrophoresis.

Differentially expressed proteins of mesenchymal stem cells derived from human cord blood (hUCB) during osteogenic differentiation.

Multipotent mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) represent promising candidates for the development of future cellular therapy strategies. MSCs have been found to be able to differentiate into various tissues. One of the primary limitations in our understanding of the biology of human MSCs is the absence of prospective markers required for the monitoring of lineage-specific differentiation. hUCB-derived MSCs have been found to have significantly greater osteogenic potential. In this study, we focused on proteins that were differentially expressed during osteogenic differentiation of hUCB-MSCs. And we analyzed the protein expression inherent to osteogenic differentiation by two-dimensional gel electrophoresis, ESI-Q-TOF, and Western blotting. Eleven differentially expressed spots were observed between the two groups (before and after differentiation) on the 2-DE map. These might also be proved as useful cytosolic biomarker proteins for osteogenesis, and might be employed in quality control of osteoblasts in cell-therapy applications.

Comparison of tear proteins between healthy and early diabetic retinopathy patients.

OBJECTIVES: To identify potential prognostic or diagnostic marker tear proteins for early diabetic retinopathy (DR) and to investigate the pathogenesis of this disease using proteomics techniques. DESIGN AND METHODS: The tear proteins expressed in patients suffering from diabetes mellitus without the retinopathy symptoms, nonproliferative diabetic retinopathy and healthy volunteers were analyzed by 2-DE. The differentially expressed proteins in patients were identified by ESI-Q-TOF and confirmed by Western blotting. RESULTS: Proteins which were differentially expressed with statistical significance (P<0.05) in two diabetic groups as compared to those in healthy group were selected and identified by ESI-Q-TOF MS/MS. Among these proteins, three proteins (LCN-1, HSP27 and B2M) were found to exhibit a progressive reduction in two disease groups. The expression levels of which might be useful as diagnostic biomarkers of DR were verified by Western blotting CONCLUSIONS: Proteomic analysis using tear is a novel approach that can provide insight into possible biomarker and the pathogenesis of early DR.

Comparative analysis of serum proteomes of patients with cardiovascular disease.

OBJECTIVES: To monitor increases or decreases in cardiovascular disease (CVD)-related proteins that will be released from the deposits or plaque on the inner wall of blood vessels. DESIGN AND METHODS: Protein profiles of sera from healthy subjects and CVD patients were determined via 2-DE. Differentially expressed spots in CVD patients were identified by ESI-Q-TOF MS/MS. Retinol binding protein 4 (RBP4), ceruloplasmin, and hemopexin were confirmed by Western blotting and RBP4 was further verified by ELISA. RESULTS: Approximately, 400 spots were detected in each gel via comparisons of the serum proteome. Among these spots, 19 spots were selected and identified by ESI-Q-TOF MS/MS (P<0.05). The expression levels of RBP4 and ceruloplasmin were higher in CVD patients by Western blotting. The level of immunoreactive RBP4 in CVD patients was higher than that in healthy subjects. CONCLUSIONS: The three proteins identified in the present study may constitute potential biomarkers for the diagnosis of CVD in patients.

Proteome analysis of serum from type 2 diabetics with nephropathy.

Diabetic nephropathy (DN) is a renal disease which develops as a consequence of diabetes mellitus. Microalbuminuria is the earliest clinical sign of DN. There are no specific diagnostic biomarkers for type 2 diabetics with nephropathy other than microalbuminuria and macroalbuminuria. However, microalbuminuria does not constitute a sole independent indicator for type 2 diabetics with nephropathy, and thus, another screening method, such as a biomarker assay, is required in order to diagnose it more correctly. Therefore, we have utilized two-dimensional electrophoresis (2-DE) to identify human serum protein markers for the more specific and accurate prediction of progressive nephropathy in type 2 diabetes patients, via comparisons of the serum proteome in three experimental groups: type 2 diabetes patients without microalbuminuria (DM, n = 30), with microalbuminuria (MA, n = 29), and with chronic renal failure (CRF, n = 31). As a result, proteins which were differentially expressed with statistical significance (p < 0.05) in MA and CRF groups as compared to those in DM group were selected and identified by ESI-Q-TOF MS/MS. Among these identified proteins, two proteins which might be useful as diagnostic biomarkers of type 2 diabetics with nephropathy were verified by Western blotting: extracellular glutathione peroxidase (eGPx) and apolipoprotein (ApoE) were found to exhibit a progressive reduction in MA and CRF groups. Notably, eGPx was further verified by ELISA using DM (n = 100) and MA (n = 96) patient samples. Collectively, our results show that the two proteins identified in this study may constitute potential biomarkers for the diagnosis of type 2 diabetics with nephropathy.

Proteome analysis of the rat hepatic stellate cells under high concentrations of glucose.

To study the change in hepatic stellate cell (HSC) function under diabetic conditions, we cultured rat HSC in the presence of 5 and 30 mM glucose, which correspond to blood glucose concentrations during the early and late stages of diabetes, respectively. The differentially expressed HSC proteins were analyzed using 2-DE and ESI-Q-TOF MS/MS and confirmed with Western blotting. The changed protein expression will provide greater understanding of glycolysis in HSC at the high concentration of glucose.

The discovery of biomarkers for type 2 diabetic nephropathy by serum proteome analysis.

Diabetic nephropathy (DN) is a serious kidney complication of diabetes, and constitutes the leading cause of end-stage renal disease. The earliest clinical evidence of DN is microalbuminuria, a term which refers to the appearance of small but abnormal amounts of albumin in the urine. However, screening methods for DN, such as biomarker assays, are yet to be developed for type 2 DN. In the present study, in an attempt to identify the biomarkers for initial diagnoses of type 2 DN, the protein profiles of human sera collected from 30 microalbuminuric type 2 diabetic patients were compared with those collected from 30 normoalbuminuric type 2 diabetic patients, via 2-DE. As a result, a total of 18 spots were determined to have different protein levels in the microalbuminuric patients. Twelve spots had lower protein levels of approximately 50%, and the other six had higher levels of approximately 100-300% as compared to the spots of normoalbuminuric patients. These spots were identified with ESI-Q-TOF (ESI-quadrupole-TOF) MS. Among the identified proteins, vitamin D-binding protein (DBP) and pigment epithelium-derived factor (PEDF) were verified by Western blotting. The results of this study indicate that the DBP may be employed as diagnostic and monitoring biomarkers of type 2 DN, contingent on further study into the matter.