C-Reactive Protein: The Most Familiar Stranger.

C-reactive protein (CRP) is a highly conserved pentraxin with pattern recognition receptor-like activities. However, despite being used widely as a clinical marker of inflammation, the in vivo functions of CRP and its roles in health and disease remain largely unestablished. This is, to certain extent, due to the drastically different expression patterns of CRP in mice and rats, raising concerns about whether the functions of CRP are essential and conserved across species and how these model animals should be manipulated to examine the in vivo actions of human CRP. In this review, we discuss recent advances highlighting the essential and conserved functions of CRP across species, and propose that appropriately designed animal models can be used to understand the origin-, conformation-, and localization-dependent actions of human CRP in vivo. The improved model design will contribute to establishing the pathophysiological roles of CRP and facilitate the development of novel CRP-targeting strategies.

Glycolysis-related biomarker TCIRG1 participates in regulation of renal cell carcinoma progression and tumor immune microenvironment by affecting aerobic glycolysis and AKT/mTOR signaling pathway.

BACKGROUND: Renal cell carcinoma (RCC) is a hypermetabolic disease. Abnormal up-regulation of glycolytic signaling promotes tumor growth, and glycolytic metabolism is closely related to immunotherapy of renal cancer. The aim of the present study was to determine whether and how the glycolysis-related biomarker TCIRG1 affects aerobic glycolysis, the tumor microenvironment (TME) and malignant progression of clear cell renal cell carcinoma (ccRCC). METHODS: Based on The Cancer Genome Atlas (TCGA, n = 533) and the glycolysis-related gene set from MSigDB, we identified the glycolysis-related gene TCIRG1 by bioinformatics analysis, analyzed its immunological properties in ccRCC and observed how it affected the biological function and glycolytic metabolism using online databases such as TIMER 2.0, UALCAN, LinkedOmics and in vitro experiments. RESULTS: It was found that the expression of TCIRG1, was significantly increased in ccRCC tissue, and that high TCIRG1 expression was associated with poor overall survival (OS) and short progression-free interval (PFI). In addition, TCIRG1 expression was highly correlated with the infiltration immune cells, especially CD4+T cell Th1, CD8+T cell, NK cell, and M1 macrophage, and positively correlated with PDCD1, CTLA4 and other immunoinhibitors, CCL5, CXCR3 and other chemokines and chemokine receptors. More importantly, TCIRG1 may regulate aerobic glycolysis in ccRCC via the AKT/mTOR signaling pathway, thereby affecting the malignant progression of ccRCC cell lines. CONCLUSIONS: Our results demonstrate that the glycolysis-related biomarker TCIRG1 is a tumor-promoting factor by affecting aerobic glycolysis and tumor immune microenvironment in ccRCC, and this finding may provide a new idea for the treatment of ccRCC by combination of metabolic intervention and immunotherapy.

Trends in under-five mortality rate in China, 1996-2020: a Joinpoint regression and correlation analysis.

OBJECTIVES: To analyse annual trends of the under-five mortality rate (U5MR) and main cause-specific U5MR in China from 1996 to 2020 and to assess the potential correlation of the healthcare system and health expenditure with the U5MR in China. DESIGN: A retrospective observational study using national data from 1996 to 2020. Joinpoint regression was employed to model U5MR trends and Pearson correlation analysis was conducted to examine the relationship between healthcare system factors, health expenditure and U5MR. SETTING: Nationwide study covering both rural and urban populations across China over a 25-year period. RESULTS: The U5MR in China experienced a three-stage decline from 1996 to 2020 with an average annual percentage rate change (AAPC) of -7.27 (p<0.001). The AAPC of the rural U5MR (-7.07, p<0.001) was higher than that in urban areas (-5.57, p<0.001). Among the five main causes, the decrease in pneumonia-caused U5MR was the fastest while the decreases in congenital heart disease and accidental asphyxia were relatively slow. The rates of hospital delivery (r=-0.981, p<0.001), neonatal visits (r=-0.848, p<0.001) and systematic health management (r=-0.893, p<0.001) correlated negatively with U5MR. The proportion of government health expenditure in the total health expenditure (THE) correlated negatively with the national U5MR (r=-0.892, p<0.001) while the proportion of out-of-pocket health expenditure in THE correlated positively (r=0.902, p<0.001). CONCLUSION: China made significant advances in reducing U5MR from 1996 to 2020. The rural-urban gap in U5MR has narrowed, though rural areas remain a key concern. To further reduce U5MR, China should focus on rural areas, pay more attention to congenital heart disease and accidental asphyxia, further improve its health policies, and continue to increase the government health expenditure.

Association of C-reactive protein and complement factor H gene polymorphisms with risk of lupus nephritis in Chinese population.

BACKGROUND: Complement overactivation is a major driver of lupus nephritis (LN). Impaired interactions of C-reactive protein (CRP) with complement factor H (CFH) have been shown as a pathogenic mechanism that contributes to the overactivation of complement in LN. However, genetic variations of neither CRP nor CFH show consistent influences on the risk of LN. AIM: To examine whether genetic variations of CRP and CFH in combination can improve the risk stratification in Chinese population. METHODS: We genotyped six CRP single nucleotide polymorphisms (SNPs) (rs1205, rs3093062, rs2794521, rs1800947, rs3093077, and rs1130864) and three CFH SNPs (rs482934, rs1061170, and rs1061147) in 270 LN patients and 303 healthy subjects. RESULTS: No linkage was found among CRP and CFH SNPs, indicating lack of genetic interactions between the two genes. Moreover, CRP and CFH SNPs, neither individually nor in combination, are associated with the risk or clinical manifestations of LN. Given the unambiguous pathogenic roles of the two genes. CONCLUSION: These findings suggest that the biological effects of most genetic variations of CRP and CFH on their expressions or activities are not sufficient to influence the disease course of LN.

Association between serum total bilirubin and diabetic kidney disease in US diabetic patients.

Background: Bilirubin has been widely reported to be a protective factor against diabetic kidney disease (DKD) in Asian populations. However, few large-sample analyses have been conducted in American populations. This study aimed to investigate the association between serum total bilirubin (STB) level and DKD in a US diabetic cohort. Methods: This cross-sectional study enrolled participants from the National Health and Nutrition Examination Survey (NHANES) 2003-2018. Univariate and multivariate logistic regression analyses were performed to assess the association between STB level and DKD. Three models were conducted to control the potential confounding factors. Subgroup analysis was carried out for further validation. Results: Among the 5,355 participants, the median age [interquartile range (IQR)] was 62 [52-71] years; 2,836 (52.96%) were male, and 1,576 (29.43%) were diagnosed with DKD. In the entire cohort, no significant association between STB level and DKD was observed in any logistic regression models (p > 0.05). Subgroup analysis revealed that, in U.S. diabetic males, STB levels > 11.98 µmol/L were associated with a nearly 30% lower risk of DKD than STB levels ≤ 8.55 µmol/L. Additionally, a moderate STB level (8.56-11.98 µmol/L) was found associated with a nearly 25% lower risk of DKD in U.S. diabetic patients over 65 years old. Conclusion: The association of STB level with DKD may depict differences across diverse populations, among which the impact of race, sex, and age requires thorough consideration and relevant inferences should be interpreted cautiously.

Oesophageal rupture after right pneumonectomy: A case report.

Oesophageal rupture after pneumonectomy is very rare. Management remains a significant challenge. This current case report describes a patient that underwent right pneumonectomy for right central squamous cell carcinoma. On the third day after the operation, food residues were observed in the thoracic tube. Considering oesophageal rupture, surgery was performed. Intraoperative oesophageal rupture was observed and intermittent suture was performed. Unfortunately, an oesophageal pleural fistula developed. Anti-infection treatment, chest drainage, thoracic irrigation, nutrition support and stent implantation were used after the operation. The patient recovered smoothly and had no discomfort when eating. Oesophageal rupture after pneumonectomy is extremely rare and treatment is very difficult. In our opinion, surgical repair is the key to the treatment of oesophageal rupture. Once an oesophageal pleural fistula occurs, enhanced nutrition, adequate irrigation and drainage and stent implantation are critical factors.

Cholesterol-binding sequence is a key regulatory motif of cellular folding and conformational activation for C-reactive protein.

Human, rat, and mouse C-reactive protein (CRP) possess distinct expression patterns, but have similar conformations and conserved in vivo functions. We have previously demonstrated that this level-function mismatch is delicately tuned by the hidden activities of unfolded CRP. The cholesterol-binding sequence (CBS; a.a. 35-47) is a major functional motif exposed on monomeric CRP, which is the unfolded and activated conformation of CRP. We replaced the CBS of rat CRP with that of either mouse or human CRP, yielding two grafting mutants with unaffected pentameric assembly. However, these mutants exhibited altered cellular foldability and conformational activation efficiency that matched those of the CRP that provided the grafted CBS. These results indicate that CBS is a critical regulatory motif, whose variation maintains the pentameric assembly of CRP but derives distinct cellular foldabilities and conformational activation efficiencies, therefore helping to ensure that CRPs with various expression patterns exhibit overall conserved functions.

"Zero ischemia" laparoscopic partial nephrectomy by high-power GreenLight laser enucleation for renal carcinoma: A single-center experience.

BACKGROUND: Laparoscopic partial nephrectomy has been widely used in renal cell carcinoma treatment. The efficacy of GreenLight laser on Laparoscopic partial nephrectomy is still unknown. AIM: To present the first series of laparoscopic partial nephrectomy (LPN) by GreenLight laser enucleation without renal artery clamping. Due to the excellent coagulation and hemostatic properties of the laser, laser-assisted LPN (LLPN) makes it possible to perform a "zero ischemia" resection. METHODS: Fifteen patients with T1a exogenous renal tumors who received high-power GreenLight laser non-ischemic LPN in our hospital were retrospectively analyzed. All clinical information, surgical and post-operative data, complications, pathological and functional outcomes were analyzed. RESULTS: Surgery was successfully completed in all patients, and no open or radical nephrectomy was performed. The renal artery was not clamped, leading to no ischemic time. No blood transfusions were required, the average hemoglobin level ranged from 96.0 to 132.0 g/L and no postoperative complications occurred. The mean operation time was 104.3 ± 8.2 min. The postoperative removal of negative pressure drainage time ranged from 5.0 to 7.0 d, and the mean postoperative hospital stay was 6.5 ± 0.7 d. No serious complications occurred. Postoperative pathological results showed clear cell carcinoma in 12 patients, papillary renal cell carcinoma in 2 patients, and hamartoma in 1 patient. The mean creatinine level was 75.0 ± 0.8 µmol/L (range 61.0-90.4 µmol/L) at 1 mo after surgery, and there were no statistically significant differences compared with pre-operation (P > 0.05). The glomerular filtration rate ranged from 45.1 to 60.8 mL/min, with an average of 54.0 ± 5.0 mL/min, and these levels were not significantly different from those before surgery (P > 0.05). CONCLUSION: GreenLight laser has extraordinary cutting and sealing advantages when used for small renal tumors (exogenous tumors of stage T1a) during LPN. However, use of this technique can lead to the generation of excessive smoke.

Purification of Recombinant Mouse C-Reactive Protein from Pichia Pastoris GS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography and P-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem.

C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p-Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands.

C-Reactive Protein Protects Against Acetaminophen-Induced Liver Injury by Preventing Complement Overactivation.

BACKGROUND AND AIMS: C-reactive protein (CRP) is a hepatocyte-produced marker of inflammation yet with undefined function in liver injury. We aimed to examine the role of CRP in acetaminophen-induced liver injury (AILI). METHODS: The effects of CRP in AILI were investigated using CRP knockout mice and rats combined with human CRP rescue. The mechanisms of CRP action were investigated in vitro and in mice with Fcγ receptor 2B knockout, C3 knockout, or hepatic expression of CRP mutants defective in complement interaction. The therapeutic potential of CRP was investigated by intraperitoneal administration at 2 or 6 hours post-AILI induction in wild-type mice. RESULTS: CRP knockout exacerbated AILI in mice and rats, which could be rescued by genetic knock-in, adeno-associated virus-mediated hepatic expression or direct administration of human CRP. Mechanistically, CRP does not act via its cellular receptor Fcγ receptor 2B to inhibit the early phase injury to hepatocytes induced by acetaminophen; instead, CRP acts via factor H to inhibit complement overactivation on already injured hepatocytes, thereby suppressing the late phase amplification of inflammation likely mediated by C3a-dependent actions of neutrophils. Importantly, CRP treatment effectively alleviated AILI with a significantly extended therapeutic time window than that of N-acetyl cysteine. CONCLUSION: Our results thus identify CRP as a crucial checkpoint that limits destructive activation of complement in acute liver injury, and we argue that long-term suppression of CRP expression or function might increase the susceptibility to AILI.

Secretory quality control constrains functional selection-associated protein structure innovation.

Biophysical models suggest a dominant role of structural over functional constraints in shaping protein evolution. Selection on structural constraints is linked closely to expression levels of proteins, which together with structure-associated activities determine in vivo functions of proteins. Here we show that despite the up to two orders of magnitude differences in levels of C-reactive protein (CRP) in distinct species, the in vivo functions of CRP are paradoxically conserved. Such a pronounced level-function mismatch cannot be explained by activities associated with the conserved native structure, but is coupled to hidden activities associated with the unfolded, activated conformation. This is not the result of selection on structural constraints like foldability and stability, but is achieved by folding determinants-mediated functional selection that keeps a confined carrier structure to pass the stringent eukaryotic quality control on secretion. Further analysis suggests a folding threshold model which may partly explain the mismatch between the vast sequence space and the limited structure space of proteins.

Celluar Folding Determinants and Conformational Plasticity of Native C-Reactive Protein.

C-reactive protein (CRP) is an acute phase reactant secreted by hepatocytes as a pentamer. The structure formation of pentameric CRP has been demonstrated to proceed in a stepwise manner in live cells. Here, we further dissect the sequence determinants that underlie the key steps in cellular folding and assembly of CRP. The initial folding of CRP subunits depends on a leading sequence with a conserved dipeptide that licenses the formation of the hydrophobic core. This drives the bonding of the intra-subunit disulfide requiring a favorable niche largely conferred by a single residue within the C-terminal helix. A conserved salt bridge then mediates the assembly of folded subunits into pentamer. The pentameric assembly harbors a pronounced plasticity in inter-subunit interactions, which may form the basis for a reversible activation of CRP in inflammation. These results provide insights into how sequence constraints are evolved to dictate structure and function of CRP.

Endosomal pH favors shedding of membrane-inserted amyloid-ß peptide.

Amyloid-ß peptides (Aßs) are generated in a membrane-embedded state by sequential processing of amyloid precursor protein (APP). Although shedding of membrane-embedded Aß is essential for its secretion and neurotoxicity, the mechanism behind shedding regulation is not fully elucidated. Thus, we devised a Langmuir film balance-based assay to uncover this mechanism. We found that Aß shedding was enhanced under acidic pH conditions and in lipid compositions resembling raft microdomains, which are directly related to the microenvironment of Aß generation. Furthermore, Aß shedding efficiency was determined by the length of the C-terminal membrane-spanning region, whereas pH responsiveness appears to depend on the N-terminal ectodomain. These findings indicate that Aß shedding may be directly coupled to its generation and represents an unrecognized control mechanism regulating the fate of membrane-embedded products of APP processing.

A redox sensitivity-based method to quantify both pentameric and monomeric C-reactive protein in a single assay.

C-reactive protein (CRP) can exist in both pentameric (pCRP) and monomeric conformation (mCRP). Though serum pCRP is an established marker of inflammation, the diagnostic significance of mCRP remains unknown largely due to the lack of a reliable assay. The power and specificity of antibody-based assays are limited by the antibody reagents used and by the degree of cross-reactivity that may exist in detecting each antigen, as mCRP is known to be formed from the pentameric and both conformations usually coexist in clinical samples. Here, we describe an assay that measures both CRP conformations in simple samples in a single assay. This assay depends on the rationale that the intra-molecular disulfide bonds in pCRP resist reduction, while those in mCRP can be readily reduced. The distinct sensitivity of pCRP and mCRP to reduction can be easily detected and separated by electrophoresis. This assay may provide a means to study clinical correlation between pCRP and mCRP in clinical samples in the future and to evaluate their respective significance as disease markers.

IFITM3 promotes bone metastasis of prostate cancer cells by mediating activation of the TGF-ß signaling pathway.

Advanced-stage prostate cancer (PCa) is often diagnosed with bone metastasis, for which there are limited therapies. Transforming growth factor ß (TGF-ß) is known to induce epithelial-mesenchymal transition (EMT), and abundance of TGF-ß in the bone matrix is one of the important growth factors contributing to bone metastasis. TGF-ß is reported as a key mediator of bone metastasis, but the underlying mechanism has not been elucidated. It was found in our study that Interferon-inducible Transmembrane Protein 3 (IFITM3) played a key role in the regulation of malignant tumor cell proliferation, invasion, and bone migration by binding to Smad4, thus activating the TGF-ß-Smads Signaling Pathway. Lentivirus-mediated short hairpin RNA (shRNA) knockdown of IFITM3 inhibited cell proliferation and colony formation, induced apoptosis and inhibited migration by reversing EMT and downregulating the expression of metastasis-related molecules including FGFs and PTHrP. Microarray analysis showed that IFITM3 knockdown could alter the MAPK pathway associated with TGF-ß-Smads signaling. By knocking down and overexpressing IFITM3, we demonstrated that IFITM3 expression level had an effect on MAPK pathway activation, and this change was more pronounced upon exogenous TGF-ß stimulation. These results suggest that IFITM3 played an oncogenic role in PCa progression and bone metastasis via a novel TGF-ß-Smads-MAPK pathway.

In vitro generation and bioactivity evaluation of C-reactive protein intermediate.

The conformational conversion of pentameric C-reactive protein (pCRP) to monomeric CRP (mCRP) has been shown to play important roles in the action of CRP in inflammation regulation. In vivo studies revealed the origin of mCRP and provided insights into how pCRP dissociation affected its functions. However, the interplay and exact bioactivities of CRP isoforms still remain uncertain due to the rapid conformational conversion and complex milieu in vivo. Herein, we have used surface-immobilization of pCRP to generate a preservable intermediate with dual antigenicity expression of both pCRP and mCRP. The intermediate has been further shown to exhibit modified bioactivities, such as a high affinity with solution-phase pCRP and an enhanced capacity of complement interaction. These results thus not only provide the conformational conversion details of CRP, but also propose a simple way in vitro to study how the functions of CRP are tuned by distinct isoforms.

Conformational folding and disulfide bonding drive distinct stages of protein structure formation.

The causal relationship between conformational folding and disulfide bonding in protein oxidative folding remains incompletely defined. Here we show a stage-dependent interplay between the two events in oxidative folding of C-reactive protein (CRP) in live cells. CRP is composed of five identical subunits, which first fold spontaneously to a near-native core with a correctly positioned C-terminal helix. This process drives the formation of the intra-subunit disulfide bond between Cys36 and Cys97. The second stage of subunit folding, however, is a non-spontaneous process with extensive restructuring driven instead by the intra-subunit disulfide bond and guided by calcium binding-mediated anchoring. With the folded subunits, pentamer assembly ensues. Our results argue that folding spontaneity is the major determinant that dictates which event acts as the driver. The stepwise folding pathway of CRP further suggests that one major route might be selected out of the many in theory for efficient folding in the cellular environment.

α-Viniferin activates autophagic apoptosis and cell death by reducing glucocorticoid receptor expression in castration-resistant prostate cancer cells.

Prostate cancer (PCa) is one of the most commonly diagnosed urological malignancies. However, there are limited therapies for PCa patients who develop biochemical recurrence after androgen deprivation therapy (ADT). In the present study, we investigated the therapeutic efficacy and mechanism of α-Viniferin (KCV), an oligostilbene of trimeric resveratrol, against human PCa cells and found that it markedly inhibited the proliferation of LNCaP, DU145, and PC-3 cancer cells in a time- and dose-dependent manner, and had a strong cytotoxicity in non-androgen-dependent PCa cells. In addition, KCV inhibited AR downstream expression in LNCaP cells, and inhibited activation of GR signaling pathway in DU145 and PC-3. Further investigation indicated that KCV could induce cancer cell apoptosis through AMPK-mediated activation of autophagy, and inhibited GR expression in castration-resistant prostate cancer(CRPC). These findings suggest that KCV may prove to be a novel and effective therapeutic agent for the treatment of CRPC.

Profiling and bioinformatics analyses of differential circular RNA expression in prostate cancer cells.

AIM: There is little knowledge about the expression profile and function of circular RNAs (circRNAs) in prostate cancer (PCa). METHODS: The expression profiles of circRNAs in RWPE-1, 22RV1 and PC3 cells were explored via high-throughput circRNAs sequencing and validated by real-time qPCR. The roles of differentially expressed circRNAs were evaluated by bioinformatics analyses. RESULTS: Altogether 9545 circRNAs were identified and hundreds of differentially expressed circRNAs were recognized. CircRNA-miRNA networks analysis showed that many circRNAs, including circSLC7A6, circGUCY1A2 and circZFP57 could cross-talk with tumor-related miRNAs such as miR-21, miR-143 and miR-200 family. CONCLUSION: The results of our bioinformatics analyses suggested that circRNAs should play critical roles in the development and progression of PCa.

PPP5C promotes cell proliferation and survival in human prostate cancer by regulating of the JNK and ERK1/2 phosphorylation.

BACKGROUND: Prostate cancer (PCa) is one of the most common malignancies and a major leading cause of cancer-related deaths in males. And it is necessary to explore new molecular targets to enhance diagnosis and treatment level of the PCa. Serine/threonine protein phosphatase 5 (PPP5C) is a vital molecule that Involve in complex cell physiological activity. PURPOSE: The objective of this study was to detecte the expression level of PPP5C in the tissue of prostate cancer patients and further discussed the PPP5C biological function and mechanisms on the PCa. METHODS: The expression level of PPP5C was analyzed by immunohistochemistry and ONCOM-INE datasets. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to silence the expression of PPP5C in prostate cancer cell. Cell viability and proliferation were measured using MTT and colony formation, and the cell cycle and apoptosis was analyszed by flow cytometry. The changes of downstream protein level and protein phosphorylation level were detected by western blot. RESULTS: PPP5C was highly expressed in PCa tissue as analyzed by immunohistochemistry and ONCOMINE datasets. PPP5C Knockdown inhibited cell proliferation and colony formation in PCa cells. Flow cytometry analysis showed that DU145, PC3 and 22RV1 PCa cells deprived of PPP5C were arrested in G0/G1 phase and became apoptotic. Western blot analysis indicated that PPP5C knockdown could promote JNK and ERK phosphorylation. CONCLUSION: Our study indicated that the PPP5C may become a new potential diagnostic biomarker and therapeutic target for the PCa.