Kidney stones are a common threat to the health of elderly patients with a high incidence of disease. However, the specific molecular mechanism of the formation of kidney stones has not been elucidated. Here, we combined signalling molecules with signalling pathways in a double positive circulation regulation model. In addition, we found that LCN2 plays a role in promoting kidney stones through regulation of the ERK signalling pathway and expression of other kidney stone-related genes. LCN2 expression was upregulated upon oxalate stimulation. P-ERK1/2 inhibition by U0126 in kidney epithelial cells resulted in decreased expression of LCN2. Furthermore, the upregulation of LCN2 not only depended on the activation of the ERK signalling pathway but also regulated the activation of the ERK signalling pathway. Importantly, upregulation of LCN2 not only caused kidney epithelial cell damage but also promoted the expression of other kidney stone-related genes. Our findings improved the understanding of LCN2 and might lead to the development of new therapeutic and prognostic markers for kidney stones.

Patients with renal failure suffer from immune disturbances, caused by uremic toxins and influenced by dialysis treatment. The aim of the present study was to reveal whether type of dialysis modality (hemodialysis, HD, versus peritoneal dialysis, PD) differentially affects the immunocompetence, particularly the expression of genes involved in the immune response.

METHODS

87 renal transplant candidates (66 HD, 21 PD) were included in the study.

METHODS

The peripheral blood RNA samples were obtained with the PAXgene Blood system just before transplantation. The gene expression of CASP3, FAS, TP53, FOXP3, IFNG, IL2, IL6, IL8, IL10, IL17, IL18, LCN2, TGFB1, and TNF was assessed with real-time PCR on custom-designed low density arrays (TaqMan). Gene expression data were analyzed in relation to pretransplant clinical parameters.

RESULTS

The mean expression of examined genes showed no significant differences between PD and HD with the exception of FAS, expression of which was 30% higher in PD patients compared to the HD group. There was nonsignificantly higher expression of proinflammatory cytokines in the PD group. The clinical inflammatory parameters (CRP, albumin, cholesterol, and hemoglobin levels) did not differ between the groups.

CONCLUSIONS

Type of renal replacement therapy exerts no differential effect on cytokine gene expression or inflammatory clinical parameters.

Myocardial ischemia/reperfusion (MI/R) injury is a complex phenomenon that causes severe damage to the myocardium. However, the potential molecular mechanisms of MI/R injury have not been fully clarified. We identified potential molecular mechanisms and therapeutic targets in MI/R injury through analysis of Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were found between MI/R injury and normal samples, and overlapping DEGs were found between GSE61592 and GSE67308. Gene Ontology (GO) and pathway analysis were performed for overlapping DEGs by Database for Annotation, Visualization and Integration Discovery (DAVID). Then, a network of protein-protein interaction (PPI) was constructed through the Search Tool for the Retrieval of Interacting Genes (STRING) database. Potential microRNAs (miRNAs) and therapeutic small molecules were screened out using microRNA.org database and the Comparative Toxicogenomics database (CTD), respectively. Finally, we identified 21 overlapping DEGs related to MI/R injury. These DEGs were significantly enriched in IL-17 signaling pathway, cytosolic DNA-sensing pathway, chemokine signaling, and cytokine-cytokine receptor interaction pathway. According to the degree in the PPI network, CCL2, LCN2, HP, CCL7, HMOX1, CCL4, and S100A8 were found to be hub genes. Furthermore, we identified potential miRNAs (miR-24-3p, miR-26b-5p, miR-2861, miR-217, miR-4251, and miR-124-3p) and therapeutic small molecules like ozone, troglitazone, rosiglitazone, and n-3 polyunsaturated fatty acids for MI/R injury. These results identified hub genes and potential small molecule drugs, which could contribute to the understanding of molecular mechanisms and treatment for MI/R injury.

Although the postoperative recurrence rate for pancreatic neuroendocrine tumors (PNETs) is reported to be 13.5%-30%, the paucity of valuable biomarkers to predict recurrence poses a problem for the early detection of relapse. Hence, this study aimed to identify new biomarkers to predict the recurrence of PNETs. We performed RNA sequencing (RNA-Seq) on RNA isolated from frozen primary tumors sampled from all localized G1/G2 PNETs resected curatively from 1998 to 2015 in our institution. We calculated differentially expressed genes (DEGs) in tumor with and without recurrence (≥3 years) for the propensity-matched cohort. Gene ontology analysis for the identified DEGs was also performed. Furthermore, we evaluated the expression levels of candidate genes as recurrence predictors via immunostaining. Comparison of transcriptional levels in tumors with and without recurrence identified 166 DEGs. Up- and downregulated genes with high significance in these tumors were mainly related to extracellular organization and cell adhesion, respectively. We observed the top three upregulated genes, C-type lectin domain family 3 member A (CLEC3A), matrix metalloproteinase-7 (MMP7), and lipocalin2 (LCN2) immunohistochemically and compared their levels in recurrent and nonrecurrent tumors. Significantly higher recurrence rate was shown in patients with positive expression of CLEC3A (P = 0.028), MMP7 (P = 0.003), and LCN2 (P = 0.040) than that with negative expression. We identified CLEC3A, MMP7, and LCN2 known to be associated with the phosphatidylinositol-3-kinase/Akt pathway, as potential novel markers to predict the postoperative recurrence of PNETs.

Female sex protects against development of acute kidney injury (AKI). While sex hormones may be involved in protection, the role of differential gene expression is unknown. We conducted gene profiling in male and female mice with or without kidney ischemia-reperfusion injury. Mice underwent bilateral renal pedicle clamping (30 min), and tissues were collected 24 hours after reperfusion. RNA-sequencing was performed on proximal tubules and kidney endothelial cells. Female mice were resistant to ischemic injury compared to males, determined by plasma creatinine and neutrophil gelatinase-associated lipocalin, histologic scores, neutrophil infiltration, and extent of apoptosis. Sham mice had sex-specific gene disparities in proximal tubule and endothelium, and male mice showed profound gene dysregulation with ischemia-reperfusion compared to females. After ischemia proximal tubules from females exhibited smaller increases compared to males in injury-associated genes Lcn2, Havcr1, and Krt18, and no upregulation of Sox9 or Krt20. Endothelial upregulation of adhesion molecules and cytokines/chemokines occurred in males, but not females. Upregulated genes in male ischemic proximal tubules were linked to tumor necrosis factor and Toll-like receptor pathways, while female ischemic proximal tubules showed upregulated genes in pathways related to transport. The data highlight sex-specific gene expression differences in male and female proximal tubule and endothelium before and after ischemic injury that may underlie disparities in susceptibility to AKI.

Keywords: acute kidney injury; sexual dimorphism; transcriptome.

Background: Primary 12α-hydroxylated bile acids (12αOH BAs) enhance intestinal iron uptake due to their ability ex vivo to chelate iron. However, no information is available on their role in vivo, especially in the liver.

Objectives: To investigate the effects and mechanisms of primary 12αOH BAs on hepatic iron concentration in vivo.

Methods: Male Wistar King A Hokkaido male rats (WKAH/HkmSlc) rats aged 4-5 weeks were fed a control diet or a diet with cholic acid (CA; 0.5 g/kg diet), the primary 12αOH BA, for 2 weeks (Study 1) or 13 weeks (Study 2). In Study 3, rats fed the same diets were given drinking water either alone or containing vancomycin (200 mg/L) for 6 weeks. The variables measured included food intake (Studies 1-3), bile acid profiles (Studies 1 and 3), hepatic iron concentration (Studies 1-3), fecal iron excretion (Studies 1 and 2), iron-related liver gene expression (Studies 2 and 3), and plasma iron-related factors (Studies 2 and 3).

Results: In Study 1, CA feed reduced the hepatic iron concentration (-16%; P = 0.005) without changing food intake or fecal iron excretion. In Study 2, we found a significant increase in the aortic plasma concentration of lipocalin 2 (LCN2; +65%; P < 0.001), an iron-trafficking protein. In Study 3, we observed no effect of vancomycin treatment on the CA-induced reduction of hepatic iron concentration (-32%; P < 0.001), accompanied by increased plasma LCN2 concentration (+72%; P = 0.003), in the CA-fed rats despite a drastic reduction in the secondary 12αOH BA concentration (-94%; P < 0.001) in the aortic plasma.

Conclusions: Primary 12αOH BAs reduced the hepatic iron concentration in rats. LCN2 may be responsible for the hepatic iron-lowering effect of primary 12αOH BAs by transporting iron out of the liver.

Keywords: 12α-hydroxylated bile acid; high-fat diet; iron deficiency; lipocalin 2; primary bile acid.

Oesophageal squamous cell carcinoma (ESCC) has a relatively unfavourable prognosis due to metastasis and chemoresistance. Our previous research established a comprehensive ESCC database (GSE53625). After analysing data from TCGA database and GSE53625, we found that PLEK2 predicted poor prognosis in ESCC. Moreover, PLEK2 expression was also related to the overall survival of ESCC patients undergoing chemotherapy. Repression of PLEK2 decreased the proliferation, migration, invasion and chemoresistance of ESCC cells in vitro and decreased tumorigenicity and distant metastasis in vivo. Mechanistically, luciferase reporter assay and chromatin immunoprecipitation assay suggested that TGF-β stimulated the process that Smad2/3 binds to the promoter sequences of PLEK2 and induced its expression. RNA-seq suggested LCN2 might a key molecular regulated by PLEK2. LCN2 overexpression in PLEK2 knockdown ESCC cells reversed the effects of decreased migration and invasion. In addition, TGF-β induced the expression of LCN2, but the effect disappeared when PLEK2 was knockdown. Moreover, AKT was phosphorylated in all regulatory processes. This study detected the major role of PLEK2 in driving metastasis and chemoresistance in ESCC by regulating LCN2, which indicates the potential use of PLEK2 as a biomarker to predict prognosis and as a therapeutic target for ESCC.

Excessive cytokine activity underlies many autoimmune conditions, particularly through the interleukin-17 (IL-17) and tumor necrosis factor-α (TNFα) signaling axis. Both cytokines activate nuclear factor κB, but appropriate induction of downstream effector genes requires coordinated activation of other transcription factors, notably, CCAAT/enhancer binding proteins (C/EBPs). Here, we demonstrate the unexpected involvement of a posttranscriptional "epitranscriptomic" mRNA modification [N6-methyladenosine (m⁶A)] in regulating C/EBPβ and C/EBPδ in response to IL-17A, as well as IL-17F and TNFα. Prompted by the observation that C/EBPβ/δ-encoding transcripts contain m⁶A consensus sites, we show that Cebpd and Cebpb mRNAs are subject to m⁶A modification. Induction of C/EBPs is enhanced by an m⁶A methylase "writer" and suppressed by a demethylase "eraser." The only m⁶A "reader" found to be involved in this pathway was IGF2BP2 (IMP2), and IMP2 occupancy of Cebpd and Cebpb mRNA was enhanced by m⁶A modification. IMP2 facilitated IL-17-mediated Cebpd mRNA stabilization and promoted translation of C/EBPβ/δ in response to IL-17A, IL-17F, and TNFα. RNA sequencing revealed transcriptome-wide IL-17-induced transcripts that are IMP2 influenced, and RNA immunoprecipitation sequencing identified the subset of mRNAs that are directly occupied by IMP2, which included Cebpb and Cebpd Lipocalin-2 (Lcn2), a hallmark of autoimmune kidney injury, was strongly dependent on IL-17, IMP2, and C/EBPβ/δ. Imp2^-/- mice were resistant to autoantibody-induced glomerulonephritis (AGN), showing impaired renal expression of C/EBPs and Lcn2 Moreover, IMP2 deletion initiated only after AGN onset ameliorated disease. Thus, posttranscriptional regulation of C/EBPs through m⁶A/IMP2 represents a previously unidentified paradigm of cytokine-driven autoimmune inflammation.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality, both in adult and pediatric populations. However, the dynamic changes of gene expression profiles following TBI have not been fully understood. In this study, we identified the differentially expressed genes (DEGs) following TBI. Remarkably, Serpina3n, Asf1b, Folr1, LOC100366216, Clec12a, Olr1, Timp1, Hspb1, Lcn2, and Spp1 were identified as the top 10 with the highest statistical significance. The weighted gene coexpression analysis (WGCNA) identified 12 functional modules from the DEGs, which showed specific expression patterns over time and were characterized by enrichment analysis. Specifically, the black and turquoise modules were mainly involved in energy metabolism and protein translation. The green yellow and yellow modules including Hmox1, Mif, Anxa2, Timp1, Gfap, Cd9, Gja1, Pdpn, and Gpx1 were related to response to wounding, indicating that expression of these genes such as Hmox1, Anxa2, and Timp1 could protect the brains from brain injury. The green yellow module highlighted genes involved in microglial cell activation such as Tyrobp, Cx3cr1, Grn, Trem2, C1qa, and Aif1, suggesting that these genes were responsible for the inflammatory response caused by TBI. The upregulation of these genes has been validated in an independent dataset. These results indicated that the key genes in microglia cell activation may serve as a promising therapeutic target for TBI. In summary, the present study provided a full view of the dynamic gene expression changes following TBI.

Myocardial infarction (MI) is a leading cause of death worldwide and requires development of efficient therapeutic strategies . Mesenchymal stem cells (MSCs) -based therapy of MI has been promising but inefficient due to undesirable microenvironment of the infarct tissue. Hence, the current study was conducted to fortify MSCs against the unfavorable microenvironment of infarct tissue via overexpression of Lipocalin 2 (Lcn2) as a cytoprotective factor. The engineered cells (Lcn2-MSCs) were transplanted to infarcted heart of a rat model of MI. According to our findings, Lcn2 overexpression resulted in increased MSCs survival in the MI tissue (p < 0.05) compared to non-engineered cells. Furthermore, the infusion of Lcn2-MSCs mitigated Left ventricle (LV) remodeling, decreased fibrosis (p < 0.0001), and reduced apoptotic death of the LVs' cells (p < 0.0001) compared to the control. Our findings suggest a potential novel therapeutic strategy for MI, however, further investigations such as safety and efficacy assessments in large animals followed by clinical trials are required.

Keywords: Apoptosis; Exogenous expression; Lipocalin 2; Mesenchymal stem cells; Myocardial infarction.

Cancer diagnosis based on serum biomarkers requires receptors of extreme sensitivity and selectivity. Tunability of aptamer selection makes them ideal for that challenge. However, aptamer characterization is a time-consuming task, not always thoroughly addressed, leading to suboptimal aptamer performance. In this work, we report on the affinity characterization and potential usage of two aptamers against a candidate cancer biomarker, the neutrophil gelatinase-associated lipocalin (NGAL). Electrochemical sandwich assays on Au electrodes and SPR experiments showed a restricted capture ability of one of the aptamers (LCN2-4) and a small detectability of the other (LCN2-2). Interestingly, a truncated version of the signaling aptamer LCN2-2 selectively binds to NGAL covalently linked to magnetic beads due to high local protein concentration. The functional affinity of this aptamer is enhanced by three-orders of magnitude using rolling circle amplification (RCA), completed in only 15 min, followed by hybridization with short complementary fluorescein-tag probes, enzyme labeling and chronoamperometric measurement. Microscale thermophoresis experiments show a poor affinity for the protein in solution, which urges the importance of a full and in-depth characterization of aptamers to be used as diagnostic reagents.

The intake of excess dietary fructose most often leads to non-alcoholic fatty liver disease (NAFLD). Fructose is metabolized mainly in the liver and its chronic consumption results in lipogenic gene expression in this organ. However, precisely how fructose is involved in NAFLD progression is still not fully understood, limiting therapy. Lipocalin-2 (LCN2) is a small secreted transport protein that binds to fatty acids, phospholipids, steroids, retinol, and pheromones. LCN2 regulates lipid and energy metabolism in obesity and is upregulated in response to insulin. We previously discovered that LCN2 has a hepatoprotective effect during hepatic insult, and that its upregulation is a marker of liver damage and inflammation. To investigate if LCN2 has impact on the metabolism of fructose and thereby arising liver damage, we fed wild type and Lcn2-/- mice for 4 or 8 weeks on diets that were enriched in fructose either by adding this sugar to the drinking water (30% w/v), or by feeding a chow containing 60% (w/w) fructose. Body weight and daily intake of food and water of these mice was then measured. Fat content in liver sections was visualized using Oil Red O stain, and expression levels of genes involved in fat and sugar metabolism were measured by qRT-PCR and Western blot analysis. We found that fructose-induced steatosis and liver damage was more prominent in female than in male mice, but that the most severe hepatic damage occurred in female mice lacking LCN2. Unexpectedly, consumption of elevated fructose did not induce de novo lipogenesis or fat accumulation. We conclude that LCN2 acts in a lipid-independent manner to protect the liver against fructose-induced damage.

Due to their dynamic characteristics, hydroxyapatite nanoparticles (HAP-NPs) have been employed numerous times in nanomedicine and in tissue engineering, particularly as diagnostic and therapeutic agents. However, there are outstanding findings from various studies that question whether these NPs are safe when they are used in the human body. Therefore, a more in-depth toxicity assessment should be carried out to give a clear answer regarding the fate of these particles. Here we aim to investigate the possible cytotoxicity, genotoxicity and inflammation induced by HAP-NPs, as well as predict the synergistic antioxidative effect of chitosan nanoparticles (CsNPs) and curcumin nanoparticles (CurNPs) in mitigating this pronounced toxicity. The present study was conducted on eighty Wistar male rats, divided into eight equal groups. The results showed that, at the molecular level, HAP-NPs significantly induced gene expression of tumor suppressor protein p53, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and also Kidney Injury Molecule-1 (KIM-1) and Lipocalin-2 (LCN2). In addition, kidney biochemical parameters (total bilirubin, urea, uric acid and creatinine) increased, but albumin levels decreased in the group treated with HAP-NPs alone. Meanwhile, co-treatment with CsNPs and/or CurNPs with HAP-NPs showed an improvement in the activities of the kidney parameters and reduced inflammation. This study shows that the nephrotoxicity mechanism of HAP-NPs may involve various signaling pathways including alterations in biochemical parameters, gene expression of KIM-1 and LCN2 and disturbing the production of cytokines and p53. Furthermore, these insights showed that the combined effect of both CsNPs and CurNPs was more pronounced than the effect of each one on its own.

Smilax china L. (SCL) is extensively used in the treatment of pelvic inflammatory disease (PID). This study aimed to clarify the potential active ingredients of SCL and mechanisms on PID. SCL was widely distributed in Japan, South Korea, and China, which was traditionally considered heat-clearing, detoxicating, and dampness-eliminating medicine. Systems pharmacology revealed that 32 compounds in SCL may interact with 19 targets for immunoenhancement, antiapoptosis, anti-inflammation, and antioxidant activity of the PID model. Molecular docking revealed that isorhamnetin, moracin M, rutin, and oxyresveratrol may have higher binding potential with prostaglandin-endoperoxide synthase 2 (PTGS2), mitogen-activated protein kinase 1 (MAPK1), siderocalin (LCN2), tumor necrosis factor (TNF), and matrix metalloprotein-9 (MMP9), respectively. Molecular dynamics simulation showed that the binding modes of moracin M-MAPK1, rutin-TNF, and oxyresveratrol-MMP9 complexes were more stable, evidenced by relatively smaller fluctuations in root mean square deviation values. Conclusively, SCL may treat PID by inhibiting inflammatory factors, antitissue fibrosis, and microbial growth.

Objective: Liver damage is a common manifestation and can be life-threatening in adult-onset Still's disease (AOSD), an autoinflammatory disease. The hallmark of AOSD is activation of neutrophils, whose infiltration in liver is suspected to promote tissue injury. Here we aimed to identify a candidate biomarker and to validate its association with liver damage in AOSD.

Methods: Transcriptome analysis of neutrophils from treatment-naïve active AOSD patients and healthy donors was performed. Lipocalin-2 (LCN2) expression was assessed in neutrophils, plasma and liver biopsies of AOSD. The correlations of LCN2 with different variables and its ability to identify liver damage from AOSD patients were analysed.

Results: LCN2, a novel biomarker in hepatic inflammation, was found to be upregulated in AOSD neutrophils by RNA sequencing and confirmed at the mRNA and protein levels. Plasma levels of LCN2 were significantly higher in AOSD patients than healthy controls, RA and SLE patients. Plasma LCN2 levels were closely correlated with inflammatory markers, systemic score, HScore and cytokines. Moreover, LCN2 levels were increased in active AOSD with liver involvement and independently associated with liver dysfunction. Enhanced expression of LCN2 was detected in liver biopsies from three patients with ongoing liver injury. Furthermore, the area under the curve value of LCN2 for identifying AOSD with liver injury from other liver diseases was 0.9694.

Conclusion: Our results reveal that neutrophils-derived LCN2 is higher in plasma and liver tissue in AOSD patients than in healthy controls, and it could serve as a potent biomarker for identifying AOSD with systemic inflammation, especially liver damage caused by hyperinflammation.

Keywords: adult-onset Still’s disease; biomarker; lipocalin-2; liver damage; neutrophil.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by different cellular and molecular mechanisms that interplay in exacerbating the progression of the pathology. No fully restorative therapies are yet available, and it is thus becoming recognized that combinatorial approaches aimed at addressing different aspects of SCI will likely results in greater functional outcomes. Here we employed packaging RNA-mediated RNA interference (pRNA-RNAi) nanotherapeutics to downregulate in situ the expression of lipocalin 2 (Lcn2), a known mediator of neuroinflammation and autocrine mediator of reactive astrogliosis, and to create a more amenable niche for the subsequent transplantation of induced neural stem cells (iNSCs). To our knowledge, this is the first approach that takes advantage of the modular and multifunctional pRNA three-way junction platform in the SCI niche, while also exploiting the therapeutic potential of immune-compatible and feasible iNSC transplants. We show the combination of such treatments in a mouse model of contusion thoracic SCI leads to significant improvement of locomotor function, albeit not better than single pRNA-RNAi treatment, and results in synergistic histopathological effects, such as reduction of glial scar volume, diminished pro-inflammatory response, and promotion of neuronal survival. Our results provide evidence for a novel combinatorial approach for treating SCI.

Keywords: RNAi; iNSCs; lipocalin 2; nanotherapeutics; neural stem cells; neuroimmunology; pRNA; regenerative neuroimmunology; spinal cord injury.

Although some effective therapies have been available for cancer, it still poses a great threat to human health and life due to its drug resistance and low response in patients. Here, we develop a ferroptosis-based therapy by combining iron nanoparticles and cancer-specific gene interference. The expression of two iron metabolic genes (FPN and LCN2) was selectively knocked down in cancer cells by Cas13a or microRNA controlled by a NF-κB-specific promoter. Cells were simultaneously treated by iron nanoparticles. As a result, a significant ferroptosis was induced in a wide variety of cancer cells. However, the same treatment had little effect on normal cells. By transferring genes with adeno-associated virus and iron nanoparticles, the significant tumor growth inhibition and durable cure were obtained in mice with the therapy. In this work, we thus show a cancer therapy based on gene interference-enhanced ferroptosis.

Hepatoblastoma (HB) is the most common pediatric liver malignant tumor. Previously, we reported co-activation of β-catenin and Yes-associated protein-1 (YAP1) in 80% of HB. Hepatic co-expression of active β-catenin and YAP1 via sleeping beauty transposon/transposase and hydrodynamic tail vein injection led to HB development in mice. Here, we identify lipocalin 2 (Lcn2) as a target of β-catenin and YAP1 in HB and show that serum Lcn2 values positively correlated with tumor burden. Lcn2 was strongly expressed in HB tumor cells in our mouse model. A tissue array of 62 HB cases showed highest LCN2 expression in embryonal and lowest in fetal, blastemal, and small cell undifferentiated forms of HB. Knockdown of LCN2 in HB cells had no effect on cell proliferation but reduced NF-κB reporter activity. Next, liver-specific Lcn2 knockout (KO) mice were generated. No difference in tumor burden was observed between Lcn2 KO mice and wild-type littermate controls after sleeping beauty transposon/transposase and hydrodynamic tail vein injection delivery of active YAP1 and β-catenin, although Lcn2 KO mice with HB lacked any serum Lcn2 elevation, demonstrating that transformed hepatocytes are the source of serum Lcn2. More blastemal areas and inflammation were observed within HB in Lcn2 KO compared with wild-type tumors. In conclusion, Lcn2 expressed in hepatocytes appears to be dispensable for the pathogenesis of HB. However, transformed hepatocytes secrete serum Lcn2, making Lcn2 a valuable biomarker for HB.

Both host and pathogen competitively manipulate coordination environments during bacterial infections. Human cells release the innate immune protein siderocalin (Scn, also known as lipocalin-2/Lcn2, neutrophil gelatinase-associated lipocalin/NGAL) that can inhibit bacterial growth by sequestering iron in a ferric complex with enterobactin (Ent), the ubiquitous Escherichia coli siderophore. Pathogenic E. coli use the virulence-associated esterase IroE to linearize the Ent cyclic trilactone to linear enterobactin (lin-Ent). We characterized lin-Ent interactions with Scn by using native mass spectrometry (MS) with hydrogen deuterium exchange (HDX) and Lys/Arg specific covalent footprinting. These approaches support 1:1 binding of both Fe(III)-lin-Ent to Scn and iron-free lin-Ent to Scn. Both ferric and non-ferric lin-Ent localized to all three pockets of the Scn calyx, consistent with Scn capture of lin-Ent both before and after Fe(III) chelation. These findings raise the possibility that Scn neutralizes both siderophores and siderophore-bound iron during infections. This integrated, MS-based approach circumvents the limitations that frustrate traditional structural approaches to examining Scn interactions with enterobactin-based ligands.

One of the key barriers for early identification and intervention of severe influenza cases is a lack of reliable immunologic indicators. In this study, we utilized differentially expressed genes screening incorporating weighted gene co-expression network analysis in one eligible influenza GEO data set (GSE111368) to identify hub genes associated with clinical severity. A total of 10 genes (PBI, MMP8, TCN1, RETN, OLFM4, ELANE, LTF, LCN2, DEFA4 and HP) were identified. Gene set enrichment analysis (GSEA) for single hub gene revealed that these genes had a close association with antimicrobial response and neutrophils activity. To further evaluate these genes' ability for diagnosis/prognosis of disease developments, we adopted double validation with (a) another new independent data set (GSE101702); and (b) plasma samples collected from hospitalized influenza patients. We found that 10 hub genes presented highly correlation with disease severity. In particular, BPI and MMP8 encoding proteins in plasma achieved higher expression in severe and dead cases, which indicated an adverse disease development and suggested a frustrating prognosis. These findings provide new insight into severe influenza pathogenesis and identify two significant candidate genes that were superior to the conventional clinical indicators. These candidate genes or encoding proteins could be biomarker for clinical diagnosis and therapeutic targets for severe influenza infection.

Keywords: differentially expressed genes; hub genes; network analysis; neutrophils; severe influenza; weighted gene co-expression network analysis.

We aimed to investigate associations among serum levels of LCN2, bone resorption marker carboxy-terminal cross-linking telopeptide of type-1 collagen (CTx), bone formation marker osteocalcin (OCN), and bone mineral densities (BMDs) in ambulatory healthy women.

This cross-sectional study analyzed 1012 previously enrolled outpatient Han Chinese women. BMDs of the lumbar spine and femoral neck were measured using dual energy X-ray absorptiometry. Serum levels of LCN2, CTx, OCN, and creatinine (Scr) were measured.

Circulating LCN2 was inversely correlated with BMDs at the lumbar spine and femoral neck (Spearman's r = -0.08, P = 0.010 and r = -0.14, P < 0.001; respectively). A significant positive correlation between LCN2 and CTx (r = 0.11, P < 0.001), OCN (r = 0.06, P = 0.047), age (r = 0.21, P < 0.001), and Scr (r = 0.24, P < 0.001) was also observed. After adjusting for age and Scr, the correlation among LCN2, BMDs and OCN disappeared, but LCN2 was still positively associated with CTx (r = 0.08, P = 0.010). The circulating concentration of LCN2 showed no significant difference between subjects with and without osteoporotic fractures (43.63 (35.29, 53.66) vs. 42.25 (34.43, 51.46) ng/ml, respectively, P = 0.111). Serum CTx concentrations rose with serum LCN2 increasing from the lowest to the highest quartile (P for trend = 0.005), even after adjusting for age and Scr (P for trend = 0.040). In multivariate regression analysis, LCN2 was one of the main determinants for changes in serum CTx (standard β = 0.061, P = 0.005).

In ambulatory healthy women, the relationships among serum LCN2 level, BMDs, and OCN were confounded by age and Scr. Although LCN2 was positively related with CTx, the correlation was very weak and may not be physiologically relevant.

Oxidative stress is a key contributor to the pathogenesis of stroke-reperfusion injury. Neuroinflammatory peptides released after ischemic stroke mediate reperfusion injury. Previous studies, including ours, have shown that lipocalin-2 (LCN2) is secreted in response to cerebral ischemia to promote reperfusion injury. Genetic deletion of LCN2 significantly reduces brain injury after stroke, suggesting that LCN2 is a mediator of reperfusion injury and a potential therapeutic target. Immunotherapy has the potential to harness neuroinflammatory responses and provides neuroprotection against stroke. Here we report that LCN2 was induced on the inner surface of cerebral endothelial cells, neutrophils, and astrocytes that gatekeep the blood-brain barrier (BBB) after stroke. LCN2 monoclonal antibody (mAb) specifically targeted LCN2 in vitro and in vivo, attenuating the induction of LCN2 and pro-inflammatory mediators (iNOS, IL-6, CCL2, and CCL9) after stroke. Administration of LCN2 mAb at 4 h after stroke significantly reduced neurological deficits, cerebral infarction, edema, BBB leakage, and infiltration of neutrophils. The binding epitope of LCN2 mAb was mapped to the β3 and β4 strands, which are responsible for maintaining the integrity of LCN2 cup-shaped structure. These data indicate that LCN2 can be pharmacologically targeted using a specific mAb to reduce reperfusion injury after stroke.

Keywords: Lipocalin-2; immunotherapy; neutrophils; oxidative stress; reperfusion injury; stroke.

Purpose: Lipocalin 2 (LCN2) is an adipokine involved in many physiological functions, including bone metabolism. We previously demonstrated its implication in mouse models of mechanical unloading-induced osteoporosis and in a cohort of bed rest volunteers. We therefore aimed at studying its involvement in postmenopausal osteoporosis.

Methods: We measured serum LCN2 and correlated its levels to Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1), Tartrate Resistant Acid Phosphatase 5B (TRAcP5B), sclerostin, urinary N-terminal telopeptide of type I collagen (NTX), serum C-terminal telopeptide of type I collagen (CTX), parathyroid hormone and vitamin K by ELISA performed in a cohort of younger (50-65 years) and older (66-90 years) osteoporotic women in comparison to healthy subjects. A cohort of male healthy and osteoarthritic patients was also included. Sobel mediation analysis was used to test indirect associations among age, LCN2 and DKK1 or NTX.

Results: LCN2 levels were unchanged in osteoporotic and in osteoarthritis patients when compared to healthy subjects and did not correlate with BMD. However, serum LCN2 correlated with age in healthy women (R = 0.44; P = 0.003) and men (R = 0.5; P = 0.001) and serum concentrations of DKK1 (R = 0.47; P = 0.003) and urinary NTX (R = 0.34; P = 0.04). Sobel mediation analysis showed that LCN2 mediates an indirect relationship between age and DKK1 (P = 0.02), but not with NTX, in healthy subjects.

Conclusions: Taken together, the results suggest a hitherto unknown association between LCN2, DKK1 and age in healthy individuals, but not in postmenopausal osteoporotic women.

Keywords: BALP, bone-specific alkaline phosphatase; BMD, bone mineral density; BMI, body mass index; CTX, C-terminal telopeptide of type I collagen; DKK1; DKK1, Dickkopf WNT Signaling Pathway Inhibitor 1; IL, interleukin; LCN2, lipocalin 2; Lipocalin-2; NGAL; NTX, N-terminal telopeptide of type I collagen; NfκB, nuclear factor kappa-B; Osteoarthritis; Osteoporosis; PTH, parathyroid hormone; RANKL, receptor activator of nuclear factor kappa-B; TNF, tumor necrosis factor; TRAcP5B, tartrate-resistant acid phosphatase 5B; Wnt.

Shiga-toxigenic Escherichia coli (STEC) infection causes severe bloody diarrhea, renal failure, and hemolytic uremic syndrome. Recent studies showed global increases in Locus for Enterocyte Effacement (LEE)-negative STEC infection. Some LEE-negative STEC produce Subtilase cytotoxin (SubAB), which cleaves endoplasmic reticulum (ER) chaperone protein BiP, inducing ER stress and apoptotic cell death. In this study, we report that SubAB induces expression of a novel form of Lipocalin-2 (LCN2), and describe its biological activity and effects on apoptotic cell death. SubAB induced expression of a novel LCN2, which was regulated by PRKR-like endoplasmic reticulum kinase via the C/EBP homologous protein pathway. SubAB-induced novel-sized LCN2 was not secreted into the culture supernatant. Increased intracellular iron level by addition of holo-transferrin or FeCl₃ suppressed SubAB-induced PARP cleavage. Normal-sized FLAG-tagged LCN2 suppressed STEC growth, but this effect was not seen in the presence of SubAB- or tunicamycin-induced unglycosylated FLAG-tagged LCN2. Our study demonstrates that SubAB-induced novel-sized LCN2 does not have anti-STEC activity, suggesting that SubAB plays a crucial role in the survival of LEE-negative STEC as well as inducing apoptosis of the host cells.