Following the publication of this article, an interested reader drew to our attention that Fig. 1C contained an important flaw. The Figure shows a western blot for LCN2, DR4, DR5, and actin, and it was noted that the identical bands shown for actin were also featured in a paper by the same authors published in 2017 [Lipocalin 2 negatively regulates cell proliferation and epithelial to mesenchymal transition through changing metabolic gene expression in colorectal cancer. Kim SL, Lee ST, Min IS, Park YR, Lee JH, Kim DG and Kim SW: Cancer Sci 108: 2176‑2186, 2017], except that the lanes for the cell lines HCT116 and SW620 were depicted the other way around in the International Journal of Oncology article. Upon investigating the matter with the authors, they were able to confirm that the lanes were labelled incorrectly in the Figure itself; moreover, the incorrect control bands were included with the Figure. The corrected version of Fig. 1 is shown opposite, including the correct control data for Fig. 1C. This error did not have an impact on the overall meaning of the paper, or on the reported conclusions of this study. The authors regret that this error was introduced into the printed version of the paper, and apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 53: 2789‑2799, 2018; DOI: 10.3892/ijo.2018.4562].

OBJECTIVE

To identify the impact of Lipocalin-2 (LCN2) gene polymorphisms on breast cancer patients in western Saudi Arabia.

METHODS

It is a case control study in which blood samples of participants from Medical Reference Clinics and King Abdulaziz University Hospital in Jeddah, Saudi Arabia have been taken between 2014 and 2016. This study recruited 128 participants (50% control, 50% patients) and used Tetra-Primer amplification-refractory mutation system-polymerase chain reaction method for the detection of missense SNP (rs11556770). The study measured LCN2 plasma protein expression by enzyme-linked immunosorbent assay technique. Results: The results have shown that 100% of the genotypes were normal allele (G/G). In contrast, the plasma level of LCN2 was considerably elevated among patients as compared to control (p=0.001), and higher in invasive ductal carcinoma patients (p=0.001). The LCN2 protein expression in plasma level was significantly elevated among patients, particularly who demonstrated invasive ductal carcinoma. Conclusion: There is no significant relationship between breast cancer patients and LCN2 gene polymorphisms (rs11556770).

Ell3 is an RNA polymerase II transcription elongation factor that acts as a negative regulator of p53 expression, and regulates cell proliferation and survival. Recent studies by our group have demonstrated that ectopic expression of Ell3 in breast cancer cell lines enhances cell proliferation, potentiates cancer stem cell properties, and promotes 5-Fluorouracil (5-FU) resistance. In the present study, the underlying mechanism for the induction of 5-FU resistance was investigated in Ell3 over-expressing MCF-7 cells (Ell3 OE cells). By comparing the gene expression profiles of Ell3 OE cells with control cells, the present data revealed that Lipocalin2 (LCN2) and Wnt signaling activity are associated with 5-FU resistance of Ell3 OE. siRNA-mediated suppression of LCN2 reversed 5-FU resistance in Ell3 OE cells. Chemical inhibition of Wnt signaling also reversed 5-FU resistance in Ell3 OE cells. Furthermore, the expression levels of survivin, which is a direct transcriptional target of Wnt/β-catenin and an inhibitor of apoptosis, were markedly elevated when Ell3 OE cells were treated with 5-FU, as detected by western blot analysis. These findings suggest that enhanced expression of LCN2 and activation of the Wnt signaling pathway may induce 5-FU resistance in Ell3 OE cells as a means of evading apoptosis.

Recent studies strongly suggested that transcription factor 7-like 1 (Tcf7l1, also known as Tcf3) is involved in the differentiation of several types of cells, and demonstrated that Tcf7l1 modulates keratinocytes physiologically through regulating lipocalin 2 (LCN2), a key regulator of cell differentiation. To reveal the potential role of Tcf7l1 in the dysregulation of keratinocyte differentiation, both Tcf7l1 and LCN2 were determined in a variety of skin disorders. The in vitro effect of Tcf7l1 on keratinocyte differentiation was studied by culturing SCC-13 cells, and the human foreskin keratinocytes (HFKs) that were transfected with vectors for overexpressing human papillomavirus E6/E7 or Tcf7l1 genes. We found that both Tcf7l1 and LCN2 were highly expressed in those diseases characterized by defective keratinocyte differentiation (especially psoriasis vulgaris, condyloma acuminatum, squamous cell carcinoma, etc). Moreover, compared with control HFKs, SCC-13 cells and E6/E7-harboring HFKs expressed more Tcf7l1 and LCN2. Tcf7l1 siRNA transfection decreased LCN2 but increased involucrin and loricrin in HFKs under calcium stimuli. Conversely, Tcf7l1 overexpression in SCC-13 cells or vector-transfected HFKs induced lower involucrin and loricrin expression and less keratinocyte apoptosis, both of which, however, were partially abrogated by LCN2 siRNA or neutralizing anti-LCN2 antibody. Interestingly, the Tcf7l1 expression in HFKs correlated positively with the MMP-2 level, and the inhibition of MMP-2 decreased the LCN2 level and even attenuated the effect of Tcf7l1 on LCN2 expression. Therefore, Tcf7l1 dysregulates keratinocyte differentiation, possibly through upregulating the LCN2 pathway in an MMP-2 mediated manner. Elucidating the interaction between Tcf7l1 and LCN2 may help understand disordered cell differentiation in some skin diseases.

The aim of this paper was to analyze the microarray data between ulcerative colitis(UC) patients and healthy people by bioinformatics technology, screen the differentially expressed genes of UC, and predict the potential Chinese medicines for UC. The GSE36807 gene expression profile was downloaded from the gene expression database(GEO) and the differentially expressed(both up-regulated and down-regulated) genes(DEGs) were analyzed by using R language software. The core genes in the DEGs were obtained by using String database, Cytoscape software and its plug-in analysis, and the gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) were used to analyze the core genes. Moreover, the core genes and the medical ontology information retrieval platform(Coremine Medical) were mapped to each other to screen the traditional Chinese medicines and its active ingredients for treating UC. A total of 648 DEGs were screened, including 397 up-regulated genes and 251 down-regulated genes. Up-regulation of DEGs yielded 15 core genes including CXCL8, IL1 B, MMP9, CXCL1, CXCL10, CXCL9, CXCL2, CXCL5, TIMP1, CXCL11, STAT1,LCN2, IL1 RN, MMP1 and IDO1. Their biological processes and pathways were mainly enriched in interleukins, chemokine ligands and cytokines, chemokine-mediated signaling pathways, and were closely related to inflammatory responses, defense responses, cell chemotaxis, secretory granules, IL17 signaling pathways, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and TNF signaling pathway. Potential Chinese medicines for the treatment of UC include Curcumae Longae Rhizoma, Coptidis Rhizoma, Scutellariae Radix, Dendrobii Caulis, Sanguisorbae Radix, Phellodendri Chinensis Cortex, Bletillae Rhizoma and Atractylodis Rhizoma. The analysis of DEGs and core genes could promote our understanding on pathogenesis of UC. This study provides potential gene targets and research ideas for the development of new drugs of Chinese medicine intervention for UC.

Keywords: Chinese medicine prediction; Kyoto encyclopedia of genes and genomes(KEGG); differentially expressed genes; gene ontology(GO); hub genes; protein-protein interaction; ulcerative colitis.

Inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease (CD), are chronic debilitating disorders of unknown etiology. Over 200 genetic risk loci are associated with IBD, highlighting a key role for immunological and epithelial barrier functions. Environmental factors account for the growing incidence of IBD, and microbiota are considered as an important contributor. Microbiota dysbiosis can lead to a loss of tolerogenic immune effects and initiate or exacerbate inflammation. We aimed to study colonic mucosal microbiota and the expression of selected host genes in pediatric UC. We used high-throughput 16S rDNA sequencing to profile microbiota in colonic biopsies of pediatric UC patients (n = 26) and non-IBD controls (n = 27). The expression of 13 genes, including five for antimicrobial peptides, in parallel biopsies was assessed with qRT-PCR. The composition of microbiota between UC and non-IBD differed significantly (PCoA, p = 0.001). UC children had a decrease in Bacteroidetes and an increase in several family-level taxa including Peptostreptococcaceae and Enterobacteriaceae, which correlated negatively with the expression of antimicrobial peptides REG3G and DEFB1, respectively. Enterobacteriaceae correlated positively with the expression siderophore binding protein LCN2 and Betaproteobacteria negatively with DEFB4A expression. The results indicate that reciprocal interaction of epithelial microbiota and defense mechanisms play a role in UC.

Keywords: gene expression; host-microbe cross-talk; inflammatory bowel disease; microbiota; ulcerative colitis.

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Study question: Which transcriptomic alterations in mid-luteal endometrial scratch biopsies, taken prior to the assisted reproductive treatment (ART) treatment cycle are associated with unsuccessful pregnancy?

Summary answer: Dysregulated interleukin-17 (IL-17) pathway components are demonstrated in women who fail to become pregnant after ART.

What is known already: Implantation failure is now recognised as a critical factor in unexplained infertility and may be an important component of failed ART.

Study design, size, duration: Using a prospective longitudinal study design, 29 nulliparous women with unexplained infertility undergoing ART were recruited between October 2016 and February 2018. Mid-luteal stage endometrium and matched serum samples were collected, and patients underwent a single embryo transfer in the subsequent cycle. RNA-seq analysis of endometrial biopsies was performed on the discovery cohort (n = 20).

Participants/materials, setting, methods: Gene set enrichment analysis of the differentially expressed genes (DEGs) was performed. Endometrium and serum were then prepared for IL-17A analysis by ELISA.

Main results and the role of chance: There were 204 differentially expressed protein-coding genes identified in tissue from women who became pregnant (n = 9) compared with tissue from women who failed to become pregnant (n = 11) (false discovery rate; P < 0.05). Of the 204 DEGs, 166 were decreased while 38 were increased in the pregnant compared to the non-pregnant groups. Gene set enrichment analysis of the DEGs identified an over-representation of IL-17 and Pl3K-Akt signalling pathways. All the DEGs within the IL-17 signalling pathway (MMP3, MMP1, IL1β, LCN2, S100A9 and FOSL1) demonstrated decreased expression in the pregnant group. Serum IL-17 protein levels were increased in the non-pregnant discovery cohort (n = 11) and these findings were confirmed a validation cohort (n = 9).

Limitations, reasons for caution: Limitations of our study include the cohort size and the lack of aneuploidy data for the embryos; however, all embryos transferred were single good or top-quality blastocysts.

Wider implications of the findings: These findings demonstrate dysregulated IL-17 pathway components in women who fail to become pregnant after ART. Elevated serum levels of the pro-inflammatory cytokine IL-17 may predict failure of ART in women with unexplained infertility. Future trials of anti-IL-17 therapies in this cohort warrant further investigation.

Study funding/competing interest(s): Funding from the UCD Wellcome Institutional Strategic Support Fund, which was financed jointly by University College Dublin and the SFI-HRB-Wellcome Biomedical Research Partnership (ref 204844/Z/16/Z), is acknowledged. The authors have no competing interests.

Trial registration number: NA.

Keywords: RNA-seq; assisted reproductive treatment; endometrium; interleukin-17; unexplained infertility.

BACKGROUND

The amount of urinary glycoprotein lipocalin 2 (LCN2) has been known to increase after kidney injury because of failed reabsorption by the proximal tubules or direct secretion from injured tissues. However, the relationship between urinary tract obstruction and the isoform diversity of LCN2 has not been examined.

METHODS

The urinary levels of LCN2 isoforms were examined in male mice after an intraperitoneal injection of lipopolysaccharide (LPS) or in a mouse model of unilateral ureter obstruction (UUO). The LCN2 levels in sera, bladder urine, renal pelvic urine, and tissue samples were also analyzed. Endo- and exoglycosidases were used to investigate the different N-glycan patterns of LCN2.

RESULTS

Two isoforms of urinary LCN2 with different molecular weights were identified in an immunoblotting analysis, and the levels of both isoforms were increased 6 h after LPS administration. The primary LCN2 isoform was the lower molecular weight 22-kDa isoform, which was detected in the serum, urine, liver and kidney. In contrast, the 24-kDa LCN2 isoform was detected only in urine. In the UUO experiments, the levels of the 24-kDa LCN2 were increased in the bladder urine but not in the urine accumulated in the renal pelvis due to UUO. The 22-kDa LCN2 was identified in the renal pelvic urine from UUO mice. The peptide-N glycosidase F digestion of the two urinary LCN2 isoforms generated a single protein. Moreover, the two urinary LCN2 proteins were sensitive to neuraminidase and resistant to endoglycosidase H (Endo H). The LCN2 in the serum, lung and kidney was resistant to Endo H, as observed in urine, whereas the LCN2 in the liver and the ureter were degraded by this enzyme.

CONCLUSIONS

These results suggest that the difference in the molecular weights of the LCN2 proteins was due to their N-glycan structure. The high molecular weight LCN2 in urine could be detected after the inflammatory response to LPS and UUO. Furthermore, the sensitivity to Endo H identified the presence of two types of carbohydrate moieties, depending on the tissue in which the LCN2 was produced. These findings are useful for widening the clinical applicability of urinary LCN2 analyses.

Functionalized giant unilamellar vesicles (GUVs) containing a fluorescence dye Rhodamine 6G is proposed as a marker in sandwich-type immunoassay for bovine serum albumin (BSA) and lipocalin-2 (LCN2). The GUVs were prepared by the electroformation method and functionalized with anti-BSA antibody and anti-LCN2 antibody, respectively. The purification of antibody-modified GUVs was achieved by conventional centrifugation and a washing step in a flow system. To antigen on an antibody slip, antibody-modified GUVs were added as a marker and incubated. After wash-out of excess reagents and lysis of the bound GUVs with Triton X-100, the fluorescence image was captured. The fluorometric immunoassays for BSA and LCN2 exhibited lower detection limits of 4 and 80 fg ml(-)(1), respectively.

Testosterone deficiency causes fat deposition, particularly in visceral fat, and its replacement might reverse fat accumulation, however, the underlying mechanisms of such processes under diet-induced adiposity are largely unknown. To gain insights into the genome-wide role of androgen on visceral adipose tissue (VAT), RNA-Seq was used to investigate testosterone deficiency induced changes of VAT in miniature pigs fed a high-fat and high-cholesterol (HFC) diet among intact male pigs (IM), castrated male pigs (CM), and castrated male pigs with testosterone replacement (CMT) treatments. The results showed that testosterone deficiency significantly increased VAT deposition and serum leptin concentrations. Moreover, a total of 1732 differentially expressed genes (DEGs) were identified between any two groups. Compared with gene expression profiles in IM and CMT pigs, upregulated genes in CM pigs, i.e., LOC100520753 (CD68), LCN2, EMR1, S100A9, NCF1 (p47phox), and LEP, were mainly involved in inflammatory response, oxidation-reduction process, and lipid metabolic process, while downregulated genes in CM pigs, i.e., ABHD5, SPP1, and GAS6, were focused on cell differentiation and cell adhesion. Taken together, our study demonstrates that testosterone deficiency alters the expression of numerous genes involved in key biological processes of VAT accumulation under HFC diet and provides a novel genome-wide view on the role of androgen on VAT deposition under HFC diet, thus improving our understanding of the molecular mechanisms involved in VAT changes induced by testosterone deficiency.

Epithelial-mesenchymal transition (EMT) is considered to serve an important role in the metastatic/invasive ability of cancer cells, in the acquisition of drug resistance, and in metabolic reprogramming. In the present study, it was hypothesized that the Klotho gene is involved in the metastatic/invasive ability of lung cancer. We previously reported an association between Klotho expression and overall survival in patients with small cell lung cancer and large cell neuroendocrine cancer. We also found that Klotho expression was associated with EMT-related molecules in lung squamous cell carcinoma. The present study aimed to analyze the function of the Klotho gene and to elucidate its relevance to the regulation of the EMT. For this purpose, GFP-Klotho plasmids were transfected into lung adenocarcinoma cells (A549) and cell lines with stable expression (A549/KL-1 and A549/KL-2) were established. A549/KL-1 cells expressed higher levels of Klotho protein by western blot analysis compared with A549/KL-2 cells. In western blotting of A549 and A549/KL-1 cells, the expression of the mesenchymal marker N-cadherin was found to be completely inhibited in A549/KL-1 cells suggesting that Klotho expression may regulate the EMT in cancer cells via the inhibition of N-cadherin. The results of the sensitivity tests demonstrated that A549/KL-1 cells were significantly more sensitive to pemetrexed compared with A549 cells (IC₅₀ A549/KL-1 vs. A549 cells, 0.1 µM vs. 0.7 µM). The results of the microarray analysis demonstrated that a very high level of lipocalin-2 (LCN2) expression was induced in the A549/KL-1 cells. Klotho overexpression completely suppressed the expression of mesenchymal markers, such as N-cadherin and Snail1 (Snail). The results of the present study suggested that there may be a new mechanism of action for the antitumor effects of pemetrexed, namely, LCN2-mediated modulation of N-cadherin expression. Klotho expression during cancer treatment has great potential as a predictor for efficacy of pemetrexed and as a factor in the selection of personalized medicine for postoperative adjuvant chemotherapy.

Keywords: Klotho; epithelial-mesenchymal transition; lipocalin-2; lung cancer; pemetrexed.

Purpose: Combined axitinib/pembrolizumab is approved for advanced renal cell carcinoma (aRCC). This exploratory analysis examined associations between angiogenic and immune-related biomarkers and outcomes following axitinib/pembrolizumab treatment.

Experimental design: Prospectively defined retrospective correlative exploratory analyses tested biospecimens from 52 treatment-naïve patients receiving axitinib and pembrolizumab (starting doses 5 mg twice-daily and 2 mg/kg every 3 weeks). Tumor tissue, serum, and whole blood samples were collected at baseline, and at cycle 2, day 1 (C2D1), and end of treatment (EOT) for blood-based samples. Clinical outcomes were objective response rate (ORR) and progression-free survival (PFS).

Results: Higher baseline tumor levels of CD8 showed a trend toward longer PFS (hazard ratio [HR] 0.4; P = 0.091). Higher baseline serum levels of CXCL10 (P = 0.0197) and CEACAM1 (P = 0.085) showed a trend toward better ORR and longer PFS, respectively. Patients for whom IL-6 was not detected at baseline had longer PFS vs patients for whom it was detected (HR 0.4; P = 0.028). At C2D1 and/or EOT, mainly immune-related biomarkers showed any association with better outcomes. The genes CA9 (P = 0.084), HIF1A (P = 0.064), and IFNG (P = 0.073) showed trending associations with ORR, and AKT3 (P = 0.0145), DDX58 (P = 0.0726), GZMA (P = 0.0666), LCN2 (NGAL; P = 0.0267), and PTPN11 (P = 0.0287) with PFS.

Conclusions: With combined axitinib/pembrolizumab treatment in patients with aRCC, mostly immune-related biomarkers are associated with better treatment outcomes. This exploratory analysis has identified some candidate biomarkers to consider in future prospective testing. ClinicalTrials.gov identifier: NCT02133742.

OBJECTIVE

HIV lipodystrophy is characterised by abnormal adipose tissue distribution and metabolism, as a result of altered adipocyte function and gene expression. The protease inhibitor ritonavir is associated with the development of lipodystrophy. Quantifying changes in adipogenic gene expression in the presence of ritonavir may help to identify therapeutic targets for HIV lipodystrophy.

METHODS

Affymetrix Mouse Genome 430 2.0 oligonucleotide microarray was used to investigate gene expression in 3T3-L1 adipocytes treated with 20 µmol/l ritonavir or vehicle control (ethanol). Pparg, Adipoq, Retn and Il6 expression were validated by real time RT-PCR. Transcriptional signalling through PPAR-γ was investigated using a DNA-binding ELISA. Changes in adipocyte function were investigated through secreted adiponectin quantification using ELISA and Oil Red O staining for triglyceride storage.

RESULTS

Expression of 389 genes was altered by more than 5-fold in the presence of ritonavir (all P < 0.001). Gene ontology analysis revealed down-regulation of genes responsible for adipocyte triglyceride accumulation including complement factor D (Cfd; 238.42-fold), Cidec (73.75-fold) and Pparg (5.63-fold). Glucose transport genes were also down-regulated including Adipoq (24.42-fold) and Glut4 (13.36-fold), while Il6 was up-regulated (10.39-fold). PPAR-γ regulatory genes Cebpa (11.33-fold) and liver-X-receptor α (Nr1h3) were down-regulated. Changes in Pparg, Adipoq and Il6 were confirmed by RT-PCR. PPAR-γ binding to its nuclear consensus site, adiponectin secretion and triglyceride accumulation were all reduced by ritonavir.

CONCLUSIONS

Ritonavir had a significant effect on expression of genes involved in adipocyte differentiation, lipid accumulation and glucose metabolism. Down-regulation of Pparg may be mediated by changes in Cebpa, Lcn2 and Nr1h3.

Acute ischemic stroke (AIS) is common in patients with cancer, and mounting clinical evidence suggests that it may shorten the survival of cancer patients. But how stroke affects the progression of cancer remains unclear. We inoculated B16 tumor cells (2 × 10⁵) subcutaneously before distal middle cerebral artery occlusion (dMCAO) or sham surgery in C57BL/6 mice and found that compared to sham operated mice, dMCAO mice developed significantly increased tumor volume and were accompanied by lower survival rate. To explore the underlying mechanism, we performed RNA-sequencing analysis of the tumor tissue from mice with or without stroke and found prominent upregulation of lipocalin 2 (LCN2) in the tumor from stroke mice compared to those from sham mice. Using quantitative reverse transcription-PCR, we confirmed increased mRNA expression of LCN2 as well as anti-inflammatory cytokines-Arg1, IL-10, and decreased mRNA level of pro-inflammatory cytokines-IL-6, IL-23 in the tumor of cancer-bearing stroke mice. Both immunofluorescence staining and flow cytometry analysis revealed that increased expression of LCN2 was mainly derived from the polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) in the tumor. We also found that stroke reduced the PMN-MDSCs in the peripheral blood, but increased PMN-MDSCs in the tumor of the cancer-bearing mice after stroke. In conclusion, cerebral ischemic stroke may exacerbate cancer progression by increasing LCN2 expression in PMN-MDSCs, which turns out to be a promising therapeutic target to suppress cancer progression after ischemic stroke.

An innovative signal-transduction tag based on cross-linked urease nanoparticles (CLENP) was designed for the development of a pH meter-based immunoassay of lipocalin-2 (LCN2). The CLENP was synthesized with a typical desolvation method using ethanol as desolvation agent, followed by functionalization with polyaspartic acid. The carboxylated CLENP were used as the signal-generation tags for the labelling of secondary antibodies via the carbodiimide coupling. Upon target LCN2 introduction, a sandwich-type immune reaction was performed between capture antibody-coated plate and the labeled secondary antibody on the CLENP. The conjugated CLENP in the microplate hydrolyzed urea into ammonia (NH₄⁺) and carbonate (CO₃^2-), resulting in the pH change of solution, which was determined with a handheld pH meter. The pH variation was proportional to target concentration in the sample. By monitoring the pH variation of the urea solution, the level of LCN2 at a concentration as low as 5.2 pg mL^-1 was evaluated. The pH meter-based electrochemical immunoassay can be utilized for mass production of miniaturized lab-on-a-chip devices with handheld pH meter, thereby opening new opportunities for protein diagnostics and biosecurity. Graphical abstract An innovative signal-transduction tag based on cross-linked urease nanoparticles was designed for high-efficiency immunoassay of lipocalin-2 with pH meter readout.

Keywords: Cross-linkage urease nanoparticles; Electrochemical immunoassay; Enzymatic hydrolysis; Signal-transduction tags; pH measurement.

The lack of Lipocalin (LCN2) provokes overwhelming endoplasmic reticulum (ER) stress responses in vitro and in acute toxic liver injury models, resulting in hepatocyte apoptosis. LCN2 is an acute phase protein produced in hepatocytes in response to acute liver injuries. In line with these findings we investigated ER stress responses of Lcn2^-/- mice in chronic ER stress using a long-term repetitive carbon tetrachloride (CCl₄) injection model. We found chronic CCl₄ application to enhance ER stress and unfolded protein responses (UPR), including phosphorylation of eukaryotic initiation factor 2α (eIF2α), increased expression of binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (GRP94). IRE1α/TRAF2/JNK signaling enhanced mitochondrial apoptotic pathways, and showed slightly higher in Lcn2^-/- mice compared to the wild type counterparts, leading to increased hepatocyte apoptosis well evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Hepatocyte injuries were confirmed by significant high serum alanine transaminase (ALT) levels in CCl₄-treated Lcn2^-/- mice. Lcn2^-/- mice furthermore developed mild hepatic steatosis, supporting our finding that ER stress promotes lipogenesis. In a previous report we demonstrated that the pharmacological agent tunicamycin (TM) induced ER stress through altered protein glycosylation and induced high amounts of C/EBP-homologous protein (CHOP), resulting in hepatocyte apoptosis. We compared TM-induced ER stress in wild type, Lcn2^-/-, and Chop null (Chop^-/-) primary hepatocytes and found Chop^-/- hepatocytes to attenuate ER stress responses and resist ER stress-induced hepatocyte apoptosis through canonical eIF2α/GADD34 signaling, inhibiting protein synthesis. Unexpectedly, in later stages of TM incubation, Chop^-/- hepatocytes resumed activation of IRE1α/JNK/c-Jun and p38/ATF2 signaling, leading to late hepatocyte apoptosis. This interesting observation indicates Chop^-/- mice to be unable to absolutely prevent all types of liver injury, while LCN2 protects the hepatocytes by maintaining homeostasis under ER stress conditions.

Keywords: CHOP; ER stress; LCN2; UPR; apoptosis; hepatocyte.

<AbstractText>Optimizing outcomes in respiratory syncytial virus (RSV) pneumonia requires accurate diagnosis and determination of severity that, in resource-limited settings, is often based on clinical assessment alone. We describe host inflammatory biomarkers and clinical outcomes among children hospitalized with RSV lower respiratory tract infection (LRTI) in Uganda and controls with rhinovirus and pneumococcal pneumonia.</AbstractText><AbstractText>58 children hospitalized with LRTI were included. We compared 37 patients with RSV, 10 control patients with rhinovirus and 11 control patients with suspected pneumococcal pneumonia.</AbstractText><AbstractText>Patients in the RSV group had significantly lower levels of C-reactive protein (CRP) and chitinase-3-like protein 1 (CHI3L1) than the pneumococcal pneumonia group (P < 0.05 for both). Among children with RSV, higher admission levels of CRP predicted prolonged time to resolution of tachypnea, tachycardia and fever. Higher levels of CHI3L1 were associated with higher composite clinical severity scores and predicted prolonged time to resolution of tachypnea and tachycardia, time to wean oxygen and time to sit. Higher levels of lipocalin-2 (<em>LCN2</em>) predicted prolonged time to resolution of tachypnea, tachycardia and time to feed. Higher admission levels of all 3 biomarkers were predictive of a higher total volume of oxygen administered during hospitalization (P < 0.05 for all comparisons). Of note, CHI3L1 and <em>LCN2</em> appeared to predict clinical outcomes more accurately than CRP, the inflammatory biomarker most widely used in clinical practice.</AbstractText><AbstractText>Our findings suggest that CHI3L1 and <em>LCN2</em> may be clinically informative biomarkers in childhood RSV LRTI in low-resource settings.</AbstractText>

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), the first immune checkpoint to be targeted clinically, has provided an effective treatment option for various malignancies. However, the clinical advantages associated with CTLA-4 inhibitors can be offset by the potentially severe immune-related adverse events (IRAEs), including autoimmune thyroid dysfunction. To investigate the candidate genes and signaling pathways involving in autoimmune thyroid dysfunction related to anti-CTLA-4 therapy, integrated differentially expressed genes (DEGs) were extracted from the intersection of genes from Gene Expression Omnibus (GEO) datasets and text mining. The functional enrichment was performed by gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Protein-protein interaction (PPI) network, module enrichment, and hub gene identification were constructed and visualized by the online Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape software. A total of 22 and 17 integrated human DEGs in hypothyroidism and hyperthyroidism group related to anti-CTLA-4 therapy were identified, respectively. Functional enrichment analysis revealed 24 GO terms and 1 KEGG pathways in the hypothyroid group and 21 GO terms and 2 KEGG pathways in the hyperthyroid group. After PPI network construction, the top five hub genes associated with hypothyroidism were extracted, including ALB, MAPK1, SPP1, PPARG, and MIF, whereas those associated with hyperthyroidism were ALB, FCGR2B, CD44, LCN2, and CD74. The identification of the candidate key genes and enriched signaling pathways provides potential biomarkers for autoimmune thyroid dysfunction related to anti-CTLA-4 therapy and might contribute to the future diagnosis and management of IRAEs for cancer patients.

Keywords: Autoimmune thyroid dysfunction; CTLA-4; Differentially expressed genes; Immune checkpoint blockade; Signaling pathway.

Low-intensity pulsed ultrasound (LIPUS) has also been reported to improve behavioral functions in Parkinson's disease (PD) animal models; however, the effect of LIPUS stimulation on the neurotrophic factors and neuroinflammation has not yet been addressed. PD rat model was built by injection of 6-hydroxydopamine (6-OHDA) in 2 sites in the right striatum. The levels of neurotrophic factors and lipocalin-2 (LCN2)-induced neuroinflammation were quantified using a western blot. Rotational test and cylinder test were conducted biweekly for 8 weeks. When the 6-OHDA + LIPUS and 6-OHDA groups were compared, the locomotor function of the 6-OHDA + LIPUS rats was significantly improved. After LIPUS stimulation, the tyrosine hydroxylase staining density was significantly increased in the striatum and substantia nigra pars compacta (SNpc) of lesioned rats. Unilateral LIPUS stimulation did not increase brain-derived neurotrophic factor in the striatum and SNpc of lesioned rats. In contrast, unilateral LIPUS stimulation increased glial cell line-derived neurotrophic factor (GDNF) protein 1.98-fold unilaterally in the SNpc. Additionally, LCN2-induced neuroinflammation can be attenuated following LIPUS stimulation. Our data indicated that LIPUS stimulation may be a potential therapeutic tool against PD via enhancement of GDNF level and inhibition of inflammatory responses in the SNpc of the brain.

Keywords: 6-OHDA; GDNF; Parkinson’s disease; neuroinflammation; ultrasound.

The exact mechanisms associated with secondary brain damage following traumatic brain injury (TBI) remain unclear; therefore, identifying the critical molecular mechanisms involved in TBI is essential. The mRNA expression microarray GSE2871 was downloaded from the Gene Expression Omnibus (GEO) repository. GSE2871 comprises a total of 31 cerebral cortex samples, including two post-TBI time points. The microarray features eight control and seven TBI samples, from 4 hours post-TBI, and eight control and eight TBI samples from 24 hours post-TBI. In this bioinformatics-based study, 109 and 66 differentially expressed genes (DEGs) were identified in a Sprague-Dawley (SD) rat TBI model, 4 and 24 hours post-TBI, respectively. Functional enrichment analysis showed that the identified DEGs were significantly enriched in several terms, such as positive regulation of nuclear factor-κB transcription factor activity, mitogen-activated protein kinase signaling pathway, negative regulation of apoptotic process, and tumor necrosis factor signaling pathway. Moreover, the hub genes with high connectivity degrees were primarily related to inflammatory mediators. To validate the top five hub genes, a rat model of TBI was established using the weight-drop method, and real-time quantitative polymerase chain reaction analysis of the cerebral cortex was performed. The results showed that compared with control rats, Tnf-α, c-Myc, Spp1, Cxcl10, Ptprc, Egf, Mmp9, and Lcn2 were upregulated, and Fn1 was downregulated in TBI rats. Among these hub genes, Fn1, c-Myc, and Ptprc may represent novel biomarkers or therapeutic targets for TBI. These identified pathways and key genes may provide insights into the molecular mechanisms of TBI and provide potential treatment targets for patients with TBI. This study was approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Nanchang University, China (approval No. 003) in January 2016.

Keywords: DEGs; Gene Ontology; Kyoto Encyclopedia of Genes and Genomes; bioinformatics; differentially expressed genes; hub genes; inflammation; molecular mechanism; traumatic brain injury.

Previous studies have shown that mutations in the tumor suppressor gene von Hippel-Lindau (VHL) can result in the overproduction of reactive oxygen species (ROS) and chronic inflammation and are a significant predisposing factor for the development of clear-cell renal cell carcinoma (ccRCC). To study VHL's role in ccRCC formation, we previously developed a novel conditional knockout mouse model that mimicked the features of kidney inflammation and fibrosis that lead to cyst formation and hyperplasia. However, due to VHL's complex cellular functions, the mechanism of this phenomenon remains unclear. Here, we used the HK-2 cells and mouse primary renal tubule cells (mRTCs) carrying VHL mutations as models to study the effects and underlying molecular mechanisms of ROS accumulation. We also studied the role of lipocalin 2 (LCN2) in regulating macrophage recruitment by HK-2 cells. We measured the level of ROS in HK-2 cells in the presence or absence of LCN2 knockdown and found that the VHL mutation caused ROS overproduction, but an LCN2 knockdown could attenuate the process. VHL was also found to mediate the in vitro and in vivo expression and secretion of LCN2. Thus, VHL likely affects ROS production in an LCN2-dependent manner. Our findings also suggest that LCN2 sensitizes the inflammatory response of HK-2 cells and the chemotactic abilities of macrophage RAW264.7 cells. By demonstrating that the loss of function of von Hippel-Lindau triggers lipocalin 2-dependent inflammatory responses in cultured and primary renal tubular cells, our results offer novel insights into a potential therapeutic approach for interfering with the development of ccRCC.