We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1.

Clinical data strongly indicate that acute kidney injury (AKI) is a critical complication in alcoholic hepatitis, an acute-on-chronic form of liver failure in patients with advanced alcoholic fibrosis. Development of targeted therapies for AKI in this setting is hampered by the lack of an animal model. To enable research into molecular drivers and novel therapies for fibrosis- and alcohol-associated AKI, we aimed to combine carbon tetrachloride (CCl4)-induced fibrosis with chronic intra-gastric alcohol feeding. Male C57BL/6J mice were administered a low dose of CCl4 (0.2ml/kg 2× week/6weeks) followed by alcohol intragastrically (up to 25g/kg/day for 3weeks) and with continued CCl4. We observed that combined treatment with CCl4 and alcohol resulted in severe liver injury, more pronounced than using each treatment alone. Importantly, severe kidney injury was evident only in the combined treatment group. This mouse model reproduced distinct pathological features consistent with AKI in human alcoholic hepatitis. Transcriptomic analysis of kidneys revealed profound effects in the combined treatment group, with enrichment for damage-associated pathways, such as apoptosis, inflammation, immune-response and hypoxia. Interestingly, Havcr1 and Lcn2, biomarkers of AKI, were markedly up-regulated. Overall, this study established a novel mouse model of fibrosis- and alcohol-associated AKI and identified key mechanistic pathways.

Novel biomarkers are being investigated to identify patients with kidney disease. We measured a panel of 13 urinary biomarkers in participants from the Offspring Cohort of the Framingham Heart Study. Using an Affymetrix chip with imputation to 2.5 M single-nucleotide polymorphisms (SNPs), we conducted a GWAS of these biomarkers (n=2640) followed by exonic sequencing and genotyping. Functional studies in zebrafish were used to investigate histologic correlation with renal function. Across all 13 biomarkers, there were 97 significant SNPs at three loci. Lead SNPs at each locus were rs6555820 (P=6.7×10(-49); minor allele frequency [MAF]=0.49) in HAVCR1 (associated with kidney injury molecule-1), rs7565788 (P=2.15×10(-16); MAF=0.22) in LRP2 (associated with trefoil factor 3 [TFF3]), and rs11048230 (P=4.77×10(-8); MAF=0.10) in an intergenic region near RASSF8 (associated with vascular endothelial growth factor). Validation in the CKDGen Consortium (n=67,093) showed that only rs7565788 at LRP2, which encodes megalin, was associated with eGFR (P=0.003). Sequencing of exons 16-72 of LRP2 in 200 unrelated individuals at extremes of urinary TFF3 levels identified 197 variants (152 rare; MAF<0.05), 31 of which (27 rare) were nonsynonymous. In aggregate testing, rare variants were associated with urinary TFF3 levels (P=0.003), and the lead GWAS signal was not explained by these variants. Knockdown of LRP2 in zebrafish did not alter the renal phenotype in static or kidney injury models. In conclusion, this study revealed common variants associated with urinary levels of TFF3, kidney injury molecule-1, and vascular endothelial growth factor and identified a cluster of rare variants independently associated with TFF3.

Evaluation of thrombogenicity is a critical component in the preclinical testing and development of blood pumps. Left ventricular assist devices (LVADs), because of their device routing, can produce thromboembolic showers to the kidney resulting in renal cortical ischemia or infarctions. Although postmortem evaluation of renal pathology can confirm ischemic events and infarctions, there are no validated and highly sensitive real-time measures of renal ischemia in the preclinical models. In this article, we report the evaluation of urinary biomarkers of ischemic tubular damage in a lamb preclinical LVAD model. We found that urinary excretion of glutathione-S-transferase-π, heat shock protein 1B, and hepatitis A virus cellular receptor 1 homologue precursor (HAVCR1/kidney injury molecule 1) were upregulated in toxic ischemic renal injury as well as in the immediate postoperative period in an LVAD-implanted lamb. These markers were consistent with both gross and histologic pathology, and proved far more sensitive for renal injury than serum blood urea nitrogen or creatinine concentrations.

Urinary biomarkers are used increasingly for sensitive prediction of kidney injury in preclinical and clinical studies. Given the frequent requirement of anesthesia in various animal models of disease, it is important to define the effects of anesthesia on kidney injury biomarkers to guide the appropriate selection of anesthetic agents and to avoid potential confounders in the interpretation of data. Therefore, we performed a prospective study using male C57BL/6J mice (n = 45) exposed to a single anesthetic episode to determine the effects several common anesthesia regimens on the urinary excretion of 2 commonly used kidney injury biomarkers: hepatitis A virus cellular receptor 1 (HAVCR1, also known as KIM1) and lipocalin 2 (LCN2, also known as NGAL). We evaluated 3 injectable regimens (ketamine-xylazine, tiletamine-zolazepam, and pentobarbital) and 2 inhalational agents (isoflurane and sevoflurane). Concentrations of HAVCR1 and LCN2 in urine collected at various time points after anesthesia were measured by using ELISA. Administration of ketamine-xylazine resulted in a significant increase in HAVCR1 levels at 6 h after anesthesia but a decrease in LCN2 levels compared with baseline. LCN2 levels steadily increased over the first 24 h after inhalant anesthesia, with a significant increase at 24 h after sevoflurane. These results suggest that injectable anesthesia had early effects on HAVCR1 and LCN2 levels, whereas inhalational agents increased these biomarkers over prolonged time.

Background: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease, affecting ∼30% of the rapidly growing diabetic population, and strongly associated with cardiovascular risk. Despite this, the molecular mechanisms of disease remain unknown.

Methods: RNA sequencing (RNAseq) was performed on paired, micro-dissected glomerular and tubulointerstitial tissue from patients diagnosed with DN [n = 19, 15 males, median (range) age: 61 (30-85) years, chronic kidney disease stages 1-4] and living kidney donors [n = 20, 12 males, median (range) age: 56 (30-70) years].

Results: Principal component analysis showed a clear separation between glomeruli and tubulointerstitium transcriptomes. Differential expression analysis identified 1550 and 4530 differentially expressed genes, respectively (adjusted P < 0.01). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted activation of inflammation and extracellular matrix (ECM) organization pathways in glomeruli, and immune and apoptosis pathways in tubulointerstitium of DN patients. Specific gene modules were associated with renal function in weighted gene co-expression network analysis. Increased messengerRNA (mRNA) expression of renal damage markers lipocalin 2 (LCN) and hepatitis A virus cellular receptor1 (HAVCR1) in the tubulointerstitial fraction was observed alongside higher urinary concentrations of the corresponding proteins neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in DN patients.

Conclusions: Here we present the first RNAseq experiment performed on paired glomerular and tubulointerstitial samples from DN patients. We show that prominent disease-specific changes occur in both compartments, including relevant cellular processes such as reorganization of ECM and inflammation (glomeruli) as well as apoptosis (tubulointerstitium). The results emphasize the potential of utilizing high-throughput transcriptomics to decipher disease pathways and treatment targets in this high-risk patient population.

Keywords: diabetic  nephropathy; chronic kidney disease; kidney biopsy; pathway analysis; transcriptomics.

The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV.IMPORTANCE HAVCR1, an HAV receptor, is expressed in different cell types, including regulatory immune cells and antigen-presenting cells. How HAV evades the immune response during a long incubation period of up to 4 weeks and the mechanism by which the subsequent necroinflammatory process clears the infection remain a puzzle that most likely involves the HAV-HAVCR1 interaction. Based on negative data, a recent paper from the S. M. Lemon and W. Maury laboratories (A. Das, A. Hirai-Yuki, O. Gonzalez-Lopez, B. Rhein, S. Moller-Tank, R. Brouillette, L. Hensley, I. Misumi, W. Lovell, J. M. Cullen, J. K. Whitmire, W. Maury, and S. M. Lemon, mBio 8:e00969-17, 2017, https://doi.org/10.1128/mBio.00969-17) suggested that HAVCR1 is not a functional HAV receptor, nor it is it required for HAV infection. However, our data, based on regain of the HAV receptor function in HAVCR1 knockout cells transfected with HAVCR1 cDNA, disagree with their findings. Our positive data show conclusively that HAVCR1 is indeed a functional HAV receptor and lays the ground for the identification of alternative HAV receptors and how they interact with HAVCR1 in cell entry and the pathogenesis of HAV.

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.

Acute kidney injury (AKI) caused by drug and toxicant ingestion is a serious clinical condition associated with high mortality rates. We currently lack detailed knowledge of the underlying molecular mechanisms and biological networks associated with AKI. In this study, we carried out gene co-expression analyses using DrugMatrix-a large toxicogenomics database with gene expression data from rats exposed to diverse chemicals-and identified gene modules associated with kidney injury to probe the molecular-level details of this disease.

We generated a comprehensive set of gene co-expression modules by using the Iterative Signature Algorithm and found distinct clusters of modules that shared genes and were associated with similar chemical exposure conditions. We identified two module clusters that showed specificity for kidney injury in that they 1) were activated by chemical exposures causing kidney injury, 2) were not activated by other chemical exposures, and 3) contained known AKI-relevant genes such as Havcr1, Clu, and Tff3. We used the genes in these AKI-relevant module clusters to develop a signature of 30 genes that could assess the potential of a chemical to cause kidney injury well before injury actually occurs. We integrated AKI-relevant module cluster genes with protein-protein interaction networks and identified the involvement of immunoproteasomes in AKI. To identify biological networks and processes linked to Havcr1, we determined genes within the modules that frequently co-express with Havcr1, including Cd44, Plk2, Mdm2, Hnmt, Macrod1, and Gtpbp4. We verified this procedure by showing that randomized data did not identify Havcr1 co-expression genes and that excluding up to 10 % of the data caused only minimal degradation of the gene set. Finally, by using an external dataset from a rat kidney ischemic study, we showed that the frequently co-expressed genes of Havcr1 behaved similarly in a model of non-chemically induced kidney injury.

Our study demonstrated that co-expression modules and co-expressed genes contain rich information for generating novel biomarker hypotheses and constructing mechanism-based molecular networks associated with kidney injury.

Mounting evidence suggests that lncRNA regulates many important diseases. However, the biological role of most lncRNAs in gastric cancer (GC) remain unclear. In this paper, we determined differential expression of lncRNAs and predicted ceRNA networks in the GC database by bioinformatics analysis and validated in GC cells. The effect of lncRNA AL139002.1 on GC cells biological function was assessed by flow cytometry, CCK-8, colony formation, wound healing assay, transwell, western blot, and qRT-PCR. And the relationship of lncRNA AL139002.1 or HAVCR1 with miR-490-3p was verified by luciferase reporter assay. The results showed that lncRNA AL139002.1 was highly expressed in GC cells and lncRNA AL139002.1 knockdown induced apoptosis, while suppressed cell proliferation, migration, invasion, and EMT. Functional examining indicated that lncRNA AL139002.1 regulated HAVCR1 expression by competitively binding miR-490-3p. In addition, lncRNA AL139002.1/miR-490-3p/HAVCR1 regulated EMT and metastasis through MEK/ERK signaling. In conclusion, lncRNA AL139002.1 was highly expressed in GC cells, and lncRNA AL139002.1/miR-490-3p/HAVCR1 functioned critically in GC by regulating MEK/ERK signaling. Our findings demonstrated that lncRNA AL139002.1 served as a potential therapeutic and anti-metastatic biotarget for GC.

Keywords: Gastric cancer; havcr1; lncrna al139002.1; miR-490-3p.

OBJECTIVE

HAVCR1 protein is the cellular receptor for hepatitis A virus (HAV). Genetic polymorphism in this gene may alter the outcome of HAV infection. In a previous study, a 6-amino acid insertion (157insMTTTVP) in HAVCR1 gene was associated with more severe disease. We decided to investigate this association further.

METHODS

We sequenced exon 4 of the HAVCR1 gene in patients with clinical hepatitis A attending our institution, and a group of healthy controls in a disease-endemic setting in India. Frequencies of different haplotypes of a genomic region with two overlapping insertion-deletion polymorphisms (indels; rs141023871 and rs139041445) were compared between patients and controls, as well as between patients with and without a severe form of disease (liver failure).

RESULTS

The gene had three haplotypes in the region of interest - a short form, an intermediate-form with a 5-amino acid 157insMTTVP insertion and a long-form with a 6-amino acid 157insMTTTVP insertion. The allele frequency (29/150 [19%] vs. 43/146 [29%]; p = ns) and haplotype frequency (29/75 [39%] vs. 39/73 [53%]; p = ns) of the 157insMTTTVP variant were similar in hepatitis A patients and healthy controls (30%). Further, the allele frequency (12/58 [21%] vs. 17/92 [18%]; p = ns) and haplotype frequency (12/29 [41%] vs.17/46 [37%]; p = ns) of the longest variant were also similar in patients with severe and mild disease.

CONCLUSIONS

In the study population, the 157insMTTTVP variant of HAVCR1 gene was not associated with more severe outcome of HAV infection. Further studies in other populations around the world are needed to assess the relation of this genetic variation with disease outcome.

Metastasis is present in approximately 30% of patients diagnosed with renal cell carcinoma (RCC) and is associated with a 5-year survival rate of < 15%. Kidney injury molecule 1 (KIM-1), encoded by the HAVCR1 gene, is a proximal tubule cell-surface glycoprotein and a biomarker for early detection of RCC, but its pathophysiological significance in RCC remains unclear. We generated human and murine RCC cell lines either expressing or lacking KIM-1, respectively, and compared their growth and metastatic properties using validated methods. Surprisingly, KIM-1 expression had no effect on cell proliferation or subcutaneous tumour growth in immune deficient (Rag1^-/-) Balb/c mice, but inhibited cell invasion and formation of lung metastasis in the same model. Further, we show that the inhibitory effect of KIM-1 on metastases was observed in both immune deficient and immune competent mice. Transcriptomic profiling identified the mRNA for the pro-metastatic GTPase, Rab27b, to be downregulated significantly in KIM-1 expressing human and murine RCC cells. Finally, analysis of The Cancer Genome Atlas (TCGA) data revealed that elevated HAVCR1 mRNA expression in the two most common types of RCC, clear cell and papillary RCC, tumours correlated with significantly improved overall patient survival. Our findings reveal a novel role for KIM-1 in inhibiting metastasis of RCC and suggests that tumour-associated KIM-1 expression may be a favourable prognostic factor.

Drug-induced nephrotoxicity is one of the most frequently observed effects in long-term pharmacotherapy. The effects of nephrotoxicity are commonly discovered later due to a lack of sensitivity in in vivo methods. Therefore, researchers have tried to develop in vitro alternative methods for early identification of toxicity. In this study, LLC-PK1 cells were exposed to gentamicin through MTT and trypan blue assay. Concentrations of 4 (low), 8 (medium) and 12 (high) mM, were used to evaluate differential gene expression. A panel of genes was selected based on gene expression changes. The search for sequences of mRNA encoding proteins previously associated with kidney damage was conducted in the databases of the National Center for Biotechnology Information (USA). RNA was extracted from the cells, and RT-qPCR was performed to evaluate differential expression profiles of the selected genes. Among the 11 analyzed genes, four proved to be differentially up-regulated in cells exposed to gentamicin: HAVcr1, caspase 3, ICAM-1 and EXOC6. According to this study's results, we suggest that these genes play an important role in the mechanism of in vitro nephrotoxicity caused by gentamicin and can be used as early in vitro biomarkers to identify nephrotoxicity when developing safer drugs.

Acute kidney injury (AKI) is a global public health concern associated with high morbidity, mortality, and health-care costs, and the therapeutic measures are still limited. This study aims to investigate crucial genes correlated with AKI, and their potential functions, which might contribute to a better understanding of AKI pathogenesis. The high-throughput data GSE52004 and GSE98622 were downloaded from Gene Expression Omnibus; four group sets were extracted and integrated. Differentially expressed genes (DEGs) in the four group sets were identified by limma package in R software. The overlapping DEGs among four group sets were further analyzed by the VennDiagram package, and their potential functions were analyzed by the GO and KEGG pathway enrichment analyses using the DAVID database. Furthermore, the protein-protein interaction (PPI) network was constructed by STRING, and the functional modules of the PPI network were filtered by MCODE and ClusterOne in Cytoscape. Hub genes of overlapping DEGs were identified by Cyto-Hubba and cytoNCA. The expression of 35 key genes was validated by quantitative real-time PCR (qRT-PCR). Western blot and immunofluorescence were performed to validate an important gene Egr1. A total of 722 overlapping DEGs were differentially expressed in at least three group sets. These genes mainly enriched in cell proliferation and fibroblast proliferation. Additionally, 5 significant modules and 21 hub genes, such as Havcr1, Krt20, Sox9, Egr1, Timp1, Serpine1, Edn1, and Apln were screened by analyzing the PPI networks. The 5 significant modules were mainly enriched in complement and coagulation cascades and Metabolic pathways, and the top 21 hub genes were mainly enriched in positive regulation of cell proliferation. Through validation, Krt20 were identified as the top 1 upregulated genes with a log2 (fold change) larger than 10 in all these 35 genes, and 21 genes were validated as significantly upregulated; Egr1 was validated as an upregulated gene in AKI in both RNA and protein level. In conclusion, by integrated analysis of different high-throughput data and validation by experiment, several crucial genes were identified in AKI, such as Havcr1, Krt20, Sox9, Egr1, Timp1, Serpine1, Edn1, and Apln. These genes were very important in the process of AKI, which could be further utilized to explore novel diagnostic and therapeutic strategies.

Keywords: acute kidney injury; differentially expressed gene; high-throughput sequencing; integrated analysis; interaction network analysis; microarray; validation.

Presence of tubulointerstitial fibrosis is predictive of progressive decline in kidney function, independent of its underlying cause. Injury to the renal microvasculature is a major factor in the progression of fibrosis and identification of factors that regulate endothelium in fibrosis is desirable as they might be candidate targets for treatment of kidney diseases. The current study investigates how loss of Angipoietin-1 (Angpt1), a ligand for endothelial tyrosine-kinase receptor Tek (also called Tie2), affects tubulointerstitial fibrosis and renal microvasculature. Inducible Angpt1 knockout mice were subjected to unilateral ureteral obstruction (UUO) to induce fibrosis, and kidneys were collected at different time points up to 10 days after obstruction. Staining for aSMA showed that Angpt1 deficient kidneys had significantly more fibrosis compared to wildtype mice 3, 6, and 10 days after UUO. Further investigation 3 days after UUO showed a significant increase of Col1a1 and vimentin in Angpt1 deficient mice, as well as increased gene expression of Tgfb1, Col1a1, Fn1, and CD44. Kidney injury molecule 1 (Kim1/Havcr1) was significantly more increased in Angpt1 deficient mice 1 and 3 days after UUO, suggesting a more severe injury early in the fibrotic process in Angpt1 deficient mice. Staining for endomucin showed that capillary rarefaction was evident 3 days after UUO and Angpt1 deficient mice had significantly less capillaries 6 and 10 days after UUO compared to UUO kidneys in wildtype mice. RNA sequencing revealed downregulation of several markers for endothelial cells 3 days after UUO, and that Angpt1 deficient mice had a further downregulation of Emcn, Plvap, Pecam1, Erg, and Tek. Our results suggest that loss of Angpt1 is central in capillary rarefaction and fibrogenesis and propose that manipulations to maintain Angpt1 levels may slow down fibrosis progression.

To compare lupus pathogenesis in disparate tissues, we analyzed gene expression profiles of human discoid lupus erythematosus (DLE) and lupus nephritis (LN). We found common increases in myeloid cell-defining gene sets and decreases in genes controlling glucose and lipid metabolism in lupus-affected skin and kidney. Regression models in DLE indicated increased glycolysis was correlated with keratinocyte, endothelial, and inflammatory cell transcripts, and decreased tricarboxylic (TCA) cycle genes were correlated with the keratinocyte signature. In LN, regression models demonstrated decreased glycolysis and TCA cycle genes were correlated with increased endothelial or decreased kidney cell transcripts, respectively. Less severe glomerular LN exhibited similar alterations in metabolism and tissue cell transcripts before monocyte/myeloid cell infiltration in some patients. Additionally, changes to mitochondrial and peroxisomal transcripts were associated with specific cells rather than global signal changes. Examination of murine LN gene expression demonstrated metabolic changes were not driven by acute exposure to type I interferon and could be restored after immunosuppression. Finally, expression of HAVCR1, a tubule damage marker, was negatively correlated with the TCA cycle signature in LN models. These results indicate that altered metabolic dysfunction is a common, reversible change in lupus-affected tissues and appears to reflect damage downstream of immunologic processes.

Background: According to the global cancer burden data released in 2020, breast cancer (BC) has become the most common cancer in the world. Similar to those of other cancers, the present methods used in clinic for diagnosing early BC are invasive, inaccurate, and insensitive. Hence, new non-invasive methods capable of early diagnosis are needed.

Methods: We applied next-generation sequencing and analyzed the messenger RNA (mRNA) profiles of plasma extracellular vesicles (EVs) derived from 14 BC patients and 6 patients with benign breast lesions. We used 3 regression models, namely support vector machine (SVM), linear discriminate analysis (LDA), and logistic regression (LR), to develop classifiers for use in making predictive BC diagnoses; and used 259 plasma samples, including those obtained from 144 patients with BC, 72 patients with benign breast lesions, and 43 healthy women, which were divided into training groups and validation groups to verify their performances as classifiers by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The area under the curve (AUC) and accuracy, sensitivity, and specificity of the classifiers were cross-validated with the leave-1-out cross-validation (LOOCV) method.

Results: Among all combinations assessed with the 3 different regression models, an 8-mRNA combination, named EXOB_mRNA, exhibited high performance [accuracy =71.9% and AUC =0.718, 95% confidence interval (CI): 0.652 to 0.784] in the training cohort after LOOCV was performed, showing the largest AUC in the SVM model. The mRNAs in EXOB_mRNA were HLA-DRB1, HAVCR1, ENPEP, TIMP1, CD36, MARCKS, DAB2, and CXCL14. In the validation cohort, the AUC of EXOB_mRNA was 0.737 (95% CI: 0.636 to 0.837). In addition, gene function and pathway analyses revealed that different levels of gene expression were associated with cancer.

Conclusions: We developed a high-performing predictive classifiers including 8 mRNAs from plasma extracellular vesicles for diagnosing breast cancer.

Keywords: Breast cancer (BC); diagnosis; extracellular vesicles (EVs); messenger RNAs (mRNAs); predictive classifier.

Stress-induced changes in viral receptor and susceptibility gene expression were measured in embryonic stem cells (ESC) and differentiated progeny. Rex1 promoter-Red Fluorescence Protein reporter ESC were tested by RNAseq after 72hr exposures to control stress hyperosmotic sorbitol under stemness culture (NS) to quantify stress-forced differentiation (SFD) transcriptomic programs. Control ESC cultured with stemness factor removal produced normal differentiation (ND). Bulk RNAseq transcriptomic analysis showed significant upregulation of two genes involved in Covid-19 cell uptake, Vimentin (VIM) and Transmembrane Serine Protease 2 (TMPRSS2). SFD increased the hepatitis A virus receptor (Havcr1) and the transplacental Herpes simplex 1 (HSV1) virus receptor (Pvrl1) compared with ESC undergoing ND. Several other coronavirus receptors, Glutamyl Aminopeptidase (ENPEP) and Dipeptidyl Peptidase 4 (DPP4) were upregulated significantly in SFD>ND. Although stressed ESC are more susceptible to infection due to increased expression of viral receptors and decreased resistance, the necessary Covid-19 receptor, angiotensin converting enzyme (ACE)2, was not expressed in our experiments. TMPRSS2, ENPEP, and DPP4 mediate Coronavirus uptake, but are also markers of extra-embryonic endoderm (XEN), which arise from ESC undergoing ND or SFD. Mouse and human ESCs differentiated to XEN increase TMPRSS2 and other Covid-19 uptake-mediating gene expression, but only some lines express ACE2. Covid-19 susceptibility appears to be genotype-specific and not ubiquitous. Of the 30 gene ontology (GO) groups for viral susceptibility, 15 underwent significant stress-forced changes. Of these, 4 GO groups mediated negative viral regulation and most genes in these increase in ND and decrease with SFD, thus suggesting that stress increases ESC viral susceptibility. Taken together, the data suggest that a control hyperosmotic stress can increase Covid-19 susceptibility and decrease viral host resistance in mouse ESC. However, this limited pilot study should be followed with studies in human ESC, tests of environmental, hormonal, and pharmaceutical stressors and direct tests for infection of stressed, cultured ESC and embryos by Covid-19.

Keywords: Covid; Embryonic stem cells; High throughput screens; Hyperosmotic stress.

Background: The 5-year overall survival rate of ovarian cancer (OC) patients is less than 40%. Hypoxia promotes the proliferation of OC cells and leads to the decline of cell immunity. It is crucial to find potential predictors or risk model related to OC prognosis. This study aimed at establishing the hypoxia-associated gene signature to assess tumor immune microenvironment and predicting the prognosis of OC.

Methods: The gene expression data of 378 OC patients and 370 OC patients were downloaded from datasets. The hypoxia risk model was constructed to reflect the immune microenvironment in OC and predict prognosis.

Results: 8 genes (AKAP12, ALDOC, ANGPTL4, CITED2, ISG20, PPP1R15A, PRDX5, and TGFBI) were included in the hypoxic gene signature. Patients in the high hypoxia risk group showed worse survival. Hypoxia signature significantly related to clinical features and may serve as an independent prognostic factor for OC patients. 2 types of immune cells, plasmacytoid dendritic cell and regulatory T cell, showed a significant infiltration in the tissues of the high hypoxia risk group patients. Most of the immunosuppressive genes (such as ARG1, CD160, CD244, CXCL12, DNMT1, and HAVCR1) and immune checkpoints (such as CD80, CTLA4, and CD274) were upregulated in the high hypoxia risk group. Gene sets related to the high hypoxia risk group were associated with signaling pathways of cell cycle, MAPK, mTOR, PI3K-Akt, VEGF, and AMPK.

Conclusion: The hypoxia risk model could serve as an independent prognostic indicator and reflect overall immune response intensity in the OC microenvironment.

There are few studies on the role of iron metabolism genes in predicting the prognosis of lung adenocarcinoma (LUAD). Therefore, our research aims to screen key genes and to establish a prognostic signature that can predict the overall survival rate of lung adenocarcinoma patients. RNA-Seq data and corresponding clinical materials of 594 adenocarcinoma patients from The Cancer Genome Atlas(TCGA) were downloaded. GSE42127 of Gene Expression Omnibus (GEO) database was further verified. The multi-gene prognostic signature was constructed by the Cox regression model of the Least Absolute Shrinkage and Selection Operator (LASSO). We constructed a prediction signature with 12 genes (HAVCR1, SPN, GAPDH, ANGPTL4, PRSS3, KRT8, LDHA, HMMR, SLC2A1, CYP24A1, LOXL2, TIMP1), and patients were split into high and low-risk groups. The survival graph results revealed that the survival prognosis between the high and low-risk groups was significantly different (TCGA: P < 0.001, GEO: P = 0.001). Univariate and multivariate Cox regression analysis confirmed that the risk value is a predictor of patient OS (P < 0.001). The area under the time-dependent ROC curve (AUC) indicated that our signature had a relatively high true positive rate when predicting the 1-year, 3-year, and 5-year OS of the TCGA cohort, which was 0.735, 0.711, and 0.601, respectively. In addition, immune-related pathways were highlighted in the functional enrichment analysis. In conclusion, we developed and verified a 12-gene prognostic signature, which may be help predict the prognosis of lung adenocarcinoma and offer a variety of targeted options for the precise treatment of lung cancer.

Keywords: Lung adenocarcinoma; gene signature; precise treatment; risk score; survival.