Prioritization of Cancer Marker Candidates Based on the Immunohistochemistry Staining Images Deposited in the Human Protein Atlas

Cancer marker discovery is an emerging topic in high-throughput quantitative proteomics. However, the omics technology usually generates a long list of marker candidates that requires a labor-intensive filtering process in order to screen for potentially useful markers. Specifically, various parameters, such as the level of overexpression of the marker in the cancer type of interest, which is related to sensitivity, and the specificity of the marker among cancer groups, are the most critical considerations. Protein expression profiling on the basis of immunohistochemistry (IHC) staining images is a technique commonly used during such filtering procedures. To systematically investigate the protein expression in different cancer versus normal tissues and cell types, the Human Protein Atlas is a most comprehensive resource because it includes millions of high-resolution IHC images with expert-curated annotations. To facilitate the filtering of potential biomarker candidates from large-scale omics datasets, in this study we have proposed a scoring approach for quantifying IHC annotation of paired cancerous/normal tissues and cancerous/normal cell types. We have comprehensively calculated the scores of all the 17219 tested antibodies deposited in the Human Protein Atlas based on their accumulated IHC images and obtained 457110 scores covering 20 different types of cancers. Statistical tests demonstrate the ability of the proposed scoring approach to prioritize cancer-specific proteins. Top 100 potential marker candidates were prioritized for the 20 cancer types with statistical significance. In addition, a model study was carried out of 1482 membrane proteins identified from a quantitative comparison of paired cancerous and adjacent normal tissues from patients with colorectal cancer (CRC). The proposed scoring approach demonstrated successful prioritization and identified four CRC markers, including two of the most widely used, namely CEACAM5 and CEACAM6. These results demonstrate the potential of this scoring approach in terms of cancer marker discovery and development. All the calculated scores are available at http://bal.ym.edu.tw/hpa/.     

Immunization of Chlamydia pneumoniae (Cpn)-Infected Apobtm2SgyLdlrtm1Her/J Mice with a Combined Peptide of Cpn Significantly Reduces Atherosclerotic Lesion Development

Objective

To investigate the antigenic effect of a peptide containing two epitopes of Chlamydia pneumoniae (Cpn) on atherosclerotic lesion formation in mice infected with Cpn.

Materials and Methods

Six-week-old Apob^tm2SgyLdlr^tm1Her/J mice were immunized using a repetitive immunization multiple-sites strategy with KLH-conjugated peptides derived from the major outer membrane protein and the putative outer membrane protein 5 of Cpn. Mice were fed a high-fat diet and infected with Cpn twice during the 10-week diet period. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants.

Results

Mice immunized with the combined Cpn peptide showed a greater reduction in lesion size compared to mice immunized with either epitope alone [54.7% vs 39.8% or 41.72%] and was also associated with a significant decrease in lesion area in descending aortas compared with those in controls (88.9% for combined Cpn peptide, 81.9% for MOMP peptide and 75.7% for Omp5, respectively). This effect was associated with a shift in the cellular composition of plaques towards decreased inflammatory cell and increased regulatory T-cell content. Additionally, the effect was also connected with decreased secretion of proinflammatory cytokines and increased production of anti-inflammatory cytokines demonstrated in plasma and in supernatant on stimulated spleen cells.

Conclusions

Atherosclerotic lesion formation may be promoted by Cpn infection in the presence of a high-fat diet, and reduced by immunization with the combined Cpn peptide. The combined peptide has more potential than either epitope alone in reducing atherosclerotic lesion development through Treg expansion.     

Cytological and Comparative Proteomic Analyses on Male Sterility in Brassica napus L. Induced by the Chemical Hybridization Agent Monosulphuron Ester Sodium

Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility in rapeseed (Brassica napus L.). To understand MES-induced male sterility in rapeseed better, comparative cytological and proteomic analyses were conducted in this study. Cytological analysis indicated that defective tapetal cells and abnormal microspores were gradually generated in the developing anthers of MES-treated plants at various development stages, resulting in unviable microspores and male sterility. A total of 141 differentially expressed proteins between the MES-treated and control plants were revealed, and 131 of them were further identified by MALDI-TOF/TOF MS. Most of these proteins decreased in abundance in tissues of MES-treated rapeseed plants, and only a few increased. Notably, some proteins were absent or induced in developing anthers after MES treatment. These proteins were involved in several processes that may be crucial for tapetum and microspore development. Down-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in defective tapetum and abnormal microspores that lead to male sterility in MES-treated plants. Accordingly, a simple model of CHA-MES-induced male sterility in rapeseed was established. This study is the first cytological and dynamic proteomic investigation on CHA-MES-induced male sterility in rapeseed, and the results provide new insights into the molecular events of male sterility.     

Altered Lipid Metabolism in Residual White Adipose Tissues of Bscl2 Deficient Mice

Mutations in BSCL2 underlie human congenital generalized lipodystrophy type 2 disease. We previously reported that Bscl2 ^−/− mice develop lipodystrophy of white adipose tissue (WAT) due to unbridled lipolysis. The residual epididymal WAT (EWAT) displays a browning phenotype with much smaller lipid droplets (LD) and higher expression of brown adipose tissue marker proteins. Here we used targeted lipidomics and gene expression profiling to analyze lipid profiles as well as genes involved in lipid metabolism in WAT of wild-type and Bscl2^−/− mice. Analysis of total saponified fatty acids revealed that the residual EWAT of Bscl2^−/− mice contained a much higher proportion of oleic_18:1n9 acid concomitant with a lower proportion of palmitic_16:0 acid, as well as increased n3- polyunsaturated fatty acids (PUFA) remodeling. The acyl chains in major species of triacylglyceride (TG) and diacylglyceride (DG) in the residual EWAT of Bscl2^−/− mice were also enriched with dietary fatty acids. These changes could be reflected by upregulation of several fatty acid elongases and desaturases. Meanwhile, Bscl2^−/− adipocytes from EWAT had increased gene expression in lipid uptake and TG synthesis but not de novo lipogenesis. Both mitochondria and peroxisomal β-oxidation genes were also markedly increased in Bscl2^−/− adipocytes, highlighting that these machineries were accelerated to shunt the lipolysis liberated fatty acids through uncoupling to dissipate energy. The residual subcutaneous white adipose tissue (ScWAT) was not browning but displays similar changes in lipid metabolism. Overall, our data emphasize that, other than being essential for adipocyte differentiation, Bscl2 is also important in fatty acid remodeling and energy homeostasis.     

Diabetic Patients Could Do As Well as Non-Diabetic Patients without Inflammation on Peritoneal Dialysis

Background

Diabetic patients on peritoneal dialysis (PD) have lower survival and are more likely complicated with inflammation than their non-diabetic counterparts. Here, we explored the interaction effects between diabetes and inflammation on the survival of PD patients.

Methods

Overall, 2,264 incident patients were enrolled from a retrospective cohort study in China. Patients were grouped according to the baseline levels of high-sensitive C-reactive protein (hsCRP, ≤3 mg/L or >3 mg/L) or serum albumin (SA, ≥38 g/L or <38 g/L). Then, several multivariable adjusted stratified Cox regression models were constructed for these groups to explore the predicted role of diabetes on all-cause or cardiovascular death under inflammatory or non-inflammatory conditions.

Results

Diabetics on PD were more likely to have inflammation than non-diabetics on PD, and they presented with elevated hsCRP (52.7% vs. 47.3%, P = 0.03) or decreased SA (77.9% vs. 62.7%, P < 0.001) levels. After stratification by size of center and controlling for confounding factors, diabetes was found to predict all-cause death in patients with hsCRP >3 mg/L or SA <38 g/L but not in patients with hsCRP ≤3 mg/L or SA ≥38 g/L. Similarly, the presence of diabetes was an indication of cardiovascular death in patients with hsCRP >3 mg/L or SA <38 g/L. However, if further adjusted by baseline cardiovascular disease, the predicted role of diabetes on death related to cardiovascular disease in patients with SA <38 g/L disappeared.

Conclusion

Diabetic patients could do as well as non-diabetic patients without inflammation on peritoneal dialysis. Active strategies should be implemented to improve inflammation status in diabetic patients on PD.     

LncRNAs Expression in Preeclampsia Placenta Reveals the Potential Role of LncRNAs Contributing to Preeclampsia Pathogenesis

Background

Long non-coding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. They are aberrantly expressed in many types of diseases. In this study, we aimed to investigate the lncRNA profiles in preeclampsia. Preeclampsia has been observed in patients with molar pregnancy where a fetus is absent, which demonstrate that the placenta is sufficient to cause this condition. Thus, we analyzed the lncRNA profiles in preeclampsia placentas.

Methodology/Principal Findings

In this study, we described the lncRNA profiles in six preeclampsia placentas (T) and five normal pregnancy placentas (N) using microarray. With abundant and varied probes accounting for 33,045 LncRNAs in our microarray, 28,443 lncRNAs that were expressed at a specific level were detected. From the data, we found 738 lncRNAs that were differentially expressed (≥1.5-fold-change) among preeclampsia placentas compared with controls. Coding-non-coding gene co-expression networks (CNC network) were constructed based on the correlation analysis between the differentially expressed lncRNAs and mRNAs. According to the CNC network and GO analysis of differentially expressed lncRNAs/mRNAs, we selected three lncRNAs to analyze the relationship between lncRNAs and preeclampsia. LOC391533, LOC284100, and CEACAMP8 were evaluated using qPCR in 40 preeclampsia placentas and 40 controls. These results revealed that three lncRNAs were aberrantly expressed in preeclampsia placentas compared with controls.

Conclusions/Significance

Our study is the first study to determine the genome-wide lncRNAs expression patterns in preeclampsia placenta using microarray. These results revealed that clusters of lncRNAs were aberrantly expressed in preeclampsia placenta compared with controls, which indicated that lncRNAs differentially expressed in preeclampsia placenta might play a partial or key role in preeclampsia development. Misregulation of LOC391533, LOC284100, and CEACAMP8 might contribute to the mechanism underlying preeclampsia. Taken together, this study may provide potential targets for the future treatment of preeclampsia and novel insights into preeclampsia biology.     

IGFBP3, a Transcriptional Target of Homeobox D10, Is Correlated with the Prognosis of Gastric Cancer

Homeobox D10 (HoxD10) plays important roles in the differentiation of embryonic cells and progression of breast cancer. Our previous report revealed that insulin-like growth factor binding protein-3 (IGFBP3) was regulated by HoxD10 in gastric cancer cells; however, the functional roles and underlying mechanisms of IGFBP3 in gastric cancer remain unclear. Here, we found that the expression of IGFBP3 were upregulated after ectopic expression of HoxD10 in gastric cancer cells. Chromatin immunoprecipitation assay showed that HoxD10 bound to three potential regions of IGFBP3 promoter. Exogenous HoxD10 significantly enhanced the activity of luciferase reporter containing these binding regions in gastric cancer cells. Further data showed that all of these binding sites had Hox binding element “TTAT”. Immunohistochemical staining results revealed that IGFBP3 expression was significantly downregulated in 86 gastric adenocarcinomas tissues relative to their adjacent non-cancerous tissues (p<0.001). Moreover, IGFBP3 expression was significantly lower in gastric tumor with lymph node metastasis compared with that without lymph node metastasis (p=0.045). Patients with high expression level of IGFBP3 showed favorable 5 year overall survival (p=0.011). Knockdown of IGFBP3 accelerated gastric cancer cell migration and invasion and induced the expression of invasive factors including MMP14, uPA and uPAR. Thus, our data suggest that HoxD10-targeted gene IGFBP3 may suppress gastric cancer cell invasion and favors the survival of gastric cancer patients.     

Increasing RpoS Expression Causes Cell Death in Borrelia burgdorferi

RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.     

Intratumoral Hepatic Stellate Cells as a Poor Prognostic Marker and a New Treatment Target?for?Hepatocellular Carcinoma

Hepatic stellate cells (HSCs), a specialized stromal cytotype in the liver, have been demonstrated to actively contribute to hepatocellular carcinoma (HCC) development. However, the previous studies were performed using HSC cell lines, and the prognostic value of intratumoral HSCs (tHSCs) was unclear. Here we isolated tHSCs from fresh human HCC tissues, and analyzed the abilities of tHSCs to promote HCC progression by using in vitro assays for cell viability, migration and invasion as well as epithelial-mesenchymal transition (EMT) phenotype. 252 HCC patients who underwent hepatectomy were enrolled for analysis of tHSCs and E-cadherin expression in tumor tissues, and 55 HCC patients for analysis of tHSCs in tumor tissues and circulating tumor cells (CTCs) in blood. Prognostic factors were then identified. The results showed that coculture of tHSCs with HCC cells had a stronger effect on HCC cell viability, migration and invasion, accompanied with the acquisition of epithelial-mesenchymal transition (EMT) phenotype. In vivo cotransplantation of HCC cells with tHSCs into nude mice more efficiently promoted tumor formation and growth. Icaritin, a known apoptosis inducer of HSCs, was demonstrated to effectively inhibit tHSC proliferation in vitro and tHSC-induced HCC-promoting effects in vivo. Clinical evidence indicated that tHSCs were rich in 45% of the HCC specimens, tHSC-rich subtypes were negatively correlated either with E-cadherin expression in tumor tissues (r = -0.256, p < 0.001) or with preoperative CTCs in blood (r = -0.287, p = 0.033), and were significantly correlated with tumor size (p = 0.027), TNM staging (p = 0.018), and vascular invasion (p = 0.008). Overall and recurrence-free survival rates of tHSC-rich patients were significantly worse than those for tHSC-poor patients. Multivariate analysis revealed tHSC-rich as an independent factor for overall and recurrence-free survival. In conclusion, tHSCs provide a promising prognostic biomarker and a new treatment target for HCC.     

32k Da protein improve ovariectomy-induced bone loss in rats

The objective of the present study was to systematically explore the effects of 32K Da protein (32KP) on postmenopausal osteoporosis. Eighty 3-mo-old female Sprague-Dawley rats were employed and randomly divided into one sham-operated group (SHAM) and five ovariectomy (OVX) subgroups as OVX (control), OVX with 17-ethinylestradiol (E2, 25 g/kg/day), OVX with 32KP of graded doses (50, 50, or 150 mg/kg/day). 32KP or E2 diet was fed on week 4 after operation, for 16 weeks. Bone mass, bone turnover and strength were evaluated by dual-energy X-ray absorptiometry (DEXA), biochemical markers and three-point bending test, respectively. Femur marrow cavity was observed by light microscopy via hematoxylin-eosin staining. It is observed that different dosage treatment of 32KP increased the body weight and prevented the loss of bone mass induced by OVX. The prevention effect against bone loss was presumably due to the altering of the rate of bone remodeling. The bone mineral density and bone calcium content in OVX rats were lower than that in the control group, suggesting that 32KP was able to prevent significant bone loss. In addition, the data from three point bending test and femur sections showed that 32KP treatment enhanced bone strength and reduced the marrow cavity of the femur in OVX rats. In the serum and urine assay, 32KP decreased urinary deoxypyridinoline and calcium concentrations; however, serum alkaline phosphatase activities were not inhibited. It suggested that amelioration of bone loss was changed via inhibition of bone reabsorption. Our findings indicated that 32KP might be a potential alternative drug for the prevention and treatment of postmenopausal osteoporosis.