Insulin-like Growth Factor II mRNA-binding Protein 3 is a Highly Sensitive Marker for Intravascular Large B-cell Lymphoma: Immunohistochemical Analysis of 152 Pathology Specimens From 88 Patients.

Intravascular large B-cell lymphoma (IVLBCL) is a rare type of aggressive extranodal large B-cell lymphoma characterized by the selective growth of lymphoma cells within the lumina of blood vessels, particularly capillaries. IVLBCL lacks mass formation, and its diagnosis can be challenging. We analyzed the utility of insulin-like growth factor II mRNA-binding protein 3 (IMP3) immunohistochemistry for IVLBCL diagnosis in various organs. Double staining with paired box 5 (PAX5) was performed for validation. Overall, 152 pathological specimens (111 positive and 41 negative for IVLBCL) obtained from 88 patients with a diagnosis of IVLBCL were stained for IMP3 and IMP3/PAX5. As negative controls, 40 pathology specimens from 38 patients with no history of IVLBCL or other B-cell lymphomas were stained for IMP3, which comprised 31 benign pathological specimens from 29 patients in whom malignancy was suspected, 7 cases of appendicitis with intravascular and/or intralymphatic lymphoid proliferations, and 2 cases of intravascular natural killer/T-cell lymphoma. All mononuclear cells with cytoplasmic staining were considered positive for IMP3 expression, but expression restricted to germinal center B cells was excluded from evaluation. All 111 IVLBCL pathological specimens were positive for IMP3 and IMP3/PAX5. In addition, 11 of the 41 specimens originally diagnosed as IVLBCL-negative showed IMP3/PAX5 double-positive cells, raising the suspicion of IVLBCL. However, of the 40 negative control samples, IMP3-positive non-germinal center B cells were detected in only 2 samples ( P = 0.0131) and no intravascular IMP3-positive B cells suspicious for IVLBCL were identified. Altogether, IMP3 immunohistochemistry is a highly sensitive marker of IVLBCL and can be a helpful adjunct for IVLBCL diagnosis.

Improved detection and consistency of RNA-interacting proteomes using DIA SILAC.

The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing single-shot analysis of the sample. As proof of concept, we determined the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells. The DIA dataset yielded similar GO term enrichment for RNA-binding proteins involved in cellular stress responses to the DDA dataset while detecting extra proteins unseen by DDA. Overall, the DIA SILAC approach improved detection of proteins over conventional DDA SILAC for generating RNA-interactome datasets, at a lower cost due to reduced machine time. Analyses are described for TRAPP data, but the approach is suitable for proteomic analyses following essentially any RNA-binding protein enrichment technique.

Surveying the global landscape of post-transcriptional regulators.

Numerous proteins regulate gene expression by modulating mRNA translation and decay. To uncover the full scope of these post-transcriptional regulators, we conducted an unbiased survey that quantifies regulatory activity across the budding yeast proteome and delineates the protein domains responsible for these effects. Our approach couples a tethered function assay with quantitative single-cell fluorescence measurements to analyze ~50,000 protein fragments and determine their effects on a tethered mRNA. We characterize hundreds of strong regulators, which are enriched for canonical and unconventional mRNA-binding proteins. Regulatory activity typically maps outside the RNA-binding domains themselves, highlighting a modular architecture that separates mRNA targeting from post-transcriptional regulation. Activity often aligns with intrinsically disordered regions that can interact with other proteins, even in core mRNA translation and degradation factors. Our results thus reveal networks of interacting proteins that control mRNA fate and illuminate the molecular basis for post-transcriptional gene regulation.

RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins.

RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.

Bile cytology diagnosis in challenging cases: Validation of diagnostic bile cytology criteria and extensive study for immunocytochemical markers.

BACKGROUND: Bile cytology is useful in diagnosing biliary tract lesions, albeit often challenging due to equivocal findings. To achieve better diagnoses for clinical decisions, we conducted cytomorphological and immunocytochemical studies of bile cytology cases. MATERIALS AND METHODS: We re-evaluated 40 bile cytology cases with initial equivocal diagnoses, taken from the cytology records of Jichi Medical University Hospital, including 1778 bile cytology specimens. First, we assessed the cases by the diagnostic bile cytology criteria of the Japanese Society of Clinical Cytology. Second, we searched for useful immunocytochemical markers by extensive immunohistochemical analyses using tissue microarray for 10 antibodies: S100P, IMP3, GLUT1, p53, S100A4, Mapsin, MUC17, CD10, MDM2, and SMAD4. Microarrays were from 257 extrahepatic bile duct carcinoma cases. To elucidate the utility of immunocytochemistry, we applied selected markers to immunocytochemical evaluation of the equivocal cases after cell transfer. RESULTS: The criteria indicated a sensitivity 60%, specificity 87%, and accuracy 70%. Irregularly overlapping (88%), arranged (96%), and shaped (76%) nuclei were more common in malignant cases, while enlarged nuclei were more frequent in benign cases (67% vs. 28%). We applied S100P and IMP3, which showed higher accuracy (88% and 77%) in tissue microarray, to immunocytochemistry. The sensitivity of S100P and IMP3 were 69% and 70%, respectively. The specificity of S100P and IMP3 were 50% and 100%, respectively. CONCLUSION: The criteria showed a certain effectiveness even in challenging cases, and some pitfalls associated with reactive changes of benign cells. Although comprehensive diagnosis including cytomorphology seems preferable, S100P and IMP3 are promising immunocytochemical markers.

Advances in the identification of long non-coding RNA binding proteins.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt without evident protein coding function. They play important regulatory roles in many biological processes, e.g., gene regulation, chromatin remodeling, and cell fate determination during development. Dysregulation of lncRNAs has been observed in various diseases including cancer. Interacting with proteins is a crucial way for lncRNAs to play their biological roles. Therefore, the characterization of lncRNA binding proteins is important to understand their functions and to delineate the underlying molecular mechanism. Large-scale studies based on mass spectrometry have characterized over a thousand new RNA binding proteins without known RNA-binding domains, thus revealing the complexity and diversity of RNA-protein interactions. In addition, several methods have been developed to identify the binding proteins for particular RNAs of interest. Here we review the progress of the RNA-centric methods for the identification of RNA-protein interactions, focusing on the studies involving lncRNAs, and discuss their strengths and limitations.

Dynamics of epigenetic modifiers and environmentally sensitive proteins in a reptile with temperature induced sex reversal†.

The mechanisms by which sex is determined, and how a sexual phenotype is stably maintained during adulthood, have been the focus of vigorous scientific inquiry. Resources common to the biomedical field (automated staining and imaging platforms) were leveraged to provide the first immunofluorescent data for a reptile species with temperature induced sex reversal. Two four-plex immunofluorescent panels were explored across three sex classes (sex reversed ZZf females, normal ZWf females, and normal ZZm males). One panel was stained for chromatin remodeling genes JARID2 and KDM6B, and methylation marks H3K27me3, and H3K4me3 (Jumonji Panel). The other CaRe panel stained for environmental response genes CIRBP and RelA, and H3K27me3 and H3K4me3. Our study characterized tissue specific expression and cellular localization patterns of these proteins and histone marks, providing new insights to the molecular characteristics of adult gonads in a dragon lizard Pogona vitticeps. The confirmation that mammalian antibodies cross react in P. vitticeps paves the way for experiments that can take advantage of this new immunohistochemical resource to gain a new understanding of the role of these proteins during embryonic development, and most importantly for P. vitticeps, the molecular underpinnings of sex reversal.

Proteomic analysis of serum in workers exposed to diesel engine exhaust.

Diesel engine exhaust (DEE) is classified as a Group 1 human carcinogen. Using a targeted proteomics approach, we aimed to identify proteins associated with DEE and characterize these markers to understand the mechanisms of DEE-induced carcinogenicity. In this cross-sectional molecular epidemiology study, we measured elemental carbon (EC) using a personal air monitor and quantified 1317 targeted proteins in the serum using the SOMAScan assay (SOMALogic) among 19 diesel exposed factory workers and 19 unexposed controls. We used linear regressions to identify proteins associated with DEE and examined their exposure-response relationship across levels of EC using linear trend tests. We further examined pathway enrichment of DEE-related proteins using MetaCore. Occupational exposure to DEE was associated with altered levels of 22 serum proteins (permutation p < .01). Of these, 13 proteins (CXCL11, HAPLN1, FLT4, CD40LG, PES1, IGHE.IGK..IGL, TNFSF9, PGD, NAGK, CCL25, CCL4L1, PDXK, and PLA2G1B) showed an exposure-response relationship with EC (p trend < .01), with serum levels of all but PLA2G1B declining with increasing air levels of EC. For instance, C-X-C Motif Chemokine Ligand 11 (CXCL11) showed the most significant association with DEE (ß = -0.25; permutation p = .00004), where mean serum levels were 4121.1, 2356.7, and 2298.8 relative fluorescent units among the unexposed, lower exposed (median, range : 56.9, 40.2-62.1 µg/m3 EC), and higher exposed (median, range of EC: 72.9, 66.9-107.7 µg/m3 EC) groups, respectively (p trend = .0005). Pathway analysis suggested that these proteins are enriched in pathways related to inflammation and immune regulation. Our study suggests that DEE exposure is associated with altered serum proteins, which play a role in inflammation and immune regulation.

Expression of LIN28A/B and Let-7 miRNAs in canine mammary carcinomas / Expressão de LIN28A/B e Let-7 miRNAs em carcinomas mamários caninos

LIN28 is a RNA-binding protein including two highly conserved homologous, LIN28A and LIN28B. Proto-oncogenes such as LIN28A and LIN28B are generally targeted by the let-7 miRNAs in different types of human cancers. Here, we determined the expression of LIN28A in canine mammary tumor samples and the LIN28/let-7 pathway in canine mammary cell lines. In those cell lines, we identified a functional LIN28/let-7 pathway which exhibited high expression of let-7 members and low expression of its targets, including LIN28A and LIN28B. However, the mammary carcinoma tissue samples showed a frequent expression of LIN28A being expressed mainly in the epithelial cells. No association was observed between LIN28A expression and histopathological classification and grade, TNM and survival time. Our results suggested a possible role of the LIN28A protein in the development of canine mammary carcinomas due to the high frequency observed in the tumor samples (28 of 32). The in vitro experiments suggested that the LIN28/let-7 pathway is active in the tumor cells evaluated. However, more studies are necessary to elucidate the exact role of LIN28/let-7 pathway in canine mammary carcinomas.

LIN28 é uma proteína de ligação ao RNA, com duas formas homólogas altamente conservadas, LIN28A e LIN28B. Os proto-oncogenes LIN28A e LIN28B são regulados pela família de miRNAs let-7 em diferentes tipos de cânceres em humanos. No presente trabalho, o objetivo foi determinar a expressão de LIN28A em amostras de tumor mamário de cadelas e a via LIN28/let-7 em linhagens celulares mamárias caninas. Nestas linhagens, através das técnicas de qPCR e RNAseq, foi identificado que a via LIN28/let-7 apresenta-se funcional, com alta expressão dos membros da família let-7 e baixa expressão de seus alvos, entre eles LIN28A e LIN28B. No entanto, as amostras de tecidos de carcinomas mamários caninos demonstraram expressão frequente de LIN28A, sendo observada principalmente em células epiteliais. Não foram observadas associações entre expressão de LIN28A com classificação e gradação histopatológicas, TNM e tempo de sobrevida. Nossos resultados sugerem uma possível relação da proteína LIN28A no desenvolvimento de carcinomas mamários caninos devido à alta frequência observada nas amostras tumorais (28 de 32). Os experimentos in vitro sugerem que a via LIN28/let-7 é ativa nas linhagens celulares caninas avaliadas. Entretanto, estudos funcionais ainda são necessários para elucidar a função exata da via LIN28/let-7 nos carcinomas mamários caninos.

[Advances in technologies for large-scale enrichment and identification of ribonucleic acid-protein complexes].

Ribonucleic acid (RNA) rarely exists alone in the cell. RNAs interact with a variety of proteins and form RNA-protein complexes (RP-complexes) in every step of their life cycle, from transcription to degradation. These RP-complexes play key roles in regulating a variety of physiological processes. Defects in the composition and function of RP-complexes have been associated with many diseases, including metabolic disorders, muscular atrophy, autoimmune diseases, and cancer. It is hence evident that deciphering the highly complex interaction network of RNA-binding proteins (RBPs) and their RNA targets will provide a better understanding of disease development and lead to the discovery of new targets for cancer therapy. Large-scale identification of RP-complexes at the omics level is a prerequisite for obtaining insights into the complex RNA-protein interaction network. As the first step in omics-wide decoding of RP-complexes, enrichment and purification of RP-complexes is a highly challenging task. Recently, intensive efforts have been undertaken to better enrich and identify RP-complexes. Generally, the enrichment strategies can be classified into two major categories: in vitro and in vivo. Although it has been successfully applied in many studies, the in vitro transcribed bait RNA lacks modifications or structural similarity compared with its natural counterpart. Further, since the proteins relocate and remodel after cell lysis, the use of cell lysates as a protein source may result in capturing false interacting proteins that bind non-physiologically with the bait RNA. Finally, weak interactions between the non-covalently bound proteins and RNA require mild washing to remove non-specific binding, which needs careful optimization. However, substantial sample loss is inevitable. To overcome the disadvantages of in vitro approaches, in vivo cross-linking strategies that "freeze" natural RNA-protein complexes in intact cells via covalent cross-linking have become increasingly popular. The in vivo methods allow RNA to interact with proteins in the intracellular environment. Therefore, the RP-complexes formed under physiological conditions are more biologically relevant than those obtained by in vitro methods. We herein summarize recent in vivo methodological advances in the large-scale enrichment and identification of RP-complexes, including cross-linking and immunoprecipitation (CLIP) and related methods, click chemistry-assisted methods, and organic phase separations. CLIP involves irradiating living cells with 254-nm ultraviolet (UV) light to establish covalent bonds between RNA and proteins. This enables CLIP to purify RNAs bound to a specific RBP under conditions that are stringent enough to prevent co-purification of nonspecifically bound proteins or free RNAs. Since the original study, multiple variant protocols have been derived to increase both efficiency and convenience. Photoactivatable ribonucleoside-enhanced-CLIP (PAR-CLIP) introduces a variation in the crosslinking strategy. Cells were preincubated with photoactivatable ribonucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which enables protein-RNA crosslinking with 365-nm UV-A irradiation. It increases the efficiency of cross-linking between RNA and RBPs and is particularly valuable for studying the interactions between RBPs and nascent RNA. Using a click chemistry-assisted strategy, an alkyne modified uridine analog, 5-ethynyluridine (EU), was incorporated into nascent RNAs via metabolic incorporation in living cells. Combined with UV irradiation-based cross-linking, the alkyne-functionalized RNA and the bound proteins were purified in a poly A-independent fashion by the highly selective bioorthogonal copper (I)-catalyzed azide-alkyne cycloaddition using azide-modified beads. Thus, full lists of both coding and non-coding RNAs with their interacting proteins can be purified, which is a major methodological advance. Organic phase separation methods exploiting the physicochemical difference between cross-linked RP-complexes and free RNA and proteins do not require metabolic-based alkyne labeling or polyA-based RNA capture. Each method has unique strengths and drawbacks, which makes it important to select optimal approaches for the biological question being addressed. We hope that this review points out the current limitations and provides future directions to facilitate further development of methods for large-scale investigation of RP-complexes.

An immunohistochemical panel of insulin-like growth factor II mRNA-binding protein 3 (IMP3), enhancer of zeste homolog 2 (EZH2), and p53 is useful for a diagnosis in bile duct biopsy.

Bile duct biopsy is being increasingly performed in number for a definite diagnosis of cholangiocarcinoma. However, difficulties are associated with a histopathological diagnosis because of the limited small amount of specimen obtained and crash artifact. The aim of the present study was to identify useful diagnostic immunohistochemical markers in bile duct biopsy that support a histological diagnosis. Fifty-one bile duct biopsy samples, including 26 samples taken from patients with cholangiocarcinoma, 11 with intraductal papillary neoplasm of the bile duct (IPNB), and 14 with benign bile duct lesions, were examined. Histology and the immunohistochemical expression of insulin-like growth factor II mRNA-binding protein 3 (IMP3), enhancer of zeste homolog 2 (EZH2), and p53 were assessed. They were then evaluated for their usefulness as diagnostic markers of malignancy. The diagnostic sensitivity and accuracy of the institutional histological diagnosis were 53.8% and 70.0%, respectively. The diagnostic sensitivity and accuracy of IMP3, EZH2, and p53 were 69.2% and 80.0%, 76.9% and 85.0%, and 50.0% and 67.5%, respectively. Immunohistochemical staining for EZH2; the combination of either 2 of IMP3, EZH2, and p53; or the combination of IMP3, EZH2, and p53 significantly increased sensitivity and accuracy over those of the institutional histological diagnosis (p<0.05). In conclusion, an immunohistochemical panel consisting of IMP3, EZH2, and p53 increases the diagnostic sensitivity and accuracy of bile duct biopsy for the diagnosis of cholangiocarcinoma.

Exploration of the potential roles of m6A regulators in the uterus in pregnancy and infertility.

Infertility is a prevalent female reproductive disease worldwide. Currently, there are many unknown etiologies of infertility. N6-methyladenosine (m6A) is the most prevalent modification of eukaryotic mRNA. This study intended to investigate the implications of m6A regulators in the uterus for pregnancy and infertility. Pregnant ICR mice on days (D) 0, 4, 6, 10, and 15 were used to monitor m6A methylation in the uterus by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then m6A methylation regulators were detected by real-time quantitative PCR (qPCR), western blot and immunohistochemistry (IHC). We found that m6A levels increased and that m6A regulators were expressed differently in the uterus during pregnancy. Then, we acquired expression data from endometrial tissue from women with infertility and recurrent pregnancy loss from the Gene Expression Omnibus (GEO) database. The expression of m6A regulators in infertility was significantly dysregulated according to the data mining technique. Specifically, the mRNA levels of METTL16 (p = 0.0147) and WTAP (p = 0.028) were lower and those of ALKBH5 (p = 0.0432) and IGF2BP2 (p = 0.0016) were higher in the endometrium of infertile patients. Meanwhile, many immunity-related pathways are abnormal in infertility, such as cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity and leukocyte transendothelial migration. In conclusion, we found that the m6A levels in the uterus increased as pregnancy progressed, and these regulators were dysregulated in the endometrium of infertility patients. These results suggest that m6A methylation may be very important in the establishment of implantation and maintenance of pregnancy and may become a new direction for research on infertility.

Prognostic value of noggin protein expression in patients with resected gastric cancer.

BACKGROUND: Noggin and RNA-binding protein for multiple splicing 2 (RBPMS2) are known to regulate the expression of smooth muscle cells, endothelial cells, and osteoblasts. However, the prognostic role of combined Noggin and RBPMS2 expression in resected gastric cancer (GC) is unclear. METHODS: A total of 163 patients with GC who underwent gastrectomy were included in this study. The expression of Noggin and RBPMS2 proteins in tumor cells at the tumor center and invasive front of resected GC was evaluated by immunohistochemistry, and in conjunction with clinicopathological parameters the patient survival was analyzed. RESULTS: RBPMS2 protein expression was high at the tumor center (n = 86, 52.8%) and low at the invasive front (n = 69, 42.3%), while Noggin protein expression was high in both tumor center (n = 91, 55.8%) and the invasive front (n = 90, 55.2%). Noggin expression at the invasive front and tumor center was significantly decreased in advanced T stage, non-intestinal-type (invasive front, P = 0.008 and P <  0.001; tumor center lesion, P = 0.013 and P = 0.001). RBPMS2 expression at the invasive front was significantly decreased in non-intestinal-type and positive lymphatic invasion (P <  0.001 and P = 0.013). Multivariate analysis revealed that high Noggin protein expression of the invasive front was an independent prognostic factor for overall survival (hazard ratio [HR], 0.58; 95% confidence interval [CI]; 0.35-0.97, P <  0.036), but not at the tumor center (HR, 1.35; 95% CI; 0.81-2.26, P = 0.251). CONCLUSIONS: Our study indicates that high Noggin expression is a crucial prognostic factor for favorable outcomes in patients with resected GC.

MRPL15 is a novel prognostic biomarker and therapeutic target for epithelial ovarian cancer.

PURPOSE: To analyze the role of six human epididymis protein 4 (HE4)-related mitochondrial ribosomal proteins (MRPs) in ovarian cancer and selected MRPL15, which is most closely related to the tumorigenesis and prognosis of ovarian cancer, for further analyses. METHODS: Using STRING database and MCODE plugin in Cytoscape, six MRPs were identified among genes that are upregulated in response to HE4 overexpression in epithelial ovarian cancer cells. The Cancer Genome Atlas (TCGA) ovarian cancer, GTEX, Oncomine, and TISIDB were used to analyze the expression of the six MRPs. The prognostic impact and genetic variation of these six MRPs in ovarian cancer were evaluated using Kaplan-Meier Plotter and cBioPortal, respectively. MRPL15 was selected for immunohistochemistry and GEO verification. TCGA ovarian cancer data, gene set enrichment analysis, and Enrichr were used to explore the mechanism of MRPL15 in ovarian cancer. Finally, the relationship between MRPL15 expression and immune subtype, tumor-infiltrating lymphocytes, and immune regulatory factors was analyzed using TCGA ovarian cancer data and TISIDB. RESULTS: Six MRPs (MRPL10, MRPL15, MRPL36, MRPL39, MRPS16, and MRPS31) related to HE4 in ovarian cancer were selected. MRPL15 was highly expressed and amplified in ovarian cancer and was related to the poor prognosis of patients. Mechanism analysis indicated that MRPL15 plays a role in ovarian cancer through pathways such as the cell cycle, DNA repair, and mTOR 1 signaling. High expression of MRPL15 in ovarian cancer may be associated with its amplification and hypomethylation. Additionally, MRPL15 showed the lowest expression in C3 ovarian cancer and was correlated with proliferation of CD8+ T cells and dendritic cells as well as TGFßR1 and IDO1 expression. CONCLUSION: MRPL15 may be a prognostic indicator and therapeutic target for ovarian cancer. Because of its close correlation with HE4, this study provides insights into the mechanism of HE4 in ovarian cancer.

Analysis of the RNA and Protein Complexome by Grad-seq.

The complexome of a cell is the entirety of its complexes. Complexome capture studies have mostly focused on protein-protein interactions, which has left a gap in our knowledge of the global interactions of RNAs. To overcome these limitations, we recently introduced gradient profiling by sequencing (Grad-seq), which analyzes in a high-throughput fashion soluble cellular complexes after their separation in a glycerol gradient by fraction-wise RNA-seq and mass spectrometry. Here, we describe a detailed Grad-seq protocol for Streptococcus pneumoniae, which should also be applicable to other bacterial species.

Aptamer-based approaches to detect nucleolin in prostate cancer.

We have investigated the expression of nucleolin (NCL) in liquid biopsies of prostate cancer (PCa) patients and healthy controls to determine its correlation with tumor prognosis. To detect NCL we used a modified AS1411 aptamer designated by AS1411-N5. In presence of NCL, AS1411-N5 increases the fluorescence by assuming a G-quadruplex (G4) structure, while in the absence of NCL the fluorescence signal remains quenched. The structural characterization of AS1411-N5 was performed by biophysical studies, which demonstrated the formation of G4 parallel conformation in the presence of 100 mM K+ and the ability to recognize NCL with high affinity (KD = 138.1 ±â€¯5.5 nM). Furthermore, the clinical relevance of NCL in PCa liquid biopsies was assessed by using an NCL-based ELISA assay. The protein was measured in the peripheral blood mononuclear cells (PBMCs) cell lysate of 158 individuals, including PCa patients and healthy individuals. The results depicted a remarkable increase of NCL levels in the PBMC's lysate of PCa patients (mean of 626.1 pg/mL whole blood) when compared to healthy individuals (mean of 198.5 pg/mL whole blood). The ELISA results also provided evidence for the usefulness of determining NCL levels in advanced PCa stages. Furthermore, a microfluidic assay showed the ability of AS1411-N5 in recognizing NCL in spiked human plasma samples.

Quantitative STAU2 measurement in lymphocytes for breast cancer risk assessment.

Although mammograms play a key role in early breast cancer detection, the test is not applicable to all women, for example, women under the age of 40. The development of a noninvasive blood test with high sensitivity and accessibility will improve the effectiveness of breast cancer screening programmes. Secretory factors released from cancer cells can induce the expression of certain genes in a large number of white blood cells (WBCs). Therefore, cancer-dependent proteins in WBCs can be used as tumour markers with high sensitivity. Five proteins (LMAN1, AZI2, STAU2, MMP9 and PLOD1) from a systemic analysis of a variety of array data of breast cancer patients were subjected to immunofluorescence staining to evaluate the presence of fixed WBCs on 96-well plates from 363 healthy females and 358 female breast cancer patients. The results revealed that the average fluorescence intensity of anti-STAU2 and the percentage of STAU2-positive T and B lymphocytes in breast cancer patients (110.50 ± 23.38 and 61.87 ± 12.44, respectively) were significantly increased compared with those in healthy females (56.47 ± 32.03 and 33.02 ± 18.10, respectively) (p = 3.56 × 10-71, odds ratio = 24.59, 95% CI = 16.64-36.34). The effect of secreted molecules from breast cancer cells was proven by the increase in STAU2 intensity in PBMCs cocultured with MCF-7 and T47D cells at 48 h (p = 0.0289). The test demonstrated 98.32%, 82.96%, and 48.32% sensitivity and 56.47%, 83.47%, and 98.62% specificity in correlation with the percentage of STAU2-positive cells at 40, 53.34 and 63.38, respectively. We also demonstrated how to use the STAU2 test for the assessment of risk in women under the age of 40. STAU2 is a novel breast cancer marker that can be assessed by quantitative immunofluorescence staining of fixed WBCs that are transportable at room temperature via mail, representing a useful risk assessment tool for women without access to mammograms.

Identification of host factors binding to dengue and Zika virus subgenomic RNA by efficient yeast three-hybrid screens of the human ORFeome.

Flaviviruses such as the dengue (DENV) and the Zika virus (ZIKV) are important human pathogens causing around 100 million symptomatic infections each year. During infection, small subgenomic flavivirus RNAs (sfRNAs) are formed inside the infected host cell as a result of incomplete degradation of the viral RNA genome by cellular exoribonuclease XRN1. Although the full extent of sfRNA functions is to be revealed, these non-coding RNAs are key virulence factors and their detrimental effects on multiple cellular processes seem to consistently involve molecular interactions with RNA-binding proteins (RBPs). Discovery of such sfRNA-binding host-factors has followed established biochemical pull-down approaches skewed towards highly abundant proteins hampering proteome-wide coverage. Yeast three-hybrid (Y3H) systems represent an attractive alternative approach. To facilitate proteome-wide screens for RBP, we revisited and improved existing RNA-Y3H methodology by (1) implementing full-length ORF libraries in combination with (2) efficient yeast mating to increase screening depth and sensitivity, and (3) stringent negative controls to eliminate over-representation of non-specific RNA-binders. These improvements were validated employing the well-characterized interaction between DDX6 (DEAD-box helicase 6) and sfRNA of DENV as paradigm. Our advanced Y3H system was used to screen for human proteins binding to DENV and ZIKV sfRNA, resulting in a list of 69 putative sfRNA-binders, including several previously reported as well as numerous novel RBP host factors. Our methodology requiring no sophisticated infrastructure or analytic pipeline may be employed for the discovery of meaningful RNA-protein interactions at large scale in other fields.

Exploiting the Catalytic Ability of Polydopamine-Remodeling Gold Nanoparticles toward the Naked-Eye Detection of Cancer Cells at a Single-Cell Level.

In this study, a catalytic polydopamine-remodeling gold nanoparticle sensitized with an antinucleolin AS1411 probe (pAu nanoprobe) is synthesized, where the surface of the gold nanoparticles (AuNPs) is modified with a spontaneous self-polymerization of a polydopamine coating that imparts the probe functionalization ability and antispecific protein binding while the intrinsic catalytic property of the AuNPs is preserved. The functionalized AS1411 probe exerts specific recognition with nucleolin protein that is found to be overexpressed on the surface of breast cancer cells (MDA-MB-231). Scanning electron microscopy (SEM) confirms that the specific binding of the pAu nanoprobe occurs at the cancer cell surface. Taking advantage of the catalytic ability of the pAu nanoprobe in reducing blue-colored methylene blue (MB) to colorless leuco-MB, a colorimetric biosensing platform is established based on the accessible catalytic active sites on the pAu nanoprobe toward MB. The specific binding inhibits the pAu nanoprobe from efficiently catalyzing the reduction of MB, resulting in a "turn-off" catalytic biosensing platform. The catalytic conversion of MB is inversely proportional to the concentration of the nucleolin protein and the cancer cells, yielding a detection limit of 15 pM of the nucleolin protein and two cancer cells. The presence of five orders of magnitude higher concentration of bovine serum albumin hardly affects the catalytic ability of the pAu nanoprobe, that is, 88% catalytic ability is still preserved, which validates the specificity of the proposed pAu nanoprobe. In particular, a distinct color contrast creates a significant signal-to-noise ratio so as to enable single-cell level detection of two cancer cells by naked-eye judgment. Moreover, the undiluted, real human serum samples spiked with the cancer cells were examined with an impressive recovery of 94 ± 0.3%, which holds great promise in cancer cell screening.

ANP32A promotes the proliferation, migration and invasion of hepatocellular carcinoma by modulating the HMGA1/STAT3 pathway.

Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) has been reported to play an essential role in the development and progression of various human cancers. However, its expression pattern and possible mechanism in human hepatocellular carcinoma (HCC) remain to be elucidated. In this study, we used western blot and immunohistochemical staining to detect protein expression. The effects of ANP32A on the proliferation, migration and invasion of HCC cells were examined using 5-ethynyl-20-deoxyuridine (EdU), colony formation, CCK-8, and transwell assays. RT-qPCR was performed to detect mRNA expression. The interaction between ANP32A and the high mobility group A1 (HMGA1) mRNA was assessed using RNA immunoprecipitation (RIP). The tumorigenicity of ANP32A was assessed by establishing a xenograft tumor model in Balb/c nude mice. We found that the ANP32A protein was expressed at high levels in patients with HCC, which was associated with a poor prognosis. Functional experiments revealed that the silencing of ANP32A inhibited the proliferation, migration, and invasion of HCC cells, whereas overexpression of ANP32A promoted these processes. Further investigations indicated that ANP32A bound the HMGA1 mRNA and maintained its stability to promote the expression of HMGA1, thereby increasing the expression and activation of STAT3. Finally, a xenograft tumor model of Balb/c nude mice confirmed the tumorigenicity of ANP32A. This study found that ANP32A is up-regulated in patients with HCC and may accelerate the proliferation, migration and invasion of HCC cells by modulating the HMGA1/STAT3 pathway.