Substituent Effects Impact Surface Charge and Aggregation of Thiophenol-Labeled Gold Nanoparticles for SERS Biosensors.

SERS immunoassay biosensors hold immense potential for clinical diagnostics due to their high sensitivity and growing interest in multi-marker panels. However, their development has been hindered by difficulties in designing compatible extrinsic Raman labels. Prior studies have largely focused on spectroscopic characteristics in selecting Raman reporter molecules (RRMs) for multiplexing since the presence of well-differentiated spectra is essential for simultaneous detection. However, these candidates often induce aggregation of the gold nanoparticles used as SERS nanotags despite their similarity to other effective RRMs. Thus, an improved understanding of factors affecting the aggregation of RRM-coated gold nanoparticles is needed. Substituent electronic effects on particle stability were investigated using various para-substituted thiophenols. The inductive and resonant effects of functional group modifications were strongly correlated with nanoparticle surface charge and hence their stability. Treatment with thiophenols diminished the negative surface charge of citrate-stabilized gold nanoparticles, but electron-withdrawing substituents limited the magnitude of this diminishment. It is proposed that this phenomenon arises by affecting the interplay of competing sulfur binding modes. This has wide-reaching implications for the design of biosensors using thiol-modified gold surfaces. A proof-of-concept multiplexed SERS biosensor was designed according to these findings using the two thiophenol compounds with the most electron-withdrawing substitutions: NO2 and CN.

Tumor Biomarker In-Solution Quantification, Standard Production, and Multiplex Detection.

The diagnosis and monitoring of cancer have been facilitated by discovering tumor "biomarkers" and methods to detect their presence. Yet, for certain cancers, we still lack sensitive and specific biomarkers or the means to quantify subtle concentration changes successfully. The identification of new biomarkers of disease and improving the sensitivity of detection will remain key to changing clinical outcomes. Patient liquid biopsies (serum and plasma) are the most easily obtained sources for noninvasive analysis of proteins that tumor cells release directly and via extracellular microvesicles and tumor shedding. Therefore, an emphasis on creating reliable assays using serum/plasma and "direct, in-solution" ELISA approaches has built an industry centered on patient protein biomarker analysis. A need for improved dynamic range and automation has resulted in the application of ELISA principles to paramagnetic beads with chemiluminescent or fluorescent detection. In the clinical testing lab, chemiluminescent paramagnetic assays are run on automated machines that test a single analyte, minimize technical variation, and are not limited by serum sample volumes. This differs slightly from the R&D setting, where serum samples are often limiting; therefore, multiplexing antibodies to test multiple biomarkers in low serum volumes may be preferred. This review summarizes the development of historical biomarker "standards", paramagnetic particle assay principles, chemiluminescent or fluorescent biomarker detection advancements, and multiplexing for sensitive detection of novel serum biomarkers.

Characterization of recombinant ß subunit of human MUC4 mucin (rMUC4ß).

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like ß-subunit. Due to the presence of several functional domains, the characterization of MUC4ß is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4ß (rMUC4ß). Purified rMUC4ß was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical ß-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4ß physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4ß that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

Acinar transformed ductal cells exhibit differential mucin expression in a tamoxifen-induced pancreatic ductal adenocarcinoma mouse model.

Pancreatic cancer (PC) is acquired postnatally; to mimic this scenario, we developed an inducible KrasG12D; Ptf1a-CreER™ (iKC) mouse model, in which Kras is activated postnatally at week 16 upon tamoxifen (TAM) administration. Upon TAM treatment, iKC mice develop pancreatic intraepithelial neoplasia (PanIN) lesions and PC with metastasis at the fourth and fortieth weeks, respectively, and exhibited acinar-to-ductal metaplasia (ADM) and transdifferentiation. Kras activation upregulated the transcription factors Ncoa3, p-cJun and FoxM1, which in turn upregulated expression of transmembrane mucins (Muc1, Muc4 and Muc16) and secretory mucin (Muc5Ac). Interestingly, knockdown of KrasG12D in multiple PC cell lines resulted in downregulation of MUC1, MUC4, MUC5AC and MUC16. In addition, iKC mice exhibited ADM and transdifferentiation. Our results show that the iKC mouse more closely mimics human PC development and can be used to investigate pancreatic ductal adenocarcinoma (PDAC) biomarkers, early onset of PDAC, and ADM. The iKC model can also be used for preclinical strategies such as targeting mucin axis alone or in combination with neo-adjuvant, immunotherapeutic approaches and to monitor chemotherapy response.

Dual delivery nanoscale device for miR-345 and gemcitabine co-delivery to treat pancreatic cancer.

A polymeric dual delivery nanoscale device (DDND) was designed for combined delivery of microRNA (miR-345) and gemcitabine (GEM) to treat pancreatic cancer (PC). This temperature and pH-responsive pentablock copolymer system was able to restore miR-345, making xenograft tumors more susceptible to GEM, the standard therapy for PC. Restoration using DDND treatment results in sonic hedgehog signaling down regulation, which decreases desmoplasia, thereby resulting in improved GEM perfusion to the tumor and better therapeutic outcomes. The release of miR-345 and GEM could be tuned by using the DDND in the form of micelles or in the form of thermoreversible gels, based on polymer concentration. The DDNDs enabled miR-345 stability and sustained co-release of miR-345 and GEM, thereby facilitating dose-sparing use of GEM. Further, enhanced in vitro cellular uptake due to amphiphilic character, and endosomal escape because of the cationic end blocks led to efficient transfection with DDNDs. The combined DDND treatment enabled efficient reduction in cell viability of Capan-1 and CD18/HPAF cells in vitro compared with either GEM or miR-345 treatment alone. Mice carrying xenograft tumors treated with DDNDs carrying both miR-345 and GEM combination therapy displayed reduced tumor growth and less metastasis in distant organs compared to individual drug treatments. Immunohistochemical analysis of the xenograft tissues revealed significant down regulation of desmoplastic reaction, SHH, Gli-1, MUC4, and Ki67 compared to control groups.

Pancreatic cancer associated with obesity and diabetes: an alternative approach for its targeting.

BACKGROUND: Pancreatic cancer (PC) is among foremost causes of cancer related deaths worldwide due to generic symptoms, lack of effective screening strategies and resistance to chemo- and radiotherapies. The risk factors associated with PC include several metabolic disorders such as obesity, insulin resistance and type 2 diabetes mellitus (T2DM). Studies have shown that obesity and T2DM are associated with PC pathogenesis; however, their role in PC initiation and development remains obscure. MAIN BODY: Several biochemical and physiological factors associated with obesity and/or T2DM including adipokines, inflammatory mediators, and altered microbiome are involved in PC progression and metastasis albeit by different molecular mechanisms. Deep understanding of these factors and causal relationship between factors and altered signaling pathways will facilitate deconvolution of disease complexity as well as lead to development of novel therapies. In the present review, we focuses on the interplay between adipocytokines, gut microbiota, adrenomedullin, hyaluronan, vanin and matrix metalloproteinase affected by metabolic alteration and pancreatic tumor progression. CONCLUSIONS: Metabolic diseases, such as obesity and T2DM, contribute PC development through altered metabolic pathways. Delineating key players in oncogenic development in pancreas due to metabolic disorder could be a beneficial strategy to combat cancers associated with metabolic diseases in particular, PC.

MUC16 as a novel target for cancer therapy.

INTRODUCTION: MUC16 is overexpressed in multiple cancers and plays an important role in tumorigenicity and acquired resistance to therapy. Area covered: In this review, we describe the role of MUC16 under normal physiological conditions and during tumorigenesis. First, we provide a summary of research on MUC16 from its discovery as CA125 to present anti-MUC16 therapy trials that are currently in the initial phases of clinical testing. Finally, we discuss the reasons for the limited effectiveness of these therapies and discuss the direction and focus of future research. Expert opinion: Apart from its protective role in normal physiology, MUC16 contributes to disease progression and metastasis in several malignancies. Due to its aberrant overexpression, it is a promising target for diagnosis and therapy. Cleavage and shedding of its extracellular domain is the major barrier for efficient targeting of MUC16-expressing cancers. Concerted efforts should be undertaken to target the noncleaved cell surface retained portion of MUC16. Such efforts should be accompanied by basic research to understand MUC16 cleavage and decipher the functioning of MUC16 cytoplasmic tail. While previous efforts to activate anti-MUC16 immune response using anti-CA125 idiotype antibodies have met with limited success, ideification of neo-antigenic epitopes in MUC16 that correlate with improved survival have raised raised hopes for developing MUC16-targeted immunotherapy.

Development and characterization of carboxy-terminus specific monoclonal antibodies for understanding MUC16 cleavage in human ovarian cancer.

MUC16 is overexpressed in ovarian cancer and plays important roles in invasion and metastasis. Previously described monoclonal antibodies against cell surface expressed MUC16 recognize the N-terminal tandemly repeated epitopes present in cancer antigen 125 (CA125). MUC16 is cleaved at a specific location, thus, releasing CA125 into the extracellular space. Recent reports have indicated that the retained carboxy-terminal (CT) fragment of MUC16 might play an important role in tumorigenicity in diverse types of cancers. However, limited data is available on the fate and existence of CT fragment on the surface of the cancer cell. Herein, we characterize two monoclonal antibodies (mAbs) showing specificity to the retained juxtamembrane region of MUC16. For the first time, we demonstrate that MUC16 is cleaved in ovarian cancer cells (NIH:OVCAR-3 [OVCAR-3]) and that the cleaved MUC16 subunits remain associated with each other. Immunohistochemical analyses on different grades of ovarian tumor tissues indicated differential reactivity of CA125 and MUC16 CT mAbs. The CA125 (M11) mAb detected 32/40 (80%), while the CT mAb (5E6) detected 33/40 (82.5%) of total ovarian cancer cases. For serous and serous papillary cases, the CA125 (M11) mAb stained 27/31 cases (87%), while CT mAb (5E6) stained 29/31 cases (93.5%). The CT mAb(s) accurately predict expression of MUC16 since their epitopes are not tandemly repeated and their reactivity may not be dependent on O-linked glycosylation. These antibodies can serve as valuable reagents for understanding MUC16 cleavage and may also serve as potential therapeutic agents for treatment of ovarian cancer.

Characterization of human mucin MUC17. Complete coding sequence and organization.

With increasing interest on mucins as diagnostic and therapeutic targets in cancers and other diseases, it is becoming imperative to characterize novel mucins and investigate their biological significance. Here, we present the completed coding sequence and genomic organization of the previously published partial cDNA sequence of MUC17. Rapid amplification of cDNA ends with PCR, sequences from the Human Genome databases, and in vitro transcription/translational assays were used for these analyses. The MUC17 gene is located within a 39-kb DNA fragment between MUC12 and SERPINE1 on chromosome 7 in the region q22.1. The full-length coding sequence of MUC17 transcribes a 14.2-kb mRNA encompassing 13 exons. Alternate splicing generates two variants coding for a membrane-anchored and a secreted form. The canonical variable number of tandem repeats polymorphism of the central tandem repeat domain of the MUC genes is not significantly detected in the MUC17 gene. In addition, we show the overexpression of MUC17 by Western blot and immunohistochemical analyses in pancreatic tumor cell lines and tumor tissues compared with the normal pancreas. The expression of MUC17 is regulated by a 1,146-bp fragment upstream of MUC17 that contains VDR/RXR, GATA, NFkappaB, and Cdx-2 response elements.

Gene expression profiling within the developing neural tube.

The developing mammalian nervous system is subject to devastating congenital malformations with clinical significance that extends into the billions of health care dollars annually worldwide. Neural tube defects (NTDs) are among the most common of all human congenital defects, yet their etiology remains poorly understood. This is largely due to the complexity of the genetic factors regulating the intricate events involved in neurulation. Using mouse model systems and the application of modern molecular biological technologies, we have recently gained a greater appreciation for the factors that not only regulate normal neural tube closure (NTC), but those genetic factors that predispose an embryo to significant birth defects such as anencephaly or spina bifida. We have selected prominent murine mutants, both spontaneous and genetically modified, as well as the use of teratogenic agents, to examine the impact of altering the normal pattern of gene expression in the developing neural tube.