Rapid, scalable assay of amylin-ß amyloid co-aggregation in brain tissue and blood.

Islet amyloid polypeptide (amylin) secreted from the pancreas crosses from the blood to the brain parenchyma and forms cerebral mixed amylin-ß amyloid (Aß) plaques in persons with Alzheimer's disease (AD). Cerebral amylin-Aß plaques are found in both sporadic and early-onset familial AD; however, the role of amylin-Aß co-aggregation in potential mechanisms underlying this association remains unknown, in part due to lack of assays for detection of these complexes. Here, we report the development of an ELISA to detect amylin-Aß hetero-oligomers in brain tissue and blood. The amylin-Aß ELISA relies on a monoclonal anti-Aß mid-domain antibody (detection) and a polyclonal anti-amylin antibody (capture) designed to recognize an epitope that is distinct from the high affinity amylin-Aß binding sites. The utility of this assay is supported by the analysis of molecular amylin-Aß codeposition in postmortem brain tissue obtained from persons with and without AD pathology. By using transgenic AD-model rats, we show that this new assay can detect circulating amylin-Aß hetero-oligomers in the blood and is sensitive to their dissociation to monomers. This is important because therapeutic strategies to block amylin-Aß co-aggregation could reduce or delay the development and progression of AD.

Aß efflux impairment and inflammation linked to cerebrovascular accumulation of amyloid-forming amylin secreted from pancreas.

Impairment of vascular pathways of cerebral ß-amyloid (Aß) elimination contributes to Alzheimer disease (AD). Vascular damage is commonly associated with diabetes. Here we show in human tissues and AD-model rats that bloodborne islet amyloid polypeptide (amylin) secreted from the pancreas perturbs cerebral Aß clearance. Blood amylin concentrations are higher in AD than in cognitively unaffected persons. Amyloid-forming amylin accumulates in circulating monocytes and co-deposits with Aß within the brain microvasculature, possibly involving inflammation. In rats, pancreatic expression of amyloid-forming human amylin indeed induces cerebrovascular inflammation and amylin-Aß co-deposits. LRP1-mediated Aß transport across the blood-brain barrier and Aß clearance through interstitial fluid drainage along vascular walls are impaired, as indicated by Aß deposition in perivascular spaces. At the molecular level, cerebrovascular amylin deposits alter immune and hypoxia-related brain gene expression. These converging data from humans and laboratory animals suggest that altering bloodborne amylin could potentially reduce cerebrovascular amylin deposits and Aß pathology.

Advancements in Oncoproteomics Technologies: Treading toward Translation into Clinical Practice.

Proteomics continues to forge significant strides in the discovery of essential biological processes, uncovering valuable information on the identity, global protein abundance, protein modifications, proteoform levels, and signal transduction pathways. Cancer is a complicated and heterogeneous disease, and the onset and progression involve multiple dysregulated proteoforms and their downstream signaling pathways. These are modulated by various factors such as molecular, genetic, tissue, cellular, ethnic/racial, socioeconomic status, environmental, and demographic differences that vary with time. The knowledge of cancer has improved the treatment and clinical management; however, the survival rates have not increased significantly, and cancer remains a major cause of mortality. Oncoproteomics studies help to develop and validate proteomics technologies for routine application in clinical laboratories for (1) diagnostic and prognostic categorization of cancer, (2) real-time monitoring of treatment, (3) assessing drug efficacy and toxicity, (4) therapeutic modulations based on the changes with prognosis and drug resistance, and (5) personalized medication. Investigation of tumor-specific proteomic profiles in conjunction with healthy controls provides crucial information in mechanistic studies on tumorigenesis, metastasis, and drug resistance. This review provides an overview of proteomics technologies that assist the discovery of novel drug targets, biomarkers for early detection, surveillance, prognosis, drug monitoring, and tailoring therapy to the cancer patient. The information gained from such technologies has drastically improved cancer research. We further provide exemplars from recent oncoproteomics applications in the discovery of biomarkers in various cancers, drug discovery, and clinical treatment. Overall, the future of oncoproteomics holds enormous potential for translating technologies from the bench to the bedside.

The association of circulating amylin with ß-amyloid in familial Alzheimer's disease.

INTRODUCTION: This study assessed the hypothesis that circulating human amylin (amyloid-forming) cross-seeds with amyloid beta (Aß) in early Alzheimer's disease (AD). METHODS: Evidence of amylin-AD pathology interaction was tested in brains of 31 familial AD mutation carriers and 20 cognitively unaffected individuals, in cerebrospinal fluid (CSF) (98 diseased and 117 control samples) and in genetic databases. For functional testing, we genetically manipulated amylin secretion in APP/PS1 and non-APP/PS1 rats. RESULTS: Amylin-Aß cross-seeding was identified in AD brains. High CSF amylin levels were associated with decreased CSF Aß42 concentrations. AD risk and amylin gene are not correlated. Suppressed amylin secretion protected APP/PS1 rats against AD-associated effects. In contrast, hypersecretion or intravenous injection of human amylin in APP/PS1 rats exacerbated AD-like pathology through disruption of CSF-brain Aß exchange and amylin-Aß cross-seeding. DISCUSSION: These findings strengthened the hypothesis of circulating amylin-AD interaction and suggest that modulation of blood amylin levels may alter Aß-related pathology/symptoms.

Recent technological advancements in stem cell research for targeted therapeutics.

Stem cells have characteristic features of self-renewal, pluripotency and differentiation, which are responsible for replenishment of tissue or organ. Stem cells are potentiated as therapeutic tool in drug targeting and regenerative medicine-from curing various neurological diseases and malignancies to congenital diseases. These technological advancements have established stem cells as future of medicine. However, due to ethico-social limitations, the use of embryonic stem cells (ESCs) has been avoided, while physiological availability of adult stem cells (ASCs) and induced pluripotent stem cells (iPSCs) has gained appropriate preference. These iPSCs are very much similar to ESCs in terms of their self-renewal and pluripotency. Here, we have summarized the technologies that have established stem cells isolation, their molecular marker and factors responsible for their maintenance. Different cellular (transcription factors, regulatory proteins, miRNA like miRNA-296, miRNA-145, etc.) and extracellular components transcend stem cell fate. Their identification and characterization involve development and efficient utilization of tools like magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS). Some of the technologies have been patented and spin-off's based on them have been commercialized. In conclusion, we present the future scope and possibilities that stem cell technologies behold for us. Graphical abstract Pictorial representation of therapeutic approaches for disease treatment using stem cell technology. Disease-specific adult stem cells are isolated along with niche cells by utilizing tools like FACS/MACS/LCM, etc. Thereafter, cells are reprogrammed through introduction of Yamanaka factors (Oct3/4, Sox2, c-myc, Klf4) to make induced pluripotent stem cell (iPSCs). The disease-specific iPSCs undergo genetic modification after delivery of therapeutic gene through retroviral vehicle. The genetically modified cells are introduced back in person with disease for therapeutic effects. FACS, fluorescence activated cell sorting; MACS, magnetic-activated cell sorting; LCM, laser capture microdissection; Oct3/4, octamer-binding transcription factor 3/4; Sox2, sex determining region Y-box 2; Klf4, Kruppel-like factor 4.

Role of PXR in Hepatic Cancer: Its Influences on Liver Detoxification Capacity and Cancer Progression.

The role of nuclear receptor PXR in detoxification and clearance of xenobiotics and endobiotics is well-established. However, its projected role in hepatic cancer is rather illusive where its expression is reported altered in different cancers depending on the tissue-type and microenvironment. The expression of PXR, its target genes and their biological or clinical significance have not been examined in hepatic cancer. In the present study, by generating DEN-induced hepatic cancer in mice, we report that the expression of PXR and its target genes CYP3A11 and GSTa2 are down-regulated implying impairment of hepatic detoxification capacity. A higher state of inflammation was observed in liver cancer tissues as evident from upregulation of inflammatory cytokines IL-6 and TNF-α along with NF-κB and STAT3. Our data in mouse model suggested a negative correlation between down-regulation of PXR and its target genes with that of higher expression of inflammatory proteins (like IL-6, TNF-α, NF-κB). In conjunction, our findings with relevant cell culture based assays showed that higher expression of PXR is involved in reduction of tumorigenic potential in hepatic cancer. Overall, the findings suggest that inflammation influences the expression of hepatic proteins important in drug metabolism while higher PXR level reduces tumorigenic potential in hepatic cancer.

Transcription regulation of nuclear receptor PXR: Role of SUMO-1 modification and NDSM in receptor function.

Pregnane & Xenobiotic Receptor (PXR) is one of the 48 members of the nuclear receptor superfamily of ligand-modulated transcription factors. PXR plays an important role in metabolism and elimination of diverse noxious endobiotics and xenobiotics. Like in case of some nuclear receptors its function may also be differentially altered, positively or negatively, by various post-translational modifications. In this context, regulation of PXR function by SUMOylation is the subject of present investigation. Here, we report that human PXR is modified by SUMO-1 resulting in its enhanced transcriptional activity. RT-PCR analysis showed that PXR SUMOylation in presence of rifampicin also enhances the endogenous expression levels of key PXR-regulated genes like CYP3A4, CYP2C9, MDR1 and UGT1A1. In addition, mammalian two-hybrid assay exhibited enhanced interaction between PXR and co-activator SRC-1. EMSA results revealed that SUMOylation has no influence on the DNA binding ability of PXR. In silico analysis suggested that PXR protein contains four putative SUMOylation sites, centered at K108, K129, K160 and K170. In addition to this, we identified the presence of NDSM (Negative charge amino acid Dependent SUMOylation Motif) in PXR. Substitution of all its four putative lysine residues along with NDSM abolished the effect of SUMO-1-mediated transactivation function of PXR. Furthermore, we show that interaction between PXR and E2-conjugation enzyme UBCh9, an important step for implementation of SUMOylation event, was reduced in case of NDSM mutant PXRD115A. Overall, our results suggest that SUMOylation at specific sites on PXR protein are involved in enhancement of transcription function of this receptor.