Confocal Endomicroscopy Characteristics of Different Intraductal Papillary Mucinous Neoplasm Subtypes

Intraductal papillary mucinous neoplasms are classified into gastric, intestinal, pancreatobiliary, and oncocytic subtypes where morphology portends disease prognosis. The study aim was to demonstrate EUS-guided needle-based confocal laser endomicroscopy imaging features of intraductal papillary mucinous neoplasm subtypes. Four subjects, each with a specific intraductal papillary mucinous neoplasm subtype were enrolled. An EUS-guided needle-based confocal laser endomicroscopy miniprobe was utilized for image acquisition. The mean cyst size from the 4 subjects (2 females; mean age = 65.3±12 years) was 36.8±12 mm. All lesions demonstrated mural nodules and focal dilation of the main pancreatic duct. EUS-nCLE demonstrated characteristic finger-like papillae with inner vascular core for all subtypes. The image patterns of the papillae for the gastric, intestinal, and pancreatobiliary subtypes were similar. However, the papillae in the oncocytic subtype were thick and demonstrated a fine scale-like or honeycomb pattern with intraepithelial lumina correlating with histopathology. There was significant overlap in the needle-based confocal laser endomicroscopy findings for the different intraductal papillary mucinous neoplasm subtypes; however, the oncocytic subtype demonstrated distinct patterns. These findings need to be replicated in larger multicenter studies

Activation of JNK Signaling Mediates Amyloid-ß-Dependent Cell Death

Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimer's neuropathology is the generation of large aggregates of Aß42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood.We employed a Drosophila eye model of AD to study how Aß42 causes cell death. Misexpression of higher levels of Aß42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. We found that blocking caspase-dependent cell death initially blocked cell death but did not lead to a significant rescue in the adult eye. However, blocking the levels of c-Jun NH(2)-terminal kinase (JNK) signaling pathway significantly rescued the neurodegeneration phenotype of Aß42 misexpression both in eye imaginal disc as well as the adult eye. Misexpression of Aß42 induced transcriptional upregulation of puckered (puc), a downstream target and functional read out of JNK signaling. Moreover, a three-fold increase in phospho-Jun (activated Jun) protein levels was seen in Aß42 retina as compared to the wild-type retina. When we blocked both caspases and JNK signaling simultaneously in the fly retina, the rescue of the neurodegenerative phenotype is comparable to that caused by blocking JNK signaling pathway alone.Our data suggests that (i) accumulation of Aß42 plaques induces JNK signaling in neurons and (ii) induction of JNK contributes to Aß42 mediated cell death. Therefore, inappropriate JNK activation may indeed be relevant to the AD neuropathology, thus making JNK a key target for AD therapies

An appraisal of pancreatic cyst fluid molecular markers

Pancreatic malignancy is the third leading cause of cancer related death in the United States with limited viable screening options. By the end of this decade, cancers are poised to become the leading cause of death with pancreatic cancer projected to be the second leading cause of cancer related mortality. Pancreatic cystic lesions (PCLs) are found in approximately 5%–14% of patients due to the increased utilization of cross-sectional imaging, with approximately 8%–10% of pancreatic cancers originating as PCLs. Current screening guidelines have shown discrepancies between morphologic characteristics of PCLs and identifying advanced pancreatic disease. Molecular analysis has emerged as a novel technology to aid in adequate diagnosis and management decisions of PCLs. Mucinous cysts including intraductal papillary mucinous neoplasms (IPMNs) or mucinous cystic neoplasms have similar oncogenic mutations including KRAS, TP53, SMAD4, PIK3CA, PTEN, or CKDN2A, while GNAS and RNF43 mutations are specific only to IPMNs. Serous cystadenomas have been associated with a loss of tumor suppressor gene VHL, while solid-psuedopapillary neoplasms have an oncogenic mutation CTNNB1. A specific molecular marker to diagnose existing high-grade dysplasia or impending malignant transformation is yet to be identified. Moving forward it is important to advance technology in isolating and identifying high-risk molecular markers from cyst fluid while considering their increased utilization in the evaluation of PCLs

Modulating <i>crb</i> levels in the Aβ42 background leads to defects in axonal targeting from retina to the brain.

<p>(A) Wild Type eye disc stained with sensory neuron marker, Chaoptin (24B10) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078717#pone.0078717-Zipursky1" target="_blank">[34]</a>, which marks only photoreceptor neurons and their axons. The photoreceptor neurons extends through the optic stalk and innervate the medulla and lamina of the larval brain. Note that misexpression of Aβ42 (GMR>Aβ42) in the eye imaginal discs, (B) there is mislocalization of 24B10 expression showing aberrant axonal targeting from retina to brain. The retinal axons fail to innervate the two layers of the brain and end abruptly. (C) Misexpression of Crb full length (FL) in the GMR>Aβ42 background (GMR>Aβ42+Crb FL) strongly enhances the neurodegeneration phenotype which results in (C) lack of axonal targeting from retina to brain. Reducing Crb levels by using (D) <i>crb^11A22</i> allele <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078717#pone.0078717-Johnson1" target="_blank">[33]</a> (GMR>Aβ42+<i>crb^11A22</i>) or (E) <i>crb</i> RNAi (VDRC) (GMR>Aβ42+RNAi) result in the significant rescue of GMR>Aβ42 mediated neurodegeneration as evident from the (D, E) restoration of retinal axon targeting.</p

Misexpression of Crb intracellular domain (ICD) can impair axonal targeting.

<p>The eye-antennal disc is stained with Elav (red), which marks the photoreceptors, and 24B10 (chaoptin; green), which stains the axons from the retina to the brain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078717#pone.0078717-Zipursky1" target="_blank">[34]</a>. (A, B) Misexpression of intact instar cellular domain ICD GMR>Aβ42+Crb^intra results in clumping of photoreceptors (Elav; A), disorganization of axonal targeting from the retina to the brain as evident from 24B10 staining. (C, D)When the JM motif using GMR>Aβ42+Crb^{intra ΔJM}, photoreceptor organization as well as the axonal targeting is restored to the wild type. Similarly, removing the (E, F) PBM motif of the ICD using GMR>Aβ42+Crb^{intra ΔPBM}, or both PBM and JM domain in GMR>Aβ42+Crb^{intra ΔJMΔPBM}, result in restoration of axonal targeting and photoreceptors. Thus, ICD domain of Crb is required for its role in neurodegeneration.</p

Downregulation of <i>crb</i> can block neurodegeneration in the Aβ42 background.

<p>TUNEL assays are commonly employed to mark the cells undergoing cell death where the cleavage of double and single stranded DNA is labeled by a Fluorescein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078717#pone.0078717-White1" target="_blank">[31]</a>. (A) Wild type eye imaginal disc showing a few TUNEL positive nuclei. (B) Misexpression of Aβ42 using GMR-Gal4 driver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078717#pone.0078717-Moses1" target="_blank">[22]</a> in the differentiating photoreceptor neurons results in induction of neurodegeneration (B) as seen by a three-fold induction of cell death as evident from number of TUNEL positive nuclei of the dying cells in comparison to (A) wild type eye imaginal disc. Misexpression of Crb full length (FL) in GMR>Aβ42 background (GMR>Aβ42+Crb FL) strongly enhances (C) the neurodegeneration phenotype which results in nearly seven fold increase in number of TUNEL positive nuclei of dying cells in comparison to wild type eye imaginal disc. (D, E) Reducing Crb levels by using (D) <i>crb^11A22</i> mutant allele <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078717#pone.0078717-Johnson1" target="_blank">[33]</a> (GMR>Aβ42+<i>crb^11A22</i>) or (E) <i>crb</i> RNAi (VDRC) (GMR>Aβ42+RNAi) result in the rescue of GMR>Aβ42 mediated neurodegeneration as evident from reduction in numbers of TUNEL positive nuclei of the dying cells. (F) Quantitatively, the number of TUNEL cells have been counted and recorded with all five constructs shown. These phenotypes of enhancement of neurodegenerative phenotype and rescue, based on the number of TUNEL positive cells, are significant as seen by the calculation of P-values based on the one-tailed t-test using Microsoft Excel 2010.</p

Intracellular domain (ICD) of Crb is required for Aβ42 mediated neurodegeneration.

<p>(A) A cartoon depicting full length type I transmembrane Crb protein and various truncated constructs used in this study. The full length Crb protein consists of an extracellular domain (ECD), transmembrane domain (TM), and a short cytoplasmic intracellular domain (ICD), which consists of the juxtamembrane Ferm-binding motif (JM) and PDZ-binding motif (PBM) domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078717#pone.0078717-Klebes1" target="_blank">[28]</a>. GMR-Gal4 driver was used for the misexpression studies in the differentiating photoreceptor neurons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078717#pone.0078717-Moses1" target="_blank">[22]</a>. (B–D) Adult eyes of (B) Wild-Type, (C) GMR>Aβ42 (GMR enhancer driving overexpression of human Aβ42 in the developing neural retina), and (D) GMR>Crb (FL) are shown as controls. (A, E–F) Misexpression of (E) Crb^intra alone, comprising of fully intact ICD, shows a severe phenotype with a small scab on the head cuticle in the adult eye, which is similar to the (F) GMR>Aβ42+Crb^intra adult eye. (G) In the GMR>Aβ42+Crb^intra eye disc big gaps and holes between photoreceptors of the ommatidia are seen, Dlg (white) marks the membrane and provide an outline of the imaginal disc and pan neural marker Elav <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078717#pone.0078717-Dhanasekaran1" target="_blank">[52]</a> marks the photoreceptors. (A, H–P) In the three other Crb constructs, one of the two domains (JM and PBM) of the ICD is either missing or both of them are missing. (H–P) When Crb is missing either (H–J) PBM, or (K–M) JM, or (N–P) both the PBM and JM domain of the ICD, the GMR>Aβ42 neurodegenerative phenotype is restored significantly with the adult eye having a larger size, higher number of ommatidia, and interommatidial bristles. Furthermore, the Elav staining in the eye-imaginal discs shows more organized photoreceptors in comparison to the GMR>Aβ42 eye imaginal disc. (H, K, N) The controls (H) GMR>Crb^{intra ΔPBM}, (K) GMR>Crb^{intra ΔJM}, and (N) GMR>Crb^{intra ΔPBM ΔJM} showed adult eye phenotypes that are significantly closer to the wild-type. (I, J) When the PBM domain (GMR>Aβ42+Crb^{intra ΔPBM}) is missing, (I) the adult eye and (J) the eye imaginal disc showed significant rescue in comparison to the GMR>Aβ42 phenotype. (L, M) When the JM domain (GMR>Aβ42+Crb^{intra ΔJM}) is missing, (L) the adult eye and (M) the eye-imaginal disc showed significant rescue in comparison to the GMR>Aβ42 phenotype. (O, P) Finally, when both PBM and JM domains of the ICD are missing (GMR>Aβ42+Crb^{intra ΔPBM ΔJM}), a significant rescue was seen in (O) the adult eye and (P) the eye imaginal disc in comparison to the GMR>Aβ42 phenotype.</p