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Although uncommon, extrahepatic cholangiocarcinoma (EHCC) is a deadly malignancy, and the treatment approaches remain controversial. While surgery remains the only cure, few patients are candidates for resection up front, and there are high rates of both local and distant failure following resection. Herein, we systematically review the available evidence regarding treatment approaches for patients with EHCC, including surgery, radiation, and chemotherapy. The evidence regarding treatment outcomes was assessed using the Population, Intervention, Comparator, Outcome, and Study design (PICOS) framework. A summary of recommendations based on the available literature is outlined for specific clinical scenarios encountered by providers in the clinic to guide the management of these patients.

Background: Multimodal treatment strategy including perioperative chemotherapy (PEC), postoperative chemoradiation therapy (POCR), and postoperative chemotherapy (POC) has been accepted as the standard of care in gastric cancer (GC). The ideal sequence and type of therapy remain undetermined. Method: The National Cancer Database was examined from 2006 to 2016 to identify patients with resectable non-cardia gastric cancer. Patient outcomes were compared based on the receipt of PEC, POCR, and POC. This comparison was repeated in a sub-group of patients who received optimal treatment. Optimal treatment was defined as initial chemotherapy within 45 days of diagnosis, resection within 45 days of diagnosis, negative margins, adjuvant chemotherapy within 90 days of resection and standard radiation dose (45 Gy). Kaplan–Meier test, log-rank test, and multivariable analysis (MVA) were performed. Results: We identified 9589 patients. Median survival was greater in the PEC group followed by POCR and POC (60.6, 42.3, and 31.2 months, respectively). On MVA, factors associated with worse overall survival included age above median (≥ 63 years), Charlson–Deyo score of ≥ 1, non-academic/research program, poorly differentiated/undifferentiated grade, positive margins, and positive lymph nodes. Both PEC and POCR were associated with improved survival when compared to POC (HR 0.78 and 0.79; p< 0.001). When compared with PEC, no significant difference was noted with POCR (HR 1.01; p= 0.987). These results were maintained in optimally treated cohort (n= 3418). Conclusion: In patients with resectable non-cardia gastric cancer, both perioperative chemotherapy and postoperative chemoradiation therapy were associated with improved survival when compared to postoperative chemotherapy. No difference was noted between perioperative chemotherapy and postoperative chemoradiation therapy. These results were maintained in the optimally treated cohort. Graphical abstract:

Background: More complex cases are being performed robotically. This study aims to characterize trends in robotic pancreatoduodenectomy (RPD) over time and assess opportunities for advanced trainees. Methods: Using the ACS-NSQIP database from 2014 to 2019, PD cases were characterized by operative approach (open-OPN, laparoscopic-LAP, robotic-ROB). Proficiency and postoperative outcomes were described by approach over time. Results: 24,268 PDs were identified, with the ROB approach increasing from 2.8% to 7.5%. Unplanned conversion increased over time for LAP (27.7–39.0%, p= 0.003) but was unchanged for ROB cases (14.8–14.7%, p= 0.257). Morbidity increased for OPN PD (35.5–36.8%, p= 0.041) and decreased for ROB PD (38.7–30.3%, p= 0.010). Mean LOS was lower in ROB than LAP/OPN (9.5 vs. 10.9 vs. 10.9 days, p< 0.00001). Approximately, 100 AHPBA, SSO, and ASTS fellows are being trained each year in North America; however, only about 5 RPDs are available per trainee per year which is far below that recommended to achieve proficiency. Conclusion: Over a 6-year period, a significant increase was observed in the use of RPD without a concomitant increase in conversion rates. RPD was associated with decreased morbidity and length of stay. Despite this shift, the number of cases being performed is not adequate for all fellows to achieve proficiency before graduation.

Background The 5-year overall survival (OS) rate for patients with metastatic gastric cancer (mGC) is 5.3%. Surgery for mGC is controversial.Methods We identified all mGC patients who received chemotherapy using the National Cancer Database (2004-2015). Patients were grouped according to surgery of: (1) the primary site (PS) only, (2) primary and distant sites (PDS), (3) distant site only (DS), or (4) no surgery (NS). A propensity score adjustment and multivariate regression was used to compare OS.Results Overall, 18,772 patients met the inclusion criteria: (1) PS (n = 962, 5.1%), (2) PDS (n = 380, 2.1%), (3) DS (n = 984, 5.2%), and 16,446 NS (87.6%). Surgery was associated with improved OS in the PS and PDS groups (hazard ratios: .489 (95% CI: .376-.636); .583 (95% CI: .420-.811), P< .001) (median OS 15.8 and 15.9 months vs 8.6 for NS patients, respectively).Conclusions Gastrectomy with or without metastasectomy is associated with improved survival in stage IV gastric cancer patients receiving chemotherapy. This warrants further prospective studies.

Glycine is an important biomarker in clinical analysis due to its involvement in multiple physiological processes. As such, the need for low-cost analytical tools for glycine detection is growing. As a neurotransmitter, glycine is involved in inhibitory and excitatory neurochemical transmission in the central nervous system. In this work, we present a 10 μM Pt-based electrochemical enzymatic biosensor based on the flavoenzyme glycine oxidase (GO) for localized real-time measurements of glycine. Among GO variants at position 244, the H244K variant with increased glycine turnover was selected to develop a functional biosensor. This biosensor relies on amperometric readouts and does not require additional redox mediators. The biosensor was characterized and applied for glycine detection from cells, mainly HEK 293 cells and primary rat astrocytes. We have identified an enzyme, GO H244K, with increased glycine turnover using mutagenesis but which can be developed into a functional biosensor. Noteworthy, a glycine release of 395.7 ± 123 μM from primary astrocytes was measured, which is ∼fivefold higher than glycine release from HEK 293 cells (75.4 ± 3.91 μM) using the GO H244K biosensor.

Neoadjuvant therapy (NAT) for T1/T2 pancreatic adenocarcinoma (PDAC) prior to pancreaticoduodenectomy remains controversial. We compared positive margin rates in patients with clinical T1&T2 tumors who did and did not receive NAT.

Remyelination in the human CNS is ascribed to progenitor cells rather than previously myelinating oligodendrocytes (OLs). The ganglioside-recognizing antibody A2B5 has been used to isolate putative progenitor cells, whose in vitro features resemble cells labeled as "pre-oligodendrocytes." Here, we compare the transcriptional profiles of adult human brain-derived A2B5 antibody-selected cells (A+) after initial isolation (day in vitro (DIV1)) and after DIV6, with nonselected (A-) cells (mature OLs), with regard to their differentiation state and functional properties. While a number of previously recognized progenitor associated genes, specifically PTPRZ1 and PDGFRα, were upregulated in the A2B5+ population, a number of such genes were comparably expressed in the mature OLs, as were mature myelin genes. Additional progenitor-related genes were upregulated in the A+ population. We show that A2B5+ cells have greater capacity to ensheath nanofibers, a model of myelination potential; consistent with this, ingenuity pathway analysis indicated that A+ cells had upregulated expression of genes within cell growth and cell signaling pathways. Differential expression of cell death/survival pathways complements previous functional studies showing their increased susceptibility to metabolic stress. At DIV6, we observed significantly fewer differentially expressed genes; suggestive of cell maturation occurring in vitro, indicating the complexity in comparing in vitro and in situ cell properties.

The ability of the mammalian brain to maintain spatial representations of external or internal information for short periods of time has been associated with sustained neuronal spiking and reverberatory neural network activity in the medial entorhinal cortex. Here, we show that conditional genetic deletion of netrin-1 or the netrin receptor deleted-in-colorectal cancer (DCC) from forebrain excitatory neurons leads to deficits in short-term spatial memory. We then demonstrate that conditional deletion of either netrin-1 or DCC inhibits cholinergic persistent firing and show that cholinergic activation of muscarinic receptors expressed by entorhinal cortical neurons promotes persistent firing by recruiting DCC to the plasma membrane. Together, these findings indicate that normal short-term spatial memory function requires the synergistic actions of acetylcholine and netrin-1.

Background: There are no currently approved methods for the screening and early detection of lung cancer. We compared the ability of conventional white-light bronchoscopy (WLB) and laser-induced fluorescence endoscopy (LIFE) to detect preneoplastic lung lesions in a randomized trial in which both the order of the procedures and the bronchoscopists were randomly assigned. Methods: The study included high-risk subjects enrolled because of a cigarette smoking history of at least 30 pack-years, an air-flow obstruction, and either an abnormal sputum cytology (n = 48) or a previous or suspected lung cancer (n = 7). LIFE and WLB were performed on all patients. Biopsy specimens were assessed for histologic abnormalities, including the presence of angiogenic squamous dysplasia. All statistical tests were two-sided. Results: A total of 391 biopsy specimens were taken from the 55 patients. Thirty-two patients (58%; 95% confidence interval [CI] = 44% to 71%) had at least one biopsy with moderate or severe dysplasia, and 19 (59%; 95 % CI = 41% to 76%) of these patients could be diagnosed based solely on the results of LIFE. LIFE was statistically significantly more sensitive than WLB for detecting moderate dysplasia or worse (68.8% versus 21.9%, respectively) (difference = 46.9%; 95% CI = 25% to 68%; P [is less than] .001). The relative sensitivities (WLB = 1.0) were 3.1 (95% CI = 1.6 to 6.3) for LIFE and 3.7 (95% CI = 1.9 to 7.3) for LIFE and WLB combined. LIFE was less specific than WLB (69.6% versus 78.3%, respectively; P = .45), but the difference was not statistically significant. The relative specificities (WLB = 1.0) were 0.9 for LIFE (95% CI = 0.6 to 1.3) and 0.6 (95% CI = 0.4 to 1.0) for LIFE and WLB combined. The results were similar regardless of the order of the procedures or the order of the bronchoscopists. Also, LIFE was better at identifying angiogenic squamous dysplasia lesions than WLB (detection ratio [DR], which indicates the relative likelihood of getting a positive result in a sample with dysplasia compared with one without, for LIFE = 1.39 [95% CI = 1.17 to 1.65] versus DR for WLB = 0.67 [95% CI = 0.38 to 1.21]). Conclusion: LIFE was more sensitive than WLB in detecting preneoplastic bronchial changes in high-risk subjects. The prognostic implication of this finding is not yet clear. [J Natl Cancer Inst 2001;93:1385-91]

Netrin-1 was initially characterized as an axon guidance molecule that is essential for normal embryonic neural development; however, many types of neurons continue to express netrin-1 in the postnatal and adult mammalian brain. Netrin-1 and the netrin receptor DCC are both enriched at synapses. In the adult hippocampus, activity-dependent secretion of netrin-1 by neurons potentiates glutamatergic synapse function, and is critical for long-term potentiation, an experimental cellular model of learning and memory. Here, we assessed the impact of neuronal expression of netrin-1 in the adult brain on behavior using tests of learning and memory. We show that adult mice exhibit impaired spatial memory following conditional deletion of netrin-1 from glutamatergic neurons in the hippocampus and neocortex. Further, we provide evidence that mice with conditional deletion of netrin-1 do not display aberrant anxiety-like phenotypes and show a reduction in self-grooming behavior. These findings reveal a critical role for netrin-1 expressed by neurons in the regulation of spatial memory formation.

Growing primary cells and tissue in long-term cultures, such as primary neural cell culture, presents many challenges. A critical component of any environment that supports neural cell growth in vivo is an appropriate 2-D surface or 3-D scaffold, typically in the form of a thin polymer layer that coats an underlying plastic or glass substrate and aims to mimic critical aspects of the extracellular matrix. A fundamental challenge to mimicking a hydrophilic, soft natural cell environment is that materials with these properties are typically fragile and are difficult to adhere to and stabilize on an underlying plastic or glass cell culture substrate. In this review, we highlight the current state of the art and overview recent developments of new artificial extracellular matrix (ECM) surfaces for in vitro neural cell culture. Notably, these materials aim to strike a balance between being hydrophilic and soft while also being thick, stable, robust, and bound well to the underlying surface to provide an effective surface to support long-term cell growth. We focus on improved surface and scaffold coating systems that can mimic the natural physicochemical properties that enhance neuronal survival and growth, applied as soft hydrophilic polymer coatings for both in vitro cell culture and for implantable neural probes and 3-D matrixes that aim to enhance stability and longevity to promote neural biocompatibility in vivo. With respect to future developments, we outline four emerging principles that serve to guide the development of polymer assemblies that function well as artificial ECMs: (a) design inspired by biological systems and (b) the employment of principles of aqueous soft bonding and self-assembly to achieve (c) a high-water-content gel-like coating that is stable over time in a biological environment and possesses (d) a low modulus to more closely mimic soft, compliant real biological tissue. We then highlight two emerging classes of thick material coatings that have successfully captured these guiding principles: layer-by-layer deposited water-soluble polymers (LbL) and silk fibroin (SF) materials. Both materials can be deposited from aqueous solution yet transition to a water-insoluble coating for long-term stability while retaining a softness and water content similar to those of biological materials. These materials hold great promise as next-generation biocompatible coatings for tissue engineers and for chemists and biologists within the biomedical field.

Die Behandlung von Magenkrebs erfordert einen multimodalen Ansatz, um das Risiko eines lokoregionalen Rezidivs und einer Fernmetastasierung zu verringern. Der optimale Zeitpunkt von Chemotherapie, Operation und Bestrahlung wird derzeit in laufenden Studien untersucht. In den USA erhalten die Patienten im Anschluss an die chirurgische Resektion häufig eine adjuvante Radiochemotherapie oder eine Kombination aus neoadjuvanter und adjuvanter Chemotherapie. Wir berichten hier über 4 Patientinnen mit reseziertem Adenokarzinom des Magens, die mit einer Kombination dieser beiden Therapieansätze behandelt wurden und eine neoadjuvante Chemotherapie gefolgt von einer adjuvanten Radiochemotherapie erhielten. Übersetzung aus Case Rep Oncol 2017;10: 308-315 (DOI:10.1159/000464280).

Axonal growth cones extend during neural development in response to precise distributions of extracellular cues. Deleted in colorectal cancer (DCC), a receptor for the chemotropic guidance cue netrin-1, directs F-actin reorganization, and is essential for mammalian neural development. To elucidate how the extracellular distribution of netrin-1 influences the distribution of DCC and F-actin within axonal growth cones, we patterned nanoarrays of substrate bound netrin-1 using lift-off nanocontact printing. The distribution of DCC and F-actin in embryonic rat cortical neuron growth cones was then imaged using total internal reflection fluorescence (TIRF) microscopy. Fluorescence fluctuation analysis viaimage cross-correlation spectroscopy (ICCS) was applied to extract the molecular density and aggregation state of DCC and F-actin, identifying the fraction of DCC and F-actin colocalizing with the patterned netrin-1 substrate. ICCS measurement of spatially segmented images based on the substrate nanodot patterns revealed distinct molecular distributions of F-actin and DCC in regions directly overlying the nanodots compared to over the reference surface surrounding the nanodots. Quantifiable variations between the populations of DCC and F-actin on and off the nanodots reveal specific responses to the printed protein substrate. We report that nanodots of substrate-bound netrin-1 locally recruit and aggregate DCC and direct F-actin organization. These effects were blocked by tetanus toxin, consistent with netrin-1 locally recruiting DCC to the plasma membrane viaa VAMP2-dependent mechanism. Our findings demonstrate the utility of segmented ICCS image analysis, combined with precisely patterned immobilized ligands, to reveal local receptor distribution and signaling within specialized subcellular compartments.

ABSTRACTMany land-use change studies have relied on geographical explanatory factors. Unfortunately, they are but a single perspective in the multidimensional process of human decision-making. This project was designed to model the social, economic, geographic, and regulatory factors at the most appropriate unit of analysis, the landowner. By examining parcelization, a window is opened into the antecedent event of land-use change. A logistic regression model determined the likelihood a tax parcel would split in three time periods between 1953 and 2007. Geographic variables showed expected relationships to a parcelization event, while economic and regulatory variables illustrated some unexpected relationships. Social variables demonstrated scale issues that challenged their efficacy. A temporal analysis showed that historic parcelization was explained more robustly than more contemporary parcelization. The results could indicate that contemporary parcelization is driven by new and more complex factors not yet represented in similar models.

Netrins are secreted proteins that direct cell migration and adhesion during development. Netrin-1 binds its receptors deleted in colorectal cancer (DCC) and the UNC5 homologs (UNC5A–D) to activate downstream signaling that ultimately directs cytoskeletal reorganization. To investigate how netrin-1 regulates the dynamic distribution of DCC and UNC5 homologs, we applied fluorescence confocal and total internal reflection fluorescence microscopy, and sliding window temporal image cross correlation spectroscopy, to measure time profiles of the plasma membrane distribution, aggregation state, and interaction fractions of fluorescently tagged netrin receptors expressed in HEK293T cells. Our measurements reveal changes in receptor aggregation that are consistent with netrin-1-induced recruitment of DCC-enhanced green fluorescent protein (EGFP) from intracellular vesicles to the plasma membrane. Netrin-1 also induced colocalization of coexpressed full-length DCC-EGFP with DCC-T-mCherry, a putative DCC dominant negative that replaces the DCC intracellular domain with mCherry, consistent with netrin-1-induced receptor oligomerization, but with no change in aggregation state with time, providing evidence that signaling via the DCC intracellular domain triggers DCC recruitment to the plasma membrane. UNC5B expressed alone was also recruited by netrin-1 to the plasma membrane. Coexpressed DCC and UNC5 homologs are proposed to form a heteromeric netrin-receptor complex to mediate a chemorepellent response. Application of temporal image cross correlation spectroscopy to image series of cells coexpressing UNC5B-mCherry and DCC-EGFP revealed a netrin-1-induced increase in colocalization, with both receptors recruited to the plasma membrane from preexisting clusters, consistent with vesicular recruitment and receptor heterooligomerization. Plasma membrane recruitment of DCC or UNC5B was blocked by application of the netrin-1 VI-V peptide, which fails to activate chemoattraction, or by pharmacological block of Src family kinase signaling, consistent with receptor recruitment requiring netrin-1-activated signaling. Our findings reveal a mechanism activated by netrin-1 that recruits DCC and UNC5B to the plasma membrane.

Netrin-1 regulates cell migration and adhesion during the development of the nervous system, vasculature, lung, pancreas, muscle, and mammary gland. It is also proposed to function as a dependence ligand that inhibits apoptosis; however, studies disagree regarding whether netrin-1 loss-of-function mice exhibit increased cell death. Furthermore, previously studied netrin-1 loss-of-function gene-trap mice express a netrin-1-β-galactosidase protein chimera with potential for toxic gain-of-function effects, as well as a small amount of wild-type netrin-1 protein. To unambiguously assess loss of function, we generated netrin-1 floxed and netrin-1 null mouse lines. Netrin-1−/−mice die earlier and exhibit more severe axon guidance defects than netrin-1 gene-trap mice, revealing that complete loss of function is more severe than previously reported. Netrin-1−/−embryos also exhibit increased expression of the netrin receptors DCC and neogenin that are proposed dependence receptors; however, increased apoptosis was not detected, inconsistent with netrin-1 being an essential dependence receptor ligand in the embryonic spinal cord.

Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique—spatial intensity distribution analysis (SpIDA)—that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.

Oligodendrocytes exhibit a limited capacity to remyelinate in multiple sclerosis. Factors present in multiple sclerosis lesions are thought to inhibit oligodendrocyte precursor cell migration, limiting their recruitment to axons requiring remyelination; however, few inhibitors have been identified. A candidate inhibitor is netrin-1, a secreted protein that repels migrating oligodendrocyte precursor cells during neural development and is expressed by myelinating oligodendrocytes in the mature rodent central nervous system. Herein, we examined the distribution of netrin-1 in adult human white matter and multiple sclerosis lesions. We detected full-length netrin-1 protein and shorter netrin-1 fragments in samples of normal white matter and of multiple sclerosis lesions from adult human brain. We demonstrate that peptides corresponding to amino terminal domains VI and V of netrin-1 repel migrating oligodendrocyte precursor cells, but lack the chemoattractant activity of full-length netrin-1. Furthermore, recombinant domains VI-V of netrin-1 disrupt the chemoattractant activity of full-length netrin-1, consistent with a competitive mechanism of action. These findings indicate that full-length and fragmented forms of netrin-1, found in multiple sclerosis lesions, have the capacity to inhibit oligodendrocyte precursor migration, identifying netrin-1 as a potential target for therapies that promote remyelination.

Remyelination in multiple sclerosis (MS) is often incomplete. In experimental models, oligodendrocyte progenitor cells (OPCs) rather than previously myelinating oligodendrocytes (OLs) are responsible for remyelination. This study compares the relative susceptibility of adult human OPCs and mature OLs to injury in actively demyelinating MS lesions and under in vitrostress conditions. In all lesions (n= 20), the number of OLs (Olig2 weak/NogoA positive) was reduced compared to control white matter (mean 38 ± 4% of control value). In 11 cases, OPC numbers (Olig2 strong; NogoA negative) were also decreased; in eight of these, the reduction was greater for OPCs than for OLs. In the other nine samples, OPC numbers were greater than control white matter, indicating ongoing OPC migration and/or proliferation. Analysis of co-cultures with rat dorsal root ganglia neurons confirmed that OPCs were more capable of contacting and ensheathing axons than OLs. In isolated culture under stress conditions (withdrawal of serum/glucose and/or antioxidants), OPCs showed increased cell death and reduced process extension compared to OLs. Under all culture conditions, OPCs up-regulated expression of genes in the extrinsic proapoptotic pathway, and had increased susceptibility to tumor necrosis factor–induced cell death as compared to OLs. Our data suggest that susceptibility of OPCs to injury within the MS lesion environment contributes to the limited remyelination in MS.

The transmembrane protein deleted in colorectal cancer (DCC) and its ligand, netrin-1, regulate synaptogenesis during development, but their function in the mature central nervous system is unknown. Given that DCC promotes cell-cell adhesion, is expressed by neurons, and activates proteins that signal at synapses, we hypothesized that DCC expression by neurons regulates synaptic function and plasticity in the adult brain. We report that DCC is enriched in dendritic spines of pyramidal neurons in wild-type mice, and we demonstrate that selective deletion of DCC from neurons in the adult forebrain results in the loss of long-term potentiation (LTP), intact long-term depression, shorter dendritic spines, and impaired spatial and recognition memory. LTP induction requires Src activation of NMDA receptor (NMDAR) function. DCC deletion severely reduced Src activation. We demonstrate that enhancing NMDAR function or activating Src rescues LTP in the absence of DCC. We conclude that DCC activation of Src is required for NMDAR-dependent LTP and certain forms of learning and memory.

Parcelization-induced forest fragmentation is eating away at private, nonindustrial forests. Parcel sizes are declining and the number of landowners is increasing. These conditions make it challenging for resource managers to maintain economical timber harvesting on smaller privately held forested parcels. Shrinking parcel sizes also aggravate landscape and habitat fragmentation. In this article, we analyze parcelization and its link to private land-use change. Reconstruction of historic tax parcels and land use in three rural towns in Bayfield County, Wisconsin, allowed us to examine the effects of parcelization over a 53-year period. Our results indicate that parcelization is a significant factor in landscape change in northern Wisconsin. This research provides empirical evidence of the necessity to manage how private land is subdivided and used in amenity and natural resource rich areas.

Remyelination, which occurs subsequent to demyelination, contributes to functional recovery and is mediated by oligodendrocyte progenitor cells (OPCs) that have differentiated into myelinating cells. Therapeutics that impact remyelination in the CNS could be critical determinants of long-term functional outcome in multiple sclerosis (MS). Fingolimod is a S1P receptor modulator in MS clinical trials due to systemic anti-inflammatory properties, yet may impact cells within the CNS by crossing the blood-brain barrier. Previous studies using isolated dissociated cultures indicate that neural cells express S1P receptors and respond to receptor engagement. Our objective was to assess the effects of fingolimod on myelin-related processes within a multicellular environment that maintains physiological cell-cell interactions, using organotypic cerebellar slice cultures. Fingolimod treatment had no impact on myelin under basal conditions. Fingolimod treatment subsequent to lysolecithin-induced demyelination enhanced remyelination and process extension by OPCs and mature oligodendrocytes, while increasing microglia numbers and immunoreactivity for the astrocytic marker glial fibrillary acidic protein. The number of phagocytosing microglia was not increased by fingolimod. Using S1P receptor specific agonists and antagonists, we determined that fingolimod-induced effects on remyelination and astrogliosis were mediated primarily through S1P3 and S1P5, whereas enhanced microgliosis was mediated through S1P1 and S1P5. Taken together, these data demonstrate that fingolimod modulates multiple neuroglial cell responses, resulting in enhanced remyelination in organotypic slice cultures that maintain the complex cellular interactions of the mammalian brain.

The name netrin is derived from the Sanskrit Netr, meaning 'guide'. Netrins are a family of extracellular proteins that direct cell and axon migration during embryogenesis. Three secreted netrins (netrins 1, 3 and 4), and two glycosylphosphatidylinositol (GPI)-anchored membrane proteins, netrins G1 and G2, have been identified in mammals. The secreted netrins are bifunctional, acting as attractants for some cell types and repellents for others. Receptors for the secreted netrins include the Deleted in Colorectal Cancer (DCC) family, the Down's syndrome cell adhesion molecule (DSCAM), and the UNC-5 homolog family: Unc5A, B, C and D in mammals. Netrin Gs do not appear to interact with these receptors, but regulate synaptic interactions between neurons by binding to the transmembrane netrin G ligands NGL1 and 2. The chemotropic function of secreted netrins has been best characterized with regard to axon guidance during the development of the nervous system. Extending axons are tipped by a flattened, membranous structure called the growth cone. Multiple extracellular guidance cues direct axonal growth cones to their ultimate targets where synapses form. Such cues can be locally derived (short-range), or can be secreted diffusible cues that allow target cells to signal axons from a distance (long-range). The secreted netrins function as short-range and long-range guidance cues in different circumstances. In addition to directing cell migration, functional roles for netrins have been identified in the regulation of cell adhesion, the maturation of cell morphology, cell survival and tumorigenesis.

Remyelination of lesions in the central nervous system contributes to neural repair following clinical relapses in multiple sclerosis. Remyelination is initiated by recruitment and differentiation of oligodendrocyte progenitor cells (OPCs) into myelinating oligodendrocytes. Simvastatin, a blood-brain barrier-permeable statin in multiple sclerosis clinical trials, has been shown to impact the in vitroprocesses that have been implicated in remyelination. Animals were fed a cuprizone-supplemented diet for 6 weeks to induce localized demyelination in the corpus callosum; subsequent return to normal diet for 3 weeks stimulated remyelination. Simvastatin was injected intraperitoneally during the period of coincident demyelination and OPC maturation (weeks 4 to 6), throughout the entire period of OPC responses (weeks 4 to 9), or during the remyelination-only phase (weeks 7 to 9). Simvastatin treatment (weeks 4 to 6) caused a decrease in myelin load and both Olig2strongand Nkx2.2strongOPC numbers. Simvastatin treatment (weeks 4 to 9 and 7 to 9) caused a decrease in myelin load, which was correlated with a reduction in Nkx2.2strongOPCs and an increase in Olig2strongcells, suggesting that OPCs were maintained in an immature state (Olig2strong/Nkx2.2weak). NogoA+ oligodendrocyte numbers were decreased during all simvastatin treatment regimens. Our findings suggest that simvastatin inhibits central nervous system remyelination by blocking progenitor differentiation, indicating the need to monitor effects of systemic immunotherapies that can access the central nervous system on brain tissue-repair processes.

Fingolimod is a sphingosine-1-phosphate (S1P) analogue that has been used in clinical trials as a systemic immunomodulatory therapy for multiple sclerosis. Fingolimod readily accesses the central nervous system, raising the issue of its direct effects on neural cells. We assessed the effects of active fingolimod on dissociated cultures of mature, myelin-producing oligodendrocytes (OLGs) derived from adult human brain. Human OLGs express S1P receptor transcripts in relative abundance of S1P5S1P3S1P1, with undetectable levels of S1P4. Low doses of fingolimod (100 pmol/L to 1 nmol/L) induced initial membrane elaboration (2 days), subsequent retraction (4 days), and recurrence of extension with prolonged treatment (8 days). Higher doses (10 nmol/L to 1 μmol/L) caused the opposite modulation of membrane dynamics. Retraction was rescued by co-treatment with the S1P3/S1P5 pathway antagonist, suramin, and was associated with RhoA-mediated cytoskeletal signaling. Membrane elaboration was mimicked using the S1P1 agonist SEW2871. Fingolimod rescued human OLGs from serum and glucose deprivation-induced apoptosis, which was reversed with suramin co-treatment and mimicked using an S1P5 agonist. High doses of fingolimod induced an initial down-regulation of S1P5 mRNA levels relative to control (4 hours), subsequent up-regulation (2 days), and recurrent down-regulation (8 days). S1P1 mRNA levels were inversely regulated compared with S1P5. These results indicate that fingolimod modulates maturity- and species-specific OLG membrane dynamics and survival responses that are directly relevant for myelin integrity.

We present a straightforward method to create spatial gradients of substrate bound protein for live cell studies using only mechanical parts. Protein concentration gradients on a micron scale can be fabricated in several minutes for a relatively low cost using a method that is generally applicable to any protein and substrate combination. We describe the details of the device construction, and provide examples of mammalian cells grown on substrates patterned with protein concentration gradients using this technique.

Pancreatic cancer has a dismal prognosis with no effective medical therapy. Therefore, there is a need to search for novel targets for cancer prevention and treatment. The lipoxygenases oxygenate arachidonic acid and other 20-carbon fatty acids and their downstream metabolites have been found to mediate several aspects of pancreatic cancer development and growth. Therapeutic agents have been developed against various targets in the lipoxygenase pathways. Many of these were first developed for their anti-inflammatory properties and were subsequently found to have anticancer effects. Such agents include lipoxygenase and 5-lipoxygenase-activating protein inhibitors, leukotriene receptor antagonists and natural products with inhibitory effects on these pathways. Dual lipoxygenase and cyclooxygenase inhibition represents an exciting area of research and drug development.

Netrins are a family of secreted proteins that guide the migration of cells and axonal growth cones during development. DCC (deleted in colorectalcancer) is a receptor for netrin-1 implicated in mediating these responses. Here, we show that DCC interacts constitutively with the SH3/SH2 adaptor Nck in commissural neurons. This interaction is direct and requires the SH3 but not SH2 domains of Nck-1. Moreover, both DCC and Nck-1 associate with the actin cytoskeleton, and this association is mediated by DCC. A dominant negative Nck-1 inhibits the ability of DCC to induce neurite outgrowth in N1E-115 cells and to activate Rac1 in fibroblasts in response to netrin-1. These studies provide evidence for an important role of mammalian Nck-1 in a novel signaling pathway from an extracellular guidance cue to changes in the actin-based cytoskeleton responsible for axonal guidance.

Background:EUS-guided fine needle aspiration (EUS-FNA) is a highly accurate method of detecting malignant lymphadenopathy. The optimal methods for performing EUS-FNA to maximize sensitivity and to minimize the number of needle passes necessary are unknown. This is a report of the results of a prospective randomized controlled trial to determine the effect of suction, the site of FNA (edge or center of lymph node), and the method of preparation of cytologic specimens on accuracy, number of needle passes needed, and specimen quality. Methods:Consecutive patients with lymphadenopathy detected by EUS underwent FNA. Each lymph node was sampled with or without suction and from the edge or center in a 2 × 2 factorial design. The samples were expressed onto slides for cytology, and the residual material in the needle was analyzed by the cytospin-cellblock technique. Each aspirate was individually characterized for a diagnosis of malignancy, cellularity, and bloodiness. Results:Forty-three patients with a total of 46 lymph nodes were evaluated. The final lymph node diagnosis was benign in 22 (48%), “suspicious for malignancy” in 6 (13%), and malignant in 18 (39%). The use of suction was associated with an increase in the cellularity of the specimen, but did not improve the likelihood of obtaining a correct diagnosis (OR 1.52: 95% CI [0.81, 2.85]). Samples obtained with suction were of worse quality because of excessive bloodiness (OR 4.7: 95% CI [1.99, 11.24]). Aspiration from the edge of the lymph node (compared with the center) did not increase the likelihood of a correct diagnosis (OR 1.16: 95% CI [0.42, 3.21]). For 78% of malignant lymph nodes, the correct diagnosis was obtained on the first needle pass and for 100% by the third pass. Cytospin-cellblock methods did not add any additional diagnostic information compared with direct smear cytology. Conclusions:The traditional method of applying suction during EUS-FNA does not improve diagnostic accuracy and worsens specimen bloodiness compared with FNA without suction. The site of FNA within the lymph node does not affect accuracy. When EUS-FNA is necessary, our recommendation is up to 3 FNAs without suction from the most convenient and safe location within abnormal-appearing lymph nodes. (Gastrointest Endosc 2001;54:441-7.)

The Akt kinase plays a crucial role in supporting Trk-dependent cell survival, whereas the p75 neurotrophin receptor (p75NTR) facilitates cellular apoptosis. The precise mechanism that p75NTR uses to promote cell death is not certain, but one possibility is that p75NTR-dependent ceramide accumulation inhibits phosphatidylinositol 3-kinase-mediated Akt activation. To test this hypothesis, we developed a system for examining p75NTR-dependent apoptosis and determined the effect of p75NTR on Akt activation. Surprisingly, p75NTR increased, rather than decreased, Akt phosphorylation in a variety of cell types, including human Niemann-Pick fibroblasts, which lack acidic sphingomyelinase activity. The p75NTR expression level required to elicit Akt phosphorylation was much lower than that required to activate the JNK pathway or to mediate apoptosis. We show that p75NTR-dependent Akt phosphorylation was independent of TrkA signaling, required active phosphatidylinositol 3-kinase, and was associated with increased tyrosine phosphorylation of p85 and Shc and with reduced cytosolic tyrosine phosphatase activity. Finally, we show that p75NTR expression increased survival in cells exposed to staurosporine or subjected to serum withdrawal. These findings indicate that p75NTR facilitates cell survival through novel signaling cascades that result in Akt activation.

Centrally located lung cancers are radiologically occult until so far advanced as to have a low cure rate or require extensive resection for cure, but at a cost of high morbidity. These cancers represent about one‐fifth of new lung cancers. Autofluorescence bronchoscopy appears to be an important tool in localizing premalignant and early malignant lesions in the large central airways, particularly when applied to high‐risk patients. Applications include studies of molecular biology of premalignancy and early malignancy, chemoprevention studies, endobronchial therapy studies, localization of synchronous tumors, estimation of the extent of field cancerization, and better estimation of resection margins. Autofluorescence bronchoscopy appears to be significantly more sensitive than white light examination but has low specificity. This technology is likely to gain widespread use when evaluation of sputum for malignant changes is both more sensitive and specific, and when its application is demonstrated to reduce mortality in this important subgroup of non‐small cell lung cancer patients.

Netrins are a family of chemotropic factors that guide axon outgrowth during development; however, their function in the adult CNS remains to be established. We examined the expression of the netrin receptors DCC and UNC5H2 in adult rat retinal ganglion cells (RGCs) after grafting a peripheral nerve (PN) to the transected optic nerve and following optic nerve transection alone. In situhybridization revealed that both Dccand Unc5h2mRNAs are expressed by normal adult RGCs. In addition, netrin-1 was found to be constitutively expressed by RGCs. Quantitative analysis using in situhybridization demonstrated that both Dccand Unc5h2were down-regulated by RGCs following axotomy. In the presence of an attached PN graft, Dccand Unc5h2were similarly down-regulated in surviving RGCs regardless of their success in regenerating an axon. Northern blot analysis demonstrated expression of netrin-1in both optic and sciatic nerve, and Western blot analysis revealed the presence of netrin protein in both nerves. Immunohistochemical analysis indicated that netrin protein was closely associated with glial cells in the optic nerve. These results suggest that netrin-1, DCC, and UNC5H2 may contribute to regulating the regenerative capacity of adult RGCs.

This study aimed to characterize the morphological and electrophysiological properties of neurons in layer V of the entorhinal cortex in the rat brain. Using the in vitro slice preparation and sharp electrode techniques, we recorded from layer V neurons located in the medial entorhinal cortex. Recorded cells were also labeled with biocytin. Based on morphological criteria, layer V of the entorhinal cortex is comprised of three categories of neurons: pyramidal cells, horizontal cells, and polymorphic cells. Horizontal cells could be easily distinguished from the pyramidal cells because the bulk of their dendritic plexus extended horizontally within layer V. Polymorphic cells vary in size and shape. Interestingly, they typically do not have apical dendrites, and some of them have dendrites that extend into the subiculum. Based on electrophysiological criteria alone, it was not possible to unequivocally distinguish the morphological cell types because they were somewhat heterogeneous with respect to several parameters including inward rectification, spike‐frequency adaptation, and intrinsic oscillations. Nevertheless, although most horizontal cells displayed time‐dependent inward rectification, most pyramidal cells displayed fast inward rectification exclusively. None of the entorhinal cortex layer V cells displayed oscillatory activity like that of neocortical layer V “bursting” cells, although neurons from all groups displayed rhythmic subthreshold membrane potential oscillations. In summary, we have found that layer V of the rat medial entorhinal cortex consists of three morphologically distinct neuronal subtypes that cannot be clearly distinguished from each other by traditional electrophysiological measures. J. Comp. Neurol. 418:457–472, 2000. © 2000 Wiley‐Liss, Inc.

A retrospective experience with preoperative lower tract evaluation, bladder rehabilitation and transplant outcome in patients with major abnormalities of the lower urinary tract is presented. From more than 1000 renal transplants during a 23‐year period, 11 patients required either augmentation cystoplasty (7 cases) or supravesical conduit diversion (4 cases) in preparation for transplantation. The causes of bladder abnormality were neurogenic, dysfunctional voiding, posterior urethral valves, congenital atresia, and acquired contracture in 4, 3, 2, 1 and 1 cases respectively. Lower tract evaluation began with voiding history and voiding cystourethrogram. Patients with incontinence or near zero bladder capacity were managed with supravesical diversion. Continent patients were studied further with serial cystometrograms via percutaneous suprapubic catheters during trials of bladder cycling to study bladder compliance. Continent patients with abnormally high bladder pressure (greater than 75 cm water) and/or low bladder capacity (less than 100 ml) underwent augmentation cystoplasty with intestinal segments. To date, 10 renal transplants have been performed in 8 patients with a cumulative graft survival of 70%, with follow‐up of 13 months to 3 1/2 yr. Two patients with bladder augmentation are awaiting transplantation. The final patient died of suspected pulmonary embolus following bladder augmentation and subsequent development of a bowel obstruction. Additional morbidity consisted of 8 episodes of urinary infection in 6 patients and 1 case of bleeding at the augmentation cystoplasty suture line. These results suggest that, with careful lower tract evaluation, patients with major bladder abnormalities may safely undergo renal transplantation into a rehabilitated bladder in most cases, or into a supravesical conduit when necessary, and can achieve satisfactory graft survival and low morbidity. A prospective plan for pre‐transplant lower tract evaluation is offered.

A three-dimensional finite element analysis treating the nonlinear viscoelastic response of laminated composites is presented. The individual plies in the laminate are modeled using 20-node isoparametric solid elements. The transient creep compliance in the viscoelastic model is represented as an exponential series plus a steady-flow term, which allows for a simplification of the numerical procedure for handling hereditary effects. Time-dependent stress and strain distributions are calculated for unnotched and notched laminates subjected to tensile loading or bending.

Heat shock inBacillus subtilis may induce as many as 66 proteins after temperature upshift from 37° to 48°C. Four induced proteins were analyzed by microsequencing techniques. These were identified as the homologues for GroEL, DnaK, enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which are heat shock proteins in other systems. The identities of GroEL and DnaK were confirmed additionally by Western blot analysis. As a control, a protein whose synthesis was repressed approximately threefold by heat shock was identified by microsequencing as flagellin.

In this study 171 consecutive patients hospitalized for prostatectomy were prospectively evaluated by urologist-performed transabdominal ultrasound. The urologist-sonographers were blinded to the results of an excretory urogram (IVP) performed and interpreted by radiologists. All significant upper tract pathology (4 renal cell carcinomas and 1 transitional cell carcinoma of the renal pelvis) identified by an IVP also was detected by ultrasonography. Of 5 clinically silent stones seen on an IVP 4 were identified by sonography. There were 13 patients (7.6%) with severe contrast medium allergies or renal failure who were readily evaluated with ultrasonography. Urologist-performed sonography can safely replace an IVP for routine evaluation of the urinary tract before prostatectomy. Moreover, sonography provided significant additional data on prostate size and post-void residual. However, the cost-effectiveness of upper tract imaging in patients before prostatectomy may be questioned.

Objectives. In 1991, all active board-certified pediatric cardiologists were polled by questionnaire to examine the relation of subspecialty training and motivational and satisfaction issues to practice characteristics.

Flexible fiberoptic technology was first applied to cystoscopy in 1973, with greatly increased usage since 1982. Most procedures formerly performed with rigid cystoscopes can be done using flexible cystoscopes with minimal or no anesthesia. Patient positioning and precystoscopy preparation and draping are simplified with the flexible fiberoptic instruments. Complete examination of the urethra and bladder can be performed with a single-lens system and with the patient in a variety of positions. Fiberoptic cystoscopy is limited in patients who are bleeding or have blood clots in their bladders. Withdrawal of irrigant or bladder drainage is cumbersome, and the fiberoptic image is currently not of the same caliber as that of the rigid-lens systems. Fiberoptic cystoscopy has become the procedure of choice for many urologists for ureteral stenting prior to extracorporeal shock-wave lithotripsy. With the advent of lithotripters that require no anesthesia, this application is likely to broaden. Future applications of flexible cystoscopy may include a flexible videocystoscope for use in diagnostic and therapeutic procedures.

The rapid extension of a penny-shaped crack under torsion is investigated. Both dynamic and quasi-static loading is considered. The wave motion is analyzed through a Green's function technique which leads to an integral equation for the stress field around the crack. Asymptotic expansions for the stress intensity and displacement rate intensity functions which are valid for a small time are obtained for the two types of loading. The propagation of the crack is analyzed through the balance of rates of energy criterion.

The primary mode of failure in structures composed of coated fabrics is often by means of propagation of a tear or crack. Fracture models used for metal structures have generally not worked well for these materials. This investigation pro vides an evaluation of several fracture models developed for continuous fiber com posites to predict failure in coated fabrics. The inherent flaw model, the point stress model, and the modified singularity model were all found to correlate well with ex perimental data from strength testing of coated fabrics.

Forty-eight first-year college students (25 women and 23 men) completed self-report measures of level of interpersonal dependency and then provided information regarding their Scholastic Aptitude Test (SAT) scores and overall high school grade point averages (GPAs). Dependent men had significantly higher high school GPAs than did nondependent men, even when scholastic aptitude was controlled for statistically. However, a parallel relationship between dependency and academic performance was not found in women. These results are discussed in the context of previous research on the dependency-academic performance relationship. The implications of these findings for studies of gender differences in dependency and for studies examining the positive, adaptive qualities of dependent personality traits are discussed.

A syndrome characterized by hypoxemia aggravated by exercise, orthodeoxia, hypocapnia, and evidence of hyperdynamic circulation, but otherwise normal indices of pulmonary air flow, volume, and distribution of ventilation has been observed as an infrequent complication of hepatic cirrhosis. An illustrative case is described, the features of which support the presence of a shunt or shunt-like mechanism consisting of low-resistance vascular communications within the lung. We suggest that this may represent the existence of a hepatopulmonary syndrome analogous to the hepatorenal syndrome.