Population-based comparison of prognostic factors in invasive micropapillary and invasive ductal carcinoma of the breast.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) is a variant of breast carcinoma with a higher propensity for lymph node metastases compared with invasive ductal carcinoma (IDC). METHODS: Retrospective analysis of 636 IMPC and 297 735 IDC cases in the Surveillance, Epidemiology and End RESULTS database comparing disease-specific survival (DSS) and overall survival (OS) between IMPC and IDC. RESULTS: A higher percentage of IMPC cases (52.0%) had nodal metastases compared with IDC cases (34.6%). The 5-year DSS and OS for IMPC was 91.8% and 82.9%, respectively compared with 88.6% and 80.5% for IDC, respectively. For both IMPC and IDC, oestrogen-receptor positivity was associated with better survival, while having four or more positive lymph nodes or larger tumour size correlated with worse survival. Radiotherapy provided a survival benefit for both histological types. CONCLUSIONS: Despite IMPC's higher propensity for lymph node metastasis, IMPC has DSS and OS that compare favourably with IDC.

Medical students' perception of what embodies an effective surgeon educator.

BACKGROUND: Effective surgeon educators likely help medical students develop competency and may inspire pursuit of surgical training. We sought to determine the qualities medical students believe embody effective surgical educators. METHODS: Mixed-methods study of nationally electronically recruited 3rd-year medical students using virtual semi-structured interviews and anonymous quantitative survey to determine the most critical and most frequently encountered qualities of effective surgical educators. Thematic analysis using grounded theory was undertaken. RESULTS: Data saturation occurred after 9 interviews. Themes of effective surgical educators included: engagement (acknowledging student, knowing their name, talking to the student), fostering a positive learning environment (non-threatening, non-shaming questioning), inclusion (giving responsibility/appropriate autonomy), and understanding how to teach a novice (teaching the student how to learn, adapt to learner). On quantitative analysis of Likert based survey, encouraging, promoting a positive learning climate, timely constructive feedback, and questioning were ranked as most critical. CONCLUSION: Students highly value positive learning climate and inclusion. Faculty Development to promote these traits may improve clerkship learning and experience.

Video. Chopstick surgery: a novel technique enables use of the Da Vinci Robot to perform single-incision laparoscopic surgery.

INTRODUCTION: Single-incision laparoscopic surgery (SILS) is limited by the coaxial arrangement of the instruments. A surgical robot with "wristed" instruments could overcome this limitation but the "arms" collide when working coaxially. This video demonstrates a new technique of "chopstick surgery," which enables use of the robotic arms through a single incision without collision. METHODS: Experiments were conducted utilizing the da Vinci S® robot (Sunnyvale, CA) in a porcine model with three laparoscopic ports (12 mm, 2-5 mm) introduced through a single "incision." Pilot work conducted while performing Fundamentals of Laparoscopic Surgery (FLS) tasks determined the optimal setup for SILS to be a triangular port arrangement with 2-cm trocar distance and remote center at the abdominal wall. Using this setup, an experienced robotic surgeon performed a cholecystectomy and nephrectomy in a porcine model utilizing the "chopstick" technique. The chopstick arrangement crosses the instruments at the abdominal wall so that the right instrument is on the left side of the target and the left instrument on the right. This arrangement prevents collision of the external robotic arms. To correct for the change in handedness, the robotic console is instructed to drive the "left" instrument with the right hand effector and the "right" instrument with the left. RESULTS: Both procedures were satisfactorily completed with no external collision of the robotic arms, in acceptable times and with no technical complications. This is consistent with results obtained in the box trainer where the chopstick configuration enabled significantly improved times in all tasks and decreased number of errors and eliminated instrument collisions. CONCLUSION: Chopstick surgery significantly enhances the functionality of the surgical robot when working through a small single incision. This technique will enable surgeons to utilize the robot for SILS and possibly for intraluminal or transluminal surgery.

A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans.

An early event in RNA interference (RNAi) is the cleavage of the initiating double-stranded RNA (dsRNA) to short pieces, 21 to 23 nucleotides in length. Here we describe a null mutation in dicer-1 (dcr-1), a gene proposed to encode the enzyme that generates these short RNAs. We find that dcr-1(-/-) animals have defects in RNAi under some, but not all, conditions. Mutant animals have germ line defects that lead to sterility, suggesting that cleavage of dsRNA to short pieces is a requisite event in normal development.

A link between RNA interference and nonsense-mediated decay in Caenorhabditis elegans.

Double-stranded RNA (dsRNA) inhibits expression of homologous genes by a process involving messenger RNA degradation. To gain insight into the mechanism of degradation, we examined how RNA interference is affected by mutations in the smg genes, which are required for nonsense-mediated decay. For three of six smg genes tested, mutations resulted in animals that were initially silenced by dsRNA but then recovered; wild-type animals remained silenced. The levels of target messenger RNAs were restored during recovery, and RNA editing and degradation of the dsRNA were identical to those of the wild type. We suggest that persistence of RNA interference relies on a subset of smg genes.

RNA editing and hypermutation by adenosine deamination.

Double-stranded RNA adenosine deaminase (dsRAD) was discovered ten years ago. In the intervening decade, research on dsRAD has progressed not only predictably, such as with the purification of the enzyme and identification of cDNAs, but also in some quite surprising ways. This review covers both areas of progress, but will concentrate on the surprises, which include the discovery that dsRAD is a member of a larger family of deaminases and the identification of RNAs that appear to be targets for these deaminases in vivo.

RNA editing. An I for editing.

In vitro editing in mammalian nuclear extracts reveals adenosine-to-inosine conversions in glutamate receptor messenger RNAs.

Binding properties of newly identified Xenopus proteins containing dsRNA-binding motifs.

BACKGROUND: Although most RNA-binding proteins recognize a complex set of structural motifs in their RNA target, the double-stranded (ds) RNA-binding proteins are limited to interactions with double helices. Recently, it has been discovered that some dsRNA-binding proteins share regions of amino-acid similarity known as dsRNA-binding motifs. RESULTS: A Xenopus ovary cDNA expression library was screened with radiolabeled dsRNA to identify previously uncharacterized dsRNA-binding proteins. The analysis of an incomplete cDNA identified during the screen led to the discovery of two longer cDNAs of related sequence. The proteins encoded by these cDNAs each contained two dsRNA-binding motifs, in glycine. The nucleic-acid-binding properties of a fusion protein containing the two dsRNA-binding motifs and the auxiliary domain were analyzed using a gel mobility shift assay. The fusion protein bound dsRNA of a variety of different sequences, and exhibited a preference for binding to dsRNA and RNA-DNA hybrids over other nucleic acids. Appropriate mRNAs, corresponding to each cDNA, were detected in polyadenylated RNA isolated from Xenopus stage VI oocytes, but translation of one of the mRNAs appeared to be masked until meiotic maturation. CONCLUSION: dsRNA-binding motifs are often found in proteins that bind dsRNA, and our results show that they can be associated with auxiliary domains rich in arginine and glycine. These motifs can confer very tight binding to dsRNA. Binding can also occur to RNA-DNA hybrids, suggesting recognition of some aspect of the A-form helical structure that is adopted by both dsRNA and RNA-DNA hybrids.

The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification.

Here we describe studies of double-stranded RNA (dsRNA) adenosine deaminase in Xenopus laevis, in particular during meiotic maturation, the period during which a stage VI oocyte matures to an egg. We show that dsRNA adenosine deaminase is in the nuclei of stage VI oocytes. Most importantly, we demonstrate that the cytoplasm of stage VI oocytes contains a factor that protects microinjected dsRNA from deamination when dsRNA adenosine deaminase is released from the nucleus during meiotic maturation. Our data suggest that the protection factor is a cytoplasmic dsRNA-binding protein or proteins that bind to dsRNA in a sequence-independent manner to occlude dsRNA from binding to dsRNA adenosine deaminase. The cytoplasmic double-stranded RNA-binding protein(s) does not bind to other nucleic acids and can be titrated at high concentrations of dsRNA. These studies raise the question of whether all dsRNA-binding proteins share endogenous substrates and also suggest potential means of regulating dsRNA adenosine deaminase in vivo.

Hepatitis delta virus RNA editing is highly specific for the amber/W site and is suppressed by hepatitis delta antigen.

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.

Caenorhabditis elegans mRNAs that encode a protein similar to ADARs derive from an operon containing six genes.

The Caenorhabditis elegans T20H4.4 open reading frame (GenBank accession no. U00037) predicted by Genefinder encodes a 367 amino acid protein that is 32-35% identical to the C-terminal domain of adenosine deaminases that act on RNA. We show that T20H4.4 cDNAs (GenBank accession no. AF051275) encode a larger 495 amino acid protein that is extended at its N-terminus to include a single double-stranded RNA-binding motif, and that T20H4.4 occupies the second position in a six-gene operon (5'-T20H4.5, T20H4.4, R151.8A, R151.8B, R151.7, R151.6-3'). Ten different spliced-leader (SL) sequences were found attached to T20H4.4 mRNAs, including SL1, SL2 and eight SL2-like leaders that include two new variants. Characterization of cDNAs derived from all six genes confirmed the essential features of C.elegans operons: intercistronic distances in the range of 104-257 nt between the upstream polyadenylation sites and the downstream trans -splice sites; SL2, or SL2-like leaders, attached to the downstream mRNAs. Polycistronic mRNA fragments revealed a 5'-untranslated region (5'-UTR) >705 nt. The 5'-UTR is removed in mature mRNAs from the first gene (T20H4.5) and replaced primarily by SL1, and to a lesser extent by SL2. Our study provides new information regarding operons and how they are processed.

A Xenopus zinc finger protein that specifically binds dsRNA and RNA-DNA hybrids.

Proteins containing C2H2 type zinc finger motifs represent one of the largest classes of nucleic acid-binding proteins found in nature. We describe a novel zinc finger protein, dsRBP-ZFa, isolated by screening an expression library with dsRNA. The dsRBP-ZFa cDNA encodes a protein containing seven zinc finger motifs and an acidic C-terminal domain. Mobility shift experiments demonstrate that dsRBP-ZFa binds dsRNA and RNA-DNA hybrids with nanomolar dissociation constants and in a sequence independent manner. We also show that DNA and single stranded RNA fail to compete with dsRNA for binding suggesting dsRBP-ZFa prefers to bind an A-form helix. Using western analyses we have localized dsRBP-ZFa primarily to the nucleus of Xenopus laevis oocytes. The identification of dsRBP-ZFa provides the first example of a zinc finger protein that is specific for dsRNA. In addition, dsRBP-ZFa does not contain the previously described dsRNA binding motif, suggesting certain zinc fingers may provide an alternative way to recognize the A-form helix.

The importance of internal loops within RNA substrates of ADAR1.

Adenosine deaminases that act on RNA (ADARs) are a family of RNA editing enzymes that convert adenosines to inosines within double-stranded RNA (dsRNA). Although ADARs deaminate perfectly base-paired dsRNA promiscuously, deamination is limited to a few, selected adenosines within dsRNA containing mismatches, bulges and internal loops. As a first step in understanding how RNA structural features promote selectivity, we investigated the role of internal loops within ADAR substrates. We observed that a dsRNA helix is deaminated at the same sites whether it exists as a free molecule or is flanked by internal loops. Thus, internal loops delineate helix ends for ADAR1. Since ADAR1 deaminates short RNAs at fewer adenosines than long RNAs, loops decrease the number of deaminations within an RNA by dividing a long RNA into shorter substrates. For a series of symmetric internal loops related in sequence, larger loops (>/=six nucleotides) acted as helix ends, whereas smaller loops (

Deriving indicators for breast conserving surgery using finite element analysis.

Breast conserving therapy (BCT), comprising a complete surgical excision of the tumour (partial mastectomy) with post-operative radiotherapy to the remaining breast tissue, is feasible for most women undergoing treatment for breast cancer. The goal of BCT is to achieve local control of the cancer, as well as to preserve a breast that satisfies a woman's cosmetic concerns. Although most women undergo partial mastectomy with satisfactory cosmetic results, in many patients the remaining breast is left with major cosmetic defects including concave deformities, distortion of the nipple-areolar complex, asymmetry and changes in tissue density characterised by excessive density associated with parenchymal scarring, as well as breast pain. There are currently no tools, other than surgical experience and judgement, which can predict the impact of partial mastectomy on the contour, the deformity of the treated breast and the mechanical stress that it induces. In this study, we use a finite element model to execute virtual surgery and carry out a sensitivity analysis on the resection location, the resection size, the breast tissue mechanical property and the different post-surgery recovery stage. We output the result in two different built-in indicators labelled as the cosmetic and the functional indicators. This study used the breast model for three women with breast cancer who have been elected to undergo BCT and are being treated at the Methodist Hospital in Houston, TX. The goal of this study was to propose a first glimpse of the key parameter leading to satisfactory post-BCT cosmetic results.

Interstitial cellular and matrix restoration of cardiac valves after cryopreservation.

OBJECTIVES: We previously characterized the porcine aortic leaflet interstitial cell phenotype as having both synthetic and contractile characteristics; that is, it is a myofibroblast. In this study we hypothesized (1) that the cryopreservation of aortic valves causes a significant reduction in cell density, (2) that it simultaneously causes alterations in representative components of extracellular matrix, and (3) that both of these processes are reversible. METHODS: Seventy-two leaflets from 24 porcine aortic valves were studied. Whole valves were subjected to variable lengths of preharvest ischemia (group 1), ischemia followed by processing analogous to clinical methods (group 2), and ischemia followed processing with an organ culture type of resuscitation (group 3). Vital dye exclusion by cells enzymatically dispersed from leaflets was used to quantify viability. Electron and light microscopy, immunohistochemical assay, and a silicone rubber substratum contractility assay were used both in dispersed cell preparations and in leaflet cross sections to examine structural, ultrastructural, and functional changes across the 3 groups through a range of preharvest ischemic times. RESULTS: Results indicated that harvest ischemic periods between 2 and 24 hours after donor death were not responsible for cell number reductions. During this interval overt dissolution of chondroitin sulfate simultaneous with a relative sparing of fibronectin was evidenced by immunohistochemical staining. Although not reduced in number, ischemic interstitial cells did show significant ultrastructural evidence of injury and suppressed monoclonal binding to vimentin and alpha-smooth muscle actin. After cryopreservation, viable cell numbers were always markedly reduced at all ischemic intervals and damage to both soluble extracellular matrix components and cell ultrastructure was increased. At all time and processing points, however, some retention of matrix secretory and cellular contractile capabilities was observed among the surviving cells. After the extended periods of preharvest ischemia (2-24 hours) followed by processing, a restitution of functioning cells was accomplished by means of whole-leaflet incubation in 15% fetal bovine serum. CONCLUSIONS: After application of the described methods, new cells within restored intact leaflets as well as in single-cell preparations demonstrated normal ultrastructure and contractile and synthetic functions (normal phenotypic expression). If functioning leaflet interstitial cells can contribute to homograft durability, bioengineering methods for pretransplantation cell repopulation could be refined with these techniques and applied to clinical valve transplantation.

Esophageal blood flow in the rabbit: response to calcium channel blockers.

Calcium channel blockers have recently been added to the therapeutic regimen for patients who have chest pain of esophageal origin. Although relief of symptoms has been reported, this has not always been associated with changes in esophageal contraction pressures or luminal pH. Myoischemia has been proposed as one possible mechanism for esophageal chest pain. We have investigated the effect of the calcium channel blockers verapamil, nifedipine, and diltiazem on esophageal blood flow in the rabbit model. Esophageal blood flow was measured three times in each rabbit with use of the radiolabeled microsphere technique after a 30-minute continuous infusion of (1) saline solution (baseline), (2) a low dose, and (3) a high dose of each agent. Esophageal mucosal blood flow significantly decreased with nifedipine but was unchanged with verapamil and diltiazem. Esophageal muscle blood flow significantly increased--approximately 100% after administration of each of the calcium channel blockers. Thus esophageal muscle blood flow is enhanced after administration of calcium channel blockers, and this may be one therapeutic mechanism of the calcium channel blockers in the relief of esophageal chest pain in some esophageal diseases.

The pancreatic duct epithelium in vitro: bile acid injury and the effect of epidermal growth factor.

BACKGROUND: Pancreatic duct epithelial cells form a barrier against parenchymal injury. The capacity of these cells to respond to injury has not been investigated. We hypothesized that epidermal growth factor (EGF), normally found in pancreatic juice, could protect the duct epithelium from damage. METHODS: An explant system of duct cell culture developed in our lab with the bovine main pancreatic duct was used. Explants were exposed to bile acid (taurodeoxycholic acid [TDCA] 0, 0.05, 0.5, and 1 mmol/L) in the presence or absence of EGF (0, 1, 10, and 100 nmol/L) for 48 hours. Epithelial proliferation, damage, and growth out from the explant edge were assessed histologically. Expression of ductal markers and the extent of cell proliferation were determined by immunohistochemistry using specific antibodies. RESULTS: Explant duct cells proliferated and demonstrated continued expression of key duct antigens in culture. TDCA produced dose-dependent mucosal damage and reduced epithelial density and growth from the edge. EGF increased cellular density in the native epithelium, but did not significantly alter growth from the edge. Mucosal damage created by TDCA exposure was significantly decreased with EGF and both growth from the edge and cell density were preserved. CONCLUSIONS: Explants created from the bovine main pancreatic duct serve as an excellent model for the study of duct epithelial cells in vitro. These cells proliferate in response to EGF and are damaged by TDCA at concentrations below those normally associated with detergent-like activity and below levels observed in bile and duodenal secretions. The ability of EGF to protect from this injury suggests a potential physiologic role in the maintenance of the pancreatic duct mucosal barrier.

H+ back diffusion interferes with intrinsic reactive regulation of esophageal mucosal blood flow.

The esophageal mucosa maintains a barrier that is relatively impermeable to glucose, H+, and other small molecules. Injury of the mucosa causes disruption of this barrier, manifest initially by increased permeability to small molecules. In the stomach the mucosa is protected from gross ulceration in the presence of bile-induced H+ back diffusion (JH+) by increases in mucosal blood flow (Qm). Qm to the esophagus during injury has never been studied. We explored the possibility that esophageal Qm would increase as a compensatory reaction to early barrier disruption. Rabbits (2 to 4 kg) were anesthetized and the in situ esophagus was luminally perfused for two 1-hour periods with subulcerogenic concentrations of bile salts, pepsin, or trypsin in the presence (pH 2) or absence (pH 7) of acid. Qm was measured with 15 mu radioactive microspheres in nine experimental groups with a total of 62 rabbits. Changes in Qm were compared with changes in permeability of the esophageal barrier to glucose, Na+, and H+. When the mucosal barrier was broken by bile salts or trypsin at a neutral pH, no acid back diffusion occurred and barrier disruption was accompanied by dramatic increases in esophageal mucosal blood flow. In contrast, barrier disruption by bile salts, pepsin, or acid during pH 2 perfusions failed to elicit increases in Qm when significant JH+ (50 microEq/hr) occurred. These results demonstrate a loss in reactive regulation of esophageal Qm in the presence of significant JH+ that may contribute to the injury seen in acid reflux esophagitis.

Somatostatin analog: effects on hypergastrinemia and hypercalcitoninemia.

A somatostatin analog (SMS 201-995) was used to treat symptomatic patients with a residual tumor burden of gastrinoma or medullary thyroid carcinoma and pathologic elevations of circulating marker peptides associated with these neuroendocrine tumors. Possible inhibitory effects of the analog on marker peptides, patients' symptoms, or tumor progression were studied in a dose-response protocol and during several months of self-injection of SMS 201-995. Both patients reported remarkable relief of secretory diarrhea and other symptoms, and serum gastrin was successfully suppressed by increasing doses of the analog. However, no effect was seen in reduction of hypercalcitoninemia. Morphologic imaging of residual tumor showed no progression of medullary thyroid carcinoma during treatment and, in the case of hepatic gastrinoma metastases, remarkable tumor regression was confirmed. No toxicity or glucose intolerance was experienced. Somatostatin analog shows promise for palliative management of endocrinologic symptoms due to neuroendocrine tumors, and an inhibitory effect can be measured in some but not all peptide markers. Further evidence of its negative trophic effect on tumor blood flow may suggest an antineoplastic potential, as well as palliative use of this new treatment.

Capsaicin-sensitive nerves mediate esophageal mucosal protection.

The esophageal mucosa is exposed to damaging agents both by ingestion and reflux. Using our in vivo rabbit model of esophagitis, we have observed that acute luminal exposure (within 1 to 5 minutes) to potentially harmful agents, such as acid, bile, or ethanol, induces a rapid increase in mucosal blood flow; whereas prolonged exposure (10 to 60 minutes) results in mucosal injury and ablation of blood flow. We have also shown that capsaicin-sensitive mucosal afferent nerves can modulate esophageal blood flow. These findings led us to hypothesize that the reactive increase in blood flow induced by luminal agents represents a mechanism of protection mediated by capsaicin-sensitive nerves. The objective of these experiments was to determine if luminal capsaicin, a specific probe for visceral afferent nerves, could both preserve mucosal blood flow and protect against ethanol injury. Rabbits were subjected to luminal instillation of 50% ethanol with or without 1% capsaicin. Blood flow was measured with microspheres at baseline and after 2 and 10 minutes. Rabbits exposed only to ethanol developed severe mucosal injury coincident with near ablation of mucosal blood flow. In contrast, rabbits exposed to ethanol with capsaicin showed protection of the epithelium with a sixfold increase in mucosal blood flow. We conclude that capsaicin-sensitive nerves in the esophagus are local effectors of mucosal protection by virtue of preserving blood flow.