Clinical Significance of the Lymphoscintigraphy in the Evaluation of Non-axillary Sentinel Lymph Node Localization in Breast Cancer

Rezumat Semnificaåia clinicã a evaluãrii limfoscintigrafice în localizarea non-axilarã a ganglionului santinelã în cancerul mamar Introducere: Identificarea aei biopsia ganglionului santinelã (SLN) în stadiile precoce ale cancerului mamar (T1-T2N0) a devenit metoda standard în tratamentul chirurgical al axilei datoritã acurateåii evaluãrii statusului ganglionilor axilari, evitându-se disecåia extensivã a axilei la pacienåii cu SLN negativ. Studiul nostru îaei propune sã evidenåieze rolul limfoscintigrafiei cu 99m-Tc nanocoloid în vizualizarea preoperatorie a SLN, în special în cazul altor localizãri decât cea axilarã aei aportul acestei tehnici în stadializarea corectã a cancerului mamar. Material aei metodã: Au fost incluaei în studiu 430 de pacienåi (vârsta 31-81 ani) cu cancer mamar (stadializare T1-T2N0), care au efectuat limfoscintigrafie pentru identificarea ganglionului santinelã în cadrul Departamentului de Medicinã Nuclearã al Institutului Oncologic "Prof. Dr. Al. Trestioreanu" Bucureaeti în perioada octombrie 2008 -iulie 2014. S-a injectat peritumoral sau intradermic periareolar 99m Tc-nanocoloid în dozã de 20-37 MBq (volum de 0,3-0,5 ml) efectuându-se apoi achiziåii dinamice aei statice postinjectare. Identificarea ganglionilor santinelã intraoperator s-a realizat utilizând sonda gamma, dupã marcajul pe piele efectuat preoperator la finalizarea limfoscintigrafiei. Rezultate: S-au identificat imagistic un numãr de 697 ganglioni santinelã la 427 din pacienåi (99%). Dintre aceaetia localizarea a fost axilarã la 364 pacienåi aei non axilarã (interpectoralã, mamara internã, supraclavicularã, intramamarã) la 48 pacienåi (11%), un numãr de 15 pacienåi (3%) având localizare multiplã (axilarã aei non-axilarã). Examenul histopatologic intraoperator a identificat un numãr de 74 ganglioni santinelã invadaåi (macrometastaze 12% aei micrometastaze 88 %). Concluzii: Identificarea aei biopsia ganglionului santinelã în stadiile I aei IIA este o practicã utilã în stadializarea corectã nu numai în cazul drenajului limfatic axilar, dar aei în alte localizãri mai rare ale extensiei ganglionare în cancerul mamar, orientând în continuare managementul acestor pacienåi dupã efectuarea intervenåiei chirurgicale. Cuvinte cheie: cancer mamar, biopsia ganglionului santinelã (SLNB), limfadenectomia axilarã (ALND) Abstract Background: Identification and biopsy of the sentinel lymph node (SLN) in early-stage breast cancer (T1-T2N0) has become the standard method in the surgical treatment of the axilla, due to its accuracy in the evaluation of axillary lymph node status, thus avoiding extensive axillary lymph node dissection in patients with negative SLN. The aim of our study is to highlight the role of Tcnanocolloid) was injected using peritumoral or periareolar intradermal technique, doses between 20-37 MBq (0.3-0.5 ml volume), followed by static and dynamic post-injection acquisitions. Intraoperative identification of the SLN was performed using a gamma-probe, guided by the skin marker performed preoperatively after completion of lymphoscintigraphy. Results: 697 sentinel lymph nodes were identified through imaging techniques in 427 patients (99%). Of them, 364 patients had axillary localization of the SLN, while 48 patients (11%) had non-axillary (pectoral, internal mammary, supraclavicular, intra-mammary) localization and 15 patients (3%) had multiple localization (axillary and non-axillary). Intraoperative histopathological exam revealed lymphatic invasion in 74 SLN (12% macrometastases and 88% micrometastases). Conclusions: The identification and biopsy of the sentinel lymph node in stages I and IIA is a useful routine for accurate breast cancer staging, suited for axillary lymphatic drainage, as well as for unusual non-axillary SLN localization, guiding the clinician for further postoperative management of these patients

Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

Retroviruses selectively package two copies of their RNA genomes in the context of a large excess of nongenomic RNA. Specific packaging of genomic RNA is achieved, in part, by recognizing RNAs that form a poorly understood dimeric structure at their 5′ ends. We identify, quantify the stability of, and use extensive experimental constraints to calculate a 3D model for a tertiary structure domain that mediates specific interactions between RNA genomes in a gamma retrovirus. In an initial interaction, two stem–loop structures from one RNA form highly stringent cross-strand loop–loop base pairs with the same structures on a second genomic RNA. Upon subsequent folding to the final dimer state, these intergenomic RNA interactions convert to a high affinity and compact tertiary structure, stabilized by interdigitated interactions between U-shaped RNA units. This retroviral conformational switch model illustrates how two-step formation of an RNA tertiary structure yields a stringent molecular recognition event at early assembly steps that can be converted to the stable RNA architecture likely packaged into nascent virions

Secondary Structure of the Mature Ex Virio Moloney Murine Leukemia Virus Genomic RNA Dimerization Domain

Retroviral genomes are dimeric, comprised of two sense-strand RNAs linked at their 5′ ends by noncovalent base pairing and tertiary interactions. Viral maturation involves large-scale morphological changes in viral proteins and in genomic RNA dimer structures to yield infectious virions. Structural studies have largely focused on simplified in vitro models of genomic RNA dimers even though the relationship between these models and authentic viral RNA is unknown. We evaluate the secondary structure of the minimal dimerization domain in genomes isolated from Moloney murine leukemia virions using a quantitative and single nucleotide resolution RNA structure analysis technology (selective 2′-hydroxyl acylation analyzed by primer extension, or SHAPE). Results are consistent with an architecture in which the RNA dimer is stabilized by four primary interactions involving two sets of intermolecular base pairs and two loop-loop interactions. The dimerization domain can independently direct its own folding since heating and refolding reproduce the same structure as visualized in genomic RNA isolated from virions. Authentic ex virio RNA has a SHAPE reactivity profile similar to that of a simplified transcript dimer generated in vitro, with the important exception of a region that appears to form a compact stem-loop only in the virion-isolated RNA. Finally, we analyze the conformational changes that accompany folding of monomers into dimers in vitro. These experiments support well-defined structural models for an authentic dimerization domain and also emphasize that many features of mature genomic RNA dimers can be reproduced in vitro using properly designed, simplified RNAs

Управління комплексною вентиляційною мережею соляної шахти Trotus

The attestation of salt extraction in these areas was located in the Geto-Dacian period at Oituz and Târgu Ocna. The oldest written testimony dates from 1380. The mining works that serve the Trotuş mine within the Tg. Ocna Salt Mine, are located in the Feţele Târgului salt massif located near the town of Tg. Ocna, on the left side of the Trotuş river. The exploitation of the salt was carried out until 1870 at the Ocnița Mine with a bell-shaped chamber and continued in the Moldova Veche and Moldova Nouă mines with trapezoidal chambers. Since 1967, the salt has been exploited with "small rooms and abandoned square pillars". The exploitation is carried out descending, on the horizons. Currently, the Trotuș Salt Mine has 14 horizons: 2 at the Pilot mine and 12 at the Trotuș mine. The specialized 3D CANVENT program was used to establish the optimal distribution of air flows. A number of 492 junctions and 697 branches were used to solve the ventilation network related to the Trotuș Salt Mine.Атестація видобутку солі в цих районах проходила в гето-дакійський період в Ойтузі та Тиргу-Очна. Найдавніші письмові свідчення датуються 1380 роком. Гірничі роботи, що обслуговують шахту Тротуш у межах Тг. Соляна шахта Окна, розташована в соляному масиві Фецеле-Тиргулуй, що знаходиться недалеко від міста Тг. Очна, з лівого боку річки Тротуш. Експлуатація солі велася до 1870 р. На шахті Окниця з дзвоноподібною камерою і продовжувалась у шахтах Молдова Вече та Молдова Ноуа з трапецієподібними камерами. З 1967 р. Сіль експлуатується в "маленьких кімнатах і занедбаних квадратних стовпах". Експлуатація здійснюється низхідно, по горизонтах. В даний час соляна шахта Тротуш має 14 горизонтів: 2 на пілотній шахті та 12 на шахті Тротуш. Для встановлення оптимального розподілу повітряних потоків була використана спеціалізована програма 3D CANVENT. Для вирішення вентиляційної мережі, що стосується соляної шахти Тротуш, було використано 492 перехрестя та 697 гілок

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs—only four nucleotides per genomic RNA—reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs

Exploring RNA Structural Codes with SHAPE Chemistry

RNA is the central conduit for gene expression. This role depends on an ability to encode information at two levels: in its linear sequence and in the complex structures RNA can form by folding back on itself. Understanding the global structure-function interrelationships mediated by RNA remains a great challenge in molecular and structural biology. In this Account, we discuss evolving work in our laboratory focused on creating facile, generic, quantitative, accurate, and highly informative approaches for understanding RNA structure in biologically important environments. The core innovation derives from our discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl in RNA is gated by local nucleotide flexibility. The 2'-hydroxyl is reactive at conformationally flexible positions but is unreactive at nucleotides constrained by base pairing. Sites of modification in RNA can be detected efficiently either using primer extension or by protection from exoribonucleolytic degradation. This technology is now called SHAPE, for selective 2'-hydroxyl acylation analyzed by primer extension (or protection from exoribonuclease). SHAPE reactivities are largely independent of nucleotide identity but correlate closely with model-free measurements of molecular order. The simple SHAPE reaction is thus a robust, nucleotide-resolution, biophysical measurement of RNA structure. SHAPE can be used to provide an experimental correction to RNA folding algorithms and, in favorable cases, yield kilobase-scale secondary structure predictions with high accuracies. SHAPE chemistry is based on very simple reactive carbonyl centers that can be varied to yield slow- and fast-reacting reagents. Differential SHAPE reactivities can be used to detect specific RNA positions with slow local nucleotide dynamics. These positions, which are often in the C2'-endo conformation, have the potential to function as molecular timers that regulate RNA folding and function. In addition, fast-reacting SHAPE reagents can be used to visualize RNA structural biogenesis and RNA-protein assembly reactions in one second snapshots in very straightforward experiments. The application of SHAPE to challenging problems in biology has revealed surprises in well-studied systems. New regions have been identified that are likely to have critical functional roles on the basis of their high levels of RNA structure. For example, SHAPE analysis of large RNAs, such as authentic viral RNA genomes, suggests that RNA structure organizes regulatory motifs and regulates splicing, protein folding, genome recombination, and ribonucleoprotein assembly. SHAPE has also revealed limitations to the hierarchical model for RNA folding. Continued development and application of SHAPE technologies will advance our understanding of the many ways in which the genetic code is expressed through the underlying structure of RNA

Studies on the Restriction of Murine Leukemia Viruses by Mouse APOBEC3

APOBEC3 proteins function to restrict the replication of retroviruses. One mechanism of this restriction is deamination of cytidines to uridines in (−) strand DNA, resulting in hypermutation of guanosines to adenosines in viral (+) strands. However, Moloney murine leukemia virus (MoMLV) is partially resistant to restriction by mouse APOBEC3 (mA3) and virtually completely resistant to mA3-induced hypermutation. In contrast, the sequences of MLV genomes that are in mouse DNA suggest that they were susceptible to mA3-induced deamination when they infected the mouse germline. We tested the possibility that sensitivity to mA3 restriction and to deamination resides in the viral gag gene. We generated a chimeric MLV in which the gag gene was from an endogenous MLV in the mouse germline, while the remainder of the viral genome was from MoMLV. This chimera was fully infectious but its response to mA3 was indistinguishable from that of MoMLV. Thus, the Gag protein does not seem to control the sensitivity of MLVs to mA3. We also found that MLVs inactivated by mA3 do not synthesize viral DNA upon infection; thus mA3 restriction of MLV occurs before or at reverse transcription. In contrast, HIV-1 restricted by mA3 and MLVs restricted by human APOBEC3G do synthesize DNA; these DNAs exhibit APOBEC3-induced hypermutation

Digital ulcers predict a worse disease course in patients with systemic sclerosis

Objective: Systemic sclerosis (SSc) is a systemic autoimmune disease with high morbidity and significant mortality. There is a great need of predictors that would allow risk stratification of patients with SSc and ultimately initiation of treatment early enough to ensure optimal clinical results. In this study, we evaluated whether a history of digital ulcers (HDU) at presentation may be a predictor of vascular outcomes and of overall clinical worsening and death in patients with SSc. Methods: Patients from the EULAR Scleroderma Trials and Research (EUSTAR) database, satisfying at inclusion the 1980 American College of Rheumatology classification criteria for SSc, who had a follow-up of at least 3 years since baseline or who have died, were included in the analysis. HDU at presentation as a predictor of disease worsening or death was evaluated by Cox proportional hazards regression analysis. Results :3196 patients matched the inclusion criteria (male sex 13.2%, 33.4% diffuse subset). At presentation, 1092/3196 patients had an HDU (34.1%). In multivariable analysis adjusting for age, gender and all parameters considered potentially significant, HDU was predictive for the presence of active digital ulcers (DUs) at prospective visits (HR (95% CI)): 2.41(1.91 to 3.03), p<0.001, for an elevated systolic pulmonary arterial pressure on heart ultrasound (US-PAPs):1.36 (1.03 to 1.80), p=0.032, for any cardiovascular event (new DUs, elevated US-PAPs or LV failure):3.56 (2.26 to 5.62), p<0.001, and for death (1.53 (1.16 to 2.02), p=0.003). Conclusions :In patients with SSc, HDU at presentation predicts the occurrence of DUs at follow-up and is associated with cardiovascular worsening and decreased survival

Vasodilators and low-dose acetylsalicylic acid are associated with a lower incidence of distinct primary myocardial disease manifestations in systemic sclerosis: results of the DeSScipher inception cohort study

OBJECTIVES: To investigate the influence of vasodilator drugs on the occurrence of features depending on myocardial ischaemia/fibrosis (ventricular arrhythmias, Q waves, cardiac blocks, pacemaker implantation, left ventricular ejection fraction (LVEF) <55%, and/or congestive heart failure and sudden cardiac death) in systemic sclerosis (SSc). METHODS: 601 patients with SSc were enrolled from 1 December 2012 to 30 November 2015 and had a second visit 0.5-4 years apart. 153 received no vasodilators; 448 received vasodilator therapy (ie, calcium channel blockers and/or ACE inhibitors or angiotensin II receptor blockers or combinations of them), 89 of them being also treated with either endothelin receptor antagonists or PDE5 inhibitors or prostanoids. Associations between the occurrence of myocardial disease manifestations and any demographic, disease and therapeutic aspect were investigated by Cox regression analysis. A Cox frailty survival model with centre of enrolment as random effect was performed. RESULTS: During 914 follow-up patient-years, 12 ventricular arrhythmias, 5 Q waves, 40 cardiac blocks, 6 pacemaker implantations and 19 reduced LVEF and/or congestive heart failure (CHF) occurred. In multivariate Cox regression analysis, vasodilator therapy was associated with a lower incidence of ventricular arrhythmias (p=0.03); low-dose acetylsalicylic acid (ASA) with a lower incidence of cardiac blocks and/or Q waves and/or pacemaker implantation (p=0.02); active disease with a higher incidence of LVEF <55% and/or CHF and cardiac blocks and/or Q waves and/or pacemaker implantation (p=0.05). CONCLUSIONS: The present study might suggest a preventative effect on the occurrence of distinct myocardial manifestations by vasodilator therapy and low-dose ASA