Experiência de 6 Anos de Cirurgia Micrográfica de Mohs num Hospital Português

Introduction: Mohs micrographic surgery (MMS) is a surgical method of treating skin tumors that consists of histological control of the tumor margins using horizontal slices in thin, freshly frozen layers. This technique is a preferred indication for cutaneous facial tumors (basal and squamous cell carcinoma, among others) associated with higher risk of recurrence, recurrent tumors, tumors with undefined clinical margins, when there is perivascular and perineural involvement or with aggressive histological subtypes. Methods: The authors carried out a retrospective study of a 6-year period (from July 2012 until June 2018) at a Portuguese hospital – Egas Moniz hospital, to define the characteristics of patients undergoing Mohs micrographic surgery and to evaluate the advantages of this technique. The following factors were analyzed: age, gender, provenience, diagnosis, tumor location, number of stages of MMS, type of reconstruction of the surgical defect, follow-up and recurrences. Results: A total of 835 tumors were excised, 459 in male patients and 376 in female patients. The mean age at surgery was 71 years old (range 23- 95 years). Most of the lesions submitted to MMS were basal cell carcinomas (87%) and the most common location was the nasal pyramid (43%). In 44% of the cases, it was necessary to perform more than one micrographic stage. Half of the surgical defect closures were performed by graft or skin flap. Considering patients with a follow up equal or superior to 3 years, the recurrence rate was 4.9%. Discussion: The most frequent diagnosis was basal cell carcinoma, reflecting the importance of MMS in this type of skin malignancy, in both primary and persistent/recurrent lesions. Compared to other European surgery reviews, our recurrence rate is within normal range. This fact is particularly relevant, if we consider that our institution receives patients sent from hospitals and dermatologists from all over the country, specially selected for tumor aggressiveness or previous relapse. More than half of these patients were referred from other centers and consisted of persistent/recurrent basal cell carcinomas. The follow-up at recurrence for MMS was 27.9 months, supporting that a long follow-up of these patients is necessary. Conclusion: MMS allows lower rates of recurrence compared to simple surgical excision, so its practice should be encouraged in selected tumors. The surgical control of the margins instead of the “blind margins” is an advantage in saving healthy tissue and allows the complete excision of the tumor in the same surgical time.Introdução: A cirurgia micrográfica de Mohs é um método cirúrgico de tratamento dos tumores cutâneos que consiste no controlo histológico das margens do tumor usando cortes horizontais em camadas finas congeladas a fresco. Esta técnica é uma indicação preferencial para tumores cutâneos da face (carcinoma basocelular e espinocelular, entre outros) associados a maior risco de recidiva, tumores recidivados, com margens clínicas indefinidas, quando existe envolvimento perivascular e perineural ou com subtipos histológicos agressivos. Métodos: Os autores realizaram um estudo retrospetivo de um período de 6 anos (de julho 2012 até junho 2018) num hospital Português – Hospital de Egas Moniz, para definir as características dos doentes submetidos a cirurgia micrográfica de Mohs e avaliar as vantagens desta técnica. Foram analisados os fatores: idade, género, proveniência, diagnóstico, localização do tumor, número de estádios da cirurgia micrográfica de Mohs, tipo de reconstrução do defeito cirúrgico, seguimento e recidiva. Resultados: Foram operados 835 tumores no total, 459 em doentes do sexo masculino e 376 em doentes do sexo feminino. A média de idades foi de 71 anos (intervalo entre 23- 95 anos). A maioria dos tumores submetidos a cirurgia micrográfica de Mohs foram carcinomas basocelulares (87%) localizados na pirâmide nasal (43%). Em 44% dos casos, houve necessidade de realizar mais de um estadio micrográfico. Metade dos encerramentos do defeito cirúrgico foram realizados por enxerto ou retalho cutâneo. Em doentes com seguimento igual ou superior a 3 anos, a taxa de recidiva foi de 4,9%. Discussão: O diagnóstico mais frequente foi o de carcinoma basocelular, o que reflete a importância da cirurgia micrográfica de Mohs, neste tipo de neoplasia cutânea, quer em lesões primárias como persistente/recorrentes. Salientamos que comparativamente a outras revisões europeias de cirurgia de Mohs, a taxa de recidiva dos tumores se encontra dentro dos parâmetros normais. Este dado é particularmente relevante, se considerarmos que o nosso serviço recebe doentes enviados de hospitais e dermatologistas de todo o país, especialmente selecionados quanto à agressividade tumoral ou já recidivados. O intervalo médio de recorrência para MMS foi de 27,9 meses, o que demonstra a necessidade de um seguimento a longo prazo destes doentes. Conclusão: A cirurgia micrográfica de Mohs permite menores taxas de recidiva comparativamente á excisão cirúrgica simples pelo que a sua prática deve ser encorajada em tumores devidamente selecionados. O controlo cirúrgico das margens em vez das “margens cegas” é uma mais-valia tanto na poupança de tecido são como na garantia da excisão completa do tumor num mesmo tempo cirúrgico

Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as “bacterial microtubules” (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening

Role of Magmas in protein transport and human mitochondria biogenesis

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of Δpam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process

A chain mechanism for flagellum growth.

Bacteria swim by means of long flagella extending from the cell surface. These are assembled from thousands of protein subunits translocated across the cell membrane by an export machinery at the base of each flagellum. Unfolded subunits then transit through a narrow channel at the core of the growing flagellum to the tip, where they crystallize into the nascent structure. As the flagellum lengthens outside the cell, the rate of flagellum growth does not change. The mystery is how subunit transit is maintained at a constant rate without a discernible energy source in the channel of the external flagellum. We present evidence for a simple physical mechanism for flagellum growth that harnesses the entropic force of the unfolded subunits themselves. We show that a subunit docked at the export machinery can be captured by a free subunit through head-to-tail linkage of juxtaposed amino (N)- and carboxy (C)-terminal helices. We propose that sequential rounds of linkage would generate a multisubunit chain that pulls successive subunits into and through the channel to the flagellum tip, and by isolating filaments growing on bacterial cells we reveal the predicted chain of head-to-tail linked subunits in the transit channel of flagella. Thermodynamic analysis confirms that links in the subunit chain can withstand the pulling force generated by rounds of subunit crystallization at the flagellum tip, and polymer theory predicts that as the N terminus of each unfolded subunit crystallizes, the entropic force at the subunit C terminus would increase, rapidly overcoming the threshold required to pull the next subunit from the export machinery. This pulling force would adjust automatically over the increasing length of the growing flagellum, maintaining a constant rate of subunit delivery to the tip

Mechanistic Insights into a Novel Exporter-Importer System of Mycobacterium tuberculosis Unravel Its Role in Trafficking of Iron

Elucidation of the basic mechanistic and biochemical principles underlying siderophore mediated iron uptake in mycobacteria is crucial for targeting this principal survival strategy vis-à-vis virulence determinants of the pathogen. Although, an understanding of siderophore biosynthesis is known, the mechanism of their secretion and uptake still remains elusive.Here, we demonstrate an interplay among three iron regulated Mycobacterium tuberculosis (M.tb) proteins, namely, Rv1348 (IrtA), Rv1349 (IrtB) and Rv2895c in export and import of M.tb siderophores across the membrane and the consequent iron uptake. IrtA, interestingly, has a fused N-terminal substrate binding domain (SBD), representing an atypical subset of ABC transporters, unlike IrtB that harbors only the permease and ATPase domain. SBD selectively binds to non-ferrated siderophores whereas Rv2895c exhibits relatively higher affinity towards ferrated siderophores. An interaction between the permease domain of IrtB and Rv2895c is evident from GST pull-down assay. In vitro liposome reconstitution experiments further demonstrate that IrtA is indeed a siderophore exporter and the two-component IrtB-Rv2895c system is an importer of ferrated siderophores. Knockout of msmeg_6554, the irtA homologue in Mycobacterium smegmatis, resulted in an impaired M.tb siderophore export that is restored upon complementation with M.tb irtA.Our data suggest the interplay of three proteins, namely IrtA, IrtB and Rv2895c in synergizing the balance of siderophores and thus iron inside the mycobacterial cell

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C

Phosphorylation Alters the Interaction of the Arabidopsis Phosphotransfer Protein AHP1 with Its Sensor Kinase ETR1

The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1−AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta

Structure-Function Relations in Oxaloacetate Decarboxylase Complex. Fluorescence and Infrared Approaches to Monitor Oxomalonate and Na+ Binding Effect

ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of α, β and γ subunits. The α subunit contains the carboxyltransferase catalytic site. characteristic of a high content of α helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of β sheet structures and a concomitant increase of α helix structures. Oxomalonate binding to αγand α subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex. alters the tryptophan environment of the β subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the α-helical as well as β-strand secondary structure elements

Purification and Activity Testing of the Full-Length YycFGHI Proteins of Staphylococcus aureus

Background: The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in Gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance. Methodology/Principal Findings: Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposomemodel indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment. Conclusions/Significance: The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies an