Actividad antioxidante y quelante de sistemas hidrocoloides mixtos formados por hidrolizados proteicos de Phaseolus vulgaris endurecido y goma modificada de flamboyán (Delonix regia) / Antioxidant and metal-chelating activities of mixed hydrocolloid systems formed by hard-to-cook Phaseolus vulgaris protein hydrolysates and flamboyant (Delonix regia) modified gum

Las proteínas y polisacáridos con frecuencia son utilizados simultáneamente en la industria de alimentos. Las interacciones entre ambos biopolímeros juegan un papel importante en la estructura y estabilidad de muchos alimentos procesados ya que pueden resultar en un sistema con propiedades bioactivas diferentes como ocurre en el caso de las funcionales. Objetivo. Evaluar los cambios en la capacidad antioxidante de un sistema hidrocoloide mixto formado por un hidrolizado enzimático proteico de frijol endurecido (P. vulgaris) y goma modificada de flamboyán (Delonix regia) (SHM). Materiales y métodos. El estudio se hizo entre febrero-octubre, 2014, en Mérida, México. Se modificó enzimáticamente el concentrado proteico de P. vulgaris con Pepsina-Pancreatina® y la goma extraída del flamboyán mediante carboximetilación, preparando dispersiones al 1% de cada uno de los biopolímeros, determinado la fluorescencia intrínseca de Trp (FIT) como indicador de la interacción entre ambos biopolímeros y la actividad antioxidante y quelante del sistema resultante. Resultados. Se obtuvo un hidrolizado proteico extensivo con 28,8% de grado de hidrólisis y una goma de flamboyán con grado de sustitución de 0,91. La mayor interacción entre ambos biopolímeros, se obtuvo empleando una relación 5:1 del SHM a pH 4 de acuerdo con el valor de FIT. Conclusiones. Los sistemas hidrocoloides mixtos preparados con hidrolizado extensivo de las proteínas Phaseolus vulgaris y goma modificada de flamboyán presentaron un incremento en la actividad antioxidante, respecto del hidrolizado dependiendo del mecanismo de oxidación, así como de las condiciones de pH en que se encuentra el sistema y la interacción entre ambos componentes(AU)

Proteins and polysaccharides are frequently used simultaneously in the food industry. The interactions between both biopolymers play an important role in the structure and stability of many processed foods since they can result in a system with different bioactive properties as in the case of functional ones. Objective. Evaluate the changes in the antioxidant capacity of mixed hydrocolloid system formed by a hard to cook bean (P. vulgaris) protein enzymatic hydrolyzate and modified flamboyant gum (Delonix regia) (SHM). Materials and methods. The study was conducted from February to October 2014 in Merida, Mexico. For this, the protein concentrate of P. vulgaris was treated with Pepsin-Pancreatin® and the gum extracted from the flamboyant were modified enzymatically by carboxymethylation, preparing 1% dispersions of each of the biopolymers, determining the intrinsic Trp fluorescence (FIT) as an indicator of the interaction between both biopolymers and the antioxidant and chelating activity of the resulting system. Results. The main results indicated that an extensive protein hydrolyzate with 28.8% degree of hydrolysis and a flamboyant gum with a substitution degree of 0.91 were obtained. The greatest interaction between both biopolymers was obtained using a 5:1 ratio of SHM to pH 4 according to the FIT value. Conclusions. The mixed hydrocolloid systems prepared with extensive hydrolyzate of the hard to cook P. vulgaris and modified flamboyant gum proteins showed an increase in antioxidant activity, compared to the hydrolyzate depending on the oxidation mechanism, as well as the pH conditions used and interaction between both component(AU)

Estudo da estabilidade e peguilação de L-asparaginase em soluções aquosas de líquidos iônicos de imidazólio / Study of the stability and pegylation of L-asparaginase in aqueous imidazole ionic liquid solutions

A enzima L-asparaginase de Escherichia coli (ASNase) é um biofármaco indicado para o tratamento de leucemia linfoblástica aguda, mas que pode causar reações de hipersensibilidade nos pacientes tratados. Na tentativa de amenizar esse efeito, foi desenvolvida a PEG-ASNase (enzima conjugada com polietilenoglicol) que apresenta a vantagem de ser menos imunogênica e ter maior meia-vida biológica. Mais recentemente, novas abordagens têm sido desenvolvidas visando aprimorar os processos de PEGuilação por meio de reações sítio dirigidas, por exemplo N-terminal, a fim de promover maior similaridade lote a lote e controle das características farmacocinéticas e farmacodinâmicas do biofármaco. Porém, existe ainda uma limitação associada à hidrólise do PEG reativo, desta forma surge a necessidade de procurar solventes alternativos para a PEGuilação que permitam manter a estabilidade das proteínas, aumentar o rendimento de PEGuilação e a estabilidade do PEG reativo. Nesse trabalho, líquidos iônicos foram investigados como solventes alternativos para a peguilação N-terminal de PEG-ASNase. Para tal, a estabilidade de ASNase em Lis foi investigada em LIs da família metil-imidazol, analisando a influência do aumento da cadeia alquílica e de diferentes ânions. A estabilidade da ASNase é favorecida quando em contato com Lis relativamente hidrofóbicos ([C2mim]Cl, [C4mim]Cl e [C6mim]Cl), mas sua a atividade é prejudicada quando o LI é muito polar, como o [C4mim][(CH3)2PO4] ou anfifílico como o [C12mim]Cl. Apesar de seu efeito desnaturante, o [C4mim][(CH3)2PO4] resultou no maior rendimento da reação de PEGuilação da ASNase (56%) quando empregado a 75% e a reação realizada em 10 min. O [C4mim]Cl resultou em rendimento semelhante ao tampão fosfato (~ 49%), mas ambos os LIs reduziram a poliPEGuilação. Portanto, os Lis [C4mim]Cl e [C4mim][(CH3)2PO4] fornecem uma alternativa viável à reação de PEGuilação pela redução na formação de espécies poliPEGuiladas, o que facilitaria os processos de purificação e permitiria maior controle lote a lote da reação, bem como pelo aumento do rendimento da reação no caso do [C4mim][(CH3)2PO4]

Escherichia coli L-asparaginase enzyme (ASNase) is a biopharmaceutical indicated for the treatment of acute lymphoblastic leukemia, but may cause hypersensitivity in the patients used. In an attempt to alleviate this effect, PEG-ASNase (polyethylene glycol conjugated enzyme) was developed, which has the advantage of being less immunogenic and having a longer biological half-life. More recently, new approaches have been applied to improve PEGylation processes through targeted sites, for example N-terminal, in order to promote greater similarity to the batch and control of the pharmacokinetic and pharmacodynamic characteristics of the biopharmaceutical. However, there is still a limitation associated with reactive PEG hydrolysis, thus increasing the need to look for alternative PEGylation solvents to maintain protein stability, increase PEGylation yield and use reactive PEG. In this work, ions were investigated as alternative solvents for the N-terminal PEG-ASNase. For example, a stability of ASNase in ILs was investigated in imidazole ILs by analyzing the influence of increased alkyl chain and different anions. ASNase stability is enhanced when in contact with relatively hydrophobic ILs ([C2min]Cl, [C4min]Cl and [C6min]Cl), but its activity is impaired when very polar ILs such as [C4min][(CH3)2PO4] or amphiphilic as [C12mim]Cl. Despite its denaturing effect, [C4min][(CH3)2PO4] resulted in higher yield of ASNase PEGylation reaction (56%) when employed at 75% and reaction performed in 10 min. [C4min]Cl yielded similar phosphate buffer yield (~ 49%), but both ILs reduced polyPEGylation. Therefore, [C4min]Cl and [C4min][(CH3)2PO4] Ils may use a viable alternative to the PEGylation reaction and reduce the formation of polyPEGylated species, or that facilitate purification processes and allow for greater batch use of the solution, as well as increased reaction yield in the case of [C4min][(CH3)2PO4]

The insertion of bioactive peptides at the C-terminal end of an 11S globulin changes the structural stability and improves the antihypertensive activity

BACKGROUND: The 11S globulin from amaranth is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding a four valinetyrosine antihypertensive peptide at its C-terminal end to improve its functionality. The new protein was named AMR5 and expressed in the Escherichia coli BL21-CodonPlus(DE3)-RIL strain using a custom medium (F8PW) designed for this work. RESULTS: The alternative medium allowed for the production of 652 mg/L expressed protein at the flask level, mostly in an insoluble form, and this protein was subjected to in vitro refolding. The spectrometric analysis suggests that the protein adopts a ß/α structure with a small increment of α-helix conformation relative to the native amaranth 11S globulin. Thermal and urea denaturation experiments determined apparent Tm and C1/2 values of 50.4°C and 3.04 M, respectively, thus indicating that the antihypertensive peptide insertion destabilized the modified protein relative to the native one. AMR5 hydrolyzed by trypsin and chymotrypsin showed 14- and 1.3-fold stronger inhibitory activity against angiotensin I-converting enzyme (IC50 of 0.034 mg/mL) than the unmodified protein and the previously reported amaranth acidic subunit modified with antihypertensive peptides, respectively. CONCLUSION: The inserted peptide decreases the structural stability of amaranth 11S globulin and improves its antihypertensive activity.

Essential Role of Protein Arginine Methyltransferase 1 in Pancreas Development by Regulating Protein Stability of Neurogenin 3

BACKGROUND: Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells. Recent studies have revealed that PRMT1 plays important roles in the development of various tissues. However, its role in pancreas development has not yet been elucidated. METHODS: Pancreatic progenitor cell-specific Prmt1 knock-out (Prmt1 PKO) mice were generated and characterized for their metabolic and histological phenotypes and their levels of Neurog3 gene expression and neurogenin 3 (NGN3) protein expression. Protein degradation assays were performed in mPAC cells. RESULTS: Prmt1 PKO mice showed growth retardation and a severely diabetic phenotype. The pancreatic size and β-cell mass were significantly reduced in Prmt1 PKO mice. Proliferation of progenitor cells during the secondary transition was decreased and endocrine cell differentiation was impaired. These defects in pancreas development could be attributed to the sustained expression of NGN3 in progenitor cells. Protein degradation assays in mPAC cells revealed that PRMT1 was required for the rapid degradation of NGN3. CONCLUSION: PRMT1 critically contributes to pancreas development by destabilizing the NGN3 protein.

Extraction and purification of NUDT9 homology domain of human transient receptor potential melastatin 2 channel / 浙江大学学报·医学版

OBJECTIVE@#To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel.@*METHODS@#After sonication and centrifuge of strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected.@*RESULTS@#The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h.@*CONCLUSIONS@#Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.

Downregulation of SIRT2 by Chronic Stress Reduces Expression of Synaptic Plasticity-related Genes through the Upregulation of Ehmt2

Silent information regulator 2 (Sirtuin2 / SIRT2) is a NAD⁺-dependent deacetylase that regulates the cellular oxidative stress response. It modulates transcriptional silencing and protein stability through deacetylation of target proteins including histones. Previous studies have shown that SIRT2 plays a role in mood disorders and hippocampus-dependent cognitive function, but the underlying neurobiological mechanism is poorly understood. Here, we report that chronic stress suppresses SIRT2 expression in the hippocampus. Molecular and biochemical analyses indicate that the stress-induced decrease in the SIRT2 expression downregulates synaptic plasticity-related genes in the hippocampus through the increase of euchromatic histone-lysine N-methyltransferase 2 (Ehmt2) (also known as G9a). shRNA-mediated knockdown of SIRT2 in the dentate gyrus alters the expression of synaptic plasticity-related genes in a way similar to those induced by chronic stress, and produces depression-like behaviors. Our results indicate that SIRT2 plays an important role in the response to stress, thereby modulating depression-like behaviors.

Purification, characterization, mode of action, and enhanced production of Salivaricin mmaye1, a novel bacteriocin from Lactobacillus salivarius SPW1 of human gut origin

Background: Emergence of antibiotic resistance among pathogenic and food spoilage bacteria such as Staphylococcus aureus, Micrococcus luteus, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus mutans, Bacillus cereus, and Listeria monocytogenes triggered the search for alternative antimicrobials. An investigation aimed at purifying, characterizing, elucidating the mode of action, and enhancing the production of salivaricin from Lactobacillus salivarius of human gut origin was conducted. Results: Salivaricin mmaye1 is a novel bacteriocin purified from L. salivarius isolated from human feces. It is potent at micromolar concentrations and has a molecular weight of 1221.074 Da as determined by MALDI-TOF mass spectrometry. It has a broad spectrum of antibacterial activity. Salivaricin mmaye1 showed high thermal and chemical stability and moderate pH stability. The proteinaceous nature of salivaricin mmaye1 was revealed by the complete loss of activity after treatment with pepsin, trypsin, α-chymotrypsin, protease, and proteinase. Salivaricin mmaye1 is cell wall associated, and adsorption­desorption of the bacteriocin from the cell wall of the producer by pH modification proved successful. It exhibited a bactericidal mode of action mediated by pore formation. Its biosynthesis is regulated by a quorum sensing mechanism. Enhanced production of salivaricin mmaye1 was achieved in a newly developed growth medium. Conclusions: A novel, cell wall adhering, highly potent bacteriocin with a broad spectrum of inhibitory activity, membrane-permeabilizing ability, and enhanced production in a newly constituted medium has been isolated. It has a quorum sensing regulatory system and possesses interesting physicochemical characteristics favoring its future use in food biopreservation. These findings pave the way for future evaluation of its medical and food applications.

Avaliação da qualidade e da estabilidade de amostras de soro humano para análise e armazenamento em biorrepositório / Evaluation of the quality and the stability of human samples for analysis and storage in biorepository

Objetivo: O Estudo das Condições de Vida e Saúde de Pomerode, SC (SHIP-Brasil), conduzido pela Universidade Regional de Blumenau, visa estratificar os fatores de risco e genéticos eventualmente existentes na gênese das mais variadas doenças. Para tal são necessários exames laboratoriais, antropométricos, genéticos e de imagem para a definição das mesmas e o acompanhamento de sua evolução. Este trabalho teve como objetivo avaliar a qualidade das amostras armazenadas no biorrepositório do estudo, que podem ser utilizadas para pesquisas de biomarcadores no futuro. Métodos: Foram avaliadas amostras de soro em jejum de 56 voluntários. Os analitos estudados foram glicose, colesterol total, creatinina e transaminase glutâmico-pirúvica (ALT), após 1, 2 e 3 anos de armazenamento a -80ºC. Os resultados foram analisados com o Teste t de Student e de acordo com o Erro Permitido Máximo para cada analito. Resultados: Não encontramos evidências de que as amostras do Estudo SHIP-Brasil possam perder a sua qualidade após serem armazenadas por longos períodos. Conclusão: Os Procedimentos Operacionais Padrão (POPs) estabelecidos estão adequados para o processamento e preservação das amostras.

Mutations of G38R and D40G cause amyotrophic lateral sclerosis by reducing Annexin A11 protein stability / 中南大学学报(医学版)

To explore the role of the mutations G38R and D40G of Annexin A11 (ANXA11) in the onset of amyotrophic lateral sclerosis (ALS).
 Methods: The plasmids expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were constructed, respectively. The recombinant plasmids were then transfected into HEK293 cells respectively followed by cycloheximide (CHX) treatment for 0, 2, 4 and 8 h. The protein expressions of ANXA11 wild type, ANXA11 G38R and ANXA11 D40G mutations were determined by Western blot. Gray analysis by Image J was performed to compare the half-life of each protein. The NSC-34 cell lines constantly expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were established. The cells were treated with NP-40 lysis buffer to examine the protein solubility by Western blot.
 Results: Both ANXA11 G38R protein and ANXA11 D40G protein showed a shorter half-life than ANXA11 wild type protein (P0.05). There was no visible insoluble substance in the NP-40 lysates for ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein.
 Conclusion: G38R and D40G mutations reduce the stability of ANXA11 protein. G38R and D40G mutations do not alter ANXA11 solubility.

The role of ubiquitination and deubiquitination in the regulation of cell junctions

Maintenance of cell junctions plays a crucial role in the regulation of cellular functions including cell proliferation, permeability, and cell death. Disruption of cell junctions is implicated in a variety of human disorders, such as inflammatory diseases and cancers. Understanding molecular regulation of cell junctions is important for development of therapeutic strategies for intervention of human diseases. Ubiquitination is an important type of post-translational modification that primarily regulates endogenous protein stability, receptor internalization, enzyme activity, and protein-protein interactions. Ubiquitination is tightly regulated by ubiquitin E3 ligases and can be reversed by deubiquitinating enzymes. Recent studies have been focusing on investigating the effect of protein stability in the regulation of cell-cell junctions. Ubiquitination and degradation of cadherins, claudins, and their interacting proteins are implicated in epithelial and endothelial barrier disruption. Recent studies have revealed that ubiquitination is involved in regulation of Rho GTPases' biological activities. Taken together these studies, ubiquitination plays a critical role in modulating cell junctions and motility. In this review, we will discuss the effects of ubiquitination and deubiquitination on protein stability and expression of key proteins in the cell-cell junctions, including junction proteins, their interacting proteins, and small Rho GTPases. We provide an overview of protein stability in modulation of epithelial and endothelial barrier integrity and introduce potential future search directions to better understand the effects of ubiquitination on human disorders caused by dysfunction of cell junctions.

New Aspects of Vascular Calcification: Histone Deacetylases and Beyond

Vascular calcification is a pathologic phenomenon in which calcium phosphate is ectopically deposited in the arteries. Previously, calcification was considered to be a passive process in response to metabolic diseases, vascular or valvular diseases, or even aging. However, now calcification is recognized as a highly-regulated consequence, like bone formation, and many clinical trials have been carried out to elucidate the correlation between vascular calcification and cardiovascular events and mortality. As a result, vascular calcification has been implicated as an independent risk factor in cardiovascular diseases. Many molecules are now known to be actively associated with this process. Recently, our laboratory found that posttranslational modification of histone deacetylase (HDAC) 1 is actively involved in the development of vascular calcification. In addition, we found that modulation of the activity of HDAC as well as its protein stability by MDM2, an HDAC1-E3 ligase, may be a therapeutic target in vascular calcification. In the present review, we overview the pathomechanism of vascular calcification and the involvement of posttranslational modification of epigenetic regulators.

Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli

Background: The acidic subunit of amarantin (AAC)-the predominant amaranth seed storage protein-has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4). Results: E. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves. Conclusions: Modifications to the third VR affect the thermal stability of amarantin acidic subunit.

Parkin promotes proteasomal degradation of p62: implication of selective vulnerability of neuronal cells in the pathogenesis of Parkinson's disease

Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson's disease (PD), respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN) and striatum (STR) regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.

In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

The effects of storage temperatures, repeated freeze-thaw cycles, or delays in separating plasma or serum from blood samples are largely unknown for heat shock protein 27 (HSP27). We evaluated (1) the imprecision of the HSP27 assay used in this study; (2) the in vitro stability of HSP27 in blood samples stored at 4℃ for up to 6 hr with immediate and delayed serum/plasma separation from cells; and (3) the in vitro stability of HSP27 in blood samples stored at -80℃ after repeated freeze-thaw cycles. The ELISA to detect HSP27 in this study showed a within-run CV of <9% and a total CV of <15%. After 4-6 hr of storage at 4℃, HSP27 concentrations remained stable when using serum tubes irrespective of sample handling, but HSP27 concentrations decreased by 25-45% when using EDTA plasma tubes. Compared with baseline HSP27, one freeze-thaw cycle had no effect on serum concentrations. However, plasma concentrations increased by 3.1-fold after one freeze-thaw cycle and by 7.3-fold after five freeze-thaw cycles. In conclusion, serum is an appropriate biological sample type for use in epidemiological and large-scale clinical studies.

F-box only protein 9 is an E3 ubiquitin ligase of PPARγ

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of carbohydrate and lipid metabolism, adipocyte differentiation and inflammatory response. Post-translational modification of PPARγ and its degradation involve several pathways, including the ubiquitin–proteasome system. Here, we identified F-box only protein 9 (FBXO9) as an E3 ubiquitin ligase of PPARγ. We screened interacting partners of PPARγ using immunoprecipitation and mass spectrometric analysis and identified FBXO9 as an E3 ubiquitin ligase of PPARγ. FBXO9 directly interacted with PPARγ through the activation function-1 domain and ligand-binding domain. FBXO9 decreased the protein stability of PPARγ through induction of ubiquitination. We found that the F-box motif of FBXO9 was required for its ubiquitination function. The activity of PPARγ was significantly decreased by FBXO9 overexpression. Furthermore, FBXO9 overexpression in 3T3-L1 adipocytes resulted in decreased levels of endogenous PPARγ and suppression of adipogenesis. These results suggest that FBXO9 is an important enzyme that regulates the stability and activity of PPARγ through ubiquitination.

Intravenous lidocaine for postmastectomy pain treatment: randomized, blind, placebo controlled clinical trial / Lidocaína intravenosa no tratamento da dor pós-mastectomia: ensaio clínico aleatório encoberto placebo controlado / Lidocaína intravenosa en el tratamiento del dolor posmastectomía: ensayo clínico aleatorizado, encubierto, placebo controlado

BACKGROUND AND OBJECTIVE: Postoperative pain treatment in mastectomy remains a major challenge despite the multimodal approach. The aim of this study was to investigate the analgesic effect of intravenous lidocaine in patients undergoing mastectomy, as well as the postoperative consumption of opioids. METHODS: After approval by the Human Research Ethics Committee of the Instituto de Medicina Integral Prof. Fernando Figueira in Recife, Pernambuco, a randomized, blind, controlled trial was conducted with intravenous lidocaine at a dose of 3 mg/kg infused over 1 h in 45 women undergoing mastectomy under general anesthesia. One patient from placebo group was. RESULTS: Groups were similar in age, body mass index, type of surgery, and postoperative need for opioids. Two of 22 patients in lidocaine group and three of 22 patients in placebo group requested opioid (p = 0.50). Pain on awakening was identified in 4/22 of lidocaine group and 5/22 of placebo group (p = 0.50); in the post-anesthetic recovery room in 14/22 and 12/22 (p = 0.37) of lidocaine and placebo groups, respectively. Pain evaluation 24 h after surgery showed that 2/22 and 3/22 patients (p = 0.50) of lidocaine and placebo groups, respectively, complained of pain. CONCLUSION: Intravenous lidocaine at a dose of 3 mg/kg administered over a period of an hour during mastectomy did not promote additional analgesia compared to placebo in the first 24 h, and has not decreased opioid consumption. However, a beneficial effect of intravenous lidocaine in selected and/or other therapeutic regimens patients cannot be ruled out. .

JUSTIFICATIVA E OBJETIVO: O tratamento da dor pós-operatória em mastectomia continua sendo um grande desafio apesar da abordagem multimodal. O objetivo deste estudo foi investigar o efeito analgésico da lidocaína intravenosa em pacientes submetidas a mastectomia, como também, o consumo de opioide pós-operatório. MÉTODOS: Após aprovação pelo comitê de ética e pesquisa em seres humanos do Instituto de Medicina Integral Prof. Fernando Figueira em Recife - Pernambuco foi realizado ensaio clínico aleatório encoberto placebo controlado com lidocaína intravenosa na dose de 3 mg/kg infundida em uma hora, em 45 mulheres submetidas a mastectomia sob anestesia geral. Excluída uma paciente do grupo placebo. RESULTADOS: Os grupos foram semelhantes quanto à idade, índice de massa corpórea, tipo de intervenção cirúrgica e necessidade de opioide no pós-operatório. Solicitaram opioide 2/22 pacientes nos grupos da lidocaína e 3/22 placebo (p = 0,50). Identificada a dor ao despertar em 4/22 no grupo lidocaína e 5/22 (p = 0,50) no grupo placebo; na sala de recuperação pós-anestésica em 14/22 e 12/22 (p = 0,37) nos grupos lidocaína e placebo respectivamente. Ao avaliar a dor 24 horas após o procedimento cirúrgico 3/22 e 2/22 (p = 0,50) das pacientes relataram dor em ambos os grupos respectivamente. CONCLUSÃO: A lidocaína intravenosa na dose de 3mg/kg administrada em um período de uma hora no transoperatório de mastectomia não promoveu analgesia adicional em relação ao grupo placebo nas primeiras 24 horas e não diminuiu o consumo de opioide. Contudo, um efeito benéfico da lidocaína intravenosa em pacientes selecionadas e/ou em outros regimes terapêuticos não pode ser descartado. .

JUSTIFICACIÓN Y OBJETIVO: El tratamiento del dolor postoperatorio en la mastectomía continúa siendo un gran reto a pesar del abordaje multimodal. El objetivo de este estudio fue investigar el efecto analgésico de la lidocaína intravenosa en pacientes sometidas a mastectomía, así como el consumo postoperatorio de opiáceos. MÉTODOS: Después de la aprobación por el Comité de Ética e Investigación en seres humanos del Instituto de Medicina Integral Prof. Fernando Figueira, en Recife, Pernambuco, se realizó un ensayo clínico aleatorizado, encubierto, placebo controlado con lidocaína intravenosa en una dosis de 3 mg/kg infundida en una hora, en 45 mujeres sometidas a mastectomía bajo anestesia general. Una paciente del grupo placebo fue excluida. RESULTADOS: Los grupos fueron similares en cuanto a la edad, índice de masa corporal, tipo de intervención quirúrgica y necesidad de opiáceos en el postoperatorio. Solicitaron opiáceos 2/22 pacientes en los grupos de la lidocaína y 3/22 placebo (p = 0,50). Fue identificado el dolor al despertar en 4/22 en el grupo lidocaína y 5/22 (p = 0,50) en el grupo placebo; en la sala de recuperación postanestésica en 14/22 y 12/22 (p = 0,37) en los grupos lidocaína y placebo, respectivamente. Al calcular el dolor 24 h después del procedimiento quirúrgico 3/22 y 2/22 (p = 0,50) de las pacientes relataron dolor en ambos grupos respectivamente. CONCLUSIÓN: La lidocaína intravenosa en una dosis de 3 mg/kg administrada en un período de una hora en el transoperatorio de mastectomía no generó analgesia adicional con relación al grupo placebo en las primeras 24 h y no disminuyó el consumo de opiáceos. Sin embargo, no puede ser descartado un efecto beneficioso de la lidocaína intravenosa en pacientes seleccionadas y/o en otros regímenes terapéuticos. .

Parotid Incidentaloma Identified by Positron Emission/Computed Tomography: When to Consider Diagnoses Other than Warthin Tumor

Introduction Parotid gland incidentalomas (PGIs) are unexpected hypermetabolic foci in the parotid region that can be found when scanning with whole-body positron emission/computed tomography (PET/CT). These deposits are most commonly due to benign lesions such as Warthin tumor. Objective The aim of this study was to determine the prevalence of PGIs identified in PET/CT scans and to assess the role of smoking in their etiology. Methods We retrospectively reviewed all PET/CT scans performed at our center in search of PGIs and identified smoking status and standardized uptake value (SUVmax) in each case. We also analyzed the database of parotidectomies performed in our department in the previous 10 years and focused on the pathologic diagnosis and the presence or absence of smoking in each case. Results Sixteen cases of PGIs were found in 4,250 PET/CT scans, accounting for 0.4% . The average SUVmax was 6.5 (range 2.8 to 16). Cytology was performed in five patients; it was benign in four cases and inconclusive in one case. Thirteen patients had a history of smoking. Of the parotidectomies performed in our center with a diagnosis of Warthin tumor, we identified a history of smoking in 93.8% of those patients. Conclusions The prevalence of PGIs on PET/CT was similar to that reported by other authors. Warthin tumor is frequently diagnosed among PGIs on PET/CT, and it has a strong relationship with smoking. We suggest that a diagnosis other than Warthin tumor should be considered for PGIs in nonsmokers. .

Estratégias para o enfrentamento do absenteísmo em consultas odontológicas nas Unidades de Saúde da Família de um município de grande porte: uma pesquisa-ação / Strategies for tackling absenteeism in dental appointments in the Family Health Units of a large municipality: action research

O objetivo deste trabalho foi analisar os motivos das faltas às consultas odontológicas em Unidades de Saúde da Família (USF) e implementar estratégias para sua redução por meio da pesquisa-ação. O estudo foi realizado em 12 USF de Piracicaba/SP, de 01 de janeiro a 31 de dezembro de 2010. A amostra se consistiu de 385 usuários, entrevistados por telefone, sobre os motivos das faltas, além de 12 cirurgiões-dentistas e 12 enfermeiras. Realizaram-se duas oficinas com os profissionais: uma para problematização dos dados coletados nas entrevistas e elaboração de estratégias; e outra após 4 meses, para avaliação. O maior motivo de faltas foi a coincidência do horário de funcionamento das unidades com o de trabalho dos usuários. Dentre as estratégias ressaltou-se a realização de palestras sobre saúde bucal, educação permanente nas reuniões de equipe, capacitação dos Agentes Comunitários de Saúde, participação em grupos terapêuticos e parcerias entre Equipe de Saúde Bucal e equipamentos sociais da comunidade. A adoção de prontuário único foi a estratégia desafiadora encontrada pelos profissionais. Concluiu-se que as estratégias implementadas levaram à diminuição das faltas em 66,6% e o caráter motivador das oficinas possibilitou a reflexão crítica para o redirecionamento da prática em saúde.

The aim of this study was to analyze the reasons for missed appointments in dental Family Health Units (FHU) and implement strategies to reduce same through action research. This is a study conducted in 12 FHUs in Piracicaba in the State of São Paulo from January, 1 to December, 31 2010. The sample was composed of 385 users of these health units who were interviewed over the phone and asked about the reasons for missing dental appointments, as well as 12 dentists and 12 nurses. Two workshops were staged with professionals: the first to assess the data collected in interviews and develop strategy, and the second for evaluation after 4 months. The primary cause for missed appointments was the opening hours of the units coinciding with the work schedule of the users. Among the strategies suggested were lectures on oral health, ongoing education in team meetings, training of Community Health Agents, participation in therapeutic groups and partnerships between Oral Health Teams and the social infrastructure of the community. The adoption of the single medical record was the strategy proposed by professionals. The strategies implemented led to a 66.6% reduction in missed appointments by the units and the motivating nature of the workshops elicited critical reflection to redirect health practices.

Sumoylation of Hes6 Regulates Protein Degradation and Hes1-Mediated Transcription

BACKGROUND: Hes6 is a transcriptional regulator that induces transcriptional activation by binding to transcription repressor Hes1 and suppressing its activity. Hes6 is controlled by the ubiquitin-proteosome-mediated degradation system. Here we investigated the sumoylation of Hes6 and its functional role in its rhythmic expression. METHODS: Hes6, SUMO, and ubiquitin were transfected into HeLa cells and the expression pattern was observed by Western blot and immunoprecipitation. To confirm the effect of sumoylation on the rhythmic expression of Hes6, we generated mouse Hes6 promoter-driven GFP-Hes6 fusion constructs and expressed these constructs in NIH 3T3 cells. RESULTS: Overexpression of SUMO led to sumoylation of Hes6 at both lysine 27 and 30. Protein stability of Hes6 was decreased by sumoylation. Moreover, expression of a Hes6 sumoylation-defective mutant, the 2KR (K27/30R) mutant, or co-expression of SUMO protease SUSP1 with native Hes6, strongly reduced ubiquitination. In addition, sumoylation was associated with both the rhythmic expression and transcriptional regulation of Hes6. Wild type Hes6 showed oscillatory expression with about 2-hour periodicity, whereas the 2KR mutant displayed a longer period. Furthermore, sumoylation of Hes6 derepressed Hes1-induced transcriptional repression. CONCLUSION: Hes6 sumoylation plays an important role in the regulation of its stability and Hes1-mediated transcription. These results suggest that sumoylation may be crucial for rhythmic expression of Hes6 and downstream target genes.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.