Molecular mechanisms of mitotane action in adrenocortical cancer based on in vitro studies

SIMPLE SUMMARY: Mitotane is the only approved drug for the treatment of advanced adrenocortical carcinoma and for postoperative adjuvant therapy. It is known that mitotane destroys the adrenal cortex impairing steroidogenesis, although its exact molecular mechanism is still unclear. However, confounding factors affecting in vitro experiments could reduce the relevance of the studies. In this review, we explore in vitro studies on mitotane effects, highlighting how different experimental conditions might contribute to the controversial findings. On this basis, it may be necessary to re-evaluate the experiments taking into account their potential confounding factors such as cell strains, culture serum, lipoprotein concentration, and culture passages, which could hide important molecular results. As a consequence, the identification of novel pharmacological molecular pathways might be used in the future to implement personalized therapy, maximizing the benefit of mitotane treatment while minimizing its toxicity. ABSTRACT: Mitotane is the only approved drug for the treatment of advanced adrenocortical carcinoma and is increasingly used for postoperative adjuvant therapy. Mitotane action involves the deregulation of cytochromes P450 enzymes, depolarization of mitochondrial membranes, and accumulation of free cholesterol, leading to cell death. Although it is known that mitotane destroys the adrenal cortex and impairs steroidogenesis, its exact mechanism of action is still unclear. The most used cell models are H295-derived cell strains and SW13 cell lines. The diverging results obtained in presumably identical cell lines highlight the need for a stable in vitro model and/or a standard methodology to perform experiments on H295 strains. The presence of several enzymatic targets responsive to mitotane in mitochondria and mitochondria-associated membranes causes progressive alteration in mitochondrial structure when cells were exposed to mitotane. Confounding factors of culture affecting in vitro experiments could reduce the significance of any molecular mechanism identified in vitro. To ensure experimental reproducibility, particular care should be taken in the choice of culture conditions: aspects such as cell strains, culture serum, lipoproteins concentration, and culture passages should be carefully considered and explicated in the presentation of results. We aimed to review in vitro studies on mitotane effects, highlighting how different experimental conditions might contribute to the controversial findings. If the concerns pointed out in this review will be overcome, the new insights into mitotane mechanism of action observed in-vitro could allow the identification of novel pharmacological molecular pathways to be used to implement personalized therapy

Estimating the mean and variance from the median, range, and the size of a sample

BACKGROUND: Usually the researchers performing meta-analysis of continuous outcomes from clinical trials need their mean value and the variance (or standard deviation) in order to pool data. However, sometimes the published reports of clinical trials only report the median, range and the size of the trial. METHODS: In this article we use simple and elementary inequalities and approximations in order to estimate the mean and the variance for such trials. Our estimation is distribution-free, i.e., it makes no assumption on the distribution of the underlying data. RESULTS: We found two simple formulas that estimate the mean using the values of the median (m), low and high end of the range (a and b, respectively), and n (the sample size). Using simulations, we show that median can be used to estimate mean when the sample size is larger than 25. For smaller samples our new formula, devised in this paper, should be used. We also estimated the variance of an unknown sample using the median, low and high end of the range, and the sample size. Our estimate is performing as the best estimate in our simulations for very small samples (n ≤ 15). For moderately sized samples (15 <n ≤ 70), our simulations show that the formula range/4 is the best estimator for the standard deviation (variance). For large samples (n > 70), the formula range/6 gives the best estimator for the standard deviation (variance). We also include an illustrative example of the potential value of our method using reports from the Cochrane review on the role of erythropoietin in anemia due to malignancy. CONCLUSION: Using these formulas, we hope to help meta-analysts use clinical trials in their analysis even when not all of the information is available and/or reported

Heterogeneous vancomycin-intermediate susceptibility in a community-associated methicillin-resistant Staphylococcus aureus epidemic clone, in a case of Infective Endocarditis in Argentina

BACKGROUND: Community-Associated Methicillin Resistant Staphylococcus aureus (CA-MRSA) has traditionally been related to skin and soft tissue infections in healthy young patients. However, it has now emerged as responsible for severe infections worldwide, for which vancomycin is one of the mainstays of treatment. Infective endocarditis (IE) due to CA-MRSA with heterogeneous vancomycin-intermediate susceptibility-(h-VISA) has been recently reported, associated to an epidemic USA 300 CA-MRSA clone. CASE PRESENTATION: We describe the occurrence of h-VISA phenotype in a case of IE caused by a strain belonging to an epidemic CA-MRSA clone, distinct from USA300, for the first time in Argentina. The isolate h-VISA (SaB2) was recovered from a patient with persistent bacteraemia after a 7-day therapy with vancomycin, which evolved to fatal case of IE complicated with brain abscesses. The initial isolate-(SaB1) was fully vancomycin susceptible (VSSA). Although MRSA SaB2 was vancomycin susceptible (≤ 2 μg/ml) by MIC (agar and broth dilution, E-test and VITEK 2), a slight increase of MIC values between SaB1 and SaB2 isolates was detected by the four MIC methods, particularly for teicoplanin. Moreover, Sab2 was classified as h-VISA by three different screening methods [MHA5T-screening agar, Macromethod-E-test-(MET) and by GRD E-test] and confirmed by population analysis profile-(PAP). In addition, a significant increase in cell-wall thickness was revealed for SaB2 by electron microscopy. Molecular typing showed that both strains, SaB1 and SaB2, belonged to ST5 lineage, carried SCCmecIV, lacked Panton-Valentine leukocidin-(PVL) genes and had indistinguishable PFGE patterns (subtype I2), thereby confirming their isogenic nature. In addition, they were clonally related to the epidemic CA-MRSA clone (pulsotype I) detected in our country. CONCLUSIONS: This report demonstrates the ability of this epidemic CA-MRSA clone, disseminated in some regions of Argentina, to produce severe and rapidly fatal infections such as IE, in addition to its ability to acquire low-level vancomycin resistance; for these reasons, it constitutes a new challenge for the Healthcare System of this country.This study was supported by the National Council for Scientific Research and Technology of Argentina (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT - PICT 01630 to JLB), Secretaría de Ciencia y Técnica-Universidad Nacional de Córdoba (SECyT-UNC) and Agencia Córdoba Ciencia.S

A genomic, transcriptomic and proteomic look at the GE2270 producer Planobispora rosea, an uncommon actinomycete

We report the genome sequence of Planobispora rosea ATCC 53733, a mycelium-forming soil-dweller belonging to one of the lesser studied genera of Actinobacteria and producing the thiopeptide GE2270. The P. rosea genome presents considerable convergence in gene organization and function with other members in the family Streptosporangiaceae, with a significant number (44%) of shared orthologs. Patterns of gene expression in P. rosea cultures during exponential and stationary phase have been analyzed using whole transcriptome shotgun sequencing and by proteome analysis. Among the differentially abundant proteins, those involved in protein metabolism are particularly represented, including the GE2270-insensitive EF-Tu. Two proteins from the pbt cluster, directing GE2270 biosynthesis, slightly increase their abundance values over time. While GE2270 production starts during the exponential phase, most pbt genes, as analyzed by qRT-PCR, are down-regulated. The exception is represented by pbtA, encoding the precursor peptide of the ribosomally synthesized GE2270, whose expression reached the highest level at the entry into stationary phase. Copyright

Regulación de la proliferación de tumores hipofisarios inducidos por la vía FGF2/FGFR1

Abstract: Dysregulation of the growth factors and their receptors could lead to abnormal growth and progression of pituitary tumors. Previously, we demonstrated an increase in the expression of Fibroblast Growth Factor 2 (FGF2) in the experimental prolactinomas development, suggesting its participation in tumor development but the molecular mechanisms that generate this effect still now unknown. The aim was to determine the role of FGF2, receptor 1 (FGFR1) and the MEK-ERK1/2 pathway on the proliferation of pituitary tumor cells. Somatolactotroph (GH3) and corticotroph (ATt20) pituitary tumor cell lines stimulated with FGF2 (10 and 100 ng / mL) were used. The expression of FGFR1 was evaluated by western blot. Cell viability was determined by MTT assay after stimulation with FGF2 for 24-48h on medium with or without 10% serum. The proliferative response was analyzed by incorporation of BrdU for 24h and the MEK-ERK1/2 participation by ERK1/2 phosphorylation by western blot, after FGF2 stimulus for 30min. Additionally, the MEK inhibitor PD 98059 (50uM) was used. Statistics: ANOVA-Post test: Tukey. The expression of FGFR1 was higher in GH3 than in ATt20. FGF2 induced a significant increase in cell viability on GH3at 24 and 48h to both doses, while an increase (p&gt; 0.05) was only observed in ATt20 after stimulation with FGF2 100ng/mL for 48h. Considering that FGF2 effect in GH3 was major, we continue working on this cell line. The expression levels of ERK1/2 phosphorylated increased after FGF2 (10 and 100ng / mL) stimulus for 30min (p˂0.05 vs Control). Cell viability and BrdU incorporation was significantly higher in the cultures treated with both doses to FGF2 in presence of serum, effect that was reversed by PD 98059. These findings show that FGF2/FGFR1/ERK1/2 pathway participates in the increase of proliferation in GH3 lactosomatotroph tumor cells, which present greater expression of FGFR1, effect that was enhanced in serum medium which would be represent part tumor microenvironment in the tissue.Resumen:&nbsp; La desregulación de factores de crecimiento y sus receptores puede conducir al crecimiento anormal y a la progresión de tumores hipofisarios. Anteriormente, demostramos un aumento en la expresión del Factor de Crecimiento Fibroblástico 2 (FGF2) en el desarrollo de prolactinomas experimentales, sugiriendo su participación en el desarrollo tumoral, siendo desconocidos los mecanismos moleculares que generan este efecto. El objetivo fue determinar el rol de FGF2, el&nbsp;receptor 1 (FGFR1) y la vía MEK- ERK1/2 sobre la proliferación de células tumorales hipofisarias. Se utilizaron cultivos de líneas tumorales hipofisarias somatolactotropa (GH3) y corticotropa (ATt20) estimulados con FGF2 (10 y 100 ng/mL). La expresión del FGFR1 fue evaluada por western blot. La viabilidad celular fue determinada por el ensayo de MTT luego del estímulo con FGF2 por 24 y/o 48h, con 10% de suero o sin suero. La respuesta proliferativa fue analizada por incorporación de BrdU por 24h y la participación de MEK-ERK1/2 mediante la fosforilación de ERK1/2 por western blot luego de 30 min con FGF2. También se utilizó el inhibidor de MEK, PD 98059 (50uM). Estadística: ANOVA-Post test: Tukey. La expresión de FGFR1 fue mayor en GH3 que en ATt20. FGF2 indujo un aumento significativo de la viabilidad celular en GH3 con ambas dosis a 24 y 48h, mientras que en ATt20 solo se observó un aumento (p&gt;0,05) luego del estímulo con FGF2 100ng/mL por 48h. Tomando en cuenta que la respuesta a FGF2 en GH3 fue mayor, continuamos trabajando en esta línea celular. Se observó un aumento notable de los niveles de expresión de ERK1/2 fosforilada inducida por FGF2 (10 y 100ng/mL) en 30min (p˂0.05 vs Control). La viabilidad celular e incorporación de BrdU fue significativamente mayor en los cultivos tratados con FGF2 con ambas dosis en presencia de suero, efecto que fue revertido por PD 98059. Estos hallazgos muestran que la vía FGF2/FGFR1/ERK1/2 participa en el aumento de la proliferación de las células hipofisarias tumorales lactosomatotropa GH3, que presentan mayor expresión del FGFR1, efecto que es potenciado en medio con suero, lo que representaría parte del microambiente en el que se encontrarían las células tumorales en un tejido. &nbsp

Effects of cleaning procedures on the long-term corrosion behavior of bronze artifacts of the cultural heritage in outdoor environment

The cleaning of metallic artworks is a crucial step for their preservation. Cleaning operations generally aim at removing deposits and corrosion layers, and all the non-stable and potentially reactive phases formed as a consequence of the interaction of the metal with the environment. Thus, all secondary-formed compounds and layers that can undermine the overall preservation of the artwork, both from the esthetic and the corrosion point of view, should be removed. On the other hand, superficial stable patinas contributing to the artistic and historic value of the objects and that may provide protection to the metallic surface should be preserved. The optimal cleaning procedure should be able to promote a long-term improvement of the corrosion resistance of the surfaces. Therefore, the long-term monitoring of the corrosion behavior of the cleaned surfaces with electrochemical techniques could provide valuable information for the selection of the optimal methodology. In this work, five cleaning procedures have been applied to five bronze sculptures. The cleaned surfaces have been characterized following a multi-analytical and non-invasive approach, and the long-term evolution of their corrosion behavior has been monitored by means of on-site non-invasive linear polarization resistance (LPR) and electrochemical impedance spectroscopy (EIS) measurements for more than 2&nbsp;years