DNA damage independent inhibition of NF-κB transcription by anthracyclines.

Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here, we show that the extensively used anthracyclines Doxorubicin, Daunorubicin, and Epirubicin decrease the transcription of nuclear factor kappa B (NF-κB)-dependent gene targets, but not interferon-responsive genes in primary mouse (Mus musculus) macrophages. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-κB subunit RelA and its DNA-binding sites. The anthracycline variants Aclarubicin, Doxorubicinone, and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis.

EDU (5-Ethynyl-2'-Deoxyuridine)-Coupled Fluorescence-Intensity Analysis: Determining Absolute Parameters of the Cell Cycle.

The principles and practice of a methodology of cell cycle analysis that allows the estimation of the absolute length (in units of time) of all cell cycle stages (G1, S, and G2) are detailed herein. This methodology utilizes flow cytometry to take full advantage of the excellent stoichiometric properties of click chemistry. This allows detection, via azide-fluorochrome coupling, of the modified deoxynucleoside 5-ethynyl-2'-deoxyuridine (EDU) incorporated into replicated DNA through incremental pulsing times. This methodology, which we designated as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, can be applied to cell types with very distinct cell cycle features, and has shown excellent agreement with established techniques of cell cycle analysis. Useful modifications to the original protocol (Pereira et al., Oncotarget, 8:40514-40,532, 2017) have been introduced to increase flexibility in data collection and facilitate data analysis.

Ricoseius loxocheles (Acari: Phytoseiidae) is not a predator of false spider mite on coffee crops: What does it eat?

Ricoseius loxocheles (De Leon) (Acari: Phytoseiidae) is often found in coffee crops and is known to feed on coffee leaf rust, Hemileia vastatrix Berkeley and Broome (Uredinales). As the occurrence of coffee leaf rust is limited primarily to the rainy season, the mite may use other food sources to survive during the periods of low pathogen prevalence. It is well known that phytoseiid mites can survive on a variety of food sources, such as herbivorous mites, fungi and pollen. We evaluated the ability of R. loxocheles to survive and reproduce on a diet of Brevipalpus phoenicis Geijskes (Acari: Tenuipalpidae), cattail pollen (Typha spp.), clover rust (Puccinia oxalidis), bee pollen (Santa Bárbara® dehydrated pollen, Santa Bárbara, MG, Brazil) and coffee leaf rust. Ricoseius loxocheles did not survive or reproduce on any B. phoenicis stages tested (egg, larva, adult). The survival and oviposition of R. loxocheles were directly affected by the presence of coffee rust urediniospores, but not by the presence of the prey. Survival and oviposition of the phytoseiid were similar when fed cattail pollen, clover rust and coffee leaf rust but was lower when fed bee pollen. Our results show that R. loxocheles is not a predator of B. phoenicis but it is able to utilize other resources besides coffee leaf rust.

Targeting TNFα in severe psoriasis-mass spectrometry reveals a time-dependent specific inhibition of Factor H in responding patients.

Drugs targeting TNFα (eg, Etanercept®) provide effective control of severe psoriasis. In absence of validated biological parameters of inflammation in psoriasis most decisions on therapeutics have relied mostly on clinical criteria, namely the "Psoriasis Area and Severity Index" (PASI). The purpose of this study was to assess by mass spectrometry alterations in concentrations of serum proteins that specifically correlated with effectiveness of Etanercept treatment. This prospective study enrolled 10 patients suffering from moderate to severe psoriasis (PASI score > 10 and < 17) and treated with Etanercept over a period of 24 weeks; 10 healthy, age-matched volunteers provided controls. Serum proteins sensitive to Etanercept treatment were identified using SELDI-TOF (surface-enhanced laser desorption and ionization - time of flight) coupled to nano LC-ESI/MS (nano liquid chromatography-electrospray ionization/tandem mass spectrometry) technologies. For comparisons between groups of individuals p-values (considered significant when < 0.01) were estimated with non-parametric tests, namely Mann-Whitney (for unpaired data) and Wilcoxon signed-rank (for paired data). In responding patients it could be shown using SELDI-TOF spectrometry that two proteins (134 kDa and 4.3 kDa) return to control levels by 24 weeks of treatment. Using nano LC-ESI/MS the 134 kDa species was identified as complement Factor H. These observations deserve further analyses utilizing larger cohorts of patients. Determination of Factor H levels may become a complementary tool to follow remission or predict the onset of relapse in the follow-up of patients under treatment with Etanercept.

Quantification of cell cycle kinetics by EdU (5-ethynyl-2'-deoxyuridine)-coupled-fluorescence-intensity analysis.

We propose a novel single-deoxynucleoside-based assay that is easy to perform and provides accurate values for the absolute length (in units of time) of each of the cell cycle stages (G1, S and G2/M). This flow-cytometric assay takes advantage of the excellent stoichiometric properties of azide-fluorochrome detection of DNA substituted with 5-ethynyl-2'-deoxyuridine (EdU). We show that by pulsing cells with EdU for incremental periods of time maximal EdU-coupled fluorescence is reached when pulsing times match the length of S phase. These pulsing times, allowing labelling for a full S phase of a fraction of cells in asynchronous populations, provide accurate values for the absolute length of S phase. We characterized additional, lower intensity signals that allowed quantification of the absolute durations of G1 and G2 phases.Importantly, using this novel assay data on the lengths of G1, S and G2/M phases are obtained in parallel. Therefore, these parameters can be estimated within a time frame that is shorter than a full cell cycle. This method, which we designate as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, was successfully applied to cell types with distinctive cell cycle features and shows excellent agreement with established methodologies for analysis of cell cycle kinetics.

Ricoseius loxocheles, a phytoseiid mite that feeds on coffee leaf rust.

One of the most important diseases of coffee plants is the coffee leaf rust fungus Hemileia vastatrix Berkeley and Broome (Uredinales). It can cause 30 % yield loss in some varieties of Coffea arabica (L.). Besides fungus, the coffee plants are attacked by phytophagous mites. The most common species is the coffee red mite, Oligonychus ilicis McGregor (Acari: Tetranychidae). Predatory mites of the Phytoseiidae family are well-known for their potential to control herbivorous mites and insects, but they can also develop and reproduce on various other food sources, such as plant pathogenic fungi. In a field survey, we found Ricoseius loxocheles (De Leon) (Acari: Phytoseiidae) on the necrotic areas caused by the coffee leaf rust fungus during the reproductive phase of the pathogen. We therefore assessed the development, survivorship and reproduction of R. loxocheles feeding on coffee leaf rust fungus and measured predation and oviposition of this phytoseiid having coffee red mite as prey under laboratory conditions. The mite fed, survived, developed and reproduced successfully on this pathogen but it was not able to prey on O. ilicis. Survival and oviposition with only prey were the same as without food. This phytoseiid mite does not really use O. ilicis as food. It is suggested that R. loxocheles is one phytoseiid that uses fungi as a main food source.

Anthracyclines induce DNA damage response-mediated protection against severe sepsis.

Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fanconi Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.

Chemical healthcare waste management in small Brazilian municipalities.

The disposal of healthcare waste (HCW) seems to have been solved in developed countries, while in most developing countries the problem persists because the disposal methods are expensive and larger than the budget of small- and medium-sized municipalities. The current study evaluates the encapsulation process for the disposal of medical chemical waste. The experiment was developed in the Piraí municipality (Rio de Janeiro State, Brazil) and the chemical wastes were produced in the local public hospital, as well as the 12 units of primary care health services. Chemical waste generated at health services units may include the liquid waste from cleaning materials and disinfectants, expired and unused pharmaceutical products, and cytotoxins. These are all considered hazardous waste products and they must be disposed of via an authorised system at approved sites (e.g. industrial landfills). The process of encapsulating chemical medical waste in concrete (cement, crushed stones and sand) followed by their disposal at sanitary landfills is a procedure that is not considered in Brazilian Legislation. Despite the oversight, this method of disposal was used in the municipality of Piraí, with the approval of the Rio de Janeiro State Agency for Environmental Control. The safety aspects of this method and the limits of its applicability, particularly in small municipalities, were evaluated in this study. The results indicate that, within certain parameters, this method may provide a viable solution for the disposal of HCW in small municipalities.

Heart rate and energy expenditure during garbage collection in Rio de Janeiro, Brazil

Physiological workload is used to estimate the physical demand of tasks in the workforce, but limited information is available for the various work activities in developing countries. Eighty-three randomly selected male workers participated in the present study, aimed at assessing the physiological workload of garbage collection (GC) in Rio de Janeiro, Brazil. Heart rate (HR) was obtained on four consecutive workdays. Energy expenditure (EE) was extrapolated from heart rate data based on individual laboratory-established heart rate/oxygen consumption curve in 70 workers. Mean HR during GC was 104.0 ± 11.7bpm (± SD), representing 56.9 ± 7.5 percent of maximum heart rate. EE was 1608.3 ± 738.5kcal for an average of 293.1 ± 103.9 minutes of work per day. Based on all measurements, work in garbage collection in Rio de Janeiro can be considered excessively heavy. These data emphasize the need to develop appropriate classification of workload to be used in health-related research and in the development of maximum acceptable work time in association with the physiological workload, particularly in developing countries.

A carga de trabalho fisiológica é utilizada para estimar a demanda física de uma tarefa na população de trabalhadores adultos; no entanto, nos países em desenvolvimento há poucos dados disponíveis sobre as diversas atividades ocupacionais. Oitenta e três trabalhadores masculinos participaram neste estudo, com o objetivo de avaliar a carga fisiológica do trabalho de coleta de lixo domiciliar no Município do Rio de Janeiro, Brasil. A freqüência cardíaca foi medida em quatro dias de trabalho consecutivos. O gasto energético foi extrapolado a partir da freqüência cardíaca, com base em dados individuais obtidos em laboratório para a curva de freqüência cardíaca/consumo de oxigênio em 70 trabalhadores. A freqüência cardíaca média durante a coleta de lixo era 104,0 ± 11,7bpm, o que representa 56,9 ± 7,5 por cento da freqüência cardíaca máxima. O gasto energético era de 1.608,3 ± 738,5kcal para uma média de 293,1 ± 103,9 minutos de trabalho por dia. Com base em todas as medidas, o trabalho de coleta de lixo domiciliar pode ser classificado como excessivamente pesado. Os dados destacam a necessidade de se desenvolver uma classificação apropriada do trabalho, a ser utilizada em pesquisas sobre saúde do trabalhador e para definir cargas máximas de horas de trabalho em relação à carga de trabalho fisiológica, particularmente nos países em desenvolvimento.

Heart rate and energy expenditure during garbage collection in Rio de Janeiro, Brazil.

Physiological workload is used to estimate the physical demand of tasks in the workforce, but limited information is available for the various work activities in developing countries. Eighty-three randomly selected male workers participated in the present study, aimed at assessing the physiological workload of garbage collection (GC) in Rio de Janeiro, Brazil. Heart rate (HR) was obtained on four consecutive workdays. Energy expenditure (EE) was extrapolated from heart rate data based on individual laboratory-established heart rate/oxygen consumption curve in 70 workers. Mean HR during GC was 104.0 +/- 11.7 bpm (+/- SD), representing 56.9 +/- 7.5% of maximum heart rate. EE was 1608.3 +/- 738.5 kcal for an average of 293.1 +/- 103.9 minutes of work per day. Based on all measurements, work in garbage collection in Rio de Janeiro can be considered excessively heavy. These data emphasize the need to develop appropriate classification of workload to be used in health-related research and in the development of maximum acceptable work time in association with the physiological workload, particularly in developing countries.