Cell-specific regulation of acetylcholinesterase expression under inflammatory conditions

Acetylcholine (ACh) has been shown to exert an anti-inflammatory function by down-modulating the expression of pro-inflammatory cytokines. Its availability can be regulated at different levels, namely at its synthesis and degradation steps. Accordingly, the expression of acetylcholinesterase (AChE), the enzyme responsible for ACh hydrolysis, has been observed to be modulated in inflammation. To further address the mechanisms underlying this effect, we aimed here at characterizing AChE expression in distinct cellular types pivotal to the inflammatory response. This study was performed in the human acute leukaemia monocytyc cell line, THP-1, in human monocyte-derived primary macrophages and in human umbilical cord vein endothelial cells (HUVEC). In order to subject these cells to inflammatory conditions, THP-1 and macrophage were treated with lipopolysaccharide (LPS) from E.coli and HUVEC were stimulated with the tumour necrosis factor α (TNF-α). Our results showed that although AChE expression was generally up-regulated at the mRNA level under inflammatory conditions, distinct AChE protein expression profiles were aurprisingly observed among the distinct cellular types studied. Altogether, these results argue for the existence of cell specific mechanisms that regulate the expression of acetylcholinesterase in inflammation.

Cellular hypomethylation is associated with impaired nitric oxide production by cultured human endothelial cells

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease, but the underlying mechanisms remain incompletely defined. Reduced bioavailability of nitric oxide (NO) is a principal manifestation of underlying endothelial dysfunction, which is an initial event in vascular disease. Inhibition of cellular methylation reactions by S-adenosylhomocysteine (AdoHcy), which accumulates during HHcy, has been suggested to contribute to vascular dysfunction. However, thus far, the effect of intracellular AdoHcy accumulation on NO bioavailability has not yet been fully substantiated by experimental evidence. The present study was carried out to evaluate whether disturbances in cellular methylation status affect NO production by cultured human endothelial cells. Here, we show that a hypomethylating environment, induced by the accumulation of AdoHcy, impairs NO production. Consistent with this finding, we observed decreased eNOS expression and activity, but, by contrast, enhanced NOS3 transcription. Taken together, our data support the existence of regulatory post-transcriptional mechanisms modulated by cellular methylation potential leading to impaired NO production by cultured human endothelial cells. As such, our conclusions may have implications for the HHcy-mediated reductions in NO bioavailability and endothelial dysfunction.

Non-coding RNAs: multi-tasking molecules in the cell

In the last years it has become increasingly clear that the mammalian transcriptome is highly complex and includes a large number of small non-coding RNAs (sncRNAs) and long noncoding RNAs (lncRNAs). Here we review the biogenesis pathways of the three classes of sncRNAs, namely short interfering RNAs (siRNAs), microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs). These ncRNAs have been extensively studied and are involved in pathways leading to specific gene silencing and the protection of genomes against virus and transposons, for example. Also, lncRNAs have emerged as pivotal molecules for the transcriptional and post-transcriptional regulation of gene expression which is supported by their tissue-specific expression patterns, subcellular distribution, and developmental regulation. Therefore, we also focus our attention on their role in differentiation and development. SncRNAs and lncRNAs play critical roles in defining DNA methylation patterns, as well as chromatin remodeling thus having a substantial effect in epigenetics. The identification of some overlaps in their biogenesis pathways and functional roles raises the hypothesis that these molecules play concerted functions in vivo, creating complex regulatory networks where cooperation with regulatory proteins is necessary. We also highlighted the implications of biogenesis and gene expression deregulation of sncRNAs and lncRNAs in human diseases like cancer.

Complementarity of conventional and molecular methods in the assessment of fungal contamination caused by Aspergillus fumigatus complex in one Portuguese composting plant

The handling of waste and compost that occurs frequently in composting plants (compost turning, shredding, and screening) has been shown to be responsible for the release of dust and air borne microorganisms and their compounds in the air. Thermophilic fungi, such as A. fumigatus, have been reported and this kind of contamination in composting facilities has been associated with increased respiratory symptoms among compost workers. This study intended to characterize fungal contamination in a totally indoor composting plant located in Portugal. Besides conventional methods, molecular biology was also applied to overcome eventual limitations.

Fungal contamination in two Portuguese wastewater treatment plants

The presence of filamentous fungi was detected in wastewater and air collected at wastewater treatment plants (WWTP) from several European countries. The aim of the present study was to assess fungal contamination in two WWTP operating in Lisbon. In addition, particulate matter (PM) contamination data was analyzed. To apply conventional methods, air samples from the two plants were collected through impaction using an air sampler with a velocity air rate of 140 L/min. Surfaces samples were collected by swabbing the surfaces of the same indoor sites. All collected samples were incubated at 27°C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. For molecular methods, air samples of 250 L were also collected using the impinger method at 300 L/min airflow rate. Samples were collected into 10 ml sterile phosphate-buffered saline with 0.05% Triton X-100, and the collection liquid was subsequently used for DNA extraction. Molecular identification of Aspergillus fumigatus and Stachybotrys chartarum was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR Detection System (Corbett). Assessment of PM was also conducted with portable direct-reading equipment (Lighthouse, model 3016 IAQ). Particles concentration measurement was performed at five different sizes: PM0.5, PM1, PM2.5, PM5, and PM10. Sixteen different fungal species were detected in indoor air in a total of 5400 isolates in both plants. Penicillium sp. was the most frequently isolated fungal genus (58.9%), followed by Aspergillus sp. (21.2%) and Acremonium sp. (8.2%), in the total underground area. In a partially underground plant, Penicillium sp. (39.5%) was also the most frequently isolated, also followed by Aspergillus sp. (38.7%) and Acremonium sp. (9.7%). Using RT-PCR, only A. fumigatus was detected in air samples collected, and only from partial underground plant. Stachybotrys chartarum was not detected in any of the samples analyzed. The distribution of particle sizes showed the same tendency in both plants; however, the partially underground plant presented higher levels of contamination, except for PM2.5. Fungal contamination assessment is crucial to evaluating the potential health risks to exposed workers in these settings. In order to achieve an evaluation of potential health risks to exposed workers, it is essential to combine conventional and molecular methods for fungal detection. Protective measures to minimize worker exposure to fungi need to be adopted since wastewater is the predominant internal fungal source in this setting.

Assessment of fungal contamination in waste sorting and incineration: case study in Portugal

Organic waste is a rich substrate for microbial growth, and because of that, workers from waste industry are at higher risk of exposure to bioaerosols. This study aimed to assess fungal contamination in two plants handling solid waste management. Air samples from the two plants were collected through an impaction method. Surface samples were also collected by swabbing surfaces of the same indoor sites. All collected samples were incubated at 27◦C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. Air samples were also subjected to molecular methods by real-time polymerase chain reaction (RT PCR) using an impinger method to measure DNA of Aspergillus flavus complex and Stachybotrys chartarum. Assessment of particulate matter (PM) was also conducted with portable direct-reading equipment. Particles concentration measurement was performed at five different sizes (PM0.5; PM1; PM2.5; PM5; PM10). With respect to the waste sorting plant, three species more frequently isolated in air and surfaces were A. niger (73.9%; 66.1%), A. fumigatus (16%; 13.8%), and A. flavus (8.7%; 14.2%). In the incineration plant, the most prevalent species detected in air samples were Penicillium sp. (62.9%), A. fumigatus (18%), and A. flavus (6%), while the most frequently isolated in surface samples were Penicillium sp. (57.5%), A. fumigatus (22.3%) and A. niger (12.8%). Stachybotrys chartarum and other toxinogenic strains from A. flavus complex were not detected. The most common PM sizes obtained were the PM10 and PM5 (inhalable fraction). Since waste is the main internal fungal source in the analyzed settings, preventive and protective measures need to be maintained to avoid worker exposure to fungi and their metabolites.

Fungal contamination assessment in Portuguese elderly care centers

Individuals spend 80-90% of their day indoors and elderly subjects are likely to spend even a greater amount of time indoors. Thus, indoor air pollutants such as bioaerosols may exert a significant impact on this age group. The aim of this study was to characterize fungal contamination within Portuguese elderly care centers. Fungi were measured using conventional as well as molecular methods in bedrooms, living rooms, canteens, storage areas, and outdoors. Bioaerosols were evaluated before and after the microenvironments' occupancy in order to understand the role played by occupancy in fungal contamination. Fungal load results varied from 32 colony-forming units CFU m(-3) in bedrooms to 228 CFU m(-3) in storage areas. Penicillium sp. was the most frequently isolated (38.1%), followed by Aspergillus sp. (16.3%) and Chrysonilia sp. (4.2%). With respect to Aspergillus genus, three different fungal species in indoor air were detected, with A. candidus (62.5%) the most prevalent. On surfaces, 40 different fungal species were isolated and the most frequent was Penicillium sp. (22.2%), followed by Aspergillus sp. (17.3%). Real-time polymerase chain reaction did not detect the presence of A. fumigatus complex. Species from Penicillium and Aspergillus genera were the most abundant in air and surfaces. The species A. fumigatus was present in 12.5% of all indoor microenvironments assessed. The living room was the indoor microenvironment with lowest fungal concentration and the storage area was highest.

Aspergillus flavus contamination in two Portuguese wastewater treatment plants

Filamentous fungi from genus Aspergillus were previously detected in wastewater treatment plants (WWTP) as being Aspergillus flavus (A. flavus), an important toxigenic fungus producing aflatoxins. This study aimed to determine occupational exposure adverse effects due to fungal contamination produced by A. flavus complex in two Portuguese WWTP using conventional and molecular methodologies. Air samples from two WWTP were collected at 1 m height through impaction method. Surface samples were collected by swabbing surfaces of the same indoor sites. After counting A. flavus and identification, detection of aflatoxin production was ensured through inoculation of seven inoculates in coconut-milk agar. Plates were examined under long-wave ultraviolet (UV; 365 nm) illumination to search for the presence of fluorescence in the growing colonies. To apply molecular methods, air samples were also collected using the impinger method. Samples were collected and collection liquid was subsequently used for DNA extraction. Molecular identification of A. flavus was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR detection system (Corbett). Among the Aspergillus genus, the species that were more abundant in air samples from both WWTP were Aspergillus versicolor (38%), Aspergillus candidus (29.1%), and Aspergillus sydowii (12.7%). However, the most commonly species found on surfaces were A. flavus (47.3%), Aspergillus fumigatus (34.4%), and Aspergillus sydowii (10.8%). Aspergillus flavus isolates that were inoculated in coconut agar medium were not identified as toxigenic strains and were not detected by RT-PCR in any of the analyzed samples from both plants. Data in this study indicate the need for monitoring fungal contamination in this setting. Although toxigenic strains were not detected from A. flavus complex, one cannot disregard the eventual presence and potential toxicity of aflatoxins.

Epigenetic and transcriptional signatures of stable versus plastic differentiation of proinflammatory gd T cell subsets

Two distinct subsets of γδ T cells that produce interleukin 17 (IL-17) (CD27(-) γδ T cells) or interferon-γ (IFN-γ) (CD27(+) γδ T cells) develop in the mouse thymus, but the molecular determinants of their functional potential in the periphery remain unknown. Here we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory networks of peripheral IFN-γ-producing or IL-17-producing γδ T cell subsets in vivo. We found that CD27(+) γδ T cells were committed to the expression of Ifng but not Il17, whereas CD27(-) γδ T cells displayed permissive chromatin configurations at loci encoding both cytokines and their regulatory transcription factors and differentiated into cells that produced both IL-17 and IFN-γ in a tumor microenvironment.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Potential pathogenic fungi assessment through molecular biology in cork industry

Portugal has been the world leader in the cork sector in terms of exports, employing ten thousands of workers. In this working activity, the permanent contact with cork may lead to the exposure to fungi, raising concerns as potential occupational hazards in cork industry. The application of molecular tools is crucial in this setting, since fungal species with faster growth rates may hide other species with clinical relevance, such as species belonging to P. glabrum and A. fumigatus complexes. A study was developed aiming at assessing fungal contamination due to Aspergillus fumigatus complex and Penicillium glabrum complex by molecular methods in three cork industries in the outskirt of Lisbon city.

Fungal burden in waste industry: an occupational risk to be solved

High loads of fungi have been reported in different types of waste management plants. This study intends to assess fungal contamination in one waste-sorting plant before and after cleaning procedures in order to analyze their effectiveness. Air samples of 50 L were collected through an impaction method, while surface samples, taken at the same time, were collected by the swabbing method and subject to further macro- and microscopic observations. In addition, we collected air samples of 250 L using the impinger Coriolis μ air sampler (Bertin Technologies) at 300 L/min airflow rate in order to perform real-time quantitative PCR (qPCR) amplification of genes from specific fungal species, namely Aspergillus fumigatus and Aspergillus flavus complexes, as well as Stachybotrys chartarum species. Fungal quantification in the air ranged from 180 to 5,280 CFU m−3 before cleaning and from 220 to 2,460 CFU m−3 after cleaning procedures. Surfaces presented results that ranged from 29 × 104 to 109 × 104 CFU m−2 before cleaning and from 11 × 104 to 89 × 104 CFU m−2 after cleaning. Statistically significant differences regarding fungal load were not detected between before and after cleaning procedures. Toxigenic strains from A. flavus complex and S. chartarum were not detected by qPCR. Conversely, the A. fumigatus species was successfully detected by qPCR and interestingly it was amplified in two samples where no detection by conventional methods was observed. Overall, these results reveal the inefficacy of the cleaning procedures and that it is important to determine fungal burden in order to carry out risk assessment.

Slaughterhouses fungal burden assessment: a contribution for the pursuit of a better assessment strategy

In slaughterhouses, the biological risk is present not only from the direct or indirect contact with animal matter, but also from the exposure to bioaerosols. Fungal contamination was already reported from the floors and walls of slaughterhouses. This study intends to assess fungal contamination by cultural and molecular methods in poultry, swine/bovine and large animal slaughterhouses. Air samples were collected through an impaction method, while surface samples were collected by the swabbing method and subjected to further macro- and micro-scopic observations. In addition, we collected air samples using the impinger method in order to perform real-time quantitative PCR (qPCR) amplification of genes from specific fungal species, namely A. flavus, A. fumigatus and A. ochraceus complexes. Poultry and swine/bovine slaughterhouses presented each two sampling sites that surpass the guideline of 150 CFU/m3. Scopulariopsis candida was the most frequently isolated (59.5%) in poultry slaughterhouse air; Cladosporium sp. (45.7%) in the swine/bovine slaughterhouse; and Penicillium sp. (80.8%) in the large animal slaughterhouse. Molecular tools successfully amplified DNA from the A. fumigatus complex in six sampling sites where the presence of this fungal species was not identified by conventional methods. This study besides suggesting the indicators that are representative of harmful fungal contamination, also indicates a strategy as a protocol to ensure a proper characterization of fungal occupational exposure.