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

From the farm to the fork: fungal occupational exposure in the swine meat supply chain

Feed production, swine and slaughterhouses were already reported as occupational environments with high fungal contamination. This condition can ultimately lead to the development of several health conditions. This study aimed to characterize the occupational exposure to fungal burden in three different settings: swine feed unit, swine units and slaughterhouse

Effects of Quercetin in transcriptional and post-transcriptional regulation of fetal hemoglobin

This project was supported by Instituto Politécnico de Lisboa under the grant IDI&CAIPL/2022/miRCa/ESTeSL and partially supported by FCT/MCTES (UIDB/05608/2020 and UIDP/05608/2020). The authors are grateful to Fernando Nunes for kindly providing the Carica papaya leaves.Hemoglobinopathies are a group of inherited blood disorders that primarily affect red blood cells. The most common type is known as sickle cell anemia (SCA). It is characterized by mutations in the HBB gene, which encodes the β-subunit of human hemoglobin, giving rise to hemoglobin S (HbS). When deoxygenated, HbS polymerizes in the red blood cell, giving it a sickle shape and making it rigid and fragile. Fetal hemoglobin (HbF) is the major genetic modulator of the hematologic and clinical features of sickle cell disease, an effect mediated by its exclusion from the sickle hemoglobin polymer. Fetal hemoglobin genes are genetically regulated, and the level of HbF and its distribution among sickle erythrocytes is highly variable. Currently, therapies that induce HbF are promising, such as hydroxyurea (HU). However, due to high costs for underdeveloped countries and the adverse side effects, it is important to test alternative products and develop new compounds, such as Quercetin, a natural flavonoid present in plants that has antioxidant and anti-inflammatory properties.info:eu-repo/semantics/publishedVersio

Dissecting the IFN-g versus IL-17-specific mRNAomes of effector gd T lymphocytes

The ability of murine γδ T cells to rapidly produce the pro-inflammatory cytokines interleukin-17 (IL-17) or interferon-γ (IFN-γ) underlies their crucial roles in several pathophysiological contexts, from infection to cancer or autoimmunity. This functional capacity stems from a complex process of ‘developmental pre-programming’ in the thymus, after which a significant fraction of γδ T cells migrate to peripheral sites already committed to producing either IL-17 (gd17) or IFN-γ (gdIFN). While several studies have studied these gd T cell subtypes using surface markers that enrich for effector function, we still lack a characterization of the mRNA transcriptomes that specifically associate with IL-17 or IFN-g production by gd T cells. To overcome this limitation, in this study, we established a double reporter IL-17-GFP:IFN-γ-YFP mouse strain, which allowed us to isolate pure IL-17+, IFN-γ+, and the remaining IL-17-IFN-g-(DN) γδ T cell populations from the peripheral lymphoid organs to perform RNA sequencing and identify the subset-specific mRNAomes. Overall, we detected the expression of 12822 genes in gd T cells, with a significant number of genes being enriched in gd17 when compared with gdIFN and gdDN cells. Among these, 936 genes were differentially expressed between the three populations, with gd17 and gdIFN cells displaying the most distinct mRNAomes, which highlights their functional specialization, and gdIFN being more similar to DN than gd17 cells. A more detailed analysis of the top 30 differentially expressed genes among the most expressed genes by gd17 and gdIFN cells revealed that the majority of the signature genes increase their expression levels in the periphery upon their egress from the thymus, suggesting that these effector subsets only terminate their differentiation process at peripheral sites. Notably, gd17-associated signature genes are specifically expressed in this subset, unlike gdIFN signature genes, which are also often expressed by gdDN T cells, thus suggesting a developmental relationship between these two subpopulations. Collectively, our data allowed us to identify distinct mRNA signatures directly associated with cytokine expression in γδ T cells, several of which we are now further studying in disease models to identify potential new roles in pathophysiology.info:eu-repo/semantics/publishedVersio

Effects of methanolic and aqueous Carica Papaya leaf extracts in transcriptional and miRNA-mediated regulation of fetal hemoglobin

This project was supported by Instituto Politécnico de Lisboa under the grant IDI&CA-IPL/2022/miRCa/ESTeSL and partially supported by FCT/MCTES (UIDB/05608/2020 and UIDP/05608/2020). The authors are grateful to Fernando Nunes for kindly providing the Carica Papaya leaves.Sickle cell disease (SCD) is a genetic blood disorder caused by mutations in β-globin gene that affect the shape and transport of red blood cells in blood vessels, leading to various clinical complications. The pharmacological reactivation of Fetal Hemoglobin (HbF) through compounds such as Hydroxyurea (HU), is one of the available treatments, however, their safety concerns and expensive cost in low- and middle-income countries limit their use. In this context, it is essential to study novel HbF-inducing compounds that have scarcer adverse effects and can be widely available, such as Carica papaya extracts, a medicinal plant with anti-oxidant and anti-inflammatory properties. Therefore, the main aim of this work is to evaluate the effects of Carica Papaya leaf extracts (CPLE) in HbF reactivation. More specifically, we started by evaluating the effect of a methanolic CPLE extract in K562 cells (human immortalized myeloid leukemia cell line) at the proliferation rate and viability of the K562 cell line, compared to HU exposure. Subsequently, we analyzed the expression levels of HBG1 and HBG2 genes and that of their transcriptional and miRNA-mediated regulators. To achieve these goals, the K562 cell line was first exposed for 72 hours to CPMLE at 500 μg/mL and for 24 hours to EMFCP (0.5; 50, and 100 μg/mL) and to HU (25 μg/mL). After exposure to natural compounds, the effects of gene expression were quantified from total RNA using RT-qPCR. The results have indicated that cell proliferation and viability were affected by CPMLE only at the concentration of 500 μg/ml, with no effects being observed at the lower concentrations analyzed. Upon analysis of the expression levels of globins (HBA, HBB, HBG1, and HBG2), HbF regulatory genes (MYB, KLF1, BCL11A, and BGLT3), and miRNAs involved in the regulation of HbF we could observe more significant differences for the lower concentrations of extracts used, namely at the concentration of 0.5 μg/ml. As such we have decided to titrate down the concentration of methanolic leaf extracts used and test the exposure to aqueous leaf extracts which possess different biological compounds that might lead to a differential regulation of the genes under analysis. Our preliminary results have revealed that at the concentrations of 0,05 ug/ml; 0,5 ug/ml and 5 ug/ml the cell viability and proliferation rates were not affected either for methanolic or aqueous extracts. We are currently analyzing the expression levels of target and regulatory genes to determine the effect of both types of leaf extracts on regulators of HbF expression.info:eu-repo/semantics/publishedVersio

Rab27a Regulates the Peripheral Distribution of Melanosomes in Melanocytes

Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Rab27a, a protein implicated in several diseases, including Griscelli syndrome, choroideremia, and the Hermansky-Pudlak syndrome mouse model, gunmetal. We studied endogenous Rab27a and overexpressed enhanced GFP-Rab27a fusion protein in several cultured melanocyte and melanoma-derived cell lines. In pigmented cells, we observed that Rab27a decorates melanosomes, whereas in nonpigmented cells Rab27a colocalizes with melanosome-resident proteins. When dominant interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. This phenotype is similar to that observed by others in melanocytes derived from the ashen and dilute mutant mice, which bear mutations in the Rab27a and MyoVa loci, respectively. We also found that myosinVa coimmunoprecipitates with Rab27a in extracts from melanocytes and that both Rab27a and myosinVa colocalize on the cytoplasmic face of peripheral melanosomes in wild-type melanocytes. However, the amount of myosinVa in melanosomes from Rab27a-deficient ashen melanocytes is greatly reduced. These results, together with recent data implicating myosinVa in the peripheral capture of melanosomes, suggest that Rab27a is necessary for the recruitment of myosinVa, so allowing the peripheral retention of melanosomes in melanocytes

Dissecting the IFN-g versus IL-17-specific transcriptomes of effector gd T lymphocytes: a new role for signalling adaptor Themis

The crucial role of murine γδ T cells in several (patho)physiological contexts stems from a complex process of ‘developmental preprogramming’ in the thymus, after which a significant fraction of γδ T cells populate peripheral sites already endowed with the capacity to secrete either IL-17 or IFN-γ1. However, despite the relevance of these γδ T cell effector subsets, we still lack knowledge on the transcriptomes that specifically associate with IL-17 or IFN-γ production. To address this, we established a double reporter IL-17-GFP:IFN-γ-YFP mouse strain, which allowed us to isolate pure peripheral IL-17-producing (γδ17) or IFN-γ-producing (γδIFN) γδ T cells to perform RNA-sequencing.info:eu-repo/semantics/publishedVersio

Microbial contamination in the coffee industry: an occupational menace besides a food safety concern?

FCT_UIDB/05608/2020. FCT_UIDP/05608/2020.Respiratory abnormalities among workers at coffee roasting and packaging facilities have already been reported; however, little is known about microbiological contamination inside coffee production facilities. This study intends to assess the microbial contamination (fungi and bacteria) in two coffee industries in Brazil with a multi-approach protocol for sampling and for subsequent analyses using four main sources of samples: filtering respiratory protection devices (FRPD) used by workers, settled dust, electrostatic dust cloths (EDC) and coffee beans. The fungal contamination in the assessed industries was also characterized through the molecular detection of toxigenic species and antifungal resistance. Total bacteria contamination presented the highest values in FRPD collected from both industries (7.45 × 104 CFU.m−2; 1.09 × 104 CFU.m−2). Aspergillus genera were widespread in all the environmental samples collected and sections with clinical relevance (Fumigati) and with toxigenic potential (Nigri and Circumdati) were recovered from FRPD. Circumdati section was observed in 4 mg/mL itraconazole. Sections Circumdati (EDC, coffee beans, and settled dust) and Nidulantes (EDC, coffee beans, and FRPD) were detected by qPCR. Some of the targeted Aspergillus sections that have been identified microscopically were not detected by qPCR and vice-versa. Overall, this study revealed that microbial contamination is a potential occupational risk in the milling stage and should be tackled when assessing exposure and performing a risk assessment. In addition, a multi-sampling campaign should be the approach to follow when assessing microbial contamination and FRPD should be included in this campaign. Occupational exposure to mycotoxins should be considered due to high fungal diversity and contamination. A One Health approach should address these issues in order to prevent the consumption of coffee crops and beans infected by fungi and, more specifically, to avoid widespread azole resistance.H&TRC—Health & Technology Research Center, ESTeSL - Escola Superior de Tecnologia e Saúde, Instituto Politécnico de Lisboa.info:eu-repo/semantics/publishedVersio

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

MicroRNA‐181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2

γδ T cells are a conserved population of lymphocytes that contributes to anti-tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL-2 or IL-15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA-181a as a key modulator of human γδ T cell differentiation. We observe that miR-181a is highly expressed in patients with prostate cancer and that this pattern is associated with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR-181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR-181a overexpression restricts their levels of NKG2D and TNF-α. Upon in silico analysis, we identify two miR-181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR-181a as a critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next-generation immunotherapies.info:eu-repo/semantics/publishedVersio