The involvement of miRNA in mesial temporal lobe epilepsy (MTLE) pathogenesis has increasingly become a focus of epigenetic studies. Despite advances, the number of known miRNAs with a consistent ...expression response during epileptogenesis is still small. Addressing this situation requires additional miRNA profiling studies coupled to detailed individual expression analyses. Here, we perform a miRNA microarray analysis of the hippocampus of Wistar rats 24 hours after intra-hippocampal pilocarpine-induced Status Epilepticus (H-PILO SE). We identified 73 miRNAs that undergo significant changes, of which 36 were up-regulated and 37 were down-regulated. To validate, we selected 5 of these (10a-5p, 128a-3p, 196b-5p, 352 and 324-3p) for RT-qPCR analysis. Our results confirmed that miR-352 and 196b-5p levels were significantly higher and miR-128a-3p levels were significantly lower in the hippocampus of H-PILO SE rats. We also evaluated whether the 3 miRNAs show a dysregulated hippocampal expression at three time periods (0h, 24h and chronic phase) after systemic pilocarpine-induced status epilepticus (S-PILO SE). We demonstrate that miR-128a-3p transcripts are significantly reduced at all time points compared to the naïve group. Moreover, miR-196b-5p was significantly higher only at 24h post-SE, while miR-352 transcripts were significantly up-regulated after 24h and in chronic phase (epileptic) rats. Finally, when we compared hippocampi of epileptic and non-epileptic humans, we observed that transcript levels of miRNAs show similar trends to the animal models. In summary, we successfully identified two novel dysregulated miRNAs (196b-5p and 352) and confirmed miR-128a-3p downregulation in SE-induced epileptogenesis. Further functional assays are required to understand the role of these miRNAs in MTLE pathogenesis.
Layered double hydroxides (LDHs) have been employed as nano-sized carriers for therapeutic/bio-active molecules, including small interfering RNAs (siRNAs). However, the potential of LDHs ...nanoparticles for an efficient and safe antisense oligonucleotide (AMO) delivery still requires studies. In this research, we have tested the suitability of a Mg–Al-LDH-based nanocarrier loaded with a miRNA-196b-5p inhibitor. LDHs (and LDH-Oligo complex) were synthesized by the coprecipitation method followed by physicochemical characterization as hydrodynamic size, surface charge, crystallinity, and chemical groups. Thymic endothelial cell line (tEnd.1) were transfected with LDH-Oligo and were evaluated for i. cell viability by MTT, trypan blue, and propidium iodide assays; ii. transfection efficiency by flow cytometry, and iii. depletion of miRNA-196b-5p by RT-qPCR. In addition, Drosophila melanogaster larvae were fed LDHs and evaluated for: i. larval motility; ii. pupation rate; iii. larval-pupal transition; iv. lethality, and v. emergence rate. We demonstrated that LDHs nanoparticles are stable in aqueous solutions and exhibit a regular hexagonal shape. The LDH-AMO complex showed a transfection efficiency of 93.95 ± 2.15 % and induced a significant depletion of miRNA-196b-5p 48h after transfection. No cytotoxic effects were detected in tEnd.1 cells at concentrations up to 50 μg/ml, as well as in Drosophila exposed up to 500 μg of LDH. In conclusion, our data suggest that LDHs are biocompatible and efficient carriers for miRNA inhibitors and can be used as a viable and effective tool in functional miRNA inhibition assays.
•Mg-Al-LDH nanoparticles are biocompatible to t.End cells and Drosophila melanogaster.•Mg-Al-LDH nanoparticles are effective in delivering microRNA inhibitors to cells.•The complex Mg-Al-LDH and miR-196b inhibitor modulate efficient gene expression knockdown.
Passenger air transport is one of the main routes for the global spread of multidrug-resistant bacteria. This may be due to airborne pathogen transmission, which may occur within the commercial ...aircraft cabin. Because of this, we performed an investigation of aerial contamination by
Staphylococcus
species in 166 commercial aircraft and analyzed the presence of antibiotic resistance and biofilm synthesis genes in the collected isolates. Bacterial identification was performed by using species-specific primers and partial sequencing of 16S rRNA and
tuf
genes. The antibiotic resistance genes screened were:
mecA
,
mecC
,
blaZ
,
ermA
,
ermB
,
ermC,
and
vanA
. For biofilm synthesis,
ica
locus genes were screened. Fourteen species and four subspecies of
Staphylococcus
were detected in the analyzed samples. Except for
mecC
and
vanA
, all other genes were detected, including the
mecA
gene in
Staphylococcus aureus
and Coagulase-negative
Staphylococcus
isolates. Only
S. epidermidis
isolates were positive for biofilm formation. To date, this is the first study to report a significant diversity of airborne
Staphylococcus
and the presence of airborne methicillin-resistant
Staphylococcus aureus
(MRSA) in the cabin environment in commercial aircraft. Our results point to the importance of indoor air quality monitoring in the cabin environment as a preventive measure for the airborne spread of clinically significant pathogens.
Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable ...normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.
The discovery of disease-specific biomarkers, such as microRNAs (miRNAs), holds the potential to transform the landscape of Amyotrophic Lateral Sclerosis (ALS) by facilitating timely diagnosis, ...monitoring treatment response, and accelerating drug discovery. Such advancement could ultimately improve the quality of life and survival rates for ALS patients. Despite more than a decade of research, no miRNA biomarker candidate has been translated into clinical practice. We conducted a systematic review and meta-analysis to quantitatively synthesize data from original studies that analyzed miRNA expression from liquid biopsies via PCR and compared them to healthy controls. Our analysis encompasses 807 miRNA observations from 31 studies, stratified according to their source tissue. We identified consistently dysregulated miRNAs in serum (hsa-miR-3665, -4530, -4745–5p, −206); blood (hsa-miR-338–3p, -183–5p); cerebrospinal fluid (hsa-miR-34a-3p); plasma (hsa-miR-206); and neural-enriched extracellular vesicles from plasma (hsa-miR-146a-5p, −151a-5p, −10b-5p, −29b-3p, and −4454). The meta-analyses provided further support for the upregulation of hsa-miR-206, hsa-miR-338–3p, hsa-miR-146a-5p and hsa-miR-151a-5p, and downregulation of hsa-miR-183–5p, hsa-miR-10b-5p, hsa-miR-29b-3p, and hsa-miR-4454 as consistent indicators of ALS across independent studies. Our findings provide valuable insights into the current understanding of miRNAs' dysregulated expression in ALS patients and on the researchers’ choices of methodology. This work contributes to the ongoing efforts towards discovering disease-specific biomarkers.
Neuropathological studies often use autopsy brain tissue as controls to evaluate changes in protein or RNA levels in several diseases. In mesial temporal lobe epilepsy (MTLE), several genes are up or ...down regulated throughout the epileptogenic and chronic stages of the disease. Given that postmortem changes in several gene transcripts could impact the detection of changes in case-control studies, we evaluated the effect of using autopsy specimens with different postmortem intervals (PMI) on differential gene expression of the Pilocarpine (PILO)induced Status Epilepticus (SE) of MTLE. For this, we selected six genes (Gfap, Ppia, Gad65, Gad67, Npy, and Tnf-α) whose expression patterns in the hippocampus of PILO-injected rats are well known. Initially, we compared hippocampal expression of naïve rats whose hippocampi were harvested immediately after death (0h-PMI) with those harvested at 6h postmortem interval (6h-PMI): Npy and Ppia transcripts increased and Tnf-α transcripts decreased in the 6h-PMI group (p<0.05). We then investigated if these PMI-related changes in gene expression have the potential to adulterate or mask RT-qPCR results obtained with PILO-injected rats euthanized at acute or chronic phases. In the acute group, Npy transcript was significantly higher when compared with 0h-PMI rats, whereas Ppia transcript was lower than 6h-PMI group. When we used epileptic rats (chronic group), the RT-qPCR results showed higher Tnf-α only when compared to 6h-PMI group. In conclusion, our study demonstrates that PMI influences gene transcription and can mask changes in gene transcription seen during epileptogenesis in the PILO-SE model. Thus, to avoid erroneous conclusions, we strongly recommend that researchers account for changes in postmortem gene expression in their experimental design.
Introduction-Aims: Fungi are ubiquitous microorganisms that are easily dispersed through the air. In healthcare environments, indoor air can favor the spread of healthcare-associated fungal ...infections, compromising mainly immunocompromised hospitalized individuals. Therefore, this study aimed to evaluate indoor air contamination in healthcare environments, investigating mainly the presence of potentially pathogenic yeasts. Method: Indoor air samples were collected from 12 healthcare environments (hospital and medical clinics). After the growth, isolation, and purification of the yeast colonies, the isolates were identified by polymerase chain reaction using species-specific primers for yeasts of the genus Candida and sequencing of D1/D2 domains of the large ribosomal subunit (LSU rRNA). Results and interpretation: Fourteen yeast species were identified, including emerging pathogens. Species of clinical importance such as Candida parapsilosis, Candida glabrata, and Rhodotorula mucilaginosa were present. C. parapsilosis was the most prevalent species, followed by Rhodotorula mucilaginosa. In addition, we report the first occurrence of Candida orthopsilosis, Trichosporon mucoides, Fereydounia khargensis, and Hortaea werneckii in indoor air samples collected in healthcare environments. The present study shows that potentially fungal pathogens were present in air samples from healthcare environments, proving the role of indoor air in spreading infections. Therefore, monitoring air quality in healthcare environments is a fundamental approach to developing infection control measures, especially those related to invasive fungal infections.
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