Delivery of miRNA-Targeted Oligonucleotides in the Rat Striatum by Magnetofection with Neuromag®

MicroRNAs (miRNAs) regulate gene expression at posttranscriptional level by triggering RNA interference. In such a sense, aberrant expressions of miRNAs play critical roles in the pathogenesis of many disorders, including Parkinson’s disease (PD). Controlling the level of specific miRNAs in the brain is thus a promising therapeutic strategy for neuroprotection. A fundamental need for miRNA regulation (either replacing or inhibition) is a carrier capable of delivering oligonucleotides into brain cells. This study aimed to examine a polymeric magnetic particle, Neuromag®, for delivery of synthetic miRNA inhibitors in the rat central nervous system. We injected the miRNA inhibitor complexed with Neuromag® into the lateral ventricles next to the striatum, by stereotaxic surgery. Neuromag efficiently delivered oligonucleotides in the striatum and septum areas, as shown by microscopy imaging of fluorescein isothiocyanate (FITC)-labeled oligos in astrocytes and neurons. Transfected oligos showed efficacy concerning miRNA inhibition. Neuromag®-structured miR-134 antimiR (0.36 nmol) caused a significant 0.35 fold decrease of striatal miR-134, as revealed by real-time quantitative polymerase chain reaction (RT-qPCR). In conclusion, the polymeric magnetic particle Neuromag® efficiently delivered functional miRNA inhibitors in brain regions surrounding lateral ventricles, particularly the striatum. This delivery system holds potential as a promising miRNA-based disease-modifying drug and merits further pre-clinical studies using animal models of PD.The present study received financial support from CAPES [Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Programa Nacional de Pós-doutorado; grant no. 3731-37/2010)], CNPq [Conselho Nacional de Desenvolvimento Científico e Tecnológico (grant no. 467467/2014-5)] and FAP-DF [Fundação de Apoio à Pesquisa do Distrito Federal (grant no. 2010/00302-9)]

BRCAness as a biomarker of susceptibility to PARP inhibitors in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common primary brain cancer. GBMs commonly acquire resistance to standard-of-care therapies. Among the novel means to sensitize GBM to DNA-damaging therapies, a promising strategy is to combine them with inhibitors of the DNA damage repair (DDR) machinery, such as inhibitors for poly(ADP-ribose) polymerase (PARP). PARP inhibitors (PARPis) have already shown efficacy and have received regulatory approval for breast, ovarian, prostate, and pancreatic cancer treatment. In these cancer types, after PARPi administration, patients carrying specific mutations in the breast cancer 1 (BRCA1) and 2 (BRCA2) suppressor genes have shown better response when compared to wild-type carriers. Mutated BRCA genes are infrequent in GBM tumors, but their cells can carry other genetic alterations that lead to the same phenotype collectively referred to as ‘BRCAness’. The most promising biomarkers of BRCAness in GBM are related to isocitrate dehydrogenases 1 and 2 (IDH1/2), epidermal growth factor receptor (EGFR), phosphatase and tensin homolog (PTEN), MYC proto-oncogene, and estrogen receptors beta (ERβ). BRCAness status identified by accurate biomarkers can ultimately predict responsiveness to PARPi therapy, thereby allowing patient selection for personalized treatment. This review discusses potential biomarkers of BRCAness for a ‘precision medicine’ of GBM patients

Rapid detection of Vancomycin-Resistant Enterococci (VRE) in rectal samples from patients admitted to intensive care units

ABSTRACT: The reduction in time required to identify vancomycin-resistant enterococci (VRE) has gained increased importance during hospital outbreaks. In the present study, we implemented a laboratory protocol to speed up the VRE screening from rectal samples. The protocol combines a medium for selective VRE isolation (VREBAC®, Probac, São Paulo) and a multiplex PCR for detection and identification of vanA and vanB resistance genes. The screening performance was analyzed in 114 specimens collected from four intensive care units. The swabs were collected at two periods: (1) during a VRE outbreak (February 2006, n=83 patients) and (2) at the post-outbreak period, after adoption of infection control measures (June 2006, n=31 patients). Forty-one/83 VRE (49.4%) and 3/31(9.7%) VRE were found at the first and second period, respectively. All isolates harbored the vanA gene. In both periods, detection of the gene vanA parallels to the minimum inhibitory concentration values of >256 µg/mL and >48 µg/mL for vancomycin and teicoplanin, respectively. Multiplex PCR and conventional methods agreed in 90.2% for enterococci identification. Besides this accuracy, we also found a remarkable reduction in time to obtain results. Detection of enterococcal species and identification of vancomycin resistance genes were ready in 29.5 hours, in comparison to 72 hours needed by the conventional methods. In conclusion, our protocol identified properly and rapidly enterococci species and vancomycin-resistance genes. The results strongly encourage its adoption by microbiology laboratories for VRE screenning in rectal samples

Molecular epidemiology and antimicrobial susceptibility of enterococci recovered from Brazilian intensive care units

ABSTRACT: We studied the antimicrobial resistance and the molecular epidemiology of 99 enterococcal surveillance isolates from two hospitals of Brasília, Brazil. Conventional biochemical tests were used to identify the enterococcal species and the disk diffusion method was used to determine their resistance profiles. Enterococcus faecalis (76%) and E. faecium (9%) were the most prevalent species. No enterococci showed the vanA or vanB vancomycin resistance phenotypes or genotypes. Only the intrinsically resistant species E. gallinarum (n=2) and E. casseliflavus (n=3) harbored the vancomycin-resistance genes vanC1 and vanC2/3, respectively. We found E. faecalis isolates with high-level resistance to gentamicin (22%) and streptomycin (8%) and both E. faecalis and E. faecium isolates with resistance to more than two antimicrobials (84% and 67%, respectively). Nine E. faecalis isolates (12%) were resistant to ampicillin; the minimal inhibitory concentration (MIC) values were 16µg/mL (n=6) and 32µg/mL (n=3). Among these ampicillin-resistant E. faecalis, seven were also resistant to gentamicin, ciprofloxacin, rifampin, penicillin, chloramphenicol, tetracycline and erythromycin. Pulsed-field gel electrophoresis classified those isolates in three different genotypes, suggesting dissemination of genetically related ampicillin-resistant E. faecalis strains among different patients

Progress in circRNA-Targeted Therapy in Experimental Parkinson’s Disease

Circular RNAs (circRNAs) are single-stranded RNA molecules often circularized by backsplicing. Growing evidence implicates circRNAs in the underlying mechanisms of various diseases, such as Alzheimer’s and Parkinson’s disease (PD)—the first and second most prevalent neurodegenerative disorders. In this sense, circSNCA, circHIPK2, circHIPK3, and circSLC8A1 are circRNAs that have been related to the neurodegenerative process of PD. Gain-of-function and loss-of-function studies on circRNAs have shed light on their roles in the pathobiology of various diseases. Gain-of-function approaches typically employ viral or non-viral vectors that hyperexpress RNA sequences capable of circularizing to form the specific circRNA under investigation. In contrast, loss-of-function studies utilize CRISPR/Cas systems, antisense oligonucleotides (ASOs), or RNAi techniques to knock down the target circRNA. The role of aberrantly expressed circRNAs in brain pathology has raised a critical question: could circRNAs serve as viable targets for neuroprotective treatments? Translating any oligonucleotide-based therapy, including those targeting circRNAs, involves developing adequate brain delivery systems, minimizing off-target effects, and addressing the high costs of treatment. Nonetheless, RNAi-based FDA-approved drugs have entered the market, and circRNAs have attracted significant attention and investment from major pharmaceutical companies. Spanning from bench to bedside, circRNAs present a vast opportunity in biotechnology for oligonucleotide-based therapies designed to slow or even halt the progression of neurodegenerative diseases

Absence of VanA- and VanB-Containing Enterococci in Poultry Raised on Nonintensive Production Farms in Brazil

We examined cloacal samples from poultry raised on nonintensive production farms in Brazil for the presence of vancomycin-resistant enterococci. No VanA- or VanB-containing enterococci were identified in a total of 200 cloacal swabs. The most prevalent species were Enterococcus gallinarum (vanC1; 13.0%) and E. casseliflavus (vanC2/3; 5.5%)

RT-rtPCR quantification of circulating microRNAs in plasma and serum samples from healthy domestic cats

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at a post-transcriptional level by silencing targeted messenger RNA (mRNA). Most studies concerning miRNA expression use solid tissue samples. However, circulating miRNAs from different body fluids have recently emerged as diagnostic and prognostic molecules, given that they hold informative value and have increased stability in cell-free form. Blood sampling of cats can be challenging given their small body size and because they often experience distress when handled. We quantified miR-20a, -192, -365, -15b-5p, and -16-5p from plasma and serum samples of 10 healthy domestic cats. Our RT-rtPCR procedure used 100 µL of either plasma or serum samples as sources of biomarker molecules. However, serum provided higher amounts of miRNA than plasma samples, with a p < 0.0001 for miR-20a and p < 0.0002 for miR-16-5p

miR-20a is upregulated in serum from domestic feline with PKD1 mutation.

Polycystic kidney disease (PKD), also known as autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous condition characterized by cysts in renal parenchyma. It is the most prevalent inherited disease of domestic cats. MicroRNAs (miRNAs or ncRNA) are short, noncoding, single-stranded RNAs that may induce PKD cytogenesis by affecting numerous targets genes as well as by directly regulating PKD gene expression. We compared the relative expression profile of miR-20a, -192, -365, -15b-5p, and -16-5p from plasma and serum samples of nine domestic cats with PKD1 mutation, detected by polymerase chain reaction (PCR), and a control group (n = 10). Blood samples from cats with PKD1 mutation provide similar concentrations of microRNAs either from plasma or serum. Serum miR-20a is upregulated in PKD group with p < 0.005; Roc curve analysis showed an AUC of 90,1% with a cut-off value sensitivity of 77.8% and specificity of 100%. This data provides important information regarding renal miRNA expression in peripheral blood sampling