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Background It is estimated that everyday over 1 million individuals contract a curable sexually transmitted infection (STI) worldwide. For an appropriate STI treatment, it is necessary to have an accurate diagnosis. The Alinity m STI assay is a multiplex RT-PCR assay that identifies four STI pathogens: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) in a single (115 min) reaction. The aim of this study was to evaluate the assay clinical performance. Methods Clinical performance of Alinity m STI assay was assessed using 201 residual clinical samples [119 urine and 82 in gynecological specimens] and compared with Abbott RealTime CT/NG assay and XGEN MULTI UP test (Mobius Life Science) for TV/MG. Precision and reproducibility were evaluated by testing panel members in contrived swab. Five panel members (PM) were tested in 12 replicates in two days: PM1=CT, PM2=NG, PM3=TV, PM4=MG and PM5=CT/NG/TV/MG at 2X claimed LoD. Results For CT, the positive (PPA) agreement and negative (NPA) agreements were 95% and 100% respectively. For NG, the PPA was 94% for urine and 100% for gynecological specimens, and NPA was 99% and 100%, respectively. For MG, PPA and NPA were 100%. For TV, NPA was 100% (no positive result obtained). Co-infection with MG was observed in 5% of CT or NG positive samples. The overall agreement for both sample types and the four organisms was 98.5% (516/524). All panel members were detected and accurately identified, individually (PM1–4) or in the presence of the other three pathogens (PM5). Conclusion The Alinity m STI assay showed excellent agreement (97–100%) between methods and streamlines laboratory workflow with simultaneous detection of 4 pathogens in a single reaction from the same sample. This assay allows rapid infection identification supporting clinicians to properly treat patients, especially when a co-infection is present.

Traumatic brain injury is an important cause of mortality and morbidity all over the world. After the initial injury there is a cascade of cellular and molecular events that ultimately lead to cell death. Therapies aim not only to counteract these mechanisms but also to replenish the lost cell population in order to achieve a better recovery. The adult mammal brain in not as plastic as the postnatal, but it has at least two neurogenic regions that maintains physiological functions in the brain; the subgranular zone of the dentate gyrus of the hippocampus, which produces neurons that integrate locally, and the subventricular zone (SVZ) of the lateral ventricles, that produces neuroblasts that migrate through the rostral migratory stream (RMS) to the olfactory bulbs. Brain injuries, as well as neurodegenerative diseases, induce the SVZ to respond by increasing cell proliferation and migration to the injured areas. Here we report that SVZ cells migrate to the injured cortex after traumatic brain injury in mice, and that the physiological RMS migration is not impaired. We also show that Prokineticin 2 (PROK2), a chemokine important for the olfactory bulb neurogenesis by promoting the directional migration of neuroblasts, is induced in the injured cortex. Using PROK2 receptor antagonist and recombinant PROK2 we show for the first time that PROK2 can directionally attract SVZ cells in vitro and in vivo. The data we present here links one more element of the inflammatory process, PROK2 secreted by microglia, to the attempt to regenerate an acutely injured mammalian cortex.AbbreviationsSGZsubgranular zoneSVZsubventricular zoneRMSrostral migratory streamPROK2Prokineticin 2

10594 Background: As germline testing in BC is becoming more accessible, a large body of evidence from distinct populations is revealing different prevalence of affected genes or recurrent and founder mutations. We evaluated a series of Brazilian BC patients from diverse regions of the country that were studied in a single laboratory facility for likely pathogenic/pathogenic variants (PV) and variants of unknown significance (VUS) in BRCA1/2 and other BC susceptibility genes (ATM, BARD1, CDH1, CHEK2, PALB2, RAD51C, RAD51D and TP53). Methods: We used multi-gene NGS panels covering from 35 to 105 genes, including copy number alterations, to perform sequencing in patients with diagnosis of BC from 2016 to 2021. With patient consent, we extracted clinical and pathological data from curated Real-World Database of Oncoclínicas Group to classify tumors according to hormone receptor (HR) and HER2 status and assess family history of cancer. Primary endpoint was prevalence of PV and VUS in BRCA1/2 and other breast cancer risk genes. Results: In total, 1,520 patients with BC were included in the analysis, 99% were female, median age at genetic testing was 46 years and 84% had family history of cancer. BC subtype was available in 717 cases (43% HR+/HER2-, 26% HER2+, 31% triple negative). Median age at genetic testing in HR+/HER2- population was 49 years and 44 years in HER2+ and triple-negative cases. Overall, 11.7% (CI95% 10.1-13.4) of the population had at least one PV in any BC risk gene: 50 in BRCA1, 49 in BRCA2, 22 in TP53 (17 had Brazilian founder p.R337H haplotype), 19 in PALB2, 18 in CHEK2, 15 in ATM, 7 in other genes (BARD1, CDH1, RAD51C, RAD51D) and 4 patients had co-existing PV in 2 BC risk genes. BRCA1/2 PVs were found in 7% of BC patients and BRCA1/2 VUS in 3%, being higher in triple-negative cases (11% PVs) than HR+/HER2- (5% PVs) and HER2+ (3%). For other BC risk genes, 5% of the patients had a PV, without differences according to BC subtype, and the prevalence of VUS was 14%. The prevalence of TP53 Brazilian founder mutation in BC was 1.2%. Median age of genetic testing was younger in population with BRCA1 PV (43 years) than BRCA2 PV (47 years) and other genes PV (46 years). Conclusions: In this real-world cohort of BC patients enriched for high-risk features such as young age at diagnosis, positive family history and triple-negative disease, the prevalence of BRCA1 and BRCA2 PV was very similar and represented only 56% of all PV detected. Different from other studies, TP53 mutations are the third most common germline alteration in the Brazilian population, with close to 80% of the cases presenting the founder mutation p.R337H. BC with epidemiologic high-risk features should be offered a germline test in Brazilian patients due to the considerable chance of a PV detection.

Background New partially or fully automated molecular diagnostic testing platforms are being developed to address the growing demand for fast, accurate, and cost-effective testing. Objectives To evaluate the analytical and clinical performance of the Alinity m system compared to the Abbott RealTime m2000 assay system in a large central molecular laboratory. Study Design Alinity m HIV-1, HCV, and HBV assay precision, reproducibility, and sensitivity were assessed using commercial customized dilution panels. Clinical performance of the Alinity m and m2000 assay systems was compared using standard lab protocols and residual, de-identified patient specimens. A workflow analysis of 1,068 samples compared turnaround times (TATs) on five m2000 systems and one Alinity m system running Alinity m HIV-1, HCV, HBV, HR HPV, and STI assays. Results The Alinity m assay system demonstrated high detectability and precision at clinical decision points and excellent correlation with Abbott RealTime assay results. Processing TAT for 100 % of results was 117 min on Alinity m. Sample onboard TAT, from sample loading to 95 % of results, was 5:15 h for Alinity m and 7:30 h for m2000. 100 % of STAT samples were processed within 4 h on Alinity m. Total TAT for 100 % of results from all five assays was 80 h for m2000 versus 9 h for Alinity m. Conclusions The Alinity m system produces assay results comparable to those of the Abbott RealTime m2000 system, but with significantly faster turnaround times due to continuous loading and the ability to run multiple assays simultaneously on the same sample.

We investigated 313 unrelated subjects who presented with hearing loss to identify the novel genetic causes of this condition in Brazil. Causative GJB2/GJB6 mutations were found in 12.7% of the patients. Among the familial cases (100/313), four were selected for exome sequencing. In one case, two novel heterozygous variants were found and were predicted to be pathogenic based on bioinformatics tools, that is, p.Ser906* (MYO6) and p.Arg42Cys (GJB3). We confirmed that this nonsense MYO6 mutation segregated with deafness in this family. Only the proband and her unaffected mother exhibited the GJB3 mutation, which is in the same amino acid of a known Erythrokeratodermia variabilis mutation. None of the patients exhibited this skin disease, but the proband exhibited a more severe hearing loss. Hence, the GJB3 mutation was considered to be a variant of uncertain significance. In conclusion, we described a novel nonsense MYO6 mutation that was responsible for the hearing loss in a Brazilian family. This mutation resides in the neck domain of myosin-VI after the motor domain. Thus, our data give further support for genotype-phenotype correlations, which state that when the motor domain of the protein is functioning, the hearing loss is milder and has a later onset. The three remaining families without mutations in the known genes suggest that there are still deafness genes to be revealed.

Brain injuries such as trauma and stroke lead to glial scar formation by reactive astrocytes which produce and secret axonal outgrowth inhibitors. Chondroitin sulfate proteoglycans (CSPG) constitute a well-known class of extracellular matrix molecules produced at the glial scar and cause growth cone collapse. The CSPG glycosaminoglycan side chains composed of chondroitin sulfate (CS) are responsible for its inhibitory activity on neurite outgrowth and are dependent on RhoA activation. Here, we hypothesize that CSPG also impairs neural stem cell migration inhibiting their penetration into an injury site. We show that DCX+ neuroblasts do not penetrate a CSPG-rich injured area probably due to Nogo receptor activation and RhoA/ROCK signaling pathway as we demonstrate in vitro with neural stem cells cultured as neurospheres and pull-down for RhoA. Furthermore, CS-impaired cell migration in vitro induced the formation of large mature adhesions and altered cell protrusion dynamics. ROCK inhibition restored migration in vitro as well as decreased adhesion size. Electronic supplementary material The online version of this article (doi:10.1007/s12035-017-0565-8) contains supplementary material, which is available to authorized users.

Made available in DSpace on 2019-10-06T17:12:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-08-03 Alternative protein-coding transcripts of the RASSF1 gene have been associated with dual functions in human cancer: while RASSF1C isoform has oncogenic properties, RASSF1A is a tumour suppressor frequently silenced by hypermethylation. Recently, the antisense long non-coding RNA RASSF1 (ANRASSF1) was implicated in a locus-specific mechanism for the RASSF1A epigenetic repression mediated by PRC2 (Polycomb Repressive Complex 2). Here, we evaluated the methylation patterns of the promoter regions of RASSF1A and RASSF1C and the expression levels of these RASSF1 transcripts in breast cancer and breast cancer cell lines. As expected, RASSF1C remained unmethylated and RASSF1A was hypermethylated at high frequencies in 75 primary breast cancers, and also in a panel of three mammary epithelial cells (MEC) and 10 breast cancer cell lines (BCC). Although RASSF1C was expressed in all cell lines, only two of them expressed the transcript RASSF1A. ANRASSF1 expression levels were increased in six BCCs. In vitro induced demethylation with 5-Aza-2ʹ-deoxicytydine (5-Aza-dC) resulted in up-regulation of RASSF1A and an inverse correlation with ANRASSF1 relative abundance in BCCs. However, increased levels of both transcripts were observed in two MECs (184A1 and MCF10A) after treatment with 5-Aza-dC. Overall, these findings indicate that ANRASSF1 is differentially expressed in MECs and BCCs. The lncRNA ANRASSF1 provides new perspectives as a therapeutic target for locus-specific regulation of RASSF1A. Department of Genetics Institute of Biosciences São Paulo State University (Unesp) Department of Senology Amaral Carvalho Hospital Department of Biostatistics Institute of Biosciences São Paulo State University (Unesp) Department of Oncology Georgetown University Medical Center Faculdades Pequeno Préncipe e Instituto de Pesquisa Pelé Pequeno Príncipe Department of Clinical Genetics University Hospital Institute of Regional Health Research University of Southern Denmark Vejle Department of Genetics Institute of Biosciences São Paulo State University (Unesp) Department of Biostatistics Institute of Biosciences São Paulo State University (Unesp)

Potentially neurogenic areas were initially identified by incorporation of bromodeoxyuridine (BrdU) in cells underlying the subventricular zone (SVZ) of the lateral ventricles wall, hippocampus and olfactory bulbs of newborn guinea pigs. Neural precursors from the SVZ were cultured in suspension, generating neurospheres (NSFs), which, upon dissociation were able to generate new NSFs. Upon culture in the absence of growth factors, cells dissociated from NSFs displayed evidence for neural differentiation, giving rise to cells from neural lineage. Flow cytometry analysis for of NSFs-derived cells after differentiation revealed approximately 13.3% nestin positive, 5.5% Beta-III-tubulin positive, 9% GFAP positive and 7.8% mGalC positive. Functional assays by measurement of calcium influx upon gamma butiric amino acid (GABA) and glutamate stimuli, revealed stimulation in differentiated cells, an indicator of neuronal differentiation. The ability of guinea pig SVZ cells to originate functional neurons in vitro is promising for research and towards a future use of neural stem cells in the therapy of neurological disorders.

Transplanting stem cells into the central nervous system is a promising therapeutic strategy. However, preclinical trials of cell-based therapies are limited by poor local cell engraftment and survival. Here, we present a polylactic acid (PLA) scaffold to support delivery of mesenchymal stem cells (MSCs) in a mouse model of stroke. We isolated bone marrow MSCs from adult C57/Bl6 mice, cultured them on PLA polymeric rough microfibrous (PLA-PRM) scaffolds obtained by rotary jet spinning, and transplanted into the brains of adult C57/Bl6 mice, carrying thermocoagulation-induced cortical stroke. Interleukins (IL4, IL6 and IL10) and tumor necrosis factor alfa (TNFα) expression levels in the brain of mice that received PRM were similar to untreated. MSCs transplantation significantly reduced the area of the lesion and PRM delivery increased MSCs retention at the injury site. We conclude that PLA-PRM scaffolds offer a promising new system to deliver stem cells to injured areas of the brain.GRAPHICAL ABSTRACTSynthetic scaffolds offer an alternative to optimize stem cell transplantation at sites of brain injury. Here, we present a rotary jet spun polylactic acid (PLA) polymer used as a scaffold to support delivery of mesenchymal stem cells (MSCs) in a mouse model of stroke. Transplantation of MSCs isolated or cultured on PRM significantly reduced the area of the lesion and PRM delivery increased MSCs retention at the injury site.

G N […] Muscle biopsy with dystrophic pattern and rimmed vacuoles: GNE myopathy in a Brazilian patient

SummaryAims Medial ganglionic eminence (MGE) progenitors give rise to inhibitory interneurons and may serve as an alternative cell source for large-scale cell transplantation for epilepsy after in vitro expansion. We investigated whether modifications in the culture medium of MGE neurospheres affect neuronal differentiation and expression of MGE-specific genes. In vivo, we compared anticonvulsant effects and cell differentiation pattern among neurospheres grown in different culture media and compared them with freshly harvested MGE cells. Methods We used four variations of cell culture: standard, containing growth factors (EGF/FGF-2) (GF); addition of retinoic acid (GF-RA); withdrawal of EGF/FGF-2 (WD); and addition of retinoic acid and withdrawal of EGF/FGF-2 (WD-RA). Based on in vitro results neurosphere-grown (WD-RA or GF conditions) or fresh MGE cells were transplanted into the hippocampus. Results In vitro WD-RA showed increased neuronal population and higher expression of Dlx1, Nkx2.1, and Lhx6 genes in comparison with GF culture condition. After transplantation, fresh MGE cells and neurospheres (GF) showed anticonvulsant effects. However, fresh MGE cells differentiated preferentially into inhibitory neurons, while GF gave rise to glial cells. Conclusion We conclude that freshly isolated and neurosphere-grown MGE cells reduced seizures by different mechanisms (inhibitory interneurons vs. astrocytes). Fresh MGE cells appear more appropriate for cell therapies targeting inhibitory interneurons for conferring anticonvulsant outcomes.

Intense activation of neurons triggers the appearance of immediate expression genes, including c-fos. This gene is related to various signal cascades involved in biochemical processes such as neuronal plasticity, cell growth and mitosis. Here we investigate the expression pattern and the refractory period of c-Fos in rats and monkey’s brains after stimulation with pentylenetetrazol. Rats and monkeys were sacrificed at various times after PTZ-induced seizure. Here we show that rats and monkeys already showed c-Fos expression at 0.5 h after seizure. Yet, the pattern of protein expression was longer in monkeys than rats, and also was not uniform (relative intensity) across different brain regions in monkeys as opposed to rats. In addition monkeys had a regional brain variation with regard to the temporal profile of c-Fos expression, which was not seen in rats. The refractory period after a second PTZ stimulation was also markedly different between rats and monkeys with the latter even showing a summatory effect on c-Fos expression after a second stimulation. However, assessment of c-Fos mRNA in rats indicated a post-transcriptional control mechanism underlying the duration of the refractory period. The difference in the protein expression pattern in rodents and primates characterizes a functional aspect of brain biochemistry that differs between these mammalian orders and may contribute for the more developed primate cognitive complexity as compared to rodents given c-Fos involvement in cognitive and learning tasks.

Therapy with mesenchymal stem cells (MSCs) has showed to be promising due to its immunomodulatory function. Traumatic brain injury (TBI) triggers immune response and release of inflammatory mediators, mainly cytokines, by glial cells creating a hostile microenvironment for endogenous neural stem cells (NSCs). We investigated the effects of factors secreted by MSCs on NSCin vitroand analyzed cytokines expressionin vitroin a TBI model. Ourin vitroresults show that MSC-secreted factors increase NSC proliferation and induce higher expression of GFAP, indicating a tendency toward differentiation into astrocytes.In vivoexperiments showed that MSC injection at an acute model of brain injury diminishes a broad profile of cytokines in the tissue, suggesting that MSC-secreted factors may modulate the inflammation at the injury site, which may be of interest to the development of a favorable microenvironment for endogenous NSC and consequently to repair the injured tissue.

Neural stem/progenitor cells (NSC) respond to injury after brain injuries secreting IL-1, IL-6, TNF-α, IL-4 and IL-10, as well as chemokine members of the CC and CXC ligand families. CXCL12 is one of the chemokines secreted at an injury site and is known to attract NSC-derived neuroblasts, cells that express CXCL12 receptor, CXCR4. Activation of CXCR4 by CXCL12 depends on two domains located at the N-terminal of the chemokine. In the present work we aimed to investigate if the N-terminal end of CXCL12, where CXCR4 binding and activation domains are located, was sufficient to induce NSC-derived neuroblast chemotaxis. Our data show that a synthetic peptide analogous to the first 21 amino acids of the N-terminal end of CXCL12, named PepC-C (KPVSLSYRCPCRFFESHIARA), is able to promote chemotaxis of neuroblasts in vivo, and stimulate chemotaxis and proliferation of CXCR4+ cells in vitro, without affecting NSC fate. We also show that PepC-C upregulates CXCL12 expression in vivo and in vitro. We suggest the N-terminal end of CXCL12 is responsible for a positive feedback loop to maintain a gradient of CXCL12 that attracts neuroblasts from the subventricular zone into an injury site.