CRISPR-mediated base editing in mice using cytosine deaminase base editor 4

BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.

Short Tandem Repeats in plants: genomic distribution and function prediction

BACKGROUND: Short Tandem repeats (STRs) existed as popular elements in both eukaryotic and prokaryotic genomes. RESULTS: In this study, we analyzed the characteristics, distributions, and motif features of STRs within whole-genomes of 140 plant species. The results showed that STR density was negatively correlated with the genome size. Hexanucleotide repeat was the most abundant type of STRs. The distribution of algae shows a preference different from that of other plants. By analyzing GC contents of STRs and genome, it was concluded that STR motif was influenced by GC contents. Analysis of the long STRs in genome (length 1000 bp) found that dicots have the more long STRs. For STR types, di- and tri-nucleotide accounted for the highest proportion. Analyzing and designing long STRs in CDS (length 500 bp) was to verify the role of long STRs in Gossypium hirsutum TM-1 and Solanum tuberosum. By comparing the long STRs found in Fragaria x ananassa with other species, some evolutionary characteristics of the long STRs were obtained. CONCLUSIONS: We got the characteristics, distribution, and motif features of STRs in the whole genome of 140 plants and obtained some evolutionary characteristics of long STRs. The study provides useful insights into STR preference, characteristics, and distribution in plants.

Transcriptional analysis of Rhazya stricta in response to jasmonic acid

BACKGROUND: Jasmonic acid (JA) is a signal transducer molecule that plays an important role in plant development and stress response; it can also efficiently stimulate secondary metabolism in plant cells. RESULTS: RNA-Seq technology was applied to identify differentially expressed genes and study the time course of gene expression in Rhazya stricta in response to JA. Of more than 288 million total reads, approximately 27% were mapped to genes in the reference genome. Genes involved during the secondary metabolite pathways were up- or downregulated when treated with JA in R. stricta. Functional annotation and pathway analysis of all up- and downregulated genes identified many biological processes and molecular functions. Jasmonic acid biosynthetic, cell wall organization, and chlorophyll metabolic processes were upregulated at days 2, 6, and 12, respectively. Similarly, the molecular functions of calcium-transporting ATPase activity, ADP binding, and protein kinase activity were also upregulated at days 2, 6, and 12, respectively. Time-dependent transcriptional gene expression analysis showed that JA can induce signaling in the phenylpropanoid and aromatic acid pathways. These pathways are responsible for the production of secondary metabolites, which are essential for the development and environmental defense mechanism of R. stricta during stress conditions. CONCLUSIONS: Our results suggested that genes involved in flavonoid biosynthesis and aromatic acid synthesis pathways were upregulated during JA stress. However, monoterpenoid indole alkaloid (MIA) was unaffected by JA treatment. Hence, we can postulate that JA plays an important role in R. stricta during plant development and environmental stress conditions.

De novo ALX4 variant detected in child with non-syndromic craniosynostosis

Current understanding of the genetic factors contributing to the etiology of non-syndromic craniosynostosis (NSC) remains scarce. The present work investigated the presence of variants in ALX4, EFNA4, and TWIST1 genes in children with NSC to verify if variants within these genes may contribute to the occurrence of these abnormal phenotypes. A total of 101 children (aged 45.07±40.94 months) with NSC participated in this cross-sectional study. Parents and siblings of the probands were invited to participate. Medical and family history of craniosynostosis were documented. Biological samples were collected to obtain genomic DNA. Coding exons of human TWIST1, ALX4, and EFNA4 genes were amplified by polymerase chain reaction and Sanger sequenced. Five missense variants were identified in ALX4 in children with bilateral coronal, sagittal, and metopic synostosis. A de novo ALX4 variant, c.799G>A: p.Ala267Thr, was identified in a proband with sagittal synostosis. Three missense variants were identified in the EFNA4 gene in children with metopic and sagittal synostosis. A TWIST1 variant occurred in a child with unilateral coronal synostosis. Variants were predicted to be among the 0.1% (TWIST1, c.380C>A: p. Ala127Glu) and 1% (ALX4, c.769C>T: p.Arg257Cys, c.799G>A: p.Ala267Thr, c.929G>A: p.Gly310Asp; EFNA4, c.178C>T: p.His60Tyr, C.283A>G: p.Lys95Glu, c.349C>A: Pro117Thr) most deleterious variants in the human genome. With the exception of ALX4, c.799G>A: p.Ala267Thr, all other variants were present in at least one non-affected family member, suggesting incomplete penetrance. Thus, these variants may contribute to the development of craniosynostosis, and should not be discarded as potential candidate genes in the diagnosis of this condition.

Occurrence of Leishmania infection in the immediate geographic region of Ji-Paraná, Rondônia State, Brazil

Abstract INTRODUCTION: This study evaluated the epidemiology of American cutaneous leishmaniasis in the immediate region of Ji-Paraná, Rondônia State. METHODS: Samples and epidemiological data were collected from 105 patients. RESULTS: Leishmania infection was observed in 58 (55.2%) patients, and Leishmania braziliensis was present in 82.9% of the 41 sequenced samples. Infected patients were predominantly male (93.1%). Leishmania infection was twice as prevalent among rural inhabitants versus urban inhabitants. Lesions were more frequent in the upper limbs (arms/hands, 41.82%). CONCLUSIONS: The present data corroborate the zoonotic profile of cutaneous leishmaniasis; this information could help to improve surveillance and control strategies.

Microfilaremia by Cercopithifilaria bainae in a dog from the central western region of Brazil: case report / Microfilaremia por Cercopithifilaria bainae em um cão da região Centro-Oeste do Brasil: relato de caso

Cercopithifilaria bainae is a nematode belonging to the family Onchocercidae that parasitizes the subcutaneous tissue of dogs. Its transmission occurs through the tick Rhipicephalus sanguineus and its geographical distribution overlaps that of this vector. The present study reports the detection of microfilaremia by C. bainae in an eight-year-old male dog that presented anorexia, hyperthermia, motor incoordination, mydriasis, a nodule in the left testicle and concomitant infection by Ehrlichia sp. Blood samples were analyzed using microscopy, PCR and DNA sequencing. Microfilariae measuring 150±5.5µm in length and 7±1.8µm in width were retrieved. The DNA sequence exhibited 98% identity with C. bainae sequences available in Genbank. This is the first report of microfilaremia by C. bainae in a dog in the central western region of Brazil.(AU)

Cercopithifilaria bainae é um nematoide pertencente à família Onchocercidae, que parasita o tecido subcutâneo de cães. Sua transmissão ocorre pelo carrapato Rhipicephalus sanguineus, e sua distribuição geográfica se sobrepõe ao espalhamento desse vetor. O presente estudo relata a detecção de microfilaremia por C. bainae em um cão macho de oito anos que apresentava anorexia, hipertermia, incoordenação motora, midríase e nódulo no testículo esquerdo e infecção concomitante por Ehrlichia sp. A coleta de sangue foi realizada, e o material analisado por meio dos exames de microscopia, PCR e sequenciamento de DNA. Microfilárias medindo 150±5,5µm de comprimento e 7±1,8µm de largura foram recuperadas. A sequência de DNA obtida mostrou 98% de identidade com sequências de C. bainae disponíveis no Genbank. Este é o primeiro relato de microfilaremia de C. bainae em um cão na região Centro-Oeste do Brasil.(AU)

Oropouche virus detection in saliva and urine

Oropouche virus (OROV) is an arthropod-borne virus of the Peribunyaviridae family, transmitted to humans primarily by Culicoides paraensis. It is one of the main arboviruses infecting humans in Brazil, primarily in the Amazon Region. Here, we report the detection of OROV in the saliva and urine of a patient whose samples were collected five days after the onset of symptoms. Nucleotide sequencing and phylogenetic analysis further confirmed the results. To our knowledge, this is the first study reporting the detection of OROV in the saliva and urine of an infected patient. In addition, the results of our study expand the current knowledge pertaining to the natural history of Oropouche fever.

Circulating microrna expression profiles in patients with stable and unstable angina

OBJECTIVES: High incidence and case fatality of unstable angina (UA) is, to a large extent, a consequence of the lack of highly sensitive and specific non-invasive markers. Circulating microRNAs (miRNAs) have been widely recommended as potential biomarkers for numerous diseases. In the present study, we characterized distinctive miRNA expression profiles in patients with stable angina (SA), UA, and normal coronary arteries (NCA), and identified promising candidates for UA diagnosis. METHODS: Serum was collected from patients with SA, UA, and NCA who visited the Department of Cardiovascular Diseases of the Meizhou People's Hospital. Small RNA sequencing was carried out on an Illumina HiSeq 2500 platform. miRNA expression in different groups of patients was profiled and then confirmed based on that in an independent set of patients. Functions of differentially expressed miRNAs were predicted using gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway analysis. RESULTS: Our results indicated that circulating miRNA expression profiles differed between SA, UA, and NCA patients. A total of 36 and 161 miRNAs were dysregulated in SA and UA patients, respectively. miRNA expression was validated by reverse transcription quantitative polymerase chain reaction. CONCLUSION: The results suggest that circulating miRNAs are potential biomarkers of UA.

Paracoccidioides brasiliensis habitat: far beyond armadillo burrows?

Paracoccidioides spp. isolation from environmental samples is rare and hardly reproducible. Molecular techniques have facilitated the fungal detection. However, it can be still difficult. Some strategies to enhance the capacity of DNA detection have been adopted, including the analysis of soil samples belonging to the habitat of animals from which Paracoccidioides spp. have already been isolated, notably armadillo burrows. To date, the detection of Paracoccidioides spp. has not yet been reported from outbreak hotspots. Clusters and outbreaks of acute paracoccidioidomycosis (PCM), usually a more severe clinical form, have currently occurred in urban areas being associated to climate changes, deforestation, and great constructions. These occurrences potentially signalise the fungus' environmental niche, a riddle not yet solved. The authors performed an environmental investigation in a deeply disturbed area, after a highway construction in Rio de Janeiro, Brazil, where a recent outbreak of acute PCM occurred. Specific DNA sequences of Paracoccidioides brasiliensis were detected in shallow soil samples around the highway, reinforcing the association between the road construction and this PCM outbreak.

Phylogenetic relationships of closely-related phlebotomine sand flies (Diptera: Psychodidae) of Nyssomyia genus and Lutzomyia subgenus

BACKGROUND The Nyssomyia genus and Lutzomyia subgenus include medical important species that are Latin American leishmaniases vectors. Little is known about the phylogenetic relationships of closely-related species in each of these taxonomic groups that are morphologically indistinguishable or differentiated by very subtle details. OBJECTIVES We inferred the phylogenetic relationships of closely-related species within both the Nyssomyia genus and the Lutzomyia subgenus using a cytochrome c oxidase subunit I (COI) fragment. METHODS The sampling was carried out from 11 Argentinean localities. For genetic analyses, we used GenBank sequences in addition to our sequences from Argentina. Kimura 2-parameter (K2P) genetic distance and nucleotide divergence (Da) was calculated between closely-related species of Nyssomyia genus, Lutzomyia subgenus and between clades of Lutzomyia longipalpis complex. FINDINGS The K2P and Da values within species of Nyssomyia genus and Lutzomyia subgenus were lower than the divergence detected between clades of Lu. longipalpis complex. The haplotype network analyses within Lutzomyia subgenus showed shared haplotypes between species, contrary to Nyssomyia genus with none haplotype shared. Bayesian inference within Nyssomyia genus presented structuring by species. MAIN CONCLUSIONS This study evidences the phylogenetic proximity among closely-related species within Nyssomyia genus and Lutzomyia subgenus. The COI sequences of Nyssomyia neivai derived from the present study are the first available in GenBank.

Newly sequenced genomes of four Bacillus Calmette Guerin vaccines

Bacillus Calmette Guerin (BCG) vaccines comprise a family of related strains. Whole genome sequencing has allowed the better characterisation of the differences between many of the BCG vaccines. As sequencing technologies improve, updating of publicly available sequence data becomes common practice. We hereby announce the draft genome of four commonly used BCG vaccines in Brazil, Argentina and Venezuela.

Determinação do nível de resistência a isoniazida em cepas de Mycobacterium tuberculosis e correlação com mutações nos genes que conferem resistência a este fármaco: katG e inhA / Determination of the level of resistance to isoniazid in strains of Mycobacterium tuberculosis and correlation with mutations in genes that confer resistance to this drug: katG and inhA

A tuberculose (TB) resistente aos fármacos é um problema mundial. As mutações mais frequentes associadas à resistência à isoniazida em Mycobacterium tuberculosis ocorrem no códon 315 do gene katG, causando níveis moderados a altos de resistência, e na região promotora do gene inhA, associadas a baixos níveis de resistência. Mutações no gene rpoB associam-se à resistência à rifampicina. Este estudo analisou mutações nos genes katG, inhA e rpoB e níveis fenotípicos de resistência à isoniazida em isolados de M. tuberculosis de pacientes com TB resistente do estado de São Paulo, no período de outubro/2008 a março/2009 e no ano de 2016. Dos 374 isolados analisados, 165 (44%) eram monorresistentes à isoniazida, 47 (13%) polirresistentes (resistentes à isoniazida+fármaco(s) de 1ª linha exceto rifampicina), 122 (33%) multirresistentes (MR), 24 (6%) pré-extensivamente resistentes (pré-XDR) e 16 (4%) XDR. Entre os isolados com a mutação Ser315Thr no katG, 99,4% apresentaram resistência intermediária ou alta à isoniazida, enquanto 73,7% dos isolados com a mutação C-15T no inhA apresentaram baixa resistência. Dos isolados com mutações no katG e inhA, 66,6% mostraram resistência alta e 32,4% resistência intermediária. Observou-se que 58,8% dos isolados tipados estavam em cluster e 43,7% deles pertenciam...(AU)

Drug-resistant tuberculosis (TB) is a problem worldwide. The most frequent mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates occur in codon 315 of the katG gene, which have been associated with moderate- to high-levels of resistance, and in the promoter region of the inhA gene, causing low-level isoniazid resistance. In its turn, the rpoB gene is associated with resistance to rifampicin. This study analyzed mutations in katG, inhA and rpoB genes and phenotypic levels of isoniazid resistance in M. tuberculosis isolates from drug-resistant TB patients from the state of São Paulo, in the period of October 2008 to March 2009 and during the year of 2016. Of the 374 M. tuberculosis isolates analyzed 165 (44%) were monoresistant to isoniazid, 47 (13%) polydrug-resistant (resistant to isoniazid+1st line drug(s) except rifampicin), 122 (33%) multidrug-resistant (MDR), 24 (6%) pre-extensively drug-resistant (pre-XDR) and 16 (4%) XDR. Among isolates with the katG Ser315Thr mutation, 99.4% had intermediate or high-level isoniazid resistance, while 73.7% of the isolates with the inhA C15T mutation had low-level resistance. Among isolates with mutations in katG and inhA, 66.6% had high-level resistance and 32.4% had intermediate resistance. It was observed that 58.8% of the isolates submitted to typing were clustered, and 43.7% of them belonged to the five most prevalent clusters: SP5, SP2c, SP12, SP5ac and SP1i. Isolates with katG 315 mutations were more frequently clustered when compared to isolates with other katG mutations or with no mutations (p= 0.002 and 0.01, respectively). Isolates with the inhA C-15T mutation were more frequently clustered when compared to isolates with katG 315 mutations (p= 0.006). Statistically significant differences were found between cure rates of isoniazidmonoresistant TB patients and MDR-TB patients (63.4% vs 47.1%, p= 0.02), as well as between isoniazid-monoresistant TB patients and XDR-TB patients (63.4% vs 12.5%, p= 0.01). Patients with polydrug-resistant TB also had higher cure rates than patients with MDR-TB (70% vs 47.1%, p= 0.02) and XDR-TB (12.5%, p= 0.004). The present study showed that katG mutations are associated with higher levels of isoniazid resistance and inhA mutations are found in isolates with lower resistance levels. In addition, drugresistant TB was transmitted among patients from the state of São Paulo, whose cure rates were relatively low. (AU)

Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

A common Salmonella Enteritidis sequence type from poultry and human gastroenteritis in Ibagué, Colombia / Un tipo de secuencia común de Salmonella Enteritidis de origen aviar y de humano con gastroenteritis en Ibagué, Colombia

Abstract Introduction: Salmonella Enteritidis is a major cause of human salmonellosis in the world, with contaminated eggs and raw chicken meat as the main routes of infection. The main Salmonella spp. serovars circulating in laying hen farms, the surface of eggs, and in raw chicken carcasses have been identified in Ibagué, Colombia. However, it is unknown whether those serovars are responsible for human gastroenteritis. Objective: To evaluate the genetic relationship between gastroenteritis and Salmonella Enteritidis isolates from poultry and humans using multilocus sequence typing (MLST). Materials and methods: Salmonella spp. was isolated from clinical cases of gastroenteritis (n=110). Antibiotic susceptibility tests, followed by serotyping and MLST were conducted and S. Enteritidis was compared to those from laying hen farms and marketed eggs. Results: Ten isolates of Salmonella spp. were obtained from the stools of people with gastroenteritis. The prevalence of Salmonella spp. in human stools was 9.09%, and S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Uganda (n=1), S. Grupensis (n=1), and S. Braenderup (n=1) were the main serotypes. MLST indicated that a common S. Enteritidis sequence type (ST11) was present in all three sources and showed the same antibiotic resistance pattern. Conclusion: Salmonella Enteritidis ST11 constitutes a link between consumption and manipulation of contaminated eggs and human gastroenteritis in Ibagué. Additional studies would be required to establish if other Salmonella serovars isolated from raw chicken meat are also associated with human gastroenteritis.

Resumen Introducción. Salmonella Enteritidis es una de las mayores causas de salmonelosis en el mundo; los huevos contaminados y la carne de pollo cruda son sus principales fuentes de infección. En Ibagué, Colombia, se han identificado los principales serovares que circulan en granjas, superficies de huevos y canales de pollo, pero se desconoce si esos serovares son responsables de la gastroenteritis. Objetivo. Evaluar la relación genética entre los aislamientos de Salmonella Enteritidis de aves de corral y de humanos con la gastroenteritis mediante tipificación de multiloci de secuencias (Multilocus Sequence Typing, MLST). Materiales y métodos. Se aisló Salmonella spp. de casos clínicos de gastroenteritis (n=110). Se hizo la prueba de sensibilidad antibiótica, así como la serotipificación y la tipificación mediante MLST, y se comparó S. Enteritidis de humanos con la hallada en granjas de gallinas ponedoras y en huevo comercializado (n=6). Resultados. Se aislaron 10 cepas de Salmonella spp. a partir de heces de humanos con gastroenteritis. Se obtuvo una prevalencia de Salmonella spp. de 9,09%, y se identificaron los serotipos S. Enteritidis (n=4), S. Typhymurium (n=2), S. Newport (n=1), S. Grupensis (n=1), S. Uganda (n=1) y S. Braenderup presentes en pacientes con gastroenteritis. Mediante la MLST, se comprobó que un tipo de secuencia común (ST11) de S. Enteritidis estuvo presente en todas las tres fuentes y presentó el mismo patrón de resistencia antibiótica. Conclusión. Salmonella Enteritidis ST11 constituye un vínculo entre el consumo y la manipulación de huevos contaminados, y la gastroenteritis en humanos en Ibagué. Se requieren estudios complementarios para conocer si otros serovares de Salmonella aislados de carne de pollo cruda también se asocian con la gastroenteritis en humanos.

Sequencing of RAS/RAF pathway genes in primary colorectal cancer and matched liver and lung metastases

Background: Mutations in the RAS/RAF pathway predict resistance to anti-epidermal growth factor receptor antibodies in colorectal cancer (CRC), and may be targets for future therapies. This study investigates concordance of BRAF, HRAS, KRAS, NRAS and PIK3CA mutation status in primary CRC with matched liver (n = 274), lung (n = 114) or combined liver and lung metastases (n = 14). Methods: Next generation sequencing was performed on DNA from formalin-fixed paraffin embedded CRC and matched liver and/or lung metastases, for recurrent mutations in BRAF, HRAS, KRAS, NRAS and PIK3CA and using the single-molecule molecular inversion probe method. Results: Paired sequencing results on all five genes were reached in 249 of the 402 cases (62%). The obtained number of unique reads was not always sufficient to confidently call the absence or presence of mutations for all regions of interest. The mutational status of matched pairs was highly concordant; 91.1% concordance for all five genes, 95.5% for KRAS, 99.1% for NRAS. Lung metastases more often harboured RAS mutations compared to liver metastases (71% vs. 48%, p < 0.001). Conclusions: In this large series of CRC we show that both primary tumors and corresponding metastases can be used to determine the mutational status for targeted therapy, given the high concordance rates. Next generation sequencing including a single molecule tags is feasible, however in combination with archival formalin-fixed paraffin embedded material is limited by coverage depth.

Description of a human Bocavirus recombinant strain in the Americas

Human bocaviruses (HBoV) are mainly associated with respiratory and gastroenteric infections. These viruses belong to the family Parvoviridae, genus Bocaparvovirus and are classified in four subtypes (HBoV1-4). Recombination and point mutation have been described as basis of parvovirus evolution. In this study three viral sequences were obtained from positives HBoV sewage samples collected in two Uruguayan cities and were characterised by different methods as recombinant strains. This recombination event was localised in the 5' end of VP1 gene and the parental strains belonged to subtypes 3 and 4. These three Uruguayan strains are identical at the nucleotide sequences in the analysed genome region of the virus. As far as we known, this study represents the first detection of HBoV recombinants strains in the Americas.

Small RNA sequencing reveals microRNAs related to neuropathic pain in rats

The present study aimed to identify microRNAs (miRNAs) that are involved in neuropathic pain and predict their corresponding roles in the pathogenesis and development process of neuropathic pain. The rat model of neuropathic pain caused by spared nerve injury (SNI) was established in Sprague-Dawley male rats, followed by small RNA sequencing of the L3-L6 dorsal root ganglion. Real-time PCR was performed to validate the differently expressed miRNAs. Functional verification was performed by intrathecally injecting the animals with miRNA agomir. A total of 72 differentially expressed miRNAs were identified in the SNI rats, including 33 upregulated and 39 downregulated miRNAs. The results of qPCR further verified the expression levels of rno-miR-6215 (P=0.015), rno-miR-1224 (P=0.030), rno-miR-1249 (P=0.038), and rno-miR-488-3p (P=0.048), which were all significantly downregulated in the SNI rats compared to the control ones. The majority of differentially expressed miRNAs were associated with phosphorylation, intracellular signal transduction, and cell death. Target prediction, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses suggested that these differentially expressed miRNAs targeted genes that are related to axon guidance, focal adhesion, and Ras and Wnt signaling pathways. Moreover, miR-1224 agomir significantly alleviated SNI-induced neuropathic pain. The current findings provide new insights into the role of miRNAs in the pathogenesis of neuropathic pain.

Optimización de una técnica de PCR convencional para detección de virus de papiloma humano tipo 16 y 18 / Optimization of a conventional PCR technique for detection of human papillomavirus type 16 and 18

El cáncer de cuello uterino es el segundo cáncer femenino más común a nivel mundial. El agente causal es el virus de papiloma humano (VPH). Se han identificado 13 tipos de virus de papiloma humano de alto riesgo oncogénico (VPH-AR), entre los cuales el VPH 16 y VPH 18 son los más frecuentemente detectados en cáncer de cuello uterino, siendo en Paraguay detectados en el 70% de casos de cáncer invasor. Por ello, el objetivo fue estandarizar y determinar el límite de detección de una técnica de PCR convencional para la detección de VPH 16 y 18. Para la detección de ADN de VPH 16 y 18, se observaron mejores resultados con 2mM de MgCl2 y 60°C para la temperatura de alineamiento. El límite de detección para las PCR fue de 14,6x10-11ng/µL para VPH 16 y 21,7x10-12ng/µL para VPH 18. Este trabajo servirá de base a otros estudios de detección e identificación de estos tipos virales por PCR, con miras a identificar un grupo de mujeres positivas para VPH-AR que poseen mayor riesgo de desarrollo de lesión y cáncer de cuello uterino y precisan de un seguimiento más cercano(AU

Cervical cancer is the second most common female cancer worldwide. It is caused by the human papilloma virus (HPV). Thirteen genotypes of high oncogenic risk human papilloma viruses (HPV-HR) have been identified, among which types 16 and 18 are the most frequently detected in cervical cancer. In Paraguay, they are detected in 70% of the invasive cancer cases. Therefore, the objective was to standardize and determine the detection limit of a conventional PCR technique for the detection of HPV 16 and 18. Better results were observed with 2mM MgCl2 and 60°C for the alignment temperature in detection of HPV 16 and 18 DNA. The limit of detection was 14.6x10-11ng/µL for HPV 16 and 21.7x10-12ng/µL for HPV 18. This work will help other studies for the detection and identification of these viral types by PCR in order to identify a group of HPV-HR positive women who have higher risk for the development of lesions and cervical cancer and need a closer follow-up(AU)

Gastroenterite aguda associada a variantes do Norovírus GII. 4 / Acute gastroenteritis associated with norovirus gii. 4 variants

ABSTRACT BACKGROUND: Norovirus (NoV) is an important etiologic agent of acute gastroenteritis and infects individuals of all ages, especially children in Brazil and worldwide. NoV GII.4 was the most prevalent genotype worldwide because of your extensive genetic diversity. In Brazil, especially in the Northeast, few studies have been developed for identify and molecularly characterize NoV. OBJECTIVE: The present study aimed to detect and describe the molecular epidemiology of NoV associated with acute gastroenteritis. METHODS: The viral RNA extracted from stool samples were subjected to Nested RT-PCR and the genotypes were determined by nucleotide sequences analysis. In total, 278 stool samples assisted at Aliança Hospital in the city of Salvador, with acute gastroenteritis were examined, between March 2009 and July 2012. RESULTS: A high NoV rate (54.2%) was identified in children under 5 years of age. We detected the circulation of different NoV GII.4 variants in Salvador, during the study period as Den Haag 2006b, New Orleans 2009 and Sydney 2012. CONCLUSION: These findings reinforce the need to study the molecular epidemiology of NoV infections in acute gastroenteritis.

RESUMO CONTEXTO: Norovírus (NoV) é o agente etiológico mais importante nas gastroenterites agudas e infecta indivíduos de todas as idades, especialmente crianças no Brasil e no mundo. O NoV GII.4 é o genótipo mais prevalente em todo o mundo devido a sua elevada diversidade genética. No Brasil, principalmente no Nordeste, poucos estudos têm sido desenvolvidos a fim de identificar e caracterizar molecularmente o NoV. OBJETIVO: O presente estudo teve como objetivo detectar e descrever a epidemiologia molecular do NoV associado com gastroenterite aguda. MÉTODOS: RNA viral extraído a de amostras de fezes foi submetido a amplificação por Nested-RT-PCR e o genótipo determinado por analise da sequência de nucleotídeos. Um total de 278 amostras de pacientes atendidos no Hospital Aliança, na cidade de Salvador, com gastroenterite aguda foram examinados, entre março de 2009 a julho de 2012. RESULTADOS: Uma alta taxa de NoV (54,2%) foi identificado em crianças de até 5 anos de idade. Detectou-se a circulação de diferentes variantes de NoV GII.4 em Salvador, durante o período do estudo, tais como Den Haag 2006b, New Orleans 2009 e Sydney 2012. CONCLUSÃO: Estes achados reforçam a necessidade de maiores estudos para esclarecer a epidemiologia molecular das infecções por NoV em casos de gastroenterite aguda.

Variabilidad genética de Aedes aegypti en el departamento de Sucre, Colombia, mediante el análisis de la secuencia de nucleótidos del gen mitocondrial ND4 / Genetic variability of Aedes aegypti in the department of Sucre, Colombia, by analysis of the nucleotide sequence of the mitochondrial ND4 gene

Resumen Introducción. Aedes aegypti es la especie de mosquito de mayor relevancia en América por transmitir los virus del dengue, del Zika, del chikungunya y de la fiebre amarilla. Tanto factores ecológicos como el control químico, pueden influir en la composición genética de las poblaciones de Ae. aegypti, por lo cual es necesaria su caracterización. Objetivo. Determinar la variabilidad genética de las poblaciones de Ae. aegypti en cuatro municipios del departamento de Sucre, Colombia. Materiales y métodos. Larvas de Ae. aegypti, recolectadas en los municipios de Sincelejo, Sampués, Corozal y Guaranda del departamento de Sucre, fueron criadas en laboratorio hasta el estado adulto. Como marcador genético, se utilizó un segmento del gen mitocondrial ND4, que codifica para la subunidad 4 de la enzima NADH-deshidrogenasa. El análisis genético incluyó la estimación de parámetros de diversidad de nucleótidos, haplotipos, de estructura genética y de flujo de genes. Resultados. Se obtuvieron 108 secuencias parciales de 357 nucleótidos y cuatro haplotipos de nucleótidos del gen ND4 de Ae. aegypti. Se encontró una diferenciación genética significativamente alta entre las poblaciones de Sampués y Guaranda mediante el índice de fijación (F ST =0,59467), las de Sincelejo y Sampués (F ST = 0,25637), y las de Corozal y Guaranda (F ST = 0,22237). Se evidenció un gran flujo de genes (Nm=infinito) entre las poblaciones de Sincelejo y Corozal. Conclusión. Existen diferencias genéticas entre las poblaciones del mosquito Ae. aegypti de los municipios del departamento de Sucre. Se registra la presencia de un nuevo haplotipo del gen mitocondrial ND4 de Ae. aegypti en Colombia, el cual fue detectado en el municipio de Sincelejo.

Abstract Introduction. Aedes aegypti is the most important mosquito species in America for the transmission of viruses of dengue, Zika, Chikungunya and yellow fever. Ecological factors as well as chemical controls can affect the genetic composition of Ae. aegypti populations, which is why its genetic characterization is necessary. Objective. To determine the genetic variability of Ae. aegypti populations in four municipalities of Sucre department, Colombia. Materials and methods. Larvae of Ae. aegypti, collected in the municipalities of Sincelejo, Sampués, Corozal and Guaranda, Sucre department, were reared under laboratory conditions to adult stage. A segment of the mitochondrial ND4 gene which codes for the subunit 4 of the enzyme NADH-dehydrogenase was used as genetic marker. The genetic analysis included the estimation of parameters of nucleotide and haplotype diversity, genetic structure and gene flow. Results. One hundred and eight partial sequences of 357 nucleotides and four nucleotide haplotypes of the ND4 gene of Ae. aegypti were obtained. A significantly high genetic differentiation was found between the Sampués and Guaranda populations (F ST =0.59467),Sincelejo and Sampués (F ST =0.25637), and Corozal and Guaranda (F ST =0.22237). A high gene flow (Nm=infinite) was observed among the populations of Sincelejo and Corozal. Conclusion. There are genetic differences between the Ae. aegypti populations from the municipalities of Sucre department. The presence of a new haplotype of the mitochondrial ND4 gene of Ae. aegypti in Colombia was recorded, detected in the municipality of Sincelejo.