Development of a cell-based reporter assay for detection of Human alphaherpesviruses.

Immunosuppressed patients can suffer from Human alphaherpesvirus (HSV) infection with fast evolution, severe atypical symptomatology, and often-fatal outcome. Thus, the development and validation of new methods in vitro and in vivo to promote an early diagnosis and effective treatment of these patients are crucial. Therefore, this work aimed to develop a cell-based reporter assay for the detection of HSV through the transfection of Vero cells with the ICP10 promoter from HSV-2 linked to the pZsGreen1-1 plasmid. The assay was evaluated on Vero cells infected with HSV-1 or HSV-2 and followed by treating them with anti-HSV agents (acyclovir, gallic acid, convallatoxin, and Uncaria sp. extract) or with no anti-HSV activity agents (Passiflora edulis extract and cardenolide derivatives). The GFP expression was increased by both HSV cellular infection, which was detected by flow cytometry and fluorescence microscopy. F2R Zsgreen1-1 cells infection with 200 and 600 PFU/mL of HSV-2 increased the fluorescence intensity, when compared to the controls, by approximately 30% and 60%, respectively. Infection with 100 and 600 PFU/mL of HSV-1 also increased the fluorescence intensity by approximately 20% and 35%, when compared to the controls, respectively. The F2R ZsGreen1-1 system revealed to be an efficient assay, which can be used for clinical diagnosis, antiviral resistance evaluation, HSV cycle studies, and new antiviral drug research.

Disinfection of needleless connectors to reduce Staphylococcus aureus bacterial load.

HIGHLIGHTS: Compare effectiveness of chemical disinfectants in reducing S. aureus. Five disinfectants reduced the bacterial load, especially chlorhexidine solutions. Focus on Brazilian clinical practice of needleless connector disinfection. PURPOSE: This study aimed to gain further knowledge about the comparative effectiveness of chemical disinfectants in reducing the bacterial load of NCs inoculated with S. aureus. METHODS: Disinfection of needleless connectors was undertaken in vitro against S. aureus comparing 70% isopropyl alcohol (IPA), 70% ethanol, 0.5% and 2% chlorhexidine in 70% IPA applied with gauze, and 70% IPA single-use cap (Site-Scrub®). RESULTS: All disinfectants reduced the bacterial load (P<0.001), especially the chlorhexidine solutions. Mechanical friction should follow guidelines. CONCLUSION: This study found that all tested disinfectants effectively reduced the bacterial load and more clinical studies must be developed with a focus on the Brazilian clinical practice of needleless connector disinfection.

Characterization of Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Santa Catarina (SC), Brazil.

The study investigated the characteristics of Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Santa Catarina. Findings revealed prevalent SCCmecII and IV, multiresistance, Leucocidin ED genes, and one ST105 isolate. The results indicated that the in-state MRSA isolates showed the same characteristics as the out-of-state isolates among the investigated features.

Trypanosoma rangeli expresses a ß-galactofuranosyl transferase.

Glycoconjugates play essential roles in cell recognition, infectivity and survival of protozoan parasites within their insect vectors and mammalian hosts. ß-galactofuranose is a component of several glycoconjugates in many organisms, including a variety of trypanosomatids, but is absent in mammalian and African trypanosomes. Herein, we describe the presence of a ß(1-3) galactofuranosyl transferase (GALFT), an important enzyme of the galactofuranose biosynthetic pathway, in Trypanosoma rangeli. The T. rangeli GALFT gene (TrGALFT) has an ORF of 1.2 Kb and is organized in two copies in the T. rangeli genome. Antibodies raised against an internal fragment of the transferase demonstrated a 45 kDa protein coded by TrGALFT was localized in the whole cytoplasm, mainly in the Golgi apparatus and equally expressed in epimastigotes and trypomastigotes from T. rangeli. Despite the high sequence similarity with Trypanosoma cruzi and Leishmania spp. orthologous TrGALFT showed a substitution of the metal-binding DXD motif, conserved amongst glycosyltransferases, for a DXE functionally analogous motif. Moreover, a reduced number of GALFT genes were present in T. rangeli when compared with other pathogenic kinetoplastid species.

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum.

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

Novel Insights into blaGES Mobilome Reveal Extensive Genetic Variation in Hospital Effluents.

Mobile genetic elements contribute to the emergence and spread of multidrug-resistant bacteria by enabling the horizontal transfer of acquired antibiotic resistance among different bacterial species and genera. This study characterizes the genetic backbone of blaGES in Aeromonas spp. and Klebsiella spp. isolated from untreated hospital effluents. Plasmids ranging in size from 9 to 244 kb, sequenced using Illumina and Nanopore platforms, revealed representatives of plasmid incompatibility groups IncP6, IncQ1, IncL/M1, IncFII, and IncFII-FIA. Different GES enzymes (GES-1, GES-7, and GES-16) were located in novel class 1 integrons in Aeromonas spp. and GES-5 in previously reported class 1 integrons in Klebsiella spp. Furthermore, in Klebsiella quasipneumoniae, blaGES-5 was found in tandem as a coding sequence that disrupted the 3' conserved segment (CS). In Klebsiella grimontii, blaGES-5 was observed in two different plasmids, and one of them carried multiple IncF replicons. Three Aeromonas caviae isolates presented blaGES-1, one Aeromonas veronii isolate presented blaGES-7, and another A. veronii isolate presented blaGES-16. Multilocus sequence typing (MLST) analysis revealed novel sequence types for Aeromonas and Klebsiella species. The current findings highlight the large genetic diversity of these species, emphasizing their great adaptability to the environment. The results also indicate a public health risk because these antimicrobial-resistant genes have the potential to reach wastewater treatment plants and larger water bodies. Considering that they are major interfaces between humans and the environment, they could spread throughout the community to clinical settings. IMPORTANCE In the "One Health" approach, which encompasses human, animal, and environmental health, emerging issues of antimicrobial resistance are associated with hospital effluents that contain clinically relevant antibiotic-resistant bacteria along with a wide range of antibiotic concentrations, and lack regulatory status for mandatory prior and effective treatment. blaGES genes have been reported in aquatic environments despite the low detection of these genes among clinical isolates within the studied hospitals. Carbapenemase enzymes, which are relatively unusual globally, such as GES type inserted into new integrons on plasmids, are worrisome. Notably, K. grimontii, a newly identified species, carried two plasmids with blaGES-5, and K. quasipneumoniae carried two copies of blaGES-5 at the same plasmid. These kinds of plasmids are primarily responsible for multidrug resistance among bacteria in both clinical and natural environments, and they harbor resistant genes against antibiotics of key importance in clinical therapy, possibly leading to a public health problem of large proportion.

Different Swine Production Systems Can Shape Slurry Resistome at Mechanism and Class Levels Based on Swine Manure Evaluation.

Antimicrobial resistance is a major threat to public health. Antimicrobial use in animal husbandry is a major concern since it can favor an increase in antimicrobial resistance among farms. Herein, we aim to better understand and characterize the main resistome profiles in microbial communities found in pig farms. Sampling of swine manure was performed in two different timepoints (October 2019 and January 2020) in each of the 14 different swine farms, located in the mesoregion of Western Santa Catarina state in Brazil, a pole of swine product production of worldwide importance. Samples were divided into three groups: farms with the opened regimen and no usage of antimicrobials (F1; n = 10), farms with the closed regimen and usage of antimicrobials (F2; n = 16), and farms with the closed regimen and no usage of antimicrobials (F3; n = 2). The metagenomic evaluation was performed to obtain and identify genetic elements related to antimicrobial resistance using nanopore sequencing. We used ResistoXplorer software to perform composition, alpha and beta diversity, and clustering analysis. In addition, PCR reactions were performed to confirm the presence or absence of seven different beta-lactamase family genes and five phosphoethanolamine transferase gene variants clinically relevant. Our findings based on the identification of resistance genes at the mechanism level showed a prevalence of alteration of the drug target (72.3%) profile, followed by drug inactivation (17.5%) and drug efflux (10.1%). We identified predominantly aminoglycosides (45.3%), tetracyclines (15.9%), and multiclass (11,2%) resistance genes. PCoA analysis indicates differences between F1 and F2 profiles. F2 samples showed increased diversity when compared to the F1 group. In addition, herein we first report the identification of mcr-4 in a slurry sample (C1F1.1) in Santa Catarina State. In general, our findings reinforce that many factors on the practices of animal husbandry are involved in the resistome profile at the mechanism and class levels. Further studies to better understand microbiome and mobilome aspects of these elements are necessary to elucidate transmission pathways between different bacteria and environments.

Detecting KPC-2 and NDM-1 Coexpression in Klebsiella pneumoniae Complex from Human and Animal Hosts in South America.

Reports of Gram-negative bacteria harboring multiple carbapenemase genes have increased in South America, leading to an urgent need for appropriate microbiological diagnosis. We evaluated phenotypic methods for detecting Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-ß-lactamase-1 (NDM-1) coexpression in members of the K. pneumoniae complex (i.e., K. pneumoniae, K. quasipneumoniae, and K. variicola) isolated from human and animal hosts, based on inhibition of ceftazidime-avibactam (CZA) and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, or avibactam (AVI). While the presence of blaKPC-2 and blaNDM-1 genes was confirmed by whole-genome sequencing, PCR, and/or GeneXpert, coexpression was successfully detected based on the following: (i) a ≥5-mm increase in the zone diameter of ATM (30 µg) disks plus AVI (4 or 20 µg) and ≥4-mm and ≥10-mm increases in the zone diameters for "CZA 50" (30 µg ceftazidime [CAZ] and 20 µg AVI) and "CZA 14" (10 µg CAZ and 4 µg AVI) disks, respectively, when we added DPA (1 mg/disk) or EDTA (5 mM) in a combined disk test (CDT); (ii) a positive ghost zone (synergism) between ATM (30 µg) and CZA 50 disks and between CZA 50 and DPA (1 mg) disks, using the double-disk synergy test (DDST) at a disk-disk distance of 2.5 cm; (iii) ≥3-fold MIC reductions of ATM and CZA in the presence of AVI (4 µg/mL), DPA (500 µg/mL), or EDTA (320 µg/mL); and (iv) immunochromatography. Although our results demonstrated that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex, additional studies are necessary to confirm the accuracy of these methodologies by testing other Gram-negative bacterial species and other KPC and NDM variants coexpressed by WHO critical priority pathogens detected worldwide. IMPORTANCE Alerts regarding the emergence and increase of combinations of carbapenemases in Enterobacterales in Latin America and the Caribbean have recently been issued by PAHO and WHO, emphasizing the importance of appropriate microbiological diagnosis and the effective and articulated implementation of infection prevention and control programs. In this study, we evaluated methods based on inhibition of ceftazidime (CAZ), ceftazidime-avibactam (CZA), and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, and avibactam (AVI) inhibitors for the identification of KPC-2- and NDM-1-coexpression in members of the K. pneumoniae complex recovered from human and animal hosts. Our results demonstrate that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex.

WHO Critical Priority Escherichia coli as One Health Challenge for a Post-Pandemic Scenario: Genomic Surveillance and Analysis of Current Trends in Brazil.

The dissemination of carbapenem-resistant and third generation cephalosporin-resistant pathogens is a critical issue that is no longer restricted to hospital settings. The rapid spread of critical priority pathogens in Brazil is notably worrying, considering its continental dimension, the diversity of international trade, livestock production, and human travel. We conducted a nationwide genomic investigation under a One Health perspective that included Escherichia coli strains isolated from humans and nonhuman sources, over 45 years (1974-2019). One hundred sixty-seven genomes were analyzed extracting clinically relevant information (i.e., resistome, virulome, mobilome, sequence types [STs], and phylogenomic). The endemic status of extended-spectrum ß-lactamase (ESBL)-positive strains carrying a wide diversity of blaCTX-M variants, and the growing number of colistin-resistant isolates carrying mcr-type genes was associated with the successful expansion of international ST10, ST38, ST115, ST131, ST354, ST410, ST648, ST517, and ST711 clones; phylogenetically related and shared between human and nonhuman hosts, and polluted aquatic environments. Otherwise, carbapenem-resistant ST48, ST90, ST155, ST167, ST224, ST349, ST457, ST648, ST707, ST744, ST774, and ST2509 clones from human host harbored blaKPC-2 and blaNDM-1 genes. A broad resistome to other clinically relevant antibiotics, hazardous heavy metals, disinfectants, and pesticides was further predicted. Wide virulome associated with invasion/adherence, exotoxin and siderophore production was related to phylogroup B2. The convergence of wide resistome and virulome has contributed to the persistence and rapid spread of international high-risk clones of critical priority E. coli at the human-animal-environmental interface, which must be considered a One Health challenge for a post-pandemic scenario. IMPORTANCE A One Health approach for antimicrobial resistance must integrate whole-genome sequencing surveillance data of critical priority pathogens from human, animal and environmental sources to track hot spots and routes of transmission and developing effective prevention and control strategies. As part of the Grand Challenges Explorations: New Approaches to Characterize the Global Burden of Antimicrobial Resistance Program, we present genomic data of WHO critical priority carbapenemase-resistant, ESBL-producing, and/or colistin-resistant Escherichia coli strains isolated from humans and nonhuman sources in Brazil, a country with continental proportions and high levels of antimicrobial resistance. The present study provided evidence of epidemiological and clinical interest, highlighting that the convergence of wide virulome and resistome has contributed to the persistence and rapid spread of international high-risk clones of E. coli at the human-animal-environmental interface, which must be considered a One Health threat that requires coordinated actions to reduce its incidence in humans and nonhuman hosts.

Genomic analysis of an extensively drug-resistant Pseudomonas aeruginosa ST312 harbouring IncU plasmid-mediated blaKPC-2 isolated from ascitic fluid.

OBJECTIVES: The Klebsiella pneumoniae carbapenemase (KPC) is disseminated worldwide mostly by plasmids. However, in Pseudomonas aeruginosa chromosomal mutations are more frequently responsible for resistance to carbapenems than the acquisition of mobile elements harbouring carbapenemases genes. Indeed, although uncommon, KPC-2-producing P. aeruginosa has appeared more frequently, including in Brazil. Here we report the first genomic analysis of a plasmid-mediated KPC-2 in an extensively drug-resistant (XDR) P. aeruginosa isolated in Santa Catarina, Brazil. METHODS: Antimicrobial susceptibility testing was performed according to CLSI 2020 guidelines. The genome was sequenced using an Illumina MiSeq platform and the data were analysed using SPAdes and Prokka. In silico predictions were fulfilled using curated bioinformatics tools. RESULTS: Pseudomonas aeruginosa strain MIMA_PA2.1 (JACGTM000000000) was classified as XDR, belongs to sequence type 312 (ST312) and harbours the blaKPC-2 gene located on a small (7975 bp) IncU plasmid. This plasmid showed 86.3% identity with a non-conjugative plasmid (KC609322) carrying the blaKPC-2 gene from a multidrug-resistant P. aeruginosa (ST1006) from Colombia isolated in 2006. Besides the blaKPC-2 gene, other resistance genes to ß-lactams, aminoglycosides, phenicol, fosfomycin and quinolones were detected, the last two also associated with mobile genetic elements other than the IncU plasmid described here. CONCLUSION: This is the first genomic report of the presence of the blaKPC-2 gene carried by Pseudomonas in Southern Brazil and highlights the adaptability of blaKPC-2 to different mobile elements. This draft genome might be useful for comparative genomic analyses to monitor the spread of plasmid-mediated blaKPC in P. aeruginosa in Latin America.

Healthcare-Associated Infections-Related Bacteriome and Antimicrobial Resistance Profiling: Assessing Contamination Hotspots in a Developing Country Public Hospital.

Hospital-built environment colonization by healthcare-associated infections-related bacteria (HAIrB) and the interaction with their occupants have been studied to support more effective tools for HAI control. To investigate HAIrB dynamics and antimicrobial resistance (AMR) profile we carried out a 6-month surveillance program in a developing country public hospital, targeting patients, hospital environment, and healthcare workers, using culture-dependent and culture-independent 16S rRNA gene sequencing methods. The bacterial abundance in both approaches shows that the HAIrB group has important representativeness, with the taxa Enterobacteriaceae, Pseudomonas, Staphylococcus, E. coli, and A. baumannii widely dispersed and abundant over the time at the five different hospital units included in the survey. We observed a high abundance of HAIrB in the patient rectum, hands, and nasal sites. In the healthcare workers, the HAIrB distribution was similar for the hands, protective clothing, and mobile phones. In the hospital environment, the healthcare workers resting areas, bathrooms, and bed equipment presented a wide distribution of HAIrB and AMR, being classified as contamination hotspots. AMR is highest in patients, followed by the environment and healthcare workers. The most frequently detected beta-lactamases genes were, bla SHV-like, bla OXA- 23 -like, bla OXA- 51 -like, bla KPC-like, bla CTX-M- 1, bla CTX-M- 8, and bla CTX-M- 9 groups. Our results demonstrate that there is a wide spread of antimicrobial resistance due to HAIrB in the hospital environment, circulating among patients and healthcare workers. The contamination hotspots identified proved to be constant over time. In the fight for patient safety, these findings can reorient practices and help to set up new guidelines for HAI control.

Compound Heterozygous Familial Hypercholesterolemia Caused by LDLR Variants. / Portador Heterozigoto Composto de Hipercolesterolemia Familiar Causada por Variantes no LDLR.

Familial hypercholesterolemia (FH) is a genetic disease caused by a primary defect in the LDL-receptor gene. Distinct variants in the same gene characterize a compound heterozygote, but little is known about the phenotypes of the carriers. Therefore, herein, we describe the cascade screening of a Brazilian family with this characteristic. The index case, a 36-year-old male, had a total cholesterol level of 360 mg/dL (9.3 mmol/L) and LDL-c value of 259 mg/dL (6.7 mmol/L), in addition to Achilles tendon xanthomas, obesity and prehypertension. Genotyping identified the variants 661G>A, 670G>A, 682G>A in exon 4 and 919G>A in exon 6. The same variant in exon 4 was found in the index case's son (7-y), who also had hypercholesterolemia and xanthomas, while the index case's daughter (9-y) had the variant in exon 6 and hyperlipidemia, without xanthomas. In summary, this report allows for a better insight into the molecular basis of FH in Brazil, a multi-racial country where a heterogeneous population is expected.

A hipercolesterolemia familiar (HF) é uma doença genética causada por um defeito primário no gene que codifica o receptor da LDL. Mutações diferentes no mesmo gene caracterizam um heterozigoto composto, mas pouco se sabe sobre o fenótipo dos portadores. Portanto, neste estudo, descrevemos o rastreamento em cascata de uma família brasileira com essa característica. O caso-índice é um homem de 36 anos, com colesterol total (CT) de 360 mg/dL (9,3 mmol/L) e concentração de LDL-c de 259 mg/dL (6,7 mmol/L), além de xantomas de tendão de Aquiles, obesidade e pré-hipertensão. A genotipagem identificou as mutações 661G>A, 670G>A e 682G>A, no exon 4, e 919G>A, no exon 6. A mesma mutação no exon 4 foi observada no filho do caso-índice (7 anos), que também tem hipercolesterolemia e xantomas tendinosos, ao passo que a filha do caso-índice (9 anos) apresenta mutação no exon 6 e hiperlipidemia, sem xantomas. Em suma, este relato permite uma melhor compreensão acerca da base molecular da HF no Brasil, um país multirracial, onde é esperada uma população heterogênea.

Genomic Analysis of SXT/R391 Integrative Conjugative Elements From Proteus mirabilis Isolated in Brazil.

Integrative conjugative elements (ICEs) are widespread in many bacterial species, often carrying antibiotic resistance determinants. In the present work, we screened a collection of Proteus mirabilis clinical isolates for the presence of type 1 SXT/R391 ICEs. Among the 76 isolates analyzed, 5 of them carry such elements. The complete sequences of these elements were obtained. One of the isolates carried the CMY-2 beta-lactamase gene in a transposon and is nearly identical to the element ICEPmiJpn1 previously described in Japan, and later shown to be present in other parts of the world, indicating global spread of this element. Nevertheless, the Brazilian isolate carrying ICEPmiJpn1 is not clonally related to the other lineages carrying the same element around the world. The other ICEs identified in this work do not carry known antibiotic resistance markers and are diverse in variable gene content and size, suggesting that these elements may be responsible for the acquisition of other advantageous traits by bacteria. Some sequences carried by these elements in Brazilian strains were not previously found in other SXT/R391 variants.

IncX4 Plasmid-Mediated mcr-1.1 in Polymyxin-Resistant Escherichia coli from Outpatients in Santa Catarina, Southern Brazil.

Plasmid-mediated polymyxin resistance has become a global health concern, not only because its dissemination has occurred drastically but also because it has begun to be reported in multidrug-resistant (MDR) pathogens. We hereby report microbiological and genomic characteristics of two mcr-1.1-positive polymyxin-resistant Escherichia coli isolates identified for the first time in community patients, in Santa Catarina, Southern Brazil. E. coli strains belonging to ST206 and ST354 and the resistome analysis revealed the presence of clinically important genes responsible for MDR profile. Interestingly, in both polymyxin-resistant E. coli strains, mcr-1.1 genes were carried by IncX4 plasmids, responsible for the worldwide dissemination of mcr-type genes. In this regard, plasmid backbones were almost identical to the first IncX4 plasmid reported in Brazil and sharing more than 99.9% identity to IncX4 plasmids from China, also lacking the ISApl1 insertion sequence upstream of mcr-1. In conclusion, these data confirm the presence of international ST206 and ST354 carrying mcr-1.1 genes and that the IncX4 plasmids have been key vectors contributing to the endemic status of mcr-1.1-positive polymyxin-resistant E. coli in Brazil. Also, we described the first known clinical isolate with the mrc1.1 gene in Santa Catarina state, Brazil, showing that plasmid-mediated polymyxin resistance has been affecting humans earlier than has been known so far.

Characterization of Trypanosoma cruzi isolated from humans, vectors, and animal reservoirs following an outbreak of acute human Chagas disease in Santa Catarina State, Brazil.

During March 2005, 24 cases of acute human Chagas disease were detected in Santa Catarina State, southern Brazil, all of them related to the ingestion of Trypanosoma cruzi-contaminated sugar cane juice. Following field studies allowed the isolation of 13 T. cruzi strains from humans, opossums (Didelphis aurita and Didelphis albiventris), and vectors (Triatoma tibiamaculata). The isolated strains were characterized by multilocus enzyme electrophoresis (MLEE) and analysis of the spliced-leader and 24Salpha rRNA genes. The assays revealed that all strains isolated from humans belong to the TcII group but revealed a TcII variant pattern for the phosphoglucomutase enzyme. Strains isolated from opossums also showed a TcI profile in all analysis, but strains isolated from triatomines revealed a mixed TcI/TcII profile by MLEE. No indication of the presence of Trypanosoma rangeli was observed in any assay. Considering that mixed strains (TcI/TcII) were isolated from triatomines in an area without active vectorial transmission to humans and that all strains isolated from humans belong to the TcII group, our results show that T. cruzi TcI and TcII groups are circulating among reservoirs and vectors in southern Brazil and indicate that selection toward TcII group in humans may occur after ingestion of a mixed (TcI/TcII) T. cruzi population.

Cloning a new cytochrome P450 isoform (CYP356A1) from oyster Crassostrea gigas.

We have cloned the full-length cDNA of the first member of a new cytochrome P450 (CYP) family from the Pacific oyster Crassostrea gigas. This new CYP gene was obtained based on an initial 331bp fragment previously identified among the list of the differentially expressed genes in oysters exposed to untreated domestic sewage. The full-length CYP has an open reading frame of 1500bp and based on its deduced amino acid sequence was classified as a member of a new subfamily, CYP356A1. A phylogenetic analysis showed that CYP356A1 is closely related to members of the CYP17 and CYP1 subfamilies. Semi-quantitative RT-PCR was performed to analyze the CYP356A1 expression in different tissues of the oyster (digestive gland, gill, mantle and adductor muscle). Results showed slightly higher CYP356A1 expression in digestive gland and mantle, than the other tissues, indicating a possible role of the CYP356A1 in xenobiotic biotransformation and/or steroid metabolism.

Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1(-) strains and the ancestry of the Amazonian group.

Assessment of the genetic variability and population structure of Trypanosoma rangeli, a non-pathogenic American trypanosome, was carried out through microsatellite and single-nucleotide polymorphism analyses. Two approaches were used for microsatellite typing: data mining in expressed sequence tag /open reading frame expressed sequence tags libraries and PCR-based Isolation of Microsatellite Arrays from genomic libraries. All microsatellites found were evaluated for their abundance, frequency and usefulness as markers. Genotyping of T. rangeli strains and clones was performed for 18 loci amplified by PCR from expressed sequence tag/open reading frame expressed sequence tags libraries. The presence of single-nucleotide polymorphisms in the nuclear, multi-copy, spliced leader gene was assessed in 18 T. rangeli strains, and the results show that T. rangeli has a predominantly clonal population structure, allowing a robust phylogenetic analysis. Microsatellite typing revealed a subdivision of the KP1(-) genetic group, which may be influenced by geographical location and/or by the co-evolution of parasite and vectors occurring within the same geographical areas. The hypothesis of parasite-vector co-evolution was corroborated by single-nucleotide polymorphism analysis of the spliced leader gene. Taken together, the results suggest three T. rangeli groups: (i) the T. rangeli Amazonian group; (ii) the T. rangeli KP1(-) group; and (iii) the T. rangeli KP1(+) group. The latter two groups possibly evolved from the Amazonian group to produce KP1(+) and KP1(-) strains.

Portador Heterozigoto Composto de Hipercolesterolemia Familiar Causada por Variantes no LDLR / Compound Heterozygous Familial Hypercholesterolemia Caused by LDLR Variants

Resumo A hipercolesterolemia familiar (HF) é uma doença genética causada por um defeito primário no gene que codifica o receptor da LDL. Mutações diferentes no mesmo gene caracterizam um heterozigoto composto, mas pouco se sabe sobre o fenótipo dos portadores. Portanto, neste estudo, descrevemos o rastreamento em cascata de uma família brasileira com essa característica. O caso-índice é um homem de 36 anos, com colesterol total (CT) de 360 mg/dL (9,3 mmol/L) e concentração de LDL-c de 259 mg/dL (6,7 mmol/L), além de xantomas de tendão de Aquiles, obesidade e pré-hipertensão. A genotipagem identificou as mutações 661G>A, 670G>A e 682G>A, no exon 4, e 919G>A, no exon 6. A mesma mutação no exon 4 foi observada no filho do caso-índice (7 anos), que também tem hipercolesterolemia e xantomas tendinosos, ao passo que a filha do caso-índice (9 anos) apresenta mutação no exon 6 e hiperlipidemia, sem xantomas. Em suma, este relato permite uma melhor compreensão acerca da base molecular da HF no Brasil, um país multirracial, onde é esperada uma população heterogênea.

Abstract Familial hypercholesterolemia (FH) is a genetic disease caused by a primary defect in the LDL-receptor gene. Distinct variants in the same gene characterize a compound heterozygote, but little is known about the phenotypes of the carriers. Therefore, herein, we describe the cascade screening of a Brazilian family with this characteristic. The index case, a 36-year-old male, had a total cholesterol level of 360 mg/dL (9.3 mmol/L) and LDL-c value of 259 mg/dL (6.7 mmol/L), in addition to Achilles tendon xanthomas, obesity and prehypertension. Genotyping identified the variants 661G>A, 670G>A, 682G>A in exon 4 and 919G>A in exon 6. The same variant in exon 4 was found in the index case's son (7-y), who also had hypercholesterolemia and xanthomas, while the index case's daughter (9-y) had the variant in exon 6 and hyperlipidemia, without xanthomas. In summary, this report allows for a better insight into the molecular basis of FH in Brazil, a multi-racial country where a heterogeneous population is expected.

Antimutagenic and antiherpetic activities of different preparations from Uncaria tomentosa (cat's claw).

Uncaria tomentosa have been used to treat viral diseases such as herpes due to multiple pharmacological effects, but its therapeutic efficacy against this virus have not been reported yet. Thus, in vitro antiherpetic activity of hydroethanolic extract from barks, purified fractions of quinovic acid glycosides and oxindole alkaloids was evaluated by plaque reduction assay, including mechanistic studies (virucidal, attachment and penetration action). Once exposure to physical agents might lead to reactivation of the herpetic infection, antimutagenic effect (pre-, simultaneous and post-treatment protocols) was also evaluated by Comet assay. The antiherpetic activity from the samples under investigation seemed to be associated with the presence of polyphenols or their synergistic effect with oxindole alkaloids or quinovic acid glycosides, once both purified fractions did not present activity when evaluated alone. Inhibition of viral attachment in the host cells was the main mechanism of antiviral activity. Although both purified fractions displayed the lowest antimutagenic activity in pre and simultaneous treatment, they provided a similar effect to that of cat's claw hydroethanolic extract in post-treatment. Given that purified fractions may result in a reduced antiherpetic activity, the use of cat's claw hydroethanolic extract from barks should be prioritized in order to obtain a synergistic effect.