[Perinatal Outcome in Relation to Chorionicity in Twin Pregnancy]. / Impacto da Corionicidade nas Complicações Perinatais da Gestação Gemelar.

INTRODUCTION: The incidence of multiple gestations is increasing worldwide and many studies have shown higher perinatal morbidity and mortality rates in monochorionic twins compared to dichorionic. The aim of this study was to assess the twin population born at a tertiary center and to evaluate the impact of chorionicity on perinatal outcomes of twin pregnancies. MATERIAL AND METHODS: Retrospective study of all twins born in a tertiary center from January 2004 to December 2013. RESULTS: In this period, 1051 twins were born, related to 540 gestations (26.7% monochorionic; 73.3% dichorionic). There was no statistical significant difference between the groups concerning obstetric complications. The monochorionic group had a higher incidence of intrauterine growth restriction (20.5 vs 11.3%, p < 0.001), lower mean maternal age (29.9 vs 31.9 years, p < 0.001), lower mean gestational age (33.4 vs 34.3 weeks, p < 0.05) and lower mean birth weight (1943 vs 2147 g, p < 0.001). Monochorionic twins had a higher incidence of hyaline membrane disease (7 vs 4%, p < 0.05), sepsis (10.3 vs 5.8%, p < 0.05) and anemia (9.5 vs 5.4%, p < 0.05). There were no statistical significant differences concerning necrotizing enterocolitis, intraperiventricular hemorrhage or retinopathy of prematurity. Perinatal mortality was higher in the monochorionic group (5.2 vs 2.9%, p < 0.05). DISCUSSION: Monochorionic twins represent considerable challenges to both obstetricians and neonatologists and should be monitored and delivered at tertiary centers. CONCLUSION: Currently gemelarity has a major impact on total births. It would be interesting to develop protocols to standardize clinical approach to twins.

Introdução: A incidência da gestação múltipla tem vindo a aumentar em todo o mundo e vários estudos têm demonstrado taxas de morbilidade e mortalidade mais elevadas nos gémeos monocoriónicos comparativamente com os bicoriónicos. Os objetivos deste trabalho foram: caracterizar a população de gémeos fruto de gravidez bifetal nascidos num hospital nível três e avaliar o impacto da corionicidade na morbimortalidade perinatal.Material e Métodos: Estudo retrospetivo de todos os gémeos fruto de gravidez bifetal nascidos num hospital nível três entre janeiro de 2004 e dezembro de 2013.Resultados: Neste período nasceram 1051 gémeos, fruto de 540 gestações (26,7% monocoriónicos; 73,3% bicoriónicos). Não houve diferença estatisticamente significativa entre os dois grupos no respeitante às complicações obstétricas. No grupo monocoriónico verificou-se uma incidência mais elevada de restrição do crescimento intra-uterino (20,5 vs 11,3%, p < 0,001), idade materna mais baixa (29,9 vs 31,9 anos, p < 0,001), idade gestacional mais baixa (33,4 vs 34,3 semanas, p < 0,05) e peso de nascimento mais baixo (1943 vs 2147 g, p < 0,001). Os gémeos monocoriónicos tiveram uma incidência mais elevada de doença de membrana hialina (7 vs 4%, p < 0,05), sépsis (10,3 vs 5,8%, p < 0,05) e anemia (9,5 vs 5,4%, p < 0,05). Não se encontrou diferença estatisticamente significativa relativamente à ocorrência de enterocolite necrotizante, hemorragia intraperiventricular ou retinopatia da prematuridade. A mortalidade perinatal foi mais elevada no grupo monocoriónico (5,2 vs 2,9%, p < 0,05).Discussão: Os gémeos monocoriónicos representam um desafio para obstetras e neonatologistas, devendo a vigilância pré-natal e o parto ser realizados em centros de referência.Conclusão: A gemelaridade tem atualmente um importante impacto nos nascimentos, pelo que seria interessante desenvolver protocolos que uniformizassem a prática clínica na abordagem a estes recém-nascidos.

N-glycosylation influences the catalytic activity of mosquito α-glucosidases associated with susceptibility or refractoriness to Lysinibacillus sphaericus.

Cqm1 and Aam1 are α-glucosidases (EC 3.2.1.20) expressed in Culex quinquefasciatus and Aedes aegypti larvae midgut, respectively. These orthologs share high sequence similarity but while Cqm1 acts as a receptor for the Binary (Bin) insecticidal toxin from Lysinibacillus sphaericus, Aam1 does not bind the toxin, rendering Ae. aegypti refractory to this bacterium. Aam1 is heavily glycosylated, contrasting to Cqm1, but little is known regarding how glycosylation impacts on its function. This study aimed to compare the N-glycosylation patterns and the catalytic activities of Aam1 and Cqm1. Mutant proteins were generated where predicted Aam1 N-glycosylation sites (N-PGS) were either inserted into Cqm1 or abrogated in Aam1. The mutants validated four N-PGS which were found to localize externally on the Aam1 structure. These Aam1 and Cqm1 mutants maintained their Bin binding properties, confirming that glycosylation has no role in this interaction. The α-glucosidase activity of both proteins was next investigated, with Aam1 having a remarkably higher catalytic efficiency, influenced by changes in glycosylation. Molecular dynamics showed that glycosylated and nonglycosylated Aam1 models displayed distinct patterns that could influence their catalytic activity. Differential N-glycosylation may then be associated with higher catalytic efficiency in Aam1, enhancing the functional diversity of related orthologs.

Non conserved residues between Cqm1 and Aam1 mosquito α-glucosidases are critical for the capacity of Cqm1 to bind the Binary toxin from Lysinibacillus sphaericus.

The Binary (Bin) toxin from the entomopathogenic bacterium Lysinibacillus sphaericus acts on larvae of the culicid Culex quinquefasciatus through its binding to Cqm1, a midgut-bound α-glucosidase. Specific binding by the BinB subunit to the Cqm1 receptor is essential for toxicity however the toxin is unable to bind to the Cqm1 ortholog from the refractory species Aedes aegypti (Aam1). Here, to investigate the molecular basis for the interaction between Cqm1 and BinB, recombinant Cqm1 and Aam1 were first expressed as soluble forms in Sf9 cells. The two proteins were found to display the same glycosilation patterns and BinB binding properties as the native α-glucosidases. Chimeric constructs were then generated through the exchange of reciprocal fragments between the corresponding cqm1 and aam1 cDNAs. Subsequent expression and binding experiments defined a Cqm1 segment encompassing residues S129 and A312 as critical for the interaction with BinB. Through site directed mutagenesis experiments, replacing specific sets of residues from Cqm1 with those of Aam1, the 159GG160 doublet was required for this interaction. Molecular modeling mapped these residues to an exposed loop within the Cqm1's structure, compatible with a target site for BinB and providing a possible explanation for its lack of binding to Aam1.

Modo de ação do biolarvicida Lysinibacillus sphaericus: identificação do epitopo da toxina Binária no receptor Cqm1 e base molecular da seletividade / Mode of action of biolarvicide Lysinibacillus sphaericus: identification of the epitope of the CQM1 Binary toxin receptor and molecular basis of selectivity

A toxina Binária (Bin) é o principal fator tóxico da bactéria entomopatógena Lysinibacillus sphaericus e sua ação em Culex quinquefasciatus depende da ligação com receptores no intestino das larvas. Os receptores são as a-glicosidases Cqm1, localizadas no epitélio, ligadas por uma âncora de glicosil-fosfatidilinositol. Larvas de Aedes aegypti são refratárias à toxina, pois, não apresentam receptores funcionais, apesar de apresentarem um gene que codifica a proteína Aam1, com alta similaridade à Cqm1. Devido às lacunas a respeito do espectro de ação da toxina Bin, o objetivo deste estudo foi identificar epitopos de ligação da toxina no receptor Cqm1 e determinar a base molecular da sua ação para estas espécies de vetores. Os resultados obtidos a partir da análise comparativa das proteínas Cqm1 e Aam1 levaram à identificação de um epitopo da toxina Bin no receptor Cqm1, situado uma alça na região N-terminal S129-A312. Este epitopo é composto pelos aminoácidos 155PATGGG160, não conservados em Aam1 (158AETGKL163), e os resíduos 159GG160 são críticos para a ligação com a Bin...

The Bin toxin is the main toxic factor of the bacterium Lysinibacillus sphaericuswhose action in Culex quinquefasciatuslarvae depends on its binding to the midgut epithelial receptors...

Novel mutations associated with resistance to Bacillus sphaericus in a polymorphic region of the Culex quinquefasciatus cqm1 gene.

Bin toxin from Bacillus sphaericus acts on Culex quinquefasciatus larvae by binding to Cqm1 midgut-bound receptors, and disruption of the cqm1 gene is the major cause of resistance. The goal of this work was to screen for a laboratory-selected resistance cqm1(REC) allele in field populations in the city of Recife, Brazil, and to describe other resistance-associated polymorphisms in the cqm1 gene. The cqm1(REC) allele was detected in the four nontreated populations surveyed at frequencies from 0.001 to 0.017, and sequence analysis from these samples revealed a novel resistant allele (cqm1(REC-D16)) displaying a 16-nucletotide (nt) deletion which is distinct from the 19-nt deletion associated with cqm1(REC). Yet a third resistant allele (cqm1(REC-D25)), displaying a 25-nt deletion, was identified in samples from a treated area exposed to B. sphaericus. A comparison of the three deletion events revealed that all are located within the same 208-nt region amplified during the screening procedure. They also introduce equivalent frameshifts in the sequence and generate the same premature stop codon, leading to putative transcripts encoding truncated proteins which are unable to locate to the midgut epithelium. The populations analyzed in this study contained a variety of alleles with mutations disrupting the function of the corresponding Bin toxin receptor. Their locations reveal a hot spot that can be exploited to assess the resistance risk through DNA screening.

The orthologue to the Cpm1/Cqm1 receptor in Aedes aegypti is expressed as a midgut GPI-anchored α-glucosidase, which does not bind to the insecticidal binary toxin.

Aedes aegypti larvae are refractory to the insecticidal binary (Bin) toxin from Bacillus sphaericus, which is not able to bind to its target tissue in the larval midgut. In contrast, Culex pipiens larvae are highly susceptible to that toxin, which targets its midgut brush border membranes (BBMF) through the binding of the BinB subunit to specific receptors, the Cpm1/Cqm1 membrane-bound α-glucosidases. The identification of an Ae. aegypti gene encoding a Cpm1/Cqm1 orthologue, here named Aam1, led to the major goal of this study which was to investigate its expression. The aam1 transcript was found in larvae and adults from Ae. aegypti and a ≈73-kDa protein was recognized by an anti-Cqm1 antibody in midgut BBMF. The Aam1 protein displayed α-glucosidase activity and localized to the midgut epithelium, bound through a GPI anchor, similarly to Cpm1/Cqm1. However, no binding of native Aam1 was observed to the recombinant BinB subunit. Treatment of both proteins with endoglycosidase led to changes in the molecular weight of Aam1, but not Cqm1, implying that the former was glycosylated. The findings from this work rule out lack of receptors in larval stages, or its expression as soluble proteins, as a reason for Ae. aegypti refractoriness to Bin toxin.

Análise da refratariedade do Aedes aegypti à toxina binária do biolarvicida Bacillus sphaericus / Analysis of Aedes aegypti refractoriness to Bin toxin of biolarvicide Bacillus sphaericus

O principal fator larvicida do Bacillus sphaericus (Bsp) para culicídeos é a protoxina Bin, produzida sob a forma de um cristal, durante a esporulação. Quando ingerido pelas larvas o cristal é processado e a toxina Bin reconhece e liga-se a receptores específicos do epitélio intestinal. O receptor em Culex quinquefasciatus é uma alfa-glicosidase de 60 kDa, ligada à membrana intestinal por uma âncora GPI, denominado Cqm1. Larvas de Aedes aegypti são consideradas refratárias ao Bsp, pois a toxina Bin não reconhece receptores no microvilli intestinal. No entanto, a análise do genoma do Ae. aegypti, revelou a presença do gene aam1, que codificaria uma proteína ortóloga e com 83 por cento de similaridade ao receptor Cqm1. O principal objetivo deste estudo foi elucidar a base molecular da refratariedade do Ae. aegypti ao Bsp, determinada pela ausência de ligação da toxina Bin ao epitélio intestinal das larvas. Para tal, foi feita uma investigação da expressão da proteína Aam1 e do perfil de alfa-glicosidases de Ae. aegypti, tendo como referência o receptor Cqm1. Os resultados mostraram que larvas e adultos de Ae. aegypti expressam uma alfa-glicosidase de membrana de 70 kDa, reconhecida pelo anticorpo anti-Cqm1, e que provavelmente trata-se da proteína Aam1. Tal proteína é expressa no microvilli intestinal das larvas em níveis superiores à Cqm1, no entanto, não apresenta capacidade de ligação à toxina Bin. Em uma segunda etapa, a avaliação de proteínas Aam1 e Cqm1 recombinantes, produzidas em lisado de reticulócitos de coelho, mostrou que ambas não foram capazes de se ligar específicamente à toxina Bin. A falha na ligação da proteína Cqm1 à toxina Bin pode ser decorrente da ausência do processamento pós-traducional adequado neste sistema de expressão, indicando que certas modificações podem ser críticas para a sua funcionalidade. O tratamento da proteína Cqm1 nativa à temperatura de 100 °C aboliu a sua capacidade de ligação à toxina Bin, indicando que a conformação da proteína pode ser essencial para a sua funcionalidade. Os resultados obtidos demonstram que, apesar dos altos níveis de expressão da Aam1 nas larvas de Ae. aegypti, a proteína não é capaz de ligar-se à toxina Bin. Tal fato estar relacionado a outros fatores críticos para sua funcionalidade, tais como diferenças conformacionais e/ou modificações pós-traducionais que determinem o status de refratariedade do Ae. aegypti