International live insect trade: a survey of stakeholders.

There are significant numbers of transboundary shipments of live insects for pollination, pest management, industrial processes, research and other uses, but data collection and analysis have proved difficult. The World Organisation for Animal Health and Collectif TIS (Technique de l'Insecte Stérile), a French think tank, carried out a stakeholder survey to understand the nature of the live insect trade and potential challenges to safety and efficiency. Target respondents had experience in the areas of biocontrol, sterile insect technique, entomological research and regulatory affairs. Although the survey was sent globally, the responses were unintentionally biased towards Europe, where interest is high, since this region is developing a comprehensive framework to promote the use of beneficial insects to replace pesticides. The survey also explored respondents' knowledge of several international agreements on the movement and risk management of beneficial or invasive insects. Knowledge of the various regulations was generally poor, and respondents highlighted a perceived lack of clarity regarding live insect shipments in the existing international regulations and guidelines. Almost two-thirds of participants reported reluctance by carriers to accept live insects for shipment, and three-quarters described occasional to systematic delays that resulted in a reduction of quality or viability. Some respondents reported that they instead hand-carry live insects, mostly in small quantities. Participants described being directly involved in trade covering 70 species of live insects and ticks transported among 37 countries, with volumes ranging from fewer than ten insects to over a million per shipment. Of these, 30% were potential vectors of pathogens to humans or animals, 42% were potential plant pest species (including some used for biocontrol), and 17% were classical biocontrol agents. The results of this survey begin to define the current scope, scale and issues for those involved in shipping live insects and ticks across political boundaries. The survey's aim is to persuade regulatory bodies and shipping operators to facilitate safety, efficiency and consistency in this underdeveloped sector.

Les insectes vivants font l'objet d'un nombre important d'expéditions transfrontalières à des fins de pollinisation, de gestion des nuisibles, d'utilisation dans des processus industriels et de recherche, parmi d'autres emplois ; or, la collecte et l'analyse de données sur le sujet se sont révélées difficiles. L'Organisation mondiale de la santé animale et le Collectif TIS (Technique de l'insecte stérile), groupe de réflexion français, ont mené une enquête auprès de parties prenantes afin de comprendre la nature du commerce international d'insectes vivants ainsi que les éventuels problèmes de sécurité et d'efficacité qui lui sont associés. Les répondants sont des acteurs expérimentés dans les domaines du contrôle biologique, de la technique de l'insecte stérile, de la recherche entomologique et des questions réglementaires. Si le questionnaire a été distribué dans toutes les régions du monde, les réponses ont toutefois présenté un biais involontaire privilégiant l'Europe, région où l'intérêt pour cette question est élevé compte tenu du cadre réglementaire qui y est actuellement mis en place pour promouvoir l'utilisation des insectes utiles en replacement des pesticides. L'enquête visait également à déterminer le niveau de connaissance des répondants sur les divers accords internationaux relatifs aux mouvements d'insectes utiles ou envahissants, ainsi que sur la gestion des risques en la matière. Il en ressort que les diverses réglementations sont généralement méconnues, les répondants mettant en avant leur perception d'un manque de clarté des réglementations et directives internationales applicables aux expéditions d'insectes vivants. Près des deux tiers des participants ont fait état des réticences des transporteurs à accepter des cargaisons d'insectes vivants et les trois quarts d'entre eux ont rapporté des retards occasionnels ou systématiques se traduisant par une perte de qualité ou de viabilité. Certains répondants ont déclaré qu'ils préféraient transporter eux-mêmes les insectes vivants, la plupart du temps en petites quantités. Les répondants ont décrit leur participation directe dans des échanges couvrant au total 70 espèces de tiques et d'insectes vivants transportés dans 37 pays, avec des volumes allant de moins de dix insectes à plus d'un million d'insectes par cargaison. Parmi les espèces transportées, 30 % étaient potentiellement vectrices d'agents pathogènes pour les humains ou les animaux, 42 % étaient des nuisibles potentiels pour les végétaux (y compris certaines espèces utilisées à des fins de lutte biologique) et 17 % étaient des agents classiques de la lutte biologique. Les résultats de cette enquête permettent d'esquisser la portée et l'échelle des expéditions transfrontalières d'insectes vivants et de tiques, ainsi que la teneur des problèmes rencontrés par ceux qui y prennent part. L'objectif de l'enquête est de convaincre les organismes chargés de la réglementation ainsi que les transporteurs internationaux de la nécessité d'apporter de meilleures conditions de sécurité et d'efficacité à ce secteur sous-développé et d'en assurer la cohérence.

Pese al importante número de envíos transfronterizos de insectos vivos que se realizan con fines de polinización, gestión de plagas, procesos industriales, investigación u otros usos, hasta ahora ha resultado difícil reunir y analizar datos al respecto. La Organización Mundial de Sanidad Animal y el grupo de reflexión francés Collectif TIS (Technique de l'Insecte Stérile) hicieron una encuesta entre las partes interesadas con el fin de conocer mejor las características del comercio de insectos vivos y los factores que pueden amenazar su seguridad y eficacia. La encuesta iba dirigida a personas con experiencia en materia de control biológico, uso de la técnica del insecto estéril, investigaciones entomológicas o aspectos reglamentarios. Aunque la encuesta fue distribuida por todo el mundo, las respuestas presentaban un involuntario sesgo «europeo¼ debido al gran interés que el tema suscita en Europa, donde se está elaborando un marco reglamentario para promover el uso de insectos beneficiosos en sustitución de los plaguicidas. La encuesta indagaba asimismo en el grado de conocimiento que tenían los participantes de varios acuerdos internacionales relativos al desplazamiento de insectos beneficiosos o invasores y a la gestión de los riesgos conexos. En términos generales, los encuestados conocían poco las diversas reglamentaciones y destacaban lo que a su juicio era falta de claridad de las vigentes normativas y directrices internacionales por lo que respecta a los cargamentos de insectos vivos. Casi dos tercios de los participantes afirmaron que los transportistas eran reacios a aceptar cargamentos de insectos vivos y tres cuartas partes refirieron demoras ocasionales o sistemáticas que mermaban la calidad o viabilidad de las remesas. Algunos dijeron haber optado a cambio por el transporte manual de insectos vivos, casi siempre en pequeñas cantidades. Los encuestados afirmaron tomar parte directamente en un comercio que mueve 70 especies de insectos o ácaros vivos por 37 países, en cantidades que van desde menos de diez insectos a más de un millón de ejemplares por envío. De esas especies, un 30% correspondía a posibles vectores de patógenos humanos o animales, un 42% a posibles plagas vegetales (algunas de ellas utilizadas con fines de control biológico) y un 17% a agentes clásicos de control biológico. Los resultados permiten empezar a acotar el alcance, la escala y la naturaleza de los problemas a que se enfrentan quienes intervienen en el envío transfronterizo de insectos y ácaros vivos. La encuesta tiene por objetivo convencer a organismos de reglamentación y transportistas de que procuren fomentar la seguridad, eficiencia y coherencia de este infradesarrollado sector.

Can there be a common, risk-based framework for decisions around live insect trade?

A network of scientists involved in shipment of live insects has met and generated a series of articles on issues related to live insect transport. The network is diverse, covering large-scale commercial interests, government operated areawide control programmes, biomedical research and many smaller applications, in research, education and private uses. Many insect species have a record of safe transport, pose minimal risks and are shipped frequently between countries. The routine shipments of the most frequently used insect model organism for biomedical research, Drosophila melanogaster, is an example. Successful large scale shipments from commercial biocontrol and pollinator suppliers also demonstrate precedents for low-risk shipment categories, delivered in large volumes to high quality standards. Decision makers need access to more information (publications or official papers) that details actual risks from the insects themselves or their possible contaminants, and should propose proportionate levels of management. There may be harm to source environments when insects are collected directly from the wild, and there may be harm to receiving environments. Several risk frameworks include insects and various international coordinating bodies, with experience of guidance on relevant risks, exist. All stakeholders would benefit from an integrated overview of guidance for insect shipping, with reference to types of risk and categories of magnitude, without trying for a single approach requiring universal agreement. Proposals for managing uncertainty and lack of data for smaller or infrequent shipments, for example, must not disrupt trade in large volumes of live insects, which are already supporting strategic objectives in several sectors.

À l'occasion d'une réunion d'experts, un réseau de chercheurs travaillant sur l'expédition d'insectes vivants a produit un ensemble d'articles traitant des questions liées au transport des insectes vivants. Ce réseau est diversifié et représente un large éventail d'intérêts privés et de programmes de lutte biologique à grande échelle menés par les pouvoirs publics, en plus du secteur de la recherche biomédicale et de nombreux acteurs intervenant dans des applications de plus petite envergure relevant de la recherche, de l'enseignement ou du secteur privé. Un grand nombre d'espèces d'insectes sont transportées en toute sécurité avec un niveau de risque minimal, y compris lors des fréquentes expéditions internationales. Un exemple de ces expéditions régulières concerne l'espèce la plus utilisée par la recherche biomédicale en tant qu'organisme modèle, à savoir Drosophila melanogaster. Les exemples réussis d'expéditions à grande échelle provenant de fournisseurs d'agents de lutte biologique et de pollinisateurs produits par le secteur privé offrent également un relevé documenté des diverses catégories d'expéditions à faible risque pour des livraisons d'insectes vivants en grandes quantités et répondant à des normes de qualité élevées. Les décideurs politiques devraient pouvoir accéder à plus d'informations (à travers des publications ou des articles officiels) décrivant en détail les risques réels associés aux insectes eux-mêmes ou à leurs contaminants éventuels, et proposer en connaissance de cause des niveaux de gestion proportionnels à ces risques. La récolte d'insectes prélevés directement de la nature peut être dommageable aussi bien pour les environnements source que pour ceux de destination. Plusieurs cadres fondés sur le risque intègrent désormais les insectes dans leurs directives. Par ailleurs, nombre d'organismes internationaux de coordination ont acquis une expérience dans l'élaboration de lignes directrices face à ces risques. Il serait bénéfique pour toutes les parties prenantes de disposer d'une vue d'ensemble intégrée des directives applicables aux expéditions d'insectes, qui recense les différents types de risque et leurs ordres de grandeur sans se prononcer sur une approche unique qui nécessiterait une adhésion universelle. Ni les propositions visant à gérer l'incertitude ni l'insuffisance des données disponibles sur les expéditions d'insectes en petits nombres ou occasionnelles ne doivent perturber les échanges commerciaux d'insectes vivants en grandes quantités, échanges qui participent aujourd'hui aux objectifs stratégiques de nombreux secteurs.

Una red de científicos relacionados de un modo u otro con el transporte de insectos vivos mantuvo un encuentro y generó una serie de artículos en torno a la cuestión. En la red, muy heterogénea, convergen desde intereses comerciales de gran calado hasta programas públicos de lucha biológica en grandes territorios, pasando por la investigación biomédica y por numerosas aplicaciones de menor dimensión en ámbitos como la investigación, la enseñanza u otros usos privados. Muchas especies de insectos cuentan con un buen historial de seguridad en el transporte, presentan un riesgo mínimo y son expedidas a menudo de un país a otro. Buen ejemplo de ello son los envíos sistemáticos de ejemplares de Drosophila melanogaster, que es el insecto utilizado con más frecuencia como organismo modelo en la investigación biomédica. Otro precedente de envíos voluminosos cuyo transporte y entrega se ajusta a los más exigentes criterios de calidad lo sientan las remesas de grandes cantidades de polinizadores y agentes de control biológico remitidos por proveedores comerciales. Los responsables de adoptar decisiones deben disponer de más y más detallada información (publicaciones o documentos oficiales) sobre los riesgos reales derivados de los propios insectos o sus posibles contaminantes para proponer a partir de ahí medidas de gestión proporcionadas. Los perjuicios ambientales pueden darse tanto en origen (cuando hay captura salvaje de insectos, o sea recolección directa en el medio) como en destino. Hay varios sistemas de determinación del riesgo que incluyen a los insectos y también existen diversos organismos de coordinación internacional que ya tienen experiencia en sentar pautas sobre los riesgos en la materia. Todos los interlocutores del sector se beneficiarían de una visión global e integrada del transporte de insectos, que remita a diferentes tipos de riesgo y categorías de magnitud, sin necesidad de buscar un sistema único que exija consenso universal. Las propuestas para gestionar la incertidumbre y la falta de información en el caso de envíos infrecuentes o de pequeñas dimensiones, por ejemplo, no deben interferir en el transporte de grandes cantidades de insectos vivos, que ya está sirviendo a objetivos estratégicos en varios sectores.

Sampling strategies for phlebotomine sand flies (Diptera: Psychodidae) in Europe.

The distribution of phlebotomine sand flies is widely reported to be changing in Europe. This can be attributed to either the discovery of sand flies in areas where they were previously overlooked (generally following an outbreak of leishmaniasis or other sand fly-related disease) or to true expansion of their range as a result of climatic or environmental changes. Routine surveillance for phlebotomines in Europe is localized, and often one of the challenges for entomologists working in non-leishmaniasis endemic countries is the lack of knowledge on how to conduct, plan and execute sampling for phlebotomines, or how to adapt on-going sampling strategies for other haematophagous diptera. This review brings together published and unpublished expert knowledge on sampling strategies for European phlebotomines of public health concern in order to provide practical advice on: how to conduct surveys; the collection and interpretation of field data; suitable techniques for the preservation of specimens obtained by different sampling methods; molecular techniques used for species identification; and the pathogens associated with sand flies and their detection methods.

A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent 'hypnozoite' forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

PfMORC protein regulates chromatin accessibility and transcriptional repression in the human malaria parasite, P. falciparum.

The environmental challenges the human malaria parasite, Plasmodium falciparum, faces during its progression into its various lifecycle stages warrant the use of effective and highly regulated access to chromatin for transcriptional regulation. Microrchidia (MORC) proteins have been implicated in DNA compaction and gene silencing across plant and animal kingdoms. Accumulating evidence has shed light into the role MORC protein plays as a transcriptional switch in apicomplexan parasites. In this study, using CRISPR/Cas9 genome editing tool along with complementary molecular and genomics approaches, we demonstrate that PfMORC not only modulates chromatin structure and heterochromatin formation throughout the parasite erythrocytic cycle, but is also essential to the parasite survival. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments suggest that PfMORC binds to not only sub-telomeric regions and genes involved in antigenic variation but is also most likely a key modulator of stage transition. Protein knockdown experiments followed by chromatin conformation capture (Hi-C) studies indicate that downregulation of PfMORC induces the collapse of the parasite heterochromatin structure leading to its death. All together these findings confirm that PfMORC plays a crucial role in chromatin structure and gene regulation, validating this factor as a strong candidate for novel antimalarial strategies.

A Potent Kalihinol Analogue Disrupts Apicoplast Function and Vesicular Trafficking in P. falciparum Malaria.

Here we report the discovery of MED6-189, a new analogue of the kalihinol family of isocyanoterpene (ICT) natural products. MED6-189 is effective against drug-sensitive and -resistant P. falciparum strains blocking both intraerythrocytic asexual replication and sexual differentiation. This compound was also effective against P. knowlesi and P. cynomolgi. In vivo efficacy studies using a humanized mouse model of malaria confirms strong efficacy of the compound in animals with no apparent hemolytic activity or apparent toxicity. Complementary chemical biology, molecular biology, genomics and cell biological analyses revealed that MED6-189 primarily targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses in P. falciparum revealed that a mutation in PfSec13, which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. The high potency of MED6-189 in vitro and in vivo, its broad range of efficacy, excellent therapeutic profile, and unique mode of action make it an excellent addition to the antimalarial drug pipeline.

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.

A strong inhibitory element down-regulates SRE-stimulated transcription of the A3 cytoplasmic actin gene of Bombyx mori.

To identify the functional regulatory elements of the promoter of the cytoplasmic actin A3 gene in Bombyx mori, transient expression of A3-LacZ mutants was assayed in cultured Lepidoptera cells. This led to the recognition of two proximal and contiguous domains exerting strong negative and positive effects, respectively on promoter activity. The negative region contains a ten-base-pair sequence that binds Bombyx silk gland cell nuclear proteins in vitro. The positive regulatory element was identified as a serum response element (SRE) by its sequence, and its in vitro binding properties. Moreover, structural analysis of posterior and median silk gland cell chromatin by dimethyl sulfate-aided LMPCR revealed that SRE is bound to its cognate factor in situ, in most, if not all, the approximately 100,000 A3 copies of the polyploid DNA stock. The regulation of the A3 promoter in the silk gland would thus result from the combined action of these two antagonist factors.

Two alternative promoters drive the expression of the cytoplasmic actin A4 gene of Bombyx mori.

By screening cDNA and genomic libraries, we have cloned A4, the fourth and last actin gene of Bombyx mori, which encodes a typical cytoskeleton actin and is expressed in all larval tissues. A4 is closely related to A3, another cytoplasmic actin gene of the silkworm, in its encoded amino-acid sequence, and the location as well as the sequence of a single intron. Both A3 and A4 have possibly arisen from the recent duplication of an intron-containing ancestral gene. The two genes display different organization of their 5' untranslated and flanking sequences. In contrast to A3, which harbours a single promoter, A4 exhibits two leader exons transcribed by the use of alternative promoters. A3 and A4 actins differ only by two amino acids at positions known to vary among cytoplasmic actins of other species, and are likely to be functionally equivalent. We speculate that transcriptional constraints are actually the target of a selective pressure that maintains two distinct cytoplasmic actin genes in insects, as well as in other animals.

Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori.

Three alleles of the sericin (Ser) 2-encoding gene (Ser2), called L, C and mC, were isolated from a Bombyx mori genomic library, and two related ones, called mCL and Cv, were also characterized in B. mori European strains. The Ser2 gene gives rise to two middle silk gland mRNAs by differential splicing. The size of a short mRNA (3.1 kb) is constant, but the length of a longer one ranges from 5 to 6.4 kb depending on the Ser2 allele. These length variations probably result from unequal recombinations in a region which contains about 30 well conserved 45-bp repeats coding for a Ser-like peptide. Furthermore, the L allele (and probably the mCL one) contains a 4.4-kb retrotransposon, resembling the copia-like ones of Drosophila.

Differential transcription of multiple copies of a silk worm gene encoding tRNA(Gly1).

Ten different tRNA(Gly1) genes from the silk worm, Bombyx mori, have been cloned and characterized. These genes were transcribed in vitro in homologous nuclear extracts from the posterior silk gland (PSG) or nuclear extracts derived from the middle silk gland or ovarian tissues. Although the transcription levels were much higher in the PSG nuclear extracts, the transcriptional efficiency of the individual genes followed a similar pattern in all the extracts. Based on the levels of in vitro transcription, the ten tRNA(Gly1) genes could be divided into three groups, viz., those which were transcribed at very high levels (e.g., clone pR8), high to medium levels (e.g., pBmi1, pBmp1, pBmh1, pBmt1) and low to barely detectable levels (e.g., pBms1, pBmj1 and pBmk1). The coding sequences of all these tRNA genes being identical, the differential transcription suggested that the flanking sequences modulate their transcriptional efficiency. The presence of positive and negative regulatory elements in the 5' flanking regions of these genes was confirmed by transcription competition experiments. A positive element was present in the immediate upstream A+T-rich sequences in all the genes, but no consensus sequences correlating to the transcriptional status could be generated. The presence of negative elements on the other hand was indicated only in some of the genes and therefore may have a role in the differential transcription of these tRNA(Gly1) genes in vivo.

Isolation of actin genes in Bombyx mori: the coding sequence of a cytoplasmic actin gene expressed in the silk gland is interrupted by a single intron in an unusual position.

To study the regulation of the gene(s) coding for the actin present in the microfilaments involved in the secretion of silk, we have probed a Bombyx mori genomic library with a Drosophila actin cDNA clone and selected 16 recombinant phages. They correspond to 3 different genomic fragments each containing a distinct actin coding sequence. Southern blots of genomic DNA probed with the cloned genes show that in Bombyx mori, there are at least 5 different actin genomic sequences. Two cloned genes A1 and A2 hybridize to a 1.7 kb long mRNA abundant in the carcass of the larva and thus probably code for muscle type actin. The third cloned gene, A3, hybridizes to two mRNAs of about 1.8 kb present in the silk gland and thus probably encodes a cytoplasmic actin. The coding sequence of this gene has been sequenced: it is almost identical to the Drosophila cytoplasmic actin genes but it has a single intron of 92 nucleotides within the codon 116, a position not observed in any other organism.

The adaptation of the silkgland cell to the production of fibroin in Bombyx mori L.

At the end of the larval life, the posterior silk gland of Bombyx mori is highly specialized in the biosynthesis of a specific protein : silk fibroin. The successive steps of fibroin production : amino supply, synthesis and secretion are described. Their analysis shows that fibroin synthesis is important enough to orient the overall cellular activities. Thus, the terminal differentiation of the posterior silk gland cell corresponds to the cells adaptation to the production of fibroin. Cytological and biochemical studies of the silk gland development show that specialization occurs discontinuously; the fourth molt, when fibroin is no more produced, is a phase of regression of the cellular adaptation whereas cell differentiation proceeds during the growth phase of the following fifth intermolt. After the spinning of the cocoon, the cells are lysed and disappear entirely at the nymphal stage. Biometrical analysis of silk production of different Bombyx strains in relation with the development of the proteosynthesis apparatus leads to the conclusion that the specific messenger RNA content determines the amount of synthetized fibroin. At maximum secretion, the mRNAF recruits almost all the cell ribosomes. The variations of the size of the proteosynthesis machinery are sufficient to explain the differences of productivity of the various silkworm strains. Different experimental factors affect silk production. Topical applications of juvenile hormone induce an increase of the RNA content and a consequent rise of the amount of secreted protein. In contrast, starvation reduces the silk production by acting at both transcriptonal and translational levels. Current researches on this system are devoted to the study of the differential gene expression, with particular interest to the regulation of the transcription of the specific fibroin messenger RNA.

A single gene produces multiple sericin messenger RNAs in the silk gland of Bombyx mori.

The sericins are a family of major cocoon proteins specifically synthesized in the middle silk gland of the silkworm Bombyx mori. The 5' part of one sericin gene had been cloned and described by Okamoto et al. (1982, J. Biol. Chem. 257, 15192-15199). Using a differential screening procedure of Bombyx genomic libraries, we obtained the 3' part of this gene. We demonstrate that it consists of a single gene extending over 24 kb, present in two allelic forms in hybrid strains. This gene encodes for four mRNAs which result from a unique transcript by an alternative splicing mechanism. This explains, at least partially, the diversity of the sericins found in the cocoon.

A high capacity in vitro assay for measuring the cytoadherence of Plasmodium falciparum-infected erythrocytes.

A simple, efficient, sensitive, reproducible and high throughput assay for measuring the cytoadhesion of Plasmodium falciparum-infected red blood cells (human malaria) is described. The assay format uses 96-well microplates, with the number of P. falciparum parasitized erythrocytes bound determined by measuring Plasmodium specific lactic dehydrogenase activity colorimetrically (absorbance at 655 nm) using the 3-acetylpyridine analog of nicotinamide adenine dinucleotide, nitro blue tetrazolium and diaphorase. The results of the described microplate assay were found to be comparable to those using microscopic analysis but much less time consuming.

Studies of Plasmodium falciparum cytoadherence using immortalized human brain capillary endothelial cells.

The cytoadherence of Plasmodium falciparum-infected erythrocytes was studied using immortalized human brain capillary endothelial cells. The immortalized cells, denoted as BB19, derived from the human brain endothelium, were transformed with the E6E7 genes of human papilloma virus and retained their endothelial nature, i.e. tubule formation occurred with Matrigel as a substratum and the cells stained positive for Factor VIII-related antigen, or vonWillebrand's factor. Surface expression of ICAM-1, VCAM, E-selectin, and CD36 was demonstrated by immunofluorescence staining with monoclonal antibodies to these ligands. Exposure to cytokines (TNF, IFN gamma, IL-1 alpha, and IL-6) and lipopolysaccharide resulted in an increase in expression of ICAM-1, VCAM, E-selectin, and CD36. The BB19 cells bound P. falciparum-infected red blood cells with both the FCR-3 and the ITO4 strains. Antibodies to CD36 and ICAM-1 partially inhibited the binding of the FCR-3 and the ITO4 lines, respectively. These findings suggest that BB19 cells may be useful in the analysis of receptor-based cytoadherence and sequestration, as well as in the cell biology of microvessel formation.

Structure and organization of the Bombyx mori sericin 1 gene and of the sericins 1 deduced from the sequence of the Ser 1B cDNA.

The sericin 1 primary transcript of the silkworm Bombyx mori is differentially spliced via a tissue- and developmentally-regulated process. From a middle silk gland cDNA library, we have elucidated the sequence of one of the four mRNAs, the 4.0 kb Ser1B mRNA. Determination of alternative or constitutive exons and intron-exon boundaries allowed us to establish the nine exon-eight intron structure of the Ser1 gene. From these and previous data, it was possible to deduce the sequence of the sericins 1 and to predict the secondary structure and physiochemical properties of the different regions of the proteins.

Chest wall pain and possible rhabdomyolysis after chloropicrin exposure. A case series.

Three cases are summarized in which persistent chest wall pain, as well as elevations of creatine phosphokinase (CK) levels, occurred after exposure to chloropicrin vapor in an agricultural chemicals facility. Both the severity of the symptoms and the degree of biochemical abnormality appeared to follow a dose-response relationship. Myoglobinuria, on the other hand, was not found. Elevation of CK after chloropicrin exposure has not previously been reported and may represent low-grade rhabdomyolysis. Workups performed after symptomatic chloropicrine exposure should include CK-level determination.

High mortality rates among inmates during the year following their discharge from a French prison.

While the poor health status of prisoners has been highlighted in Western countries, the surveillance of their mortality has been neglected. We studied the mortality of 1305 prisoners released during 1997 from a French prison. Vital status after release was obtained for 86.4% of them. Compared with the general population, ex-prisoners non-natural mortality rates were significantly increased both in the 15-34 and 35-54 age categories (3.5-fold and 10.6-fold respectively) and the risk of death due to overdose was 124 and 274 times higher in the same categories respectively. Prevention and care should be reinforced in the pre-release period without waiting more epidemiological data.

[Knowledge and attitudes of prison staff towards HIV/AIDS: a European study]. / Connaissances et attitudes du personnel de surveillance pénitentiaire face au VIH et/ou SIDA: une enquête européenne.

The goal of this European pilot study was to evaluate the knowledge, attitudes and beliefs of prison staff from five countries towards HIV infection and to identify factors related to the potential discrimination of HIV-positive inmates. The survey revealed that the levels of knowledge with regard to HIV transmission and the degrees of tolerance varied significantly between prisons. A large proportion of staff overestimated the prevalence of HIV in their prison and feared being contaminated. The willingness of the staff to know the inmates' HIV seropositive status was negatively correlated to their level of tolerance; however, it was positively correlated to their knowledge of the modes of HIV transmission. This study underlines the necessity to improve HIV/AIDS prevention policy for prison staff in order to strengthen good practice in terms of managing the risk of contamination and hindering discrimination.