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Background: Burkholderia pseudomallei is a Tier 1 overlap select agent and subject to the select agent regulations (42 CFR §73 and 9 CFR §121). It is a gram-negative, motile, soil saprophyte, and the etiologic agent of melioidosis. B. pseudomallei infection can produce systemic illness and can be fatal in the absence of appropriate treatment. Laboratory exposures involving this organism may occur when appropriate containment measures are not employed. Current disease treatment inadequacies and the risk factors associated with melioidosis make this an agent of primary concern in research, commercial, and clinical laboratory environments. Results: This study presents data reported to Centers for Disease Control and Prevention (CDC), Division of Select Agents and Toxins for releases involving B. pseudomallei in 2017-2019 that occurred in Biosafety Level (BSL)-2 and BSL-3 laboratories. Fifty-one Animal and Plant Health Inspection Service (APHIS)/CDC Form 3 release reports led to the medical surveillance of 275 individuals. Entities offered post-exposure prophylaxis to â¼76% of the individuals impacted in the presented events. Summary: Laboratory safety can be improved by implementing appropriate safety precautions to minimize exposures. Most of the incidents discussed in this evidence-based report occurred during work conducted in the absence of primary containment. None of the releases resulted in illness, death, or transmission to or among workers, nor was there transmission outside of a laboratory into the surrounding environment or community. Effective risk assessment and management strategies coupled with standard and special microbiological policies and procedures can result in reduced exposures and improved safety at facilities.
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The gene vermilion encodes tryptophan 2,3-dioxygenase, part of the ommochrome pathway, and is responsible for the dark pigmented eyes in some insects, including beetles. Using RNA interference, we targeted the vermilion gene ortholog in embryos and pupae of the yellow mealworm, Tenebrio molitor, resulting in larvae and adults, respectively, that lacked eye pigment. RNA-Seq was used to analyze the impact of vermilion-specific RNA interference on gene expression. There was a 425-fold reduction in vermilion gene expression (p = 0.0003), as well as significant (p < 0.05) differential expression of 109 other putative genes, most of which were downregulated. Enrichment analysis of Gene Ontology terms found in the differentially expressed data set included genes known to be involved in the ommochrome pathway. However, enrichment analysis also revealed the influence of vermilion expression on genes involved in protein translocation to the endoplasmic reticulum, signal transduction, G-protein-coupled receptor signaling, cell-cycle arrest, mannose biosynthesis, and vitamin transport. These data demonstrate that knockdown of vermilion in T. molitor results in complete loss of eye color (white-eyed phenotype) and identify other interrelated genes in the vermilion metabolic pathway. Therefore, a dominant marker system based on eye color can be developed for the genetic manipulation of T. molitor to increase the value of mealworms as an alternative food source by decreasing negative traits, such as disease susceptibility, and increasing desired traits, such as protein content and vitamin production.
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Tenebrio/genética , Triptofano Oxigenase/genética , Criação de Animais Domésticos , Animais , Cor de Olho/genética , Expressão Gênica , Marcadores Genéticos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/genética , Pigmentação/genética , Pupa/genética , Interferência de RNA , Tenebrio/enzimologia , Tenebrio/crescimento & desenvolvimentoRESUMO
The Tribolium castaneum vermilion gene encodes tryptophan 2,3-dioxygenase, a pivotal enzyme in the ommochrome pathway that is required for proper pigmentation of the eye. A white-eyed mutant strain of T. castaneum, vermilionwhite (vw), lacks eye pigmentation due to a deletion of unknown size that removes all but the 3'-end of the vermilion gene. To create a more defined mutation in vermilion, the CRISPR/Cas9-nuclease system was used to target wild type vermilion in preblastoderm T. castaneum embryos. As adults, all injected beetles had wild type (black) eye pigmentation; however, when outcrossed to vw mates, one cross produced 19% white-eyed offspring. When the vermilion locus of these offspring was analyzed by target-site sequencing, it was determined that white-eyed individuals had a 2â¯bp deletion that resulted in a frame-shift mutation, presumably producing a nonfunctional enzyme. Interestingly, some of their black-eyed siblings also had a small deletion of 6â¯bp, but the resultant loss of two amino acids had no apparent impact on enzyme function. To establish a mutant strain homozygous for the CRISPR-induced knock-out allele, a CRISPR positive G0 male was crossed to wild type females. Their progeny were self-crossed, and white-eyed progeny were used to establish the new strain. This mutant strain is herein named vermilionICE and will be used in future work in addition to or in place of vw.
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Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Proteínas de Insetos/genética , Pigmentação/genética , Tribolium/fisiologia , Triptofano Oxigenase/genética , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Masculino , Redes e Vias Metabólicas , Fenômenos Fisiológicos Oculares , Filogenia , Alinhamento de Sequência , Tribolium/genética , Tribolium/crescimento & desenvolvimento , Triptofano Oxigenase/química , Triptofano Oxigenase/metabolismoRESUMO
Proanthocyanidins (PA) crosslink wheat gluten, increasing its polymer size and strength. However, mechanisms behind these interactions are unknown. This study used PA of different MW profiles (mean degree of polymerization 8.3 and 19.5) to investigate how PA polymerize gluten. The higher MW PA had greater binding affinity for both glutenins and gliadins than lower MW PA, whereas both PA precipitated glutenins more efficiently than gliadins. The PA preferentially bound the largest of the protein fractions available: high MW glutenin subunits (HMW-GS) over low MW-GS, and ω-gliadins over α- and γ-gliadins. Furthermore, within the HMW-GS, PA bound more of the larger x-type than the smaller y-type. Proanthocyanidins reduced gluten solubility in urea and decreased surface hydrophobicity of glutenins, but not gliadins. The PA appear to preferentially crosslink HMW-GS via hydrophobic interactions and hydrogen bonding, whereas their interaction with gliadins is dominated by hydrogen bonding and is relatively weaker.
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Glutens/química , Proantocianidinas/química , Triticum/química , Gliadina/química , Glutens/metabolismo , Peso Molecular , Proantocianidinas/metabolismo , Solubilidade , Ureia/químicaRESUMO
Proanthocyanidins (PA) cross-link proteins and could expand wheat gluten functionality; however, how the PA MW or gluten profile affect these interactions is unknown. Effect of PA MW profile (sorghum versus grape seed PA) on dough rheology of high versus low insoluble polymeric protein (IPP) wheat flour was evaluated using mixograph, large (TA.XT2i) and small (HAAKE Rheostress 6000) deformation rheometry. Sorghum PA (93% polymeric) more effectively (p < 0.05) strengthened both glutens than grape seed PA (45% polymeric), without reducing gluten extensibility. These effects were higher in low IPP (weak gluten) flour, e.g., sorghum PA doubled IPP, increased mix time by 75%, dough elasticity by 82%, and peak angle by 17° versus control. Grape seed PA increased IPP by 75% and elasticity by 36%, but reduced peak angle by 15°, indicating reduced mixing tolerance. Sorghum PA, but not grape seed PA, increased (p < 0.05) all above parameters in high IPP dough. Polymeric PA more effectively strengthened gluten than oligomeric PA, likely via more efficient protein cross-linking to overcome strong antioxidant effect of PA. High MW PA may be useful natural gluten strengtheners for diverse applications.
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Farinha/análise , Glutens/química , Proantocianidinas/química , Reologia , Triticum/química , Pão , Cromatografia Líquida de Alta Pressão , Elasticidade , Fluorescência , Extrato de Sementes de Uva/química , Peso Molecular , Sorghum/química , Espectrofotometria InfravermelhoRESUMO
Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi) may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA) in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs.
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Flagelliform spider silk is the most extensible silk fiber produced by orb weaver spiders, though not as strong as the dragline silk of the spider. The motifs found in the core of the Nephila clavipes flagelliform Flag protein are GGX, spacer, and GPGGX. Flag does not contain the polyalanine motif known to provide the strength of dragline silk. To investigate the source of flagelliform fiber strength, four recombinant proteins were produced containing variations of the three core motifs of the Nephila clavipes flagelliform Flag protein that produces this type of fiber. The as-spun fibers were processed in 80% aqueous isopropanol using a standardized process for all four fiber types, which produced improved mechanical properties. Mechanical testing of the recombinant proteins determined that the GGX motif contributes extensibility and the spacer motif contributes strength to the recombinant fibers. Recombinant protein fibers containing the spacer motif were stronger than the proteins constructed without the spacer that contained only the GGX motif or the combination of the GGX and GPGGX motifs. The mechanical and structural X-ray diffraction analysis of the recombinant fibers provide data that suggests a functional role of the spacer motif that produces tensile strength, though the spacer motif is not clearly defined structurally. These results indicate that the spacer is likely a primary contributor of strength, with the GGX motif supplying mobility to the protein network of native N. clavipes flagelliform silk fibers.