Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth.

ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection.

The SARS-CoV-2 virus emerged in December 2019 and has caused a worldwide pandemic due to the lack of any pre-existing immunity. Accurate serology testing is urgently needed to help diagnose infection, determine past exposure of populations and assess the response to a future vaccine. The landscape of antibody responses to SARS-CoV-2 is unknown. In this study, we utilized the luciferase immunoprecipitation system to assess the antibody responses to 15 different SARS-CoV-2 antigens in patients with COVID-19. We identified new targets of the immune response to SARS-CoV-2 and show that nucleocapsid, open reading frame (ORF)8 and ORF3b elicit the strongest specific antibody responses. ORF8 and ORF3b antibodies, taken together as a cluster of points, identified 96.5% of COVID-19 samples at early and late time points of disease with 99.5% specificity. Our findings could be used to develop second-generation diagnostic tests to improve serological assays for COVID-19 and are important in understanding pathogenicity.

Author Correction: ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome expansion by a CRISPR trimmer-integrase.

CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity1. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient2-5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation6,7, but many CRISPR-Cas systems lack Cas48. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited9,10 exonucleases for faithful acquisition of new CRISPR immune sequences.

Diversity, duplication, and genomic organization of homeobox genes in Lepidoptera.

Homeobox genes encode transcription factors with essential roles in patterning and cell fate in developing animal embryos. Many homeobox genes, including Hox and NK genes, are arranged in gene clusters, a feature likely related to transcriptional control. Sparse taxon sampling and fragmentary genome assemblies mean that little is known about the dynamics of homeobox gene evolution across Lepidoptera or about how changes in homeobox gene number and organization relate to diversity in this large order of insects. Here we analyze an extensive data set of high-quality genomes to characterize the number and organization of all homeobox genes in 123 species of Lepidoptera from 23 taxonomic families. We find most Lepidoptera have around 100 homeobox loci, including an unusual Hox gene cluster in which the lab gene is repositioned and the ro gene is next to pb A topologically associating domain spans much of the gene cluster, suggesting deep regulatory conservation of the Hox cluster arrangement in this insect order. Most Lepidoptera have four Shx genes, divergent zen-derived loci, but these loci underwent dramatic duplication in several lineages, with some moths having over 165 homeobox loci in the Hox gene cluster; this expansion is associated with local LINE element density. In contrast, the NK gene cluster content is more stable, although there are differences in organization compared with other insects, as well as major rearrangements within butterflies. Our analysis represents the first description of homeobox gene content across the order Lepidoptera, exemplifying the potential of newly generated genome assemblies for understanding genome and gene family evolution.

High-Energy-Density Material with Magnetically Modulated Ignition.

Preparation of a redox-frustrated high-energy-density energetic material is achieved by gentle protolysis of Mn[N(SiMe3)2]2 with the perchlorate salt of the tetrazolamide [H2NtBuMeTz]ClO4 (Tz = tetrazole), yielding the Mn6N6 hexagonal prismatic cluster, Mn6(µ3-NTztBuMe)6(ClO4)6. Quantum mechanics-based molecular dynamics simulations of the decomposition of this molecule predict that magnetic ordering of the d5 Mn2+ ions influences the pathway and rates of decomposition, suggesting that the initiation of decomposition of the bulk material might be significantly retarded by an applied magnetic field. We report here experimental tests of the prediction showing that the presence of a 0.5 T magnetic field modulates the ignition onset temperature by +10.4 ± 3.9 °C (from 414 ± 4 °C), demonstrating the first example of a magnetically modulated explosive.

Opsin Gene Duplication in Lepidoptera: Retrotransposition, Sex Linkage, and Gene Expression.

Color vision in insects is determined by signaling cascades, central to which are opsin proteins, resulting in sensitivity to light at different wavelengths. In certain insect groups, lineage-specific evolution of opsin genes, in terms of copy number, shifts in expression patterns, and functional amino acid substitutions, has resulted in changes in color vision with subsequent behavioral and niche adaptations. Lepidoptera are a fascinating model to address whether evolutionary change in opsin content and sequence evolution are associated with changes in vision phenotype. Until recently, the lack of high-quality genome data representing broad sampling across the lepidopteran phylogeny has greatly limited our ability to accurately address this question. Here, we annotate opsin genes in 219 lepidopteran genomes representing 33 families, reconstruct their evolutionary history, and analyze shifts in selective pressures and expression between genes and species. We discover 44 duplication events in opsin genes across ∼300 million years of lepidopteran evolution. While many duplication events are species or family specific, we find retention of an ancient long-wavelength-sensitive (LW) opsin duplication derived by retrotransposition within the speciose superfamily Noctuoidea (in the families Nolidae, Erebidae, and Noctuidae). This conserved LW retrogene shows life stage-specific expression suggesting visual sensitivities or other sensory functions specific to the early larval stage. This study provides a comprehensive order-wide view of opsin evolution across Lepidoptera, showcasing high rates of opsin duplications and changes in expression patterns.

Harmonized Multisite MRI-Based Quantification of Human Liver Fat and Stiffness: A Pilot Study.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of end-stage liver disease. NAFLD diagnosis and follow-up relies on a combination of clinical data, liver imaging, and/or liver biopsy. However, intersite imaging differences impede diagnostic consistency and reduce the repeatability of the multisite clinical trials necessary to develop effective treatments. PURPOSE/HYPOTHESIS: The goal of this pilot study was to harmonize commercially available 3 T magnetic resonance imaging (MRI) measurements of liver fat and stiffness in human participants across academic sites and MRI vendors. STUDY TYPE: Cohort. SUBJECTS: Four community-dwelling adults with obesity. FIELD STRENGTH/SEQUENCE: 1.5 and 3 T, multiecho 3D imaging, PRESS, and GRE. ASSESSMENT: Harmonized proton density fat fraction (PDFF) and magnetic resonance spectroscopy (MRS) protocols were used to quantify the FF of synthetic phantoms and human participants with obesity using standard acquisition parameters at four sites that had four different 3 T MRI instruments. In addition, a harmonized magnetic resonance elastography (MRE) protocol was used to quantify liver stiffness among participants at two different sites at 1.5 and 3 T field strengths. Data were sent to a single data coordinating site for postprocessing. STATISTICAL TESTS: Linear regression in MATLAB, ICC analyses using SAS 9.4, one-sided 95% confidence intervals for the ICC. RESULTS: PDFF and MRS FF measurements were highly repeatable among sites in both humans and phantoms. MRE measurements of liver stiffness in three individuals at two sites using one 1.5 T and one 3 T instrument showed repeatability that was high although lower than that of MRS and PDFF. CONCLUSIONS: We demonstrated harmonization of PDFF, MRS, and MRE-based quantification of liver fat and stiffness through synthetic phantoms, traveling participants, and standardization of postprocessing analysis. Multisite MRI harmonization could contribute to multisite clinical trials assessing the efficacy of interventions and therapy for NAFLD. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2.

Fascial plane mapping for superficial tumor resection in dogs. Part III: Hindlimb and pelvis.

OBJECTIVES: To categorize the fascial planes and the intersections of these fascial planes in the hindlimb of the dog to facilitate preoperative planning for superficial cancers. STUDY DESIGN: Qualitative anatomical study. SAMPLE POPULATION: Four male and five female mixed breed dogs, weighing ~15-35 kg. METHODS: Skin and subcutaneous fat were removed, and fascial planes were explored to determine borders and quality. Fascia was categorized as type I (discrete sheets), type II (adhered to thin muscles), type III (adhered to thick muscles), or type IV (associated with periosteum). Digital modification of specimen photographs was performed to map tissues. RESULTS: Differences in dogs were noted due to either size or sex but were sufficiently minor to allow mapping. Fasciae of the hindlimb were predominantly type II or III, with type I fascia primarily at the lateral gluteal region, fascia lata, and lateral crus. Type IV fascia was seen at the iliac wing, ischium, patella, tibial tuberosity, medial tibia, distal crus, and pes. Fascia for surgical use was thin or absent at the ischiorectal fossa, femoral triangle, extensor mechanism, medial and distal crus, and pes. Intersections and tissues at the ventral perineum may also pose challenges for maintenance of a deep margin. CONCLUSION: Fascial types and integrity of the hindlimb varied with location, with the perineum, cranial stifle, and distal limb presenting the greatest challenges. CLINICAL SIGNIFICANCE: These images may be used to guide both therapeutic decision-making and intraoperative excision of superficial tumors of the hindlimb and pelvis.

B2 cells contribute to hypertension and natural killer cell activation possibly via AT1-AA in response to placental ischemia.

Preeclampsia, new onset hypertension during pregnancy, is associated with activated T helper cells (Th) and B cells secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1-AA). The reduced uterine perfusion pressure (RUPP) model of placental ischemia recapitulates these characteristics. We have shown that Th-B cell communication contributes to AT1-AA and symptoms of preeclampsia in the RUPP rat. B2 cells are classical B cells that communicate with Th cells and are then transformed into memory B cells. We hypothesize that B2 cells cause hypertension, natural killer (NK) cell activation, and complement activation during pregnancy through the production of AT1-AA. To test this hypothesis, total splenic B cells and B2 cells were isolated from normal pregnant (NP) or RUPP rats on gestational day (GD)19 and adoptively transferred into GD12 NP rats. A group of recipient rats was treated with a specific inhibitor peptide of AT1-AA. On GD19, mean arterial pressure was measured, tissues were collected, activated NK cells were measured by flow cytometry, and AT1-AA was measured by cardiomyocyte assay. NP recipients of RUPP B cells or RUPP B2 cells had increased mean arterial pressure, AT1-AA, and circulating activated NK cells compared with recipients of NP B cells. Hypertension in NP recipients of RUPP B cells or RUPP B2 was attenuated with AT1-AA blockade. This study demonstrates that B cells and B2 cells from RUPP rats cause hypertension and increased AT1-AA and NK cell activation in response to placental ischemia during pregnancy.NEW & NOTEWORTHY This study demonstrates that placental ischemia-stimulated B2 cells induce hypertension and circulating natural killer cell activation and angiotensin II type 1 receptor production in normal pregnant rats.

Microbiome Structure of a Wild Drosophila Community along Tropical Elevational Gradients and Comparison to Laboratory Lines.

Variation along environmental gradients in host-associated microbial communities is not well understood compared to free-living microbial communities. Because elevational gradients may serve as natural proxies for climate change, understanding patterns along these gradients can inform our understanding of the threats hosts and their symbiotic microbes face in a warming world. In this study, we analyzed bacterial microbiomes from pupae and adults of four Drosophila species native to Australian tropical rainforests. We sampled wild individuals at high and low elevations along two mountain gradients to determine natural diversity patterns. Further, we sampled laboratory-reared individuals from isofemale lines established from the same localities to see if any natural patterns are retained in the lab. In both environments, we controlled for diet to help elucidate other deterministic patterns of microbiome composition. We found small but significant differences in Drosophila bacterial community composition across elevation, with some notable taxonomic differences between different Drosophila species and sites. Further, we found that field-collected fly pupae had significantly richer microbiomes than laboratory-reared pupae. We also found similar microbiome composition in both types of provided diet, suggesting that the significant differences found among Drosophila microbiomes are the products of surrounding environments with different bacterial species pools, possibly bound to elevational differences in temperature. Our results suggest that comparative studies between lab and field specimens help reveal the true variability in microbiome communities that can exist within a single species. IMPORTANCE Bacteria form microbial communities inside most higher-level organisms, but we know little about how the microbiome varies along environmental gradients and between natural host populations and laboratory colonies. To explore such effects on insect-associated microbiomes, we studied the gut microbiome in four Drosophila species over two mountain gradients in tropical Australia. We also compared these data to individuals kept in the laboratory to understand how different settings changed microbiome communities. We found that field-sampled individuals had significantly higher microbiome diversity than those from the lab. In wild Drosophila populations, elevation explains a small but significant amount of the variation in their microbial communities. Our study highlights the importance of environmental bacterial sources for Drosophila microbiome composition across elevational gradients and shows how comparative studies help reveal the true flexibility in microbiome communities that can exist within a species.

Experimental heatwaves facilitate invasion and alter species interactions and composition in a tropical host-parasitoid community.

As mean temperatures increase and heatwaves become more frequent, species are expanding their distributions to colonise new habitats. The resulting novel species interactions will simultaneously shape the temperature-driven reorganization of resident communities. The interactive effects of climate change and climate change-facilitated invasion have rarely been studied in multi-trophic communities, and are likely to differ depending on the nature of the climatic driver (i.e., climate extremes or constant warming). We re-created under laboratory conditions a host-parasitoid community typical of high-elevation rainforest sites in Queensland, Australia, comprising four Drosophila species and two associated parasitoid species. We subjected these communities to an equivalent increase in average temperature in the form of periodic heatwaves or constant warming, in combination with an invasion treatment involving a novel host species from lower-elevation habitats. The two parasitoid species were sensitive to both warming and heatwaves, while the demographic responses of Drosophila species were highly idiosyncratic, reflecting the combined effects of thermal tolerance, parasitism, competition, and facilitation. After multiple generations, our heatwave treatment promoted the establishment of low-elevation species in upland communities. Invasion of the low-elevation species correlated negatively with the abundance of one of the parasitoid species, leading to cascading effects on its hosts and their competitors. Our study, therefore, reveals differing, sometimes contrasting, impacts of extreme temperatures and constant warming on community composition. It also highlights how the scale and direction of climate impacts could be further modified by invading species within a bi-trophic community network.

Latitudinal and anthropogenic effects on the structuring of networks linking blood-feeding flies and their vertebrate hosts.

Biting flies (Diptera) transmit pathogens that cause many important diseases in humans as well as domestic and wild animals. The networks of feeding interactions linking these insects to their hosts, and how they vary geographically and in response to human land-use, are currently poorly documented but are relevant to understanding cross-species disease transmission. We compiled a database of biting Diptera-host interactions from the literature to investigate how key interaction network metrics vary latitudinally and with human land-use. Interaction evenness and H2' (a measure of the degree of network specificity) did not vary significantly with latitude. Compared to near-natural habitats, interaction evenness was significantly lower in agricultural habitats, where networks were dominated by relatively few species pairs, but there was no evidence that the presence of humans and their domesticated animals within networks led to systematic shifts in network structure. We discuss the epidemiological relevance of these results and the implications for predicting and mitigating future spill-over events.

Ancillary investigations for death determination in infants and children: a systematic review and meta-analysis.

PURPOSE: We performed a systematic review and meta-analysis to determine the diagnostic test accuracy of ancillary investigations for declaration of death by neurologic criteria (DNC) in infants and children. SOURCE: We searched MEDLINE, EMBASE, Web of Science, and Cochrane databases from their inception to June 2021 for relevant randomized controlled trials, observational studies, and abstracts published in the last three years. We identified relevant studies using Preferred Reporting Items for Systematic Reviews and Meta-Analysis methodology and a two-stage review. We assessed the risk of bias using the QUADAS-2 tool, and applied Grading of Recommendations Assessment, Development, and Evaluation methodology to determine the certainty of evidence. A fixed-effects model was used to meta-analyze pooled sensitivity and specificity data for each ancillary investigation with at least two studies. PRINCIPAL FINDINGS: Thirty-nine eligible manuscripts assessing 18 unique ancillary investigations (n = 866) were identified. The sensitivity and specificity ranged from 0.00 to 1.00 and 0.50 to 1.00, respectively. The quality of evidence was low to very low for all ancillary investigations, with the exception of radionuclide dynamic flow studies for which it was graded as moderate. Radionuclide scintigraphy using the lipophilic radiopharmaceutical 99mTc-hexamethylpropyleneamine oxime (HMPAO) with or without tomographic imaging were the most accurate ancillary investigations with a combined sensitivity of 0.99 (95% highest density interval [HDI], 0.89 to 1.00) and specificity of 0.97 (95% HDI, 0.65 to 1.00). CONCLUSION: The ancillary investigation for DNC in infants and children with the greatest accuracy appears to be radionuclide scintigraphy using HMPAO with or without tomographic imaging; however, the certainty of the evidence is low. Nonimaging modalities performed at the bedside require further investigation. STUDY REGISTRATION: PROSPERO (CRD42021278788); registered 16 October 2021.

RéSUMé: OBJECTIF: Nous avons réalisé une revue systématique et une méta-analyse pour déterminer la précision des tests diagnostiques des examens auxiliaires pour la déclaration du décès selon des critères neurologiques (DCN) chez les nourrissons et les enfants. SOURCES: Nous avons effectué des recherches dans les bases de données MEDLINE, EMBASE, Web of Science et Cochrane de leur création jusqu'en juin 2021 pour trouver des études randomisées contrôlées, des études observationnelles et des résumés pertinents publiés au cours des trois dernières années. Nous avons identifié les études pertinentes utilisant la méthodologie PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) et une revue en deux étapes. Nous avons évalué le risque de biais en utilisant l'outil QUADAS-2 et appliqué la méthodologie GRADE (Grading of Recommendations Assessment, Development and Evaluation) afin d'évaluer la certitude des données probantes. Un modèle à effets fixes a été utilisé pour méta-analyser les données de sensibilité et de spécificité regroupées pour chaque examen auxiliaire avec au moins deux études. CONSTATATIONS PRINCIPALES: Trente-neuf manuscrits admissibles évaluant 18 examens auxiliaires uniques (n = 866) ont été identifiés. La sensibilité et la spécificité variaient de 0,00 à 1,00 et de 0,50 à 1,00, respectivement. La qualité des données probantes était faible à très faible pour tous les examens auxiliaires, à l'exception des études de circulation nucléaire dynamique, pour lesquelles elle a été classée comme modérée. La scintigraphie nucléaire à l'aide du produit radiopharmaceutique lipophile 99mTc- hexa-méthyl-propylène amine oxime (HMPAO) avec ou sans imagerie tomographique était à la base des examens auxiliaires les plus précis, avec une sensibilité combinée de 0,99 (intervalle de densité le plus élevé [IDE] à 95 %, 0,89 à 1,00) et une spécificité de 0,97 (IDE à 95 %, 0,65 à 1,00). CONCLUSION: L'examen auxiliaire pour un DCN chez les nourrissons et les enfants offrant la plus grande précision semble être la scintigraphie nucléaire utilisant le HMPAO avec ou sans imagerie tomographique; cependant, la certitude des données probantes est faible. Les modalités sans imagerie réalisées au chevet du patient nécessitent un examen plus approfondi. Enregistrement de l'étude: PROSPERO (CRD42021278788); enregistrée le 16 octobre 2021.

A brain-based definition of death and criteria for its determination after arrest of circulation or neurologic function in Canada: a 2023 clinical practice guideline.

This 2023 Clinical Practice Guideline provides the biomedical definition of death based on permanent cessation of brain function that applies to all persons, as well as recommendations for death determination by circulatory criteria for potential organ donors and death determination by neurologic criteria for all mechanically ventilated patients regardless of organ donation potential. This Guideline is endorsed by the Canadian Critical Care Society, the Canadian Medical Association, the Canadian Association of Critical Care Nurses, Canadian Anesthesiologists' Society, the Canadian Neurological Sciences Federation (representing the Canadian Neurological Society, Canadian Neurosurgical Society, Canadian Society of Clinical Neurophysiologists, Canadian Association of Child Neurology, Canadian Society of Neuroradiology, and Canadian Stroke Consortium), Canadian Blood Services, the Canadian Donation and Transplantation Research Program, the Canadian Association of Emergency Physicians, the Nurse Practitioners Association of Canada, and the Canadian Cardiovascular Critical Care Society.

RéSUMé: Ces Lignes directrices de pratique clinique 2023 Lignes directrices de pratique clinique dicale du décès basée sur l'arrêt permanent de la fonction cérébrale qui s'applique à toute personne, ainsi que des recommandations pour la détermination du décès par des critères circulatoires pour des donneurs d'organes potentiels et des recommandations pour la détermination du décès par des critères neurologiques pour tous les patients sous ventilation mécanique, indépendamment de leur potentiel de donneur d'organes. Les présentes Lignes directrices sont approuvées par la Société canadienne de soins intensifs, l'Association médicale canadienne, l'Association canadienne des infirmiers/infirmières en soins intensifs, la Société canadienne des anesthésiologistes, la Fédération des sciences neurologiques du Canada (représentant la Société canadienne de neurologie, la Société canadienne de neurochirurgie, la Société canadienne de neurophysiologie clinique, l'Association canadienne de neurologie pédiatrique, la Société canadienne de neuroradiologie et le Consortium neurovasculaire canadien), la Société canadienne du sang, le Programme de recherche en don et transplantation du Canada, l'Association canadienne des médecins d'urgence, l'Association des infirmières et infirmiers praticiens du Canada, et la Société canadienne de soins intensifs cardiovasculaires (CANCARE) et la Société canadienne de pédiatrie.

Buccal transposition flap for closure of maxillary lip defects in 5 dogs.

OBJECTIVE: To describe the technique and outcomes of the closure of maxillary lip defects using a buccal transposition flap and to identify potential routes of vascular supply to the flap. ANIMALS: Five dogs treated clinically and 1 cadaveric dog head. STUDY DESIGN: Short case series and cadaveric study. METHODS: A left maxillary labial defect and a buccal transposition flap were created on a cadaver head. Iodinated contrast was injected into the left common carotid artery and computed tomography was performed to assess the vascular supply. Medical records were reviewed for all dogs that underwent tumor excision with maxillary lip resection, reconstructed with a buccal transposition flap. RESULTS: The buccal transposition flap was perfused by branches of the angularis oris artery and superior labial artery. Five dogs were included in this study. All flaps survived. Three dogs developed postoperative complications, including oronasal fistula (n = 2) and partial flap dehiscence (n = 1). The cosmetic and functional outcomes were considered satisfactory in all cases. CONCLUSION: Buccal transposition flaps for the closure of large maxillary lip defects provided adequate functional and cosmetic outcomes. The buccal transposition flap had vascular contributions from the angularis oris artery and the superior labial artery.

Abundant small RNAs in the reproductive tissues and eggs of the honey bee, Apis mellifera.

BACKGROUND: Polyandrous social insects such as the honey bee are prime candidates for parental manipulation of gene expression in offspring. Although there is good evidence for parent-of-origin effects in honey bees the epigenetic mechanisms that underlie these effects remain a mystery. Small RNA molecules such as miRNAs, piRNAs and siRNAs play important roles in transgenerational epigenetic inheritance and in the regulation of gene expression during development. RESULTS: Here we present the first characterisation of small RNAs present in honey bee reproductive tissues: ovaries, spermatheca, semen, fertilised and unfertilised eggs, and testes. We show that semen contains fewer piRNAs relative to eggs and ovaries, and that piRNAs and miRNAs which map antisense to genes involved in DNA regulation and developmental processes are differentially expressed between tissues. tRNA fragments are highly abundant in semen and have a similar profile to those seen in the semen of other animals. Intriguingly we also find abundant piRNAs that target the sex determination locus, suggesting that piRNAs may play a role in honey bee sex determination. CONCLUSIONS: We conclude that small RNAs may play a fundamental role in honey bee gametogenesis and reproduction and provide a plausible mechanism for parent-of-origin effects on gene expression and reproductive physiology.

Trait-mediated competition drives an ant invasion and alters functional diversity.

The assumption that differences in species' traits reflect their different niches has long influenced how ecologists infer processes from assemblage patterns. For instance, many assess the importance of environmental filtering versus classical limiting-similarity competition in driving biological invasions by examining whether invaders' traits are similar or dissimilar to those of residents, respectively. However, mounting evidence suggests that hierarchical differences between species' trait values can distinguish their competitive abilities (e.g. for the same resource) instead of their niches. Whether such trait-mediated hierarchical competition explains invasions and structures assemblages is less explored. We integrate morphological, dietary, physiological and behavioural trait analyses to test whether environmental filtering, limiting-similarity competition or hierarchical competition explain invasions by fire ants on ant assemblages. We detect both competition mechanisms; invasion success is not only explained by limiting similarity in body size and thermal tolerance (presumably allowing the invader to exploit different niches from residents), but also by the invader's superior position in trait hierarchies reflecting competition for common trophic resources. We find that the two mechanisms generate complex assemblage-level functional diversity patterns-overdispersion in some traits, clustering in others-suggesting their effects are likely missed by analyses restricted to a few traits and composite trait diversity measures.

Localization of EccA3 at the growing pole in Mycobacterium smegmatis.

BACKGROUND: Bacteria require specialized secretion systems for the export of molecules into the extracellular space to modify their environment and scavenge for nutrients. The ESX-3 secretion system is required by mycobacteria for iron homeostasis. The ESX-3 operon encodes for one cytoplasmic component (EccA3) and five membrane components (EccB3 - EccE3 and MycP3). In this study we sought to identify the sub-cellular location of EccA3 of the ESX-3 secretion system in mycobacteria. RESULTS: Fluorescently tagged EccA3 localized to a single pole in the majority of Mycobacterium smegmatis cells and time-lapse fluorescent microscopy identified this pole as the growing pole. Deletion of ESX-3 did not prevent polar localization of fluorescently tagged EccA3, suggesting that EccA3 unipolar localization is independent of other ESX-3 components. Affinity purification - mass spectrometry was used to identify EccA3 associated proteins which may contribute to the localization of EccA3 at the growing pole. EccA3 co-purified with fatty acid metabolism proteins (FAS, FadA3, KasA and KasB), mycolic acid synthesis proteins (UmaA, CmaA1), cell division proteins (FtsE and FtsZ), and cell shape and cell cycle proteins (MurS, CwsA and Wag31). Secretion system related proteins Ffh, SecA1, EccA1, and EspI were also identified. CONCLUSIONS: Time-lapse microscopy demonstrated that EccA3 is located at the growing pole in M. smegmatis. The co-purification of EccA3 with proteins known to be required for polar growth, mycolic acid synthesis, the Sec secretion system (SecA1), and the signal recognition particle pathway (Ffh) also suggests that EccA3 is located at the site of active cell growth.