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Coating high-touch surfaces with inorganic agents, such as metals, appears to be a promising long-term disinfection strategy. However, there is a lack of studies exploring the effectiveness of copper-based products against viruses. In this study, we evaluated the cytotoxicity and virucidal effectiveness of products and materials containing copper against mouse hepatitis virus (MHV-3), a surrogate model for SARS-CoV-2. The results demonstrate that pure CuO and Cu possess activity against the enveloped virus at very low concentrations, ranging from 0.001 to 0.1% (w/v). A greater virucidal efficacy of CuO was found for nanoparticles, which showed activity even against viruses that are more resistant to disinfection such as feline calicivirus (FCV). Most of the evaluated products, with concentrations of Cu or CuO between 0.003 and 15% (w/v), were effective against MHV-3. Cryomicroscopy images of an MHV-3 sample exposed to a CuO-containing surface showed extensive damage to the viral capsid, presumably due to the direct or indirect action of copper ions., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The sacred ayahuasca brew, utilized by indigenous communities in the Amazon and syncretic religious groups in Brazil, primarily consists of a decoction of two plants: (i) the Amazonian liana known as Mariri or Jagube (Banisteriopsis caapi), and (ii) the shrub referred as Chacrona or Rainha (Psychotria viridis). While Chacrona leaves are rich in N,N-Dimethyltryptamine (DMT), a potent psychedelic, the macerated vine of Mariri provides beta-carboline alkaloids acting as monoamine oxidase inhibitors, preventing DMT's degradation. This study sequenced, assembled, and analyzed the complete genome of B. caapi's mitochondrion, yielding a circular structure spanning 503,502 bp. Although the mtDNA encompasses most plant mitochondrial genes, it lacks some ribosomal genes, presents some atypical genes, and contains plastid pseudogenes, suggesting gene transfer between organelles. The presence of a 7-Kb repetitive segment containing copies of the rrnL and trnfM genes suggests mitogenome isomerization, supporting the hypothesis of dynamic mitogenome maintenance in plants. Phylogenetics and phylogenomics across 24 Malpighiales confirms the sample's placement in the "Tucunacá" ethnovariety, aligning with morphological identification. This study spearheads efforts to decode the genome of this esteemed Malpighiaceae.

We report 4 highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b viruses in samples collected during June 2023 from Royal terns and Cabot's terns in Brazil. Phylodynamic analysis revealed viral movement from Peru to Brazil, indicating a concerning spread of this clade along the Atlantic Americas migratory bird flyway.

Psychotria viridis (Rubioideae: Rubiaceae), popularly known as chacrona , is commonly found as a shrub in the Amazon region and is well-known to produce psychoactive compounds, such as the N,N-dimethyltryptamine (DMT). Together with the liana Banisteropsis caapi , P. viridis is one of the main components of the Amerindian traditional, entheogenic beverage known as ayahuasca. In this work, we assembled and annotated the organellar genomes (ptDNA and mtDNA), presenting the first genomics resources for this species. The P. viridis ptDNA exhibits 154,106 bp, encoding all known ptDNA gene repertoire found in angiosperms. The Psychotria genus is a complex paraphyletic group, and according to phylogenomic analyses, P. viridis is nested in the Psychotrieae clade. Comparative ptDNA analyses indicate that most Rubiaceae plastomes present conserved ptDNA structures, often showing slight differences at the junction sites of the major four regions (LSC-IR-SSC). For the mitochondrion, assembly graph-based analysis supports a complex mtDNA organization, presenting at least two alternative and circular mitogenomes structures exhibiting two main repeats spanning 24 kb and 749 bp that may symmetrically isomerize the mitogenome into variable arrangements and isoforms. The circular mtDNA sequences (615,370 and 570,344 bp) encode almost all plant mitochondrial genes (except for the ccmC , rps7 , rps10 , rps14 , rps19 , rpl2 and rpl16 that appears as pseudogenes, and the absent genes sdh3, rps2, rsp4, rsp8, rps11, rpl6 , and rpl10 ), showing slight variations related to exclusive regions, ptDNA integration, and relics of previous events of LTR-RT integration. The detection of two mitogenomes haplotypes is evidence of heteroplasmy as observed by the complex organization of the mitochondrial genome using graph-based analysis. Taken together, these results elicit the primary insights into the genome biology and evolutionary history of Psychotria viridis and may be used to aid strategies for conservation of this sacred, entheogenic species., Competing Interests: The authors declare that they have no competing interests., (© 2022 Varani et al.)

Background and Aims: The entangled relationship of chromosome number and genome size with species distribution has been the subject of study for almost a century, but remains an open question due to previous ecological and phylogenetic knowledge constraints. To better address this subject, we used the clade Maxillariinae, a widely distributed and karyotypically known orchid group, as a model system to infer such relationships in a robust methodological framework., Methods: Based on the literature and new data, we gathered the chromosome number and genome size for 93 and 64 species, respectively. We built a phylogenetic hypothesis and assessed the best macroevolutionary model for both genomic traits. Additionally, we collected together ecological data (preferences for bioclimatic variables, elevation and habit) used as explanatory variables in multivariate phylogenetic models explaining genomic traits. Finally, the impact of polyploidy was estimated by running the analyses with and without polyploids in the sample., Key Results: The association between genomic and ecological data varied depending on whether polyploids were considered or not. Without polyploids, chromosome number failed to present consistent associations with ecological variables. With polyploids, there was a tendency to waive epiphytism and colonize new habitats outside humid forests. The genome size showed association with ecological variables: without polyploids, genome increase was associated with flexible habits, with higher elevation and with drier summers; with polyploids, genome size increase was associated with colonizing drier environments., Conclusions: The chromosome number and genome size variations, essential but neglected traits in the ecological niche, are shaped in the Maxillariinae by both neutral and adaptive evolution. Both genomic traits are partially correlated to bioclimatic variables and elevation, even when controlling for phylogenetic constraints. While polyploidy was associated with shifts in the environmental niche, the genome size emerges as a central trait in orchid evolution by the association between small genome size and epiphytism, a key innovation to Neotropical orchid diversification., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite the clear circumscription of tribe Sobralieae (Orchidaceae), its internal relationships are still dubious. The recently delimited genus Brasolia , based on previous Sobralia species, is now assumed to be paraphyletic, with a third genus, Elleanthus , nested in it. The morphology of these three genera is significantly different, indicating the necessity of new data for a better genera delimitation. Though morphology and molecular data are available, cytogenetics data for Sobralieae is restricted to two Sobralia and one Elleanthus species. Aiming to evaluate the potential of cytogenetic data for Brasolia-Elleanthus-Sobralia genera delimitation, we present chromosome number and genome size data for 21 and 20 species, respectively, and used such data to infer the pattern of karyotype evolution in these genera. The analysis allowed us to infer x = 24 as the base chromosome number and genome size of average 1C-value of 5.0 pg for the common ancestor of Brasolia-Elleanthus-Sobralia . The recurrent descending dysploidy in Sobralieae and the punctual genome upsize suggest a recent diversification in Sobralieae but did not allow differing between Brasolia and Sobralia . However, the basal position of tribe Sobralieae in the subfamily Epidendroideae makes this tribe of interest to further studies clarifying the internal delimitation and pattern of karyotype evolution.

Murine hepatitis virus (MHV) strain 3, one of the most important inducers of viral hepatitis, has been extensively studied as an organism to gain a better understanding of coronavirus biology and pathogenesis. Only one sequence is currently available. Another representative isolate has now been sequenced and added to the arsenal of MHV-3 variants., (Copyright © 2021 Garcia et al.)

Ploidy level and genome size (GS) could affect the invasive capacity of plants, although these parameters can be contradictory. While small GS seems to favor dispersion, polyploidy-which increases the GS-also seems to favor it. Using a phylogenetic path analysis, we evaluated the effects of both factors on the environmental tolerance and invasive success of plants. We selected 99 invasive plant species from public online databases and gathered information about invasive capacity (number of non-original countries in which each species occurs), tolerance to environmental factors, ploidy level, and GS. The invasive capacity varied depending on the ploidy level and tolerance to environmental factors. Polyploids and species with increased tolerance to elevated temperatures and rainfall values exhibited high invasive capacity. We found no evidence that GS affects the invasive capacity of plants. We suggest that the genetic variability provided by polyploidization has a positive impact on plant competitiveness, which may ultimately lead to an increased ability to colonize new environments. In a global warming scenario, integrative approaches using phenotypic, genetic, epigenetic, and ecological traits should be a productive route to unveil the aspects of invasive plants.

Objective: To assess childhood-onset systemic lupus erythematosus-related antiphospholipid syndrome(cSLE-APS) in a large Brazilian population., Methods: A retrospective observational cohort study was carried-out in 27 Pediatric Rheumatology university centers, including 1519 cSLE patients., Results: cSLE-APS was observed in 67/1519 (4%) and was diagnosed at disease onset in 39/67 (58%). The median disease duration was 4.9 (0-17) years. Thrombosis recurrences were evidenced in 18/67 (27%) cSLE-APS patients. The most frequent thrombosis sites in cSLE-APS patients were: venous thrombosis in 40/67 (60%), especially deep vein thrombosis in 29/40 (72%); arterial thrombosis in 35/67 (52%), particularly stroke; small vessels thrombosis in 9/67 (13%) and mixed thrombosis in 3/67 (4%). Pregnancy morbidity was observed in 1/67 (1%). Non-thrombotic manifestation associated to cSLE-APS occurred in 21/67 (31%), mainly livedo reticularis in 14/67 (21%), valvar thickening in 4/67 (6%) and valvar vegetations not related to infections in 2/67 (3%). None of them had catastrophic APS. Further analysis demonstrated that the median of SLICC/ACR-DI [1(0-5) vs. 0(0-7),p < 0.0001] was significantly higher in cSLE-APS patients compared to cSLE without APS. The frequencies of cerebrovascular disease (40% vs. 1%,p < 0.0001), polyneuropathy (9% vs. 1%,p < 0.0001), SLICC/ACR-DI ≥1 (57% vs. 27%, p < 0.0001) and intravenous cyclophosphamide use (59% vs. 37%, p < 0.0001) were significantly higher in the former group., Conclusions: Our large multicenter study demonstrated that cSLE-APS was a rare condition, occurring during disease course with a high accrual damage. Central and peripheral neuropsychiatric involvements were distinctive features of this autoimmune thrombosis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although the evolution of differentiated thyroid cancer (DTC) is usually indolent, some tumors grow fast, metastasize, and may be fatal. Viruses have been associated with many human tumors, especially the Epstein-Barr virus (EBV), which shows a high viral load in DTC. In order to evaluate the ability of the virus to cause morphological and molecular changes in neoplastic thyroid cell lines TPC-1, BCPAP, and 8505C, a viral adaptation was performed for the analysis of EBV cytopathic effect (CPE), viral kinetics and gene expression analysis of oncogenes KRAS, NRAS, HRAS, and TP53. Comparison of inoculated cells with non-inoculated control cells showed that all tumor cell lines were permissive to the virus. The virus caused CPE in the TPC-1 and 8505C, but not in BCPAP cells. Viral kinetic was similar in both BCPAP and 8505C with a point of eclipse at 24 h post infection. TPC-1 cell line displayed a decreasing growth curve, with highest viral load right after inoculation, which decreased over time. There was hyperexpression of TP53 and NRAS in BCPAP cell (p = 0.012 and p = 0.0344, respectively). The 8505C cell line presented NRAS hyperexpression (p = 0.0255), but lower TP53 expression (p = 0.0274). We concluded that neoplastic thyroid cell lines are permissive to EBV that the virus presents different viral kinetic patterns in different cell lines and produces a CPE on both well-differentiated and undifferentiated thyroid cell lines. We also demonstrated that EBV interferes in oncogene expression in thyroid neoplastic cell lines, suggesting that these effects could be related to different tumor progression patterns.

Utricularia belongs to Lentibulariaceae, a widespread family of carnivorous plants that possess ultra-small and highly dynamic nuclear genomes. It has been shown that the Lentibulariaceae genomes have been shaped by transposable elements expansion and loss, and multiple rounds of whole-genome duplications (WGD), making the family a platform for evolutionary and comparative genomics studies. To explore the evolution of Utricularia , we estimated the chromosome number and genome size, as well as sequenced the terrestrial bladderwort Utricularia reniformis (2 n = 40, 1C = 317.1-Mpb). Here, we report a high quality 304 Mb draft genome, with a scaffold NG50 of 466-Kb, a BUSCO completeness of 87.8%, and 42,582 predicted genes. Compared to the smaller and aquatic U. gibba genome (101 Mb) that has a 32% repetitive sequence, the U. reniformis genome is highly repetitive (56%). The structural differences between the two genomes are the result of distinct fractionation and rearrangements after WGD, and massive proliferation of LTR-retrotransposons. Moreover, GO enrichment analyses suggest an ongoing gene birth-death-innovation process occurring among the tandem duplicated genes, shaping the evolution of carnivory-associated functions. We also identified unique patterns of developmentally related genes that support the terrestrial life-form and body plan of U. reniformis . Collectively, our results provided additional insights into the evolution of the plastic and specialized Lentibulariaceae genomes.

Massive occurrence of trichodinids is frequently accompanied by serious disease in fish farms. In this study, trichodinid species from the gills and skin of Nile tilapia (Oreochromis niloticus) farmed in the central-western region of Brazil (state of Goiás) were morphologically characterized. Dried slides were prepared from the parasites and were impregnated with silver nitrate (2%). Morphometric characteristics were determined and schematic drawings of the denticles were made using photomicrographs produced from the slides. Seven species of trichodinid ectoparasites (Protozoa: Ciliophora: Trichodinidae) were found parasitizing the gills: four of the genus Trichodina Ehrenberg, 1838; one of Tripartiella Lom, 1959; one of Paratrichodina Lom, 1963; and one of Trichodinella Ṧrámek-Huṧek, 1953. On the body surface, three specimens of the genus Trichodina were identified. This study presents new geographical records of trichodinids in Brazil, thus confirming that Trichodina centrostrigeata, Trichodina compacta, Trichodina heterodentata, Paratrichodina africana and Tripartiella orthodens are widely distributed worldwide. Additionally, the first record of the genus Trichodinella in Brazil is presented.

Backgound: Leg length discrepancy (LLD) can be related to different pathologies, due to an inadequate distribution of mechanical loads, as well as gait kinematics asymmetries resulted from LLD., Research Question: To validate a model to predict anatomical LLD (ALLD) based on gait kinematics., Methods: Gait of 39 participants with different lower limb pathologies and mild discrepancy were collected. Pelvic, hip, knee and ankle kinematics were measured with a 3D motion analysis system and ALLD, femur discrepancy (FD) and tibia discrepancy (TD) were measured by a computerized digital radiograph. Three multiple linear regression models were used to identify the ability of kinematic variables to predict ALLD (model 1), FD (model 2) and TD (model 3)., Results: Difference between peak knee and hip flexion of the long and short lower limb was selected by models 1 (p < 0.001) and 2 (p < 0.001). Hip adduction was selected as a predictor only by model 1 (p = 0.05). Peak pelvic obliquity and ankle dorsiflexion were not selected by any model and model 3 did not retain any dependent variable (p > 0.05). Regression models predicted mild ALLD with moderate accuracy based on hip and knee kinematics during gait, but not ankle strategies. Excessive hip flexion of the longer limb possibly occurs to reduce the limb to equalize the LLD, and discrepancies of the femur and tibia affects gait cycle in a different way., Significance: This study showed that kinematic variables during gait could be used as a screening tool to identify patients with ALLD, reducing unnecessary x-ray exposure and assisting rehabilitation programs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Species of the genera Psychotria and Palicourea are sources of indole alkaloids, however, the distribution of alkaloids within the plants is not known. Analysing the spatial distribution using desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) has become attractive due to its simplicity and high selectivity compared to traditional histochemical techniques., Objectives: To apply DESI-MSI to visualise the alkaloid distribution on the leaf surface of Psychotria prunifolia and Palicourea coriacea and to compare the distributions with HPLC-MS and histochemical analyses., Methodology: Based upon previous structure elucidation studies, four alkaloids targeted in this study were identified using high resolution mass spectrometry by direct infusion of plant extracts, and their distributions were imaged by DESI-MSI via tissue imprints on a porous Teflon surface. Relative quantitation of the four alkaloids was obtained by HPLC-MS/MS analysis performed using multiple-reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer., Results: Alkaloids showed distinct distributions on the leaf surfaces. Prunifoleine was mainly present in the midrib, while 10-hydroxyisodeppeaninol was concentrated close to the petiole; a uniform distribution of 10-hydroxyantirhine was observed in the whole leaf of Psychotria prunifolia. The imprinted image from the Palicourea coriacea leaf also showed a homogeneous distribution of calycanthine throughout the leaf surface., Conclusion: Different distributions were found for three alkaloids in Psychotria prunifolia, and the distributions found by MSI were in complete accordance with HPLC-MS analysis and histochemical results. The DESI-MSI technique was therefore demonstrated to provide reliable information about the spatial distribution of metabolites in plants. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background & Aims: Both green tea and metformin are used as adjuvants to treat and prevent complications associated with obesity; however, studies comparing their action and interaction in non-diabetic overweight women have not been reported. Thus, the current study evaluated the effects of green tea extract and metformin, both individually and in combination, on type 2 diabetes risk factors in non-diabetic overweight women., Methods: A total of 120 overweight women were randomly assigned in a double-blind manner to 1 of 4 groups, as follows: control (n = 29; 1 g of cellulose), green tea (n = 32; 1 g of dry green tea extract), metformin (n = 28; 1 g of metformin), and green tea + metformin (n = 31; 1 g of dry green tea extract + 1 g of metformin). Each group took the indicated capsules daily for 12 weeks. Anthropometric measurements, body composition, and fasting blood samples were evaluated., Results: Although no significant interactions were observed in glycaemic control (p = 0.07), green tea in the absence of metformin reduced fasting glucose (-4.428 ± 2.00; p = 0.031), but when combined the lowering effect was nullified. In contrast, metformin increased HbA1c concentration (0.048 ± 0.189%; p = 0.017) and also reduced body weight (-1.318 ± 0.366 kg; p = 0.034) and LM (lean mass) (-1.249 ± 0.310; p = 0.009). Regarding lipid parameters, green tea significantly reduced total cholesterol and LDL-c., Conclusions: Green tea was superior to metformin in improving glycaemic control and lipid profile in non-diabetic overweight women and, therefore, green tea extract is a promising alternative for reducing type 2 diabetes risk in overweight women., (Copyright © 2017 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

The asymmetry indexes have helped cytotaxonomists to interpret and classify plant karyotypes for species delimitation efforts. However, there is no consensus about the best method to calculate the intrachromosomal asymmetry. The present study aimed to compare different intrachromosomal asymmetry indexes in order to indicate which are more efficient for the estimation of asymmetry in different groups of orchids. Besides, we aimed to compare our results with the Orchidaceae phylogenetic proposal to test the hypothesis of Stebbins (1971). Through a literature review, karyotypes were selected and analyzed comparatively with ideal karyotypes in a cluster analysis. All karyotypes showed some level of interchromosomal asymmetry, ranging from slightly asymmetric to moderately asymmetric. The five tested intrachromosomal asymmetry indexes indicated Sarcoglottis grandiflora as the species with the most symmetrical karyotype and Christensonella pachyphylla with the most asymmetrical karyotype. In the cluster analysis, the largest number of species were grouped with the intermediary ideal karyotypes B or C. Considering our results, we recommend the combined use of at least two indexes, especially Ask% or A1 with Syi, for cytotaxonomic analysis in groups of orchids. In an evolutionary perspective, our results support Stebbins' hypothesis that asymmetric karyotypes derive from a symmetric karyotypes.

Orchidaceae is a widely distributed plant family with very diverse vegetative and floral morphology, and such variability is also reflected in their karyotypes. However, since only a low proportion of Orchidaceae has been analysed for chromosome data, greater diversity may await to be unveiled. Here we analyse both genome size (GS) and karyotype in two subtribes recently included in the broadened Maxillariinea to detect how much chromosome and GS variation there is in these groups and to evaluate which genome rearrangements are involved in the species evolution. To do so, the GS (14 species), the karyotype - based on chromosome number, heterochromatic banding and 5S and 45S rDNA localisation (18 species) - was characterised and analysed along with published data using phylogenetic approaches. The GS presented a high phylogenetic correlation and it was related to morphological groups in Bifrenaria (larger plants - higher GS). The two largest GS found among genera were caused by different mechanisms: polyploidy in Bifrenaria tyrianthina and accumulation of repetitive DNA in Scuticaria hadwenii. The chromosome number variability was caused mainly through descending dysploidy, and x=20 was estimated as the base chromosome number. Combining GS and karyotype data with molecular phylogeny, our data provide a more complete scenario of the karyotype evolution in Maxillariinae orchids, allowing us to suggest, besides dysploidy, that inversions and transposable elements as two mechanisms involved in the karyotype evolution. Such karyotype modifications could be associated with niche changes that occurred during species evolution., (© 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.)

The karyotype is shaped by different chromosome rearrangements during species evolution. However, determining which rearrangements are responsible for karyotype changes is a challenging task and the combination of a robust phylogeny with refined karyotype characterization, GS measurements and bioinformatic modelling is necessary. Here, this approach was applied in Heterotaxis to determine what chromosome rearrangements were responsible for the dysploidy variation. We used two datasets (nrDNA and cpDNA, both under MP and BI) to infer the phylogenetic relationships among Heterotaxis species and the closely related genera Nitidobulbon and Ornithidium. Such phylogenies were used as framework to infer how karyotype evolution occurred using statistical methods. The nrDNA recovered Ornithidium, Nitidobulbon and Heterotaxis as monophyletic under both MP and BI; while cpDNA could not completely separate the three genera under both methods. Based on the GS, we recovered two groups within Heterotaxis: (1) "small GS", corresponding to the Sessilis grade, composed of plants with smaller genomes and smaller morphological structure, and (2) "large GS", corresponding to the Discolor clade, composed of plants with large genomes and robust morphological structures. The robust karyotype modeling, using both nrDNA phylogenies, allowed us to infer that the ancestral Heterotaxis karyotype presented 2n = 40, probably with a proximal 45S rDNA on a metacentric chromosome pair. The chromosome number variation was caused by ascending dysploidy (chromosome fission involving the proximal 45S rDNA site resulting in two acrocentric chromosome pairs holding a terminal 45S rDNA), with subsequent descending dysploidy (fusion) in two species, H. maleolens and H. sessilis. However, besides dysploidy, our analysis detected another important chromosome rearrangement in the Orchidaceae: chromosome inversion, that promoted 5S rDNA site duplication and relocation., Competing Interests: The authors have declared that no competing interests exist.

Purpose: To investigate the impact of organizational characteristics and processes of care on hospital mortality and resource use in patients with cancer admitted to intensive care units (ICUs)., Patients and Methods: We performed a retrospective cohort study of 9,946 patients with cancer (solid, n = 8,956; hematologic, n = 990) admitted to 70 ICUs (51 located in general hospitals and 19 in cancer centers) during 2013. We retrieved patients' clinical and outcome data from an electronic ICU quality registry. We surveyed ICUs regarding structure, organization, staffing patterns, and processes of care. We used mixed multivariable logistic regression analysis to identify characteristics associated with hospital mortality and efficient resource use in the ICU., Results: Median number of patients with cancer per center was 110 (interquartile range, 58 to 154), corresponding to 17.9% of all ICU admissions. ICU and hospital mortality rates were 15.9% and 25.4%, respectively. After adjusting for relevant patient characteristics, presence of clinical pharmacists in the ICU (odds ratio [OR], 0.67; 95% CI, 0.49 to 0.90), number of protocols (OR, 0.92; 95% CI, 0.87 to 0.98), and daily meetings between oncologists and intensivists for care planning (OR, 0.69; 95% CI, 0.52 to 0.91) were associated with lower mortality. Implementation of protocols (OR, 1.52; 95% CI, 1.11 to 2.07) and meetings between oncologists and intensivists (OR, 4.70; 95% CI, 1.15 to 19.22) were also independently associated with more efficient resource use. Neither admission to ICUs in cancer centers compared with general hospitals nor annual case volume had an impact on mortality or resource use., Conclusion: Organizational aspects, namely the implementation of protocols and presence of clinical pharmacists in the ICU, and close collaboration between oncologists and ICU teams are targets to improve mortality and resource use in critically ill patients with cancer., (© 2016 by American Society of Clinical Oncology.)

Statement of Problem: Direct and indirect techniques are used for intracanal impression and fabrication of cast metal posts. However, whether those techniques affect the accuracy of cast metal posts is unknown., Purpose: The purpose of this randomized clinical trial was to evaluate the accuracy of cast metal posts depending on tooth position and impression technique., Material and Methods: Endodontically treated teeth (anterior or posterior) planned to receive cast metal posts and complete crowns were randomized according to impression technique (direct or indirect). Impressions were made by 2 operators, and time for the impression was recorded. All impressions and cast metal posts were photographed to assess any possible differences between techniques in length of the cast metal post., Results: All cast metal posts were shorter than the impressions. The mean reduction for the metal posts was 2.3% for direct in anterior teeth, 5.7% for direct in posterior teeth, 6.3% for indirect in anterior teeth, and 7.2% for indirect in posterior teeth (all P<.05). Statistically significant differences were found between time of technique and tooth position (P=.031), with the direct technique more time consuming than the indirect technique (P<.001) for both tooth positions. For the indirect technique, the impression times for both tooth groups were similar (P=.459)., Conclusions: Both of the intracanal impression techniques resulted in cast posts that were shorter than the impressed post space. The discrepancy was greatest for the indirect technique. Nevertheless, all posts were considered clinically acceptable and were cemented., (Copyright © 2016 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

Osteosarcoma (OS) is the most common primary bone cancer in childhood. OS is an aggressive disease, and metastatic patients evolve with very poor clinical outcomes. Genetically, OSs are extremely complex tumors, and the related metastatic process is not well understood in terms of the biology of the disease. In this context, long non-coding RNAs (lncRNAs) have emerged as an important class of gene expression regulators that play key roles in the invasion and metastasis of several human tumors. Here, we evaluated the expression of HULC, which is an lncRNA that is associated with the tumor metastatic process, and assessed its potential role as a prognostic marker in OS. HULC expression was evaluated in primary OS samples using real-time RT-PCR. HULC expression status was determined by receiver operating characteristic (ROC) analysis, and its association with survival was assessed using the Kaplan-Meier method. The HULC expression level was not significantly associated with the clinicopathological characteristics of the OS patients. However, our data demonstrated that higher levels of expression of HULC were associated with lower survival rates in OS patients, both in terms of overall and event-free survival. Elevated HULC expression was associated with poor clinical outcomes among the OS patients, which suggests that HULC could be a potential prognostic biomarker in OS.

Aim: To evaluate the potential for electromagnetic interference (EMI) of electronic apex locators (EALs) and a gutta-percha heating device (HD) in patients with implantable cardiac pacemakers (ICPs) or cardioverter-defibrillators (ICDs)., Methodology: Two types of EALs (Romiapex A-15 and Novapex) and a HD (Touch'n Heat) were tested in patients followed in an outpatient clinic for cardiac arrhythmias. The heart rhythm was monitored on a computer screen during all experimental phases. After baseline data collection, the patient held each appliance (turned on) for 30 s, simulating their clinical use. If background noise was detected on the cardiac monitor, the sensitivity of the ICP/ICD was lowered by the cardiologist to evaluate the intensity of the detected EMI., Results: Twelve patients were evaluated (5 female and 7 male), and in nine instances, background noise in their cardiac devices related to the use of the endodontic devices was detected (6 patients). After lowering the sensitivity of the cardiac implants, three patients had more severe EMI in six instances, including pauses in ICP function. The presence of a symptomatic or asymptomatic pause was related to the patient's underlying heart rhythm. The HD device produced background noise more often compared to EALs. These were associated with more severe types of EMI., Conclusion: The EALs and gutta-percha HD were capable of causing background noise detection or pauses in cardiac implants in vivo. The use of electronic dental devices nearby patients with cardiac implants should be carefully considered in clinical practice., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Purpose: To compare the cytotoxicity, gelatinolytic activity, and protein levels (MMP-2 and MMP-9) produced by 3T3 fibroblasts cells after stimulation with GuttaFlow 2 and AH Plus., Methods: 3T3 fibroblasts were incubated with elutes of GuttaFlow 2 and AH Plus for 24 h. The cytotoxicity of tested materials was determined using the MTT and the LDH assay. Supernatants of cell cultures incubated with sealers were collected to determine the levels of MMP-2 and MMP-9 gelatinolytic activity by gelatin zymography. Cell lysates were used to determine MMP-2 and MMP-9 protein levels by Western Blot. Data were analyzed using ANOVA and Tukey test (P&lt;0.05)., Results: AH Plus showed significantly less cell viability (mitochondrial activity of cells) than GuttaFlow 2 (P&lt;0.01). Moreover, GuttaFlow 2 was noncytotoxic, showing no statistically significant difference in LDH leakage levels compared to the control group (P&gt;0.05). Specific characterization of MMPs demonstrated that GuttaFlow 2 seemed not to affect MMP-2 levels compared with the control group, while AH Plus had elevated gelatinolytic activity and protein levels of MMP-2 as confirmed by quantitative measurements. No detectable gelatinolytic activity or protein levels of MMP-9 (92 kDa) was observed in any tested group., Conclusions: GuttaFlow 2 did not showed cytotoxic effects and did not induce MMP-2 or MMP-9 expression.

This study aimed to verify whether transient inflammatory reactions incited by the administration of intra-articular platelet-rich plasma (PRP) affected joint components through short- and long-term in vivo evaluation of inflammatory biomarkers and extracellular matrix degradation products in synovial fluid. The effects of PRP were analyzed in a short phase protocol (SPP) and in a prolonged phase protocol (PPP), using saline-injected joints as controls. In the SPP, higher white blood cell counts and prostaglandin E2 and total protein concentrations were observed in the synovial fluid of PRP-treated joints (P < 0.05). There were no differences between the interleukin-1β, interleukin-1 receptor antagonist protein, tumor necrosis factor-α, chondroitin sulfate, or hyaluronic acid concentrations between PRP and saline injected joints. In the PPP, there were no differences in evaluated parameters between groups. PRP injection elicits a mild and self-limiting inflammatory response shortly after administration, without long-term deleterious effects on joint homeostasis.

This study had the aims of evaluating the antimicrobial characteristics of Stomoxys calcitrans (Diptera: Muscidae) larvae against the fungal isolates CG138, CG228 and ESALQ986 of Beauveria bassiana sensu lato (Balsamo-Crivelli) Vuillemin, 1912 (Hypocreales: Cordycipitaceae). S. calcitrans eggs, larvae and pupae were exposed to these same isolates. Statistical analysis showed that the immature stages of S. calcitrans were not susceptible to the fungal isolates used, regardless of the exposure method. Diffusion test on solid culture medium reveled that macerated S. calcitrans larvae exposed to isolate CG138 reduced CG138 fungal development. The analysis of the chromatographic profiles indicated that the macerate or mucus of larvae of the control group and the groups exposed to the isolate CG138 presented different profiles. Reduced development of the isolate CG138 on the larvae cuticle was observed by means of scanning electron microscopy.

This experimental controlled study was performed to evaluate the composition of autologous processed plasma (APP), and the effects of APP intra-articular injection into healthy equine metacarpophalangeal joints. The effects on joints were analysed with a short-phase protocol and a prolonged-phase protocol using saline-injected joints as controls. For the short protocol, horses received one intra-articular APP injection. Synovial fluid samples were collected prior to the injection and 3, 6, 24, 48, and 16 h after treatment. For the prolonged protocol, the joints received three weekly injections of APP, and samples were collected at 0, 7, 14, 21, and 28 days before APP administration. IL1-ra level was found to be increased in APP compared to plasma. Upon intra-articular administration of APP, transient (up to 24 h) increases in white blood cell (WBC) counts along with elevated protein and prostaglandin E2 (PGE2) concentrations were observed in the treated joints. Over the 28-day observation period, APP did not elicit changes relative to baseline levels, but WBC counts, PGE2 and chondroitin sulphate concentrations were lower than those found in the control. In conclusion, APP intra-articular injection induced a mild and transitory inflammatory response but no inflammation reaction was observed over a longer period of treatment and observation.

Aeschynomene falcata is an important forage species; however, because of low seed production, it is underutilized as forage species. Aeschynomene is a polyphyletic genus with a challenging taxonomic position. Two subgenera have been proposed, and it is suggested that Aeschynomene can be split in 2 genera. Thus, new markers, such as microsatellite sequences, are desirable for improving breeding programs for A. falcata. Based on transferability and in situ localization, these microsatellite sequences can be applied as chromosome markers in the genus Aeschynomene and closely related genera. Here, we report the first microsatellite library developed for this genus; 11 microsatellites were characterized, with observed and expected heterozygosities ranging from 0.0000 to 0.7143 and from 0.1287 to 0.8360, respectively. Polymorphic information content varied from 0.1167 to 0.7786. The departure from Hardy-Weinberg equilibrium may have resulted from frequent autogamy, which is characteristic of A. falcata. Of the 11 microsatellites, 9 loci were cross-amplified in A. brevipes and A. paniculata and 7 in Dalbergia nigra and Machaerium vestitum. Five of these 7 cross-amplified microsatellites were applied as probes during the in situ hybridization assay and 2 showed clear signals on A. falcata chromosomes, ensuring their viability as chromosome markers.

The microbiota present in Stomoxys calcitrans larvae may assist their survival in contaminated environments through production of inhibitory substances. Bacteriological identification methods, the polymerase chain reaction (PCR) and scanning electron microscopy (SEM) were used to detect a bacterium naturally present in mucus and macerated S. calcitrans larvae. The antifungal activity was determined based on the results from disk diffusion tests on an artificial solid medium. The bacterium was identified as Stenotrophomonas maltophilia and presented antifungal activity against Beauveria bassiana sensu lato isolates CG 138, CG 228 and ESALQ 986. This result suggests that the larval microbiota is a factor that can compromise the use of B. bassiana s.l. fungus for biological control of S. calcitrans larvae.

Indole alkaloids and synthetic indole derivatives are well known for their therapeutic importance. In fact, preclinical and clinical studies had already demonstrated several pharmacological activities for these compounds. Here, we overview the multifunctional potential of these molecules for the inhibition of enzymes related to neurodegenerative disease: acetylcholinesterase (AChE), butyrylcholinesterase (BChE), monoamine oxidases A and B (MAO-A and MAO-B). A focus will be given on Psychotria L. genus, considering its reported central effects. Finally, three Psychotria alkaloids, namely desoxycordiofoline (61), bahienoside A (64) and bufotenine (65), along with the synthetic indole derivatives (5S)- 5-(1H-indol-3-ylmethyl)imidazolidine-2,4-dione (66), 5-(1H-indol-3-ylmethyl)-2-thioxoimidazolin-4-one (67), 5-(1Hindol- 3-ylmethyl)-3-methyl-2-thioxoimidazolidin-4-one (68), and methyl 2-(aminoN-(2-(4-methylcyclohex-3-enyl)propan- 2-yl)methanethioamino)-3-(1H-indol-3-yl)propanoate (69), were evaluated in vitro regarding their interactions with AChE, BChE, MAO-A and MAO-B. It was observed that 66 and 68 were able to inhibit MAO-A activity with IC50 value of 8.23 and 0.07 μM. Molecular docking calculations were performed in order to understand the interactions between both ligands (66 and 68) and MAO-A. It was observed that the indole scaffold of both compounds bind into the MAO-A active site in the same orientation, establishing van der Waals contacts with lipophilic amino acids. Additionally, the hydantoin ring of 66 is able to interact by hydrogen bonds with two conserved water molecules in the MAO-A active site, while the methyl-thiohydantoin ring of 68 is within hydrogen bond distance from the hydrogen atom attached to the (N-5) of FAD cofactor. Taking together, our findings demonstrate that the indolyl-hydantoin and indolylmethyl-thiohydantoin rings might consists of good scaffolds for the development of new MAO-A inhibitors possessing neuroprotective properties.

Interspecific hybridization is a primary cause of extensive morphological and chromosomal variation and plays an important role in plant species diversification. However, the role of interploidal hybridization in the formation of hybrid swarms is less clear. Epidendrum encompasses wide variation in chromosome number and lacks strong premating barriers, making the genus a good model for clarifying the role of chromosomes in postzygotic barriers in interploidal hybrids. In this sense, hybrids from the interploidal sympatric zone between E. fulgens (2n = 2x = 24) and E. puniceoluteum (2n = 4x = 56) were analyzed using cytogenetic techniques to elucidate the formation and establishment of interploidal hybrids. Hybrids were not a uniform group: two chromosome numbers were observed, with the variation being a consequence of severe hybrid meiotic abnormalities and backcrossing with E. puniceoluteum. The hybrids were triploids (2n = 3x = 38 and 40) and despite the occurrence of enormous meiotic problems associated with triploidy, the hybrids were able to backcross, producing successful hybrid individuals with broad ecological distributions. In spite of the nonpolyploidization of the hybrid, its formation is a long-term evolutionary process rather than a product of a recent disturbance, and considering other sympatric zones in Epidendrum, these events could be recurrent.

Background: Maternal mortality and morbidity are among the top public health priorities in Brazil, being quite high, especially among the most disadvantage women. A case control study was developed to identify risk factors for severe maternal morbidity in Sao Luis, one of the poorest Brazilian State Capitals., Methods: The case-control study was carried out between 01/03/2009 and 28/02/2010 in two public high-risk maternities facilities and in two intensive care units (ICUs) for referral of obstetric cases. All cases hospitalized due to complications during gestation period, childbirth or up to 42 days of puerperium and who fulfilled any of Mantel's and/or Waterstone's criteria were identified. Two controls per case were randomly selected among patients of the same clinics discharged for other reasons. Data were obtained through a structured interview as well as from medical charts and prenatal cards and included sociodemographic variables, clinical and obstetric histories, behavioral factors and exposure to stress factors during pregnancy, pre-natal assistance and obstetric complication and childbirth care., Results: In the final model of the unconditional logistic regression analysis, being older than 35 years (OR=3.11; 95% CI:1.53-6.31), previous hypertension (OR=2.52; 95% CI:1.09-5.80), history of abortion (OR=1.61; 95% CI:0.97-2.68), 4-5 pre-natal consultations (OR=1.78; 95% CI:1.05-3.01) and 1-3 pre-natal consultations (OR=1.89; 95% CI:1.03-3.49) were independently associated with severe maternal morbidity., Conclusions: The results corroborate the importance of reproductive healthcare, of identifying a high-risk pregnancy and of a qualified and complete prenatal care to prevent severe morbid events.

The preservation of teeth to support an attachment-retained overdenture is an appropriate and stable alternative to extractions and complete dentures. A key to success is the strategic selection of teeth for retention. This clinical report discusses a method for fabricating an overdenture based on a combination of direct and indirect impression techniques. This technique is a simple and time-efficient alternative to traditional methods that aids in overcoming difficulties in fabricating overdentures., (Copyright © 2013 The Editorial Council of the Journal of Prosthetic Dentistry. Published by Mosby, Inc. All rights reserved.)

Rhipicephalus microplus (Canestrini) is an ectoparasite accountable for great economic losses. The use of entomopathogenic fungi to control arthropods has shown promising responses. The present study evaluated the virulence of Isaria farinosa (Holmsk.) Fr., Isaria fumosorosea (Wize) Brown and Smith, and Purpureocillium lilacinum (=Paecilomyces lilacinus) (Thom.) Samson to engorged females, eggs, and larvae of R. microplus. There were four treatment groups (10(5), 10(6), 10(7), and 10(8) conidia ml(-1)) and the control group (water and Tween 80, 0.1% v/v). The treatment was based on immersion of the specimen in 1 ml of the suspension or control solution. The study observed changes in egg viability and larval mortality after treatment. The results showed that I. farinosa, P. lilacinum, and I. fumosorosea caused alterations in the biological parameters of R. microplus ticks. I. fumosorosea presented the greatest potential to control R. microplus engorged females in vitro, causing a 49% decrease in nutritional index. All fungal isolates presented significant reduction in the egg production index. I. farinosa reduced the hatching percentage if the eggs were treated with the two highest conidial concentrations. All conidial concentrations of I. fumosorosea were able to reduce the hatching percentage significantly. All tested isolates showed pathogenicity toward unfed R. microplus larvae. As far as we know, this is the first study reporting the effect in vitro of I. farinosa, I. fumosorosea, and P. lilacinum to different developmental stages of R. microplus ticks.

Our objectives were to characterize the urinary excretion of glycosaminoglycans (GAGs) in horse osteoarthritis, and to investigate the effects of chondroitin sulfate (CS) and glucosamine (GlcN) upon the disease. Urinary GAGs were measured in 47 athletic horses, 20 healthy and 27 with osteoarthritis. The effects of CS and GlcN were investigated in mild osteoarthritis. In comparison to normal, urinary GAGs were increased in osteoarthritis, including mild osteoarthritis affecting only one joint. Treatment with CS+GlcN led to a long lasting increase in the urinary CS and keratan sulfate (KS), and significant improvement in flexion test of tarsocrural and metacarpophalangeal joints was observed. In conclusion, urinary CS and KS seems to reflect the turnover rates of cartilage matrix proteoglycans, and the measurement of these compounds could provide objective means of evaluating and monitoring joint diseases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The aim of this study was to assess the effect of triazine incorporation to denture materials on biofilm formation of saliva derived from microcosms of patients who are positive for Candida albicans. Biofilms were formed on microwave-cured acrylic resin, one hard denture liner, and two soft denture liners containing 0, 2.5, 5, and 10% triazine. For experimental subset (n = 10), mechanical properties of the materials and colony-forming unit counts from the biofilms formed on the materials were assessed. Flexural strength and modulus decreased with the addition of 2.5% triazine (p < 0.01). In general, the addition of 5 and 10% triazine leaded to more soluble materials (p < 0.001). Saliva donor with candidiasis resulted in higher counts of total microorganisms (p = 0.0294) and Streptococci (p = 0.0008). Soft denture liners showed the highest counts for total microorganisms, Streptococci, and Candida species (p < 0.001). The addition of triazine directly to denture materials was not beneficial in reducing biofilm formation in a complex biofilm model., (Copyright © 2012 Wiley Periodicals, Inc.)

Context and Objective: Evaluation of severe maternal morbidity has been used in monitoring of maternal health. The objective of this study was to estimate its incidence and main causes in São Luís, Maranhão, Brazil., Design and Setting: Prospective longitudinal study, carried out in two public high-risk maternity hospitals and two public intensive care units (ICUs) for referral of obstetric cases from the municipality., Methods: Between March 1, 2009, and February 28,2010, all cases of severe maternal morbidity according to the Mantel and Waterstone criteria were identified. The sociodemographic and healthcare characteristics of the extremely severe cases were compared with the less severe cases, using the Fisher, χ(2), Student t and Mann-Whitney tests, with a significance level of < 0.05., Results: 127 cases of severe maternal morbidity were identified among 8,493 deliveries, i.e. an incidence of 15.0/1000 deliveries. Out of 122 cases interviewed, 121 cases were within the Waterstone criteria and 29 were within the Mantel criteria, corresponding to incidences of 14.1/1000 and 3.4/1000 deliveries, respectively. These rates were lower than those described in the literature, possibly due to case loss. The main causes were hypertension during pregnancy, which was more frequent in less severe cases (P = 0.001) and obstetric hemorrhage, which was more common among extremely severe cases (P = 0.01)., Conclusions: Direct obstetric disorders were the main causes of severe maternal morbidity in São Luís, Maranhão. Investigation and monitoring of severe morbidity may contribute towards improving obstetric care in the municipality.

The genus Citrus is well-known for its economic importance and complex taxonomy. Only three to six Citrus taxa are considered true biological species, among them is Citrus medica, the citron. Previous studies based on chromomycin A3 (CMA)/4',6-diamidino-2-phenylindole staining showed that the citron has a homomorphic karyotype, consisting of four distinct chromosome types according to the CMA(+) heterochromatin distribution. Based on the pattern of CMA(+) bands and the hybridization of 25 bacterial artificial chromosomes (BACs) from a genomic library of Poncirus trifoliata, a closely related species to the genus Citrus, we were able to identify each chromosome pair and build a comparative cytogenetic map for C. medica. The data showed a high degree of sequence conservation between these genera, enabling heterologous hybridization of BACs, and the establishment of chromosomal homeologies. It was thus possible to visualize changes in the position of some BACs in relation to CMA(+) bands. Since no breakdown of synteny was observed between these species, expansions and contractions in repetitive DNA sequences seem to be the major driving force of chromosomal evolution since the separation of these two genera.

Rhipicephalus microplus, known as the cattle tick, causes serious economic losses in the Brazilian cattle industry each year. Traditional parasite control is primarily based on the use of chemical acaricides, which unfortunately have many negative side effects. Biological control is seen as a promising alternative to chemical acaricide use. This study evaluates the entomopathogenic fungus Lecanicillium lecanii for effectiveness in controlling engorged females, eggs, and larvae of R. microplus. Conidial formulations of L. lecanii, isolate CG 420, were prepared in both oil (15% mineral oil) and aqueous suspensions. Ticks were immersed in a 1ml oil-based conidial suspension at 1x10(8)conidiaml(-1) or one of several aqueous conidial suspensions at 1x10(5), 10(6), 10(7) or 10(8)conidiaml(-1). The control groups were immersed in water or oil solutions with no conidia. Treatments with aqueous conidial suspensions were conducted with 10 ticks per group (1x10(5), 10(6), 10(7), 10(8)conidiaml(-1) or control) whereas the oil treatments used 30 ticks per group (1x10(8)conidiaml(-1) or control). Bioassays were repeated twice on different days with different batches of conidia. After treatment, the biological parameters of engorged females were evaluated, while eggs and larvae were evaluated taking into consideration hatchability and mortality, respectively. The results indicate that L. lecanii has the potential to control engorged females, eggs and larvae of R. microplus. Better results were observed when conidial oil-based suspension was used. In general, engorged females treated with 1x10(8)conidiaml(-1) oil suspensions died before laying eggs, resulting in 97.6% of tick control. As far as we know, this is the first report of the effects of L. lecanii on R. microplus tick.

Allopolyploids represent natural experiments in which DNA sequences from different species are combined into a single nucleus and then coevolve, enabling us to follow the parental genomes, their interactions and evolution over time. Here, we examine the fate of satellite DNA over 5 million yr of divergence in plant genus Nicotiana (family Solanaceae). We isolated subtelomeric, tandemly repeated satellite DNA from Nicotiana diploid and allopolyploid species and analysed patterns of inheritance and divergence by sequence analysis, Southern blot hybridization and fluorescent in situ hybridization (FISH). We observed that parental satellite sequences redistribute around the genome in allopolyploids of Nicotiana section Polydicliae, formed c. 1 million yr ago (Mya), and that new satellite repeats evolved and amplified in section Repandae, which was formed c. 5 Mya. In some cases that process involved the complete replacement of parental satellite sequences. The rate of satellite repeat replacement is faster than theoretical predictions assuming the mechanism involved is unequal recombination and crossing-over. Instead we propose that this mechanism occurs with the deletion of large chromatin blocks and reamplification, perhaps via rolling circle replication.

Amaiouine, a cyclopeptide alkaloid, was isolated from the ethanolic extract of the leaves of Amaioua guianensis. The structure was elucidated by NMR spectroscopy and confirmed by X-ray crystallography.

Stomoxys calcitrans control is primarily based on the use of chemical products; however, new methods for control have been investigated. Biological control has been proposed as a promising alternative to traditional chemicals. The current study evaluated three methods in vitro to treat S. calcitrans eggs with the fungus Metarhizium anisopliae and assessed the virulence of this fungus to immature stages of this fly. In the first method of egg exposure, eggs were immersed in conidial suspensions and transferred to Petri plates with high humidity. A high mortality was observed in both treated and control groups. In the second method, the eggs were transferred to rearing medium right after immersion in conidial suspensions; the suspensions with highest conidial concentrations, 10(7) and 10(8) conidia mL(-1), caused 96.25% and 100% mortality, respectively. In the third method, the eggs were sprayed with conidial suspensions on the rearing medium. A high mortality was observed at 10(7) and 10(8) conidia mL(-1), 78.3% and 100%, respectively. The second method had the lowest mortality in the control group and was considered the most useful method for evaluating the effect of entomopathogenic fungi on S. calcitrans eggs. While eggs were very susceptible to M. anisopliae infection, larvae and pupae were not.