The effectiveness of phototherapy for surface decontamination against SARS-Cov-2. A systematic review.

COVID-19 appeared in December 2019, needing efforts of science. Besides, a range of light therapies (photodynamic therapy, ultraviolet [UV], laser) has shown scientific alternatives to conventional decontamination therapies. Investigating the efficacy of light-based therapies for environment decontamination against SARS-CoV2, a PRISMA systematic review of Phototherapies against SARS-CoV or MERS-CoV species discussing changes in viral RT-PCR was done. After searching MEDLINE/PubMed, EMBASE, and Literatura Latino-Americana e do Caribe em Ciências da Saúde we have found studies about cell cultures irradiation (18), blood components irradiation (10), N95 masks decontamination (03), inanimate surface decontamination (03), aerosols decontamination (03), hospital rooms irradiation (01) with PDT, LED, and UV therapy. The best quality results showed an effective low time and dose UV irradiation for environments and inanimate surfaces without human persons as long as the devices have safety elements dependent on the surfaces, viral charge, humidity, radiant exposure. To interpersonal contamination in humans, PDT or LED therapy seems very promising and are encouraged.

Intraspecific variation in ultrastructure and secretion of the resin canals in Anacardium humile (Anacardiaceae).

The study combines a range of light and electron microscopy methods to access variation in secretion and ultrastructure in the secretory canals in the above- and belowground stems of Anacardium humile, which here serves as a model system. The aboveground stem canals show epithelial cells with ultrastructural characteristics typical of cells active in secretion, while in the belowground stems, the subcellular characteristics are typical of cells with low rates of metabolism. The secretory canals of the belowground stems show uniformity in size and shape, a large central vacuole, a cytoplasm reduced to a thin layer at the cell periphery, and a reduced population of organelles. The aboveground stem canals had voluminous nuclei with evident nucleoli, a very dense cytoplasm with free ribosomes, polyribosomes, mitochondria with developed cristae, and ellipsoid plastids with electron-opaque droplets surrounded by a periplastid reticulum. The vacuoles were of different sizes and often had membranous contents and the dictyosomes were very developed with dilated ends to the cisternae, rough endoplasmic reticulum, and numerous vesicles. The results show that particularities in above- and belowground environment have significant implications for ultrastructural morphology and functioning of secretory canals in the stems of A. humile.

Azadirachtin-based insecticide impairs testis morphology and spermatogenesis of the southern armyworm Spodoptera eridania (Lepidoptera: Noctuidae).

BACKGROUND: In the search for alternative tools for integrated pest management, azadirachtin, a botanical insecticide, has been used with the most promising activity against Spodoptera spp., but the mechanism of cytotoxicity on reproductive organs remains unclear. Spodoptera eridania (Stoll, 1782) is a polyphagous pest with great economic importance that has become an important target to elucidate the action of azadirachtin on the reproductive organs of insect pests, helping to understand the deleterious effects caused by its exposure. This study evaluated the effects of chronic exposure to azadirachtin on the morphology and ultrastructure of S. eridania larval testes as well as larval development. RESULTS: Azadirachtin exposure (6 or 18 mg a.i. L-1 ) caused a progressive increase in cumulative mortality and reduced gain in body mass after 5 days. Testicular structure indicated a reduction in their size with internal morphological changes such as spermatogonia, spermatogonial, spermatocytes and spermatid cysts in degeneration. The occurrence of cell death in germ and somatic cells was evidenced by the TUNEL technique. Electron microscopy revealed changes in cystic cells, such as cytoplasmic membrane rupture and cytoplasmic vacuolization. Chromatin compaction, changes in the rough endoplasmic reticulum and Golgi complex cisternae were observed in germ cells. Apoptotic bodies occurred between germ cell cysts. CONCLUSION: Azadirachtin damaged the testes of S. eridania larvae, and these changes compromised spermatogenesis and consequently the development of the reproductive potential of this specimen, making azadirachtin a promising botanical insecticide for application in integrated pest management programs. © 2022 Society of Chemical Industry.

Antifungal activity of liriodenine on clinical strains of Cryptococcus neoformans and Cryptococcus gattii species complexes.

Background: Cryptoccocal meningitis continues to present high incidence among AIDS patients. The treatment of choice is the synergistic combination of flucytosine (5-FC) with amphotericin B deoxycholate (AmBd) or its lipid formulations. However, 5-FC is unavailable in many countries and AmB demands hospitalization. The combination of AmB with the fungistatic fluconazole (FLC) or the use of high FLC daily doses alone became the choice. Nonetheless, sterilization of cerebrospinal fluid is delayed with FLC monotherapy, mainly with high fungal burden. These findings suggest the search for new antifungal compounds, such as liriodenine. Methods: Liriodenine antifungal activity was evaluated by three procedures: determining the minimum inhibitory concentration (MIC) on 30 strains of the Cryptococcus neoformans (C. neoformans) complex and 30 of the Cryptococcus gattii (C. gattii) complex, using EUCAST methodology and amphotericin B deoxycholate as control; performing the time-kill methodology in two strains of the C. neoformans complex and one of the C. gattii complex; and injury to cryptococcal cells, evaluated by transmission electron microscopy (TEM). Liriodenine absorption and safety at 0.75 and 1.50 mg.kg-1 doses were evaluated in BALB/c mice. Results: Liriodenine MICs ranged from 3.9 to 62.5 µg.mL-1 for both species complexes, with no differences between them. Time-kill methodology confirmed its concentration-dependent fungicidal effect, killing all the strains below the limit of detection (33 CFU.mL-1) at the highest liriodenine concentration (32-fold MIC), with predominant activity during the first 48 hours. Liriodenine induced severe Cryptococcus alterations - cytoplasm with intense rarefaction and/or degradation, injury of organelles, and presence of vacuoles. Liriodenine was better absorbed at lower doses, with no histopathological alterations on the digestive tract. Conclusion: The fungicidal activity confirmed by time-kill methodology, the intense Cryptococcus injury observed by TEM, the absorption after gavage administration, and the safety at the tested doses indicate that the liriodenine molecule is a promising drug lead for development of anticryptococcal agents.

Midgut and fat body: Multisystemic action of pyriproxyfen on non-target organism Ceraeochrysa claveri (Neuroptera: Chrysopidae).

Morphological tools can assist in the evaluation of effects of insecticides on non-target insects. Pyriproxyfen, a juvenile hormone analog, is known to interfere with growth and metamorphosis of insects. However, there are studies showing indirect effects on natural enemies, including green lacewings. Few prior studies describe morphological effects of pyriproxyfen on target insect organs, especially on natural enemies. Through morphological tools, this study aimed to characterize the midgut and fat body, both important organs of digestion and great metabolic activity respectively, of the predator Ceraeochrysa claveri after chronic exposure to pyriproxyfen. Larvae of C. claveri were fed Diatraea saccharalis egg clusters treated with pyriproxyfen in solution of 50 or 100 mg a.i. L-1 throughout the larval stage. The biological data revealed significant increases in development time, especially in the third instar, and in cumulative mortality from the prepupal into the pupal stage. Morphological analysis of adult midgut (≤24 h old) showed damage including formation of epithelial folds, intercellular spaces, emission of cytoplasmic protrusions. Both fat body regions presented decrease of lipid droplets, vacuolization of trophocytes and mitochondrial injury featuring a multisystemic action. In both organs, pyriproxyfen exposure induced significant oxidative stress by mitochondrial superoxide production. Cytoprotective responses were induced in midgut and fat body cells by augmenting the number of cytoplasmic granules containing calcium and expression of HSP 90. Both organs proved to be efficient in presenting histopathological alterations, showing the sensitivity and applicability of this morphological tool for evaluating other insecticides in non-target organisms.

Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides.

BACKGROUND: Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. RESULTS: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 µg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 µg.mL-1), including C. krusei (250 µg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 µg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). CONCLUSION: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.

Blocking drug-induced autophagy with chloroquine in HCT-116 colon cancer cells enhances DC maturation and T cell responses induced by tumor cell lysate.

Autophagy is an important mechanism for tumor escape, allowing tumor cells to recover from the damage induced by chemotherapy, radiation therapy, and immunotherapy and contributing to the development of resistance. The pharmacological inhibition of autophagy contributes to increase the efficacy of antineoplastic agents. Exposing tumor cells to low concentrations of select autophagy-inducing antineoplastic agents increases their immunogenicity and enhances their ability to stimulate dendritic cell (DC) maturation. We tested whether the application of an autophagy-inhibiting agent, chloroquine (CQ), in combination with low concentrations of 5-fluorouracil (5-FU) increases the ability of tumor cells to induce DC maturation. DCs sensitized with the lysate of HCT-116 cells previously exposed to such a combination enhanced the DC maturation/activation ability. These matured DCs also increased the allogeneic responsiveness of both CD4+ and CD8+ T cells, which showed a greater proliferative response than those from DCs sensitized with control lysates. The T cells expanded in such cocultures were CD69+ and PD-1- and produced higher levels of IFN-γ and lower levels of IL-10, consistent with the preferential activation of Th1 cells. Cocultures of autologous DCs and lymphocytes improved the generation of cytotoxic T lymphocytes, as assessed by the expression of CD107a, perforin, and granzyme B. The drug combination increased the expression of genes related to the CEACAM family (BECN1, ATGs, MAPLC3B, ULK1, SQSTM1) and tumor suppressors (PCBP1). Furthermore, the decreased expression of genes related to metastasis and tumor progression (BNIP3, BNIP3L, FOSL2, HES1, LAMB3, LOXL2, NDRG1, P4HA1, PIK3R2) was noted. The combination of 5-FU and CQ increases the ability of tumor cells to drive DC maturation and enhances the ability of DCs to stimulate T cell responses.

Ultrastructural characterization of damage in the basement membrane of facial melasma.

The pathogenesis of melasma is not fully understood, and the role of skin basement membrane zone (BMZ) alterations in disease development and the maintenance of hypermelanogenesis are also poorly known. We performed a comparative study to characterize the ultrastructural alterations that occur in BMZ in melasma and adjacent normal skin, as well as we discuss the implications of these changes in the physiopathology of the disease. Pairs of facial skin biopsies (2 mm) from 10 women with melasma and normal skin (< 2 cm apart) were processed by Transmission Electronic Microscopy or immunohistochemistry for Melan-A counterstained with Periodic acid-Schiff stain. Cytoplasmic organelles (from keratinocyte or melanocyte), BMZ damage were assessed and melanocyte counting (total and pendulous) was done. There was greater amount of cytoplasmic organelles inside basal keratinocytes and melanocytes in melasma, as well as structural damaged areas in the lamina densa (disruptions, gaps, lower density and thinning) and anchoring fibrils (lamina lucida), compared to healthy adjacent skin. Areas with pendulous melanocytes are characterized by discontinuity of BMZ ultrastructure. The prominence of cytoplasmic organelles from melanocytes and keratinocytes evidences the involvement of both cell groups in melasma. The damage in the lamina densa and lamina lucida suggest the role of upper dermis injury/repair process in the pathogenesis of the disease.

Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides

Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. Methods: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. Results: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 µg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 µg.mL-1), including C. krusei (250 µg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 µg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). Conclusion: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.(AU)

The comet assay in Ceraeochrysa claveri (Neuroptera: Chrysopidae): A suitable approach for detecting somatic and germ cell genotoxicity induced by agrochemicals.

Some agrochemicals are genotoxic to several organisms. Nevertheless, few protocols are currently available for measuring the toxicogenetic effects of these compounds in target and non-target field-collected species of insects important to agriculture. Herein, we used the species Ceraeochrysa claveri (Neuroptera: Chrysopidae), a non-target predator insect, to investigate the ability of an azadirachtin-based biopesticide (Azamax™) to induce DNA damage. The alkaline version of the comet assay was standardized to evaluate genetic instability caused by the toxicant in somatic (gut) and germ (nurse cells and oocytes) cells of C. claveri. For this, C. claveri larvae were distributed into three groups (10/each) and treated with Azamax™ at 0, 0.3% or 0.5% throughout the larval stage. DNA damage (tail intensity) was measured in adult insects, four days after emerged. The data showed that both doses of Azamax™ (0.3% and 0.5%) were able to significantly (p < 0.05) increase DNA damage in somatic and germ cells of C. claveri. In conclusion, C. claveri (intestinal and ovarian cells) was a sensitive bioindicator for identifying Azamax™ genotoxic potential, whereas the comet assay was a useful tool for detecting the genotoxic hazard of the pesticide in the field-collected insect species. Given that estimation of adverse effects of pollutants on ecosystems is an essential component of environmental risk assessment, the approach used can be recommended to estimate the ecotoxicity of agricultural chemicals.

Negative impact of Novaluron on the nontarget insect Bombyx mori (Lepidoptera: Bombycidae).

Due to increased use of agrochemicals and growing concerns about ecotoxicology, the development of new insecticides, moving away from those with neurotoxic and broad spectrum effects towards insecticides that are safer for the environment and nontarget beneficial species, has been a research priority. Novaluron stands out among these newer insecticides, is an insect growth regulator that is used for the control of insect pests in crops grown close to mulberry plantations. Mulberry serves as food for the silkworm Bombyx mori, which is a nontarget insect of great economic importance to silk production. We investigated the lethal and sublethal effects of Novaluron on the development of B. mori. Larvae were segregated into experimental groups: the control groups (CGs) and the treatment groups (TGs), which were treated with the Novaluron concentration of 0.15 mL/L. Following exposure, we analyzed: larval mortality, changes in the insect life cicle and cytotoxic effects on the midgut cells. This is the first report about the Novaluron's effects on B.mori. We detected rupture in the integument, complete cessation of feeding, late development, incomplete ecdysis and production of defective cocoons. After 240 h of exposure, there was 100% mortality in TG larvae exposed in the 3rd instar and 20% mortality from larvae exposed in the 5th instar. Cytotoxic effects was observed, such as dilation of cells, emission of cytoplasmic protrusions, extreme rarefaction of the cytoplasm and nuclei, dilation of the endoplasmic reticulum in addition to changes in mitochondria, the presence of large digestive vacuoles and intercellular spaces and the presence of active caspase. Novaluron exposure impairs the midgut and may affect the physiological functions of this organ. Novaluron additionally compromises several phases of insect development, indicating the importance of toxicology studies that utilize different life stages of nontarget species to evaluate the safe use of insecticides.

Pest and natural enemy: how the fat bodies of both the southern armyworm Spodoptera eridania and the predator Ceraeochrysa claveri react to azadirachtin exposure.

The effects of biopesticides on insects can be demonstrated by morphological and ultrastructural tools in ecotoxicological analysis. Azadirachtin-based products are widely used as biopesticides, affecting numerous insect populations. Through morphological biomarkers, this study aimed to characterize the fat bodies of both the southern armyworm Spodoptera eridania and the predator Ceraeochrysa claveri after chronic exposure to azadirachtin. Larvae of S. eridania and C. claveri were fed with fresh purple lettuce leaves (Lactuca sativa) and egg clusters of Diatraea saccharalis treated with azadirachtin solution of 6 mg active ingredient (a.i.)/L and 18 mg a.i./L for 7 days, respectively. The biological data showed a significant reduction in survival and body mass in S. eridania and cytotoxic effects in the parietal and perivisceral fat bodies in both species. Ultrastructural cell damage was observed in the trophocytes of both species such as dilated cisternae of the rough endoplasmic reticulum and swollen mitochondria. Trophocytes of S. eridania and C. claveri of the parietal and perivisceral layers responded to those injuries by different cytoprotective and detoxification means such as an increase in the amount of cytoplasmic granules containing calcium, expression of heat shock protein (HSP)70/HSP90, and development of the smooth endoplasmic reticulum. Despite all the different means of cytoprotection and detoxification, they were not sufficient to recover from all the cellular damages. Azadirachtin exhibited an excellent performance for the control of S. eridania and a moderate selectivity for the predator C. claveri, which presents better biological and cytoprotective responses to chronic exposure to azadirachtin.

Can exposure to neem oil affect the spermatogenesis of predator Ceraeochrysa claveri?

Novel biological control methods and integrated pest management strategies are basic requirements for the development of sustainable agriculture. As a result, there is a growing demand for research on the use of plant extracts and natural enemies such as the green lacewing, Ceraeochrysa claveri, as natural pest control methods. Studies have shown that although natural compounds such as neem oil (Azadirachta indica) are effective as pest control strategies, they also cause sublethal effects on nontarget insects, such as C. claveri. The aim of this study was to examine the effects of neem oil on C. claveri testes. C. claveri larvae were fed Diatraea saccharalis eggs, which were pretreated with 0.5%, 1%, and 2% neem oil. Testes were collected from larvae, pupae, and adults and analyzed using light and electron (transmission and scanning) microscopy. Changes in cellular stress and possible cell death were also determined by TUNEL assay and the marker HSP-70. The results showed that neem oil affects the organization and distribution of cysts in the testes and the normal sequence of cyst development, causing a delay in spermatogenesis in the testes of treated insects. Tests for cellular stress and DNA fragmentation indicated there was no cellular alteration in the treated groups. Although neem oil does not induce cell death or changes in HSP-70 expression, this biopesticide negatively impacts the process of spermatogenesis and could decrease the perpetuation of this species in the agroecosystem, indicating that the use of neem oil in association with green lacewings as a biological control should be carefully evaluated.

Nanoskin® to treat full thickness skin wounds.

This experimental study evaluated 40 guinea pigs that received Nanoskin®. A full thickness skin rectangle measuring 2x4 cm was removed from the median dorsal region and the wound was covered by a 2X2 cm fragment of uncoated Nanoskin® graft (uncoated group) or Nanoskin® coated with gelatin (coated group) and sutured in the caudal region and a 2x2 cm fragment of autologous skin sutured in the cranial aspect of the surgical wound served a control. The animals were examined daily by ectoscopy and euthanized at 7, 30, 90 and 180 days postoperatively. Immediately after euthanasia, the operated area was shaved, documented with photos and removed, and prepared for morphological, morphometric and ultrastructural exam. It was found that the full thickness skin wound healed in a centripetal pattern. The healing process was similar between groups, with a more pronounced inflammatory reaction initially that gradually decreased over time. The conclusion is that the uncoated Nanoskin® or Nanoskin® coated with gelatin is a good material to treat full thickness skin wound. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res B Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 724-732, 2019.

Nanoskin® Subcutaneous Implant in Guinea Pigs.

PURPOSE: To evaluate the suitability of Nanoskin, a biomaterial derived from bacterial cellulose, as an alternate material for repairing the posterior lamellae of the eyelids. METHODS: Forty guinea pigs received a fragment of Nanoskin subcutaneously in the dorsal region. The animals received Nanoskin (group 1) or Nanoskin coated with a gelatin layer (group 2). The animals were observed daily for over 7, 30, 90, or 180 days with the subcutaneous implants in place. The animals were euthanized at a designated time and the Nanoskin was removed for histologic, morphometric, and transmission electron microscopy evaluation. RESULTS: The results were similar for both groups. The subcutaneously implanted Nanoskin resulted in good tissue integration, induced mild inflammation, and was surrounded by a delicate pseudocapsule. CONCLUSION: The good tissue integration of Nanoskin in study indicates that it may be a potential material for application as a substitute of posterior eyelid lamellar tissue. Further clinical studies are required to confirm the preliminary outcomes of this experimental study.

A unique heterologous fibrin sealant (HFS) as a candidate biological scaffold for mesenchymal stem cells in osteoporotic rats.

BACKGROUND: The injection of mesenchymal stem cells (MSCs) directly into the bone of osteoporotic (OP) patients for rapid recovery has been studied worldwide. Scaffolds associated with MSCs are used to maintain and avoid cell loss after application. A unique heterologous fibrin sealant (HFS) derived from snake venom was evaluated for the cytotoxicity of its main components and as a three-dimensional biological scaffold for MSCs to repair a critical femur defect in osteoporotic rats. METHODS: The cytotoxicity of HFS was assessed using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay and transmission electron microscopy. The cells were cultured, characterized by flow cytometry and differentiated into the osteogenic lineage. Two-month-old rats underwent ovariectomy to induce OP. After 3 months, a 5 mm critical bone defect was made in the distal end of the rat femurs and filled with HFS; HFS + MSCs; and HFS + MSCs D (differentiated into the osteogenic lineage) to evaluate the effects. An injury control group (injury and no treatment) and blank control group (no injury and no treatment) were also included. The animals were observed at days 14 and 28 by microtomographic (micro-CT) analyses, histologic and biochemical analysis, as well as scanning electron microscopy. RESULTS: The results revealed that one of the compounds of HFS, the thrombin-like enzyme extracted from snake venom, had no cytotoxic effects on the MSCs. OP was successfully induced, as demonstrated by the significant differences in the levels of 17ß-estradiol, Micro-CT analyses and alkaline phosphatase between the ovariectomized (OVX) and non-ovariectomized (NOVX) groups. The histological data revealed that at 14 days after surgery in both the OVX and NOVX animals, the HFS + CTMs and HFS + CTMsD showed a higher formation of bone cells at the site in relation to the control group (without treatment). Collagen formation was evidenced through bone neoformation in all treated and control groups. No morphological differences in the femurs of the NOVX and OVX animals were observed after the surgical procedure. Scanning electron microscopy (SEM) confirmed the histological analysis. CONCLUSIONS: The new HFS composed of two non-toxic components for MSCs showed capacity to promote the recovery of the bone lesions in OVX and NOVX animals at 14 days after surgery. In addition, the HFS enabled the differentiation of MSCs into MSCs D in the group treated with HFS + MSCs. Using the MSCs and/or MSCs D together with this biopharmaceutical could potentially enable significant advances in the treatment of osteoporotic fractures. Future clinical trials will be necessary to confirm these results.

Ampullae infection of the malpighian tubules of Bombyx mori by Alphabaculovirus / Infecção de células da ampola dos túbulos de malpighi de Bombyx mori pelo Alphabaculovirus

Alphabaculovirus is a genus of the entomopathogenic virus, whose species Bombyx mori nucleopolyhedrovirus (BmNPV) infects the silkworm, Bombyx mori, which is an important insect in the sericulture industry. A geographic isolate of BmNPV was identified in the state of Paraná, Brazil. It was infecting B. mori larvae and various organs and target tissues were identified, however, there was no information about the infection of Malpighian tubules (MT). The MT comprises the excretory system of B. mori and acts in the elimination of toxic substances and in hydroelectrolytic homeostasis. Thus, the present study examined the susceptibility and cytopathology of B. mori MT to BmNPV. To this end, hybrid fifth instar larvae were inoculated with a virus suspension at different days post-inoculation (dpi). MT segments were collected and divided into the ampullae, proximal, medial and distal regions. These were processed for light microscopy and transmission electron analysis. The MT regions revealed differences in susceptibility to BmNPV and the ampullae in its transition area was infected from the sixth dpi; the other regions did not reveal any evidence of infection. The transition area of the ampullae has not been previously described in Lepidoptera and its cytopathology revealed a hypertrophic nucleus with viroplasm, followed by the formation and development of viral polyhedra, which are common characteristics of infections by Alphabaculovirus. Thus, infection of the ampullae of the MT of B. mori by BmNPV, together with other known targets, compromises the metabolic balance of the insect, which results in consequences for silk production and damage to the sericulture sector.

Alphabaculovirus é um gênero de vírus entomopatogênico, cuja espécie Bombyx mori nucleopolyhedrovirus (BmNPV) infecta o bicho-da-seda, Bombyx mori, inseto importante na indústria sericícola. Um isolado geográfico do BmNPV foi identificado no estado do Paraná, Brasil, infectando lagartas de B. mori e vários órgãos e tecidos alvos foram identificados; entretanto, não há informações sobre a infecção do túbulo de Malpighi (TM). O TM compõe o sistema excretor de B. mori, atuando na eliminação de substâncias tóxicas e na homeostase hidroeletrolítica. Assim, o presente estudo, analisou a susceptibilidade e a citopatologia do TM de B. mori ao BmNPV. Para tanto, lagartas híbridas de 5° instar foram inoculadas com uma suspensão viral e em diferentes dias pós-inoculação (dpi), segmentos do TM foram coletados e subdivididos nas regiões da ampola, proximal, média e distal; sendo processados para análises em microscopias de luz e eletrônica de transmissão. As regiões do TM revelaram diferenças na susceptibilidade ao BmNPV e a ampola, na sua área de transição, foi infectada a partir do 6° dpi, já as demais regiões não revelaram quaisquer indícios de infecção. A área de transição da ampola, ainda não havia sido descrita em lepidópteros e sua citopatologia revelou núcleo hipertrófico com viroplasma, seguido da formação e desenvolvimento dos poliedros virais, características comuns das infecções pelo Alphabaculovirus. Assim, a infecção da ampola do TM de B. mori ao BmNPV, somada a de outros alvos conhecidos, compromete o equilíbrio metabólico do inseto, com consequências na produção de seda e prejuízos ao setor sericícola.

Neem oil (Azadirachta indica A. Juss) affects the ultrastructure of the midgut muscle of Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae).

Cytomorphological changes, by means of ultrastructural analyses, have been used to determine the effects of the biopesticide neem oil on the muscle fibers of the midgut of the predator Ceraeochrysa claveri. Insects, throughout the larval period, were fed eggs of Diatraea saccharalis treated with neem oil at a concentration of 0.5%, 1% or 2%. In the adult stage, the midgut was collected from female insects at two stages of adulthood (newly emerged and at the start of oviposition) and processed for ultrastructural analyses. In the newly emerged insects obtained from neem oil treatments, muscle fibers showed a reduction of myofilaments as well as swollen mitochondria and an accumulation of membranous structures. Muscular fibers responded to those cellular injuries with the initiation of detoxification mechanisms, in which acid phosphatase activity was observed in large vesicles located at the periphery of the muscle fiber. At the start of oviposition in the neem oil treated insects, muscle fibers exhibited signs of degeneration, containing vacant areas in which contractile myofilaments were reduced or completely absent, and an accumulation of myelin structures, a dilatation of cisternae of sarcoplasmic reticulum, and mitochondrial swelling and cristolysis were observed. Enzymatic activity for acid phosphatase was present in large vesicles, indicating that mechanisms of lytic activity during the cell injury were utilized but insufficient for recovery from all the cellular damage. The results indicate that the visceral muscle layer is also the target of action of neem oil, and the cytotoxic effects observed may compromise the function of that organ.

Scanning Electron Microscopy of the Presbylarynx.

OBJECTIVE: To describe the findings on the presbylarynx under scanning electron microscopy. STUDY DESIGN: Cadaver study. SETTING: Universidade Estadual Paulista (Botucatu, São Paulo, Brazil). SUBJECTS AND METHODS: Sixteen vocal folds were removed during necropsies and distributed into 2 age groups: control (n = 8; aged 30-50 years) and elderly (n = 8; aged 75-92 years). The right vocal fold was dissected, fixed in glutaraldehyde 2.5%, and prepared for scanning electron microscopy. The thickness of the epithelium was measured using a scandium morphometric digital program. RESULTS: In the control group, the epithelium had 5 to 7 overlapped cell layers, rare desquamation cells, and little undulation with protruding intercellular junctions. The lamina propria showed a uniform network of collagen and elastic fibers in the superficial layer. A dense network of collagen was identified in the deeper layer. In the elderly group, the epithelium was atrophic (2-3 cells), with more desquamation cells and intercellular junctions delimited by deep sulci. The epithelial thickness was lower in elderly than in controls (mean [SD], 221.64 [145.90] µm vs 41.79 [21.40] µm, respectively). The lamina propria had a dense and irregular distribution of collagen and elastic fibers in the superficial layer. In the deep layers, the collagen fibers formed a true fibrotic and rigid skeleton. CONCLUSION: Scanning electron microscopy identified several changes in the elderly larynx, differentiating it from the controls. These alterations are probably related to the aging process of the vocal folds. However, the exact interpretation of these findings requires additional studies, even to the molecular level, having the fibroblasts as targets.

Cytotoxic effects of neem oil in the midgut of the predator Ceraeochrysa claveri.

Studies of morphological and ultrastructural alterations in target organs have been useful for evaluating the sublethal effects of biopesticides regarded as safe for non-target organisms in ecotoxicological analyses. One of the most widely used biopesticides is neem oil, and its safety and compatibility with natural enemies have been further clarified through bioassays performed to analyze the effects of indirect exposure by the intake of poisoned prey. Thus, this study examined the cellular response of midgut epithelial cells of the adult lacewing, Ceraeochrysa claveri, to neem oil exposure via intake of neem oil-contaminated prey during the larval stage. C. claveri larvae were fed Diatraea saccharalis eggs treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval stage. The adult females obtained from these treatments were used at two ages (newly emerged and at the start of oviposition) in morphological and ultrastructural analyses. Neem oil was found to cause pronounced cytotoxic effects in the adult midgut, such as cell dilation, emission of cytoplasmic protrusions, cell lysis, loss of integrity of the cell cortex, dilation of cisternae of the rough endoplasmic reticulum, swollen mitochondria, vesiculated appearance of the Golgi complex and dilated invaginations of the basal labyrinth. Epithelial cells responded to those injuries with various cytoprotective and detoxification mechanisms, including increases in cell proliferation, the number of calcium-containing cytoplasmic granules, and HSP 70 expression, autophagic processes and the development of smooth endoplasmic reticulum, but these mechanisms were insufficient for recovery from all of the cellular damage to the midgut. This study demonstrates that neem oil exposure impairs the midgut by causing sublethal effects that may affect the physiological functions of this organ, indicating the importance of studies of different life stages of this species and similar species to evaluate the safe and compatible integrated use of biopesticides.