The Association of Nanostructured Carbonated Hydroxyapatite with Denatured Albumin and Platelet-Rich Fibrin: Impacts on Growth Factors Release and Osteoblast Behavior.

Platelet-rich Fibrin (PRF), a second-generation blood concentrate, offers a versatile structure for bone regeneration due to its composition of fibrin, growth factors, and cytokines, with adaptations like denatured albumin-enriched with liquid PRF (Alb-PRF), showing potential for enhanced stability and growth factor dynamics. Researchers have also explored the combination of PRF with other biomaterials, aiming to create a three-dimensional framework for enhanced cell recruitment, proliferation, and differentiation in bone repair studies. This study aimed to evaluate a combination of Alb-PRF with nanostructured carbonated hydroxyapatite microspheres (Alb-ncHA-PRF), and how this association affects the release capacity of growth factors and immunomodulatory molecules, and its impact on the behavior of MG63 human osteoblast-like cells. Alb-PRF membranes were prepared and associated with nanocarboapatite (ncHA) microspheres during polymerization. MG63 cells were exposed to eluates of both membranes to assess cell viability, proliferation, mineralization, and alkaline phosphatase (ALP) activity. The ultrastructural analysis has shown that the spheres were shattered, and fragments were incorporated into both the fibrin mesh and the albumin gel of Alb-PRF. Alb-ncHA-PRF presented a reduced release of growth factors and cytokines when compared to Alb-PRF (p < 0.05). Alb-ncHA-PRF was able to stimulate osteoblast proliferation and ALP activity at lower levels than those observed by Alb-PRF and was unable to positively affect in vitro mineralization by MG63 cells. These findings indicate that the addition of ncHA spheres reduces the biological activity of Alb-PRF, impairing its initial effects on osteoblast behavior.

Effects of rotor angle and time after centrifugation on the biological in vitro properties of platelet rich fibrin membranes.

This study evaluated the impact of rotor angle and time of storage after centrifugation on the in vitro biological properties of platelet-rich fibrin (PRF) membranes. Blood samples (n = 9) were processed with a vertical fixed-angle (V) or a swing-out horizontal (H) centrifuge, with 20-60 min of sample storage after centrifugation. Leukocytes, platelets, and red blood cells were counted, and fibrin architecture was observed by scanning electron microscopy (SEM). The release of FGF2, PDGFbb, VEGF, IL-6, and IL-1ß was measured after incubation on culture media for 7-21 days. Cell content was equivalent in all experimental groups (p > .05). The fibrin matrix was similar for fixed-angle and horizontal centrifugation. Horizontal centrifugation induced a twofold increase in PDGF and 1.7× increase on FGF release as compared to V samples, while IL-1ß was significantly reduced (p < .05). No significant difference was observed on the release of growth factors and cytokines at different times after centrifugation (p < .05). These data suggest that both angles of centrifugation produce PRF membranes with similar structure and cellularity, but horizontal centrifugation induces a higher release of growth factors. Higher times of storage after centrifugation did not impact on cell content and the release of growth factors.

Molecular signatures of sexual communication in the phlebotomine sand flies.

Phlebotomine sand flies employ an elaborate system of pheromone communication wherein males produce pheromones that attract other males to leks (thus acting as an aggregation pheromone) and females to the lekking males (sex pheromone). In addition, the type of pheromone produced varies among populations. Despite the numerous studies on sand fly chemical communication, little is known of their chemosensory genome. Chemoreceptors interact with chemicals in an organism's environment to elicit essential behaviors such as the identification of suitable mates and food sources. Thus, they play important roles during adaptation and speciation. Major chemoreceptor gene families, odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) together detect and discriminate the chemical landscape. Here, we annotated the chemoreceptor repertoire in the genomes of Lutzomyia longipalpis and Phlebotomus papatasi, major phlebotomine vectors in the New World and Old World, respectively. Comparison with other sequenced Diptera revealed a large and unique expansion where over 80% of the ~140 ORs belong to a single, taxonomically restricted clade. We next conducted a comprehensive analysis of the chemoreceptors in 63 L. longipalpis individuals from four different locations in Brazil representing allopatric and sympatric populations and three sex-aggregation pheromone types (chemotypes). Population structure based on single nucleotide polymorphisms (SNPs) and gene copy number in the chemoreceptors corresponded with their putative chemotypes, and corroborate previous studies that identified multiple populations. Our work provides genomic insights into the underlying behavioral evolution of sexual communication in the L. longipalpis species complex in Brazil, and highlights the importance of accounting for the ongoing speciation in central and South American Lutzomyia that could have important implications for vectorial capacity.

Morphological and ultrastructural analysis of an important place of sexual communication of Rhodnius prolixus (Heteroptera: Reduviidae): the Metasternal Glands.

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the etiological agent of Chagas disease. Insect adults have a pair of Metasternal Glands (MGs) and the secretion emitted by these glands acts as sex pheromone. Recent studies have focused on the chemical composition of this pheromone, electrophysiological responses to MGs compounds and mating behavior assays. Morphological studies of these glands are still scarce. Thus, considering the relevance of MGs in the sex pheromone biosynthesis, we investigated the morphology and ultrastructure of R. prolixus MGs. The glandular apparatus presents a tubular structure containing secretory cells with canalicules that fuse with the central duct which conducts the secretion to a pear-shaped reservoir connected to the exterior by a droplet-shape orifice. The secretory cells are classified as class III, they present a well-developed rough and smooth endoplasmic reticulum. Smooth endoplasmic reticulum is a site of lipid biosynthesis that may be involved in the mevalonate pathway, a probable route of the sex pheromone biosynthesis in this insect. The presence of rough endoplasmic reticulum indicates a possible peptides/proteins secretions site which were still not characterized in MGs. Several mitochondria are scattered in the cytoplasm that may suggest a high metabolic activity. Further studies should be carried out to correlate these data with the sex pheromone biosynthesis in this vector.

Impact of crystallinity and crystal size of nanostructured carbonated hydroxyapatite on pre-osteoblast in vitro biocompatibility.

Nanostructured carbonated hydroxyapatite (nCHA) is a promising biomaterial for bone tissue engineering due to its chemical properties, similar to those of the bone mineral phase and its enhanced in vivo bioresorption. However, the biological effects of nCHA nanoparticles on cells and tissues are not sufficiently known. This study assessed the impact of exposing pre-osteoblasts to suspensions with high doses of nCHA nanoparticles with high or low crystallinity. MC3T3-E1 pre-osteoblasts were cultured for 1 or 7 days in a culture medium previously exposed to CHA nanoparticles for 1 day. Control groups were produced by centrifugation for removal of bigger nCHA aggregates before exposure. Interaction of nanoparticles with the culture medium drastically changed medium composition, promoting Ca, P, and protein adsorption. Transmission Electron microscopy revealed that exposed cells were able to internalize both materials, which seemed concentrated inside endosomes. No cytotoxicity was observed for both materials, regardless of centrifugation, and the exposure did not induce alterations in the release of pro-and anti-inflammatory cytokines. Morphological analysis revealed strong interactions of nCHA aggregates with cell surfaces, however without marked alterations in morphological features and cytoskeleton ultrastructure. The overall in vitro biocompatibility of nCHA materials, regardless of physicochemical characteristics such as crystallinity, encourages further studies on their clinical applications.

Origens do(s) sexo(s) e a rainha dos problemas da biologia evolutiva / Origins of sex(es) and the queen of problems of evolutionary biology

A reprodução sexuada já foi considerada universal, e posteriormente, a forma mais perfeita de reprodução. Todavia, a partir de meados do século XIX, pesquisas no nível celular colocaram em xeque a ideia de que tipos de reprodução assexuadas fossem primitivos ou inferiores. Ao longo do século XX, e adentrando no XXI, hipóteses foram levantadas para explicar as vantagens da reprodução sexuada sobre a assexuada assim como o que permitiria a reprodução sexuada se manter quando seria mais vantajoso se reproduzir de forma assexuada. A mais importante e conhecida é a hipótese da Rainha Vermelha. Paralelamente, vários trabalhos procuraram entrever as pressões ecológicas que permitiram e favoreceram o aparecimento da reprodução sexuada em um cenário situado há cerca de dois bilhões de anos. O objetivo desse trabalho é revisar respostas históricas que marcaram o estudo da origem, da evolução e da manutenção da reprodução sexuada, identificando algumas das principais questões que a comunidade científica elaborou nos últimos duzentos anos.

Sexual reproduction has already been considered universal, and subsequently, the most perfect form of reproduction. However, since the mid-nineteenth century, research at the cellular level has questioned the idea that asexual reproduction types are primitive or inferior. During the twentieth century, and entering the XXI, hypotheses were raised to explain the advantages of sexual reproduction over the asexual as well as what would allow sexual reproduction to be maintained when it would be more advantageous to reproduce asexually. The most important and known is the Red Queen hypothesis. At the same time, several studies have sought to understand the ecological pressures that allowed and favored the appearance of sexual reproduction in a scenario that was around two billion years ago. The aim of this work is to review historical responses that marked the study of the origin, evolution and maintenance of sexual reproduction, identifying some of the main questions that the scientific community has elaborated over the last two hundred years.

Teaching Cell Biology to Dental Students with a Project-Based Learning Approach.

Although the discipline of cell biology (CB) is part of the curricula of predoctoral dental schools, students often fail to recognize its practical relevance. The aim of this study was to assess the effectiveness of a practical-theoretical project-based course in closing the gaps among CB, scientific research, and dentistry for dental students. A project-based learning course was developed with nine sequential lessons to evaluate 108 undergraduate dental students enrolled in CB classes of a Brazilian school of dentistry during 2013-16. To highlight the relevance of in vitro studies in the preclinical evaluation of dental materials at the cellular level, the students were challenged to complete the process of drafting a protocol and performing a cytocompatibility assay for a bone substitute used in dentistry. Class activities included small group discussions, scientific database search and article presentations, protocol development, lab experimentation, and writing of a final scientific report. A control group of 31 students attended only one laboratory class on the same theme, and the final reports were compared between the two groups. The results showed that the project-based learning students had superior outcomes in acknowledging the relevance of in vitro methods during biocompatibility testing. Moreover, they produced scientifically sound reports with more content on methodological issues, the relationship with dentistry, and the scientific literature than the control group (p<0.05). The project-based learning students also recognized a higher relevance of scientific research and CB to dental practice. These results suggest that a project-based approach can help contextualize scientific research in dental curricula.

The Lutzomyia longipalpis complex: a brief natural history of aggregation-sex pheromone communication.

In this paper we review the natural history of pheromone communication and the current diversity of aggregation-sex pheromones in the sand fly Lutzomyia longipalpis. This species complex is the main vector of Leishmania infantum, the agent of visceral leishmaniasis in the Americas. The identification of variation in pheromone chemotypes combined with molecular and sound analyses have all contributed to our understanding of the extent of divergence among cryptic members of this complex. The importance of chemical signals as pre-mating barriers and drivers of speciation is discussed. Moreover, the importance of aggregation-sex pheromones as sexually selected signals is highlighted with evidence from the literature suggesting their potential role in species and mate recognition as well as mate assessment. The distinct evolutionary forces possibly involved are briefly reviewed and discussed in the context of this intriguing insect.

Fine structure of the male reproductive system and reproductive behavior of Lutzomyia longipalpis sandflies (Diptera: Psychodidae: Phlebotominae).

BACKGROUND: The male reproductive system of insects can have several tissues responsible for the secretion of seminal fluid proteins (SFPs), such as testes, accessory glands, seminal vesicles, ejaculatory duct and ejaculatory bulb. The SFPs are transferred during mating and can induce several physiological and behavioral changes in females, such as increase in oviposition and decrease in sexual receptivity after copulation. The phlebotomine Lutzomyia longipalpis is the main vector of visceral leishmaniasis. Despite its medical importance, little is known about its reproductive biology. Here we present morphological aspects of the male L. longipalpis reproductive system by light, scanning and transmission electron microscopy, and compare the mating frequency of both virgin and previously mated females. RESULTS: The male L. longipalpis reproductive system is comprised by a pair of oval-shaped testes linked to a seminal vesicle by vasa deferentia. It follows an ejaculatory duct with an ejaculatory pump (a large bulb enveloped by muscles and associated to tracheas). The terminal endings of the vasa deferentia are inserted into the seminal vesicle by invaginations of the seminal vesicle wall, which is composed by a single layer of gland cells, with well-developed endoplasmic reticulum profiles and secretion granules. Our data suggest that the seminal vesicle acts both as a spermatozoa reservoir and as an accessory gland. Mating experiments support this hypothesis, revealing a decrease in mating frequency after copulation that indicates the effect of putative SFPs. CONCLUSION: Ultrastructural features of the L. longipalpis male seminal vesicle indicated its possible role as an accessory gland. Behavioral observations revealed a reduction in mating frequency of copulated females. Together with transcriptome analyses from male sandfly reproductive organs identifying ESTs encoding orthologs of SFPs, these data indicate the presence of putative L. longipalpis SFPs reducing sexual mating frequency of copulated females.

Pheromone gland development and pheromone production in lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae).

The sand fly Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae: Phlebotominae) is the main vector of American visceral leishmaniasis. Adult males produce a terpenoid sex pheromone that in some cases also acts as male aggregation pheromone. We have analyzed the correlation between male pheromone production levels and pheromone gland cell morphogenesis after adult emergence from pupae. The abdominal tergites of L. longipalpis males were dissected and fixed in glutaraldehyde for transmission electron microscopy, or the pheromone was extracted in analytical grade hexane. Pheromone chemical analysis was carried out at 3- to 6-h intervals during the first 24 h after emergence and continued daily until the seventh day. All extracts were analyzed by gas chromatography. For the morphological analysis, we used insects collected at 0-6, 9-12, 12-14, and 96 h after emergence. Ultrastructural data from 0- to 6-h-old adult males revealed smaller pheromone gland cells with small microvilli at the end apparatus. Lipid droplets and peroxisomes were absent or very rare, but a large number of mitochondria could be seen. Lipid droplets started to appear in the gland cells cytoplasm approximately 9 h after adult emergence, and their number and size increased with age, together with the presence of several peroxisomes, suggesting a role for these organelles in pheromone biosynthesis. At 12-15 h after emergence, the lipid droplets were mainly distributed near the microvilli but were smaller than those in mature older males (4 d old). Pheromone biosynthesis started around 12 h after emergence and increased continuously during the first 3 d, stabilizing thereafter, coinciding with the period when males are more able to attract females.

Introducing DNA concepts to Swiss high school students based on a Brazilian educational game.

Subjects such as techniques for genetic diagnosis, cloning, sequencing, and gene therapy are now part of our lives and raise important questions about ethics, future medical diagnosis, and such. Students from different countries observe this explosion of biotechnological applications regardless of their social, academic, or cultural backgrounds, although they are not usually familiar with their theoretical genetic bases. To introduce some molecular biology concepts for high school students, we developed a new problem for the Brazilian board game "Discovering the cell" ("Célula Adentro©" in Portuguese), a pedagogic tool based on inquiry-, cooperative-, and problem-based learning. This problem (Case) is based on the forensic DNA, which represents an interesting theme for students, as it recurrently appears on newspapers and television series. In this work, we tested this game with secondary students and teachers from Switzerland. Our results indicate that the game "Discovering the cell" is well accepted by both students and teachers and may represent a good pedagogical approach to help teaching complex themes in molecular biology, even with students from different socioeconomical, cultural, and academic backgrounds.

Structure and distribution of sensilla on maxillary palps and labella of Lutzomyia longipalpis (Diptera: Psychodidae) sand flies.

The typology, number, size, and distribution pattern of labellar and palpal sensilla of male and female Lutzomyia longipalpis (Lutz and Neiva, 1912) sand flies were analyzed by scanning electron and light microscopy, contributing to the construction of a gustatory and olfactory map in this important vector species. About 35 trichoid sensilla, sorted into three different classes according to their lengths, are present on the two labellar lobes. Five distinct structures are observed in the maxillary palps, which could be classified as noninnervated (microtrichia and scales) or the following innervated structures: 1) campaniform sensilla, found near the distal end of palpal segments II and III; 2) multiporous capitate peg sensilla, found in reduced number (1-6) on segment II, but located in a group of 15-25 on the distal third part of segment III; and 3) trichoid sensilla, distributed linearly on segment IV and spirally on segment V. Light microscopy analysis of silver-stained specimens indicated the presence of pores in the trichoid sensilla and their chemosensory function is discussed, as well as the possible function of the other sensilla.

Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae: Phlebotominae).

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium-potassium iodide (Os-KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis.

Ultrastructure of male sex pheromone glands in abdominal tergites of five Lutzomyia sandfly species (Diptera: Psychodidae).

The fine structure of male sex pheromone disseminating structures on abdominal segments of five species of Lutzomyia sandflies (Diptera: Psychodidae) was analyzed. Scanning electron microscopy showed that in Lutzomyia cruzi [Mem. Inst. Oswaldo Cruz 33 (1938) 349] and Lutzomyia longipalpis [Mem. Inst. Oswaldo Cruz 4 (1912) 84], the disseminating structures are located in pale spots on abdominal tergites IV or III and IV and are morphologically similar, appearing as small round cuticular elevations with a central pore. Observation of abdominal tergites of L. longipalpis pupae showed that the spots, but not the structures, are already present in this stage. In Lutzomyia lenti [Mem. Inst. Oswaldo Cruz 33 (1938) 349] and Lutzomyia carmelinoi [Mem. Inst. Oswaldo Cruz 81 (1986) 323] adult males, the disseminating structures are present on tergal segments V and VI, where pale spots could not be observed, and appear as apple-like elevations with a central pore. In Lutzomyia renei, a single disseminating structure is found at the anterior region of tergal segment VI. Transmission electron microscopy was used to analyze the gland cell fine structure in L. cruzi. Several class III gland cells are located side by side in the fourth abdominal segment. Each epidermal secretory cell contains a small reservoir and a short outlet channel through the cuticle.