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In nearly every species of insect, embryonic development takes place outside of the mother's body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus . We discovered that Sec16 is strongly expressed in vitellogenic females' ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45-which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rios, Bomfim, Pereira, Miranda, Majerowicz, Pane and Ramos.)

Among the most malignant cancers, oral squamous cell carcinoma (OSCC) stands out as the most common malignant head and neck tumor. Despite advances in the field of treatment, the prognosis of patients with OSCC remains poor. Aiming to overcome the limitations of the currently existing therapies against OSCC, the present work aims to investigate the potential of photodynamic therapy (PDT) with phenothiazine derivatives used alone or in combination. The incorporation of methylene blue (MB) and toluidine blue (TB) was evaluated in OSCC cell lines (HSC-3 and SCC-9) and a nontumor cell line (Hfib). Both compounds exhibited concentration and time-dependent incorporation, with higher rates observed in tumor cells. Regarding dark-phase cytotoxic activity, SCC-9 cells were the most sensitive cell line with an IC 50 value of 362.6 µM and 41.4 µM for MB and TB, respectively. Using PDT, all lineages showed greater sensitivity, presenting lower IC 50 values when compared to the dark phase values. The combination index values of 0.69 (dark phase) and 0.73 (clear phase) associated with concave isobolograms, in both phases, revealed that MB and TB have synergistic effects when combined against SCC-9 cells. These findings suggest that MB or TB assisted with PDT holds promise for OSCC treatment.

Neglected tropical diseases (NTDs) constitute a group of diseases that generally develop in tropical or subtropical climatic conditions and are related to poverty. Within the spectrum of NTDs, diseases caused by protozoa such as malaria, Chagas disease, and leishmaniasis exhibit elevated mortality rates, thereby constituting a substantial public health concern. Beyond their protozoan etiology, these NTDs share other similarities, such as the challenge of control and the lack of affordable, safe, and effective drugs. In view of the above, the need to explore novel diagnostic predictors and therapeutic targets for the treatment of these parasitic diseases is evident. In this context, galectins are attractive because they are a set of lectins bound to β-galactosides that play key roles in a variety of cellular processes, including host-parasite interaction such as adhesion and entry of parasites into the host cells, and participate in antiparasitic immunity in either a stimulatory or inhibitory manner, especially the galectins-1, -2, -3, and -9. These functions bestow upon galectins significant therapeutic prospects in the context of managing and diagnosing NTDs. Thus, the present review aims to elucidate the potential role of galectins in the diagnosis and treatment of malaria, leishmaniasis, and Chagas disease.

Introduction: To describe CT findings in patients with confirmed Covid-19 infection and correlate them with the disease evolution stages., Methods: This is a historical cohort observational analytical study carried out with outpatients, inpatients, and emergency patients from a private hospital in Maceió/AL, Brazil. The final sample consisted of 390 patients with positive RT-PCR for Covid-19 with available laboratory tests and chest CT results., Results: The most frequent initial symptoms were cough, fever, dyspnea and headache. The most commonly found comorbidities were hypertension, diabetes mellitus and obesity. A total of 22% of the CT scans showed no alterations; ground-glass opacity was the most frequently found one. There was a significant association between age, comorbidities, pulmonary involvement, ground-glass opacity, mosaic attenuation and percentage of pulmonary involvement with death. The analysis of the disease stages showed a significant association with laboratory data (CRP and platelet levels), ground-glass opacity and mosaic attenuation with the disease evolution stages in relation to the days since symptom onset., Conclusion: The disease evolution of Covid-19 occurs in stages, and this study describes tomographic findings in patients with confirmed Covid-19 infection and shows they vary depending on the disease evolution stages., Implications for Practice: This paper provides important addition to the various records that have been accumulated through the Covid-19 pandemic., (Copyright © 2023 The College of Radiographers. Published by Elsevier Ltd. All rights reserved.)

Autophagy and the ubiquitin-proteasome system (UPS) are important cellular mechanisms that coordinate protein degradation essential for proteostasis. P62/SQSTM1 is a receptor cargo protein able to deliver ubiquitinated targets to the proteasome proteolytic complex and/or to the autophagosome. In the insect vector of Chagas disease, Rhodnius prolixus, previous works have shown that the knockdown of different autophagy-related genes (ATGs) and ubiquitin-conjugating enzymes resulted in abnormal oogenesis phenotypes and embryo lethality. Here, we investigate the role of the autophagy/UPS adaptor protein p62 during the oogenesis and reproduction of this vector. We found that R. prolixus presents one isoform of p62 encoded by a non-annotated gene. The predicted protein presents the domain architecture anticipated for p62: PB1 (N-term), ZZ-finger, and UBA (C-term) domains, and phylogenetic analysis showed that this pattern is highly conserved within insects. Using parental RNAi, we found that although p62 is expressed in the ovary, midgut, and fat body of adult females, systemic silencing of this gene did not result in any apparent phenotypes under in-house conditions. The insects' overall levels of blood meal digestion, lifespan, yolk protein production, oviposition, and embryo viability were not altered when compared to controls. Because it is known that autophagy and UPS can undergo compensatory mechanisms, we asked whether the silencing of p62 was triggering adaptative changes in the expression of genes of the autophagy, UPS, and the unfolded protein response (UPR) and found that only ATG1 was slightly up regulated in the ovaries of silenced females. In addition, experiments to further investigate the role of p62 in insects previously silenced for the E1-conjugating enzyme (a condition known to trigger the upregulation of p62), also did not result in any apparent phenotypes in vitellogenic females., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Pereira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rhodnius prolixus is a hematophagous insect, vector of Chagas disease. After feeding, as blood is slowly digested, amino acids are used as substrates to fuel lipid synthesis, and adult females accumulate lipids in the fat body and produce eggs. In order to evaluate the importance of de novo fatty acid synthesis for this insect metabolism, we generated acetyl-CoA carboxylase (ACC) deficient insects. The knockdown (AccKD) females had delayed blood digestion and a shorter lifespan. Their fat bodies showed reduced de novo lipogenesis activity, did not accumulate triacylglycerol during the days after blood meal, and had smaller lipid droplets. At 10 days after feeding, there was a general decrease in the amounts of neutral lipids and phospholipids in the fat body. In the hemolymph, no difference was observed in lipid composition at 5 days after blood meal, but at day ten, there was an increase in hydrocarbon content and a decrease in phospholipids. Total protein concentration and amino acid composition were not affected. The AccKD females laid 60% fewer eggs than the control ones, and only 7% hatched (89% for control), although their total protein and triacylglycerol contents were not different. Scanning electron microscopy of the egg surface showed that chorion (eggshell) from the eggs laid by the AccKD insects had an altered ultrastructural pattern when compared to control ones. These results show that ACC has a central role in R. prolixus nutrient homeostasis, and its appropriate activity is important to digestion, lipid synthesis and storage, and reproductive success., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Moraes, Braz, Santos-Araujo, Oliveira, Bomfim, Ramos and Gondim.)

Community testing is a crucial tool for the early identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission control. The emergence of the highly mutated Omicron variant (B.1.1.529) raised concerns about its primary site of replication, impacting sample collection and its detectability by rapid antigen tests. We tested the performance of the Panbio antigen rapid diagnostic test (Ag-RDT) using nasal and oral specimens for COVID-19 diagnosis in 192 symptomatic individuals, with quantitative reverse transcription-PCR (RT-qPCR) of nasopharyngeal samples as a control. Variant of concern (VOC) investigation was performed with the 4Plex SARS-CoV-2 screening kit. The SARS-CoV-2 positivity rate was 66.2%, with 99% of the positive samples showing an amplification profile consistent with that of the Omicron variant. Nasal Ag-RDT showed higher sensitivity (89%) than oral (12.6%) Ag-RDT. Our data showed good performance of the Ag-RDT in a pandemic scenario dominated by the Omicron VOC. Furthermore, our data also demonstrated that the Panbio COVID-19 antigen rapid diagnostic test does not provide good sensitivity with oral swabs for Omicron Ag-RDT detection. IMPORTANCE This study showed that the antigen rapid test for COVID19 worked fine using nasal swabs when it was utilized in patients infected with the Omicron variant, showing a concordance with PCR in 93% of patients tested. The nasal swab yielded more reliable results than the oral swab when an antigen rapid diagnosis test (the Panbio COVID-19 antigen rapid diagnostic test) was used in patients infected with the Omicron variant.

In insects, the follicle cells (FCs) give rise to a single-layered tissue of binucleated professional secretory cells that surround the oocytes during oogenesis. In the latest stage of oocyte development, the FCs rapidly synthesize and secrete the chorion (eggshell) immediately before degenerating through apoptosis. Here, we used RT-qPCR, electron microscopy, and RNAi silencing to explore the role of the main unfolded protein response (UPR) receptors IRE1 and PERK, as well as the ultrastructure dynamics of the FCs during oogenesis of the insect vector of Chagas disease Rhodnius prolixus. We found that IRE1 and PERK mRNAs are highly expressed in the ovaries of vitellogenic females. Interestingly, we observed that IRE1 and PERK, as well as different isoforms of the chaperones Bip and PDI, have their FCs gene expression levels decreased during the vitellogenesis to choriogenesis transition. Using transmission electron microscopy, we observed that the downregulation of the UPR gene expression is accompanied by dramatic changes in the FCs ultrastructure, with an 80% reduction in the mean area of the ER tubules, and circularization and enlargement of the mitochondria. Additionally, we found that parental RNAi silencing of both IRE1 and PERK resulted in minor changes in the chorion protein composition and ultrastructure, accessed by urea extraction of the chorion proteins and scanning electron microscopy, respectively, but did not impact the overall levels of oviposition and F1 embryo development., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rhodnius prolixus is an obligatorily hematophagous insect known as an important vector of Chagas disease. Autophagy is a conserved cellular mechanism that acts in response to nutrient starvation, where components of the cytoplasm are sequestered by a double membrane organelle, named autophagosome, which is targeted to fuse with the lysosome for degradation. Lipophagy is the process of lipid degradation by selective autophagy, where autophagosomes sequester lipid droplets and degrade triacylglycerol (TAG) generating free fatty acids for β-oxidation. Here, two essential genes of the autophagic pathway, Atg6/Beclin1 (RpAtg6) and Atg8/LC3 (RpAtg8), were silenced and the storage of lipids during starvation in Rhodnius prolixus was monitored. We found that RNAi knockdown of both RpAtg6 and RpAtg8 resulted in higher levels of TAG in the fat body and the flight muscle, 24 days after the blood meal, as well as a larger average diameter of the lipid droplets in the fat body, as seen by Nile Red staining under the confocal fluorescence microscope. Silenced starved insects had lower survival rates when compared to control insects. Accordingly, when examined during the starvation period for monitored activity, silenced insects had lower spontaneous locomotor activity and lower forced flight rates. Furthermore, we found that some genes involved in lipid metabolism had their expression levels altered in silenced insects, such as the Brummer lipase (down regulated) and the adipokinetic hormone receptor (up regulated), suggesting that, as previously observed in mammalian models, the autophagy and neutral lipolysis machineries are interconnected at the transcriptional level. Altogether, our data indicate that autophagy in the fat body is important to allow insects to mobilize energy from lipid stores., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell., (© 2020 Federation of American Societies for Experimental Biology.)

Follicular atresia is the mechanism by which the oocyte contents are degraded during oogenesis in response to stress conditions, allowing the energetic resources stored in the developing oocytes to be reallocated to optimize female fitness. Autophagy is a conserved intracellular degradation pathway where double-membrane vesicles are formed around target organelles leading to their degradation after lysosome fusion. The autophagy-related protein 8 (ATG8) is conjugated to the autophagic membrane and has a key role in the elongation and closure of the autophagosome. Here we identified one single isoform of ATG8 in the genome of the insect vector of Chagas Disease Rhodnius prolixus (RpATG8) and found that it is highly expressed in the ovary during vitellogenesis. Accordingly, autophagosomes were detected in the vitellogenic oocytes, as seen by immunoblotting and electron microscopy. To test if autophagosomes were important for follicular atresia, we silenced RpATG8 and elicited atresia in vitellogenic females by Zymosan-A injections. We found that silenced females were still able to trigger the same levels of follicle atresia, and that their atretic oocytes presented a characteristic morphology, with accumulated brown aggregates. Regardless of the difference in morphology, RpATG8-silenced atretic oocytes presented the same levels of protein, TAG and PolyP, as detected in control atretic oocytes, as well as the same levels of acidification of the yolk organelles. Because follicular atresia has the ultimate goal of restoring female fitness, we tested if RpATG8-silenced atresia would result in female physiology and behavior changes. Under insectarium conditions, we found that atresia-induced control and RpATG8-silenced females present no changes in blood meal digestion, survival, oviposition, TAG content in the fat body, haemolymph amino acid levels and overall locomotor activity. Altogether, we found that autophagosomes are formed during oogenesis and that the silencing of RpATG8 impairs autophagosome biogenesis in the oocytes. Nevertheless, regarding major macromolecule degradation and adaptations to the fitness costs imposed by triggering an immune response, we found that autophagic organelles are not essential for follicle atresia in R. prolixus., Competing Interests: The authors have declared that no competing interests exist.

In oviparous animals, the egg yolk is synthesized by the mother in a major metabolic challenge, where the different yolk components are secreted to the hemolymph and delivered to the oocytes mostly by endocytosis. The yolk macromolecules are then stored in a wide range of endocytic-originated vesicles which are collectively referred to as yolk organelles and occupy most of the mature oocytes cytoplasm. After fertilization, the contents of these organelles are degraded in a regulated manner to supply the embryo cells with fundamental molecules for de novo synthesis. Yolk accumulation and its regulated degradation are therefore crucial for successful development, however, most of the molecular mechanisms involved in the biogenesis, sorting and degradation of targeted yolk organelles are still poorly understood. ATG6 is part of two PI3P-kinase complexes that can regulate the recruitment of the endocytic or the autophagy machineries. Here, we investigate the role of RpATG6 in the endocytosis of the yolk macromolecules and in the biogenesis of the yolk organelles in the insect vector Rhodnius prolixus. We found that vitellogenic females express high levels of RpATG6 in the ovaries, when compared to the levels detected in the midgut and fat body. RNAi silencing of RpATG6 resulted in yolk proteins accumulated in the vitellogenic hemolymph, as a consequence of poor uptake by the oocytes. Accordingly, the silenced oocytes are unviable, white (contrasting to the control pink oocytes), smaller (62% of the control oocyte volume) and accumulate only 40% of the yolk proteins, 80% of the TAG and 50% of the polymer polyphosphate quantified in control oocytes. The cortex of silenced oocytes present atypical smaller vesicles indicating that the yolk organelles were not properly formed and/or sorted, which was supported by the lack of endocytic vesicles near the plasma membrane of silenced oocytes as seen by TEM. Altogether, we found that RpATG6 is central for the mechanisms of yolk accumulation, emerging as an important target for further investigations on oogenesis and, therefore, reproduction of this vector., Competing Interests: The authors have declared that no competing interests exist.

Most vectors of arthropod-borne diseases produce large eggs with hard and opaque eggshells. In several species, it is still not possible to induce molecular perturbations to the embryo by delivery of molecules using microinjections or eggshell permeabilization without losing embryo viability, which impairs basic studies regarding development and population control. Here we tested the properties and permeability of the eggshell of R. prolixus, a Chagas disease vector, with the aim to deliver pharmacological inhibitors to the egg cytoplasm and allow controlled molecular changes to the embryo. Using field emission scanning and transmission electron microscopy we found that R. prolixus egg is coated by three main layers: exochorion, vitelline layer and the plasma membrane, and that the pores that allow gas exchange (aeropiles) have an average diameter of 10 μm and are found in the rim of the operculum at the anterior pole of the egg. We tested if different solvents could permeate through the aeropiles and reach the egg cytoplasm/embryo and found that immersions of the eggs in ethanol lead to its prompt penetration through the aeropiles. A single five minute-immersion of the eggs/embryos in pharmacological inhibitors, such as azide, cyanide and cycloheximide, solubilized in ethanol resulted in impairment of embryogenesis in a dose dependent manner and DAPI-ethanol solutions were also able to label the embryo cells, showing that ethanol penetration was able to deliver those molecules to the embryo cells. Multiple immersions of the embryo in the same solutions increased the effect and tests using bafilomycin A1 and Pepstatin A, known inhibitors of the yolk proteolysis, were also able to impair embryogenesis and the yolk protein degradation. Additionally, we found that ethanol pre-treatments of the egg make the aeropiles more permeable to aqueous solutions, so drugs diluted in water can be carried after the eggs are pre-treated with ethanol. Thus, we found that delivery of pharmacological inhibitors to the embryo of R. prolixus can be performed simply by submersing the fertilized eggs in ethanol with no need for additional methods such as microinjections or electroporation. We discuss the potential importance of this methodology to the study of this vector developmental biology and population control.

A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P. discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.