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Fiorillo, B. S., Zama, U., Lino-Neto, J. and Báo, S. N. 2010. Structural and ultrastructural studies of male reproductive tract and spermatozoa in Xylocopa frontalis (Hymenoptera, Apidae). — Acta Zoologica (Stockholm) 91: 176–183. In Xylocopa frontalis the reproductive tract is composed of testes, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. Each testis comprises four testicular tubules in which multiple cysts are present containing approximately 64 spermatozoa per cyst. The seminal vesicle consists of an epithelium, a thick basement lamina and a muscular external sheet. In the luminal region some vesicles can be observed; however, the epithelial cells of the seminal vesicle do not display morphological features associated with secretory functions. The spermatozoa, measuring approximately 260 µm long, are similar to the hymenopteran pattern. The head region consists of an acrosome with an inner perforatorium that penetrates an asymmetrical nuclear tip. The nucleus is linear, electron-dense and its posterior tip projects into the beginning of the axoneme. The centriolar adjunct is asymmetric with many electron-lucent lacunae interspersed throughout. The axoneme has the 9 + 9 + 2 pattern of microtubules and in the posterior region the central microtubules finish first, followed by the doublets and finally the accessory microtubules. The mitochondrial derivatives are asymmetric in both length and diameter with paracrystalline material present only in the larger one. These features may be useful characters for taxonomy and phylogenetic studies. [ABSTRACT FROM AUTHOR]

Although multiple approaches have been suggested, treating mild-to-severe fibrosis in the context of metabolic dysfunction associated with liver disease (MASLD) remains a challenging area in drug discovery. Pathogenesis of liver fibrosis is multifactorial, and pathogenic mechanisms are deeply intertwined; thus, it is well accepted that future treatment requires the development of multitarget modulators. Harnessing the 3,4,5-trisubstituted isoxazole scaffold, previously described as a key moiety in Farnesoid X receptor (FXR) agonism, herein we report the discovery of a novel class of hybrid molecules endowed with dual activity toward FXR and the leukemia inhibitory factor receptor (LIFR). Up to 27 new derivatives were designed and synthesized. The pharmacological characterization of this series resulted in the identification of 3a as a potent FXR agonist and LIFR antagonist with excellent ADME properties. In vitro and in vivo characterization identified compound 3a as the first-in-class hybrid LIFR inhibitor and FXR agonist that protects against the development of acute liver fibrosis and inflammation.

Serotonin [5-hydroxytryptamine (5-HT)] acts via 13 different receptors in humans. Of these receptor subtypes, all but 5-HT 1e R have confirmed roles in native tissue and are validated drug targets. Despite 5-HT 1e R's therapeutic potential and plausible druggability, the mechanisms of its activation remain elusive. To illuminate 5-HT 1e R's pharmacology in relation to the highly homologous 5-HT 1F R, we screened a library of aminergic receptor ligands at both receptors and observe 5-HT 1e R/5-HT 1F R agonism by multicyclic drugs described as pan-antagonists at 5-HT receptors. Potent agonism by tetracyclic antidepressants mianserin, setiptiline, and mirtazapine suggests a mechanism for their clinically observed antimigraine properties. Using cryo-EM and mutagenesis studies, we uncover and characterize unique agonist-like binding poses of mianserin and setiptiline at 5-HT 1e R distinct from similar drug scaffolds in inactive-state 5-HTR structures. Together with computational studies, our data suggest that these binding poses alongside receptor-specific allosteric coupling in 5-HT 1e R and 5-HT 1F R contribute to the agonist activity of these antidepressants.

The farnesoid-x-receptor (FXR) and the G protein bile acid activated receptor (GPBAR)1 are two bile acid activated receptors highly expressed in entero-hepatic, immune, adipose and cardiovascular tissues. FXR and GPBAR1 are clinically validated targets in the treatment of metabolic disorders and FXR agonists are currently trialled in patients with non-alcoholic steato-hepatitis (NASH). Results of these trials, however, have raised concerns over safety and efficacy of selective FXR ligands suggesting that the development of novel agent designed to impact on multiple targets might have utility in the treatment of complex, multigenic, disorders. Harnessing on FXR and GPBAR1 agonists, several novel hybrid molecules have been developed, including dual FXR and GPBAR1 agonists and antagonists, while exploiting the flexibility of FXR agonists toward other nuclear receptors, dual FXR and peroxisome proliferators-activated receptors (PPARs) and liver-X-receptors (LXRs) and Pregnane-X-receptor (PXR) agonists have been reported. In addition, modifications of FXR agonists has led to the discovery of dual FXR agonists and fatty acid binding protein (FABP)1 and Leukotriene B4 hydrolase (LTB4H) inhibitors. The GPBAR1 binding site has also proven flexible to accommodate hybrid molecules functioning as GPBAR1 agonist and cysteinyl leukotriene receptor (CYSLTR)1 antagonists, as well as dual GPBAR1 agonists and retinoid-related orphan receptor (ROR)γt antagonists, dual GPBAR1 agonist and LXR antagonists and dual GPBAR1 agonists endowed with inhibitory activity on dipeptidyl peptidase 4 (DPP4). In this review we have revised the current landscape of FXR and GPBAR1 based hybrid agents focusing on their utility in the treatment of metabolic associated liver disorders., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Background and Aim: Drug-induced liver injury (DILI) is a common disorder that involves both direct liver cell toxicity and immune activation. The bile acid receptor, G-protein-coupled bile acid receptor 1 (GPBAR1; Takeda G-protein-coupled receptor 5 [TGR5]), and cysteinyl leukotriene receptor (CYSLTR) 1 are G-protein-coupled receptors activated by bile acids and leukotrienes, exerting opposite effects on cell-to-cell adhesion, inflammation, and immune cell activation. To investigate whether GPBAR1 and CYSLTR1 mutually interact in the development of DILI, we developed an orally active small molecule, CHIN117, that functions as a GPBAR1 agonist and CYSLTR1 antagonist., Approach and Results: RNA-sequencing analysis of liver explants showed that acetaminophen (APAP) intoxication positively modulates the leukotriene pathway, CYSLTR1, 5-lipoxygenase, and 5-lipoxygenase activating protein, whereas GPBAR1 gene expression was unchanged. In mice, acute liver injury induced by orally dosing APAP (500 mg/kg) was severely exacerbated by Gpbar1 gene ablation and attenuated by anti-Cysltr1 small interfering RNA pretreatment. Therapeutic dosing of wild-type mice with CHIN117 reversed the liver damage caused by APAP and modulated up to 1300 genes, including 38 chemokines and receptors, that were not shared by dosing mice with a selective GPBAR1 agonist or CYSLTR1 antagonist. Coexpression of the two receptors was detected in liver sinusoidal endothelial cells (LSECs), monocytes, and Kupffer cells, whereas combinatorial modulation of CYSLTR1 and GPBAR1 potently reversed LSEC/monocyte interactions. CHIN117 reversed liver damage and liver fibrosis in mice administered CCl 4 ., Conclusions: By genetic and pharmacological approaches, we demonstrated that GPBAR1 and CYSLTR1 mutually interact in the development of DILI. A combinatorial approach designed to activate GPBAR1 while inhibiting CYSLTR1 reverses liver injury in models of DILI., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Compounds featuring a 1,2,4-oxadiazole core have been recently identified as a new chemotype of farnesoid X receptor (FXR) antagonists. With the aim to expand this class of compounds and to understand the building blocks necessary to maintain the antagonistic activity, we describe herein the synthesis, the pharmacological evaluation, and the in vitro pharmacokinetic properties of a novel series of 1,2,4-oxadiazole derivatives decorated on the nitrogen of the piperidine ring with different N-alkyl and N-aryl side chains. In vitro pharmacological evaluation showed compounds 5 and 11 as the first examples of nonsteroidal dual FXR/Pregnane X receptor (PXR) modulators. In HepG2 cells, these compounds modulated PXR- and FXR-regulated genes, resulting in interesting leads in the treatment of inflammatory disorders. Moreover, molecular docking studies supported the experimental results, disclosing the ligand binding mode and allowing rationalization of the activities of compounds 5 and 11 .

Retinoic acid receptor-related orphan receptor γ-t (RORγt) and GPBAR1, a transmembrane G-protein-coupled receptor for bile acids, are attractive drug targets to develop clinically relevant small modulators as potent therapeutics for autoimmune diseases. Herein, we designed, synthesized, and evaluated several new bile acid-derived ligands with potent dual activity. Furthermore, we performed molecular docking and MD calculations of the best dual modulators in the two targets to identify the binding modes as well as to better understand the molecular basis of the inverse agonism of RORγt by bile acid derivatives. Among these compounds, 7 was identified as a GPBAR1 agonist (EC 50 5.9 μM) and RORγt inverse agonist (IC 50 0.107 μM), with excellent pharmacokinetic properties. Finally, the most promising ligand displayed robust anti-inflammatory activity in vitro and in vivo in a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis., Competing Interests: The authors declare the following competing financial interest(s): S. F. and A. Z. have filed a patent application in the name of PRECISION BIO-THERAPEUTICS S.R.L., a spinoff of the University of Perugia, on the same compounds described in this paper., (© 2023 The Authors. Published by American Chemical Society.)

G Protein-Coupled Receptors (GPCRs) are a large family of membrane proteins with pluridimensional signaling profiles. They undergo ligand-specific conformational changes, which in turn lead to the differential activation of intracellular signaling proteins and the consequent triggering of a variety of biological responses. This conformational plasticity directly impacts our understanding of GPCR signaling and therapeutic implications, as do ligand-specific kinetic differences in GPCR-induced transducer activation/coupling or GPCR-transducer complex stability. High-resolution experimental structures of ligand-bound GPCRs in the presence or absence of interacting transducers provide important, yet limited, insights into the highly dynamic process of ligand-induced activation or inhibition of these receptors. We and others have complemented these studies with computational strategies aimed at characterizing increasingly accurate metastable conformations of GPCRs using a combination of metadynamics simulations, state-of-the-art algorithms for statistical analyses of simulation data, and artificial intelligence-based tools. This minireview provides an overview of these approaches as well as lessons learned from them towards the identification of conformational states that may be difficult or even impossible to characterize experimentally and yet important to discover new GPCR ligands., 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 Salas-Estrada, Fiorillo and Filizola.)

Fatty acid amide hydrolase (FAAH) plays a key role in the control of cannabinoid signaling and it represents a promising therapeutic strategy for the treatment of a wide range of diseases, including neuropathic pain and chronic inflammation. Starting from kinetics experiments carried out in our previous work for the most potent inhibitor 2-amino-3-chloropyridine amide (TPA14), we have investigated its non-competitive mechanism of action using molecular dynamics, thermodynamic integration and QM-MM/GBSA calculations. The computational studies highlighted the impact of mutations on the receptor binding pockets and elucidated the molecular basis of the non-competitive inhibition mechanism of TPA14, which prevents the endocannabinoid anandamide (AEA) from reaching its pro-active conformation. Our study provides a rationale for the design of non-competitive potent FAAH inhibitors for the treatment of neuropathic pain and chronic inflammation.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are two highly prevalent human diseases caused by excessive fat deposition in the liver. Although multiple approaches have been suggested, NAFLD/NASH remains an unmet clinical need. Here, we report the discovery of a novel class of hybrid molecules designed to function as cysteinyl leukotriene receptor 1 (CysLT 1 R) antagonists and G protein bile acid receptor 1 (GPBAR1/TGR5) agonists for the treatment of NAFLD/NASH. The most potent of these compounds generated by harnessing the scaffold of the previously described CystLT 1 R antagonists showed efficacy in reversing liver histopathology features in a preclinical model of NASH, reshaping the liver transcriptome and the lipid and energy metabolism in the liver and adipose tissues. In summary, the present study described a novel orally active dual CysLT 1 R antagonist/GPBAR1 agonist that effectively protects against the development of NAFLD/NASH, showing promise for further development., Competing Interests: The authors declare the following competing financial interest(s): SF and AZ have filed the Italian patent application no. 102020000019210 and the corresponding PCT application (PCT/IB 2021/057,131) in the name of PRECISION BIO-THERAPEUTICS S.R.L, a spinoff of the University of Perugia, on same the compounds described in this paper. The remaining 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 Fiorucci, Rapacciuolo, Fiorillo, Roselli, Marchianò, Di Giorgio, Bordoni, Bellini, Cassiano, Conflitti, Catalanotti, Limongelli, Sepe, Biagioli and Zampella.)

The angiotensin-converting enzyme II (ACE2) is a key molecular player in the regulation of vessel contraction, inflammation, and reduction of oxidative stress. In addition, ACE2 has assumed a prominent role in the fight against the COVID-19 pandemic-causing virus SARS-CoV-2, as it is the very first receptor in the host of the viral spike protein. The binding of the spike protein to ACE2 triggers a cascade of events that eventually leads the virus to enter the host cell and initiate its life cycle. At the same time, SARS-CoV-2 infection downregulates ACE2 expression especially in the lung, altering the biochemical signals regulated by the enzyme and contributing to the poor clinical prognosis characterizing the late stage of the COVID-19 disease. Despite its important biological role, a very limited number of ACE2 activators are known. Here, using a combined in silico and experimental approach, we show that ursodeoxycholic acid (UDCA) derivatives work as ACE2 activators. In detail, we have identified two potent ACE2 ligands, BAR107 and BAR708, through a docking virtual screening campaign and elucidated their mechanism of action from essential dynamics of the enzyme observed during microsecond molecular dynamics calculations. The in silico results were confirmed by in vitro pharmacological assays with the newly identified compounds showing ACE2 activity comparable to that of DIZE, the most potent ACE2 activator known so far. Our work provides structural insight into ACE2/ligand-binding interaction useful for the design of compounds with therapeutic potential against SARS-CoV-2 infection, inflammation, and other ACE2-related diseases.

G-protein-coupled receptors (GPCRs) are the molecular target of 40% of marketed drugs and the most investigated structures to develop novel therapeutics. Different members of the GPCRs superfamily can modulate the same cellular process acting on diverse pathways, thus representing an attractive opportunity to achieve multitarget drugs with synergic pharmacological effects. Here, we present a series of compounds with dual activity toward cysteinyl leukotriene receptor 1 (CysLT 1 R) and G-protein-coupled bile acid receptor 1 (GPBAR1). They are derivatives of REV5901─the first reported dual compound─with therapeutic potential in the treatment of colitis and other inflammatory processes. We report the binding mode of the most active compounds in the two GPCRs, revealing unprecedented structural basis for future drug design studies, including the presence of a polar group opportunely spaced from an aromatic ring in the ligand to interact with Arg79 2.60 of CysLT 1 R and achieve dual activity.

The severe acute respiratory syndrome (SARS)-CoV-2 is the pathogenetic agent of Corona Virus Induced Disease (COVID)19. The virus enters the human cells after binding to the angiotensin converting enzyme (ACE)2 receptor in target tissues. ACE2 expression is induced in response to inflammation. The colon expression of ACE2 is upregulated in patients with inflammatory bowel disease (IBD), highlighting a potential risk of intestinal inflammation in promoting viral entry in the human body. Because mechanisms that regulate ACE2 expression in the intestine are poorly understood and there is a need of anti-SARS-CoV-2 therapies, we have settled to investigate whether natural flavonoids might regulate the expression of Ace2 in intestinal models of inflammation. The results of these studies demonstrated that pelargonidin activates the Aryl hydrocarbon Receptor (AHR) in vitro and reverses intestinal inflammation caused by chronic exposure to high fat diet or to the intestinal braking-barrier agent TNBS in a AhR-dependent manner. In these two models, development of colon inflammation associated with upregulation of Ace2 mRNA expression. Colon levels of Ace2 mRNA were directly correlated with Tnf-α mRNA levels. Molecular docking studies suggested that pelargonidin binds a fatty acid binding pocket on the receptor binding domain of SARS-CoV-2 Spike protein. In vitro studies demonstrated that pelargonidin significantly reduces the binding of SARS-CoV-2 Spike protein to ACE2 and reduces the SARS-CoV-2 replication in a concentration-dependent manner. In summary, we have provided evidence that a natural flavonoid might hold potential in reducing intestinal inflammation and ACE2 induction in the inflamed colon in a AhR-dependent manner., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The primary cilium is a microtubule-based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X-linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin-proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G-protein-coupled receptor (GPCR)-cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2-UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non-phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

The coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by the severe acute respiratory syndrome coronavirus (SARS)-CoV-2. In light of the urgent need to identify novel approaches to be used in the emergency phase, we have embarked on an exploratory campaign aimed at repurposing natural substances and clinically available drugs as potential anti-SARS-CoV2-2 agents by targeting viral proteins. Here we report on a strategy based on the virtual screening of druggable pockets located in the central β-sheet core of the SARS-CoV-2 Spike's protein receptor binding domain (RBD). By combining an in silico approach and molecular in vitro testing we have been able to identify several triterpenoid/steroidal agents that inhibit interaction of the Spike RBD with the carboxypeptidase domain of the Angiotensin Converting Enzyme (ACE2). In detail, we provide evidence that potential binding sites exist in the RBD of the SARS CoV-2 Spike protein and that occupancy of these pockets reduces the ability of the RBD to bind to the ACE2 consensus in vitro . Naturally occurring and clinically available triterpenoids such as glycyrrhetinic and oleanolic acids, as well as primary and secondary bile acids and their amidated derivatives such as glyco-ursodeoxycholic acid and semi-synthetic derivatives such as obeticholic acid reduces the RBD/ACE2 binding. In aggregate, these results might help to define novel approaches to COVID-19 based on SARS-CoV-2 entry inhibitors., (Copyright © 2020 Carino, Moraca, Fiorillo, Marchianò, Sepe, Biagioli, Finamore, Bozza, Francisci, Distrutti, Catalanotti, Zampella and Fiorucci.)

This study focuses on the parasitic associations of mites and ticks infesting reptiles and amphibians through a multifocal approach. Herein, reptiles (n= 3,596) and amphibians (n= 919) were examined to ensure representativeness of the Brazilian herpetofauna megadiversity. The overall prevalence was calculated to better understand which were the preferred hosts for each order of Acari (Trombidiformes, Mesostigmata and Ixodida), as well as to determine which orders frequently parasitize reptiles and amphibians in Brazil, and their host specificity. Infestation rates were calculated [prevalence, mean intensity (MI) and mean abundance (MA)] for each order and species, determining which mites and ticks are more likely to be found parasitizing the ectothermic tetrapod fauna. Parasitic niches and preferred locations were recorded to help identify specific places exploited by different Acari, and to determine the host-parasite adaptations, specificity, and relationships in terms of co-evolution. In total 4,515 reptiles and amphibians were examined, of which 170 specimens were infested by mites and ticks (overall prevalence of 3.8%). Trombidiformes mites were prevalent in lizards (55.3%), followed by Ixodida on snakes (24.7%). Mesostigmata mites were the less prevalent, being identified only on Squamata reptiles (4.3% on snakes, 2.4% on lizards). In amphibians, Ixodida ticks were the most prevalent (63.2%), followed by Trombidiformes (34.6%), and lastly Oribatida (2%). From the 13 species of Trombidiformes identified, Eutrombicula alfreddugesi (19.9%) was the most abundant in terms of number of host species and infested individuals. Specimens of Ixodida, yet more common, showed low preferred locations and different values of infestation rates. Co-infestations were recorded only on snakes. Lizard mites generally adhered to the ventral celomatic area (Pterygosomatidae), and some species to the pocket-like structures (Trombiculidae). Lizards, at variance from snakes, have adapted to endure high parasitic loads with minimum effects on their health. The high number of mites recorded in the digits of toads (Cycloramphus boraceiensis, Corythomantis greening, Cycloramphus dubius, Leptodactylus latrans, Melanophryniscus admirabilis) could lead to avascular necrosis. Frogs were often infested by Hannemania larvae, while Rhinella toads were likely to be infested by Amblyomma ticks. Of note, Rhinella major toad was found infested by an oribatid mite, implying first a new parasitic relationship. The effect of high parasitic loads on critically endangered species of anurans deserves further investigation. Our results add basic knowledge to host association of mites and ticks to Brazilian reptiles and amphibians, highlighting that routine ectoparasite examination is needed in cases of quarantine as well as when for managing reptiles and amphibians in captivity given the wide diversity of Acari on the Brazilian ectothermic tetrapod fauna., Competing Interests: Declaration of competing interest "Host association of mites and ticks to reptiles and amphibians in Brazil”. This manuscript has not been submitted or accepted for publication elsewhere. There are no financial or other relationships that might lead to a conflict of interest. All authors have approved the manuscript and have contributed significantly to the work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: A transcanal endoscopic infracochlear surgical approach to the internal auditory canal (IAC) in a human temporal bone model has previously been described. However, the proportion of patients with favorable anatomy for this novel surgical technique remains unknown. Herein, we perform a quantitative analysis of the transcanal endoscopic infracochlear corridor to the IAC based on computed tomography., Materials and Methods: High resolution computed tomography scans of adult temporal bones were measured to determine the accessibility of the IAC when using an endoscopic transcanal, cochlear-sparing surgical corridor., Results: This approach to the IAC was feasible in 92% (35 of 38) specimens based on a minimum distance of 3mm between the basilar turn of the cochlear and the great vessels (jugular bulb and carotid artery)., Conclusions: Infracochlear access to the IAC is feasible in the majority of adult temporal bones and has implications for future hearing preservation drug delivery approaches to the IAC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Perceptual performance in persons with hearing loss, especially those using devices to restore hearing, is not fully predicted by traditional audiometric measurements designed to evaluate the status of peripheral function. The integrity of auditory brainstem synapses may vary with different forms of hearing loss, and differential effects on the auditory nerve-brain interface may have particularly profound consequences for the transfer of sound from ear to brain. Loss of auditory nerve synapses in ventral cochlear nucleus (VCN) has been reported after acoustic trauma, ablation of the organ of Corti, and administration of ototoxic compounds. The effects of gradually acquired forms deafness on these synapses are less well understood. We investigated VCN gross morphology and auditory nerve synapse integrity in DBA/2J mice with early-onset progressive sensorineural hearing loss. Hearing status was confirmed using auditory brainstem response audiometry and acoustic startle responses. We found no change in VCN volume, number of macroneurons, or number of VGLUT1-positive auditory nerve terminals between young adult and older, deaf DBA/2J. Cell-type specific analysis revealed no difference in the number of VGLUT1 puncta contacting bushy and multipolar cell body profiles, but the terminals were smaller in deaf DBA/2J mice. Transmission electron microscopy confirmed the presence of numerous healthy, vesicle-filled auditory nerve synapses in older, deaf DBA/2J mice. The present results suggest that synapses can be preserved over a relatively long time-course in gradually acquired deafness. Elucidating the mechanisms supporting survival of central auditory nerve synapses in models of acquired deafness may reveal new opportunities for therapeutic intervention., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The morphological aspects of male reproductive tract, spermiogenesis and spermatozoa are typical for each species and reflect its evolution, establishing a unique source of characters, which has been used to help solve phylogenetic problems. In Hypanthidium foveolatum the reproductive tract is composed of the testes comprising 28 testicular tubules, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. The differentiation of spermatids occurs within cysts of up to 128 germ line cells each one. During the early spermatid phase, the nucleus resembles that of somatic cells. There follows a gradual chromatin condensation with an increase in nuclear electron density. In the spermatozoon, the nucleus contains heterogeneous chromatin with a loose appearance. The acrosome, shaped with the active participation of the Golgi complex, shows an electron-dense perforatorium involved by four electron-lucent acrosomal vesicle projections. The sperm tail presents an axoneme with a 9+9+2 microtubule pattern and two mitochondrial derivatives, which appear with different sizes. A dense crystalloid is formed initially in the mitochondrial matrix of the large derivative. The mitochondrial derivatives' differentiation occurs concomitantly with an axoneme outgrowth. The centriolar adjunct is observed near the axoneme, anterior to the smaller mithocondrial derivative and exhibits an approximately triangular shape in cross-sections. Microtubules were observed around the head region and flagellar components during spermiogenesis.

The three Pegoscapus species present the same internal reproductive tract features comprising testes with a single testicular tubule, seminal vesicles, vasa deferentia, accessory glands and an ejaculatory duct. The seminal vesicle shows two morphologically distinct portions although they do not resemble the separate chambers found in other Chalcidoidea. The anterior portion of the seminal vesicle shows a prominent epithelium and stores the mature spermatozoa, while the posterior region is formed by a thicker muscular sheath that participates on ejaculation. The sexual maturation in Pegoscapus is achieved at emergence, when the testicular degeneration occurs. The spermatozoa of Pegoscapus reveal a basic structure similar to that of other Chalcidoidea. In Pegoscapus sp1. and Pegoscapus sp2. they present the same features, whereas Pegoscapus tonduzi comprises some different characteristics. It measures approximately 160 microm in Pegoscapus sp1. and Pegoscapus sp2., while in P. tonduzi the spermatozoa measure about 360 microm. The extracellular sheath thickness is another difference among the species. While Pegoscapus sp1. and Pegoscapus sp2. show a thick extracellular sheath, in P. tonduzi this sheath is very thin resulting in a large space intervening between the extracellular sheath and the nucleus. Despite these differences, the three species analyzed share some characteristics that allow the establishment of an identity to the spermatozoon of the genus Pegoscapus: the seminal vesicle not divided in chambers; the absence of acrosomal structures in the spermatozoa; the length of the extracellular sheath; the central microtubules being the firsts to terminate in the sequence of microtubular cutoff at the final axonemal portion.

The spermatozoa in Halictidae are sometimes observed in spermatodesmata in the seminal vesicle. They are linear, long, slender and their lengths vary from 213 microm to about 330 pm. The head region consists in the anterior acrosomal complex, formed by a conical acrosomal vesicle that shows an inner paracrystalline perforatorium extending into the nucleus. The nucleus, measuring about 16 microm to 46 microm, is linear and strongly electron-dense, however some electron-lucent lacunae with electron-dense granules homogeneously organized were observed. The nucleus is attached to the flagellum by the centriolar adjunct, which is compact and electron-dense. It begins at the nuclear base and finishes just above the smaller mitochondrial derivative. The flagellum consists of two mitochondrial derivatives, an axoneme and two accessory bodies. Halictidae have an axoneme with 9+9+2 microtubule pattern which gradually disorganizes towards the final region. The mitochondrial derivatives are asymmetric in both length and diameter and only the larger presents the paracrystalline region. The typical pattern for Halictidae spermatozoa here described may provide useful additional information for future phylogenetic analysis of the superfamily Apoidea.