The Key Role of Lysosomal Protease Cathepsins in Viral Infections.

Cathepsins encompass a family of lysosomal proteases that mediate protein degradation and turnover. Although mainly localized in the endolysosomal compartment, cathepsins are also found in the cytoplasm, nucleus, and extracellular space, where they are involved in cell signaling, extracellular matrix assembly/disassembly, and protein processing and trafficking through the plasma and nuclear membrane and between intracellular organelles. Ubiquitously expressed in the body, cathepsins play regulatory roles in a wide range of physiological processes including coagulation, hormone secretion, immune responses, and others. A dysregulation of cathepsin expression and/or activity has been associated with many human diseases, including cancer, diabetes, obesity, cardiovascular and inflammatory diseases, kidney dysfunctions, and neurodegenerative disorders, as well as infectious diseases. In viral infections, cathepsins may promote (1) activation of the viral attachment glycoproteins and entry of the virus into target cells; (2) antigen processing and presentation, enabling the virus to replicate in infected cells; (3) up-regulation and processing of heparanase that facilitates the release of viral progeny and the spread of infection; and (4) activation of cell death that may either favor viral clearance or assist viral propagation. In this review, we report the most relevant findings on the molecular mechanisms underlying cathepsin involvement in viral infection physiopathology, and we discuss the potential of cathepsin inhibitors for therapeutical applications in viral infectious diseases.

DNA fragmentation and morphometric studies in sperm of stallions supplemented with maca (Lepidium meyenii).

The reproductive performances of livestock play an essential role in the economic management of the farm. The improvement of semen quantity and quality through the use of food supplements that lack substances which are forbidden in animal feeding, or that may have detrimental effects, is an important goal. Maca (Lepidium meyenii) is a plant that has been used for centuries in the Andes for nutrition and fertility enhancement in humans and animals. The aim of this study was to evaluate the effects of food supplementation of stallions with maca during the breeding season on spermatozoa parameters such as DNA fragmentation and shape, which are two predictive indexes of spermatozoa functionality. For this purpose, ejaculate volume, semen gel-free volume, sperm concentration and motility, total sperm count, sperm DNA fragmentation and sperm head parameters (length, width, perimeter, area, shape factor, roughness) were measured in four stallions. Maca food supplementation in stallions during breeding reduced the percentage of spermatozoa with fragmented DNA, increased significantly sperm concentration and exerted an elongation of the spermatozoa head, a condition that is believed to improve spermatozoa functionality, suggesting that food supplementation of maca could be useful in horse breeding during the breeding season.

Heparan Sulfate Proteoglycans in Viral Infection and Treatment: A Special Focus on SARS-CoV-2.

Heparan sulfate proteoglycans (HSPGs) encompass a group of glycoproteins composed of unbranched negatively charged heparan sulfate (HS) chains covalently attached to a core protein. The complex HSPG biosynthetic machinery generates an extraordinary structural variety of HS chains that enable them to bind a plethora of ligands, including growth factors, morphogens, cytokines, chemokines, enzymes, matrix proteins, and bacterial and viral pathogens. These interactions translate into key regulatory activity of HSPGs on a wide range of cellular processes such as receptor activation and signaling, cytoskeleton assembly, extracellular matrix remodeling, endocytosis, cell-cell crosstalk, and others. Due to their ubiquitous expression within tissues and their large functional repertoire, HSPGs are involved in many physiopathological processes; thus, they have emerged as valuable targets for the therapy of many human diseases. Among their functions, HSPGs assist many viruses in invading host cells at various steps of their life cycle. Viruses utilize HSPGs for the attachment to the host cell, internalization, intracellular trafficking, egress, and spread. Recently, HSPG involvement in the pathogenesis of SARS-CoV-2 infection has been established. Here, we summarize the current knowledge on the molecular mechanisms underlying HSPG/SARS-CoV-2 interaction and downstream effects, and we provide an overview of the HSPG-based therapeutic strategies that could be used to combat such a fearsome virus.

Chemical Analysis of Lepidium meyenii (Maca) and Its Effects on Redox Status and on Reproductive Biology in Stallions †.

The present study was conducted to assess the chemical composition of Yellow Maca (Lepidium meyenii) and its biological activity on stallions following oral administration of hypocotyl powder. Maca was subjected to methanolic extraction and the chemical analysis was carried out by LC-MS-QTOF (liquid chromatography-mass spectrometry). Our results showed that Maca contains some effective antioxidants, a high percentage of glucosinolates, and other important components with a high antioxidant capacity. To evaluate the plant biological activity in stallions fed with Maca powder for 60 days, the redox status and some reproductive parameters were investigated. Blood and semen samples were collected at 0, 30, 60, and 90 days from the beginning of this study. Blood samples showed a decrease of the reactive oxygen metabolites, evaluated by d-ROMs test, and an increase of the antioxidant barrier in terms of biological antioxidant potential (BAP test), powerful oxidant capacity (OXY-Adsorbent test), and thiols evaluation (-SHp test). Furthermore, semen samples showed a positive trend during Maca administration in the following parameters: ejaculate volumes and sperm concentrations, total and progressive motility, and acrosome integrity.

Expression of orexin B and its receptor 2 in rat testis.

The peptides orexin A (OxA) and orexin B (OxB) deriving from a common precursor molecule, prepro-orexin, by proteolytic cleavage, bind the two G-coupled OX1 and OX2 receptors. While OX1 selectively binds OxA, OX2 shows similar affinity for both orexins. Firstly discovered in the hypothalamus, orexins and their receptors have been found in other brain regions as well as in peripheral tissues of mammals, thus resulting involved in the regulation of a broad variety of physiological functions. While the functional localization of OxA and OX1 in the mammalian genital tract has been already described, the expression of OxB and OX2 and their potential role in the reproductive functions remain to be explored. Here, we investigated the presence of OxB and OX2 in the rat testis by immunohistochemical and biochemical analyses. The results definitely demonstrated the localization of OxB and OX2 in pachytene and second spermatocytes as well as in spermatids at all stages of the cycle of the seminiferous epithelium. The expression of both OX2 mRNA and protein in the rat testis was also established by RT-PCR and Western blotting, respectively. The analysis of the molecular mechanism of action of OxB in the rat testis showed that OxB, in contrast with OxA, is unable to promote steroidogenesis. These results translate into the regulation of diverse biological actions by OxA and OxB in the male gonad.

Expression and potential role of the peptide orexin-A in prostate cancer.

The peptides orexin-A and orexin-B and their G protein-coupled OX1 and OX2 receptors are involved in multiple physiological processes in the central nervous system and peripheral organs. Altered expression or signaling dysregulation of orexins and their receptors have been associated with a wide range of human diseases including narcolepsy, obesity, drug addiction, and cancer. Although orexin-A, its precursor molecule prepro-orexin and OX1 receptor have been detected in the human normal and hyperplastic prostate tissues, their expression and function in the prostate cancer (PCa) remains to be addressed. Here, we demonstrate for the first time the immunohistochemical localization of orexin-A in human PCa specimens, and the expression of prepro-orexin and OX1 receptor at both protein and mRNA levels in these tissues. Orexin-A administration to the human androgen-dependent prostate carcinoma cells LNCaP up-regulates OX1 receptor expression resulting in a decrease of cell survival. Noteworthy, nanomolar concentrations of the peptide counteract the testosterone-induced nuclear translocation of the androgen receptor in the cells: the orexin-A action is prevented by the addition of the OX1 receptor antagonist SB-408124 to the test system. These findings indicate that orexin-A/OX1 receptor interaction interferes with the activity of the androgen receptor which regulates PCa onset and progression, thus suggesting that orexin-A and its receptor might represent novel therapeutic targets to challenge this aggressive cancer.

G Protein-Coupled Estrogen Receptor (GPER) and ERs Are Modulated in the Testis-Epididymal Complex in the Normal and Cryptorchid Dog.

There is growing evidence by the literature that the unbalance between androgens and estrogens is a relevant condition associated with a common canine reproductive disorder known as cryptorchidism. The role of estrogens in regulating testicular cell function and reproductive events is supposedly due to the wide expression of two nuclear estrogen receptors (ERs), ER-alpha and ER-beta and a trans-membrane G protein-coupled estrogen receptor (GPER) in the testis. In this study, immunohistochemistry, Western blotting and qRT-PCR were used to assess the distribution and expression of GPER in the testis-epididymal complex in the normal and cryptorchid dog. ER-alpha and ER-beta were also evaluated to better characterize the relative abundances of all three receptors. In addition, in these tissues, the expression level of two proteins as SOD1 and Nrf2 normally associated with oxidative stress was investigated to evaluate a possible relationship with ERs. Our data revealed changes in the distribution and expression of the GPER between the normal and cryptorchid dog. In particular, dogs affected by cryptorchidism showed an upregulation of GPER at level of the examined reproductive tract. Also considering the obtained result of a modulation of SOD1 and Nrf2 expression, we could hypothesize the involvement of GPER in the cryptorchid condition. Further studies are, however, necessary to characterize the role of GPER and its specific signaling mechanisms.

Expression of orexin A and its receptor 1 in the human prostate.

The peptides orexin A (OXA) and orexin B, deriving from the cleavage of the precursor molecule prepro-orexin, bind two G-coupled transmembrane receptors, named as receptor 1 (OX1R) and receptor 2 for orexin, showing different affinity-binding properties. First discovered in the rat hypothalamus, orexins and their receptors have been also found in many peripheral tissues where they exert neuroendocrine, autocrine and paracrine functions. Because inconclusive data on their localization in the mammalian prostate are reported, the aim of this study was to investigate the presence of prepro-orexin, OXA and OX1R in the human normal and hyperplastic gland. Immunohistochemistry revealed the localization of both OXA and OX1R in the cytoplasm of the follicular exocrine epithelium of all tested normal and hyperplastic prostates. Positive immunostaining was mainly observed in the basal cells of the stratified epithelium, and only rarely in the apical cells. The expression of mRNAs coding for prepro-orexin and OX1R and of proteins in the tissues was also ascertained by polymerase chain reaction and Western blotting analysis, respectively. In order to gain insights into the functional activity of OXA in the prostate, we administered different concentrations of OXA to cultured prostatic epithelial cells PNT1A. We first demonstrated that PNT1A cells express OX1R. The addition of OXA did not affect PNT1A cell proliferation, while it enhanced cAMP synthesis and Ca(2+) release from intracellular storage. Overall, our results definitely demonstrate the expression of OXA and OX1R in the human prostate, and suggest an active role for them in the metabolism of the gland.

Evaluation of Muscle Proteins for Estimating the Post-Mortem Interval in Veterinary Forensic Pathology.

Postmortem cadaveric changes are commonly used to estimate the postmortem interval (PMI) in humans and animals. However, these modifications have been poorly investigated in animals of interest to veterinary forensic pathology. The aim of this study was to investigate the potential use of muscle proteins (desmin and dystrophin) as biomarkers for estimating the PMI in dogs. For this study, 10 dead adult dogs were evaluated for 4 days in a temperature-controlled room at 19 ± 1 °C. For each animal, at 3, 24, 48, 72, and 96 h after death, a 1 × 1 × 1 cm cube of muscle tissue was removed from the vastus lateralis and triceps brachii. Protein expression levels were analyzed by immunohistochemical examination and immunoblot analysis. The obtained results showed rapid dystrophin degradation, with complete disappearance at 72 h after death. In contrast, desmin-positive fibers and desmin protein bands detected by immunoblot were observed on all 4 days of observation. Our findings suggest the potential use of muscle proteins as biomarkers for estimating the PMI in dogs.

Expression and role of receptor 1 for orexins in seminiferous tubules of rat testis.

Orexins (OxA and OxB) and their receptors (Ox1R and Ox2R), originally detected in the hypothalamus, have also been localized in multiple cerebral areas and peripheral organs. Thus, in addition to their central function in the regulation of food intake, arterial blood pressure, heart rate, sleep/wake cycle, sexual behaviour, arousal, and hypothalamic/hypophyseal axis, these neuropeptides may exert a local action in various peripheral organs and tissues. Emerging evidence suggests a main role of OxA and its highly specific receptor Ox1R in the male genital tract of mammals. We previously demonstrated OxA localization in Sertoli cells and spermatids of rat testis. Here, we show positive stainings of Ox1R in developing spermatocytes, and spermatids of rat testis by immunohistochemistry. The expression of Ox1R mRNA and the protein in the tissue was also established by reverse-transcription polymerase chain reaction and Western blotting respectively. The addition of OxA to fresh testis slices significantly increased testosterone (T) secretion which, conversely, was inhibited by Mullerian inhibiting substance (MIS). The sequential treatment of testis samples with the two substances highlighted an antagonizing activity of OxA versus MIS in regulating T levels. Furthermore, the stimulating effect on T production by OxA was prevented by the addition of the selective Ox1R inhibitor SB-408124. Overall, our findings suggest that locally secreted OxA interacting with Ox1R activates signals which antagonize MIS action in the control of T levels in mammalian testis.

Effect of Aqueous Extract of Maca Addition to an Extender for Chilled Canine Semen.

Antioxidant supplementation has been proposed as a new strategy to improve the long-term preservation of semen. The aim of this study was to evaluate the effect of Maca supplementation of semen extender on quality-related canine semen parameters during cooling. Ejaculates from nine dogs were cooled for 7 days in the absence (control group) or in the presence of 10, 20 and 50 µL/mL of an aqueous extract of Maca. Sperm were evaluated for sperm viability, motility, DNA fragmentation and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage. The addition of 10 µL/mL of Maca preserved sperm DNA and plasma membrane integrity at 3 h and increased sperm curvilinear velocity after 24 h. Treatment with 20 and 50 µL/mL of Maca increased the percentage of hyperactivated sperm after 3 h. Moreover, semen treated with 20 µL/mL of Maca decreased lipid peroxidation at 24 h. A significant reduction of sperm DNA and plasma membrane integrity as well as of kinetics parameters between 3 and 24 h of refrigerated storage with the higher concentration tested was observed. Although Maca was not able to protect canine semen with extended refrigeration storage time, it increased hyperactivation and preserved DNA integrity in short-term storage.

Antioxidants in assisted reproductive technologies: An overview on dog, cat, and horse.

Assisted reproductive technologies (ARTs) are widely used as a tool to improve reproductive performance in both humans and animals. In particular, in the veterinary field, ARTs are used to improve animal genetics, recover endangered animals, and produce offspring in the event of subfertility or infertility in males or females. However, the use of ARTs did not improve the fertilization rate in some animals due to various factors such as the difficulty in reproducing an anatomical and humoral substrate typical of the natural condition or due to the increase in catabolites and their difficult elimination. The in vitro environment allows the production and increase in the concentration of substances, including reactive oxygen species (ROS), which could be harmful to gametes. If produced in high concentration, the ROS becomes deleterious, both in vitro and in vivo systems. It has been seen that the use of antioxidants can help neutralize or counteract the production of ROS. The present study aims to report the latest findings regarding the use of antioxidants in ARTs of some domestic species, such as dogs, cats, and horses, compared to other animal species, such as cattle, in which ARTs have instead developed more widely.

Lepidium meyenii (Maca) in male reproduction.

Lepidium meyenii (Maca) is an edible root plant that grows in the Andean region of Peru. For centuries, the plant has been used as a dietary supplement for its nutritional and therapeutic properties. Maca are rich in high value nutritional elements and secondary metabolites (macaridine, macamides and glucosinolates) with high biological activity. Several studies demonstrated various biological effects of Maca mainly in the field of fertility. The aim of this review is to summarize the state of knowledge on the properties of Maca on male reproduction. Literature data was performed in PubMed with researches published from 2000 to 2019. The research showed results related to the effects of Maca on the quality and quantity of the semen, sexual behaviour and disorders of the male genital tract. Despite the numerous studies carried out on different animal species, further research is needed to clarify the mechanisms of action of Maca.

Assessment of living conditions in wild boars by analysis of oxidative stress markers.

This study demonstrated that it is possible to differentiate wild boars living in habitats with different animal densities by the measurement of oxidative stress markers. Therefore, reactive oxygen metabolites, the antioxidant barrier, i.e., the biological antioxidant potential and the antioxidative power (OXY-Adsorbent), as well as cortisol were measured in freely ranging wild boars. In two different areas of a State Forest in the Campania Region (Italy), 42 freely ranging, managed wild boars were captured with a corral trap, and blood samples were collected. The wild boars were divided by age (>1 year old and <1 year old) and sex (male and female). Animals in one area showed significantly higher values of oxidative stress parameters than those living in the other area. The annual boar censuses of areas highlighted a higher population density in the first area than in the second one, and this would explain the results obtained. Since the health or nutritional status of animals is reflected by oxidative stress, wild boars in areas with higher animal densities may live under worse conditions than animals in areas with lower densities which becomes evident by measuring oxidative stress markers. As cortisol in blood is only reflecting a short period, the measurement of oxidative stress level may be a better indicator to evaluate the living conditions of wild boars.

Effects of Oral Administration of Lepidium meyenii on Morphology of Mice Testis and Motility of Epididymal Sperm Cells After Tetrahydrocannabinol Exposure.

Background: Tetrahydrocannabinol (THC) administration is associated with testicular damage and reduced semen quality. Oral administration of Lepidium Meyenii (maca) improves spermatogenesis and sperm motility and count and reduces spermatogenic damage. Objectives: The aim of this study was to evaluate the effect of administration of THC, maca, and their combination on testicular tissue and semen parameters. Materials and Methods: Thirty-six-week-old male mice were classified into control, THC, Maca, and THC + Maca groups. The mice were subjected to Eco Color Doppler ultrasound examination of the testicles before and after treatment. After euthanasia, the epididymis, testes, liver, and kidney were collected for histological examination. For morphometry of the testis, tubular diameters and seminiferous epithelium height were measured. Sperm concentration and sperm motilities were assessed. Differences among the groups were assessed using the Kruskal-Wallis and Dunn's post-hoc test. Results: In all the groups, there were no significant changes in testicular morphology before and after treatment. Histological assessment of the testes showed no alterations in control, no significant alterations in Maca, mild to moderate alterations in THC, and mild alterations in THC + Maca groups. Histological examination of the other organs showed no significant differences among the groups. Tubular diameter showed significantly increased thickening for THC and THC + Maca compared with that for Maca and control. Moreover, seminiferous epithelium height decreased for THC compared with that in the control, Maca, and THC + Maca groups. No statistically significant reduction in the spermatogenic index was observed for THC compared with that for Maca and THC + Maca. Epididymal cross-sections of the groups showed no significant alterations. Sperm concentration and motility were higher for control and THC + Maca groups than in group THC and Maca. Conclusion: In vivo maca administration reduced the deleterious effect of THC on testicular parenchyma and semen production.

Antimicrobial activity of harzianic acid against Staphylococcus pseudintermedius.

The emerging concern about the increase of antibiotic resistance has encouraged research efforts to develop effective alternatives to counteract bacterial infections. Herein, we studied a new perspective to therapeutic treatment against Staphylococcus pseudintermedius, an opportunistic pathogen documented as the major cause of skin, ear, and post-operative bacterial infections in dogs and cats. Antimicrobial activity of secondary metabolites produced by selected microbial strains belonging to Trichoderma, Talaromyces, Clonostachys and Coniothyrium fungal genera has been tested against S. pseudintermedius. Several extracts, particularly those obtained from Trichoderma harzianum E45 and ET45, showed a significant antimicrobial activity towards S. pseudintermedius methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains. Bioassay-guided fractionation of E45 and ET45 extracts allowed to isolate harzianic acid as the major compound responsible for biological activities (e.g. antimicrobial, antibiofilm formation and biofilm disaggregation).

Integrin receptors play a role in the internalin B-dependent entry of Listeria monocytogenes into host cells.

Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host's E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain (DeltainlA) carrying a deletion in the gene coding for inlA. The DeltainlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-beta(3)- and anti-beta(1)-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-beta(2)- and anti-beta(4)-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial uptake, showing different potencies correlated to their selective affinity for the beta(3)- and beta(1)-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes DeltainlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings provide the first evidence that beta(3)- and beta(1)-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells.

Human-Animal Relationship Dysfunction: A Case Study of Animal Hoarding in Italy.

"Animal hoarding" or "compulsive hoarding of animals" is a psychiatric disease, which has important social implications and a profound influence on animal welfare. To date, this phenomenon has been little investigated and largely unexplored. The present study aims to systematically describe a case of animal hoarding, which remains unresolved. The report refers to a case of a woman suffering from animal hoarding that emerged in 2005. From March 2014 to December 2019, 450 animals were seized over nine different occasions. This disease had significant implications on the welfare of the animals collected, which lived in poor housing and hygiene conditions that frequently led to their death. Since animal hoarding cases involve sanitary, legal, and veterinary aspects, we believe that a multidisciplinary approach is necessary in order to prevent a recurrence and a new accumulation of animals. A holistic approach should be taken according to the One Health principle that involves different stakeholders at every level in order to adopt an efficient solution.

Expression of the serotonin transporter SERT in the genital tract of cattle.

Both prostate and vestibular glands of mammals contain neuroendocrine cells which synthesize, store and release growth factors including neuropeptides and biogenic amines such as serotonin. An increase of the secretory products by these cells has been correlated to tumour progression and poor prognosis. Serotonin mediates a wide range of physiological functions by binding to multiple receptors on cell surface. However, the entire serotonergic system is mainly regulated by the serotonin transporter SERT which modulates serotonin concentration in extracellular fluid. Primarily located in serotonergic neurons, SERT is also expressed in various cell types in the periphery. In this study, we found a wide distribution of SERT in the parenchymal cells of both the prostate and the vestibular glands of cattle using immunohistochemistry. The expression of SERT mRNA transcripts was assessed by reverse-transcription polymerase chain reaction, thus suggesting that SERT is locally synthesized. Furthermore, Western blotting analysis showed the presence of two isoforms of the protein (70 and 140 kDa), probably corresponding to the high mannose-type SERT and its dimeric form. Our results provide the first evidence for SERT expression in the mammalian genital tract, thus highlighting a new potential target for the therapy of the genital tract cancers.